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WO2025186660A1 - Protéines de spicule mutées en tant que vaccins contre le sars-cov-2 - Google Patents

Protéines de spicule mutées en tant que vaccins contre le sars-cov-2

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Publication number
WO2025186660A1
WO2025186660A1 PCT/IB2025/052041 IB2025052041W WO2025186660A1 WO 2025186660 A1 WO2025186660 A1 WO 2025186660A1 IB 2025052041 W IB2025052041 W IB 2025052041W WO 2025186660 A1 WO2025186660 A1 WO 2025186660A1
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seq
cov
sars
protein
nucleic acid
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WO2025186660A8 (fr
Inventor
Rino Rappuoli
Emanuele ANDREANO
Giuseppe Maccari
Giulia REALINI
Piero Pileri
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Fondazione Toscana Life Sciences
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Fondazione Toscana Life Sciences
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to second-generation vaccines against COVID-19 to abrogate or diminish the binding of the SARS-CoV-2 spike (S) protein, or part of it, to the human angiotensin-converting enzyme 2 (hACE2).
  • S SARS-CoV-2 spike
  • hACE2 human angiotensin-converting enzyme 2
  • Such vaccines present several advantages as to avoid pathways of immune dysregulation activated following S protein/hACE2 interaction while maintaining the ability to elicit a strong and robust antibody neutralization response to SARS-CoV-2.
  • the invention relates also to the use of such vaccines in the prevention or treatment of SARS-CoV-2 infection or conditions or disorders resulting from such infection.
  • Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible and pathogenic coronavirus that emerged in late 2019 and has caused a pandemic of acute respiratory disease, named ‘coronavirus disease 2019’ (CO VID-19), which threatens human health and public safety.
  • coronavirus disease 2019 (CO VID-19)
  • WHO World Health Organization
  • SARS-CoV- 2 infection starts with the interaction between the viral glycoprotein spike (S) and the human angiotensin-converting enzyme 2 (hACE2).
  • the S protein attaches hACE2 on the host cell through the receptor binding domain (RBD) positioned in the SI domain.
  • RBD receptor binding domain
  • TMPRSS2 receptor binding domain
  • TMPRSS2 TMPRSS2 to cleave the S protein, leading to activation of the S2 domain and consequent fusion of the viral membrane with the host cell membrane.
  • hACE2 was previously considered only as a component of the renin-angiotensin system, recent reports highlight the widespread distribution of hACE2 in different tissues and organs and its possible contribution to extrapulmonary manifestations of COVID-19 (Baldari et al.
  • SARS-CoV-2 Spike-ACE2 Emerging roles of SARS-CoV-2 Spike-ACE2 in immune evasion and pathogenesis. Trends in Immunology 44, 424-434, 2023). Among the different pathways that involve hACE2, recent findings have shown that SARS-CoV-2 S protein exploits hACE2 signaling to suppress immunological synapse assembly and CD8 + cytotoxic T lymphocyte-mediated killing (Onnis et al. SARS-CoV-2 Spike protein suppresses CTL-mediated killing by inhibiting immune synapse assembly. Journal of Experimental Medicine 220, e20220906, 2022). Similar effects could also be used to subvert different activities of the innate and adaptive immune response highlighting a key role of the S protein/hACE2 axis in the SARS- CoV-2 immune evasion.
  • the present invention relates to second-generation vaccines against COVID-19 to abrogate or diminish the binding of the SARS-CoV-2 S protein, or part of it, to ACE2, in particular the human ACE2.
  • Such vaccines present several advantages as to avoid pathways of immune dysregulation activated following S protein/hACE2 interaction while maintaining the ability to elicit a strong and robust antibody neutralization response to SARS-CoV-2.
  • current vaccines are based on the wild-type sequence of SARS-CoV-2 S protein allowing the binding of this protein to hACE2. This interaction has been associated with mechanisms of immune evasion which can exacerbate the severity of CO VID-19.
  • the in silico prediction data were experimentally validated, while for the remaining thirty sequences only data on the prediction of loss of binding and stability were evaluated.
  • the seven S protein mutants experimentally validated have been all recombinantly expressed through CHO transient transfection for in vitro evaluation. Specifically, the inventors performed two different ELISA assays to characterize the antigens. The first ELISA was used to confirm that the quaternary structure of the newly generated S protein mutants was not modified by the introduction of selected mutations.
