ES2210244T3 - RETROVIRUS HIV-3 strains and their use. - Google Patents

Abstract

NEW RETROVIRUS HIV-3 LINES DESCRIBED IN THEIR NUCLEIC ACID SEQUENCE CONTAIN A R REGION AND A U3 REGION THAT SHOW A HOMOLOGY OF MORE THAN 70% WITH THE NUCLEIC ACID SEQUENCE AS REPRESENTED IN SEQ ID NO 3 AND ENTEND THAT SUCH RETROVIRUS LINES HAVE AT LEAST A NUCLEOTID DIFFERENCE IN THEIR SEQUENCE COMPARED TO THE RETROVIRUS HIV-3 LINE (ANT 70) DEPOSITED UNDER ECACC N * V88060301. IN ADDITION ANTIGENS OBTAINED FROM VIRUS, PARTICULARLY PROTEINS P12, P16, P25 AND GLICOPROTEINS GP41 AND GP120, ARE DESCRIBED FOR USE IN THE ARC DIAGNOSIS OR AIDS CAUSED BY HIV-3. IMMUNOGENIC COMPOSITIONS TO BE USED AS VACCINES CONTAIN AN HIV-3 GLICOPROTEIN COVER AS GP41 OR GP120.

Description

HIV-3 retrovirus strains and their use.

The present invention relates to the field of human immunodeficiency virus.

More particularly, the present invention is refers to new variants of the retrovirus strain HIV-3 deposited in the European Collection of Animal  Cell Cultures (European Animal Cell Culture Collection, ECACC, with No. V880 60301 and the others variants characterized in the claims.

Substantial progress has been made in our understanding of acquired immunodeficiency syndrome or AIDS. It has been shown that the main agent that causes it is a non-transforming retrovirus with a tropism for T4 lymphocytes auxiliaries / inductors (1, 2) and it has been estimated that they have already been Infected millions of people worldwide. Infection with this virus drives at least a significant percentage of cases, to a progressive decrease of T4 lymphocytes with a increased concomitant susceptibility to infections opportunists that are characteristic of the disease.

Epidemiological studies indicate that the type 1 (HIV-1) of the human immunodeficiency virus, the etiologic agent responsible for most cases of AIDS and that is normally the most widely disseminated HIV, had probably its origins in Central Africa (3). He discovery of this virus did not necessarily imply existence of other types of human immunodeficiency virus. Without However, a second group of retroviruses associated with human immunodeficiency Type 2 was identified (HIV-2) of human immunodeficiency virus in West Africa (4, 5). A virus is described HIV-2 in EPO-85/00548. An HIV-1 virus is described in the document EPO-0 239 425. Other retroviruses have been isolated similar, but not identical, of apes (virus Apes immunodeficiency, SIV), such as green monkeys Africans (6, 7) and macaques (8, 9). It has been seen that Apes isolates are more genetically related to HIV-2 than with HIV-1, but they are, without However, different (10).

A characteristic of human immunodeficiency viruses that complicates their comparison is their genetic variability, genetic variants occur spontaneously and with high frequency. A comparison of the different HIV-1 isolates revealed that some regions of the genome are very variable, while others are reasonably well preserved (11-16). Similar polymorphisms have been observed in HIV-2 (17). The regions with the greatest genetic stability are presumably the regions that encode regions of viral proteins that are structurally and enzymatically essential. The viral genes with the greatest overall genetic stability are the gag and pol genes, while other regions of the env gene and the genes encoding regulatory proteins, such as art , tat , sor and 3'orf, exhibit a high degree of variability. Some of the main structural characteristics of gag and pol gene products are apparently shared, not only by all variants of a particular type of HIV, but have been conserved among virus types, at least to some extent. The antiserum produced against HIV-1 reacts cross-linking with the products of the gag and pol genes of HIV-2, although with a lower affinity than for the corresponding HIV-1 gene products. However, despite the demonstrable immunological cross-reaction, at the nucleic acid level there is a small sequence homology and no significant hybridization can be detected between these two viruses, except under very strict conditions (17).

A greater degree of kinship between SIVagm (STLV-III agm, almost or completely identical to type 4 of human lymphotropic virus (15)) and HIV-2 can be demonstrated. The immunological cross-reaction is not only limited to the products of gag and pol genes, but also extends to the products of the env gene. However, genomic analysis of SIVagm and HIV-2 showed that they were genetically distinguishable (19). DNA probes specific for HIV-2, although capable of hybridizing with SIVagm sequences, preferentially hybridize with HIV-2 (18).

We report in EP-A 0 345 375 the isolation and partial characterization of a new human immunodeficiency virus from a Cameroonian woman and her partner. Geographically, the virus comes from a region of Africa located between West Africa, where HIV-2 is endemic, and from East Central Africa, where HIV-1 is endemic. It is immunologically seen that this isolation is more antigenically related to HIV-1, than to HIV-2, since an analysis of the products of partial fragmentation, obtained by chemical fragmentation of the products of the gag and pol genes demonstrates that This isolation is neither HIV-1 nor HIV-2. This new isolation could represent an evolutionary link between HIV-1 and HIV-2. This new virus will be referred to hereinafter as HIV-3.

Therefore, the invention relates to variants of the HIV-3 virus deposited in the ECACC with the nº V88060301.

Virus isolation was performed from the blood of an asymptomatic Cameroonian woman, who is the couple of an HIV seropositive man with generalized lymphadenopathy. He The woman's serum was moderately positive (O.D./cut ratio 4.5) in the test of immunoabsorbent substances bound to enzymes (EIA, Organon Teknika) and had a low titer (1/40) in the immunofluorescent antibody assay against HIV.1, but gave ambiguous results in the immunoblot test HIV-1 Western with clear bands on p33, P53 / 55 and p64, but very weak bands in p24, gp41 and gp120. Virus was isolated  by co-culture of women's lymphocytes with PHA stimulated lymphocytes from healthy donors not infected in a medium consisting of RPMI 1640 buffered with 20 mM HEPES (hydroxyethylpiperazine ethanesulfonate) and supplemented with 15% fetal calf serum, 5 µg / ml of hydrocortisone, 75 u / ml interleukin-2 (IL-2) and 2 µg / ml of polybrene.

After 52 days of culture, the virus in the culture due to the presence of syncytia and based in the positive immunofluorescence observed when an laboratory reference anti-HIV antiserum with Acetone-fixed cells from culture. It was detected also the presence of reverse transcriptase in the supernatant of the culture (10 4 cpm / ml, 27 x depth). The culture cell-free supernatant to transfer the virus to newly obtained lymphocytes. After 15 days, they were observed of new cytopathic effects and reverse transcriptase was detected in the supernatant The virus continued to spread in lymphocytes stimulated with PHA from donors with healthy blood and transferred to continuous cell lines of leukemic origin. He tested in the supernatant containing the viruses in parallel with crop supernatants that were known to contain HIV-1 by the differential capture assay of antigens, described in detail below. The results of this comparison indicated that the new isolation was not HIV-1

Then the new virus was characterized with regarding its protein antigens and its nucleic acids. The Cell lines used to spread the virus can be, depending on the case, lines of the types CEM, HUT, Molt-4 or MT4; or any other cell line immortalized to carry the T4 receiver on its surface mobile.

A preferred cell line for propagation Continuous HIV-3 is Molt-4. He deposited Molt-4 cells infected with HIV-3 at ECACC on June 3, 1988 with the number V88060301. The establishment of a cell line chronically infected can, for example, be carried out as follow:

The cells are co-cultured Mol-4 (10 6 / ml) and preferably the cells Molt-4 of clone 8 (obtained from N. Yamamoto, Yamaguchi, Japan) with infected human lymphocytes (10 6 / ml) in RPMI 1640 culture medium buffered with 20 mM HEPES and containing 10% fetal calf serum. In one or two weeks, it observes a cytopathic effect on the culture, which follows the cell death A fraction of the cells in the culture survive the infection and produces virus continuously. With the crop Continuously, these cells increase in number and can be transferred. The supernatants of these cells can be used as a virus source

In addition, the invention relates to a retrovirus purified that has morphological and immunological properties Essentials described below. In many cases, the unique features of HIV-3 variants of the present invention can be better appreciated by comparison with the same type of characteristics that refer to the others human immunodeficiency virus, HIV-1 and HIV-2

1. Morphology

MT4 cell electron microscopy infected with HIV-3 revealed the presence of extracellular viral particles, which have a diameter of approximately 120 nm and consisting of an outer shell, which surrounds an elongated inner core, which has a diameter of approximately 20 to 40 nm and that appears in some thin sections which is slightly cone-shaped, in contrast to the appearance more or less cylindrical of the core of HIV-1. Without However, HIV-3 is morphologically very similar to HIV-1 and HIV-2, but it is distinguished easily from the other human retroviruses, such as HTLV-I and HTLV-II.

2. Protein and glycoprotein antigens

The virus present in the culture supernatant of Mol-4 cells infected with HIV-3 was concentrated by precipitation with polyethylene glycol (mean molecular weight 6000) followed by centrifugation. The resulting particle was resuspended in phosphate buffered saline solution, covered on top with a 20% sucrose pad and re-sedimented at 100,000 g for 1.5 hours. The particle virus was then dissociated in 62.5 mM Tris, pH 6.7, containing 2% 2-mercaptoethanol, 1% sodium dodecyl sulfate and 10% glycerol and the main viral antigens were separated by electrophoresis on a polyacrylamide gel (12.5%) under denaturing conditions. Molecular weight markers were included in the same gel, to provide a basis for estimating molecular weights. Once separated, the molecules were transferred by electrophoresis to nitrocellulose paper (Western blot), which was then incubated with an antiserum derived from a person infected with HIV. In the initial experiments, a high titre antiserum from an individual who was infected with HIV-1 and who had previously been seen to cross-react with HIV-2 gag and pol derived proteins was used. In this way, the molecular weights of the gag and pol gene products could be compared with those of HIV-1 and HIV-2.

