[go: up one dir, main page]

WO2007107597A2 - Construction immunogene et procede pour le traitement prophylactique ou therapeutique du sida - Google Patents

Construction immunogene et procede pour le traitement prophylactique ou therapeutique du sida Download PDF

Info

Publication number
WO2007107597A2
WO2007107597A2 PCT/EP2007/052715 EP2007052715W WO2007107597A2 WO 2007107597 A2 WO2007107597 A2 WO 2007107597A2 EP 2007052715 W EP2007052715 W EP 2007052715W WO 2007107597 A2 WO2007107597 A2 WO 2007107597A2
Authority
WO
WIPO (PCT)
Prior art keywords
hiv
seq
sequence
amino acid
ectodomain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2007/052715
Other languages
English (en)
Other versions
WO2007107597A3 (fr
Inventor
Joachim Denner
Reinhard Kurth
Uwe Fiebig
Rayk Behrendt
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Robert Koch Institute
Federal Government of Germany
Original Assignee
Robert Koch Institute
Federal Government of Germany
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Robert Koch Institute, Federal Government of Germany filed Critical Robert Koch Institute
Publication of WO2007107597A2 publication Critical patent/WO2007107597A2/fr
Publication of WO2007107597A3 publication Critical patent/WO2007107597A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/686—Anti-idiotype
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the invention relates to an immunogenic construct and a pharmaceutical composition comprising it for the induction of antiviral neutraiizing antibodies in humans and animals.
  • the invention also relates to a method for the prophylactic or therapeutic treatment of AIDS.
  • a number of therapies for viral diseases such as HIV infections or AIDS are known in the prior art.
  • combination therapies especially using reverse transcriptase inhibitors and protease inhibitors such as AZT and nevirapin, or a combination therapy of a nti retroviral substances with immunomodulators and other substances such as interferon, interleukins and/or thymostimulin have been described.
  • a cure in a classical sense i.e., elimination of the virus, is not possible.
  • the development of an HIV vaccine is critical.
  • the technical problem forming the basis of the present invention was to provide an immunogenic construct, which has an improved efficacy in neutralizing HIV and is able to protect safely.
  • an effective vaccine should elicit cell-mediated and neutralizing humoral immune responses.
  • Subject-matter of the invention is an immunogenic construct comprising an ectodomain of a transmembrane envelope protein from a gammaretrovirus, or variants, mutants, parts of the ectodomain or at least 80 % homologous sequences having the same function.
  • the amino acid sequence E1 at the N-terminal end, which comprises parts of the fusion peptide and of the N-helical region (NHR) is substituted by a sequence E1 from the ectodomain of the transmembrane envelope protein of a HIV, and wherein at least parts of an amino acid sequence E2 at the C-terminal end, which comprises parts of the C-he!ical region (CHR) are substituted by a sequence E2 comprising several epitope sequences from the ectodomain of the HIV transmembrane envelope protein, and wherein the epitopes are linked via a spacer sequence of the ectodomain of the envelope protein from the gammaretrovirus.
  • a construct comprising a DNA encoding the respective immunogenic construct, and in this case as well, it is the presence of the two regions in the coding sequence that matters. In respect of selecting the amino acid sequences or DNA sequences, the combination of both regions is crucial for the immunogenic potential of the construct.
  • an immunogenic construct comprising a backbone of a transmembrane envelope protein of a gammaretrovirus having a spacer sequence of the gammaretrovirus within an E2 sequence insert of HIV together with an E1 sequence of HIV significantly enhances the accessibility of epitopes interrupted thereby.
  • Higher titers of neutralizing antibodies are induced by the inventive construct along with a retroviral protection of every subject of such species capable of being immunized.
  • Neutralization relates to a mechanism which prevents the infection and propagation of a retrovirus,.
  • the neutralizing antibodies are especially directed against the vira! structures which induced them.
  • the complete anti-viral protection achieved herewith is of clear advantage over the partial immunization subset if using the constructs of prior art.
  • the inventive immunogenic construct causes high titers of neutralizing antibodies within short periods of immunization along with an effective reduction of the number of HIV.
  • the high neutralizing antibody titers are reflected by a high dilution of serum which is obtained after immunization and used in neutralization assays. Simultaneously, adverse effects which could be caused by other serum components are largely reduced due to their diluted presence.
  • transmembrane envelope proteins has a homologous structure.
  • transmembrane envelope proteins comprise an ectodomain, an anchor domain
  • transmembrane region also termed transmembrane region
  • a fusion domain and a cytoplasmatic portion
  • the ectodomain comprises two alpha-helical structures, the N-terminal helical region (NHR) and the C-terminal helical region (CHR), respectively, and a cysteine-cysteine-loop.
  • NHR N-terminal helical region
  • CHR C-terminal helical region