  • mAbs monoclonal antibodies
  • the second ELISA allowed us to evaluate the binding activity of our new mutated S proteins to hACE2.
  • the results obtained by the inventors showed that produced mutated S proteins maintained their quaternary structure while showing up to 3.3 -fold binding reduction to hACE2 measured by ELISA.
  • the invention provides an immunogenic polypeptide comprising or consisting of a receptor-binding domain (RBD) of the SARS-CoV-2 spike protein wherein one or more of the residues of said receptor-binding domain (RBD) in positions 417, 449, 477, 487, 489, 493, 494, 500, 502, and 505 are mutated, wherein the numbering of said positions refers to SEQ ID NO: 1.
  • RBD receptor-binding domain
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes the immunogenic polypeptide according to any one of the embodiments herein disclosed and a vector comprising said nucleic acid molecule.
  • the invention provides an immunogenic composition
  • an immunogenic composition comprising the immunogenic polypeptide or the nucleic acid molecule or a vector according to any one of the embodiments herein disclosed, optionally comprising one or more adjuvants or excipients.
  • the invention provides the immunogenic polypeptides and immunogenic compositions herein disclosed for use in a prophylactic and/or therapeutic treatment of the SARS-CoV-2 infection or conditions or disorders resulting from such infection, in particular COVID-19 disease.
  • the invention provides the immunogenic polypeptides and immunogenic compositions herein disclosed for use as a vaccine against SARS-CoV-2 infection.
  • Fig- 1 Conservation analysis of the RBD domain in the SARS-CoV-2 S protein. Over 11 million sequences were downloaded from GISAID and aligned to identify the frequency of mutation of each residue composing the SARS-CoV-2 RBD domain. hACE2 interacting residues are highlighted in purple, the Receptor Binding Motif (RBM) is highlighted in red. Fig. 2. Prediction results for the binding affinity (A) and the protein stability (B). Binding affinity measures the AAG score by comparing the WT free energy with the mutated one. In the same way, stability is measured by comparing the WT RBD monomer against the mutated monomer.
  • Fig. 4. ELISA results.
  • Fig- 5 Schematic representation of the SARS-CoV-2 S protein RBD. The illustration shows the four main anchors of interactions between SARS-CoV-2 RBD and hACE2 that were manually identified by the experts through three-dimensional visualization.
  • Fig- 6 Serum virus neutralization against SARS-CoV-2 Wuhan strain.
  • the graph shows the inhibition of virus infectivity in cell culture in the presence of serum neutralizing antibodies. Technical triplicates were performed for the experiment.
  • Fig. 7 ELISA results of Second-Round mutated SARS-CoV-2 S-proteins.
  • Graph shows the ability of mutated SARS-CoV-2 S-proteins to bind hACE2, and their binding fold-change reduction compared to corresponding WT SARS-CoV-2 S protein.
  • Technical triplicates were performed for the experiment. Mean and standard deviation are denoted on the graph.
  • polypeptide generally refer to a polymer of amino acid residues and are not limited to a minimum length of the product.
  • Polypeptides of the invention can be prepared in many wayse.g.by chemical synthesis (at least in part), by digesting longer polypeptides using proteases, by translation from RNA, by purification from cell culture (e.g. from recombinant expression), from the organism itself.
  • Biological methods are in general restricted to the production of polypeptides based on L- amino acids, but manipulation of translation machinery (e.g.
  • polypeptide encompasses native or artificial proteins, protein fragments and polypeptide analogues of a protein sequence, i.e. isolated or purified polypeptide.
  • a polypeptide may be monomeric or polymeric.
  • isolated protein is a protein, polypeptide that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature.
  • a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components.
  • a protein may also be rendered substantially free of naturally-associated components by isolation, using protein purification techniques well known in the art.
  • a protein or polypeptide is "substantially pure”, “substantially homogeneous”, or “substantially purified” when at least about 60 to 75% of a sample exhibits a single polypeptide.
  • the polypeptide or protein may be monomeric or multimeric.
  • a substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80% or 90% W/W of a protein sample, more usually about 95%, and preferably will be over 99% pure.
  • Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.
  • polypeptide fragment refers to a polypeptide that has an aminoterminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally occurring sequence.
  • SARS-CoV-2 refers to severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2), the type of coronavirus that causes coronavirus disease 2019 (COVID-19).
  • polynucleotide as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide.
  • the term includes single and double stranded forms.
