DE3856564T2 - HIV-3 strains of retrovirus and their use - Google Patents

Description

The present invention relates to the area of human immunodeficiency viruses.

In particular, the present concerns Invention of new variants of the HIV-3 retrovirus strain, which is used by the European Collection of Animal Cell Cultures (ECACC) under the deposit number V880 60301 was deposited, as well as others characterized in the claims Embodiments.

Our understanding of AIDS (acquired immunodeficiency syndrome) has made significant progress. It has been shown that the essential causative agent with a non-transforming retrovirus a tropism for T4 helper / inducer lymphocytes (1,2). According to estimates millions of people are already infected worldwide. The infection with this virus leads at least in a significant percentage of cases, too progressive exhaustion of the T4 lymphocyte population with a simultaneous increase in susceptibility across from opportunistic infections that are characteristic of this condition are.

Epidemiological studies indicate out that human immunodeficiency virus Type 1 (HIV-1), which is the etiological agent for the majority of the AIDS cases is responsible, and that is currently the most widespread HIV is believed to have originated in Central Africa (3). The discovery of this virus did not necessarily imply that Existence of other types of human immunodeficiency viruses. However, a second group of retroviruses has been identified in West Africa, that are associated with human immunodeficiency, that is human immunodeficiency virus Type 2 (HIV-2) (4.5). An HIV-2 virus is disclosed in EPO-85/00548. An HIV-1 virus is disclosed in EPO-239 425. Other, similar, but not identical, retroviruses are also from monkeys (monkey immunodeficiency virus, SIV) like African green vervet monkeys (6.7) and macaque (8.9). It has been shown that the monkey isolates genetically closer are related to HIV-2 than HIV-1, but still different (10).

A characteristic of human immunodeficiency viruses that makes their comparison difficult is their genetic variability; genetic variants occur spontaneously and with great frequency. A comparison of the numerous HIV-1 isolates showed that some regions of the genome are very variable, while others are fairly well preserved (11-16). Similar polymorphisms have also been observed for HIV-2 (17). The regions with the greatest genetic stability are probably the regions that code for the regions of the viral proteins that are structurally or enzymatically essential. The viral genes with the greatest overall genetic stability are the gag and pol genes, while some regions of the env gene and the genes coding for regulatory proteins such as art , tat , sor and 3'orf have a high degree of variability. Some of the essential structural features of the gene products from gag and pol are obviously not only present in all variants of a certain HIV type, but are, at least to a certain extent, also conserved between different virus types. Antiserum raised against HIV-1 cross-reacts with the gene products gag , and pol of HIV-2, albeit with a lower affinity than for the corresponding gene products of HIV-1. Despite the detectable immunological cross-reaction, there is little sequence homolougie at the nucleic acid level. No remarkable hybridization can be detected between these two viruses except under very stringent conditions (17).

A closer relationship can be shown between SIVagm (STLV-III agm, almost or completely identical to the human lymphotropic virus type 4 (15)) and HIV-2. Immunological cross-reactions are not only limited to the gag and pol gene products, but also extend to the env gene products. Nevertheless, genomic analyzes of SIVagm and HIV-2 showed that they are genetically distinguishable (19). DNA probes specific for HIV-2 preferably hybridize with HIV-2, even if they can hybridize with SIVagm sequences (18).

We reported in EP-AO 345 375 on the isolation and partial characterization of a new human immunodeficiency virus from a woman and her partner from Cameroon. Geographically, this virus comes from a region in Africa that lies between West Africa, where HIV-2 is endemic, and East-Central Africa, where HIV-1 is endemic. It is shown immunologically that this isolate is antigenically more closely related to HIV-1 than to HIV-2. However, analysis of the products of partial cleavage obtained by chemical cleavage of the gene products of gag and pol shows that this isolate is neither HIV-I nor HIV-2. This new isolate could represent an evolutionary link between HIV-1 and HIV-2. This new virus is hereinafter referred to as HIV-3. Accordingly, the invention relates to variants of the HIV-3 virus, which are deposited with the ECACC under the number V 88060301.

Virus isolation was obtained from the blood of a symptom-free woman from Cameroon, whose partner is an HIV-seropositive man with general lymphadenopathy. The woman's serum was moderately positive (ratio OD / cutoff of 4.5) in an enzyme-linked immunoadsorption test (EIA, Organon Teknika). It had a low titer (1/40) in the immunofluorescent antibody test for HIV-1, but gave ambiguous results in the HIV-1 Western blot test with clear bands at p33, p53 / 55 and p64 but very weak bands at p24, gp41 and gp120. The virus was co-cultured from female lymphocytes with PHA stimulated lymphocytes from healthy, non-infected donors in a medium derived from RPMI 1640 stood, buffered with 20 mM HEPES (hydroxyethylpiperazinethanesulfonate) and supplemented with 15% fetal calf serum, 5 μg / ml hydrocortisone, 75 U / ml interleukin-2 (IL-2) and 2 μg / ml polybrene, isolated.

After 52 days in the culture, the virus was detected in the culture due to the presence of syncytia and on the basis of positive immunofluorescence, observed when a reference anti-HIV antiserum from the laboratory was incubated with acetone-fixed cells from the culture. The presence of reverse transcriptase in the culture supernatant (10 ⁴ cpm / ml, 27-fold background) was also detected. Cell-free culture supernatant was used to pass the virus through fresh lymphocytes. A CPE was again observed after 15 days and reverse transcriptase was detected in the supernatant. The virus was further propagated in lymphocytes stimulated with PHA from healthy blood donors and transferred to permanent cell lines of leukemic origin. Virus-containing supernatant was examined simultaneously with culture supernatants known to contain HIV-1 in the differential antigen capture test described in more detail below. The results of this comparison showed that the new isolate was not HIV-1.

The new virus then became related to its Protein antigens and nucleic acids characterized. The cell lines used to multiply the virus can dependent on from individual cases the cell lines CEM, HUT, Molt-4 or MT4 or any other immortalized cell lines that target the T4 receptor their cell surface wear.

A preferred cell line for the continuous proliferation of HIV-3 is the Molt-4 cell line. Molt-4 cells infected with HIV-3 were deposited with the ECACC on June 3, 1988 under number V 88060301. Establishing a chronically infected cell line can e.g. For example, Molt-4 cells (10 ⁶ / ml), and preferably cells of Molt-4 clone 8 (obtained from N. Yamamoto, Yamaguchi, Japan), are analyzed together with infected human lymphocytes (10 ⁶ / ml ) in RPMI 1640 culture medium, buffered with 20 mM HEPES and containing 10% fetal calf serum. A cytopathic effect is observed in the culture within a week or two, followed by cell death. A fraction of the cells in the culture survive the infection and continuously produce the virus. With continued cultivation, the number of cells increases and the cells can be subjected to passages. Supernatants from these cells can be used as the source of the virus.

The invention also relates to the purified retrovirus with the essential morphological and immunological Properties described below. Leave in many cases the unique properties of the HIV-3 variants of the present Invention by comparison with the same kind of properties of the other human immunodeficiency viruses HIV-1 and HIV-2 am best estimate.

1. Morphology

Electron microscopy from with HIV-3 infected MT4 cells showed the presence of extracellular virus particles with a diameter of approximately 120 nm, which consist of an outer shell, the one inner, elongated Core with a diameter of about 20 to 40 nm surrounds the in some thin cuts in contrast to the more or less cylindrical appearance of the Cores of HIV-1 slightly conical appears. Still, HIV-3 is morphologically very similar to HIV-1 and HIV-2, let yourself but easily from other human retroviruses like HTLV-I and HTLV-II differentiate.

2. Protein and glycoprotein antigens

The virus present in the culture supernatant of HIV-3 infected Molt-4 cells was concentrated by means of precipitation with polyethylene glycol (average molecular weight: 6,000) and then centrifuged. The resulting pellet was resuspended in phosphate buffered saline, layered on a 20% sucrose pad, and pelleted at 100,000 x g for one and a half hours. The pelleted virus was then digested in 62.5 mM Tris, pH 6.7, containing 2% 2-mercaptoethanol, 1% sodium dodecyl sulfate and 10% glycerol, and the essential viral antigens were electrophoresed on a polyacrylamide gel (12 , 5%) separated under denaturing conditions. Molecular weight standards were run on the same gel to provide a basis for estimating molecular weights. Once the proteins were separated, they were transferred electrophoretically onto nitrocellulose paper (Western blot). The nitrocellulose paper was then incubated with an antiserum derived from a person infected with an HIV virus. In the initial experiments, a high titer antiserum from a person infected with HIV-1 was used, the antiserum having previously been shown to cross-react with gag and pol- derived proteins from HIV-2. In this way the molecular weights of the gene products of gag and pol of HIV-3 could be compared with those of HIV-1 and HIV-2.

The apparent molecular weights observed for the HIV-3 proteins are similar to those observed for both HIV-1 and HIV-2. Still, they are small, but reproductive visible, differences in molecular weights between the proteins of HIV-3, HIV-1 and HIV-2 are also evident.

The protein blots showed that HIV-3 like HIV-1 and HIV-2 has three core proteins. In the case of HIV-3 it was found that this Proteins have molecular weights of approximately 12,000, 16,500 and 25,000, respectively. Usually proteins become common with a "p" for protein or with "gp" for glycoprotein and a subsequent number multiplied by a factor of 1000, the approximate Molecular weight of the polypeptide indicates. The three main core proteins of HIV-3 are hereinafter referred to as p12, p16 and p25.

