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WO2024240848A1 - Ldlr derived polypeptides for anti-viral uses - Google Patents

Ldlr derived polypeptides for anti-viral uses Download PDF

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Publication number
WO2024240848A1
WO2024240848A1 PCT/EP2024/064152 EP2024064152W WO2024240848A1 WO 2024240848 A1 WO2024240848 A1 WO 2024240848A1 EP 2024064152 W EP2024064152 W EP 2024064152W WO 2024240848 A1 WO2024240848 A1 WO 2024240848A1
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egfa
la6x
la4x
ldlr
egfbx
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Ali MIRAZIMI
Moritz HORN
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Jlp Health GmbH
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Jlp Health GmbH
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Priority claimed from EP23174811.2A external-priority patent/EP4467563A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the present invention relates to an LDLR derived polypeptide that binds a Bunyavirales virus and is thus useful in the prophylaxis and/or treatment of a Bunyavirales virus infection in a subject, preferably a CCHV infection.
  • LDLR derived polypeptides comprising an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain, which bind a Bunyavirales virus.
  • LDLR derived polypeptides comprising a multimer of an LDLR ectodomain (SEQ ID NO 18), or of a functional fragment of said LDLR ectodomain, or of an amino acid variant of said LDLR ectodomain, or of an amino acid variant of said functional fragment of said LDLR ectodomain, wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said monomers.
  • the LDLR polypeptides here described are particularly useful against Bunyavirales virus infections. Further, the LDLR polypeptides can be used for diagnostic detection of Bunyavirales virus infection, particularly CCHFV infection, and quality control testing of related vaccines. A method to identify new gene factors involved in an infection by a Bunyavirales virus is also provided herein.
  • CHFV Crimean-Congo hemorrhagic fever virus
  • CCHF Crimean-Congo hemorrhagic fever
  • CCHF is transmitted by Hyalomma ticks and through direct contact with the blood and other bodily fluids of patients or infected animals.
  • the CCHF incubation period is 2-14 days and correlates with the type of transmission and viral load.
  • the clinical manifestations can be from subclinical illness to acute infection with haemorrhage such as pulmonary haemorrhage, intra-abdominal bleeding, or haematuria, and multiorgan failure.
  • CCHF chronic myemorrhagic fever
  • CCHFV CCHFV is the most widespread haemorrhagic fever, being endemic in certain regions of Africa and in Asia but also in Europe.
  • CCHFV is a tick-borne pathogen that is also capable of interpersonal transmission.
  • CCHFV CCHFV complex protein kinase
  • CCHFV enters cells through clathrin-mediated endocytosis and uses the endosomal pathway to release viral RNA segments.
  • CCHFV enters cells through clathrin-mediated endocytosis and uses the endosomal pathway to release viral RNA segments.
  • many different cell types can be infected with CCHFV, suggesting the existence of either a widely distributed receptor or several redundant entry receptors.
  • nucleoside analogue ribavirin is the only direct-acting anti-viral that has been widely used clinically in patients with CCHF.
  • ribavirin is controversial, with continued debate about whether treatment improves outcome.
  • antibody-based therapies have also been evaluated for treatment of CCHFV, however large- scale trials have not been performed so far.
  • RNA viruses tend to generate vaccine-resistant escaper strains through mutations of antibody-binding sites.
  • a host-related decoy receptor such as a LDLR-derived polypeptide as disclosed herein would require a gain-of-function mutation switching to another host cell receptor.
  • Such a mutation is less likely to appear so that there is a decreased risk of generating resistant virus strains in response to treatment with a LDLR- derived polypeptide according to this invention compared to vaccine approaches.
  • LDLR polypeptides comprising soluble LDL receptors, functional fragments thereof, or single amino acid variants thereof, which can be used for preventing and/or treating a Bunyavirales virus infection, preferably a CCHFV infection.
  • a method to identify new factors involved in a CCHFV infection is also provided herein.
  • an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain.
  • an amino acid variant of an LDLR ectodomain refers to a single amino acid variant of an LDLR ectodomain. In some embodiments, an amino acid variant of an LDLR ectodomain refers to a multiple amino acid variant of an LDLR ectodomain.
  • a preferred embodiment of present invention relates to LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection in a human, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain.
  • said LDLR derived polypeptide for use comprises an LDLR ectodomain having amino acid sequence SEQ ID NO 18, or a functional fragment thereof, or an amino acid variant thereof.
  • said LDLR derived polypeptide for use comprises a dimer, a trimer, or a multimer of an LDLR ectodomain monomer having amino acid sequence SEQ D NO 18, or of a functional fragment thereof, or of an amino acid variant thereof. More preferred said LDLR derived polypeptide comprises a dimer or trimer, still more preferred a dimer, still more preferred is a homodimer or a homotrimer, and still more preferred is a homodimer.
  • said LDLR derived polypeptide for use comprises a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41 ), OLSD (SEQ ID NO 36), LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA1 - LA2-LA3-LA4
  • an amino acid variant of a functional fragment of an LDLR ectodomain refers to a single amino acid variant of a functional fragment of an LDLR ectodomain. In some embodiments, an amino acid variant of an LDLR ectodomain refers to a multiple amino acid variant of a functional fragment of an LDLR ectodomain.
  • said LDLR derived polypeptide for use further comprises a peptide linker connecting said fragments or monomers.
  • said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava-Belgrade hantavirus (DOBV), Haantan virus (HTNV), Website
  • CATV Longquan hantavirus
  • LQUV Longquan hantavirus
  • PUUV Puumala hantavirus
  • SANGV Sangassou hantavirus
  • SEOV Sin Nombre hantavirus
  • SNV Sin Nombre hantavirus
  • TAAIV Tula hantavirus
  • NW New York hantavirus
  • RVFV Rift Valley fever virus
  • NSDV Sandfly Fever Naples virus
  • said Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
  • said LDLR derived polypeptide for use comprises an LDLR ectodomain having amino acid sequence SEQ ID NO 18, and said Bunyavirales virus is CCHFV.
  • a further embodiment of the invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising the LDLR derived polypeptide for use described above, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent.
  • Another embodiment of the invention is directed to a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an LDLR derived polypeptide for use described above.
  • a particular embodiment of the invention is directed to an LDLR derived polypeptide comprising a functional fragment of an LDLR ectodomain selected from the group comprising or consisting of:
  • LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41 ), OLSD (SEQ ID NO 36), LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA1 - LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA1 - LA2-
  • LA2-LA3-LA4X-LA5-LA6X-LA7 LA2-LA3-LA4X-LA5-LA6, LA2-LA3-LA4X- LA5, LA2-LA3-LA4, LA2-LA3, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX- EGFCX-OLSD, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFB, LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFB, LA3-LA4X-LA5-LA6X-LA7-EGFA, LA3-LA4X-LA5-LA6X-LA7, LA3-LA4X-LA5-LA6X-LA7, LA3-LA
  • a further particular embodiment of the invention is directed to LDLR derived polypeptide comprising a multimer of monomers, wherein each monomer is selected from the group comprising: an LDLR ectodomain (SEQ ID NO 18), a functional fragment of said LDLR ectodomain, an amino acid variant of said LDLR ectodomain, an amino acid variant of said functional fragment of said LDLR ectodomain, and wherein said multimer comprises between 2 and 8 monomers, preferably 2 or 3 monomers, and wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said monomers; and wherein said multimer is a homomultimer or a heteromultimer.
  • a more particular embodiment of the invention is directed to an LDLR derived polypeptide comprising a functional fragment of an LDLR ectodomain as described above, or comprising a multimer of monomers as described above, wherein said LDLR derived polypeptide is for use in the prophylaxis and/or treatment of a virus infection, wherein said virus is preferably a Bunyavirales virus.
  • a further particular embodiment of the invention is directed to a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an LDLR derived polypeptide comprising a functional fragment of an LDLR ectodomain or comprising a multimer of monomers as described above.
  • a particular embodiment of the invention is directed to an in vitro screening method to identify a gene that modulates a Bunyavirales virus infection, the method comprising:
  • step B) performing chemical random mutagenesis in said cells of step A) in order to obtain mutagenized cells
  • step B) infecting the mutagenized cells of step B) with a Bunyavirales virus or with a Bunyavirales virus pseudotyped RNA viral particle;
  • step D culturing the infected mutagenized cells of step C) to obtain resistant colonies; E) isolating the resistant colonies of step D);
  • step E) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate a Bunyavirales virus infection
  • the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof
  • the pseudotyped RNA viral particle is a HIV particle or a MSV particle or a VSV particle, wherein the pseudotyped RNA viral particle express a Bunyavirales virus specific glycoprotein Gc and/or Gn
  • the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hant
  • whole exome sequencing refers to the sequencing of all exons in a specific cell or tissue type, wherein the exons are the protein coding regions of a gene. Therefore, in “whole exome sequencing” only the protein-coding DNA regions are sequenced as compared to whole genome sequencing.
  • whole transcriptome sequencing refers to sequencing of expressed transcripts at the mRNA level to obtain sequence information on all mRNAs made in a cell or cell pool at a given time
  • the pseudotyped RNA viral particle is preferably a VSV particle.
  • performing chemical random mutagenesis of step B) comprises incubating the haploid cells with N-Ethyl-N-nitrosourea (ENU).
  • the inventors were able to identify host factors important for CCHFV infections by using a novel method based on haploid murine stem cells mutagenized with N- Ethyl-N-nitrosourea (ENU), which induces single nucleotide variants into the genome, and subsequent infection with a CCHFV glycoprotein pseudotyped vesicular stomatitis virus (VSV-CCHF_G) ( Figure 1).
  • ENU N- Ethyl-N-nitrosourea
  • VSV-CCHF_G CCHFV glycoprotein pseudotyped vesicular stomatitis virus
  • Haploid cells containing only a single genomic copy allow for direct translation of the introduced genetic changes such as gene knockouts and point mutations to a respective phenotype.
  • LDLR Low Density Lipoprotein Receptor
  • Table 2 Experiments using a soluble LDLR, ldlr knockout cells, and both a laboratory strain and a patient isolate of CCHFV, confirmed that LDLR mediates CCHFV infection ( Figure 2 and 4). Furthermore, experiments based on Bioluminescence Resonance Energy Transfer (BRET) technique confirmed that LDLR is a receptor for CCHFV, as the viral glycoprotein Gc directly attaches to LDLR ( Figures 3 and 8).
  • BRET Bioluminescence Resonance Energy Transfer
  • the low-density lipoprotein receptor gene family encodes for a class of structurally related cell surface receptors also named low density lipoprotein receptor-related proteins (LRP) that fulfil diverse biological functions in different organs, tissues, and cell types, including the regulation of lipid metabolism, protection against atherosclerosis, neurodevelopment, and transport of nutrients and vitamins.
  • LRP low density lipoprotein receptor-related proteins
  • LDLR low-density lipoprotein receptor
  • VLDLR very low-density lipoprotein receptor
  • ApoER2 also known as low density lipoprotein receptor-related protein- 8, LRP8;
  • LRP4 low density lipoprotein receptor-related protein 4, LRP4, also known as multiple epidermal growth factor (EGF) repeat-containing protein, MEGF7;
  • LRP1 LDLR-related protein 1
  • LRP1 b LDLR-related protein 1b
  • LDL is one of the major cholesterol-carrying lipoprotein of plasma, acting to regulate cholesterol homeostasis in mammalian cells.
  • the LDL receptor binds and transports LDL particles into cells by acidic endocytosis. Once inside the cell, the LDLR separates from its ligand, which is degraded in the lysosomes, while the receptor returns to the cell surface.
  • the internal dissociation of the LDLR with its ligand is mediated by proton pumps within the walls of the endosome that lower the pH.
  • the LDLR is a multi-domain protein, containing the following five domains:
  • LA repeats 40-amino acid LDLR class A cysteine-rich repeats, also named LA repeats, each of which contains a coordinated calcium ion and six cysteine residues involved in disulphide bond formation.
  • LA1 40-amino acid LDLR class A cysteine-rich repeats
  • LA7 a ligand-binding domain containing seven 40-amino acid LDLR class A cysteine-rich repeats, also named LA repeats, each of which contains a coordinated calcium ion and six cysteine residues involved in disulphide bond formation.
  • LDLR class B repeats LB1 , LB2, LB2, LB3, LB4, LB5, and LB6
  • EGFC EGF repeat
  • the six LDLR class B repeats each contain a conserved YWTD motif and are predicted to form a [3-propeller structure, and are thus referred herein with the term "PROP”. This region is critical for ligand release and recycling of the receptor.
  • LDLR ectodomain module or "LDLR module” or simply “module” is used herein to refer to the individual repeats and domains: LA1 , LA2, LA3, LA4, LA5, LA6, LA7, EGFA, EGFB, LB1 , LB2, LB2, LB3, LB4, LB5, LB6, PROP, EGFC, and OLSD.
  • amino sequences of the "LDLR ectodomain modules" used in the present invention are:
  • SEQ ID NO 36 OLSD Clustered O-linked oligosaccharides
  • Soluble low-density lipoprotein receptor is the circulating LDLR ectodomain of transmembrane LDLR comprising the domains I) - III) described above, i.e. the LDLR derived polypeptide consisting of LA1-LA7 repeats, EGFA, EGFB, PROP, EGFC, and OLSD.
  • LDLR ectodomain refers to the soluble form of the low-density lipoprotein receptor (sLDLR), comprising the "modules" LA1-LA7,
  • LDLR ectodomain refers to the soluble form of the low-density lipoprotein receptor (sLDLR) that binds a Bunyavirales virus, and which comprises LA1-LA7, EGFA,
  • the soluble "LDLR ectodomain” corresponds to the extracellular portion of 766 amino acid residues of the LDL receptor, which is purified to homogeneity from impurities. More in detail, the soluble "LDLR ectodomain” corresponds to the extracellular portion of mature LDL receptor, which has an amino acid sequence SEQ ID NO 18 corresponding to amino acid residue Ala22 to Arg788 of the mature LDL receptor as deposited at Uniprot ID P01130.
  • LDLR derived polypeptide is used herein to refer to the following polypeptides (also referred to as “polypeptides of the disclosure”): an "LDLR ectodomain” having amino acid sequence SEQ ID NO 18; a “functional fragment” of said "LDLR ectodomain” having amino acid sequence SEQ ID NO 18; a “single or multiple amino acid variant” of said "LDLR ectodomain” having amino acid sequence SEQ ID NO 18; a single or multiple amino acid variant of said "functional fragment of said LDLR ectodomain” having amino acid sequence SEQ ID NO 18; a multimer of a "LDLR ectodomain” having amino acid sequence SEQ ID NO 18, wherein said multimer is a dimer, or a trimer or comprises more repetitions of said "LDLR ectodomain", and wherein said multimer is a homomultimer of said "LDLR ectodomain”; a multimer of a functional fragment of a "LDLR ectodomain”
  • the multimer as disclosed herein is preferably a dimer or trimer, still more preferred a dimer, still more preferred is a homodimer or a homotrimer, and still more preferred is a homodimer.
  • the present invention provides an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain.
  • a preferred embodiment of present invention relates to LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection in a human, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain.
  • An embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain having amino acid sequence SEQ ID NO 18, or a functional fragment thereof, or an amino acid variant thereof.
  • amino acid variant refers to an analog of the natural soluble LDL receptor (LDLR ectodomain) or of a functional fragment thereof in which one or more amino acid residues of the natural soluble LDL receptor is replaced by a different amino acid residue or is deleted, so that the resulting LDLR derived polypeptide has an ability of binding to a Bunyavirales virus about equal to or greater than that of the natural LDLR ectodomain.
  • LDLR ectodomain the natural soluble LDL receptor
  • amino acid variants are prepared by known synthesis and/or by site-directed mutagenesis techniques, or any other known technique suitable therefor.
  • amino acid variant comprises "single amino acid variant” and "multiple amino acid variant”.
  • single amino acid variant refers to an analog of the natural soluble LDL receptor (LDLR ectodomain) or of a functional fragment thereof in which one amino acid residue of the natural soluble LDL receptor is replaced by a different amino acid residue, or is added, or is deleted, so that the resulting LDLR derived polypeptide has an ability of binding to a Bunyavirales virus about equal to or greater than that of the natural LDLR ectodomain.
  • the term “multiple amino acid variant” refers to an analog of the natural soluble LDL receptor (LDLR ectodomain) or of a functional fragment thereof in which multiple amino acid residues, i.e. at least two amino acid residues of the natural soluble LDL receptor are replaced by different amino acid residues or deleted, or at least two amino acid residues are added compared to the natural soluble LDL receptor, so that the resulting LDLR derived polypeptide has an ability of binding to a Bunyavirales virus about equal to or greater than that of the natural LDLR ectodomain.
  • a “multiple amino acid variant of an LDLR ectodomain or of a functional fragment thereof” comprises an amino acid sequence that shows high aminoacidic sequence identity to the parent LDLR ectodomain (SEQ ID NO 18) or a fragment thereof, such as it shows more than 60%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity with the parent sequence and is preferably characterised by similar properties of the parent sequence, namely binding to a Bunyavirales virus.
  • the percentage of "sequence identity” is determined by comparing two optimally aligned amino acid sequences over a “comparison window” on the full length of the reference sequence.
  • a “comparison window” as used herein refers to the optimal alignment between the reference and variant sequence after that the two sequences are optimally aligned, wherein the variant amino acid sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment.
  • Identity percentage is calculated by determining the number of positions at which the identical amino acids occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the full length in amino acid residues) and multiplying the results by 100 to yield the percentage of sequence identity.
  • Two amino acid sequences are said to be "identical” if the sequence of amino acid residues in the two sequences is the same when optimally aligned as described above.
  • the term "functional fragment of an LDLR ectodomain” refers to a fragment of the LDLR ectodomain comprising one or more "LDLR ectodomain modules" (i.e. LA1 to LA7, EGFA, EGFB, PROP, EGFC, and OLSD), provided that said fragment has an ability to bind to a Bunyavirales virus about equal to or greater than that of the natural LDLR ectodomain.
  • LDLR ectodomain modules i.e. LA1 to LA7, EGFA, EGFB, PROP, EGFC, and OLSD
  • the term “functional fragment” refers to a fragment of the LDLR ectodomain, wherein said “functional fragment” comprises one or more "LDLR ectodomain modules", wherein said "LDLR ectodomain modules” have the same order in the resulting "functional fragment” as in the natural LDLR ectodomain (SEQ ID NO 18), provided that said fragment has an ability to bind to a Bunyavirales virus about equal to or greater than that of the natural LDLR ectodomain.
  • the term "functional fragment of an LDLR ectodomain” is selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41 ), OLSD (SEQ ID NO 36), LA1-LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROPX-EGFCX-OLSD, LA1 -LA2-LA3-LA4X-LA5-LA6X-OLSD, LA
  • an embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41),
  • said LDLR derived polypeptide for use is a homomultimer of functional fragment monomers, wherein each monomer is a functional fragment of an LDLR ectodomain, it is preferred that said homomultimers comprises between 2 and 10 monomers, 2 and 12 monomers, 2 and 14 monomers, 2 and 16 monomers, 2 and 18 monomers, 2 and 20 monomers, 2 and 8 monomers, 2 and 6 monomers, 2 and 4 monomers. More preferably, When said LDLR derived polypeptide for use is a homomultimer of monomers, wherein each monomer is a functional fragment, it is preferred that said homomultimers comprises between 2 and 8 monomers, preferably 2 or 3 monomers.
  • a monomer in said LDLR derived polypeptide for use, can be a functional fragment of an LDLR ectodomain or an amino acid variant thereof.
  • An exemplary LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises a homomultimer of a functional fragment of an LDLR ectodomain selected from the group reported above, has the following composition: (LA2-LA3-LA4) (LA2-LA3-LA4) (LA2-LA3-LA4) (LA2-LA3-LA4) (LA2-LA3-LA4) (LA2-LA3-LA4), or (LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP) (LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFBX-PROP), and similars. Multimers
  • a further embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises a dimer, a trimer, a heteromultimer, or a homomultimer of an LDLR ectodomain monomer having amino acid sequence SEQ ID NO 18, or of a functional fragment thereof, or of an amino acid variant thereof. More preferred is a dimer or trimer, still more preferred a dimer, still more preferred is a homodimer or a homotrimer, and still more preferred is a homodimer.
  • a monomer is thus selected from the group comprising: an LDLR ectodomain (SEQ ID NO 18), a functional fragment of said LDLR ectodomain, an amino acid variant of said LDLR ectodomain, an amino acid variant of said functional fragment of said LDLR ectodomain.
  • said LDLR derived polypeptide for use is a homomultimer of the monomers defined above, it is preferred that said homomultimers comprises between 2 and 10 monomers, 2 and 12 monomers, 2 and 14 monomers, 2 and 16 monomers, 2 and 18 monomers, 2 and 20 monomers, 2 and 8 monomers, 2 and 6 monomers, 2 and 4 monomers. More preferably, said LDLR derived polypeptide for use comprises between 2 and 8 monomers as defined above, preferably 2 or 3 monomers.
  • homomultimer of an LDLR ectodomain refers to a multimeric polypeptide consisting of two or more identical monomers, wherein each monomer is an LDLR ectodomain having amino acid sequence SEQ ID NO 18, or an amino acid variant thereof. Therefore, the term “homomultimer of an LDLR ectodomain” also refers to a multimeric polypeptide consisting of two or more identical monomers, wherein each monomer is an amino acid variant of LDLR ectodomain having amino acid sequence SEQ ID NO 18.
  • the term "homomultimer of an LDLR ectodomain” refers to a dimer or trimer of an LDLR ectodomain monomer, wherein each LDLR ectodomain monomer has amino acid sequence SEQ ID NO 18, or an amino acid variant thereof. Therefore, the term “homomultimer of an LDLR ectodomain” also refers to a dimer or trimer of an LDLR ectodomain monomer, wherein each monomer is an amino acid variant of LDLR ectodomain having amino acid sequence SEQ ID NO 18.
  • the term "homomultimer of an LDLR ectodomain functional fragment” refers to a multimeric polypeptide consisting of two or more identical monomers, wherein each monomer is an LDLR ectodomain functional fragment, and wherein each functional fragment is selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41 ), OLSD (SEQ ID NO 36), LA1 -LA2-LA3-LA4X-
  • LA1-LA2-LA3-LA4X-LA5-LA6X-LA7 LA1 -LA2-LA3-LA4X-LA5-LA6, LA1- LA2-LA3-LA4X-LA5, LA1 -LA2-LA3-LA4, LA1-LA2-LA3, LA1-LA2, LA2-LA3-LA4X- LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA2-LA3-LA4X-LA5-LA6X- LA7-EGFA-EGFBX-PROPX-EGFC, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROPX-EGFC, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROP, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA2-LA3-LA4X-LA5-LA6X
  • LA4X-LA5-LA6X-LA7-EGFA LA4X-LA5-LA6X-LA7
  • LA4X-LA5-LA6X-LA7 LA4X-LA5-LA6, LA4X- LA5, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD
  • LA5-LA6X-LA7-EGFA-EGFBX-PROP LA5-LA6X- LA7-EGFA-EGFB
  • LA5-LA6X-LA7-EGFA LA5-LA6X-LA7, LA5-LA6X-LA7, LA5-LA6X-LA7, LA5-LA6, LA6X-LA7- EGFA-EGFBX-PROPX-EGFCX-OLSD
  • LA6X-LA7-EGFA-EGFBX-PROPX-EGFC LA6X-LA7-EGFA-EGFBX-PROPX-EGFC
  • the disclosed dimer of an LDLR ectodomain monomer comprising two monomers, wherein each monomer is an LDLR ectodomain or an LDLR ectodomain functional fragment or an amino acid variant thereof, has a binding capacity for Bunyavirales virus that is about equal to or greater than the binding capacity of the single LDLR ectodomain monomers.
  • the disclosed trimer of an LDLR ectodomain monomer comprising three monomers, wherein each monomer is an LDLR ectodomain or an LDLR ectodomain functional fragment or an amino acid variant thereof, has a binding capacity for Bunyavirales virus that is about equal to or greater than the binding capacity of the single LDLR ectodomain monomer.
  • the disclosed homomultimer of an LDLR ectodomain monomer comprising a plurality of monomers, wherein each monomer is an LDLR ectodomain or an LDLR ectodomain functional fragment or an amino acid variant thereof, has a binding capacity for Bunyavirales virus, preferably CCHFV or RVFV and most preferably CCHFV, that is about equal to or greater than the binding capacity of an LDLR ectodomain monomer.
  • each monomer or functional fragments or single amino acid variant thereof may be linked directly to the adjacent monomer or functional fragments or single amino acid variant thereof.
  • the N-terminus of the second or successive monomer e.g. LDLR ectodomain of SEQ ID NO 18, or a functional fragment thereof or an amino acid variant thereof
  • the C-terminus of the first or previous monomer of the disclosure e.g., LDLR ectodomain of SEQ ID NO 18, or a functional fragment thereof or an amino acid variant thereof.
  • the LDLR ectodomain monomers, functional fragment thereof or single amino acid variant thereof can be associated in any suitable manner to form an "LDLR derived polypeptide" or an homomultimer forming an "LDLR derived polypeptide".
  • Said various monomers or functional fragments may be covalently associated, e.g., by means of a peptide or disulfide bond.
  • the polypeptide fragments or monomers can thus be directly fused.
  • the resulting fusion protein representing the LDLR derived polypeptide of the invention can include any suitable number of modified bonds, e.g., isosteres, within or between the polypeptide fragments or monomers.
  • the LDLR derived polypeptide can include a "peptide linker" or "peptide linker sequence” between any two polypeptide fragments or monomers that includes one or more amino acid sequences not forming part of the biologically active peptide portions.
  • Any suitable peptide linker can be used.
  • Such a linker can be of any suitable size.
  • the linker predominantly may comprise or consist of neutral amino acid residues. If separation of peptide fragments or monomers is desirable a linker that facilitates separation can be used.
  • Flexible linkers typically composed of combinations of glycine and/or serine residues, or combinations of glycine, proline, serine and alanine can be advantageous. Examples of such linkers are described in "Table 1".
  • linker sequences are used in the "LDLR derived polypeptides" to connect different components of the "LDLR derived polypeptide".
  • the amino terminal end of the linker sequence is joined by a peptide bond to a first (or previous) LDLR ectodomain monomer or fragment and the carboxy terminal end of the linker sequence is joined by a peptide bind to a second (or successive) LDLR ectodomain monomer or fragment.
  • a particular embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain, and wherein said LDLR derived polypeptide further comprises a peptide linker connecting said fragments.
  • a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1)
  • a further embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises a dimer, a trimer, or a multimer of an LDLR ectodomain monomer having amino acid sequence SEQ ID NO 18, or of a functional fragment thereof, or of an amino acid variant thereof, and wherein said LDLR derived polypeptide further comprises a peptide linker connecting said fragments or monomers.
  • a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1).
  • said multimer comprises between 2 and 8 monomers, preferably 2 or 3 monomers.
  • a further embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ
  • said homomultimer comprises between
  • This invention further concerns a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain or of a functional fragment thereof.
  • nucleic acid molecule as used herein includes genomic DNA, cDNA, synthetic DNA, synthetic RNA, mRNA, ssDNA, dsDNA, self-amplifying RNA, dsRNA, and combinations thereof.
  • nucleic acid molecule preferably includes synthetic DNA, synthetic RNA, and mRNA.
  • the "nucleic acid molecule” is a “synthetic delivery RNA”.
  • the “nucleic acid molecule” is a "therapeutic RNA” for delivery into a mammal cell.
  • RNA or "RNA molecule” or “ribonucleic acid molecule” refers to a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides).
  • DNA or "DNA molecule” or “deoxyribonucleic acid molecule” refers to a polymer of deoxyribonucleotides.
  • DNA and RNA can be synthesized naturally e.g., by DNA replication or transcription of DNA, respectively. RNA can be post-transcriptionally modified. DNA and RNA can also be chemically synthesized. DNA and RNA can be single-stranded (i.e.
  • mRNA or “messenger RNA” is a single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains.
  • isolated RNA or isolated DNA refers to RNA or DNA molecules which are substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • the polypeptides and recombinant nucleic acid molecules of interest are "purified” to essential homogeneity, i.e., contaminant species cannot be detected in the composition by conventional detection methods.
  • Purity and homogeneity can be determined using a number of techniques well known in the art, such as agarose or polyacrylamide gel electrophoresis of a protein or nucleic acid sample, followed by visualization upon staining, or using a high-resolution technique, such as high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • Synthetic delivery RNA as described herein can refer to a synthetic RNA for delivery into the nucleus of a cell.
  • the invention also concerns replicable expression vectors containing said nucleic acid molecules, hosts transformed therewith and protein produced by expression of such transformed hosts.
  • the production of the recombinant LDLR ectodomain or of the functional fragment thereof may be carried out by different techniques well known in the prior art.
  • a DNA molecule, nucleic acid molecule or polypeptide is "recombinant" when it is artificial or engineered or derived from an artificial or engineered protein or nucleic acid.
  • LDLR derived polypeptides of the invention Recombinant methods for producing and isolating LDLR derived polypeptides of the invention are those described in the prior art.
  • the LDLR derived polypeptides may be produced by direct peptide synthesis using solid-phase techniques. Peptide synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.) in accordance with the instructions provided by the manufacturer. For example, sub-sequences may be chemically synthesized separately and combined using chemical methods to provide LDLR derived polypeptides of the invention or functional fragments thereof. Alternatively, such sequences may be ordered from a company or from a protein production facility that specialize in production of polypeptides.
  • a recombinant method for producing and isolating polypeptides of the invention comprises introducing into a population of cells any "recombinant nucleic acid molecule" described herein, which is operatively linked to a regulatory sequence (e.g. a promoter) effective to produce the encoded polypeptide, culturing the cells in a culture medium to produce the polypeptide, and isolating the polypeptide from the cells or from the culture medium.
  • a regulatory sequence e.g. a promoter
  • An amount of nucleic acid sufficient to facilitate uptake by the cells (transfection) and/or expression of the polypeptide is utilized.
  • Appropriate cell culture media are known to those of skill in the art.
  • the nucleic acid is introduced into such cells by any delivery method known in the prior art, including, e.g., injection, needleless injection device, gene gun, transfection, transfection with lipofectamine, retroviral gene delivery, electroporation transdermal delivery, passive uptake, etc.
  • the nucleic acid of the invention may be part of a vector, such as a recombinant expression vector, including a DNA plasmid vector, viral vector, or any suitable vector known in the prior art.
  • the nucleic acid or vector comprising a nucleic acid of the invention may be prepared and formulated as known in the prior art. Such a nucleic acid or expression vector may be introduced into a population of in vitro cultured cells.
  • a "vector” may be any agent that is able to deliver or maintain a nucleic acid in a host cell and includes, for example, but is not limited to, plasmids (e.g., DNA plasmids), naked nucleic acids, viral vectors, viruses, nucleic acids complexed with one or more polypeptide or other molecules, as well as nucleic acids immobilized onto solid phase particles.
  • plasmids e.g., DNA plasmids
  • naked nucleic acids e.g., viral vectors, viruses, nucleic acids complexed with one or more polypeptide or other molecules, as well as nucleic acids immobilized onto solid phase particles.
  • a vector can be useful as an agent for delivering or maintaining an exogenous gene, nucleic acid, and/or protein in a host cell.
  • a vector may be capable of transducing, transfecting, or transforming a cell, thereby causing the cell to replicate or express nucleic acids and/or proteins other than those native to the cell or in a manner not native to the cell.
  • a vector may include materials to aid in achieving entry of a nucleic acid into the cell, such as a viral particle, liposome, protein coating, or the like. Any method of transferring a nucleic acid into the cell may be used; unless otherwise indicated, the term vector does not imply any particular method of delivering a nucleic acid into a cell or imply that any particular cell type is the subject of transduction.
  • expression vector typically refers to a nucleic acid construct or sequence, generated recombinantly or synthetically, with a series of specific nucleic acid elements that permit transcription of a particular "nucleic acid molecule" in a host cell.
  • the expression vector typically includes a nucleic acid to be transcribed operably linked to a promoter.
  • expression includes any step involved in the production of the polypeptide (i.e. the LDLR ectodomain polypeptide, or a functional fragment thereof or an amino acid variant thereof) including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and/or secretion.
  • the nucleic acids can also be modified to include one or more codons that provide for optimum expression in an expression system (e.g., mammalian cell or mammalian expression system), while, if desired, said one or more codons still encode the same amino acid(s).
  • an expression system e.g., mammalian cell or mammalian expression system
  • said one or more codons still encode the same amino acid(s).
  • a DNA sequence encoding a desired linker sequence may be inserted between, and in the same reading frame as, for example, two adjacent DNA sequences encoding two monomers of the LDLR ectodomain using conventional techniques known in the art.
  • a chemically synthesized oligonucleotide encoding the linker may be ligated between sequences encoding the first and second monomer, or the second and third monomer, or the third and fourth monomer, and fourth monomer and fifth monomer, etc.
  • subject includes, but is not limited to, an organism or animal, including mammals and non-mammals.
  • a mammal includes, e.g., but is not limited to, a human, non-human primate (e.g., baboon, orangutan, monkey, gorilla), mouse, dog, pig, cow, goat, cat, rabbit, rat, guinea pig, hamster, horse, sheep, or other non-human mammal.
  • a non-mammal includes, e.g., but is not limited to, a non-mammalian invertebrate and non-mammalian vertebrate, such as a bird (e.g., a chicken or duck) or a fish.
  • Bunyavirales order encompasses nine families of enveloped viruses containing a single-stranded negative-sense RNA genome divided into three segments.
  • the small (S) and large (L) segments encode proteins participating in genome replication in the infected cell cytoplasm.
  • Bunyavirales are vector-borne viruses transmitted mostly by bites of arthropods such as mosquitoes, ticks, flies, or livestock animals, with the exception of the viruses from the Hantaviridae family, which are transmitted by infectious excreta or bites of rodents and other small mammals. In certain cases, human-to-human transmission can occur due to close contact with infected blood or other bodily fluids.
  • Bunyavirales are found throughout the world and are known to resist to adverse climate changes allowing for seasonal and persistent occurrence of the diseases. Bunyavirales are endemic in certain regions of the globe, such as Africa, the Middle East and Asia. In addition, outbreaks of Bunyavirales are often reported in both animals and humans.
  • viruses of the order Bunyavirales are described as being human pathogens, and particularly the viruses of the genus Orthohantavirus (Hantaviridae family, also named Hantavirus) such as Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava-Belgrade hantavirus (DOBV), Haantan virus (HTNV), Vietnamese Negra hantavirus (LANV), Longquan hantavirus (LQIIV), Puumala hantavirus (PUIIV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TLILV), New York hantavirus (NYV), viruses of the genus Orthonairovirus such as Crimean-Congo hemorrhagic fever virus (CCHFV), Dugbe virus (DLIGV), Amsterdam sheep disease virus (CC
  • Bunyavirales viruses cause a broad spectrum of clinical illnesses: self-limited febrile disease, respiratory and pulmonary syndromes, encephalitis, and life-threatening hemorrhagic fevers.
  • CCHFV is a tripartite RNA genome virus with negative polarity of the genus Orthonairovirus and family Nairoviridae.
  • the nairoviruses are predominantly tick- borne viruses.
  • the genome of this virus is composed of the three different RNA segments small (S), medium (M) and large (L).
  • S small segment ‘S’ encoding nucleoprotein (NP) is around 1.6kb long.
  • the medium segment ‘M’ encoding glycoproteins is around 5.5kb long.
  • the large segment ‘L’ encoding RNA - dependent RNA polymerase (RdRp or "L protein”) is around 12 kb long.
  • the untranslated regions (UTRs) on 5' and 3' of the S, M and L segments are necessary for viral transcription, replication and packaging.
  • the M segment generally encodes two structural glycoproteins, hereafter referred to as Gn and Gc, based on their location relative to the N and C termini of the (M segment encoded) polyprotein.
  • the precursor of GP is cleaved and modified to generate the structural proteins Gn and Gc and the non-structural proteins GP38 and NSm.
  • Gn and Gc structural glycoproteins
  • NSm non-structural proteins
  • the tri-segmented viral genome is coated with the viral NP and bound by the L protein.
  • the viral proteins produce positive-sense viral RNA using the genomic negative-sense viral RNA as a template to initiate viral protein production and replication.
  • the viral glycoproteins Gn and Gc are found on the virion surface and are responsible for receptor binding and viral entry.
  • the present invention particularly relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain, and wherein said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Website Georgia Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou
  • the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Rift Valley fever virus (RVFV), Kenya sheep virus (NSDV), and Sandfly Fever Naples virus (SFNV), more preferably the Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
  • CCHFV Crimean-Congo Haemorrhagic Fever Virus
  • RVFV Rift Valley fever virus
  • NSDV Baltimore sheep virus
  • SFNV Sandfly Fever Naples virus
  • the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Rift Valley fever virus (RVFV), Kenya sheep virus (NSDV), and Sandfly Fever Naples virus (SFNV), more preferably the Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
  • CCHFV Crimean-Congo Haemorrhagic Fever Virus
  • RVFV Rift Valley fever virus
  • NSDV Baltimore sheep virus
  • SFNV Sandfly Fever Naples virus
  • the present invention more particularly relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an amino acid variant of an LDLR ectodomain, and wherein said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Website Madison Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre han
  • An embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain having amino acid sequence SEQ ID NO 18, or a functional fragment thereof, or an amino acid variant thereof, and wherein said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Website Website), Georgia Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hanta
  • the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Rift Valley fever virus (RVFV), Kenya sheep virus (NSDV), and Sandfly Fever Naples virus (SFNV), more preferably the Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
  • CCHFV Crimean-Congo Haemorrhagic Fever Virus
  • RVFV Rift Valley fever virus
  • NSDV Baltimore sheep virus
  • SFNV Sandfly Fever Naples virus
  • said LDLR derived polypeptide further comprises a peptide linker connecting said fragments. More preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1)
  • An alternative embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an amino acid variant of an LDLR ectodomain having amino acid sequence SEQ ID NO 18, or a functional fragment thereof, and wherein said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Website Website), Georgia Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hantavirus
  • a further embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, wherein said LDLR derived polypeptide comprises a dimer, a trimer, or a multimer of an LDLR ectodomain monomer having amino acid sequence SEQ ID NO 18, or of a functional fragment thereof, or of an amino acid variant thereof, and wherein said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Website Georgia Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumal
  • the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Rift Valley fever virus (RVFV), Kenya sheep virus (NSDV), and Sandfly Fever Naples virus (SFNV), more preferably the Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
  • CCHFV Crimean-Congo Haemorrhagic Fever Virus
  • RVFV Rift Valley fever virus
  • NSDV Baltimore sheep virus
  • SFNV Sandfly Fever Naples virus
  • a further alternative embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, wherein said LDLR derived polypeptide comprises a dimer, a trimer, or a multimer of an amino acid variant of an LDLR ectodomain monomer having amino acid sequence SEQ ID NO 18, or of a functional fragment thereof, and wherein said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Website Georgia Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala
  • said LDLR derived polypeptide further comprises a peptide linker connecting said fragments. More preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1). More preferably, said multimer comprises between 2 and 8 monomers of an LDR ectodomain, or of a functional fragment thereof or of an amino acid variant thereof.
  • a further embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41),
  • the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Rift Valley fever virus (RVFV), Kenya sheep virus (NSDV), and Sandfly Fever Naples virus (SFNV), more preferably the Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
  • CCHFV Crimean-Congo Haemorrhagic Fever Virus
  • RVFV Rift Valley fever virus
  • NSDV Baltimore sheep virus
  • SFNV Sandfly Fever Naples virus
  • a further alternative embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an amino acid variant of a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (
  • said LDLR derived polypeptide further comprises a peptide linker connecting said fragments. More preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1) Moreover, the present invention particularly relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain, and wherein said Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
  • the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain having amino acid sequence SEQ ID NO 18, or a functional fragment thereof, or an amino acid variant thereof, and wherein said Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
  • said LDLR derived polypeptide further comprises a peptide linker connecting said fragments. More preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1)
  • a further embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, wherein said LDLR derived polypeptide comprises a dimer, a trimer, a homodimer, a homotrimer, or a multimer of an LDLR ectodomain monomer having amino acid sequence SEQ ID NO 18, or of a functional fragment thereof, or of an amino acid variant thereof, and wherein said Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
  • said LDLR derived polypeptide further comprises a peptide linker connecting said fragments. More preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1). More preferably, said multimer comprises between 2 and 8 monomers, preferably 2 or 3 monomers of an LDLR ectodomain, or of a functional fragment thereof or of an amino acid variant thereof.
  • a further embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41),
  • said LDLR derived polypeptide further comprises a peptide linker connecting said fragments. More preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1). More preferably, said multimer comprises between 2 and 8 monomers of an LDR ectodomain, or of a functional fragment thereof or of an amino acid variant thereof.
  • the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain having amino acid sequence SEQ ID NO 18, and wherein said Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
  • the present invention relates to a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain, and wherein said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Website Website, Website, Website, Georgia Negra hantavirus (LANV),
  • the present invention relates to a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an amino acid variant of an LDLR ectodomain or a functional fragment thereof, and wherein said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Website Georgia Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (CCHF
  • An embodiment of the present invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising the LDLR derived polypeptide described herein, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent.
  • composition refers to a composition suitable for pharmaceutical use in a subject, including an animal or human.
  • a pharmaceutical composition typically comprises an effective amount of an active agent and a carrier, excipient, or diluent.
  • the carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient or diluent, respectively.
  • compositions of the invention are prepared for administration by mixing the LDLR derived polypeptide, with physiologically acceptable carriers, stabilizers, and excipients, and prepared in dosage form, e.g. by lyophilization in dosage vials.
  • a specific embodiment of the present invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent.
  • the present invention is also directed to a pharmaceutical composition
  • a pharmaceutical composition comprising the LDLR derived polypeptide as disclosed herein together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent for use in the prophylaxis and/or treatment of an infection by a Bunyavirus.
  • the present invention is directed to a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an LDLR derived polypeptide as disclosed herein for use in the prophylaxis and/or treatment of an infection by a Bunyavirus.
  • said LDLR derived polypeptide further comprises a peptide linker connecting said fragments. More preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1)
  • Said Bunyavirales virus is preferably selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Website
  • LANV Longquan hantavirus
  • LQUV Longquan hantavirus
  • PUUV Puumala hantavirus
  • SANGV Sangassou hantavirus
  • SEOV Sin Nombre hantavirus
  • SNV Sin Nombre hantavirus
  • TAAIV Tula hantavirus
  • NW New York hantavirus
  • RVFV Rift Valley fever virus
  • NSDV Sandfly Fever Naples virus
  • SFTSV Severe fever with thrombocytopenia syndrome virus
  • SFTSV Sandfly Fever Naples virus
  • An alternative embodiment of the present invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an amino acid variant of an LDLR ectodomain, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent, and wherein said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Website Georgia Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala
  • a more specific embodiment of the present invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain having amino acid sequence SEQ ID NO 18, or a functional fragment thereof, or an amino acid variant thereof.
  • said LDLR derived polypeptide further comprises a peptide linker connecting said fragments. More preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1)
  • Said Bunyavirales virus is preferably selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Website
  • LANV Longquan hantavirus
  • LQUV Longquan hantavirus
  • PUUV Puumala hantavirus
  • SANGV Sangassou hantavirus
  • SEOV Sin Nombre hantavirus
  • SNV Sin Nombre hantavirus
  • TAAIV Tula hantavirus
  • NW New York hantavirus
  • RVFV Rift Valley fever virus
  • NSDV Sandfly Fever Naples virus
  • SFTSV Severe fever with thrombocytopenia syndrome virus
  • SFTSV Sandfly Fever Naples virus
  • a specific embodiment of the present invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent, and wherein said LDLR derived polypeptide comprises a dimer, a trimer, a homodimer, a homotrimer, or a multimer of an LDLR ectodomain monomer having amino acid sequence SEQ ID NO 18, or of a functional fragment thereof, or of an amino acid variant thereof.
  • said LDLR derived polypeptide further comprises a peptide linker connecting said fragments. More preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1). More preferably, said multimer comprises between 2 and 8 monomers of an LDR ectodomain, or of a functional fragment thereof or of an amino acid variant thereof.
  • Said Bunyavirales virus is preferably selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Website
  • LANV Longquan hantavirus
  • LQUV Longquan hantavirus
  • PUUV Puumala hantavirus
  • SANGV Sangassou hantavirus
  • SEOV Sin Nombre hantavirus
  • SNV Sin Nombre hantavirus
  • TAAIV Tula hantavirus
  • NW New York hantavirus
  • RVFV Rift Valley fever virus
  • NSDV Sandfly Fever Naples virus
  • SFTSV Severe fever with thrombocytopenia syndrome virus
  • SFTSV Sandfly Fever Naples virus
  • a specific embodiment of the present invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent, and wherein said LDLR derived polypeptide comprises a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID
  • said LDLR derived polypeptide further comprises a peptide linker connecting said fragments. More preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1). More preferably, said multimer comprises between 2 and 8 monomers of an LDR ectodomain, or of a functional fragment thereof or of an amino acid variant thereof.
  • Said Bunyavirales virus is preferably selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Website
  • LANV Longquan hantavirus
  • LQUV Longquan hantavirus
  • PUUV Puumala hantavirus
  • SANGV Sangassou hantavirus
  • SEOV Sin Nombre hantavirus
  • SNV Sin Nombre hantavirus
  • TAAIV Tula hantavirus
  • NYV New York hantavirus
  • RVFV Rift Valley fever virus
  • NSDV Sandfly Fever Naples virus
  • SFTSV Severe fever with thrombocytopenia syndrome virus
  • SFNV Sandfly Fever Naples virus
  • a specific embodiment of the present invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain having amino acid sequence SEQ ID NO 18, and wherein said Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
  • the term "effective amount" refers to a dosage (or dose) or amount of a substance sufficient to produce a desired result.
  • the desired result may comprise an objective or subjective improvement in the recipient of the dosage or amount.
  • the desired result may comprise a measurable, or detectable inhibition of a virus infection in a subject to whom a dosage or amount of a particular LDLR ectodomain or a fragment thereof has been administered.
  • a “prophylactic treatment” is a treatment administered to a subject who does not display signs or symptoms of, or displays only early signs or symptoms of, a disease, pathology, or disorder, such that treatment is administered for the purpose of preventing or decreasing the risk of developing the disease, pathology, or disorder.
  • a prophylactic treatment functions as a preventative treatment against a disease, pathology, or disorder, or as a treatment that inhibits or reduces further development or enhancement of a disease, pathology or disorder.
  • a “prophylactic activity” is an activity of an agent that, when administered to a subject who does not display signs or symptoms of, or who displays only early signs or symptoms of, a pathology, disease, or disorder, prevents or decreases the risk of the subject developing the pathology, disease, or disorder.
  • a “prophylactically useful” agent refers to an agent that is useful in preventing development of a disease, pathology, or disorder, or useful in inhibiting or reducing further development or enhancement of a disease, pathology or disorder.
  • a “therapeutic treatment” is a treatment administered to a subject who displays symptoms or signs of pathology, disease, or disorder, in which treatment is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms.
  • a “therapeutic activity” is an activity of an agent that eliminates or diminishes signs or symptoms of pathology, disease or disorder when administered to a subject suffering from such signs or symptoms.
  • a “therapeutically useful” agent means the agent is useful in decreasing, treating, or eliminating signs or symptoms of a disease, pathology, or disorder.
  • LDLR derived polypeptide of functional fragments or of a multimer thereof LDLR derived polypeptide of functional fragments or of a multimer thereof.
