CA1340875C - Variants of lav viruses their dna- and protein- components and their uses, particularly for diagnostic purposes and for the preparation of immunogenic compositions - Google Patents

Abstract

The invention relates to two variants of LAV
viruses capable of causing acquired immunosuppressive syndrome (AIDS), which virus variants have been designated as LAV ELI and LAV MAL. There DNAs and antigens can be used for the diagnostic of AIDS or pre-AIDS.

Description

VARIANTS OF LAV VIRUSES, THEIR DNA- AND PROTEIN-COMPONENTS AND THEIR USES. PARTICULARLY FOR DIAGNOSTIC
PURPOSES AND FOR THE PREPARATION
OF IMMLUNOGENIC COMPOSITIONS
The pre~;ent invention relates to viruses ca-pable of inducing lymphadenopathies (denoted below by the abbreviation LAS) acquired immuno-depressive syn-dromes (denoted below by the abbreviation AIDS), to antigens of said viruses, particularly in a purified form, and to processes for producing these antigens, particularly antigens of the envelopes of these viruses.
The invention also relates to polypeptides, whether,gly-cosylated or not, encoded by said DNA sequences.
The invention also relates to cloned DNA se quences hybridizable to genomic RNA and DNA of the new lymphadenopathy associated viruses (LAV) disclosed here after, to processes for their preparation and their uses. It relates more particularly to stable probes in cluding a DNA sequence which can be used for the detec Lion of the new LAV viruses or related viruses or DNA
proviruses in any medium, particularly biological, sam-ples, containing of any them.
An important genetic polymorphism has been re cognized for the human retrovirus at the origin of the acquired immune deficiency syndrome (AIDS) and other diseases, like lymphadenopathy syndrome (LAS), AIDS-related complex (ARC) and probably some encephalopathies (for review see Weiss, 1984). Indeed all of the isolates analyzed until now have a distinct restriction map, even if recovered from the same place and time (BENN et al., 1985). Identical restriction maps have only been observed fo:r the first two isolates designated lymphadenopat:hy-associated virus, LAV (ALIZON et al., 1984) and human T-cell lymphotropic virus type 3, HTLV-3 (HAHN et al., 1984) and thus appears as an exception.

. -- 13408?5 The genetic polymorphism of the AIDS virus was better assessed after the determination of the complete nucleo-tide sequence of hAV (WAIN-HOBSON et al., 1985), HTLV-3 (RATNER et al., 1985 ; MUESING et al., 1985) and of a third isolate designated AIDS-associated retrovirus, ARV
(SANCHE2-PESC,ADOR et al., 1985). In particular it ap-peared that, besides the nucleic acid variations respon-sible for the restriction map polymorphism, isolates could differ significantly at the protein level, espe-cially in the envelope (up to 13 so of difference between ARV and LAV), by both amino-acids substitutions and re-ciprocal insertions-deletions (RABSON and MARTIN, 1985).
Nevertheless the differences mentioned above do not go as far as to destroy a level of immunological relationship sufficient, as evidenced by the capabili ties of similar proteins, i. e. core proteins of similar nature, such as the p25 proteins, or of similar envelope glycoproteins, such as the 110-120 kD glycoproteins, to immunologically cross-react. Accordingly the proteins of any of said L,AV viruses can be used for the in vitro de-tection of ,antibodies induced in vivo and present in biological fluids obtained from individuals infected with the other LAV variants. Therefore these viruses are grouped in ,a class of LAV viruses, hereafter generally said to belong to the class of LAV-1 viruses.
The invention stems from the discovery of new viruses which although held as responsible of diseases which are clinically related to AIDS and still belonging to the class of "LAV-1 viruses", differ genetically to a much larger extent from the above mentioned LAV va-riants.
The new viruses are basically characterized by their DNA sequences.
The invention further relates to variants of _.

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the new viruses t:he RNAs of which or the related cDNAs derived from said F:NAs are hybridizable to corresponding parts of the cDNAs of either LAVELI or LAVM~.
The invention also relates to the DNAs them selves of said viruses, including DNA fragments derived therefrom hybridiz;able with the genomic RNA of either LAV~I or LAVAL. F~articularly said DNAs consist of said cDNAs or cDNA fragments or of recombinant DNAs contai ning said cDNAs or cDNA fragments.
It further relates to DNA recombinants contai-ning DNAs or cDNA fragments of either LAVELI or LAV~h or of related viruses. It is of course understood that fragments which would include some deletions or muta-tions which would not substantially alter their capabi-~5 lity of also hybridizing with the retroviral genomes of LAVELI or LAV,MAL are to be considered as forming obvious equivalents of th.e DNAs or DNA fragments more specifi-cally referred to hereabove.
The invention also relates more specifically 20 to cloned probes which can be made starting from any DNA
fragment according to the invention, thus to recombinant DNAs containing such fragments, particularly any plas mids amplifiable in procaryotic or eucaryotic cells and carrying said fragments.
25 Using the cloned DNA containing a DNA fragment of LAVELI or .of LAVM~ as a molecular hybridization pro-be - either by marking with radionucleotides or with fluorescent :reagents - LAV virion RNA may be detected directly e. g. in the blood, body fluids and blood pro-30 ducts (e. g. o:E the antihemophylic factors such as Factor VIII concentrates). A suitable method for achieving that detection comprises immobilizing virus onto said a sup-port e.g. n.itrocellulose filters, etc., disrupting the virion and hybridizing with labelled (radiolabelled or 35 "cold" fluor~=scent- or enzyme-labelled) probes. Such 1310 ~'~~

an approach has already been developed for Hepatitis B
virus in peripheral. blood (according to SCOTTO J. et al.
Hepatology (1983), 3, 379-384).
Probes according to the invention can also be used for rapid screening of genomic DNA derived from the tissue of pa.tienta with LAV related symptoms, to see if the proviral DNA or RNA present in host tissue and other tissues are related to LAVELI or LAVM~ .
A method which can be used for such screening comprise the following steps . extraction of DNA from tissue, restriction enzyme cleavage of said DNA, elec trophoresis of the fragments and Southern blotting of genomic DNA from tissues, subsequent hybridization with labelled cloned LA,V provival DNA. Hybridization in situ can also be used.
Lymphatic' fluids and tissues and other non-lymphatic tissues of humans, primates and other mamma-lian species can also be screened to see if other evo-lutionnary related retrovirus exist. The methods referred to hereabove can be used, although hybridiza-tion and washing, would be done under non stringent conditions.
The DNA according to the invention can be used also for achieving the expression of LAV viral antigens for diagnostic purposes as well as far the production of a vaccine against L~AV. Fragments of particular advantage in that respect will be discussed later.
The methods which can be used are multifold .
a) DNA can be transfected into mammalian cells with appropriate selection markers by a variety of tec-hniques, calcium phosphate precipitation, polyethylene glycol, protoplast-fusion, etc..
b) :DNA fragments corresponding to genes can be cloned into expression vectors for _E, coli , yeast- or mammalian cells and the resultant proteins purified.

