WO2005075679A2

WO2005075679A2 - Antigenic hiv-1 peptides

Info

Publication number: WO2005075679A2
Application number: PCT/IT2005/000058
Authority: WO
Inventors: Massimo Amicosante; Giulia Cappelli; Vittorio Colizzi; Roberta D'arrigo; Francesco Dieli; Carlo Federico Perno; Alessandra Sacchi; Alfredo Salerno; Luc Montagner; Henri Chenal; Thomas Toni; Jacques Simpore
Original assignee: Universita'degli Studi Di Roma 'tor Vergata' (70%); Universita'degli Studi Di Palermo (20%); Consiglio Nazionale Delle Ricerche (10%)
Priority date: 2004-02-09
Filing date: 2005-02-08
Publication date: 2005-08-18
Also published as: WO2005075679A3; ITRM20040066A1

Abstract

The invention refers to a method of antigenic peptide identification and to the relative use for the preparation of a specific vaccine against HIV-1 for the populations of sub-Saharan Africa.

Description

METHOD OF ANT1GEN1C PEPTIDE IDENTIFICATION AND RELATIVE USE FOR THE PREPARATION OF A VACCINE ANTI HIV-1

The present patent concerns a method of antigenic peptide identification and the relative use for the preparation of a specific vaccine against HIV-1. Specifically, the invention refers to a method of antigenic peptide identification and to the relative use for the preparation of a specific vaccine against HIV-1 for the populations of sub-Saharan Africa. The virus of the human acquired immunodeficiency syndrome

(HIV) belongs to the family of the Retroviridae (sub-family of the lentivirus).

Like all the retroviruses, HIV has three essential structural genes for the replication of the virus, named: gag (group specific antigen), pol (polymarase) and env (envelope). Moreover, in its genome the information are present for codify other proteins with regulatory functions (tat, rev, nef, vif, vpr, vpu). In particular, the gag gene codes for a precursors protein that is subsequently cut through mechanisms of proteolysis in various structural components of the virion. The genes nef, tat, vpr and vpu codify for proteins with regulatory functions whose protein are respective of the 25- 27 kDa, 14-16 kDa, 10-15 kDa and 16 kDa. They present multiple functions that are essential to the vital cycle of the virus. In the control of the HIV-1 infection a fundamental role is played by the CD4+ and CD8+ T-lymphocytes specific for the HIV-1 virus. In fact, the CD8+ T-cells play an important function in the control of viremia (McMichael AJ, Rowland-Jones SL. Cellular immune response to HIV. Nature 2001 , 410:980-987), and also in the prevention of the infection in subjects with high degree of exposure to HIV-1 virus (Kaul R, Plummer AGO, Kimani J, Dong T, Kiama P, Rostron T, Njagi and, MacDonald KS, Bwayo JJ, McMichael AJ, Rowland-Jones SL. J Immunol. 2000; 164: 1602-11.). These cells represent the main cellular population with CTL activity able to remove the cells infected with the virus of the HIV. The complete maturation and activation of these lymphocytes, therefore the cytotoxic activity itself, is closeiy dependent from the "help" supplied by the activation of CD4+ T-lymphocytes. The correct and complete activation of both these T-cell populations is therefore at the base of an adequate control of the HIV infection. In fact, HIV-positive subjects defined "long term not progressors", although resulting chronically infected with the virus do not progress towards the AIDS, present an elevated capability to respond in vitro to HIV-1 antigen both with CD4 and CD8 T-cells (Gea-Banacloche JC, Migueles KNOWS, Martino L, Shupert WL, McNeil AC, Sabbaghian MS, Ehler L, Prussin C, Stevens R, Lambert L, Altman J, Hallahan CW, de Quiros JC, Connors M. J Immunol 2000, 165:1082-1092). This CD4+ and CD8+ immune response against the HIV is present also in the subjects that, in spite of the elevated degree of exposure to virus the HIV, do not remain infected (Rowland-Jones SL, Dong T, Fowke KR, Kimani J, Krausa P, Newell H, Blanchard T, Ariyoshi K, Oyugi J, Ngugi E, Bwayo J, MacDonald KS, McMichael AJ, Plummer FA. J Clin Invest 1998, 102:1758-1765). Therefore, the correct and complete activation of the CD4+ and CD8+ lymphocytes immune response directed against HIV virus represents an useful instrument for the control of the progression of the disease and the prevention of the transmission. T-lymphocytes recognizes their antigen in the context of the histocompatibility molecules (MHC-restriction). In fact, the minimal condition for a peptide to be defined as T-lymphocytes antigen, thus able to be recognized and to activate T-cells, is its ability to bind to the Major Histocompatibility Complex (MHC) molecules. Therefore in the design and selection of whichever antigen a strong limitation is determined by the restriction imposed from the HLA of the subject, so that the subject itself can develop or not an immune response (McMichael AJ, Rowland-Jones SL: Cellular immune response to HIV. Nature 2001 , 410:980-987; Walker BD, Korber BT: Immune control of HIV: the obstacles of HLA and viral diversity. Nature Immunol 2001 , 2:473-475). The genes coding for the molecules of human MHC molecules are the most polymorphic in the genome and almost all the the differences between the single alleles of these molecules are localized in one area that interact with the antigenic peptide, determining the binding properties of the different HLA molecules. Moreover, in the context of the specific immune response of

