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WO2009024789A1 - Peptides antigéniques du vih-2 - Google Patents

Peptides antigéniques du vih-2 Download PDF

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Publication number
WO2009024789A1
WO2009024789A1 PCT/GB2008/002831 GB2008002831W WO2009024789A1 WO 2009024789 A1 WO2009024789 A1 WO 2009024789A1 GB 2008002831 W GB2008002831 W GB 2008002831W WO 2009024789 A1 WO2009024789 A1 WO 2009024789A1
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WIPO (PCT)
Prior art keywords
hiv
polypeptide
infection
nucleic acid
gag
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PCT/GB2008/002831
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English (en)
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Jones Sarah Rowland
Aleksandra Leligdowicz
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Isis Innovation Limited
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Publication of WO2009024789A1 publication Critical patent/WO2009024789A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—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
    • 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

Definitions

  • This invention relates to novel HIV peptides, particularly HIV-2 peptides and to their use in pharmaceutical compositions
  • HIV infection in humans can be caused by two related yet distinct viruses: HIV-I and HIV-2.
  • Infection with HIV-2 is associated with a reduced rate of progression to AIDS (1) and significantly lower levels of plasma viral RNA (2, 3) in spite of similar proviral load (4) and a 30-60% sequence homology between the two viruses (5) . Consequently, the majority of HIV-2 -infected individuals are asymptomatic and die of causes unrelated to immunodeficiency (1) .
  • HIV-2 infection has no effect on survival in most adults, individuals who do progress to AIDS are clinically indistinguishable from those infected with HIV-I (6, 7) , demonstrating that HIV-2 is not simply an attenuated virus.
  • HIV-I infection antigen-specific CD8 T cells are important in controlling viremia (14-16) and comprehensive epitope analysis established that virus-specific immune responses can be mounted to the entire HIV-I proteome (17-23) .
  • the contribution of the cellular immune system to viremia control is controversial with evidence to support either a positive (18, 19), a negative (14, 24-26), or no correlation (17, 21, 22) with HIV-I plasma viral load (VL) .
  • HIV vaccines Using an ex vivo IFN- ⁇ enzyme-linked immunospot (ELISpot) assay and a 3 -dimensional matrix of overlapping peptides spanning the HIV-2 proteome, the current inventors have studied the in vivo frequency, relative dominance, breadth, and specificity of HIV-2 -specific T cell responses. Using this approach, they have identified a number of highly immunogenic HIV-2 peptides which may be used to prevent or treat HIV infection.
  • IFN- ⁇ enzyme-linked immunospot enzyme-linked immunospot
  • an isolated polypeptide consisting of 9 to 149 contiguous amino acids of the HIV Gag polypeptide sequence shown in at least one of SEQ ID NOS: 2 to 6 or a functional equivalent thereof, or a sequence showing at least 60% identity to said polypeptide.
  • the sequence shows at least 70, 80, 85, 90 or 95% identity to the sequence of at least one of SEQ ID NOS: 2 to 6 or a functional equivalent thereof.
  • the polypeptide consists of 9 to 21 contiguous amino acids of the polypeptide sequence shown in SEQ ID NOS : 2 to 6 or a functional equivalent thereof, or a sequence showing at least 60% identity to said polypeptide. More preferably, the polypeptide consists of between 9 and 18 contiguous amino acids.
  • HIV-I comprises clades A-H all of which have differing Gag protein sequences and which show different prevalence in different regions of the world.
  • the term at least 90% identical thereto includes sequences that range from 90 to 99.99% identity to the indicated sequences and includes all ranges in between.
  • the term at least 90% identical thereto includes sequences that are 91, 91.5, 92, 92.5, 93, 93.5. 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.5 percent identical to the indicated sequence.
  • the term "at least 60% identical” includes sequences that range from 60 to 99.99% identical, with all ranges in between. The determination of percent identity is determined using the algorithms described herein.
  • the polypeptide consists of 9 to 21 contiguous amino acids of the polypeptide sequence shown in any one of SEQ ID NOS: 7 to 11 or a functional equivalent thereof or a sequence showing at least 60% identity to said polypeptide.