Protein immunoblotting revealed that HIV-3, same as HIV-1 and HIV-2 has three nuclear proteins. If of HIV-3, it was found that these proteins had molecular weights of approximately 12,000, 16,500 and 25,000 respectively. Conventionally, it is done frequently reference to proteins by a "p" per protein or "gp" by glycoprotein, followed by a number, which, when multiply by 1,000, give the approximate molecular weight of polypeptide. Hereinafter referred to as the three largest proteins nuclear weapons of HIV-3 p12, p16, and p25, respectively.

Weights values are expected molecular, as determined, are correct and do not exceed the 10% of true values. However, there is a lot of confusion with respect to the molecular weight values of proteins, since the structure of electrophoresis devices used and the origin of the components of the buffers varies from Laboratory to laboratory Therefore, it is necessary when compare the apparent molecular weights of protein antigens of HIV-3 with respect to those of HIV-1 or HIV-2, submitting to All samples to electrophoresis on the same gel. It can see such gel, for example, in Fig. 5. In particular, it is evident that while in the case of the major nuclear protein, they are very close the values of molecular weights of proteins homologous of the three HIVs, the protein derived from HIV-1 is the smallest. The major nuclear protein of HIV-2 is somewhat larger than that of HIV-1, as previously reported. The HIV-3 homologous protein is slightly more larger than the nuclear protein greater than HIV-2. He give in Table 1 the calculated molecular weight of these proteins

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one

Similarly, the molecular weight differences between the three HIVs with respect to second nuclear protein, which has, in most strains of HIV-1, a molecular weight of 18,000. However, it have documented differences between strains in molecular weight of this protein, in the case of HIV-1, and the weight molecular of this protein can be 17,000 in some insulations. The homologous HIV-2 protein has a molecular weight of approximately 16,000 while the protein of HIV-3 has an intermediate molecular weight of approximately 16,500.

By analogy with HIV-1 and HIV-2, HIV-3 also has two virally encoded reverse transcriptase enzyme forms. These two types also differ slightly in the molecular weight of the corresponding types of HIV-1 and of HIV-2 and are characteristic of HIV-3 These weights are also summarized in Table 1 molecular.

HIV-3 has an additional polypeptide derived from the pol gene, which is an endonuclease with an apparent molecular weight of 31,000 and does not differ significantly in the molecular weight of homologous HIV-1 and HIV-2 proteins.

When protein immunoblotting, containing HIV-3 proteins, is incubated with serum obtained from an individual infected with this virus, two additional proteins can be seen. These proteins are derived from the env gene and are the viral envelope glycoproteins. The smallest protein, which is the transmembrane protein, migrates in the form of a broad band with an apparent molecular weight between 40,000 and 45,000. This protein will be referred to hereafter as gp41 with the understanding that the protein exhibits some intrinsic heterogeneity with respect to its apparent molecular weight and its migration on polyacrylamide gels. The largest protein, which is the outer membrane protein, is similarly somewhat diffuse on polyacrylamide gels and has a molecular weight of approximately 120,000. This protein is referred to hereafter as gp120. It should be noted that the apparent heterogeneous migration of these two types on the polyacrylamide gel is not due to heterogeneity in the polypeptide chain, but rather to post-translational glycosylation. In particular, gp120 is very glycosylated and the apparent molecular weight observed is influenced to some extent by the cell line used to produce the virus.

In addition to Western blotting, protein antigens can also be visualized by radioimmunoprecipitation assay (RIPA). For this purpose, viral proteins can be radioactively labeled in vivo metabolically, by culturing HIV-3 infected cells in the presence of 35S-cysteine and 35S-methionine (200 µCi / ml) in RPMI 1640 medium devoid of these two amino acids and supplemented with fetal dialyzed calf serum. After 16 hours, the labeled virus from the culture supernatant is collected by centrifugation in a 20% sucrose pad at 100,000 g for 1.5 hours. The resulting sedimented virus is then resuspended in RIPA buffer (20 mM triethanolamine, pH 8.00, 0.5 M NaCl, 0.5% Nonidet P40, 0.1% sodium deoxycholate, and phenylmethylsulfonyl fluoride 1 mM).

Alternatively, viruses can be marked radioactively with 125 I, using chloramine T, by means of Family technique for subject matter experts. In this case, it purifies the virus from the supernatant of infected cells by sedimentation of the virus through a sucrose pad at 20%, re-suspending the virus in buffered saline and obtaining its band pattern by ultracentrifugation in a 20 to 50% sucrose gradient at 60,000 g for 12 hours. He you can locate the virus in bands in the fractionated gradient, or by the reverse transcriptase assay, or by the assay of antigen capture. Fractions containing viruses are combined and Triton X-100 is added at a concentration of 0.5%.  Then the lysate virus can be iodized with Triton X-100

For immunoprecipitations, it is done react 100,000-200,000 cpm of viral proteins labeled in RIPA buffer with 5 microliters of a test serum in a volume of 200 microliters for 16 hours at 4 ° C. Then the resulting immune complexes bind Protein A-Sepharose (Pharmacia), washed extensively, and bound proteins are eluted with a sample buffer of electrophoresis containing 1% SDS. The antigens are analyzed subsequently by electrophoresis followed by autoradiography

Protein antigens can be characterized of HIV-3 with respect to those of HIV-1 and HIV-2 using two different but related approaches. On the one hand, you can characterize the antigens based on their ability to react in crossbreeding with antisera from people infected with HIV-1 and HIV-2. On the other hand, it they can use the antisera of people infected with HIV-3, which contain antibodies produced in response to HIV-3 antigens, to test cross-reactivity to HIV-1 proteins and HIV-2 Relationships are illustrated substantially antigens between HIV-3, and HIV-1 and HIV-2, in the examples given below.

The results of these experiments indicate that HIV-3 is only closely related to HIV-2, since cross-reactivity is observed only with respect to viral nuclear proteins and pol gene products. No cross-reactivity of the env gene products could be observed when anti-HIV-2 antiserum was incubated with HIV-3 proteins or when anti-HIV-3 antiserum was incubated with HIV-2 proteins.

In contrast to this, HIV-3 is more closely related to HIV-1, since the anti-HIV-3 antiserum reacts cross-breeding not only with the products of the gag and pol genes of HIV-1, but also to a certain extent. point with the products of the gene env gp41 and gp120, although with a lower affinity. Similarly, it reacts cross-linking with all HIV-3 protein antigens, but with a lower affinity than with HIV-1 proteins.

In the examples that follow, it is shown that HIV-3 is substantially different, from the point of antigenic view of HIV-1 on the basis of 1.) a different model of reactivity with antiserum anti-HIV-1 than what is observed for HIV-1, 2.) a drastically reduced capacity if recognized by mouse monoclonal antibodies produced against HIV-1 p24 and p17 nuclear proteins, and 3.) preferential recognition of proteins from HIV-3, including cover proteins, on HIV-1 proteins by antisera from individuals infected with HIV-3.

Despite the characteristic genetic variation of human immunodeficiency viruses, a trial that is based, for example, in HIV-1 proteins derived from a particular strain will work satisfactorily to determine antibodies produced in response to other variants of HIV-1 This can also be seen in particular in the example in which monoclonal antibodies were tested about its ability to react with antigens derived from isolates of HIV-1, HIV-2 and HIV-3 In this case, antibodies were produced monoclonal against the nuclear proteins of HIV-IIIB strain 1, and reacted still very intensely with derived proteins of HIV-1 strain SF4. In contrast to this, these same monoclonal antibodies react only weakly or not react at all with the nuclear proteins of HIV-3 This indicates that antigenic differences between HIV-1 and HIV-3 are of such magnitude, that immunological tests based on the use of HIV-1 proteins will not be appropriate to assay sera from individuals infected with HIV-3.

Finally, in the examples given below, differences in the number and / or positions of methionine and tryptophan residues have been seen in the more conserved products of the gag and pol genes.

3. HIV-3 nucleic acids A. HIV-3 viral RNA

When the RNA of the HIV-3 on a Hybond-H filter (Amersham) according to the "punctual immunoblot" technique, did not hybridize with HIV-1 DNA under conditions strict hybridization.

By "strict conditions" or "conditions not strict "means the conditions under which performs the real hybridization and / or subsequent stages of washed.

Hybridizations of punctual immunoblotting by marking dilutions of the strain SF4 of HIV-1, of HIV-2 rod, of strain ANT 70 of HIV-3 and strain ANT 70 NA of HIV-3 on Hybond-H filters.

The dilution series for each virus corresponded to the viral RNA sedimented from the equivalent of 5, 2.5, 1.25 and 0.62 milliliters of culture supernatant. RNA was fixed on the filter by UV irradiation for 2 min, and subjected to hybridization by putting the filter in contact with a 32P-labeled DNA probe. The probe chosen was derived from the HIV-1 sequence that encompassed nucleotides 487-4652 (sac I-Eco R1) and includes a portion of the long terminal repeat of the 5 'end, the entire gag region and most of the pol gene, subcloned into the pUC 13 vector. Hybridization of the 32P labeled probe was performed with the filter under strict conditions in 3 X SSC, 0.5% milk powder, 1% SDS, 10 dextran sulfate %, formed at 50% (volume / volume) at 42 ° C for 18 hours (1 X SSC corresponds to 0.15 M NaCl, 0.015 M sodium citrate). Subsequent washing steps were performed under strict conditions in 0.1 X SSC and 0.1% SDS at 65 ° C (washings of 2-30 minutes). The filter was then dried and autoradiographed with intensifying screens at -70 ° C. After autoradiography, only spots corresponding to HIV-1 viral RNA were visible. No hybridization was observed with HIV-2 or with either of the two strains of HIV-3. Therefore, it seems that HIV-3 is distantly related to HIV-1.