  • isolated polynucleotide as used herein means a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated polynucleotide” (1) is not associated with all or a portion of a polynucleotides with which the "isolated polynucleotide” is found in nature, (2) is operably linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.
  • nucleic acid is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogues of the DNA or RNA generated using nucleotide analogues.
  • the nucleic acid can be single-stranded or doublestranded.
  • nucleotides as used herein includes deoxyribonucleotides and ribonucleotides.
  • modified nucleotides as used herein includes nucleotides with modified or substituted sugar groups and the like.
  • oligonucleotide linkages referred to herein includes oligonucleotides linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like. See e.g., LaPlanche et al., Nucl. Acids Res.
  • expression control sequence means polynucleotide sequences that are necessary to affect the expression and processing of coding sequences to which they are ligated.
  • Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion.
  • control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence.
  • control sequences is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • vector as used herein, means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a plasmid, i.e., a circular double stranded piece of DNA into which additional DNA segments may be ligated.
  • the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • the vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • the vectors e.g., non- episomal mammalian vectors
  • recombinant host cell means a cell into which a recombinant expression vector has been introduced. It should be understood that "recombinant host cell” and “host cell” mean not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
  • sequence identity in the context of nucleotide or aminoacidic sequences means the residues in two sequences that are the same when aligned for maximum correspondence.
  • the length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides.
  • polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs available, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132: 185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996); Pearson, J Mol. Biol 276:71-84 (1998); incorporated herein by reference).
  • nucleic acid or fragment thereof, or aminoacidic when referring to a nucleic acid or fragment thereof, or aminoacidic means that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.
  • the term "substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights as supplied with the programs, share at least 70%, 75% or 80% sequence identity, preferably at least 90% or 95% sequence identity, and more preferably at least 97%, 98% or 99% sequence identity.
  • residue positions that are not identical differ by conservative amino acid substitutions.
  • a "conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well- known to those of skill in the art. See, e.g., Pearson, Methods Mol. Biol. 243:307-31 (1994).
  • Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic- hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulphur-containing side chains: cysteine and methionine.
  • Conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.
  • a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al, Science 256: 1443-45 (1992), incorporated herein by reference.
  • a “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix. Sequence identity for polypeptides is typically measured using sequence analysis software.
  • Protein analysis software matches sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions.
  • GCG contains programs such as "Gap” and "Bestfit” which can be used with default parameters as specified by the programs to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof.
  • the expression “to reduce the binding to ACE2” means that the one or more mutations of the receptor-binding domain (RBD) are designed to diminish or decrease the binding of the polypeptide of the invention comprising or consisting of said mutated RBD, or part of it, to angiotensin-converting enzyme 2 (ACE2), in particular to human ACE2 (hACE2), with respect to a polypeptide comprising or consisting of a non-mutated or native RBD.
  • RBD receptor-binding domain
  • the binding activity of a polypeptide to ACE2 can be determined and characterized by means of any of the techniques or assays known to the person skilled in the art, such as, in particular, an ELISA assay.
  • the ELISA assay is performed as disclosed in paragraph 1.7 of the experimental section of the present specification.
  • immunogenic polypeptide or nucleic acid molecules or immunogenic compositions of the present invention provide the induction of an immune response in a subject or host (human or non-human) but they avoid the activation of ACE2 signalling thanks to their reduced binding to ACE2.
  • the selective induction of an immune response in a subject or host (human or non-human animal) by the immunogenic products described herein may be determined and characterized by methods described herein and routinely practiced in the art. These methods include in vivo assays, such as animal immunization studies. A number of in vitro assays, such as immunochemistry methods for detection and analysis of antibodies, including Western immunoblot analysis, ELISA, immunoprecipitation, radioimmunoassay, and the like, and combinations thereof. Other methods and techniques that may be used to analyse and characterize an immune response include neutralization assays (such as a plaque reduction assay or an assay that measures cytopathic effect (CPE) or any other neutralization assay practiced by persons skilled in the art). These and other assays and methods known in the art can be used to characterize immunogens and variants thereof.
  • CPE cytopathic effect
  • an "immunogenic composition” or “vaccine” or “vaccine formulation” as used herein refers to a composition that comprises an antigenic molecule, where administration of the composition to a subject results in the development in the subject of a humoral and/or a cellular immune response to the antigenic/immunogenic molecule of interest.
