AU608294B2 - Peptides having immunological properties 2-hiv-2 - Google Patents

Peptides having immunological properties 2-hiv-2 Download PDF

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AU608294B2
AU608294B2 AU12250/88A AU1225088A AU608294B2 AU 608294 B2 AU608294 B2 AU 608294B2 AU 12250/88 A AU12250/88 A AU 12250/88A AU 1225088 A AU1225088 A AU 1225088A AU 608294 B2 AU608294 B2 AU 608294B2
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peptide
ile
hiv
aat
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AU1225088A (en
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Marc Alizon
Lisa Chakrabarti
Francois Clavel
Ronald Desrosiers
Denise Guetard
Mireille Guyader
Luc Montagnier
Pierre Sonigo
Pierre Tiollais
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Institut Pasteur de Lille
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Priority claimed from US07/003,764 external-priority patent/US5051496A/en
Priority claimed from FR8701739A external-priority patent/FR2610632B1/en
Priority claimed from FR8705398A external-priority patent/FR2614025B1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • 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
    • 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
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • 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
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2
    • G01N2333/162—HIV-1, HIV-2 env, e.g. gp160, gp110/120, gp41, V3, peptid T, DC4-Binding site
    • 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

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Abstract

Peptides having immunological properties in common with those of the peptidic skeleton of peptides of viruses of the family HIV-2, particularly the envelope glycoprotein of HIV-2, characterized in that they have also a peptidic structure in common with the peptidic skeleton of peptides of SIV, particularly the envelope glycoprotein of SIV. The invention also relates to diagnosis compositions capable of detecting an infection due to HIV-2 and to vaccine compositions.

Description

_!AUlAN AUG 1983 PATENT OFFICE AU- AL 5/8 ORGANISATION MON LAe REINTg C DEMANDE INTERNATIONALE PUBLIEE EN VERTU DU TRAITE DE COOPERATION-EN MATIERE DE BREVETS (PCT[) (51) Classification internationale des brevets 4 Numero de publication internationale: WO 88/ 05440 C07K 7/10, 7/06, C12N 15/00 Al(4)Dtdepbiainitrainl:2 ult1982.08) GOIN 33/569, A61K 39/21, 37/02(4)Dtdepbiainitraiae:2jult1982.78) (21) Numnro de la demande internationale: PCT/FR88/00025 (72) Inventeurs; et Inventeurs/Deposants (US seulement) :ALIZON, Marc [FR/ (22) Date de dep6t international: 15janvier 1988 (15.01.88) FR]; 71, rue du Cardinal-Lemoine, F-75005 Paris (FR).
MONTAGNIER, Luc [FR/FR]; 21, rue de Malabry, F- 92350 Le-Plessis-Robinson GUETARD, Denise [FR/ (31) Numiros des demnandes prioritaires: 003,764 FR]; 4 B, rue Anselme-Payen, F-75015 Paris CLA- 87/01739 VEL, Frangois [FR/US]; 12103 Portree Drive, Rockville, 87/05398 MD 20852 SONIGO, Pierre [FR/FR]; 23, rue Gutenberg, F-75015 Paris GUYADER, Mireille [FR/FR]; (32) Dates de prioriti: 16 janvier 1987 (16.01.8 7) 68, rue Laugier, F-75017 Paris TIOLLAIS, Pierre [FR/ I I f~vrier 1987 (11.02.87) FR]; 16, rue de la Glaci~re, F-75013 Paris CHAKRAavril 1987 (15.04.87) BARTI, Lisa [FR/FR]; 16, rue des 3 Portes, F-75005 Paris DESROSIERS, Ronald [US/US]; 13 Causeway Street, (33) Pays de prioriti: us Udson, MA 01749 (US).
FR
FR (74) Mandataires: GUTMANN Ernest etc.; S.C. Ernest Gutmann Yves Plasserand, 67, boulevard Haussmann, F-75008 Paris
(FR).
Brevet ou demnande principal(e) (63) Apparent par continuation us 013,477 (CIP) (81) Etats disign~s: AU, DK, JP, KR, US.
D~pos~e le I11 f~vrier 1987 (11.02.87) Publiie (71) Diposant (pour tous les Etats d~sign~s .vauf US): INSTITUT PASTEUR [FR/FR]; 25-28, rue du Dr.-Roux, F-75015 Paris Avec rapport de recherche inhernationale.
A vant /'expiration dui d~lai pr~u pour/la modification des revendications, sera republiie si de tel/es modifications sont repues, &DJP 15 SEP 1988 (54) Title: PEPTIDES HAVING IMMUNOLOGICAL PROPERTIES 2-HIV-2 (54) Titre: PEPTIDES AYANT DES PROPRIETES IMMUNOLOGIQUES 2-HIV-2 (57) Abstract Peptides having immunological properties in common with those of the peptidic skeleton of peptides of viruses of the family HIV-2, particularly the envelope glycoprotein of HIV-2, characterized in that they have also a peptidic structure in common with the peptidic skeleton of peptides of SIV, particularly the envelope glycoprotein of SIV. The invention also relates to diagnosis compositions capable of detecting an infection due to HIV-2 and to vaccine compositions.
(57) Abr~gi Peptides ayant des proprik~s immunologiques en commun avec celles de l'ossature peptidique des peptides des virus de la classe HIV-2, notamnment de la glycoprot~ine d'enveloppe de HIV-2, caract~ris~s en ce qu'ils ont 6galement une structure peptidique en commun avec l'ossature peptidique des peptides de Sly, notamment de la glycoprot~ine d'enveloppe de SIV. L'invention concerne des compositions de diagnostic capable de d~tecter une infection due d HIV-2 et des compositions de vaccin.
This document contins Tl amen drats made uinder"I Sc-tionl 49 and is ccirect for printing.I p.- .7] 1 PEPTIDES CAPABLE OF BEING RECOGNIZED BY ANTIBODIES INDUCED AGAINST RETRO- VIRUSES OF HUMAN IMMUNODEFICIENCY (HIV VIRUSES), THEIR USES FOR THE DIAGNOSIS OF INFECTIONS DUE TO SOME OF THESE VIRUSES AND, WHERE APPROPRIATE FOR VACCINATION AGAINST AIDS.
The present invention relates to peptides having immunological, even immunogenic, properties in common with antigens which can be obtained in a purified form from viruses capable of causing lymphadenopathies which, in man, may subsequently degenerate into the acquired immunodeficiency syndrome (AIDS).
The invention relates in particular to antigenic peptides capable of being recognized by antibodies induced in man by viruses designated by the abbreviation HIV according to the nomenclature defined in NATURE. The invention also relates to peptides having immunogenic properties or which are capable of being rendered immunogenic in vivo, this immunogenicity manifesting itself by the induction in vivo of antibodies recognizing antigens characteristic of the viruses HIV-2 and even, at least in the case of certain of these peptides, antigens derived from HIV-1.
The invention relates in addition to uses of these peptides for the mass production of compositions for the in vitro diagnosis in man of the latent existence of certain forms of AIDS and, in the case of some of the AIDS viruses, for the production of immunogenic compositions and of compositions to be used as vaccines against the HIV retroviruses.
I Similarly, the invention relates to the uses for the same purpose of the antibodies capable of being induced in vivo by the immunogenic S peptides or peptides made immunogenic and, in the case of some of these antibodies, to their uses for the production of medicinal active f principles against these human AIDS.
The invention also relates to the utilization of some of these peptides in procedures for the in vitro diagnosis in man of some forms of AIDS as well as their use for making up diagnostic "kits".
The first retrovirus designated LAV-1 or HIV-1 was isolated and described in the patent application GB.83/24.800 and in the application EP.84/401.834 of 14/09/84. This virus has also been described by F. Barre Sinoussi et al. in Science; 220 No. 45-99, 20 pages 868-871.
/4 2 1 Variants of this HIV-1 virus designated as LAV ELI and LAV MAL have also been isolated, characterized and described in the patent application EP.84/401.834.
The HIV-1 viruses and their variants possess the following properties: their preferred targets are the human Leu3 cells (or T4 lymphocytes) and the "immortalized" cells derived from them.
they have a reverse transcriptase activity requiring the presence of Mg 2 ions and exhibit a marked activity toward poly(adenylate-oligodeoxythymidylase) poly(A)-oligo(dT)12-18).
they have a density of 1.16 to 1.17 on a sucrose gradient, they have an average diameter of 139 nanometers and a nucleus with an average diameter of 41 nanometers, the lysates of these viruses contain a protein p25 (core protein) Swhich immunologically does not cross-react with protein p24 of HTLV-1, 15 they contain a protein p42 as a constituent of their envelope, they also contain a glycoprotein gpllO of molecular weight 110,000 Sin their envelope.
The isolation and characterization of retroviruses belonging to a Sdistinct class which are immunologically related to those just mentioned only to a limited extent have been described in the European patent application No. 87/400.151.4. These retroviruses which have been grouped together under the designation HIV-2 were isolated from several African patients exhibiting symptoms of a lymphadenopathy or an AIDS.
The retroviruses of the HIV-2 type like the retroviruses of the HIV-1 type are characterized by a tropism for the human T4 lymphocytes and by a t cytopathogenic effect on these lymphocytes as a result of multiplying within them, thus giving rise either to generalized and persistent polyadenopathies or an AIDS.
More generally, the retroviruses purified by HIV-2 possess the following 3 properties: the preferred target of the HIV-2 retroviruses is constituted by human Leu3 cells (or T4 lymphocytes) and for "immortalized" cells derived from these T4 lymphocytes; they are cytotoxic for human T4 lymphocytes 3 e* t Vt,' 4 t 4 4 4 l* 4I 1 $e rtr 0 they have a reverse transcriptase activity requiring the presence of 2+ Mg ions and exhibit a marked activity for poly(adenylate-oligodeoxythymidylase) (poly(A)-oligo(dT) 12-18); they have a density of 1.16 in a sucrose gradient; they have a mean diameter of 140 nanometers and a nucleushaving a mean diameter of 41 nanometers; they can be cultured in permanent cell lines of the HUT type or those expressing the T4 protein; they do not infect T8 lymphocytes; the lysates of these viruses contain a protein p26 which immunologically does not cross-react with the protein p24 of the virus HTLV-I or of the virus HTLV-II; in addition, these lysates contain a protein p16 which is not recognized immunologically by the protein p19 of HTLV-I or of HTLV-II in radioimmunoprecipitation assays.
in addition, they contain a glycoprotein having a molecular weight of the order of 130,000-140,000 in their envelope which immunologically does not cross-react with the gpllO of the HIV-1, but which, in contrast, does cross-react immunologically with the glycoprotein gpl40 of the envelope of STLV-III (a virus isolated from monkeys); these lysates also contain antigens which can be labelled with 3 S-cysteine, the molecular weights of which range between 32,000 and 42,000-45,000: they include in particular an antigen having a molecular weight of the order of 36,000 and an antigen having the molecular weight of the order of 42,000, one of these antigens (p36 or p42) probably constituting a transmembrane glycoprotein of the virus HIV-2; the genomic RNA of the UIV-2 does not hybridize with the genomic RNA of HIV-1 under stringent conditions; under non-stringent conditions, the genomic RNA of HIV-2 hybridizes neither with the env gene and the LTR which is next to it of HIV-1 nor with the sequences of the pol region of the genome of HIV-1; under non-stringent conditions, it hybridizes weakly with nucleotide sequences of the region of HIV-1.
Another retrovirus designated SIV-1, this designation replacing the earlier one of STLV III, was isolated from the rhesus macaque monkey D. Daniel et al., Science 228, 1201,(1985); N. L. Letwin et al., 4~ SScience 230, 71 (1985) under the name "STLV-IIImac").
Another retrovirus, designated "STLV-IIIAG M (or SIVA was isolated from wild green monkeys. However, in contrast to the virus present in the rhesus macaque monkeys, the presence of "STLV-III AGM" does not seem to induce a disease of the AIDS type in the African green monkey.
A strain of the SIV-lmac retrovirus was deposited with the CNCM on 7th February 1986 under the No. 1-521. Studies have shown that the retrovirus SIV-1 contains some proteins which are immunologically related to structural proteins or glycoproteins which can be isolated from 3 HIV-2 under similar conditions. This retrovirus SIV-1, the infectious nature of which has been observed in monkeys, has been designated as STLV-III by the research scientists who have isolated it (literature references cited above).
For semantic convenience, these viruses will be referred to in the i 15 following discussion only by the expression SIV (the expression SIV is San abbreviation for "Simian Immunodeficiency Virus") which may be followed by an abbreviation indicating the species of monkey from which it was derived, for example MAC (or mac) for the macaque or AGM for the African green monkey.
2-1 Using the same methods as those mentioned above, it has been observed that the SIV-1 mac also contains: a major nuclear protein p27 having a molecular weight of the order of 27 kilodaltons, a major glycoprotein, gpl40, in its envelope, a protein p32, probably transmembrane, "which is hardly observed in RIPA when the virus has been labelled beforehand with S-cysteine, but which can be observed in the form ofbroad bands on Western blots.
More precise studies have been carried out on the HIV-2 and SIV 4 viruses. The continuation of the study of the HIV-2 retroviruses has also led to the preparation of complementary DNA sequences (cDNA) of the RNAs of their genomes. The complete nucleotide sequence of the cDNA of a representative retrovirus of the HIV-2 class (HIV-2 ROD) was deposited on 21/02/1986 with the CNCM under No. 1-522 and the reference LAV-II ROD).
This nucleotide sequence and the open-reading frames which it contain 3 are indicated in the Figure 1A.
5' 1 Furthermore, continuation of the study of other retroviruses has also made it possible to obtain their complete nucleotide sequences. This is the case in particular for the cDNA derived from the genomic RNA of SIV.
The cloning and sequencing of the SIV-lmac virus which led to the elucidation of its nucleotide sequence were carried out under the following conditions: The DNA of HUT 78 cells infected with the SIV virus (isolate STLV-III mac 142-83 described by Daniel et al. (1985) Science, 228, p. 1201-1204, partially digested by the restriction enzyme Sau3A was cloned at the BamHI site of the bacteriophage vector lambda ELBL3 in order to constitute a genomic library.The2million recombinant phages of the genomic library thu constituted were screened in situ under P3 conditions of security with the aid of sequences of the HIV-2 virus, derived from clones of lambda-ROD4, lambda-ROD35 and E2 (Clavel et al., 1986, Nature, 324, p. 691.) and nicktranslated.
Hybridization was carried out in 5 x SSC at 50 C and washings in 2 x SSC at 50 C. A single clone containing all of the viral sequences was obtained. This clone was designated as lan:' la-SIV-1. The insert of the phage lambda-SIV-1 measures a total of 16.5 kb and includes ;D an integrated provirus which lacks only the first 250 bases of the leftside of LTR whereas the right-side of LTR is complete.
The integrated provirus was sequenced by the dideoxynucleotide c method after subcloning of random fragments in the phage M13mp8. 300 subclones were analyzed.
Fragments of cDNA derived from the clone lambda-SIV-1 inserted into plasmids pSIV-1.1 and pSIV-1.2 were deposited with the CNCM on 15th April 1987 under the numbers 1-658 (pSIV-1.1) and 1-659 (pSIV-1.2).
The results are mentioned in the figures described below.
S* Figure 1B presents the nucleotide sequence of the viral genome of SIV and the sequences which have been deduced from it for the.viral proteins corresponding to the gene products gag, pol, env, Q, X, R, tat, art, F.
The Figures 3 to 11 and the Figure 1C present comparisons between HIV- 2 and SIVmac of the theoretical products of the viral genes and the LTR ('SIV-1).
I I 6
L.
1 The invention also relates to fragments of cDNA deduced from the cDNA derived from the entire genome of SIV-1, these fragments containing one or more sequences derived from the complete sequence of cDNA and which code for interesting peptides of the invention. These sequences are indicated in Figure lB and that relating to the LTR sequence of the virus in Figure 1C.
The nucleotide sequences of the cDNA of SIV were matched with the nucleotide sequences of the virus HIV-2 ROD with respect to the LTR sequence (Figure IC). This type of match which is made for the entire genome by comparing Figure 1B with Figures 3 to 11 makes it possible to locate or deduce the nucleotide sequences of the two viruses which have common essential structural elements.
The invention naturally also relates to the use of the cDNAs derived from SIV or their fragments (or recombinants containing them) both as 15 probes for diagnosing the presence or absence of HIV-2 viruses in serum samples or other liquids or biological tissues obtained from patients o oo suspected to be carriers of the HIV-2 virus. These probes are preferably also labelled (radioactive, enzymatic, fluorescent markers, .oop. etc). Probes of particular interest for the application of the diagnostic procedure for the HIV-II virus or a variant of HIV-2 may be characterized in that they contain the totality or a fraction of the cDNA complementary to the gene of the SIV virus or particularly the recombinant fragments contained in various clones.
0o00 The probes used in this diagnostic procedure for the HIV-2 virus and in the diagnostic kits are in no way limited to the probes described 0 "above. On the contrary, they comprise all of the nucleotide sequences derived from the genome of the SIV virus, of a variant of SIV or of a virus closely related in structure as long as they make it possible to 0',Ot detect in the biological fluids of persons suspected of developing an AIDS,antibodies directed against a HIV-2 or a virus which is closely related to it.
The detection may be carried out by any known method. It may comprise the placing in contact of these probes either with the nucleic acids obtained from cells containedinthese sera or other biological fluids, for example cerebrospinal fluid, saliva, etc It may also
U
p.Li i I Il 7 involve placing these probes in contact with these fluids themselves once their nucleic acids have been made accessible to hybridization with these probes under conditions permitting hybridization between these probes and these nucleic acids to occur. The final step of in vitro diagnosis thus comprises the detection of the hybridization which may have occurred. The above-mentioned diagnostic procedure involving hybridization reactions may also be carried out with the aid of mixtures of probes derived from a HIV-2 and a SIV-I or a HIV-l, a HIV-2 an( i SIV, respective4, if it is not necessary to distinguish between the type if virus searched.
Generally speaking, the diagnostic procedure for the presence or absence of the HIV-2 virus or a variant in serum samples or other liquids or tissues obtained from patients suspected of being carriers of the HIV-2 virus comprise the following steps: 1) at least one hybridization step conducted under stringent conditions by placing in contact the DNA of the cells of the sample taken from the suspected patient with one of the above-mentioned labelled probes on an appropriate membrane, 2) the washing of the said membrane with a solution which maintains chese stringent conditions of hybridization, 3) the detection of the presence or absence of the HIV-2 virus by a method of immunodetection.
In another preferred embodiment of the procedure according to the invention the above-mentioned hybridization is conducted under nonstringent conditions and the washing of the membrane is carried out under conditions suited to those of the hybridization.
It will be obvious that the invention relates to the nucleic acids corresponding to sequences located in analogous regions oif variants of SIV as well as to all of the nucleic acids in which modification would make it possible to take advantage of thr. degeneracy of the genetic code.
The comparative studies which have also enabled results'to be obtained relating to the core proteins, hereafter termed "gag proteins" and to proteins of the envelope, hereafter referred to as "env proteins", have also been reported in the European patent application No. 87/400.151.4 already mentioned. These results show that the core proteins (gag proteins) in HIV-2 exhibit less marked differences from those of the HIV-l |rr I t I 4$ I l 4 i 441 U 1a4 14 I L -8viruses than the protein:s of the envelope (env proteins). Overall, the env proteins of HIV-2 have been found to be immunologically related with the corresponding env proteins of the HIV-1 viruses to only a very slight degree or not at all.
On the other hand, comparative studies on the structures of the cDNA sequences of the HIV-2 and SIV viruses have demonstrated certain common properties which make their appearance at the level of the proteins.
Overall, the proteins of HIV-2 and SIV-1 show a considerable degree of immunological relatedness.
S-The major glycoprotein of the envelope of HIV-2 has been shown to be more closely related immunologically to the major glycoprotein of the envelope ofSIV than to the major glycoprotein of the envelope of HIV-1.
These observations can be affirmed not only with regard to the molecular weights: 130-140 kilodaltons for the major glycoproteins of I 15 HIV-2 and SIV compared with about 110 kilodaltons for the major glycoprotei oo of the envelope of HIV-1, but also with respect to their immunological Sproperties since sera taken from patients infected with HIV-2, and more particularly antibodies formed against the gpl40 of HIV-2 recognize the of SIV-lmac, whereas in similar assays the same sera and the same 27* antibodies of HIV-2 do not recognize the gpllO of HIV-1. However, the anti-HIV-1 sera which have never reacted with the gpl40 of HIV-2 precipitatE a protein of 26 Kdal labelled with 3S-cysteine contained in the extracts of HIV-2.
,oo The major core protein of HIV-2 seems to possess an average molecular weight (about 26,000) intermediate between that of the p25 of HIV-1 and the St". p27 of SIV.
These observations derive from assays carried out with viral extracts obtained from a HIV-2 isolated from one of the patients mentioned above.
I Similar results have been obtained with viral extracts of a HIV-2 isolated from another patient.
More extensive studies have led the inventors to recognize an initial class of peptides having sequences of amino acids which are either identical or very similar to sequences contained in the interior of the structures of the gag and env proteins of HIV-2 or SIV, or even HIV-1. These L uj\ ^gi i I -9peptides are particularly useful for the diagnosis of an infection by the HIV-2 virus or one of its variants in man.
In this respect, the present invention also relates to diagnostic procedures and compositions for the in vitro detection of antibodies directed against a HIV-2 virus or its variants, more particularly in biological samples, particularly in the sera of patients who have become infected with the HIV-2 virus, since some of these peptides make possible a particularly fine distinction between the infections due to HIV-2 viruses and those due to HIV-1 viruses.
13 These more exhaustive studies have also led to the possibility of synthesizing immunogenic peptides or peptides capable of being made immunogenic which exhibit structural characteristics enabling them to induce in vivo the production of antibodies capable of recognizing env proteins both of HIV-1 and HIV-2 and, at least in the case of some of these peptides, of binding both to HIV-1 viruses and to HIV-2 viruses, and resulting more particularly in their neutralization. The use of these latter types of peptides is thus particularly indicated for the production of active principles of vaccines against the HIV viruses, and hence against the AIDS.
2 In order to specify in the subsequent discussion the amino acid residues which constitute the peptides according to the invention, resort will be had to the code shown in the table which follows for those 2 amino acids having an unequivocal meaning in the international nomenclature which designates each naturally occurring amino acid by a single letter (capital letter): M Methionine L Leucine I Isoleucine V Valine F Phenylalanine S Serine P Proline T Threonine A Alanine Y Tyrosine z, !i
II
'I
i r r 10 1 H Histidine Q Glutamine N Asparagine K Lysine D Aspartic acid E Glutamic acid C Cysteine W Tryptophan R A'rginine iO G Glycine When, on account of its position within the chain of amino acids characteristic of a specific peptide, an amino acid can assume several meanings, it will be designated either by a dash if it can be any amino acid, or by a lower case letter when this amino acid can assume a limited number of preferred meanings, this number being, however, always higher than one. In this latter case, the possible meanings of this Slower case letter will always be specified in relation to the peptide Sto which it belongs.
In order to facilitate reading, these peptides will be designated 2D by an abbreviation env or gag followed by a number referring to sequences of amino acids containing either in the env proteins or in the gag proteins of certain HIV-1, HIV-2 or SIV. Reference will also be made to them in the ensuing discussion.