c) The provival DNA can be "shot-gunned"
(fragmented) into procaryotic expression vectors to ge-nerate fusion pol~~peptides. Recombinant producing anti-genically competent. fusion proteins can be identified by 5 simply screening the recombinants with antibodies against LAVELI or L~AV~L antigens.
Particular reference in that respect is made to those portions of the genomas of LAV~I and LAVAL
which, in the drawings, are shown to belong to open reading frames ands which encode the products having the polypeptidic backbones shown.
Methods disclosed in European application 0 178 978 and in PCT application PCT/EP 85/00548 filed on Oct.lB, 1985 are applicable for the production of s-uetr peptides from the corresponding viruses.
The present invention further aims at provi ding polypep~tides containing sequences in common with polypeptides comprising antigenic determinants included in the proteins encoded and expressed by the LAVELI or of LAVM~ genome. An additional object of the invention is to further provide means for the detection of pro-teins related to these LAV viruses, particularly for the diagnosis of AIDS or pre-AIDS or, to the contrary, for the detection of antibodies against the LAV virus or proteins related therewith, particularly in patients afflicted with AIDS or pre-AIDS or more generally in asymtomatic carriers and in blood-related products.
Finally the invention also aims at providing immunogenic polypeptides, and more particularly protective polypep-tides for use in the preparation of vaccine compositions against AIDS or related syndroms.
The invention relates also to polypeptide fragments having lower molecular weights and having 13~08~~

peptide sequencEa or frac~nents in camnn with those encoded by LAV~T
and I~AVr,~ I~~. Frac~:nts of smaller sizes may be obtained by resorting to known techniques. For instance such a method comprises cleaving the original larger polyp.°_ptide by enzymes capable of cleaving it at specific sites. By way of examples of such proteins, may be mentioned the enzyme of ytaphvlococcvus s~reus V8, a-chymotrypsi:ne, "mouse sub-maxillary gland protease"
marketed by the BOF:HRINGER company, Vibrio alQinolvticus ~hemovar ~OPhacrus collagenase, which specifically re-cognizes said peptides Gly-Pro and Gly-Ala, etc.
Other features of this invention will appear in the following disclosure of the data obtained starting from LAVES and LAVM~ , in relation to the drawings in which .
- Figs 1A and 1B provide restriction maps of the genomas of LAV ~ I and LAV C~ L as compared to LAVE RU ( a known LAV
isolate deposited at CNCM under number I-232 on July 15th, 1983) ;
- Fig. 2 shows i~he comparative maps setting forth the relative position~~ of the open reading frames of the above genomas ;
- Figs. 3A-3F (sometimes also designated globally here after by fig. 3) indicate the relative correspondance between the proteins (or glycoproteins) encoded by the open reading frames, whereby aminoacid residues of protein sequences of LAV and LAV are in vertical alinment with corres~~iding a~~oacid residues (numbered) of corresponding or homologous proteins or glycoproteins of LAV ;
- Figs. 4A-9EH (some~es also designated globally here-after by fig. 4D provide for quantitation of the se-quence divergence )between homologous proteins of LAVB ~, LAV and LAV ;;
- Fi~I 5 shows~~agrammatically the degree of divergence of the different viLrus enveloppe proteins ;
- Figs. 6A and 6B (or Fig. 6 when viewed altogether) render apparent i~he direct repeats which appear in the ~.34087~
proteins of the different AIDS virus isolates.
- Figs. 7A-7I and 8A-8I show the full nucleotidic sequences o1: LAVELI and LAVAL respectively.
RESULTS
Clharacterization and molecular cloning of two African isolLates.
Tlhe different AIDS virus isolates concerned are designated by three letters of the patients name, LAVH ~ referring to the prototype AIDS virus isolated in 1983 from a French homosexual patient with LAS and thought to have been infected in USA in the preceding years (Harr~~-Sino~ussi et al., 1983). Hoth of the African patients originated from Zaire ; LAV ~I was recovered in 1983 from a 24 year old woman with AIDS, and LAVA L in 1985 from a 7 year old boy with ARC, probably infected in 1981 after a blood-transfusion in Zaire, since his parents were LAV-seronegative.
Recovery and purification of each of the two viruses wer<~ performed according to the method disclosed in European Patent Aplication 84 401834/138 667 filed on September 9" 1984.
L,AVELI and LAVM~ are indistinguishable from the previously characterized isolates by their struc-tural and biological properties ~ vitro. Virus meta-bolic labelling and immune precipitation by patients ELI
and MAL sera, as well as reference sera, showed that the proteins of LAV ~I and LAVM ~ had the same molecular weight (Mw) and cross-reacted immunologically with those of prototype AIOiS virus (data not shown) of the 'LAV 1' class.
RE:ference is again made to European Application 178.978 published on April 23, 1986 and International Application ~a0/8602383 published on April 24, 1986 as concerns thE: purification, mapping and sequencing proce-dures used herein. See also "experimental procedures"
and "legend: of the figures" hereafter.

134n~~~5 a Primary restriction enzyme analysis of LAVELI
and LAVM~ g~enomea was done by southern blot with total DNA derived from acutely infected lymphocytes, using cloned LAVBR.U Complete genome as probe. Overall cross-hybridization was observed under stringent conditions, but the restriction profiles of the Zairian isolates were clearly different. Phage lambda clones carrying the complete viral genetic information were obtained and further chara~cteri:~ed by restriction mapping and nucleo-tide sequence analysis ; clone E-H12 is derived from LAVELI infected cells and contains an integrated provirus with 5' flanking cellular sequences but a truncated 3' lone terminal repeat (LTR) ; clone M-H 11 was obtained by cornplete HindIII restriction of DNA from ~5 LAVA L infected cells, taking advantage of the existence of a unique HindIII site in the LTR. M-H 11 is thus probably derived from unintegrated viral DNA since that species was at 7_east ten times more abundant than integrated proviru~; .
Figure 1B gives a comparaison of the restric tion maps of LAVELI' LAVM~ and prototype LAVBRU
all three being derived from their nucleotide sequences, as well of three Zairian isolates previously mapped for seven restriction enzymes (Benn et al., 1985). Despite this limited number, all of the profiles are clearly different (out of: the 23 sites making up the map of LAVBRU only seven are present in all six maps presented), confirming the genetic polymorphism of the AIDS virus. No obvious relationship is apparent between the five Zairian maps, and all of their common sites are also found in LAVBR.U' Conservation of the genetic organization.
Th e, genetic organization of LAVELI and LAVAL
as deduced from the complete nucleotide sequences of their cloned genomes is identical to that found in other 4340 l5 isolates, i.e. 5'gag-pol-central region-env-F3'. Most noticeable is the conservation of the "central region"
(fig. 2), located between the pol and env genes, which is composed of a series of overlapping open reading frames (orf) we had previously designated Q, R, S, T, and U after observing a similar organization in the ovine lentivirus visna (Sonigo et al., 1985). The product of orf S (also designated "tat") is implicated in the transactiva.tion of virus expression (Sodroski et al., 1985 ; Arya et al., 1985) ; the biological role of the product of orf Q (also designated "sor" or orf A) is still unknown (Lee et al., 1986 ; kang et al., 1986). Of the three other orf's (R, T, and U), only orf R is likely to be a seventh viral gene, for the following reasons .
the exact conservation of its relative position with respect to Q and S (fig. 2), the constant presence of a possible splice acceptor and of a consensus AUG
initiator cod~on, its similar codon usage with respect to viral genes, and finally the fact that the variation of its protein sequence within the different isolates is comparable to that of gag, pol and Q (see fig. 4).
Also conserved are the sizes of the U3, R and U5 elements of the LTR (data not shown), the location and sequence of their regulatory elements such as TATA
box and AATAAA polyadenylation signal, and their flanking sequences i.e. primer binding site (PBS) complementary to 3' end of tRNALYS and polypurine tract (PPT). Most of the genetic variability within the LTR is located in the 5' half of U3 (which encodes a part of orf F) while the 3' end of U3 and R, which carry most of the cis-acting regulatory elements . promoter, enhancer and trans-activating factor receptor (Rosen et al., 1985), as well as the U5 element are well-conserved.
Overall, it clearly appears that the Zairian ~34p~~~

isolates belong to the same type of retrovirus as the previously sequenced isolates of American or European orlgln.
Vax-iabil:Lty of the viral proteins .
Desk>ite their identical genetic organization, these isolates show .substantial differences in the primary structure of their proteins. The amino acid sequences of LAVELZ and LAVN~, proteins are presented in figures 3A-3F
(to be examined in conjunction with Figs. 7A-7J and 8A-8I), aligned with those of LAVBRU and ARV 2. Their divergence was quantified as the percentage of amino-acids substitutions in two-by-two alignments (Fig. 4). We have also scored the number of insertions and deletions that had to be introduced in each of these alignments.
Three general observations can be made. First, the protein sequences of the African isolates are more divergent from LAVBRU than are those of HTLV-3 and ARV 2 (Fig. 4A); similar results are obtained if ARV 2 is taken as reference (not. shown). The range of genetic polymorphism between isolates of the AIDS virus is considerably greater than previously observed. Second, our two sequences confirm that the envelope is more variable than the gag and pol genes. Here again, the relatively small difference observed between the env of LAVHRU and HTLV-3 appears as an exception. Third, the mutual divergence of the two African isolates (Fig. 4B) is comparable to that between LAVBRU and either of them; as far as we ca.n extrapolate from only three sequenced isolates from the US.A and Europe and two from Africa, this is indicative of a wider evolution of the AIDS virus in Africa.
The HIV-1 variant viruses of this invention present an overall percent of substitution, with regard to 4340 l~
- l0a -the known HIV-1 variants, of at least 3.4o in Gag, at least 3.1% in Pol and at least l3.Oo even at least 20.70 in Env amino acid sequences.
_a~ag and Col . Their greater degree of conservation compared to the envelope is consistent with their encoding important structura=L or enzymatic activities.
~..'~A