CD4 and CD8 T-lymphocytes in HIV infection is important to consider that the same antigenic peptide (also named epitope) of the HIV-1 recognized in the context of an allele of the HLA is not always recognised by al! the subjects that carry that allele, while the immune response answer can be directed against others epitopes recognised in the context of the same allele or recognised by others HLA aileli of the subject (Betts MR, Casazza JP, Patterson BA, Waldrop S, Trigona W, Was TM, Kern F, Picker LJ, Koup RA. J Virol 2000, 74:9144-9151). Moreover, almost the totality of the HIV-1 infected subjects presents a specific CD8+ T-cell response directed against various proteins of HIV virus recognised in the context of all the subject's HLA class I loci alleles (HLA-A, HLA-B, HLA-C) (Casazza JP, Betts MR, Picker LJ, Koup RA. J Virol 2001 , 75:6508-6516; Gray C, Lawrence J, Schapiro JM, Altman JD, Winters MA, Crompton M, Loi M, Kundu SK, David MM, Merigan TC. J Immunol 1999, 162:1780-1788; Brander C, Goulder PJR. In: HIV molecular immunology database. Edited by Korber BTM, Brander C, Haynes B, Koup RA, Kuiken C, Moore J, Walker B, Watkins D. Los Alamos National Laboratory, Los Alamos, N.Mex. 2000; 11-13). Beyond to this limit imposed by the immunogenetic background of the host, or of the population target in its complex, for the development of a vaccine directed to the control of the HIV-1 infection, the situation is ulteriorly complicated from the elevated variability of HIV-1 virus. In fact, under the pressure of the immune system, the virus is in continuous evolution and mutation with new emergent strains, particularly in those areas where the spread of the infection is greater and the control guaranteed from the pharmaco-therapy is limited or absent like in the underdeveloped nations. At the present, it is not available a system to induce an effective immuno-protecting response against HIV. In particular this is not available for the area where the virus is most diffused such as the populations of the western sub-Saharan area. In the context of the problems exposed above, it appears evident the need of new antigenic peptides able to induce a specific CD4+ and CD8+ protective immune response to be used in the preparation of an effective and specific anti-HIV-1 vaccine. The authors of the present invention have put into effect a method for the identification of antigenic peptides, everyone containing one or more epitopes that can be used in the preparation of a vaccine against HIV-1 circumventing the limits described before due to the variability of the virus and the immunogenetic background of the host. The protein sequences of the HIV-1 virus reported in the invention are subject to one reduced variability for their structural/functional critic role in the biological cycle of HIV-1. Therefore, using the conserved areas of the HIV-1 viruses circulating in the geographic area in which it is wanted to design the vaccine, it is possible to define the aminoacidic sequences that can be able to induce an effective immunity response against the virus, avoiding the classics escape mechanisms that the HIV-1 virus puts in action in order to circumvent the immune response of the host. In order to obtain a width and accurate activation of the specific CD4+ and CD8+ T-cells directed against the HIV-1 virus, it is necessary to use the highest number possible of peptide epitopes, ideally able to cover all the peptide set that can be presented by the HLA alleles of a single subject. The greater problems in the design of immunogenes able of inducing a CD4 and CD8 specific immune response against HIV-1 virus, or more in a generalized manner against viruses or the other pathogens that present a high degree of genomic variability in the species, are represented by the restriction of the subject's HLA and from the variability of HIV-1. The method for the identification of immunogenes based on the invention can be summarised in following steps: a) identification of the conserved genomic areas of the etiological agent in examination; b) identification of CD4 and CD8 T-cell epitopes in the conserved genomic areas of the etiological agent in examination; c) design of the antigenic peptides based on the allelic frequency of the target population. About phase a), since the HIV-1 viruses can be grouped in several clades based on the genomic sequences of the virus in examination, it is possible to identify inside the clades some areas of the genome highly conserved to the aim to identify effective immunogens. In fact, the immunogens designed in these areas will have a reduced probability to endure the phenomena of "escape", a typical process due to mutations in the areas containing the peptide fragments that work as T-cell epitopes. For this purpose, the direct sequencing of the viral genome(s) from biological samples coming from the geographic area where the vaccine is destined is of fundamental importance for the identification of circulating variants of the virus in the area of interest. The sequencing data obtained can be analyzes by bio-informatic software for the analysis of the sequences. In particular, the nucleotidic sequences can re-assembled and translated into the aminoacidics sequences for each gene in examination, which can be aligned through CLUSTALW algorithm by using the Antheprot software (http://antheprot-pbil.ibcp.fr) in order to identify the most conserved areas of the proteins. As pointed out before, the phase b) of the method for the preparation of immunogens based on the invention consists in the identification of CD4 and CD8 T-cell epitopes in the most conserved genomic areas of the etiologic agent in evaluation. The biological role of the HLA molecules is to bind peptides derived from cellular proteins (HLA class I) or from the degradation of protein ingested from the cell (HLA class II molecules) and to "present" these peptide fragments to CD8 and CD4 T-cells. Therefore, the minimal condition so that a peptide can be considered antigenic is that it is able to bind to one HLA molecule. Such binding ability depends on the relative affinity of HLA allelic variant for the peptide (also called "the peptide binding motif). Therefore, using the most conserved areas of proteins coming from the analyses carried out in the phase a), the authors of the present invention have identified in these protein sequences the presence of "peptide binding motif for HLA class I and II by using the "Epitope motif search facility" of the "HiV-lmmunology Database" (http://hiv-web.lanl.gov/immunology/) for the some serotypes HLA of class I and II corresponding to 281 different alleles of the HLA class I and 236 alleles of HLA class II. Subsequently, these most conserved areas containing the greater peptide number of "binding motifs" can be analysed through algorithms of quantitative type, such as SYFPEITHI (http://www.syfpeithi.de);BIMAS(http://bimas.dctr.nih.gov/molbio/hla_bi nd/);Propred(http://www.imtech.res.in/raghava/propred/);Propredll(http:

//www.imtech.res.in/raghava/propredl/); HLA Ligand/motif database (http://hlaligand.ouhsc.edu/prediction.htm) with the aim to predict and supply a peptide binding score for each single epitope for all the HLA class I and II alleles present in the five databases. Herein we defined the most conserved area of one protein as a region in which the amino-acidic positions that present mutations are less than 5-10% and the mutations presents have a degree of homology of at least 50% (based on the specific characteristics of the aminoacids). Once carried out the identification of T-cell epitopes, the analysis proceeds with phase c) of the identification method, selecting the epitopes based on the distribution of the HLA alleles most frequents in the population target of the vaccine. To this end, taking into account the distribution of the HLA class I and II alleles in the target, is possible to design peptides of 15-

17 amino acids everyone containing one or more than one HLA class I and ll epitope(s). Since each one of these epitopes can bind to HLA molecules with several degree of affinity, is fundamental that: (i) the single chosen peptides presents an ability to bind a determined HLA allele with at least 30% of the maximum ability to the binding for that allele; (ii) that in theirs all the peptides together cover at least 90% of the allelic variants presents in the population target of the vaccine. The maximum binding ability of one HLA allele is defined as the maximum result supplied from a test algorithm in predicting the binding of a peptide for that determining allele. All the others results for that HLA allele can be expressed like the percentage of this maximum binding capability. Finally, it is fundamental to estimate the antigenicity of selected peptides. In order to be used as immunogens in the human population for inducing a CD4 and CD8 HIV-1 specific T-cell response, these peptides cannot be evaluated for their antigenic property in animal models. An indirect method to determine the immunogenicity of these selected peptides is to evaluate if they are recognized in subjects coming from the same geographic area target of the vaccine carrying the infection with the etiologic agent in examination. Therefore, according to the method of the present invention, the designed peptides have been used to determine the number of peptide(s)-specific CD4+ or CD8+ T-cells (able to produce IFN-gamma as a result of stimulation) through specific tests such as Intracellular Staining (ICS). In order to set up an antigen able to activate the immune response of CD4+ and CD8+ T-cells specific for HIV-1 in particular in populations of Western sub-Saharan Africa, the authors of the present invention have carried out a study with the end to identify peptides of the HIV-1 gene products gag, nef, tat, vpr and vpu suitable to the be used for the activation/identification of HIV-1 specific CD4+ and CD8+ T-cells. Thus, it is object of the present invention a method for the identification of immunogenanti-HIV-1 antigenic peptides for a target population, said method comprising the following steps: a) identification of the most conserved genomic areas of the HIV-1 virus of the Hiv-1 , belonging to the proteins gag, nef tat, vpr and vpu, by using, amplification of the genome sequences and subsequent nucleic acid sequencing; b) identification of CD4 and CD8 T-cell epitopes in such genomic areas; c) construction, on the basis of the distribution of the HLA class I and ll allelic variants in the target population, of antigenic peptides comprising at least one CD4 and CD8 T-cell epitope, said antigenic peptides being characterized by a binding capability at least equal to 30% of the maximum binding ability for that HLA allele.

In a particular embodiment of the aforesaid method, can be addressed as an example to a target population such as the population of sub-Saharan and central western Africa. In this specific case, the genomic amplification of the phase a) of the method according to the invention can be done by using at least one couple of primers selected from the following group of sequences:

SEQ ID No 1 : 5'GCC TCA ATA AAG CTT GCC TT 3';

SEQ ID No 2: 5'CCA ATT CCC CCT ATC ATT TTT 3';

SEQ ID No 3: 5'CCC TAA AAA ATT AGC CTG TCT 3';

SEQ ID No 4: 5'CAA TTT TAA AAG AAA AGG GGG GAT 3'; SEQ ID No 5: 5'CCA TGT TAT TTT TCC ACA TGT TAAA 3';

SEQ ID No 6: 5'GAA AGG TGA AGG GGC AGT A 3';

SEQ ID No 7: 5'GCT TCT TCC TGC CAT AGG AGA T 3';

SEQ ID No 8: 5OTT TTT AAA AGA AAA GGG GGG A 3'; SEQ ID No 9: 5'CTC AAG GCA AGC TTT ATT GA 3'; SEQ ID No 10: 5'GCT GCT TAT ATG CAG CAT CT 3' or complementary sequences thereof. In the phase a) of the method of the present invention addressing the population of sub-Saharan Africa, the sequencing can be performed by using at least one couple of primers selected from the following group of sequences:

SEQ ID No 11: 5' GAC TAG CGG AGG CTA GAA 3';