  • a second aspect of the present invention is directed to an LDLR derived polypeptide comprising a functional fragment of an LDLR ectodomain selected from the group LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41), OLSD (SEQ ID NO 36), LA1-LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX- PROPX-EGFCX-OLSD, LA1 -LA2-LA3-LA
  • amino acid variant refers to a single amino acid variant or to a multiple amino acid variant as previously defined.
  • An embodiment of the second aspect is directed to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a virus infection, wherein said virus is preferably a Bunyavirales virus, wherein said LDLR derived polypeptide comprises a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41 ), OLSD (SEQ ID NO 36), LA1-
  • Said Bunyavirales virus is preferably selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Website
  • CATV Longquan hantavirus
  • LQUV Longquan hantavirus
  • PUUV Puumala hantavirus
  • SANGV Sangassou hantavirus
  • SEOV Sin Nombre hantavirus
  • SNV Sin Nombre hantavirus
  • TAAIV Tula hantavirus
  • NW New York hantavirus
  • RVFV Rift Valley fever virus
  • NSDV Sandfly Fever Naples virus
  • Said Bunyavirales virus is more preferably CCHFV or RVFV and most preferably CCHFV.
  • a particular embodiment of the second aspect is directed to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a virus infection, wherein said virus is preferably a RVF virus (RVFV), wherein said LDLR derived polypeptide comprises an amino acid variant of a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID
  • a further aspect of the present invention is directed to a LDLR derived polypeptide comprising a multimer of monomers, wherein each monomer is selected from the group comprising: an LDLR ectodomain (SEQ ID NO 18), a functional fragment of said LDLR ectodomain, an amino acid variant of said LDLR ectodomain, an amino acid variant of said functional fragment of said LDLR ectodomain, and wherein said multimer comprises between 2 and 8 monomers, and wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said monomers; and wherein said multimer is a homomultimer or a heteromultimer.
  • amino acid variant refers to a single amino acid variant or to a multiple amino acid variant as previously defined.
  • a particular further aspect is directed to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a virus infection, wherein said virus is preferably a Bunyavirales virus, wherein said LDLR derived polypeptide comprises a multimer of monomers, wherein each monomer is selected from the group comprising: an LDLR ectodomain (SEQ ID NO 18), a functional fragment of said LDLR ectodomain, an amino acid variant of said LDLR ectodomain, an amino acid variant of said functional fragment of said LDLR ectodomain, and wherein said multimer comprises between 2 and 8 monomers, and wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said monomers; and wherein said multimer is a homomultimer or a heteromultimer.
  • Said Bunyavirales virus is preferably selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Website
  • HTNV Haantan virus
  • LQLIV Longquan hantavirus
  • PIIV Puumala hantavirus
  • SEOV Seoul hantavirus
  • Sin Nombre hantavirus SNV
  • Thailand hantavirus TAAIV
  • Tula hantavirus TILV
  • NYV New York hantavirus
  • RVFV Rift Valley fever virus
  • NSDV Sandfly Fever Naples virus
  • Said Bunyavirales virus is more preferably a CCHF virus or RVFV and most preferably CCHFV.
  • a more particular further aspect is directed to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a virus infection, wherein said virus is preferably a RVF virus, wherein said LDLR derived polypeptide comprises a multimer of monomers, wherein each monomer is selected from the group comprising: an amino acid variant of an LDLR ectodomain (SEQ ID NO 18), an amino acid variant of a functional fragment of said LDLR ectodomain, and wherein said multimer comprises between 2 and 8 monomers, preferably 2 or 3 monomers, and wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said monomers; and wherein said multimer is a homomultimer or a heteromultimer.
  • SEQ ID NO 18 amino acid variant of an LDLR ectodomain
  • said multimer comprises between 2 and 8 monomers, preferably 2 or 3 monomers
  • said LDLR derived polypeptide optionally comprises a peptide linker connecting said
  • the present invention also discloses a pharmaceutical composition
  • a pharmaceutical composition comprising said LDLR derived polypeptide, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent, wherein said LDLR derived polypeptide comprises a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41), OLSD (SEQ ID NO 36), LA1 -LA2-LA
  • the present invention also discloses a pharmaceutical composition
  • a pharmaceutical composition comprising said LDLR derived polypeptide, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent, wherein said LDLR derived polypeptide comprises a multimer of monomers, wherein each monomer is selected from the group comprising: an LDLR ectodomain (SEQ ID NO 18), a functional fragment of said LDLR ectodomain, an amino acid variant of said LDLR ectodomain, an amino acid variant of said functional fragment of said LDLR ectodomain, and wherein said multimer comprises between 2 and 8 monomers, preferably 2 or 3 monomers, and wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said monomers; and wherein said multimer is a homomultimer or a heteromultimer.
  • the invention also contemplates a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an LDLR derived polypeptide, wherein said LDLR derived polypeptide comprises a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41), OLSD (SEQ ID NO 36), LA1 -LA2-LA3-LA4X-LA5
  • the invention also contemplates a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an LDLR derived polypeptide, wherein said LDLR derived polypeptide comprises a multimer of monomers, wherein each monomer is selected from the group comprising: an LDLR ectodomain (SEQ ID NO 18), a functional fragment of said LDLR ectodomain, an amino acid variant of said LDLR ectodomain, an amino acid variant of said functional fragment of said LDLR ectodomain, and wherein said multimer comprises between 2 and 8 monomers, preferably 2 or 3 monomers, and wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said monomers; and wherein said multimer is a homomultimer or a heteromultimer.
  • the inventors were able to identify LDLR as involved in CCHF virus infection by using a screening method based on infection of randomly mutagenized murine haploid cells with a CCHFV pseudotyped VSV, selection of mutant resistant clones and whole exome sequencing to individuate the gene mutations responsible for CCHFV resistance ( Figure 1a-c, Table 2).
  • a diploid cell contains two copies of genomic DNA.
  • a point mutation introduced by chemical mutagenesis always affects only one allele, while the second copy remains intact.
  • One intact copy of a host cell factor is typically sufficient for a virus to hijack the cellular machinery and replicate.
  • a haploid cell is a cell that only carries a single copy of the genome. Therefore, a point mutation introduced in a haploid cell alters 100% of the cellular protein. In case of a mutation interfering with viral entry or replication, this would completely block infection in the haploid cell and allow its identification in a screening setup. In sum, recessive mutations with regards to the viral infection can be identified.
  • diploid is used herein to specifically refer to a cell or cell line including a genome wherein the cell is diploid for one or more specific genomic loci, or for the full genome, i.e. "fully diploid" cells.
  • haploid as used herein shall specifically refer to a cell or cell line including a single copy of the genome. Haploidy may be determined or tested by known methods, e.g. spectral karyotyping, comparative genomic hybridization or comparative propidium iodide staining.
  • near-haploid cell is a cell in which no more than 5 chromosomes are present in two or more copies. Near-haploid cells were found to maintain their status several months in culture.
  • An exemplary near-haploid somatic human cell is of a KBM-7 cell line, which is haploid for most chromosomes with the exception of chromosome 8, and optionally a portion of chromosome 15, and is a non-adherent cell line.
  • a further example of a near-haploid cell line is the HAP1 cell line, which is an adherent cell line obtained by engineering the KBM-7 cell line, which has lost the second copy of chromosome 8, and is hence “more haploid” than its KBM-7 parent, but still retains a portion of chromosome 15 and can therefore not be considered fully haploid.
  • Further near-haploid cell lines may be derived from a cancer patient, or from a patient suffering from leukemia.
  • HAP2 cell line which is obtained by engineering HAP1 cells through excision of the portion of chromosome 15 that retained its diploidy in the HAP1 cell line, thus, is considered truly or fully haploid. It turned out that the HAP2 cell line comprises the complete set of human chromosomes in the monosomic state.
  • the eHAP1 cell line is a human fully haploid cell line deriving from HAP1 cells, which have been genetically engineered to delete the diploid portion of chromosome 15.
  • eHAP1 is commercially available (e.g. Horizon Discovery (SKU: c669).
  • mutagenesis as used in the context of the present invention shall refer to a method of providing mutants of a sequence, e.g. through insertion, deletion and/or substitution of one or more nucleotides or amino acids, so to obtain variants thereof. Mutagenesis may be through random, semi-random or site directed mutation.
  • haploid murine stem cells refers to haploid murine stem cells that can be obtained and cultured as described in the prior art (Eiling, et al., Nat Protoc. 2019 Jul 1 ; 14(7): 1991-2014), or can be stabilized cell lines such as AN3-12, H129-2, H129-1. Therefore, in some embodiments, haploid murine stem cells are selected from the group comprising primary haploid murine stem cells, AN3-12, H129-2, H129-1 , and derivatives thereof.
  • haploid human stem cells refers to haploid human stem cells that can be obtained and cultured as described in the prior art, e.g in Sagi I, et al. Identification and propagation of haploid human pluripotent stem cells. Nat Protoc. 2016 11(11 ): 2274-2286.
  • haploid human stem cells refers to haploid human stem cells of different origins, such as for example, haploid human embryonic stem cells (hESCs), haploid human spermatogonial stem cells, haploid hematopoietic stem cell,
  • PV pseudovirus
  • pp pseudoparticles
  • pseudotyped virus refers to a virus expressing heterologous viral envelope proteins.
  • a pseudotyped VSV has the envelope protein of a heterologous virus assembled into the VSV membrane.
  • VSV is a bullet-shaped, negative polarity enveloped RNA virus in the Rhabdoviridae family.
  • VSV has a genome size of 11 kb that contains five main viral proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and large polymerase protein (L).
  • VSV-AG Recombinant virus VSV in which the glycoprotein (G) gene is deleted (rVSV-AG) (Whitt, J Virol Methods. 2010 Nov; 169(2): 365-374.) has been used to produce VSV pseudotypes containing the envelope glycoproteins of heterologous viruses, including viruses that require high-level biocontainment, such as Bunyavirales.
  • rVSV-AG pseudotypes The advantage of rVSV-AG pseudotypes is that it performs only one single round of replication, so that the experiments can be performed using biosafety level 2 (BSL-2) containment.
  • rVSV-AG expressing heterologous envelope glycoproteins can be used to analyse genetic factors involved in infection of viral pathogens without the need for specialized containment facilities.
  • RNA viral particles pseudotyped with Bunyavirales glycoproteins can be obtained by using a method described in the prior art, such as for example in Cegolon et al 2021 .
  • Preferred viruses are HIV, MSV, and VSV.
  • Random mutagenesis can be performed by a method selected from chemical random mutagenesis, UV mediated random mutagenesis, transposon mutagenesis, or error prone PCR.
  • random mutagenesis is performed by chemical random mutagenesis, and more preferably by incubating the haploid cells with Ethylnitrosurea (ENU) as described in the prior art.
  • Ethylnitrosurea Ethylnitrosurea
  • Bunyavirales glycoproteins Gn and Gc are derived from a precursor polyprotein by host cell proteases. Bunyavirales glycoproteins have been described in the prior art, for example in Hulswit, RJC et al, Viruses. 2021 Feb; 13(2): 353.
  • whole exome sequencing refers to the sequencing of all exons in a specific cell or tissue type, wherein the exons are the protein coding regions of a gene. Therefore, in “whole exome sequencing” (WES) only the protein-coding DNA regions are sequenced as compared to whole genome sequencing (WGS).
  • the term “whole transcriptome sequencing” refers to sequencing of expressed transcripts at the mRNA level to obtain sequence information on all mRNAs made in a cell or cell pool at a given time
  • NGS next generation sequencing
  • NGS refers to a group of platforms to perform sequencing of millions of small fragments of DNA in parallel. NGS can be used for whole genome sequencing, whole exome sequencing, or whole transcriptome sequencing.
  • NGS can be used for whole genome sequencing, whole exome sequencing, or whole transcriptome sequencing.
  • the terms “next generation sequencing”, “massively parallel sequencing", and “deep sequencing” are related terms and can be used interchangeably.
  • a third aspect of the present invention is directed to an in vitro screening method to identify a gene that modulates a Bunyavirales virus infection, the method comprising:
  • step B) performing chemical random mutagenesis in said cells of step A) in order to obtain mutagenized haploid or near-haploid cells;
  • step B) infecting the mutagenized cells of step B) with a Bunyavirales virus or with a Bunyavirales virus pseudotyped RNA viral particle;
  • step D) culturing the infected mutagenized cells of step C) to obtain resistant colonies
  • step E) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate said Bunyavirales virus infection
  • the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof
  • the pseudotyped RNA viral particle is a HIV particle or a MSV particle or a VSV particle
  • the pseudotyped RNA viral particle express a Bunyavirales virus specific glycoprotein Gc and /or Gn
  • the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade han
  • step B) of performing chemical random mutagenesis in said haploid or near-haploid cells of step A) in order to obtain mutagenized haploid or near-haploid cells preferably comprises incubating the haploid or near-haploid cells with N-Ethyl-N-nitrosourea (ENU).
  • ENU N-Ethyl-N-nitrosourea
  • the present invention is directed to an in vitro screening method to identify a gene that modulates a Bunyavirales virus infection, the method comprising:
  • step B) performing chemical random mutagenesis in said cells of step A) in order to obtain mutagenized haploid or near-haploid cells;
  • step B) infecting the mutagenized cells of step B) with a Bunyavirales virus or with a Bunyavirales virus pseudotyped RNA viral particle;
  • step D) culturing the infected mutagenized cells of step C) to obtain resistant colonies
  • the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof, wherein the pseudotyped RNA viral particle is a HIV particle or a MSV particle or a VSV particle, and wherein the pseudotyped RNA viral particle express a Bunyavirales virus specific glycoprotein Gc and /or Gn, and wherein the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Bel
  • step B) of performing chemical random mutagenesis in said haploid or near-haploid cells of step A) in order to obtain mutagenized haploid or near-haploid cells preferably comprises incubating the haploid or near-haploid cells with N-Ethyl-N-nitrosourea (ENU).
  • ENU N-Ethyl-N-nitrosourea
  • An alternative embodiment of the present invention is directed to an in vitro screening method to identify a gene that modulates a Bunyavirales virus infection, the method comprising:
  • step B) performing chemical random mutagenesis in said cells of step A) in order to obtain mutagenized haploid or near-haploid cells;
  • step B) infecting the mutagenized cells of step B) with a Bunyavirales virus or with a Bunyavirales virus pseudotyped RNA viral particle;
  • step D) culturing the infected mutagenized cells of step C) to obtain resistant colonies
  • step E) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate said Bunyavirales virus infection
  • the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof
  • the pseudotyped RNA viral particle is a HIV particle or a MSV particle or a VSV particle
  • the pseudotyped RNA viral particle express a Bunyavirales virus specific glycoprotein Gc and /or Gn
  • the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade han
  • step B) of performing chemical random mutagenesis in said haploid or near-haploid cells of step A) in order to obtain mutagenized haploid or near-haploid cells preferably comprises incubating the haploid or near-haploid cells with N-Ethyl-N-nitrosourea (ENU).
  • ENU N-Ethyl-N-nitrosourea
  • the present invention is directed to an in vitro screening method to identify a gene that modulates a Bunyavirales virus infection, the method comprising:
  • step B) performing chemical random mutagenesis in said haploid or near- haploid cells of step A) in order to obtain mutagenized haploid or near- haploid cells;
  • step B) infecting the mutagenized haploid or near-haploid cells of step B) with a Bunyavirales virus or with a Bunyavirales virus pseudotyped RNA viral particle;
  • step D) culturing the infected mutagenized haploid or near-haploid cells of step C) to obtain resistant colonies;
  • step E) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate said Bunyavirales virus infection
  • the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof
  • the pseudotyped RNA viral particle is a HIV particle or a MSV particle or a VSV particle
  • the pseudotyped RNA viral particle express a Bunyavirales virus specific glycoprotein Gc and /or Gn
  • the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Rift Valley fever virus (RVFV), Kenya sheep virus (NSDV), and Sandfly Fever Naples virus (SFNV), preferably
  • step B) of performing chemical random mutagenesis in said haploid or near-haploid cells of step A) in order to obtain mutagenized haploid or near-haploid cells preferably comprises incubating the haploid or near-haploid cells with N-Ethyl-N-nitrosourea (ENU).
  • ENU N-Ethyl-N-nitrosourea
  • the present invention is directed to in vitro screening method to identify a gene that modulates a Bunyavirales virus infection, the method comprising:
  • step B) performing chemical random mutagenesis in said haploid or near- haploid cells of step A) in order to obtain mutagenized haploid or near- haploid cells;
  • step C) infecting the mutagenized haploid or near-haploid cells of step B) with a Bunyavirales virus
  • step D) culturing the infected mutagenized haploid or near-haploid cells of step C) to obtain resistant colonies;
  • step F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate said Bunyavirales virus infection, and wherein the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof, wherein the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Website Georgia Negra hantavirus (LANV), Longquan hantavirus (LQIIV), Puumala hantavirus (PUIIV), Sangas
  • step B) of performing chemical random mutagenesis in said haploid or near-haploid cells of step A) in order to obtain mutagenized haploid or near-haploid cells preferably comprises incubating the haploid cells with N-Ethyl- N-nitrosourea (ENU).
  • ENU N-Ethyl- N-nitrosourea
  • the present invention is directed to in vitro screening method to identify a gene that modulates a Bunyavirales virus infection, the method comprising:
  • step B) performing chemical random mutagenesis in said haploid or near- haploid cells of step A) in order to obtain mutagenized haploid or near- haploid cells;
  • step C) infecting the mutagenized haploid or near-haploid cells of step B) with a Bunyavirales virus pseudotyped RNA viral particle;
  • step D) culturing the infected mutagenized haploid or near-haploid cells of step C) to obtain resistant colonies;
  • step E) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate said Bunyavirales virus infection
  • the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof
  • the pseudotyped RNA viral particle is a HIV particle or a MSV particle or a VSV particle
  • the pseudotyped RNA viral particle express a Bunyavirales virus specific glycoprotein Gc and /or Gn
  • the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hant
  • step B) of performing chemical random mutagenesis in said haploid or near-haploid cells of step A) in order to obtain mutagenized haploid or near-haploid cells preferably comprises incubating the haploid cells with N-Ethyl- N-nitrosourea (ENU).
  • ENU N-Ethyl- N-nitrosourea
  • the present invention is directed to an in vitro screening method to identify a gene that modulates a Bunyavirales virus infection, the method comprising:
  • step B) performing chemical random mutagenesis in said haploid or near- haploid cells of step A) in order to obtain mutagenized haploid or near- haploid cells;
  • step C) infecting the mutagenized haploid or near-haploid cells of step B) with a Bunyavirales virus pseudotyped RNA viral particle;
  • step D) culturing the infected mutagenized haploid or near-haploid cells of step C) to obtain resistant colonies;
  • step F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate said Bunyavirales virus infection, wherein the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof, wherein the pseudotyped RNA viral particle is a VSV particle, and wherein the pseudotyped RNA viral particle express a Bunyavirales virus specific glycoprotein Gc and /or Gn, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
  • the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from
  • step B) of performing chemical random mutagenesis in said haploid or near-haploid cells of step A) in order to obtain mutagenized haploid or near-haploid cells preferably comprises incubating the haploid or near-haploid cells with N-Ethyl-N-nitrosourea (ENU).
  • ENU N-Ethyl-N-nitrosourea
  • the present invention is directed to an in vitro screening method to identify a gene that modulates a Bunyavirales virus infection, the method comprising:
  • step B) performing chemical random mutagenesis in said haploid or near- haploid cells of step A) in order to obtain mutagenized haploid or near- haploid cells;
  • step C) infecting the mutagenized haploid or near-haploid cells of step B) with a Bunyavirales virus pseudotyped RNA viral particle;
  • step D) culturing the infected mutagenized haploid or near-haploid cells of step C) to obtain resistant colonies;
  • step E) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate said Bunyavirales virus infection
  • the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof
  • the pseudotyped RNA viral particle is a VSV particle
  • the pseudotyped RNA viral particle express a Bunyavirales virus specific glycoprotein Gc and /or Gn
  • the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (CCHFV
  • step B) of performing chemical random mutagenesis in said haploid or near-haploid cells of step A) in order to obtain mutagenized haploid or near-haploid cells preferably comprises incubating the haploid or near-haploid cells with N-Ethyl-N-nitrosourea (ENU).
  • ENU N-Ethyl-N-nitrosourea
  • the third aspect of the invention is directed to an in vitro screening method to identify a gene that modulates a CCHFV infection, the method comprising:
  • step B) performing chemical random mutagenesis in said haploid or near- haploid cells of step A) in order to obtain mutagenized haploid or near- haploid cells;
  • step B) infecting the mutagenized haploid or near-haploid cells of step B) with a CCHFV or with a CCHFV glycoprotein pseudotyped RNA viral particle;
  • step D) culturing the infected mutagenized haploid or near-haploid cells of step C) to obtain resistant colonies;
  • step F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate a CCHFV infection
  • the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof, wherein the pseudotyped RNA viral particle is a HIV particle or a MSV particle or a VSV particle, and wherein said CCHFV glycoprotein is CCHFV glycoprotein Gc and/or Gn, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
  • performing chemical random mutagenesis in said haploid or near- haploid cells of step B) in order to obtain mutagenized haploid or near-haploid cells comprises incubating the haploid cells with N-Ethyl-N-nitrosourea (ENU).
  • ENU N-Ethyl-N-nitrosourea
  • a particular embodiment is directed to an in vitro screening method to identify a gene that modulates a CCHFV infection, the method comprising:
  • step B) performing chemical random mutagenesis in said haploid or near- haploid cells of step A) in order to obtain mutagenized haploid or near- haploid cells;
  • step B) infecting the mutagenized haploid or near-haploid cells of step B) with a CCHFV glycoprotein pseudotyped RNA viral particle;
  • step D) culturing the infected mutagenized haploid or near-haploid cells of step C) to obtain resistant colonies;
  • step F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate a CCHFV infection, wherein the pseudotyped RNA viral particle is a VSV particle, and wherein said CCHFV glycoprotein is CCHFV glycoprotein Gc and/or Gn, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
  • a further embodiment is directed to an in vitro screening method to identify a gene that modulates a CCHFV infection, the method comprising:
  • step B) performing chemical random mutagenesis in said haploid or near- haploid cells of step A) in order to obtain mutagenized haploid or near- haploid cells by incubating the haploid or near-haploid cells with N- Ethyl-N-nitrosourea (ENU);
  • ENU N- Ethyl-N-nitrosourea
  • step B) infecting the mutagenized haploid or near-haploid stem cells of step B) with a CCHFV glycoprotein pseudotyped RNA viral particle;
  • step D) culturing the infected mutagenized haploid or near-haploid cells of step C) to obtain resistant colonies;
  • step F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate a CCHFV infection
  • the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof, wherein the pseudotyped RNA viral particle is a HIV particle or a MSV particle or a VSV particle, and wherein said CCHFV glycoprotein is CCHFV glycoprotein Gc and/or Gn, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
  • step B) of performing chemical random mutagenesis in said haploid or near-haploid cells of step A) in order to obtain mutagenized haploid or near-haploid cells preferably comprises incubating the haploid or near-haploid cells with N-Ethyl-N-nitrosourea (ENU).
  • ENU N-Ethyl-N-nitrosourea
  • a further particular embodiment is directed to an in vitro screening method to identify a gene that modulates a CCHFV infection, the method comprising:
  • step B) performing chemical random mutagenesis in said cells of step A) in order to obtain mutagenized haploid or near-haploid cells by incubating said cells with N-Ethyl-N-nitrosourea (ENU);
  • ENU N-Ethyl-N-nitrosourea
  • step B) infecting the mutagenized haploid or near-haploid cells of step B) with a CCHFV glycoprotein pseudotyped RNA viral particle;
  • step D) culturing the infected mutagenized haploid or near-haploid cells of step C) to obtain resistant colonies;
  • step F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate a CCHFV infection
  • the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof, wherein the pseudotyped RNA viral particle is a VSV particle, and wherein said CCHFV glycoprotein is CCHFV glycoprotein Gc and/or Gn, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
  • a further more particular embodiment is directed to an in vitro screening method to identify a gene that modulates a CCHFV infection, the method comprising:
  • step B) infecting the mutagenized haploid or near-haploid cells of step B) with CCHFV;
  • step F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate a CCHFV infection, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
  • step B) of performing chemical random mutagenesis in said haploid or near-haploid cells of step A) in order to obtain mutagenized haploid or near-haploid cells preferably comprises incubating the haploid or near-haploid cells with N-Ethyl-N-nitrosourea (ENU).
  • ENU N-Ethyl-N-nitrosourea
  • an embodiment is directed to an in vitro screening method to identify a gene that modulates a CCHFV infection, the method comprising:
  • step B) performing chemical random mutagenesis in said haploid or near- haploid cells of step A) in order to obtain mutagenized haploid or near- halpoid cells by incubating the said cells with N-ethyl-N-nitrosourea (ENU);
  • ENU N-ethyl-N-nitrosourea
  • step B) infecting the mutagenized haploid or near-haploid cells of step B) with a CCHFV;
  • step D) culturing the infected mutagenized haploid or near-haploid cells of step C) to obtain resistant colonies;
  • step F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate a CCHFV infection, wherein the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
  • a more specific embodiment is directed to an in vitro screening method to identify a gene that modulates a CCHFV infection, the method comprising:
  • step B) performing chemical random mutagenesis in said cells of step A) in order to obtain mutagenized haploid murine stem cells
  • step B) infecting the mutagenized cells of step B) with a Bunyavirales virus or with a Bunyavirales virus pseudotyped RNA viral particle;
  • step D) culturing the infected mutagenized cells of step C) to obtain resistant colonies
  • step F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate a Bunyavirales virus infection, wherein the pseudotyped RNA viral particle is a HIV particle or a MSV particle or a VSV particle, wherein the pseudotyped RNA viral particle express a Bunyavirales virus specific glycoprotein Gc and/or Gn, and wherein the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Website Website), Georgia Negra hantavirus (LANV), Longquan hantavirus (LQIIV), Puumala hantavirus (PUIIV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre
  • the pseudotyped RNA viral particle is preferably a VSV particle.
  • step B) comprises incubating the haploid murine stem cells with N-Ethyl-N-nitrosourea (ENU).
  • ENU N-Ethyl-N-nitrosourea
  • step B) performing chemical random mutagenesis in said cells of step A) in order to obtain mutagenized haploid murine stem cells
  • step B) infecting the mutagenized cells of step B) with a Bunyavirales virus or with a Bunyavirales virus pseudotyped RNA viral particle;
  • step D) culturing the infected mutagenized cells of step C) to obtain resistant colonies
  • step F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate a Bunyavirales virus infection, wherein the pseudotyped RNA viral particle is a VSV particle, wherein the pseudotyped VSV particle express a Bunyavirales virus specific glycoprotein Gc and/or Gn, and wherein the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Website Website, Longquan hantavirus (LANV), Longquan hantavirus (LQIIV), Puumala hantavirus (PUIIV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand han
  • step B) comprises incubating the haploid murine stem cells with N-Ethyl-N-nitrosourea (ENU).
  • ENU N-Ethyl-N-nitrosourea
  • the binding of the LDLR derived polypeptide can also be used for the detection of a Bunyavirales virus infection. Therefore, the present application is also directed to the use of an LDLR derived polypeptide in the diagnosis of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain as disclosed herein and wherein said binding diagnoses the infection by the Bunyavirales virus.
  • the binding of the LDLR derived polypeptide can also be used for the quality control of vaccines produced to prevent or ameliorate Bunyavirales virus infection, and especially CCHFV infection. Therefore, the present application is also directed to the use of an LDLR derived polypeptide in quality control of a Bunyavirales virus vaccine, especially a CCHFV vaccine, wherein said LDLR derived polypeptide binds to vaccine ingredients, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain as disclosed herein and wherein said binding assesses the quality of that vaccine to prevent or ameliorate a Bunyavirales virus infection.
  • Figure 1 shows generation of VSV-CCHF_G and haploid cells screening.
  • a Schematic representation of the methods used to produce the VSV- CCHF_G pseudotyped virus in HEK293T cells.
  • b Validation of resistant clones obtained in the primary haploid screen with VSV- CCHF_G. Each clone (1-13) was isolated, amplified and assessed for infection with the CCHFV lbAR10200 laboratory strain (MOI 0.1). The data show the level of infection for each clone compared to wild-type haploid cells (AN3-12) as determined by RT-PCR for CCHFV and RNase P RNA 24hpi.
  • c Deep exome sequencing identified three distinct single point mutations in the Ldlr gene in three resistant clones (5, 8, 10) resulting in stop codons or a missense mutation.
  • Figure 2 shows CCHFV infections in Ldlr knockout cells
  • a Level of infections in control wild type AN3-12 haploid and sister knock out (KO) cells infected with VSV-CCHF_G, CCHFV lbAr10200 (MOI 0.1 , 48hpi). Level of infection was assessed by RT-qPCR for viral and RNase P RNA.
  • b Levels of infection of lbAr10200 CCHFV in wild type (WT) and two different LDLR KO (clones C2 and C12) Vero cells and c, in three different clones of LDLR KO (clones C8, C10 and C11) A549 cells.
  • Figure 3 shows that recombinant CCHFV Gc protein binds to and induces the internalization of LDLR.
  • a Illustration depicting the BRET-based binding assay that was used to measure the interaction of BODIPY-FL labelled LDL and soluble BODIPY-FL labelled CCHFV Gc with the LDLR.
  • Figure 4 shows inhibition of CCHFV infections by soluble LDLR.
  • a Levels of VSV- CCHF_G infections in human SW13 cells, treated with the indicated range of soluble LDLR concentrations or left untreated (mock-treated) (MOI 0.01 , 6hpi)
  • b Levels of lbAr10200 CCHFV infections in SW13 cells, treated with a range of soluble LDLR concentrations (MOI 0.01 , 24hpi).
  • c Level of VSV infections in SW13 cells, treated with the indicated concentrations of soluble LDLR (MOI 0.01 , 6hpi).).
  • Figure 5 shows CCHFV infections in human BVOs and Ldlr mutant mice, a, Scheme representing blood vessel organoids made from LDLR+ and LDLR- iPSC cells that were dissociated and seeded as 2D monolayer, b, Level of CCHFV (lbAr10200) infection of BVO-derived vascular cells generated from wild type (WT) and LDLR KO iPSCs. Levels of infections were determined by RT-PCR at 1day post-infection (1dpi) and 3 days post-infection (3dpi) (MOI 0.1 ). P values were calculating using unpaired student’s t-tests.
  • c-e CCHFV (lbAr10200) infections of wild type or Ldlr KO mice.
  • n 6 fenescence mice per group (400 PFU/mouse).
  • c Weight loss was assessed the day of infection (day 0) and the day of euthanasia (day 4 post-infection),
  • d Numbers of CCHFV RNA copies in serum, liver and spleen of wild type and Ldlr KO mice determined the day of euthanasia (day 4 post-infection). P values were calculating using unpaired student’s t-tests comparing two groups. All graphs are show as mean values ⁇ SD.
  • Figure 6 shows validation of LDLR with a CCHFV patient isolate.
  • CCHFV was isolated from the serum of a Turkish patient and this clinical isolate used for all subsequent experiments shown in Figure 6.
  • a-c Levels of CCHFV infections (MOI 0.01 , 24hpi) of SW13 cells treated with the indicated concentrations of a, sLDLR, b, sLRP8, and c, sVLDLR.
  • d Levels of infection with clinical CCHFV in wild type and LDLR KO (clones C2 and C12) Vero cells and in wild type and LDLR KO (clones C8, C10 and C11 ) A549 cells (MOI 0.1 , 24hpi).
  • Graphs show mean value ⁇ SD.
  • n 3 independent experiments. P values were calculating using unpaired student’s t-tests.
  • hpi hour post-infection.
  • Figure 7 shows generation and validation of Ldlr knockout in A549 and Vero cells, a, Schematic of CRISPR-Cas9 editing strategy. The extracellular region of LDLR was targeted, leading to putative N-terminally truncated proteins not displayed on the cell surface for entry, b, Schematic of editing and a-LDLR sorting procedure, c, PE intensity from a-LDLR- PE staining is shown. a-LDLR-PE staining was evaluated on single cells. Event densities were smoothened and are displayed as absolute counts or as counts normalization to the mode. Numbers indicate the percentage of single cells defined as a-LDLR-PE negative, d, Non- reactive and stained Tb1-Lu cells were used as negative control.
  • PE intensity from a-LDLR-PE staining is shown. Event density was smoothened by normalization to the mode. e, Bulk sorting after transfection and transient Puromycin selection of a-LDLR-PE stained A549 or Vero cells, edited or unmodified (control) via CRISPR-Cas9. Event densities were smoothened and are displayed as counts normalization to the mode, f, Flow-cytometry result from A549-wild type and edited A549 clone 10 cells. PE intensity from a-LDLR-PE staining is shown. Event density was smoothened by normalization to the mode. Numbers indicate the percentage of single cells defined as a- LDLR-PE positive for A549 clone 10 and unmodified WT cells. Event densities were smoothened and are displayed as absolute counts.
  • Figure 8 shows that ligand selectivity at LDLR drives receptor internalization.
  • Figure 9 shows creation and validation of NC8 cells knocked out for LDLR.
  • a Gating strategy and PE intensity from a-LDLR-PE staining are shown.
  • a-LDLR-PE staining was evaluated on single cells
  • b Sorting results from bulk NC8 iPSC after Cas9 LDLR editing.
  • PE intensity from a-LDLR-PE staining is shown for cells targeted with an LDLR guide RNA or for unmodified control cells. Event densities were smoothened and are displayed as absolute counts or as counts normalization to the mode, c, Qualitative flow-cytometry result of selected clones stained with an a-LDLR-PE antibody.
  • FIG. 11 Immunofluorescence assays a, Immunofluorescence staining of CCHFV in wild-type and LDLR KO cells. P values were calculating using two tailed student t-test. **P ⁇ 0.01. b, Immunofluorescence staining of CCHFV in SW13 cells infected with CCHFV mock or sLDLR treated.
  • CCHFV produced on Hyalomma tick cells (a) and CCHFV from human patient serum (b) were tested for blocking with sLDLR or sLRP8 (MOI 0.01 , 24hpi).
  • Graphs show mean value ⁇ SD.
  • n 3 independent experiments. P values were calculating using two tailed student t-test. **P ⁇ 0.01 ; Non significant: p>0.05.
  • FIG. 14 Protective effect of LDLR, LDLR-fragments, and multimers against CCHF-VLP infection CCHF-VLP infection assays in Huh-7 cells. Luminescence was normalized to infection control. VLPs were incubated with 10nM purified LDLR-fragments or commercially available rhLDLR (RnD System; Cat.- No. 2148-HP) or rhLDLR-mFc (SinoBiological; Cat.-No. 10231-H05H). Graph shows mean value + SEM; n > 3 independent experiments; p- values were calculated using two tailed student t-test: * p ⁇ 0.05, ** p ⁇ 0.01 , *** p ⁇ 0.001.
  • sLDLR-sLRP8 expressed and purified from E.coli concentration ⁇ 5nM: sLDLR-sLRP8 (2.4nM), sLDLR-sVLDLR (1.7nM), sLRP8-sLDLR (4.4nM), sVLDLR-sLDLR (1.4nM).
  • s soluble (receptor ectodomain without the transmembrane anchor)
  • HEK293 ATCC®, CRL-1573
  • HEK293T/17 HEK293T, ATCC® CRL-11268TM
  • A549 ATCC® CCL-185
  • Vero cells ATCC® CCL-81
  • All cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Life Technologies, Italy), supplemented with 10 % v/v of heat-inactivated Fetal Bovine Serum (FBS, Life Technologies) and incubated at 37° C with 95 % humidity and 5% CO2.
  • DMEM Modified Eagle’s Medium
  • FBS heat-inactivated Fetal Bovine Serum
  • SW13 epidermal cells from adrenal gland, ATCC® CCL-105 cells were maintained in Leibovitz’s L15 medium (Thermofisher) at 37° C without CO2.
  • Haploid mouse Stem-Cells mSCs, clone AN3-12 were maintained in standard embryonic stem-cell medium, supplemented with 10% (v/v) fetal bovine serum (Hyclone), recombinant mouse Leukemia Inhibitory Factor (LIF) and [3-mercaptoethanol at 37°C with 95 % humidity and 5% CO2. All cell lines were regularly tested for mycoplasma contamination.
  • VSV-CCHF_G was produced as described below.
  • CCHFV lbAr10200 strain was cultured on SW13 cells.
  • CCHFV clinical strain was isolated on SW13 cells from a Turkish patient serum sampled as a part of another project. Ethical clearance has been obtained (Nr: 2017/1712-31/2) as well as fully informed patient consents.
  • D-PBS, DMEM, Trypsin, PBS, penicillin/streptomycin, fetal bovine serum were from Gibco (ThermoFisher Scientific, Waltham, MA, USA).
  • Polyethylenimine (PEI) was purchased from Alfa Aesar (ThermoFisher Scientific, Waltham, MA, USA).
  • Unlabeled low-density lipoprotein (LDL) from human plasma and BOPIDY FL complexed LDL were purchased from ThermoFisher Scientific (Waltham, MA, USA).
  • CCHFV Gc Human Fc-tagged CCHFV Gc, 6xHis-tagged CCHFV Gn and BODIPY FL complexed CCHFV Gc were purchased from Native Antigen (Kidlington, UK). Coelenterazine h was purchased from Nanolight Technologies (Pinetop, AZ, USA). NanoBRET Nano-Gio substrate was purchased from Promega (Madison, Wl, USA). Trizol was purchased from ThermoFisher (ThermoFisher Scientific, Waltham, MA, USA). Anti-IFN type I receptor antibody (MAR1 -5A3) was purchased from Leinco (MAR1 -5A3 [5A3]; Leinco Technologies, Inc.).
  • Soluble LDLR, VLDLR and LRP8 were purchased from R&D systems.
  • This soluble LDLR (Catalog #: 2148-LD R&D systems) corresponds to human LDLR protein (Uniprot P01130) Ala22-Arg788;
  • soluble VLDLR (Catalog #: 8444-VL-025 R&D systems) corresponds to human VLDLR protein (UniProt: P98155.1) Thr25- Ser797;
  • soluble LRP8 Catalog #: 3520-AR-050, R&D systems) corresponds to human LRP8 protein (UniProt: Q14114.4) Asp35-Lys818 (Ala262Val).
  • the plasmid pC-G (Shtanko et al, 2014, Crimean-Congo hemorrhagic fever virus entry into host cells occurs through the multivesicular body and requires ESCRT regulators.
  • VSV-G Vesicular Stomatitis Virus glycoprotein
  • pVSV-G The plasmid expressing the Vesicular Stomatitis Virus (VSV) glycoprotein (pVSV-G) was previously described (Salata et al., 2009, vOX2 glycoprotein of human herpesvirus 8 modulates human primary macrophages activity. J Cell Physiol. 2009 Jun;219(3):698-706).
  • VSVAG-GFP The recombinant VSV encoding the GFP in place of the VSV-G gene
  • CCHFV-Gn/Gc pseudotyped VSVAG-GFP (CCHFV-pseudotyped virus) was generated as previously described.
  • HEK293T cells were seeded in T75 flask and 24 h later transfected by calcium-phosphate protocol with 20 pg of pC-G plasmid and, 24 h later, infected with the recombinant VSVAG-GFP virus at the multiplicity of infection (MOI) of 4 fluorescent focus-forming units (FFU)/cell.
  • MOI multiplicity of infection
  • FFU fluorescent focus-forming units
  • virus particles were pelleted by ultracentrifugation on a 20% p/v a sucrose cushion (27,000 rpm for 150 min at 4° C) in a Beckmann (Beckman Coulter Italia, Italy) SW 28 Ti swingingbucket rotor. Pellets were resuspended in 1 mL of ice-cold PBS1X/tube and mixed. Subsequently, the virus was aliquoted and stored at -80° C until use. Virus titer was determined by immunofluorescence on Vero cells seeded on 96-well plates. Viral stock was 10-fold serially diluted in DMEM and inoculated on confluent Vero cells for 1 h at 37° C.
  • VSV-M [clone 23H12], Kerafast) antibody Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibodies (ThermoFisher). The fluorescent foci were counted and viral titer was expressed as FFU/mL.
  • Chemical mutagenesis of haploid stem-cells Chemical mutagenesis using Ethylnitrosurea (ENU) was performed treating haploid AN3-12 cells for 2h with 0.1 mg/ml ENU in full medium while in suspension and under constant agitation. Cells were washed 5 times and transferred to a culture dish. Cells were left to recover for 48h, singled using Trypsin/EDTA and frozen in 10% DMSO, 40% FBS and 50% full medium. ENU libraries as well as untreated control libraries were used in screening experiments with VSV-CCHFV.
  • ENU Ethylnitrosurea
  • Haploid cells screens and analysis 50 million haploid mSCs were thawn and infected with VSV-CCHF_G at a MOI of 10 in 5 ml of ES medium without FBS. One hour after infection, the cells were supplemented with complete ES medium and incubated at 37°C with 5% CO2. After outgrowth of virus resistant cells, cell clones were picked separately and cultured before being validated by infection assay with CCHFV lbAr10200. Briefly, cells (AN3-12 wild-type and potentially resistant clones) were seeded at 5x10 4 cells per well in DMEM 5% FBS for 24h.
  • A549 (ATCC CCL-185) and Vero (CCL-81) cells were grown in complete DMEM medium (DMEM High Glucose supplemented with 10% Fetal bovine serum (Gibco), 1x MEM-NEAA (Gibco), 1x Glutamax (Gibco), 1 mM Sodium Pyruvate (Gibco), 100 ll/rnl Penicillin-Streptomycin (Gibco)).
  • the day before transfection 1 .05x10 5 cells were seeded per well of a 24- well plate in 0.5 complete DMEM medium.
  • culture medium was replaced with fresh complete DMEM medium and transfected with a liposome:DNA mixture composed of 50pl Opti-MEM I (Gibco), 500ng of PX459 v2.0 plasmid coding (Addgene Plasmid #62988, Puro resistant), 1.5 pl Lipofectamine 3000 reagent and 1.0 pl P3000 reagent.
  • Opti-MEM I Gibco
  • PX459 v2.0 plasmid coding Additional Plasmid #62988, Puro resistant
  • 1.5 pl Lipofectamine 3000 reagent 1.5 pl Lipofectamine 3000 reagent
  • Several sgRNA were derived from CRISPick (https://portals.broadinstitute.org/gppx/crispick/public) using SpCas9 Cas9 knockout and the human LDLR gene as input.
  • the final guide LDLR RNA sequence used for knockout studies was gATGAACAGGATCCACCACGA (SEQ ID NO 1 ), where lower latter g denotes preceding Guanosine to enhance transcription from the U6 Promoter.
  • medium was replaced with complete DMEM supplemented with 1 pg/ml Puromycin for transient selection. 60 hours post transfection, each well containing selected A549, or Vero cells were expanded to one well of a 6-well plate in complete DMEM medium. Once cell reached 80% confluency, they were dissociated with 500 l with TrypLE Express enzyme solution (Gibco) for 5 minutes and collected in FACS Buffer (D-PBS containing 5% FBS).
  • LDLR-PE Antibody R&D Systems FAB2148P
  • 10 pl of a-LDLR-PE Antibody per 1.0 x 10 6 cells were added and stained for 1 h on ice in the dark. Unmodified cells were used as controls. After one hour of staining, cells were collected by centrifugation and washed twice in FACS Buffer. Finally, cells were resuspended in 1 ml of FACS Buffer and LDLR-negative cells were sorted into individual wells of a 96-well plate. LDLR-negative cells were defined as single cells displaying no PE fluorescence.
  • Soluble LDLR, VLDLR and LRP8 assays SW13 were seeded at a density of 5.0 x 10 4 cells per well in a 48-well plate. 24h post-seeding, cells were counted to define the quantity of virus needed for an infection at a MOI of 0.01 . The virus was then mixed in 1.5 ml tubes (Sarstedt) with the appropriate quantity of sLDLR, sVLDR or sLRP8 in L15 medium containing 0.5% FBS. The tubes were the incubated for 30 min under shaking (75rpm) at 37°C.
  • Plasmid DNA constructs for BRET assay To generate LDLR-Rlucll, codon- optimized LDLR was synthesized as a gBIock (Integrated DNA Technologies) and subcloned by Gibson assembly in pcDNA3.1/Hygro(+) GFP 10 -F?lucll db v.2 that had been linearized by PCR to exclude GFP 10 .
  • To generate Nluc-LDLR codon- optimized LDLR from LDLR-Rlucll was amplified by PCR and subcloned by Gibson assembly in pcDNA3.1 NIuc-synFZDs that had been linearized by PCR to exclude FZDs.
  • rGFP-FYVE (Namkung et al., 2016. Monitoring G protein-coupled receptor and (3-arrestin trafficking in live cells using enhanced bystander BRET. Nat Commun. 11 ;7:12178) has been described previously. All plasmid constructs were verified by Sanger sequencing.
  • HEK293 cells were propagated in plastic flasks and grown at 37°C in 5% CO2 and 90% humidity. Cells (350,000 in 1 ml) were transfected in suspension with 1.0 pg of plasmid DNA complexed with linear polyethyleneimine (PEI; MW 25,000, 3:1 PEI:DNA ratio). All cell lines were regularly tested for mycoplasma contamination.
  • PEI linear polyethyleneimine
  • BRET assays Receptor trafficking-.
  • HEK293 cells were transfected with LDLR-Rlucll and rGFP-FYVE and seeded in 6-well plates (7.0 x 10 5 cells/well). After a 48-hour incubation, cells were washed once with HBSS, detached and resuspended in HBSS containing 0.1 % BSA before distribution into white 96-well plates containing serial dilutions of LDL, CCHFV Gc or CCHFV Gn and returned to the incubator for 45 min at 37°C. Prior to BRET measurements, cells were incubated with coelenterazine h (10 min).
  • NanoBRET binding assay To monitor the binding of fluorescent ligands to LDLR, HEK293 cells were transfected with Nluc-LDLR and seeded in white 96-well plates (3.5 x 10 4 cells/well). After a 48-hour incubation, cells were washed once with HBSS and maintained in the same buffer. Prior to BRET measurements, cells were incubated with NanoBRET Nano-Gio substrate (6 min) and then stimulated with either BODIPY FL LDL or BODIPY FL Gc for 90 min following a baseline measurement of 3 cycles.
  • BRET measurements Plates were read on a Tecan Spark multimode microplate reader (Mannedorf, Switzerland) equipped with a double monochromator system to measure the emission of the Rlucll/rGFP donor-acceptor pair in receptor trafficking experiments [430-485 nm (donor) and 505-590 nm (acceptor)] or the Nluc/BODIPY FL donor-acceptor pair in the NanoBRET binding assay [445-470 nm (donor) and 520-575 nm (acceptor)].
  • NC8 iPSC male, pericyte derived
  • Matrigel hESC qualified, Coming
  • Stemflex medium Gibco + 1 :100 Antibiotic-Antimycotic (Gibco) (Invivogen).
  • Cells were passaged using 0.5mM EDTA at a ratio of 1 :6 every 3 to 4 days.
  • iPSCs were dissociated into single cells using TrypLE select (Gibco) and seeded at 5.0 x 10 4 cells per 1-well of a rhLaminin521 (Gibco) coated 24-well plate in complete Stemflex medium supplemented with 1 :100 RevitaCell (Gibco).
  • Blood-vessel organoids from NC8 clone 10 (LDLR+) and clone 4 (LDLR-) were produced as previously described (Wimmer et al, Generation of blood vessel organoids from human pluripotent stem cells. Nat Protoc. 2019; 14(11 ):3082-3100).
  • the cells were collected through centrifugation (300xg, 5 min) and replated in PureCol (Advanced BioMatrix, 30pg/ml in PBS for 1 h at RT) coated T-25 flasks at 30840 cells/cm 2 in Sprouting media.