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Of the three mature gag proteins, the p25 which was the first recognized immunogenic protein of LAV (Barre-Sinoussi et al., 1983) is also the better conserved (fig. 3). In gag and pol, differences between isolates are principally due to point mutations, and only a small number of insertional or deletional events is observed.
Among these, we must note the presence in the over-lapping part of gag and pol of LAVBRU of an insertion of 12 aminoacids (AP,) which is encoded by the second copy of a 36 by direct repeat present only in this isolate and in HTLV-3. This. duplication was omitted because of a computing error in the published sequence of LAVBRU
(position 1712, Wai.n-Hobson et al., 1985) but was indeed present in the HT'LV-3 sequences (Ratner et al., 1985 ;
Muesing et al., 1985).
env . Three segments can be distinguished in the envelope glycoprot:ein precursor (Allan et al., 1985 ;
Montagnier et al., 1985 ; DiMarzoVeronese et al., 1985).
The first is the a;ignal peptide (positions 1-33 in fig.
3)~ and its sequence appears as variable ; the second segment (pos. 34-530) forms the outer membrane protein (OMP or gp110) and carries most of the genetic variations, and in particular almost all of the numerous reciprocal insertions and deletions ; the third segment (531-877) is separated from the OMP by a potential cleavage site following a constant basic stretch (Arg-Glu-Lys-Arg) and forms the transmembrane protein (TMP or gp 41) responsible for the anchorage of the envelope glycoprotesin in the cellular membrane. A better conservation of the TMP than the OMP has also been observed between the different murine leukemia viruses (MLV, Koch et al., 1983), and could be due to structural constraints.
From the alignment of figure 3 and the graphical representation of the envelope variability .~340~y~

shown in fugure 5, we clearly see the existence of conserved domains,, withlittle or no genetic variation, and hypervariable domains, in which even the alignment of the different sE~quences is very difficult, because of the existence of a large number of mutations and of reciprocal insert:ions and deletions. We have not included the sequence of the envelope of the HTLV-3 isolate since it so close to that of LAVBRU ( cf . fig .
4), even in the hypervariable domains, that it did not add anything to the analysis. While this graphical representation wi:Ll be refined by more sequence data, the general profile is already apparent, with three hypervariable: dom<~ins (Hyl, 2 and 3) all being located in the OMP, and separated by three well-conserved stretches (residuea 37-130, 211-289, and 488-530 of fig.
3 alignment) probably associated with important biolo-gical functions.
In spitE~ of the extreme genetic variability, the folding pattern of the envelope glycoprotein is probably cor,~stant. Indeed the position of virtually all of the cy:cteine residues is conserved within the different isolates (fig. 3 and 5), and the only three variable cy~~teinea fall either in the signal peptide or in the very C-terminal part of the TMP. The hyper-variable domains of the OMP are bounded by conserved cysteines, suggesting that they may represent loops attached to the common folding pattern. Also the calculated h.ydropathic profiles (Kyte and Doolittle, 1982) of the different envelope proteins are remarkably conserved (notshown).
About half of the potential N-glycosylation sites, Asn-~s:-Ser/7Chr, found in the envelopes of the Zairian isolates map to the same positions in LAVBRU
( 17/26 for LA,VELI and 17/28 for LAVAL) . The other sites appear to fall within variable domains of env, 13408~l~

suggesting the exi~;tence of differences in the extent of envelope glycosylat.ion between different isolates.
Other viral proteins . Of the three other identified viral proteins, t:he p27 encoded by orf F, 3' of env (Allan et al., 1985b) is the most variable (fig. 4). The proteins encoded by orfs Q and S of the central region are remarkable by their absence of insertions/deletions.
Surprisingly, a high frequency of aminoacids substitu-tions, comparable to that observed in env, is found for the product of orf S (trans-activating factor). On the other hand, the protein encoded by orf Q is no more variable than gag. Also noticeable is the lower variation of the proteins encoded by the central regions of LAVELI and LAVMA,L .
DISCUSSION
With the availability of the complete nucleo-tide sequence from five independant isolates, some general features of the AIDS virus genetic variability are now emerging. Firstly, its principal cause are point mutations very often resulting in amino-acid substitu-tions, and which are more frequent in the 3' part of the genome (orf S, env and orf F). Like all RNA viruses, the retroviruses are thought to be highly subject to mutations caused by errors of the RNA polymerases during their replication, since there is no proofreading, of this step (Holland et al., 1982 ; Steinhauer and Holland, 1986).
Another source of genetic diversity are insertions/deletions. From the figure 3 alignments, insertional events seem to be implicated in most of the cases, since otherwise deletions should have occurred in independant isolates at the precisely the same location.
Furthermore, upon analyzing these insertions, we have observed that they most often represent one of the two copies of a direct repeat (fig. 6). Some are perfectly 13~O~rl conserved like the 36 by repeat in the gag-pol overlap of LAV BR U (fig. 6-a) ; others carry point mutations resulting in aminoacid substitutions, and as a consequence, they are more difficult to observe, though clearly present, in the hypervariable domains of env (cf. fig. 6-g and -h). As noted for point mutations, env gene and orf F also appear as more susceptible to that form of genetic ariation than the rest of the genome.
The degree of conservation of these repeats must be related to their date of occurrence in the analyzed sequences . the more degenerated, the more ancient. A
very recent divergence of LAVBRU and HTLV3 is suggested by with extremely low number of mismatched AA between their homologous proteins. However, one of the LAVBRU
repeats (located in the Hyl domain of env, fig. 6-f) is not present inn HTLV3, indicating that this generation of tandem repeats isa rapid source of genetic diversity. We have found Sao traces of such a phenomenon, even when comparing very closely related viruses, such as the Mason-Pfizer monkey virus, MPMV (Sonigo et al., 1986), and an immu:nosuppressive simian virus, SRV-1 (Power et al., 1986). Insertion or deletion of one copy of a direct repeat have been occasionally reported in mutant retroviruses (Shimotohno and T emin, 1981 ; Darlix, 1986), but the extent at which we observe this pheno-menon is unprecedented.
The molecular basis of these duplications is unclear, but could be the "copy-choice" phenomenon, resulting from the diploidy of the retroviral gesnome (Varmus and Swanstrom, 1984 ; Clark and Mak, 1983). During the synthesis of the first-strand of the viral DNA, jumps are known to occur from one RNA
molecule to another, especially when a break or a stable secondary structure is present on the template ; an inaccurate re--initiation on the other RNA template could 13108'~~
result in the generation (or the elimination) of a short direct repeat.
Genetic variability, and subsequent antigenic modifications, have often been developed by micro s organisms as a means to escape the host's immune res ponse, either by modifying their epitopes during the course of tike infection, as in trypanosomes (Borst and Cross, 1982), or by generating a large repertoire of antigens, as observed in influenza virus (Webster et 10 al., 1982). As the human AIDS virus is related to animal lentiviruses (Sonigo et al., 1985 ; Chiu et al., 1985), its genetic variability could be a source of antigenic variation, as can be observed during the course of the infection by the ovine lentivirus visna (Scott et al., 15 199 ~ Clements et al., 1980) or by the equine infec-tious anemia virus (EIAV, Montelaro et al., 1984).
However, a major discrepancy with these animal models is the extremely low, if any, neutralizing activity of the sera of indi~,riduals infected by the AIDS virus, whether they are healthy carriers, displaying minor symptoms or afflicted with AIDS (Weiss et al., 1985 ; Clavel, et al., 1985). Furthermore, even for the visna virus the exact role of antigenic variation in the pathogenesis is unclear (Thormar et al., 1983 ; Lutley et al., 1983). We rather feel i~hat genetic variation represents a general selective advantage for lentiviruses by allowing an adaptation to different environments, for example by modifying their tissue or host tropisms. In the particu-lar case of the AIDS virus, rapid genetic variations are tolerated, especially in the envelope ; they could allow the virus to get adapted to different "micro-environ-ments" of the membrane of their principal target cells, namely the ".C4 lymphocytes. These "micro-environments"
could result from the immediate vicinity of the virus receptor to polymorphic surface proteins, differring I3~pg~~