SEQ ID No 12: 5' GGG GTG GCT CCC TCT GAT AA 3'; SEQ ID No 13: 5' CCA GAA GTA ATA CCC ATG TT 3'; SEQ ID No 14: 5' CCT GAC ATG CTG TCATCAT 3'; SEQ ID No 15: 5' GAT GGA AAG AGC CCC AGA 3'; SEQ ID No 16: 5' GCT TCT TCC TGC CAT AGG AGAT 3"; SEQ ID No 17: 5' GAG CCC TGG AAC CAC CCA 3'; SEQ ID No 18: 5' CTG TGG GTA CAC AGG CAT 3'; SEQ ID No 19: 5' CTT TTT AAA AGA AAA GGG GGG A 3'; SEQ ID No 20: 5' GCT GCT TAT ATG CAG CAT CT 3'; or complementary sequences thereof. According to a preferred embodiment of the method of the present invention, the HLA class I allelic variants correspond to the following serotypes: A1 , A10, A11 , A19, A2, A2.1 , A23, A24, A25, A26, A28, A29, A3, A3.1 , A30, A31 , A32, A33, A34, A35, A36, A43, A66, A68, A69, A74, A9, B12, B13, B14, B15, B16, B17, B18, B22, B24, B27, B35, B37, B38, B39, B40, B41 , B42, B44, B45, B46, B47, B48, B49, B5, B50, B51 , B52, B53, B54, B55, B56, B57, B58, B59, B60, B61 , B62, B63, B64, B65, B67, B7, B70, B71 , B72, B73, B75, B76, B77, B78, B8, B81 , Bw52, Bw57, Bw60, Bw62, C4, Cw1 , Cw10, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7, Cw8, Cw9; and the HLA class II allelic variants correspond to the following serotypes: DPw1 , DPw2, DPw3, DPw4, DPw5, DPw6, DQ1 , DQ2, DQ3, DQ4, DQ5, DQ6, DQ7, DQ8, DQ9, DR1 , DR10, DR11 , DR12, DR13, DR14, DR15, DR16, DR17, DR18, DR2, DR3, DR4, DR51 , DR52, DR53,

DR6, DR7, DR8, DR9. The method for the identification of antigenic peptides against

HIV-1 can further comprises a phase d) of determination of the immunogenicity of the designed antigenic peptides from phase c) through indirect ex vivo and in vitro tests in HIV-1 infected subjects. The HIV-1 antigenic peptides, that can be identifies by the method described in the present invention, constitute ulterior object of the present invention: SEQ ID No 21: EKIRLRPGGKKKYRL;

SEQ ID No 22: PGGKKKYRLKHLVWA;

SEQ ID No 23: KKYRLKHLVWASREL;

SEQ ID No 24: RELERFALNPGLLET;

SEQ ID No 25: EELKSLYNTIATLWC; SEQ ID No 26: SLYNTIATLWCVHQR;

SEQ ID No 27: SPEVIPMFTALSEGA;

SEQ ID No 28: QDLNMMLNIVGGHQA;

SEQ ID No 29: MMLNIVGGHQAAMQML;

SEQ ID No 30: GEIYKRWIVLGLNKI; SEQ ID No 31 : IVRMYSPVSILDIRQ;

SEQ ID No 32: SDIAGTTSTLQEQIG;

SEQ ID No 33: TLQEQIGWMTSNPPI;

SEQ ID No 34: EQIGWMTSNPPIPVG;

SEQ ID No 35: SILDIKQGPKEPFRD; SEQ ID No 36: EPFRDYVDRFFKTLRA;

SEQ ID No 37: RFFKTLRAEQATQEV;

SEQ ID No 38: DLWVYHTQGFFPDWQ;

SEQ ID No 39: FFPDWQNYTPGPGTRF;

SEQ ID No 40: NYTPGPGTRFPLTFG; SEQ ID No 41 : GTRFPLTFGWCFKLVP;

SEQ ID No 42: ANEGENNCLLHPVCQ;

SEQ ID No 43: NCYCKMCCWHCQLCF;

SEQ ID No 44: CCWHCQLCFLTKGLG; SEQ ID No 45: LCFLNKGLGISYGRK;

SEQ ID No 46: LGISYGRKKRRRRRG;

SEQ ID No 47: SLEIAAIVGLVVAFIA;

SEQ ID No 48: GLVVAFIAAIVVWTl; SEQ ID No 49: EYRKIRKQKKIDKLL;

SEQ ID No 50: DRIRERAEDSGNESDG;

SEQ ID No 51 : KHEAVRHFPRPWLHGL;

SEQ ID No 52: DTWEGVEAIIRILQQLL;

SEQ ID No 53: TYGDTWEGVEAIIRI; SEQ ID No 54: LFVHFRIGCQHSRIGI. The aforesaid antigenic peptides comprise in the sequence at least one CD4 and CD8 T-cell epitope with a binding ability at least equal to 30% of the maximum binding ability for the HLA class I and II allelic variants. According to a further aspect, the present invention has for object the use of antigenic peptides for the preparation of a anti-HlV-1 specific vaccine, preferentially a paediatric vaccine, for the population of sub-Saharan Africa. Finally it is object of present invention an anti-HIV 1 vaccine identifiable by using the above-mentioned method, comprising at least 16 antigenic peptides with one or more pharmacologically acceptable adjuvants and/or excipients. Preferentially, the anti-HlV-1 vaccine is specific for the population of sub-Saharan and western central Africa and can comprise from 16 (SEQ ID No 21-26, SEQ ID No 29-30, SEQ ID No 38-41 , SEQ ID

No 44-45, SEQ ID No 47 and SEQ ID No 52) to 34 (SEQ ID No 21-54) of selected antigenic peptides from the group that comprises SEQ ID No 21-

54, like active principles, with one or more pharmacologically acceptable adjuvants and/or excipients. The optimal formulation of the vaccine based on the present invention comprises 24 antigenic peptides, preferentially SEQ ID No 21-