  • cysteine-cysteine-loop cysteine-cysteine-loop.
  • transmembrane region, and cytoplasmatic portion are dispensable for inducing neutralizing antibodies.
  • the ectodomain is characterized by the induction of neutralizing antibodies in several species and a better solubility compared to the total transmembrane envelope protein. Due to its small size, the ectodomain is cost-efficiently produced.
  • Starting point of the inventive construct is an ectodomain of a transmembrane envelope protein from a gammaretrovirus, especially from a p15E, or variants, mutants, parts of the ectodomain or homologous sequences having the same function.
  • a couple of methods are known to the skilled artisan to generate equivalent ectodomains, i.e. proteins which are analog in function to those of the inventive teaching. Therefore, the invention also contains the aforementioned modifications.
  • mutants can be generated by substitution, deletion, insertion, translocation, inversion and/or addition of at least a single amino acid. It is known that certain amino acids exhibit similar physicochemical characteristics making the substitution among each other possible.
  • Variants of the ectodomain can be arise from modifications, such as alkylation, arylation or acetylation of at least a single amino acid, from incorporation of enantiomers and/or from fusion of the ectodomain with a single or multiple amino acids, a peptide or a protein. It is preferred in the meaning of the invention that the ectodomain is fused to a purification tag for affinity chromatography. Parts of the ectodomain relates to a restriction to those regions which are sufficient for the expression of a specific function. Ai! alterations are inevitably limited by the requirement of preserving the function.
  • the parts of the ectodomain can be very small due to the characterization of epitopes which also induce neutralizing antibodies as peptides.
  • the homology between a natural ectodomain and a derivative thereof having the same features amounts to at least 60 %, more preferably 80 %, most preferably 95 %.
  • the homology is to be considered if the aforementioned part of any size is altered to a variant or mutant.
  • the ectodomain contains highly conserved regions which do not mutate during virus replication, thereby representing the principal antibody target.
  • sequence E1 and E2 have been identified for several ectodomains.
  • sequences E1 and E2 are anti-parallei arranged to present the E2 sequence in such a suitable conformation that makes the induction of neutralizing antibodies possible.
  • another primary sequence denotes a sequence having different primary structure, i.e. the degree of homology between the exchanged sequences is considered as low.
  • the homology amounts to less than 50 %, more preferably less than 30 %, most preferably less than 10 %.
  • the matching sequences E1 within the ectodomains of the envelope proteins from the gammaretrovirus and HIV show a homology concerning their secondary and/or tertiary structure enabling sequence allocation and the specific incorporation at a certain position. The same is applicable for the E2 sequences.
  • the HIV to be attacked is exclusively determined by the E2 sequence.
  • This sequence comprises at least two epitopes, preferably just two epitopes. Such epitopes are designated E2', E2" and so on.
  • the epitopes of an E2 sequence are usually separated by a spacer sequence.
  • a sequence E2a which comprise at least one epitope is positioned in front of the spacer and a sequence E2b which comprise also at least one epitope is positioned downstream of the spacer. It has been shown that the kind (e.g. composition, length etc.) and location of the spacer affect the immune response.
  • the spacer peptide of the selected E2 region is conveniently mutated, e.g. as described in the course of the specification.
  • a spacer peptide comprises at least a single amino acid.
  • the preferred length of the spacer peptide is between 2 and 10 amino acids, more preferably 3-8 amino acids, most preferably 4 amino acids.
  • the backbone and the spacer sequence have the same retroviral origin, i.e. the spacer sequence is originated from the ectodomain of the envelope protein from the gammaretrovirus. That means, in the course of the substitution procedure several partial sequences corresponding to the epitopes are replaced, whereas the original spacer remained unaltered.
  • the complete E2 sequence is exchanged by HIV and the spacer peptide is subsequently re-mutated to the initial spacer sequence as found in the ectodomain of the envelope protein from the gammaretrovirus.
  • a construct can comprise the backbone from the ectodomain of an envelope protein from the gammaretrovirus, the sequence E1 and the sequence E2 from an ectodomain of a HIV envelope protein, wherein E2 is separated in two HIV parts E2a and E2b and a spacer of the gammaretrovirus peptide.
  • the gammaretroviral transmembrane envelope protein is considered as the backbone of the immunogenic construct. It is preferred to use p15E of a gammaretrovirus, such as for instance PERV p15E, FeLV p15E or KoRV p15E.
  • the KoRV p15E ectodomain is applied as backbone.
  • the backbone can be mutated, preferably in CHR, in order to enhance the physical/chemicai properties and/or immune response, but the final homology should amount to at ieast 80 %, preferably at least 90 %.