  • PureCol Advanced BioMatrix, 30pg/ml in PBS for 1 h at RT
  • mice In vivo experiments. In the current study were used six female C57BL/6J mice (Charles River, Germany) and six female B6.129S7Ldlrtm1 Her/J (LdLr KO) (stock#002207), (Jackson Laboratory, USA). All mice were 10 weeks old at the time of infection. The animals were housed according to Karolinska Institute ethical rules and observed daily.
  • mice were challenged with 400 focus forming units (FFU) of CCHFV lbAr10200 in 100 pl via i.p injection.
  • FFU focus forming units
  • the mice were monitored daily with respect to clinical signs of disease, and their overall well-being.
  • FFU focus forming units
  • mice were euthanized independent on clinical signs.
  • Blood was collected in microcontainer tubes for serum separation and serum was inactivated with Trizol for subsequent qRT-PCR analysis.
  • liver, spleen and kidney were collected, a part in Trizol for qRT-PCR and a part were fixed in PFA 4% for histopathological analyzes.
  • the experimenters were not blinded to the identity of the animals. However, the pathologist who analyzed livers as well as the scientist who runned the RT-qPCRs and the subsequent analysis were blinded.
  • RNase P RNA was used as an endogenous control for normalization (Fwd: AGATTTGGACCTGCGAGCG (SEQ ID NO 12), Rev: GAGCGGCTGTCTCCACAAGT (SEQ ID NO 13), Probe: FAM- TTCTGACCTGAAGGCTCTGCGCG-MGB (SEQ ID NO 14)).
  • CCHFV RNA Absolute quantification of CCHFV RNA for mice samples was performed by RT- qPCR.
  • the standard synthetic RNA was solubilised in RNase-free water and the copy number calculated after quantification by nanodrop.
  • SW13 were seeded at a density of 5.0 x 10 4 cells per well in a 48-well plate. 24h post-seeding, cells were counted to define the quantity of virus needed for an infection at a MOI of 0.01 .
  • CCHFV was then mixed in 1.5 ml tubes (Sarstedt) with different concentration of LDL (Thermofisher, #L3486) or BSA (Saveen & Werner AB, #A1391 ) in L15 medium containing 0.5% FBS. Cells were rinsed once with PBS before being infected with virus only or with the mix virus/LDL or virus/BSA for 1 hour at 37°C.
  • Hyalomma anatolicum embryo-derived cell lines HAE/CTVM9 were grown L- 15/M EM medium (equal volumes of L-15 and Minimal Essential Medium with Hank’s salts supplemented with 10% TPB), both supplemented with 2 mM l-glutamine, 20% FBS, and incubated in sealed flasks at 28°C, 0% CO2.
  • CCHF L and N gene were seeded into each well of 24-well plates. Once cells attached, transfection of CCHF L and N gene (strain lbAr10200) was performed using GeneJammer Transfection Reagent (Agilent; Cat.-No. 204130). 16h post transfection indicator cells were infected with an 10Oul inoculum containing CCHF- VLPs (NanoLuc) after 1h pre-incubation with a protein-fragment of interest where applicable. 24hpi the wells were washed with PBS and analyzed using Nano-Gio® Luciferase Assay System (Promega; Cat.-No. N1110) according to manufacturer’s protocol. Luminescence was quantified with a plate reader (BMG Clario Star).
  • Example 1 Haploid cells screening highlighted LDLR as an essential protein for VSV-CCHFGP/AG entry
  • ENU mutagenized mES cells were infected with a viral RNA replication competent vesicular stomatitis virus pseudotyped with the glycoproteins of the Crimean-Congo hemorrhagic fever virus (VSV-CCHF_G) (see Figure 1A).
  • VSV-CCHF_G Crimean-Congo hemorrhagic fever virus
  • Table 2 Sequencing data of the three clones highlighting LDLR.
  • Example 2 Validation of LDLR in CCHFV infections
  • Ldlr mutant haploid mouse embryonic cells were assessed. These Lcf/r-knockout cells and wild-type sister cells were infected with VSV-CCHF_G and CCHFV. These murine Ldlr- knockout cells displayed more than a 90% decrease in infection rates compared to the wild type cells (Fig. 2a).
  • Example 3 Gc binds to LDLR and induces endocytosis
  • a bioluminescence resonance energy transfer (BRET) assay was developed to assess receptor-ligand interactions.
  • BRET bioluminescence resonance energy transfer
  • the inventors genetically engineered and expressed an LDLR that carries an N-terminal bioluminescent probe (NanoLuc or NIuc) in HEK293 cells. Addition of fluorescent ligands then allow to measure direct interaction through BRET (Fig. 3a).
  • BODIPY-FL-labelled LDL was initially used to assess receptor binding, resulting in the expected concentration-dependent ligandbinding BRET signal in cells expressing Nluc-LDLR (Fig. 3b).
  • sLDLR soluble LDLR
  • VSV and VSV-CCHF_G infected cells have to be assessed at early time points post-infection to avoid a second round of infection.
  • the cells were harvested and the levels of infection determined by qRT-PCR.
  • sLDLR blocked the infection of rVSV-CCHF (Fig. 4a) and, importantly, CCHFV (Fig. 4b) in a dose-dependent manner.
  • sLDLR was also able to inhibit VSV (Fig. 4c)
  • sLRP8 is a one chain LRP8 Asp35-Lys818 (Ala262Val) fragment and sVLDLR a one chain VLDLR Thr25-Ser797 fragment.
  • sLDLR neither sVLDLR nor sLRP8 decoys provided protection from CCHFV infections (Fig. 4d, e), while they were active against VSV infection (Fig. 4f, g). This data indicates that soluble LDLR can prevent CCHFV infections.
  • Example 5 Infection of blood-vessels organoids
  • Blood vessels are key target cells for viral tropism involved in hemorrhaging.
  • Human blood vessel organoids were generated using the iPSC line NC8. Ldlr in the iPSCs was deleted using CRISPR/Cas9-mediated genome editing. Knockout iPSCs for Ldlr were validated by flow-cytometry (Fig. 9a - e). For infection were generated Ldlr mutant and wild type blood vessel organoids (BVOs), containing self-organizing bona fide capillaries formed by pericytes and endothelial cells.
  • BVOs wild type blood vessel organoids
  • the BVOs containing mature human capillaries were disaggregated, and the pericytes and endothelial cells were further cultured as monolayers in collagen-coated flasks (Fig. 5a). These cultures were subsequently infected with CCHFV. Knockout of Ldlr, using two different mutant iPSC clones, resulted in a significant reduction in CCHFV infections, detected one and three days post-infection (Fig. 5b). These data show that LDLRs are also involved in infections of human blood vessels.
  • Example 6 LDLR KO protects mice against CCHF or LDLR mutant/KO mice are protected from CCHF
  • C57BL/6J wild type and Ldlr-I- mice are naturally resistant to CCHF, but blockade or knockout of IFNa receptors render these mice susceptible to the infection, as previously reported. Therefore wild type and Ldlr-I- mice were treated with 2.5mg of anti-IFNa receptor antibodies at the time of CCHFV infection (400 PFU per mouse).
  • All mice were euthanized, and their serum, liver, and spleen were analyzed for viral RNA and livers were assayed for pathologies using histology.
  • Ldlr-I- mice did not show any weight loss or other macroscopic signs of disease (weakness, swollen eyes, marked incoordination, piloerection, light bleeding around the marking hole in the ear) (Fig. 5c). Ldlr-I- mice also exhibited significantly reduced level of viral RNA in the serum, with two mice even showing undetectable levels of circulating virus (Fig 5d). The mutant mice also exhibited reduced virus load in liver and spleen (Fig. 5d). Histopathological analysis of livers of CCHFV-infected wild type mice revealed midzonal necrosis (Fig. 5e upper left panel), periportal coagulative necrosis (Fig.
  • Example 7 LDLR is also a receptor for CCHFV isolate
  • LDLR mutant Vero and A549 cells challenged with the CCHFV patient isolate showed significantly reduced infection rates as compared to their respective LDLR expressing wild type control cells (Fig. 6d). This data confirms that LDLR also acts as a receptor for patient-derived CCHFV isolates.
  • Example 8 Analysis of binding of LDLR fragments, and homo- and heteromultimers thereof to viruses.
  • the LDLR fragments of the LDLR ectodomain as described above, and homo- and heteromultimers thereof covalently linked with or without a peptide linker, are expressed in bacteria, insect cells, or mammalian cells. Following purification, fragments are tested by: a) cell-based assays for their ability to block virus infections, preferably Bunyavirus infections, and more preferred, CCHFV infections. To this end wildtype virus strains, pseudotyped viruses, or viruses and pseudotyped viruses expressing marker proteins such as GFP can be used for infection of relevant cell lines.
  • LDLR fragments Infection rates in the presence and absence of the LDLR fragments will be assessed by quantitative analysis of viral genomes or protein, including artificially expressed marker proteins such as GFP.
  • biochemical binding assays for their ability to bind virus glycoproteins, preferred bunyavirus glycoproteins, more preferred Gc and Gn protein.
  • Virus envelope gylcoproteins will be expressed and purified or purchased and then immobilized on suitable plates.
  • LDLR fragments, and homo- and heteromultimers thereof will be extended by a peptide Tag such as a polyhistidine tag (6xH), a FLAG tag (DYKDDDDK), or a human influenza hemagglutinin (HA) tag (YPYDVPDYA), expressed and purified. Purified fragments and multimers thereof will be incubated with the immobilized glycoproteins. After several wash steps, high affinity binding peptides will be identified via antibody-based detection methods.
  • VLPs transcription competent virus-like-particles
  • the CCHFV L and N genes were co-transfected into the indicator cells 16h prior to infection.
  • Pre-incubation of VLPs with full length recombinant human LDLR rhLDLR, RnD Systems; Cat.-No.
  • LDLR purified soluble LDLR expressed from Hek293 cells
  • CTR VLPs only
  • fragments of LDLR containing only the ligand-binding domains LA1-7 or LA4-7 show protective effects against infection of Huh-7 cells with CCHF-VLPs. Multimerization of LDLR fragments containing a linker (I) between repetitive domains such as LDLR LA1-7I1-7 and LDLR LA2-3I2-3I2-3 also ameliorate infection, while a single LDLR LA2-3 fragment does not reach significance.
  • Combinations of sLDLR fused to other LDLR family members (LRP8 and VLDLR) increases its potency to protect against CCHF-VLP infection (note that concentrations ⁇ 5nM were applied here).
  • the closely related LDLR family member VLDLR alone shows significant protection against infection with CCHF-VLPs (Fig. 14).
  • LDLR Nucleotide Sequence: LDLR (2-3) (SEQ ID NO 199) ATGGGACCATGGGGATGGAAGCTGCGCTGGACTGTGGCCCTTCTGCTTGCGGCGGCAGGAA CcgcaGTGGGTGACCGGGGCAGCGGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGG CGGAGTAACCTGCAAATCGGGAGACTTCAGCTGTGGTGGACGGGTTAACCGCTGTATACCTC AGTTTTGGCGGTGTGACGGACAAGTTGACTGTGATAATGGCTCAGATGAGCAGGGGTGCCCG CCAAAGACCTGCAGTCAGGACGAATTCCGATGCCACGATGGAAAATGTATTAGCCGTCAATTC GTTTGTGACTCGGACCGCGACTGTCTGGATGGTTCAGATGAAGCTTCGTGCCCAGTAgGAGA CGCTTCGgattacaaagacgatgacgataagCTGGAAGTTCTGTTCCAGGGGCCCGGTCATCACCACCA TCATCACCA
  • LDLR (2-3) (SEQ ID NO 200) MGPWGWKLRWTVALLLAAAGTAVGDRGSGVSGWRLFKKISGGVTCKSGDFSCGGRVNRCIPQF
  • sLDLR (SEQ ID NO 202) MGPWGWKLRWTVALLLAAAGTAVGDRGSGVSGWRLFKKISGGAVGDRCERNEFQCQDGKCISY KWVCDGSAECQDGSDESQETCLSVTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGC PPKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPVLTCGPASFQCNSSTCIPQLWACD NDPDCEDGSDEWPQRCRGLYVFQGDSSPCSAFEFHCLSGECIHSSWRCDGGPDCKDKSDEEN CAVATCRPDEFQCSDGNCIHGSRQCDREYDCKDMSDEVGCVNVTLCEGPNKFKCHSGECITLDK VCNMARDCRDWSDEPIKECGTNECLDNNGGCSHVCNDLKIGYECLCPDGFQLVAQRRCEDIDEC QDPDTCSQLCVNLEGGYKCQCEEGFQLDPHTKACKAVGSIAYLF
  • VLDLR (SEQ ID NO 209)
  • VLDLR (SEQ ID NO 210)
  • sLDLR-sVLDLR (SEQ ID NO 218) MGPWGWKLRWTVALLLAAAGTAVGDRGSGVSGWRLFKKISGGSGAVGDRCERNEFQCQDGKC ISYKWVCDGSAECQDGSDESQETCLSVTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDE QGCPPKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPVLTCGPASFQCNSSTCIPQLW ACDNDPDCEDGSDEWPQRCRGLYVFQGDSSPCSAFEFHCLSGECIHSSWRCDGGPDCKDKSD EENCAVATCRPDEFQCSDGNCIHGSRQCDREYDCKDMSDEVGCVNVTLCEGPNKFKCHSGECIT LDKVCNMARDCRDWSDEPIKECGTNECLDNNGGCSHVCNDLKIGYECLCPDGFQLVAQRRCEDI
  • sLRP8-sLDLR (SEQ ID NO 220) MGPWGWKLRWTVALLLAAAGTAVGDRGSGVSGWRLFKKISGGSGHLAAAAADPLLGGQGPAKD CEKDQFQCRNERCIPSVWRCDEDDDCLDHSDEDDCPKKTCADSDFTCDNGHCIHERWKCDGEE ECPDGSDESEATCTKQVCPAEKLSCGPTSHKCVPASWRCDGEKDCEGGADEAGCATSLGTCRG DEFQCGDGTCVLAIKHCNQEQDCPDGSDEAGCLQVPPTFLGNRRRPRGLNECLHNNGGCSHICT DLKIGFECTCPAGFQLLDQKTCGDIDECKDPDACSQICVNYKGYFKCECYPGYEMDLLTKNCKAA AGKSPSLIFTNRHEVRRIDLVKRNYSRLIPMLKNVVALDVEVATNRIYWCDLSYRKIYSAYMDKASD PKEQEVLIDEQLHSPEGLAVDWVHKHI
  • HiBiT-Tag VSGWRLFKKIS (SEQ ID NO 224) s: soluble (receptor ectodomain without the transmembrane anchor) *: stop codon encoded by the last triplet of the nucleotide sequence.
  • Example 9 A phage-display approach to identify high affinity binding LDLR fragments and variants thereof to viral glycoproteins in a high-throughput format (Fig. 10).
  • a viral glycoprotein of interest (preferred from Bunyaviridae, more preferred from CCHFV) is expressed, purified, and immobilized on a surface.
  • a DNA pool encoding LDLR family fragments, and single and multiple amino acid variants thereof is cloned into a phagemid, i.e. an expression vector that expresses the cloned peptides as part of a bacteriophage envelope protein.
  • the phagemids are transfected into bacteria cells to produces phages, wherein each phage displays a different peptide or set or peptides on its surface, wherein each peptide is a different LDLR fragment or an amino acid variant thereof.
  • Display phages are incubated on the immobilized glycoprotein and will attach in dependence of the interaction strength between glycoprotein and LDLR fragment on phages.
  • Example 10 Immunofluorescence stains confirm requirement of LDLR for CCHFV infections.
  • Example 11 LDL outcompetes CCHFV for cell entry via the LDLR.
  • CCHFV glycoproteins can bind to LDLR.
  • a competition assay using CCHFV and LDL, the natural ligand for LDLR was conducted.
  • LDL effectively competed with CCHFV infection in a dose-dependent manner for cell entry via LDLR.
  • Bovine Serum Albumin (BSA) was used as a control and showed no impact on CCHFV infection.
  • BSA Bovine Serum Albumin
  • CCHFV lbAr10200 was initially cultured on a Hyalomma tick cell line, the natural vector, and the potential inhibitory effects of sLDLR and sLPR8 were evaluated.
  • sLDLR demonstrated the ability to block the virus, while sLRP8 did not (Fig. 13a).
  • a patient isolate that was cultivated on tick cells (see Example 7).
  • the capacity of LDLR and LRP8 to block CCHFV was examined using virus present in patient serum without prior propagation on tick or human cells. As illustrated in Fig. 13b, both sLDLR and sLRP8 were effective in blocking the virus from this direct patient serum.
  • LDLR LDLR family members like LRP8 can also interfere with virus spreading, potentially due to an altered protein decoration of the virus as compared to virus propagated in vitro.
  • Example 13 Quality control of Bunyavirus vaccines.
  • LDLR is used to assess Bunyavirus vaccine integrity or affinity of antigens for diagnostic purposes and especially to assess CCHFV vaccine integrity or affinity of antigens for diagnostic purposes.
  • LDLR or LDLR fusion proteins or LDLR variants can be used to assess a bunyavirus vaccine, preferably a CCHFV vaccine, quality or the integrity of a diagnostic antigen, e.g. from patient samples.
  • LDLR is a receptor that directly interacts with multiple bunyaviruses, such as CCHFV, it can be used to physically assess the quality and integrity of a bunyavirus vaccine, which is important to define lot-to-lot variation in commercial production. Further, LDLR can be used to detect diagnostic antigens, e.g. in a lateral flow immunoassay.
  • Bunyavirus vaccine or diagnostic antigens can include, but are not limited to, live viruses from patient samples, live-attenuated viruses, virus-like particles, viral structural proteins, viral glycoproteins, or nucleic acids or vectors producing these viral proteins or particles.
  • the format of the integrity assay can include immobilizing LDLR or LDLR fusion proteins (e.g. LDLR-Fc), or LDLR variants on a surface (e.g., microtiter well plate, chip, or slide) followed by incubation with the bunyavirus vaccine or diagnostic antigen and detection with a monoclonal or polyclonal anti-vaccine I anti- diagnostic antigen antibody.
  • detection can be achieved via LDLR-Fc itself.
  • the bunyavirus vaccine or diagnostic antigen can be captured on the surface (directly or indirectly) and then incubated with LDLR or an LDLR fusion protein or an LDLR variant for binding analysis.
  • the assay could be performed by detection methods suitable to assess binding including, but not limited to ELISA, biolayer interferometry and surface plasmon resonance.

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Abstract

The present invention relates to an LDLR derived polypeptide that binds a Bunyavirales virus and is thus useful in the prophylaxis and/or treatment of a Bunyavirales virus infection in a subject, preferably a CCHV infection. Described herein are LDLR derived polypeptides comprising an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain, which bind a Bunyavirales virus. Also described are LDLR derived polypeptides comprising a multimer of an LDLR ectodomain (SEQ ID NO 18), or of a functional fragment of said LDLR ectodomain, or of an amino acid variant of said LDLR ectodomain, or of an amino acid variant of said functional fragment of said LDLR ectodomain, wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said monomers. The LDLR polypeptides are particularly useful against Bunyavirales infections. Further, the LDLR polypeptides can be used for diagnostic detection of Bunyavirales virus infection, particularly CCHFV infection, and quality control testing of related vaccines. A method to identify new gene factors involved in a CCHFV infection is also disclosed.

Description

LDLR derived polypeptides for anti-viral uses
Specification
The present invention relates to an LDLR derived polypeptide that binds a Bunyavirales virus and is thus useful in the prophylaxis and/or treatment of a Bunyavirales virus infection in a subject, preferably a CCHV infection. Described herein are LDLR derived polypeptides comprising an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain, which bind a Bunyavirales virus. Moreover, herein are also described LDLR derived polypeptides comprising a multimer of an LDLR ectodomain (SEQ ID NO 18), or of a functional fragment of said LDLR ectodomain, or of an amino acid variant of said LDLR ectodomain, or of an amino acid variant of said functional fragment of said LDLR ectodomain, wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said monomers. The LDLR polypeptides here described are particularly useful against Bunyavirales virus infections. Further, the LDLR polypeptides can be used for diagnostic detection of Bunyavirales virus infection, particularly CCHFV infection, and quality control testing of related vaccines. A method to identify new gene factors involved in an infection by a Bunyavirales virus is also provided herein.
Background of the invention
Crimean-Congo hemorrhagic fever virus (CCHFV) is the causative agent of Crimean-Congo hemorrhagic fever (CCHF), an emerging infectious disease that can lead to severe cases and death, with a mortality of up to 40%. Currently, there are no approved vaccines or specific therapeutics available against CCHFV.
CCHF is transmitted by Hyalomma ticks and through direct contact with the blood and other bodily fluids of patients or infected animals. The CCHF incubation period is 2-14 days and correlates with the type of transmission and viral load. The clinical manifestations can be from subclinical illness to acute infection with haemorrhage such as pulmonary haemorrhage, intra-abdominal bleeding, or haematuria, and multiorgan failure.
CCHF is the most widespread haemorrhagic fever, being endemic in certain regions of Africa and in Asia but also in Europe. CCHFV is a tick-borne pathogen that is also capable of interpersonal transmission. The critical lack of approved interventions against CCHFV, either prophylactic or therapeutic, combined with its increasing spread and new autochthonous introductions, constitutes a serious public health threat for many regions.
Despite intensive research in last years, much of the molecular pathogenesis of CCHFV is still unknown, including the identity of its receptor(s). Prior art shows that CCHFV enters cells through clathrin-mediated endocytosis and uses the endosomal pathway to release viral RNA segments. In vitro, many different cell types can be infected with CCHFV, suggesting the existence of either a widely distributed receptor or several redundant entry receptors.
To date, the nucleoside analogue ribavirin is the only direct-acting anti-viral that has been widely used clinically in patients with CCHF. However, the use of ribavirin is controversial, with continued debate about whether treatment improves outcome.
Although ribavirin, favipiravir, and 2'-deoxy-2'-fluorocytidine are all thought to exert antiviral activity through catastrophic mutagenesis or inhibition of the viral replicase, additional small molecules acting through distinct mechanisms have been reported effective against CCHFV in vitro.
In addition to small molecules, antibody-based therapies have also been evaluated for treatment of CCHFV, however large- scale trials have not been performed so far.
To date, multiple vaccine platforms have been evaluated in animal models for CCHFV such as inactivated virus preparations, subunit vaccines, VLPs, recombinant live-attenuated viruses, replication-deficient viral-vectored vaccines, and nucleic acid-based vaccines, many with promising efficacy. However, the vaccine landscape for CCHFV is complex: vaccine-induced neutralizing antibodies seem neither necessary nor sufficient for protection, several viral antigens can confer protection, and yet the same viral antigens expressed from different vaccine platforms can confer distinctly different levels of efficacy.
Moreover, RNA viruses tend to generate vaccine-resistant escaper strains through mutations of antibody-binding sites. Using a host-related decoy receptor such as a LDLR-derived polypeptide as disclosed herein would require a gain-of-function mutation switching to another host cell receptor. Such a mutation is less likely to appear so that there is a decreased risk of generating resistant virus strains in response to treatment with a LDLR- derived polypeptide according to this invention compared to vaccine approaches. In sum, there is a need for the development of a novel and efficient strategy to prevent and/or treat CCHFV infections.
It is the objective of the present invention to provide LDLR polypeptides comprising soluble LDL receptors, functional fragments thereof, or single amino acid variants thereof, which can be used for preventing and/or treating a Bunyavirales virus infection, preferably a CCHFV infection. A method to identify new factors involved in a CCHFV infection is also provided herein.
The objective of the present invention is solved by the teaching of the independent claims. Further advantageous features, aspects and details of the invention are evident from the dependent claims, the description, the figures, and the examples of the present application.
Brief description of the invention
The objective of the present invention is solved by an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain.
In some embodiments, an amino acid variant of an LDLR ectodomain refers to a single amino acid variant of an LDLR ectodomain. In some embodiments, an amino acid variant of an LDLR ectodomain refers to a multiple amino acid variant of an LDLR ectodomain.
A preferred embodiment of present invention relates to LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection in a human, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain.
In some embodiments, said LDLR derived polypeptide for use comprises an LDLR ectodomain having amino acid sequence SEQ ID NO 18, or a functional fragment thereof, or an amino acid variant thereof.
In some embodiments, said LDLR derived polypeptide for use comprises a dimer, a trimer, or a multimer of an LDLR ectodomain monomer having amino acid sequence SEQ D NO 18, or of a functional fragment thereof, or of an amino acid variant thereof. More preferred said LDLR derived polypeptide comprises a dimer or trimer, still more preferred a dimer, still more preferred is a homodimer or a homotrimer, and still more preferred is a homodimer.
In some embodiments, said LDLR derived polypeptide for use comprises a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41 ), OLSD (SEQ ID NO 36), LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA1 - LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA1 -LA2-LA3- LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFB, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA, LA1 -LA2-LA3-LA4X- LA5-LA6X-LA7, LA1 -LA2-LA3-LA4X-LA5-LA6, LA1 -LA2-LA3-LA4X-LA5, LA1-LA2- LA3-LA4, LA1-LA2-LA3, LA1-LA2, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX- PROPX-EGFCX-OLSD, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX- EGFC, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA2-LA3-LA4X- LA5-LA6X-LA7-EGFA-EGFB, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA, LA2-LA3- LA4X-LA5-LA6X-LA7, LA2-LA3-LA4X-LA5-LA6, LA2-LA3-LA4X-LA5, LA2-LA3- LA4, LA2-LA3, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA3-LA4X-LA5-LA6X- LA7-EGFA-EGFBX-PROP, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA3-LA4X- LA5-LA6X-LA7-EGFA, LA3-LA4X-LA5-LA6X-LA7, LA3-LA4X-LA5-LA6, LA3- LA4X-LA5, LA3-LA4, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA4X-LA5-LA6X- LA7-EGFA-EGFBX-PROP, LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA4X-LA5-LA6X- LA7-EGFA, LA4X-LA5-LA6X-LA7, LA4X-LA5-LA6, LA4X-LA5, LA5-LA6X-LA7- EGFA-EGFBX-PROPX-EGFCX-OLSD, LA5-LA6X-LA7-EGFA-EGFBX-PROPX- EGFC, LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA5-LA6X-LA7-EGFA-EGFB, LA5- LA6X-LA7-EGFA, LA5-LA6X-LA7, LA5-LA6, LA6X-LA7-EGFA-EGFBX-PROPX- EGFCX-OLSD, LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA6X-LA7-EGFA- EGFBX-PROP, LA6X-LA7-EGFA-EGFB, LA6X-LA7-EGFA, LA6X-LA7, LA7- EGFA-EGFBX-PROPX-EGFCX-OLSD, LA7-EGFA-EGFBX-PROPX-EGFC, LA7- EGFA-EGFBX-PROP, LA7-EGFA-EGFB, LA7-EGFA, EGFA-EGFBX-PROPX- EGFCX-OLSD, EGFA-EGFBX-PROPX-EGFC, EGFA-EGFBX-PROP, EGFA- EGFB, EGFBX-PROPX-EGFCX-OLSD, EGFBX-PROPX-EGFC, EGFBX-PROP, or amino acid variants thereof, or a homomultimer thereof, preferably a homodimer or a homotrimer thereof.
In some embodiments, an amino acid variant of a functional fragment of an LDLR ectodomain refers to a single amino acid variant of a functional fragment of an LDLR ectodomain. In some embodiments, an amino acid variant of an LDLR ectodomain refers to a multiple amino acid variant of a functional fragment of an LDLR ectodomain.
In some embodiments, said LDLR derived polypeptide for use further comprises a peptide linker connecting said fragments or monomers.
In a particular aspect of the invention, said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava-Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TULV), New York hantavirus (NW), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV), and preferably CCHFV, RVFV, Hantavirus, NSDV, and SFNV.
In a more particular aspect of the invention, said Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
In a still more particular aspect of the invention, said LDLR derived polypeptide for use comprises an LDLR ectodomain having amino acid sequence SEQ ID NO 18, and said Bunyavirales virus is CCHFV.
A further embodiment of the invention is directed to a pharmaceutical composition comprising the LDLR derived polypeptide for use described above, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent.
Another embodiment of the invention is directed to a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an LDLR derived polypeptide for use described above. A particular embodiment of the invention is directed to an LDLR derived polypeptide comprising a functional fragment of an LDLR ectodomain selected from the group comprising or consisting of:
LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41 ), OLSD (SEQ ID NO 36), LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA1 - LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA1 -LA2-LA3- LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFB, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA, LA1 -LA2-LA3-LA4X- LA5-LA6X-LA7, LA1 -LA2-LA3-LA4X-LA5-LA6, LA1 -LA2-LA3-LA4X-LA5, LA1-LA2-LA3-LA4, LA1-LA2-LA3, LA1-LA2, LA2-LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFBX-PROPX-EGFCX-OLSD, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROPX-EGFC, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA2-LA3-LA4X-LA5-LA6X-LA7-
EGFA, LA2-LA3-LA4X-LA5-LA6X-LA7, LA2-LA3-LA4X-LA5-LA6, LA2-LA3-LA4X- LA5, LA2-LA3-LA4, LA2-LA3, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX- EGFCX-OLSD, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFB, LA3-LA4X-LA5-LA6X-LA7-EGFA, LA3-LA4X-LA5-LA6X-LA7, LA3-LA4X-LA5-LA6, LA3-LA4X-LA5, LA3-LA4, LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROPX-EGFCX-OLSD, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX- EGFC, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA4X-LA5-LA6X-LA7-EGFA-
EGFB, LA4X-LA5-LA6X-LA7-EGFA, LA4X-LA5-LA6X-LA7, LA4X-LA5-LA6, LA4X-LA5, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA5-LA6X- LA7-EGFA-EGFBX-PROPX-EGFC, LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA5-LA6X-LA7-EGFA-EGFB, LA5-LA6X-LA7-EGFA, LA5-LA6X-LA7, LA5-LA6, LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA6X-LA7-EGFA-EGFBX- PROPX-EGFC, LA6X-LA7-EGFA-EGFBX-PROP, LA6X-LA7-EGFA-EGFB, LA6X- LA7-EGFA, LA6X-LA7, LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA7-EGFA- EGFBX-PROPX-EGFC, LA7-EGFA-EGFBX-PROP, LA7-EGFA-EGFB, LA7- EGFA, EGFA-EGFBX-PROPX-EGFCX-OLSD, EGFA-EGFBX-PROPX-EGFC, EGFA-EGFBX-PROP, EGFA-EGFB, EGFBX-PROPX-EGFCX-OLSD, EGFBX- PROPX-EGFC, EGFBX-PROP, or amino acid variants of said functional fragment, and wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said fragments.
A further particular embodiment of the invention is directed to LDLR derived polypeptide comprising a multimer of monomers, wherein each monomer is selected from the group comprising: an LDLR ectodomain (SEQ ID NO 18), a functional fragment of said LDLR ectodomain, an amino acid variant of said LDLR ectodomain, an amino acid variant of said functional fragment of said LDLR ectodomain, and wherein said multimer comprises between 2 and 8 monomers, preferably 2 or 3 monomers, and wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said monomers; and wherein said multimer is a homomultimer or a heteromultimer.
A more particular embodiment of the invention is directed to an LDLR derived polypeptide comprising a functional fragment of an LDLR ectodomain as described above, or comprising a multimer of monomers as described above, wherein said LDLR derived polypeptide is for use in the prophylaxis and/or treatment of a virus infection, wherein said virus is preferably a Bunyavirales virus.
A further particular embodiment of the invention is directed to a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an LDLR derived polypeptide comprising a functional fragment of an LDLR ectodomain or comprising a multimer of monomers as described above.
A particular embodiment of the invention is directed to an in vitro screening method to identify a gene that modulates a Bunyavirales virus infection, the method comprising:
A) providing haploid or near-haploid cells;
B) performing chemical random mutagenesis in said cells of step A) in order to obtain mutagenized cells;
C) infecting the mutagenized cells of step B) with a Bunyavirales virus or with a Bunyavirales virus pseudotyped RNA viral particle;
D) culturing the infected mutagenized cells of step C) to obtain resistant colonies; E) isolating the resistant colonies of step D);
F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate a Bunyavirales virus infection, wherein the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof, wherein the pseudotyped RNA viral particle is a HIV particle or a MSV particle or a VSV particle, wherein the pseudotyped RNA viral particle express a Bunyavirales virus specific glycoprotein Gc and/or Gn, and wherein the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TULV), New York hantavirus (NW), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV), wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
The term "whole exome sequencing" refers to the sequencing of all exons in a specific cell or tissue type, wherein the exons are the protein coding regions of a gene. Therefore, in “whole exome sequencing” only the protein-coding DNA regions are sequenced as compared to whole genome sequencing.
The term “whole transcriptome sequencing” refers to sequencing of expressed transcripts at the mRNA level to obtain sequence information on all mRNAs made in a cell or cell pool at a given time
In the in vitro screening method described above, the pseudotyped RNA viral particle is preferably a VSV particle. In the in vitro screening method described above, it is preferred that performing chemical random mutagenesis of step B) comprises incubating the haploid cells with N-Ethyl-N-nitrosourea (ENU).
Description of the invention
The inventors were able to identify host factors important for CCHFV infections by using a novel method based on haploid murine stem cells mutagenized with N- Ethyl-N-nitrosourea (ENU), which induces single nucleotide variants into the genome, and subsequent infection with a CCHFV glycoprotein pseudotyped vesicular stomatitis virus (VSV-CCHF_G) (Figure 1).
Haploid cells containing only a single genomic copy allow for direct translation of the introduced genetic changes such as gene knockouts and point mutations to a respective phenotype.
By using this novel and advantageous method, the inventors surprisingly identified Low Density Lipoprotein Receptor (LDLR) as an essential host factor for CCHFV entry (Table 2). Experiments using a soluble LDLR, ldlr knockout cells, and both a laboratory strain and a patient isolate of CCHFV, confirmed that LDLR mediates CCHFV infection (Figure 2 and 4). Furthermore, experiments based on Bioluminescence Resonance Energy Transfer (BRET) technique confirmed that LDLR is a receptor for CCHFV, as the viral glycoprotein Gc directly attaches to LDLR (Figures 3 and 8). Knockout of Ldlr in BVOs resulted in a significant reduction of CCHFV infection in BVO-derived vascular cells (Fig. 5a, b). Most importantly, Ldlr-/- mice were protected against the disease following CCHFV infection (Figure 5c,d,e). The role of sLDLR acting as receptor for CCHFV was confirmed using a clinical CCHFV isolate (Figures 6a-d).
LDLR Ectodomain and functional fragments thereof
The low-density lipoprotein receptor gene family encodes for a class of structurally related cell surface receptors also named low density lipoprotein receptor-related proteins (LRP) that fulfil diverse biological functions in different organs, tissues, and cell types, including the regulation of lipid metabolism, protection against atherosclerosis, neurodevelopment, and transport of nutrients and vitamins.
There are several members of the LDLR family in mammals, namely:
1 ) low-density lipoprotein receptor, LDLR (Uniprot P01130);
2) very low-density lipoprotein receptor, VLDLR; 3) ApoER2, also known as low density lipoprotein receptor-related protein- 8, LRP8;
4) low density lipoprotein receptor-related protein 4, LRP4, also known as multiple epidermal growth factor (EGF) repeat-containing protein, MEGF7;
5) LDLR-related protein 1 (LRP1 );
6) LDLR-related protein 1b (LRP1 b);
7) Megalin also known as LRP2;
8) LRP5;
9) LRP6.
LDL is one of the major cholesterol-carrying lipoprotein of plasma, acting to regulate cholesterol homeostasis in mammalian cells. The LDL receptor binds and transports LDL particles into cells by acidic endocytosis. Once inside the cell, the LDLR separates from its ligand, which is degraded in the lysosomes, while the receptor returns to the cell surface. The internal dissociation of the LDLR with its ligand is mediated by proton pumps within the walls of the endosome that lower the pH.
The LDLR is a multi-domain protein, containing the following five domains:
I) a ligand-binding domain containing seven 40-amino acid LDLR class A cysteine-rich repeats, also named LA repeats, each of which contains a coordinated calcium ion and six cysteine residues involved in disulphide bond formation. Thus, the terms "LA1", "LA2", "LA3", "LA4", "LA5", "LA6", "LA7" are used herein to refer to each of the seven LA repeats of the LDLR ligand binding domain.
II) a conserved region containing two EGF repeats ("EGFA" and "EGFB"), followed by six LDLR class B repeats (LB1 , LB2, LB2, LB3, LB4, LB5, and LB6), and another EGF repeat ("EGFC"). The six LDLR class B repeats each contain a conserved YWTD motif and are predicted to form a [3-propeller structure, and are thus referred herein with the term "PROP". This region is critical for ligand release and recycling of the receptor.
III) a third domain rich in serine and threonine residues and containing clustered O-linked carbohydrate chains, and is referred herein as "OLSD".
IV) a fourth domain being the hydrophobic transmembrane region.
V) a fifth domain being the cytoplasmic tail that directs the receptor to clathrin- coated pits. The term "LDLR ectodomain module" or "LDLR module" or simply "module" is used herein to refer to the individual repeats and domains: LA1 , LA2, LA3, LA4, LA5, LA6, LA7, EGFA, EGFB, LB1 , LB2, LB2, LB3, LB4, LB5, LB6, PROP, EGFC, and OLSD.
The amino sequences of the "LDLR ectodomain modules" used in the present invention are:
SEQ ID NO Abbreviation Name
SEQ ID NO 18 LDLR ECD LDLR ectodomain
SEQ ID NO 19 LA1 LDL-receptor class A 1
SEQ ID NO 20 LA2 LDL-receptor class A 2
SEQ ID NO 21 LA3 LDL-receptor class A 3
SEQ ID NO 22 LA4 LDL-receptor class A 4
SEQ ID NO 37 LA4X LA4 and amino acids between LA4 and LA5
SEQ ID NO 23 LA5 LDL-receptor class A 5
SEQ ID NO 24 LA6 LDL-receptor class A 6
SEQ ID NO 38 LA6X LA6 and amino acid between LA6 and LA7
SEQ ID NO 25 LA7 LDL-receptor class A 7
SEQ ID NO 26 EG FA EGF-like 1
SEQ ID NO 27 EGFB EGF-like 2; calcium-binding
SEQ ID NO 39 EGFBX EGFB and amino acids between EGFB and LB1
SEQ ID NO 28 LB1 LDL-receptor class B 1
SEQ ID NO 29 LB2 LDL-receptor class B 2
SEQ ID NO 30 LB3 LDL-receptor class B 3
SEQ ID NO 31 LB4 LDL-receptor class B 4
SEQ ID NO 32 LB5 LDL-receptor class B 5
SEQ ID NO 33 LB6 LDL-receptor class B 6
SEQ ID NO 34 PROP LB1 to LB6
SEQ ID NO 40 PROPX PROP and amino acids between PROP and EGFC
SEQ ID NO 35 EGFC EGF-like 3
SEQ ID NO 41 EGFCX EGFC and amino acids between EGFC and OLSD
SEQ ID NO 36 OLSD Clustered O-linked oligosaccharides
Soluble low-density lipoprotein receptor (sLDLR) is the circulating LDLR ectodomain of transmembrane LDLR comprising the domains I) - III) described above, i.e. the LDLR derived polypeptide consisting of LA1-LA7 repeats, EGFA, EGFB, PROP, EGFC, and OLSD. Thus, the term "LDLR ectodomain" as used herein refers to the soluble form of the low-density lipoprotein receptor (sLDLR), comprising the "modules" LA1-LA7,
EGFA, EGFB, PROP, EGFC, and OLSD. Preferably, the term "LDLR ectodomain" refers to the soluble form of the low-density lipoprotein receptor (sLDLR) that binds a Bunyavirales virus, and which comprises LA1-LA7, EGFA,
EGFB, PROP, EGFC, and OLSD. Therefore, the soluble "LDLR ectodomain" corresponds to the extracellular portion of 766 amino acid residues of the LDL receptor, which is purified to homogeneity from impurities. More in detail, the soluble "LDLR ectodomain" corresponds to the extracellular portion of mature LDL receptor, which has an amino acid sequence SEQ ID NO 18 corresponding to amino acid residue Ala22 to Arg788 of the mature LDL receptor as deposited at Uniprot ID P01130.
The term "LDLR derived polypeptide" is used herein to refer to the following polypeptides (also referred to as "polypeptides of the disclosure"): an "LDLR ectodomain" having amino acid sequence SEQ ID NO 18; a "functional fragment" of said "LDLR ectodomain" having amino acid sequence SEQ ID NO 18; a "single or multiple amino acid variant" of said "LDLR ectodomain" having amino acid sequence SEQ ID NO 18; a single or multiple amino acid variant of said "functional fragment of said LDLR ectodomain" having amino acid sequence SEQ ID NO 18; a multimer of a "LDLR ectodomain" having amino acid sequence SEQ ID NO 18, wherein said multimer is a dimer, or a trimer or comprises more repetitions of said "LDLR ectodomain", and wherein said multimer is a homomultimer of said "LDLR ectodomain"; a multimer of a functional fragment of a "LDLR ectodomain", wherein said multimer is a dimer, or a trimer or comprises more repetitions of said functional fragment of a "LDLR ectodomain", and wherein said multimer is a homomultimer or a heteromultimer of said functional fragment of a "LDLR ectodomain"; a multimer of a single or multiple amino acid variant of a "LDLR ectodomain", wherein said multimer is a dimer, or a trimer or comprises more repetitions of said single amino acid variant of a "LDLR ectodomain", and wherein said multimer is a homomultimer or a heteromultimer of said single amino acid variant of a "LDLR ectodomain"; a multimer of a single or multiple amino acid variant of a functional fragment of a "LDLR ectodomain", wherein said multimer is a dimer, or a trimer or comprises more repetitions of said single amino acid variant of a functional fragment of a "LDLR ectodomain", and wherein said multimer is a homomultimer or a heteromultimer of said single amino acid variant of a functional fragment of a "LDLR ectodomain".
The multimer as disclosed herein is preferably a dimer or trimer, still more preferred a dimer, still more preferred is a homodimer or a homotrimer, and still more preferred is a homodimer.
The terms "functional fragments" of a "LDLR ectodomain", "single amino acid variants", "multiple amino acid variants" are defined in the following sections.
The present invention provides an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain.
A preferred embodiment of present invention relates to LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection in a human, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain.
An embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain having amino acid sequence SEQ ID NO 18, or a functional fragment thereof, or an amino acid variant thereof.
As used herein the term "amino acid variant" refers to an analog of the natural soluble LDL receptor (LDLR ectodomain) or of a functional fragment thereof in which one or more amino acid residues of the natural soluble LDL receptor is replaced by a different amino acid residue or is deleted, so that the resulting LDLR derived polypeptide has an ability of binding to a Bunyavirales virus about equal to or greater than that of the natural LDLR ectodomain. These variants are prepared by known synthesis and/or by site-directed mutagenesis techniques, or any other known technique suitable therefor. The term "amino acid variant" comprises "single amino acid variant" and "multiple amino acid variant".
Thus, as used herein, the term "single amino acid variant" refers to an analog of the natural soluble LDL receptor (LDLR ectodomain) or of a functional fragment thereof in which one amino acid residue of the natural soluble LDL receptor is replaced by a different amino acid residue, or is added, or is deleted, so that the resulting LDLR derived polypeptide has an ability of binding to a Bunyavirales virus about equal to or greater than that of the natural LDLR ectodomain.
Thus, as used herein, the term "multiple amino acid variant" refers to an analog of the natural soluble LDL receptor (LDLR ectodomain) or of a functional fragment thereof in which multiple amino acid residues, i.e. at least two amino acid residues of the natural soluble LDL receptor are replaced by different amino acid residues or deleted, or at least two amino acid residues are added compared to the natural soluble LDL receptor, so that the resulting LDLR derived polypeptide has an ability of binding to a Bunyavirales virus about equal to or greater than that of the natural LDLR ectodomain.
In one aspect, a "multiple amino acid variant of an LDLR ectodomain or of a functional fragment thereof" comprises an amino acid sequence that shows high aminoacidic sequence identity to the parent LDLR ectodomain (SEQ ID NO 18) or a fragment thereof, such as it shows more than 60%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity with the parent sequence and is preferably characterised by similar properties of the parent sequence, namely binding to a Bunyavirales virus.
The percentage of "sequence identity" is determined by comparing two optimally aligned amino acid sequences over a "comparison window" on the full length of the reference sequence. A "comparison window" as used herein, refers to the optimal alignment between the reference and variant sequence after that the two sequences are optimally aligned, wherein the variant amino acid sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment. Identity percentage is calculated by determining the number of positions at which the identical amino acids occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the full length in amino acid residues) and multiplying the results by 100 to yield the percentage of sequence identity. Two amino acid sequences are said to be "identical" if the sequence of amino acid residues in the two sequences is the same when optimally aligned as described above.
As used herein, the term "functional fragment of an LDLR ectodomain" refers to a fragment of the LDLR ectodomain comprising one or more "LDLR ectodomain modules" (i.e. LA1 to LA7, EGFA, EGFB, PROP, EGFC, and OLSD), provided that said fragment has an ability to bind to a Bunyavirales virus about equal to or greater than that of the natural LDLR ectodomain. More in detail, the term "functional fragment" refers to a fragment of the LDLR ectodomain, wherein said "functional fragment" comprises one or more "LDLR ectodomain modules", wherein said "LDLR ectodomain modules" have the same order in the resulting "functional fragment" as in the natural LDLR ectodomain (SEQ ID NO 18), provided that said fragment has an ability to bind to a Bunyavirales virus about equal to or greater than that of the natural LDLR ectodomain.
Still more detailed, the term "functional fragment of an LDLR ectodomain" is selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41 ), OLSD (SEQ ID NO 36), LA1-LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROPX-EGFCX-OLSD, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROPX-EGFC, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX- PROP, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA1 -LA2-LA3-LA4X- LA5-LA6X-LA7-EGFA, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7, LA1 -LA2-LA3-LA4X- LA5-LA6, LA1-LA2-LA3-LA4X-LA5, LA1 -LA2-LA3-LA4, LA1-LA2-LA3, LA1-LA2, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA2-LA3- LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA2-LA3-LA4X-LA5-LA6X- LA7-EGFA-EGFBX-PROP, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA2- LA3-LA4X-LA5-LA6X-LA7-EGFA, LA2-LA3-LA4X-LA5-LA6X-LA7, LA2-LA3-LA4X- LA5-LA6, LA2-LA3-LA4X-LA5, LA2-LA3-LA4, LA2-LA3, LA3-LA4X-LA5-LA6X- LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA3-LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROPX-EGFC, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA3- LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA3-LA4X-LA5-LA6X-LA7-EGFA, LA3-LA4X- LA5-LA6X-LA7, LA3-LA4X-LA5-LA6, LA3-LA4X-LA5, LA3-LA4, LA4X-LA5-LA6X- LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA4X-LA5-LA6X-LA7-EGFA-EGFBX- PROPX-EGFC, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA4X-LA5-LA6X- LA7-EGFA-EGFB, LA4X-LA5-LA6X-LA7-EGFA, LA4X-LA5-LA6X-LA7, LA4X-LA5- LA6, LA4X-LA5, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA5-LA6X-LA7-EGFA-EGFB, LA5-LA6X-LA7-EGFA, LA5-LA6X-LA7, LA5-LA6, LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA6X-LA7-EGFA-EGFBX- PROPX-EGFC, LA6X-LA7-EGFA-EGFBX-PROP, LA6X-LA7-EGFA-EGFB, LA6X- LA7-EGFA, LA6X-LA7, LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA7-EGFA- EGFBX-PROPX-EGFC, LA7-EGFA-EGFBX-PROP, LA7-EGFA-EGFB, LA7- EGFA, EGFA-EGFBX-PROPX-EGFCX-OLSD, EGFA-EGFBX-PROPX-EGFC, EGFA-EGFBX-PROP, EGFA-EGFB, EGFBX-PROPX-EGFCX-OLSD, EGFBX- PROPX-EGFC, EGFBX-PROP, or a homomultimer thereof, preferably a homodimer or a homotrimer thereof.