  • the immunogenic composition can be introduced directly into a recipient subject, such as by injection, inhalation, oral, intranasal or any other parenteral, mucosal, or transdermal (e.g., intra-rectally or intra-vaginally) route of administration.
  • a first aspect of the present invention provides an immunogenic polypeptide comprising or consisting of a receptor-binding domain (RBD) of the SARS-CoV-2 spike protein wherein one or more of the residues of said RBD in positions 417, 449, 477, 487, 489, 493, 494, 500, 502, and 505 are mutated, wherein the numbering of said positions refers to SEQ ID NO: 1.
  • said immunogenic polypeptide is mutated in at least 2, 3, or all said positions.
  • said one or more mutations in the RBD are capable to reduce the binding of said immunogenic polypeptide to ACE2, in particular to human ACE2, with respect to a polypeptide comprising or consisting of a non-mutated or native RBD.
  • the invention provides an immunogenic polypeptide comprising or consisting of the SARS-CoV-2 spike protein having SEQ ID NO: 1, wherein one or more residues of the receptor-binding domain (RBD) of said protein in positions 417, 449, 477, 487, 489, 493, 494, 500, 502, and 505 are mutated, wherein the numbering of said positions refers to said SEQ ID NO: 1.
  • RBD receptor-binding domain
  • one or more of said RBD residues are mutated in alanine or leucine or methionine or lysine or glycine or glutamine.
  • the invention provides an immunogenic polypeptide comprising or consisting of a RBD of the SARS-CoV-2 spike protein having one or more of the following mutations: K417A, K417L, K417Q, Y449K, Y449N, Y449W, Y449M, S477Q, S477Y, S477N, N487M, N487G, N487A, N487I, N487P, N487S, N487K, N487L, Y489K, Y489V, Y489R, Q493Y, Q493A, Q493W, Q493I, S494I, T500V, T500I, G502T, G502I, G502A, G502S, G502F, Y505K, Y505Q, and Y505G, wherein the numbering of said positions refers to said SEQ ID NO: 1.
  • the immunogenic polypeptide comprises a mutated RBD having at least 2, 3, or 4 of such mutations.
  • the immunogenic polypeptide comprises or consists of a RBD of the SARS-CoV-2 spike protein having one of the following groups of mutations: (1) N487M and Y505K; or
  • N487P N487P, Q493 Y, Y449N, and T500I; or (33) N487A, Q493W, Y449M, and G502S; or
  • N487I, Q493I, Y449W, and T500I wherein the numbering of said positions refers to said SEQ ID NO: 1.
  • the immunogenic polypeptide comprises or consists of a RBD of the SARS-CoV-2 spike protein, which comprises or consists of a sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID N0:8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40
  • immunogenic polypeptides and variants thereof may be produced synthetically or recombinantly.
  • the immunogenic polypeptides may be expressed from a polynucleotide that is operably linked to an expression control sequence, such as a promoter, in a nucleic acid expression construct.
  • an expression control sequence such as a promoter
  • they may be expressed in mammalian cells, yeast, bacteria, insect or other cells under the control of appropriate expression control sequences.
  • Cell-free translation systems may also be employed to produce such coronavirus proteins using nucleic acids, including RNAs, and expression constructs.
  • Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are routinely used by persons skilled in the art and are described, for example, by Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989) and Third Edition (2001), and may include plasmids, cosmids, shuttle vectors, viral vectors, and vectors comprising a chromosomal origin of replication as disclosed therein.
  • nucleic acid molecules in certain aspects, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes the immunogenic polypeptide according to any one of the embodiments herein disclosed.
  • said isolated nucleic acid molecule comprises or consists of a nucleic acid sequence selected from SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
  • said nucleic acid molecules are DNA or mRNA sequences.
  • RNA instead of DNA for genetic vaccination, the risk of undesired genomic integration and generation of anti- DNA antibodies is minimized or avoided.
  • said mRNA is within lipid nanoparticles.
  • Lipid nanoparticles formed from cationic lipids with other lipid components have been used to block degradation of the RNAs in plasma and facilitate the cellular uptake of the oligonucleotides.
  • Procedures to manufacture said mRNA vaccine are known in the art see for example in US 10,576,146, US 10,485,884, and US 9,950,065 herein incorporated by reference.
  • the invention provides a vector comprising a nucleic acid molecule coding the immunogenic polypeptide according to any of the embodiments herein discloses.
  • the invention provides a vector for use as vaccine or as expression vector to produce said immunogenic polypeptides.