4. t Finally, in the definitions which follow S 25 the X groups represant either a free NH 2 -group or one amidated in S, particular by one or two alkyl groups containing from 1 to 5 carbon atoms, or a peptide group comprising from 1 to 5 amino acids, the N-terminal amino acid of which itself possesses a free NH 2 group or one amidated as II previously mentioned, and t i 30 the Z groups represent either a free -OH group or an alkoxy. group contain ing an alkyl group comprising from 1 to 5 carbon atoms, or a peptide group comprising from 1 to 5 amino acids, the C-terminal amino acid of which itself possesses a free -OH group or an alkoxy group as previously indicated, the groups of from 1 to 5 amino acids contained,as appropriate, in X or Z or in both at once being such that their presence is for the i tI -1 1 most part not incompatible with the preservation of the immunological or even immunogenic, properties of the peptides which do not contain them.
The peptides according to the invention which have immunological properties in common with antigens of HIV-2 and, in certain cases, also with antigens of HIV-l or its variants are characterized in that they also have a peptide structure in common with the antigens of SIV. In an advantageous manner, these peptides normally contain a maximum of amino acid residues.
:0 The following are preferred peptides: erivi XRV-AI EKYL-DQA-LN-WGCAFRQVCZ -*oftenv2
X-LE-AQI-QQEKNMYELQKLNZ
eriv3 t4 XELGDYKLVEITPIG-APT--KR Z t9 9 env4 X VTV-YGVP-WK-AT-LFCA-Z X QE---L-NVTE-F--W-NZ env6 4 9 -S-KPCVKLTPLCV--Z 't 0:1"env7 X N-S-IT--C-K Z env8 X-I YC-P-G-A-L-C-N-TZ env9 a 0 X A-C W--Z envl0 X-G-DPE NC-GEF-YCN NZ env 11 -I KQ -Y Z
I:
I
I
12 1 More particularly, the invention relates to the following peptides: envl
XRV-AIEKYL-DQA--LN-WGCAFRQVCZ
env2
X-LE-AQIQQEKNMYELQKLNZ
env3 XELGDYKLVEITPIG-APT--KR Z env4 X VTV-YGVP-W--AT--LFCA-Z X E L -NVT E -W NZ env6 XL S-KPCVKL-PLC Z env7 *0 env8 O env9 X A-C W--Z envl0 X-G-DPE NC-GEF-YC NZ envi11 X C-I-Q-I G YZ Useful peptides corresponding to those just cited above are presented 0 in the formulas which follow: env 1 O 030 XRVTAIEKYLQDQARLNSWGCAFRQVCZ, or
XRVTAIEKYLKDQAQLNAWGCAFRQVCZ
env2 XSLEQAQIQQEKNMYELQKLNSWZ, or
XLLEEAQIQQEKNMYELQKLNSWZ
env3 XELGDYKLVEITPIGFAPTKEKRYSSAHZ, or C. XELGDYKLVEITPIGLAPTNVKRYTTG-Z 27 -13 I I 4141 1 4 4 4 1~ r II 14 I I I 11 14141 I I (It will be noticed that the peptides envi, env2, env3 testify to the very close relationship between HIV-2 and SIV-1. In fact, the first peptide is included in the genome of HIV-2 and the second in that of Sly-1).
e nv 4 in which the letters from a to j may have the following meanings: a is C, E or D b is T, K, D, N or I c is 0 or L d is Y or W e is F or Y f is T, V or A g is N or E h is I or T i is P or T j is T or S o is K or R XabcoEdeLfNVTEgFhiWiNZr in which the letters from a to jmay have the following meanings: a is D or P b is D or N c is Y or P d is I, V, I or L eis T, V, E or A f is V, G or E org is A, N, G or S h is D or N i is A or M~ j is N, K or E o is Q or S env6 XLabcSdKPCVKLOPLCuefKZ, I A54I Ill 1 I I 1 44 I I I £14 ~i II I~ i r 14 1 in which the letters from a to f may have the following meanings: a is F or W b is E or D c is T or Q d is Ior L e is A, S or T f is M or L o is T or S u is V or I env7 XabCNxSyIocdCeKfghiZ, in which the letters from a to i and x and y may have the following meanings: a is N or T or I b is H or S or N c is E or Q d is S, A or C e is D or P f is H, V or D 3 g is Y or S Sh is W or F Si is D or E i «s 4 x is T or R y is V or A o is T or Q env8 XalbcdYCxPeGfAgLhCiNjTZ, 4 44* in which the letters from a to k and x may have the following meanings: a is A or P S" 30 b is R or P c is F, I or C d is R or H 4 is P or A f is Y or F g is L or I h is R or K 1 1 is-or N j is D or K x is A or T env9 XwabcxyAdCefghizWjkZ, in which the letters from a to k and x to z may have the following meanings: a is K or or E b is R or c is P or Mor I d is W or H or Y e is W or N or T or R f is F or I g is K or S or N or G is h is G or R or E i is or A or T j is K or N or D or S s..k is D or A or N or K or E 2J w is N, D or I x i s R o r G or K S..y i s Q o r K o r R z is K o r E or Q or N Xa~bDpEcdefghNCiGEFjYCokxlmlNZ, in which the letters from a to n and x may have the following meanings: 0a is K or or G 0:0b is S or G or c is V or I 30 d is Aor Vor T e is Y or T or M or F f is M or H g gis 4or S h is T or F 3 i is Ror G w 16 j is L or F o is N or K k is M or S 1 is W or Q or K or G m is F or L n is L or F x is T or S or N env1 1 XabcdwCeIoQfIxgyhizGjklYZ, in which the letters from a to 1 and w to z may have the following meanings: a is R or T or S or N b is N or I c is Y or T 15 d is A or L or V e is H or R f is I or F t g is T or M h is H or Q or A 2 i is K or E i r l j is R or K k is N or A S 1 is V or H w is P or Q x is N or K y is W or V 44* z is V or T or K o is K or R The structure of the antigenic peptide coded by the gene gag and S* designated by gag 1 is also shown below: XDCKLVLKGLGaNPTLEEMLTAZ, in which the letter a specifies M or T.
SIt will be noticed that, generally speaking, the amino acids having an unequivocal meaning (hence represented by a capital letter in accordance with the international nomenclature) which appear in the definitions which 21
I-I-
1 17 precede'of the peptides according to the invention are found to correspond to identical amino acids placed in the same order in the corresponding env or gag sequences of the env or gag protein of at least one of the HIV or of SIV-1.
The positions of the sequences are underlined and located within the sequences of amino acids of the env proteins of HIV-2 ROD (CNCM No. 1-532) and HIV-1 BRU (CNCM No. 1-232) shown in Figure 2. Moreover, the alignments of the amino acids of the env and gag proteins of SIV-lmac (CNCM No. 1-521) and of HIV-2 ROD are presented in Figures 3 and 4.
The continuous lines which replace individual dashes at certain locations in these sequences is intended to underline the fact that certain amino acids contained in these.sequences have been deliberately omitted from the figure in order to allow identical amino acids to be aligned (in that case marked with an asterisk) or for two points to be aligned vertically in the sequences of the corresponding proteins of HIV-1 and HIV-2 on the one hand, and SIV and HIV-2 on the other.
In addition to the peptides already mentioned, the invention also relates to peptides modified by insertion and/or deletion and/or substitution of one or more amino acids provided that the antigenic or immunogenic properties of the said peptides are not modified and that the recognition properties of the antigen and the antibody for these peptides are not substantially modified.
In a particularly preferred embodiment the invention relates to peptides having immunological properties in common with the peptide skeleton of the glycoprotein of the envelope of the viruses of the class HIV-2, these peptides containing a number of amino acid residues not exceeding These preferred peptides according to the invention have the following sequences: tirt i i Li I rt C 4 *r iS i I 1" di C 1 i -18envi
RVTAIEKYLQDQARLNSWGCAFRQVC
AIEKYLQDQ
RVSAI EKYLKbDQAQLNAWGCAFRQVC
AIEKYLKDQ
env2 SLEQAQ IQQEKNMYELQKIINSW
QIQQEKN
LLEEAQIQQEKNMYELQKLNSW
env3 ELGDYKLVEITP IGFAPTKEKRYS SAH
YKLVEITPIGFAPTKEK
ELGDYKLVEITPIGLAPTNVKRYTTG
YKLVEITPIGLAPTNVK
is env4 CTQYVTVFYGVPTWKNAT IPLFCAT VT VFYG VPT WKN AT CIQYVTVFYGVPAWRNATI PLFCAT VT V FYG VPAWRN AT 20 EKLWVTVYYG3VPVWKEATTTLFCAS
VTVYYGVPVWKEAT
EDL WVT VYYG V PV WKEAT TTLFCAS VT VYYG VP VWKEAT DN LWVT14 2 YYG VP VWK EAT TTL FCAS VTV YYGVPVWKEAT q 4*4 4 44 4 4 q~ 0~ *4 4 4 4640 4 4 4*4* 4*44 C 4, 4* 4 04 4 444 0
DDYQEITL-NVTEAFDAWNN
L-NVTEAF
DDYSELAL-NVTESFDAWEN
L-NVTESF
PN PQE VVL VNVT ENFNMWKN
LVNVTENF
PNPQEI ELENVTEGFNMWKN
LENVTEGF
PNPQEI ALENVTENFNMWKN LEN VT ENF Sl lie -19env6 1 ETSIKPCVKLTPLCVAMK
ETSIKPCVKLSRLCITMR
DQSLKPCVKLTPLCVSL(
DQSLKPCVKLTPLCVTLN
PCVKLTPLCV
env7
NH-CNTSVITESCD
NTSVIT
NHC NT SVI QECCD
NTSVIQ
TSCNTSVITQACP
NTSVIT
INCNTSVITQACP
NTSVIT
INCNTSAITQACP
NTSAIT
env8 YCAP P(YALLRC -NDT
YCAPAGFAILKCNNKT
YCAPAGFAILKCNDKK
YCAPAGF ,ATLKCRDKK I t env9 it IL NKRPRQAWCWFKG-KWKD
NERPKQAWCRFGG-NWKE
N- -MRQAHCNISRAKWNA 0- -IRRAYCTINETEWDK "ttitI--IGQAHCNISRAQWSK 14c4 t envl0
KGSDIPEVAYMWTNCRGEFLYCNMTWFLN
NCRGEFLYCN
GG -DPEVTFMWTNCRGEFLYCKMNWFLN
NCRGEFLYC(
l~t(4,-GGDPEIVTHSFNCGGEFFYCNsTQLFN
NCGGEFFYCN
-GGDPEITTHSFNCRGEFFYCNTSKLFN
N CRG EF FY CN r A -GGDPEITTHSFNCGGEFFYCNTSGLFIJ
NCGGEFFYCN
A-
20 S- 21 1 The peptides according to the invention may also advantageously be prepared by the standard methods applied in the field of peptide synthesis.
This synthesis may be carried out in homogeneous solution or on a solid phase.
For example, use can be made of the methods of synthesis in homogeneous solution described by HOUBENWEYL in the monograph entitled "Methoden der Organischen Chemie", edited by E. Wunsch, vol. 15-1 and II, THIEME, Stuttgart, 1974.
These methods of synthesis consist of condensing successive amino acids two at a time in the required order, or of condensing amino acids with fragments already formed and already containing several amino acids in the appropriate order or, again, of condensing several fragments previously prepared in this way, it being understood that precautions will be taken to protect beforehand all of the reactive functions present on these amino acids or fragments with the exception of the amino function of one and the carboxyl function of the other which must be free to enter into the formation of the peptide bond, particularly after activation of the carboxyl function according to the well-known methods of peptide synthesis. As an alternative, recourse may be had to coupling reaction 20 making use of standard coupling reagents of the carbodiimide type such as, I -for example, l-ethyl-3-(3-dimethyl-' aminopropyl) carbodiimide.
f When the amino acid used possesses an additional acid function (particularl in the case of glutamic acid) such functions must be protected by t-bustyl ester groups, for example.
In the case of step-wise synthesis in which one amino acid is added at a time, the synthesis starts preferably with the condensation of the J C-terminal amino acid with the amino acid which corresponds to the neighbouring aminoacyl in the desired sequence and it continues in this cC manner until the N-terminal amino acid is reached. According to another 30 preferred method of the invention use may be made of the procedure described by R. D. MERRIFIELD in the article entitled "Solid phase peptide synthesis" Am. Soc., 45, 2149-2154).
For the preparation of a peptide chain according to the procedure of MERRIFIELD it is necessary to make use of a very porous polymeric resin to which is attached the first amino acid, the C-terminal residue of i 22- 1 the chain. This amino acid is attached to the resin through its carboxyl group and its amino function is protected by the t-butoxycarbonyl group, for example.
When the, C-terminal acid is attached to the resin-as the first amino acid, the protecting group of the amine function is removed by washing the resin with an acid. In the case in which the protecting group of the amine function is the t-butoxycarbonyl group, it can be removed by treatment of the resin with trifluoroacetic acid.
Subsequently, the second amino acid, which furnishes the second aminoacyl of the desired sequence counting from the C-terminal aminoacyl residue, is coupled to the deprotected amine function of the Cterminal amino acid, the first amino acid attached to the resin. The carboxyl function of this second amino acid is activated preferably for example with dicyclohexylcarbodiimide and its amine function is protected 15 for example by means of the t-butoxycarbonyl group.
T I In this way, the first part of the desired piptide chain is obtained Sconsisting of two amino acids, the terminal amine function of which is C t t t T« protected. The amine function is deprotected as previously described and subsequently the third aminoacyl is coupled under conditions analogous 20 to those used for the addition of the amino acid next to the C-terminal amino acid. In this way, one after the other, each of the amino acids which will constitute the peptide chain is coupled to the deprotected amine group of the portion of the peptide chain already formed and which is attached to the resin.
When the desired peptide chain has been-assembled in its entirety, Sthe protecting group of the different amino acids constituting the peptide chain are removed and the peptide is cleaved from the resin by lit 4 means of hydrogen fluoride, for example.
iThe invention also relates to water-soluble oligomers of the abovementioned peptide monomers. Oligomerization can bring about an increase Sof the immunogenicity of the peptide monomers according to the invention.
It may be mentioned that these oligomers may, for example, contain from two to ten monomeric units without implying that this number is to be considered as limiting.
CIs ~D -1 23 1 The monomeric units forming this oligomer are either all constituted of the polypeptide of sequence 1 or by the polypeptide of sequence 2 or by both of .hese polypeptides.
For the preparation of the oligomer use may be made of any method of polymerization commonly used in the field of peptides, this polymerization reaction being continued until an oligomer or a polymer is obtained which contains the number of monomeric units required for the acquisition of the desired immunogenicity.
One method of oligomerization or polymerization of the monomer consists in allowing the latter to react with a cross-linking agent such as glutaraldehyde.
Use may also be made of other methods of oligomerization or coupling, for example that involving successive coupling of monomeric units through their terminal carboxyl and amine functions in the presence of homo- or hetero-bifunctional coupling agents.
For the production of molecules containing one or more sequences of 17 amino acids such as those defined above, use may also be made of genetic engineering techniques using micro-organisms transformed by a specific nucleic acid containing the corresponding, appropriate nucleotide sequences.
The invention also relates to the nucleic acids containing one or more sequences derived from the sequence of the cDNA of the virus HIV-2 ROD.
These sequences, located by the numbers shown on the sequence previously described, code for some interesting peptides of the invention.
(*44 Sequence coding for envl nucleotides 7850 to 7927 env2 8030 to 8095 env3 7601 to 7636 env4 6170 to 6247 n" e 6294 to 6349 S"env6 6392 to 6445 env7 6724 to 6763 env8 6794 to 6838 env9 7112 to 7162 env1 7253 to 7336 "env 7358 to 7426 A, i 1 1535 to 1597 .B m a-L. uIt- uing sucn cnat tneir presence is for the ii f I 24 4,, 4*.
44 *1 6 4 4O 25 4444 44 4 4 44 7 0 0 Finally, the invention relates to nucleic acids corresponding to the SIV virus containing one or more sequences derived from the cDNA of the virus SIV-1. These sequences coding for the peptides env 1 to env 11 and gag 1 can be located on Figure 3 by comparison with the corresponding sequences described for HIV-2.
It will be obvious that the invention relates to the nucleic acid corresponding to sequences located in analogous regions of the cDNA derived from variants of HIV-2 ROD or SIV as well as all of the other nucleic acids representing modifications of those already described which result from exploiting the degeneracy of the genetic code.
The invention also concerns the conjugates obtained by covalently coupling the peptides according to the invention (or the above-mentioned oligomers) to carrier molecules (naturally occurring or synthetic), physiologically acceptable and non-toxic, through the intermediary of complementary reactive groupings situated on the carrier molecule and the peptide. Examples of appropriate groupings are illustrated in what follows: As examples of carrier molecules or-macromoleclar supports constituting part of the conjugates according to the invention, mention will be made of naturally occurring proteins such as tetanus anatoxin ovalbumin, serum albumins, hemocyanins, etc As examples of synthetic macromolecular supports mention may be made of the polylysines or poly(D,L-alanine)-poly(L-lysine).
Other types of macromolecular supports which can be used are mentioned in the literature; usually they have a molecular weight higher than 20,000.
In order to synthesize the conjugates-according to the invention use may be made of known procedures such as that described by FRANTZ and ROBERTSON in Infect. and Immunity, 33, 193-198 (1981) or that described in Applied and Environmental Microbiology, (October 1981), vol. 42, No. 4, 611-614 by P. E. KAUFFMAN by using the peptide and the appropriate carrier molecule.
In practice, and without implying any restriction on the use of others, it is advantageous to use the following compounds as coupling agents: glutaraldehyde, ethyl chloroformate, water-soluble carbodiimides /N-ethyl-N'(3-dimethylamino-propyl) carbodiimide, diisocyanates,.
bis-diazobenzidine, di- and trichloro-s-triazines, cyanogen bromide as well as the coupling agents mentioned 4 n Scand. J. Immunol., (1978), vol. 8, p. 7-23 (AVRAMEAS, TERNYNCK, GUESDON).
4 *o- S- 1 It is possible to use any other coupling procedure which causes one or more reactive functions of the peptide, on the one hand, to react with one or more reactive functions of the support molecules, on the other.
Advantageously, these reactive functions are carboxyl and amine functions which can undergo a coupling reaction in the presence of a coupling agent of the type used in the synthesis of proteins, for example, l-ethyl-3- (3-dimethylamino-propyl)-carbodiimide, N-hydroxybenzotriazole, etc Use may again be made of glutaraldehyde, particularly when it is required to link amino groups to each other which are situated on the peptide and the support molecule respectively.
The peptides according to the invention possess antigenic properties.
They may thus be used in diagnostic procedures for the detection of an infection by the HIV-2 virus.
As has already been mentioned, studies have made it possible to distinguish between two groups of peptides which can be used in procedures for the detection of antibodies to the HIV-2 virus in a human biological fluid, particularly serum or cerebrospinal fluid.
The first group includes the gag 1 peptides. These peptides recognize anti-HIV-2 antibodies and are thus capable of detecting infection by HIV-2. To a certain extent they also recognize anti-HIV-1 antibodies.
The second group (II) includes peptides which correspond more especially to those which are located in the transmembrane portion and at the terminus of the external part of the protein of the envelope.
'It' 25 These peptides are those previously designated by env 1, env 2 and env 3.
SThey make possible the specific recognition of the presence of antibodies to HIV-2 and thus make it possible to distinguish in a given individual It past or present infections due to a HIV, more particularly between those caused by a HIV-2 and those caused by a HIV-1.
The invention also relates to a composition containing at least one C. of the above-mentioned peptides or at least one oligomer of this peptide, characterized in that it has the capacity to be recognized by sera of human origin containing antibodies against the HIV-2 virus.
The invention relates to an in vitro diagnostic procedure involving one or more peptides according to the invention for the detection of NLI antibodies to HIV-2 in biological fluids, in particular in human sera.
is /I I C1 i- r 1 26- 1 Generally speaking, the above in vitro diagnostic procedure comprises the following steps: the placing in contact of this biological fluid with the said peptides, the detection of the possible presence of an antibody-peptide complex by physical or chemical methods in the said biological fluid.
In a preferred embodiment of the invention, the detection of the antibody-antigen complex is carried out by means of immunoenzymatic assays (of the ELISA type), immunofluorescence assays (of the IFA type), radioimmunological assays (of the RIA type) or radioimmunoprecipitation assays (of the RIPA type).
In this sense, the invention also relates to any peptide according to the invention labelled by means of an appropriate marker of the enzymatic, fluorescence, radioactive, etc type.
Such methods comprise for example the following steps: deposition of defined amounts of a peptide composition according I, to the invention in the wells of a microtitre plate, introduction into the said wells of increasing dilutions of the serum requiring diagnosis, 20 incubation of the microtitre plate, SL- repeated rinsing of the microtitre plate, introduction into the wells of the microtitre plate of labelled antibodies against immunoglobulins of the blood, the antibodies having been labelled with an enzyme chosen from among those which are capable t 25 of hydrolyzing a substrate, thus leading fo"a change in the absorption spectrum of the latter at at least one specific wavelength, detection of the amount of substrate hydrolyzed in comparison Swith a control.
The invention also relates to kits for the in vitro diagnosis of the presence of antibodies to the HIV-2 viruses and, in certain cases, the HIV-1 viruses in a biological fluid. The kits contain: a peptide composition according to the invention, the reagent necessary for constituting a medium appropriate for performing the immunological reaction, the reagents used to detect the antibody-antigen complex produced XN ?rUj 1.
t II 27 by the immunological reaction. These reagents may also comprise a marker or be capable of being recognized in turn by a labelled reagent, more particularly in the case in which the above-mentioned polypeptide composition is not labelled, a. reference biological tissue fluid not containing antibodies recognized by the above-mentioned polypeptide composition.
The invention relates to the antibodies themselves formed against the peptides of the invention. It will be obvious that these antibodies are not limited to polyclonal antibodies. The invention also relates to any monoclonal antibody produced by any hybridoma capable of being formed by standard methods from the spleen cells of an animal,in particular of a mouse or a rat immunized against one of the peptides of the invention, on the one hand, and the cells of an appropriate myeloma cell line on the other, and of being selected by its capacity to produce monoclonal antibodies recognizing the peptide C t rinitially used for the immunization of the animals.
The invention also relates to immunogenic compositions for the production of vaccines, the active principle of which is constituted by at least one peptide according to the invention, or an oligomer of this 2D peptide or a peptide in the form of a conjugate with a carrier molecule, Scharacterized in that they induce the production of antibodies against the above-mentioned peptides in amounts sufficient to inhibit the proteins of the HIV-2 retrovirus as well, and even indeed the HIV-2 retrovirus in combination with a pharmaceutically acceptable vehicle.
I 25 The immunogenic compositions for the production of vaccines contain Smore particularly in an advantageous manner at least one of the peptides previously designated as env4, env5, env6, env7, env8, env9, envlO, envll Sand even mixtures of them.
Of the peptides suitable for constituting active principles of vaccines some are particularly preferred because they possess a basic amino acid structure corresponding to regions of the glycoproteins of the envelope which exhibit a considerable degree of conservation, not only in the HIV-2 and SIV viruses, but also in the HIV-1 viruses. These particularly preferred peptides are the peptides designated as env4, and certain peptides of env5, env6 and envlO.
7 t; 4 It 28 Irr I In a preferred embodiment of the invention the immunogenic peptides (or fragments of these peptides), suitable for constituting active principles of vaccines are chosen from among those, the formulas of which correspond to sequences which, in the glycoproteins of the envelopes of HIV-2, SIV and HIV-1, exhibit an amino acid sequence homology greater than 50%, belong to the external part of the envelope of the virus, show no or almost no deletions and contain cysteine residues favourable to the stabilization ,of bonds and to the constitution of bonding loops.