  • the extent of mutations is inevitably limited by the requirement of protein folding into a tertiary structure which promotes the region arrangement and exposure. Possible mutations include deletion, insertion, substitution, modification and/or addition of at least one amino acid, as well as the fusion with another protein.
  • the immune suppressive domain isu can be deleted in the N-termina! helical region and/or the peptide K can be deleted in the C-terminal helical region.
  • the portion of the transmembrane envelope protein of the gammaretrovirus represents the ectodomain, although remains of flanking sequences may be present.
  • the skilled artisan knows data bases of molecular biology, such as Swiss-Prot, in order to extract sequences to be applied herein.
  • the KoRV p15E ectodomain comprises the amino acid sequence of SEQ ID NO: 1 (amino acids 464 to 627 Swiss-Prot Q9TTC0).
  • the variants, mutants, parts of the amino acid sequence or at least 80 % homologous sequences having the same function are also included.
  • the backbone sequence of the KoRV p15E ectodomain is used for a preferred immunogenic construct.
  • This backbone sequence comprises the amino acids 500 to 579 or 575 respectively (Swiss-Prot account Q9TTC0) - SEQ ID NO: 12.
  • the preferred spacer sequence from the KoRV p15E ectodomain is the amino acid sequence of SEQ ID NO: 5, or variants, mutants, parts of the amino acid sequence or at least 80 % homologous sequences having the same function.
  • the spacer peptide is preferably derived from the KoRV p15E ectodomain, more preferably being WYEG (SEQ ID NO: 5). This spacer peptide is especially situated between both HIV epitopes.
  • these include the group of amino acids (a) glycine, alanine, valine, leucine and/or isoleucine; or the amino acids (b) serine and threonine; the amino acids (c) asparagine and glutamine; the amino acids (d) aspartic acid and glutamic acid; the amino acids (e) lysine and arginine; as well as the group of aromatic amino acids (f) phenylalanine, tyrosine and/or tryptophan.
  • Amino acids within one and the same group (a-f) can be replaced with one another.
  • the amino acids can be replaced by modified amino acids or specific enantiomers.
  • amino acid sequences, sequence fragments or structures comprising sequences designed using the methods mentioned for example in PNAS USA 1998, Oct. 13, 9521 :12179-84 - starting from an amino acid sequences of the invention - are sequences in the meaning of the invention and included in the teaching according to the invention, provided they accomplish the object of the invention, particularly of generating neutralizing antibodies.
  • the regions E1 and E2 which are to be incorporated, are derived from transmembrane enveiope proteins of HIV.
  • the regions are selected from HIV gp41 , such as the gp41 ectodomain from HIV-1 or HIV-2, especially HIV-1 gp41.
  • the first region or E1 which terms are used interchangeably herein essentially comprises the stretch of 533 to 546 of the aforementioned gp41 embodiment.
  • the numbers refer to the amino acid position in the transmembrane envelope protein according to well-known sequence data bases, e.g. the NCBI data base, and match within any HIV subtype.
  • the E1 stretch consists of the sequence TSMTLTVQARQLLS (SEQ ID NO: 2), or variants, mutants, parts of the amino acid sequence or at least 80 % homologous sequences having the same function. It shall be understood that the sequence of HIV subtypes may vary.
  • the second region or E2 which terms are used interchangeably herein essentially comprises the stretch of 662 to 676 in case of HIV gp41.
  • the stretch is composed of several components being the epitope for 2F5, a spacer peptide and the epitope for 4E10, whereby each component is required.
  • the spacer peptide is preferably originated from the backbone of the gammaretroviral protein and framed by the 2F5 and 4E10 epitopes.
  • E2 consists of the sequence ELDKWASLWNWFNIT (SEQ ID NO: 7) or variants, mutants or at least 80 % homologous sequences having the same function.
  • sequence E2b comprises the amino acid sequence of SEQ ID NO: 4 (WFNIT of the HIV-1 gp41 ectodomain) corresponding to the epitope 4E10, or variants, mutants, parts of the amino acid sequence or at least 80 % homologous sequences having the same function.
  • the stretch can be altered in the inventive manner explained below, whereby the induction efficiency of neutralizing antibodies is clearly improved.
  • a specified epitope region can appear repeatedly, such as in the line of an epitope doubling which is preferred.
  • the 2F5 epitope occurs twice in the inventive construct, in this case, the sequence E2a comprises the amino acid sequence of SEQ ID NO: 3 (ELDKWASELDKWAS, derived from the HIV-1 gp41 ectodomain), or variants, mutants, parts of the amino acid sequence or at least 80 % homologous sequences having the same function.
  • the immunogenic construct comprises the amino acid sequence of SEQ iD NO: 6 (TSMTLTVQARQLLSLTSLQIAMDTDLRALQDSiSKLEDSLTSLSEWLQNRRGLDLLFLKEGGLCAALK EECCFYVDHSGAVRDSMRRLKELDKWASELDKWAS WYEGYJFN IT) representing the structure of HIV-1 epitope (E1 ) - KoRV p15E backbone - doubled HIV-1 epitope 2F5 (E2a) - KoRV p15E spacer - HIV-1 epitope 4E10 (E2b).
  • SEQ iD NO: 6 TMTLTVQARQLLSLTSLQIAMDTDLRALQDSiSKLEDSLTSLSEWLQNRRGLDLLFLKEGGLCAALK EECCFYVDHSGAVRDSMRRLKELDKWASELDKWAS WYEGYJFN IT