B. cDNA and subclones of this cDNA derived from HIV-3

The conditions under the conditions are described below. which was synthesized the cDNA corresponding to the sequences of HIV-3 and was cloned. HIV-3 was precipitated  (strain ANT 70) from 1 liter of culture with polyethylene glycol 6000, was dissolved again in buffered saline solution with phosphate, and sediment through a sucrose pad at twenty%. The resulting virus pellet was dissolved dissolved in 6 M guanidinium chloride in 20 mM dithiothreitol and Nonidet 0.5% P-40, CsCl was added at a concentration 2 molar and the solution containing fragmented virus was placed on a 1.2 milliliter pad of 5.7 M CsCl containing EDTA 0.1 M. The viral RNA was precipitated by centrifugation for 20 hours at 25,000 rpm in a Beckman SW28 centrifuge at 15 ° C. He dissolved the precipitated RNA again, extracted with phenol and precipitated with ethanol and 2M LiCl.

A fifth of the viral RNA was used, prepared as as described above, to direct the first phase in the cDNA synthesis, which employed a oligonucleotide primer (dT) that served to launch the synthesis of the first chain of cDNA

A commercially available kit supplied by Amersham was used for the preparation of HIV-3 cDNA and an exogenously added reverse transcriptase was used to synthesize the first chain. The synthesis of the second chain was performed using E. coli DNA polymerase I in the presence of RNAase H to digest the RNA chain of the RNA / DNA hybrid by digestion.

Synthesis of the second chain was performed in the presence of 32P-dCTP to label the cDNA. The resulting cDNA was treated with T4 DAN polymerase to create blunt ends, the cDNA was mixed to protect possible EcoRI internal fragmentation sites, and then joined the EcoRI bonds, also supplied by Amersham. The EcoRI restriction sites were then broken and the cDNA was chopped on a 1.2% agarose gel. The region of the gel corresponding to a cDNA length of 500 to 2000 base pairs was cut and the cDNA eluted and cloned into the pUC13 vector that had been fragmented with EcoRI and dephosphorylated. The DNA was then used to transform competent cells of E. coli MC1016 (lambda). The resulting colonies were transferred to Pall membranes (Pall Biodyne), lysed and denatured with 1.5 M NaCl, 0.5 M NaOH and neutralized with 3 M NaOAc, pH 5.5. Investigation of colonies that had an HIV-3 insert was carried out under moderately stringent conditions in a buffer containing 5 X SSC, 5 X Denhardt solution, 0.2% SDS, 250 mg / ml DNA of Denatured salmon sperm was left overnight at 65 ° C, using 32P-labeled plasmid containing the Sacl-EcoRI fragment of HIV-1 discussed above. After hybridization, the filters were washed as follows:

1.

1 hour in 2 X SSC, 0.1% SDS at room temperature.

two.

30 minutes in 0.1 X SSC, 0.1% SDS at room temperature.

3.

20 minutes in 2 X SSC, 0.1% SDS at 42 ° C.

Four.

20 minutes in 0.1 X SSC, 0.1% SDS at 65 ° C.

After the autoradiography of the filter, several weakly positive colonies were identified, which were grown for analysis, the positive signal was expected, or was due to weak homology with the gag or pol regions of HIV-1, or was due to some sequence homology with the R region of the LTR.

C. Sequences contained in the cDNA of the HIV-3

For sequence analysis, a clone that carried the largest insertion, which was found to be of approximately 1600 base pairs in length and at which called iso 70-11. Several subclones were prepared of the insertion by digestion of the insertion with different restriction enzymes and subcloned of the resulting fragments in the pUC 13 vector. Sequence determinations were performed according to the dideoxy method, described by Sanger, (Proc. Natl. Acad. Sci. USA 74: 5463-5467, 1977) using a kit provided by Boehringer, which uses M13 primers of 17 units. Sequence analysis of the iso cDNA clone 70-11 revealed that the insertion corresponded to 3 'end of the viral genome, which possessed a poly (A) chain at the 3 'end.

The HIV-3 retrovirus contains a LTR 3 ', which is composed of a U3 region, as well as an R region. Similar to the 3 'LTR region of HIV-1, the clone iso 70-11 contains a polyadenylation signal AATAAA located approximately 23 nucleotides from the 3 'end of the R region. The sequence analysis of the HIV-3 revealed approximately 70% homology with the corresponding 3 'LTR sequence of HIV-1 and less than 55% homology with the corresponding sequence of HIV-2

The retrovirus as deposited in the ECACC with the number V88060301 contains in its env gene a sequence that corresponds to the following nucleotide sequence:

two

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HIV-3 sequence exhibits less than 60% homology with the corresponding sequence in the HIV-1 and in HIV-2. The sequence is derives from a part of the HIV-3 genome that encodes the transmembrane protein of the shell.

It was found that the sequences contained in the RNA of the new virus do not hybridize with either the HIV-1 env gene, nor with the sequences contained in the LTR region of the HIV-1 genome. The Sal I-BgI II restriction fragment, which contains sequences located in the HIV-3 env gene, of the iso 70-11 clone DNA was cut, labeled with 32P and hybridized under strict conditions with a filter containing DNA genomic isolate of cells infected with HIV-1 (strain SF2) and HIV-3 (strain ANT 70). Hybridization was observed only with DNA corresponding to cells infected with HIV-3. Similarly, when the entire iso 70-11 plasmid was labeled and used as a probe under strict hybridization conditions, the only hybridization detected was with DNA from cells infected with HIV-3. This demonstrates that HIV-3 is related, only distantly, with HIV-1 and that the iso 70-11 clone is suitable for use as a hybridization probe to distinguish HIV-3 from the other two known viruses of human immunodeficiency

Conversely, hybridization using HIV-1 gag - pol sequences as the labeled probe to detect cross-hybridization with HIV-3 RNA did not reveal detectable hybridization when hybridization was performed under strict conditions. This again indicates that viruses are only related in a distant way and that a distinction can be made between HIV-1 and HIV-3 at the level of nucleic acids in the region of the genome comprising the gag and pol genes. However, this same labeled probe hybridized with RNA derived from strain SF4 of HIV-1.

In addition, the invention relates to a composition comprising at least one antigen, in particular a protein or glycoprotein of HIV-3 retrovirus variants of the present invention. You can use that composition in the methods to detect antibodies and in kits to perform such methods

It has been shown that the virus HIV-3 is usable as a source of antigens to detect antibodies in people who have been in contact with  HIV-3 As such, viruses can grow and concentrate by the methods already described and prepare a lysate treating the virus with an appropriate detergent. A detergent Preferred to prepare a total viral lysate is Triton X-100, used at a concentration of 0.5%. Other Preferred detergent is Nonidet P-40 (NP-40), also used at a concentration of 0.5%

Alternatively, the viral protein can be purified from the lysate of the virus. A preferred method to purify these proteins is affinity chromatography. For example, viral antigens can be separated on a preparative polyacrylamide gel and the individual antigens eluted in purified form. These can continue to be used to produce antisera in, for example, rabbits, which is specific for individual viral proteins. The IgG fraction derived from the serum of the immune rabbit can be bound to a solid phase, such as Sepharose 48 activated with CNBr (Pharmacia) and used to selectively remove individual viral antigens from individual lysates. Then, these proteins can be eluted from the affinity support using a low pH buffer and further purified using standard chromatography techniques, of which an example is given in Montelaro et al. , J. of Virology (1982) 42: 1029-1030.

The invention generally relates to any composition that can be used for the diagnosis of HIV-3 infection or for trials that have a forecast value.

The invention also relates to any composition, in which any viral lysate of the HIV-3, in combination with proteins prepared from similarly derived from HIV-1 and / or HIV-2, for the general diagnosis of infection or contact with human immunodeficiency virus, without considering absolute identity of the virus that is being detected. For example, such compositions could consist of a mixture of lysates of the HIV-1, HIV-2 and HIV-3

The present invention also relates to labeled extracts of the variant of HIV-3 or a compositions, as previously described. The market It can be of any type, such as enzymatic, chemical, fluorescent or radioactive.

In addition, the invention relates to a kit for anti-HIV-3 antibody detection in biological fluids, which comprises a lysate of HIV-3 or a composition, as described above and a means to detect immune complexes formed.

In the case of kits designed to detect specific antibodies by immunoenzymatic methods, a kit of this type would include:

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A lysate of HIV-3 or composition of one of the types already described, preferably in purified form, and attached preferably to a solid support, such as a plate of microtiter

-

A conjugate between an enzyme and an immunoglobulin fraction, which is capable of bind to the antibodies to be detected, or a conjugate between  an enzyme and bacterial protein A or protein G.

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An antigen of control, which does not have epitopes that are shared with another virus human immunodeficiency

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Appropriate buffers To perform the test.

-

A substrate appropriate for the enzyme.

The kits for antibody detection Specific ones that use labeled antigens would include:

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An antigen properly labeled or combination of antigens of the types already described.

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Protein A or united human anti-human immunoglobulins preferably to an insoluble support, such as Protein A-Sepharose 4B (Pharmacia) or a support equivalent.

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Control antigen, that is not recognized by antisera anti-HIV-3.

-

Appropriate buffers to perform the test,

-

If appropriate, substrates for detection of labeled antigen enzymatically

The invention also relates to acids nuclei, optionally labeled, which are derived in part from the less, of RNA from an HIV-3 retrovirus or variants of this virus.

Similarly, the invention also relates to the use of cDNA or parts of the cDNA or of the recombinants that contain them, which are characterized by containing at least a part of the cDNA corresponding to the entire genomic RNA of the HIV-3 retrovirus. Such cDNAs can be used as probes for the specific detection of HIV-3 sequences in biological fluids, tissues and cells. The probes, preferably, are also labeled, either radioactively or chemically, alternatively enzymatic, fluorescent or chemiluminescent markers are used, which allow the probes to be detected. Preferred probes for the specific detection of HIV-3 and diagnosis of HIV-3 infection are probes containing all or a part of the cDNA complementary to the HIV-3 genome. In this context, a particularly advantageous probe can be characterized as one that contains, in particular, the nucleic acid sequence contained in the iso 70-11 clone and that includes the LTR-R viral sequence, which is located at both ends. 'and 3' of the viral genomic RNA and at the two 5 'and 3' ends of the integrated proviral DNA. In addition, sequences corresponding to the viral env gene and which are also included in the iso 70-11 clone are also particularly advantageous.