  • polypeptide consists of the amino acid sequence shown in at least one of SEQ ID NOS: 7-21 or a functional equivalent thereof, or a sequence showing at least 60% identity to said polypeptides.
  • the sequence shows at least 70, 80, 85, 90 or 95% identity to the sequence of any one of SEQ ID NOS: 7 to 21 or a functional equivalent thereof.
  • the isolated polypeptide consists of the amino acid sequence shown in SEQ ID NO: 12.
  • an isolated polypeptide which comprises a biologically active fragment of the HIV-2 Gag polypeptide, wherein said fragment consists of the amino acid sequence shown in at least one of SEQ ID NO: 7 or 12 to 16, or a sequence showing at least 60% identity to at least one of said sequences.
  • the sequence shows at least 70, 80, 85, 90 or 95% identity to the sequence of any one of SEQ ID NOS: 7 or 12 to 16.
  • the nucleic acid is DNA or RNA.
  • nucleic acid may be an artificial synthesised nucleic acid, produced by any suitable means known to the skilled man.
  • a vector comprising the nucleic acid of the third aspect.
  • the vector is an expression vector and the nucleic acid is operably linked to a control sequence which is capable of providing expression of the nucleic acid in a host cell.
  • suitable vectors may include viruses (e.g. vaccinia virus, adenovirus, etc., baculovirus) ; yeast vectors, phage, chromosomes, artificial chromosomes, plasmids, cosmid DNA and lipososmes, polyplexes, or cells (e.g. mesenchymal stem cells, macrophages) .
  • a host cell transformed with a vector according the fourth aspect may be used for expression of the nucleic acid of the invention.
  • Suitable host cells for use in the invention may be prokaryotic or eukaryotic. They include bacteria, e.g. E. coli, yeast, insect cells and mammalian cells. Mammalian cell lines which may be used include but are not limited to, Chinese hamster ovary (CHO) cells, baby hamster kidney cells, NSO mouse melanoma cells, monkey and human cell lines and derivatives thereof and many others .
  • a method of producing a polypeptide according to any of aspects one to three comprising culturing a host cell transformed with a vector according to the fourth aspect under conditions which permit expression of said polypeptide and recovering the expressed polypeptide.
  • a vaccine composition comprising at least one polypeptide of aspects one to three or the nucleic acid of the fourth aspect and a pharmaceutically acceptable diluent and/or carrier and optionally an excipient and/or adjuvant.
  • a vaccine according to the present invention may be a prophylactic vaccine for preventing HIV infection, or a therapeutic vaccine for treating individuals previously infected with HIV-I and/ or HIV-2.
  • vaccines formed according to the invention may target simultaneously a plurality of epitopes of HIV.
  • the vaccine comprises a polypeptide having the sequence of SEQ ID NO: 12.
  • the pharmaceutical composition includes at least one pharmaceutically acceptable excipient.
  • polypeptide according to any of aspects one to three or the nucleic acid according to the fourth aspect for use in therapy.
  • a ninth aspect there is provided a polypeptide according to any of aspects one to three or the nucleic acid according to the fourth aspect for use in the prevention or treatment of HIV infection.
  • the HIV infection is HIV-2 infection.
  • a composition comprising the polypeptide of aspects one to three or the nucleic acid according to the fourth aspect in the manufacture of a medicament for the prevention or treatment of HIV infection.
  • the HIV infection is HIV-I and/or HIV-2 infection.
  • vaccines/ medicaments may be formulated into pharmaceutical dosage forms, together with suitable pharmaceutically acceptable carriers, such as diluents, fillers, salts, buffers, stabilizers, solubilizers, etc.
  • suitable pharmaceutically acceptable carriers such as diluents, fillers, salts, buffers, stabilizers, solubilizers, etc.
  • the dosage form may contain other pharmaceutically acceptable excipients for modifying conditions such as pH, osmolarity, taste, viscosity, sterility, lipophilicity, solubility etc.
  • Suitable dosage forms include solid dosage forms, for example, tablets, capsules, powders, dispersible granules, cachets and suppositories, including sustained release and delayed release formulations. Powders and tablets will generally comprise from about 5% to about 70% active ingredient. Suitable solid carriers and excipients are generally known in the art and include, e.g. magnesium carbonate, magnesium stearate, talc, sugar, lactose, etc. Tablets, powders, cachets and capsules are all suitable dosage forms for oral administration.