The determined molecular weight values are expected to be correct within a 10% deviation from the true values. However, there is much confusion regarding the molecular weight values of proteins, since the structure of the electrophoresis apparatus used and the source of the buffer components vary from laboratory to laboratory. Therefore, in order to compare the apparent molecular weights of the protein antigens of HIV-3 compared to those of HIV-1 or HIV-2, it is necessary to subject all samples to electrophoresis on the same gel. Such a gel can e.g. B. in 5 to be viewed as. In particular, it is apparent that while in the case of the main core protein the molecular weight values of the homologous proteins of the three HIV viruses are very close, the protein derived from HIV-1 is the smallest. The main core protein of HIV-2 is slightly larger than that of HIV-1 as recently described. The homologous protein of HIV-3 is a little bit larger than the main core protein of HIV-2. The calculated molecular weights of these proteins are given in Table 1.

Differences are similar in molecular weight also between the three HIV viruses in terms of second core protein obvious. This has in most strains HIV-1 has a molecular weight of 18,000. Differences in molecular weight however, this protein from strain to strain is in the case of HIV-1 have been documented. The molecular weight of this protein can be in some isolates are 17,000. The homologous protein of HIV-2 has a molecular weight of approximately 16,000 while the protein of HIV-3 has an intermediate molecular weight of owns about 16,500.

Analogous to HIV-1 and HIV-2, HIV-3 also has two forms of the virally encoded enzyme reverse transcriptase. These two species also differ slightly in molecular weight from the corresponding ones species in HIV-1 and HIV-2. They are therefore characteristic of HIV-3. These molecular weights are also shown in Table 1.

HIV-3 has one more Polypeptide derived from the pol gene, which is an endonuclease with an obvious Molecular weight is 31,000 and that is not essential in the Molecular weight differs from the homologous proteins from HIV-1 or HIV-2.

When protein blots containing HIV-3 proteins are incubated with serum obtained from a person infected with this virus, two additional proteins can be seen. These proteins come from the env gene and are the viral envelope glycoproteins. The smallest protein, which is the transmembrane protein, migrates as a broad band with an apparent molecular weight of between 40,000 and 45,000. This protein is hereinafter referred to as gp41, it being understood that the protein has a certain heterogeneity inherent to it in terms of its apparent molecular weight and its migration in polyacrylamide gels. The larger 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 shown below referred to as gp120. It should be noted that the obvious heterogeneous migration of both species on a polyacrylamide gel not in the heterogeneity of the Polypeptide chain established is, but rather has its cause in post-translational glycosylation. In particular, the gp120 is heavily glycosylated, and the obvious one Molecular weight that can be observed depends to some extent on the cell line that has been used to produce the virus.

In addition to the Western blot can viral protein antigens also by means of the radio immune precipitation test (RIPA) can be made visible. To this end, viral Proteins are metabolically radioactively labeled in vivo by using HIV-3 infected cells in the presence of 35 S-cysteine and 35 S-methionine (200 μCi / ml) in RPMI 1640 medium without these two amino acids, supplemented with dialyzed fetal Calf serum, be cultivated. After 16 hours, the labeled virus will go out the culture supernatant by centrifugation over a 20% sucrose pillow harvested at 100,000 g for an hour and a half. The resulting pelleted virus is then placed in RIPA buffer (20 mM triethanolamine, pH 8.0, 0.5 M NaCl, 0.5% Nonidet P40, 0.1% Sodium deoxycholate and 1 mM phenylmethylsulfonyl fluoride) resuspended.

On the other hand, the virus can with 125I be radiolabelled by chloramine T using the technique which is familiar to the person skilled in the art becomes. In this case, the virus is made from the supernatant of infected cells purified by pelleting the virus through a pillow of 20% sucrose, resuspended in saline buffered with phosphate and by means of Ultracentrifugation on a 20 to 50% sucrose gradient Concentrated in bands at 60,000 g for 12 hours. This in Gangs of concentrated virus can be found in the fractional gradient either by means of the reverse transcriptase test or by means of an antigen capture test be localized. The fractions containing the virus are combined, and Triton X-100 is added to a concentration of 0.5%. The one with Triton X-100 lysed virus can then be iodized.

For immunoprecipitations are labeled with 100,000 to 200,000 cpm of viral protein in the RIPA buffer with 5 μl of a test serum in a volume of 200 μl for 16 hours at 4 ° C. The resulting immune complexes are then attached to Protein A Sepharose (Pharmacia) bound, washed extensively, and the bound proteins are eluted with electrophoresis sample buffer containing 1% SDS. The Antigens are subsequently analyzed by electrophoresis. An autoradiography follows.

The protein antigens of HIV-3 can be found at Use of two different but similar approaches in the Comparison to the antigens of HIV-1 and HIV-2 can be characterized. On the one hand, they can Antigens based on their ability to interact with antisera from with HIV-1 and HIV-2 to cross-react infected persons. on the other hand can Antisera from people infected with HIV-3 who are antibodies Response to the HIV-3 antigens used to contain the Cross-reactivity across from HIV-1 and HIV-2 proteins to test. The antigenic relationships between HIV-3 and HIV-1 and HIV-2 are essentially in the examples given below demonstrated.

The results of these experiments show that HIV-3 is only distantly related to HIV-2 because cross-reactivity is observed only with pol viral core proteins and gene products. No cross-reactivity of the env gene products was 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, HIV-3 is more closely related to HIV-1 because antiserum directed against HIV-3 not only cross-reacts with the gene products of gag and pol from HIV-1, but also to a certain extent with the gp41 and gp120 env Gene products cross-reacted, albeit with a lower affinity. Similarly, anti-HIV-1 antiserum cross-reacts with all of the protein antigens of HIV-3, but with a lower affinity than for the proteins of HIV-1.

The following examples show that HIV-3 antigenically is substantially different from HIV-1. This is done based on 1. a different reactivity pattern with anti-HIV-1 antiserum compared to the pattern observed for HIV-1; 2. a drastically reduced ability to be recognized by mouse monoclonal antibodies raised against the core proteins p24 and p17 of HIV-1, and 3. preferential recognition of the HIV-3 proteins, including the envelope proteins, in comparison to the HIV-1 proteins by antisera from people infected with HIV-3.

Despite that for human immunodeficiency viruses genetic variation characteristic is a test based on e.g. B. on HIV-1 proteins, derived from a special strain, satisfactory for the detection of antibodies work that generates in response to other variants of HIV-1 have been. This can be seen in particular in the example in the monoclonal antibody on their ability for reaction with antigens derived from HIV-1, HIV-2 and HIV-3 isolates have been tested. In this case, the monoclonal antibody against the core proteins of HIV-1 strain III B. Yet they react very strongly to each other Proteins derived from HIV-1 of the SF4 strain. In contrast the same monoclonal antibodies react weakly or at all not with the core proteins of HIV-3. This suggests that the antigenic differences between HIV-1 and HIV-3 from one Size are that immunological Tests based on the use of HIV-1 proteins do not are suitable for testing sera from people infected with HIV-3.

Finally, the examples given below show differences in the number and / or positions of methionine and tryptophan residues in the most conserved gene products gag and pol .

3. Nucleic acids from HIV-3

A. HIV-3 viral RNA

The RNA of HIV-3 hybridized under stringent hybridization conditions not with DNA from HIV-1, if after the "Dot Blot "technique on a Hybond-H (Amersham) filter was applied.

Under "stringent conditions" or "non-stringent Conditions " understood the conditions under which the actual hybridization and / or the following washing steps are carried out. Dot blot hybridizations were executed by dilutions of viral RNA from HIV-1 of the SF4 strain, from HIV-2rod and HIV-3 from ANT 70 strain and HIV-3 ANT 70 NA strain were spotted on Hybond-H filters.

The dilution series for each virus corresponded to viral RNA pelleted by equivalents of 5, 2.5, 1.25 and 0.62 ml culture supernatant. The RNA was fixed on the filter by UV irradiation for 2 minutes and subjected to hybridization by contacting the filter with a 32P-labeled DNA probe. The probe selected was from a sequence of HIV-1 which comprised nucleotides 487-4,652 (SacI - EcoRI). It comprises part of the long 5 'terminal repeat, the entire region of gag and most of the pol gene, subcloned in the vector pUC13. Hybridization of the 32 P-labeled probe to the filter was carried out under stringent conditions in 3 × SSC, 0.5% milk powder, 1% SDS, 10% dextran sulfate, 50% formamide (vol./vol.) For 18 hours at 42 ° C performed (1 × SSC corresponds to 0.15 M NaCl, 0.015 M sodium citrate). The subsequent washing steps were carried out under stringent conditions in 0.1 × SSC and 0.1% SDS at 65 ° C. (washing cycles 2-30 minutes). The filter was then dried and autoradiographed with reinforcing films at -70 ° C. After autoradiography, only points were visible that corresponded to HIV-1 viral RNA. No hybridization with HIV-2 or either of the strains of HIV-3 was observed. It therefore appears that HIV-3 is only distantly related to HIV-1.

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

The conditions under which cDNA, according to the sequences of HIV-3, synthesized and cloned are described below. HIV-3 (strain ANT 70) of 1 liter Culture was precipitated with polyethylene glycol 6000, again with phosphate buffered saline solved and pelleted through a 20% sucrose pillow. The resulting The virus was pelleted in 6 M guanidinium chloride in 20 mM dithiothreitol and 0.5% Nonidet P-40 dissolved. CsCl was added to a concentration of 2 M and the the solution containing the broken virus was placed on a 1.2 ml cushion layered of 5.7 M CsCl that was 0.1 in EDTA. Viral RNA was through 20 hours Centrifugation at 25,000 rpm in a Beckman SW28 rotor Pelleted at 15 ° C. The pelleted RNA was redissolved, extracted with phenol and precipitated with ethanol and 2 M LiCl.

A fifth of the viral RNA that, as described above was used for the first Step in the synthesis of cDNA using an oligo (dT) primer which served to prime the synthesis of the first strand of cDNA.