Thus, an embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41), OLSD (SEQ ID NO 36), LA1 -LA2-LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA1 -LA2-LA3-LA4X-LA5-LA6X- LA7-EGFA-EGFBX-PROPX-EGFC, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROP, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA1 -LA2-LA3- LA4X-LA5-LA6X-LA7-EGFA, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7, LA1 -LA2-LA3- LA4X-LA5-LA6, LA1 -LA2-LA3-LA4X-LA5, LA1 -LA2-LA3-LA4, LA1-LA2-LA3, LA1- LA2, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA2- LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA2-LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROP, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA, LA2-LA3-LA4X-LA5-LA6X-LA7, LA2-LA3- LA4X-LA5-LA6, LA2-LA3-LA4X-LA5, LA2-LA3-LA4, LA2-LA3, LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA3-LA4X-LA5-LA6X-LA7-EGFA, LA3- LA4X-LA5-LA6X-LA7, LA3-LA4X-LA5-LA6, LA3-LA4X-LA5, LA3-LA4, LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROPX-EGFC, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA4X-LA5- LA6X-LA7-EGFA-EGFB, LA4X-LA5-LA6X-LA7-EGFA, LA4X-LA5-LA6X-LA7, LA4X-LA5-LA6, LA4X-LA5, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA5-LA6X-LA7-EGFA- EGFBX-PROP, LA5-LA6X-LA7-EGFA-EGFB, LA5-LA6X-LA7-EGFA, LA5-LA6X- LA7, LA5-LA6, LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA6X-LA7-EGFA-EGFBX-PROP, LA6X-LA7- EGFA-EGFB, LA6X-LA7-EGFA, LA6X-LA7, LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA7-EGFA-EGFBX-PROPX-EGFC, LA7-EGFA-EGFBX-PROP, LA7- EGFA-EGFB, LA7-EGFA, EGFA-EGFBX-PROPX-EGFCX-OLSD, EGFA-EGFBX- PROPX-EGFC, EGFA-EGFBX-PROP, EGFA-EGFB, EGFBX-PROPX-EGFCX- OLSD, EGFBX-PROPX-EGFC, EGFBX-PROP, or amino acid variants thereof, or a homomultimer thereof, preferably a homodimer or a homotrimer thereof.
When said LDLR derived polypeptide for use is a homomultimer of functional fragment monomers, wherein each monomer is a functional fragment of an LDLR ectodomain, it is preferred that said homomultimers comprises between 2 and 10 monomers, 2 and 12 monomers, 2 and 14 monomers, 2 and 16 monomers, 2 and 18 monomers, 2 and 20 monomers, 2 and 8 monomers, 2 and 6 monomers, 2 and 4 monomers. More preferably, When said LDLR derived polypeptide for use is a homomultimer of monomers, wherein each monomer is a functional fragment, it is preferred that said homomultimers comprises between 2 and 8 monomers, preferably 2 or 3 monomers.
In said LDLR derived polypeptide for use, a monomer can be a functional fragment of an LDLR ectodomain or an amino acid variant thereof.
An exemplary LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises a homomultimer of a functional fragment of an LDLR ectodomain selected from the group reported above, has the following composition: (LA2-LA3-LA4) (LA2-LA3-LA4) (LA2-LA3-LA4) (LA2-LA3-LA4) (LA2-LA3-LA4), or (LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP) (LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFBX-PROP), and similars. Multimers
A further embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises a dimer, a trimer, a heteromultimer, or a homomultimer of an LDLR ectodomain monomer having amino acid sequence SEQ ID NO 18, or of a functional fragment thereof, or of an amino acid variant thereof. More preferred is a dimer or trimer, still more preferred a dimer, still more preferred is a homodimer or a homotrimer, and still more preferred is a homodimer.
A monomer is thus selected from the group comprising: an LDLR ectodomain (SEQ ID NO 18), a functional fragment of said LDLR ectodomain, an amino acid variant of said LDLR ectodomain, an amino acid variant of said functional fragment of said LDLR ectodomain.
When said LDLR derived polypeptide for use is a homomultimer of the monomers defined above, it is preferred that said homomultimers comprises between 2 and 10 monomers, 2 and 12 monomers, 2 and 14 monomers, 2 and 16 monomers, 2 and 18 monomers, 2 and 20 monomers, 2 and 8 monomers, 2 and 6 monomers, 2 and 4 monomers. More preferably, said LDLR derived polypeptide for use comprises between 2 and 8 monomers as defined above, preferably 2 or 3 monomers.
The term "homomultimer of an LDLR ectodomain" refers to a multimeric polypeptide consisting of two or more identical monomers, wherein each monomer is an LDLR ectodomain having amino acid sequence SEQ ID NO 18, or an amino acid variant thereof. Therefore, the term "homomultimer of an LDLR ectodomain" also refers to a multimeric polypeptide consisting of two or more identical monomers, wherein each monomer is an amino acid variant of LDLR ectodomain having amino acid sequence SEQ ID NO 18.
In some embodiments, the term "homomultimer of an LDLR ectodomain" refers to a dimer or trimer of an LDLR ectodomain monomer, wherein each LDLR ectodomain monomer has amino acid sequence SEQ ID NO 18, or an amino acid variant thereof. Therefore, the term "homomultimer of an LDLR ectodomain" also refers to a dimer or trimer of an LDLR ectodomain monomer, wherein each monomer is an amino acid variant of LDLR ectodomain having amino acid sequence SEQ ID NO 18. In some embodiments, the term "homomultimer of an LDLR ectodomain functional fragment" refers to a multimeric polypeptide consisting of two or more identical monomers, wherein each monomer is an LDLR ectodomain functional fragment, and wherein each functional fragment is selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41 ), OLSD (SEQ ID NO 36), LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA1 - LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA1 -LA2-LA3- LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFB, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA, LA1 -LA2-LA3-LA4X- LA5-LA6X-LA7, LA1 -LA2-LA3-LA4X-LA5-LA6, LA1 -LA2-LA3-LA4X-LA5, LA1-LA2- LA3-LA4, LA1-LA2-LA3, LA1-LA2, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX- PROPX-EGFCX-OLSD, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX- EGFC, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA2-LA3-LA4X- LA5-LA6X-LA7-EGFA-EGFB, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA, LA2-LA3- LA4X-LA5-LA6X-LA7, LA2-LA3-LA4X-LA5-LA6, LA2-LA3-LA4X-LA5, LA2-LA3- LA4, LA2-LA3, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA3-LA4X-LA5-LA6X- LA7-EGFA-EGFBX-PROP, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA3-LA4X- LA5-LA6X-LA7-EGFA, LA3-LA4X-LA5-LA6X-LA7, LA3-LA4X-LA5-LA6, LA3- LA4X-LA5, LA3-LA4, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA4X-LA5-LA6X- LA7-EGFA-EGFBX-PROP, LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA4X-LA5-LA6X- LA7-EGFA, LA4X-LA5-LA6X-LA7, LA4X-LA5-LA6, LA4X-LA5, LA5-LA6X-LA7- EGFA-EGFBX-PROPX-EGFCX-OLSD, LA5-LA6X-LA7-EGFA-EGFBX-PROPX- EGFC, LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA5-LA6X-LA7-EGFA-EGFB, LA5- LA6X-LA7-EGFA, LA5-LA6X-LA7, LA5-LA6, LA6X-LA7-EGFA-EGFBX-PROPX- EGFCX-OLSD, LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA6X-LA7-EGFA- EGFBX-PROP, LA6X-LA7-EGFA-EGFB, LA6X-LA7-EGFA, LA6X-LA7, LA7- EGFA-EGFBX-PROPX-EGFCX-OLSD, LA7-EGFA-EGFBX-PROPX-EGFC, LA7- EGFA-EGFBX-PROP, LA7-EGFA-EGFB, LA7-EGFA, EGFA-EGFBX-PROPX- EGFCX-OLSD, EGFA-EGFBX-PROPX-EGFC, EGFA-EGFBX-PROP, EGFA- EGFB, EGFBX-PROPX-EGFCX-OLSD, EGFBX-PROPX-EGFC, EGFBX-PROP,or wherein each monomer is an amino acid variant of a functional fragment selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ D NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41 ), OLSD (SEQ ID NO 36), LA1-LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX- PROPX-EGFCX-OLSD, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX- PROPX-EGFC, LA1-LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA1- LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-
EGFA, LA1-LA2-LA3-LA4X-LA5-LA6X-LA7, LA1 -LA2-LA3-LA4X-LA5-LA6, LA1- LA2-LA3-LA4X-LA5, LA1 -LA2-LA3-LA4, LA1-LA2-LA3, LA1-LA2, LA2-LA3-LA4X- LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA2-LA3-LA4X-LA5-LA6X- LA7-EGFA-EGFBX-PROPX-EGFC, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROP, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA2-LA3-LA4X-LA5- LA6X-LA7-EGFA, LA2-LA3-LA4X-LA5-LA6X-LA7, LA2-LA3-LA4X-LA5-LA6, LA2- LA3-LA4X-LA5, LA2-LA3-LA4, LA2-LA3, LA3-LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROPX-EGFCX-OLSD, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX- PROPX-EGFC, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFB, LA3-LA4X-LA5-LA6X-LA7-EGFA, LA3-LA4X-LA5-LA6X- LA7, LA3-LA4X-LA5-LA6, LA3-LA4X-LA5, LA3-LA4, LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROPX-EGFCX-OLSD, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX- EGFC, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA4X-LA5-LA6X-LA7-EGFA-
EGFB, LA4X-LA5-LA6X-LA7-EGFA, LA4X-LA5-LA6X-LA7, LA4X-LA5-LA6, LA4X- LA5, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA5-LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA5-LA6X- LA7-EGFA-EGFB, LA5-LA6X-LA7-EGFA, LA5-LA6X-LA7, LA5-LA6, LA6X-LA7- EGFA-EGFBX-PROPX-EGFCX-OLSD, LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA6X-LA7-EGFA-EGFBX-PROP, LA6X-LA7-EGFA-EGFB, LA6X-LA7-EGFA, LA6X-LA7, LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA7-EGFA-EGFBX- PROPX-EGFC, LA7-EGFA-EGFBX-PROP, LA7-EGFA-EGFB, LA7-EGFA, EGFA- EGFBX-PROPX-EGFCX-OLSD, EGFA-EGFBX-PROPX-EGFC, EGFA-EGFBX- PROP, EGFA-EGFB, EGFBX-PROPX-EGFCX-OLSD, EGFBX-PROPX-EGFC, EGFBX-PROP.
The disclosed dimer of an LDLR ectodomain monomer comprising two monomers, wherein each monomer is an LDLR ectodomain or an LDLR ectodomain functional fragment or an amino acid variant thereof, has a binding capacity for Bunyavirales virus that is about equal to or greater than the binding capacity of the single LDLR ectodomain monomers. The disclosed trimer of an LDLR ectodomain monomer comprising three monomers, wherein each monomer is an LDLR ectodomain or an LDLR ectodomain functional fragment or an amino acid variant thereof, has a binding capacity for Bunyavirales virus that is about equal to or greater than the binding capacity of the single LDLR ectodomain monomer.
The disclosed homomultimer of an LDLR ectodomain monomer comprising a plurality of monomers, wherein each monomer is an LDLR ectodomain or an LDLR ectodomain functional fragment or an amino acid variant thereof, has a binding capacity for Bunyavirales virus, preferably CCHFV or RVFV and most preferably CCHFV, that is about equal to or greater than the binding capacity of an LDLR ectodomain monomer.
Connection between fragments or monomers
In the LDLR derived polypeptide comprising two or more LDLR ectodomains monomers or two or more functional fragments thereof or two or more single amino acid variant thereof, each monomer or functional fragments or single amino acid variant thereof, may be linked directly to the adjacent monomer or functional fragments or single amino acid variant thereof. For example, the N-terminus of the second or successive monomer (e.g. LDLR ectodomain of SEQ ID NO 18, or a functional fragment thereof or an amino acid variant thereof) may be covalently fused directly to the C-terminus of the first or previous monomer of the disclosure (e.g., LDLR ectodomain of SEQ ID NO 18, or a functional fragment thereof or an amino acid variant thereof).
In other words, the LDLR ectodomain monomers, functional fragment thereof or single amino acid variant thereof, can be associated in any suitable manner to form an "LDLR derived polypeptide" or an homomultimer forming an "LDLR derived polypeptide". Said various monomers or functional fragments may be covalently associated, e.g., by means of a peptide or disulfide bond. The polypeptide fragments or monomers can thus be directly fused. The resulting fusion protein representing the LDLR derived polypeptide of the invention can include any suitable number of modified bonds, e.g., isosteres, within or between the polypeptide fragments or monomers.
Alternatively or additionally, the LDLR derived polypeptide can include a "peptide linker" or "peptide linker sequence" between any two polypeptide fragments or monomers that includes one or more amino acid sequences not forming part of the biologically active peptide portions. Any suitable peptide linker can be used. Such a linker can be of any suitable size. The linker predominantly may comprise or consist of neutral amino acid residues. If separation of peptide fragments or monomers is desirable a linker that facilitates separation can be used. Flexible linkers typically composed of combinations of glycine and/or serine residues, or combinations of glycine, proline, serine and alanine can be advantageous. Examples of such linkers are described in "Table 1".
Said linker sequences are used in the "LDLR derived polypeptides" to connect different components of the "LDLR derived polypeptide". Thus, the amino terminal end of the linker sequence is joined by a peptide bond to a first (or previous) LDLR ectodomain monomer or fragment and the carboxy terminal end of the linker sequence is joined by a peptide bind to a second (or successive) LDLR ectodomain monomer or fragment.
Consequently, a particular embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain, and wherein said LDLR derived polypeptide further comprises a peptide linker connecting said fragments. Preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1)
A further embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises a dimer, a trimer, or a multimer of an LDLR ectodomain monomer having amino acid sequence SEQ ID NO 18, or of a functional fragment thereof, or of an amino acid variant thereof, and wherein said LDLR derived polypeptide further comprises a peptide linker connecting said fragments or monomers. Preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1). Preferably, said multimer comprises between 2 and 8 monomers, preferably 2 or 3 monomers. A further embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41), OLSD (SEQ ID NO 36), LA1 -LA2-LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA1 -LA2-LA3-LA4X-LA5-LA6X- LA7-EGFA-EGFBX-PROPX-EGFC, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROP, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA1 -LA2-LA3- LA4X-LA5-LA6X-LA7-EGFA, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7, LA1 -LA2-LA3- LA4X-LA5-LA6, LA1 -LA2-LA3-LA4X-LA5, LA1 -LA2-LA3-LA4, LA1-LA2-LA3, LA1- LA2, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA2- LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA2-LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROP, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA, LA2-LA3-LA4X-LA5-LA6X-LA7, LA2-LA3- LA4X-LA5-LA6, LA2-LA3-LA4X-LA5, LA2-LA3-LA4, LA2-LA3, LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA3-LA4X-LA5-LA6X-LA7-EGFA, LA3- LA4X-LA5-LA6X-LA7, LA3-LA4X-LA5-LA6, LA3-LA4X-LA5, LA3-LA4, LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROPX-EGFC, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA4X-LA5- LA6X-LA7-EGFA-EGFB, LA4X-LA5-LA6X-LA7-EGFA, LA4X-LA5-LA6X-LA7, LA4X-LA5-LA6, LA4X-LA5, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA5-LA6X-LA7-EGFA- EGFBX-PROP, LA5-LA6X-LA7-EGFA-EGFB, LA5-LA6X-LA7-EGFA, LA5-LA6X- LA7, LA5-LA6, LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA6X-LA7-EGFA-EGFBX-PROP, LA6X-LA7- EGFA-EGFB, LA6X-LA7-EGFA, LA6X-LA7, LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA7-EGFA-EGFBX-PROPX-EGFC, LA7-EGFA-EGFBX-PROP, LA7- EGFA-EGFB, LA7-EGFA, EGFA-EGFBX-PROPX-EGFCX-OLSD, EGFA-EGFBX- PROPX-EGFC, EGFA-EGFBX-PROP, EGFA-EGFB, EGFBX-PROPX-EGFCX- OLSD, EGFBX-PROPX-EGFC, EGFBX-PROP, or amino acid variants thereof, or a homomultimer thereof, and wherein said LDLR derived polypeptide further comprises a peptide linker connecting said functional fragments or monomers. Preferably, a peptide linker connecting said functional fragments is selected from the group comprising
Linked to Linker76 (Table 1). Preferably, said homomultimer comprises between
2 and 8 monomers, preferably 2 or 3 monomers.
Table 1 : Linker List
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Recombinant nucleic acid molecules
This invention further concerns a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain or of a functional fragment thereof.
The term "nucleic acid molecule" as used herein includes genomic DNA, cDNA, synthetic DNA, synthetic RNA, mRNA, ssDNA, dsDNA, self-amplifying RNA, dsRNA, and combinations thereof. The term "nucleic acid molecule" preferably includes synthetic DNA, synthetic RNA, and mRNA. In some alternatives described herein, the "nucleic acid molecule" is a "synthetic delivery RNA". In some alternatives described herein, the "nucleic acid molecule" is a "therapeutic RNA" for delivery into a mammal cell.
The term "RNA" or "RNA molecule" or "ribonucleic acid molecule" refers to a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides). The term "DNA" or "DNA molecule" or "deoxyribonucleic acid molecule" refers to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally e.g., by DNA replication or transcription of DNA, respectively. RNA can be post-transcriptionally modified. DNA and RNA can also be chemically synthesized. DNA and RNA can be single-stranded (i.e. , ssRNA and ssDNA, respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA and dsDNA, respectively). "mRNA" or "messenger RNA" is a single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains.
As used herein, the term "isolated RNA" or "isolated DNA" refers to RNA or DNA molecules which are substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
Preferably, the polypeptides and recombinant nucleic acid molecules of interest are "purified" to essential homogeneity, i.e., contaminant species cannot be detected in the composition by conventional detection methods. Purity and homogeneity can be determined using a number of techniques well known in the art, such as agarose or polyacrylamide gel electrophoresis of a protein or nucleic acid sample, followed by visualization upon staining, or using a high-resolution technique, such as high performance liquid chromatography (HPLC).
For example, a purified polypeptide or recombinant nucleic acid molecule is at least about 50% pure, usually at least about 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more pure (%=percent by weight on a molar basis).
"Synthetic delivery RNA" as described herein can refer to a synthetic RNA for delivery into the nucleus of a cell.
The invention also concerns replicable expression vectors containing said nucleic acid molecules, hosts transformed therewith and protein produced by expression of such transformed hosts. The production of the recombinant LDLR ectodomain or of the functional fragment thereof may be carried out by different techniques well known in the prior art.
A DNA molecule, nucleic acid molecule or polypeptide is "recombinant" when it is artificial or engineered or derived from an artificial or engineered protein or nucleic acid.
Recombinant methods for producing and isolating LDLR derived polypeptides of the invention are those described in the prior art. In addition to recombinant production, the LDLR derived polypeptides may be produced by direct peptide synthesis using solid-phase techniques. Peptide synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.) in accordance with the instructions provided by the manufacturer. For example, sub-sequences may be chemically synthesized separately and combined using chemical methods to provide LDLR derived polypeptides of the invention or functional fragments thereof. Alternatively, such sequences may be ordered from a company or from a protein production facility that specialize in production of polypeptides.
A recombinant method for producing and isolating polypeptides of the invention comprises introducing into a population of cells any "recombinant nucleic acid molecule" described herein, which is operatively linked to a regulatory sequence (e.g. a promoter) effective to produce the encoded polypeptide, culturing the cells in a culture medium to produce the polypeptide, and isolating the polypeptide from the cells or from the culture medium. An amount of nucleic acid sufficient to facilitate uptake by the cells (transfection) and/or expression of the polypeptide is utilized. Appropriate cell culture media are known to those of skill in the art. The nucleic acid is introduced into such cells by any delivery method known in the prior art, including, e.g., injection, needleless injection device, gene gun, transfection, transfection with lipofectamine, retroviral gene delivery, electroporation transdermal delivery, passive uptake, etc. The nucleic acid of the invention may be part of a vector, such as a recombinant expression vector, including a DNA plasmid vector, viral vector, or any suitable vector known in the prior art. The nucleic acid or vector comprising a nucleic acid of the invention may be prepared and formulated as known in the prior art. Such a nucleic acid or expression vector may be introduced into a population of in vitro cultured cells.
A "vector" may be any agent that is able to deliver or maintain a nucleic acid in a host cell and includes, for example, but is not limited to, plasmids (e.g., DNA plasmids), naked nucleic acids, viral vectors, viruses, nucleic acids complexed with one or more polypeptide or other molecules, as well as nucleic acids immobilized onto solid phase particles. A vector can be useful as an agent for delivering or maintaining an exogenous gene, nucleic acid, and/or protein in a host cell. A vector may be capable of transducing, transfecting, or transforming a cell, thereby causing the cell to replicate or express nucleic acids and/or proteins other than those native to the cell or in a manner not native to the cell. A vector may include materials to aid in achieving entry of a nucleic acid into the cell, such as a viral particle, liposome, protein coating, or the like. Any method of transferring a nucleic acid into the cell may be used; unless otherwise indicated, the term vector does not imply any particular method of delivering a nucleic acid into a cell or imply that any particular cell type is the subject of transduction.
The term "expression vector" typically refers to a nucleic acid construct or sequence, generated recombinantly or synthetically, with a series of specific nucleic acid elements that permit transcription of a particular "nucleic acid molecule" in a host cell. The expression vector typically includes a nucleic acid to be transcribed operably linked to a promoter. The term "expression" includes any step involved in the production of the polypeptide (i.e. the LDLR ectodomain polypeptide, or a functional fragment thereof or an amino acid variant thereof) including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and/or secretion. The nucleic acids can also be modified to include one or more codons that provide for optimum expression in an expression system (e.g., mammalian cell or mammalian expression system), while, if desired, said one or more codons still encode the same amino acid(s). Procedures for making variants of nucleic acids by using nucleic acid substitutions, deletions, insertions, and additions, and degenerate codons, are routine in the art, and nucleic acid variants encoding polypeptides having the desired properties described herein are readily identified using the assays known in the prior art. Such nucleic acid changes might provide for certain advantages in their therapeutic or prophylactic use or administration, or diagnostic application.
A DNA sequence encoding a desired linker sequence, such as one listed in Table 1, may be inserted between, and in the same reading frame as, for example, two adjacent DNA sequences encoding two monomers of the LDLR ectodomain using conventional techniques known in the art. For example, a chemically synthesized oligonucleotide encoding the linker may be ligated between sequences encoding the first and second monomer, or the second and third monomer, or the third and fourth monomer, and fourth monomer and fifth monomer, etc.
The term "subject" as used herein includes, but is not limited to, an organism or animal, including mammals and non-mammals. A mammal includes, e.g., but is not limited to, a human, non-human primate (e.g., baboon, orangutan, monkey, gorilla), mouse, dog, pig, cow, goat, cat, rabbit, rat, guinea pig, hamster, horse, sheep, or other non-human mammal. A non-mammal includes, e.g., but is not limited to, a non-mammalian invertebrate and non-mammalian vertebrate, such as a bird (e.g., a chicken or duck) or a fish.
Bunyavirales
The Bunyavirales order encompasses nine families of enveloped viruses containing a single-stranded negative-sense RNA genome divided into three segments. The small (S) and large (L) segments encode proteins participating in genome replication in the infected cell cytoplasm. Typically, Bunyavirales are vector-borne viruses transmitted mostly by bites of arthropods such as mosquitoes, ticks, flies, or livestock animals, with the exception of the viruses from the Hantaviridae family, which are transmitted by infectious excreta or bites of rodents and other small mammals. In certain cases, human-to-human transmission can occur due to close contact with infected blood or other bodily fluids. Bunyavirales are found throughout the world and are known to resist to adverse climate changes allowing for seasonal and persistent occurrence of the diseases. Bunyavirales are endemic in certain regions of the globe, such as Africa, the Middle East and Asia. In addition, outbreaks of Bunyavirales are often reported in both animals and humans.
Several viruses of the order Bunyavirales are described as being human pathogens, and particularly the viruses of the genus Orthohantavirus (Hantaviridae family, also named Hantavirus) such as Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava-Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQIIV), Puumala hantavirus (PUIIV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TLILV), New York hantavirus (NYV), viruses of the genus Orthonairovirus such as Crimean-Congo hemorrhagic fever virus (CCHFV), Dugbe virus (DLIGV), Nairobi sheep disease virus (NSDV), viruses of the genus Orthobunyavirus such as Bunyamwera virus (BLINV), Ngari virus (NRIV), Bwamba bunyavirus (BWAV), California encephalitis virus (CEV), Jamestown Canyon virus (JCV), Keystone virus (KEYV), La Crosse virus (LACV), Oropouche virus (OROV), and viruses of the genus Phlebovirus such as Heartland virus (HRTV), Punta Toro virus (PTV), Sandfly fever Naples virus (SFNV), Rift Valley fever virus (RVFV), Toscana virus (TOSV), and Severe fever with thrombocytopenia syndrome virus (SFTSV).
In human infections, Bunyavirales viruses cause a broad spectrum of clinical illnesses: self-limited febrile disease, respiratory and pulmonary syndromes, encephalitis, and life-threatening hemorrhagic fevers.
CCHFV is a tripartite RNA genome virus with negative polarity of the genus Orthonairovirus and family Nairoviridae. The nairoviruses are predominantly tick- borne viruses. The genome of this virus is composed of the three different RNA segments small (S), medium (M) and large (L). The small segment ‘S’ encoding nucleoprotein (NP) is around 1.6kb long. The medium segment ‘M’ encoding glycoproteins is around 5.5kb long. The large segment ‘L’ encoding RNA - dependent RNA polymerase (RdRp or "L protein") is around 12 kb long. The untranslated regions (UTRs) on 5' and 3' of the S, M and L segments are necessary for viral transcription, replication and packaging.
In detail, the M segment generally encodes two structural glycoproteins, hereafter referred to as Gn and Gc, based on their location relative to the N and C termini of the (M segment encoded) polyprotein. The precursor of GP is cleaved and modified to generate the structural proteins Gn and Gc and the non-structural proteins GP38 and NSm. Recently it was demonstrated that during CCHF virus infection, the mature Gn (37-kDa) and Gc (75-kDa) proteins form the predominant structural glycoprotein components of the virus.
As in other bunyaviruses, the tri-segmented viral genome is coated with the viral NP and bound by the L protein. On entry in host cells, the viral proteins produce positive-sense viral RNA using the genomic negative-sense viral RNA as a template to initiate viral protein production and replication. The viral glycoproteins Gn and Gc are found on the virion surface and are responsible for receptor binding and viral entry.
The present invention particularly relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain, and wherein said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TULV), New York hantavirus (NW), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV).
In preferred embodiments the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), and Sandfly Fever Naples virus (SFNV), more preferably the Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
In alternative embodiments the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), and Sandfly Fever Naples virus (SFNV), more preferably the Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
Therefore, the present invention more particularly relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an amino acid variant of an LDLR ectodomain, and wherein said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TULV), New York hantavirus (NYV), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV).
An embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain having amino acid sequence SEQ ID NO 18, or a functional fragment thereof, or an amino acid variant thereof, and wherein said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TULV), New York hantavirus (NYV), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV).
In preferred embodiments the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), and Sandfly Fever Naples virus (SFNV), more preferably the Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
Preferably, said LDLR derived polypeptide further comprises a peptide linker connecting said fragments. More preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1)
An alternative embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an amino acid variant of an LDLR ectodomain having amino acid sequence SEQ ID NO 18, or a functional fragment thereof, and wherein said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TULV), New York hantavirus (NYV), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV).
A further embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, wherein said LDLR derived polypeptide comprises a dimer, a trimer, or a multimer of an LDLR ectodomain monomer having amino acid sequence SEQ ID NO 18, or of a functional fragment thereof, or of an amino acid variant thereof, and wherein said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TLILV), New York hantavirus (NYV), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV).
In preferred embodiments the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), and Sandfly Fever Naples virus (SFNV), more preferably the Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
A further alternative embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, wherein said LDLR derived polypeptide comprises a dimer, a trimer, or a multimer of an amino acid variant of an LDLR ectodomain monomer having amino acid sequence SEQ ID NO 18, or of a functional fragment thereof, and wherein said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TULV), New York hantavirus (NYV), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV), preferably CCHFV or RVFV and most preferably CCHFV.
Preferably, said LDLR derived polypeptide further comprises a peptide linker connecting said fragments. More preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1). More preferably, said multimer comprises between 2 and 8 monomers of an LDR ectodomain, or of a functional fragment thereof or of an amino acid variant thereof. A further embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41), OLSD (SEQ ID NO 36), LA1 -LA2-LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA1 -LA2-LA3-LA4X-LA5-LA6X- LA7-EGFA-EGFBX-PROPX-EGFC, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROP, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA1 -LA2-LA3- LA4X-LA5-LA6X-LA7-EGFA, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7, LA1 -LA2-LA3- LA4X-LA5-LA6, LA1 -LA2-LA3-LA4X-LA5, LA1 -LA2-LA3-LA4, LA1-LA2-LA3, LA1- LA2, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA2- LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA2-LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROP, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA, LA2-LA3-LA4X-LA5-LA6X-LA7, LA2-LA3- LA4X-LA5-LA6, LA2-LA3-LA4X-LA5, LA2-LA3-LA4, LA2-LA3, LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA3-LA4X-LA5-LA6X-LA7-EGFA, LA3- LA4X-LA5-LA6X-LA7, LA3-LA4X-LA5-LA6, LA3-LA4X-LA5, LA3-LA4, LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROPX-EGFC, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA4X-LA5- LA6X-LA7-EGFA-EGFB, LA4X-LA5-LA6X-LA7-EGFA, LA4X-LA5-LA6X-LA7, LA4X-LA5-LA6, LA4X-LA5, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA5-LA6X-LA7-EGFA- EGFBX-PROP, LA5-LA6X-LA7-EGFA-EGFB, LA5-LA6X-LA7-EGFA, LA5-LA6X- LA7, LA5-LA6, LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA6X-LA7-EGFA-EGFBX-PROP, LA6X-LA7- EGFA-EGFB, LA6X-LA7-EGFA, LA6X-LA7, LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA7-EGFA-EGFBX-PROPX-EGFC, LA7-EGFA-EGFBX-PROP, LA7- EGFA-EGFB, LA7-EGFA, EGFA-EGFBX-PROPX-EGFCX-OLSD, EGFA-EGFBX- PROPX-EGFC, EGFA-EGFBX-PROP, EGFA-EGFB, EGFBX-PROPX-EGFCX- OLSD, EGFBX-PROPX-EGFC, EGFBX-PROP, or an amino acid variant thereof, or a multimer thereof, and wherein said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TULV), New York hantavirus (NW), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV).
In preferred embodiments the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), and Sandfly Fever Naples virus (SFNV), more preferably the Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
A further alternative embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an amino acid variant of a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41 ), OLSD (SEQ ID NO 36), LA1-LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA1 -LA2-LA3- LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFB, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA, LA1 -LA2-LA3-LA4X- LA5-LA6X-LA7, LA1 -LA2-LA3-LA4X-LA5-LA6, LA1 -LA2-LA3-LA4X-LA5, LA1-LA2- LA3-LA4, LA1-LA2-LA3, LA1-LA2, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX- PROPX-EGFCX-OLSD, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX- EGFC, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA2-LA3-LA4X- LA5-LA6X-LA7-EGFA-EGFB, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA, LA2-LA3- LA4X-LA5-LA6X-LA7, LA2-LA3-LA4X-LA5-LA6, LA2-LA3-LA4X-LA5, LA2-LA3- LA4, LA2-LA3, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA3-LA4X-LA5-LA6X- LA7-EGFA-EGFBX-PROP, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA3-LA4X- LA5-LA6X-LA7-EGFA, LA3-LA4X-LA5-LA6X-LA7, LA3-LA4X-LA5-LA6, LA3- LA4X-LA5, LA3-LA4, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA4X-LA5-LA6X- LA7-EGFA-EGFBX-PROP, LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA4X-LA5-LA6X- LA7-EGFA, LA4X-LA5-LA6X-LA7, LA4X-LA5-LA6, LA4X-LA5, LA5-LA6X-LA7- EGFA-EGFBX-PROPX-EGFCX-OLSD, LA5-LA6X-LA7-EGFA-EGFBX-PROPX- EGFC, LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA5-LA6X-LA7-EGFA-EGFB, LA5- LA6X-LA7-EGFA, LA5-LA6X-LA7, LA5-LA6, LA6X-LA7-EGFA-EGFBX-PROPX- EGFCX-OLSD, LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA6X-LA7-EGFA- EGFBX-PROP, LA6X-LA7-EGFA-EGFB, LA6X-LA7-EGFA, LA6X-LA7, LA7- EGFA-EGFBX-PROPX-EGFCX-OLSD, LA7-EGFA-EGFBX-PROPX-EGFC, LA7- EGFA-EGFBX-PROP, LA7-EGFA-EGFB, LA7-EGFA, EGFA-EGFBX-PROPX- EGFCX-OLSD, EGFA-EGFBX-PROPX-EGFC, EGFA-EGFBX-PROP, EGFA- EGFB, EGFBX-PROPX-EGFCX-OLSD, EGFBX-PROPX-EGFC, EGFBX-PROP, or an amino acid variant thereof, or a multimer thereof, preferably a dimer or trimer, more preferably a dimer, still more preferably a homodimer or a homotrimer, and still more preferably a homodimer, and wherein said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TULV), New York hantavirus (NW), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV), preferably CCHFV or RVFV and most preferably CCHFV.
Preferably, said LDLR derived polypeptide further comprises a peptide linker connecting said fragments. More preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1) Moreover, the present invention particularly relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain, and wherein said Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
More particularly, the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain having amino acid sequence SEQ ID NO 18, or a functional fragment thereof, or an amino acid variant thereof, and wherein said Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
Preferably, said LDLR derived polypeptide further comprises a peptide linker connecting said fragments. More preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1)
A further embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, wherein said LDLR derived polypeptide comprises a dimer, a trimer, a homodimer, a homotrimer, or a multimer of an LDLR ectodomain monomer having amino acid sequence SEQ ID NO 18, or of a functional fragment thereof, or of an amino acid variant thereof, and wherein said Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
Preferably, said LDLR derived polypeptide further comprises a peptide linker connecting said fragments. More preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1). More preferably, said multimer comprises between 2 and 8 monomers, preferably 2 or 3 monomers of an LDLR ectodomain, or of a functional fragment thereof or of an amino acid variant thereof. A further embodiment of the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41), OLSD (SEQ ID NO 36), LA1 -LA2-LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA1 -LA2-LA3-LA4X-LA5-LA6X- LA7-EGFA-EGFBX-PROPX-EGFC, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROP, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA1 -LA2-LA3- LA4X-LA5-LA6X-LA7-EGFA, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7, LA1 -LA2-LA3- LA4X-LA5-LA6, LA1 -LA2-LA3-LA4X-LA5, LA1 -LA2-LA3-LA4, LA1-LA2-LA3, LA1- LA2, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA2- LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA2-LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROP, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA, LA2-LA3-LA4X-LA5-LA6X-LA7, LA2-LA3- LA4X-LA5-LA6, LA2-LA3-LA4X-LA5, LA2-LA3-LA4, LA2-LA3, LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA3-LA4X-LA5-LA6X-LA7-EGFA, LA3- LA4X-LA5-LA6X-LA7, LA3-LA4X-LA5-LA6, LA3-LA4X-LA5, LA3-LA4, LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROPX-EGFC, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA4X-LA5- LA6X-LA7-EGFA-EGFB, LA4X-LA5-LA6X-LA7-EGFA, LA4X-LA5-LA6X-LA7, LA4X-LA5-LA6, LA4X-LA5, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA5-LA6X-LA7-EGFA- EGFBX-PROP, LA5-LA6X-LA7-EGFA-EGFB, LA5-LA6X-LA7-EGFA, LA5-LA6X- LA7, LA5-LA6, LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA6X-LA7-EGFA-EGFBX-PROP, LA6X-LA7- EGFA-EGFB, LA6X-LA7-EGFA, LA6X-LA7, LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA7-EGFA-EGFBX-PROPX-EGFC, LA7-EGFA-EGFBX-PROP, LA7- EGFA-EGFB, LA7-EGFA, EGFA-EGFBX-PROPX-EGFCX-OLSD, EGFA-EGFBX- PROPX-EGFC, EGFA-EGFBX-PROP, EGFA-EGFB, EGFBX-PROPX-EGFCX- OLSD, EGFBX-PROPX-EGFC, EGFBX-PROP, or an amino acid variant thereof, or a multimer thereof, preferably a dimer or trimer, more preferably a dimer, still more preferably a homodimer or a homotrimer, and still more preferably a homodimer, and wherein said Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
Preferably, said LDLR derived polypeptide further comprises a peptide linker connecting said fragments. More preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1). More preferably, said multimer comprises between 2 and 8 monomers of an LDR ectodomain, or of a functional fragment thereof or of an amino acid variant thereof.
In the specific, the present invention relates to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain having amino acid sequence SEQ ID NO 18, and wherein said Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
In another aspect, the present invention relates to a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain, and wherein said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TULV), New York hantavirus (NW), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) or Sandfly Fever Naples virus (SFNV), and preferably wherein said Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV. In another aspect, the present invention relates to a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an amino acid variant of an LDLR ectodomain or a functional fragment thereof, and wherein said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TULV), New York hantavirus (NYV), Rift Valley fever virus (RVFV); Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) or Sandfly Fever Naples virus (SFNV), and preferably wherein said Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
"Pharmaceutical composition"
An embodiment of the present invention is directed to a pharmaceutical composition comprising the LDLR derived polypeptide described herein, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent.
The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a subject, including an animal or human. A pharmaceutical composition typically comprises an effective amount of an active agent and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient or diluent, respectively.
The pharmaceutical compositions of the invention are prepared for administration by mixing the LDLR derived polypeptide, with physiologically acceptable carriers, stabilizers, and excipients, and prepared in dosage form, e.g. by lyophilization in dosage vials.
Thus, a specific embodiment of the present invention is directed to a pharmaceutical composition comprising an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent.
The present invention is also directed to a pharmaceutical composition comprising the LDLR derived polypeptide as disclosed herein together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent for use in the prophylaxis and/or treatment of an infection by a Bunyavirus.
Moreover, the present invention is directed to a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an LDLR derived polypeptide as disclosed herein for use in the prophylaxis and/or treatment of an infection by a Bunyavirus.
Preferably, said LDLR derived polypeptide further comprises a peptide linker connecting said fragments. More preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1)
Said Bunyavirales virus is preferably selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TULV), New York hantavirus (NW), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV). Said Bunyavirales virus is more preferably CCHFV or RVFV and most preferably CCHFV.
An alternative embodiment of the present invention is directed to a pharmaceutical composition comprising an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an amino acid variant of an LDLR ectodomain, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent, and wherein said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TULV), New York hantavirus (NW), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV). Said Bunyavirales virus is more preferably CCHFV or RVFV and most preferably CCHFV.
A more specific embodiment of the present invention is directed to a pharmaceutical composition comprising an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain having amino acid sequence SEQ ID NO 18, or a functional fragment thereof, or an amino acid variant thereof.
Preferably, said LDLR derived polypeptide further comprises a peptide linker connecting said fragments. More preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1)
Said Bunyavirales virus is preferably selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TULV), New York hantavirus (NW), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV). Said Bunyavirales virus is more preferably CCHFV or RVFV and most preferably CCHFV.
A specific embodiment of the present invention is directed to a pharmaceutical composition comprising an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent, and wherein said LDLR derived polypeptide comprises a dimer, a trimer, a homodimer, a homotrimer, or a multimer of an LDLR ectodomain monomer having amino acid sequence SEQ ID NO 18, or of a functional fragment thereof, or of an amino acid variant thereof.
Preferably, said LDLR derived polypeptide further comprises a peptide linker connecting said fragments. More preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1). More preferably, said multimer comprises between 2 and 8 monomers of an LDR ectodomain, or of a functional fragment thereof or of an amino acid variant thereof.
Said Bunyavirales virus is preferably selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TULV), New York hantavirus (NW), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV). Said Bunyavirales virus is more preferably CCHFV or RVFV and most preferably CCHFV.
A specific embodiment of the present invention is directed to a pharmaceutical composition comprising an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent, and wherein said LDLR derived polypeptide comprises a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41), OLSD (SEQ ID NO 36), LA1 -LA2-LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA1 -LA2-LA3-LA4X-LA5-LA6X- LA7-EGFA-EGFBX-PROPX-EGFC, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROP, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA1 -LA2-LA3- LA4X-LA5-LA6X-LA7-EGFA, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7, LA1 -LA2-LA3- LA4X-LA5-LA6, LA1 -LA2-LA3-LA4X-LA5, LA1 -LA2-LA3-LA4, LA1-LA2-LA3, LA1- LA2, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA2- LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA2-LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROP, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA, LA2-LA3-LA4X-LA5-LA6X-LA7, LA2-LA3- LA4X-LA5-LA6, LA2-LA3-LA4X-LA5, LA2-LA3-LA4, LA2-LA3, LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA3-LA4X-LA5-LA6X-LA7-EGFA, LA3- LA4X-LA5-LA6X-LA7, LA3-LA4X-LA5-LA6, LA3-LA4X-LA5, LA3-LA4, LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROPX-EGFC, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA4X-LA5- LA6X-LA7-EGFA-EGFB, LA4X-LA5-LA6X-LA7-EGFA, LA4X-LA5-LA6X-LA7, LA4X-LA5-LA6, LA4X-LA5, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA5-LA6X-LA7-EGFA- EGFBX-PROP, LA5-LA6X-LA7-EGFA-EGFB, LA5-LA6X-LA7-EGFA, LA5-LA6X- LA7, LA5-LA6, LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA6X-LA7-EGFA-EGFBX-PROP, LA6X-LA7- EGFA-EGFB, LA6X-LA7-EGFA, LA6X-LA7, LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA7-EGFA-EGFBX-PROPX-EGFC, LA7-EGFA-EGFBX-PROP, LA7- EGFA-EGFB, LA7-EGFA, EGFA-EGFBX-PROPX-EGFCX-OLSD, EGFA-EGFBX- PROPX-EGFC, EGFA-EGFBX-PROP, EGFA-EGFB, EGFBX-PROPX-EGFCX- OLSD, EGFBX-PROPX-EGFC, EGFBX-PROP,or an amino acid variant thereof, or a homomultimer thereof, preferably a homodimer or a homotrimer thereof.
Preferably, said LDLR derived polypeptide further comprises a peptide linker connecting said fragments. More preferably, a peptide linker connecting said functional fragments is selected from the group comprising Linked to Linker76 (Table 1). More preferably, said multimer comprises between 2 and 8 monomers of an LDR ectodomain, or of a functional fragment thereof or of an amino acid variant thereof.
Said Bunyavirales virus is preferably selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TULV), New York hantavirus (NYV), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV). Said Bunyavirales virus is more preferably CCHFV or RVFV and most preferably CCHFV.
A specific embodiment of the present invention is directed to a pharmaceutical composition comprising an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain having amino acid sequence SEQ ID NO 18, and wherein said Bunyavirales virus is CCHFV or RVFV and most preferably CCHFV.
The term "effective amount" refers to a dosage (or dose) or amount of a substance sufficient to produce a desired result. The desired result may comprise an objective or subjective improvement in the recipient of the dosage or amount. For example, the desired result may comprise a measurable, or detectable inhibition of a virus infection in a subject to whom a dosage or amount of a particular LDLR ectodomain or a fragment thereof has been administered.
A "prophylactic treatment" is a treatment administered to a subject who does not display signs or symptoms of, or displays only early signs or symptoms of, a disease, pathology, or disorder, such that treatment is administered for the purpose of preventing or decreasing the risk of developing the disease, pathology, or disorder. A prophylactic treatment functions as a preventative treatment against a disease, pathology, or disorder, or as a treatment that inhibits or reduces further development or enhancement of a disease, pathology or disorder. A "prophylactic activity" is an activity of an agent that, when administered to a subject who does not display signs or symptoms of, or who displays only early signs or symptoms of, a pathology, disease, or disorder, prevents or decreases the risk of the subject developing the pathology, disease, or disorder. A "prophylactically useful" agent (e.g., LDLR ectodomain or a fragment thereof) refers to an agent that is useful in preventing development of a disease, pathology, or disorder, or useful in inhibiting or reducing further development or enhancement of a disease, pathology or disorder.
A "therapeutic treatment" is a treatment administered to a subject who displays symptoms or signs of pathology, disease, or disorder, in which treatment is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms. A "therapeutic activity" is an activity of an agent that eliminates or diminishes signs or symptoms of pathology, disease or disorder when administered to a subject suffering from such signs or symptoms. A "therapeutically useful" agent means the agent is useful in decreasing, treating, or eliminating signs or symptoms of a disease, pathology, or disorder.
LDLR derived polypeptide of functional fragments or of a multimer thereof.
A second aspect of the present invention is directed to an LDLR derived polypeptide comprising a functional fragment of an LDLR ectodomain selected from the group LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41), OLSD (SEQ ID NO 36), LA1-LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX- PROPX-EGFCX-OLSD, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX- PROPX-EGFC, LA1-LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA1- LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7- EGFA, LA1-LA2-LA3-LA4X-LA5-LA6X-LA7, LA1 -LA2-LA3-LA4X-LA5-LA6, LA1- LA2-LA3-LA4X-LA5, LA1 -LA2-LA3-LA4, LA1-LA2-LA3, LA1-LA2, LA2-LA3-LA4X- LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA2-LA3-LA4X-LA5-LA6X- LA7-EGFA-EGFBX-PROPX-EGFC, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROP, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA2-LA3-LA4X-LA5- LA6X-LA7-EGFA, LA2-LA3-LA4X-LA5-LA6X-LA7, LA2-LA3-LA4X-LA5-LA6, LA2- LA3-LA4X-LA5, LA2-LA3-LA4, LA2-LA3, LA3-LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROPX-EGFCX-OLSD, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX- PROPX-EGFC, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFB, LA3-LA4X-LA5-LA6X-LA7-EGFA, LA3-LA4X-LA5-LA6X- LA7, LA3-LA4X-LA5-LA6, LA3-LA4X-LA5, LA3-LA4, LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROPX-EGFCX-OLSD, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX- EGFC, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA4X-LA5-LA6X-LA7-EGFA- EGFB, LA4X-LA5-LA6X-LA7-EGFA, LA4X-LA5-LA6X-LA7, LA4X-LA5-LA6, LA4X- LA5, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA5-LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA5-LA6X- LA7-EGFA-EGFB, LA5-LA6X-LA7-EGFA, LA5-LA6X-LA7, LA5-LA6, LA6X-LA7- EGFA-EGFBX-PROPX-EGFCX-OLSD, LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA6X-LA7-EGFA-EGFBX-PROP, LA6X-LA7-EGFA-EGFB, LA6X-LA7-EGFA, LA6X-LA7, LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA7-EGFA-EGFBX- PROPX-EGFC, LA7-EGFA-EGFBX-PROP, LA7-EGFA-EGFB, LA7-EGFA, EGFA- EGFBX-PROPX-EGFCX-OLSD, EGFA-EGFBX-PROPX-EGFC, EGFA-EGFBX- PROP, EGFA-EGFB, EGFBX-PROPX-EGFCX-OLSD, EGFBX-PROPX-EGFC, EGFBX-PROP,or amino acid variants of said functional fragment, and wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said fragments.
Said "amino acid variant" refers to a single amino acid variant or to a multiple amino acid variant as previously defined.