  • Liquid dosage forms include solutions, suspensions and emulsions.
  • Liquid form preparations may be administered by intravenous, intracerebral, intraperitoneal, parenteral or intramuscular injection or infusion.
  • Sterile injectable formulations may comprise a sterile solution or suspension of the active agent in a non-toxic, pharmaceutically acceptable diluent or solvent.
  • Suitable diluents and solvents include sterile water, Ringer's solution and isotonic sodium chloride solution, etc.
  • Liquid dosage forms also include solutions or sprays for intranasal administration.
  • Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be combined with a pharmaceutically acceptable carrier, such as an inert compressed gas .
  • dosage forms for transdermal administration including creams, lotions, aerosols and/or emulsions. These dosage forms may be included in transdermal patches of the matrix or reservoir type, which are generally known in the art.
  • compositions may be conveniently prepared in unit dosage form, according to standard procedures of pharmaceutical formulation.
  • the quantity of active compound per unit dose may be varied according to the nature of the active compound and the intended dosage regime.
  • the active agents are to be administered to human subjects in "therapeutically effective amounts" , which is taken to mean a dosage sufficient to provide a medically desirable result in the patient.
  • the exact dosage and frequency of administration of a therapeutically effective amount of active agent will vary, depending on such factors as the nature of the active substance, the dosage form and route of administration.
  • an eleventh aspect there is provided a method of treating a patient infected with HIV comprising administering a therapeutically effective amount of the polypeptide of aspects one to three or the nucleic acid according to the fourth aspect to a patient in need thereof.
  • the patient is infected with HIV-2.
  • a method of immunising an individual against HIV infection comprising administering to an individual an effective amount of the polypeptide of aspects one to three or the nucleic according to the fourth aspect .
  • the HIV is HIV-I and/or HIV-2.
  • an antibody characterised that it is capable of selectively reacting with the polypeptide of aspects one to three.
  • antibody encompasses purified or isolated naturally occurring antibodies of any isotype having the required immunological specificity, as well as synthetically produced antibodies or structural analogs thereof. Preparations of antibody can be polyclonal or monoclonal. Reference to such an “antibody” as described above includes not only complete antibody molecules, but also fragments thereof which retain substantial antigen binding capability. It is not necessary for any effector functions to be retained in such fragments, although they may be included. Suitable antibody fragments which may be used include, inter alia, F(ab') 2 fragments, scAbs, Fv, scFv fragments and nanoantibodies etc.
  • Antibody fragments which contain the idiotype of the molecule can be generated by known techniques, for example, such fragments include but are not limited to the F(ab')2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent .
  • Other antibody fragments with the required antigen binding activity can be prepared by recombinant expression techniques generally known in the art.
  • Chimeric humanized and fully humanized monoclonal antibodies can be made by recombinant engineering. By addition of the human constant chain to F(ab')2 fragments it is possible to create a humanized monoclonal antibody which is useful in immunotherapy applications where patients making antibodies against the mouse Ig would otherwise be at a disadvantage. Breedveld F. C. Therapeutic Monoclonal Antibodies. Lancet 2000 Feb 26; 335, P735-40. Recombinant therapeutic monoclonal antibodies may be advantageously prepared by recombinant expression in mammalian host cells (e.g. CHO cells) .
  • mammalian host cells e.g. CHO cells
  • Monoclonal antibodies with immunological specificity for HIV Gag can be prepared by immunisation of a suitable host animal (e.g. mouse or rabbit) with a suitable challenging antigen.
  • a suitable host animal e.g. mouse or rabbit
  • Figure 1 shows the proportion of patients with positive IFN- ⁇ ELISpot responses to HIV-2 gene products
  • Figure 2 shows the relationship between HIV-2 -specific IFN- ⁇ immune response magnitude and HIV-2 plasma viral load.
  • Proteome magnitude is the sum of responses in pools 1-24 and Gag magnitude is the sum of responses to pools 1-4 in the first matrix dimension.
  • Figure 3 shows the relative dominance of each gene product for every member of the two groups (plasma viral load ⁇ 100 or ⁇ IOO copies/ml) .