  • the vector according to any of the embodiments herein disclosed optionally comprises an expression control sequence operably linked to the nucleic acid molecule.
  • said vector is selected from RNA virus vectors, DNA virus vectors, plasmid viral vectors, adenovirus vectors, adenovirus associated virus vectors, herpes virus vectors and retrovirus vectors.
  • said vectors are Adenoviral vectors, and specifically simian adenoviral vectors, which are generally known in the art.
  • a number of replication-defective recombinant simian adenoviruses are described for example in WO 2005/071093.
  • Vectors comprising these nucleic acid molecules can be used for transfection or transformation of a suitable mammalian, plant, bacterial or yeast host cell. Transfection or transformation can be by any known method for introducing polynucleotides into a host cell.
  • Methods for introduction of heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, lipid nanoparticles and direct microinjection of the DNA into nuclei.
  • nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art (see, e.g., U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455, incorporated herein by reference).
  • the invention provides an immunogenic composition or a vaccine comprising an immunogenic polypeptide or a nucleic acid molecule or a vector according to any one of the embodiments herein disclosed, optionally comprising one or more adjuvants and/or excipients.
  • each composition or vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and the type and amount of adjuvant used. An optimal amount for a particular vaccine may be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Generally, it is expected that each dose will comprise l-1000pg of immunogen, for example l-200pg, or 10-100pg. A typical dose will contain 10-50pg, for example 15- 25pg, suitably about 20pg of immunogen. Alternatively, a "dose-sparing" approach may be used, for example in a pandemic situation.
  • each human dose may contain a significantly lower quantity of immunogen, for example from 0.1 to lOpg, or 0.5 to 5pg, or 1 to 3pg, suitably 2pg protein per dose.
  • human dose is meant a dose which is in a volume suitable for human use. Generally, this is between 0.3 and 1.5 ml. In one embodiment, a human dose is 0.5 ml.
  • subjects typically receive a boost after a 2 to 4 weeks interval, for example a 3 -week interval, optionally followed by repeated boosts for as long as a risk of infection exists.
  • a single-dose vaccination schedule is provided, whereby one dose of immunogenic compound in combination with adjuvant is sufficient to provide protection against the SARS CoV-2, without the need for any boost after the initial vaccination.
  • the immunogenic compositions of the invention may be provided by any of a variety of routes such as oral, topical, subcutaneous, mucosal (typically intravaginal), intravenous, intramuscular, intranasal, sublingual, intradermal and via suppository.
  • routes such as oral, topical, subcutaneous, mucosal (typically intravaginal), intravenous, intramuscular, intranasal, sublingual, intradermal and via suppository.
  • immunogenic compositions according to any of the embodiments disclosed in the present specification can thus be in any form suitable for administration by oral, topical, subcutaneous, mucosal (typically intravaginal), intravenous, intramuscular, intranasal, sublingual, intradermal or suppository route.
  • Immunisation can be prophylactic or therapeutic.
  • the invention described herein is primarily but not exclusively concerned with prophylactic vaccination against SARS-COV-2.
  • Appropriate pharmaceutically acceptable carriers or excipients for use in the invention are well known in the art and include for example water or buffers.
  • Vaccine preparation is generally described in Pharmaceutical Biotechnology, Vol.61 Vaccine Design - the subunit and adjuvant approach, edited by Powell and Newman, Plenum Press New York, 1995. New Trends and Developments in Vaccines, edited by Voller et al, University Park Press, Baltimore, Maryland, U.S.A. 1978.
  • the immunogenic compositions of the invention may further comprise one or more adjuvants, such for example oil-in-water emulsion, mineral containing compositions, saponin formulations and other adjuvant known in the state of the art.
  • adjuvants such for example oil-in-water emulsion, mineral containing compositions, saponin formulations and other adjuvant known in the state of the art.
  • Methods of producing oil-in-water emulsions are well known to the person skilled in the art. Commonly, the method comprises mixing the oil phase (optionally comprising a tocol) with a surfactant such as a PBS/TWEEN80TM solution, followed by homogenisation using a homogenizer.
  • Mineral containing compositions suitable for use as adjuvants in the disclosure include mineral salts, such as aluminium salts and calcium salts.
  • the disclosure includes mineral salts such as hydroxides (e.g.