It is understood, however, that the probes that can be used for the diagnosis of HIV-3 infection are in no way limited to the probes described above, and that the invention incorporates all sequences originating from the HIV-3 genome. or of its natural variants and includes sequences that encode viral nuclear proteins ( gag gene), the two forms of reverse transcriptase and endonuclease ( pol gene), as well as the two viral envelope glycoproteins ( env gene).

The invention also relates to sequences of HIV-3 nucleic acid, which have been incorporated into a recombinant nucleic acid, which comprises a nucleic acid coming from a vector, and having said cDNA or part of said cDNA inserted there. Such construction could be used to replicate the viral cDNA or its fragments in an organism or cell different from the natural host, in such a way that it provides sufficient quantities of the probe to be used for purposes Diagnostics

A probe generated from such can be used way in a diagnostic test for specific detection of HIV-3 incorporating the following stages essential:

-

Probe marking generated as described above, by the methods described above.

-

Put the probe in contact, under strict hybridization conditions, with DNA from the infected cells or viral RNA from infected cells or fluids biological, once said DNA or RNA has been applied preferably to a membrane and has been made accessible to the probe.

-

Washing the membrane with a buffer in circumstances in which they are maintained strict conditions.

-

Detection of the labeled probe, preferably by autoradiography, in the cases in which the probe has been radioactively marked, or by a appropriate immunodetection technique, in case the probe is has chemically marked.

The invention also relates to a procedure for the production of variant retrovirus HIV-3, characterized by T4 lymphocyte culture human or human lymphocytic cell lines of origin leukemia, which carry the T4 + phenotype, with lymphocytes or lines cell phones that have previously been infected with an isolation of HIV-3 retrovirus, as well as recovery and purification of the retrovirus from the culture medium.

Brief description of the figures

In Figures 1 to 16, the designations HIV-3 (ANT 70) and HIV-3 (ANT 70 NA) they refer to two strains of a new HIV-3 virus isolated from a Cameroonian woman and her partner, of which HIV-3 (ANT 70) has been deposited in the ECACC with the number V88060301.

Figure 1 shows a procedure for Prepare fragmentation maps of viral proteins.

Figure 2 shows the differential capture of antigens in culture supernatants containing viruses. He performed differential antigen capture as described in the text. The continuous line represents the results obtained using an anti-HIV-1 IgG of broad spectrum, while the dashed line represents the results obtained using an IgG, which was quite HIV-1 specific. The qualifications that are shown in panels A-E are typical for HIV-1 Panel F shows the result obtained with HIV-3 (ANT 70).

Figure 3 shows differential capture of antigens in HIV-1 supernatants and HIV-3 (ANT 70 NA). Differential capture was performed of antigens as described in the text. The line continues represents the results obtained on plates coated with IgG broad-spectrum anti-HIV-1, while the dashed line represents the results obtained on plates coated with IgG, which shows less cross reactivity with HIV types other than HIV-1.

Figure 4 shows the reactivity of antisera anti-HIV in Western blotting strips of HIV-1 and HIV-2. The reactivities of 3 different sera on strips of Western blotting of HIV-1 and HIV-2 Serums: 1. anti-HIV, 2. anti-HIV-3 (ANT 70), 3. anti-HIV-2 (isolation 53). The Molecular weights indicated are those given by the manufacturer (Dupont Biotech)

Figure 5 refers to the comparison of gag and pol proteins of various isolates of HIV-1, HIV-2rod and HIV-3 (ANT 70). The proteins were separated by electrophoresis and immunoblotted as described. Immunoblotting was incubated with a broad-spectrum anti-HIV antiserum, followed by alkaline phosphatase-labeled conjugate / (anti-human IgG) to visualize the proteins.

A. HIV-2rod, B. an isolation of HIV-1 laboratory, C. HIV-3 (ANT 70), D. a laboratory isolate of HIV-1, E. HIV-1 (SF4).

Figure 6 shows a comparison of HIV-3 (ANT 70) and HIV-3 proteins (ANT 70 NA). The proteins were separated by electrophoresis and immunoblotted as described. The immunoblot with the BSR antiserum, followed by conjugate alkaline phosphatase: anti-human IgG, for Visualize proteins. Zone 1: HIV-3 (ANT 70 NA), Zone 2: HIV-3 (ANT 70), Zone 3: HIV-1 (SF4). The apparent intensity difference between zones 1 and 2 is caused by the difference in quantity of loaded material.

Figure 7 refers to the ability to different anti-HIV-1 sera humans to capture viral antigens. Several sera were diluted 1: 1000 humans and coated directly on plates of microwells Supernatants from cultures treated with detergent containing HIV-1 (SF4), HIV-3 (ANT 70), HIV-2rod or HIV-2 (isolation 53) and the antigen was detected bound using a conjugate (anti-HIV) / peroxidase of broad spectrum horseradish. Sera 1-7 were of African origin, while the sera 8-11 were from Europeans. It can be explained, in part, the greater ability of African sera to capture antigen not of HIV, for its highest anti-p24 titres (data not revealed).

Figure 8 shows the effect of dilution of Coating IgG on the binding of HIV isolates. He they made double successive dilutions of four different sera, starting with a dilution of 1: 1000 and were used to coat microwell plates. Supernatants treated with HIV-1 detergent (SF4), HIV-3 (ANT 70), HIV-2rod and HIV-2 (isolation 53) to give approximately the same optical density on plates coated with the antiserum shown in panel B with a dilution of 1: 1000. Bound antigen was detected using conjugated (anti-HIV) / horseradish peroxidase  spread spectrum.

Figure 9 shows the capture of antigens from virus isolates using human polyclonal antibodies and anti-HIV-1 monoclonal antibodies mouse

The wells were coated and incubated such As described in the text. The IgGs used are the following:

1. Polyclonal anti-HIV IgG human, 2. Monoclonal antibody CLB 59, 3. Monoclonal antibody CLB 21, 4. Monoclonal antibody CLB 64, 5. Monoclonal antibody CLB 14, 6. Monoclonal antibody CLB 16, 7. Monoclonal antibody 47, 8. CLB 13.6 monoclonal antibody (anti-p18), 9. CLB monoclonal antibody 19,7, 10. CLB monoclonal antibody 13.4 (anti-p18).

Figure 10 is a comparison of reactivity of human anti-HIV antisera with different types of HIV

Lysate electrophoresis were separated from HIV-1 (SF4), HIV-3 (ANT 70), strain of HIV-2 and HIV-2 (isolation 53) on SDS-polyacrylamide gels, it immunoblotted onto nitrocellulose, and incubated with a high anti-HIV-1 antiserum title (panel A), an antiserum lower-grade anti-HIV-1 (panel B), serum of the woman from which the HIV-3 (ANT 70) (panel C), of your partner of which isolated HIV-3 (ANT 70 NA) (panel D) and antiserum anti-HIV-2 of the person from which HIV-2 (isolation 53) was isolated (panel E).

Figure 11 shows the titrations of sera anti-HIV by enzyme immunoassay.

Microwell plates were coated with lysates of HIV-1 (SF4), ANT 70 and HIV-2 (isolation 53). They were titled serum from a European infected with HIV-1 (panel left), antiserum with respect to HIV-3 (ANT 70 NA) (central panel) and antiserum with respect to HIV-2 (isolation 53) (right panel) in double dilutions starting with a 1: 100 dilution over all Three coated layers.

Figure 12 shows the positions of the methionine and tryptophan residues in the products of the gag and pol viral genes. The amino acid positions for p17 gag proteins are given starting from the first methionine in the coding sequence. Positions for the p24 gag protein are given starting at the p17 / p24 proteolytic fragmentation site. Positions for the pol gene are shown after alignment with the tryptophan doublet very conserved in the HIV-1 sequence at positions 556 and 557. The positions of a conserved protease sequence, the place of protease fragmentation, are indicated. / reverse transcriptase and the reverse transcriptase / endonuclease fragmentation site. In this case, the terms p24 and p17 in the genetic sense are used to refer to the viral nuclear proteins of larger size and second in size respectively. The term "HIV-2 (LAV-2)" is a synonym for HIV-2rod.

Figure 13 is a comparison of the products of partial fragmentation of the products of the gag and pol genes of HIV-1 (SF4) (HIV-1 in the figure), HIV-3 (ANT 70) (isolation 70 in the figure ) (ANT 70), HIV-2rod (HIV-2 (LAV-2) in the figure) and HIV-2 (isolation 53) (isolation 53 in the figure). The terms p24 and p17 are used in the genetic sense to indicate the largest and second largest viral nuclear proteins respectively.

Figure 14 shows the hybridization of the probes of cDNA with viral RNA.

RNA was sprayed on a membrane filter viral from HIV-1 (SF4), HIV-2rod, and HIV-3 (ANT 70), such as It is described in the Materials and Methods section. They hybridized filters or in non-strict conditions (A) or in strict conditions and they autoradiographed.

Figure 15 shows cDNA hybridization (iso 70-11) of HIV-3 (ANT 70) with DNA genomic from uninfected and infected cells with HIV.

The genomic DNA from the cell lines listed below to enzyme fragmentation of restriction, separated by electrophoresis and immunoblotted. Hybridizations were performed as Describe in Materials and Methods. The samples were as follows:

Band DNA Digestion by enzymes of restriction TO lambda Hind III B iso 19 (HIV-1) Pst I C iso 19 (HIV-1) Pst I, EcoRI D lambda Hind III AND HIV-3 (ANT 70) Hind III F HIV-3 (ANT 70) Pst I G HIV-3 (ANT 70) Pst I, EcoRI H HIV-1 (SF4) Hind III I HIV-1 (SF4) Pst I J HIV-1 (SF4) Pst I, EcoRI K MT4 (no infected) Pst I L HUT 78 (no infected) Pst I

Panel A: Iso hybridization 70-11

Panel B: Hybridization with the iso fragment 70-11 Bgl II / Salt I.