  • Pie charts display mean relative dominance values for each gene product expressed as a percentage of the total IFN- ⁇ immune response.
  • Figure 4 shows the relationship between breadth of ELISpot responses and HIV-2 viral load.
  • Breadth of ELISpot responses was defined as the average number of pools recognized per matrix dimension (range 0-24) .
  • Figure 5 shows the peptide 46-specific immune responses.
  • Peptide 46-specific IFN- ⁇ responses can be blocked with anti -HLA-DR antibodies
  • portion and “fragment” are used interchangeably to refer to parts of a polypeptide, nucleic acid, or other molecular construct.
  • Polypeptide and “protein” are used interchangeably herein to describe protein molecules that may comprise either partial or full-length proteins.
  • peptide is used to denote a less than full-length protein or a very short protein unless the context indicates otherwise.
  • proteins As is known in the art, “proteins”, “peptides,” “polypeptides” and “oligopeptides” are chains of amino acids (typically L-amino acids) whose alpha carbons are linked through peptide bonds formed by a condensation reaction between the carboxyl group of the alpha carbon of one amino acid and the amino group of the alpha carbon of another amino acid.
  • amino acids making up a protein are numbered in order, starting at the amino terminal residue and increasing in the direction toward the carboxy terminal residue of the protein.
  • nucleic acid is a polynucleotide such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) .
  • the term is used to include single-stranded nucleic acids, double-stranded nucleic acids, and RNA and DNA made from nucleotide or nucleoside analogues.
  • vector refers to a nucleic acid molecule that may be used to transport a second nucleic acid molecule into a cell.
  • the vector allows for replication of DNA sequences inserted into the vector.
  • the vector may comprise a promoter to enhance expression of the nucleic acid molecule in at least some host cells.
  • Vectors may replicate autonomously (extrachromasomal) or may be integrated into a host cell chromosome.
  • the vector may comprise an expression vector capable of producing a protein derived from at least part of a nucleic acid sequence inserted into the vector.
  • identity refers to sequence identity between two amino acid sequences or between two nucleic acid sequences. Percent identity can be determined by aligning two sequences and refers to the number of identical residues (i.e., amino acid or nucleotide) at positions shared by the compared sequences. Sequence alignment and comparison may be conducted using the algorithms standard in the art ⁇ e.g. Smith and Waterman, 1981, Adv. Appl. Math. 2:482; Needleman and Wunsch, 1970, J. MoI. Biol. 48:443; Pearson and Lipman, 1988, Proc . Natl. Acad. Sci . , USA 1 85:2444) or by computerized versions of these algorithms (Wisconsin Genetics Software Package
  • BLAST and FASTA may be used for sequence comparison.
  • ENTREZ available through the National Institutes of Health, Bethesda MD, may be used for sequence comparison.
  • BLASTN available at the Internet site for the National Center for Biotechnology Information
  • the percent identity of two sequences may be determined using GCG with a gap weight of 1, such that each amino acid gap is weighted as if it were a single amino acid mismatch between the two sequences.
  • the ALIGN program version 2.0
  • GCG Accelrys, San Diego, CA
  • an “effective amount” means the amount of an agent that is effective for producing a desired effect in a subject.
  • the term “therapeutically effective amount” denotes that amount of a drug or pharmaceutical agent that will elicit therapeutic response of an animal or human that is being sought.
  • the actual dose which comprises the effective amount may depend upon the route of administration, the size and health of the subject, the disorder being treated, and the like.
  • pharmaceutically acceptable carrier may refer to compounds and compositions that are suitable for use in human or animal subjects, as for example, for therapeutic compositions administered for the treatment of a disorder or disease of interest.
  • composition that may be administered to a mammalian host, e.g., orally, parenterally, topically, by inhalation spray, intranasally, or rectally, in unit dosage formulations containing conventional non-toxic carriers, diluents, adjuvants, vehicles and the like.
  • parenteral includes subcutaneous injections, intravenous, intramuscular, intracisternal injection, or infusion techniques.
  • a “stable” formulation is one in which the polypeptide or protein therein essentially retains its physical and chemical stability and biological activity upon storage.
  • Various analytical techniques for measuring protein stability are available in the art and are reviewed in
  • Stability can be measured at a selected temperature for a selected time period.