  • 12 out of 12 rats produced neutralizing sera after immunization with the construct of the invention, in detail, antibodies neutralizing laboratory and primary strains of HIV-1 were induced if using hybrid proteins containing a gammaretrovirus backbone and gp41 derived domains.
  • the high neutralizing antibody titers are reflected by a high dilution of serum which is obtained after immunization and used in neutralization assays.
  • the above-mentioned rat sera were still neutralizing at a 60-foid dilution. Adverse effects which could be caused by other serum components are largely reduced due to their diluted presence.
  • a stable titer over a period of at least six month has been found.
  • subject matter of the invention are furthermore immunogenic constructs which comprise a backbone of an ectodomain of a transmembrane envelope protein from a gammaretrovirus, or variants, mutants, parts of the ectodomain or at least 80 % homologous sequences having the same function, preferably a backbone of an ectodomain of p15E, and which further comprise an amino acid sequence E1 at the N-terminal end and an amino acid sequence E2 at the C-terminal end which are characterized by the sequences selected from the following groups, and wherein the spacer is at least a single amino acid of the gammaretrovirus which links HIV epitope sequences of E2:
  • E1 FLGFLGAAGSTMGAASITLTVQARQLLS - SEQ ID NO: 13
  • E2 NEQDLLALDKWASLWNWFDiTNWLWYIK - SEQ ID NO: 14
  • E1 FLGFLGAAGSTMGAASITLTVQARQLLS - SEQ ID NO: 15
  • E2 NEQDLLALDKWANLWNWFDISNWLWYiK - SEQ ID NO: 16
  • HIV-1A2 E1 : FLGFLGAAGSTMGAASITLTVQARQLLS - SEQ ID NO: 17 E2: NEQDLLALDKWAXLWNWFXITXWLWYIR - SEQ iD NO: 18
  • E1 FLGFLGAAGSTMGAASMTLTVQARQLLS - SEQ ID NO: 19
  • E2 NEQELLELDKWASLWNWFDITNWLWYIK - SEQ ID NO: 20
  • E1 LLGFLGAAGSTMGAASITLTVQARQLLS - SEQ ID NO: 27
  • E2 NEQXLLALDKWDNLWSWFSITNWLWYIK - SEQ ID NO: 28
  • HIV-1 G FLGFLGAAGSTMGAASITLTVQVRQLLS - SEQ ID NO: 29
  • E2 NEQDLLALDKWASLWXWFDITKWLWYIK - SEQ iD NO: 30
  • E1 FLGFLGAAGSTMGAASITLTVQARQLLS - SEQ ID NO: 31
  • E2 NEQDLLALDKWASLWNWFSITNWLWYIK - SEQ ID NO: 32
  • E1 FLGVLSAAGSTMGAAATALXVQTHTLMK - SEQ ID NO: 33
  • E2 NEKKLLELDEWASIWNWLDITKWLWYIK - SEQ ID NO: 34
  • E1 FLGFLGAAGSTMGAASITLTVQARTLLS - SEQ ID NO: 35
  • E2 TNEKSLLELDQWDSLWSWFGITKWLWYiK - SEQ ID NO: 36
  • the present invention also relates to DNA encoding the immunogenic constructs for the induction of antiviral neutralizing antibodies in humans and animais.
  • the prior teaching of the present specification concerning the amino acid sequences and derivatives thereof is also valid and applicable without restrictions to the underlying DNA.
  • Antibodies neutralizing H1V-1 were induced by DNA vaccination of these constructs.
  • the construct is recombinantly expressed and purified. Therefore, the construct can be fused with a tag for affinity chromatography, such as Strep-tag, His-tag, GST-tag, Arg-tag or the calmodulin binding protein, preferably the calmodulin binding protein (CBP).
  • CBP calmodulin binding protein
  • the tag does not affect the induction of neutralizing antibodies.
  • the DNA encoding the protein sequences can be obtained, amplified, altered or synthesized with techniques known to the skilled artisan. Subsequently, the DNA can be introduced into a vector and transcribed and translated in cells.
  • Another object of the invention is a pharmaceutical agent comprising at least one of the immunogenic constructs and/or the polynucleotide according to the invention, optionally together with pharmaceutically tolerable adjuvants.
  • a pharmaceutical agent in the meaning of the invention is any agent in the field of medicine, which can be used in prophylaxis, diagnosis, therapy, follow-up or aftercare of patients who have come in contact with enveloped viruses, including retroviruses, in such a way that a pathogenic modification of their overall condition or of the condition of particular regions of the organism could establish at least temporarily.
  • the pharmaceutical agent in the meaning of the invention can be a vaccine or an immunotherapeutic agent.
  • the pharmaceutical agent in the meaning of the invention may include e.g. an acceptable salt or components thereof.
  • these can be salts of inorganic acids such as phosphoric acid or salts of organic acids.
  • the respective dose or dosage range for administering the pharmaceutical agent according to the invention is sufficiently high in order to achieve the desired prophylactic or therapeutic effect of forming neutralizing antibodies.
  • the specific dose level, frequency and period of administration of any particular mammal will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet time of administration, route of administration, rate of excretion, drug combination and the severity of the specific therapy.
  • the exact dose can be determined by a person skilled in the art, e.g. by determining the antibody titer as a function of dosage or as a function of the inoculation regime or pharmaceutical carrier and the iike.
  • the agent can be either administered to prevent the infection of a mammal with a retrovirus and the outbreak of the disease in advance, or to treat the disease caused by the infectious agent. Particularly, later stages of virus internalization can be prevented.
  • monitoring is considered as kind of treatment provided that the pharmaceutical combined composition is sequentially administered, e.g. in order to booster the immune response and eradicate the retrovirus and the arisen symptoms completely.
  • the retroviral disease AIDS caused by HiV can be successfully combated by applying the inventive composition