An embodiment of the second aspect is directed to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a virus infection, wherein said virus is preferably a Bunyavirales virus, wherein said LDLR derived polypeptide comprises a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41 ), OLSD (SEQ ID NO 36), LA1-LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA1 -LA2-LA3- LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFB, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA, LA1 -LA2-LA3-LA4X- LA5-LA6X-LA7, LA1 -LA2-LA3-LA4X-LA5-LA6, LA1 -LA2-LA3-LA4X-LA5, LA1-LA2- LA3-LA4, LA1-LA2-LA3, LA1-LA2, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX- PROPX-EGFCX-OLSD, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX- EGFC, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA2-LA3-LA4X- LA5-LA6X-LA7-EGFA-EGFB, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA, LA2-LA3- LA4X-LA5-LA6X-LA7, LA2-LA3-LA4X-LA5-LA6, LA2-LA3-LA4X-LA5, LA2-LA3- LA4, LA2-LA3, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA3-LA4X-LA5-LA6X- LA7-EGFA-EGFBX-PROP, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA3-LA4X- LA5-LA6X-LA7-EGFA, LA3-LA4X-LA5-LA6X-LA7, LA3-LA4X-LA5-LA6, LA3- LA4X-LA5, LA3-LA4, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA4X-LA5-LA6X- LA7-EGFA-EGFBX-PROP, LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA4X-LA5-LA6X- LA7-EGFA, LA4X-LA5-LA6X-LA7, LA4X-LA5-LA6, LA4X-LA5, LA5-LA6X-LA7- EGFA-EGFBX-PROPX-EGFCX-OLSD, LA5-LA6X-LA7-EGFA-EGFBX-PROPX- EGFC, LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA5-LA6X-LA7-EGFA-EGFB, LA5- LA6X-LA7-EGFA, LA5-LA6X-LA7, LA5-LA6, LA6X-LA7-EGFA-EGFBX-PROPX- EGFCX-OLSD, LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA6X-LA7-EGFA- EGFBX-PROP, LA6X-LA7-EGFA-EGFB, LA6X-LA7-EGFA, LA6X-LA7, LA7- EGFA-EGFBX-PROPX-EGFCX-OLSD, LA7-EGFA-EGFBX-PROPX-EGFC, LA7- EGFA-EGFBX-PROP, LA7-EGFA-EGFB, LA7-EGFA, EGFA-EGFBX-PROPX- EGFCX-OLSD, EGFA-EGFBX-PROPX-EGFC, EGFA-EGFBX-PROP, EGFA- EGFB, EGFBX-PROPX-EGFCX-OLSD, EGFBX-PROPX-EGFC, EGFBX-PROP, or amino acid variants of said functional fragment, and wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said fragments.
Said Bunyavirales virus is preferably selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TULV), New York hantavirus (NW), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV).
Said Bunyavirales virus is more preferably CCHFV or RVFV and most preferably CCHFV. A particular embodiment of the second aspect is directed to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a virus infection, wherein said virus is preferably a RVF virus (RVFV), wherein said LDLR derived polypeptide comprises an amino acid variant of a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41 ), OLSD (SEQ ID NO 36), LA1 -LA2-LA3-LA4X-LA5-LA6X- LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROP, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA1 -LA2-LA3- LA4X-LA5-LA6X-LA7-EGFA, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7, LA1 -LA2-LA3- LA4X-LA5-LA6, LA1 -LA2-LA3-LA4X-LA5, LA1 -LA2-LA3-LA4, LA1-LA2-LA3, LA1- LA2, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA2- LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA2-LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROP, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA, LA2-LA3-LA4X-LA5-LA6X-LA7, LA2-LA3- LA4X-LA5-LA6, LA2-LA3-LA4X-LA5, LA2-LA3-LA4, LA2-LA3, LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA3-LA4X-LA5-LA6X-LA7-EGFA, LA3- LA4X-LA5-LA6X-LA7, LA3-LA4X-LA5-LA6, LA3-LA4X-LA5, LA3-LA4, LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROPX-EGFC, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA4X-LA5- LA6X-LA7-EGFA-EGFB, LA4X-LA5-LA6X-LA7-EGFA, LA4X-LA5-LA6X-LA7, LA4X-LA5-LA6, LA4X-LA5, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA5-LA6X-LA7-EGFA- EGFBX-PROP, LA5-LA6X-LA7-EGFA-EGFB, LA5-LA6X-LA7-EGFA, LA5-LA6X- LA7, LA5-LA6, LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA6X-LA7-EGFA-EGFBX-PROP, LA6X-LA7- EGFA-EGFB, LA6X-LA7-EGFA, LA6X-LA7, LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA7-EGFA-EGFBX-PROPX-EGFC, LA7-EGFA-EGFBX-PROP, LA7- EGFA-EGFB, LA7-EGFA, EGFA-EGFBX-PROPX-EGFCX-OLSD, EGFA-EGFBX- PROPX-EGFC, EGFA-EGFBX-PROP, EGFA-EGFB, EGFBX-PROPX-EGFCX- OLSD, EGFBX-PROPX-EGFC, EGFBX-PROP, and wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said fragments.
A further aspect of the present invention is directed to a LDLR derived polypeptide comprising a multimer of monomers, wherein each monomer is selected from the group comprising: an LDLR ectodomain (SEQ ID NO 18), a functional fragment of said LDLR ectodomain, an amino acid variant of said LDLR ectodomain, an amino acid variant of said functional fragment of said LDLR ectodomain, and wherein said multimer comprises between 2 and 8 monomers, and wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said monomers; and wherein said multimer is a homomultimer or a heteromultimer.
Said "amino acid variant" refers to a single amino acid variant or to a multiple amino acid variant as previously defined.
A particular further aspect is directed to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a virus infection, wherein said virus is preferably a Bunyavirales virus, wherein said LDLR derived polypeptide comprises a multimer of monomers, wherein each monomer is selected from the group comprising: an LDLR ectodomain (SEQ ID NO 18), a functional fragment of said LDLR ectodomain, an amino acid variant of said LDLR ectodomain, an amino acid variant of said functional fragment of said LDLR ectodomain, and wherein said multimer comprises between 2 and 8 monomers, and wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said monomers; and wherein said multimer is a homomultimer or a heteromultimer.
Said Bunyavirales virus is preferably selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQLIV), Puumala hantavirus (PUIIV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TLILV), New York hantavirus (NYV), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV).
Said Bunyavirales virus is more preferably a CCHF virus or RVFV and most preferably CCHFV.
A more particular further aspect is directed to an LDLR derived polypeptide for use in the prophylaxis and/or treatment of a virus infection, wherein said virus is preferably a RVF virus, wherein said LDLR derived polypeptide comprises a multimer of monomers, wherein each monomer is selected from the group comprising: an amino acid variant of an LDLR ectodomain (SEQ ID NO 18), an amino acid variant of a functional fragment of said LDLR ectodomain, and wherein said multimer comprises between 2 and 8 monomers, preferably 2 or 3 monomers, and wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said monomers; and wherein said multimer is a homomultimer or a heteromultimer.
Thus, the present invention also discloses a pharmaceutical composition comprising said LDLR derived polypeptide, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent, wherein said LDLR derived polypeptide comprises a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41), OLSD (SEQ ID NO 36), LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFBX-PROPX-EGFCX-OLSD, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROP, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA1 -LA2-LA3- LA4X-LA5-LA6X-LA7-EGFA, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7, LA1 -LA2-LA3- LA4X-LA5-LA6, LA1 -LA2-LA3-LA4X-LA5, LA1 -LA2-LA3-LA4, LA1-LA2-LA3, LA1- LA2, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA2- LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA2-LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROP, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA, LA2-LA3-LA4X-LA5-LA6X-LA7, LA2-LA3- LA4X-LA5-LA6, LA2-LA3-LA4X-LA5, LA2-LA3-LA4, LA2-LA3, LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA3-LA4X-LA5-LA6X-LA7-EGFA, LA3- LA4X-LA5-LA6X-LA7, LA3-LA4X-LA5-LA6, LA3-LA4X-LA5, LA3-LA4, LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROPX-EGFC, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA4X-LA5- LA6X-LA7-EGFA-EGFB, LA4X-LA5-LA6X-LA7-EGFA, LA4X-LA5-LA6X-LA7, LA4X-LA5-LA6, LA4X-LA5, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA5-LA6X-LA7-EGFA- EGFBX-PROP, LA5-LA6X-LA7-EGFA-EGFB, LA5-LA6X-LA7-EGFA, LA5-LA6X- LA7, LA5-LA6, LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA6X-LA7-EGFA-EGFBX-PROP, LA6X-LA7- EGFA-EGFB, LA6X-LA7-EGFA, LA6X-LA7, LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA7-EGFA-EGFBX-PROPX-EGFC, LA7-EGFA-EGFBX-PROP, LA7- EGFA-EGFB, LA7-EGFA, EGFA-EGFBX-PROPX-EGFCX-OLSD, EGFA-EGFBX- PROPX-EGFC, EGFA-EGFBX-PROP, EGFA-EGFB, EGFBX-PROPX-EGFCX- OLSD, EGFBX-PROPX-EGFC, EGFBX-PROP,or amino acid variants of said functional fragment, and wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said fragments.
Thus, the present invention also discloses a pharmaceutical composition comprising said LDLR derived polypeptide, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent, wherein said LDLR derived polypeptide comprises a multimer of monomers, wherein each monomer is selected from the group comprising: an LDLR ectodomain (SEQ ID NO 18), a functional fragment of said LDLR ectodomain, an amino acid variant of said LDLR ectodomain, an amino acid variant of said functional fragment of said LDLR ectodomain, and wherein said multimer comprises between 2 and 8 monomers, preferably 2 or 3 monomers, and wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said monomers; and wherein said multimer is a homomultimer or a heteromultimer.
Finally, the invention also contemplates a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an LDLR derived polypeptide, wherein said LDLR derived polypeptide comprises a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41), OLSD (SEQ ID NO 36), LA1 -LA2-LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA1 -LA2-LA3-LA4X-LA5-LA6X- LA7-EGFA-EGFBX-PROPX-EGFC, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROP, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA1 -LA2-LA3- LA4X-LA5-LA6X-LA7-EGFA, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7, LA1 -LA2-LA3- LA4X-LA5-LA6, LA1 -LA2-LA3-LA4X-LA5, LA1 -LA2-LA3-LA4, LA1-LA2-LA3, LA1- LA2, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA2- LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA2-LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROP, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA, LA2-LA3-LA4X-LA5-LA6X-LA7, LA2-LA3- LA4X-LA5-LA6, LA2-LA3-LA4X-LA5, LA2-LA3-LA4, LA2-LA3, LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA3-LA4X-LA5-LA6X-LA7-EGFA, LA3- LA4X-LA5-LA6X-LA7, LA3-LA4X-LA5-LA6, LA3-LA4X-LA5, LA3-LA4, LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROPX-EGFC, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA4X-LA5- LA6X-LA7-EGFA-EGFB, LA4X-LA5-LA6X-LA7-EGFA, LA4X-LA5-LA6X-LA7, LA4X-LA5-LA6, LA4X-LA5, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA5-LA6X-LA7-EGFA- EGFBX-PROP, LA5-LA6X-LA7-EGFA-EGFB, LA5-LA6X-LA7-EGFA, LA5-LA6X- LA7, LA5-LA6, LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA6X-LA7-EGFA-EGFBX-PROP, LA6X-LA7- EGFA-EGFB, LA6X-LA7-EGFA, LA6X-LA7, LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA7-EGFA-EGFBX-PROPX-EGFC, LA7-EGFA-EGFBX-PROP, LA7- EGFA-EGFB, LA7-EGFA, EGFA-EGFBX-PROPX-EGFCX-OLSD, EGFA-EGFBX- PROPX-EGFC, EGFA-EGFBX-PROP, EGFA-EGFB, EGFBX-PROPX-EGFCX- OLSD, EGFBX-PROPX-EGFC, EGFBX-PROP, or amino acid variants of said functional fragment, and wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said fragments.
Thus, the invention also contemplates a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an LDLR derived polypeptide, wherein said LDLR derived polypeptide comprises a multimer of monomers, wherein each monomer is selected from the group comprising: an LDLR ectodomain (SEQ ID NO 18), a functional fragment of said LDLR ectodomain, an amino acid variant of said LDLR ectodomain, an amino acid variant of said functional fragment of said LDLR ectodomain, and wherein said multimer comprises between 2 and 8 monomers, preferably 2 or 3 monomers, and wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said monomers; and wherein said multimer is a homomultimer or a heteromultimer.
Screening of gene factors of a Bunyavirales infection.
The inventors were able to identify LDLR as involved in CCHF virus infection by using a screening method based on infection of randomly mutagenized murine haploid cells with a CCHFV pseudotyped VSV, selection of mutant resistant clones and whole exome sequencing to individuate the gene mutations responsible for CCHFV resistance (Figure 1a-c, Table 2).
The use of chemical mutagenesis screens in haploid cells offers novel and unique opportunities for the discovery of host cell factors required for viral infection.
A diploid cell contains two copies of genomic DNA. A point mutation introduced by chemical mutagenesis always affects only one allele, while the second copy remains intact. One intact copy of a host cell factor is typically sufficient for a virus to hijack the cellular machinery and replicate. A haploid cell is a cell that only carries a single copy of the genome. Therefore, a point mutation introduced in a haploid cell alters 100% of the cellular protein. In case of a mutation interfering with viral entry or replication, this would completely block infection in the haploid cell and allow its identification in a screening setup. In sum, recessive mutations with regards to the viral infection can be identified. Other screening systems such as CRISPR/Cas9 or transposon or gene trap-based mutagenesis frequently target both copies of a gene and do therefore not require the use of haploid cells to identify viral host cell factors. However, most cellular factors hijacked by viruses constitute ancient proteins that are in many cases essential for cellular fitness and survival. Since the above-mentioned screening approaches fully remove the respective protein, cells would not die from the viral infection, but from the loss of the essential protein, so they remain invisible in the screen. By introducing only subtle changes to the host cell factors such as point mutations, a residual activity of these factors can typically be maintained allowing for cell survival while preventing or strongly reducing viral infection and replication. Therefore, applying chemical mutagenesis to a haploid cell system provides an inventive tool to study virus-host cell factor interactions in a forward genetic screening setup.
The term “diploid” is used herein to specifically refer to a cell or cell line including a genome wherein the cell is diploid for one or more specific genomic loci, or for the full genome, i.e. "fully diploid" cells.
The term “haploid” as used herein shall specifically refer to a cell or cell line including a single copy of the genome. Haploidy may be determined or tested by known methods, e.g. spectral karyotyping, comparative genomic hybridization or comparative propidium iodide staining.
The term “near-haploid” cell is a cell in which no more than 5 chromosomes are present in two or more copies. Near-haploid cells were found to maintain their status several months in culture. An exemplary near-haploid somatic human cell is of a KBM-7 cell line, which is haploid for most chromosomes with the exception of chromosome 8, and optionally a portion of chromosome 15, and is a non-adherent cell line. A further example of a near-haploid cell line is the HAP1 cell line, which is an adherent cell line obtained by engineering the KBM-7 cell line, which has lost the second copy of chromosome 8, and is hence “more haploid” than its KBM-7 parent, but still retains a portion of chromosome 15 and can therefore not be considered fully haploid. Further near-haploid cell lines may be derived from a cancer patient, or from a patient suffering from leukemia.
A specific example of a fully haploid somatic human cell line is the HAP2 cell line which is obtained by engineering HAP1 cells through excision of the portion of chromosome 15 that retained its diploidy in the HAP1 cell line, thus, is considered truly or fully haploid. It turned out that the HAP2 cell line comprises the complete set of human chromosomes in the monosomic state.
The eHAP1 cell line is a human fully haploid cell line deriving from HAP1 cells, which have been genetically engineered to delete the diploid portion of chromosome 15. eHAP1 is commercially available (e.g. Horizon Discovery (SKU: c669).
The term “mutagenesis” as used in the context of the present invention shall refer to a method of providing mutants of a sequence, e.g. through insertion, deletion and/or substitution of one or more nucleotides or amino acids, so to obtain variants thereof. Mutagenesis may be through random, semi-random or site directed mutation.
The term "haploid murine stem cells" refers to haploid murine stem cells that can be obtained and cultured as described in the prior art (Eiling, et al., Nat Protoc. 2019 Jul 1 ; 14(7): 1991-2014), or can be stabilized cell lines such as AN3-12, H129-2, H129-1. Therefore, in some embodiments, haploid murine stem cells are selected from the group comprising primary haploid murine stem cells, AN3-12, H129-2, H129-1 , and derivatives thereof.
The term "haploid human stem cells" refers to haploid human stem cells that can be obtained and cultured as described in the prior art, e.g in Sagi I, et al. Identification and propagation of haploid human pluripotent stem cells. Nat Protoc. 2016 11(11 ): 2274-2286. The term "haploid human stem cells" refers to haploid human stem cells of different origins, such as for example, haploid human embryonic stem cells (hESCs), haploid human spermatogonial stem cells, haploid hematopoietic stem cell,
The term "pseudovirus" (abbreviation: PV), or “pseudoparticles” (pp) or "pseudotyped virus" refers to a virus expressing heterologous viral envelope proteins. For example, a pseudotyped VSV has the envelope protein of a heterologous virus assembled into the VSV membrane.
"Pseudoviruses" are primarily derived from retroviruses such as HIV, Murine Leukemia Virus (MLV), and rhabdoviruses (VSV), and are used to investigate the function of viral fusion proteins in enveloped viruses. VSV is a bullet-shaped, negative polarity enveloped RNA virus in the Rhabdoviridae family. VSV has a genome size of 11 kb that contains five main viral proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and large polymerase protein (L).
Recombinant virus VSV in which the glycoprotein (G) gene is deleted (rVSV-AG) (Whitt, J Virol Methods. 2010 Nov; 169(2): 365-374.) has been used to produce VSV pseudotypes containing the envelope glycoproteins of heterologous viruses, including viruses that require high-level biocontainment, such as Bunyavirales.
The advantage of rVSV-AG pseudotypes is that it performs only one single round of replication, so that the experiments can be performed using biosafety level 2 (BSL-2) containment.
As such, rVSV-AG expressing heterologous envelope glycoproteins can be used to analyse genetic factors involved in infection of viral pathogens without the need for specialized containment facilities.
RNA viral particles pseudotyped with Bunyavirales glycoproteins can be obtained by using a method described in the prior art, such as for example in Cegolon et al 2021 . Preferred viruses are HIV, MSV, and VSV.
Random mutagenesis can be performed by a method selected from chemical random mutagenesis, UV mediated random mutagenesis, transposon mutagenesis, or error prone PCR. Preferably, random mutagenesis is performed by chemical random mutagenesis, and more preferably by incubating the haploid cells with Ethylnitrosurea (ENU) as described in the prior art.
Bunyavirales glycoproteins Gn and Gc are derived from a precursor polyprotein by host cell proteases. Bunyavirales glycoproteins have been described in the prior art, for example in Hulswit, RJC et al, Viruses. 2021 Feb; 13(2): 353.
The term "whole exome sequencing" refers to the sequencing of all exons in a specific cell or tissue type, wherein the exons are the protein coding regions of a gene. Therefore, in “whole exome sequencing” (WES) only the protein-coding DNA regions are sequenced as compared to whole genome sequencing (WGS).
The term “whole transcriptome sequencing” refers to sequencing of expressed transcripts at the mRNA level to obtain sequence information on all mRNAs made in a cell or cell pool at a given time The term "next generation sequencing" (NGS) refers to a group of platforms to perform sequencing of millions of small fragments of DNA in parallel. NGS can be used for whole genome sequencing, whole exome sequencing, or whole transcriptome sequencing. The terms "next generation sequencing", "massively parallel sequencing", and "deep sequencing" are related terms and can be used interchangeably.
A third aspect of the present invention is directed to an in vitro screening method to identify a gene that modulates a Bunyavirales virus infection, the method comprising:
A) providing haploid or near-haploid cells;
B) performing chemical random mutagenesis in said cells of step A) in order to obtain mutagenized haploid or near-haploid cells;
C) infecting the mutagenized cells of step B) with a Bunyavirales virus or with a Bunyavirales virus pseudotyped RNA viral particle;
D) culturing the infected mutagenized cells of step C) to obtain resistant colonies;
E) isolating the resistant colonies of step D);
F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate said Bunyavirales virus infection, wherein the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof, wherein the pseudotyped RNA viral particle is a HIV particle or a MSV particle or a VSV particle, and wherein the pseudotyped RNA viral particle express a Bunyavirales virus specific glycoprotein Gc and /or Gn, and wherein the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQIIV), Puumala hantavirus (PUIIV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TLILV), New York hantavirus (NYV), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV) preferably CCHFV or RVFV and most preferably CCHFV, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
In the above method, step B) of performing chemical random mutagenesis in said haploid or near-haploid cells of step A) in order to obtain mutagenized haploid or near-haploid cells preferably comprises incubating the haploid or near-haploid cells with N-Ethyl-N-nitrosourea (ENU).
More in detail, the present invention is directed to an in vitro screening method to identify a gene that modulates a Bunyavirales virus infection, the method comprising:
A) providing haploid or near-haploid cells;
B) performing chemical random mutagenesis in said cells of step A) in order to obtain mutagenized haploid or near-haploid cells;
C) infecting the mutagenized cells of step B) with a Bunyavirales virus or with a Bunyavirales virus pseudotyped RNA viral particle;
D) culturing the infected mutagenized cells of step C) to obtain resistant colonies;
E) isolating the resistant colonies of step D);
F) performing whole exome sequencing of the resistant colonies of step E) in order to identify the genes that modulate said Bunyavirales virus infection, wherein the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof, wherein the pseudotyped RNA viral particle is a HIV particle or a MSV particle or a VSV particle, and wherein the pseudotyped RNA viral particle express a Bunyavirales virus specific glycoprotein Gc and /or Gn, and wherein the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQIIV), Puumala hantavirus (PUIIV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TLILV), New York hantavirus (NYV), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV) preferably CCHFV or RVFV and most preferably CCHFV.
In the above method, step B) of performing chemical random mutagenesis in said haploid or near-haploid cells of step A) in order to obtain mutagenized haploid or near-haploid cells preferably comprises incubating the haploid or near-haploid cells with N-Ethyl-N-nitrosourea (ENU).
An alternative embodiment of the present invention is directed to an in vitro screening method to identify a gene that modulates a Bunyavirales virus infection, the method comprising:
A) providing haploid or near-haploid cells;
B) performing chemical random mutagenesis in said cells of step A) in order to obtain mutagenized haploid or near-haploid cells;
C) infecting the mutagenized cells of step B) with a Bunyavirales virus or with a Bunyavirales virus pseudotyped RNA viral particle;
D) culturing the infected mutagenized cells of step C) to obtain resistant colonies;
E) isolating the resistant colonies of step D);
F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate said Bunyavirales virus infection, wherein the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof, wherein the pseudotyped RNA viral particle is a HIV particle or a MSV particle or a VSV particle, and wherein the pseudotyped RNA viral particle express a Bunyavirales virus specific glycoprotein Gc and /or Gn, and wherein the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQIIV), Puumala hantavirus (PUIIV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TLILV), New York hantavirus (NYV), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV), preferably CCHFV or RVFV and most preferably CCHFV, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
In the above method, step B) of performing chemical random mutagenesis in said haploid or near-haploid cells of step A) in order to obtain mutagenized haploid or near-haploid cells preferably comprises incubating the haploid or near-haploid cells with N-Ethyl-N-nitrosourea (ENU).
In particular, the present invention is directed to an in vitro screening method to identify a gene that modulates a Bunyavirales virus infection, the method comprising:
A) providing haploid or near-haploid cells;
B) performing chemical random mutagenesis in said haploid or near- haploid cells of step A) in order to obtain mutagenized haploid or near- haploid cells;
C) infecting the mutagenized haploid or near-haploid cells of step B) with a Bunyavirales virus or with a Bunyavirales virus pseudotyped RNA viral particle;
D) culturing the infected mutagenized haploid or near-haploid cells of step C) to obtain resistant colonies;
E) isolating the resistant colonies of step D);
F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate said Bunyavirales virus infection, wherein the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof, wherein the pseudotyped RNA viral particle is a HIV particle or a MSV particle or a VSV particle, and wherein the pseudotyped RNA viral particle express a Bunyavirales virus specific glycoprotein Gc and /or Gn, and wherein the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), and Sandfly Fever Naples virus (SFNV), preferably CCHFV or RVFV and most preferably CCHFV, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
In the above method, step B) of performing chemical random mutagenesis in said haploid or near-haploid cells of step A) in order to obtain mutagenized haploid or near-haploid cells preferably comprises incubating the haploid or near-haploid cells with N-Ethyl-N-nitrosourea (ENU).
More in particular, the present invention is directed to in vitro screening method to identify a gene that modulates a Bunyavirales virus infection, the method comprising:
A) providing haploid or near-haploid cells;
B) performing chemical random mutagenesis in said haploid or near- haploid cells of step A) in order to obtain mutagenized haploid or near- haploid cells;
C) infecting the mutagenized haploid or near-haploid cells of step B) with a Bunyavirales virus;
D) culturing the infected mutagenized haploid or near-haploid cells of step C) to obtain resistant colonies;
E) isolating the resistant colonies of step D);
F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate said Bunyavirales virus infection, and wherein the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof, wherein the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQIIV), Puumala hantavirus (PUIIV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TLILV), New York hantavirus (NYV), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) or Sandfly Fever Naples virus (SFNV), preferably CCHFV or RVFV and most preferably CCHFV, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
In the above method, step B) of performing chemical random mutagenesis in said haploid or near-haploid cells of step A) in order to obtain mutagenized haploid or near-haploid cells preferably comprises incubating the haploid cells with N-Ethyl- N-nitrosourea (ENU).
Still more in particular, the present invention is directed to in vitro screening method to identify a gene that modulates a Bunyavirales virus infection, the method comprising:
A) providing haploid or near haploid cells;
B) performing chemical random mutagenesis in said haploid or near- haploid cells of step A) in order to obtain mutagenized haploid or near- haploid cells;
C) infecting the mutagenized haploid or near-haploid cells of step B) with a Bunyavirales virus pseudotyped RNA viral particle;
D) culturing the infected mutagenized haploid or near-haploid cells of step C) to obtain resistant colonies;
E) isolating the resistant colonies of step D);
F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate said Bunyavirales virus infection, wherein the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof, wherein the pseudotyped RNA viral particle is a HIV particle or a MSV particle or a VSV particle, and wherein the pseudotyped RNA viral particle express a Bunyavirales virus specific glycoprotein Gc and /or Gn, wherein the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQLIV), Puumala hantavirus (PUIIV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TLILV), New York hantavirus (NYV), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) or Sandfly Fever Naples virus (SFNV), preferably CCHFV or RVFV and most preferably CCHFV, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
In the above method, step B) of performing chemical random mutagenesis in said haploid or near-haploid cells of step A) in order to obtain mutagenized haploid or near-haploid cells preferably comprises incubating the haploid cells with N-Ethyl- N-nitrosourea (ENU).
Specifically, the present invention is directed to an in vitro screening method to identify a gene that modulates a Bunyavirales virus infection, the method comprising:
A) providing haploid or near-haploid cells;
B) performing chemical random mutagenesis in said haploid or near- haploid cells of step A) in order to obtain mutagenized haploid or near- haploid cells;
C) infecting the mutagenized haploid or near-haploid cells of step B) with a Bunyavirales virus pseudotyped RNA viral particle;
D) culturing the infected mutagenized haploid or near-haploid cells of step C) to obtain resistant colonies;
E) isolating the resistant colonies of step D);
F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate said Bunyavirales virus infection, wherein the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof, wherein the pseudotyped RNA viral particle is a VSV particle, and wherein the pseudotyped RNA viral particle express a Bunyavirales virus specific glycoprotein Gc and /or Gn, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing. In the above method, step B) of performing chemical random mutagenesis in said haploid or near-haploid cells of step A) in order to obtain mutagenized haploid or near-haploid cells preferably comprises incubating the haploid or near-haploid cells with N-Ethyl-N-nitrosourea (ENU).
More specifically, the present invention is directed to an in vitro screening method to identify a gene that modulates a Bunyavirales virus infection, the method comprising:
A) providing haploid or near-haploid cells;
B) performing chemical random mutagenesis in said haploid or near- haploid cells of step A) in order to obtain mutagenized haploid or near- haploid cells;
C) infecting the mutagenized haploid or near-haploid cells of step B) with a Bunyavirales virus pseudotyped RNA viral particle;
D) culturing the infected mutagenized haploid or near-haploid cells of step C) to obtain resistant colonies;
E) isolating the resistant colonies of step D);
F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate said Bunyavirales virus infection, wherein the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof, wherein the pseudotyped RNA viral particle is a VSV particle, and wherein the pseudotyped RNA viral particle express a Bunyavirales virus specific glycoprotein Gc and /or Gn, wherein the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQIIV), Puumala hantavirus (PUIIV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TLILV), New York hantavirus (NYV), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) or Sandfly Fever Naples virus (SFNV), preferably CCHFV or RVFV and most preferably CCHFV, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
In the above method, step B) of performing chemical random mutagenesis in said haploid or near-haploid cells of step A) in order to obtain mutagenized haploid or near-haploid cells preferably comprises incubating the haploid or near-haploid cells with N-Ethyl-N-nitrosourea (ENU).
Moreover, the third aspect of the invention is directed to an in vitro screening method to identify a gene that modulates a CCHFV infection, the method comprising:
A) providing haploid or near-haploid cells;
B) performing chemical random mutagenesis in said haploid or near- haploid cells of step A) in order to obtain mutagenized haploid or near- haploid cells;
C) infecting the mutagenized haploid or near-haploid cells of step B) with a CCHFV or with a CCHFV glycoprotein pseudotyped RNA viral particle;
D) culturing the infected mutagenized haploid or near-haploid cells of step C) to obtain resistant colonies;
E) isolating the resistant colonies of step D);
F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate a CCHFV infection, wherein the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof, wherein the pseudotyped RNA viral particle is a HIV particle or a MSV particle or a VSV particle, and wherein said CCHFV glycoprotein is CCHFV glycoprotein Gc and/or Gn, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
Preferably, performing chemical random mutagenesis in said haploid or near- haploid cells of step B) in order to obtain mutagenized haploid or near-haploid cells comprises incubating the haploid cells with N-Ethyl-N-nitrosourea (ENU). A particular embodiment is directed to an in vitro screening method to identify a gene that modulates a CCHFV infection, the method comprising:
A) providing haploid or near-haploid cells;
B) performing chemical random mutagenesis in said haploid or near- haploid cells of step A) in order to obtain mutagenized haploid or near- haploid cells;
C) infecting the mutagenized haploid or near-haploid cells of step B) with a CCHFV glycoprotein pseudotyped RNA viral particle;
D) culturing the infected mutagenized haploid or near-haploid cells of step C) to obtain resistant colonies;
E) isolating the resistant colonies of step D);
F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate a CCHFV infection, wherein the pseudotyped RNA viral particle is a VSV particle, and wherein said CCHFV glycoprotein is CCHFV glycoprotein Gc and/or Gn, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
A further embodiment is directed to an in vitro screening method to identify a gene that modulates a CCHFV infection, the method comprising:
A) providing haploid or near-haploid cells;
B) performing chemical random mutagenesis in said haploid or near- haploid cells of step A) in order to obtain mutagenized haploid or near- haploid cells by incubating the haploid or near-haploid cells with N- Ethyl-N-nitrosourea (ENU);
C) infecting the mutagenized haploid or near-haploid stem cells of step B) with a CCHFV glycoprotein pseudotyped RNA viral particle;
D) culturing the infected mutagenized haploid or near-haploid cells of step C) to obtain resistant colonies;
E) isolating the resistant colonies of step D);
F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate a CCHFV infection, wherein the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof, wherein the pseudotyped RNA viral particle is a HIV particle or a MSV particle or a VSV particle, and wherein said CCHFV glycoprotein is CCHFV glycoprotein Gc and/or Gn, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
In the above method, step B) of performing chemical random mutagenesis in said haploid or near-haploid cells of step A) in order to obtain mutagenized haploid or near-haploid cells preferably comprises incubating the haploid or near-haploid cells with N-Ethyl-N-nitrosourea (ENU).
A further particular embodiment is directed to an in vitro screening method to identify a gene that modulates a CCHFV infection, the method comprising:
A) providing haploid or near-haploid cells;
B) performing chemical random mutagenesis in said cells of step A) in order to obtain mutagenized haploid or near-haploid cells by incubating said cells with N-Ethyl-N-nitrosourea (ENU);
C) infecting the mutagenized haploid or near-haploid cells of step B) with a CCHFV glycoprotein pseudotyped RNA viral particle;
D) culturing the infected mutagenized haploid or near-haploid cells of step C) to obtain resistant colonies;
E) isolating the resistant colonies of step D);
F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate a CCHFV infection, wherein the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof, wherein the pseudotyped RNA viral particle is a VSV particle, and wherein said CCHFV glycoprotein is CCHFV glycoprotein Gc and/or Gn, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
A further more particular embodiment is directed to an in vitro screening method to identify a gene that modulates a CCHFV infection, the method comprising:
A) providing haploid or near-haploid cells; B) performing chemical random mutagenesis in said haploid or near- haploid cells of step A) in order to obtain mutagenized haploid or near- haploid cells;
C) infecting the mutagenized haploid or near-haploid cells of step B) with CCHFV;
D) culturing the infected mutagenized haploid or near-haploid cells of step
C) to obtain resistant colonies;
E) isolating the resistant colonies of step D);
F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate a CCHFV infection, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
In the above method, step B) of performing chemical random mutagenesis in said haploid or near-haploid cells of step A) in order to obtain mutagenized haploid or near-haploid cells preferably comprises incubating the haploid or near-haploid cells with N-Ethyl-N-nitrosourea (ENU).
Moreover, an embodiment is directed to an in vitro screening method to identify a gene that modulates a CCHFV infection, the method comprising:
A) providing haploid or near-haploid cells;
B) performing chemical random mutagenesis in said haploid or near- haploid cells of step A) in order to obtain mutagenized haploid or near- halpoid cells by incubating the said cells with N-ethyl-N-nitrosourea (ENU);
C) infecting the mutagenized haploid or near-haploid cells of step B) with a CCHFV;
D) culturing the infected mutagenized haploid or near-haploid cells of step C) to obtain resistant colonies;
E) isolating the resistant colonies of step D);
F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate a CCHFV infection, wherein the haploid cells are selected from the group comprising human haploid stem cells, or murine haploid stem cells, or human eHAP1 , or human HAP2, or preferably murine haploid stem cells or wherein the near-haploid cells are selected from the group comprising KBM-7 cells, HAP1 cells, and derivatives thereof, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
A more specific embodiment is directed to an in vitro screening method to identify a gene that modulates a CCHFV infection, the method comprising:
A) providing haploid murine stem cells;
B) performing chemical random mutagenesis in said cells of step A) in order to obtain mutagenized haploid murine stem cells;
C) infecting the mutagenized cells of step B) with a Bunyavirales virus or with a Bunyavirales virus pseudotyped RNA viral particle;
D) culturing the infected mutagenized cells of step C) to obtain resistant colonies;
E) isolating the resistant colonies of step D);
F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate a Bunyavirales virus infection, wherein the pseudotyped RNA viral particle is a HIV particle or a MSV particle or a VSV particle, wherein the pseudotyped RNA viral particle express a Bunyavirales virus specific glycoprotein Gc and/or Gn, and wherein the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQIIV), Puumala hantavirus (PUIIV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TLILV), New York hantavirus (NYV), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV), preferably CCHFV or RVFV and most preferably CCHFV, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
In the in vitro screening method described above, the pseudotyped RNA viral particle is preferably a VSV particle.
In the in vitro screening method described above, it is preferred that performing chemical random mutagenesis of step B) comprises incubating the haploid murine stem cells with N-Ethyl-N-nitrosourea (ENU). Thus, a still more specific embodiment is directed to an in vitro screening method to identify a gene that modulates a CCHFV infection, the method comprising:
A) providing haploid murine stem cells;
B) performing chemical random mutagenesis in said cells of step A) in order to obtain mutagenized haploid murine stem cells;
C) infecting the mutagenized cells of step B) with a Bunyavirales virus or with a Bunyavirales virus pseudotyped RNA viral particle;
D) culturing the infected mutagenized cells of step C) to obtain resistant colonies;
E) isolating the resistant colonies of step D);
F) performing sequencing of the resistant colonies of step E) in order to identify the genes that modulate a Bunyavirales virus infection, wherein the pseudotyped RNA viral particle is a VSV particle, wherein the pseudotyped VSV particle express a Bunyavirales virus specific glycoprotein Gc and/or Gn, and wherein the Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQIIV), Puumala hantavirus (PUIIV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TLILV), New York hantavirus (NYV), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) and Sandfly Fever Naples virus (SFNV), preferably CCHFV or RVFV and most preferably CCHFV, wherein sequencing of step f) comprises whole genome sequencing or whole exome sequencing or whole transcriptome sequencing.
In the in vitro screening method described above, it is preferred that performing chemical random mutagenesis of step B) comprises incubating the haploid murine stem cells with N-Ethyl-N-nitrosourea (ENU).
Moreover, the binding of the LDLR derived polypeptide can also be used for the detection of a Bunyavirales virus infection. Therefore, the present application is also directed to the use of an LDLR derived polypeptide in the diagnosis of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain as disclosed herein and wherein said binding diagnoses the infection by the Bunyavirales virus.
Further, the binding of the LDLR derived polypeptide can also be used for the quality control of vaccines produced to prevent or ameliorate Bunyavirales virus infection, and especially CCHFV infection. Therefore, the present application is also directed to the use of an LDLR derived polypeptide in quality control of a Bunyavirales virus vaccine, especially a CCHFV vaccine, wherein said LDLR derived polypeptide binds to vaccine ingredients, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain as disclosed herein and wherein said binding assesses the quality of that vaccine to prevent or ameliorate a Bunyavirales virus infection.
Description of the Figures
Figure 1 shows generation of VSV-CCHF_G and haploid cells screening. a, Schematic representation of the methods used to produce the VSV- CCHF_G pseudotyped virus in HEK293T cells. b, Validation of resistant clones obtained in the primary haploid screen with VSV- CCHF_G. Each clone (1-13) was isolated, amplified and assessed for infection with the CCHFV lbAR10200 laboratory strain (MOI 0.1). The data show the level of infection for each clone compared to wild-type haploid cells (AN3-12) as determined by RT-PCR for CCHFV and RNase P RNA 24hpi. c, Deep exome sequencing identified three distinct single point mutations in the Ldlr gene in three resistant clones (5, 8, 10) resulting in stop codons or a missense mutation.
Figure 2 shows CCHFV infections in Ldlr knockout cells, a, Level of infections in control wild type AN3-12 haploid and sister knock out (KO) cells infected with VSV-CCHF_G, CCHFV lbAr10200 (MOI 0.1 , 48hpi). Level of infection was assessed by RT-qPCR for viral and RNase P RNA. b, Levels of infection of lbAr10200 CCHFV in wild type (WT) and two different LDLR KO (clones C2 and C12) Vero cells and c, in three different clones of LDLR KO (clones C8, C10 and C11) A549 cells. All mutant clones in b-c were generated using CRISPR/cas9. Mutant haploid clones were from our previously reported Haplobank. All infections of diploid cells were done at a MOI of 0.1 for 24h. Graphs show mean ± SD. n= 3 independent experiments. P values were calculating using unpaired student’s t-tests. Significance values are shown; non-significant: p>0.05.
Figure 3 shows that recombinant CCHFV Gc protein binds to and induces the internalization of LDLR. a, Illustration depicting the BRET-based binding assay that was used to measure the interaction of BODIPY-FL labelled LDL and soluble BODIPY-FL labelled CCHFV Gc with the LDLR. b, Cells expressing Nluc-LDLR (donor) were stimulated with vehicle or increasing concentrations of BODIPY-FL-labelled ligand (acceptor) for 90 min during which the BRET was measured continuously. Data are represented as the mean area under the curve ± SEM (n=3-5). c, Schematic of the internalization assay to assess the ligand-dependent accumulation of LDLR in early endosomes, d, Cells expressing LDLR-Rlucll (donor) and rGFP-FYVE (acceptor) were stimulated with vehicle or increasing concentrations of LDL or recombinant CCHFV Gc for 45 min prior to BRET measurements. Data are represented as the mean ± SEM (n=4). Binding and internalization were assessed by comparing the top and bottom parameters from nonlinear regression in the extra sum-of-squares F-test (P < 0.05). **P < 0.01 ; ****P < 0.0001 (one-tailed extra sum-of-squares F test).
Figure 4 shows inhibition of CCHFV infections by soluble LDLR. a, Levels of VSV- CCHF_G infections in human SW13 cells, treated with the indicated range of soluble LDLR concentrations or left untreated (mock-treated) (MOI 0.01 , 6hpi) b, Levels of lbAr10200 CCHFV infections in SW13 cells, treated with a range of soluble LDLR concentrations (MOI 0.01 , 24hpi). c, Level of VSV infections in SW13 cells, treated with the indicated concentrations of soluble LDLR (MOI 0.01 , 6hpi).). d, Levels of lbAr10200 CCHFV infections in SW13 cells, treated with the indicate concentrations of soluble LRP8 (MOI 0.01 , 24hpi). e, Levels of lbAr10200 CCHFV infections in SW13 cells, treated with soluble VLDLR decoys (MOI 0.01 , 24hpi) f, Levels of VSV infections in SW13 cells, treated with soluble LRP8 decoys. (MOI 0.01 , 6hpi) g, Levels of VSV infections in human SW13 cells, treated with soluble VLDLR (MOI 0.01 , 6hpi). Graphs show mean ± SD. n= 3 independent experiments. One-way ANOVA. Significance values are shown; non significant: p>0.05.
Figure 5 shows CCHFV infections in human BVOs and Ldlr mutant mice, a, Scheme representing blood vessel organoids made from LDLR+ and LDLR- iPSC cells that were dissociated and seeded as 2D monolayer, b, Level of CCHFV (lbAr10200) infection of BVO-derived vascular cells generated from wild type (WT) and LDLR KO iPSCs. Levels of infections were determined by RT-PCR at 1day post-infection (1dpi) and 3 days post-infection (3dpi) (MOI 0.1 ). P values were calculating using unpaired student’s t-tests. c-e, CCHFV (lbAr10200) infections of wild type or Ldlr KO mice. n=6 femelle mice per group (400 PFU/mouse). c, Weight loss was assessed the day of infection (day 0) and the day of euthanasia (day 4 post-infection), d, Numbers of CCHFV RNA copies in serum, liver and spleen of wild type and Ldlr KO mice determined the day of euthanasia (day 4 post-infection). P values were calculating using unpaired student’s t-tests comparing two groups. All graphs are show as mean values ± SD. e, Histopathological analysis (H&E staining) of livers from wild type (WT) and Ldlr KO (KO) mice showing little-to-no pathology in Ldlr KO mice mice infected with CCHFV and analysed on day 4 after infection. Midzonal necrosis, periportal coagulative necrosis, as well as sporadic necrosis of single cells in livers of wild type mice. Livers of Ldlr KO mice showed little-to-no pathology. Scales are noted on each picture.
Figure 6 shows validation of LDLR with a CCHFV patient isolate. CCHFV was isolated from the serum of a Turkish patient and this clinical isolate used for all subsequent experiments shown in Figure 6. a-c, Levels of CCHFV infections (MOI 0.01 , 24hpi) of SW13 cells treated with the indicated concentrations of a, sLDLR, b, sLRP8, and c, sVLDLR. d, Levels of infection with clinical CCHFV in wild type and LDLR KO (clones C2 and C12) Vero cells and in wild type and LDLR KO (clones C8, C10 and C11 ) A549 cells (MOI 0.1 , 24hpi). Graphs show mean value ± SD. n= 3 independent experiments. P values were calculating using unpaired student’s t-tests. hpi = hour post-infection.
Figure 7 shows generation and validation of Ldlr knockout in A549 and Vero cells, a, Schematic of CRISPR-Cas9 editing strategy. The extracellular region of LDLR was targeted, leading to putative N-terminally truncated proteins not displayed on the cell surface for entry, b, Schematic of editing and a-LDLR sorting procedure, c, PE intensity from a-LDLR- PE staining is shown. a-LDLR-PE staining was evaluated on single cells. Event densities were smoothened and are displayed as absolute counts or as counts normalization to the mode. Numbers indicate the percentage of single cells defined as a-LDLR-PE negative, d, Non- reactive and stained Tb1-Lu cells were used as negative control. PE intensity from a-LDLR-PE staining is shown. Event density was smoothened by normalization to the mode. e, Bulk sorting after transfection and transient Puromycin selection of a-LDLR-PE stained A549 or Vero cells, edited or unmodified (control) via CRISPR-Cas9. Event densities were smoothened and are displayed as counts normalization to the mode, f, Flow-cytometry result from A549-wild type and edited A549 clone 10 cells. PE intensity from a-LDLR-PE staining is shown. Event density was smoothened by normalization to the mode. Numbers indicate the percentage of single cells defined as a- LDLR-PE positive for A549 clone 10 and unmodified WT cells. Event densities were smoothened and are displayed as absolute counts.
Figure 8 shows that ligand selectivity at LDLR drives receptor internalization. a,b, Specificity of the BRET-based binding assay was determined through coadministration of increasing concentrations of labelled LDL in the presence of excess unlabelled LDL to cells expressing Nluc-LDLR. Data are represented as the mean area under the curve ± SEM (n=5). c,d, LDLR exhibits ligand selectivity as evidenced by the absence of increased receptor internalization following the addition of the receptor binding domain from SARS-CoV-2. Data are represented as the mean ± SEM (n=3). Binding and internalization were assessed by comparing the top and bottom parameters from non-linear regression in the extra sum-of-squares F-test (P < 0.05). ns non-significant (one-tailed extra sum-of-squares F test).
Figure 9 shows creation and validation of NC8 cells knocked out for LDLR. a, Gating strategy and PE intensity from a-LDLR-PE staining are shown. a-LDLR-PE staining was evaluated on single cells, b, Sorting results from bulk NC8 iPSC after Cas9 LDLR editing. PE intensity from a-LDLR-PE staining is shown for cells targeted with an LDLR guide RNA or for unmodified control cells. Event densities were smoothened and are displayed as absolute counts or as counts normalization to the mode, c, Qualitative flow-cytometry result of selected clones stained with an a-LDLR-PE antibody. Shown is the PE intensity from a-LDLR- PE staining from gated single cells of LDLR- or LDLR+ iPSC clones, d, Flow-cytometry result of the studied LDLR-KO or wild-type LDLR iPSC clones (clone 4, clone 10). Shown is the overlayed mode- normalized density of PE intensity from a-LDLR-PE staining for clone 10 and 4. The legend percentages indicate the fraction of a-LDLR-PE negative stained single cells.
Figure 10 Phage display
Schematic of a phage-display approach to identify high-affinity binding LDLR fragments and variants thereof to viral glycoproteins in a high- throughput format.
Figure 11 Immunofluorescence assays a, Immunofluorescence staining of CCHFV in wild-type and LDLR KO cells. P values were calculating using two tailed student t-test. **P < 0.01. b, Immunofluorescence staining of CCHFV in SW13 cells infected with CCHFV mock or sLDLR treated.
Figure 12 LDL outcompetes CCHFV from cellular entry via LDLR a, Competition assay between CCHFV and LDL in SW13 cells (MOI 0.01 , 24hpi). b, BSA was used as control. Data are represented as the mean ± SD. n= 3 independent experiments. P values were calculating using One-way ANOVA. *P<0.05, **P < 0.01 ; non-significant: p>0.05.