  • oxyhydroxides phosphates (e.g.hydroxyphoshpates, orthophosphates), sulphates, etc. (e.g. See chapters 8 & 9 of Vaccine desigmthe subunit and adjuvant approach (1995) Powell & Newman. ISBN 0-306-44867-X), or mixtures of different mineral compounds, with the compounds taking any suitable form (e.g. Gel, crystalline, amorphous, etc.), and with adsorption being preferred.
  • the mineral containing compositions may also be formulated as a particle of metal salt (See WO00/23105). Medical uses
  • polypeptides, the nucleic acid molecules, the vectors, and immunogenic compositions described herein can be used in a prophylactic and/or a therapeutic treatment of the SARS- CoV-2 infection or conditions or disorders resulting from such infection. Immunisation can thus be prophylactic or therapeutic.
  • polypeptides, nucleic acid molecules, vectors or immunogenic compositions as described herein are used for prophylactic vaccination against SARS-COV-2.
  • polypeptides, the nucleic acid molecules, the vectors and immunogenic compositions according to any of the embodiments described herein may be provided by any of a variety of routes such as oral, topical, subcutaneous, mucosal (typically intravaginal), intravenous, intramuscular, intranasal, sublingual, intradermal and via suppository.
  • routes such as oral, topical, subcutaneous, mucosal (typically intravaginal), intravenous, intramuscular, intranasal, sublingual, intradermal and via suppository.
  • the polypeptides, the nucleic acid molecules, the vectors and immunogenic compositions described herein are for use as a vaccine against SARS-CoV-2 infection, in particular for use in a prophylactic or therapeutic treatment of the SARS-CoV- 2 infection or conditions or disorders resulting from such infection, more in particular for use in the prevention of COVID-19 disease.
  • the objective of this study is to reduce the interaction strength between the RBD domain of the SARS-CoV-2 S-protein and the human hACE2 receptor.
  • the RBD residues interacting with hACE2 were identified by analysing the crystallised structure of RBD-hACE2 complex downloaded from the PDB database (PDB ID: 6M0J) and all residues within 5 A from the protein interface were identified as interacting.
  • residues 417, 446, 447, 449, 453, 455, 456, 473, 475, 476, 484, 486, 487, 489, 493, 496, 498, 500, 501, 502, and 505 were selected for mutagenesis.
  • Rosetta Flex ddG Rosetta Ensemble-Based Estimation of Changes in Protein Protein Binding Affinity upon Mutation. The Journal of Physical Chemistry B 122, 5389- 5399, 2018).
  • Rosetta Flex ddG is the current state-of-the art method for predicting changes in protein-protein and protein-ligand binding free energy. Rosetta is a software suite for macromolecular modelling and design that uses all-atom mixed physics- and knowledgebased potentials and provides a diverse set of protocols to perform specific tasks, such as structure prediction, molecular docking and homology modelling.
  • the Flex ddG protocol has been found to perform better than machine learning methods and comparably to molecular dynamics methods when tested on a large dataset of ligand binding free energy changes upon protein mutation.
  • the S protein RBD-hACE2 complex structure was downloaded from PDB database (PDB ID: 6M0J).
  • the structure was relaxed using Rosetta FastRelax Mover (Tyka, M.D., Jung, K. & Baker, D. Efficient sampling of protein conformational space using fast loop building and batch minimization on highly parallel computers. Journal of Computational Chemistry 33, 2483-2491, 2012). Resfiles describing individual point mutation were generated for each selected residue in the relaxed structure, and backrub sampling was applied around the mutation site.
  • AAG AAG
  • Rosetta Flex ddG was employed to predict the change in stability (AAG) of the monomeric RBD protein caused by single point mutations.
  • AAG the change in stability
  • the predicted stability change is given as the difference in predicted energy between the modelled wild-type and mutant structures.
  • the predicted substitutions were then analysed by the inventors that based on their expertise and their analysis of the 3D structure of the molecule decided the amino acid substitutions that would be tested experimentally.
  • the list of single point mutations was analysed in order to define the combination of mutations with high overall predicted binding affinity and stability compared to the WT protein. Specifically, all possible combinations of two, three and four mutations were computed and the combined score for affinity and stability was calculated as separated sets. For each set of mutations, the pareto optimal solutions were identified and further analysed, with a final number of pareto optimal solution for two, three and four mutations of 5, 22 and 42 respectively ( Figure 3).
  • the list of candidates underwent manual inspection via Pymol (Rigsby, R.E. & Parker, A.B. Using the PyMOL application to reinforce visual understanding of protein structure.