The following peptides belong to this class of preferred peptides.
env4
XVTV-YGVP-W--ATZ
XL-NVTE-FZ
env6
XKPCVKL-PLC-Z
env7
XN-S-I-Z
envO1
XNC-GEF-YC-Z
env11
XC-I-Q-IZ
Advantageous pharmaceutical compositions are constituted by solutions, suspensions or injectable liposomes containing an efficacious dose of at least one product according to the invention. These solutions, suspensions and liposomes are prdferably prepared in a sterilized isotonic aqueous phase, preferably saline or a glucose solution The invention relates more particularly to those suspensions, solutions and liposomes which are suitable for administration by intradermal, intramuscular and subcutaneous injections or even by scarifications.
The invention also relates to pharmaceutical compositions which can be administered by other routes, in particular by the oral route.
The pharmaceutical compositions according to the invention which can be used as vaccines to stimulate the production of antibodies against the HIV-2 virus can, for example, be administered at doses varying I(IcIt 25 t 29 between 10 and 500 pg/kg, and preferably between 50 and 100 pg/kg, of peptide according to the invention.
These doses are cited as examples but do not imply any restriction on the dose which may be used.
As has already been indicated above the various peptides which have been specified may contain modifications which do not have the effect of modifying fundamentally their immunological properties. The peptide equivalents which result form part of the claims which follow.
As examples of peptide equivalents, mention will be made of those with structures corresponding to equivalent regions in the cDNAs of other variants of HIV-2, SIV or HIV-1 when these regions are aligned under conditions similar to those which were mentioned above with regard to HIV-2 ROD, SIV and HIV-1 BRU. As other examples of these peptides mention will be made of those with structures corresponding to equivalent regions in the cDNAs which were deposited with the CNCM in particular those under the numbers 1-502, 1-642 (HIV-2 IRMO), 1-643, (HIV-2 EHO) as well as, in appropriate cases, variants of HIV-1 which were deposited with the CNCM under the numbers 1-232, 1-240, 1-241, 1-550, 1-551.
The neptides according to the invention can also be defined by the 23 following formulae (in which X, Z and the dashes have the meanings indicated above): t I 30 XRV-AIEKYL -DQA-LN -WGCAFRQVC-Z XAI ERYL -DZ X-LE-AQ IQQEKNMYELQKLNSWZ XQrQQEKNZ XELGDYKLVEITPIG-APT--KR Z
XYKLVEITPIG-APT--KRZ
X VTV-YGVP-W--AT--LFCA-Z
XVTV-YGVP-W--ATZ
L -NVTE -F W -N Z
XL-NVTE-FZ
XL---S-KPCVKL-PLC Z
XKPCVKL-PLC-Z
XS -KPCVKL-PLC-Z 20
XN-S-I-Z
XYC- P-G-A-L-C-N-TZ 25 X A-C
W--Z
NKRPRQAWCWFKG -KWKD X-G-DPE NC-GEF-YC NZ
YZ
t e
I
t~ t t I I V I- Vt I *~4t I 4 ,~4 I t I. V e I a 1 I I $4 I'~4 V LI Ii 4* 'a V I i: 31 t t ~*2 ia I: S 4 4t The invention also relates in addition to the peptides of SIV already described to the proteins coded by the cDNA of the SIV virus.
It also relates to the proteins of any virus immunologically closely related to SIV-lmac, in particular any virus, the proteins and the glycoproteins of the envelope of which cross-react immunologically and the cDNAs of which exhibit a sequence homology of at least 95% and preferably at least 98%.
In particular, the invention relates to: 1) the proteins and glycoproteins of the envelope encoded in the env gene and shown in Figure 3, 2) the protein GAG shown in Figure 4, 3) the protein POL shown in Figure 4) the protein 0 shown in Figure 6, the protein R shown in Figure 7, 6) the protein X shown in Figure 8, 7) the protein F shown in Figure 9, 8) the protein TAT shown in Figure The amino acids of the previously mentioned proteins of SIV have been presented in alignment with the sequences of the amino acids of tba corresponding proteins of the HIV-2 virus; the points aligned vertically between the two sequences indicate amino acids common to the proteins of the two viruses.
The cDNA sequences coding for the previously mentioned proteins appear in the Figure lB. In addition to the nucleic acid sequences previously mentioned, the invention relates to any modified nucleic acid sequence which also codes for the proteins of the retrovirus SIV or one of its variants.
These cDNA sequences indicated by numbers shown on the sequences previously described (Figure 1B) are the following: I I
I
*L
41 rr
I
32 -sequence coding for M I* GAG, nucleotides
POL,
Q,
X,
R,
F,
TAT-1 ART-1 TAT-2 ART-2
ENV
551 to 2068 1726 to 4893 4826 to 5467 5298 to 5633 5637 to 5939 8569 to 9354 5788 to 6084 6014 to 6130 8296 to 8391 8294 to 8548 6090 to 8732 j 20
'I
I 21 ,xLI tt ei t
I
Thus, the invention quite naturally relates to the proteins previously described when they are isolated from the SIV virus or when they are prepared by a method of synthesis, in particular by one of the methods already cited in connection with the synthesis of shorter peptides.
The invention also relates to the use of the preceding proteins for the diagnosis of the possible presence of antibodies directed against the proteins of HIV-2, and even against the whole HIV-2 virus, and, in some cases, to their use for the purposes of diagnosis of an infection due to one of the HIV viruses. Thus, the peptide GAG encoded by the corresponding gene can be used to locate the possible presence of anti- HIV-1 or anti-HIV-2 antibodies. The env proteins are used preferably for the specific diagnosis of an infection -de to HIV-2 or one of its variants, and sometimes for the diagnosis of an infection due to HIV-2 or HIV-1.
Thus, the invention also relates to an in vitro diagnostic procedure for the detection of antibodies against HIV-2 and possibly against HIV-1 in biological fluids and in particular in human sera.
Such procedures entailing the use of the proteins of SIV previously mentioned for diagnostic purposes have already been described in the present invention.
The invention also relates to "kits" for the in vitro diagnosis of the presence of antibodies against the HIV-2 virus and, in certain cases, fI 33 against HIV-1 in a biological fluid. Such kits employing the peptides previously mentioned have also been described in the present invention.
The invention also relates to immunogenic compositions for the production of vaccines, the active principle of which is constituted advantageously by at least a part of the ENV protein of the SIV virus and this protein may exist as a conjugate formed with a carrier molecule. These immunogenic compositions induce the production of antibodies against the above-mentioned peptide in sufficient amounts to inhibit the proteins of the retrovirus HIV-2, and even the retrovirus HIV-2 itself.
However, the use for diagnostic purposes of the proteins of SIV is in no way limited only to the proteins ENV and GAG. Other proteins among those described may be considered for the preparation of compositions for diagnosis and even as vaccines.
1 I 8 i t t t
S
st t 41 C1^ II t

Claims (6)

1. Peptides having immunological with those of the peptide backbone of the envelope of the viruses of characterized in that they contain a residues not exceeding properties in common the glycoprotein of the HIV-2 class, number of aminoacid t t it t tt C t t t Ci I S A. L lar~rrrc~nc~ c
34- 1) Peptides having immunological properties in com those of the peptide backbone of the glycopro o0 the envelope of the viruses of the HIV-2 clas aracterized in that it also has a peptide structure on with the peptide backbone of the glycoprotein of 2) Peptide having immunological properties in common with those of the peptide backbone of the glycoprotein of the envelope of the viruses of the HIV-2 class, these peptides containing a number of amino acid IC residues not exceeding 40, characterized in that it also has a peptide structure in common with the peptide backbone of the glycoprotein of SIV-1. 3) Peptide according to Claim 2 characterized by one of the formulae: XRV-AIEKYL-DQA-LN-WGCAFRQVCZ XAIEKYL-DZ in which X and Z are OH and NH 2 groups or, in cases in which the immunological properties of the peptide not containing these groups are found to be not substantially modified, they may represent groups containing from 1 to 5 amino acid residues and each of the dashes S4' 2: corresponds to an aminoacyl residue chosen from among those which enable 0 the peptide defined above to conserve the immunological properties of one of the following sequences: 'RVTAIEKYLQDQARLNSWGCAFRQVC AIEKYLQDQ RVSAIEKYLKDQAQLNAWGCAFRQVC AIEKYLKDQ 4) Peptide according to Claim 2 characterized by one of the formulae: X-LE-AQIQQEKNMYELQKLNSWZ S I XQIQQEKNZ in which X and Z are OH and NH 2 groups or, in cases in which the "immunological properties of the peptide not containing these groups are found to be not substantially modified, they may represent groups containing from 1 to 5 amino acid residues and each of the dashes corresponds to an aminoacyl residue chosen from among those which-enable the peptide defined above to conserve the immunological properties of one ®o C 35 1 of the following sequences: SLEQAQIQQEKNMYELQKLNSW QIQQEKN LLEEAQIQQEKNMYELQKLNSW 5) Peptide according to Claim 2 characterized by one of the formulae: XELGDYKLVEITPIG-APT--KR-----Z XYKLVEITPIG-APT--KRZ in which X and Z are OH and NH 2 groups or, in cases in which the immunological properties of the peptide not containing these groups are found to be not substantially modified, they may represent groups containing from 1 to 5 amino acid residues and each of the dashes corresponds to an aminoacy]. residue chosen from among those which enable the peptide defined above to conserve the immunological properties of one of the following sequences: ELGDYKLVEITPIGFAPTKEKRYSSAH YKLVEITPIGFAPTKEK ELGDYKLVEITPIGLAPTNVKRYTTG- YKLVEITPIGLAPTNVK S6) Peptide according to Claim 2 characterized by one of the formulae: X----VTV-YGVP-W--AT--LFCA-Z XVTV-YGVP-W--ATZ in which X and Z are OH and NH 2 groups or, in cases in which the immunological properties of the peptide not containing these groups are found to be not substantially modified, they may represent groups containing from 1 to 5 amino acid residues and each of the dashes corresponds to an aminoacyl residue chosen from among those which i'enable the peptide defined above to conserve the immunological properties c' of one of the following sequences: CTQYVTVFYGVPTWKNATIPLFCAT VTVFYGVPTWKNAT CIQYVTVFYGVPAWRNATIPLFCAT SVTVFYGVPAWRNAT EKLWVTVYYGVPVWKEATTTLFCAS VTVYYGVPVWKEAT ;A~l/i 1 i i 36 7) Peptide according to Claim 6 characterized by ono of the formulae: CTQYVTVFYGVPTWKNATIPLFCAT VTVFYGVPTWKNAT CIQYVTVFYGVPAWRNATIPLFCAT VTVFYGVPAWRNAT EKLWVTVYYGVPVWKEATTTLFCAS VTVYYGVPVWKEAT EDLWVTVYYGVPVWKEATTTLFCAS VTVYYGVPVWKEAT DNLWVTVYYGVPVWKEATTTLFCAS VTVYYGVPVWKEAT 8) Peptide according to Claim 2 characterized by one of the formulae: X----E--L-NVTE-F--W-NZ XL-NVTE-FZ in which X and Z are OH and NH 2 groups or, in cases in which the immunological properties of the peptide not containing these groups are At A found to be not substantially modified, they may represent groups Scontaining from 1 to 5 amino acid residues and each of the dashes corresponds to an aminoacyl residue chosen from among those which enable the peptide defined above to conserve the immunological properties of one of the following sequences: DDYQEITL-NVTEAFDAWNN L-NVTE DDYSELAL-NVTESFDAWEN PNPQEVVLVNVTENFNMWKN I I LVNVTE 9) Peptide according to Claim 8 characterized by one of the formulae: DDYQEITL-NVTEAFDAWNN 3 L-NVTEAF DDYSELAL-NVTESFDAWEN r L-NVTESF PNPQEVVLVNVTENFNMWKN LVNVTENF PNPQEIELENVTEGFNMWKN <1 k~~tl 37 LENVTEGF PNPQEIALENVTENFNMWKN LENVTENF 10) Peptide according to Claim 2 characterized by one of the formulae: XL---S-KPCVKL-PLC----Z XKPCVKLTPLCVZ XS-KPCVKLTPLCVZ in which X and Z are OH and NH 2 groups or, in cases in which the immunological properties of the peptide not containing these groups are found to be not substantially modified, they may represent groups containing from 1 to 5 amino acid residues and each of the dashes corresponds to an aminoacyl residue chosen from among those which enable the peptide defined above to conserve the immunological properties of one of the following sequences: LFETSIKPCVKLTPLCVAMK LFETSIKPCVKLSPLCITMR LWDQSLKPCVKLTPLCVSLK KPCVKLTPLCV KPCVKLSPLCI SLKPCVKLTPLCV S11) Peptide according to Claim 10 characterized by one of the following structures: LFETSIKPCVKLTPLCVAMK LFETSIKPCVKLSPLCITMR LWDQSLKPVKLTPLCVSLK till' LWDQSLKPCVKLTPLCVTLN PCVKLTPLCV KPCVKLSPLCI t, I 3CI 12) Peptide according to Claim 2 characterized in that it contains the basic structure: XN-S-I-Z I 38 1144 I A( 4) r.4 4 4 2 9r in which X and Z are OH and NH 2 groups or, in cases in which the immunological properties of the peptide not containing these groups are found to be not substantially modified, they may represent groups containing from 1 to 5 amino acid residues and each of the dashes corresponds to an aminoacyl residue chosen from among those which enable the peptide defined above to conserve the immunological properties of one of the following sequences: NHCNTSVITESCD NTSVIT NHCNTSVIQECCD NTSVIQ TSCNTSVITQACP NTSVIT 13) Peptide according to Claim 12 characterized by one of the following formulae: NHCNTSVITESCD NTSVIT NHCNTSVIQECCD NTSVIQ TSCNTSVITQACP NTSVIT INCNTSVITQACP NTSVIT INCNTSAITQACP NTSAIT 14) Peptide according to Claim 2 characterized by the following formula: XYC-P-G-A-L-C-N-TZ in which X and Z are OH and NH 2 groups or, in cases in which the immunological properties of the peptide not containing these groups are found to be not substantially modified, they may represent groups containing from 1 to 5 amino acid residues and each of the dashes corresponds to an aminoacyl residue chosen from among those which 4 I .9 *9 *r 4 A s B 39 enable the peptide defined above to