either between individuals or betwwen clones of lymphocytes.
Conserved domains in the AIDS virus envelope.
Since the proteins of most of the isolates are antigenically cross-reactive, the genotypic differences do not seem to affect the sensitivity of actual diagnos tic tests, based upon the detection of antibodies to the AIDS virus and using purified virions as antigens. They nevertheless have to be considered for the development of the "second-generation" tests, that are expected to be more specific, and will use smaller synthetic or genetically-engineered viral antigens. The identifi-cation of conserved domains in the highly immunogenic envelope glycoprot:ein, and also the core structural 1 5 proteins ( gag ) , i.s very important for these tests . The conserved stretch found at the end of the OMP and the beginning of the TMP (490-620, fig. 3) could be a good candidate, since a bacterial fusion protein containing this domain was well-detected by AIDS patients sera (Chang et al., 198Fi).
The envelope, specifically the OMP, mediates the interaction between a retrovirus and its specific cellular receptor (DeLarco and Todaro, 1976 ; Robinson et al., 1980). In the case of the AIDS virus, in vitro binding assays have shown the interaction of the envelope glycoprotein gp110 with the T4 cellular surface antigen (McDougal et al., 1986), already thought to be, or closely associated to, the virus receptor (Klatzmann et al., 1984 ; Dagleish et al., 1984). Identification of the AIDS virus envelope domains that are responsible for this interaction (receptor-binding domains) appears as fundamental for understanding of the host-viral interactions, bust also for designing a protective vaccine, since an immune response against these epitopes could possibly e:Licit neutralizing antibodies. As the . 13~0~7j AIDS virus recepi~or is at least partly formed of a constant structure', the T4 antigen, the binding site of the envelopes is unlikely to be exclusively encoded by domains undergoing drastic genetic changes between isolates, even if i=hese could be implicated in some kind of an "adap~tatiorv" . One, or several of the conserved domains of the 0MP (residues 37-130, 211-289, and 488-530 of fig. 3 alignment) brought together by the folding of the protein, must play a part in the virus-receptor interaction, and this can be explored with synthetic or genetically-engineered peptides derived from these domain;, either by direct binding assays, or indirectly by assaying the neutralizing activity of specific antibodie~~ raised against them.
-African AIDS viruses Zaire a.nd the neighbouring countries of Central Africa are' considered as an area of endemic for the AIDS virus infection, and the possibility that the virus has emerged in Africa has became a subject of intense controversy (see Norman, 1985). From the present study, it is clear that the genetic organization of Zairian isolates is the same as that of american isolates, thereby indicating a common origin. The very important sequence differences observed between the proteins are con~;istent with a divergent evolutionary process. In addition, the two African isolates are mutually more divergent than the American isolates already analyzed ; as far as that observation can be extrapolated, it suggests a longer evolution of the virus in Africa, and is also consistent with the fact that a larger fraction of the population is exposed than in developed countries.
A novel human retrovirus with morphology and biologocal properties (cytopathogenicity, T4 tropism) similar to those of LAV, but nevertheless clearly 1340a~~

genetically a.nd ani:igenically distinct from that latter, was recently isolated from two patients with AIDS
originating from Guinea Bissau, West-Africa (Clavel et al., 1986). In the neighbouring Senegal the population seems exposed to a retrovirus also distinct from LAV, but apparently non pathogenic (Barin et al., 1985 ;
Kanki et al., 1986). Both of these novel African retroviruses seem to be antigenically related to the simian T-cell lymphotropic virus, STLV-III, shown to be widely present in healthy African green monkeys and other simian species (Kanki et al. 1985). This raises the possibility of a large group of African primate lentiviruses, ranging from the apparently non-pathogenic simian viruses to the LAV-type viruses. Their precise relationship will only be known after their complete genetic characterization, but it is already very likely that they have evolved from a common progenitor. The important genetic variability we have observed between isolates of the AIDS virus in Central Africa is probably a hallmark of this entire group, and may account for the apparently important genetic divergence between its members (loss of cross-antigenicity in the envelopes).
In this sense the conservation of the tropism for the T4 lymphocytes suggests that it is a major advantage squired by these re~troviruses.
EXPERIMENTAL PROCEDURES
Virus isolations LAVELI and LAVAL were isolated from the peripheral blood lymphocytes of the patients as des cribed (Barre-Sinoussi et al., 1983) ; briefly, the lymphocytes were fractionated and co-cultivated with phytohaemagglutinin-stimulated normal human lymphocytes in the presence o~f interleukin 2 and anti-alpha inter-feron serum. Viral production was assessed by cell-free reverse transcriptase (RT) activity assay in the cultures and by electron microscopy.
llol~ecular cloning Normal donor lymphocytes were acutely infected (104 cpm of RT activity/106 cells) as described (Barre Sinoussi et al., 1983), and total DNA was extracted at the beginning of the RT activity peak. For LAVELI' a lambda library using the L47-1 vector (Loenen and Brammar, 19:32) was constructed by partial HindIII
digestion of the DNA as already described (Alizon et al., 1984). For LAVA L, DNA from infected cells was digested to completion with HindIII and the 9-10kb fraction was selected on 0.8 °-° low melting point agarose gel and ligated into L47-1 HindIII arms. About 5.105 plaques for LAVELI and 2.105 for LAVM~, obtained by in vitro packaging (Amersham) were plated on E. coli LA101 and screened in situ under stringent conditions, using the 9 kb Sac~I insert of the clone lambda J19 (Alizon et al., 1984) c<irrying most of the LAVBRU genome as probe.
Clones displ<~ying positive signals were plaque-purified and propagated on E. coli C600 recBC, and two recombi-nant phages carrying the complete genetic information of LAV~I (E-H1a?) and LAVAL (M-H11 ) were further charac-terized by restriction mapping.
l~ucJleotia~e sequence strategy Viral fragments derived from E-H12 and M-H11 were sequenced by t:he dideoxy chain terminator procedure (Sanger et al., 1977) after "shotgun" cloning in the M13mp8 vector (Measing and Viera, 1982), as previously described (Sonigo et al., 1985). The viral genome of LAVELI is 91 ~'6 nuc:leotides, that of LAVAL 9229 nucleo-tides long. Each nucleotide was determined from more than 5 independent: clones on average. Complete nucleotide sequences are not ;presented in this article for obvious reasons of ;pace limitation but are freely available upon request to 'the authors, until they are released I3408'~5 through sequence data banks.
LEGEND OF THE FIGURES
Figure 1 . Restrict:ion map analysis of AIDS virus isolate~~ .
5 A/ Restriction maps of the inserts of phage lambda clones derived from cells infected with LAVELI
(E-H12) and LAVMAL (M-H11). The schematic genetic organization of the AIDS virus has been drawn above the maps. T'he LTRs are indicated by solid boxes.
10 A:Aval-B:Bam HI-Bg:BgIII-E:EcoRI - H:HindIII - Hc:HincII
- K:KpnI-N:Nde I-P:PstI-S:SacI-X:XbaI. Asterisks indicate the HincIIII cloning sites in lambda L47-1 vector.
B/Comparison of the sites for seven restric-15 tion enzymes in si.x isolates . the prototype AIDS virus LAVBRU' LAVMAL and LAVELI ; Z1, Z2, Z3 are Zairian isolates with published restriction maps (Benn et al., 1985). Restriction sites are represented by the following symbols . HglII ; EcoRI ; HincII ; HindIII ;
20 KpnI ; NdeI ; SacI.
Figure 2 . Co:nservation of the genetic organization of the central region in AIDS virus isolates.
Stop codons in each _phse are represented as vertical bars. Vertical arrows indicate possible AUG
initiation codons. Splice acceptor (A) and donor (D) sites identified in subgenomic viral mRNA (Muesing et al., 1985) .are shown below the graphic of LAVBRU' and corresponding sites in LAVELI and LAWM~ are indicated.
PPT indicates the repeat of the polypurine tract flan king the 3'I~TR. As observed in LAVBRU (Wain-Hobson et al., 1985), the PPT is repeated 256 nucleotides 5' to the end of 'the pol gene in both our sequences, but this repeat is degenerated at two positions in LAVELI' Figure 3 . Al:ignment of the protein sequences of four AIDS virus isolates.