  • the formulation of interest may be kept at 40° C for 1 week to 1 month, at which time stability is measured.
  • the extent of aggregation following lyophilization and storage can be used as an indicator of peptide and/or protein stability.
  • a “stable" formulation is one wherein less than about 10% and preferably less than about 5% of the polypeptide or protein is present as an aggregate in the formulation.
  • a "stable" lyophilized formulation may be one wherein the increase in aggregate in the lyophilized formulation is less than about 5% or less than about 3%, when the lyophilized formulation is incubated at 40 °C for at least one week. Stability of the fusion protein formulation may be measured using a biological activity assay such as a binding assay as described herein.
  • HIV screening was performed using the Murex ICE HIV- 1.2.0 capture enzyme immunoassay (Murex Diagnostics) . Reactive sera were confirmed using an immunochromatographic rapid test for viral differentiation (HEXAGON HIV, Human GmbH) . Weakly positive HIV-I or HIV-2 tests or dually positive results were tested by a synthetic peptide-based strip method, (Pepti-Lav 1-2, Sanofi Diagnostics Pasteur) . Indeterminate results were subjected to HIV-I and HIV-2 - specific PCR using nested primers based on long terminal repeat regions specific for the respective virus (7, 47) .
  • HIV-I OR (mo 023) : GCGCCACTGCTAGAGATTTT SEQ ID NO: 91 HIV-I LTR OF (mo 034) : TGAGCCTGGGAGCTCTCTG SEQ ID NO: 92
  • HIV-I IF (mo 024) : AACCCACTGCTTAAGCCTCA SEQ ID NO: 93
  • HIV-I LTR IR (mo 035) : GTCTGAGGGATCTCTAGKTACCAG SEQ ID NO: 94
  • HIV-2 Primers HIV-2 OF (mo 026) : GCTGGCAGATTGAGCCCTG SEQ ID NO: 95
  • HIV-2 OR (mo 027) : AAGGGTCCTAACAGACCAGGG SEQ ID NO: 96
  • HIV-2 IF (mo 028) : CAGCACTAGCAGGTAGAGCCTGGG SEQ ID NO: 97
  • HIV-2 IR (mo 029) : GGCGGCGACTAGGAGAGATGG SEQ ID NO: 98
  • HIV-2 plasma viral load was quantified by RT-PCR using specific LTR primers (47) .
  • HIV-2 -specific LTR primers used for quantification of HIV-2 plasma viral load by RT-PCR:
  • H2SO ATTGAGCCCTGGGAGGTTCTCTCTCCA SEQ ID NO: 99
  • Antisense primer (H2AOB) Bio-TTCGGGCGCCAACCTGCTAGGGATTTT SEQ ID NO: 100
  • CD4 count analysis was carried out using a manual total white blood cell (WBC) count and differential lymphocyte count based on freshly collected blood. CD4 percentage analysis was done using BD MultiTest reagents and Multiset software (BD Immunocytometry Systems) on whole blood stabilized in a 5:1 ratio with TransFixTM (Cytomark) . CD4 counts were calculated using the following formula: (total WBC)x(%lymphocytes)x (CD4%) . A summary of the cohort's clinical and demographic parameters is presented in Table 1
  • HIV-2 gene products (Gag, Pol, Vif, Tat, Rev, Vpr, Vpx, Env, Nef ) .
  • the consensus sequence was used to generate 424 peptides overlapping by 10 amino acids, which were 15 to 19 amino acids in length (PeptGen, http : //hiv-web . lanl . gov) .
  • Peptides were synthesized by the PEPscreen service from
  • Overlapping peptides spanning the HIV-2 proteome were arranged in a 3 -dimensional matrix of 24 pools per dimension with 13-20 peptides per pool. This peptide configuration was deduced by the "Deconvolute This! software (48) as the most optimal matrix design based on the number of expected positive peptides. Since there is no information on HIV-2 specific immune responses to the entire HIV-2 expressed genome, the expected number of positive peptides was derived based on data from HIV-1-specific IFN- ⁇ responses that suggest that approximately 18 epitopic regions are recognized (17) . Past data show that the mean magnitude of responses to homologous HIV-I and HIV-2 Gag, Tat, and Env gene products is either similar (29) or lower when compared to HIV-1-specific responses (27) . Therefore, recognition of 18 or less epitopic regions would be expected per HIV-2- positive subject.