conserve the immunological properties of one of the following sequences: YCAPPGYALLRC-NDT YCAPAGFAILKCNNKT 15) Peptide according to Claim 14 characterized by one of the formulae: YCAPPGYALLRC-NDT YCAPAGFAILKGNNKT YCAPAGFAILKCNDKK YCAPAGFAILKCRDKK 16) Peptide according to Claim 2 characterized by the formula: in which X and Z are OH and NH groups or, in cases in which the immunological properties of the peptide not containing these groups are found to be not substantially modified, they may represent groups containing from 1 to 5 amino acid residues and each of the dashes corresponds to an aminoacyl residue chosen from among those which enable the peptide defined above to conserve the immunological properties of Sone of the following sequences: NKRPRQAWCWFKG-KWKD NERPKQAWCRFGG-NWKE N--MRQAHCNISRAKWNA 17) Peptide according to Claim 16 characterized by the following Sformulae: NKRPRQAWCWFKG-KWKD NERPKQAWCRFGG-KWKE N--MRQAHCNISRAKWNA D--IRRAYCTINETEWDK I--IGQAHCNISRAQWSK 3D 18) Peptide according to Claim 2 characterized by the following formulae: SXNC-GEF-YC-Z in which X and Z are OH and NH 2 groups or, in cases in which the immunological properties of the peptide not containing these groups. I 40 1 are found to be not substantially modified, they may represent groups containing from 1 to 5 amino acid residues and each of the dashes corresponds to an aminoacyl residue chosen from among those which enable the peptide defined above to conserve the immunological properties of one of the following sequences: KGSDPEVAYMWTNCRGEFLYCNMTWFLN NCRGEFLYCN GG-DPEVTFMWTNCRGEFLYCKMNWFLN NCRGEFLYCK -GGDPEIVTHSFNCGGEFFYCNSTQLFN NCGGEFFYCN 19) Peptide according to Claim 18 characterized by one of the following structures: KGSDPEVAYMWTNCRGEFLYCNMTWFLN NCRGEFLYCN GG-DPEVTFMWTNCRGEFLYCKMNWFLN NCRGEFLYCK -GGDPEIVTHSFNCGGEFFYCNSTQLFN S. NCGGEFFYCN °I -GGDPEITTHSFNCRGEFFYCNTSKLFN NCRGEFFYCN -GGDPEITTHSFNCGGEFFYCNTSGLFN NCGGEFFYCN 20) Peptide according to Claim 2 characterized by one of the following formulae: S, XC-I-Q-IZ in which X and Z are OH and NH 2 groups or, in cases in which the immunological properties of the peptide not containing these groups t. are found to be not substantially modified, they may reDresent groups containing from 1 to 5 amino acid residues and each of the dashes corresponds to an aminoacyl residue chosen from among those which enable .5 the peptide defined above to conserve the immunological properties of 1\ one of the following sequences: iR ,11 i 41 RNYAPCHIKQIINTWHKVGRNVY CHIKQII RNYVPCHIRQIINTWHKVGKNVY CHIRQII TITLPCRIKQFPINMWQEVGKAMY CRIKQFI 21) Peptide according to Claim structures: RNYAPCHIKQIINTWHKVGRNVY CHIKQII RNYVPCHIRQIINTWHKVGKNVY CHIRQII TITLPCRIKQFINMWQEVGKAMY CRIKQFI SITLPCRIKQIINMWQKTCKAMY CRIKQII NITLQCRIKQIIKMVAGR-KAIY CRIKQII 20 characterized by one of the following 4(D t i 22) Antigenic peptide gagl, characterized by one of the basic structures: XDCKLVLKGLGMNPTLEEMLTAZ XDCKLVLKGLGTNPTLEEMLTAZ in which X and Z are OH and NH 2 groups or, in cases in which the immunological properties of the peptide not containing these groups are found to be not substantially modified, they may represent groups containing from 1 to 5 amino acid residues and each of the dashes corresponds to an aminoacyl residue chosen from among those which enable the peptide defined above to conserve the immunological properties of one or other of the following sequences: DCKLVLKGLGMNPTLEEMLTA DCKLVLKGLGTNPTLEEMLTA 23) Nucleotide sequence characterized iri that it contains all or part of the sequence of the nucleic acids defined in-Figure IB. -qE i I -I~-x1. 42 24) Nucleotide sequence characterized in that it contains all or part of the sequence of the nucleic acids defined in Figure IC. Nucleotide sequence according to Claim 23, characterized in that it comprises the nucleotide sequences: GAG extending from POL nucl6otides TAT-1 ART-1 TAT-2 ART-2 LTR 550 to 2068 1726 to 4893 4826 to 5467 5298 to 5633 5637 to 5939 8569 to 9354 5788 to 6084 6014 to 6130 8296 to 8391 8294 to 8548 8950 to 9468 and 1 to 316 6090 to 8732 ENV 26) Peptide having a peptide structure in common with the peptide backbone of SIV-1, characterized in that it comprises all or part of the following amino acid sequences: ENV shown in figure 3 GAG 4 POL TAT ART 11 27) Recombinant nucleic acid characterized in that it comprises the totality or part of a cDNA according to any one of the Claims 23 to inserted in a nucleic acid derived from a vector. 28) Recombinant nucleic acid according to Claim 27, characterized in that it is labelled. I 43 1 29) Antigenic composition containing the gag peptide according to Claim 26 or 27, at least one gagl peptide according to Claim 22 or at least an oligomer of this peptide, characterizedin that\the composition has the capacity to be recognized by biological fluids of human origin, in particular by sera containing anti-HIV-2 antibodies and, to a certain extent,anti-HIV-1 antibodies. Antigenic composition containing the env peptide according to Claim 26 or at least a peptide according to Claims 3, 4 and 5 or at least an oligomer of this peptide, characterized in that the composition specifically recognizes the presence of antibodies against HIV-2. 31) Immunogenic composition containing all or part of the env peptide according to Claim 26 or at least a peptide or at least an oligomer of this peptide or this peptide in the form of a conjugate with a carrier molecule according to Claims 6 to 21 in combination with a pharmaceutically acceptable vehicle for the production of vaccines, characterized in that the composition induces the production of antibodies against the above-mentioned peptides in sufficient amounts for them to be able to inhibit in an efficacious manner the proteins of the retrovirus S 20 HIV-2, or even the entire HIV-2 retrovirus, 32) Immunogenic composition according to Claim 31 characterized in that Sit contains peptides, the formulae of which correspond to sequences in the glycoproteins of the envelope of HIV-2, SIV-1 and HIV-1 which exhibit an amino acid sequence homology greater than 33) Immunogenic composition according to one of the Claims 31 or 32, characterized in that it contains at least one peptide or at least one oligomer of this peptide or this peptide in the form of a conjugate with a carrier molecule chosen from among env4, env5, env6 and envl0. S' 34) In vitro diagnostic procedure for an infection by HIV-2 in a 3 t 0 biological fluid comprising: the placing in contact of this biological fluid with at least one peptide according to one of the Claims 1, 2, 3, 4, 5, 22 or a conjugate of these peptides with a carrier molecule, or with the peptides ga or env according to Claim 26. UA c 44 1 the detection of the possible presence of an antibody-antigen complex by physical or chemical methods in the said biological fluid. In vitro diagnostic procedure for an infection by HIV-2 in a biological fluid according to Claim 34, characterized in that the antibody- antigen complex which may have formed is detected by means of immuno- enzymatic assays (of the ELISA type), immunofluorescence assay (of the IFA type), radioimmunological assays (of the RIA type) or radioimmuno- precipitation assays (of the RIPA type). 36) Kit for the in vitro diagnosis of an infection by HIV-2 in a biological fluid characterized in that it comprises: a peptide composition containing a peptide according to one of the Claims 1 to 5, 22 or a mixture of these peptides or a conjugate of these peptides with a carrier molecule, or the peptides gag or env according to Claim 26, a reagent for constituting an appropriate medium for the performance of an immunological reaction, one or more reagents, possibly labelled, for the detection of the antibody-antigen complex formed as a result of the immunological reaction, a reference biological fluid not containing antibodies recognized by the above-mentioned peptide composition. 1/35 F IG.A .1 RIV2 .ROD R GGCTCTGCGGAGAGGCTGGCAGATTGAGCCCTGGGAGGTTCTCTCCAGCACTAGCAGG TAGAGCC TGGGTGTTCCCTGCTAGACTCTCACCAGCACTTGGCCGGTGCTGGGCAGACGG 100 9 1US CCC CACGCTTGCTTGCTTAAAAACCTCTTAATAAAGCTGCCAGTTAGAAGCAAGTTAAGT GTGTGCTCCCATCTCTCCTAGTCGCCGCCTGGTCATTCGGTGTTCACCTCAGTAACAAGA *200 1 "CCTGGTCTGTTAGGACCCTTCTTGCTTTGGGAAACCGACGCAGGAAAATCCCTAGCAGG *300 TTGGCGCC TCAACAGGGACTTGAAGAAGACTGAGAAGTCTTGGAACACGCTGAGTGAAG GCAGTAAGGGCGGCAGGAACAAACCACGACGGAGTGCTCCTAGAAAGGC"GCGGGCCGAGG I I I 400 ACCAAAGGCAGCGTGTGGAGCGGGAGGAGAAGAG-CCTCGGT-laA,'GGTAAGTACCTA CACCAAAAACTGTAGCCGAAAGGGCTTGC;TTCTAX-TTAO Y'AGGTAGAAGATTGTG *500 1 Me;lArg~eusnroGeGraL us~rgcGLvLy 3 .~"AiIeA1Trp"I!*,L'-Arlals CAGGTTACGGCCCGGCGGAAAGAAAAAGTAC AGGCTAtiA AATTTGTGTGCVGCAGCGAA LysLeuAspArgPheGly~euAlaGluSerLeuLeuGiU'SerLys~lu~lyCyGlnLys rAAATTGGACAGAuTTCGGATTAGCAGAGAGCCTGTTGGAGTCAAAkAGAGGCTTG~tlAAA 700 IleLeuThVaLeuAsp~roMetVaProThrG..ySerG31uaisnLeuLy,,sSerLeuPhe AATTCTTACAGTTTTAGATCCAATGGTACCGACAGGWTTOAGAAAATTTAA.AAAGTCTTTT 4~AI AstiThrValCysValIleTrpCysIleHisAlaGluG~uLysValLysAspThrGluGly TAATACTGTCTGCGTCATTTGGTGCATACACGCAGAAGAGAAAGTGAAAGATACIGAAGG t iAlaLysGlnI [eVa lArgArgHisLeuValAlaGluThrGlyThrAla~luLyaMetPro AGCAAAACAAATAGTGCGGAGACATCTAGTGGCAGAAACAGCAACTGCAGAGAAAATGCC FIG. 1A TRPTRP 2/35 SerThrSerArgProThrAlaProSerSerG luLysG lyGlyAsnTyrProValGlnHis AAGCACAAGTAGACCAACAGCACCArCTAGCGAGAAGG4,AGGAAATTACCCAGTGCAACA ValGlyGlyAsnTyrThrliislleProLeuSerProArgThrLeuAsnAlarrpValLys TGTAGGCGGCAACTACACCCATATACCGCTGAGTCCCCGAACCCTAAATGCCTGGGTAAA 1000 LeuValGluGluLysLys~heGlyAlaGlIuValValProGlyPheGlnAlaLeuSerGlu ATTAGTAGAGGAAAAAAAGTTCGGGGCAGAAGTAGTGCCAGGATTTCAGGCACTCTCAGA Gl1y Cy sTb rP roT yr As p Ile Asn G.n Met L euAs nC y sVa1G ly Asp His G 11AIa A 1.a AGGCTGCACGCCCTATGATATCAACCAAATGCTTAATTGTGTGGGCGACCATCAAGCAGC 1100 MetGlnIleIleArgGluIleIleAsnGluGluAlaAlaGluTrpAsp~alGlnHiaPro CATGCAGATAATCAGGGAGATTATCAATGAGGAAGCAGCAGAATGGGATGTGCAACATCC 1200 I leProGlyProLeuPro~laG lyGlnLeuArgGluProArgGlySer~spIleAlaG ly AATACCAGGCCCCTTACCAGCGGGGCAGCTTAGAGAGCCAAGGGGATCTGACATAGCAGG ThrThrSerThrValGluluGlnlleGlnlrpMetPheArgProGlnAsnProValPro GACAACAAGCACAGTAGAAGAACAGATCCAGTGGATGTTTAGGCCACAAAATCCTGTACC **1300 Va1GlyAsnIleTyrArg~rgTrpIleG*InIleGlyLeuG~nLyaCysValArgMetTyr AGTAGGAAACATCTATAGAAGATGGATCCAGATAGGATTGCAGAAGTGTGTCAGGATGTA AsnProThrAsnlleLeu~sp IleLysGinGlyProLysGiuProPheG inSerTyrVa 1 CAACCCGACCAACATCCTAGACATAAAACAGGGACCAAAGGAGCCGTTCCAAAGCTATGT 1400 0 AspArgPheTyx-LysSerLeuArgAlaGluGlnThrAspProAlaValLysAsnTrpMet AGATAGATTCTACAAAAGCTTGAGGGCAGAACAAACAGATCCAGCAGTGAAGAATTGGAT *1500 ThrGlnThrLeuLeuVal~lnAsnAlaAsnProAspCysLysLeuValLeuLyIGlyLeu GACCCAAACACTGCTAGTACAAAATGCCAACCCAGACTGTAAATTAGTGCTAAAAGGACT GlyMetAsnProThrLeuGluGluMetLeuThrAlaCysGlnGlyValGlyGlyProGly AGGGATGAACCCTACCTTAGAAGAGATGCTGACCGCCTGTCAGGGGGTAGGTGGGCCAGG 1600 GlnLysAlaArgLeuMetA1'aGluAlaL uLyaGluVa1IleGlyProAlaProIlePro CCAGAAAGCTAGATTAATGGCAGAGGCCCTGAAAGAGGTCATAGGACCTGCCCCTATCCC PheAlaAlaAlaGlnGln~rgLysAlaPheLysCysTrpAmnCysGly~yaGluGlyH2iS ATTCGCAGCAGCCCAGCAGAGAAAGGCATTTAAATGCTGGAACTGTGGAAAGGAAGGGCA 1700 SerAlaArgGl~nCyArgAlaProArgArgGlnGlyCyITrpLysCyBGlyLysProGiy CTCGGCAAGACAATGCCGAGCACCTAGAAGGCAGGCCTGCTGGAAGTGTGGTAACCCAGG 0 1 1800 ThrGlyArgPhePheArgThrG lyProLeuGly HisIleMetThrAsneysProAspArgGlnAlaGlyPheLeuGlyLeuGlyProTrpGly ACACATCATGACAAACTGCCCAGATAGACAGGCAGGTTTTTTAGGACTGGGCCCTTGGGG LysGluAla~roGlnLeuProArgGlyProSerSerAlaGlyAlaAspThrAsnSerThr LysLysProArgAsnPheProValAlaGlnValPraGlnGlyLeuTbrProThrAlaPro AAAGAAGCCCCGCAACTTCCCCGTGGCCCAAGTTCCGCAGGGGCTGACACCAACAGCACC *1900 ProSerGlySerSerSerGlySerThrGlyGluIletyrAlaAlaArgGluLysThrGlu ProValAspProAlaValAspLeuLeuGluLysTyrMetGlnGlGlyLyArgGllArg CCCAGTGGATCCAGCAGTGGATCTACTGGAGAAATATATGCAGCAAGGGAAAAGACAGAG ArgAlaG lu~rgG luThrI leG luG lySerAspArgG ly~euThrAlaProArgAlaGly GluGlbnArgGluArgProTyrLyuG luVa lTbrGluAapLeuLeu~isLeuGluGllGly AGAGCAGAGAGAGAiGACCATACAAGGAAGTGACAGAGGA.VTTACTGCACCTCGAGCAGGG (fig. lA-suite 1) 7 '1 444, 4 6 1444 I6 #4 6 444 46 4 44 a ti 4 o *4 6 44 4 #t 4~64 (4 4 *444 4 4 4 A 44-
044. 44 *4 j 4 44 4 a 444 6 44 04 4 4 4 3,35 GlyAspThr IleGlnG IyAlaThrAsnArSG lyLeuAlaAlaProG In? he SerLeuTrp G luThrF roTy rArgG luProProThrG luA s pLeu LeuH i sLeuAs niS erLeuP heG ly GGAGACACCATACAGGGAGCCACCAACAGAGGACTTGCTGCACCTCAATTCTCTCTTTGG 4 2100 Lys ArgProV-a Va lThrAiaTy I eGuG yGn Pr Va GluVa LeuLeuAspThr LysAspG In AAAAGA CC A GTAGTCACAGCATA CATT GAG GGTCA GC CAG TA GAAG TC TTGTTAGACAC A G lyAlaAsp~spSer I IeVa lAlaG ly IleGluLeuG lysflAsTyrSerProLys 11; GGGGCTGACGACTCAATAGTAGCAGGAATAGAGTTAGGGAACAATTATAGCCCAAAAATA 2200 Val GlyGlyi leGlyGlyPhe I eAsnThrLysG IuTyr Lys AsnVa lGlu IleGluVal1 GTA GGGGGAATAGGGGGATT CA TAAATAC CAAG GAATATAAAAATGTA GAAATAGAAGTT LeuAsnLys ysVa lArgA iaThr I leMe I ThrG I yAspT-hrPro I IleA n I I ePheGly CTAAATAAAAAGGTACGGGCCACCATAATGACAGGCGACACCCCAATCAACATTTTTGGC 2300 A rgAsn Ile~euThrAl aLeuG ly~et SerLeuAsflLeuProValIA laLYsVa lGluPro AGAAATATTCTGACAGCCTTAGGCATGTCATTAAATCTACCAGTCGCCAAAGTAGAGCCA 2400 Il ILys I lelle tLeuLys ProClyLysAs pG I yProLy s LuArgG lnTrpP raLeuThr ATAAAAATAATGCTAAAGCCAGGGAAAGATGGACCAAAACTGAGACAATGGCCCTTAACA Lys G I Lys I eG uAlaLuLysG u1eCysG uLys~etGluLysG iuG yGluLeU' AAkAGAAAAAATAGAAGCAC TAAAAGAAAT CTGTGAAAAAATGGAAAAAGAAG GC CAG CTA 2500 G luG uA a roProThrAsnP roTyrAsnThrP roThrPheAl a 1eLys LysLysAsp GAGGAAGCACCTCCAACTAATCCTTATAATACCCC CACATTTGCAATCAAGAAAAAGGAC LysAsn Lys rpArgMe tLeulI leAspPheArgG luLeuAsflLys Va 1ThrG laAapPhe AAAAACAAAT GGAGGAT GC TAATAGAT'T CAGAGAA CTAAA CAAGGTAAC T CAAGATT TC 2600 TbrG lu~leG InLeuG 11 leProHisProA IaG lyLeuAlaLysLysArgArg IleThr ACAGAAATTCAGTTAGGAATTCCACACCCAGCAGGGTTGGCCAAGAAGAGAAGAATTACT 2700 Va1LeuApp~alGlyAspAiaTyrPheSerIleProLeuHL3GluAspPheArgProTyr GTACTAGATGTAGGGGATGCTTACTTTTCCATACCACTACATGAGGACTTTAGACCATAT 4 0~uro~~lsns*l~l~ol~y~g~ll~ry ACTGCATTTACTCTACCATCAGTGAACAATGCAGAACCAGGAAAAAGATACATATATAAA 2800 ValLeuPro~inG lyTrpLyaGlySerPro,&laIlePeGllHisThrHetArgGllVal GTCTTGCCACAGGGATGGAAGGGATCACCAGCAATTTTTCAACACACAATGAGACAGGTA LeuGluPro~beArgLysAlaAsuLysAspVal~leIleIleGlnlTyrMetASpAspIle TTAGAACCAT'C CAGAAAAGCAAACAAGGATGTCATTATCATTCAGTACATGGATGATATC