• 1. 1 Stunde in 2 × SSC, 0,1 % SDS bei Raumtemperatur.
• 2. 30 Minuten in 0,1 × SSC, 0,1 % SDS bei Raumtemperatur.
• 3. 20 Minuten in 2 × SSC, 0,1 % SDS bei 42 °C.
• 4. 20 Minuten in 0,1 × SSC, 0,1 % SDS bei 42 °C.

A kit commercially available from Amersham was used to prepare the HIV-3 cDNA. In addition, an exogenously added reverse transcriptase was used so that the first strand was synthesized. This second strand synthesis was performed using E. coli DNA polymerase I in the presence of RNase H so that the RNA strand of the RNA / DNA hybrid was digested away. Second strand synthesis was performed in the presence of 32P-dCTP to label the cDNA. The resulting cDNA was treated with T4 DNA polymerase to produce blunt ends. The cDNA was methylated to protect any internal EcoRI cleavage sites. Then it was coupled to EcoRI linkers, also supplied by Amersham. The recognition sites for EcoRI were then digested and the cDNA was size-separated on a 1.2% agarose gel. The region in the gel corresponding to the length of the cDNA from 500 to 2,000 bp was excised, and the cDNA was eluted and cloned into the vector pUC13, which had previously been digested with EcoRI and dephosphorylated. The DNA was then used to transform competent cells from 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. Colonies were screened for those containing an insert of HIV-3 under moderately stringent conditions in a buffer overnight at 65 ° C using 32P-labeled plasmid containing the above-described SacI-EcoRI fragment of HIV- 1, performed, the buffer containing 5 × SSC, 5 × Denhardt's solution, 0.2% SDS, 250 mg / ml denatured salmon sperm DNA. After hybridization, the filters were washed as follows:

• 1. 1 hour in 2 × SSC, 0.1% SDS at room temperature.
• 2. 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.
• 4. 20 minutes in 0.1 x SSC, 0.1% SDS at 42 ° C.

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

C. Sequences contained in HIV-3 cDNA One of the largest insertion carrying clone, which was found to be approximately 1,600 bp in length, and which was given the designation iso 70-11, was used for sequence analysis selected. A number of subclones of the insert were made by digested the insertion with various restriction enzymes and the resulting fragments were subcloned into the vector pUC13. The sequences were determined by the dideoxy method, described by Sanger (Proc. Natl. Acad. Sci. USA 74: 5463-5467, 1977) using a kit sold by Boehringer, the 17mer long M13 primer used. The sequence analysis of the cDNA clones iso 70-11 showed that the Insertion at the 3 'end corresponded to the viral genome that had a poly A chain at its 3 'end.

The HIV-3 retrovirus contains one 3'-LTR that out a U3 region and an R region. As the 3 'LTR region of Contains HIV-1 the clone iso 70-11 also has an AATAAA polyadenylation signal 23 nucleotides from the 3 'end the R region. Analysis of the HIV-3 sequence showed one Homology of approximately 70% with the corresponding 3'-LTR sequence of HIV-1 and homology less than 55% with the corresponding sequence of HIV-2.

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

The HIV-3 sequence has homology of less than 60% to the corresponding sequences in HIV-1 and HIV-2 on. The sequence comes from part of the genome of HIV-3, the for the transmembrane coat protein coded.

The sequences contained in the RNA of the new virus were found not to hybridize with either the env gene of HIV-1 or the sequences contained in the LTR region of the genome of HIV-1. The SaII / BgIII restriction fragment, which contains sequences located in the env gene of HIV-3, was cut out from the DNA of the clone iso 70-11, labeled with 32P and hybridized under stringent conditions with a filter which contained genomic DNA, which was isolated from cells infected with HIV-1 (strain SF2) and HIV-3 (strain ANT 70). Hybridization was observed only with DNA, corresponding to the cells infected with HIV-3. Similarly, the only detectable hybridization was that with DNA from cells infected with HIV-3 when the entire plasmid iso 70-11 labeled and used as a probe under stringent hybridization conditions. This shows that HIV-3 is only distantly related to HIV-1 and that clone iso 70-11 is suitable as a hybridization probe to distinguish HIV-3 from the other two known human immunodeficiency viruses.

Conversely, hybridizations using the sequences of HIV-1's gag - pol as the labeled probe for detection of cross-hybridization with HIV-3 RNA showed no detectable hybridization when hybridization was performed under stringent conditions. This in turn shows that the viruses are only distantly related 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 that gag and pol includes the genes. However, the same labeled probe hybridized to RNA derived from the SF4 strain of HIV-1.

The invention also relates to a composition comprising at least one antigen. This antigen is in particular a protein or a glycoprotein of the HIV-3 retrovirus variants of the present invention. Such a composition can in methods for the detection of antibodies and in kits for carrying out such Procedures are used.

The HIV-3 virus has become a source for antigens for the detection of antibodies useful in people who have come into contact with HIV-3 proved. As such, the virus can be grown and after the already described methods are concentrated. A lysate can pass through Treatment of the virus with a suitable detergent become. A preferred detergent for the manufacture of a total lysate of the virus is Triton X-100 in a concentration of 0.5%. On another preferred detergent is Nonidet P-40 (NP-40), which is also is used in a concentration of 0.5%.

Alternatively, viral protein can be purified from lysates of the virus. A preferred method Affinity chromatography is used to purify these proteins. For example can the viral antigens on a preparative polyacrylamide gel separated and the individual antigens are eluted in purified form. these can continue to be used, for. B. to generate antisera in rabbits, the for the respective viral proteins are specific. The derived from the rabbit immune serum IgG fraction can attach to a solid phase such as CNBr-activated Sepharose 48 (Pharmacia) coupled and for the selective removal of individual viral antigens from viral lysates can be used. These proteins can then eluted from the affinity support using a low pH buffer and using standard chromatography techniques can be further cleaned. An example of such a technique is in Montelaro et al., J. of Virology (1982) 42: 1029-1030. The invention relates generally to any composition that for the Diagnosing an infection with HIV-3 or for testing with prognostic Value can be used.

The invention also relates to each Composition containing a viral lysate of HIV-3 in association with on similar Proteins made from HIV-1 and / or HIV-2 for general use Diagnosing an infection or contact with a human immunodeficiency virus is used without the absolute identity the virus is detected. Such compositions could e.g. B. consist of a mixture of lysates of HIV-1, HIV-2 and HIV-3.

The present invention relates to Moreover labeled extracts of HIV-3 variants or compositions as previously described. The marking can be of any type, e.g. B. enzymatic, chemical, fluorescent or radioactive.

The invention also relates to a kit for the detection of antibodies directed against HIV-3 in biological fluids, which is a lysate of HIV-3 or a composition as described above, and a device for the detection of the immunological complexes formed includes.

• – Ein Lysat von HIV-3 oder eine Zusammensetzung einer der schon beschriebenen Typen, vorzugsweise in gereinigter Form, vorzugsweise an einen festen Träger wie eine Mikrotiter-Platte gekoppelt.
• – Ein Konjugat zwischen einem Enzym und einer Fraktion von Immunglobulinen, die an die nachzuweisenden Antikörper binden kann, oder ein Konjugat zwischen einem Enzym und dem bakteriellen Protein A oder G.
• – Ein Antigen zur Kontrolle, das keine Epitope besitzt, die von irgendeinem menschlichen Immunschwächevirus geteilt werden.
• – Geeignete Puffer zur Durchführung des Tests.
• – Ein geeignetes Substrat für das Enzym.

In the case of kits for the detection of specific antibodies by means of immuno-enzymatic methods, such a kit would contain:

• - A lysate of HIV-3 or a composition of one of the types already described, preferably in purified form, preferably coupled to a solid support such as a microtiter plate.
• - A conjugate between an enzyme and a fraction of immunoglobulins that can bind to the antibodies to be detected, or a conjugate between an enzyme and the bacterial protein A or G.
• A control antigen that has no epitopes shared by any human immunodeficiency virus.
• - Suitable buffers for performing the test.
• - A suitable substrate for the enzyme.

• – Ein geeignet markiertes Antigen oder eine Kombination von Antigenen der bereits beschriebenen Typen.
• – Protein A oder gegen menschliche Immunglobuline gerichtete Antikörper, vorzugsweise gekoppelt an einen unlöslichen Träger wie Protein A-Sepharose 4B (Pharmacia) oder einen gleichwertigen Träger.
• – Ein Antigen zur Kontrolle, das von gegen HIV-3 gerichteten Antiseren nicht erkannt wird.
• – Geeignete Puffer zur Durchführung des Tests.
• – Falls geeignet, Substrate zum Nachweis des enzymatisch markierten Antigens.

Kits for the detection of specific antibodies using labeled antigen would include the following:

• - A suitably labeled antigen or a combination of antigens of the types already described.
• - Protein A or antibodies directed against human immunoglobulins, preferably coupled to an insoluble carrier such as Protein A-Sepharose 4B (Pharmacia) or an equivalent carrier.
• - A control antigen that is not recognized by anti-HIV-3 antisera.
• - Suitable buffers for performing the test.
• - If appropriate, substrates for the detection of the enzymatically labeled antigen.

The invention also relates to nucleic acids which possibly marked and some of them at least from RNA of the retrovirus HIV-3 or derived from variants of this virus.

The invention also relates to the use of cDNA or parts of the cDNA or the recombinants containing them, which are characterized in that they contain at least a part of the cDNA which correspond to the entire RNA genome of the retrovirus HIV-3. Such cDNAs can be used as probes for the specific detection of sequences of HIV-3 in biological fluids, tissues and cells. The probes are preferably also labeled, either radioactive or chemical, alternatively using enzymatic, fluorescent or chemical luminescent labels that enable the detection of the probes. Preferred probes for the specific detection of HIV-3 and for the diagnosis of infection with HIV-3 are probes which contain all or part of the cDNA which is complementary to the genome of HIV-3. In this context, a particularly advantageous probe can be characterized as one which contains in particular the nucleic acid sequence which is contained in the clone iso 70-11 and which contains the viral sequence LTR-R which is linked to the 5 'and 3 'ends of the viral, genomic RNA and at the 5' and 3 'ends of the integrated, proviral DNA. In addition, sequences which correspond to the viral env gene and which are also contained in the clone iso 70-11 are particularly advantageous.