Figure 16 refers to the location of several restriction enzyme fragmentation sites in the HIV-3 insertion contained in iso DNA 70-11

The fragment does not contain fragmentation sites internal for EcoRI, Bam HI, Hinc II, or Xba I.

Examples Materials and methods Virus and cell culture to. Virus strains and cell lines

HUT-78 cells chronically infected with ARV-4 (HIV-1 SF4), originally isolated by J. Levy, San Francisco, USA (20) and uninfected HUT-78 cells were kindly provided by S. Sprecher, Brussels, Belgium; Strain LAV-2 originally from L. Montagnier, Paris, and CEM cells were obtained from J. De Smeyter, Leuven, Belgium, isolation 53, an isolation of HIV-2 was isolated in this laboratory (21).

b. Virus isolates

Virus isolates were performed in a manner similar to that described by Levy and Shimabukuro (22), with modifications. Lymphocytes were isolated from patients and healthy heparinized whole blood donors on Lymphoprep (Nyegaard and Co., Oslo, Norway) and were grown in RPMI 1640 which contained 20 mM HEPES, 15% fetal calf serum (Gibco), 5 µg / ml hydrocortisone (Merck), 75 U / ml of IL-2 and 2 µg / ml of polybrene (Aldrich).

Lymphocytes from healthy donors were stimulated with 2 µg / ml phytohemagglutinin PHA, Wellcome) for 3 days before use. Lymphocytes stimulated with fresh PHA were added to virus isolation cultures every 3 to 4 days. Cultures were monitored for cytopathic effect, immunofluorescence, using a polyclonal reference antiserum of broad specificity (23), and the presence of antigen in culture supernatants (Innotest VCA-HIV, Innogenetics). The broad specificity reference antiserum (BRS) used was derived from an HIV-1 infected donor and was found experimentally to have an exceptionally high titer ? 1,000,000 in a protein-based enzyme immunoassay p24 of recombinant HIV-1) and which reacts strongly cross-linked with the products of the gag and pol genes of other types of HIV, in particular with HIV-2. Reverse transcriptase was also tested, essentially as described in (24).

To establish permanent cell lines, chronically infected, they were co-cultivated primary lymphocytes infected with virus with Molt 4 cells of the clone 8 (25), kindly provided by N. Yamamoto, Japan, and monitored to see cell growth. The virus production by reverse transcriptase assay, as well as the capture of antigens.

Differential capture of antigens

A test system was developed, whereby a distinction can be made between HIV-1 and others Human immunodeficiency virus related. The system is based in comparing the capacity of two polyclonal IgG preparations different, one with a broad specificity anti-HIV, which is due to its title exceptionally elevated, particularly against the largest nuclear protein, and the another with a lower title, which react preferentially with HIV-1, to capture detergent treated viruses in crop supernatants. Antigen detection is achieved captured using a conjugate (wide IgG specificity / horseradish peroxidase).

The trial detects primarily, but not exclusively, the p24 nuclear protein.

Monoclonal antibodies against HIV-1

The antibody panel has been described monoclonal used (26). Antibodies were prepared against native viral proteins in HIV-1 preparations fragmented with Triton X-100.

Protein analysis to. Electrophoresis

Viral protein electrophoresis was performed with polyacrylamide gel essentially as described by Maizel (27).

b. Protein immunoblot

The immunoblot was carried out, either in a transimmunoblot cell from Bio-Rad to 400 mA for 4 hours using the carbonate buffer described by Dunn (28), or using the immunoblotting device semi-dry KLB at 0.8 mA / cm2 for 1 hour at 48 mM Tris, 39 mM glycine, 0.0375% sodium dodecyl sulfate (SDS) and 20% methanol.

c. Generation of partial fragmentation products

Viral proteins were analyzed by technique shown in Figure 1. The fact that the corresponding proteins of the different isolates have similar molecular weights. The proteins were separated in gels from 12.5 percent SDS-polyacrylamide, along with a ARV-4 protein marker band, which was cut after electrophoresis, it was immunoblotted and incubated with an anti-HIV antiserum to reveal positions of viral proteins. The marker stain was used in turn to locate the approximate positions in the part of the stained gel with Coomassie blue of the viral proteins that are going to fragment The horizontal gel bands that were cut they contained the proteins, were transferred to glass tubes and were underwent chemical fragmentation.

1. Fragmentation with cyanogen bromide

The gel band was incubated with 10 ml of a freshly prepared solution of 40 mg / ml CNBr (Merck) in 0.3 N HCl for 3 hours at room temperature in a steam hood. After incubation, the gel band was equilibrated with buffer SDS sample for two-dimensional electrophoresis.

2. Fragmentation with BNPS-escatol

The gel band was incubated with 10 ml of a freshly prepared saturated solution of 2- (2'-nitrophenylsulfenyl) -3-methyl-3'-bromoindolinine (BNPS-eschatol, Pierce) in 70 acetic acid by percent, 30% H2O, containing 0.1% phenol, for 3 hours at room temperature, it was protected from light. After the incubation, the gel band was equilibrated by repeated washing in  SDS sample buffer for electrophoresis.

After fragmentation, the individual bands of the gel bands, turned 90 ° and placed on top of a gradient gel of SDS-polyacrylamide from 10 to 20 percent. To the finish the electrophoresis the gel was immunoblotted on nitrocellulose (Schleicher and Schuell) and was blocked with PBS that It contained 1 mg / ml casein (Merck). They could only visualize the products of fragmentation with molecular weights above 10 kD, since peptides with molecular weights Minors do not bind nitrocellulose effectively. He incubated immunoblotting with an antiserum broad-spectrum anti-HIV, followed by a conjugate of goat anti-human IgG: alkaline phosphatase (Promise) The products of the partial fragmentation by reaction with phosphate of 5-bromo-4-chloro-3-indolyl and tetrazolium nitro blue (Sigma).

Viral nucleic acids to. Hybridization with viral RNA

The virus was collected from crop supernatants by sedimentation through 20% sucrose pads by centrifugation at 26,500 rpm for 1.5 hours at 4 ° C and fragmented into 10 mM Tris, pH 7.4, 10 mM NaCl, 10 mM EDTA containing 0.5% sodium dodecyl sulfate. Aliquots of the fragmented virus were sprayed on a Hybond H (Amersham) membrane in amounts corresponding to 5, 2.5, 1.25 and 0.62 ml of crop supernatants originals The RNA deposited on the filter was fixed to the membrane by irradiation with ultraviolet light for 2 min. Next, the RNA bound to the filter was subjected to hybridization with an HIV-1 cDNA probe, which had been labeled by false translation with 32P-dCTP. It has been made Hybridization under strict conditions in 3 X SSC, milk powder 0.5%, 1% SDS, 10% dextran sulfate and 50% formamide at 42 ° C for 18 hours. After hybridization, the filter was washed twice under strict conditions in 0.1 X SSC and 0.1% SDS for 30 minutes Hybridization was detected by autoradiography at -70ºC with booster screens. Hybridization of similarly using a probe derived from the env region of the HIV-2

Hybridizations were also performed in Non-strict conditions in 5 X SSC, 25% formamide, 5 X solution of Denhardt, 10% dextran sulfate, and 100 µg / ml of DNA from denatured salmon sperm at 37 ° C overnight. The filter was then washed 4 times for 15 minutes in 5 X SSC, 0.1% SDS at room temperature and autoradiographed.

b. Preparation of ANT 70 cDNA

The virus was sedimented from 1 liter of culture supernatant using polyethylene glycol 6000, it dissolved again in PBS and sedimented through a pad 20% sucrose. The resulting blood cell was fragmented in 6 M guanidinium chloride in 20 mM sodium phosphate buffer, pH 6.5,  containing 20 mM dithiothreitol and 0.5% NP-40. He added solid CsCl at a concentration of 2 molar. The solution containing fragmented virus on a pad of 5.7 M CsCl containing 0.1 M EDTA and viral RNA was sedimented by centrifugation at 25,000 in a Beckman SW 28 centrifuge at 15 ° C for 20 hours. After centrifugation, it was again dissolve the RNA, extracted with phenol and precipitated with ethanol and LiCl 2 M.

A fifth of the prepared RNA was used to direct the first stage in cDNA synthesis using a kit provided by Amersham. CDNA synthesis was primed using oligo (dT).

The synthesis was carried out using the reverse transcriptase supplied with the kit. The synthesis of the second chain was performed using E. coli DNA polymerase in the presence of RNAase H to digest the RNA chain of the RNA / DNA hybrid. Synthesis of the second chain was performed in the presence of 32P-dCTP to label the cDNA. The resulting cDNA was treated with T4 DNA polymerase to create blunt ends, the cDNA was mixed to protect possible EcoRI internal fragmentation sites and then bound to EcoRI linkers (Amersham). The EcoRI sites in the linkers were then fragmented and the 1.2% agarose gel cDNA was chopped. The region of the gel corresponding to a cDNA length of 500 to 2000 base pairs was cut, and the cDNA eluted and cloned into the pUC13 vector, which had previously been fragmented with EcoRI, and dephosphorylated. After ligation, the DNA was used to transform competent cells of E. coli MC1016 (lambda). The resulting colonies were transferred to Pall membrane filters (Pall Biodyne), lysed and denatured with 1.5 M NaCl, 0.5 M NaOH and neutralized with 3 M NaOAc, pH 5.5. Evaluation of colonies that had an HIV-3 insert was performed by hybridization under moderately stringent conditions in 5 X SSC, 5 X Denhardt solution, 0.2% SDS, 250 mg / ml denatured salmon sperm DNA overnight at 65 ° C. Hybridization was performed using the Sacl-EcoRI fragment of HIV-1. After hybridization, the filters were washed as follows:

1.

1 hour. in 2 X SSC, 0.1% SDS at room temperature.

two.

30 minutes in 0.1 X SSC, 0.1% SDS at room temperature.

3.

20 minutes in 2 X SSC, 0.1% SDS at 42 ° C.

Four.

20 minutes in 0.1 X SSC, 0.1% SDS at 65 ° C.