  • Peptides in the first matrix dimension were arranged in sequential order, allowing the calculation of IFN- ⁇ responses to individual gene products (Gag, Pol,
  • PBMC freshly isolated PBMC were used in ex vivo IFN- ⁇ ELISpot assays. Assays were carried out in 96-well Multiscreen filter plates (Millipore) coated with 15 ⁇ g/ml of anti-IFN- ⁇ monoclonal antibody (1-DIK, MABTECH) . PBMC were added at
  • HlO medium RPMI 1640 (Sigma) , 10% Human AB serum, 2mM L-glutamine, 50U/ml penicillin/streptomycin
  • 20 ⁇ l of lO ⁇ g/ml peptide pools final concentration 2 ⁇ g/ml per peptide
  • PHA phytohemagglutinin
  • Anti-HLA-DR L243 blocking antibodies were used at lO ⁇ g/ml and lOO ⁇ g/ml under the same assay conditions. Plates were incubated for 16 hours at 37 * C, 5% CO 2 . Spot enumeration was performed with an AID ELISpot reader system (Autoimmun Diagnostika GmbH) . To quantify antigen-specific responses, mean spots of the control wells were subtracted from the positive wells and results were expressed as SFU per 10 6 PBMC. Responses were regarded as positive if at least three times the mean of the quadruplicate negative control wells and over 50 SFU/10 6 PBMC. If background wells were more than 30 SFU/l0 G PBMC or if both positive control wells (PHA or FEC stimulation) were negative, the assay was excluded from further analysis.
  • Gag is the most frequently recognized HIV-2 gene product and Nef responses are infrequent
  • the IFN- ⁇ responses to the first ELISpot matrix dimension were used to quantify the total number of functional antigen- specific T cells.
  • the IFN- ⁇ immune response to the entire HIV-2 proteome (pools 1-24) was examined in relation to plasma viral load, a significant negative correlation was found, see Figure 2a. More specifically, this relationship was due to immune responses targeted to the Gag region (70 peptides present in pools 1-4) of the proteome, see Figure 2b.
  • the cohort was next divided into two groups according to HIV-2 plasma viral load (VL ⁇ 100 and VL ⁇ IOO copies/ml) to evaluate the role of the strength of gene product - specific IFN- ⁇ immune responses in the control of plasma viremia see Figure 2c.
  • SFU spot-forming units
  • the strength of a protein-specific immune response as a proportion of the entire virus-specific response is defined as the relative dominance (protein-specific IFN- ⁇ magnitude/proteome IFN- ⁇ magnitude xlOO) .
  • Analysis of relative dominance identified a hierarchy in HIV-2 protein recognition, with Gag-specific IFN- ⁇ secretion dominating the immune response (66%), followed by Env (16.3%), Pol (13.7%), Nef (2.3%), and Accessory proteins (1.7%) .
  • the relative dominance of Gag-specific T cell responses is particularly striking considering that Gag peptides constituted only 16.5% of the total peptides used in the ELISpot matrix, see Table II.
  • the average number of matrix pools with positive ELISpot responses was calculated by dividing the total number of positive peptide pools by the number of dimensions used in the ELISpot assay (range 0-
  • the most frequently recognized peptide was peptide 46 (Gag 29 8-3i5, YVDRFYKSLRAEQTDPAV, SEQ ID NO: 12) and a response to this peptide was present in 31% of the cohort.
  • peptide 46-specific responses were inversely related to HIV-2 plasma viral load see Figure 5a.
  • CD8 + and CD8 " PBMC fractions were used in an IFN- ⁇ ELISpot assay and IFN- ⁇ intracellular staining to determine T cell subtype restriction of these responses.
  • peptide 46 responses can be CD4 and CD8 T cell-restricted (data not shown) .
  • Anti -HLA-DR antibodies were next used in an IFN- ⁇ ELISpot assay in the presence of fresh PBMC from a peptide 46 responder stimulated with peptide 46. This eliminated the antigen-specific response, see Figure 5b, indicating that peptide .46 responses can be restricted by HIJA-DR, thus supporting the potential role of CD4 T cells in the restriction of this epitope-containing peptide.