13~0~'~5 Isolate LAVBRU (Wain-Hobson et al., 1985) is taken as reference ; only differences with LAVBRU are noted for AIEtV2 (Sanchez-Pescador et al., 1985) and the two Zairian iaolates LAV ~L and LAVELI' A minimal number of gaps (-) was introduced in the alignments. The NH2-termini of ;p25gag and pl8gag are indicated (Sanchez-Pescador, 19.95). The potential cleavage sites in the envelope pre~~ursor (Allan et al., 1985a ; diMarzo-Veronese, 19.95) separating the signal peptide (SP), the outer membrane protein (OMP) and the transmembrane protein (TMl?) are indicated as vertical arrows ;
conserved cysteine;s are indicated by black circles and variable cysteines are boxed. The one letter code for amino acids is . A:Ala ; C:Cys ; D:Asp ; E:Glu ; F:Phe ;
G:Gly ; H :Hia ; I:Ile ; K:Lys ; L:Leu ; M:Met ; N:Asn ;
P:Pro ; Q:Gln ; R:A:rg ; S:Ser ; T:Thr ; V:Val; W:Trp ;
Y:Tyr.
Figure ~ . Quantitation of the sequence divergence between Izomologous proteins of different isolates.
Part: A of each table gives results deduced from two-by-two alignments using the proteins of LAVBRU as reference, part B those of LAVEr~I as reference .
Sources: Muesing et al., 1985 for HTLV-3 ; Sanchez-Pescador et al., 1!385 for ARV 2 and Wain-Hobson et al., 1985 for LAVBRU. 7For each case of the tables, the size in amino-acids of t:he protein (calculated from the first methionine reaidue, or from the beginning of the orf for pol) is given at the upper left part. Below are given 0 the number c>f de_Letions (left) and insertions (right) necessary for the alignment. The large numbers in bold face represent they percentage of amino-acids substitu-tions (insert:ions/deletions being excluded). Two by two alignments were dome with computer assistance Wilburg and Lipman, 1983), using a gag penalty of 1, K-tuple of 13~0~~~~

1, and window o:E 20, except for the hypervariable domains of env, where the number of gaps was made minimum, and which are essentially aligned as shown in fig. 3. The ~;equence of the predicted protein encoded by orf R of HThV-3 has not been compared because of a pre-mature termination relative to all other isolates.
Figure 5 . Variabi:Lity of the AIDS virus envelope protein.
For eactl position x of the alignment of env (Fig. 3), variability V(x) was calculated as number of: different amino-acids at position x V(x) -fre:quency of the most abundant amino-acid at pow;ition x.
Gaps in the alignments are considered as another amino-acid.. For an alignment of 4 proteins, V(x) ranges from 1 (identical AA in the 4 sequences) to 16 (4 different AA). Thia type of representation has previous ly been used in a compilation of the AA sequence of immunoglobuli.ns variable regions (Wu and Kabat, 1970).
Vertical arrows indicate the cleavage sites ; asterisks represent ~~otential N-glysosylation sites (N-X-S/T) conserved in all four isolates ; black triangles re-present conserved cysteine residues. Black lozanges mark the three major hydlrophobic domains. OMP . outermembrane protein ; TMIP . transmembrane protein ; signal . signal peptide ; Hyl, 2, 3, . hypervariable domains.
Figure 6 . Direct repeats in the proteins of different AIDS virus isolates.
These examples are derived from the aligned sequences of gag (a, b), F (c,d) an env (e, f, g, h) shown in figure 3. The two elements of the direct repeat are. boxed, while degenerated positions are underlined.
The invention thus pertains more specifically to the proteins, polypeptides or glycoproteins including the polypeptidic strucutres shown in the drawings. The .F 13~U~~1~

first and last amino-acid residues of these proteins, polypeptides or c~lycoproteins carry numbers computed from a first acninoacid of the open-xeading frames concerned, although these numbers do not correspond exactly to those of the LAVELI or LAVM~ proteins concerned, rather to those of the LAVBRU corresponding proteins or sequences shown in figs. 3A, 3B and 3C.
Thus a number corresponding to a "first amino-acid residue" of a LAVELI protein corresponds to the number of the first amino-acyl residue of the corresponding LAVBRU protein which, in any of figs. 3A, 3B or 3C is in direct alignment with the corresponding first amino-acid of the LAV ~I prot.ein. Thus the sequences concerned can be read f rom figs . 7A-7I and 8A -8I , to the extent where they do not appear with sufficient clarity from Figs.
3A-3F.
The preferred protein sequences of this invention extend from the corresponding "first" and "last" amino-acid residues (reference is also made to the protein(s)- or glycoprotein(s)-portions including part of the sequences which follow .
OMP or gp~110 proteins, including precursors .
1 to 530 OMP or gp110 without precursor .

Sequence carrying the TMP or gp41 protein .
531-877, particularly well conserved stretches of OMP .
37-130, 211-289 and well conserved stretch found at the end of the OMP and the beginning of TMP .
490-620.

13~0~~~5 Proteins containing or consisting of the "well conserved stretches"are of particular interest for the production c>f imrnunogenic compositions and (preferably in relation to t:he stretches of the env protein) of vaccine compositicans against the LAV-viruses of class 1 as above-defined.
The invention concerns more particularly all the DNA fragments which have been more specifically referred to in the drawings and which correspond to open reading frames. 7:t will be understood that the man skilled in the art: will be able to obtain them all, for instance by cleaving an entire DNA corresponding to the complete genome of either LAVELI °r of LAVAL, such as by cleavage by a partial or complet digestion thereof with a suitable restriction enzyme and by the subsequent recovery of the relevant fragments. The different DNAs disclosed above can be resorted to also as a source of suitable fragment;. The techniques disclosed in PCT li application for the isolation of the fragments which can then be included in suitable plasmids are applicable here too.
Of course other methods can be used. Some of them have been ex:amplified in European Application Nr.
178,978 filed on September 17, 1985. Reference is for instance made to th.e following methods.
a) DNA can be transfected into mammalian cells with appropriate selection markers by a variety of tech-niques, calcium phosphate precipitation, polyethylene glycol, protoplast-fusion, etc..
b) DNA fragments corresponding to genes can be cloned into expression vectors for E. coli, yeast- or mammalian cells and the resultant proteins purified.
c) The provival DNA can be "shot-gunned"
(fragmented) into procaryotic expression vectors to generate fusion polypeptides. Recombinant producing antigenically competent fusion proteins can be identi-fied by simply screening the recombinants with antibodies against LAV antigens.
The invention further refers more specifically 5 to DNA recombinants, particularly modified vectors, including any of the preceding DNA sequences and adapted to transform corresponding microorganisms or cells, particularly eucaryotic cells such as yeasts, for instance saccharomyces cerevisiae, or higher eucaryotic 10 Cells, particularly cells of mammals, and to permit ex pression of said DNA sequences in the corresponding microorganisms or cells. General methods of that type have been recalled in the abovesaid PCT international patent aplic,~tion PCT/EP 85/00548 filed on October 18, 15 1985.
More particularly the invention relates to such modified DNA recombinant vectors modified by the abovesaid DNA sequences and which are capable of trans-forming higher eucaryotic cells particularly mammalian 20 Cells. Preferably any of the abovesaid sequences are placed under the direct control of a promoter contained in said vectors and which is recognized by the poly-merases of aaid cells, such that the first nucleotide codons expressed correspond to the first triplets of the 25 above-defined DNA-sequences. Accordingly this invention also relates to the corresponding DNA fragments which can be obtained from genom~s of LAVELI or LAVM~ or corresponding cDNAs by any appropriate method. For instance such a method comprises cleaving said LAV ge-nomas or cDNAs by restriction enzymes preferably at the level of restriction sites surrounding said fragments and close to the opposite extremities respectively thereof, recovering and identifying the fragments sought according to sizes, if need be checking their restric-tion maps oz- nucleotide sequences (or by reaction with ~3~~~~,~