Nevertheless, the invention is to be understood so that the probes that can be used to diagnose HIV-3 infection are by no means limited to the probes described above. Rather, the invention encompasses all sequences derived from the genome of HIV-3 or its naturally occurring variants. All sequences encoding the viral core proteins ( gag gene ), the two forms of reverse transcriptase and the endonuclease ( pol gene ) and the two viral envelope glycoproteins ( env gene ) are included.

The invention also relates to sequences of nucleic acids of HIV-3 that in a recombinant nucleic acid have been incorporated which comprises a nucleic acid of a vector and into which the cDNA or part of this cDNA has been inserted. Such a construction can replicate the viral cDNA or its fragments in one Organism or a cell that is different from the natural Host are used so that sufficient amounts are used for diagnostic Purpose of the probe to be used.

• – Markierung der wie oben beschriebenen hergestellten Sonde mittels der zuvor beschriebenen Verfahren.
• – Inkontaktbringen der Sonde mit DNA aus infizierten Zellen oder mit viraler RNA aus infizierten Zellen oder biologischen Flüssigkeiten unter stringenten Hybridisierungsbedingungen, nachdem die DNA oder RNA vorzugsweise auf eine Membran aufgebracht worden ist und für die Sonde zugänglich gemacht worden ist.
• – Waschen der Membran mit einem Puffer unter Bedingungen, bei denen die stringenten Bedingungen aufrecht erhalten werden.
• – Nachweis der markierten Sonde, vorzugsweise mittels Autoradiographie in den Fällen, in denen die Sonde radioaktiv markiert worden ist, oder mittels einer geeigneten Immunnachweistechnik in den Fällen, in denen die Sonde chemisch markiert worden ist.

A probe produced in this way can be used for a diagnostic test for the specific detection of HIV-3, the test comprising the following essential steps:

• - Labeling the probe prepared as described above using the methods described above.
• Contacting the probe with DNA from infected cells or with viral RNA from infected cells or biological fluids under stringent hybridization conditions after the DNA or RNA has preferably been applied to a membrane and has been made accessible to the probe.
• - Washing the membrane with a buffer under conditions in which the stringent conditions are maintained.
• - Detection of the labeled probe, preferably by means of autoradiography in the cases in which the probe has been radiolabelled or by means of a suitable immune detection technique in the cases in which the probe has been chemically labeled.

The invention also relates to a process for the production of HIV-3 retrovirus variants, which is characterized in that human T4 lymphocytes or lymphocyte cell lines from humans of leukemic origin, which carry the phenotype T4 ⁺ , with lymphocytes or cell lines which have previously been infected with an isolate of the retrovirus HIV-3, cultured and that the retrovirus is obtained from the culture medium and purified.

BRIEF DESCRIPTION OF THE CHARACTERS

In the 1 to 16 The terms HIV-3 (ANT 70) and HIV-3 (ANT 70 NA) refer to two strains of a new HIV-3 virus isolated from a woman from Cameroon and her partner, by which the strain HIV-3 (ANT 70) has been deposited under ECACC V88060301.

1 shows a method to generate cleavage patterns of viral proteins.

2 shows differential antigen capture on virus-containing culture supernatants. Differential antigen capture was performed as described below. The solid line represents the results obtained using a broad spectrum anti-HIV-1 IgG, while the dashed line represents the results obtained using IgG which was quite specific for HIV-1. The titrations shown in columns AE are typical for HIV-1. Column F shows the result obtained with the supernatant containing HIV-3 (ANT 70).

3 shows differential antigen capture on HIV-1 and HIV-3 (ANT 70 NA) supernatants. Differential antigen capture was performed as described below. The solid line represents the results obtained using plates coated with the broad spectrum anti-HIV IgG, while the dashed line represents the results obtained using plates coated with an IgG that shows less cross-reactivity with types of HIV other than HIV-1.

4 shows the reactivity of anti-HIV sera against HIV-1 and HIV-2 western blot strips. The reactivities of three different sera give HIV-1 and HIV-2 western blot strips are shown. Sera: 1. Anti-HIV-1, 2. Anti-HIV-3 (ANT 70), 3. Anti-HIV-2 (Isolate 53). The molecular weights displayed are those specified by the manufacturer (Dupont Biotech).

5 concerns the comparison of the proteins gag and pol from different HIV-1 isolates, HIV-2rod and HIV-3 (ANT 70). The proteins were separated electrophoretically and blotted as described later. The blot was first incubated with a broad spectrum anti-HIV antiserum followed by an alkaline phosphatase labeled conjugate with anti-human IgG antibody so that the proteins were visualized.

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

6 shows a comparison of the proteins of HIV-3 (ANT 70) and HIV-3 (ANT 70 NA). Proteins were separated electrophoretically and blotted as described later. The blot was first incubated with the BSR antiserum. The blot was then incubated with an alkaline phosphatase-labeled conjugate with anti-human IgG so that the proteins were visualized. Lane 1: HIV-3 (ANT 70 NA), Lane 2: HIV-3 (ANT 70), Lane 3: HIV-1 (SF4). The apparent difference in intensities in lanes 1 and 2 is caused by different amounts of material applied.

7 refers to the ability of various human anti-HIV-1 sera to capture viral antigens. A number of human sera were diluted 1: 1000 and coated directly on microtiter plates. Detergent-treated culture supernatants containing HIV-1 (SF4), HIV-3 (ANT 70), HIV-2rod or HIV-2 (isolate 53) were incubated and the bound antigen was removed using a broad spectrum conjugate anti-HIV / horseradish peroxidase detected. Sera 1-7 were of African origin, while sera 8-11 were of European origin. The greater ability of African sera to capture non-HIV-1 antigen can be partly explained by their higher anti-p24 titers (data not shown).

8th shows the effect of coating an IgG dilution on binding the HIV isolates. Successive 2-fold dilutions were made from four different sera, starting at 1: 1000 dilution. These dilutions were used to coat microtiter plates. Detergent treated supernatants of HIV-1 (SF4), HIV-3 (ANT 70), HIV-2rod and HIV-2 (isolate 53) were diluted to approximately the same optical density on plates coated with antiserum were obtained as shown in lane B at a dilution of 1: 1000. Bound antigen was detected using the (broad-spectrum anti-HIV-IgG) / horseradish peroxidase conjugate.

9 shows antigen capture by virus isolates using human mouse polyclonal and anti-HIV-1 monoclonal antibodies.

The wells were coated and incubated as described in the text. The IgGs used were the following: 1. HIV polyclonal IgG of man, 2nd MAb CLB 59, 3rd MAb CLB 21, 4th MAb CLB 64, 5th MAb CLB 14, 6th MAb CLB 16, 7th MAb CLB 47, B. MAb CLB 13.6 (Anti-p18), 9. MAb CLB 19.7, 10. MAb CLB 13.4 (Antip18).

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

The lysates of HIV-1 (SF4), HIV-3 (ANT 70), HIV-2rod and HIV-2 (isolate 53) were electrophoresed on SDS polyacrylamide gels, blotted on nitrocellulose and with a high level of antiserum against HIV-1 Titer (lane A), antiserum to HIV-1 with a low titer (lane B), serum from the woman from which HIV-3 (ANT 70) had been isolated (lane C), serum from her partner from the HIV -3 (ANT 70 NA) had been isolated (lane D) and antiserum to HIV-2 from the person from whom HIV-2 (isolate 53) had been isolated (Lane E), incubated

11 shows the titrations of anti-HIV sera using an enzyme immunoassay.

Microtiter plates were made with lysates coated by HIV-1 (SF4), ANT 70 and HIV-2 (isolate 53). serum from a European infected with HIV-1 (left lane), antiserum against HIV-3 (ANT 70 NA) (middle lane) and antiserum against HIV-2 (Isolate 53) (right lane) were diluted twice, starting with a dilution of 1: 100, titrated on all three coated plates.

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

13 is a comparison of partial cleavage products of the gag and pol gene products of HIV-1 (SF4) (HIV-1 in the figure), HIV-3 (ANT 70) (isolate 70 in the figure) (ANT 70), HIV-2rod ( HIV-2 (LAV-2) in the figure) and HIV-2 (isolate 53) (isolate 53 in the figure). The terms p24 and p17 are used in the genetic sense to refer to the largest and the second largest viral core protein.

14 shows the hybridization of cDNA probes with viral RNA.

Viral RNA from HIV-1 (SF4), HIV-2rod and HIV-3 (ANT 70) as described in the Materials and Methods described are spotted on a membrane filter. The Filters were either under non-stringent (A) or under stringent Conditions hybridized and autoradiographed.

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

Genomic DNA from the cell lines listed below was subjected to restriction enzyme cleavage, electrophoretically separated and blotted. Hybridizations were performed as described in the Materials and Methods section. The samples are the following:

Lane A: Hybridization with iso 70-11.

Lane B: Hybridization with the BglII / SaII fragment from iso 70-11.

16 relates to the location of a number of restriction enzyme cleavage sites in the HIV-3 insert contained in the iso 70-11 DNA.

The fragment contains no internal cleavage sites for EcoRI, BamHI, HincII, or XbaI.