  • the agent of the invention is produced in a known way using the usual solid or liquid carriers, diluents and/or additives and the common adjuvants for pharmaceutical engineering and with an appropriate dosage depending on the intended mode of application.
  • These pharmaceutically acceptable excipie ⁇ ts comprise salts, buffers, fillers, chelating agents, antioxidants, solvents, bonding agents, iubricants, tablet coatings, flavor additives, flavors, preservatives and suspending agents, in the meaning of the invention, an adjuvant denotes every substance which enables, intensifies or modifies a specific immune response against the inventive agent if administered simultaneously, contemporarily or sequentially.
  • adjuvants for vaccines are for example aluminum compositions, such as aluminum hydroxide or aluminum phosphate, saponins, such as QS21 , muramyldipeptide or muramyitripeptide, proteins, such as gamma-interferon or TNF, M59 ; squalen or polyols.
  • the co-application of egg albumin in complete Freund's adjuvant also increases cell-mediated immunity, thereby supporting the effect of neutralizing antibodies.
  • Preferred adjuvants are Freund's adjuvant, Leucogen adjuvant containing Quil-A and aluminum hydroxide, or Montanide, such as Montanide ISA 720.
  • the amount of excipient material that is combined with the active ingredient(s) to produce a single dosage form varies depending upon the host treated and the particular mode of administration.
  • Possible formulations of the inventive agent are those forms which are suitable for oral administration, such as tablets, film tablets, lozenges, capsules, pills, powders, solutions, dispersions, suspensions or depot forms thereof, for transdermal administration, such as solutions, suspensions, creams, emulsions or band-aids, for parental administration, such as suppositories, and for intravenous infusion, subcutaneous injection or intramuscular administration, examples for the latter three are solutions and suspensions.
  • said pharmaceutical agent is a vaccine or vaccine-adjuvant.
  • the term vaccine composition relates to any composition which can be used as a vaccine.
  • a vaccine means a therapeutic or prophylactic use of the medicament which attacks retroviruses.
  • the vaccination is advantageously performed in such a way that an active protection is provided in the mammal.
  • the initial immunization can be boostered by subsequent inoculations.
  • the inoculation can be administered before or following an infection once or several times acting as therapy.
  • the forms or methods for manufacturing vaccine compositions according to the present invention are not particularly limited, and a composition in a desired form can be prepared by applying a single method available in the field of the art or methods in an appropriate combination.
  • aqueous media such as distilled water for injection and physiological saline, as well as one or more kinds of pharmaceutical additives available in the field of the art can be used.
  • buffering agents, pH adjusting agents, solubilizing aids, stabilizing agents, soothing agents, antiseptics, and the like can be used, and specific ingredients thereof are well known to those skilled in the art.
  • the vaccine composition can also be prepared as a solid preparation such as a lyophiiized preparation, and then prepared as an injection by adding a solubilizing agent such as distilled water for injection before use.
  • the inventive agent can be used as vaccine adjuvant to enhance the protection afforded by animai or human vaccines that are considered weak, i.e. by providing diminished protection in terms of level, extent, and/or duration.
  • the ingredients as vaccine-adjuvant will normally be administered separately from the vaccine, although it may be administered in combination with the vaccine as well.
  • the administration of the inventive agent as vaccine-adjuvant can be performed in subcutaneous, intravenous, parenteral, intramuscular, or in any other acceptable method.
  • the vaccine-adjuvant is administered prior to the administration of the vaccine and at the same site where the vaccine is to be administered.
  • the formulations and pharmaceutical compositions contemplated by the above dosage forms can be prepared with conventional pharmaceutically acceptable excipients, and by using conventional techniques. Other adjuvants may be administered either with the vaccine or together with the inventive pharmaceutical agent.
  • Still another object of the invention concerns a neutralizing antibody produced by immunization using the immunogenic construct or a combination of the immunogenic constructs.
  • a neutralizing antibody produced by immunization using the immunogenic construct or a combination of the immunogenic constructs.
  • at least one pharmaceutical agent of the invention is exposed to a mammal that it is capable to induce antibodies which are directed against the active substances of the agent and obtained by routine procedures known to those skilled in the art. Any mammal is chosen which produces high antibody titers.
  • the neutralizing antibodies are used for prophylactic or therapeutic treatment and/or monitoring of retroviral diseases, preferably a HIV infection.
  • the neutralizing effect of the antibodies is demonstrated by inhibiting the viral infection, the formation of syncytiums or the fusion between virus and target membrane, or everything thereof, or by reducing or stabilizing the propagation rate of a retrovirus in a mammal.