  • the role of CD4 T cell help in the function of CD8 T cells remains incompletely defined.
  • CD4 T cells The fact that this frequently targeted region can be restricted by CD4 T cells (as shown by ELISpot assays done using CD8 -depleted T cell, by using HLA-DR blocking in ELISpot assays, and by intracellular cytokine staining using flow cytometry) suggests that the function of CD4 T cells specific for this region may offer insight into the correlates of protection associated with beneficial HIV-specific CD4 immune responses.
  • HIV-2 infection is a relatively neglected model of naturally attenuated HIV infection with infected subjects falling into two broad groups: progressors, who are clinically indistinguishable from people infected with HIV-I, and non- progressors.
  • progressors who are clinically indistinguishable from people infected with HIV-I
  • non- progressors As the majority of HIV-2 -positive patients have a low or undetectable viral load and are long-term non- progressors, this infection serves as a human model for controlled retroviral infection. The question of what antigen-specific immune responses could account for long- term non-progression in HIV-2 infection has never previously been addressed.
  • a more complete understanding of the interplay between the immune system and HIV-2 replication may provide information about correlates of immune protection pertinent to HIV-I infection. This data has been difficult to acquire as nearly all HIV-I patients progress to disease.
  • the current work which included 64 patients from a well- characterized community-based cohort with follow-up exceeding 17 years, is the largest study to date examining HIV-2 -specific T cells and the only study to do so outside clinical cohorts that are dominated by the minority of HIV- 2 -positive patients who progress to AIDS. It is also the first comprehensive characterization of cellular immune responses against the entire HIV-2 proteome using a novel 3- dimensional peptide matrix in an ex vivo IFN- ⁇ ELISpot assay. Using this approach, it was demonstrated that antigen- specific T cell responses can be mounted to all HIV- 2 proteins. HIV-2 -specific immune responses were unevenly distributed across the expressed genome which is similar to HIV-1-specific responses (17, 18, 20, 33) .
  • Gag is among the most immunogenic region of the virus (20) and preferential targeting of Gag (18) , especially by CD8 T cells (33, 34) , is associated with enhanced control of viral replication.
  • the region of HIV-I Gag corresponding to peptide 46 (SEQ ID NOS: 18-21) contributes to the total immune response in HIV-I infection in terms of frequency of recognition (35) , immunodominance (18, 36), and avidity (22) .
  • This frequently targeted and highly immunogenic region of Gag can be restricted by both CD4 (21, 37) and CD8 (35) T cells as well as by a variety of HLA haplotypes (38) . However this is the first time that this region has been shown to be associated with control of viral replication.
  • Gag 298 - 3 i 5 in HIV-2 is located in area of the virus equivalent to the HIV-I capsid (CA) protein. More precisely, the 18 amino acid-long sequence of the peptide overlaps with the Major Homology Region (MHR) , a conserved stretch of 20 amino acids found in the carboxyl-terminal domain of the capsid protein.
  • MHR Major Homology Region
  • the MHR is highly conserved across all retroviruses and is essential for viral assembly, maturation, and infectivity and its deletion impairs membrane binding, vira . l particle formation, and correct assembly of the viral core (39-41) .
  • a vital aim of HIV vaccine design is eliciting antigen- specific CD8 T cell responses, thus it is important that vaccine constructs include areas of the viral genome that are often recognized by antigen-specific T cells, are restricted by various HLA alleles, and are resistant to escape mutations.
  • the capsid region of Gag represents such an area and since peptide 46 is located in this region, it is crucial to further examine the function and phenotype of peptide 46-specific T cells in the context of natural infection and to include this peptide in future CTL- inducing vaccines.
  • Comprehensive analysis of HIV-2 -specific immune responses has demonstrated that a narrow part of the proteome is most often targeted by functional, IFN- ⁇ -producing T cells.
  • the capsid region of Gag may be better processed by the host and more effectively presented on MHC molecules relative to other areas of the viral proteome. If this is the case, it would support the prior finding that CTL effectiveness critically depends upon epitope density (44) . It is also possible that antigens from this region may be more available for presentation since capsid is expressed at higher levels compared to other parts of viral genome (45) . Also, the ample capsid from incoming virions may provide abundant substrate for antigen processing without requiring de novo protein synthesis early after viral entry (46) . Regardless, the capsid area of HIV-I and HIV-2 Gag represents an equilibrium between the host immune response and the virus functional constraints.