After washing, the autoradiographs were filters at -70 ° C using intensifying screens.

Hybridizations were also performed in non-strict conditions used for non-strict hybridization of the HIV-1 and HIV-2 probe.

c. Analysis of cDNA clones

Colonies that were grown for analysis were grown They gave a positive hybridization signal. Plasmids were isolated, they were fragmented with EcoRI and subjected to gel electrophoresis of agarose to confirm the presence of an insert and to Determine its size. Of the 96 colonies analyzed, 17 were found They contained inserts. Five were taken for further analysis and they were ordered in sizes from approximately 800 to 1600 pairs of bases in length.

d. Sequence determinations

Sequence determinations were performed. of nucleotides according to the Sanger dideoxynucleotide method (Proc. Natl. Acad. Sci. 74: 5463-6467, 1977), using a kit supplied by Boehringer. Sequencing was carried out using M12 primers of 17 units.

and. ANT 70 cDNA hybridization with viral RNA from HIV-1 and HIV-2

The ANT 70 cDNA clone containing the largest insert (iso 70-11) for the hybridization with the filter on which the Viral RNAs.

Hybridization was performed under conditions Strict in 3 X SSC, 0.5% milk powder, 1% SDS, sulfate 10% dextran, and 50% formamide at 42 ° C for 18 hours. After hybridization, the filter was washed with 0.1 X SSC, SDS at 0.1% at 65 ° C (washings of 2-30 minutes) after which was autoradiographed the filter at -70 ° C with a screen intensifier

Genomic DNA was also prepared from HIV-1 infected cells (SF4), ANT 70 isolation, as well as with uninfected control cells. After fragmentation with restriction enzymes, it was separated The genomic DNA on a 0.8% agarose gel was transferred to a Hybond N (Amersham) membrane and then hybridized with DNA labeled with 32P from clone iso 70-11 in strict conditions in 50% formamide, dextran sulfate at 10%, 3 X SSPE (1 X SSPE is 180 mM NaCl, 10 mM NaH2PO4, pH 7.4, 1 mM EDTA, pH 7.4), 1% SDS and 0.5% milk powder. After hybridization, the filter was washed as follows way:

15 minutes with 2 X SSC, SDS at 0.1% at room temperature.

15 minutes with 0.5 X SSC, SDS 0.1% at room temperature.

15 minutes with 0.1 X SSC, SDS 0.1% at room temperature.

30 minutes with 0.1 X SSC, SDS 0.1% at 52 ° C.

Next, the filter was autoradiographed at -70ºC using an intensifying screen.

Results Virus isolation

As part of a continuous study on the heterosexual transmission of HIV, isolation of the virus from a Cameroonian woman and her partner. The women the couple of an HIV-positive man with lymphadenopathy generalized The woman's serum was moderately positive. (O. D. / cut ratio of 4.5) in the immunosorbent assay linked to enzyme (EIA, for its acronym in English, Organon Teknika) and had a low titer (1/40) in the immunofluorescent antibody assay for HIV-1, but it gave ambiguous results in the Western immunoblotting assay of HIV-1, with light bands on p33, P53 / 55 and p64, but very weak bands on p24, gp41 and gp120. The woman had elevated serum levels of IgG and IgM and a CD4 / CD8 ratio of 0.46. The virus was isolated by co-culture of lymphocytes in women with PHA stimulated lymphocytes from healthy donors without infecting After 52 days in culture, the virus was detected in the crop due to the presence of syncytia and based on the positive immunofluorescence observed when an antiserum laboratory reference anti-HIV was incubated with cells fixed with acetone from the culture. It was detected also the presence of reverse transcriptase in the supernatant of the culture (10 4 cpm / ml, 27 X background). The supernatant was used of cell-free culture to pass the virus to recent lymphocytes. After 15 days, cytopathic effects were observed again and detected reverse transcriptase in the supernatant. However, a comparison of detergent treated culture supernatant from this isolation, with other insulations by differential antigen capture revealed that this isolation does not It was HIV-1.

These results are illustrated in Figure 2. It is evident that due to the low values of O. D. (optical density), that ANT 70, unlike the other insulations, is poorly recognized for HIV-1 specific IgG, but, as the other isolates, it was easily captured by IgG wide specificity (panel F). The other insulations, which were seen at then they were all strains of HIV-1, using a specific monoclonal antibody of HIV-1 (CLB MAb 14), gave all more values high levels of O. D. on plates coated with specific IgG that on plates coated with the specificity reference IgG wide.

An attempt was made to transfer the virus to a permanent cell line by co-culture of ANT 70 infected primary lymphocytes with clone 8 cells from Molt-4. In the initial phase of the infection, it observed an extensive cytopathic effect with syncytium formation and cell death In several weeks a growth was detected mobile. The cells gave a positive immunofluorescence when were tested using a broad spectrum anti_HIV antiserum and the presence of antigen and transcriptase was easily detectable inverse in the culture supernatant.

The virus was similarly isolated from the couple of the woman from whom ANT 70 was isolated (strain ANT 70 NA). The man He suffered from lymphadenopathy and was classified as class 3, according to CDC classification system. The man also had levels high levels of IgM and IgG in serum and a CD4 / CD8 ratio of 0.4. He detected the virus in the culture supernatant on day 18. It also analyzed the detergent treated supernatant containing the virus by differential capture of antigens and it was found that It reacted in a similar manner as ANT 70 (Figure 3). The Union of antigen derived from this isolation was again less than with HIV-1 specific IgG than with wide IgG specificity

The serum of the person from which this isolation was derived was stripped of Western blotting of HIV-1 and HIV-2 (Biotech). Additional strips were also incubated with serum from a donor infected with HIV-1, as well as serum from the person from whom HIV-2 was isolated (isolation 53). These results are shown in Figure 4. The serum of the person infected with ANT NA reacted in a significant way with virtually all HIV-1 proteins, including the envelope proteins. In contrast, the serum of the HIV-2 infected individual reacted cross-linked only with gag p24 protein, p34 endonuclease and p68 reverse transcriptase. The anti-HIV-1 serum reacted only with the HIV-2 p26 gag protein, while the ANT 70 NA isolation carrier serum recognizes this protein and HIV-2 reverse transcriptase.

Characterization of viral proteins

The virus was precipitated in the supernatant of the culture using polyethylene glycol 6000 (Merck) and returned to dissolve the resulting material and sediment through a 15 percent sucrose pad. Virus dissociated sedimented in SDS sample buffer and analyzed by polyacrylamide gel electrophoresis followed by protein immunoblot. The immunoblot was incubated, shown in Figure 5, with anti-HIV serum of broad specificity to reveal viral proteins.

In addition to reacting with all HIV-1 viral proteins, the BSR antiserum also reacted cross-reactively with the HIV-2 gag and pol gene products. This antiserum clearly recognizes also the products of the gag and pol genes of ANT 70. It is evident that the molecular weights of the products of the ANT 70 genes differ from those of HIV-1 or HIV-2. The molecular weights of the different viral proteins are summarized in Table 1. The variability in HIV-1 p17 / p18 protein is due to a 6-amino acid insert that is present in some strains between positions 120 and 121 in the sequence. HXB2 of HIV-1. A comparison of the ANT 70 and ANT 70 NA proteins is shown in Figure 6. The molecular weights of all ANT 70 NA proteins are identical to those of ANT 70.

To further investigate the relationship of antigens between HIV-1, HIV-2 and ANT 70, it diluted 1: 1000 a series of sera African and European anti-HIV-1 and it  used to coat microwell plates to capture antigens

Supernatants from treated cultures were diluted with detergent containing HIV-1, ANT 70, HIV-2 (LAV-2rod) and HIV-2 (isolation 53) and the ability to analyze Each antiserum captures the four different isolates. He show the representative results in Figure 7. You can see by this experiment, that the capacity of these different sera of capturing HIV-1 is not in any way related to its ability to capture or HIV-2 or ANT 70. On the contrary, the ability of these sera to capture LAV-2rod, the prototype of the strain of HIV-2, is highly correlated with the ability to these sera capture the isolation 53, which is also a strain of HIV-2, but it is an independent isolation. These data indicate that ANT 70 is neither HIV-1 nor HIV-2

In a series of capture experiments of related antigens, four African sera were chosen anti-HIV-1 to investigate your ability to join HIV-1, ANT 70, HIV-2 (LAV-2rod) and HIV-2 (isolation 53) when IgGs were coated at different dilutions. The culture supernatants were diluted in such a way to give approximately the same optical density when they were captured on plates coated with the IgG used in panel B of Figure 8. The dilutions of the four sera and the supernatant containing the virus was added. He supposed that similar viruses should give similar curves of Title. It is true, that Figure 5, LAV-2rod and isolation 53 reacted both similarly with IgGs coated. On the contrary, ANT 70 gave more intense signals to higher dilutions of IgG than any of the isolates of HIV-2 and the shapes of the curves obtained with ANT 70 are more similar to the curves obtained for HIV-1, except that the optical densities are consistently lower.

Cross-reactivity of mouse monoclonal antibodies directed against the p24 nuclear protein of HIV-1

A panel of monoclonal antibodies of mouse (MAbs) prepared against the p24 nuclear protein of HIV-1 to see your ability to react crossed with ANT 70 and HIV-2 isolates. In principle, any antibody panel can be used monoclonal of p24 anti-HIV-1, provided that the series includes monoclonal antibodies that react with different epitopes on the p24 molecule of HIV-1 The ascites fluid containing it was diluted antibodies and was used to coat the plates of microwells They were then added to the coated wells detergent treated supernatants containing viruses. He detected bound antigen using BSR-HIV IgGs conjugated with horseradish peroxidases. The results obtained in Figure 9.