  • the effect of the antibodies is not restricted to the elimination of a retrovirus, but comprises the entire spectrum of advantageous effects in prophylaxis and therapy.
  • the invention relates to a method for inducing an antibody response, wherein the inventive immunogenic construct and/or polynucleotide is administered, thereby inducing the production of neutralizing antibodies.
  • the skilled artisan can set the concentration and the mode of application by routine experiments as already mentioned in the course of the specification.
  • the administration is performed prophylactically or therapeutically.
  • the composition is injected. It is the intention of a prophylactic inoculation to prevent the infection with a retrovirus after the infiltration of singie viral representatives, e.g. into a wound, such that the subsequent propagation of the virus is strictly diminished, or it is even completely inactivated. If an infection of the patient is already given, a therapeutic induction of an immune response is performed in order to inactivate the retrovirus being present in the body or to stop its propagation.
  • Monocional antibodies can be produced and used, such as in humans foiiowing humanization. In doing so, it is also possible to obtain antibody-producing cells from inoculated or infected individuals which neutralizing antibodies are directed against the inventive composition and applied as monoclonal antibodies during passive immunization. That mode of immunization does not cause a patient's own immune reaction to certain viruses, but the antibodies are introduced in the body as healing sera. The approach aims at a prompt effect, i.e. to cure the given infection sickness as quickly as possible or to protect against a virai infection immediately. Vaccination schedules are known to those skilled in the art and can be easily adapted to specific retroviruses which are to be attacked by the combined composition of the present invention. Preferably, monoclonal antibodies are used for passive immunization. Their use in a combination therapy is of special benefit.
  • the invention relates to the use of the immunogenic construct according to the invention, the polynucleotide according to the invention and/or the antibodies according to the invention for the prophylactic or therapeutic treatment and/or monitoring of retroviral diseases, preferably HIV.
  • retroviral diseases preferably HIV.
  • the prior teaching of the present invention and embodiments thereof is considered as valid and applicable without restrictions to the use of the above- mentioned components if expedient.
  • an immunoassay for the detection of H1V-1 and/or HIV-2 in a biological sample comprising
  • Another immunoassay is suitable for the detection of viral antigens in a biological sample comprising the steps of
  • the invention relates to an immunogenic construct comprising a backbone from the transmembrane envelope protein of a gammaretrovirus and two domains preferred derived from gp41 of HIV (HIV-1, all clades and combination of them, but E1 and E2 were always derived from the same clade, whereas in E2 is a spacer sequence of the gammaretrovirus which links the HIV epitope sequences in E2) for the induction of neutralizing antibodies in humans (prophylactic immunization).
  • monoclonal or polyclonal antibodies may be generated for use in therapeutic treatment of AiDS.
  • Fig. 1 shows the principle of generating hybrid proteins for the induction of antibodies, which neutralize HIV.
  • Fig, 2a shows the composition of the hybrid Il antigen.
  • Fig. 2b shows the composition of antigens derived from KoRV p15E. Notice: Only UR004 (SEQ ID NO: 6) is able to induce neutralizing antibodies, at! other proteins were not able.
  • Fig. 3 shows the sequence homology in the E2 region of HIV and KoRV. Identical amino acids are unlined; the cursive marked sequence 583-590 was deleted to guarantee an identical distance of the 2F5 epitope from the 4E10 epitope as exhibited in HIV-1 gp41. Arginine (R577, bold) was deleted to enhance the sequence homology. The epitopes of the antibodies 2F5 and 4E10 are framed.
  • Fig. 4 shows an ELlSA analysis indicating that antibodies in goat sera 53 and 63 bind to peptides E1 and E2. Both goats were immunized with the UR004 antigen derived from KoRV p15E ⁇ SEQ ID NO: 6).
  • Fig. 5 shows the proof that the serum of goat 63 immunized with the UR004 antigen derived from KoRV p15E is directed against the epitopes within HIV-1 gp41 being relevant for neutralization.
  • the epitope mapping was performed with overlapping peptides corresponding to the complete UR004 sequence; the epitopes are bold.
  • the antibodies bind to the epitopes for 2F5 (ELDKWAS) and 4E19 (NWFNIT).
  • Fig. 6 shows the proof that the serum of goat 44 immunized with the hybrid Ii antigen derived from PERV p15E is directed against the epitopes within HIV-1 gp41 being relevant for neutralization.
  • the epitope mapping was performed with overlapping peptides corresponding to the complete HIV-1 gp41 sequence; the epitopes are bold.
  • the antibodies bind to the epitopes for 2F5 (ELDKWAS) and 4E19 (NWFNIT).
  • Fig. 7 shows the proof that the serum of a rat immunized with the hybrid Il antigen derived from PERV p15E is directed against the epitopes within HIV-1 gp41 being relevant for neutralization.