monoclonal antibodies specifically directed against epitopes carried by the polypeptides encoded by said DNA
fragments), and further if need be, trimming the extremities of the fragment, for instance by an exonucleolytic enzyme such as Ba131 , for the purpose of controlling the desired nucleotide-sequences of the ex-tremities of said DNA fragments or, conversely, repairing aaid extremities with Klenow enzyme and possibly. lig<<ting the latter to synthetic polynucleotide fragments designed to permit the reconstitution of the nucleotide e~xtrem.ities of said fragments. Those frag-ments may then be inserted in any of said vectors for causing the expression of the corresponding polypeptide by the cell. transformed therewith. The corresponding polypeptide can then be recovered from the transformed cells, if need be after lysis thereof, and purified, by methods such as electrophoresis. Needless to say that all conventional methods for performing these operations can be resori:ed to .
2Q The' invE~ntion also relates more specifically to cloned probes which can be made starting from any DNA
fragment according to this invention, thus to recombi-nant DNAs containing such fragments, particularly any plasmids amX>lifiable in procaryotic or eucaryotic cells and carrying said :fragments.
Using the cloned DNA fragments as a molecular hybridization probe - either by labelling with radio-nucleotides or with fluorescent reagents - LAV virion RNA may be detecited directly in the blood, body fluids and blood products (e. g. of the antihemophylic factors such as Factor VIII concentrates) and vaccines, i.e.
hepatitis B vaccine It has already been shown that whole virus can be dei_ected in culture supernatants of LAV
producing cells. A suitable method for achieving that detection camprisE~s immobilizing virus onto a support, 13~Q~~' e.g. nitrocellulose filters, etc., disrupting the virion and hybridizing with labelled (radiolabelled or "cold"
fluorescent- or enzyme-labelled) probes. Such an approach has already been developed for Hepatitis B
virus in peripheral. blood (according to SCOTTO J. et al.
Hepatology (1983), 3, 379-384).
Probes according to the invention can also be used for rapid screening of genomic DNA derived from the tissue of pa.tient:~ with LAV related symptoms, to see if the proviral DNA or RNA present in host tissue and other tissues can be related to that of LAVELI or LAVMAL.
A method which can be used for such screening comprises the following steps . extraction of DNA from tissue, restriction enzyme cleavage of said DNA, elec-trophoresis of the fragments and Southern blotting of genomic DNA from tissues, subsequent hybridization with labelled cloned LF~V proviral DNA. Hybridization in situ can also be used.
Lymphatic' fluids and tissues and other non lymphatic tissues of humans, primates and other mamma lian species can also be screened to see if other evolutionnary related retrovirus exist. The methods referred to herea~bove can be used, although hybridi zation and washings would be done under non stringent Conditions.
The DNA; or DNA fragments according to the invention ca.n be used also for achieving the expression of viral antigen; of LAVELI or LAVAL for diagnostic purposes.
The invention relates generally to the poly-peptides themselves, whether synthetrized chemically °<
isolated from viral preparation or expressed by the different DNAs o1: the inventions, particularly by the ORFs or fragments thereof, in appropriate hosts, particularly procaryotic or eucaryotic hosts, after I34D~t~J

transformation thereof with a suitable vector previously modified by the corresponding DNAs.
MorE~ generally, the invention also relates to any of the polypevptide fragments (or molecules, parti cularly gly<~oproteins having the same polypeptidic backbone as the polypeptides mentioned hereabove) bearing an ~~pitope characteristic of a protein or glycoprotein of L.AV~I or LAVAL, which polypeptide or molecule then has N-terminal and C-terminal extremities respectively either free or, independently from each other, covalently bond to aminoacids other than those which are normally associated with them in the larger polypeptides or glycoproteins of the LAV virus, which last mentioned aminoacids are then free or belong to another po:Lypeptidic sequence. Particularly the invention re:Lates to hybrid polypeptides containing any of the epitope-bearing-polypeptides which have been defined more specifically hereabove, recombined with other polypeptides fragments normally foreign to the LAV
proteins, haring sizes sufficient to provide for an increased immunogenicity of the epitope-bearing-poly peptide yet, said foreign polypeptide fragments either being immuno~~enically inert or not interfering with the immunogenic properties of the epitope-bearing-poly peptide.
Such hybrid polypeptides which may contain from 5 up to 150, even 250 aminoacids usually consist of the expression products of a vector which contained ab initio a nu~~leic acid sequence expressible under the control of a auitable promoter or replicon in a suitable host, which nucleic acid sequence had however beforehand been modified by insertion therein of a DNA sequence encoding said epitope-bearing-polypeptide.
Sai<i epitope-bearing-polypeptides, particular-ly those whose N-terminal and C-terminal aminoacids are free, are also accessible by chemical synthesis, accord-ing to technics well known in the chemistry of proteins.
The synthesis of peptides in homogeneous solution and :in solid phase is well known.
In this respect, recourse may be had to the method of synthesis in homogeneous solution described by Houbenweyl in the work entitled "Methoden der Orga-nischen Chemie" (Methods of Organic Chemistry) edited by E. WUNSCH., vol. 15-I and II, THIEME, Stuttgart 1974.
Thi:~ method of synthesis consists of successively condensing either the successive aminoacids in twos, in the appropriate order or successive peptide fragments previously available or formed and containing already several aminoacyl residues in the appropriate order respectively. Except for the carboxyl and amino-groups which will be engaged in the formation of the peptide bond:, care must be taken to protect beforehand all other re~activ~~ groups borne by these aminoacyl groups or fr<~gments.However, prior to the formation of the peptide bonds, the carboxyl groups are advantageously activated, according to methods well known in the syn-thesis of peptides. Alternatively, recourse may be had to coupling reactions bringing into play conventional coupling reagents, for instance of the carbodiimide type, such as 1-ethyl-3-(3-dimethyl-aminopropyl)-carbo-diimide. When the aminoacid group used carries an additional amine group (e. g. lysine) or another acid function (e.c~. glutamic acid), these groups may be protected by carbolbenzoxy or t-butyloxycarbonyl groups, as regards the amine groups, or by t-butylester groups, as regards the' carboxylic groups. Similar procedures are available for the protection of other reactive groups.
for example, SH group (e.g. in cysteine) can be protected by an acetamidomethyl or paramethoxybenzyl group.