EXAMPLES

MATERIALS AND METHODS

Virus and cell culture

a) virus strains and cell lines

HUT-78 cells chronically associated with ARV-4 (HIV-1 SF4) are infected and were originally developed by J. Levy, San Francisco, USA (20) and non-infected HUT-78 cells were kindly provided by S. Speaker, Brussels, Belgium. LAV-2rod, originally by L. Montagnier, Paris, and CEM cells were developed by J. De Smeyter, Leuven, Belgium. Isolate 53, an isolate of HIV-2, was isolated in this laboratory (21).

b) virus isolations

Virus isolations have been similar Performed way as described by Levy and Shimabukuro (22), with changes introduced were. Lymphocytes from patients as well as from healthy donors were derived from heparinized whole blood on lymphoprep (Nyegaard and Co., Oslo, Norway) isolated and in RPMI 1640 containing 20 mM HEPES, 15% fetal calf serum (Gibco), 5 µg / ml Hydrocortisone (Merck), 75 U / ml IL-2 and 2 μg / ml Polybren (Aldrich), cultured.

Lymphocytes from healthy donors were stimulated with 2 µg / ml phytohemagglutinin (PHA, Wellcome) for 3 days before use. Fresh lymphocytes stimulated with PHA were added to the cultures for virus isolation every 3-4 days. The cultures were examined for cytopathic effect and immunofluorescence using a polyclonal comparison antiserum with broad specificity (23) and for the presence of antigen in the culture supernatants (Innotest VCA-HIV, Innogenetics). The broad specificity control antiserum (BSR) used was from a donor infected with HIV-1. It was shown experimentally that it had an extremely high titer (≥ 1,000,000 in an enzyme immunoassay based on a recombinant p24 protein of HIV-1) and that it was compatible with the gene products of gag and pol of the other types of HIV , especially of HIV-2, strongly cross-reactive. Reverse transcriptase was also examined, essentially as described (24).

To chronically infected, permanent Establishing cell lines have been virus-infected primary lymphocytes co-cultivated with cells from clone 8 of Molt 4 (25). These cells were kindly provided by N. Yamamoto, Yamaguchi, Japan disposal posed and observed for cell growth. The production of virus was done using the reverse transcriptase test and capture observed by antigen.

differential Antigen capture

A test system was developed by means of which a distinction between HIV-1 and other relatives human immunodeficiency viruses can be hit. The system is based on a comparison of the ability of two different polyclonal preparations from IgG, one of these preparations has a broad specificity against HIV due to its exceptionally high level Titers, especially opposite the main core protein, the other preparation has a lower titer and reacts preferentially with HIV-1, capture a virus treated with detergent in culture supernatants. The proof of the captured antigen is achieved in that a conjugate of broad specificity IgG with horseradish peroxidase is used.

The test mainly shows, but not exclusively, the core protein p24 after.

Against HIV-1 monoclonal antibodies

The spectrum of monoclonal used antibody has been described (26). The antibodies were raised against native, viral Proteins prepared in HIV-1 preparations broken up with Triton X-100.

Protein Analysis

a) Electrophoresis

Polyacrylamide gel electrophoresis viral proteins, essentially as described in Maizel (27), carried out.

b) protein blotting

Blotting was performed either in a Bio-Rad transblot cell at 400 mA for 4 hours using the carbonate buffer as described by Dunn (28) or using the LKB semi-dry blotting apparatus at 0.8 for 1 hour mA / cm ² in 48 mM Tris, 39 mM glycine, 0.0375% sodium dodecyl sulfate (SDS) and 20% methanol.

c) Generation of partial fission products

Viral proteins were identified in the 1 shown technique analyzed. The advantage was taken that corresponding proteins from the different isolates have similar molecular weights. The proteins were separated on 12.5% SDS-polyacrylamide gels together with a marker trace of proteins from ARV-4 which had been cut out after electrophoresis, blotted and incubated with an anti-HIV antiserum, so that the Positions of the viral proteins were recognizable. The marker blot, in turn, was used to locate the approximate positions of the viral proteins to be cleaved in the part of the gel stained with Coomassie blue. Horizontal gel slices containing the proteins were cut out, transferred into glass tubes and subjected to chemical cleavage.

1. Cleavage with cyanogen bromide

The gel disk was washed with 10 ml of a freshly made solution of 40 mg / ml CNBr (Merck) in 0.3 N HCl for 3 hours at room temperature incubated in a hood. After the incubation, the gel disc was also used SDS sample buffer for electrophoresis equilibrated in the second dimension.

2. BNPS-Skatol cleavage

The gel disk was washed with 10 ml of a freshly made saturated solution of 2- (2'-nitrophenylsulfenyl) -3-methyl-3'-bromoindolinine (BNPS-Skatol, Pierce) 3 hours at room temperature with the exclusion of light in 70% acetic acid; 30 Water containing 0.1% phenol incubated. After incubation the gel disk was washed repeatedly in SDS electrophoresis sample buffer equilibrated.

After the split, the individual Cut out traces from the gel slices, turned 90 ° and cut to a 10 to Given 20% SDS-polyacrylamide gradient gel. After completing the The gel was electrophoresed on nitrocellulose (Schleicher and Schuell) blotted and with PBS containing 1 mg / ml casein (Merck) blocked. Only fission products with molecular weights over 10 kD can be made visible because peptides with lower molecular weights do not bind effectively to nitrocellulose. The blots were made with a anti-HIV broad spectrum antiserum and then with a conjugate of antibodies directed against human IgG which Goat incubated with alkaline phosphatase (Promega). Partial fission products were then by reaction with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (Sigma) made visible.

Viral nucleic acids

a) Hybridization against viral RNA

Virus from culture supernatants was harvested by pelleting by centrifugation at 26,500 rpm over a period of 1 1/2 hours at 4 ° C through pillows of 20% sucrose and the virus in 10 mM Tris, pH 7.4, 10 mM NaCl , 10 mM EDTA containing 0.5% SDS was broken up. Aliquots of the broken virus were spotted on a Hybond H (Amersham) membrane in amounts corresponding to 5, 2.5, 1.25 and 0.62 ml of the original culture supernatants. The RNA deposited on the filter was fixed to the membrane by irradiation with ultraviolet light over a period of 2 minutes. The RNA bound to the filter was then subjected to hybridization with a cDNA probe from HIV-1 which had been labeled with 32P-dCTP by nick translation. The hybridization was carried out under stringent conditions performed in 3 × SSC, 0.5% milk powder, 1% SDS, 10% dextran sulfate and 50% formamide at 42 ° C for 18 hours. After hybridization, the filter was washed twice under stringent conditions in 0.1 × SSC and 0.1% SDS for 30 minutes. Hybridization was demonstrated by autoradiography at -70 ° C with intensifying screens. Similarly, hybridizations were also performed using a probe derived from the env region of HIV-2.

Hybridizations have also been under non-stringent conditions in 5 × SSC, 25% formamide, 5 × Denhardt's solution, 10 % Dextran sulfate and 100 μg / ml denatured salmon sperm DNA performed at 37 ° C overnight. The The filter was subsequently 4 × 15 Minutes in 5 × SSC, 0.1% SDS washed at room temperature and autoradiographed.

b) Preparation of the cDNA from ANT 70

Virus was made from 1 liter of culture supernatant pelleted using polyethylene glycol 6000, dissolved in PBS and by a 20% sucrose pillow is pelleted. The resulting pellet the virus was dissolved in 6 M guanidinium chloride in 20 mM sodium phosphate buffer, pH 6.5, containing 20 mM dithiothreitol and 0.5% NP-40, was broken up. Solid CsCl was added to a concentration of 2M. The solution containing the broken virus was placed on a cushion of 5.7 M CsCl containing 0.1 M EDTA layered. The viral RNA was by centrifugation at 25,000 for 20 hours at 15 ° C in one SW 28 rotor from Beckman pellets. After centrifugation, the RNA was turned again solved, extracted with phenol and precipitated with ethanol and 2 M LiCl.

A fifth of the viral produced RNA was used to take the first step in the synthesis of Control cDNA using a kit supplied by Amersham.

• 1. 1 Stunde in 2 × SSC, 0,1 % SDS bei Raumtemperatur.
• 2. 30 Minuten in 0,1 × SSC, 0,1 % SDS bei Raumtemperatur.
• 3. 20 Minuten in 2 × SSC, 0,1 % SDS bei 42 °C.
• 4. 20 Minuten in 0,1 × SSC, 0,1 % SDS bei 65 °C.

The cDNA synthesis was started using oligo (dT). The synthesis was carried out using the reverse transcriptase included in the kit. The second strand was synthesized using E. coli DNA polymerase I in the presence of RNase N so that the RNA strand of the hybrid was digested from RNA / DNA. Second strand synthesis was performed in the presence of ₃₂ P-dCTP so that the cDNA was labeled. The resulting cDNA was treated with T4 DNA polymerase to produce blunt ends. The cDNA was methylated to protect possible internal cleavage sites for EcoRI and was then coupled to EcoRI linkers (Amersham). The cleavage sites for EcoRI in the linker were then cleaved and the cDNA was size-separated on a 1.2% agarose gel. The region of the gel, which corresponds to a length of the cDNA of 500 to 2,000 base pairs, was cut out. The cDNA was eluted and cloned into the vector pUC13, which had previously been digested with EcoRI and dephosphorylated. After ligation, the DNA was used to transform competent cells MC1016 (lambda) from E. coli. 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. The screening of the colonies containing an insert of HIV-3 was performed by hybridization under moderately stringent conditions in 5x SSC, 5x Denhardt's solution, 0.2% SDS, 250 mg / ml denatured salmon sperm DNA at 65 ° C performed overnight. Hybridization was carried out using the SacI-EcoRI fragment of HIV-1. After hybridization, the filters were washed as follows:

• 1. 1 hour in 2 × SSC, 0.1% SDS at room temperature.
• 2. 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.
• 4. 20 minutes in 0.1 x SSC, 0.1% SDS at 65 ° C.