  • the epitope mapping was performed with overlapping peptides corresponding to the complete HIV-1 gp41 sequence.
  • the antibodies bind to E1 (peptides 3 and 4) and E2 (peptides 51-57).
  • the epitopes 2F5 (ELDKWAS) and 4E10 (NWFNIT) are framed, p marks the peptide used in an ELISA; a marks the HiV sequence incorporated into the p15E sequence.
  • the serum showed a positive reaction in the ELISA using both peptides.
  • Fig. 8 exhibits the results of neutralizing assays using sera from different rats immunized with hybrid Il (neutralizing 2 of 4) and the preferred construct UR004 (neutralizing 4 of 4) depicted in Fig. 2b.
  • the inhibition of HIV-1 Bill and HIV-1-Ba-L by 2F5 is presented for comparative reasons.
  • the inhibition of the infection of Ba-L by the rat sera is shown here.
  • Sera from 6 rats proved to be very effective in neutralizing the primary HIV-1-Ba-L.
  • Fig. 9 shows the composition of the constructs used for DNA immunization.
  • the constructs were inserted into the pDisplay expression vector.
  • the genes contain a CMV promoter, the lgk leader and the PDGF transmembrane region. Two sequences bear a stop codon (STOP); they use the transmembrane region of gp41 or p15E, respectively.
  • Fig. 10 shows the analysis of the neutralizing features of rat sera immunized with the DNA constructs of Fig. 9. Only the sera of animals immunized with the construct that corresponds to hybrid II (PERV-Hyb II) demonstrated neutralizing features. Control animals were immunized with the empty vector (pDisplay).
  • Fig. 11 summarizes immunization experiments using 168 different synthetic peptides or recombinant proteins all containing the E1 and E2 region in a different context. The results of the neutralization assays are shown here.
  • hybrid Il which contained a domain derived from the N-terminal end of gp41, designated E1 (LTVQARQLLSSDIVQQQ - SEQ ID NO. 8), and a domain derived from the C-terminai membrane proximal external region (MPER) containing the epitopes of 2F5 and 4E10 and designated E2 (ELDKWASLWNWFNITNWLWY - SEQ ID NO. 9) (Fig. 2a).
  • E1 LTVQARQLLSSDIVQQQQ - SEQ ID NO. 8
  • MPER C-terminai membrane proximal external region
  • UR001 One construct, UR001, consisted of an HlV-derived E2' domain containing two 2F5 epitopes (ELDKWASELDKWAS - SEQ ID NO: 3, twice 662-668); the E1 domain was left from the original KoRV sequence (Fig. 2b).
  • UR002 contained the same HlV-derived E2' domain as in UR001 , but in addition an HlV-derived E1' (TSMTLTVQARQLLS, - SEQ ID NO: 2, 533-546) domain replaced the original KoRV E1 domain (Fig. 2b).
  • UR003 contained the original E1 domain of the KoRV and a different E2 domain (E2"), containing two 2F5 epitopes (ELDKWASELDKWAS - SEQ ID NO: 3, twice 662-668), a linker corresponding to the original p15E sequence (WYEG 1 - SEQ ID NO: 5, 591-594) and the 4E10 core epitope (WFNIT - SEQ ID NO: 4, 672-676).
  • E2 E2 domain
  • E2 2F5 epitopes
  • hybrid Il the backbone was p15E from PERV
  • UR004 it was p15E from the KoRV (500-579)
  • the EV domain was shorter (SDIVQQQ - compared with LTVQARQLLSSDIVQQQ - SEQ ID NO: 8)
  • third, in UR004 two 2F5 epitopes were present fourth, the E2" domain is shorter (reduced by NWLWY) and fifth, in UR004 four amino acids from the KoRV backbone remained (WYEG) between the two 2F5 epitopes and the 4E10 epitope.
  • One of the 2F5 epitopes represents a mutated sequence in p15E of the KoRV (ERLDKRQ - SEQ ID NO: 10:), after deletion of R(577) and exchange of two other amino acids (shown in bold).
  • the sequence 583-590 of p15E was removed (Fig. 3).
  • DNA immunization induced higher titers of neutralizing antibodies when compared to immunization with the hybrid protein
  • the pDisplay vector was used and sequences (i) corresponding to hybrid Ii, (ii) to the gp41 of HIV-1 sequence 629-683 which corresponds to the sequence of the dpi 78 domain, (iii) to the sequence 530-598, which corresponds to the sequence of the dpi 07 domain, (iv) to the sequence 512-850, which corresponds to the entire gp41 were inserted (Fig. 8). Animals were immunized and sera were tested for neutralizing antibodies. Only sera from animals immunized with the hybrid Il protein showed neutralizing activity (Fig. 10).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Veterinary Medicine (AREA)
  • Biotechnology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Organic Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Communicable Diseases (AREA)
  • AIDS & HIV (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne une construction immunogène comprenant un squelette de protéine d'enveloppe transmembranaire d'un gammarétrovirus et deux domaines E1 et E2 du HIV1, de préférence dérivés de gp41 du HIV (HIV-1, tous les clades et association de ceux-ci, mais E1 et E2 étaient toujours dérivés du même clade);par ailleurs, une séquence d'espacement du gammarétrovirus dans E2 lie les séquences d'épitopes HIV de E2, pour induire la neutralisation des anticorps chez les humains (immunisation prophylactique). L'utilisation des constructions selon l'invention permet de produire des anticorps monoclonaux ou polyclonaux destinés à être utilisés dans le cadre du traitement thérapeutique du SIDA.
PCT/EP2007/052715 2006-03-21 2007-03-21 Construction immunogene et procede pour le traitement prophylactique ou therapeutique du sida Ceased WO2007107597A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US78426506P 2006-03-21 2006-03-21
US60/784,265 2006-03-21