In control wells coated with IgGs polyclonal broad spectrum, all supernatants that they contained viruses gave optical densities that exceeded Limits of the microwell plate reader. Nevertheless, when s tested in wells coated with the different monoclonal antibodies, a quite different model appeared. Previous studies indicated that all MAbs tested react against different epitopes in the p24 molecule with the except for the CLB 59 and CLB 21 MAbs, which have been seen to recognize The same epitope. These two MAbs react strongly with the HIV-1, as expected, and also give a signal measurable with ANT 70, but do not react with either of the two strains of HIV-2. Two other MAbs, CLB 64 and CLB 14, joined well to HIV-1 and showed a weak affinity for ANT 70, as well as for the two isolates of HIV-2. In particular, it has been seen that MAb CLB 14 recognizes all HIV-1 isolates (> 150 tested). By so much, this Mab has to join a very well preserved epitope, remains of which can also be detected in other viruses of human immunodeficiency The other MAbs with respect to p24 (CLB 16, 47 and 19.7) and the other two that were produced against the p18 protein of HIV-1 (CLB 13.4 and CLB 13.6), neither recognized nor ANT 70 or the other two isolates of HIV-2, but yes they captured the corresponding antigens from HIV-1

Reaction of human anti-HIV antisera with viral proteins

Protein immunoblotting was prepared of viral proteins from HIV-1 (ARV 4), ANT 70 and HIV-2 (LAV-2rod) after electrophoresis of detergent solubilized extracts and incubated with different human sera (Figure 10). Panel A shows the wide laboratory reference serum reaction specificity with the three virus isolates. In panel B, it incubated an anti-HIV antiserum with the immunoblot and preferentially recognize the proteins of the HIV-1 They were tested in the serum of the woman of the which was isolated ANT 70 (panel C) and that of your partner (panel D) to see its ability to recognize other viral isolates. Both sera preferentially recognize ANT 70 including the protein of the gp120 cover of this virus. The couple's serum has a title higher than the serum from the woman from which it was isolated ANT 70 and recognizes HIV-1 gp41. These two sera have a higher affinity for ANT 70 than for HIV-1 or HIV-2 isolates. On the contrary, the serum of the person from whom the Isolation 53 of HIV-2 binds preferentially to HIV-2 proteins and recognizes the protein of the gp120 cover of HIV-2 of this virus, as well as the transmembrane go41 protein (panel E). Does not react with HIV-1 or ANT 70 glycoproteins. These results also indicate that ANT 70 is different from HIV-1 and of HIV-2.

Immunosorbent assays of enzymes using viral protein titers coated with anti-HIV-1 sera, anti-ANT 70 and anti-HIV-2 on plates microwells coated with viral lysates of HIV-1 (ARV-4), ANT 70 and HIV-2 (isolation 53). Dilutions were tested doubles of each serum, starting with an initial dilution of 1: 100, to see its ability to bind to the coated antigen. It was detected antibody bound using a peroxidase conjugate Spicy-labeled anti-human IgG from goat. These results are shown in Figure 11. The serum anti-HIV-1 preferentially recognized HIV-1 proteins, but shows an amount significant cross-reaction with ANT 70 proteins. Only HIV-2 proteins were detected. For him on the contrary, the anti-ANT 70 serum recognized preferably ANT 70 proteins, showed a reactivity crossed with respect to HIV-1 proteins, and reacted better with coated wells of HIV-2 than what serum did anti-HIV-1, as evidenced the highest values obtained from the optical density. Serum anti-HIV-2 had a very low title,  but nevertheless it reacted very well with the proteins of HIV-2 No detectable signal was observed in the wells coated with HIV-1 or ANT 70. The inability to detect cross reaction in this case is related no doubt with the low anti-HIV title of this serum.

Analysis of partial chemical fragmentation products of viral proteins

The two reagents used for chemical fragmentation, cyanogen bromide and BNPS-escatol, were chosen because of their high specificities with methionine (29) and tryptophan (30), respectively. These two amino acids are also quite hydrophobic and are therefore also less likely to be located in epitopes on the outer surface of protein molecules (31). Examination of published amino acid sequences of the gag and pol gene products of HIV-1 (32-36), HIV-2 (19), SIVagm (10), SIVmac (9), equine infectious anemia virus (EIAV, 37) and Visna (38) reveals that while there is poor amino acid homology between some of these different isolates, many of the positions of methionine residues in these proteins are conserved notably (Figure 12), and even in A greater extent, tryptophan residues. In addition, the intraspecific variation in these residues is minimal or absent, at least in the case of HIV-1 (36) and is probably true for all human and ape immunodeficiency retroviruses.

The partial digestion models of the gag and pol gene products of HIV-1, ANT 70, HIV-2 (LAVrod) and HIV-2 (isolation 53) are shown in Figure 13.

Inspection of fragmentation models with CNBr of p24 protein from the four isolates reveals than the models generated for HIV-2 (LAV-2rod) and HIV-2 (isolation 53) They are identical.

However, different models for HIV-1 and for ANT 70 have been observed. Thus, there are significant differences in the locations of methionine residues in the main nuclear protein of HIV-1, ANT 70 and HIV-2. In the case of the p17 nuclear protein, differences are observed between the two HIV-2 isolates. Inspection of the published sequence for the HIV-2 strain indicates that there is a methionine located 18 amino acids from the carboxyl terminal of this protein. We conclude that this methionine must be absent in the corresponding protein of isolation 53. From the fragmentation model, it is also possible to deduce the presence of a methionine near (10-15 amino acids) from one of the terminals of p16 of ANT 70. CNBr fragmentation of the retrovirus reverse transcriptase reveals that once again the proteins of the two HIV-2 isolates are identical, while different models for HIV-1 and ANT 70 proteins are observed. In the case of endonuclease p31 derived from the 3 'part of the pol gene, similarities can be deduced among all isolates, although some small differences are apparent.

Fragmentation with BNPS-eschatol of the p24 proteins from the four isolates gives as result surprisingly similar models. It is evident from of Figure 8, that this is what is expected, since the Tryptophan positions in this protein are very good conserved, particularly for retroviruses of human origin and ape. We conclude that tryptophan positions in protein p25 of the ANT 70 also agree with this model. Without However, inspection of the models reveals that they can be observed small differences, not in all aspects of the model, but more well in the apparent molecular weights of the species generalized by fragmentation. In particular, they are detected differences in apparent molecular weights of stains central of each model. As expected, they are identical models for HIV-2 (strain LAV-2) and HIV-2 (isolation 53). However, the stain central in the model for ANT 70 has a higher molecular weight apparent while the central spot for HIV-1 (ARV-4) has a lower molecular weight. With regard to p16, the tryptophan positions in the ANT protein 70 seem closer to the tryptophan positions found in the HIV-2 protein. The p17 of HIV-1 has a tryptophan located at 16 amino acids of the amino terminal of the protein and causes a stain additional not seen in the ANT 70 and HIV-2 after fragmentation with BNPS-eschatol It is preserved in all isolates the tryptophan corresponding to that of position 36 in the sequence of p17 of HIV-1.

The models generated by fragmentation of the reverse transcriptase from the four isolates are complex, but it is again apparent that the two isolates of HIV-2 are identical. Models are obtained for ANT 70, which does not correspond to any model obtained or for the model of HIV-1 nor for the HIV-2 model. Differences in apparent molecular weights are also observed. of the p31 endonuclease fragmentation products, but the generated models of the corresponding proteins of HIV-1, ANT 70, and HIV-2 show also common characteristics, which suggests a structure preserved.

Viral nucleic acids to. Hybridization of HIV-1 cDNA and HIV-2 with viral RNAs

Cross-hybridization of nucleic acids between HIV-1 and RNA from HIV-2 and ANT 70 viruses was evaluated by hybridization with the HIV-1 restriction fragment Sacl-BgIII, which had been inserted into the pUC13 vector. This fragment contains a part of the 5'LTR, including the R region, the entire gag gene and most of the HIV-1 pol gene. Under strict hybridization conditions, only hybridization was observed between this probe and the HIV-1-derived RNA (SF4). No hybridization was observed between the probe and HIV-2 or ANT 70 (Figure 14). This indicates that the gag and pol regions of HIV-1 and ANT 70 are significantly different from the corresponding HIV-1 region.

The HIV-2 probe used contains a sequence of approximately 100 base pairs derived from the HIV-2 env gene. This probe hybridized only with HIV-2 RNA under strict hybridization conditions and no hybridization was observed with either HIV-1 or HIV-3.

b. Homology between the ANT 70 cDNA and the sequences of HIV-1 and HIV-2

The iso 70 cDNA clone was used as a probe to determine the degree of homology between the different virus isolates The filter in which they had been subjected was submitted deposited aliquots of HIV-1, HIV-2 and ANT 70 to hybridization under conditions strict. The results are also shown in Figure 14. The experiment shows that, under strict hybridization conditions, no cross hybridization was detected between any of the viral isolates. The ANT 70 only hybrid derived probe with ANT 70. DNA from cDNA of ANT 70 iso clone was used 70-11 for hybridization with immunoblotting of restriction enzyme digestions of cellular genomic DNA from cells infected with HIV-1 and ANT 70, as well as DNA from uninfected cells. The results are displayed. in Figure 15. Hybridization was not observed in any area different from areas containing digested genomic DNA from infected ANT 70 cells. This also indicates that the 70-11 insert is not derived from sequences cell phones.

Additional hybridization was performed using the Sacl-BgIII fragment from the insertion iso 70-11, which corresponds to the leftmostmost away from the sequences presumably contained within the LTR and that, by analogy with HIV-1 and HIV-2, should correspond to the sequences of the env gene Hybridization with this fragment produced the same bands that hybridized with the entire fragment.

c. Iso clone sequence analysis 70-11

The locations of a number of restriction enzyme sites in the ANT insert 70 iso 70-11. The subclones of the insert were made in pUC13 and the sequences using the method of dideoxy nucleotide. The presence of a tail of poly A confirmed that the iso70-11 insert is derived of the 3 'end of the viral RNA: adjacent to the tail of poly A is the sequence corresponding to the R region of the 3 'viral RTL. He show below the sequence contained in the ANT 70 cDNA and the viral sequences to which they correspond:

1. HIV-3 LTR (Table II)

3

2. 3'ORF sequences of HIV-3 (Table III)

4

Discussion

As described in the document EP-A 0 345 375, we have isolated a new retrovirus associated with the human immunodeficiency of a Cameroonian woman (ANT 70) and your partner (ANT 70 NA). At the time the original virus isolation, the woman was only slightly HIV positive, gave ambiguous results in the trial of Western immunoblot and was clinically asymptomatic. TO From that moment, the woman has begun to develop some of the symptoms of the AIDS-related complex (ARC, for its acronym in English). On the contrary, your partner, of which also we were able to isolate a virus with the same characteristics as the original isolation, I was suffering from lymphadenopathy and since Then he has developed other characteristic symptoms of AIDS. Therefore, this new isolation can be considered to be a virus of human immunodeficiency. The fact that this same virus could be isolated from sexual partners, also suggests a way of transmission that is similar to that of human retroviruses.