After washing the filters using reinforcing foils autoradiographed at –70 ° C.

Hybridizations were also under the non-stringent conditions as used for non-stringent hybridization the probe of HIV-1 and HIV-2 was used.

c) Analysis of the cDNA clones

Colonies showing a positive hybridization signal surrendered for pulled up the analysis. Plasmids were isolated, digested with EcoRI and subjected to agarose gel electrophoresis so that the present of an insertion confirmed and determined its size has been. Of 96 colonies analyzed, 17 were inserted found. five were for taken further analysis and were in a size range of about 800 to 1,600 base pairs in length.

d) determinations of sequences

Determinations of nucleotide sequences were carried out according to the Sanger di-deoxynucleotide method (Proc. Natl. Acad. Sci. USA 74: 5463-6467, 1977) using a kit supplied by Boehringer. Sequencing was carried out using 17mer M12 primers.

e) hybridizations between ANT 70 cDNA and HIV-1 and HIV-2 viral RNA

The cDNA clone of ANT 70, which the largest insertion contains (iso 70-11) for the Hybridization with the filter on which the viral RNAs are applied had been used.

The hybridization was under stringent Conditions in 3 × SSC, 0.5% milk powder, 1% SDS, 10% dextran sulfate and 50% formamide 18 hours at 42 ° C carried out. After hybridization, the filter was washed with 0.1 × SSC, 0.1 % SDS at 65 ° C (Washes from 2 - 30 Minutes), then the filter at -70 ° C with an intensifying screen was autoradiographed.

• – 15 Minuten mit 2 × SSC, 0,1 % SDS bei Raumtemperatur
• – 15 Minuten mit 0,5 × SSC, 0,1 % SDS bei Raumtemperatur
• – 15 Minuten mit 0,1 × SSC, 0,1 % SDS bei Raumtemperatur
• – 30 Minuten mit 0,1 × SSC, 1 % SDS bei 52 °C.

Genomic DNA was also made from cells infected with HIV-1 (SF4) isolate ANT 70 and also from uninfected control cells. After cleavage with a restriction enzyme, the genomic DNA was separated on a 0.8% agarose gel, transferred to a Hybond N membrane (Amersham) and then with ₃₂ P-labeled DNA from clone iso 70-11 under stringent conditions in 50% Formamide, 10% dextran sulfate, 3 × SSPE (1 × SSPE = 180 mM NaCl, 10 mM NaHP ₂ O ₄ , pH 7.4, 1 mM EDTA, pH 7.4), 1% SDS and 0.5% milk powder hybridized , After hybridization, the filter was washed as follows:

• - 15 minutes with 2 × SSC, 0.1% SDS at room temperature
• - 15 minutes with 0.5 × SSC, 0.1% SDS at room temperature
• - 15 minutes with 0.1 × SSC, 0.1% SDS at room temperature
• - 30 minutes with 0.1 × SSC, 1% SDS at 52 ° C.

The filter was then used an intensifying screen autoradiographed at –70 ° C.

RESULTS Virus isolation

Virus isolation from the blood of a woman from Cameroon and her partner was carried out as part of an ongoing study of heterosexual transmission of HIV. The woman is the partner of an HIV-seropositive man with general lymphadenopathy. The serum of the woman was moderately positive (ratio OD / cut-often 4.5) in the enzyme-linked immunoadsorption test (EIA, Organon Teknika) and had a low titer (1/40) in the immunofluorescence antibody test for HIV-1, but gave ambiguous results in the HIV-1 western blot test with clear bands at p33, p53 / 55 and p64, but with very weak bands at p24, gp41 and gp120. The woman had elevated serum IgG and IgM and a CD4 / CD8 ratio of 0.46. Virus was isolated by coculturing female lymphocytes with PHA-stimulated lymphocytes from healthy, non-infected donors. After 52 days in the culture, virus was detected in the culture due to the presence of syncytia and based on positive immunofluorescence, which was observed when an anti-HIV antiserum was incubated as a laboratory comparison with cells from the culture fixed with acetone was determined. The presence of reverse transcriptase in the culture supernatant (10 ⁴ cpm / ml, 27-fold background) was also detected. Cell-free culture supernatant was used to pass the virus through fresh lymphocytes. A CPE was again observed after 15 days and reverse transcriptase was detected in the supernatant. However, comparison of a detergent-treated culture supernatant of this isolate (ANT 70) with other isolates using differential antigen capture showed that this isolate was not HIV-1.

These results are in 2 shown. Due to the lower OD values, it is evident that, in contrast to the other isolates, ANT 70 was only moderately recognized by the IgG specific for HIV-1, like the other isolates, but was easily captured by IgG of broad specificity (lane F) , The other isolates, all of which were then shown to be strains of HIV-1 using an HIV-1 specific MAb (CLB MAb 14), all gave higher OD values on the plates coated with specific IgG in the Compared to plates that had been coated with broad specificity reference IgG.

An attempt was made the virus is infected by coculturing primary lymphocytes with ANT 70, with cells from clone 8 of Molt-4 in a permanent cell line. In The initial stage of infection had a significant cytopathic effect observed with formation of syncytium and cell death. Within several Cell growth was noted for weeks. The cells gave one positive immunofluorescence when used against HIV-directed broad-spectrum antiserums have been tested and the presence of antigen and reverse transcriptase was in the culture supernatant easily detectable.

Virus was similarly isolated from the partner of the woman from whom ANT 70 had been isolated (strain ANT 70 NA). The man suffered from lymphadenopathy and was classified according to the CDC system classified in class 3. The man also had elevated serum IgM and IgG and a CD4 / CD8 ratio of 0.4. On day 18, virus was detected in the culture supernatant. Detergent-treated supernatant containing this virus was also analyzed using differential antigen capture. It was found that he reacted in a manner similar to ANT 70 ( 3 ). Binding of the antigen derived from this isolate was again less with HIV-1 specific IgG than with broad specificity IgG.

The serum of the person from whom the isolate was obtained was incubated with Western blot strips from HIV-1 and HIV-2 (Biotech). Additional strips were also incubated with serum from a donor infected with HIV-1 and with serum from the person from whom HIV-2 (isolate 53) had been isolated. These results are in 4 shown. The serum from the person infected with ANT 70 NA reacted to a considerable extent with practically all proteins of HIV-1 including the envelope proteins. In contrast, the serum of the person infected with HIV-2 only cross-reacted with the protein gag p24, the endonuclease p34 and the reverse transcriptase p68. The anti-HIV-1 serum recognized only the protein gag p26 of HIV-2, while the serum of the carrier of the isolate ANT 70 NA recognized this protein and the reverse transcriptase of HIV-2.

Viral characterization proteins

Virus in the culture supernatant was precipitated using polyethylene glycol 6000 (Merck). The resulting material was redissolved and pelleted through a 15% sucrose cushion. The pelleted virus was dissolved in SDS sample buffer and analyzed by means of polyacrylamide gel electrophoresis and subsequent protein blotting. The blot, shown in 5 was incubated with a broad specificity anti-HIV serum so that the viral proteins were detected.

The BSR antiserum not only reacts with all viral proteins of HIV-1, but also cross-reacts with the gene products of the gag and pol genes of HIV-2. This antiserum also clearly recognizes the gene products of the gag and pol genes of ANT 70. It is clear that the molecular weights of the ANT 70 gene products are different from the molecular weights of both HIV-1 and HIV-2. The molecular weights of the various viral proteins are summarized in Table 1. The variability of the p17 / p18 protein of HIV-1 is based on an insertion of 6 amino acids, which occurs in some strains between positions 120 and 121 in the HXB2 sequence of HIV-1. A comparison of the proteins of ANT 70 and ANT 70 NA is shown in 6 shown. The molecular weights of all ANT 70 NA proteins are identical to the ANT 70 molecular weights.

To also the antigenic relationship between To further investigate HIV-1, HIV-2 and ANT 70 has been a number of African and European anti-HIV-1 sera diluted 1: 1000 and used to coat Microtiter plates for capture antigens.

Detergent-treated culture supernatant containing HIV-1, ANT 70, HIV-2 (LAV-2rod) and HIV-2 (isolate 53) was diluted and the ability of each antiserum to capture the four different isolates was examined. Representative results are in 7 shown. It can be seen from this experiment that the ability of the various sera to capture HIV-1 is in no way related to their ability to capture either HIV-2 or ANT 70. On the contrary, the ability of these sera to capture LAV-2rod, the prototype strain of HIV-2, correlates strongly with the ability of these sera to capture isolate 53, which is also a strain of HIV-2 but is an independent isolate is. These data indicate that ANT 70 is neither HIV-1 nor HIV -2.

In a series of similar antigen capture experiments, four African anti-HIV-1 sera were selected to bind their ability to bind HIV-1, ANT 70, HIV -2 (LAV-2rod) and HIV-2 (isolate 53) if the IgGs were coated in different dilutions. The culture supernatants were diluted so that approximately the same optical density was obtained when capturing plates coated with the IgG as in Lane B of 8th have been used. Dilutions of the four sera were coated and virus-containing supernatant was added. The assumption was made that similar viruses should give similar titration curves. Indeed react in 5 LAV-2rod and isolate 53 with the coated IgGs in a similar manner. On the other hand, ANT 70 gave more intense signals at higher dilutions of the IgGs than both isolates of HIV-2, and the shape of the curves obtained with ANT 70 is more similar to the curves obtained for HIV-1, except that the optical densities are consistently lower.