31 , SEQ ID No 38-41 , SEQ ID No 43-49 and SEQ ID No 51-52; such vaccine being particularly indicated for paediatric use. In a preferable formulation of the vaccine the antigenic peptides used as active principles of the vaccine itself, according to the present invention, can be conjugated to apolar and lipophil compounds or with glucids. In fact, the use of peptide conjugates to lipophil or apolar compounds or with glucids determine increase probability that such peptides are presented by antigen presenting cells and to reduce the clearance in the site of the inoculums (Andrieu M, Desoutter JF, Loing E,

Gaston J, Hanau D, Guillet JG, Hosmalin A. Andersen P, Munk ME, Pollock JM, Doherty TM: Two human immunodeficiency virus vaccinal lipopeptides follow different cross-presentation pathways in human dendritic cells. J Virol. 2003; 77: 1564-70; Hertz CH, Kiertscher SM, Godowski PJ, Bouis DA, Norgard MV, Roth MD, Modlin RL: Microbial Lipopeptides stimulate dendritic sell maturation via toll-like receptor2. J. Immun 2001 , 166 : 2444-2456). The present invention will be now described at illustrative but not limitative title, based on its preferred form of realization, with particular reference to the figures of the designs enclosed, in which: figure 1 shows the outline of the amplifications and the sequencing carried out on the viral variant of HIV-1 prevailing of in the HIV-seropositive subjects coming from sub-Saharan and western centre Africa; figure 2 shows the capability from part of T-lymphocytes obtained from HIV-positive subject from Western and sub-Saharan Africa to respond to the selects peptides in example 1.

Example 1 : Identification of antigenic peptides for the anti-HlV vaccination for sub-Saharan Africa

The complete nucleotidic sequence of the viral variant prevalent in the subject under examination for the genes gag and to the regulatory genes tat, vpr, vpu and nef it has been obtained in HIV-1 positive subjects coming from centre-western and sub-Saharan Africa through retrotrascription and amplification of the viral RNA by RT-PCR and subsequent sequencing of the genomic fragments amplified as outlined in figure 1 with primers for amplification and the sequencing reported in table

1.

The primers used in the PCR reactions and sequencing are not described in literature and have been purposely design for the determination of all the sequence of the genes target of the anti-HIV-1 vaccine based on antigenic peptides.

In particular, for their construction have been considered various parameters:

- the choice of the regions to use as primer it has been estimated evaluating the areas mainly conserved in the genome of HIV-1 as well as the sequences of various seropositive subjects for the virus and belonging to different subtypes than that of laboratory clones (HXB2 and NL43 strains of HIV deposited in the data bank of HIV of the laboratories of Los Alamos) in the external areas of the genes target of the anti-HIV vaccine; - the starting sequence of the primer always presents a guanine or a cytosine in order to have a more efficient process of annealing;

- the percentage of GC bases in every primer is always comprised between the 40 and 60% in order to render the chemically synthetized oligonucleotide sequence itself more stable; - the primers have been designs with a number of bases always comprised between 18 and 30 to the aim of obtaining temperatures of melting and annealing not too much high and not too much similar between them;

- in every oligonucleotide has been avoided both at the 5' and at the 3' complementary sequences to diminish the formation of dimers and secondary structures that can interfere in obtaining the PCR products and in the reaction of sequencing. More specifically:

- the area of the gag gene has been amplified by using the primers SEQ ID No 1 and SEQ ID No 2 (1857 bps in the sequence of reference of Hiv-

1), and in case of insufficient amplification re-amplified through primer SEQ ID 1 and SEQ ID 3 (1557 bps in the sequence of reference of Hiv-1) by using an high fidelity DNA polymerase (errors below to 1x10^"5 amplified bases) in order to do not introduce ulterior variability in the amplicon

(figure 1 and table 1 ).

- The obtained amplicon has been sequenced with the primer of sequence SEQ ID No 11 , SEQ ID No 12, SEQ ID 13 and SEQ ID 14 in order to cover all the amplified area and reading to the DNA strand in the two senses as an ulterior control (figure 1 and table 1 ).

- The area comprising the genes tat, vpr and vpu has been amplified with primer SEQ ID No 4 and SEQ ID No 5 (1700 bps in the HIV-1 reference sequence), and in case of insufficient amplification re-amplified through primer SEQ ID 6 and SEQ ID 7 (990 bps in the HIV-1 reference sequence) by using an high fidelity DNA polymerase (errors below to 1x10^"5 amplified bases) in order to do not introduce ulterior variability in the amplicon (figure 1 and table 1).

- The obtained amplicon has been sequenced with the primers of sequence SEQ ID No 15, SEQ ID No 16, SEQ ID 17 and SEQ ID 18 in order to cover all the amplified area and reading to the DNA strand in the two senses as an ulterior control (figure 1 and table 1 ).

- the area of the gene nef has been amplified with primer SEQ ID No 8 and SEQ ID No 9 (550 bps in the HIV-1 reference sequence), and in case of insufficient amplification re-amplified through primer SEQ ID 8 and SEQ ID 10(430 bps in the HIV-1 reference sequence) by using an high fidelity DNA polymerase (errors below to 1x10^"5 amplified bases) in order to do not introduce ulterior variability in the amplicon (figure 1 and table 1).

- The obtained amplified has been sequenced with primers of sequence SEQ ID 19 and SEQ ID 20 in order to cover all the amplified area and reading to the DNA strand in the two senses as an ulterior control (figure 1 and table 1).

Table 1 reports the sequences of the primer are for the RT-PCR amplification and sequencing; the numbers between parenthesis close to every primer of sequencing indicate the start nucleotide of the primer referred to the HIV-1 reference sequence.