2900.. LeulleAla~erAspArgThrAspLeuG IuHisAspArg~alValLeuGlflLeuLysGlu TTAATAGCTAGTGACAGGACAGATTTAGAACATGATAGGGTAGTCCTGCAGCTCAAGGAA *3000 LeuLeuAsnG lyLeuG lyPheSerThrProAspG luLyspheGlnLysAspProProTyr CTTCTAAATGGCCTAGGATTTTCTACCCCAGATGAGAAGTTCCAAAAAGACCCTCCATAC HisTrp~et~lyTyrGluLeuTrpPraTbrLysTrpLys~euGllLysIleGflnLeuPro CACTGGATGGGCTATGAACTATGGCCAACTAAATGGAAGTTGCAGAAAATACAGTTGCCC 3100 GlnLysGluIleTrpThrValAsnAspIleGlnLyaLeuValGlyValLeuAsflTrpAla CAAAAAGAAATATGG^ACAGTCAATGACATCCAGAAGCTAGTGGGTGTCCTAAATTGGGCA %fig.lA-suite 2) 0 4/35 A Ia GluLeuTyrPrQGly I leLy aThr Lys Hi aLeuCyrs Avg LeuI eArgGlyLy aMet GCACAACTCTACCCAGGGATAAAGACCAAACACTTATGTAGGTTAATCAGAGGAAAAATG 3200 0 ThrLeuThrG luG luVaiG lnTrpThrG luLeuAlaG luAlaG luLeuG luG luAsnArg ACACTCACAGAAGAAGTACAGTGGACAGAATTAGCAGAAGCAGAGCTAGAAGAAAACAGA a 0 3300 IleIleLeuSerGlGuG~nGuGy~iTyrTyGlG1uGluLyIGiuLeuGluAla A TTA T.CC-!rAAGC CA GGAA CAA GA GGGACAC TATTAC CAL GAAGAAAAAGAG C TAGAAGCA Th rValGinLys~spGluGluAsnGluTrpThrTyrLysleHisGlnGluluLystle AGAGTCCAAAAGGATCAAGAGAATGAGTGGACATATAAAATACACCAGGAAGAAAAAATT LeuLysVa lGlyLysTyrAlaLysValLysAsnThrHiSThrAoflGlyIleArgLeuLeu C TAAAA GTA GGAAAATATGCAAAGG TGAAAAA CACC ATA CC AATG GAAT CAGAT TG TTA GCACAGGTAGTTCAGAAA ATAGGAAAAGAAGCACTAGTCATTTGGGGACGAATACCAAAA 3500 P heHisLeuP roVaiG luArgG lu IleTrpG luG lnTrp~rpAspAsnTy rTrpG inVa 1 TTTCACCTACCAGTAGAGAGAGAAATCTGGGAGCAGTGGTGGGATAACTACTGGCAAGTG *3600 ThrTrpIleProAspTrpAspPheValSerThrProPro~euVa'lArgLeuAlaPheAsn ACATGGATCCCAGACTGGGACTTCGTGTCTACCCCACCACTGGTCAGGTTAGCGTTTAAC LeuValGlyAspProIleProGlyAlaGluThrPheTyrThrAspGlySerCysAsflArg C TGGTAGGGGATCCTATACCAGGTGCAGAGACCTTCTACACAGATGGATCCTGCAATAGG o 3700 Gin SerLysG luG lyLysAlaG 2yTyr Va lThrAspArgG lyLysAspLys Va iLys Lys C AAT CAAAAGAA GGAAAAGC AG GATATG TAA CA GATAGAGG GAAAGA CAAGGCTAAAGAAA LeuGluGln~!hrThrAsnGlnGlnAlaGluLeuGluAlaPheAlaMetAlaLeuThrAsp CTAGAGCAAACTACCAATCAGCAAGCAGAACTAGAAGCCTTTGCGATGGCACTAACAGAC 3800 SerGlyProLysValAsnIleIleValAspSerGlflTyr~alMetGlyIleSerAlaSer TCGGGTCCAAAAGTTAATATTATAGTAGACTCACAGTATGTAATGGGGATCAGTGCAAGC 3900 GlnProThrGluSerGluSerLys IleValAsaG InIleIleGluG luMetlleLysLys C AA CCAA CAGAG TCAG AAAG TAAAATAG TGAA CCAGAT CATAGAAGAAATGATAAAA.G GluAlaIleTyrValAlaTrpValProAlaiLySGlyleGyGlyAUG inGluVal GAAGCAATCTATGTTGCATGGGTCCCAGCCCACAAAGGCATAGGGGGAAACCAGG&AGTA 4000 AspliisLeuValSerGlnGlyIleArgGllValLeuPheLeuGluLysIleGluProAla GACTTATATA GTTCAGACAAGTGTTGTTCC.TGGAAAAAATAGAGCCC GOT GlnGluGlu~isGluLysT;y7,'isSSrAoflValLysGlu~euSerHisLoysPheGlyIle CAGGAA GAA CAT GAAAAATA ICA TAG CAL G TAAAAGAAC TGTC TCATAAAT TTG GALTA ProAsnLeuValAlakrgGl IleVaADSerCysAlGICysGlnlul~yaGlyGlu C CCAATTTAGTGGCAAGGICAAATAGTAAACTCATGTGCCCAATGTCAACAGAAAGGGGAA *4200 Alalle~isGyGlValAsAlaGluLeiGlyThrTrPG ln~et~tspCysThrudisLeu GCTATACATGGGCAAGTAAATGCAGAACTAGGCACTTGGCAAATGGACtGCACACATTTA G luGlyLyslleIleleValAlaValHiValAlSerGlyPheIleGluAlaGluVaI GAAGGAAAGATCATTATAGTAGCAGTACATGTTGCAAGTGGATTTATAGAAGCAGAAGTC 0 0 0 4300 I1leProGluGluSerGlyArgGlnThrAlaLeuPheLeuLeuLy9LeuAl8SerArgTrp ATCCCACAGGAATCAGGAAGACAAACAGCACTCTTCCTATTGAAACTGGCAAGTAGGTGG (fig.rA-suite 3) 5/35 ProlleThrliisLeuuumThrAspAaaGlyAlaAsnPheThrSerclnGluValr;y.Met CCAATAACACAC TTGCATACAGATAATGGTGC CAACTTCACTTCACAGGAG GTGAAGATG 0 4400 ValAlaTrprrplleGlylleGluGlnSerPheGlyValproTyrAsnP'roGlnSerGlu* GTAGCATGGTGGATAGGTATAGAACAATCCTTTGGAGTACCTTACAATCCACAGAGCCAA 9 4500 G GAGTAGTAGAAGCAATGAATCACCATCTAAAAAACCAMATAAGTGAAACAATAGTACTA HetAlal leliuCyaMetAsnPheLysArgArgc lyG ly~leG 1yAspMetThrProSer ATGGCAATTCATTGCATGAATTTTAAAAGAGGGGGGGAATAGGGGATATGACTCCATCA 0 4600 GluArgLeuIieAsnMetIleThrThrGluGlnGluIleGlnPheLeuGlnAlaLysAan GAAAGATTAATCAATATGATCACCACAGAACAAGAGATACAATTCCTC CAAGCCAAAAAT S erLys Leu~ysAspF heArg VaiTy rP heArgG luG lyA rgA BpG lnLeuTrpLysG ly TCAAAATTAAAAGATTTCccoorc- j. .TCAGAGAAGGCAGAGATCAGTTGTGGAAACCA 4700* P roG lyG lu~euLeuTrpLys GlyG luG lyAl aVa iLeuVa iLys VaIG lyThrAsp le C C TGGGAAC TACGOTG GAAAGGAGAAGGAGCAGTCC TAGTCAAG OTAG GAACAGACATA *4800 Lysllelle~roArArgLysAlaLysIlelleArAsp~yrolyGlyArgolnoluMet Me toluG luAs pLys Arg Trp AAAATAATACCAAGAAGGAAAGCCAAGATCATCAGAGACTATGGAGGAAGACAAGAGATG -ft* AspSerGly~er~isLeuGluGlyAlaArgGluAspolyoluMetAla IleValValProThrTrpArgValProolyArgMetoluLysTrpuisSerLeuValLys GATAGTGGTTCCCACCTGGAGGGTGCCAGGGAGGATGGAGAAATGGCATAGCC TTGTCAA 4900 TyrLeuLysTyrLysThr~ysAspLeuG luLysValCysTyrVa iProHisliisiysVal GTATCTAAAATACAAAACAAAGGATCTAGAAAAGGTGTGCTATGTTCCCCACCATAAGGT GlyTrpAlaTrpTrpThrcysSerArgValllePheProLeuLysoly~snSerHisLeu GGGATGGGCATGGTGGACTTGCAGCAGGGTAATATTCCCATTAAAAGGAAACAGTCATCT 5000 GlulleGlnAlaTyrTrpAsnLeurhrProoluLysolyTrpLeuSerSerTyrSerVal AGAGATACAGGCATATTGGAACTTAACACCAGAAAAAGGATGGCTCTCCTCTTATTCAGT 5100 ArgIleh 1y~rl~ah~phrs~lh~ba~sl~pa AAGAATAACTTGGTACACAGAAAAGTTCTGGACAGATGTTACCCCAGACTGTGCAGATGT i L tLeulleliisSerThrlyr~hePro'CysPheThrAlaG lyGluValArgArgAlaIleArg CCTAATACATAGCACTTATTTCCCTTGCTTTACAGCAGGTGAAGTAAGAAGAGCCATCAG 200 G lyGluLysLeuLeuSerCyuCysAsuryrProArgAlaHisArg'AlaGlnzValProSer AGGGGAAAAGTTATTGTCCTGCTGCAATTATCCCCGAGCTCATAGAGCCCAGGTACCGTC LeuGlnPheLeuAlaLeuvalValValolnolnAsnAspArgProalnArgAspSerThr MetThrAspProArgG luThrVa IPro ACTTCAATTTCTGGCCTTAGTGGTAGTGCAACAAAATGACAGACCCCAGAGAGACAGTAC 5300 ThrArgLysGlnArgArgArgAspTyrArg,(rgolyLeuArgLeuAla~ysGonAspSer ProGlyAsnSerGlyGluGluThrIleGlyGlt AlaPheAlaTrpLeuAsuArgThrVal CACCAGGAAACAGCGGCGAAGAGACTATCGGAGAGGCC TTCGCCTGGCTAAACAGGACAG 5400 ArgSeriLyaGlnArgSerSerGluSerProThr ProArgThrryrPheProGlyVal GluAlalleAmnArgGluAlaValAauHisLeuProArgGluLeullPheGlnVAITrp TAGAAGCCCATAAACAGAGAAGCAGTGAATCACCTAC CC CGAGAACTTATTTTCCAGGTGT (fig.lA-suite 4) AlaGlu.Va iLeuG luIleLeuAla 63 GlnArgSerTrpArgTyrTrpliiaAspGluGlnGly~etSerGluSerTyrThrLyaTyr G GCAGAG GTCC TGGAGATACTGGCATGATGAACAAGGGATGTCAGAAAG TTACACAAAGT 5500 ArgTyrLeuCysIleIleGlnLysAlaValTyrMetI~isValArgLysGlyCyaThrCye ATAGATATTTGTGCATAATACAGAAAGCAGTGTACATGCATGTTAGGAAAGGGTGTACTT LeuG lyArgG lyHisG lyProG lyG ly~rpArgProG lyProProProProProProP ro GCCTGGGGAGGGGACATGGGCCAGGAGGGTGGAGACCAGCGCCTCCTCCTCCTCCCCCTC 5600 0 MetAlaGluAlaProTbrGluLeuProProValAspGlyThrProLeu G lyLeuVal*** CAGGTCTGGTCTAATGGCTGAAGCACCAACAGAGCTCCCCCCGGTGGATGGGACCCCACT ArgGluProGlyAspGluTrpIleIleGluIleLeuArgGluIleLysGluGluAlaLeu GAGGGAGCCAGGGGATGAGTGATAATAGAAATCTTGAGAGAAATAAAAGAAGAAGCTTT LyslisPheAspProArg~euLeuleAlaLeuGlyLysTyrleTyrrhrArguisGly MetG lu AAAGCATTTTGACCCTCGCTTGCTAATTGCTCTTGGCAAATATATCTATACTAGACATGG 5800 AspThrLeuGluGlyAla~irgGluLeuIleLysValLeuGlnArgAlaLeuPheThrHis ThrProLeuLysAlaProGluSerSerLeuLysSerCysAsnGluProPheSerArgThr AGACACCCTTGAAGGCGCCAGAGAGCTCATTAAAGTCCTGCAACGAGCCCTTTTCACGCA il4 PheArgAlaGlyCysGly~isSerArglleGlyGlnThrArgGlyGly~snProLeuSer SerGluGlnAspValAlaThrGlnGluLeuAlaArgGlnGlyGluGluIleLeuSerGln CTTCAGAGCAGGATGTGGCCACTCAAGAATTGGCCAGACAAGGGGAGGAAATCCTCTCTC 5900* AlalleProThrProArgAonMetGln LeuTyrArgProLeuG luThrCysAsnAsnSerCysTyrCyoLysArgCysCysTyrHis AGCTATACCGACCCCTAGAAACATGCAATAACTCATGCTATTGTAAGCGATGCTGCTACC *6000 MetAsnG luArgAlaAsp CysGlnMetCysPheLeuAsnLysclyLeuGlyl eCysTyrG luArgLyeGlyArgArg ATTGTCAGATGTGTTTTCTAAACAAGGGGCTCGGGATATGTTATGAACGAAAGGGCAGAC GluGluGly~euGlnArgLysLeuArgLeuIleArgLeu~euiGnThrSe-GluTyr Met 4 1 ArgArgThrProLysLysThrLysTbrnisProSerProTh. ProAspLys GAAGAAGGACTCCAAAGAAAACTAAGACTCATCCGTCTCCTACACCAGACAAGTGAGTAT 4 ~1 6100 AspGluSer~laAlaTyrCysllisPhelleSer Me tAsnG inLeuLeulleAla IleLeuLeuA laSerAlaCys Leu Va lTyrCysThrG in GATGAATCAGCTGCTTATTGCCATTTTATTAGCTAGTGCTTGCTTAGTATATTGCACCCA TyrVa lThr Va lPheTyrGlyVa lPxroThrTr.pLysAsnAlaThrlleProLeuPheCys ATATGTAACTGTTTTCTATGGCGTACCACGTGGAAAAATGCAACCATTCCCCTCTTTTG 41 6200 AlaThrArgAsnArgksprhrTrpGlyThrlleG lnCysLeuProAspAsnAspkspTyr TGCAACCAGAAATAGGGATACTTGGGGAACCATACAGTGCTTGCCTGACAATGATGATTA 6300 GlnGlulleTbrLeuAsnValThrGluAlaPbeAspAlaTrpAsnAsnThrValThrGlu T-CAGGAAATAAC TTTGAATGTAACAGAGGCTTTTGATGCATGGAATAATACAGTAACAGA GlnAlaIleGluAspValTrpHisLeuPheGluThrSerIleLysProCyuValLyuLeu ACAAGCAATAGAAGATGTC TGGCATCTATTCGAGACATCAATAAAACCATGTGT.CAAA&CT ~(fig.lA-stifte 5)40 7/35 ThrProLeuCysValAls~etLyaCyuSerSerThrGluSerSerThrGlyAsnkunTbr AACACC TTTATGTGTAGCAATGAAATGCAGCAGCACAGAGAGCAGCACAGGGAACAACAC ThrSerLysSerThrSer~hrThrThrThrTbrProThrAspGlnGlu InGlulleSer AACC TCAAAGAGCACAAGCACAACCACAACCACACCCACAGACCAGGAGCAAGAGATAAG 6500 GluAspTbrProCysAlaArgAlaAspAsnCyaSerGlyLeuGlyGluluCluThrIle TGAGGATACTCCATGCGCACGCGCAGACAACTGCTCAGGATT-GGGAGAGGAAGAAACGAT 4 6600 AsnCysGlnPheAsnMetThrGlyLeuGluArgAspLysLyeLysGlnTyrAsnGluThr C AAT TGC CAGTT CAATATGAC AG GATTA GAAAG AGATAAGAAAAAA CAG TATAATG AAAC TrpTyrSertyeAspVal~alCysG luThrAunAunSerTbrksnGln~ThrGluCysTyr ATGGTACTCAAAAGATGTGGTTTGTGAGACAAATAATAGCACAAATCAGACCCAGTGTTA 6700 14etAsnuisCysAsnThr~erVallleThroluSerCysAspLysllis~yrTrpAspAla CATGAACCATTGCAACACATCAGTCATCACAGAATCATGTGACAAGCACTATTGGGATGC I IeArgPheArgTyrCys~l aProProG lyTyrAlaLeuLeuArgCys~snAspThrAsn TATAAGGTTTAGATACTGTGCACCACCGGGTTATGCCCTATTAAGATGTAATGATACCAA 6800 TyrSerGlyPheAlaProAsnCysSerLysValValAlaSerThrCys~hrArglietMet TTATTCAGGCTTTGCACCCAACTGTTCTAAAGTAGTAGCTTCTACATGCACCAGGATGAT *6900 GluThrGlnThrSerThr~rpPheGlyPheAsnGlyThrArgAlaGluAsnArgThrTyr GGAAACGCAAACTTCCACATGGTTTGGCTTTAATGGCACTAGAGCAGAGAATAGAACATA I leTyrTrpHisG IyArgAjpAsnArgThr IlelleSerLeuAsnLys TyrTyrAsnLeu TAT CTATTGGCATGGCAGAGATAATAGAACTATCATCAGCTTAAACAAATATTATAATCT 7000 SerLeuliisCysLysArgProGlyAsnLyaThrValLysGlnhleMet~euMetSerGly CAGTTTGCATTGTAAGAGGCCAGGGAATAAGACAGTGAAACAAATAATGCTTATGTCAGG HisValPhefliaSeruis~yrGlnProlleAsnLysArgProArgGln~laTrpCysTrp ACATGTGTTTCACTCCCACTACCAGCCGATCAATAAAAGACCCAGACAAGCATGGTGCTG 7100 0 PheLyaG lyLysTrpLysAspAlaHetGlnG luValLysGluThrLeullaLysRisPro GTTCAAAGGCAAATGGAAAGACGCCATGCAGGAGGTGAAGGAAACCCTTGCAAAACATCC *7200 (lit ArgTyrArgGlyThrAsnispThrArgisnhleSerPheAlaAlaProGlyLyaGIySer CAGGTATAGAGGAACCAATGACACAAGGAATATTAGCTTTGCAGCGCCAGGAAAAGGCTC AspProGluiVa lAlaTyr~etTrpThrAsnCysArgGlyGluPheLeuTyrCysAsnliet AGACCCAGAAGTAGCATACATGTGGACTAACTGCAGAGGAGAGTTTCTCTACTGCAACAT 7300 ThrTrpPheLeuAsuTrplleGluAsnLysThrliisArgAsnTyrAlaProCys~islle GACTTGGTTCCTCAATTGGATAGAGAATAAGACACACCGCAATTATGCACCGTGCCATAT LysGlnleleAsnThr~rpliisLysValGlyArgAsnValTyrLeu~roProArgGlu AAAGCAAATAATTAACACAT GGCATAAGGTAGGGAGAAATGTATATTTG CC TCCCAGGGA *7400 GlyGluLeuSerCysAsnSerThrValThrSerIleIleAlaAsnhle~apTrpGlnAsu AGGGGAGCTGTCCTGCAACTCAACAGTAACCAGCATAATTGCTAACATTGACTGGCAAAA *7500 AsnAsnGluTbrAsnlleThrPhe SerAlaG IuValAla(luLeuTyrArgLeuG luLeu CAATAATCAGACAAACATTACCTTTAGTGCAGAGGTGGCAGAACTATACAGATTGGAGTT GlyAspTyzLyuLeuVal~lulleThrProlleGlyPbAlaProTbr~ysGluLysArg GGGAGATTATAAATTGGTAGAAATAACACCAATTGGCTTCGCACCTACAAAAGAAAAAAG ~~fig.lA-sui~e 6) 8/35 TyrSerSerAlaHisGlyArg~iaThrArgGlyVa iPheValLeuGlyPheLeuGlyPhe ATACTCCTCTGCTCACGGGAGACATACAAGAGGTGTGTTCGTGCTAGGGTTCTTGGGTTT LeuAlaThrAla, lySerAla~etG lyAlaAlaSerLeuThrValSerAlaGlnSerArg "'CTCGCAACAGCAGGTTCTGCAATGGGCGCGGCGTCCCTGACCGTGTCGGCTCAGTCCCG 7700 ThrLeuLeuAlaGlyIleValGlrlGlnGlnGlnGlnLcuLeuAspValvalLysArgGlo GACTTTACTGGCCGGGATAGTGCAGCAACAGCAACAGCTGTTGGACGTGGTCAAGAGACA 0 7800 G uG luLeuLeuArgLeuihr Va lTrpG lyThrLysAanLeuG lnAlaAig Va lThrAla ACAAGAACTGTTGCGACTGACCGTCTGGGGAACGALAAACCTCCAGGCAAGAGTCACTGC IleGluLysTyrLeuGln~spG lnAlaArgLeuAsnSerTrpGlyCys~laPheArgGln TATAGAGAAGTACCTACAGGACCAGGCGCGGCTA.ATTCATGGGGATGTGCGTTTAGACA *7900 0 Va1C ys HisThrThrVa 1ProTrpVa lAsnAsp Ser LeuA 1aP roAspTrpAspAsn~e t AGTCTGCCACACTACTGTACCATGGGTTAATGATTCCTTAGCACCTGACTGGGACAATAT ThrTrpG lnG lu TrpG lu~yaG InVa lArgTyrLeuG luAl aAsn 11 e~er Lys S erLeu GACGTGGCAGGAATGGGAAAAACAAGTCCGCTACCTGGAGGCAAATATCAGTAAAAGTTT 8000 f;luG lnA 1aGin ln 1eG lnG lnG luLysAsnMe tTyrG lu LeuG lnLys euAsnS e rTrp A GA ACAGGCACAAATTCAGCAAGAGAAAAATATGTATGAACTACAAAAATTAAATAGCTG 8100 *As pl11ePheG lyAsnrrp~heAs pleuTr Se rrpValLys Tyrl leG lnTy rG lyVa 1 GCTTATAATAGTAGCAGTAATAGCTTTAAGAATAGTGATATATGTAGTACAAATGTTAAG 8200 AlaCysPbeLeuPbeProProArgLeuTyrProThrAsp Ar~ur~sl~rr~oa~e~re~or~yy~el~nl GlyLeuGluArgAlalleGlyLeuPheSerLeuProProProVammmeSerAsnArgSer TAGGCTTAGAAAGGGCTATAGGCCTGTTTTCTCTTCCCCCCCCGGTTATATCCAACAGAT P roTyrPro~inG lyProGlyThrAlaSeGlnArgArg~snArgArgArgArgTrpLys HialleliiLy8AspArgGlyGlnProAlaAsuGluGluThrGluGluAspGlyGlySer GlnArgTrp~rgGlnhleLeuAlaLeuAlaApSerleTyrThrPheProAspProPro AsnGlyGlyAspArgTyrTrpProTrpProlleAlaTyrl leHisPheLeuIleArgGln ThrVaiG luThrAapTbrG lyProG lyArg CAACGGTGGAGACAGATACTGGCCC TGGCCGATAGCATATATACATTTCCTGATCCGCCA *8400 AlaAspSer~roLeuAspGlnThrIleGlnHisLeuGlu~lyLeuThrleG lnGluLeu LeulleArgLeuLeuThrArgLeuTyrSerIleCysArgAspLeuLeuSerArgSerPhe 44 GCTGATTCGCCTCTTGACCAGACTATACAGCATCTGCAGGGACTTACTATCCAGGAGCTT ProAspProProThrllisLeuProGluSerGlnArgLeu~laG luThr LeuTbrLeuGlnLeulleTyrGlnAsnLeuArsapTrpLeuArgLeuArgThrAlaPhe CCTGACCCTCCAACTCATCTACCAGAATCTCAGAGACTGGCTGAGACTTAGAACAGCCTT 8500 LeuGlnTyrGlyCyaGluTrpleGlncluAlaPhecmDAlaAlaAlaArgAlaThrArg MetGlyAlaSerGlySerLysLyaiaSerArgProProArgG lyLeuGinG lu CTTGCAATATGGGTGCGAGTGGATCI"AAGAAGCATTCCAGGCCGCCGCGAGGGCTACAAG (fig.lA-suite 7) 9/35 GluThrLeuAlaGlyAlaCysArgGlyLeuTrpArgValLeuGluArgIleGlyArgGly ArgLeuLeuArgAlaArgAlaG lyAlaCysGlyGlyTyrTrpAsnG luSerGlyG ly~lu AGAGACTCTTGCGGGCGCGTGCAGGGGCTTGTGGAGGGTATTGGAACGAATCGGGAGGGG 0 8600 0 0 IleLeuAlaValProArgArgIleArgGlnGlyAlaGluIleAlaLeuLeu TyrSerArgPheGlnG luGlySerAspArgGluG inLyaSerProSerCyaG luG lyArg AATACTCGCGGTTCCAAGAAGGATCAGACAGGGAGCAGAAATCGCCCTCCTGTGAGGGAC *8700 GinTyrGinG lnGlyAap;PheHetAsn~hrProTrpLyaAspProAlaAlaG luArgG lu GGCAGTATCAGCAGGGAGACTTTATGAATACTCCATGGAAGGACCCAGCAGCAGAAAGGG LysAsnLeuTyrArgGlnG lnAsnMet~spAspVa lAapSerAspAspAspAspGln~ai AGAAAAATTTGTACAGGCAACAAAATATGGATGATGTAGATTCAGATGATGATGACCAAG