Cross reactivity of monoclonal antibodies Mouse directed against HIV-1 core protein p24

A spectrum of mouse monoclonal antibodies (MAbs) produced against HIV-1 core protein p24 was tested for the ability to cross-react with isolates ANT 70 and HIV-2. Basically, any spectrum of monoclonal antibodies to HIV-1 p24 can be used as long as the series includes monoclonal antibodies that react with different epitopes on the HIV-1 p24 molecule. ace Zites fluid containing the antibodies was diluted and used to coat microtiter plates. Detergent treated virus containing supernatants were then added to the coated wells. Bound antigen was detected using BSR-HIV IgGs conjugated to horseradish peroxidase. The results obtained are in 9 shown.

In control wells with a wide range of polyclonal IgGs had been coated, all of the virus-containing supernatants were optical Densities that exceeded the limits of the microtiter plate reader. However, if in the wells that with the different monoclonal antibodies were coated, tested, there was quite a different one Template. Previously done Studies indicated that everyone tested MAbs reacted against different epitopes on the p24 molecule. Exceptions were the MAbs CLB 59 and CLB 21, of which shown was that she recognize the same epitope. These two MAbs react as expected strong with HIV-1 and also give a measurable signal with ANT 70. However, they do not react with the HIV-2 strains. Two other MAbs, CLB 64 and CLB 14 bound HIV-1 well and showed weak affinity for ANT 70 and for the two isolates of HIV-2. In particular, it has been shown that the MAb CLB 14 recognizes all isolates from HIV-1 well (> 150 tested). This MAb must therefore bind to a very well preserved epitope, the remainder of which also in other human immunodeficiency viruses can. The other MAbs against p24 (CLB 16, 47 and 19.7) and two others, which are directed against the protein p18 of HIV-1 (CLB 13.4 and CLB 13.6), recognize neither ANT 70 nor the two isolates of HIV-2, but capture the corresponding antigens from HIV-1.

Response from human anti-HIV anti-viral proteins

Protein blots of viral proteins from HIV-1 (ARV 4), ANT 70 and HIV-2 (LAV2rod) were prepared after electrophoresis of extracts which had been detergent-soluble and mixed with various human sera ( 10 ) incubated. Lane A shows the reaction of the broad specificity reference serum from the laboratory with the three virus isolates. In lane B, an antiserum directed against HIV-1 was incubated with the blot and preferably recognizes the proteins of HIV-1. Serum from the woman from which ANT 70 was isolated (lane C) and her partner (lane D) were tested for their ability to recognize other viral isolates. These two sera preferably recognize ANT 70 including the gp120 coat protein of this virus. The partner's serum has a higher titer than the woman's serum from which ANT 70 was isolated and recognizes the gp41 of HIV-1. Both of these sera have a higher affinity for ANT 70 than for the isolates of HIV-1 or HIV-2. In contrast, the serum from the person from which isolate 53 of HIV-2 was isolated preferentially binds to HIV-2 proteins and recognizes the HIV-2 coat protein gp120 of this virus as well as the transmembrane protein gp41 (lane E ). It does not react with glycoproteins from HIV-1 or ANT 70. These results also indicate that ANT 70 is different from both HIV-1 and HIV-2.

Enzyme immunosorption tests using anti-HIV-1, anti-70 and anti-HIV-2 sera for titration of coated viral proteins were performed in microtiter plates containing viral lysates of HIV-1 (ARV-4), ANT 70 and HIV-2 (Isolat 53) had been coated. Two-fold dilutions of each serum, based on an initial 1: 100 dilution, were tested for their ability to bind the coated antigen. Bound antibody was detected using a horseradish peroxidase labeled goat anti-human IgG conjugate. These results are in 11 shown. The anti-HIV-1 serum preferentially recognized the proteins of HIV-1, but shows a significant cross-reaction with the proteins of ANT 70. The proteins of HIV-2 have hardly been detected. In contrast, the anti-70 serum preferentially recognized the proteins of ANT 70, showed cross-reactivity to the proteins of HIV-1 and reacted better with the wells coated with HIV-2 than did the anti-HIV-1 serum, which became clear due to the higher optical density values obtained. The anti-HIV-2 serum had a very low titer but still responded best with the HIV-2 proteins. No detectable signal was observed with the wells coated with HIV-1 or ANT 70. The inability to detect cross-reactions in this example is undoubtedly due to the low titer of this anti-HIV serum.

Analysis of Products of partial chemical cleavage of viral proteins

The two reagents used for chemical cleavage, cyanogen bromide and BNPS-Skatol, were chosen for their high specificities for methionine (29) and tryptophan (30). These two amino acids are also quite hydrophobic and therefore less likely to be found in epitopes on the outer surfaces of the protein molecule (31). The investigation of published amino sequences of the gene products of gag and pol from HIV-1 (32-36), HIV-2 (19), SIVagm (10), SIVmac (9), the infectious equine anemia virus (EIAV, 37) and Visna (38) shows that many of the positions of the Methio nin residues in these proteins and to an even greater extent the tryptophan residues are remarkably conserved ( 12 ), although there is little amino acid homology between some of these different isolates. In addition, the variation in these residues is very small or even nonexistent within the species, which is true at least for the case of HIV-1 (36) and probably also for all human and monkey immunodeficiency retroviruses.

The partial digestion patterns of the products of the gag and pol genes of HIV-1, ANT 70, HIV-2 (LAVrod) and HIV-2 (isolate 53) are shown in 13 shown.

Examination of the CNBr fission pattern for the Protein p24 from the four isolates shows that those for HIV-2 (LAV-2rod) and HIV-2 (Isolat 53) generated patterns are identical.

However, different patterns are observed for HIV-1 and for ANT 70. Thus there are significant differences in the positions of the methionine residues of the main core protein of HIV-1, ANT 70 and HIV-2. In the case of the core protein p17, differences between the two isolates of HIV-2 are observed. Analysis of the published sequence for HIV-2rod indicates that there is a methionine 18 amino acids away from the carboxyl end of this protein. We conclude that this methionine must be absent from isolate 53 in the corresponding protein. It is also possible to deduce the presence of a methionine near (10-15 amino acids) one of the ends of p16 of ANT 70 from the cleavage pattern. Cleavage of the retroviral reverse transcriptase with CNBr shows that the proteins from the two isolates of HIV-2 are again identical, while different patterns are observed for both the proteins of HIV-1 and those of ANT 70. In the case of endonuclease p31, derived from the 3 'part of the pol gene, similarities between all isolates can be inferred, although some minor differences are evident.

The cleavage of the proteins p24 of the four isolates with BNPS-Skatol results in very similar patterns. It will be out 8th clear that this is to be expected since the tryptophan positions in this protein are very highly conserved, especially for the retroviruses of human and monkey origin. We conclude that the tryptophan positions in the p25 protein of ANT 70 also correspond to this pattern. Examination of the samples, however, shows that minor differences can be observed, not in the overall appearance of the sample, but rather in the apparent molecular weights of the species produced by cleavage. In particular, differences in the apparent molecular weights of the central points in each sample are detected. As expected, the patterns for HIV-2 (LAV-2rod) and HIV-2 (isolate 53) are identical. However, the central point in the pattern for ANT 70 has a larger apparent molecular weight, while the central point for HIV-1 (ARV-4) has a lower molecular weight. Regarding p16, the positions of the tryptophans in the ANT 70 protein appear to be more similar to the positions of the tryptophans found in the HIV-2 protein. The protein p17 of HIV-1 has a tryptophan 16 amino acids from the amino terminus of the protein and gives an additional point which is not observed in the ANT 70 and HIV-2 patterns after cleavage with BNPS-Skatol. The tryptophan, corresponding to that at position 36 in the p17 protein of HIV-1, is conserved in all isolates.

That by splitting the reverses Transcriptase generated patterns from the four isolates are complex. However, it is again evident that the two isolates of HIV-2 are identical. For ANT 70 samples obtained that are neither the pattern obtained for HIV-1 nor the pattern for HIV-2 was obtained correspond. There will also be differences in the apparent molecular weights of the endonuclease cleavage products p31 observed, but those from the corresponding proteins of HIV-1, ANT 70 and HIV-2 generated patterns also show common features, which suggest a conserved structure.

Viral nucleic acids

a) Hybridization of HIV-1 and HIV-2 cDNA with viral RNAs

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

The HIV-2 probe used contains a sequence of approximately 100 base pairs from the env gene of HIV-2. Under stringent hybridization conditions, this probe hybridized only with RNA from HIV-2. Hybridization was not 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 70-11 cDNA clone was used as a probe to assess the level of homology at the nucleic acid level between the various isolates of the virus. The filter, on which aliquots of HIV-1, HIV-2 and ANT 70 had been applied, was subjected to hybridization under stringent conditions. The results are also in 14 shown. The experiment shows that cross-hybridization cannot be detected between any of the virus isolates under stringent hybridization conditions. The probe derived from ANT 70 hybridizes only with ANT 70. DNA of the cDNA clone iso 70-11 from ANT 70 was used for the hybridization of blots of restriction enzyme cleavages of cellular, genomic DNA from cells infected with HIV-1 and ANT 70 and from DNA from non-infected cells used. The results are in 15 shown. Hybridization was not observed in any trace other than those containing cleavages of genomic DNA from cells infected with ANT 70. This also indicates that the insert iso 70-11 does not originate from cellular sequences.