The obtained nucleotidic sequences have been re-assemblated and translated into amino-acidic sequences for each one of the gene in examination, and have been aligned through CLUSTALW algorithm using the Antheprot software (http://antheprot-pbil.ibcp.fr). δ The most conserved areas of the proteins Gag, Nef, Tat, Vpr and Vpu, have been therefore analysed for the presence of "peptide binding motif for HLA class I and II by using the "Epitope motif search facility" of the "HIV-lmmunology Database" (http://hiv- web.lanl.gov/immunology/) for following the 94 sierotipi HLA of class I:0 - A1 , A10, A11 , A19, A2, A2.1 , A23, A24, A2δ, A26, A28, A29, A3, A3.1, A30, A31 , A32, A33, A34, A3δ, A36, A43, A66, A68, A69, A74, A9, B12, B13, B14, B15, B16, B17, B18, B22, B24, B27, B35, B37, B38, B39, B40, B41 , B42, B44, B45, B46, B47, B48, B49, Bδ, BδO, B51 , B52, B53, Bδ4, B55, Bδ6, Bδ7, Bδ8, Bδ9, B60, B61 ,5 B62, B63, B64, B65, B67, B7, B70, B71 , B72, B73, B7δ, B76, B77, B78, B8, B81 , Bw52, Bw57, Bw60, Bw62, C4, Cw1 , Cw10, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7, Cw8, Cw9; and for the 36 sierotipi HLA of class II: - DPw , DPw2, DPw3, DPw4, DPw5, DPwδ, DQ1 , DQ2, DQ3, DQ4,0 DQ5, DQ6, DQ7, DQ8, DQ9, DR1 , DR10, DR11 , DR12, DR13, DR14, DR15, DR16, DR17, DR18, DR2, DR3, DR4, DR51 , DRδ2, DRδ3, DR6, DR7, DR8, DR9; correspondents to 281 different alleli of the HLA of class and the 236 alleli of class II. Consequently, the most conserved areas of the proteinsδ containing the greater number of "peptide binding motifs" have been analysed through algorithms for "peptide binding motifs" of quantitative type: - SYFPEITHI (http://www.svfpeithi.de); - BIMAS (http://bimas.dctr.nih.gov/molbio/hla bind/)0 - Propred (http://www.imtech.res.in/raghava/propred/) - Propred II (http://www.imtech.res.in/raqhava/propred1/) - HLA Ligand/motif database (http://hlaligand.ouhsc.edu/prediction.htm) With the end to predict and supply one binding score to every epitopes for all the HLA class I and II alleles present in the δ databases. In table 2 they are listed the CD8 and CD4 T-cell epitopes (restricted for HLA class I and II respectively) present in the antigenic peptides selected for the anti-HIV-1 vaccine.

Table 2

2δ

A subsequence analysis using the PAProC software (http://www.paproc.de) has been carried out in order to identify the areas of cut of the cellular proteosome with the aim to design in the most correct way the overlapping peptides. At the end of the analysis, taking into account the allelic distribution of the HLA class I and II in the sub-Saharan populations, it has been designed 24 peptides of 16-17 amino acids everyone containing one or more epitope(s) putatively able to bind HLA class I and/or II, with a minimum of 30% of the maximum ability to the binding for every allele evaluated, that in theirs entirety cover at least a frequency of the 90% of the allelic variants presents in the Sub-Saharian and west African populations. The details of the antigenic peptides including their binding property to the HLA coming from several algorithms used in the selection process are reported in table 3.

Table 3

Example 2: Procedure of the citofluorimetric test for the determination of the number of precursors specific for the pool of peptides to be used in the anti-HIV vaccine in HIV-positive subjects. Systems able to induce an effective and protective immune response against HIV-1 object are not available yet. Thus, it is not possible to carry out a comparison of the performances of antigenic peptides of the present invention with other vaccine systems. In order to demonstrate the antigenicity of the peptide mixture object of the invention, it has been proceeded to the survey for the quantification of the specific CD4+ and CD8+ T-cells specific for the peptide mixture by using IntraCellular Staining (ICS) in HIV-positive subjects of Western and sub-Saharan Africa (figure 2). Although this test does not represent the proof of the peptide mixture ability to induce protection from the HIV-1 infection, having found the presence of lymphocytes that specifically recognize these peptides in HIV-positive subjects from the Western sub-Saharan area is index of their immunogenicity, indispensable minimal characteristic so that a vaccine can be effective. More specifically, the designed peptides have been synthesized and purified through inverse phase chromatography to a purity >90% and re-suspended in dimetilsulfoxid (DMSO) at the concentration of 10 mg/ml for each peptide. A mixture of antigenic peptides has been prepared containing an equi-concentration of the 24 synthesized peptides. Therefore, the peptide mixture therefore has been used to determine the number of HIV-specific CD4+ or CD8+ T cells able to produce IFN-gamma as a result of stimulation, in HIV-positive subjects of sub-Saharan and western Africa. Figure 2 shows an example of the number of lymphocytes able to respond to the peptide mixture in a HIV-positive subject respect to a subject HIV-negative used as control. In HIV-positive subjects a greater number of CD4+ and CD8+