Figure 13 Role of LDLR and LRP8 in the infection of CCHFV from ticks or human serum
CCHFV produced on Hyalomma tick cells (a) and CCHFV from human patient serum (b) were tested for blocking with sLDLR or sLRP8 (MOI 0.01 , 24hpi). Graphs show mean value ± SD. n= 3 independent experiments. P values were calculating using two tailed student t-test. **P < 0.01 ; Non significant: p>0.05.
Figure 14 Protective effect of LDLR, LDLR-fragments, and multimers against CCHF-VLP infection CCHF-VLP infection assays in Huh-7 cells. Luminescence was normalized to infection control. VLPs were incubated with 10nM purified LDLR-fragments or commercially available rhLDLR (RnD System; Cat.- No. 2148-HP) or rhLDLR-mFc (SinoBiological; Cat.-No. 10231-H05H). Graph shows mean value + SEM; n > 3 independent experiments; p- values were calculated using two tailed student t-test: * p < 0.05, ** p < 0.01 , *** p < 0.001. f: expressed and purified from E.coli
Figure imgf000080_0001
concentration < 5nM: sLDLR-sLRP8 (2.4nM), sLDLR-sVLDLR (1.7nM), sLRP8-sLDLR (4.4nM), sVLDLR-sLDLR (1.4nM). s: soluble (receptor ectodomain without the transmembrane anchor) rh: recombinant human protein from commercial provider I: linker (GGGAAAGGAAGGGAAAGGAA, SEQ. ID NO 225 with n = 2)
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the scope of the invention.
Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the scope of the invention as described in the following claims. Examples
Materials and Methods
Cells and viruses. The cell lines used were HEK293 (ATCC®, CRL-1573), HEK293T/17 (HEK293T, ATCC® CRL-11268™), A549 (ATCC® CCL-185) and Vero cells (ATCC® CCL-81 ). All cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Life Technologies, Italy), supplemented with 10 % v/v of heat-inactivated Fetal Bovine Serum (FBS, Life Technologies) and incubated at 37° C with 95 % humidity and 5% CO2. SW13 (epithelial cells from adrenal gland, ATCC® CCL-105) cells were maintained in Leibovitz’s L15 medium (Thermofisher) at 37° C without CO2. Haploid mouse Stem-Cells (mSCs, clone AN3-12) were maintained in standard embryonic stem-cell medium, supplemented with 10% (v/v) fetal bovine serum (Hyclone), recombinant mouse Leukemia Inhibitory Factor (LIF) and [3-mercaptoethanol at 37°C with 95 % humidity and 5% CO2. All cell lines were regularly tested for mycoplasma contamination.
VSV-CCHF_G was produced as described below. CCHFV lbAr10200 strain was cultured on SW13 cells. CCHFV clinical strain was isolated on SW13 cells from a Turkish patient serum sampled as a part of another project. Ethical clearance has been obtained (Nr: 2017/1712-31/2) as well as fully informed patient consents.
Biosafety. All our experiments involving VSV-CCH_G were done in biosafety level 2 laboratory and experiments involving CCHFV were done in biosafety level 4 laboratory in compliance with the Swedish Public Health Agency guidelines (Folkhalsomyndigheten, Stockholm).
Reagents. D-PBS, DMEM, Trypsin, PBS, penicillin/streptomycin, fetal bovine serum were from Gibco (ThermoFisher Scientific, Waltham, MA, USA). Polyethylenimine (PEI) was purchased from Alfa Aesar (ThermoFisher Scientific, Waltham, MA, USA). Unlabeled low-density lipoprotein (LDL) from human plasma and BOPIDY FL complexed LDL were purchased from ThermoFisher Scientific (Waltham, MA, USA). Human Fc-tagged CCHFV Gc, 6xHis-tagged CCHFV Gn and BODIPY FL complexed CCHFV Gc were purchased from Native Antigen (Kidlington, UK). Coelenterazine h was purchased from Nanolight Technologies (Pinetop, AZ, USA). NanoBRET Nano-Gio substrate was purchased from Promega (Madison, Wl, USA). Trizol was purchased from ThermoFisher (ThermoFisher Scientific, Waltham, MA, USA). Anti-IFN type I receptor antibody (MAR1 -5A3) was purchased from Leinco (MAR1 -5A3 [5A3]; Leinco Technologies, Inc.). Soluble LDLR, VLDLR and LRP8 were purchased from R&D systems. This soluble LDLR (Catalog #: 2148-LD R&D systems) corresponds to human LDLR protein (Uniprot P01130) Ala22-Arg788; soluble VLDLR (Catalog #: 8444-VL-025 R&D systems) corresponds to human VLDLR protein (UniProt: P98155.1) Thr25- Ser797; soluble LRP8 (Catalog #: 3520-AR-050, R&D systems) corresponds to human LRP8 protein (UniProt: Q14114.4) Asp35-Lys818 (Ala262Val).
Pseudotyped virus production and titration. The plasmid pC-G (Shtanko et al, 2014, Crimean-Congo hemorrhagic fever virus entry into host cells occurs through the multivesicular body and requires ESCRT regulators. PLoS Pathog. 2014 Sep 18; 10(9):e1004390) expressing the CCHFV glycoproteins Gn and Gc (strain lbAr10200) was kindly provided by Robert A. Davey (Texas Biomedical Research Institute, San Antonio, Texas, USA). The plasmid expressing the Vesicular Stomatitis Virus (VSV) glycoprotein (pVSV-G) was previously described (Salata et al., 2009, vOX2 glycoprotein of human herpesvirus 8 modulates human primary macrophages activity. J Cell Physiol. 2009 Jun;219(3):698-706). The recombinant VSV encoding the GFP in place of the VSV-G gene (VSVAG-GFP) was kindly provided by Michael Whitt, University of Tennessee, USA. CCHFV-Gn/Gc pseudotyped VSVAG-GFP (CCHFV-pseudotyped virus) was generated as previously described. Briefly, HEK293T cells were seeded in T75 flask and 24 h later transfected by calcium-phosphate protocol with 20 pg of pC-G plasmid and, 24 h later, infected with the recombinant VSVAG-GFP virus at the multiplicity of infection (MOI) of 4 fluorescent focus-forming units (FFU)/cell. Sixteen hours p.i., cell culture supernatants were harvested and cell debris were cleared by centrifugation (2,300 rpm for 7 min at 4° C). Thereafter, virus particles were pelleted by ultracentrifugation on a 20% p/v a sucrose cushion (27,000 rpm for 150 min at 4° C) in a Beckmann (Beckman Coulter Italia, Italy) SW 28 Ti swingingbucket rotor. Pellets were resuspended in 1 mL of ice-cold PBS1X/tube and mixed. Subsequently, the virus was aliquoted and stored at -80° C until use. Virus titer was determined by immunofluorescence on Vero cells seeded on 96-well plates. Viral stock was 10-fold serially diluted in DMEM and inoculated on confluent Vero cells for 1 h at 37° C. Cells were then washed and DMEM supplemented with 10% FBS was added. After 18 h, cells were fixed in chilled methanol/aceton and stained with anti-VSV-M protein (VSV-M [clone 23H12], Kerafast) antibody Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibodies (ThermoFisher). The fluorescent foci were counted and viral titer was expressed as FFU/mL.
Chemical mutagenesis of haploid stem-cells. Chemical mutagenesis using Ethylnitrosurea (ENU) was performed treating haploid AN3-12 cells for 2h with 0.1 mg/ml ENU in full medium while in suspension and under constant agitation. Cells were washed 5 times and transferred to a culture dish. Cells were left to recover for 48h, singled using Trypsin/EDTA and frozen in 10% DMSO, 40% FBS and 50% full medium. ENU libraries as well as untreated control libraries were used in screening experiments with VSV-CCHFV.
Haploid cells screens and analysis. 50 million haploid mSCs were thawn and infected with VSV-CCHF_G at a MOI of 10 in 5 ml of ES medium without FBS. One hour after infection, the cells were supplemented with complete ES medium and incubated at 37°C with 5% CO2. After outgrowth of virus resistant cells, cell clones were picked separately and cultured before being validated by infection assay with CCHFV lbAr10200. Briefly, cells (AN3-12 wild-type and potentially resistant clones) were seeded at 5x104 cells per well in DMEM 5% FBS for 24h. They were then infected with CCHFV at a MOI of 0.1 and the cells were recovered 24 hours post-infection in Trizol before being analysed by qRT-PCR. All clones being fully or partly resistant to CCHFV infection were subjected to DNA extraction using the Centra Puregene Tissue Kit (Qiagen). Paired end, 150 bp whole exome sequencing was performed on an Illumina Novaseq 6000 instrument after precapture-barcoding and exome capture with the Agilent SureSelect Mouse All Exon kit. For data analysis, raw reads were aligned to the reference genome mm9. Variants were identified and annotated using GATK and snpEff. CCHFV resistance causing alterations were identified by allelism only considering variants with moderate or high effect on protein and a read coverage > 20.
Generation of LDLR Knockout cells. A549 (ATCC CCL-185) and Vero (CCL-81) cells were grown in complete DMEM medium (DMEM High Glucose supplemented with 10% Fetal bovine serum (Gibco), 1x MEM-NEAA (Gibco), 1x Glutamax (Gibco), 1 mM Sodium Pyruvate (Gibco), 100 ll/rnl Penicillin-Streptomycin (Gibco)). The day before transfection, 1 .05x105 cells were seeded per well of a 24- well plate in 0.5 complete DMEM medium. The next day, culture medium was replaced with fresh complete DMEM medium and transfected with a liposome:DNA mixture composed of 50pl Opti-MEM I (Gibco), 500ng of PX459 v2.0 plasmid coding (Addgene Plasmid #62988, Puro resistant), 1.5 pl Lipofectamine 3000 reagent and 1.0 pl P3000 reagent. Several sgRNA were derived from CRISPick (https://portals.broadinstitute.org/gppx/crispick/public) using SpCas9 Cas9 knockout and the human LDLR gene as input. The final guide LDLR RNA sequence used for knockout studies was gATGAACAGGATCCACCACGA (SEQ ID NO 1 ), where lower latter g denotes preceding Guanosine to enhance transcription from the U6 Promoter. The next day, medium was replaced with complete DMEM supplemented with 1 pg/ml Puromycin for transient selection. 60 hours post transfection, each well containing selected A549, or Vero cells were expanded to one well of a 6-well plate in complete DMEM medium. Once cell reached 80% confluency, they were dissociated with 500 l with TrypLE Express enzyme solution (Gibco) for 5 minutes and collected in FACS Buffer (D-PBS containing 5% FBS). After one wash with FACS Buffer, 10 pl of a-LDLR-PE Antibody (R&D Systems FAB2148P) per 1.0 x 106 cells were added and stained for 1 h on ice in the dark. Unmodified cells were used as controls. After one hour of staining, cells were collected by centrifugation and washed twice in FACS Buffer. Finally, cells were resuspended in 1 ml of FACS Buffer and LDLR-negative cells were sorted into individual wells of a 96-well plate. LDLR-negative cells were defined as single cells displaying no PE fluorescence. Individual clones were expended and analyzed: unmodified A549 or Vero cells, as well as bat Tb-1 Lu cells (ATCC CCL-88) were used as positive and negative controls respectively. When individual cells grew to 85% confluency, they were expanded onto 24-well plates. After expansion, LDLR gene editing was verified by Flow-Cytometry analysis using the a-LDLR-PE antibody as described above and genotyped using the forward and reverse primer F: CTAACCAGTTCCTGAAGC (SEQ ID NO 2) R: GCACCCAGCTTGACAGAG (SEQ ID NO 3). For genotyping, 5.0 x 104 cells were collected and resuspended in 100 pl of Nuclease free water. 100 pl of DNA QuickExtract Lysis solution (Lucigen) was added and incubated for 5 minutes at 65°C, 5 minutes at 95°C. Two microliters (2 pl) of the lysis were used per 20 pl of PCR reaction containing 1x Kapa Kapa HiFi HotStart ReadyMix (Roche) and 0.5 pM of each forward and reverse primer. PCR was performed with an initial 3-m inute 95°C denaturation step, followed by 35 cycles of 98° C for 10 seconds, annealing at 58° C for 20 seconds, extension at 1 minute at 72° C, and a final extension for 2 minutes at 72° C. PCR products were purified and subjected to Sanger sequencing for verification. Cells which showed Cas9 editing at the LDLR locus and negative a-LDLR staining were used as knockout for entry studies (Figure 7).
Cells infection. For all infections involving AN3-12, A549 and Vero cells, 5.0 x 104 cells per well were seeded in 48-well plates (Sarstedt). 24h post-seeding, cells were infected with either VSV, VSV-CCHF_G, or CCHFV (lbAr10200 or isolate) at a MOI of 0.1 for one hour in corresponding medium containing 2% FBS. After one hour, cells were washed once with PBS and fresh medium containing 5% FBS was added. 24h (A549 and Vero) or 48h (AN3-12) post-infection, cells were washed three times with PBS and lysed with Trizol. RNA was extracted and analyzed by qRT-PCR as described below.
Soluble LDLR, VLDLR and LRP8 assays. SW13 were seeded at a density of 5.0 x 104 cells per well in a 48-well plate. 24h post-seeding, cells were counted to define the quantity of virus needed for an infection at a MOI of 0.01 . The virus was then mixed in 1.5 ml tubes (Sarstedt) with the appropriate quantity of sLDLR, sVLDR or sLRP8 in L15 medium containing 0.5% FBS. The tubes were the incubated for 30 min under shaking (75rpm) at 37°C. After 30 min, cells were rinsed once with PBS before being infected with virus only or with the mix virus/sLDLR, virus/sLRP8 or virus/VLDLR for 1 hour at 37°C. After one hour, inoculums were removed, cells washed once with PBS and L15 medium containing 5% FBS was added to each well. VSV and VSV-CCHF_G entering cells and replicating very fast, cells infected with these viruses were recovered 6h postinfection while cells infected with CCHFV were recovered 24h post-infection. At the time of recovery, cells were washed three times with PBS and lysed with Trizol. RNA was extracted and analyzed by qRT-PCR as described below.
Plasmid DNA constructs for BRET assay. To generate LDLR-Rlucll, codon- optimized LDLR was synthesized as a gBIock (Integrated DNA Technologies) and subcloned by Gibson assembly in pcDNA3.1/Hygro(+) GFP10-F?lucll db v.2 that had been linearized by PCR to exclude GFP10. To generate Nluc-LDLR, codon- optimized LDLR from LDLR-Rlucll was amplified by PCR and subcloned by Gibson assembly in pcDNA3.1 NIuc-synFZDs that had been linearized by PCR to exclude FZDs. rGFP-FYVE (Namkung et al., 2016. Monitoring G protein-coupled receptor and (3-arrestin trafficking in live cells using enhanced bystander BRET. Nat Commun. 11 ;7:12178) has been described previously. All plasmid constructs were verified by Sanger sequencing.
Cell culture and transfection for BRET assay. HEK293 cells were propagated in plastic flasks and grown at 37°C in 5% CO2 and 90% humidity. Cells (350,000 in 1 ml) were transfected in suspension with 1.0 pg of plasmid DNA complexed with linear polyethyleneimine (PEI; MW 25,000, 3:1 PEI:DNA ratio). All cell lines were regularly tested for mycoplasma contamination.
BRET assays. Receptor trafficking-. To monitor the trafficking of LDLR to early endosomes, HEK293 cells were transfected with LDLR-Rlucll and rGFP-FYVE and seeded in 6-well plates (7.0 x 105 cells/well). After a 48-hour incubation, cells were washed once with HBSS, detached and resuspended in HBSS containing 0.1 % BSA before distribution into white 96-well plates containing serial dilutions of LDL, CCHFV Gc or CCHFV Gn and returned to the incubator for 45 min at 37°C. Prior to BRET measurements, cells were incubated with coelenterazine h (10 min). NanoBRET binding assay: To monitor the binding of fluorescent ligands to LDLR, HEK293 cells were transfected with Nluc-LDLR and seeded in white 96-well plates (3.5 x 104 cells/well). After a 48-hour incubation, cells were washed once with HBSS and maintained in the same buffer. Prior to BRET measurements, cells were incubated with NanoBRET Nano-Gio substrate (6 min) and then stimulated with either BODIPY FL LDL or BODIPY FL Gc for 90 min following a baseline measurement of 3 cycles.
BRET measurements: Plates were read on a Tecan Spark multimode microplate reader (Mannedorf, Switzerland) equipped with a double monochromator system to measure the emission of the Rlucll/rGFP donor-acceptor pair in receptor trafficking experiments [430-485 nm (donor) and 505-590 nm (acceptor)] or the Nluc/BODIPY FL donor-acceptor pair in the NanoBRET binding assay [445-470 nm (donor) and 520-575 nm (acceptor)].
Generation of LDLR Knockout iPSC. NC8 iPSC (male, pericyte derived) were grown on Matrigel (hESC qualified, Coming) coated dishes in complete Stemflex medium (Gibco) + 1 :100 Antibiotic-Antimycotic (Gibco) (Invivogen). Cells were passaged using 0.5mM EDTA at a ratio of 1 :6 every 3 to 4 days. The day before transfection, iPSCs were dissociated into single cells using TrypLE select (Gibco) and seeded at 5.0 x 104 cells per 1-well of a rhLaminin521 (Gibco) coated 24-well plate in complete Stemflex medium supplemented with 1 :100 RevitaCell (Gibco). The next day, culture medium was replaced with Opti-MEM I (Gibco) + 1 :00 RevitaCell and transfected with a liposome:DNA mixture composed of 50pl Opti- MEM (Gibco), 500ng of PX459 v2.0 plasmid with LDLR guide sequence (SEQ ID NO 1 ) cloned in (Addgene Plasmid #62988, Puro resistant), 1.5pl Lipofectamine 3000 reagent and 1 l P3000 reagent. After four hours, the transfection mixture was removed and fresh complete Stemflex medium was added. After 48 hours post transfection, complete Stemflex medium with 0.5pg/ml Puromycin was added for transient selection. 60 hours post transfection, selection medium was removed, and cells were expanded to one well of a 6-well plate. Once cells reached 85% confluency, iPSC were dissociated into single cells using TrypLE select enzyme (Gibco) and resuspended in iPSC FACS Buffer (D-PBS+1 % KOSR+1 :100 RevitaCell+0.5mM EDTA). Anti-LDLR staining was done as described for A549. LDLR-negative as well as LDLR-positive cells were sorted into rhLaminin521 coated 96-well plates containing 150pl of complete Stemflex medium + 1 :100 RevitaCell. Four days post sorting, medium was replaced with complete Stemflex medium until cells reached confluency. Individual clones were expanded and analysed as described for A549 cells.
Preparation of Blood-vessel organoid derived 2D-Monolayer for infection. Blood-vessel organoids (BVO) from NC8 clone 10 (LDLR+) and clone 4 (LDLR-) were produced as previously described (Wimmer et al, Generation of blood vessel organoids from human pluripotent stem cells. Nat Protoc. 2019; 14(11 ):3082-3100). To prepare the BVO for infections, they were cut out of the matrix on day 11 of the procedure and cultured in Sprouting Media (StemPro-34 SFM medium (Gibco), 1X StemPro-34 nutrient supplement (Gibco), 0.5 ml Glutamax (Gibco), 15% FCS, 100 ng/ml VEGF-A (Peprotech) and 100 ng/ml FGF-2 (Miltenyi Biotec)) for five additional days with media changes every other day. To dissociate the organoids, 25 mature blood vessel organoids per genotype were washed twice with PBS and were transferred into a prefiltered and prewarmed enzymatic dissociation mix consisting of 4 mg Liberase TH (Sigma Aldrich), 30 mg Dispase II (Life Technologies) dissolved in 10 ml of PBS. The organoid containing enzymatic mix was incubated for 25 min at 37°C followed by trituration 15 times with a 10 ml stripette. The 37°C incubation and trituration were repeated for 10min twice more. The dissociated organoids were passed through a 70pm cell strainer into 5 ml of ice-cold DMEM/F12 medium. Following filtering, the cells were collected through centrifugation (300xg, 5 min) and replated in PureCol (Advanced BioMatrix, 30pg/ml in PBS for 1 h at RT) coated T-25 flasks at 30840 cells/cm2 in Sprouting media.
In vivo experiments. In the current study were used six female C57BL/6J mice (Charles River, Germany) and six female B6.129S7Ldlrtm1 Her/J (LdLr KO) (stock#002207), (Jackson Laboratory, USA). All mice were 10 weeks old at the time of infection. The animals were housed according to Karolinska Institute ethical rules and observed daily.
Antibody treatment and challenge. To make the mice susceptible to CCHFV infection, all animals received an intraperitoneal (i.p) injection of 2.5 mg anti-IFN type I receptor antibody at the time of infection. Each mouse was challenged with 400 focus forming units (FFU) of CCHFV lbAr10200 in 100 pl via i.p injection. The mice were monitored daily with respect to clinical signs of disease, and their overall well-being. When the wild type mice reached the pre-determined humane endpoint, all mice were euthanized independent on clinical signs. Blood was collected in microcontainer tubes for serum separation and serum was inactivated with Trizol for subsequent qRT-PCR analysis. In addition, liver, spleen and kidney were collected, a part in Trizol for qRT-PCR and a part were fixed in PFA 4% for histopathological analyzes.
The experimenters were not blinded to the identity of the animals. However, the pathologist who analyzed livers as well as the scientist who runned the RT-qPCRs and the subsequent analysis were blinded.
Histopathology. PFA fixed livers were cut in section of 3-4pm thin and stained for Haematoxylin-Eosin (HE). The stained sections were analyzed by a pathologist at BioVet, a laboratory of animal medicine (BioVet, Sollentuna, Sweden). RT-qPCR analysis. All RNA extractions were performed using Direct-zol RNA extraction kit (Zymo Research). Quantitative real time PCR reactions were performed using a TaqMan Fast Virus 1-step Master Mix (Thermofisher) and run on an Applied Biosystems machine. Following primers were used in this study to detect CCHFV L gene Fwd: GCCAACTGTGACKGTKTTCTAYATGCT (SEQ ID NO 4), Rev1 : CGGAAAGCCTATAAAACCTACCTTC (SEQ ID NO 5), Rev2: CGGAAAGCCTATAAAACCTGCCYTC (SEQ ID NO 6), Rev3: CGGAA AGCCTAAAAAATCTGCCTTC (SEQ ID NO 7), probe: FAM- CTGACAAGYTCAGCAAC-MGB (SEQ ID NO 8), VSV M gene Fwd: TGATACAGTACAATTA TTTTGGGAC (SEQ ID NO 9); Rev: GAGACTTTCTGTTACGGGATCTGG (SEQ ID NO 10); Probe: FAM-ATGATGCA TGATCCAGC-MGB (SEQ ID NO 11 ). RNase P RNA was used as an endogenous control for normalization (Fwd: AGATTTGGACCTGCGAGCG (SEQ ID NO 12), Rev: GAGCGGCTGTCTCCACAAGT (SEQ ID NO 13), Probe: FAM- TTCTGACCTGAAGGCTCTGCGCG-MGB (SEQ ID NO 14)).
Absolute quantification of CCHFV RNA for mice samples was performed by RT- qPCR. A 120bp synthetic RNA corresponding to nucleotides 9625 to 9744 of CCHFV Ibar 10200 L segment (GenBank MH483989.1 ) was produced by Integrated DNA Technologies. The standard synthetic RNA was solubilised in RNase-free water and the copy number calculated after quantification by nanodrop. The efficiency and linearity of the RT-qPCR reaction using the primers forward GCCAACTGTGACKGTKTTCTAYATGCT (SEQ ID NO 15) and reverse CGGAAAGCCTAAAAAATCTGCCTTC (SEQ ID NO 16) with probe FAM- CTGACAAGYTCAGCAAC-MGB (SEQ ID NO 17) with the standard RNA was validated over serial 10-fold dilutions. This standard curve RT-qPCR was then performed simultaneously with RNA samples in order to quantify the absolute copy number of CCHFV RNA.
LDL competition assays.
SW13 were seeded at a density of 5.0 x 104 cells per well in a 48-well plate. 24h post-seeding, cells were counted to define the quantity of virus needed for an infection at a MOI of 0.01 . CCHFV was then mixed in 1.5 ml tubes (Sarstedt) with different concentration of LDL (Thermofisher, #L3486) or BSA (Saveen & Werner AB, #A1391 ) in L15 medium containing 0.5% FBS. Cells were rinsed once with PBS before being infected with virus only or with the mix virus/LDL or virus/BSA for 1 hour at 37°C. After one hour, inoculums were removed, cells washed once with PBS and L15 medium containing 5% FBS was added to each well. Cells were recovered 24h post-infection. At the time of recovery, cells were washed three times with PBS and lyzed with Trizol. RNA was extracted and analyzed by qRT-PCR as described below.
Tick cell culture
The Hyalomma anatolicum embryo-derived cell lines HAE/CTVM9 were grown L- 15/M EM medium (equal volumes of L-15 and Minimal Essential Medium with Hank’s salts supplemented with 10% TPB), both supplemented with 2 mM l-glutamine, 20% FBS, and incubated in sealed flasks at 28°C, 0% CO2.
Protein purification (related to Fig. 14)
30ug of expression vector coding for the respective LDLR-fragment were transfected into HEK293 cells. After 3-4 days the medium was harvested and secreted proteins were purified using Ni Sepharose™ 6 Fast Flow beads (Cytiva; Cat.-No. 10291210). Medium and beads were incubated for 16h before washing and elution of the protein from the beads. The concentration of HiBiT-tagged LDLR-fragments was determined using Nano-Gio® HiBiT Lytic Detection System (Promega; Cat.-No. N3040) with HiBiT Control Protein (Promega; Cat.-No. N3010) as a standard.
CCHF-VLP infection assay (related to Fig. 14)
5.0 x 104 Huh-7 cells were seeded into each well of 24-well plates. Once cells attached, transfection of CCHF L and N gene (strain lbAr10200) was performed using GeneJammer Transfection Reagent (Agilent; Cat.-No. 204130). 16h post transfection indicator cells were infected with an 10Oul inoculum containing CCHF- VLPs (NanoLuc) after 1h pre-incubation with a protein-fragment of interest where applicable. 24hpi the wells were washed with PBS and analyzed using Nano-Gio® Luciferase Assay System (Promega; Cat.-No. N1110) according to manufacturer’s protocol. Luminescence was quantified with a plate reader (BMG Clario Star).
Transient expression and secretion of LDLR-fragments into supernatant (related to Fig. 14)
3.0 x 105 HEK293 cells were seeded into wells of 6-well plates. On the next day, 2ug of expression vector were transfected and medium was changed on the following day. The supernatant was collected 72h post transfection and LDLR- fragment quantity was determined using Nano-Gio® HiBiT Lytic Detection System (Promega; Cat.-No. N3040) with HiBiT Control Protein (Promega; Cat.-No. N3010) as a standard. Statistical analyses. All analyses were done using the data from at least three independent experiments and are shown as mean ± SD in GraphPad Prism (Version 9.4.1). One-way ANOVA analyses (multiple comparisons. Dunnett corrections) and two-tailed student t-test were used as indicated in figure legends.
Example 1 : Haploid cells screening highlighted LDLR as an essential protein for VSV-CCHFGP/AG entry
The inventors sought to identify potential host factors involved in CCHFV infection by employing a screening method using ENU mutagenized murine haploid embryonic cells (AN3-12). ENU mutagenized mES cells were infected with a viral RNA replication competent vesicular stomatitis virus pseudotyped with the glycoproteins of the Crimean-Congo hemorrhagic fever virus (VSV-CCHF_G) (see Figure 1A). This virus lacks the region coding for any glycoproteins, and therefore produces non-infectious particles unless reconstituted with a novel surface glycoprotein, i.e. in our screen with the Gc glycoprotein of the CCHFV.
Infections with VSV-CCHF_G efficiently killed the haploid cells. Genome wide, single amino acid mutagenesis in haploid cells resulted in the emergence of resistant colonies to VSV-CCHF_G mediated killing (Fig. 1b). These resistant colonies were individually selected, expanded and rescreened using the infectious CCHFV lbAr10200 laboratory strain (Fig. 1c). Subsequently, whole exome sequencing was conducted on the resistant clones. Three clones that showed nearly 100% resistant to CCHFV, namely clones 5, 8, and 10 (Fig. 1c), displayed mutations in the gene encoding Low Density Lipoprotein Receptor (Ldlr) (Table 2). The mutations occurred at different locations in the Ldlr gene, likely resulting in gene knockout. Of note, protein coding mutations were not observed in the other resistant colonies, suggesting that these mutations might be in regulatory gene regions. Thus, these data identify LDLR as a candidate gene for CCHFV infections.
Table 2: Sequencing data of the three clones highlighting LDLR.
Figure imgf000090_0001
Example 2: Validation of LDLR in CCHFV infections
To verify the role of LDLR for CCHFV entry, the inventors first assessed Ldlr mutant haploid mouse embryonic cells and their respective Haplobank wild type sister clones, as previously described. These Lcf/r-knockout cells and wild-type sister cells were infected with VSV-CCHF_G and CCHFV. These murine Ldlr- knockout cells displayed more than a 90% decrease in infection rates compared to the wild type cells (Fig. 2a).
The following experiments aimed to confirm the data on murine haploid cells using African green monkey kidney epithelial cells (Vero) and human lung epithelial A549 cells, both being susceptible to CCHFV infection. The Ldlr gene was mutated in these cell lines using CRISPR-Cas9 editing (Fig. 7). Both Ldlr mutant A549 as well as Vero cells showed a marked reduction in CCHFV infections as compared to their respective LDLR expressing control cells, which was determined by viral RNA detection 24 hours post-infection (Fig. 2b, c). These genetic deletion data validate the role of the LDLR in CCHFV infections across host species.
Example 3: Gc binds to LDLR and induces endocytosis
To test whether the LDLR can directly bind to the surface Gc glycoprotein of CCHFV, i.e. the protein expressed by VSV-CCHF_G, a bioluminescence resonance energy transfer (BRET) assay was developed to assess receptor-ligand interactions. To achieve this, the inventors genetically engineered and expressed an LDLR that carries an N-terminal bioluminescent probe (NanoLuc or NIuc) in HEK293 cells. Addition of fluorescent ligands then allow to measure direct interaction through BRET (Fig. 3a). BODIPY-FL-labelled LDL was initially used to assess receptor binding, resulting in the expected concentration-dependent ligandbinding BRET signal in cells expressing Nluc-LDLR (Fig. 3b). Unlabelled LDL competed for receptor binding with the labelled LDL, resulting in a nearly complete loss of the BRET signal (Fig. 8a, b). After establishing the assay, the inventors cloned and engineered a BODIPY-FL-labelled soluble Gc ligand. Indeed, addition of BODIPY-FL-labelled soluble Gc to NIuc-LDLR-expressing cells resulted in a marked increase in the BRET signal in a concentration-dependent manner (Fig. 3a, b). These data indicate that Gc can directly bind to the LDLR.
It is well known that removal of circulating LDL from the bloodstream and subsequent LDL hydrolysis is achieved through its uptake by the LDLR via receptor-mediated endocytosis. To investigate the mechanism by which LDLR facilitates CCHFV infection, the inventors next engineered LDLR to express Rlucll, a bioluminescent donor, at its C-terminus. A fluorescent acceptor, rGFP, was cloned to the FYVE domain of the human endofin as an early endosome marker (Namkung, Y. et al. 2016. Monitoring G protein-coupled receptor and fi-arrestin trafficking in live cells using enhanced bystander BRET. Nat Com mu n 7, 12178), and then co-expressed with LDLR-Rlucll to measure the degree of internalized LDLR (Fig. 3c). Exposure of cells to LDL led to a concentration-dependent increase in BRET between LDLR-Rlucll and rGFP-FYVE, confirming that LDLR traffics to endosomes upon binding to LDL (Fig. 3d). Importantly, addition of the CCHFV surface glycoprotein Gc also triggered endocytosis and enrichment of LDLR in early endosomes (Fig. 3d). As a control, the recombinant receptor binding domain (RBD) from SARS-CoV-2 was unable to elicit LDLR endocytosis and trafficking to early endosomes (Fig. 8c, d), demonstrating specific LDLR internalization upon CCHFV Gc binding. Taken together, these results demonstrate that CCHFV Gc directly binds to human LDLR and exploits its endocytic pathway to infect cells.
Example 4: Soluble LDLR blocks CCHFV infections
To investigate the potential of soluble LDLR (sLDLR) as a molecular decoy to inhibit CCHFV infections, sLDLR was added at varying concentrations to VSV- CCHF_G or CCHFV for 30 minutes prior to cell infection. sLDLR is a one chain LDLR Ala22-Arg788 fragment (SEQ ID NO 18). VSV was used as a positive control due to its known reliance on LDLR, but also LRP8 (Low Density Lipoprotein Receptor Related Protein 8) and VLDLR (Very Low Density Lipoprotein Receptor), as its receptors. Because the VSV life cycle in cells, from entry to new virus egress, is extremely rapid, VSV and VSV-CCHF_G infected cells have to be assessed at early time points post-infection to avoid a second round of infection. Thus, at 6 hours post-infection for VSV and VSV-CCHF_G, and at 24 hours post-infection for CCHFV, the cells were harvested and the levels of infection determined by qRT-PCR. Importantly, sLDLR blocked the infection of rVSV-CCHF (Fig. 4a) and, importantly, CCHFV (Fig. 4b) in a dose-dependent manner. As expected, sLDLR was also able to inhibit VSV (Fig. 4c)
In addition, we tested the ability of other members of the LDLR family, namely VLDLR and LRP8, to block CCHFV as well control VSV infections using soluble decoys. sLRP8 is a one chain LRP8 Asp35-Lys818 (Ala262Val) fragment and sVLDLR a one chain VLDLR Thr25-Ser797 fragment. Unlike sLDLR, neither sVLDLR nor sLRP8 decoys provided protection from CCHFV infections (Fig. 4d, e), while they were active against VSV infection (Fig. 4f, g). This data indicates that soluble LDLR can prevent CCHFV infections. Example 5: Infection of blood-vessels organoids
Blood vessels are key target cells for viral tropism involved in hemorrhaging. Human blood vessel organoids were generated using the iPSC line NC8. Ldlr in the iPSCs was deleted using CRISPR/Cas9-mediated genome editing. Knockout iPSCs for Ldlr were validated by flow-cytometry (Fig. 9a - e). For infection were generated Ldlr mutant and wild type blood vessel organoids (BVOs), containing self-organizing bona fide capillaries formed by pericytes and endothelial cells. As the culture conditions for the BVO were not compatible with virus infection, the BVOs containing mature human capillaries were disaggregated, and the pericytes and endothelial cells were further cultured as monolayers in collagen-coated flasks (Fig. 5a). These cultures were subsequently infected with CCHFV. Knockout of Ldlr, using two different mutant iPSC clones, resulted in a significant reduction in CCHFV infections, detected one and three days post-infection (Fig. 5b). These data show that LDLRs are also involved in infections of human blood vessels.
Example 6: LDLR KO protects mice against CCHF or LDLR mutant/KO mice are protected from CCHF
The inventors next investigated whether the absence of LDLR can protect against in vivo CCHFV infections and CCHF disease manifestations using C57BL/6J wild type and Ldlr-I- mice. C57BL/6J mice are naturally resistant to CCHF, but blockade or knockout of IFNa receptors render these mice susceptible to the infection, as previously reported. Therefore wild type and Ldlr-I- mice were treated with 2.5mg of anti-IFNa receptor antibodies at the time of CCHFV infection (400 PFU per mouse). Four days post-infection, all mice were euthanized, and their serum, liver, and spleen were analyzed for viral RNA and livers were assayed for pathologies using histology.
Whereas wild type mice lost 15% of their starting body weights, Ldlr-I- mice did not show any weight loss or other macroscopic signs of disease (weakness, swollen eyes, marked incoordination, piloerection, light bleeding around the marking hole in the ear) (Fig. 5c). Ldlr-I- mice also exhibited significantly reduced level of viral RNA in the serum, with two mice even showing undetectable levels of circulating virus (Fig 5d). The mutant mice also exhibited reduced virus load in liver and spleen (Fig. 5d). Histopathological analysis of livers of CCHFV-infected wild type mice revealed midzonal necrosis (Fig. 5e upper left panel), periportal coagulative necrosis (Fig. 5d upper middle panel), as well as sporadic necrosis of single cells (Fig. 5e upper right panel), accompanied by severe vascular congestion with low to moderate numbers of intravascular macrophages, neutrophils, and occasional fibrin thrombi (Fig. 5e upper panel). Interestingly, livers from CCHFV-infected Ldlr knockout mice showed little-to-no evidence of these pathologies (Fig. 5e, lower panel), indicating that Ldlr knockout protects mice CCHFV infections and liver damage.
Example 7: LDLR is also a receptor for CCHFV isolate
To investigate whether LDLR acts as a receptor for clinical isolates of CCHFV, experiments were conducted using a virus isolated from a Turkish patient sample and cultured on tick cells, differently from previous experiments that utilized the CCHFV lbAr10200 laboratory strain.
Consistent with the results using the laboratory strain lbAr10200, addition of sLDLR, but not sLRP8 nor sVLDLR, reduced the infection of human cells exposed to the clinical CCHFV isolate in a dose dependent fashion (Fig. 6a-c). In addition, LDLR mutant Vero and A549 cells challenged with the CCHFV patient isolate showed significantly reduced infection rates as compared to their respective LDLR expressing wild type control cells (Fig. 6d). This data confirms that LDLR also acts as a receptor for patient-derived CCHFV isolates.
Example 8: Analysis of binding of LDLR fragments, and homo- and heteromultimers thereof to viruses.
In these experiments, the inventors analyse the LDLR fragments with highest virus affinity. The LDLR fragments of the LDLR ectodomain as described above, and homo- and heteromultimers thereof covalently linked with or without a peptide linker, are expressed in bacteria, insect cells, or mammalian cells. Following purification, fragments are tested by: a) cell-based assays for their ability to block virus infections, preferably Bunyavirus infections, and more preferred, CCHFV infections. To this end wildtype virus strains, pseudotyped viruses, or viruses and pseudotyped viruses expressing marker proteins such as GFP can be used for infection of relevant cell lines. Infection rates in the presence and absence of the LDLR fragments will be assessed by quantitative analysis of viral genomes or protein, including artificially expressed marker proteins such as GFP. b) biochemical binding assays for their ability to bind virus glycoproteins, preferred bunyavirus glycoproteins, more preferred Gc and Gn protein. Virus envelope gylcoproteins will be expressed and purified or purchased and then immobilized on suitable plates. LDLR fragments, and homo- and heteromultimers thereof will be extended by a peptide Tag such as a polyhistidine tag (6xH), a FLAG tag (DYKDDDDK), or a human influenza hemagglutinin (HA) tag (YPYDVPDYA), expressed and purified. Purified fragments and multimers thereof will be incubated with the immobilized glycoproteins. After several wash steps, high affinity binding peptides will be identified via antibody-based detection methods.
The protective effect of LDLR, LDLR-fragments, and fragment multimers on infection of Huh-7 cells was tested by incubating them with transcription competent virus-like-particles (VLPs), where the viral CCHF genome was replaced by NanoLuc luciferase. Upon infection, cells express NanoLuc luciferase, which serves as a measure of infection rates. To boost the luminescent signal, the CCHFV L and N genes (strain lbAr10200) were co-transfected into the indicator cells 16h prior to infection. Pre-incubation of VLPs with full length recombinant human LDLR (rhLDLR, RnD Systems; Cat.-No. 2148-HP) or purified soluble LDLR expressed from Hek293 cells (sLDLR) leads to a clear reduction of infection compared to VLPs only (CTR) at 24 hours post infection (Fig. 14). Furthermore, fragments of LDLR containing only the ligand-binding domains LA1-7 or LA4-7, show protective effects against infection of Huh-7 cells with CCHF-VLPs. Multimerization of LDLR fragments containing a linker (I) between repetitive domains such as LDLR LA1-7I1-7 and LDLR LA2-3I2-3I2-3 also ameliorate infection, while a single LDLR LA2-3 fragment does not reach significance. A dimerization utilizing an lgG1 Fc-domain fused to the C-terminus of full length recombinant human LDLR (rhLDLR-mFc, SinoBiological; Cat.-No. 10231-H05H) or fused to only the ligand-binding domains LA1-7 (LDLR (1-7)-hFc) shows strong protective effect. Combinations of sLDLR fused to other LDLR family members (LRP8 and VLDLR) increases its potency to protect against CCHF-VLP infection (note that concentrations < 5nM were applied here). In this assay, also the closely related LDLR family member VLDLR alone shows significant protection against infection with CCHF-VLPs (Fig. 14).