  • Immunodominance hierarchy of HIV-I epitopes is determined early in infection and is seldom modified in chronic infection (23) . Thus it is likely that responses to conserved parts of the genome, such as the capsid region of HIV-2 Gag, are made early in the course of infection and are maintained for many years post -infection. To address the question of maintenance of antigen-specific responses over time, individual peptide responses after 6-12 months were first examined. It was found that responses observed in the initial screen were preserved in the follow-up sample collection. Whether the length of infection influences the ' specificity of immune responses was then tested.
  • the Gag and Nef proteins are consistently identified as the most frequently targeted parts of the HIV-I proteome and responses to Nef contribute to almost one-third of the response to the expressed genome (17, 18, 20) . Therefore, the paucity of HIV-2 Nef-specific immune responses observed in this study was unexpected. In fact, the only difference in HIV-I and HIV-2 immune response specificity is in the recognition of Nef (29) . To rule out the possibility that the sequence of peptides used in this study did not accurately represent the Nef sequences of circulating viral strains, Nef sequences generated from HIV-2 -infected members of the cohort (Jerome Feldmann, manuscript in preparation) were compared to the Nef consensus sequence used for the synthesis of overlapping peptides.
  • HIV-2 Nef is longer (257aa and 34kDa versus 208aa and 27kDa) and the sequence homology between the two viral consensus sequences has only a 30.9% identity (http://hiv-web.lanl.gov) . It is possible the sequence variation could affect a combination of epitope processing, MHC binding and epitope presentation, or T cell receptor recognition of the peptide-MHC complex thereby altering HIV-2 Nef-specific immune responses. An alternate possibility is that the level of Nef in controlled HIV-2 infection is insufficient for adequate immunogenicity.
  • IFN- ⁇ secretion is antigen-specific, it is not the only cytokine secreted upon antigen exposure. Therefore, further studies are being conducted to determine the functional profile of antigen-specific cells identified during the cross-sectional analysis of HIV-2 -specific immune responses.
  • the IFN- ⁇ ELISpot assay does not discriminate the subset of lymphocytes that contribute to the antigen- specific cytokine secretion.
  • the inventors have shown that in HIV-2 infection, robust IFN- ⁇ immune responses made by antigen- specific T cells are an important determinant of control of viral replication and disease outcome. Furthermore, immune responses mounted to a narrow and highly conserved region of the HIV-2 capsid region of Gag are a strong marker for control of HIV-2 viremia. It has been demonstrated that both the recognition of this region as well as the magnitude of the IFN- ⁇ response mounted by antigen-specific T cells significantly correlate with low viral loads in HIV-infected subjects. The findings suggest that cellular mediated immunity is far better preserved in non-progressive HIV-2 -positive patients, implying that the immune .system strives to control chronic HIV-2 infection. References
  • Plasma RNA viral load predicts the rate of CD4 T cell decline and death in HIV-2-infected patients in West Africa. Aids 14:339- 344.
  • HIV-I anti-human immunodeficiency virus type 1

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Abstract

L'invention porte sur un polypeptide isolé constitué de 9 à 149 acides aminés contigus de la séquence polypeptidique Gag du VIH représentée dans la SEQ ID NO : 2 à 6 ou sur un équivalent fonctionnel de celui-ci, ou sur une séquence présentant une identité d'au moins 60 % audit polypeptide.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012016713A1 (fr) * 2010-08-05 2012-02-09 Ruprecht-Karls-Universität Heidelberg Ciblage de tumeurs à l'aide de polypeptides
WO2015127201A1 (fr) * 2014-02-21 2015-08-27 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Acides nucléiques du vih-2 et leurs procédés de détection

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012016713A1 (fr) * 2010-08-05 2012-02-09 Ruprecht-Karls-Universität Heidelberg Ciblage de tumeurs à l'aide de polypeptides
WO2015127201A1 (fr) * 2014-02-21 2015-08-27 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Acides nucléiques du vih-2 et leurs procédés de détection

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