8800. ArgValSerValThrProLysValPro~euArgProMetrhrlli8ArgLeuAlalleAap TAACAGTTTCTGTCACACCAAAAGTA CCACTAAGACCAATGACACATAGATTGGCAATAG MetSerHisLeulleLysThrArgGly~lyLeuGluGlyMetPheTyrSerGluArg~rg ATATGTCACATTTAATAAAAACAAGGCGGGGACTGGAAGGGATGTTTTACAGTGAAAGAA 8900 HisLysIleLeuAsnIleTyrLeuGlu~ysGluGluGiyIldIleAlaAspTrpGln~sn GACATAAAATCTTAAATATATACTTAGAAAAGGAAG;.AGGGATAATTGCAGATTGGCAGA 9000 *44TyrThrH eG lyProG lyVa lArgTyr~rofle PhePheC lyT rpLeuTrpLys LeuVa 1 ACTACAC TCATGGGCCAGGAGTAAGATACCCAATGTTCTTTGGGTGGCTATGGAAGCTAG ProValAapValProGluGluG1lyGlu~spThrGluThrHisCysLeuValHisProAla TACCAGTAGATGTCCCACAAGAAGGGGAGGACACTGAGACTCACTGCTTAGTACATCCAG *9100 G InThrSerLya PheA spAspP roHisaGlyG luThrLeuVa lTrpG luPheAopP roLeu CACAAACAAGCAAGTTTGATGACCCGCATGGGGAGACACTAGTCTGGGAGTTTGATCCCT LeuAlaTyrSerTyrGluAlaPhelle~rgTyr~roGluGluPheGlyHisLysSer~ly TGCTGGCTTATAGTTACGAGGCTTTTATTCGGTACCCAGAGGAATTTGGGCACAAGTCAG 9200 0 LeuProGluGluGluTrpLysAlaArgLeuLyaAlaArgGlylleProPbeSer GCCTGCCAGAGGAAGAGTGGAAGGCGAGACTGAAAGCAAGAGGAATACCATTTAGTTAAA *0 1 9300 (too GACAGGAACAGC TATACTTGGTCAGGGCAGGAAGTAACTAACAGAAACAGCTGAGACTGC t4* tAGG GA CTTTC CAGAAG GG GCTGTAAC CAAG GGAG GGACATGG GAG GAGC TGGTGG GGAAC 9400 GCCCTCATATTCTCTGTATAAATATACCCGCTAGCTTGCATTGTACTTCGGTCGCTCUTGC GGAGAGGCTGGCAGATTGAGCCCTGGGAGGTTCTCTCCAGCAGTAGCAGGTAGAGCCTGG GTGTTCCCTGCTAGACTCTCACCAGCACTTGGCCGGTGC.TGGGCAGACGGCCCCIACGCTT 0 0 9600 GC TTG CTTAAAAACC TCC TTAA TA AAGC TGC CAGT TAG AAGCA Cfig.lA-suite 8) FIG 1B AGTCGCTCTGCGGAGAGGCTGGCAGATTGAGCCCTtGGAGGTTCTCTCCAGCACTAGCAG GTAGAGCC TGGGTGTTCCCTGCTAGACTCTCACC AGCACTTGGCCGGTGCTGGGCAGAGT *10 0 GGCTCCACGCTTGCTTGC TTAAAGACCTCTTCAATAAAGCTGCCATTTAGAAGTAAGCTA GTGTGTGTTCCCATCTCTCCTAGTCGCCGCCTGGTCAACTCGGTACTCGGTAATAAAAAG 200 0 ACCCTGGTCTGTTAGGACCCTGGTCTGTTAGGACCCTTTCTGCTTTGGGAAACCGAAGCA *300 GGAAAATCCCT AGCAGATTGGCGCCCGAACAGGGACTTGAAGGAGAGTGAGAGACTCCTG AGTACGGCTGAGTGAAGGCAGTAAGGGCGGCAGGAACCAACC ACGACGGAGTGCTCCTAG ',00 AAAGGCGCGGGTCGGTACCAGACGGCGTGAGGAGCGGGGAGAGAAGAGGCCTCCTGGTTG CAGGTAAGTGCAACACAAAAAGGAAATAGCTGTCTTTTATCCAGGAAGGGATAATAAGAT 1500 0 GAG DMETGLY AL AARG ASN SERVALLEUSERGLYLYSLYS ALAASP GLUL EUGLU AGAG TGGGA GAT GGGC GCGCAGAA AC TCCGT CT TGT CAG GGAAG AAAGC AGATGAATTAGA *600 LYS I LEARGLEUARG PR OGLYGLYLYSL;SLYSTYRMETLEUL YSHI SYAL VALTRP ALA A AAA ATT AGA CTACG AC CCGGCG GA AAGA AA AAG TAC ATGT TGA AGC ATGCTAGT AI' C GC ALAASNGLULEUASPARGPHEGLYLEUALAGLUSERLEULEUGLUASNLYSGLUGLYCyS AGCAAATGAATTAGATAGATTTGGATTAGCACAAAGCCTGTTGGAGAACAAAGAACGATG **700 0 GLNLY SILELEUSERVALLEUALAPROLEUVALPROTHRGLYSERGLUASNLEULYSSER TCAAAAAATACTTTCGGTCTTAGCTCCATTAGTGCCAACAGGCTCAGAAAATTTAAAAAG LEUTYRASNTHRVALCYS; AL ILETRP CYS I LEHI SALAGLUGLULYS;ALLYSHI STHR CCTTTATAATACTGTCTGCGTCATCTGGTGCATTCACGCAGAAGAGAAAGTCAAACACAC 800 0 0 GLUGL UALALY SGLN I LEVAL GLNARGH ISLE UVALMETGLUTHRGL YTHRALAGLUT R TGAGGAAGCAAAACAGATAGTGCAGAGACACCTAGTGATGGAAACAGGAACAGCACAAAC 900 PIET PROLYSTHRSERAR GPROTHRALAPRIJPHE SERGLYARGGLYGLYASNTYRPROVAL .44 TATGCCAAAAACAAGTAGACCAACAGCACCATTTAGCGGCAGAGGAGCAAATTACCCAGT GLNGLNILEGLYGLYASNTYRTHRHISLEUPROLEUSE-RPR-OARGTHRLEUASNALATRP 4. ACAACAAATAGGTGGTAACTATACCCACCTACCATTAAGCCCGAGAACATTAAATGCCTG 1000 a 6 VALLYSLEU ILEGLUGLULYSLYSP HEGLYALAGLUVALVAL SERGLYPHEGLNALALEU GGTAAAATTAATAGAGGAGAAGAAATTTGGAGCAGAAGTAGTGTCAGGATTTCAGGCACT SERGLUGLYCYSLEUPROTYRASPILEANGLNMETLEASNCYSVALLYASPISGLN GTCAGAAGGCTGCCTCCCCT ATGACATTAATC AGAT GTTAAATT GTGT GGGAGACC ATCA 1100 0 S ALAALAMETGLN ILEIL EARGA SP ILIE ILEAS NGLIJGLUAL AALAAS PTRPAS PLEUGLN AGCG GCT ATGCAGATC ATCAGAG ATATTATA AATCGAGGAGCTGCACATTGG GACTTGCA 1200 HI SPROGLNGLNALAPROGLNGLNGLYGLNLEUARGGLUPROSERCLYSERASPILEALA GCACCC ACAACAAGCTCC AC AACA AGGACAGC TTAGGGAGCC GTCAGGATC AGATATTGC GLY THRTH; SE RTHRVAL;L~l. UGLNLEGLNTRPIETTYRARGGLNGLNASNPROILE AGGAACAACTAGTACAGTAGAAGAACAAATCCAGTGGATGTACAGACA ACAGAACCCCAT 1300 Fir,. 1B~- PRO VAL GLY AS NILE TYRA RGARGTRP I LEGLNLEUGLYLEUGLNLYSCYS VAL ARGMET ACCAGT A GCAAC ATTT ACA GG AGAT GGATCC A ACT GGGGTTGCAAAA AT GTGTC AGA AT TY RAS NPR;THRASN I LEL EUA SP VAIL ;S GLNGLY PR;LYS GLUPROPHE GINSERTYR G TAT AAC CC AACAAAC AT TCT AGA TGTA AAA CA AGGG CC AAAAGAGCC AT TTCAGAGCT A 1400 VAL AS PAR;GPHETYRLYS SERLEUARG ALA GLUGLNTHRASP PROAL AVAIIYSASNTRP TGT AGACAGGT TCT AC AAAA GTTT AAGAGC AGAACA AACAG ATCC AGCAGTA AAGAATTG 1500 ME T THR GINTH R LEU LEU; L E GINA SN A LA A S N PRO AS PC Y SLY SL Eu yALL E ULVS G LV GAT GACTC AAA CA CTGCTG AT TC AAAA TGCT AACCCAG AT TGC AAGCTAGTGCTGAAGGG LEUGIVTHRAS NPROTHRL EUGL'GLUMETLEUTHRALACVSGLNGLYVAI GIYGIY PRO GCTGGGTACG AATCCC ACCC TAGAAGAA ATGC TGACGGCCTGTCAAGGAGTAGGGGGGCC *.1600 GLY GLNLYSALA ARGL EUAET AL AGLUAIAIEUIYSGIUALALEUA A PRO ALA PRO ILE AGGACAGAAGGCT A GATT AA TGGC AGA AG CCCTG AAA GAGGCCCTCGCACCAGCGCC AAT P OL VALLE UGI ULE UTR P PRO PHEALAALAALAGLNGLNLYS GIYPRO ARGLYS PRO ILEL YSCYSTRP ASNCYSGLY C CC TTTT GCAGC AGC CC AACAGA AGGGACC AAGA AAGCC AAT TAAG TGT TGG AAT TGTGG 1700 0 GL UGLY ARGTHRLEU CYS LYS AL AM ETGL NSE R P ROL Y SLY S THRGL Y ME TLEUGLUMET LYS GLUGLYH IS SERAL A ARGGLNCYSARG ALAPROAR GAR GGLNGLYCYSTRPLYSCVS GAAGG A AGGAC ACT CTGC AAGGC AAT GCAGA GCC CC A AGA AGACAGCO'GAT GCTGGAAATG 1800 TRPL YSAS NGLYPROCVSTV RGLYGLNMETP ROLIYSGNHRGLGLY PHEP HEAR GPRO GLYIYS ME TAS PHI SVALMET ALA LYS CSPROASNARGGNALAGL PHELEUGLYE U #iTGGA AA A AT GGA CCAT GTT AT GGCC AAATGC CCA AAC AGAC AGGCGGGTT TTTTAGGCCT 6 0 ~~TR P PROLE U;LYLYSGLU ALAPR OGLNPH PROH IS GLY;ERSE RALA SERGLYALAASP GLY PROTRPGIVI YSLY SPRO ARGASNPHE PRIJMETALAGLNVALH I SGINGLVIEUTHR TGGC CC TTGGGGAAAGAAGCCCC GCA AT TT CCCCATGGCT CA AGTG CATC AGGGGCTGAC 0 1900 0 ALA AS NCYS SERPRCA RG ARGTHRSERCYSGLYSERALALVSGLULEUH ISALALEUGLY PRIJTHRALA PRO PROGL UGLUPROAL AVAL ASPLEULE UL YSASNTYRMETH I SLEUGLY GCCAACTGCT CCCCC AGAAGAACC AGCTGTGGATCTGCTAAAGAACT ACATGCACTTGGG GINALAAIAGLUARGLVS GLNARGGLUALALEUGLNGLYGLVASPARGGLYPHEAIAAIA LYS GLNGLNARGGLU SE RARGGLYLY S PRO T RLVS GLU VAI.THRGLUA SP LEUL EUHI S CAAGCAGC AGAGAGAAAGCAGAGGGAAGCCTTACAAGGAGGT GACAGAGGATTTGCTGCA 2000 a a 0 PROGLNPHESERIEUTRP ARGARG PROVALVALTHRALAHI S ILEGLUGLYGLNPROVAL LEUASNSERLEUPHEGLYGLYASPGLNO 9 CCTCAATTCTCTCTTTGGAGGAGACCAGTAGTCACTGCTCATATTGAAGGACAGCCTGTA 0 a 0 a 2100 GIUV ALLEULEUASPTHRGLYALAAS PAS PSER ILE VALTHRGLY ILECGLUL EUGLY PRO GAAGTATTAT TAGATACAGGGGCTGATGATTCTATTGT AACAGGAATAGAGTTAGGTCCA 4 A1 HI STYRTHRPRJLYS ILE VALGIVGLY IIEGLYGLYPHEILEASNTHRLVSGLuTYRLYS CATTATACCCC AAAAATAGT AGGAGGAATAGGAGGTTTTATTAATACT AAAGAATe 1 CAAA 2200 a ASNVAIGLUILEGLUVALL;UGLYLYSARGI LELYSGLYTH R LEMETTHRGIYASPTHR 9 AATGTAGAAATAGAAGTTTTAGGC AAAAGGA7TAAAGGGACA ATCATGACAGGGGACACC PRO ILEASN; LEP HE GLAGA SNLEULETH RALALE ULYME TSERLUA SNLEUPRO CCG ATT AACAT TTTTGGT AGAAATTT ACT AAC AGCTCT GGGGATGTCTCTAAATCTTCCC a 2300 1 LEALALVYSVALGLUPRO VALL YS SE RPROLE ULVSPROGLYLYSASPGLYPROLYSL EU ATAGCTAAGGTAGAGCCTGT AAAGTCGCCCTTAAAGCC AGGAAAGGATGGACCAAAATTG 9 6 24,00 LYSGLNTRP PROLEUSERLYSGIULYS IIEVALAL:ALEUARGGLUI LECYSGLULYSMET AAGCAGTGGCC ATTATCAAAAGAAAAGATAGTTGCATTAAGAGAAATCTGTGAAAAGATG 2 r 12/35. 00 GL UL YS AS PGLY CL NIEUGL UGL UAL APR OPROTHR AS N PROT YRASN THRPROTHRP HE CA AA AAG ATGGTC AG TTG GAGG AAGC TCC CC CGACC AAT CCAT AT AACACCC CCACAT TT 2500 A LA ILE LYSLYSLYS ASP L;SASNLYSTRPARGMETLEU ILEAS PPHE ARGGLULEUASN GCT ATAAAGAAAAAG GAT AAAAAC AAA TGGAGAATGCT GAT AGATTTT AGGGAACTAAAT A RGVALTHRGLNASPP HE THRGLUVALGLNLE UGLY ILEPROH I SPROALAGLYLEUALA A GGGTCAC TCA AGACTTT ACGGAAGTCC AAT TAG GA AT ACC ACACCCTGCAGG ACTAGCA 2600 a 0 LYSARGLYSARG ILE THRVALLEUASP ILEG LYAS PALA TYRPHESER ILE PROLEUASP A AAAGGA AA AGG AT TAC AGT AC TGGAT AT AGGTG AC GCAT AT TTCTCT AT ACC TCT AGA T 0 0 0 0 2700 GLUGL.UPHEARGGLN TYRTHRAL APHE THRLEUPROSE RVAL AS NASNALAGLUPROGLY GAAGAATTTAGGCAGTACACTGCCTTTACTTTACCATC AGTAAATAATGCAGAGCC AGGA LY SARGTYR I LET YRI YS VALL EU PROGLNG IYTRPLY SGLY SE RPROAL A I LEPHE GLN A A ACG ATACAT TTAT AA GGTTCTGCCTC AGGG AT GGAAGGGGTCACCAGCC ATCTTCC AA 2800 TY RTHRMETARGH ISVALLEUGLUPROPHE ARGLYSALAASN PROASP V;LTHRLEUVAL T ACA CTAT G AGAC AT GTGCT AGAACCCT TC AGG AA GGC AAATCCAG AT GTGACCTTAGTC GLNTYRMETASP ASP ILELEU ILE ALA SERA SPARGTHRASPLEUGLUH ISAS PARGVAL CAGTATATGGATGACATCTTAATAGCTAGTGACAGGACAGACCTGGAACATGACAGGGTA 2900 a 0 0. VALLEUGLNLEULYSGLULEULEUASNSER! LEGLYPH-ESERSERPROGLUGLULYSPHE GTTTTACAGTTAAAAGAACTCTTAAATAGCATAGGGTTTTCATCCCCAGAAGAGAAATTC GLNLY AS 3000 GL L S A PPRO PROP HE GI NT RP METGL;TYRGLULE UTRP PRaTHRIY STRPLYSIEU C A A AAA G ATCCC CCAT TT C AAT G GATGG GG TAC GA AT TG TG GC C GACA AA A T GGA ACTT G GLINLYS I LEGIULEUPROGLNARGGLUTHRT RPTHRV AIASNASP ILE GINLYSLEUVAZ C A AAAG A TAGAG T TGC CAC AAAG A GA GAC CT GG A CAGT GAA T GATAT A CA GA AGT TACT A 3100 GLYVAILEUASNTRP ALA ALAGLNNI LETYRPROGLY ILELYS TH RLYSHJ SLEUCYSARG G GA G TA TTAA ATTGG G CAGC TC A AA TT TA TC CAG G TAT A AA A AC CAAACA T C TCTG T AGG LE U ILE A RGGLYLYS MET TH RL EUT TIRG LUGIU V AL GLN TR P T HRGLUMET ALAGLUALA T TAA T TAGAGG AA A AAT GAC TCT AA CAG A GG AAG T TCA GT G GAC TGAGATGG CA GA AG CA 3200 GLUT YRGLUGLUASNIYS IE IILELE USE RGL NGLUGL N;LUGLYCYST;RTYRGINGL; G AAT AT GA GG AA AAT AAA ATAA TTCT CA GT C AGG AAC AAG AA G GAT GTTA T TACC AAG AA *3300 7SERLYS PROLEUGLUALAT;RV AL ILELYS SE RGLNAS PASNGLNTRP SERT YRLYS ILE AGC AAG CCATTAGCA AGC CAC GGTGCA TA AAG AGT C A GGA CAAT CA GT GGTCTT AT AAAAT T H IS GLN GIUASPLY S IL E LiULY S VALGILY S PHEALA Y S I LELY S A;N T HRH I STH CA CC AAG AAG AC AAAAT AC T GAA ACTA G GA AAAT TTG C AAAG ATAA A GAA TA CAC ATAC C 34,00 AS NGLYVAIARGIEUIEUALAHl SVAL I EGINIYS I EGIYLYSGLUAIA ILEVAL ILE A AT G GAGT TA GA CTATT AGC ACAT GT AATAC A GA AAA TAGGA A AGG AAGCA A TAG T GAT C TR P GLY GL N;AL PR OIYS P;EH ISLE U P R VALGL ULY SA;SP VALT RP GI UG LNTRP TR T G GGGAC AGGT CCC A AAA TT CCAC TT A CCAG TT GA G AAGG ATG TAT GGGAAC A GT GGT GG a 3500 0 a T HRA SP T YRT RP GLN VAL THRTR P IL EP R 0GLUT R PAS P PHE IILESER TH R PR 0PROL EU AC A GACT ATTGG CAG G TAAC CT G GAT AC CGG AAT G GGA TT T CA TCT CAAC A CCAC CAT TA U U 3600 V A LA RGL EU VAL PHE A S NL;UVALL YS AS P RO I LE GLULY GILUGLU T R T YRT Y RVAL G T AACAT T AGTC TT CA ATCT AGT GA AGG ACC C TAT A GAGG GA GAAGA AAC CTA T TATGT A A S P GLY SERC;YS S ERLY S LN S ERLY SGLUG LYL YS AL AGLYT YRIILE THA 5P ARGGLY GA T GGATC AT GT AGTAAA CAGT C AAA AGAAG GAA AAGC AGGAT ATATC AC AGA CAGGGGC (f ig.IB-suite 2 t a 13 3700 LYS ASP LYS VALL YS VALIE UGLUGLNTHRT HR ASNGLNGLNALAGLULE UGLUALAP HE A AAG AC AAGGT AAA AGTGTTAGAACAG ACT ACT AATC AAC AAGC AGAATTGGAAGC ATTT LE UME TALALEUTHRASP 5ERGLYPROLYSA LA ASN ILE ;LEVYALASP SERGLNTYRVAL CTC ATGCCATTGACAGACTC AGGGCC A iAGGC AAA TAT TAT AGTAGACTC ACAATATGTT 3800 ME TGLYILE ILETHRGLYCYSPROTHRGLUSERGLUSERARGLEUVAL A;NGLN ILEI L; AT GGG AAT AAT AACAGG ATGCC CT ACAG AAT CAGAGAGCAGGC7AGTTAAC CAAATAA TA 0 3900 GL UGLUME T ILE LYSLYSTHRGLU I LET YRVALALATR P VAL PR OALAH ISL YSGLY ILE GAAGAA AT GAT CA AAAAGAC AGA AATTT AT GTGGC AT GGGT ACC AG CA CACA AAGGTATA GLYGLY AS N;LNG LU ILEA;SPHI SLEUVALSERG LNG LY ILEARGGLNVALL EUPHELEU GGA GG A AACC AAGA AAT AGA CC ACCT AGTTAGTC AAGG GAT TA GAC AAGTTC TCTTCTTG 0 0 40000 GLULYS TLEGLUPROALAGLNGLUGLUH IS SE RLYSTY RHIS SE RASN ILEL YSGLULEU G AAAA GAT AGACPCC A GC ACA AGA AGAAC AT AGT A AAT ACC ATAGTAAC AT AAAAGAATTG VAL PHELYSP HE GLYLEU PRARGLEUVALALALYSGLNI LEV ALAS PTHRCYSAS PLY; GT AT TC AAATTT GGAT T ACCCAGACT AG TGGC CA AAC AGAT A GT AGAC ACAT GTG AT AAA 4100 0 CYSH I SGLNIYSGLYGLUAL A ILEHI SGLYGLNV ALAS NSER AS PLEUGLYTHRTRPGLN TGTC AT CAAAA AGG AG A AGC TAT AC ATGGG CA GGTAA ATT CAGACC TAAGGACTTGGCA A S 4200 ME TAS PCY S;HRH I SLE UGLUGLYLYS IE VAL ILE VALALAVALH IS VALALA SE RGLY AT GGAT TGTACCC ATCT AGA GGG AAAAATAG TCAT AGT TGCAGTACATGTAGC TAGTGGA PH E I LEG LU;LAGLUVAL ILEPROGLNGLUTHRGLYARGCLNTHRALALE UP HELEULEU TT CAT AGAAGCAGAAGT AAT TCCAC AAGAAAC AGGA A GAC AGACAG CA CTAT TTCTGTTA 4300 LYSLEU ALA SERARGTRP P;01 LETHRH I SLE UH ISTHRASP AS NGLYALA A SPHEALA A AAT TGGC AAGC AG ATGG CC TATTAC AC AT CTGC ACAC AGAT AATGGTGC TAACTTTGCT SERGLNGLUVALLYSMETYALALATRPTRPALAGLYILEGLUHISTHRPHEGLYVALPRO TCGC AAGAAGT AAA GAT GGT TGC ATGGTGGGC AGGG AT AGA GCACACCTTTGGGGTACC A S 4400 0 0 0 TYRA SNPRQ;LNSERGLNGLYVALVALGLUAL AMETA SNH!