T-cells that responds to the peptide mixture compared to control subject confirming the immunogenicity of the selected peptides. The following specific monoclonal antibodies (Becton Dickinson Immunocytometry Systems, Saint Jose, CA) have been used in the test: anti-CD28 and anti-CD49d purified for cell culture; anti-IFN-gamma conjugated with fluorescein (FITC); anti-CD3 conjugated with phycoeritrina (PE); anti-CD4 or anti-CD8 conjugated with the cianina-5-phycoeritrina (Cy-δ); and an isotypic control (lgG1) conjugated with FITC. The antibodies have been used at a concentration of 0,26 μg/ml. Peripheral blood mononuclear cells (PBMC) have been isolated from 10 millilitre of vein blood through gradient of density centrifugation (Ficoll-Hypaque, Pharmacia, Uppsala, Sweden). After 3 washings in Phosphate Buffer Saline (PBS) 1x, the cell pellet has been re-suspended to a concentration of 1x10⁶/ml in complete medium (RPMI 1640 with HEPES 25mM, 10% v/v FCS, 2mM L-Glutammina, 10 U/ml penicillina/streptomicina). Two test tubes have been prepared: One control (not stimulated and with mitogenic stimulus) and one containing the peptide mixture. In order to control the spontaneous production of cytokines, cells incubated with the co-stimuli anti-CD28 and -CD49d has been included in every test (control not stimulated). The release of IFN-gamma induced from PMA (50 ng/ml) plus ionomicina (10 μg/ml) has been used as positive control. The sample have been incubated at 37 °C for an hour and subsequently have been incubate for all the night in presence of 10 μg/ml of Brefeldin-A (Sigma, St. Louis, MO), an inhibitor of the cellular secretion. In order to execute the immunofluorescence staining the control and peptide mixture stimulated samples have been washed in buffer containing PBS 1 % of albumin fraction V (bovine serum albumin, BSA) and 0.1% of sodium azide. The cells have been washed two times in PBS, BSA 1 % and sodium azide 0.1% and stained and with the specific monoclonal antibodies mixture for the membrane antigens (CD3 and CD4 or CD8) previously described for 15 minutes at 4°C. The samples have been then fixed in paraformaldeide 1 % for 10 minutes at room temperature and incubate with the specific antibody against IFN-gamma diluted in a buffered solution composed by PBS 1X, BSA 1 % and saponine 0,5%. Finally, the cells have been washed two times in PBS 1X, BSA 1 %, saponine 0.1 % and re-suspended in PBS before being acquired through a citofluorimeter. The control of the not specific staining has been executed through monoclonal antibodies of the same isotype and the eventual presence of not specific staining has been subtracted from the results. The samples have been analysed with a citofluorimeter using a constant instrumental setting, obtained by means of daily gauging of the instrument with fluorescent beads. For each samples has been acquired in the region of the lymphocytes 2x10⁵ events, in order to guarantee an adequate representation of all the above cellular sub-populations (CD4 and CD8), allowing therefore to carry out statistical analysis.

Claims

1. Method for the identification of immunogen anti-HIV antigenic peptides for a target population, said method comprising the following steps: a) identification of the most conserved genomic areas of the HIV- 1 virus by using amplification of the genome sequences and the subsequent sequencing; b) identification of CD4 and CD8 T-cell epitopes in those genomic areas; c) construction, on the basis of the distribution of the HLA class I and II allelic variants in the target population, of antigenic peptides comprising at least one CD4 and CD8 T-cell epitope, said antigenic peptides being characterized by a binding capability at least equal to 30% of the maximum binding ability to that HLA allele.

2. Method according to Claim 1 , wherein the most conserved genomic areas are pertaining to proteins gag, nef, tat, vpr and vpu.

3. Method according to anyone of the claims 1 and 2, wherein said population target is the population of sub-Saharan and central- western Africa.

4. Method according to claim 3, wherein the amplification of the phase a) is made by using at least one couple of primers selected from the following group of sequences: SEQ ID No 1 : 5'GCC TCA ATA AAG CTT GCC TT 3'; SEQ ID No 2: δ'CCA ATT CCC CCT ATC ATT TTT 3'; SEQ ID No 3: δ'CCC TAA AAA ATT AGC CTG TCT 3'; SEQ ID No 4: δ'CAA TTT TAA AAG AAA AGG GGG GAT 3'; SEQ ID No δ: δ'CCA TGT TAT TTT TCC ACA TGT TAAA 3'; SEQ ID No 6: δ'GAA AGG TGA AGG GGC AGT A 3'; SEQ ID No 7: δ'GCT TCT TCC TGC CAT AGG AGA T 3'; SEQ ID No 8: δ'CTT TTT AAA AGA AAA GGG GGG A 3'; SEQ ID No 9: δ'CTC AAG GCA AGC TTT ATT GA 3';

SEQ ID No 10: 5'GCT GCT TATATG CAG CAT CT 3' or complementary sequences thereof. δ. Method according to anyone of claims 3 and 4, wherein the sequencing of the phase a) is made by using at least one couple of δ primers selected from the following group of sequences: SEQ ID No 11: 5' GAC TAG CGG AGG CTA GAA 3'; SEQ ID No 2: 5' GGG GTG GCT CCC TCT GATAA 3'; SEQ ID No 13: 5' CCA GAA GTAATA CCC ATG TT 3'; SEQ ID No 14: 5' CCT GAC ATG CTG TCA TCA T 3';0 SEQ ID No 15: 5' GAT GGAAAG AGC CCC AGA 3'; SEQ ID No 16: 5' GCT TCT TCC TGC CAT AGG AGA T 3'; SEQ ID No 17: 5' GAG CCC TGG AAC CAC CCA 3'; SEQ ID No 18: 5' CTG TGG GTA CAC AGG CAT 3'; SEQ ID No 19: 5' CTT TTT AAAAGA AAA GGG GGG A 3';5 SEQ ID No 20: 5' GCT GCT TATATG CAG CAT CT 3'; or complementary sequences thereof.