  • GeneArt Strings DNA Fragments were resuspended with water and amplified using Q5® High-Fidelity DNA Polymerase (NEB), with primer forward 5’- CAGAGAGCATCGTACGATTTCCAAAC-3’ and reverse 5’- GGTATTGGTCCCGGGGGTGATCACAG-3’, following manufacturer’s protocol.
  • PCR products were purified using GenEluteTM PCR Clean-Up kit (Sigma-Aldrich), according to the manufacturer's instructions. Amplified DNA fragments were digested with BsiWI-HF and Xmal restriction enzymes (NEB) and purified using GenEluteTM PCR Clean-Up kit.
  • S protein expressing plasmids were digested with BsiWI-HF and Xmal restriction enzymes and purified from agarose gel, using QIAquick Gel Extraction Kit (QIAGEN). Digested DNA fragments were ligated to S protein expressing plasmid using T4 DNA Ligase (NEB). Ligation products were used to transformation One Shot® TOP 10 Chemically Competent E. coli (InvitrogenTM), following manufacturing instructions. Selected colonies were analysed by plasmid isolation/restriction and sequencing. Large-scale isolation of plasmid DNA from recombinant A.
  • Coli cultures was performed using NucleoBond® Xtra Midi/Maxi - High copy plasmid purification (MACHEREY-NAGEL), according to the manufacturer's instructions. Plasmids were quantified by NanoDropTM and stored at - 20°C prior to use.
  • Plasmid encoded mutated SARS-CoV-2 S proteins were transiently transfected into ExpiCHOTM Expression System (GibcoTM), according to manufacturer’s instructions. Following harvest and clarification by centrifugation, cell culture supernatants were filtered through a 0.22-pm filter and then purified through immobilized metal affinity chromatography using HisTrapTMHP His tag protein purification columns (Cytiva). Chromatography was conducted at room temperature using the AKTA goTM chromatography system (Cytiva).
  • the culture supernatant of S protein cell culture was applied to a single 5 mL HisTrapTMHP column, previously equilibrated in Buffer A (20 mM NaftPCU, 500 mM NaCl + 30 mM imidazole pH 7.4).
  • Buffer A 20 mM NaftPCU, 500 mM NaCl + 30 mM imidazole pH 7.4
  • the column was washed in Buffer A for 3 column volumes (CV) with all 3 CV collected as the column wash.
  • Recombinant proteins were eluted from the column applying an isocratic elution of 5 CV of 60% Buffer B (20mM Na ⁇ PCU, 500mM NaCl + 500 mM imidazole pH 7.4). Elution steps were collected in 1 mL fractions.
  • Eluted fractions were analysed by SDS-PAGE and appropriate fractions containing recombinant proteins were pooled. Protein-containing fractions were dialyzed in PBS buffer overnight at 4°C. Final protein concentrations were determined by NanoDropTM. Purified proteins were stored at -80°C prior to use.
  • 384-well plates (microplate clear, Greiner Bio-one) were coated with 3 pg/ml of streptavidin (Thermo Fisher) diluted in carbonate-bicarbonate buffer (Bethyl Laboratories) and incubated at room temperature overnight. The next day, 3 pg/mL of S proteins diluted in PBS were added and incubated for Ih at RT. Plates were then saturated with 50 pl/well of blocking buffer (phosphate-buffered saline and 1% BSA) for 1 h at 37 °C.
  • streptavidin Thermo Fisher
  • carbonate-bicarbonate buffer Bethyl Laboratories
  • mAbs monoclonal antibodies
  • samples buffer phosphate-buffered saline, 1% BSA, 0.05% Tween-20
  • Goat Anti-Human IgG-AP diluted 1 :20’000 in sample buffer was then added and incubated for 1 h at 37 °C. Plates were incubated with Alkaline Phosphatase Yellow (pNPP) (Sigma) for 20-30 minutes.
  • pNPP Alkaline Phosphatase Yellow
  • the optical density (OD) values were identified using the Varioskan Lux Reader (Thermo Fisher Scientific) at 405 nm.
  • FIG. 4A shows the results for the binding of tested mAbs to mutated SARS-CoV-2 S proteins. All mAbs bind to the seven mutated SARS-CoV-2 S proteins, confirming the maintaining of their quaternary structure, except, as expected, for the unrelated mAb.