In the case of progressive synthesis, amino-acid by aminoacid, the synthesis starts preferably by the condensation of the C-terminal aminoacid with the aminoacid which corresponds to the neighboring aminoacyl group in the desired sequence and so on, step by step, up to the N-terminal aminoacid. Another preferred tech nique can be relied upon is that described by R.D.
Merrifield in "solid phase peptide synthesis" (J. Am.
Chem. Soc., 9~5, 2149-2154).
In accordance with the Merrifield process, the first C-terminal aminoacid of the chain is fixed to a suitable porous polymeric resin, by means of its carbo xylic group, the amino group of said aminoacid then being protected, l:or example by a t-butyloxycarbonyl group.
When thES first C-terminal aminoacid is thus fixed to the resin, the protective group of the amine group is removed b5r washing the resin with an acid, i.e.
trifluoroacet:ic ac=id, when the protective group of the amine group i.s a t--butyloxycarbonyl group.
Then the carboxylic group of the second aminoacid which is to provide the second aminoacyl group of the des~.red ~peptidic sequence, is coupled to the deprotected amine group of the C-terminal aminoacid fixed to the resin. Preferably, the carboxyl group of this second aminoac=id has been activated, for example by dicyclohexyl--carbodiimide, while its amine group has been protected, :Eor example by a t-butyloxycarbonyl group. The first part of the desired peptide chain, which comprising the first two aminoacids, is thus obtained. As previously, the amine group is then de-protected, and one can further proceed with the fixing of the next ~iminoa~cyl group and so forth until the whole peptide sought is obtained.
.15 The' protective groups of the different side ~~~o~~~

groups, if any, of the peptide chain so formed can then be removed. T he peptide sought can then be detached from the resin, for example, by means of hydrofluoric acid, and finally recovered in pure form from the acid solution according to conventional procedures.
As regards the peptide sequences of smallest size and bearing a.n epitope or immunogenic determinant, and more particularly those which are readily accessible by chemical synthesis, it may be required, in order to Increase their in vivo immunogenic character, to couple or "conjugate" them covalently to a physiologically acceptable and non toxic carrier molecule.
By way of examples of carrier molecules or macromolecular supports which can be used for making the conjugates according to the invention, will be mentioned natural proteins, such as tetanic toxoid, ovalbumin, serum-albumins, hemocyanins, etc.. Synthetic macromole-cular carriers, for example polysines or poly(D-L-alanine)-poly(L-lys~ine)s, can be used too.
Other types of macromolecular carriers which can be used, which generally have molecular weights higher than 20,000, are known from the literature.
The conjugates can be synthesized by known processes, such as described by Frantz and Robertson in ~~Infect. and Immunity", 33, 193-198 (1981), or by P.E.
Kauffman in "Applied and Environmental Microbiology", October 1981 Vol. 42, n° 4, 611-614.
For instance the following coupling agents can be used . glutaric aldehyde, ethyl chloroformate, water-soluble carbodiimides (N-ethyl-N'(3-dimethylamino propyl) carbodiimide, HC1), diisocyanates, bis-diazoben zidine, di- and tri.chloro-s-triazines, cyanogen bromides, benzaquinone, as well as coupling agents mentioned in "Scand. J. Immunol., 1978, vol. 8, p. 7-23 (Avrameas, Ternynck, Guesdon).

_ 13~O~r~~

Any coupling process can be used for bonding one or several reactive groups of the peptide, on the one hand, and one or several reactive groups of the carrier, on the other hand. Again coupling is advanta-geously achieved between carboxyl and amine groups carried by t:he peptide and the carrier or vice-versa in the presence of a coupling agent of the type used in protein synthesis, i.e. 1-ethyl-3-(3-dimethylaminopro-pyl)-carbodiimide, N-hydroxybenzotriazole, etc..
Coupling between amine groups respectively borne by the peptide and the carrier can also be made with gluta ralc3ehyde, for instance, according to the method des cribed by BO~~UET, P. et al. (1982) Molec. Lmmunol., 19, 1441-1549, when the carrier is hemocyanin.
The immunogenicity of epitope-bearing-peptides can also be reinforced,by oligomerisation thereof, for example in the presence of glutaraldehyde or any other suitable coupling agent. In particular, the invention relates to the water soluble immunogenic oligomers thus obtained, comprising particularly from 2 to 10 monomer units.
The glycoproteins, proteins and polypeptides (generally designated hereafter as "antigens" of this invention, whether obtained (by methods such as dis-closed in the earlier patent applications referred to above) in a purified state from LAVELI °r LAVAL virus preparations or - as concerns more particularly the peptides - by chemical synthesis, are useful in pro-cesses for the dietection of the presence of anti-LAV
antibodies in biological media, particularly biological fluids such as sera from man or animal, particularly with a view of possibly diagnosing LAS or AIDS.
Particularly the invention relates to an in vitro process of diagnosis making use of an envelope glYcoprotein (or of a polypeptide bearing an epitope of this glycoprotein of LAVELI or LAVM~ for the detection of anti-LAV antibodies in the serums of persons who carry them. Other polypeptides - particular those carrying an epitopea of a core protein - can be used too.
A preferred embodiment of the process of the invention corn~prise:>
- depositing a predetermined amount of one or several of said antigens in the cups of a titration microplate ;
- introducing of increasing dilutions of the biological fluid, i.e. serum t:o be diagnosed into these cups ;
- incubating the mi.croplate ; .
- washing carefully the microplate with an appropriate buffer ;
- adding into the cups specific labelled antibodies directed against blood immunoglobulins and - detecting the antigen-antibody-complex formed, which is then indicative of the presence of LAV antibodies in the biological fluid.
Advantageously the labelling of the anti immunoglobulin antibodies is achieved by an enzyme selected from among those which are capable of hydro lysing a substrate, which substrate undergoes a modi fication of its radiation-absorption, at least within a predetermined band of wavelenghts. The detection of the substrate, preferably comparatively with respect to a control, then provides a measurement of the potential risks or of the effective presence of the disease.
Thus preferred methods immuno-enzymatic or also immunofluorescent detections, in particular according to the ELISA technique. Titrations may be determinations by immunofluorescence or direct or indirect immuno-enzymatic determinations. Quantitative titrations of antibodies on the serums studied can be made.
The invention also relates to the diagnostic kits themselves for the in vitro detection of antibodies against the LAV virus, which kits comprise any of the polypeptides ideni~ified herein, and all the biological and chemical_ reagents, as well as equipment, necessary for peforminc~ diagnostic assays. Preferred kits comprise all reagent:> required for carrying out ELISA assays.
Thus preferred kiia will include, in addition to any of said polype~ptidea, suitable buffers and anti-human immunoglobuli.ns, which anti-human immunoglobulins are labelled either by an immunofluorescent molecule or by an enzyme. In the .Last instance preferred kits then also comprise a substrate hydrolysable by the enzyme and providing a signal, particularly modified absorption of a radiation, at least in a determined wavelength, which signal is then ind_Lcative of the presence of antibody in the biological fluid to be assayed with said kit.
It can of course be of advantage to use seve-ral proteins or po:Lypeptides not only of both LA,VELI and LAVM ~, but also of any or both of them together with homologous proteina or polypeptides of earlier described viruses, e.g. of LAVBRU or HTLVIII °r ARV, etc..
The' invention also relates to vaccine composi-tions whose active principle is to be constituted by any of the antigen, i.a. the hereabove disclosed polypeptid~
whole antigens, of either LAVELI or LAVAL, or both, particularly the purified gp110 or immunogenic fragments thereof, fu~;ion polypeptides or oligopeptides in asso-ciation with a suitable pharmaceutical or physiolo-gically acceptable carrier.
A first type of preferred active principle is the gp110 immunogen of said immunogens.
Other preferred active principles to be con-sidered in that fields consist of the peptides con-taining less, than 250 aminoacid units, preferably less than 150, particu:Larly from 5 to 150 aminocid residues, as deducible for the complete genomas of LAVELI and LAVM~and even more preferably those peptides which contain one or more groups selected from Asn-X-Ser and Asn-X-Ser as defined above. Preferred peptides for use in the production of vaccinating principles are peptides (a) to (f) as defined above. By way of example having no limitative charac;ter, there may be mentioned that suitable dosages of the vaccine compositions are those which are effective: to elicit antibodies in vivo, in the host, particularly a human host. Suitable doses range from 10 to 500 micrograms of polypeptide, protein or glycoprotein per kg, for instance 50 to 100 micrograms per kg.
The different peptides according to this in vention can also be used themselves for the production of antibodies, preferably monoclonal antibodies specific of the different peptides respectively. For the produc tion of hybridomas secreting said monoclonal antibodies, conventional production and screening methods are used.
These monoclonal antibodies, which themselves are part of the invention then provide very useful tools for the identification and even determination of relative proportions of the different polypeptides or proteins in biological samples., particularly human samples contain-lng LAV or related viruses.
The invention further relates to the hosts (procaryotic or eucaryotic cells) which are transformed by the above mentioned recombinants and which are capable of expressing said DNA fragments.
Finally the invention also concerns vectors for the transformation fo eucaryotic cells of human origin, particularly lymphocytes, the polymerase of which are capable of recognizing the LTRs of LAV.
Particularly said vectors are characterized by the presence of a LAV LTR therein, said LTR being then .~~40~ l~