6. A method according to anyone of claims from 3 to 5, wherein said HLA allelic variants of class I correspond to the following serotypes: A1 , A10, A11 , A19, A2, A2.1 , A23, A24, A25, A26, A28, A29, A3, A3.1 , A30,0 A31 , A32, A33, A34, A35, A36, A43, A66, A68, A69, A74, A9, B12, B13, B14, B15, B16, B17, B18, B22, B24, B27, B35, B37, B38, B39, B40, B41 , B42, B44, B4δ, B46, B47, B48, B49, B5, BδO, B51 , B52, B63, B54, Bδδ, Bδ6, B57, Bδδ, B59, B60, B61 , B62, B63, B64, B65, B67, B7, B70, B71 , B72, B73, B76, B76, B77, B78, B8, B81 , Bwδ2, Bwδ7, Bw60, Bw62, C4, Cw1 , Cw10, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7, Cw8, Cw9.

7. A method according to anyone of the claims from 3 to 6, wherein said HLA allelic variants of class II correspond to the following serotypes: DPw1 , DPw2, DPw3, DPw4, DPwδ, DPw6, DQ1 , DQ2, DQ3, DQ4, DQ5, DQ6, DQ7, DQ8, DQ9, DR1 , DR10, DR11 , DR12, DR13, DR14, DR15, DR16, DR17, DR18, DR2, DR3, DR4, DR51 , DR62, DR53, DR6, DR7, DR8, DR9.

8. A method according to anyone of the previous claims, comprising a further phase d) of determination of the immunogenicity of the antigenic anti-HIV-1 peptides obtained in phase c).

9. Antigenic anti-HIV-1 peptides, identifiable by using the method δ according to anyone of the claims from 3 to 8, having aminoacid sequence: SEQ ID No 21 : EKIRLRPGGKKKYRL; SEQ ID No 22: PGGKKKYRLKHLVWA; SEQ ID No 23: KKYRLKHLVWASREL; 0 SEQ ID No 24: RELERFALNPGLLET; SEQ ID No 26: EELKSLYNTIATLWC; SEQ ID No 26: SLYNTIATLWCVHQR; SEQ ID No 27: SPEVIPMFTALSEGA; SEQ ID No 28: QDLNMMLNIVGGHQA; 5 SEQ ID No 29: MMLNIVGGHQAAMQML; SEQ ID No 30: GEIYKRWIVLGLNKI; SEQ ID No 31 : IVRMYSPVSILDIRQ; SEQ ID No 32: SDIAGTTSTLQEQIG; SEQ ID No 33: TLQEQIGWMTSNPPI;0 SEQ ID No 34: EQIGWMTSNPPIPVG; SEQ ID No 36: SILDIKQGPKEPFRD; SEQ ID No 36: EPFRDYVDRFFKTLRA; SEQ ID No 37: RFFKTLRAEQATQEV; SEQ ID No 38: DLWVYHTQGFFPDWQ;5 SEQ ID No 39: FFPDWQNYTPGPGTRF; SEQ ID No 40: NYTPGPGTRFPLTFG; SEQ ID No 41 : GTRFPLTFGWCFKLVP; SEQ ID No 42: ANEGENNCLLHPVCQ; SEQ ID No 43: NCYCKMCCWHCQLCF; SEQ ID No 44: CCWHCQLCFLTKGLG; SEQ ID No 45: LCFLNKGLGISYGRK; SEQ ID No 46: LGISYGRKKRRRRRG; SEQ ID No 47: SLEIAAIVGLVVAFIA; SEQ ID No 48: GLVVAFIAAIVVWTI;

SEQ ID No 49: EYRKIRKQKKIDKLL;

SEQ ID No 50: DRIRERAEDSGNESDG;

SEQ ID No 51 : KHEAVRHFPRPWLHGL; SEQ ID No 52: DTWEGVEAIIRILQQLL;

SEQ ID No 53: TYGDTWEGVEAIIRI;

SEQ ID No 54: LFVHFRIGCQHSRIGI; said antigenic peptides comprising at least one CD4 and CD8 T-cell epitope and having a binding ability at least equal to 30% of the maximum binding ability of said HLA class I or II alleles.

10. Use of antigenic peptides according to claim 9 for the preparation of a vaccine specific against HIV-1 for the population of sub-Saharan and western Central Africa.

11. Use according to claim 10, wherein the vaccine is a paediatric vaccine.

12. Anti-HIV-1 vaccine comprising at least 16 antigenic peptides identifiable by using the method defined in claims 1 and 2, as active principles, in association with one or more pharmacological acceptable adjuvants and/or excipients.

13. Anti-HIV-1 vaccine comprising from 16 to 34 antigenic peptides as defined in claim 9, as active principles, in association with one or more pharmacological acceptable adjuvants and/or excipients.

14. Vaccine according to claim 13, wherein 16 antigenic peptides are SEQ ID No 21-26, SEQ ID No 29-30, SEQ ID No 38-41 , SEQ ID No 44-45, SEQ ID No 47 and SEQ ID No 52.

15. Vaccine according to claim 13, wherein antigenic peptides are 24.

16. Vaccine according to claim 15, wherein the 24 antigenic peptides are SEQ ID No 21-31 , SEQ ID No 38-41 , SEQ ID No 43-49 and SEQ ID No 51-52.

17. Vaccine according to claims from 12 to 16, wherein the vaccine is for paediatric use.

18. Anti-HIV-1 vaccine according to claims from 12 to 17, wherein the antigenic peptides are conjugated with apolar or lipophil compounds or glucids.