Related sequences:
Nucleotide Sequence: LDLR (2-3) (SEQ ID NO 199) ATGGGACCATGGGGATGGAAGCTGCGCTGGACTGTGGCCCTTCTGCTTGCGGCGGCAGGAA CcgcaGTGGGTGACCGGGGCAGCGGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGG CGGAGTAACCTGCAAATCGGGAGACTTCAGCTGTGGTGGACGGGTTAACCGCTGTATACCTC AGTTTTGGCGGTGTGACGGACAAGTTGACTGTGATAATGGCTCAGATGAGCAGGGGTGCCCG CCAAAGACCTGCAGTCAGGACGAATTCCGATGCCACGATGGAAAATGTATTAGCCGTCAATTC GTTTGTGACTCGGACCGCGACTGTCTGGATGGTTCAGATGAAGCTTCGTGCCCAGTAgGAGA CGCTTCGgattacaaagacgatgacgataagCTGGAAGTTCTGTTCCAGGGGCCCGGTCATCACCACCA TCATCACCATCACTAA
Amino Acid Sequence: LDLR (2-3) (SEQ ID NO 200) MGPWGWKLRWTVALLLAAAGTAVGDRGSGVSGWRLFKKISGGVTCKSGDFSCGGRVNRCIPQF
WRCDGQVDCDNGSDEQGCPPKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPVGDA
SDYKDDDDKLEVLFQGPGHHHHHHHH*
Nucleotide Sequence: sLDLR (SEQ ID NO 201)
ATGGGACCATGGGGATGGAAGCTGCGCTGGACTGTGGCCCTTCTGCTTGCGGCGGCAGGAA
CcgcaGTGGGTGACCGGGGCAGCGGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGG
CGGAGCAGTAGGCGACAGATGCGAGCGTAACGAATTCCAATGCCAAGACGGTAAGTGTATTA
GTTACAAATGGGTTTGTGACGGCAGTGCCGAATGCCAAGACGGTTCGGATGAATCTCAGGAA
ACGTGTCTGTCGGTAACCTGCAAATCGGGAGACTTCAGCTGTGGTGGACGGGTTAACCGCTG
TATACCTCAGTTTTGGCGGTGTGACGGACAAGTTGACTGTGATAATGGCTCAGATGAGCAGG
GGTGCCCGCCAAAGACCTGCAGTCAGGACGAATTCCGATGCCACGATGGAAAATGTATTAGC
CGTCAATTCGTTTGTGACTCGGACCGCGACTGTCTGGATGGTTCAGATGAAGCTTCGTGCCCA
GTATTAACTTGCGGACCGGCGAGTTTTCAATGCAATTCCAGCACGTGTATTCCCCAACTCTGG
GCTTGCGACAACGACCCGGATTGCGAGGACGGCTCTGACGAGTGGCCGCAGAGATGCCGTG
GGTTATACGTGTTTCAAGGAGACAGCTCACCGTGCAGCGCTTTCGAGTTTCATTGTCTTAGCG
GCGAGTGTATCCACTCATCATGGCGCTGCGATGGTGGGCCTGATTGTAAGGACAAGAGCGAC
GAGGAGAATTGTGCTGTTGCCACGTGTCGACCAGACGAATTTCAATGTAGCGACGGTAATTGT
ATCCATGGATCTAGACAATGCGACCGTGAATACGATTGTAAAGACATGTCCGACGAGGTTGGC
TGTGTGAATGTGACTTTGTGCGAGGGACCGAATAAGTTTAAGTGCCACTCAGGCGAATGCATA
ACACTTGACAAAGTGTGTAACATGGCACGCGACTGCCGTGATTGGTCAGACGAGCCAATAAA
GGAATGCGGTACCAACGAGTGCCTGGACAACAATGGCGGCTGTAGTCATGTTTGCAATGACC
TTAAGATCGGTTACGAGTGTCTCTGCCCAGATGGTTTCCAGTTGGTTGCTCAACGCCGATGCG
AAGACATAGACGAATGCCAAGATCCGGACACATGCTCCCAACTGTGCGTCAATTTGGAAGGC
GGATACAAGTGCCAATGCGAGGAAGGCTTCCAGTTGGATCCGCACACGAAGGCCTGCAAAGC
GGTGGGTAGTATTGCTTACTTATTCTTCACGAACCGACACGAAGTTCGGAAAATGACTTTGGA
TAGATCCGAGTACACATCACTTATACCTAACCTTCGTAATGTAGTCGCACTTGATACCGAGGTA
GCAAGTAACCGCATCTATTGGTCTGACTTGTCCCAGCGCATGATATGTTCTACTCAATTGGATC
GTGCACACGGAGTTAGCTCGTATGACACCGTAATATCACGTGACATACAGGCACCGGATGGC
CTGGCGGTTGACTGGATTCATAGCAATATTTACTGGACAGACTCCGTGCTGGGCACAGTGAGT
GTTGCTGATACGAAAGGGGTTAAACGTAAGACACTGTTTCGGGAAAATGGTAGCAAGCCGCG
CGCTATAGTGGTTGACCCGGTACATGGATTCATGTACTGGACGGACTGGGGTACACCAGCCA
AGATCAAGAAGGGTGGTCTGAACGGAGTCGACATATACAGCCTGGTCACGGAGAATATACAG
TGGCCGAACGGGATTACTTTGGACCTTCTGAGTGGACGCTTGTACTGGGTGGATAGCAAGCT
GCATTCTATCTCCTCTATTGATGTGAACGGTGGAAACCGGAAGACTATATTGGAAGATGAGAA
GAGATTGGCCCACCCGTTCTCTCTCGCGGTCTTTGAGGACAAGGTATTCTGGACGGACATAAT
TAACGAGGCTATCTTCTCTGCCAACCGTCTGACTGGCTCTGACGTGAATTTACTGGCGGAGAA
TTTGCTGAGTCCTGAGGATATGGTATTGTTTCACAACTTAACGCAACCGCGGGGCGTGAACTG
GTGTGAGCGTACGACGTTAAGCAACGGTGGCTGTCAGTACCTGTGTTTACCTGCGCCGCAAA
TAAATCCTCACTCTCCGAAGTTTACCTGCGCTTGTCCAGACGGCATGCTTCTTGCCCGCGACA
TGCGTTCATGCTTAACGGAAGCTGAAGCTGCCGTTGCTACACAAGAAACGAGCACCGTGAGA
TTAAAGGTGAGCTCAACCGCCGTCCGCACACAACACACCACAACACGCCCTGTCCCGGACAC
CTCGCGGTTACCGGGTGCGACCCCCGGATTAACCACCGTTGAGATCGTAACCATGTCACACC
AAGCCCTCGGCGATGTGGCTGGCCGTGGCAATGAGAAGAAACCTTCCAGTGTCCGGGGAGA
CGCTTCGGATtacaaagacgatgacgataagCTGGAAGTTCTGTTCCAGGGGCCCGGTCATCACCACC ATCATCACCATCAC
Amino Acid Sequence: sLDLR (SEQ ID NO 202) MGPWGWKLRWTVALLLAAAGTAVGDRGSGVSGWRLFKKISGGAVGDRCERNEFQCQDGKCISY KWVCDGSAECQDGSDESQETCLSVTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGC PPKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPVLTCGPASFQCNSSTCIPQLWACD NDPDCEDGSDEWPQRCRGLYVFQGDSSPCSAFEFHCLSGECIHSSWRCDGGPDCKDKSDEEN CAVATCRPDEFQCSDGNCIHGSRQCDREYDCKDMSDEVGCVNVTLCEGPNKFKCHSGECITLDK VCNMARDCRDWSDEPIKECGTNECLDNNGGCSHVCNDLKIGYECLCPDGFQLVAQRRCEDIDEC QDPDTCSQLCVNLEGGYKCQCEEGFQLDPHTKACKAVGSIAYLFFTNRHEVRKMTLDRSEYTSLI PNLRNVVALDTEVASNRIYWSDLSQRMICSTQLDRAHGVSSYDTVISRDIQAPDGLAVDWIHSNIY
WTDSVLGTVSVADTKGVKRKTLFRENGSKPRAIVVDPVHGFMYWTDWGTPAKIKKGGLNGVDIYS LVTENIQWPNGITLDLLSGRLYWVDSKLHSISSIDVNGGNRKTILEDEKRLAHPFSLAVFEDKVFWT DIINEAIFSANRLTGSDVNLLAENLLSPEDMVLFHNLTQPRGVNWCERTTLSNGGCQYLCLPAPQIN PHSPKFTCACPDGMLLARDMRSCLTEAEAAVATQETSTVRLKVSSTAVRTQHTTTRPVPDTSRLP GATPGLTTVEIVTMSHQALGDVAGRGNEKKPSSVRGDASDYKDDDDKLEVLFQGPGHHHHHHHH
Nucleotide Sequence: LDLR (1-7) (SEQ ID NO 203)
ATGGGACCATGGGGATGGAAGCTGCGCTGGACTGTGGCCCTTCTGCTTGCGGCGGCAGGAA CcgcaGTGGGTGACCGGGGCAGCGGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGG CGGATccggtGACAGATGCGAGCGTAACGAATTCCAATGCCAAGACGGTAAGTGTATTAGTTAC AAATGGGTTTGTGACGGCAGTGCCGAATGCCAAGACGGTTCGGATGAATCTCAGGAAACGTG TCTGTCGGTAACCTGCAAATCGGGAGACTTCAGCTGTGGTGGACGGGTTAACCGCTGTATAC CTCAGTTTTGGCGGTGTGACGGACAAGTTGACTGTGATAATGGCTCAGATGAGCAGGGGTGC
CCGCCAAAGACCTGCAGTCAGGACGAATTCCGATGCCACGATGGAAAATGTATTAGCCGTCA ATTCGTTTGTGACTCGGACCGCGACTGTCTGGATGGTTCAGATGAAGCTTCGTGCCCAGTATT AACTTGCGGACCGGCGAGTTTTCAATGCAATTCCAGCACGTGTATTCCCCAACTCTGGGCTTG CGACAACGACCCGGATTGCGAGGACGGCTCTGACGAGTGGCCGCAGAGATGCCGTGGGTTA TACGTGTTTCAAGGAGACAGCTCACCGTGCAGCGCTTTCGAGTTTCATTGTCTTAGCGGCGAG TGTATCCACTCATCATGGCGCTGCGATGGTGGGCCTGATTGTAAGGACAAGAGCGACGAGGA
GAATTGTGCTGTTGCCACGTGTCGACCAGACGAATTTCAATGTAGCGACGGTAATTGTATCCA TGGATCTAGACAATGCGACCGTGAATACGATTGTAAAGACATGTCCGACGAGGTTGGCTGTGT GAATGTGACTTTGTGCGAGGGACCGAATAAGTTTAAGTGCCACTCAGGCGAATGCATAACACT TGACAAAGTGTGTAACATGGCACGCGACTGCCGTGATTGGTCAGACGAGCCAATAAAGGAAT GCGGAGACGCTTCGGATtacaaagacgatgacgataagCTGGAAGTTCTGTTCCAGGGGCCCGGTCA TCACCACCATCATCACCATCAC
Amino Acid Sequence: LDLR (1-7) (SEQ ID NO 204)
MGPWGWKLRWTVALLLAAAGTAVGDRGSGVSGWRLFKKISGGSGDRCERNEFQCQDGKCISYK WVCDGSAECQDGSDESQETCLSVTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGCP PKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPVLTCGPASFQCNSSTCIPQLWACDN DPDCEDGSDEWPQRCRGLYVFQGDSSPCSAFEFHCLSGECIHSSWRCDGGPDCKDKSDEENC AVATCRPDEFQCSDGNCIHGSRQCDREYDCKDMSDEVGCVNVTLCEGPNKFKCHSGECITLDKV CNMARDCRDWSDEPIKECGDASDYKDDDDKLEVLFQGPGHHHHHHHH*
Nucleotide Sequence: LDLR (4-7) (SEQ ID NO 205)
ATGGGACCATGGGGATGGAAGCTGCGCTGGACTGTGGCCCTTCTGCTTGCGGCGGCAGGAA CcgcaGTGGGTGACCGGGGCAGCGGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGG CGGATccggtTTAACTTGCGGACCGGCGAGTTTTCAATGCAATTCCAGCACGTGTATTCCCCAAC TCTGGGCTTGCGACAACGACCCGGATTGCGAGGACGGCTCTGACGAGTGGCCGCAGAGATG CCGTGGGTTATACGTGTTTCAAGGAGACAGCTCACCGTGCAGCGCTTTCGAGTTTCATTGTCT TAGCGGCGAGTGTATCCACTCATCATGGCGCTGCGATGGTGGGCCTGATTGTAAGGACAAGA GCGACGAGGAGAATTGTGCTGTTGCCACGTGTCGACCAGACGAATTTCAATGTAGCGACGGT AATTGTATCCATGGATCTAGACAATGCGACCGTGAATACGATTGTAAAGACATGTCCGACGAG GTTGGCTGTGTGAATGTGACTTTGTGCGAGGGACCGAATAAGTTTAAGTGCCACTCAGGCGA
ATGCATAACACTTGACAAAGTGTGTAACATGGCACGCGACTGCCGTGATTGGTCAGACGAGC CAATAAAGGAATGCGGAGACGCTTCGGATtacaaagacgatgacgataagCTGGAAGTTCTGTTCCAG GGGCCCGGTCATCACCACCATCATCACCATCAC
Amino Acid Sequence: LDLR (4-7) (SEQ ID NO 206)
MGPWGWKLRWTVALLLAAAGTAVGDRGSGVSGWRLFKKISGGSGLTCGPASFQCNSSTCIPQL
WACDNDPDCEDGSDEWPQRCRGLYVFQGDSSPCSAFEFHCLSGECIHSSWRCDGGPDCKDKS DEENCAVATCRPDEFQCSDGNCIHGSRQCDREYDCKDMSDEVGCVNVTLCEGPNKFKCHSGEC ITLDKVCNMARDCRDWSDEPIKECGDASDYKDDDDKLEVLFQGPGHHHHHHHH*
Nucleotide Sequence: LDLR (2-3I2-3I2-3) (SEQ ID NO 207)
ATGGGACCATGGGGATGGAAGCTGCGCTGGACTGTGGCCCTTCTGCTTGCGGCGGCAGGAA
CcgcaGTGGGTGACCGGGGCAGCGGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGG CGGATGTACaGTAACCTGCAAATCGGGAGACTTCAGCTGTGGTGGACGGGTTAACCGCTGTA
TACCTCAGTTTTGGCGGTGTGACGGACAAGTTGACTGTGATAATGGCTCAGATGAGCAGGGG TGCCCGCCAAAGACCTGCAGTCAGGACGAATTCCGATGCCACGATGGAAAATGTATTAGCCG TCAATTCGTTTGTGACTCGGACCGCGACTGTCTGGATGGTTCAGATGAAGCTTCGTGCCCAGT
AGGTGGgGGAGCCGCGGCCGGTGGAGCAGCCGGTGGTGGAGCAGCTGCGGGTGGTGCTGC CGTAACCTGCAAATCGGGAGACTTCAGCTGTGGTGGACGGGTTAACCGCTGTATACCTCAGT TTTGGCGGTGTGACGGACAAGTTGACTGTGATAATGGCTCAGATGAGCAGGGGTGCCCGCCA AAGACCTGCAGTCAGGACGAATTCCGATGCCACGATGGAAAATGTATTAGCCGTCAATTCGTT
TGTGACTCGGACCGCGACTGTCTGGATGGTTCAGATGAAGCTTCGTGCCCAGTAGGTGGgGG AGCCGCGGCCGGTGGAGCAGCCGGTGGTGGAGCAGCTGCGGGTGGTGCTGCCGTAACCTG CAAATCGGGAGACTTCAGCTGTGGTGGACGGGTTAACCGCTGTATACCTCAGTTTTGGCGGT GTGACGGACAAGTTGACTGTGATAATGGCTCAGATGAGCAGGGGTGCCCGCCAAAGACCTGC
AGTCAGGACGAATTCCGATGCCACGATGGAAAATGTATTAGCCGTCAATTCGTTTGTGACTCG GACCGCGACTGTCTGGATGGTTCAGATGAAGCTTCGTGCCCAGTAttAACcgGAGACGCTTCGg attacaaagacgatgacgataagCTGGAAGTTCTGTTCCAGGGGCCCGGTCATCACCACCATCATCACC ATCAC
Amino Acid Sequence: LDLR (2-3I2-3I2-3) (SEQ ID NO 208)
MGPWGWKLRWTVALLLAAAGTAVGDRGSGVSGWRLFKKISGGCTVTCKSGDFSCGGRVNRCIP QFWRCDGQVDCDNGSDEQGCPPKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPVG GGAAAGGAAGGGAAAGGAAVTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGCPPKT CSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPVGGGAAAGGAAGGGAAAGGAAVTCKS
GDFSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGCPPKTCSQDEFRCHDGKCISRQFVCDSDR DCLDGSDEASCPVLTGDASDYKDDDDKLEVLFQGPGHHHHHHHH*
Nucleotide Sequence: VLDLR (SEQ ID NO 209)
ATGGGACCATGGGGATGGAAGCTGCGCTGGACTGTGGCCCTTCTGCTTGCGGCGGCAGGAA
CcgcaGTGGGTGACCGGGGCAGCGGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGG CGGATccggtGGGAGAAAAGCCAAATGTGAACCCTCCCAATTCCAGTGCACAAATGGTCGCTGT ATTACGCTGTTGTGGAAATGTGATGGGGATGAAGACTGTGTTGACGGCAGTGATGAAAAGAAC TGTGTAAAGAAGACGTGTGCTGAATCTGACTTCGTGTGCAACAATGGCCAGTGTGTTCCCAGC
CGATGGAAGTGTGATGGAGATCCTGACTGCGAAGATGGTTCAGATGAAAGCCCAGAACAGTG CCATATGAGAACATGCCGCATACATGAAATCAGCTGTGGCGCCCATTCTACTCAGTGTATCCC AGTGTCCTGGAGATGTGATGGTGAAAATGATTGTGACAGTGGAGAAGATGAAGAAAACTGTG
GCAATATAACATGTAGTCCCGACGAGTTCACCTGCTCCAGTGGCCGCTGCATCTCCAGGAACT
TTGTATGCAATGGCCAGGATGACTGCAGCGATGGCAGTGATGAGCTGGACTGTGCCCCGCCA
ACCTGTGGCGCCCATGAGTTCCAGTGCAGCACCTCCTCCTGCATCCCCATCAGCTGGGTATG
CGACGATGATGCAGACTGCTCCGACCAATCTGATGAGTCCCTGGAGCAGTGTGGCCGTCAGC
CAGTCATACACACCAAGTGTCCAGCCAGCGAAATCCAGTGCGGCTCTGGCGAGTGCATCCAT
AAGAAGTGGCGATGTGATGGGGACCCTGACTGCAAGGATGGCAGTGATGAGGTCAACTGTCC
CTCTCGAACTTGCCGACCTGACCAATTTGAATGTGAGGATGGCAGCTGCATCCATGGCAGCA
GGCAGTGTAATGGTATCCGAGACTGTGTCGATGGTTCCGATGAAGTCAACTGCAAAAATGTCA
ATCAGTGCTTGGGCCCTGGAAAATTCAAGTGCAGAAGTGGAGAATGCATAGATATCAGCAAAG
TATGTAACCAGGAGCAGGACTGCAGGGACTGGAGTGATGAGCCCCTGAAAGAGTGTCATATA
AACGAATGCTTGGTAAATAATGGTGGATGTTCTCATATCTGCAAAGACCTAGTTATAGGCTACG
AGTGTGACTGTGCAGCTGGGTTTGAACTGATAGATAGGAAAACCTGTGGAGATATTGATGAAT
GCCAAAATCCAGGAATCTGCAGTCAAATTTGTATCAACTTAAAAGGCGGTTACAAGTGTGAAT
GTAGTCGTGGCTATCAAATGGATCTTGCTACTGGCGTGTGCAAGGCAGTAGGCAAAGAGCCA
AGTCTGATCTTCACTAATCGAAGAGACATCAGGAAGATTGGCTTAGAGAGGAAAGAATATATC
CAACTAGTTGAACAGCTAAGAAACACTGTGGCTCTCGATGCTGACATTGCTGCCCAGAAACTA
TTCTGGGCCGATCTAAGCCAAAAGGCTATCTTCAGTGCCTCAATTGATGACAAGGTTGGTAGA
CATGTTAAAATGATCGACAATGTCTATAATCCTGCAGCCATTGCTGTTGATTGGGTGTACAAGA
CCATCTACTGGACTGATGCGGCTTCTAAGACTATTTCAGTAGCTACCCTAGATGGAACCAAGA
GGAAGTTCCTGTTTAACTCTGACTTGCGAGAGCCTGCCTCCATAGCTGTGGACCCACTGTCTG
GCTTTGTTTACTGGTCAGACTGGGGTGAACCAGCTAAAATAGAAAAAGCAGGAATGAATGGAT
TCGATAGACGTCCACTGGTGACAGCGGATATCCAGTGGCCTAACGGAATTACACTTGACCTTA
TAAAAAGTCGCCTCTATTGGCTTGATTCTAAGTTGCACATGTTATCCAGCGTGGACTTGAATGG
CCAAGATCGTAGGATAGTACTAAAGTCTCTGGAGTTCCTAGCTCATCCTCTTGCACTAACAATA
TTTGAGGATCGTGTCTACTGGATAGATGGGGAAAATGAAGCAGTCTATGGTGCCAATAAATTC
ACTGGATCAGAGCTAGCCACTCTAGTCAACAACCTGAATGATGCCCAAGACATCATTGTCTAT
CATGAACTTGTACAGCCATCAGGTAAAAATTGGTGTGAAGAAGACATGGAGAATGGAGGATGT
GAATACCTATGCCTGCCAGCACCACAGATTAATGATCACTCTCCAAAATATACCTGTTCCTGTC
CCAGTGGGTACAATGTAGAGGAAAATGGCCGAGACTGTCAAAGTACTGCAACTACTGTGACTT
ACAGTGAGACAAAAGATACGAACACAACAGAAATTTCAGCAACTAGTGGACTAGTTCCTGGAG
GGATCAATGTGACCACAGCAGTATCAGAGGTCAGTGTTCCCCCAAAAGGGACTTCTGGAGGT
CATCACCACCATCATCAC
Amino Acid Sequence: VLDLR (SEQ ID NO 210)
MGPWGWKLRWTVALLLAAAGTAVGDRGSGVSGWRLFKKISGGSGGRKAKCEPSQFQCTNGRCI
TLLWKCDGDEDCVDGSDEKNCVKKTCAESDFVCNNGQCVPSRWKCDGDPDCEDGSDESPEQC
HMRTCRIHEISCGAHSTQCIPVSWRCDGENDCDSGEDEENCGNITCSPDEFTCSSGRCISRNFVC
NGQDDCSDGSDELDCAPPTCGAHEFQCSTSSCIPISWVCDDDADCSDQSDESLEQCGRQPVIHT
KCPASEIQCGSGECIHKKWRCDGDPDCKDGSDEVNCPSRTCRPDQFECEDGSCIHGSRQCNGIR
DCVDGSDEVNCKNVNQCLGPGKFKCRSGECIDISKVCNQEQDCRDWSDEPLKECHINECLVNNG
GCSHICKDLVIGYECDCAAGFELIDRKTCGDIDECQNPGICSQICINLKGGYKCECSRGYQMDLAT
GVCKAVGKEPSLIFTNRRDIRKIGLERKEYIQLVEQLRNTVALDADIAAQKLFWADLSQKAIFSASID
DKVGRHVKMIDNVYNPAAIAVDWVYKTIYWTDAASKTISVATLDGTKRKFLFNSDLREPASIAVDPL
SGFVYWSDWGEPAKIEKAGMNGFDRRPLVTADIQWPNGITLDLIKSRLYWLDSKLHMLSSVDLNG
QDRRIVLKSLEFLAHPLALTIFEDRVYWIDGENEAVYGANKFTGSELATLVNNLNDAQDIIVYHELVQ PSGKNWCEEDMENGGCEYLCLPAPQINDHSPKYTCSCPSGYNVEENGRDCQSTATTVTYSETKD
TNTTEISATSGLVPGGINVTTAVSEVSVPPKGTSGGHHHHHH*
Nucleotide Sequence: LDLR (1-711-7) (SEQ ID NO 211)
ATGAAACATCACCATCACCATCACTCCGCGGGTCTGGAAGTTCTGTTCCAGGGGCCCGACAG
ATGCGAGCGTAACGAATTCCAATGCCAAGACGGTAAGTGTATTAGTTACAAATGGGTTTGTGA
CGGCAGTGCCGAATGCCAAGACGGTTCGGATGAATCTCAGGAAACGTGTCTGTCGGTAACCT
GCAAATCGGGAGACTTCAGCTGTGGTGGACGGGTTAACCGCTGTATACCTCAGTTTTGGCGG
TGTGACGGACAAGTTGACTGTGATAATGGCTCAGATGAGCAGGGGTGCCCGCCAAAGACCTG
CAGTCAGGACGAATTCCGATGCCACGATGGAAAATGTATTAGCCGTCAATTCGTTTGTGACTC
GGACCGCGACTGTCTGGATGGTTCAGATGAAGCTTCGTGCCCAGTATTAACTTGCGGACCGG
CGAGTTTTCAATGCAATTCCAGCACGTGTATTCCCCAACTCTGGGCTTGCGACAACGACCCGG
ATTGCGAGGACGGCTCTGACGAGTGGCCGCAGAGATGCCGTGGGTTATACGTGTTTCAAGGA
GACAGCTCACCGTGCAGCGCTTTCGAGTTTCATTGTCTTAGCGGCGAGTGTATCCACTCATCA
TGGCGCTGCGATGGTGGGCCTGATTGTAAGGACAAGAGCGACGAGGAGAATTGTGCTGTTGC
CACGTGTCGACCAGACGAATTTCAATGTAGCGACGGTAATTGTATCCATGGATCTAGACAATG
CGACCGTGAATACGATTGTAAAGACATGTCCGACGAGGTTGGCTGTGTGAATGTGACTTTGTG
CGAGGGACCGAATAAGTTTAAGTGCCACTCAGGCGAATGCATAACACTTGACAAAGTGTGTAA
CATGGCACGCGACTGCCGTGATTGGTCAGACGAGCCAATAAAGGAATGCGGAGGAGGAGCA
GCAGCGGGCGGAGCAGCGGGAGGAGGGGCAGCGGCAGGTGGTGCGGCTGACAGATGCGA
GCGTAACGAATTCCAATGCCAAGACGGTAAGTGTATTAGTTACAAATGGGTTTGTGACGGCAG
TGCCGAATGCCAAGACGGTTCGGATGAATCTCAGGAAACGTGTCTGTCGGTAACCTGCAAAT
CGGGAGACTTCAGCTGTGGTGGACGGGTTAACCGCTGTATACCTCAGTTTTGGCGGTGTGAC
GGACAAGTTGACTGTGATAATGGCTCAGATGAGCAGGGGTGCCCGCCAAAGACCTGCAGTCA
GGACGAATTCCGATGCCACGATGGAAAATGTATTAGCCGTCAATTCGTTTGTGACTCGGACCG
CGACTGTCTGGATGGTTCAGATGAAGCTTCGTGCCCAGTATTAACTTGCGGACCGGCGAGTTT
TCAATGCAATTCCAGCACGTGTATTCCCCAACTCTGGGCTTGCGACAACGACCCGGATTGCGA
GGACGGCTCTGACGAGTGGCCGCAGAGATGCCGTGGGTTATACGTGTTTCAAGGAGACAGCT
CACCGTGCAGCGCTTTCGAGTTTCATTGTCTTAGCGGCGAGTGTATCCACTCATCATGGCGCT
GCGATGGTGGGCCTGATTGTAAGGACAAGAGCGACGAGGAGAATTGTGCTGTTGCCACGTGT
CGACCAGACGAATTTCAATGTAGCGACGGTAATTGTATCCATGGATCTAGACAATGCGACCGT
GAATACGATTGTAAAGACATGTCCGACGAGGTTGGCTGTGTGAATGTGACTTTGTGCGAGGG
ACCGAATAAGTTTAAGTGCCACTCAGGCGAATGCATAACACTTGACAAAGTGTGTAACATGGC
ACGCGACTGCCGTGATTGGTCAGACGAGCCAATAAAGGAATGCtaa
Amino Acid Sequence: LDLR (1-711-7) (SEQ ID NO 212)
MKHHHHHHSAGLEVLFQGPDRCERNEFQCQDGKCISYKWVCDGSAECQDGSDESQETCLSVTC
KSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGCPPKTCSQDEFRCHDGKCISRQFVCDS
DRDCLDGSDEASCPVLTCGPASFQCNSSTCIPQLWACDNDPDCEDGSDEWPQRCRGLYVFQGD
SSPCSAFEFHCLSGECIHSSWRCDGGPDCKDKSDEENCAVATCRPDEFQCSDGNCIHGSRQCD
REYDCKDMSDEVGCVNVTLCEGPNKFKCHSGECITLDKVCNMARDCRDWSDEPIKECGGGAAA
GGAAGGGAAAGGAADRCERNEFQCQDGKCISYKWVCDGSAECQDGSDESQETCLSVTCKSGD
FSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGCPPKTCSQDEFRCHDGKCISRQFVCDSDRDCL
DGSDEASCPVLTCGPASFQCNSSTCIPQLWACDNDPDCEDGSDEWPQRCRGLYVFQGDSSPCS
AFEFHCLSGECIHSSWRCDGGPDCKDKSDEENCAVATCRPDEFQCSDGNCIHGSRQCDREYDC
KDMSDEVGCVNVTLCEGPNKFKCHSGECITLDKVCNMARDCRDWSDEPIKEC*
Nucleotide Sequence: LDLR (1-7)-hFc (SEQ ID NO 213) ATGGGACCATGGGGATGGAAGCTGCGCTGGACTGTGGCCCTTCTGCTTGCGGCGGCAGGAA CcgcaGTGGGTGACCGGGGCAGCGGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGG CGGATccggtGACAGATGCGAGCGTAACGAATTCCAATGCCAAGACGGTAAGTGTATTAGTTAC AAATGGGTTTGTGACGGCAGTGCCGAATGCCAAGACGGTTCGGATGAATCTCAGGAAACGTG TCTGTCGGTAACCTGCAAATCGGGAGACTTCAGCTGTGGTGGACGGGTTAACCGCTGTATAC CTCAGTTTTGGCGGTGTGACGGACAAGTTGACTGTGATAATGGCTCAGATGAGCAGGGGTGC CCGCCAAAGACCTGCAGTCAGGACGAATTCCGATGCCACGATGGAAAATGTATTAGCCGTCA ATTCGTTTGTGACTCGGACCGCGACTGTCTGGATGGTTCAGATGAAGCTTCGTGCCCAGTATT AACTTGCGGACCGGCGAGTTTTCAATGCAATTCCAGCACGTGTATTCCCCAACTCTGGGCTTG CGACAACGACCCGGATTGCGAGGACGGCTCTGACGAGTGGCCGCAGAGATGCCGTGGGTTA TACGTGTTTCAAGGAGACAGCTCACCGTGCAGCGCTTTCGAGTTTCATTGTCTTAGCGGCGAG TGTATCCACTCATCATGGCGCTGCGATGGTGGGCCTGATTGTAAGGACAAGAGCGACGAGGA GAATTGTGCTGTTGCCACGTGTCGACCAGACGAATTTCAATGTAGCGACGGTAATTGTATCCA
TGGATCTAGACAATGCGACCGTGAATACGATTGTAAAGACATGTCCGACGAGGTTGGCTGTGT GAATGTGACTTTGTGCGAGGGACCGAATAAGTTTAAGTGCCACTCAGGCGAATGCATAACACT TGACAAAGTGTGTAACATGGCACGCGACTGCCGTGATTGGTCAGACGAGCCAATAAAGGAAT GCGGTGGCGGATccggtggaGACAAAACTCACACATGCCCACCGTGCCCAGgtaagccagcccaggcct cgccctccagctcaaggcgggacaggtgccctagagtagcctgcatccagggacaggccccagccgggtgctgacacgtccacctcc atctcttcctcagCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGG ACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAA GACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA GCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCAC
CAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC CATCGAGAAAACCATCTCCAAAGCCAAAGgtgggacccgtggggtgcgagggccacatggacagaggccggctcg gcccaccctctgccctgagagtgactgctgtaccaacctctgtccctacagGGCAGCCCCGAGAACCACAGGTGTACA CCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACC GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCT GCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGGTGGTTCAGGCGGCC ATCACCACCATCATCACCATCAC
Amino Acid Sequence: LDLR (1-7)-hFc (SEQ ID NO 214)
MGPWGWKLRWTVALLLAAAGTAVGDRGSGVSGWRLFKKISGGSGDRCERNEFQCQDGKCISYK WVCDGSAECQDGSDESQETCLSVTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGCP PKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPVLTCGPASFQCNSSTCIPQLWACDN DPDCEDGSDEWPQRCRGLYVFQGDSSPCSAFEFHCLSGECIHSSWRCDGGPDCKDKSDEENC AVATCRPDEFQCSDGNCIHGSRQCDREYDCKDMSDEVGCVNVTLCEGPNKFKCHSGECITLDKV CNMARDCRDWSDEPIKECGGGSGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGSGGH HHHHHHH
Nucleotide Sequence: sLDLR-sLRP8 (SEQ ID NO 215)
ATGGGACCATGGGGATGGAAGCTGCGCTGGACTGTGGCCCTTCTGCTTGCGGCGGCAGGAA CcgcaGTGGGTGACCGGGGCAGCGGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGG CGGATccggtGCAGTAGGCGACAGATGCGAGCGTAACGAATTCCAATGCCAAGACGGTAAGTG TATTAGTTACAAATGGGTTTGTGACGGCAGTGCCGAATGCCAAGACGGTTCGGATGAATCTCA
GGAAACGTGTCTGTCGGTAACCTGCAAATCGGGAGACTTCAGCTGTGGTGGACGGGTTAACC
GCTGTATACCTCAGTTTTGGCGGTGTGACGGACAAGTTGACTGTGATAATGGCTCAGATGAGC
AGGGGTGCCCGCCAAAGACCTGCAGTCAGGACGAATTCCGATGCCACGATGGAAAATGTATT
AGCCGTCAATTCGTTTGTGACTCGGACCGCGACTGTCTGGATGGTTCAGATGAAGCTTCGTG
CCCAGTATTAACTTGCGGACCGGCGAGTTTTCAATGCAATTCCAGCACGTGTATTCCCCAACT
CTGGGCTTGCGACAACGACCCGGATTGCGAGGACGGCTCTGACGAGTGGCCGCAGAGATGC
CGTGGGTTATACGTGTTTCAAGGAGACAGCTCACCGTGCAGCGCTTTCGAGTTTCATTGTCTT
AGCGGCGAGTGTATCCACTCATCATGGCGCTGCGATGGTGGGCCTGATTGTAAGGACAAGAG
CGACGAGGAGAATTGTGCTGTTGCCACGTGTCGACCAGACGAATTTCAATGTAGCGACGGTA
ATTGTATCCATGGATCTAGACAATGCGACCGTGAATACGATTGTAAAGACATGTCCGACGAGG
TTGGCTGTGTGAATGTGACTTTGTGCGAGGGACCGAATAAGTTTAAGTGCCACTCAGGCGAAT
GCATAACACTTGACAAAGTGTGTAACATGGCACGCGACTGCCGTGATTGGTCAGACGAGCCA
ATAAAGGAATGCGGTACCAACGAGTGCCTGGACAACAATGGCGGCTGTAGTCATGTTTGCAAT
GACCTTAAGATCGGTTACGAGTGTCTCTGCCCAGATGGTTTCCAGTTGGTTGCTCAACGCCGA
TGCGAAGACATAGACGAATGCCAAGATCCGGACACATGCTCCCAACTGTGCGTCAATTTGGAA
GGCGGATACAAGTGCCAATGCGAGGAAGGCTTCCAGTTGGATCCGCACACGAAGGCCTGCA
AAGCGGTGGGTAGTATTGCTTACTTATTCTTCACGAACCGACACGAAGTTCGGAAAATGACTT
TGGATAGATCCGAGTACACATCACTTATACCTAACCTTCGTAATGTAGTCGCACTTGATACCGA
GGTAGCAAGTAACCGCATCTATTGGTCTGACTTGTCCCAGCGCATGATATGTTCTACTCAATT
GGATCGTGCACACGGAGTTAGCTCGTATGACACCGTAATATCACGTGACATACAGGCACCGG
ATGGCCTGGCGGTTGACTGGATTCATAGCAATATTTACTGGACAGACTCCGTGCTGGGCACA
GTGAGTGTTGCTGATACGAAAGGGGTTAAACGTAAGACACTGTTTCGGGAAAATGGTAGCAAG
CCGCGCGCTATAGTGGTTGACCCGGTACATGGATTCATGTACTGGACGGACTGGGGTACACC
AGCCAAGATCAAGAAGGGTGGTCTGAACGGAGTCGACATATACAGCCTGGTCACGGAGAATA
TACAGTGGCCGAACGGGATTACTTTGGACCTTCTGAGTGGACGCTTGTACTGGGTGGATAGC
AAGCTGCATTCTATCTCCTCTATTGATGTGAACGGTGGAAACCGGAAGACTATATTGGAAGAT
GAGAAGAGATTGGCCCACCCGTTCTCTCTCGCGGTCTTTGAGGACAAGGTATTCTGGACGGA
CATAATTAACGAGGCTATCTTCTCTGCCAACCGTCTGACTGGCTCTGACGTGAATTTACTGGC
GGAGAATTTGCTGAGTCCTGAGGATATGGTATTGTTTCACAACTTAACGCAACCGCGGGGCGT
GAACTGGTGTGAGCGTACGACGTTAAGCAACGGTGGCTGTCAGTACCTGTGTTTACCTGCGC
CGCAAATAAATCCTCACTCTCCGAAGTTTACCTGCGCTTGTCCAGACGGCATGCTTCTTGCCC
GCGACATGCGTTCATGCTTAACGGAAGCTGAAGCTGCCGTTGCTACACAAGAAACGAGCACC
GTGAGATTAAAGGTGAGCTCAACCGCCGTCCGCACACAACACACCACAACACGCCCTGTCCC
GGACACCTCGCGGTTACCGGGTGCGACCCCCGGATTAACCACCGTTGAGATCGTAACCATGT
CACACCAAGCCCTCGGCGATGTGGCTGGCCGTGGCAATGAGAAGAAACCTTCCAGTGTCCG
GGGCGGATccggtCATCTTGCGGCGGCAGCGGCTGATCCGCTGCTCGGCGGCCAAGGGCCGG
CCAAGGATTGCGAAAAGGACCAATTCCAGTGCCGGAACGAGCGCTGCATCCCCTCTGTGTGG
AGATGCGACGAGGACGATGACTGCTTAGACCACAGCGACGAGGACGACTGCCCCAAGAAGA
CCTGTGCAGACAGTGACTTCACCTGTGACAACGGCCACTGCATCCACGAACGGTGGAAGTGT
GACGGCGAGGAGGAGTGTCCTGATGGCTCCGATGAGTCCGAGGCCACTTGCACCAAGCAGG
TGTGTCCTGCAGAGAAGCTGAGCTGTGGACCCACCAGCCACAAGTGTGTACCTGCCTCGTGG
CGCTGCGACGGGGAGAAGGACTGCGAGGGTGGAGCGGATGAGGCCGGCTGTGCTACCTCA
CTGGGCACCTGCCGTGGGGACGAGTTCCAGTGTGGGGATGGGACATGTGTCCTTGCAATCAA
GCACTGCAACCAGGAGCAGGACTGTCCAGATGGGAGTGATGAAGCTGGCTGCCTACAGGTTC
CGCCAACATTCCTGGGAAACAGGAGGAGGCCCAGGGGGCTGAACGAGTGTCTGCACAACAA TGGCGGCTGCTCACACATCTGCACTGACCTCAAGATTGGCTTTGAATGCACGTGCCCAGCAG GCTTCCAGCTCCTGGACCAGAAGACCTGTGGCGACATTGATGAGTGCAAGGACCCAGATGCC TGCAGCCAGATCTGTGTCAATTACAAGGGCTATTTTAAGTGTGAGTGCTACCCTGGCTACGAG ATGGACCTACTGACCAAGAACTGCAAGGCTGCTGCTGGaAAGAGCCCATCCCTAATCTTCACC
AACCGGCACGAGGTGCGGAGGATCGACCTGGTGAAGCGGAACTATTCACGCCTCATCCCCAT GCTCAAGAATGTCGTGGCACTAGATGTGGAAGTTGCCACCAATCGCATCTACTGGTGTGACCT CTCCTACCGTAAGATCTATAGCGCCTACATGGACAAGGCCAGTGACCCGAAAGAGCAGGAGG TCCTCATTGACGAGCAGTTGCACTCTCCAGAGGGCCTGGCAGTGGACTGGGTCCACAAGCAC
ATCTACTGGACTGACTCGGGCAATAAGACCATCTCAGTGGCCACAGTTGATGGTGGCCGCCG ACGCACTCTCTTCAGCCGTAACCTCAGTGAACCCCGGGCCATCGCTGTTGACCCCCTGCGAG GGTTCATGTATTGGTCTGACTGGGGGGACCAGGCCAAGATTGAGAAATCTGGGCTCAACGGT GTGGACCGGCAAACACTGGTGTCAGACAATATTGAATGGCCCAACGGAATCACCCTGGATCT
GCTGAGCCAGCGCTTGTACTGGGTAGACTCCAAGCTACACCAACTGTCCAGCATTGACTTCA
GTGGAGGCAACAGAAAGACGCTGATCTCCTCCACTGACTTCCTGAGCCACCCTTTTGGGATA GCTGTGTTTGAGGACAAGGTGTTCTGGACAGACCTGGAGAACGAGGCCATTTTCAGTGCAAA TCGGCTCAATGGCCTGGAAATCTCCATCCTGGCTGAGAACCTCAACAACCCACATGACATTGT CATCTTCCATGAGCTGAAGCAGCCAAGAGCTCCAGATGCCTGTGAGCTGAGTGTCCAGCCTA
ATGGAGGCTGTGAATACCTGTGCCTTCCTGCTCCTCAGATCTCCAGCCACTCTCCCAAGTACA CATGTGCCTGTCCTGACACAATGTGGCTGGGTCCAGACATGAAGAGGTGCTACCGAGCACCT CAATCTACCTCAACTACGACGTTAGCTTCTACCATGACGAGGACAGTACCTGCCACCACAAGA GCCCCCGGGACCACCGTCCACAGATCCACCTACCAGAACCACAGCACAGAGACACCAAGCCT
GACAGCTGCAGTCCCAAGCTCAGTTAGTGTCCCCAGGGCTCCCAGCATCAGCCCGTCTACCC TAAGCCCTGCAACCAGCAACCACTCCCAGCACTATGCAAATGAAGACAGTAAGATGGGCTCAA CAGTCACTGCCGCTGTTATCGGGATCATCGTGCCCgGAGACGCTTCGgattacaaagacgatgacgata agCTGGAAGTTCTGTTCCAGGGGCCCGGTCATCACCACCATCATCACCATCACTAA
Amino Acid Sequence: sLDLR-sLRP8 (SEQ ID NO 216)
MGPWGWKLRWTVALLLAAAGTAVGDRGSGVSGWRLFKKISGGSGAVGDRCERNEFQCQDGKC
ISYKWVCDGSAECQDGSDESQETCLSVTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDE
QGCPPKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPVLTCGPASFQCNSSTCIPQLW
ACDNDPDCEDGSDEWPQRCRGLYVFQGDSSPCSAFEFHCLSGECIHSSWRCDGGPDCKDKSD
EENCAVATCRPDEFQCSDGNCIHGSRQCDREYDCKDMSDEVGCVNVTLCEGPNKFKCHSGECIT
LDKVCNMARDCRDWSDEPIKECGTNECLDNNGGCSHVCNDLKIGYECLCPDGFQLVAQRRCEDI
DECQDPDTCSQLCVNLEGGYKCQCEEGFQLDPHTKACKAVGSIAYLFFTNRHEVRKMTLDRSEY TSLIPNLRNVVALDTEVASNRIYWSDLSQRMICSTQLDRAHGVSSYDTVISRDIQAPDGLAVDWIHS NIYWTDSVLGTVSVADTKGVKRKTLFRENGSKPRAIVVDPVHGFMYWTDWGTPAKIKKGGLNGV DIYSLVTENIQWPNGITLDLLSGRLYWVDSKLHSISSIDVNGGNRKTILEDEKRLAHPFSLAVFEDKV
FWTDIINEAIFSANRLTGSDVNLLAENLLSPEDMVLFHNLTQPRGVNWCERTTLSNGGCQYLCLPA
PQINPHSPKFTCACPDGMLLARDMRSCLTEAEAAVATQETSTVRLKVSSTAVRTQHTTTRPVPDT SRLPGATPGLTTVEIVTMSHQALGDVAGRGNEKKPSSVRGGSGHLAAAAADPLLGGQGPAKDCE KDQFQCRNERCIPSVWRCDEDDDCLDHSDEDDCPKKTCADSDFTCDNGHCIHERWKCDGEEEC PDGSDESEATCTKQVCPAEKLSCGPTSHKCVPASWRCDGEKDCEGGADEAGCATSLGTCRGDE
FQCGDGTCVLAIKHCNQEQDCPDGSDEAGCLQVPPTFLGNRRRPRGLNECLHNNGGCSHICTDL
KIGFECTCPAGFQLLDQKTCGDIDECKDPDACSQICVNYKGYFKCECYPGYEMDLLTKNCKAAAG
KSPSLIFTNRHEVRRIDLVKRNYSRLIPMLKNWALDVEVATNRIYWCDLSYRKIYSAYMDKASDPK EQEVLIDEQLHSPEGLAVDWVHKHIYWTDSGNKTISVATVDGGRRRTLFSRNLSEPRAIAVDPLRG FMYWSDWGDQAKIEKSGLNGVDRQTLVSDNIEWPNGITLDLLSQRLYWVDSKLHQLSSIDFSGGN RKTLISSTDFLSHPFGIAVFEDKVFWTDLENEAIFSANRLNGLEISILAENLNNPHDIVIFHELKQPRA
PDACELSVQPNGGCEYLCLPAPQISSHSPKYTCACPDTMWLGPDMKRCYRAPQSTSTTTLASTM
TRTVPATTRAPGTTVHRSTYQNHSTETPSLTAAVPSSVSVPRAPSISPSTLSPATSNHSQHYANED
SKMGSTVTAAVIGIIVPGDASDYKDDDDKLEVLFQGPGHHHHHHHH*
Nucleotide Sequence: sLDLR-sVLDLR (SEQ ID NO 217)
ATGGGACCATGGGGATGGAAGCTGCGCTGGACTGTGGCCCTTCTGCTTGCGGCGGCAGGAA
CcgcaGTGGGTGACCGGGGCAGCGGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGG
CGGATccggtGCAGTAGGCGACAGATGCGAGCGTAACGAATTCCAATGCCAAGACGGTAAGTG
TATTAGTTACAAATGGGTTTGTGACGGCAGTGCCGAATGCCAAGACGGTTCGGATGAATCTCA
GGAAACGTGTCTGTCGGTAACCTGCAAATCGGGAGACTTCAGCTGTGGTGGACGGGTTAACC
GCTGTATACCTCAGTTTTGGCGGTGTGACGGACAAGTTGACTGTGATAATGGCTCAGATGAGC
AGGGGTGCCCGCCAAAGACCTGCAGTCAGGACGAATTCCGATGCCACGATGGAAAATGTATT
AGCCGTCAATTCGTTTGTGACTCGGACCGCGACTGTCTGGATGGTTCAGATGAAGCTTCGTG
CCCAGTATTAACTTGCGGACCGGCGAGTTTTCAATGCAATTCCAGCACGTGTATTCCCCAACT
CTGGGCTTGCGACAACGACCCGGATTGCGAGGACGGCTCTGACGAGTGGCCGCAGAGATGC
CGTGGGTTATACGTGTTTCAAGGAGACAGCTCACCGTGCAGCGCTTTCGAGTTTCATTGTCTT
AGCGGCGAGTGTATCCACTCATCATGGCGCTGCGATGGTGGGCCTGATTGTAAGGACAAGAG
CGACGAGGAGAATTGTGCTGTTGCCACGTGTCGACCAGACGAATTTCAATGTAGCGACGGTA
ATTGTATCCATGGATCTAGACAATGCGACCGTGAATACGATTGTAAAGACATGTCCGACGAGG
TTGGCTGTGTGAATGTGACTTTGTGCGAGGGACCGAATAAGTTTAAGTGCCACTCAGGCGAAT
GCATAACACTTGACAAAGTGTGTAACATGGCACGCGACTGCCGTGATTGGTCAGACGAGCCA
ATAAAGGAATGCGGTACCAACGAGTGCCTGGACAACAATGGCGGCTGTAGTCATGTTTGCAAT
GACCTTAAGATCGGTTACGAGTGTCTCTGCCCAGATGGTTTCCAGTTGGTTGCTCAACGCCGA
TGCGAAGACATAGACGAATGCCAAGATCCGGACACATGCTCCCAACTGTGCGTCAATTTGGAA
GGCGGATACAAGTGCCAATGCGAGGAAGGCTTCCAGTTGGATCCGCACACGAAGGCCTGCA
AAGCGGTGGGTAGTATTGCTTACTTATTCTTCACGAACCGACACGAAGTTCGGAAAATGACTT
TGGATAGATCCGAGTACACATCACTTATACCTAACCTTCGTAATGTAGTCGCACTTGATACCGA
GGTAGCAAGTAACCGCATCTATTGGTCTGACTTGTCCCAGCGCATGATATGTTCTACTCAATT
GGATCGTGCACACGGAGTTAGCTCGTATGACACCGTAATATCACGTGACATACAGGCACCGG
ATGGCCTGGCGGTTGACTGGATTCATAGCAATATTTACTGGACAGACTCCGTGCTGGGCACA
GTGAGTGTTGCTGATACGAAAGGGGTTAAACGTAAGACACTGTTTCGGGAAAATGGTAGCAAG
CCGCGCGCTATAGTGGTTGACCCGGTACATGGATTCATGTACTGGACGGACTGGGGTACACC
AGCCAAGATCAAGAAGGGTGGTCTGAACGGAGTCGACATATACAGCCTGGTCACGGAGAATA
TACAGTGGCCGAACGGGATTACTTTGGACCTTCTGAGTGGACGCTTGTACTGGGTGGATAGC
AAGCTGCATTCTATCTCCTCTATTGATGTGAACGGTGGAAACCGGAAGACTATATTGGAAGAT
GAGAAGAGATTGGCCCACCCGTTCTCTCTCGCGGTCTTTGAGGACAAGGTATTCTGGACGGA
CATAATTAACGAGGCTATCTTCTCTGCCAACCGTCTGACTGGCTCTGACGTGAATTTACTGGC
GGAGAATTTGCTGAGTCCTGAGGATATGGTATTGTTTCACAACTTAACGCAACCGCGGGGCGT
GAACTGGTGTGAGCGTACGACGTTAAGCAACGGTGGCTGTCAGTACCTGTGTTTACCTGCGC
CGCAAATAAATCCTCACTCTCCGAAGTTTACCTGCGCTTGTCCAGACGGCATGCTTCTTGCCC
GCGACATGCGTTCATGCTTAACGGAAGCTGAAGCTGCCGTTGCTACACAAGAAACGAGCACC
GTGAGATTAAAGGTGAGCTCAACCGCCGTCCGCACACAACACACCACAACACGCCCTGTCCC
GGACACCTCGCGGTTACCGGGTGCGACCCCCGGATTAACCACCGTTGAGATCGTAACCATGT
CACACCAAGCCCTCGGCGATGTGGCTGGCCGTGGCAATGAGAAGAAACCTTCCAGTGTCCG
GGGCGGATccggtGGGAGAAAAGCCAAATGTGAACCCTCCCAATTCCAGTGCACAAATGGTCG
CTGTATTACGCTGTTGTGGAAATGTGATGGGGATGAAGACTGTGTTGACGGCAGTGATGAAAA GAACTGTGTAAAGAAGACGTGTGCTGAATCTGACTTCGTGTGCAACAATGGCCAGTGTGTTCC CAGCCGATGGAAGTGTGATGGAGATCCTGACTGCGAAGATGGTTCAGATGAAAGCCCAGAAC AGTGCCATATGAGAACATGCCGCATACATGAAATCAGCTGTGGCGCCCATTCTACTCAGTGTA TCCCAGTGTCCTGGAGATGTGATGGTGAAAATGATTGTGACAGTGGAGAAGATGAAGAAAACT GTGGCAATATAACATGTAGTCCCGACGAGTTCACCTGCTCCAGTGGCCGCTGCATCTCCAGG AACTTTGTATGCAATGGCCAGGATGACTGCAGCGATGGCAGTGATGAGCTGGACTGTGCCCC
GCCAACCTGTGGCGCCCATGAGTTCCAGTGCAGCACCTCCTCCTGCATCCCCATCAGCTGGG TATGCGACGATGATGCAGACTGCTCCGACCAATCTGATGAGTCCCTGGAGCAGTGTGGCCGT CAGCCAGTCATACACACCAAGTGTCCAGCCAGCGAAATCCAGTGCGGCTCTGGCGAGTGCAT CCATAAGAAGTGGCGATGTGATGGGGACCCTGACTGCAAGGATGGCAGTGATGAGGTCAACT GTCCCTCTCGAACTTGCCGACCTGACCAATTTGAATGTGAGGATGGCAGCTGCATCCATGGC AGCAGGCAGTGTAATGGTATCCGAGACTGTGTCGATGGTTCCGATGAAGTCAACTGCAAAAAT
GTCAATCAGTGCTTGGGCCCTGGAAAATTCAAGTGCAGAAGTGGAGAATGCATAGATATCAGC AAAGTATGTAACCAGGAGCAGGACTGCAGGGACTGGAGTGATGAGCCCCTGAAAGAGTGTCA TATAAACGAATGCTTGGTAAATAATGGTGGATGTTCTCATATCTGCAAAGACCTAGTTATAGGC TACGAGTGTGACTGTGCAGCTGGGTTTGAACTGATAGATAGGAAAACCTGTGGAGATATTGAT GAATGCCAAAATCCAGGAATCTGCAGTCAAATTTGTATCAACTTAAAAGGCGGTTACAAGTGT GAATGTAGTCGTGGCTATCAAATGGATCTTGCTACTGGCGTGTGCAAGGCAGTAGGCAAAGA
GCCAAGTCTGATCTTCACTAATCGAAGAGACATCAGGAAGATTGGCTTAGAGAGGAAAGAATA TATCCAACTAGTTGAACAGCTAAGAAACACTGTGGCTCTCGATGCTGACATTGCTGCCCAGAA ACTATTCTGGGCCGATCTAAGCCAAAAGGCTATCTTCAGTGCCTCAATTGATGACAAGGTTGG TAGACATGTTAAAATGATCGACAATGTCTATAATCCTGCAGCCATTGCTGTTGATTGGGTGTAC AAGACCATCTACTGGACTGATGCGGCTTCTAAGACTATTTCAGTAGCTACCCTAGATGGAACC AAGAGGAAGTTCCTGTTTAACTCTGACTTGCGAGAGCCTGCCTCCATAGCTGTGGACCCACTG
TCTGGCTTTGTTTACTGGTCAGACTGGGGTGAACCAGCTAAAATAGAAAAAGCAGGAATGAAT GGATTCGATAGACGTCCACTGGTGACAGCGGATATCCAGTGGCCTAACGGAATTACACTTGA CCTTATAAAAAGTCGCCTCTATTGGCTTGATTCTAAGTTGCACATGTTATCCAGCGTGGACTTG AATGGCCAAGATCGTAGGATAGTACTAAAGTCTCTGGAGTTCCTAGCTCATCCTCTTGCACTA ACAATATTTGAGGATCGTGTCTACTGGATAGATGGGGAAAATGAAGCAGTCTATGGTGCCAAT AAATTCACTGGATCAGAGCTAGCCACTCTAGTCAACAACCTGAATGATGCCCAAGACATCATT
GTCTATCATGAACTTGTACAGCCATCAGGTAAAAATTGGTGTGAAGAAGACATGGAGAATGGA GGATGTGAATACCTATGCCTGCCAGCACCACAGATTAATGATCACTCTCCAAAATATACCTGTT CCTGTCCCAGTGGGTACAATGTAGAGGAAAATGGCCGAGACTGTCAAAGTACTGCAACTACT GTGACTTACAGTGAGACAAAAGATACGAACACAACAGAAATTTCAGCAACTAGTGGACTAGTT CCTGGAGGGATCAATGTGACCACAGCAGTATCAGAGGTCAGTGTTCCCCCAAAAGGGACTTC TGGAGGTCATCACCACCATCATCACtaA
Amino Acid Sequence: sLDLR-sVLDLR (SEQ ID NO 218) MGPWGWKLRWTVALLLAAAGTAVGDRGSGVSGWRLFKKISGGSGAVGDRCERNEFQCQDGKC ISYKWVCDGSAECQDGSDESQETCLSVTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDE QGCPPKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPVLTCGPASFQCNSSTCIPQLW ACDNDPDCEDGSDEWPQRCRGLYVFQGDSSPCSAFEFHCLSGECIHSSWRCDGGPDCKDKSD EENCAVATCRPDEFQCSDGNCIHGSRQCDREYDCKDMSDEVGCVNVTLCEGPNKFKCHSGECIT LDKVCNMARDCRDWSDEPIKECGTNECLDNNGGCSHVCNDLKIGYECLCPDGFQLVAQRRCEDI