-SH I SLEULYS AS NGLN ILE T ACA ATCC ACAGAGTCAGGGAGTAGTGGAAGC AATGAATCACCACCTGAAAA I TCAAATA q.a 4500 AS PARG ILEARGGLUGL.NALAASNSERVALGLUTHR ILE VALLE UMET ALA VALH I SCYS GATA G AAT CAGGGAAC AAGC AAA TTCAGTAG A AACC AT AGTATTAATGGCAGTTC ATTC META SIPHELYSARGARGGLYGLY I LEGLY ASP MET THRPROALAGLU A;GLEU ILEA S; AT G AATTTTA AAA GAAGGGGAGGAATAGGGG AT ATGACTCC AGCAG A AAGAT TAATTAAC 0 0 0 4f600 0 MET I LE THRT HRGLUGLtjGLUJILE GLNP HEGL NGLNSE RLYSASNSERLY SPHELYSASN A TGA T CA CT A CA GA AC AAGA A AT ACA AT T TCA AC A ATC AA AA A ACT C AA AA T TT AA AAAT P H* TYR S S S G SYGLU 'k700 0 T GG AA A GGG G AAG GAG CA G TC AT CT TA AA G GTA GG A AC A GA C AT TAA G GT AG T AC CC AGG S S 4800 ARGLYS ALALYS ILE ILE LYSA.SPTYRGL;GLYGLYLY S;LUME TASP SERSERSERHI S QMETGLUGLUGLULYSARGTRP ILEVALVALPROTHR AGAAAGGCTAAAATTATCAAAG~fTATGGAG GAGGAAAAGAGATGGATAGTAGTTCCC AC ME TOLUA SPT NRCLY CLU ALAARGCGLU VALAL A TRP ARC ILE PROGLUARGILEUGLUARGTRPH IS SEFLEU ILELYSTY RLIEULYSTYRLYS AT CGAGGATACCCCACAGGCTAGAGACCTGCCATAGCCTC ATAAAATAYTTCAAATATAA *a '.9000 ~f is.1B -s uito- 3 14/35 THRLYSASPLEUGLNLYSALACYSTYRVAL PROIIISHI SLYSVALGLYTRPALATRPTRP AACTAAAGATCTACAAAAGGCTTGCTATGTGCCCCATCATAAGGTCGGATGGGCATGGTG THRCYSSERARGVALILEPHEPROLEUGNGLUGLYSEI-ISLEUGLUVALGLNGLYTYR GACCTGCAGCAGAGTAATCTTCCCACTACAGGAAGGAAGCCATTTAGAAGTACAAGGGTA 5000 TRPASNLEUTHRPROGLUARGGLYTRPLEUSERTHRTYRALAVALARGILETHRTRPTYR TTGGAATTTGACACCAGAAAGAGGGTGGCTCAGTACTTATGCAGTG AGGATAACCTGGTA 0 0 5100 SERLYSASPPH-ETRPTHRASPVALTHRPROGLUTYRALAASPILELEULEUHISSERTHR CTCAAAGGACTTTTGGACAGATGTAACACCAGAATATGCAGATATTTTACTGCATAGCAC TYRPHE PROCYSPHETHRALAGLYGLUVALARGARGALA ILEARGGLYCLUARGLEULEU TTATTTCCCTTGCTTTACAGCGGGAGAAGTGAGAAGGGCCATCAGGGGAGAACGACTGCT 5200 0 CTCTTGCTGCAGGTTCCC AAGAGCTC AT AAGCACCAGGTACC A ATCTACAGTACTT AGC LEUARGVALVALSER-II SVALARGSERGLNGLYGLUASNPROTHRTRPLYSGLNTRPARG X METSERASPPROARGGLUARGILEPROPROGLYASNSERGLYGLU ACTGAGAGTAGTAAGTCATGTCAGATCCCAGGGAGAGAATCCCACCTGGAAACAGTGGAG 5300 0 a ARGASPASNARGARGSERLEUARGVALALALYSGLNASNSERARGGLYASPLYSGLNARG GLUTHRILEGLYGLUALAPHEGLUTRPLEUASNARGTHRVALGLUG UILEASNARGGLU AAG AGACAAT AGGAGAAGCCTT CG AGTGGCT AAACAGAACAGTAGAGGAGATAAACAGAG 0 GLYGLYLYSPROPROTHRGLUGLYALAASNPHEPROGLYLEUALALYSVALLEUGLYILE ALAVALASNHISLEUPROARGGLULEUILEPHEGLNVALTRPGLNARGSERTRPGLUTYR AG GCGGT AAA CC ACCTAC CGAG(.'GAGCT AAT TTT CC AGGTTTGG CA AAGGTCTTGGGA AT r LEUALA TR PHI SAS PGLUGLNGLY ME TSERGLN SE RTYRTHRLYSTY RARGTYRLEUCYSLEU ILE ACT GGCATGATG AACAAGGG AT GTCACAAAGCTATAC AAAATACAG AT ACTTGTGTTTAA 5500 GLNLYSALALEUPHEMETHISCYSLYSLYSGLYCYSARGCYSLEUGLYGLUGLYHISGLY TACAAAAGGCTTTATTTATGCATTGCAAGAAAGGCTGTAGATGTCTAGGGGAAGGACACG ALAGLYGLYTRPARG PROGLY PRO PROPR 0PROPRO P;OPROGLYL EUALA R ETGLU GGGCAGGGGGATGGAGACCAGGACCTCCTCCTCCTCCCCCTCCAGGACTAGCATAAATGG 5600 0 GLUARGPROPROGLUASNGLUGLYPROGLNARGGLUPROTRPASPGLUTRPVALVALGLU AAGAAAGACCTCCAGAAAATGAAGGCCC ACAAAGGGAACC ATGGGATGAGTGGGTAGTGG 5700 721 ALLEULYSGLULEULYSGLUGLUALALE ULYSHI SHE ASP PROARGLEULEUTHRALA AAGTTCTGAAAGAACTGAAAGAAGAAGCTTTAAAGCATTTTGATCCTCGGCTTCTAACCG TA.Ti METGLUTHRPROLEUARGGLUGLNGLUASNSER LEUGLYASNHIS ILETYRASNARGHI SGLYASPTHRLEUGLUGLYALAGLYGLULEUILE CA CT TGGT AAT CAT ATCT AT AAT AGACAT GGA GACACC CTTGAGGGAGC AGGAGA ACT CA 0 0 0 5800 a LEUGLUSER SERASNGLUARGSERSERTYRILESERGLUALAALAALAALA ILE PROGLU ARGILELEUGLNAtRGALALEUPHE [LEHI SPHEARGSERGLYCYSSERHI SSERARGILE TTAGAATCCTCCAAC(cAAGCGCTCTTCATACATTTCAGAAGCGGCTGCAGCCATTCCAGAA SE RALAAS NLEUGL YGLUGL U!LELE US ERGLNI EUTY RARG PROLE UGLUAL AC YSTYR GLYGLNPR0GLYGLY'GLYASNPROLEUSE RTH RILE PROPRO SERARGSERMETLEU TCGGCC AACCTGGG GGAG GAAATCCT CT CT CAACT ATACCGCCC TC TAGAAGCATGCT AT a 5900 a ASNTHRCYSTYRCYSLYSLYSCYSCYSTYRH ISCYSGLNPHECYSPHELEULYSLYSGLY AACACATGCTATTGCAAAAAGTGTTGCT ACCATTGCCAGTTTTGTTTTCTTAAAAAGGGC 6000 LE&JGLY ILESE RTYRGLULYSSE RHI SARGA RGARGARGTHRPROLYSLYSALALY SALA ART1IETARGSERIISTHRGLYGLUGLUGLULEUARGARtGARtGLEUARkGLEU (fig.1B-suite 4) j I ~t 1. I I I I I I 15/35 TT GGAT AAG TTAT GAG AACT CAC ACAGGAG AAGAAG AACT CC GA AGAAGGCT AAGGCT AS NTHR SE R ERALA SER AS NCLU I LEHI SL.EULEUH I SGLNTHRSERLYSTYRCLYLE USE RTRPLYS SERALAAL AT YRARG ENV METCLYCYSLEUGLYAS NGLNLE ULEUI LE ALA AATACATCTTCTGCATCAAACGAGTAAGTATCGGTTGTCTTGGAAATCAGCTGCTTATCG 61.00 NISLEUIEU I LECYS SE RLYS CYSL EUTRP ILE ILE CYSIL EGLNTYRVAL THRVALPHETYRCLYVAL CCATCTCCTCTAAGTCTCTATGGATTATTTGTATTCAATATCTCACAGTCTTTTATGGTG PROALATRPARGASNALATHRILEPROLEUPHECYSALATHRLYSASNARGASPTHRRP TACCAGCTTGCAGGAATGCGACAATTCCCCTCTTCTGTGCAACCAACAATAGGGATACTT 6200 CLYTHRTHRGLNCYSLEU PR OASPAS NAS P ASP TYR SERGLULEUA.LALEUASNVALTHR GGAACAACTCAGTGCCTACCAGATAATGATGATTATTCAGAATTGGCCCT TAATCTTA 6300 CL USE RPrIEASPALAT RPGL UA SNTHRVA THRCLU LNA LA ILE LUA SPVALTRPCLN CAGAAAGCTTTGATGCTTGGGAGAATACAGTCACACAACACGCAATAGAGGACGTATGCC LEUPHECLUTHRSERILELYSPROCYSYVALLYSLEUSERPROLEUCYsILETHRMETARC. AACTC TTYGA GAC CTC AATAAAGCCT TCTGT AAAATT ATCCCC ATT AT GCAT TACT AT GA 64.00 CYSASNLYSSERCLUTHRASPLYSTRPGLYLEUTHRLYSSERSERTHRTHRTHRALASER GATCCAATAAAAGTCACACACATAAATGCGCATTCACA AAATCATCA*CAACAACAGCAT THRTH-RTHRTHRTHRTHRALALYSSERVALCLUTHRARCASPILEVALASNGLUTHRSER CAACAACAACAACAACAACAGCAAAATCAGTACAGACAACAGACATACTCAATGAGACTA 6500 PROCYSVALVALHI SASPASNCYSTHRGLYIEUGLUGLNGLUPROMET ILE SERCYSLYS GTCCTTGTGTAGITTCATGATAATTCCACAGGCTTGCAACAACAGCCAATCATAACCTGTA 0 0 6600 PHEASNDMETTHRGLYLEULYSARGASPLYSLYSLYSGLUTYRASNCLUTHRTRPTYRSER AATTCAACATGACACGGTTAAAAAGAGACAACAAAAAGGACTACAATGAAACTTGGTACT ALAASPLEUYALCYSGLUCLNGLYASNSERTHRGLYASNGLUSERARGCYSTYRMETASN CTGCAGATCTCCTTTCTCAACAAGGGAATAG CACTGGTAATGAAAGTAGATGTTACATCA 6700 HI SCYS ASNTHRSERV AL ILEGLNCLUCY SCYSASPLYSA SPTYRTRPAS PALA ILEARG ATCACTGTAATACTTCTCTTATCCAAGAGTG TTCT AC AAAGATTATTGCCATCCTATTA CYSARGTYRCYSALAPROPROCI.YTYRALALEULEUARCCYSASNASPTHRASNTYRSERt GATGTAGATATTGTGCACCTCCAGGTTATGCCTTCCTT AGATGTAATCACAC AAATTATT 6800 0 0 a GLYPHEMETPROASNCYSSERLYSVALVALYALSERSERCYSTHRARGMETMETGLUTHR CAGGCTTTATGCCT AACTCTTCTAAGGTAGT CGT CTCTTCATCCACAAGCATCATCCAGA 6900 CLNTHR SERTHRTRP PH; ARG PHE AS N;LYTHRA RGALAGLUA SNARGTHRTYR ILE TYR CACAGACTTCTACTTGGTTTCGGTTTAATGCAACTAGAGCAAAAATAGAACCT ATATTT TRPHISCLYARGASPASNARGTHRILE ILESERtLEUASNLYSHI sTYRASNLEUTHRMET ACTCCCATGGTACAGATAATACCACTATAATTACTCT AAATAACCATTAT AATCTAACAA 0 s 7000 LYSCYSARCARGPRUCLYASNLYSTHRVALLEUPROVALTHRTLEMETSERALALEUYAL T GAAAT CT ACAAGA CC ACCAAAT AA GACACT T TTA CC ACTCAC CAT TATGCTC TGCATT CC PHEHI S SERLNPROVALASNCLUARtG;ROLYSGLNALATRPCYSARGPHECLYCLYASN TTTTCCACTCACAACC AGTCAATGACAGCCCAAACCAOCCATGCTGTACCTTT6GAGGAA 7100 TRPLYSCGLUALA ILELYS GLUVALLYSLNTHR LE VLLYSHII SPROARCTYRTHRGLY A TTG GA AGCACCCAATAAAAGAGGT GAAGCAG ACC ATT GTCA AACATC CC AGGTAT AC TG 7200 THRA SNASNTHRASPL Y; ILEA SNLEUTH RALAPiROARCCLYGLYAS; PR OCLUVALTHR CAACTAACAATACTCATAAAATCAATTTCACGCCTCCT ACAGCAGCAGATCC CCAAGTTA (fig.1B-suite 16/35 V -1 PHE METRPTHRASNCY;A RGGLYGLUPHELEUTYRC; SLY SME TAS;TRP PHELEUASN CC TTCAT GTGG ACA AATT GCAGAGG AGAG TT TCTCT AC TGTA A AATG AAT TGGTTTCT AA 7300 TRPV ALCLASP ARGSERLE U'HRTH RGLNLY SPROLYSGLUARGH ISLY SARGASNTYR A TTGGGT AGAA GAT AG GAG TCT AACTACCCAGAAGCC AAAGGAACGGC AT AAAAGGAATT VALPROC;SH IS ILE AR;GLN ILE ILEA;SNTHRTRPHISLYS VAL GLYLYSASN VAL;TYR ACGT ACC ATGT CAT AT TAGAC AAAT AAT CA ACACTTGG CA TAA AGT AGGCAA AAATGTTT 74.00 0 a LE UPROPR OAR GGLUGLY ASP LE UTH RCY SAS NSE RTHRVALTIIRSE RLEU ILE ALA;ASN ATTTGCCTCCAAGAGAGGG AGACCTCACGTGTAACT CC ACAGTGACCAGTCT CAT AGC AA 0 a a 0 7500 I LEAS NYRPTHR AS PGLY AS NGL NTH RSE R I LET HRMET SERALAGLUVALALAGLULEU AC AT AA ATTGGACTG AT GG AAACC AAACTAGT ATC ACC ATG AGTGCAG AGGT GGC AGAAC TY RARGLEUGLULEUGLY ASP TYRLY SLEU VAL GLU ILETHRPRO I EGL YLE UAL A RO T GT ATC GATT GG AAT TGGGAG AT TATA AAT T AGTAGA AAT CAC TCC AA TTGG CTTGGCCC 0 7600 THRASNVALLYSARGTYRTHRTHR ULYGLYTHRSERARGASNLY SAR;GLYVALP HE 4L CC AC AAATGTG A AGAGGT AC ACT ACT GGTGGC ACCTC AAG AAAT AAAAGAGGGGTCTTTG LE UGLY 4ELEUGLYP H;LEUAL ATHR ALAGL YSER ALAME TrLY A LAL ASE RLEU;H R TGCTAGGG TTCTTGGGTT TTCTCGC AACGGCAGGTTCTGCA AT GGGCGCGGCGTCGTTGA 7700 VALTHRALAGLNSERARGTHRLEULEUALAGLYILEVALGLNGLNGLNGLNGLNLEULEU CC GTGACCGCTCAGTCCC GG ACT TT ATT GGCTGGG ATAGTGCAGC AAC AGCA ACAGCTGT 0 7800 AS PVALVAL LYSARGGLNGLNGLULEULE U ARGLEUTARVALTR PGLYTHRLYS AS NLEJ TGGACGTGGTC AAGAGAC A ACA AGA AT TGTT GCGAC TG ACCGTCTGGGGAAC AAAGAACC GL NTH RARGVALSERALA ILE GLULYS1,YRLEULYS A;PGLNALAGL;LEUASN ALA;TRP TCC AGACTAGGGTCTCTG CC AT CG AGAAGTACTTAAAGGA CC AGGCGCAGCT AAA TGCTT 7900 GLYCYS ALAP HE ARGGLN VAL CYSH I STHRTHRVALP ROT RP PRO ASNALASERLEUTHR GGGG A tGTGCGTTTAGACAAGTCTGTCACACT ACT GT ACC ATGGCCAAATGC AAGTCTAA PR OAS PTRPASNAS NGLUTH RTRPG;LN;LUTRPGLU ARGLYSVALASPPHEIEUGLUALA CACCAG AT TGGAAC AATGAG ACTTGGCA AGA GTGGGAGCGGAAGGTTGACTTCTTGGAGG 8000 a a 0 AS NILE THRALALEULEUGLUGLUALAGLN I LEGLNGLNGLULYS AS NMETTYRGLULEU CA AAT AT AACGGC CC TCC TAG AAGA GGC AC A AAT TC AAC AAGA GAAG AAC AT GTATGA AT 0 8100 GLNLYSLEIJASNSERTR; ASP VAL PHE;LY AS NTRP P;EASPLEUTHR SERTRP ILELYS TAC AAAA GTT GAATAGCT GG GAT GTGTTTGGC AATTGGTTTGACCTTACTTCTTGG AT AA TYR ILE GLNTYRGLY I iTYR ILE ILE; ALGLY VAL I ELEULEUAR I LEVAL ILETYR AGT ATAT ACA ATAT GGAATTT AT ATAATTG TAGGAGTAATACTGTTAAGAAT AGTG AT CT 0 0 C 5200 0 1 LEVAL GLN1METLEUALA ARGL EUARGGLNGLYTYRARG PRO VA LP HESE RSE RPRO PRO AT ATAGTACAAATGCT AGCT AGGTT AAGA CA GGGGTATAGGCCAGTGTTCTCTTCC CCAC 0TAT2ARG PROILE PROA SNARGI LEAR GIEUCYSGLN PROLY SLYS ALA ART2VALAS PPROTYRPROTHRGLYSERGLYSERALAASNGLNARGARGGLN SERTY RPHECLN***THRHI STHRGLNGLNASP PRO ALALEUPROTHRLYSGLUGLYLYS CC TCTTATTT CC AGT AGA CC CAT ACCC AAC AGG ATCCGGCYCTG CC AACC AAA GAAG6 CA 0 8300 a a LYS LYSGLUT HRVALGLUAL AALA VALALA T HRALA PROGLYLE UGLY ARG OTAT(f in) LYSARGARGARGTRP ARGGLNARGTRPGLNGLNLEULEUALALEUALAASPARGILE TYR SERTRP PROTRPGLNILEGLUTYRILE AAAAAGGAGACGGTGGAGGC ACGGTGGCAAC AGCTCCTG6CCTTGGCAGATAGAATATA 0 4 0 a~oo (flig.lB-suite 6) 17/35 SERP HE PROAS PPRO PROT HR ASPTHRPROLEUA SPLEUAL AI LEGLMGLNCVUGLNiSN H ISP HE LEU ILEAR GGLNLEU ILE ARGLEULE UTHRTRPLEUPHE SE RAS NCYSARGTHR TT CAT TTCCTG AT CCGCC A ACT GAT ACGCCTCTT GAC TTGGC T ATT CAGC AACT GCAGAA LE UAL A ILE GLUSER ILEPRO ASP PRO PROT HR ASN I LEPROGLUALALEUCYS AS PLEU LEULEUSERARGALATYRGIN I LELEUGLNPRO ILE PHE GLNARGLEUSERALATHRTYR CC TTGCT ATCGAGAGCAT ACCAGATCCTCC AACC AATATT CC AGAGGCTC TCTGCG ACCT 8500 F METGLYGLYALA ARGARGILEARGARGSERPROGLNALA .ART2 (fin) GLYGLUPHEGLYGLUV ALLE UARGLE UGLULE UTIRTY RLEUGLNTYRGLYTRPSER TYR ACGGAGAATTCGGAGAAGTCCTCAGGCTTGAACTGACCTACCTACAATATGGGTGGAGCT I LE SERLYSLYSARGSERLYSPROPROGLUILECYSASPARGASPSERCYSGLYARGVAL PHEGLNGLUALAVALGLNALAALAARGASPLEUARGGLNARGLEULEUARGALAARGGLY ATTTCCAAGAAGCGGTCCAAGCCGCCAGAGATCTGCGACAGAGACTCTTGCGGGCGCGTG GLYARGASNTYRGLYARGLEUPHELYSGLYVALGLUASPGLY SERSERGLNSERLEUGL; GLULYSLEUTRPGLUALALEUGLNARGGLYGLYARGTRP ILELEUALA ILE PROARGARG GGGAGAAATTAT GGGAGGCTCTTCAAAGGGGTGGAAGATGGATCCTCGCA ATCCCTAGGA 8700 GLYLE UA SPLYSGLYLEU S;RSERLE USE;C YSGLUGL Y;LNLY STY RAS NGLNGLYGLU !LEARGGLNdLYLEUGLULEUTHRLEULEU 0 GGATTAGACAAGGGCTTGAGCTCACTCTCTTGTGAGGGCCAAAAATACAATCAGGGAGAA TYRMETASN;HRPROTRPARGASN PROALAGLUGLUARGLYSLYSLOEUPROTYRARGLYS TACATGAATACTCCATGGAGAAACCCAGCTGAAGAGAGGAAAAAATTACC ATACAGAAAA 8800 GLNASNILEASPASPILEASPGLUGLUASPASPASPLEUVALGLYILEPROVALGLUAL; CAAAATATAGATGATATAGA.TGAGGAAGATGATGACTTGGTAGGGATACCAGTTGAGGCC ARGVALPROLEUARGTHRMETSERTYRLYSLEUALA IL E;SPMiETSERHI SPHE ILELY; AGAGTTCCCCT AAGAACAATGAGTTACAAATTGGCAATAGATATGTCTCATTTTATAAAA 8900 GLULYSGLYGLYLEUGLUGLYILETYRTYRSERALAARGARGH! SARG!LELEUASP IL; GAAAAGGGGGGACTGGAAGGGATTTATTACAGTGCAAGAAGACATAGAATCTTAGACATA *9000 TYRLEUGLULYSGLUGLUGLYILE ILEPROASPTRPGLNI LEHI SSERGLYPROGLYILE TACTTAGAAAAGGAAGAAGGCATC ATACCAGATTGGCAGATACACTCCGGACCAGGAATT ARGTYRLEULYS METP HE GLYTRPLEUT'RPYSLEU ILE PROVALASNV;LSERASPGL; AGATACCTAAAGATGTTTGGCTGGCTATGGAAATTAATCCCTGTAAATGTATCAGATGAG a 9100 ALAGLNGLUASPGLUGLUHI STYRLEUVAL11 SPROALAGLNTHRSERGLNTRP AS PAS; GCACAGGAGGAT GAGGAGCATTATTTAGTGCACCCAGCTCAAACTTCCCAGTGGGATGAC PROT RPGLY;LUvALLEUALAT RPLYSPHEAS PPROTIRLEUALAT YRTHRTYRGLUALA CCTTGGGGAGAGGTTCTAGCATGGAAGTTTGATCCAACTCTAGCCTACACTTATGAGGCA 9200 *4 TYRItI LE ARGTYRPROGLUGL UP HEGL YSE RLY SSERGLYLE USERGLUL;SGLUVALLY; TATATTAG AT ACCC AGAAGAGTTT GGAAGCA AGTCAGGCCTG TCAG AGAAAGAGGTTAAA ARGARGLEUALAALAARGGL YL EULE UGL;METALAAS P ARGLY SGLUTHRSER AGAAGGCT AGCC GC AAGA GGCC TTCTTG AAA TGGCTGACAGGAAGG A AACT AGCTGAG AC AGCAGGGACTTTCCACAAGGGGAT GTCATGGGGAGGTACT GGGGAGGAGCCGGTTGGGAA 49'00 4 4 CACCCACTTTC TTGATGTA4AAAT AT CACTGC AT TTCGCTCT GTATTC AGTC GC TCTGCG GAGAGGCTGGCAGATTGAGCCCTGGGAGGTT CTCTCC AGCACT AGCAGGT AGAGCCTGGG *1 9500 0 TGTTCCCTGCTAGACTCTCACC AGC ACT TGGCCGGTGCTGGGCAGAGT GGCT CC ACGCTT
9600. (fig.lB-suite 7) 18 FIG. 1C s~quence LTR CIVET vers us HIV-2 ROD x 8960 8970 8980 8990 9000 9010 TGGAAGGGATTTATTACAGTGCAAGAAGACATAGAATCTTAGACATATACTTAGAAAAGG TGGAAGGGATGTTTTACAGTGAAAGAAGACATAAAATCTTAAATATATACTTAGAAAAGG X 8950 8960 8970 8980 84190 9020 9030 9040 9050 9060 AAGAAGGCATCATACCAGATTGGCAGATACACTCCGGA---CCAGGAATTAGATACCTAA AAGAAGGGATAATTGCAGATTGGCAGAACTACACTCATGGGCC AGGAL;TAAGATACCCAA 9010 9020 9030 9040O 9050 9080 9090 9100 9110 9120 AGATGTTTGGCTGGCTATGGAAATTAATCCCTGTAAATGTATCAGATGAkGGCACAGGAGG TGTTCTTTGGGTGGCTATGGAAGCTAGTACCAGTAGATGTCCC ACAAGA.AGGGGAGGACA 9070 9080 9090 9100 9110 9140 9150 9160 9170 11180 ATGAGGAGCATTATTTAGTGCACCCAGCTCAAACTTCCCAGTGGGATGACCCTTGGGGAG CTGAGACTCACTGCTTAGTACATCCAGCACAAACAAGCAAGTTTGATGACCCGCATCGGGG 9130 91',0 9150 9160 9110 9200 9210 9220 9230 9240 AGGTTCTAGCATGGAAGTTTGATCC AACTCTAGCCT.ACACTTATGAGGCATATATTAGAT AGACACTAGTC TGGGAGTTTGATCCCTTGCTGGCTTATAGTTACGAGGCTTTT ATTCGGT 9190 9200 9210 9220 9230 9260 9270 9280 9290 9300 ACCCAGAAGAGTTTGGAAGCAAGTCAGGCCTGTCAGAGAAAGAGGTTAAAAGAAGGCTAG ACC;CAGAGGAATTTGGGCACAAGTCAGGCCTGCCAGAGGAAGAGTGGAAGGCGAGACTGA 9250 9260 9270 9280 9290 9320 9330 9340 9350 CCGCAAGAGGCCTTCTTGAAATGGCT-GACAGGAAGGAAACT---------------- AAGCAAGAGGAA.TACCATTTAGTTAAAGACAGGAACAGCTATACTTGGTCAGGGCAGGAA 9310 9320 .9330 9340 9350 FIG. 1C I I (II, 1.111 I I It I 19/35 9360 9370 9380 9390 AGCTGAGACAGCAGGGACTTTCCACAAGGGGATGTCATG--GGGA GTAAC TAACAGAAACAGCTGAGACTGCAGGGACTTTCCAGAAGGGGCTGTAACCAAGGGA 9370 9380 9390 940O0 9410 94,00 9410 9420 9430 9440 9450 GGTACTGGGGAGGAGCCGGTTGGGAACACCCACTTTCTTGATGTATAAATATCACTGCAT GGGACATGGGAGGAGCTGGTGGGGAACGCCCTCATATTCTCTGTATAAATATACCCGCTA 9430 9440 9450 9460 9470 9460 XX 10 20 30 TTCGCTCTGTA--TTCTGGAAGGGATTTATTACAGTGCAAGAAGACATAGAATCTTAGAC GCTTGCATTGTACTTCTGGAAGGGATGTTTTACAGTGAAAGAAGACATAAAATCTTAAAT 9490 XX 10 20 30 60 70 80 ATATACTTAGAAAAGGAAGAAGGCATCATACCAGATTGGCAGATACACTCCGGA---CCA ATATACTTAGAAAAGGAAGAAGGGATAATTGCAGATTGGCAGAACTACACTCATGGGCCA 60 70 80 90 100 110 120 130 140 GGAATTAGATACCTAAAGATGTTTGGCTGGCTATGG AAATTAATCCCTGTAAA TGTATCA GGAGTAAGATACCCAATGTTCTTTGGGTGGCTATGGAAGCTAGTACCAGTAGATGTCCCA 110 120 130 140 150 160 17TO 180 190 200 210 GATGAGGCACAGGAGGATGAGGAGCATVATTTAGTGCACCCAGCTCAAACTTCCCAGTGG CAAGAAGGGGAGGACACTGAGACTC AC'TGCTTAGTACATCC AGCACAAACAAGCAAGTTT d170 180 1910 200 210 220 230 240 250 260 270 GATGACCCTTGGGGAGAGGTTCT hGCATGGAAGTTTGATCC AACTCTAGCCTACACTTAT GATGACCCGCATGGGGAGACACTAGTCTGOGAGTTTGATCCCTTGCTGGCTTATAGTTAC 230 240 250 260 270 280 290 300 310 GAG GC ATATAT TAG AT AC CCAGA AG AGTTT GGA AGC A C AG GC TTTT ATTC GG 290 (fig.lC-suite 1) 7 20/3 (HIV-2.P FIG. 2 (versus HIV-1.F ijlI 1-1RVIZ EKYQ PLFCAIRN TLFCASDA TEQAIEDV VEQI4HEDI .5(4 4 4454, *4 4 4*1 St 4 4 4, 4 4,5 S 4 S 44 (~s 4, 4444 S 4444 4444 44 4 4 44 44 4 445 4 4* 4~ 44 4 5 HIVI---- TTTPTD S S S GEMMM 2 Y NET-14 P IDNDTTS 2 Y CAP PGYA YCAPAGFA 3 GTRAE- GSLAEZEV 1ILMS-GF RIQRGPGY :3 env4 ~1JQLAILLA-SACLV .C-(NYVTV-,v GVPTIt212,AT1. 'iL WRWGW1lWGT1K LLGILI4ICSA TEi',LWVTVYY CVPVWKEATT '70 foenv5 90 100 R- -DT- liG TIQCLPDNDD YOEITL-NVT EAFDAWNNTV KA YDTEVHNVWA TH-ACVFTDPN PQEVVLVNVT ENFNX!WKNDM 120 env 6 130 140 150 WE LFETSiPCV KLTPLCV.AN!