Publications (2)

Publication Number Publication Date
WO2007107597A2 true WO2007107597A2 (fr) 2007-09-27
WO2007107597A3 WO2007107597A3 (fr) 2007-12-21

Family

ID=38515456

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2007/052715 Ceased WO2007107597A2 (fr) 2006-03-21 2007-03-21 Construction immunogene et procede pour le traitement prophylactique ou therapeutique du sida

Country Status (1)

Country Link
WO (1) WO2007107597A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2205256A4 (fr) * 2007-09-26 2012-10-24 Dana Farber Cancer Inst Inc Réactifs destinés à induire une réponse immunitaire
WO2021185851A1 (fr) * 2020-03-17 2021-09-23 INSERM (Institut National de la Santé et de la Recherche Médicale) Compositions de vaccin pour la prévention et le traitement du vih

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005021574A2 (fr) * 2003-08-26 2005-03-10 Brd, Vertreten Durch Das Bundesministerium Für Gesundheit Und Soziale Sicherung, Letztvertreten Durch Den Präsidenten Des Robert-Koch-Institutes Induction d'anticorps neutralisants antiviraux chez les hommes et les animaux
EP1754715A1 (fr) * 2005-08-19 2007-02-21 Bundesrepublik Deutschland vertreten durch das Bundesminsterium für Gesundheit, dieses vertr. durch das Robert-Koch-Institut Vaccin à base des anticorps neutralisant un virus

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2205256A4 (fr) * 2007-09-26 2012-10-24 Dana Farber Cancer Inst Inc Réactifs destinés à induire une réponse immunitaire
WO2021185851A1 (fr) * 2020-03-17 2021-09-23 INSERM (Institut National de la Santé et de la Recherche Médicale) Compositions de vaccin pour la prévention et le traitement du vih

Also Published As

Publication number Publication date
WO2007107597A3 (fr) 2007-12-21

Similar Documents

Publication Publication Date Title
EP2237794B1 (fr) Protéines hybrides vih chimèriques utilisées en tant que vaccins
EP0939835A1 (fr) Compositions et procedes destines au traitement d'infections virales
US9381237B2 (en) Neutralizing immunogen (NimIV) of rhinovirus and its uses for vaccine applications
US20250109175A1 (en) Recombinant gp120 protein with v1-loop deletion
JP2023519837A (ja) コロナウイルスを処置するためのワクチン組成物
US20230149537A1 (en) Vaccines, adjuvants, and methods of generating an immune response
US20230192813A1 (en) Antibody that binds specifically to the sars cov 2 spike protein, and methods for its manufacture
WO2007107597A2 (fr) Construction immunogene et procede pour le traitement prophylactique ou therapeutique du sida
Xiao et al. Multiepitope vaccines intensively increased levels of antibodies recognizing three neutralizing epitopes on human immunodeficiency virus‐1 envelope protein
CN118076646A (zh) 一种可诱导广谱中和活性重组多组分新冠病毒三聚体蛋白疫苗的制备及应用
CA2548483A1 (fr) Induction d'anticorps neutralisants antiviraux chez les hommes et les animaux
US20190185548A1 (en) Antiviral polyclonal antibodies against ebola virus and the uses thereof
US20240189418A1 (en) Virus vaccine based on virus surface engineering providing increased immunity
US20240366753A1 (en) Viral-like particles for the treatment or prevention of an infection by a coronaviridae virus
WO2007023189A2 (fr) Composition pharmaceutique et utilisation dans le traitement prophylactique ou therapeutique de maladies retrovirales
WO2024130061A1 (fr) Protéines de fusion de virus ebola immunogènes et procédés associés
EP3254691A1 (fr) Anticorps polyclonal pour une utilisation dans la prévention et/ou le traitement de la maladie du virus d'ebola
JP3725899B2 (ja) Ｈｉｖに対して使用するための多分岐ペプチド構築物
EP4577239A1 (fr) Compositions et méthodes immunogènes contre le sars-cov-2
JP3725899B6 (ja) Ｈｉｖに対して使用するための多分岐ペプチド構築物

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07727191

Country of ref document: EP

Kind code of ref document: A2