DECQDPDTCSQLCVNLEGGYKCQCEEGFQLDPHTKACKAVGSIAYLFFTNRHEVRKMTLDRSEY TSLIPNLRNVVALDTEVASNRIYWSDLSQRMICSTQLDRAHGVSSYDTVISRDIQAPDGLAVDWIHS NIYWTDSVLGTVSVADTKGVKRKTLFRENGSKPRAIVVDPVHGFMYWTDWGTPAKIKKGGLNGV DIYSLVTENIQWPNGITLDLLSGRLYWVDSKLHSISSIDVNGGNRKTILEDEKRLAHPFSLAVFEDKV
FWTDIINEAIFSANRLTGSDVNLLAENLLSPEDMVLFHNLTQPRGVNWCERTTLSNGGCQYLCLPA
PQINPHSPKFTCACPDGMLLARDMRSCLTEAEAAVATQETSTVRLKVSSTAVRTQHTTTRPVPDT SRLPGATPGLTTVEIVTMSHQALGDVAGRGNEKKPSSVRGGSGGRKAKCEPSQFQCTNGRCITLL
WKCDGDEDCVDGSDEKNCVKKTCAESDFVCNNGQCVPSRWKCDGDPDCEDGSDESPEQCHM RTCRIHEISCGAHSTQCIPVSWRCDGENDCDSGEDEENCGNITCSPDEFTCSSGRCISRNFVCNG
QDDCSDGSDELDCAPPTCGAHEFQCSTSSCIPISWVCDDDADCSDQSDESLEQCGRQPVIHTKC PASEIQCGSGECIHKKWRCDGDPDCKDGSDEVNCPSRTCRPDQFECEDGSCIHGSRQCNGIRDC
VDGSDEVNCKNVNQCLGPGKFKCRSGECIDISKVCNQEQDCRDWSDEPLKECHINECLVNNGGC SHICKDLVIGYECDCAAGFELIDRKTCGDIDECQNPGICSQICINLKGGYKCECSRGYQMDLATGVC KAVGKEPSLIFTNRRDIRKIGLERKEYIQLVEQLRNTVALDADIAAQKLFWADLSQKAIFSASIDDKV GRHVKMIDNVYNPAAIAVDWVYKTIYWTDAASKTISVATLDGTKRKFLFNSDLREPASIAVDPLSGF
VYWSDWGEPAKIEKAGMNGFDRRPLVTADIQWPNGITLDLIKSRLYWLDSKLHMLSSVDLNGQDR RIVLKSLEFLAHPLALTIFEDRVYWIDGENEAVYGANKFTGSELATLVNNLNDAQDIIVYHELVQPSG KNWCEEDMENGGCEYLCLPAPQINDHSPKYTCSCPSGYNVEENGRDCQSTATTVTYSETKDTNT TEISATSGLVPGGINVTTAVSEVSVPPKGTSGGHHHHHH*
Nucleotide Sequence: sLRP8-sLDLR (SEQ ID NO 219)
ATGGGACCATGGGGATGGAAGCTGCGCTGGACTGTGGCCCTTCTGCTTGCGGCGGCAGGAA
CcgcaGTGGGTGACCGGGGCAGCGGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGG
CGGATccggtCATCTTGCGGCGGCAGCGGCTGATCCGCTGCTCGGCGGCCAAGGGCCGGCCA AGGATTGCGAAAAGGACCAATTCCAGTGCCGGAACGAGCGCTGCATCCCCTCTGTGTGGAGA
TGCGACGAGGACGATGACTGCTTAGACCACAGCGACGAGGACGACTGCCCCAAGAAGACCT GTGCAGACAGTGACTTCACCTGTGACAACGGCCACTGCATCCACGAACGGTGGAAGTGTGAC
GGCGAGGAGGAGTGTCCTGATGGCTCCGATGAGTCCGAGGCCACTTGCACCAAGCAGGTGT
GTCCTGCAGAGAAGCTGAGCTGTGGACCCACCAGCCACAAGTGTGTACCTGCCTCGTGGCG
CTGCGACGGGGAGAAGGACTGCGAGGGTGGAGCGGATGAGGCCGGCTGTGCTACCTCACT GGGCACCTGCCGTGGGGACGAGTTCCAGTGTGGGGATGGGACATGTGTCCTTGCAATCAAG
CACTGCAACCAGGAGCAGGACTGTCCAGATGGGAGTGATGAAGCTGGCTGCCTACAGGTTCC
GCCAACATTCCTGGGAAACAGGAGGAGGCCCAGGGGGCTGAACGAGTGTCTGCACAACAAT GGCGGCTGCTCACACATCTGCACTGACCTCAAGATTGGCTTTGAATGCACGTGCCCAGCAGG
CTTCCAGCTCCTGGACCAGAAGACCTGTGGCGACATTGATGAGTGCAAGGACCCAGATGCCT GCAGCCAGATCTGTGTCAATTACAAGGGCTATTTTAAGTGTGAGTGCTACCCTGGCTACGAGA
TGGACCTACTGACCAAGAACTGCAAGGCTGCTGCTGGaAAGAGCCCATCCCTAATCTTCACCA ACCGGCACGAGGTGCGGAGGATCGACCTGGTGAAGCGGAACTATTCACGCCTCATCCCCAT
GCTCAAGAATGTCGTGGCACTAGATGTGGAAGTTGCCACCAATCGCATCTACTGGTGTGACCT CTCCTACCGTAAGATCTATAGCGCCTACATGGACAAGGCCAGTGACCCGAAAGAGCAGGAGG
TCCTCATTGACGAGCAGTTGCACTCTCCAGAGGGCCTGGCAGTGGACTGGGTCCACAAGCAC
ATCTACTGGACTGACTCGGGCAATAAGACCATCTCAGTGGCCACAGTTGATGGTGGCCGCCG
ACGCACTCTCTTCAGCCGTAACCTCAGTGAACCCCGGGCCATCGCTGTTGACCCCCTGCGAG GGTTCATGTATTGGTCTGACTGGGGGGACCAGGCCAAGATTGAGAAATCTGGGCTCAACGGT
GTGGACCGGCAAACACTGGTGTCAGACAATATTGAATGGCCCAACGGAATCACCCTGGATCT
GCTGAGCCAGCGCTTGTACTGGGTAGACTCCAAGCTACACCAACTGTCCAGCATTGACTTCA
GTGGAGGCAACAGAAAGACGCTGATCTCCTCCACTGACTTCCTGAGCCACCCTTTTGGGATA GCTGTGTTTGAGGACAAGGTGTTCTGGACAGACCTGGAGAACGAGGCCATTTTCAGTGCAAA
TCGGCTCAATGGCCTGGAAATCTCCATCCTGGCTGAGAACCTCAACAACCCACATGACATTGT CATCTTCCATGAGCTGAAGCAGCCAAGAGCTCCAGATGCCTGTGAGCTGAGTGTCCAGCCTA ATGGAGGCTGTGAATACCTGTGCCTTCCTGCTCCTCAGATCTCCAGCCACTCTCCCAAGTACA
CATGTGCCTGTCCTGACACAATGTGGCTGGGTCCAGACATGAAGAGGTGCTACCGAGCACCT
CAATCTACCTCAACTACGACGTTAGCTTCTACCATGACGAGGACAGTACCTGCCACCACAAGA
GCCCCCGGGACCACCGTCCACAGATCCACCTACCAGAACCACAGCACAGAGACACCAAGCCT
GACAGCTGCAGTCCCAAGCTCAGTTAGTGTCCCCAGGGCTCCCAGCATCAGCCCGTCTACCC
TAAGCCCTGCAACCAGCAACCACTCCCAGCACTATGCAAATGAAGACAGTAAGATGGGCTCAA
CAGTCACTGCCGCTGTTATCGGGATCATCGTGCCCGGCGGAGCAGTAGGCGACAGATGCGA
GCGTAACGAATTCCAATGCCAAGACGGTAAGTGTATTAGTTACAAATGGGTTTGTGACGGCAG
TGCCGAATGCCAAGACGGTTCGGATGAATCTCAGGAAACGTGTCTGTCGGTAACCTGCAAAT
CGGGAGACTTCAGCTGTGGTGGACGGGTTAACCGCTGTATACCTCAGTTTTGGCGGTGTGAC
GGACAAGTTGACTGTGATAATGGCTCAGATGAGCAGGGGTGCCCGCCAAAGACCTGCAGTCA
GGACGAATTCCGATGCCACGATGGAAAATGTATTAGCCGTCAATTCGTTTGTGACTCGGACCG
CGACTGTCTGGATGGTTCAGATGAAGCTTCGTGCCCAGTATTAACTTGCGGACCGGCGAGTTT
TCAATGCAATTCCAGCACGTGTATTCCCCAACTCTGGGCTTGCGACAACGACCCGGATTGCGA
GGACGGCTCTGACGAGTGGCCGCAGAGATGCCGTGGGTTATACGTGTTTCAAGGAGACAGCT
CACCGTGCAGCGCTTTCGAGTTTCATTGTCTTAGCGGCGAGTGTATCCACTCATCATGGCGCT
GCGATGGTGGGCCTGATTGTAAGGACAAGAGCGACGAGGAGAATTGTGCTGTTGCCACGTGT
CGACCAGACGAATTTCAATGTAGCGACGGTAATTGTATCCATGGATCTAGACAATGCGACCGT
GAATACGATTGTAAAGACATGTCCGACGAGGTTGGCTGTGTGAATGTGACTTTGTGCGAGGG
ACCGAATAAGTTTAAGTGCCACTCAGGCGAATGCATAACACTTGACAAAGTGTGTAACATGGC
ACGCGACTGCCGTGATTGGTCAGACGAGCCAATAAAGGAATGCGGTACCAACGAGTGCCTGG
ACAACAATGGCGGCTGTAGTCATGTTTGCAATGACCTTAAGATCGGTTACGAGTGTCTCTGCC
CAGATGGTTTCCAGTTGGTTGCTCAACGCCGATGCGAAGACATAGACGAATGCCAAGATCCG
GACACATGCTCCCAACTGTGCGTCAATTTGGAAGGCGGATACAAGTGCCAATGCGAGGAAGG
CTTCCAGTTGGATCCGCACACGAAGGCCTGCAAAGCGGTGGGTAGTATTGCTTACTTATTCTT
CACGAACCGACACGAAGTTCGGAAAATGACTTTGGATAGATCCGAGTACACATCACTTATACC
TAACCTTCGTAATGTAGTCGCACTTGATACCGAGGTAGCAAGTAACCGCATCTATTGGTCTGA
CTTGTCCCAGCGCATGATATGTTCTACTCAATTGGATCGTGCACACGGAGTTAGCTCGTATGA
CACCGTAATATCACGTGACATACAGGCACCGGATGGCCTGGCGGTTGACTGGATTCATAGCA
ATATTTACTGGACAGACTCCGTGCTGGGCACAGTGAGTGTTGCTGATACGAAAGGGGTTAAAC
GTAAGACACTGTTTCGGGAAAATGGTAGCAAGCCGCGCGCTATAGTGGTTGACCCGGTACAT
GGATTCATGTACTGGACGGACTGGGGTACACCAGCCAAGATCAAGAAGGGTGGTCTGAACGG
AGTCGACATATACAGCCTGGTCACGGAGAATATACAGTGGCCGAACGGGATTACTTTGGACCT
TCTGAGTGGACGCTTGTACTGGGTGGATAGCAAGCTGCATTCTATCTCCTCTATTGATGTGAA
CGGTGGAAACCGGAAGACTATATTGGAAGATGAGAAGAGATTGGCCCACCCGTTCTCTCTCG
CGGTCTTTGAGGACAAGGTATTCTGGACGGACATAATTAACGAGGCTATCTTCTCTGCCAACC
GTCTGACTGGCTCTGACGTGAATTTACTGGCGGAGAATTTGCTGAGTCCTGAGGATATGGTAT
TGTTTCACAACTTAACGCAACCGCGGGGCGTGAACTGGTGTGAGCGTACGACGTTAAGCAAC
GGTGGCTGTCAGTACCTGTGTTTACCTGCGCCGCAAATAAATCCTCACTCTCCGAAGTTTACC
TGCGCTTGTCCAGACGGCATGCTTCTTGCCCGCGACATGCGTTCATGCTTAACGGAAGCTGA
AGCTGCCGTTGCTACACAAGAAACGAGCACCGTGAGATTAAAGGTGAGCTCAACCGCCGTCC
GCACACAACACACCACAACACGCCCTGTCCCGGACACCTCGCGGTTACCGGGTGCGACCCC
CGGATTAACCACCGTTGAGATCGTAACCATGTCACACCAAGCCCTCGGCGATGTGGCTGGCC
GTGGCAATGAGAAGAAACCTTCCAGTGTCCGGGGAGACGCTTCGGATtacaaagacgatgacgataag
CTGGAAGTTCTGTTCCAGGGGCCCGGTCATCACCACCATCATCACCATCACTAA
Amino Acid Sequence: sLRP8-sLDLR (SEQ ID NO 220) MGPWGWKLRWTVALLLAAAGTAVGDRGSGVSGWRLFKKISGGSGHLAAAAADPLLGGQGPAKD CEKDQFQCRNERCIPSVWRCDEDDDCLDHSDEDDCPKKTCADSDFTCDNGHCIHERWKCDGEE ECPDGSDESEATCTKQVCPAEKLSCGPTSHKCVPASWRCDGEKDCEGGADEAGCATSLGTCRG DEFQCGDGTCVLAIKHCNQEQDCPDGSDEAGCLQVPPTFLGNRRRPRGLNECLHNNGGCSHICT DLKIGFECTCPAGFQLLDQKTCGDIDECKDPDACSQICVNYKGYFKCECYPGYEMDLLTKNCKAA AGKSPSLIFTNRHEVRRIDLVKRNYSRLIPMLKNVVALDVEVATNRIYWCDLSYRKIYSAYMDKASD PKEQEVLIDEQLHSPEGLAVDWVHKHIYWTDSGNKTISVATVDGGRRRTLFSRNLSEPRAIAVDPL RGFMYWSDWGDQAKIEKSGLNGVDRQTLVSDNIEWPNGITLDLLSQRLYWVDSKLHQLSSIDFSG GNRKTLISSTDFLSHPFGIAVFEDKVFWTDLENEAIFSANRLNGLEISILAENLNNPHDIVIFHELKQP RAPDACELSVQPNGGCEYLCLPAPQISSHSPKYTCACPDTMWLGPDMKRCYRAPQSTSTTTLAS TMTRTVPATTRAPGTTVHRSTYQNHSTETPSLTAAVPSSVSVPRAPSISPSTLSPATSNHSQHYAN EDSKMGSTVTAAVIGIIVPGGAVGDRCERNEFQCQDGKCISYKWVCDGSAECQDGSDESQETCL SVTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGCPPKTCSQDEFRCHDGKCISRQFV CDSDRDCLDGSDEASCPVLTCGPASFQCNSSTCIPQLWACDNDPDCEDGSDEWPQRCRGLYVF QGDSSPCSAFEFHCLSGECIHSSWRCDGGPDCKDKSDEENCAVATCRPDEFQCSDGNCIHGSR QCDREYDCKDMSDEVGCVNVTLCEGPNKFKCHSGECITLDKVCNMARDCRDWSDEPIKECGTN ECLDNNGGCSHVCNDLKIGYECLCPDGFQLVAQRRCEDIDECQDPDTCSQLCVNLEGGYKCQCE EGFQLDPHTKACKAVGSIAYLFFTNRHEVRKMTLDRSEYTSLIPNLRNVVALDTEVASNRIYWSDL SQRMICSTQLDRAHGVSSYDTVISRDIQAPDGLAVDWIHSNIYWTDSVLGTVSVADTKGVKRKTLF RENGSKPRAIVVDPVHGFMYWTDWGTPAKIKKGGLNGVDIYSLVTENIQWPNGITLDLLSGRLYW VDSKLHSISSIDVNGGNRKTILEDEKRLAHPFSLAVFEDKVFWTDIINEAIFSANRLTGSDVNLLAEN LLSPEDMVLFHNLTQPRGVNWCERTTLSNGGCQYLCLPAPQINPHSPKFTCACPDGMLLARDMR SCLTEAEAAVATQETSTVRLKVSSTAVRTQHTTTRPVPDTSRLPGATPGLTTVEIVTMSHQALGDV AGRGNEKKPSSVRGDASDYKDDDDKLEVLFQGPGHHHHHHHH*
Nucleotide Sequence: sVLDLR-sLDLR (SEQ ID NO 221)
ATGGGACCATGGGGATGGAAGCTGCGCTGGACTGTGGCCCTTCTGCTTGCGGCGGCAGGAA CcgcaGTGGGTGACCGGGGCAGCGGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGG CGGATccggtGGGAGAAAAGCCAAATGTGAACCCTCCCAATTCCAGTGCACAAATGGTCGCTGT ATTACGCTGTTGTGGAAATGTGATGGGGATGAAGACTGTGTTGACGGCAGTGATGAAAAGAAC TGTGTAAAGAAGACGTGTGCTGAATCTGACTTCGTGTGCAACAATGGCCAGTGTGTTCCCAGC CGATGGAAGTGTGATGGAGATCCTGACTGCGAAGATGGTTCAGATGAAAGCCCAGAACAGTG CCATATGAGAACATGCCGCATACATGAAATCAGCTGTGGCGCCCATTCTACTCAGTGTATCCC AGTGTCCTGGAGATGTGATGGTGAAAATGATTGTGACAGTGGAGAAGATGAAGAAAACTGTG GCAATATAACATGTAGTCCCGACGAGTTCACCTGCTCCAGTGGCCGCTGCATCTCCAGGAACT TTGTATGCAATGGCCAGGATGACTGCAGCGATGGCAGTGATGAGCTGGACTGTGCCCCGCCA ACCTGTGGCGCCCATGAGTTCCAGTGCAGCACCTCCTCCTGCATCCCCATCAGCTGGGTATG CGACGATGATGCAGACTGCTCCGACCAATCTGATGAGTCCCTGGAGCAGTGTGGCCGTCAGC CAGTCATACACACCAAGTGTCCAGCCAGCGAAATCCAGTGCGGCTCTGGCGAGTGCATCCAT AAGAAGTGGCGATGTGATGGGGACCCTGACTGCAAGGATGGCAGTGATGAGGTCAACTGTCC CTCTCGAACTTGCCGACCTGACCAATTTGAATGTGAGGATGGCAGCTGCATCCATGGCAGCA GGCAGTGTAATGGTATCCGAGACTGTGTCGATGGTTCCGATGAAGTCAACTGCAAAAATGTCA ATCAGTGCTTGGGCCCTGGAAAATTCAAGTGCAGAAGTGGAGAATGCATAGATATCAGCAAAG TATGTAACCAGGAGCAGGACTGCAGGGACTGGAGTGATGAGCCCCTGAAAGAGTGTCATATA AACGAATGCTTGGTAAATAATGGTGGATGTTCTCATATCTGCAAAGACCTAGTTATAGGCTACG AGTGTGACTGTGCAGCTGGGTTTGAACTGATAGATAGGAAAACCTGTGGAGATATTGATGAAT GCCAAAATCCAGGAATCTGCAGTCAAATTTGTATCAACTTAAAAGGCGGTTACAAGTGTGAAT GTAGTCGTGGCTATCAAATGGATCTTGCTACTGGCGTGTGCAAGGCAGTAGGCAAAGAGCCA
AGTCTGATCTTCACTAATCGAAGAGACATCAGGAAGATTGGCTTAGAGAGGAAAGAATATATC
CAACTAGTTGAACAGCTAAGAAACACTGTGGCTCTCGATGCTGACATTGCTGCCCAGAAACTA
TTCTGGGCCGATCTAAGCCAAAAGGCTATCTTCAGTGCCTCAATTGATGACAAGGTTGGTAGA
CATGTTAAAATGATCGACAATGTCTATAATCCTGCAGCCATTGCTGTTGATTGGGTGTACAAGA
CCATCTACTGGACTGATGCGGCTTCTAAGACTATTTCAGTAGCTACCCTAGATGGAACCAAGA
GGAAGTTCCTGTTTAACTCTGACTTGCGAGAGCCTGCCTCCATAGCTGTGGACCCACTGTCTG
GCTTTGTTTACTGGTCAGACTGGGGTGAACCAGCTAAAATAGAAAAAGCAGGAATGAATGGAT
TCGATAGACGTCCACTGGTGACAGCGGATATCCAGTGGCCTAACGGAATTACACTTGACCTTA
TAAAAAGTCGCCTCTATTGGCTTGATTCTAAGTTGCACATGTTATCCAGCGTGGACTTGAATGG
CCAAGATCGTAGGATAGTACTAAAGTCTCTGGAGTTCCTAGCTCATCCTCTTGCACTAACAATA
TTTGAGGATCGTGTCTACTGGATAGATGGGGAAAATGAAGCAGTCTATGGTGCCAATAAATTC
ACTGGATCAGAGCTAGCCACTCTAGTCAACAACCTGAATGATGCCCAAGACATCATTGTCTAT
CATGAACTTGTACAGCCATCAGGTAAAAATTGGTGTGAAGAAGACATGGAGAATGGAGGATGT
GAATACCTATGCCTGCCAGCACCACAGATTAATGATCACTCTCCAAAATATACCTGTTCCTGTC
CCAGTGGGTACAATGTAGAGGAAAATGGCCGAGACTGTCAAAGTACTGCAACTACTGTGACTT
ACAGTGAGACAAAAGATACGAACACAACAGAAATTTCAGCAACTAGTGGACTAGTTCCTGGAG
GGATCAATGTGACCACAGCAGTATCAGAGGTCAGTGTTCCCCCAAAAGGGACTTCTGGCGGA
GCAGTAGGCGACAGATGCGAGCGTAACGAATTCCAATGCCAAGACGGTAAGTGTATTAGTTA
CAAATGGGTTTGTGACGGCAGTGCCGAATGCCAAGACGGTTCGGATGAATCTCAGGAAACGT
GTCTGTCGGTAACCTGCAAATCGGGAGACTTCAGCTGTGGTGGACGGGTTAACCGCTGTATA
CCTCAGTTTTGGCGGTGTGACGGACAAGTTGACTGTGATAATGGCTCAGATGAGCAGGGGTG
CCCGCCAAAGACCTGCAGTCAGGACGAATTCCGATGCCACGATGGAAAATGTATTAGCCGTC
AATTCGTTTGTGACTCGGACCGCGACTGTCTGGATGGTTCAGATGAAGCTTCGTGCCCAGTAT
TAACTTGCGGACCGGCGAGTTTTCAATGCAATTCCAGCACGTGTATTCCCCAACTCTGGGCTT
GCGACAACGACCCGGATTGCGAGGACGGCTCTGACGAGTGGCCGCAGAGATGCCGTGGGTT
ATACGTGTTTCAAGGAGACAGCTCACCGTGCAGCGCTTTCGAGTTTCATTGTCTTAGCGGCGA
GTGTATCCACTCATCATGGCGCTGCGATGGTGGGCCTGATTGTAAGGACAAGAGCGACGAGG
AGAATTGTGCTGTTGCCACGTGTCGACCAGACGAATTTCAATGTAGCGACGGTAATTGTATCC
ATGGATCTAGACAATGCGACCGTGAATACGATTGTAAAGACATGTCCGACGAGGTTGGCTGTG
TGAATGTGACTTTGTGCGAGGGACCGAATAAGTTTAAGTGCCACTCAGGCGAATGCATAACAC
TTGACAAAGTGTGTAACATGGCACGCGACTGCCGTGATTGGTCAGACGAGCCAATAAAGGAA
TGCGGTACCAACGAGTGCCTGGACAACAATGGCGGCTGTAGTCATGTTTGCAATGACCTTAA
GATCGGTTACGAGTGTCTCTGCCCAGATGGTTTCCAGTTGGTTGCTCAACGCCGATGCGAAG
ACATAGACGAATGCCAAGATCCGGACACATGCTCCCAACTGTGCGTCAATTTGGAAGGCGGA
TACAAGTGCCAATGCGAGGAAGGCTTCCAGTTGGATCCGCACACGAAGGCCTGCAAAGCGGT
GGGTAGTATTGCTTACTTATTCTTCACGAACCGACACGAAGTTCGGAAAATGACTTTGGATAGA
TCCGAGTACACATCACTTATACCTAACCTTCGTAATGTAGTCGCACTTGATACCGAGGTAGCAA
GTAACCGCATCTATTGGTCTGACTTGTCCCAGCGCATGATATGTTCTACTCAATTGGATCGTG
CACACGGAGTTAGCTCGTATGACACCGTAATATCACGTGACATACAGGCACCGGATGGCCTG
GCGGTTGACTGGATTCATAGCAATATTTACTGGACAGACTCCGTGCTGGGCACAGTGAGTGTT
GCTGATACGAAAGGGGTTAAACGTAAGACACTGTTTCGGGAAAATGGTAGCAAGCCGCGCGC
TATAGTGGTTGACCCGGTACATGGATTCATGTACTGGACGGACTGGGGTACACCAGCCAAGA
TCAAGAAGGGTGGTCTGAACGGAGTCGACATATACAGCCTGGTCACGGAGAATATACAGTGG
CCGAACGGGATTACTTTGGACCTTCTGAGTGGACGCTTGTACTGGGTGGATAGCAAGCTGCA
TTCTATCTCCTCTATTGATGTGAACGGTGGAAACCGGAAGACTATATTGGAAGATGAGAAGAG ATTGGCCCACCCGTTCTCTCTCGCGGTCTTTGAGGACAAGGTATTCTGGACGGACATAATTAA CGAGGCTATCTTCTCTGCCAACCGTCTGACTGGCTCTGACGTGAATTTACTGGCGGAGAATTT GCTGAGTCCTGAGGATATGGTATTGTTTCACAACTTAACGCAACCGCGGGGCGTGAACTGGT GTGAGCGTACGACGTTAAGCAACGGTGGCTGTCAGTACCTGTGTTTACCTGCGCCGCAAATA AATCCTCACTCTCCGAAGTTTACCTGCGCTTGTCCAGACGGCATGCTTCTTGCCCGCGACATG CGTTCATGCTTAACGGAAGCTGAAGCTGCCGTTGCTACACAAGAAACGAGCACCGTGAGATTA AAGGTGAGCTCAACCGCCGTCCGCACACAACACACCACAACACGCCCTGTCCCGGACACCTC GCGGTTACCGGGTGCGACCCCCGGATTAACCACCGTTGAGATCGTAACCATGTCACACCAAG CCCTCGGCGATGTGGCTGGCCGTGGCAATGAGAAGAAACCTTCCAGTGTCCGGGGAGACGC TTCGGATtacaaagacgatgacgataagCTGGAAGTTCTGTTCCAGGGGCCCGGTCATCACCACCATC ATCACCATCACTAA
Amino Acid Sequence: sVLDLR-sLDLR (SEQ ID NO 222)
MGPWGWKLRWTVALLLAAAGTAVGDRGSGVSGWRLFKKISGGSGGRKAKCEPSQFQCTNGRCI TLLWKCDGDEDCVDGSDEKNCVKKTCAESDFVCNNGQCVPSRWKCDGDPDCEDGSDESPEQC HMRTCRIHEISCGAHSTQCIPVSWRCDGENDCDSGEDEENCGNITCSPDEFTCSSGRCISRNFVC NGQDDCSDGSDELDCAPPTCGAHEFQCSTSSCIPISWVCDDDADCSDQSDESLEQCGRQPVIHT KCPASEIQCGSGECIHKKWRCDGDPDCKDGSDEVNCPSRTCRPDQFECEDGSCIHGSRQCNGIR DCVDGSDEVNCKNVNQCLGPGKFKCRSGECIDISKVCNQEQDCRDWSDEPLKECHINECLVNNG GCSHICKDLVIGYECDCAAGFELIDRKTCGDIDECQNPGICSQICINLKGGYKCECSRGYQMDLAT GVCKAVGKEPSLIFTNRRDIRKIGLERKEYIQLVEQLRNTVALDADIAAQKLFWADLSQKAIFSASID DKVGRHVKMIDNVYNPAAIAVDWVYKTIYWTDAASKTISVATLDGTKRKFLFNSDLREPASIAVDPL SGFVYWSDWGEPAKIEKAGMNGFDRRPLVTADIQWPNGITLDLIKSRLYWLDSKLHMLSSVDLNG QDRRIVLKSLEFLAHPLALTIFEDRVYWIDGENEAVYGANKFTGSELATLVNNLNDAQDIIVYHELVQ PSGKNWCEEDMENGGCEYLCLPAPQINDHSPKYTCSCPSGYNVEENGRDCQSTATTVTYSETKD TNTTEISATSGLVPGGINVTTAVSEVSVPPKGTSGGAVGDRCERNEFQCQDGKCISYKWVCDGSA ECQDGSDESQETCLSVTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGCPPKTCSQD EFRCHDGKCISRQFVCDSDRDCLDGSDEASCPVLTCGPASFQCNSSTCIPQLWACDNDPDCEDG SDEWPQRCRGLYVFQGDSSPCSAFEFHCLSGECIHSSWRCDGGPDCKDKSDEENCAVATCRPD
EFQCSDGNCIHGSRQCDREYDCKDMSDEVGCVNVTLCEGPNKFKCHSGECITLDKVCNMARDC RDWSDEPIKECGTNECLDNNGGCSHVCNDLKIGYECLCPDGFQLVAQRRCEDIDECQDPDTCSQ LCVNLEGGYKCQCEEGFQLDPHTKACKAVGSIAYLFFTNRHEVRKMTLDRSEYTSLIPNLRNWAL DTEVASNRIYWSDLSQRMICSTQLDRAHGVSSYDTVISRDIQAPDGLAVDWIHSNIYWTDSVLGTV SVADTKGVKRKTLFRENGSKPRAIVVDPVHGFMYWTDWGTPAKIKKGGLNGVDIYSLVTENIQWP NGITLDLLSGRLYWVDSKLHSISSIDVNGGNRKTILEDEKRLAHPFSLAVFEDKVFWTDIINEAIFSAN RLTGSDVNLLAENLLSPEDMVLFHNLTQPRGVNWCERTTLSNGGCQYLCLPAPQINPHSPKFTCA CPDGMLLARDMRSCLTEAEAAVATQETSTVRLKVSSTAVRTQHTTTRPVPDTSRLPGATPGLTTV EIVTMSHQALGDVAGRGNEKKPSSVRGDASDYKDDDDKLEVLFQGPGHHHHHHHH*
Nucleotide Sequence HiBiT-Tag: GTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC (SEQ ID NO 223)
Amino acid sequence HiBiT-Tag: VSGWRLFKKIS (SEQ ID NO 224) s: soluble (receptor ectodomain without the transmembrane anchor) *: stop codon encoded by the last triplet of the nucleotide sequence. Example 9: A phage-display approach to identify high affinity binding LDLR fragments and variants thereof to viral glycoproteins in a high-throughput format (Fig. 10).
A viral glycoprotein of interest (preferred from Bunyaviridae, more preferred from CCHFV) is expressed, purified, and immobilized on a surface.
A DNA pool encoding LDLR family fragments, and single and multiple amino acid variants thereof is cloned into a phagemid, i.e. an expression vector that expresses the cloned peptides as part of a bacteriophage envelope protein.
The phagemids are transfected into bacteria cells to produces phages, wherein each phage displays a different peptide or set or peptides on its surface, wherein each peptide is a different LDLR fragment or an amino acid variant thereof.
Display phages are incubated on the immobilized glycoprotein and will attach in dependence of the interaction strength between glycoprotein and LDLR fragment on phages.
Weak binders are washed away.
Strong binders are eluted and will be sequenced to identify highest affinity binders. If the enrichment is not strong enough use the eluted phage to infect bacteria and amplify the selected pool for a 2nd or 3rd round of enrichment, then sequence for identification
These experiments are performed using standard and mutator bacteria strains. The latter produce a lot of point mutations allowing to generate fragments with multiple amino acid variants that potentially confer stronger binding to the target glycoprotein.
Example 10: Immunofluorescence stains confirm requirement of LDLR for CCHFV infections.
Immunofluorescence data of wild-type and LDLR KO SW13 cells infected with CCHFV are presented in Fig. 11. Knockout of LDLR as well as treatment with a soluble receptor decoy strongly decrease infection rates as visualized by immunofluorescence staining.
Example 11: LDL outcompetes CCHFV for cell entry via the LDLR.
We demonstrated that CCHFV glycoproteins can bind to LDLR. To validate this interaction for the virus, a competition assay using CCHFV and LDL, the natural ligand for LDLR, was conducted. As depicted in Fig. 12, LDL effectively competed with CCHFV infection in a dose-dependent manner for cell entry via LDLR. Bovine Serum Albumin (BSA) was used as a control and showed no impact on CCHFV infection. Example 12: LDLR and LRP8 decrease CCHFV infection in a patient context.
To investigate the involvement of LDLR and the closely related LRP8 in natural infections, CCHFV lbAr10200 was initially cultured on a Hyalomma tick cell line, the natural vector, and the potential inhibitory effects of sLDLR and sLPR8 were evaluated. As anticipated, sLDLR demonstrated the ability to block the virus, while sLRP8 did not (Fig. 13a). The same applies for a patient isolate that was cultivated on tick cells (see Example 7). Subsequently, the capacity of LDLR and LRP8 to block CCHFV was examined using virus present in patient serum without prior propagation on tick or human cells. As illustrated in Fig. 13b, both sLDLR and sLRP8 were effective in blocking the virus from this direct patient serum. These results underscore the importance of LDLR as a critical receptor during both the transmission of the virus from ticks to humans as well as the virus spreading throughout the human body. Further, the data show that in the human context, LDLR family members like LRP8 can also interfere with virus spreading, potentially due to an altered protein decoration of the virus as compared to virus propagated in vitro.
Example 13: Quality control of Bunyavirus vaccines.
LDLR is used to assess Bunyavirus vaccine integrity or affinity of antigens for diagnostic purposes and especially to assess CCHFV vaccine integrity or affinity of antigens for diagnostic purposes.
The following example shows that LDLR or LDLR fusion proteins or LDLR variants can be used to assess a bunyavirus vaccine, preferably a CCHFV vaccine, quality or the integrity of a diagnostic antigen, e.g. from patient samples.
Because LDLR is a receptor that directly interacts with multiple bunyaviruses, such as CCHFV, it can be used to physically assess the quality and integrity of a bunyavirus vaccine, which is important to define lot-to-lot variation in commercial production. Further, LDLR can be used to detect diagnostic antigens, e.g. in a lateral flow immunoassay.
Bunyavirus vaccine or diagnostic antigens can include, but are not limited to, live viruses from patient samples, live-attenuated viruses, virus-like particles, viral structural proteins, viral glycoproteins, or nucleic acids or vectors producing these viral proteins or particles. The format of the integrity assay can include immobilizing LDLR or LDLR fusion proteins (e.g. LDLR-Fc), or LDLR variants on a surface (e.g., microtiter well plate, chip, or slide) followed by incubation with the bunyavirus vaccine or diagnostic antigen and detection with a monoclonal or polyclonal anti-vaccine I anti- diagnostic antigen antibody. Optionally detection can be achieved via LDLR-Fc itself.
Alternatively, the bunyavirus vaccine or diagnostic antigen can be captured on the surface (directly or indirectly) and then incubated with LDLR or an LDLR fusion protein or an LDLR variant for binding analysis. The assay could be performed by detection methods suitable to assess binding including, but not limited to ELISA, biolayer interferometry and surface plasmon resonance.

Claims

Claims
1 . An LDLR derived polypeptide for use in the prophylaxis and/or treatment of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain.
2. The LDLR derived polypeptide for use of claim 1 , wherein said LDLR derived polypeptide comprises an LDLR ectodomain having amino acid sequence SEQ ID NO 18, or a functional fragment thereof, or an amino acid variant thereof.
3. The LDLR derived polypeptide for use of claim 1 , wherein said LDLR derived polypeptide comprises a dimer, a trimer, or a multimer of an LDLR ectodomain monomer having amino acid sequence SEQ ID NO 18, or of a functional fragment thereof, or of an amino acid variant thereof.
4. The LDLR derived polypeptide for use of any one of the claims 1 - 3, wherein said LDLR derived polypeptide comprises a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41 ), OLSD (SEQ ID NO 36), LA1-LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA1 - LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA1 -LA2-LA3-LA4X- LA5-LA6X-LA7-EGFA-EGFB, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7, LA1 -LA2-LA3-LA4X-LA5-LA6, LA1 -LA2- LA3-LA4X-LA5, LA1 -LA2-LA3-LA4, LA1-LA2-LA3, LA1-LA2, LA2-LA3-LA4X- LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA2-LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA2-LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFBX-PROP, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA2- LA3-LA4X-LA5-LA6X-LA7-EGFA, LA2-LA3-LA4X-LA5-LA6X-LA7, LA2-LA3- LA4X-LA5-LA6, LA2-LA3-LA4X-LA5, LA2-LA3-LA4, LA2-LA3, LA3-LA4X- LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA3-LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROP, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA3-LA4X-LA5- LA6X-LA7-EGFA, LA3-LA4X-LA5-LA6X-LA7, LA3-LA4X-LA5-LA6, LA3- LA4X-LA5, LA3-LA4, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROP, LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA4X- LA5-LA6X-LA7-EGFA, LA4X-LA5-LA6X-LA7, LA4X-LA5-LA6, LA4X-LA5, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA5-LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA5- LA6X-LA7-EGFA-EGFB, LA5-LA6X-LA7-EGFA, LA5-LA6X-LA7, LA5-LA6, LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA6X-LA7-EGFA-EGFBX- PROPX-EGFC, LA6X-LA7-EGFA-EGFBX-PROP, LA6X-LA7-EGFA-EGFB, LA6X-LA7-EGFA, LA6X-LA7, LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA7-EGFA-EGFBX-PROPX-EGFC, LA7-EGFA-EGFBX-PROP, LA7-EGFA- EGFB, LA7-EGFA, EGFA-EGFBX-PROPX-EGFCX-OLSD, EGFA-EGFBX- PROPX-EGFC, EGFA-EGFBX-PROP, EGFA-EGFB, EGFBX-PROPX- EGFCX-OLSD, EGFBX-PROPX-EGFC, EGFBX-PROP, or an amino acid variant thereof, or a homomultimer thereof.
5. The LDLR derived polypeptide for use of any one of the claims 1 - 4, wherein said LDLR derived polypeptide further comprises a peptide linker connecting said fragments or monomers.
6. The LDLR derived polypeptide for use of any one of the claims 1 - 5, wherein said Bunyavirales virus is selected from the group comprising or consisting of Crimean-Congo Haemorrhagic Fever Virus (CCHFV), Hantavirus, Andes hantavirus (ANDV), Black Creek Canal hantavirus virus (BCCV), Dobrava- Belgrade hantavirus (DOBV), Haantan virus (HTNV), Laguna Negra hantavirus (LANV), Longquan hantavirus (LQUV), Puumala hantavirus (PUUV), Sangassou hantavirus (SANGV), Seoul hantavirus (SEOV), Sin Nombre hantavirus (SNV), Thailand hantavirus (THAIV), Tula hantavirus (TULV), New York hantavirus (NYV), Rift Valley fever virus (RVFV), Nairobi sheep virus (NSDV), Severe fever with thrombocytopenia syndrome virus (SFTSV) or Sandfly Fever Naples virus (SFNV).
7. The LDLR derived polypeptide for use of claim 6, wherein said Bunyavirales virus is CCHFV.
8. The LDLR derived polypeptide for use of claim 7, wherein said LDLR derived polypeptide comprises an LDLR ectodomain having amino acid sequence SEQ ID NO 18, and wherein said Bunyavirales virus is CCHFV.
9. A pharmaceutical composition comprising the LDLR derived polypeptide for use according to any one of the claims 1 - 8, together with at least one pharmaceutically acceptable vehicle, excipient and/or diluent.
10. A recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an LDLR derived polypeptide for use according to any one of the claims 1 - 8.
11. An LDLR derived polypeptide comprising a functional fragment of an LDLR ectodomain selected from the group comprising LA1 (SEQ ID NO 19), LA2 (SEQ ID NO 20), LA3 (SEQ ID NO 21 ), LA4 (SEQ ID NO 22), LA4X (SEQ ID NO 37), LA5 (SEQ ID NO 23), LA6 (SEQ ID NO 24), LA6X (SEQ ID NO 38), LA7 (SEQ ID NO 25), EGFA (SEQ ID NO 26), EGFB (SEQ ID NO 27), EGFBX (SEQ ID NO 39), PROP (SEQ ID NO 34), PROPX (SEQ ID NO 40), EGFC (SEQ ID NO 35), EGFCX (SEQ ID NO 41 ), OLSD (SEQ ID NO 36), LA1-LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA1 - LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA1 -LA2-LA3-LA4X- LA5-LA6X-LA7-EGFA-EGFB, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA, LA1 -LA2-LA3-LA4X-LA5-LA6X-LA7, LA1 -LA2-LA3-LA4X-LA5-LA6, LA1 -LA2- LA3-LA4X-LA5, LA1 -LA2-LA3-LA4, LA1-LA2-LA3, LA1-LA2, LA2-LA3-LA4X- LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA2-LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA2-LA3-LA4X-LA5-LA6X-LA7- EGFA-EGFBX-PROP, LA2-LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA2- LA3-LA4X-LA5-LA6X-LA7-EGFA, LA2-LA3-LA4X-LA5-LA6X-LA7, LA2-LA3- LA4X-LA5-LA6, LA2-LA3-LA4X-LA5, LA2-LA3-LA4, LA2-LA3, LA3-LA4X- LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA3-LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA3-LA4X-LA5-LA6X-LA7-EGFA- EGFBX-PROP, LA3-LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA3-LA4X-LA5- LA6X-LA7-EGFA, LA3-LA4X-LA5-LA6X-LA7, LA3-LA4X-LA5-LA6, LA3- LA4X-LA5, LA3-LA4, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX- OLSD, LA4X-LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFC, LA4X-LA5- LA6X-LA7-EGFA-EGFBX-PROP, LA4X-LA5-LA6X-LA7-EGFA-EGFB, LA4X- LA5-LA6X-LA7-EGFA, LA4X-LA5-LA6X-LA7, LA4X-LA5-LA6, LA4X-LA5, LA5-LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA5-LA6X-LA7- EGFA-EGFBX-PROPX-EGFC, LA5-LA6X-LA7-EGFA-EGFBX-PROP, LA5- LA6X-LA7-EGFA-EGFB, LA5-LA6X-LA7-EGFA, LA5-LA6X-LA7, LA5-LA6, LA6X-LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA6X-LA7-EGFA-EGFBX- PROPX-EGFC, LA6X-LA7-EGFA-EGFBX-PROP, LA6X-LA7-EGFA-EGFB, LA6X-LA7-EGFA, LA6X-LA7, LA7-EGFA-EGFBX-PROPX-EGFCX-OLSD, LA7-EGFA-EGFBX-PROPX-EGFC, LA7-EGFA-EGFBX-PROP, LA7-EGFA- EGFB, LA7-EGFA, EGFA-EGFBX-PROPX-EGFCX-OLSD, EGFA-EGFBX- PROPX-EGFC, EGFA-EGFBX-PROP, EGFA-EGFB, EGFBX-PROPX- EGFCX-OLSD, EGFBX-PROPX-EGFC, EGFBX-PROP, or amino acid variants of said functional fragment, and wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said fragments.
12. An LDLR derived polypeptide comprising a multimer of monomers, wherein each monomer is selected from the group comprising: an LDLR ectodomain (SEQ ID NO 18), a functional fragment of said LDLR ectodomain, an amino acid variant of said LDLR ectodomain, an amino acid variant of said functional fragment of said LDLR ectodomain, and wherein said multimer comprises between 2 and 8 monomers, and wherein said LDLR derived polypeptide optionally comprises a peptide linker connecting said monomers; and wherein said multimer is a homomultimer or a heteromultimer.
13. An LDLR derived polypeptide according to claim 11 or 12 for use in the prophylaxis and/or treatment of a virus infection, wherein said virus is preferably a Bunyavirales virus.
14. A recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an LDLR derived polypeptide according to any of claims 11 - 13.
15. An LDLR derived polypeptide for use in the diagnosis of a Bunyavirales virus infection, wherein said LDLR derived polypeptide binds to said Bunyavirales virus, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain according to claim 11 or 12 and wherein said binding diagnoses the infection by the Bunyavirales virus.
16. An LDLR derived polypeptide for use in quality control of a Bunyavirales virus vaccine, wherein said LDLR derived polypeptide binds to vaccine ingredients, and wherein said LDLR derived polypeptide comprises an LDLR ectodomain or a functional fragment of an LDLR ectodomain or an amino acid variant of an LDLR ectodomain according to claim 11 or 12 and wherein said binding assesses the quality of that vaccine to prevent or ameliorate a Bunyavirales virus infection.
17. The LDLR derived polypeptide for use according to claim 15, wherein the
Bunyavirales virus is CCHFV or according to claim 16, wherein the Bunyavirales virus vaccine is a CCHFV vaccine.
PCT/EP2024/064152 2023-05-23 2024-05-22 Ldlr derived polypeptides for anti-viral uses Pending WO2024240848A1 (en)

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