Retroviral strains of the non-M, non-O HIV-1 group, in particular a strain designated YBE30, its fragments and also its uses as a diagnostic reagent and as an immunogenic agent.The HIV-1 viruses which differ both from the M group and the O group exhibit the following characteristics: little or no serological reactivity with regard to the proteins of the M and O groups and strong serological reactivity with regard to the proteins which are derived from the strain YBF30 according to the invention or the strain CPZGAB SIV; absence of genomic amplification when using primers from the env and gag regions of the M and O HIV-1 groups; genomic amplification in the presence of primers which are derived from the YBF30 strain according to the invention; and homology of the products of the envelope gene which is greater than 70% with regard to the YBF30 strain.

Description of the Invention

The present invention relates to retroviral strains of the non-M, non-O HIV-1 group, in particular a strain designated YBF30, to its fragments and to its uses as a diagnostic reagent and as an immunogenic agent.

The human acquired immunodeficiency viruses HIV-1 and HIV-2 are retrolentiviruses, which are viruses found in a large number of African primates. All these viruses appear to have a common ancestor; however, it is very difficult to prejudge the period at which these different viruses became separated from this precursor. Other viruses which are more distant, but which nevertheless belong to the same group, are found in other mammals (ungulates and felines).

All these viruses are associated with long infections; an absence of symptoms is the rule in monkeys which are infected naturally.

While the origin of HIV-2 appears to be clear on account of its strong homology with the Sooty Mangabey (West Africa) virus, no virus which is closely related to HIV-1 has been found in monkeys. The most closely related viruses are viruses found in two chimpanzees (CPZGAB SIV, ANT SIV).

All the lentiviruses have been found to exhibit substantial genetic variability, and the phylogenetic study of these variants, obtained from a large number of different geographic locations, has enabled 8 subtypes (clades) of HIV-1 to be distinguished, all of which are equidistant from each other. The clades are only a mathematical representation of the expression of the variability: phenetic analysis, which is based on the amino acids rather than on the nucleic acids, gives different results (Korber et al., 1994).

The demonstration of subtypes is in accord with a phylogenetic analysis which does not, to date, have any pathophysiological correlation but, instead, a geographical correspondence. This is because each subtype is mainly found in a particular geographical area. The B subtype is predominant in Europe and the United States whereas two subtypes, i.e. E and B, are found in Thailand and there is a strong correlation between the mode of transmission which, in actual fact, corresponds to a particular population and the subtype found. All the clades have been found in Africa and their distribution across the rest of the world reflects a probability of encounter between persons indulging in high-risk behaviour. The main clade, which is the main one because it is present in substantial proportions in Africa, is clade A. A very great degree of variability has been found in some African countries (G. Myers, 1994; P. M. Sharp et al., 1994). Several subtypes have been characterized in the western central African countries such as the Central African Republic (Murphy et al., 1993) and Cameroon (Nkengasong et al., 1994).

Finally, patients have been characterized who are carriers of viral variants of HIV-1, whose sera have posed detection problems for particular kits which are sold on the French market and whose confirmatory Western blots have been atypical (Loussert-Ajaka et al., 1994; Simon et al., 1994; PCT International Application WO 96/27013).

Analysis of these variants has confirmed the fact that the type 1 HIV viruses should be subdivided into two groups, i.e. the M (major) group and an O (outlier) group, which includes these isolates, as Charneau et al., 1994 had proposed. Analysis of the synonymous mutations/non-synonymous mutations ratio carried out on the sequences of the known O group viruses indicates that this new group is also ancient, even if no more ancient than the M group (Loussert-Ajaka et al., 1995). Its low prevalence to date, i.e. 8% of patients infected with HIV-1 in Cameroon (Zekeng et al., 1994) and 18 cases characterized in France, is thought to be due to factors which are purely epidemiological.

These two groups of HIV-1 form a tree which is in the shape of a double star (FIGS. 9 to 19, see Original Patent). Two isolates, i.e. CPZGAB SIV, characterized from a chimpanzee from Gabon (Huet et al., 1990) and CPZANT SIV, characterized from a chimpanzee in the Antwerp Zoo, possess sequences and genetic organizations which are very closely related to HIV-1 but which do not fall within either of these two groups and form two new branches on the phylogenetic tree.

The demonstration of new variants is important for developing sufficiently sensitive and specific reagents for detecting HIV infections, that is to say reagents which do not lead to false-negative or false-positive results, and for developing compositions which are protective in regard to subtypes which do not belong either to the M group or to the O group.

Consequently, the applicant has set itself the objective of providing a non-M, non-O strain, as well as sequences derived from this strain, which are suitable for detecting non-M and non-O HIV-1 variants and which do not lead to false-negative or false-positive results being obtained. In order to do this, the inventors have, in particular, established an algorithm for differentiating between, and confirming, group M and group O HIV-1 infections, thereby enabling them to select non-M, non-O variants.

The present invention relates to a non-M, non-O HIV-1 strain which exhibits the morphological and immunological characteristics of the retrovirus which was deposited on 2 Jul. 1996 under number I-1753 (designated YBF30) in the Collection Nationale de Cultures de Microorganismes (National Collection of Microorganism Cultures, 28 rue du Docteur Roux, 75724 Paris Cedex 15), kept by the Pasteur Institute.

A non-M, non-O variant is understood as meaning a type 1 HIV which cannot serologically and molecularly be recognized as belonging to either of these groups.

The present invention also relates to the complete nucleotide sequence of the strain as defined above (SEQ ID NO: 1) as well as to nucleic acid fragments which are at least 10 nucleotides in size and which are derived from the said strain.