active as a promoter enabling the efficient transcrip-tion and translation in a suitable host of a DNA insert coding for a determined protein placed under its controls.
Needless to say that the invention extends to all variants of ge~nomes and corresponding DNA fragments (ORFs) having substantially equivalent properties, all of said genomes belonging to retroviruses which can be considered as equivalents of LAV.
It must be understood that the claims which follow are also intended to cover all equivalents of the products (glycoproteins, polypeptides, DNAs, etc..) whereby an equivalent is a product, i.e. a polypeptide which may distin<~uish from a determined one defined in ~5 any of said claims, say through one or several amino-acids, while s1ti11 having substantially the same immunological or immunogenic properties. A similar rule of equivalency s11a11 apply to the DNAs, it being understood that th<~ rule of equivalency willthen be tied 20 to the rule of equivalency pertaining to the polypeptides which they encode.
It should further be mentioned that the invention further relates to immunogenic compositions containing preferably not only any of the polypepdides more specif~_cally identified above and which have the aminoacid-seduence;s of LAVELI and LAVAL which have been identified, but corresponding peptidic sequences to previously defined LAV proteins too.
In that respect the invention relates more ~r .~3~~~~

particularly to the particular polypeptides which have the sequences corresponding more specifically to the LAVBRU sequences which have been referred to earlier, i.e. the sequences extending between the following first and last ami:noacids, of the LAVBRU proteins themselves, i.e. the polypeptides having sequences contained in the LAVBRU OMP or LAVBRU TMP or sequences extending over both, particularly those extending from between the following positions of the aminoacids included in the env open reading frame of the LAVBRU genome, and more preferably 531-877, particularly 490-620.
These different sequences can be used for any of the above defined purposes and in any of the compo-sitions which have been disclosed.
Finally the invention also relates to the different antibodies which can be formed specifically against the different peptides which have been disclosed herein, particularly to the monoclonal antibodies which recognize them sF>ecifically. The corresponding hybri-domas which can be formed starting from spleen cells previously immuni2;ed with such peptides which are fused with appropriate myeloma cells and selected according to standard procedure; also form part of the invention.
Phage ~ clone E-H12 derived from LAVELI
infected cells has been deposited at the "Collection Nationale des Cultures de Micro-organismes" (National Collection of Cultures of Microorganisms) (CNCM) of the Pasteur Institute of Paris France, under n' I-550 on May 9th, 1986.
Phage a clone M-H11 derived from LAV ~L
infected cells has been deposited at the CNCM under n' I-551 on May 9th, 1986.

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Claims (21)

1. An isolated LAV ELI virus whose RNA
corresponds to the cDNA of figs. 7A-7J.

2. An isolated LAV MAL virus whose RNA
corresponds to the cDNA of figs. 8A-81.

3. The cDNA of figs. 7A-7J or parts thereof.

4. The cDNA of figs. 8A-8I or parts thereof.

5. DNA recombinants containing at least part of the cDNA according to claim 3 or 4.

6. A probe comprising a fragment from the cDNA according to claim 3 or 4.

7. A method for identifying the presence in a host tissue of a virus or provirus related to either LAV ELI or LAV MAL which comprises hybridizing DNA obtained from said tissue with a probe according to claim 6 and detecting the presence of said virus or provirus in said tissue according to whether hybridization with said probe is detected or not.

8. An isolated or synthetic peptide, protein, or parts thereof encoded by open reading frames of the DNA sequences according to claim 3 or fragments thereof.

9. An isolated or synthetic peptide, protein, or parts thereof encoded by open reading frames of the DNA sequences of claim 4 or fragments thereof.

10. A peptide according to claim 8 which corresponds to any of the stretches extending respectively from aminoacyl residue 37 to aminoacyl residue 130, or from aminoacyl residue 211 to aminoacyl residue 289, or from aminoacyl residue 488 to aminoacyl residue 530, of fig. 3.

11. A peptide according to claim 8 which corresponds to the stretch extending from the aminoacyl residue 490 to the aminocyl residue 620 of fig. 3.

12. An isolated or synthetic portion of a protein or glycoprotein of the LAV ELI or LAV MAL viruses shown in figures 7A-7J and 8A-8I respectively, whose aminoacid sequence includes all or part of the sequences which follow:
OMP or gp110 proteins, including precursors:
1 to 530 OMP or gp110 without precursor:

Sequence carrying the TMP or gp41 protein:
531-877, or well conserved stretches of OMP:
37-130, 211-289 and well conserved stretch found at the end of the OMP and the beginning of TMP:
430-620.

13. A method for the in nitro detection of the presence of antibodies directed against LAV ELI or LAV MAL or against related viruses in human body fluids which comprises contacting said body fluids with antigens obtained from the viruses of claims 1 or 2 or consisting of peptides according to any of claims 8 to 11 and detecting the immunological reaction between said antigens and said antibodies.

14. A method according to claim 13 which comprises:
- depositing a predetermined amount of one or several of said antigens in the cups of a titration microplate;

- introducing said human body fluids into said cups;
- incubating said microplate;
- washing carefully said microplate with an appropriate buffer;
- adding into said cups specific labelled antibodies directed against blood immunoglobulins and - detecting the antigen-antibody-complex formed, which is then indicative of the presence of LAV antibodies in the body fluid.

15. A diagnostic kit for the in vitro detection of antibodies against the viruses of claims 1 or 2 or viruses related therewith, which comprises an isolated or synthetic peptide according to any one of claims 8 to 12 and a reagent for detecting the formation of peptide/antibody complex.

16. An immunogenic composition comprising an antigen of the viruses of claim 1 or 2, or both or of any immunogenic peptide encoded by the RNAs of said viruses or by part thereof in association with a suitable pharmaceutically or physiologically acceptable carrier.

17. An immunogenic composition according to claim 16 wherein said peptide is the gp110 envelope glycoprotein or part thereof of the LAV ELI and LAV MAL
viruses shown in figures 7A-7J and 8A-8I respectively.

18. An immunogenic composition according to claim 16 which contains a protein or glycoprotein of the LAV ELI or LAV MAL viruses shown in figures 7A-7J and 8A-8J respectively whose aminoacid sequence includes all or part of any of the sequences which follow:
OMP or gp110 proteins, including precursors:
1 to 530 OMP or gp110 without precursor:

Sequence carrying the TMP or gp41 protein:

531-877, particularly well conserved stretches of OMP:
37-130, 211-289 and well conserved stretch found at the end of the OMP and the beginning of the TMP:

19. Cells transformed with a DNA recombinant according to claim 5.

20. A purified HIV1 variant virus, wherein said HIV1 variant virus differs genetically from the HIV1-IIIB, HIV1-BRU and HIV1-ARV2 viruses in such a way that its amino acid sequences have respectively greater than 3.4% amino acid substitution in Gag, greater than 3.1% amino acid substitution in Pol and greater than 13.0% amino acid substitution in Env with respect to each of the homologous sequences of the viruses consisting of HIV1-IIIB, HIV1-BRU and HIV1-ARV2, and wherein antibodies in AIDS patients sera bind to Gag, Pol and Env polypeptides of said HIV1-variant virus, and wherein the genetic structure comprises 5'-gag-pol-central region-env-F-3' genes.

21. A purified HIV1 variant virus, wherein said HIV1 variant virus differs genetically from the HIV1-IIIB, and HIV1-ARV2 viruses in such a way that its amino acid sequences have respectively greater than 3.4% amino acid substitution in Gag, greater than 3.1% amino acid substitution in Pol and greater than 13.0% amino acid substitution in Env and differs genetically from HIV1-BRU by at least 3.4% amino acid substitution in Gag, at least 3.1% amino acid substitution in Pol, and at least 20.7% amino acid substitution in Env with respect to each of the homologous sequences of the viruses consisting of HIV-IIIB, HIV1-BRU and HIV1-ARV2, and wherein antibodies in AIDS patient sera bind to Gag, Pol or Env polypeptides of said HIV1-variant virus, and wherein the genetic structure comprises 5'-gag-pol-central region-env-F-3' genes.