  • FIG. 4B shows the results for the binding of mutated SARS-CoV-2 S-proteins to hACE2. All mutated SARS-CoV-2 S-proteins showed a binding decrease to ACE2 compared to wildtype SARS-CoV-2 S-protein, particularly mut 2S-MK, mut 4S-LMKG and 4S-QMKK, with a fold change reduction of -1.8x, -3. lx and -3.3x, respectively.
  • the middle high anchor was formed by residues Y453, Q493 and S494, while the middle low anchor was formed by residues Y449 and R498.
  • the bottom anchor was constituted by residues T500, Y501, G502 and H505( Figure 5).
  • the first strategy was based on the mutation of a single residue per each of the four anchors.
  • the second strategy aimed to mutate at least two residues per each anchor.
  • the third strategy had the objective to mutated up to four residues in the same anchor.
  • Each of these strategies has undergone virtual screening, where the results from single point mutations were combined and analyzed in all possible combinations. This process resulted in a total of 1,563,852, 2,607,142, and 137,541 candidates for the first, second, and third strategies, respectively. A score was calculated for each candidate by combining the results of the single point mutations. Pareto-optimal solutions were then identified to select combinations with high stability and decreased affinity.
  • Balb/c mice were immunized with 25 pL of AddaVaxTM (Squalene-based oil-in-water adjuvant, Catalog code: vac-adx-10, InvivoGen) and 5 pg of recombinant protein diluted in PBS (WT, mut 2S-MK, mut 4S- LMKG and mut 4S-QMKK), to reach a final volume of 50 pL.
  • AddaVaxTM Squalene-based oil-in-water adjuvant, Catalog code: vac-adx-10, InvivoGen
  • PBS PBS
  • mut 2S-MK mut 4S-LMKG
  • mut 4S-QMKK mut 4S-QMKK
  • Terminal blood sample were collected via exsanguination in anesthetized animals using isofluorane. Sacrifice was carried out by cervical dislocation. Targeted volume for terminal blood collection was approximately 800 pl. Serum was obtained from the whole blood placed in the tubes by centrifugation. All serum samples were stored in vials at -20°C until usage.
  • Wild type SARS-CoV-2 authentic viruses’ neutralization assays were all performed in the biosafety level 3 (BSL3) laboratories at Toscana Life Sciences in Siena (Italy). BSL3 laboratories are approved by a Certified Biosafety Professional and are inspected every year by local authorities.
  • the humoral immune response was evaluated by a virus neutralization test, where mice sera were all tested in a cytopathic effect-based neutralization assay (CPE- MN). In brief, serum samples were heat-inactivated for 30 minutes at 56°C; two-fold serial dilutions, starting from 1 : 10, were then mixed with an equal volume of viral solution containing 100 median tissue culture infectious dose (lOOTCIDso) of SARS-CoV-2 virus.
  • CPE- MN cytopathic effect-based neutralization assay
  • ELISA assay to evaluate the Second-Round mutated S proteins
  • the ELISA assay was performed as previously described in 1.6 paragraph, using the Second- Round mutated SARS-CoV-2 S-, adding to the plates, after blocking, 25 pl/well of hACE2 diluted at 3 pg/mL in samples buffer.
  • Figure 7 shows the results for the binding of mutated SARS-CoV-2 S-proteins to hACE2. All mutated SARS-CoV-2 S-proteins showed a complete binding abrogation to hACE2 receptor compared to wildtype SARS-CoV-2 S-protein.

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Abstract

La présente invention concerne un vaccin de seconde génération contre la COVID-19 permettant d'abroger ou de diminuer la liaison de la protéine de spicule (S) du SARS-CoV-2, ou d'une partie de celle-ci, à l'enzyme de conversion de l'angiotensine 2 humaine (hACE2). De tels vaccins présentent plusieurs avantages, tels que le fait d'éviter des voies de dérégulation immunitaire activées à la suite d'une interaction protéine S/hACE2 tout en maintenant la capacité d'induire une réponse de neutralisation d'anticorps forte et robuste au SARS-CoV-2. L'invention concerne également l'utilisation de la protéine S pour des utilisations thérapeutiques et pour des vaccins dans la prévention ou le traitement d'une infection ou d'états pathologiques ou de troubles du SARS-CoV-2 résultant d'une telle infection.
PCT/IB2025/052041 2024-03-05 2025-02-26 Protéines de spicule mutées en tant que vaccins contre le sars-cov-2 Pending WO2025186660A1 (fr)

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