Additional hybridization was performed using the SacI-BgIII fragment from the iso 70-11 insert, which corresponds to the left end of the insert that is furthest from the sequences believed to be contained in the LTR. In analogy to HIV-1 and HIV-2, this fragment should correspond to the sequences of the env gene. Hybridization with this fragment gave the same bands that hybridization with the whole fragment gave.

c) Sequence analysis of the Clones iso 70-11

2. Sequenzen vom 3'-ORF von HIV-3 (Tabelle III)

The location of a number of restriction enzyme sites in the iso 70-11 insert of ANT 70 is shown in 16 shown. Subclones of the insert in pUC13 were generated and sequenced using the dideoxynucleotide method. The presence of a poly A tail confirmed that the iso 70-11 insert is from the 3 'end of the viral RNA. Adjacent to the poly A tail is the sequence corresponding to the R region of the 3 'LTR viral. The sequence contained in the ANT 70 cDNA and the viral sequences corresponding to it are shown below: 1. HIV-3 LTR (Table II)

2. Sequences from the 3'-ORF of HIV-3 (Table III)

DISCUSSION

As described in EP-A 0 345 375, we have a new retrovirus associated with human immunodeficiency from a woman from Cameroon (ANT 70) and her partner (ANT 70 NA) isolated. At the time when virus isolation was originally done the woman was only slightly seropositive, revealed in Western blot studies ambiguous results and was clinically symptom free. since At that time, the woman started experiencing some of the symptoms related to AIDS Develop complex (ARC). In contrast, her partner suffered which we also have a virus with the same properties as the original Isolate could isolate from lymphadenopathy and has had others since then for AIDS characteristic symptoms developed. This new isolate can therefore as a human immunodeficiency virus be considered. The fact that the same virus from sexual partners could also be isolated, suggests a type of transmission that is similar to that for human Is retroviruses.

The virus was first recognized as different from HIV-1 based on its altered ability to be captured in a differential antigen capture test. This test has proven to be extremely reliable in distinguishing between strains of HIV-1 and non-HIV-1 strains. The protein level also shows that this isolate is not HIV-1. This is due to 1) different molecular weights of the viral proteins, 2) different patterns of cross-reactivity with anti-HIV-1 antiserum compared to HIV-1, 3) a drastically reduced ability of mouse monoclonal antibodies against the core proteins p24 and p17 were produced by HIV-1 to be recognized, 4) a preferred recognition of proteins of ANT 70 compared to proteins of HIV-1 by antisera from the virus carrier, and 5) patterns of partial cleavage of the four highest - Preserved viral proteins that do not match the patterns obtained when HIV-1 proteins are subjected to the same treatment. Nevertheless, the sera of the two individuals infected with this virus recognize the gp41 coat protein of HIV 1. Based on the same criteria as set out above, it is also clear that ANT 70 is not HIV-2. In fact, the antigenic differences between ANT 70 and HIV-1 are less than the differences between HIV-2 and HIV-1. This is shown in particular in the 8th and 10 presented results clearly.

Further clear indications that ANT 70 is a unique virus different from HIV-1 and HIV-2, come from partial peptide mapping. We have shown that it significant differences in the most conserved viral Proteins there. The two isolates of HIV-2 used for comparison used essentially identical slit patterns, except in the case of cleavage with CNBr of the core protein p17. It should however, it is found that the Core protein p17 has greater variability than the protein p24, which is at least true in the strains of HIV-1 (34). Whether this is also for HIV-2 applies, remains until the determination of the sequence of more strains than have been analyzed so far, to be seen. Given the fact that ANT 70 closer to the antigen level is related to HIV-1 than to HIV-2 because of its higher grade the cross reactivity, even referring to the envelope protein gp41 extends, it can be seen, it was necessary to determine that ANT 70 is more than just a simple genetic variant of HIV-1. This was made possible that the Localization of some of the highest preserved amino acids in a number of viral proteins that have a genetic variation least accessible are examined. The fact that there are significant differences found in the split patterns indicates that HIV-1, HIV-2 and ANT 70 are three genetically different viruses. on the other hand the same series of experiments also showed similarities between them Viruses, which can mean all three from a common precursor descended.

The hybridization data also confirm the finding that ANT 70 is fundamentally different from both HIV-1 and HIV-2. As long as the conditions under which the hybridization is carried out are stringent, a distinction can be made between the three virus types be easily hit.

Analysis of the sequences of the cDNAs showed that the Insertion from the 3 'end of the viral Genome. An analysis of the homology between these sequences and the sequence of HIV-1 and HIV-2 shows that ANT 70, especially in the LTR sequences (approximately 70% homology), somewhat more closely related to HIV-1. The differences are nevertheless of such a size that the possibility it can be excluded that ANT 70 is simply a genetic variant of HIV-1. The 3'-LTR of ANT 70 also contains signal sequences the typical of are retroviral LTRs.

Sequences corresponding to the env gene of ANT 70 have been found to be even less related to the sequences of HIV-1 or HIV-2. While it is true that retroviral env genes often have genetic variability, the differences in sequence between the variants are rarely more than 10%. However, the degree of homology between ANT 70 on the one hand and HIV-1 and HIV-2 on the other hand turned out to be less than 60%. Such a difference in homology, along with the results of the hybridizations, shows that ANT 70 belongs to a new class of human immunodeficiency retroviruses.

The existence of a third type of human immunodeficiency virus has immediate epidemiological implications and implications for blood bank testing. Antibodies from humans infected with this virus have been shown to react preferentially with this virus, although these antibodies also cross-react with HIV-1 proteins. While it was possible to detect a positive response in enzyme immunoassays, immunofluorescence tests and Western blot tests based on proteins of HIV-1, the fact that the positive signal was due to a cross reaction necessarily implies that the sensitivity of such tests is Antibodies produced in response to this virus will be lower. This was done using the results of the enzyme immunoassay ( 11 ) clearly shown. In addition, a criterion for the seropositive reaction in the Western blot test is the presence of detectable antibodies against the protein gag and / or the protein pol and one of the coat proteins. Since a cross-reaction with the p24 and the envelope proteins of HIV-1 was observed in the case of the two persons infected with ANT 70 and ANT 70 NA, the conclusion which is drawn without exception is that these people are indeed infected with HIV-1 but for some reason do not develop high titers against HIV-1. It is therefore possible that this virus is more widespread than is currently perceived. From an epidemiological point of view, it is essential to develop specific diagnostic tests for this virus to determine the boundaries of the geographical area in Africa where the virus can be found and the extent to which this virus has been spread.

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Claims (15)

Variants of the HIV-3 retrovirus deposited with the European Collection of Animal Cell Cultures (ECACC) under the number V88060301, the variants having the following essential morphological and immunological properties: the virus shows a tropism for T4 lymphocytes; - the virus is cytotoxic for the lymphocytes infected with it; The virus has a diameter of approximately 120 nm; - The virus has a magnesium-dependent reverse transcriptase activity; - It can be cultivated in immortalized cell lines carrying T4 receptors; - Lysates of the virus contain a p25 protein which differs immunologically from the p19 protein from HTLV-I and the p24 proteins from HIV-1 and HIV-2, as by Western blot analysis or partial CNBr cleavage was determined; Lysates of the virus contain a gp120 protein which is immunologically different from the gp110 protein from HTLV-1, the gp120 from HIV-1 and the gp120 from HIV-2, as determined by Western blot analysis; - The lysate of the virus additionally contains a gp41 glycoprotein with a molecular weight of 40,000-45,000; - The genomic RNA of the HIV-3 variant does not hybridize with the sequences of HIV-1 or with the sequences of HIV-2 under stringent hybridization conditions; Lysates of the virus contain a p16 protein that is different from the p17 of HIV-1 and HIV-2, as determined by partial CNBr cleavage; Virus lysates contain a p31 endonuclease different from the p31 endonucleases of HIV-1 and HIV-2 as determined by partial CNBr cleavage; and wherein the variants have CNBr and BNPS-Skatol cleavage patterns of the p25 protein, the p16 protein, the p31 protein and the reverse transcriptase, as in 13 shown. HIV-3 retrovirus variant strain according to claim 1, that in its nucleic acid sequence comprises a consecutive R-range and a U3-range sequence, which are stringent Hybridization conditions with those as in Tables II and III nucleic acid sequences shown hybridized. Nucleic acid molecule that at least contains part of the cDNA, the entire RNA genome of an HIV-3 retrovirus variant strain according to claim 1 or 2 or the complementary strand thereof and that specifically with the nucleotide sequence of the under ECACC no. V88060301 deposited HIV-3 strain under stringent hybridization conditions hybridized. Nucleic acid molecule according to claim 3, which comprises the sequences which the entire RNA genome of the HIV-3 retrovirus strain according to claim 1 or 2. Part of the nucleic acid molecule cDNA of the entire RNA genome of the deposited under ECACC No. V88060301 HIV-3 retrovirus strain or the complementary strand thereof and that specifically with the nucleotide sequence of the HIV-3 strain below stringent hybridization conditions hybridized. Probe which comprises the nucleic acid molecule according to any one of claims 3 to 5 includes, which is optionally marked. Vector which comprises the nucleic acid molecule according to any one of claims 3 to 5 includes. The vector of claim 7, which is a Expression vector. Host cell containing a vector according to claim 7 or 8 is transformed. Process for the production of an HIV-3 retrovirus strain according to claim 1 or 2, characterized by culturing human T4 lymphocytes or derived permanent ones bearing the T4 phenotype Cell lines with lymphocytes or with previously with the HIV-3 retrovirus strain infected cell lines, and by extracting and purifying the retrovirus from the culture medium. Composition containing a total extract or lysate of the HIV-3 retrovirus strain comprises according to claim 1 or 2 or according to the method of claim 10 is manufactured. The composition of claim 11, which further a lysate of HIV-1, HIV-2, or a mixture of both. Kit containing the probe of claim 6 or a composition according to claim 11 or 12. Procedure for the detection of an HIV-3 retrovirus strain or its RNA in a biological fluid or tissue, marked by (a) contacting nucleic acids which in the biological fluid or tissue are included with the probe of claim 6 below stringent conditions, (b) washing the hybrid formed with a solution which maintains the stringent conditions, and (c) Evidence of the hybrid formed. Use of a nucleic acid molecule according to any one of claims 3 to 5, a probe according to claim 6 or one, the nucleic acid molecule or the Probe comprehensive kits for in-vitro detection of HIV-3 or for in-vitro diagnosis infection with HIV-3.