First, the virus was recognized as different of HIV-1 based on its impaired capacity to be captured in a differential capture test of antigens This has shown that it is a very safe trial, which is able to distinguish between strains of HIV-1 and not of HIV-1 That this isolation is not HIV-1 is deduced at the protein level by 1.) different molecular weights of viral proteins. 2.) a different cross-reaction model with antiserum anti-HIV-1 that HIV-1 3.) a drastically reduced capacity of be recognized by mouse monoclonal antibodies produced against the nuclear proteins p24 and p17 of HIV-1. 4.) preferential recognition of ANT 70 proteins over HIV-1 proteins by antisera from virus carrier, and 5.) partial fragmentation models of four of the best conserved viral proteins, which do not marry the models obtained when HIV-1 proteins They undergo the same treatment. However, the sera of the two individuals infected with this virus recognize the protein in the gp41 cover of HIV-1. By the same criteria mentioned above, it is also clear that ANT 70 is not HIV-2 It is true that antigenic differences between ANT 70 and HIV-1 are lower than those between HIV-2 and HIV-1. This is particularly evident with the results presented in the Figures 8 and 10.

The additional evident evidence that ANT 70 is a virus different from HIV-1 and HIV-2 It comes from partial peptide maps. We have shown that there are significant differences in viral proteins more preserved. The two isolates of MIV-2 that used to compare, gave fragmentation models essentially identical, except in the case of fragmentation by CNBr of nuclear protein p17. However, it must be done emphasize that the p17 nuclear protein exhibits more variability than p24 protein, at least in HIV-1 strains (3. 4). If this is true or not for HIV-2 you will have to wait for the sequence determination of more strains than  So far they have been analyzed.

In light of the fact that ANT 70 is antigenically more related to HIV-1 than with HIV-2, as evidenced by a greater degree of cross reactivity, which extends even to the protein of cover gp41. It was essential to establish that ANT 70 was more than simply a genetic variant of HIV-1. This it was possible by investigating locations of some of the best conserved amino acids in a number of proteins viral, which are at least subject to genetic variation. That you will notice these main differences in the models of fractionation indicates that HIV-1, HIV-2 and ANT 70 are three viruses genetically distinct Moreover, the same series of experiments also revealed similarities between viruses, which It may indicate that all three come from a common parent.

Hybridization data also support the idea that ANT 70 is fundamentally different from HIV-1 and of HIV-2. As long as the conditions under which hybridization is performed, you can Easily make a distinction between the three types of viruses.

Analysis of cDNA sequences revealed that the insert is derived from the 3 'end of the viral genome. A Homology analysis between these sequences and the sequence of HIV-1 and HIV-2 reveals, that ANT 70 is somehow more related to HIV-1, particularly in LTR sequences (approximately 70% of homology). However, the differences are of such magnitude, that the possibility that ANT 70 is simply a genetic variant of HIV-1. The 3'LTR of ANT 70 it also contains the signal sequences that are typical of LTRs retroviral

It is found that the sequences corresponding to the env gene of ANT 70 are even less related to the HIV-1 or HIV-2 sequences. While it is true that retroviral env genes frequently exhibit genetic variability, differences in sequence between variants is rarely more than 10%. However, it was found that the degree of homology between ANT 70, HIV-1 and HIV-2 is less than 60%. Such a difference in homology, together with the results of hybridization, indicates that ANT 70 belongs to a new type of human immunodeficiency retrovirus.

The existence of a third type of human immunodeficiency virus has immediate epidemiological implications and consequences for blood bank trials. As it has been seen, the antibodies of people infected with this virus react preferentially with this virus, although these antibodies also react in crossbreeding with HIV-1 proteins. While this was possible to detect a positive reaction in enzyme immunoassays, immunofluorescence assays and Western blotting assays based on HIV-1 proteins, the fact that the positive signal was due to a cross-reaction inevitably implies the sensitivity of Such assays will be lower for antibodies produced in response to this virus. This was demonstrated extensively by the results of enzyme immunoassay (Figure 11). In addition, a criterion for seropositivity in the Western blot assay is the presence of detectable antibodies to a gag and / or pol protein and one of the envelope proteins. Since in the case of the two individuals infected with ANT 70 and ANT 70 NA, respectively, cross-reaction was observed in the two p24 and HIV-1 envelope proteins, the conclusion invariably drawn is that these individuals are infected with HIV-1, but for some reason they don't develop high titles. Therefore it is possible that this virus is more widespread than is usually considered. From an epidemiological point of view, it is essential to develop specific diagnostic tests for this virus to assess the boundaries of the geographic area in Africa in which the virus can be found, and to assess the extent to which the virus has spread.

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Claims (15)

1. Variant of the HIV-3 retrovirus deposited in the European Collection of Animal Cell Culture (European Animal Cell Culture Collection, ECACC for its acronym in English) with the number V88060301, said variants having  following essential morphological and immunological properties:

-

The virus exhibits a tropism towards T4 lymphocytes;

-

The virus is cytotoxic with respect to the lymphocytes it infects;

-

The virus has a diameter of approximately 120 nm.

-

The virus has a magnesium-dependent reverse transcriptase activity;

-

It can be grown in immortalized cell lines that have receptors from T4;

-

The lysates of viruses contain a p25 protein, which is immunologically distinct of p19 HTLV-1 protein and p24 proteins of HIV-1 and HIV-2, as determined by Western blot analysis and partial fragmentation by CNBr, respectively;

-

The lysates of viruses contain a gp120 protein that is immunologically different from the gp110 protein of HTLV-1, from the gp120 of HIV-1 and gp120 of HIV-2, as determined by analysis with Western blotting;

-

The virus lysate It also contains a gp41 glycoprotein with a molecular weight of 40,000-45,000.

-

The genomic RNA of the non-hybrid HIV-3 variant or with the sequences of HIV-1, nor with the sequences of HIV-2 under strict conditions of hybridization;

-

The lysates of viruses contain a p16 protein that differs from the p17 of HIV-1 and HIV-2, as determined by partial fragmentation with CNBr;

-

The lysates of viruses contain a p31 endonuclease, which differs from those HIV-1 and HIV-2 endonucleases, as determined by partial fragmentation with CNBr;

and having said variants models of fragmentation with CNBr and BNPS-eschatol from the p25 protein, p16 protein, p31 protein and reverse transcriptase, as illustrated in Fig. 13. 2. A strain of a retrovirus variant HIV-3 of claim 1, comprising nucleic acid sequence a contiguous R region and a sequence of the U3 region that hybridizes, under strict conditions of hybridization, with nucleic acid sequences, as they represent in Tables II and III. 3. A process to prepare an acid molecule nucleic that contains at least a part of the cDNA that corresponds to the entire RNA genome of a strain of a variant of HIV-3 retrovirus of claim 1 or 2 or its complementary chain and that hybridizes specifically with the nucleotide sequence of the strain of HIV-3 deposited with No. V88060301 of the ECACC under conditions strict hybridization, said process comprising isolating said nucleic acid molecule of a variant of claim 1 or two. 4. The process of claim 3, wherein said nucleic acid molecule comprises the sequences that correspond to the entire RNA genome of the retrovirus strain HIV-3 of claim 1 or 2. 5. A process to prepare a part of a cDNA nucleic acid molecule that corresponds to all the RNA genome of HIV-3 retrovirus strain deposited with No. V88060301 in the ECACC or its chain complementary and that hybridizes specifically with the sequence of nucleotides of said HIV-3 under strict conditions of hybridization, said process comprising isolating said part of nucleic acid molecule of said retrovirus strain HIV-3 6. A process to prepare a probe that comprises the nucleic acid molecule of any one of the claims 3 to 5, which comprises marking said molecule of nucleic acid. 7. A process to prepare a vector recombinant, which comprises isolating the nucleic acid molecule from any one of claims 3 to 5 in a vector. 8. The process of claim 6, wherein said vector is an expression vector. 9. A process to transform a cell host, comprising incorporating a vector of the claim 7 or 8 in said cell. 10. A process for the production of an HIV-3 retrovirus strain of claim 1 or 2, characterized by culturing human T4 lymphocytes, or permanent cell lines derived therefrom carrying the T4 phenotype, with lymphocytes or cell lines that are they have previously infected with said HIV-3 retrovirus strain, as well as by recovering and purifying the retrovirus from the culture medium. 11. A process to prepare a composition that comprises an extract or total lysate of the retrovirus strain HIV-3 of claim 1 or 2, comprising said process to grow said virus and treat it with a detergent appropriate. 12. The process of claim 11, which it also comprises a lysate of HIV-1, HIV-2, or a mixture of two. 13. A process to prepare a kit, which comprises the process of claim 6 or a process of the claim 11 or 12. 14. A method for the detection of a strain of an HIV-3 retrovirus or its RNA in a biological liquid or tissue, characterized by a) contact contained nucleic acids in said biological liquid or tissue with the probe of the claim 6 under strict hybridization conditions, b) wash the hybrid formed with a solution that retains these strict conditions, and c) detect the hybrid formed. 15. Use of a nucleic acid molecule of any one of claims 3 to 5, a probe of claim 6 or a kit comprising said nucleic acid molecule or probe for in vitro detection of HIV-3 or in vitro diagnosis of infection with HIV-3.