(U SSTESSTGN NTTSKSTSTT IS LWDQSLKPCV KLTPLCVSLK CTDL Gl ATNTNSSNTN 60 170 IS0 190 200 QE QEISEDTFCA RADNCSGLGE TEETINCQFNM TGLERDKIKKQ EK GEIK- -lCSFNIS TSIRGKVQKE YAFFYKLDII 10 220 f 3 0 env7 240 4' 250 YS KVVCETNNST NQTQCY1HN1HC 11TSVITESCD KHYW I I RF YT .TS C NTSVITQACP KVSFEFIIH 60 eriv8 270 280 290 300 LL RC-NDt-NYS GFAPNCSKVV ASTCTR2*IET QTSTWF-GFN IL KCNNKTFNGT GP-CTNVS TVQCTUGIRP VVSTQLLL-N 10 320 330 340 350 1; RTY'IYWHiGRD N-RT!I-SLN KYYI;LSLHCK RPGIIKTVKQI 'V RSANFT-D NAKTIIVQL1N QSVE--INCT 'itPNN 14TR rS I 3 k 0 env9 VF LAF HSFYQPI!IKRPROAU7CWFKG_-IKDA IQEV VTIGKIGII,-- 14RQAiC'NSR AKW'liAT-L KETLAKHPRY KQIASKLRE-Q FIG. 2 r' I 11"1 f le r r :t 1 t 21/35 HIV2- IvI HIV2---i-- 10 I HIV2 Bivi RGTNDT: FGIINK T WSTECSI TSIIANI TGLLLTI env3 KERRYSS KAKRR-- LAGIVQQ LSGIVQ 410 4 420 RNIS FAAPGKGSDP -II FKQSS-GGDP 460 470 -EN KT RNYAPCH t* NNTE GSDTITLPCR 510 520 IDWQ NNNQTNITFS IDCG NNNNGSEIFR 560 570 AUG RUTRGVFVLG *VVQ. REKRAVGI-G 610 620 !QQQ LLDVVKRQQE 1* QNN LLAIEAQQH 660 670 pVC HTTVPW- :LIC TTtV21;A1Td. 710 erv2, 720 .QQE KNMYELQKLN w IQQE KREQELLELD envl0 430 440 EVAY):WTNCR GEFLYCNMTII FL U WI-- u EIVTHSFNCG envil 480 IKOIINTWHK IKQFINXWQE 530 AEVAELYRL- FCC CDMRDNW 580 -F GFLATA ALFLGFLGAA 630 LLRLTVWGTK LLQLTVWGIK 680 VNDSLAD WD S INK S LEQ I W 1 730 SWD IFGNWFD KWASLWWFN u* C GEFFYCNSTQ LFSTWFNST 490 500 VCRNVY(PF.R EGELSCNSTV VGKAMYAPPI SCQIRCSSNI 540 550 -ELGDYKLV EITPIGFAPT RSELYKYKVV KIEPLGVAPT 590 600 GSAMGAAS- LTVSAQSRTL GSTNGARSMT LTVQA--RQL 640 envi 650 NLQARVTAIE KYLQDOARLN QLQARILAVE RYLKDQQLLG 690 700 NMTWQEWEKQ VRYLEANISK ~I'ITW: EDRE IUNYTSLIS 740 750 LTSWVKYIQY GVLIIVAVIA ITNWLWYIKI FIIIIVGGLVG HIVI I SWGCAFR IWGCSGK SLEQA21 j LIEESQN (fig.2 suite 1) LL_ 1 ,1 I 22/35 t It 44 4 .4 l S S.. eqleq 4 4 4: 5 10 15 760 LRIVIYVVQ LRIVFAVLSI B10 GGSNGCDRYW CGERDRDRSI 860 LQLIYONLRD VELLO--RRG 910 -AGACRG IEVVQACRA 770 LSRLRKGYRP VNRVRQGYSP 820 PWPIAYIHFL RLVNGSLLA-L 870 WLRLRTA-F WEALKYWWNL 920 LWRVLERIGR 780 V-FSSPGYI LSFQT 830 IRQLIRLLT- IWDDLRSLCL 880 LQYGCEWIQE LQYWSQELKN 930 GILAVPRRIR -IRRIPRRIR 790 QQIHIHKDRC -HLPTPRG 840 LYSIC FSYHRL- 890 AFQ -AAA SAVSLLNATA 938 QGAEIALL QGLERILL B00 QPANEETEED PDRPEGIEEE 850 RDLLSRSFLT RDLLLIVTRI 900 RATRETL- IAVAEGTDRV 4* 44 4 0 (fig. 2 suite 2) 23/35 CENV-mac FIG.3 Cversus ENV-?ROD 20 30 40 MGCLGNQLLIAIC--SKCLWJICIQYVTVFYGVPAWRNATIPLFCATKNRDTWGTTOCL MM---NQLLI AILLAS ACLVY-CTQYVTVFYGVPTWKNAT IPLFCATRNRDTWGTI QCL 20 30 It 0 so 70 80 90 100 110 P ONDDYSEL ALN VTESF DA WENT VTEOAIED VWQLFETSIKPC VKLS PLCITMiRCNK SET P D NDD YOE ITLN VTE A FDA WNNT VT EOA IE D VWHLF ETS IK P CVKLT P LCVAM KC SS T ES 70 80 90 100 110 120 130 140 150 160 170 D K WG LTK SSTT TAST T TTTTAK S VE TR D IVNETS PC VVHDNCT GLEQOE PM!I S CK FN M STGNNTTSKST--STTTTTP T-DEOEISEDTPCARADNCSGLGEEETINCQFNM ft1~120 130 140 150 160 t t It ft180 190 200 210 220 230 ft ftTGLKRDKKKEYNETWYSADLVCEOGNSTGNESRCYMNIICNTSVIQECCDKDYWDAIRCRY T GL ER DK KKOY NETWY SKDV VCE TN NST-NOT CYM NHC NTSV I TE SCDKHYW DA IRF RY 170 180 190 200 210 220 240 250 260 270 280 290 C AP P GYALL RC NDT NYS GF MP NCSKV VY S SCTRMIME TQTST WF R FNG TRAE NRTY I Y WHG Ct f AP P GYALL RCN DT NYS GFAP NCSK VVA STC TRMME TOT STWF GFNG TRAE NR TYIY WHG ft230 240 250 260 270 280 300 310 320 330 340 350 4 RDNRTIISLNKHYNLTMKCRRPGNKTVLPVTIMSALVFHS--QPVNERPKQAWCRFGGNW RDNRTI ISLNKYYNLSLHCKRPGNKTVKQIMLMSGHVFHSHYOPINKRPROAWCWFKGKW 290 300 i$10 320 330 340 360 370 380 390 400 KEAIKEVKQTI VKHPRYTGTNNTOK INLTAPRGG-DPEVTFMWTNCRGEFLYCKMNWFLN KDAMQEVKETLAKHPRYRGTNDTRN IS FAAPGKGSD PEVAYPMWTNCRGEFLYCNMTWFLN 350 360 370 380 390 400 F 10' 3J I I I 24/35 1,20 '3 0 /1 .O0'.50 'eb0 WVEDReLTTOKPKERHKRNYVPCHIRQ1INTWHKVGKNVYLPPREGDLTCNSTVTSL!AN WIEIN---------KT-H-RNYAPCHIKQIINTWHKVGRNVYLPPREGELSCNSTVTSTIAN '.20 '.30 '.40 480 '.90 500 510 520 INWTDGNQTSITMSAEVAELYRLELGDYKLVEITPIGLAPTNVKRYTTG-GTSRNKRGVF IDW0NNN0TNITFSAEVAELYRLELGDYKLVEITPIGFAPTKEKRY'SAHG--RHTRGvF l160 '.70 '.80 '.90 500 510 540O 550 560 570 580 VLGFLGFLATAGSAliCAASLTVTAQSRTLLAGI VQQQQQLLOVVKRQQELLRLTVWGTKN VLGFLGFLATAGSAMGAASLTVSAQSRTLLAGI VQQQQOLLDVVKRQQELLRLTVWTKN 520 530 540 550 560 570 600 610 620 630 640 LOTRVSAIEKYLKDOAQLNAWGCM-'ROVCHTTVPWP NASLTPDWNNETWOEWERKVOFLE LOARVTAIEKYLQOQARLNSWGCAF ROVCHTTVPWVNOSLAPDWDNMTWOEWEKQVRYLE 580 590 600 610 620 630 660 670 680 690 700 ANITALLEEAQIQQEKNMYOELgKLNSWDVFGNWFDLTSWIKYIQYGIYIIVGVILLR IVI ANISKSL,.EQAQIQQEKNMYELOKLNSWOIFGNWFDLTSWVKYIQYGVLIIVAVIALRIVI 640 650 660 670 680 690 I:,720 730 740 750 760 YI VQMLARLRQGYRPVFSSPPSYFQ*ThiTQQDPALPTKEGKKGDGGGSGGNSSWPWQIEY 9c YVVQMLSRLRKGYRPVFSSPPGYIQQIHIHKDRGQPANEETEEDGGSNGGDRYWPWP lAY Co700 710 720 73 0 740 750 cc 780 790 800 810 820 IHFLIRQL IRLLTWLFSNCRTLLSRAY~QTLQPIFORLSATYGEFGEVLRLELTYLQYGWS IHFLIROLI RLLTRLYSICRDLLSRSFL1'LQLIYQNLRDW LRLRTAFLQYGCE 760 770 780 790 600 840 850 860 870 880 YFQEAVOAA-RDLRQRLLRA-RGEKLWEALQRGGRW IIAIPRRIRQGLELTLL WI QEAFOAAARATRETLAGACRG--LWRVLERIGRGILAVPRRIRQGAEIALL 810 820 830 840 850 (fig. 3-suite 1) 25/35 (GAG-mac FIG. 4 (versus (GAG-ROD 20 30 4.0 VOHKKEIAVFYPGRDNKIEWEMGARNSVLSGKKADELEKIRLRPGGKKKYMLKHVVWAAN MGARNSVLRGKKADELERIRLRPGGKKKYRLKHIVWAAN 20 80 90 100 110 ELDRFGLAESLLENKEGCQKILSVLAPLVPTGSENLKSLYNTVCVIWCIHAEEKVKHTEE KLDRFGLAESLLESKEGCQKILTVLPVPTGSENLKSLFNTVCVIWC14-AEEKVKDTEG 50 60 70 80 AKQIVQRHLVMETGTAETMPKTSRPTAPFSGRGGNYPVQQIGGNYTHLPLSPRTLNAWVK AKO IVRRH-LVAETGTAEKMPSSRPTAPSSEKGGNYPVQHVGGNYTHIPLSPRTLNAWVK 100 110 120 130 14.0 150 190 200 210 220 230 LIEEKKFGAEVVSGFQALSEGCLPYDINMLNCGD OAAMiQIIRDIINEEAADWDLQHP LVEEKKFGAEVVPGFQALSEGCTPYDINQPMLNCV$DHQAAMQ!IREIINEEAAEWDV0HP 160 170 180 190 200 210 250 260 270 280 290 QQAPQQ-GOLREPSGSDI AGTTSTYEEQI QWMYRQQNPIPVGNIYRRW IQLGLQKCVRMY IPGPLPAGQLREPRGSDIAGTTSTVEEQIQWtIFRPONPVPVGNIYRRWIQIGLOKCVRMY 220 230 24.0 250 260 270 300 310 320 330 34.0 350 NPTNILDVKQGPKE PFQSYYDRFYKSLRAEQ1'DPAKNWM'TQTIL IQNANPDCKLVLKGL NPTNILDIKQGPKEPFOSYVDRFYKSLRAEQTDPAVKNWMTQTLLVQNANPDCKLVLKGL 280 290 300 310 320 330 360 370 380 390 1,00 4.10 GTNPTLEEMLTACQGVGGPGQKARLMAEALKEALAP API PFAAAQQKGPRKP IKCWNCGK GMNPTLEEMLTACQGVGGPGQKARLMAEALKEVIGPAPIPFAAAQO--RKAFKCWNCGK 340 350 360 370 380 390 FIG. 4 I T t# 4 t I 4$ I I 4 I 4£ 4 I I I a. a 4 Ii 1$ at,, I E It 4 44 1 III t 26/35 20 It 30 4410 4.50 4.60 EGHSAROCRAPRRQGCWKCGKMDHVMAKCPNROAGeLGLGPWGKKPRNFPMAQVHQGLTP EGHSAR0CRAPRRQGCWKCGKPGHIMTNCPDR0AGFLGLGPWGKKPRNFPVAQVPQGLTP 4$00 4$10 '420 '430 '450 4.80 4$90 500 510 TAPPEEPAVDLLKNYMHLGKQQRESRGKPYKEV1TEDLLHL-------------------- NS rAPPVDPAVDLLEKYMQQGKRQREQRERPYKEVTEDLLHLEQGETPYRE PPTEDLLHLNS 4.60 4.70 4.80 4,90 500 510 (fig.4 suite 1) 27/35 (POL-mac F IG.5 versus (POL-ROD 20 30 i0 VLELWEGRTLCKAMOSPKKTGMLEMWKNGPCYGOMPKQTGGFFRPWPLGKEAPUFPHGSS TGRFFRTGPLGKEAPOLPRGPS 80 90 100 ASGADANCSPRRTSCGSAKELHALGQAAERKOREALOGGDRGF------------------- SAGADTNSTPSGSSSGSTGEIYAAREKTERAERETIOGSDRGLTAPRAGGDTIQGATNRG 40 50 60 70 0110 120 130 140 150 160 -AAPOFSLWRRPVVTAHIEGQPVEVLLDTGADDSIVTGI ELGPHYTPKIVGGI GGF INTK LAAPQFSLWKRPVVTAYIEGQPVEVLLDTGADDSIVAGIELGNNYSPKIVGGIGGF INTK 100 110 120 130 140 170 180 190 200 210 220 EYKNVEIEVLGKRIKGTIMTGDTPINIFGRNLLTALGIMSLNLPIAKVEPVKSPLKPGKDG EYKNVEIEVLNKKVRATIMTGDTPINIFGRNILTALGMSLNLPVAKYEPIKIMLKPGKDG 160 170 180 190 200 230 240 250 260 270 280 PKLKOWPLSKEKIVALRE ICEKMEKDGQLEEAPPTNPYJTPTFA IKKKDKNKWRMLIDFR PKLRQWPLTKEKIEALKE ICEKMEKEGQLEEAPPTNPYNTPTFAIKKKKNKWRML IDFR 210 220 230 240 250 260 290 300 310 320 330 340O ELNRVTQDFTEVOLGIPHPAGLAKRKRITVLDIGDAYFSIPLDEEFRQYTAFTL'SVNNA ELNKVTQDFTEIQLGIPHPAGLAKKRRITVLDVGDAYFSIPLHEDFRFPrTAFTLPSVNNA 270 280 290 300 310 320 350 360 370 380 390 400 EPGKRY IYKVLPQGWKGSPAIFQYTMRHVLEPFRKANPDVTLYQYMDOILIASDRTDLEH EPGKRYIYKVLPQGWdKGSPAIFQHTIIROVLEPFRKANKBVII IQYMDOILIASDRTDLEH 330 340 350 360 370 380 28/35 '.20 4.30 4',0 '.50 4i60 OR VVLQLKELLNS IGFSSPEEKF OKDPPF OWMGYELWPTKWKLOK IELPORETkTYND IQ DRY VLOLKELLNGLGFSTPOEKF QKDPPYHWMGYELWPTKWKLOK I LPQKE IWTVND 390 4.00 '.10 1.20 '.30 '.80 1,90 500 510 520 KLVGVLNWAAQIYPGIKTKHLCRLIRGKMTLTEEVOWTEMAEAEYEENKIILSOEOEGCY KLVGVLNWAAOLYPGIKTKHLCRLIRGKITLTEEVOWTELAEAELEENRIJLSQEOECHY '.60 '70 '.80 '.90 500 530 54.0 550 560 570 580 YQESKPLEATVIKSQDNQWdSYKIHQEDKILKVGKFAKIKNTHTNGVRLLAHV!0KIGKEA YQEEKELEATVQKDOENOWTYKIHQEEKILKVGKYAKVKNTHTNGIRLLAQVVQKIGKEA 510 520 530 54.0 550 560 590 600 610 620 630 640 IVIWGQVPKFHLPVEKDVWEOWWTOYWQVTWIPEWDFISTPPLVRLVFNLVKDPIEGEET LVIWlGR IPKFIILPVERE IWEQWWDNYWVTWI POWOF VSIPPLVRLAFNLVGDP IPGAET 570 580 590 600 610 620 650 660 670 680 690 700 YYVDGSCSKOSKEGKAGY ITDRGKDKVKVLEOTTNQQAELE AFL MAL TOSGPKAN I I VOS FYTDGSCNRQSKEGKAGYVTDRGKDKVKKLEQTTNQQAELEAFAMALTDSGPKVNII VOS 630 640 650 660 670 680 710 720 730 740 750 760 QYVMGIITGCPTESESRLVNQIIEEMIKKTEIYVAWVPAHKGIGGNQEIOHLVSQGIRQV QYVMGISASOPTESESKIYNQI IEEMIKKEAIYVAWVPAHKGIGGNOEVDHLVSOGIRQV 690 700 710 720 730 740 770 780 790 800 810 820 LFLEKIEPAQEEHSKYHSNIKELVFKFGLPRLVAKQ IVD'iCDKCHOKGEAIHGQVNSOLG 4 LFLEKJEPAQF.EHEKYHSNVKELSHKFGIPNLVARIVNSCACQKGEAIHGQVNAELG 750 760 170 780 790 800 830 8140 850 860 870 880 TWQMDCTHLE ',1VIVAVFVASGFIEAEVIP0ETGR0TALFLLKLASRWPITHLHTDNGA TWOMDCTHLEGKIIIVAVHVASGFIEAEVIPOESGROTALFLLKLASRWPITHLHTDNGA J810 820 830 81 0 850 860 890 900 910 920 930 940 NFASCEVKMiVAWWAGI EITFGVP YNPQSQGYYEAMNHHLk'NQI DRIREQANSVET!YLMA NFTSQEYKIVAWW IGI EQSFGVPYNPQSQGYVEAMNHHLKNOI SRI REQANTIETIVLMA 870 880 890 900 910 920 950 960 970 980 990 VHCMNFKRRGGIGDMTPAERL INMITTEQEIQFQQSKNSKFKNFRVYYREGRDQLWKGPG IHCMNFKRRGG!GOMTPSERL INiI TTEQEIQFLOAKNSKLKDFRVYFREGRDQLWKGPG 930 940 950 960 970 980 1010 1020 1030 -14 1050 ELLWKGEGAVILKVCTDIKVVPRRKAKI IKDYGC'GKEMDSSSHMEDTGEAREVA 1) 29/35 ELLWKGEGAVLVKVGTDIKI IPRRKAKI IRDYGGRQEMDSGSHLEGAREDGEMA 990 1000 1010 1020 1030 (fig. S-suite 2) 4 i r I A 30/35 (Q.mac FIG.6 (versus (Q.ROD 20 30 1050 MEEEKRWI'-YPTWRI PERLERWHSLIKYLKYKTKDL0KACY~eYHHKVGWAWWTCSRV IFP MEEDKRWIVVPTWRVPGRMEKWHSLVKYLKYKTKDLEKVCYWPHH-K GWAWT SRVIFP 20 30 'tO 80 90 100 110 LOEGSHLEVOGYWNLTPERGWLSTYAVRITWYSKDF WTDVTPEYAD ILLHSTYFPCFTAG 'I ~LKG NSH-LE IQAYWNLTPEKGWLSSYSVR ITWYTEKFWTDVTPDCADVLTHSTYFPCFTAG 80 90 100 110 130 14,0 150 160 170 EVRRAIRGERLLSCCRFPRAHKHQVPSLOYLALRVVSHV-RSQGENPTWKQWRRDNRRSL EVRRAIRGEKLLSCCNYPRAHRAQVPSLOFLALVVVQQNDRPORDSTTRKQRRRDYRRGL 130 14,0 150 160 170 180 190 200 210 RVAKQNSRGDKQRGGKPPTEGANFPGLAKVLGILA RLAKQDSRSHKQRSSESP1PRTYFPGVAEVLEILA 190 200 210 II I t FIG. 6 I, 31/35 (R.mac FIG. 7( versus (R.ROD 20 30 450 ME---E RP PE NEGP ORE PWDE WVVEYLKELKEEAL KHFOP RLLTAL GNHI YNRHGDTLE MAEAPTELPPVDGTPLREPGDEWI IEILREIKEEALKH-FDPRLLIALGKYIYTRHGDTLE 10 20 30 4~0 60 70 80 90 100 GAGEL IR ILQRALF IHF RSGC SHSRIGOP GGGNPLS TIPP SRS ML GARELIKVLORALFTHFRAGCGHSRIGQTRGGNPLSAIPTPRNMQ 70 80 90 100 4* 4. 4 4 4 4. 4 I 4. *4 t4 4 4 4 4 '4. 4. 44 4 1 1 1 I I 4144 4. a 4.1 4.4 414>4 I 4.4 It I 4. 14. *1 1 III I FIG. 7 q 32/35 X.tnac FIG.8 (versus (X.ROD tf tm I f~ I g4~ 4t j t S t It 1 I t t4 20 30 40 MSDPRERIPPGNSGEETI GEAFEWLNRTVEEINREAVNHLPRELIFQVVQRSWEYWHDE0 MTOPRETVPPGNSGEETI GEAFAWLNRTVEAINREAVNHLPRELIFQVWQRSWRYWHOEO 10 20 30 'i0 70 80 90 100 110 GMSOSYTKYRYLCLIQKALFMHCKKGCRCLGEGHGAGGWRPGPPPPP PPGLA GMSESYTrKYRYLCI IQKAVYMHVRKGCTCLGRGHGPGGWRPGPPPPPPPGLV 80 90 100 110 t~ I 4,1 I SI I I IS. I I t 4,14, 1 33 (F.mac FIG.9 (versus (F.ROD 20 30 '40 MGGAI SKKRSKPPEI CD-ROSCGRVGRNYGRLFK-GVEDGSSOSLGGLDKGLSSLSCEGQ MGASGSKKHSRP PRGLOERLLRARAGACGGYWNESGGEYSRFQE-GSDREQKSPSCEGR 20 30 40 70 80 90 100 110 KYNOGEYMNTPWRNPAEERKKLPYRKQNIDDIDEEDODLVGIPVEARVPLRTMSYKLAID QYOOGDFMNTPWKDPAAEREKNLYRQQNMODDSDDDOVRVSVTPKVPLRPMTHRLAID 70 80 90 100 110 120 130 140 150 160 170 Ik SFIKEKGGLEGIYYSARRHRILIYLEKEEGIIPDWQI-HSGPGIRYLKMFGWLWKL MSHLIKTRGGLEGMFYSERRHKILNIYLEKEEGJADWQNYTH-GPGVRYPFFGWLdKL 120 130 1',0 150 160 170 180 190 200 210 220 230 IPVNYSDEAQEDEEHYLVHPAQTSQWDOPWGEVLAWKFDPTLAYTYEAYIRYPEEFGSKS VPVDVPQEGEDTETHCLVHPAQTSKFDDPHGETLVWEFDPLLAYSYEAF IRYPEEFGHKS 180 190 200 210 220 230 21t0 250 260 GLS EK EVKR RL AARGLL EM1A0RKET S GLPEWKARLKARGIPFS' 250 FIG. 9 34/35 (TAT.rnac (versus (TAT. ROD 20 30 40 MET PLREQENSLE SSNERSSY I SEAAAA IPESANLGEE ILSQLYRPLEACYNTCYCKKCC METPLKAPESSLKSCNEPFSRTSEQDVATQELARQGE EILSQLRPL"EfCNNSCYCKRCC 20 30 4~0 so 80 90 100 110 YHCOFCFLKKGLGI SYEKSHRRRRTPKKAKANTSSASNERP--- IPNRI RLCOPKKAKKE YHCQMCFLNKGLGICYERKGRRRRTPKKTKTH-PSPT PDKS ISTRTGOSOPTKKQKK 80 90 100 110 120 130 TYE AAVA TA PGLGR. TVEATVETDTGP GR 120 130 FIG. 35/35 CART.mac FIG.1l versus CART.'RQD 20 30 io MRSHT GEEELRRRLRL IHLLHOTSKYGL SWKSAAYRHLLVDPYPTGSG SANORRQKRRRW MNERADEEGLORKLRLIRLLHOTN PYPQGPGTASQRRNRRRRW 20 3041 70 80 90 100 110 RORWOQLLALAORIYSFPOPPTDTPLDLAIQQLQNLAIES !POPPTNIPEALCDLRRIRR KOR WRQ I LALADS T YTF POP PAOSPLDQT I OHLOGLT I QELPDP PTHL P E SQRLAET 50 60 70 80 90 100 SPOA. 'it, S itt I, e a L~j ti a t I I ii tt i t t ~t t It t Ii tt~ I j 4 t FIG. 11
AU12250/88A 1987-01-16 1988-01-15 Peptides having immunological properties 2-hiv-2 Expired AU608294B2 (en)

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US003764 1987-01-16
US07/003,764 US5051496A (en) 1986-01-22 1987-01-16 Peptides related to human immunodeficiency virus II (HIV-2)
FR8701739A FR2610632B1 (en) 1987-02-11 1987-02-11 CHARACTERISTIC PEPTIDES OF HUMAN IMMUNODEFICIENCY RETROVIRUSES (HIV VIRUSES) THEIR APPLICATIONS IN THE DIAGNOSIS OF INFECTIONS DUE TO CERTAIN OF THESE VIRUSES AND, IF NECESSARY, IN VACCINATION AGAINST AIDS
FR8705398A FR2614025B1 (en) 1987-04-15 1987-04-15 PEPTIDES LIKELY TO BE RECOGNIZED BY ANTIBODIES INDUCED AGAINST HUMAN IMMUNODEFICIENCY RETROVIRUSES (HIV VIRUSES) THEIR APPLICATIONS IN THE DIAGNOSIS OF INFECTIONS DUE TO CERTAIN VIRUSES AND, IF NECESSARY, IN VACCINATION AGAINST AIDS
FR8705398 1987-04-15
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ATE154808T1 (en) 1997-07-15
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DE3855947T2 (en) 1997-12-11
EP0283327B1 (en) 1997-06-25
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GR3024823T3 (en) 1998-01-30
JP2862810B2 (en) 1999-03-03
JP2948823B2 (en) 1999-09-13
EP0283327A2 (en) 1988-09-21
JPH07300498A (en) 1995-11-14
AU1225088A (en) 1988-08-10
DK513388A (en) 1988-11-16
ES2104556T3 (en) 1997-10-16
DK174705B1 (en) 2003-09-29
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JPH11322792A (en) 1999-11-24
DE3855947D1 (en) 1997-07-31

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