Fragments of this type which may be mentioned are -- see Original Patent.
Such sequences can be used in the specific identification of a non-M, non-O HIV-1, and as diagnostic reagents, either alone or pooled with other reagents, for the differential identification of any HIV-1.

These sequences may, in particular, be employed in diagnostic tests which comprise either a direct hybridization with the viral sequence to be detected or an amplification of the said viral sequence, with these tests using, as primers or as probes, an oligonucleotide which comprises at least 10 nucleotides and which is included in any one of the above sequences, in particular one of the abovementioned sequences, SEQ ID NOs: 21-57.

The present invention also relates to HIV-1 viruses which are characterized in that they differ both from the M group and from the O group and exhibit the following characteristics: little or no serological reactivity with regard to proteins of the M and O groups and strong serological reactivity with regard to proteins which are derived from the YBF30 strain or the CPZGAB SIV strain; absence of genomic amplification when using primers from the env and gag regions of HIV-1 viruses of the M and O groups; genomic amplification in the presence of primers which are derived from the YBF30 strain, as defined above; and homology of the products of the envelope gene which is >70% with regard to the YBF30 strain.

The invention also relates to the use of the above described sequences for implementing a method of hybridization and/or of gene amplification of nucleic acid sequences of the HIV-1 type, with these methods being applicable to the in-vitro diagnosis of the potential infection of an individual with a virus of the non-M, non-O HIV-1 type.

This in-vitro diagnostic method is carried out using a biological sample (serum or circulating lymphocyte) and comprises: a step of extracting the nucleic acid which is to be detected and which belongs to the genome of the virus, which virus may possibly be present in the biological sample, and, where appropriate, a step of treating the nucleic acid using a reverse transcriptase, if this nucleic acid is in RNA form, at least one cycle comprising the steps of denaturing the nucleic acid, of hybridizing with at least one sequence in accordance with the invention and, where appropriate, extending the hybrid, which has been formed, in the presence of suitable reagents (polymerizing agent, such as DNA polymerase and dNTP), and a step of detecting the possible presence of the nucleic acid belonging to the genome of a virus of the non-M, non-O HIV-1 group type.

The following conditions are employed for the PCR using the primers derived from the YBF30 strain: extracting the lymphocytic DNA by means of the phenol/chloroform technique and quantifying it by spectrophotometry at a wavelength of 260 nm. All the amplifications are carried out using a Perkin Elmer 2400 thermocycler. the long (9 kb) PCRs are carried out using an XL PCR kit (Perkin Elmer) in accordance with the manufacturer's conditions and using the dNTP's, the buffers provided and Perkin Elmer's "hot start"; the amplification cycles of this long PCR are: 1 cycle of denaturation for 2 minutes at 94.degree. C., then 16 cycles: 15 seconds at 94.degree. C., 15 seconds at 55.degree. C., 8 minutes at 68.degree. C., then 24 cycles: 15 seconds at 94.degree. C., 15 seconds at 55.degree. C., 8 minutes at 68.degree. C., adding a further 15 seconds (incrementation) to each cycle. the nested PCRs are carried out on the amplification products of the long PCRs. The conditions for carrying out the nested PCRs are as follows: "Expand High Fidelity PCR System" Taq polymerase buffer and enzyme from Boehringer Mannheim in accordance with the manufacturer's instructions, dNTP and "hot start" from Perkin Elmer, 200 .mu.mol of each dNTP, 20 pmol of each primer in accordance with the invention, 5 .mu.l of DNA, 10 .mu.l of 10 .times.PCR buffer and 2.6 units of Taq polymerase in a volume of 100 .mu.l, amplification: one cycle of 2 minutes at 940.degree. C. followed by 38 cycles: 15 seconds at 940.degree. C., 15 seconds at 55.degree. C., a time of elongation at 72.degree. C. which varies in accordance with the size of the PCR product to be amplified (from 30 seconds to 2 minutes) and a final elongation cycle of 10 minutes at 72.degree. C.

The amplified product is preferably detected by direct sequencing.

The invention also relates to a peptide or a peptide fragment which is characterized in that it can be expressed by a non-M, non-O HIV-1 strain or using a nucleotide sequence as defined above, and in that it is capable: (1) of being recognized by antibodies which are induced by a non-M, non-O HIV-1 virus, as defined above, in particular the YBF30 strain or a variant of this strain, and which are present in a biological sample which is obtained following an infection with a non-M, non-O HIV-1 strain, and/or (2) of inducing the production of anti-non-M, non-O HIV-1 antibodies.

Peptides of this type which may be mentioned are, in particular, those which are derived from the YBF30 strain, in particular: that which is expressed by the gag gene (SEQ ID NO: 4), that which is expressed by the pol gene (SEQ ID NO: 6), that which is expressed by the vif gene (SEQ ID NO: 8), that which is expressed by the vpr gene (SEQ ID NO: 10), that which is expressed by the vpu gene (SEQ ID NO: 12), that which is expressed by the tat gene (SEQ ID NO: 14), that which is expressed by the rev gene (SEQ ID NO: 16), that which is expressed by the env gene (SEQ ID NO: 18), or one of its fragments such as a fragment of the V3 loop region, i.e. CTRPGNNTGGQVQJGPAMTFYNIEKIVGDIRQAYC (SEQ ID NO: 58), and that which is expressed by the nef gene (SEQ ID NO: 20), or a fragment of these peptides which are capable of recognizing the antibodies which are produced during an infection with a non-M, non-O HIV-1 as defined above.

The invention also relates to immunogenic compositions which comprise one or more translation products of the nucleotide sequences according to the invention and/or one of the peptides as defined above, obtained, in particular, by synthetic means.

The invention also relates to the antibodies which are directed against one or more of the above-described peptides and to their use for implementing methods for the in-vitro, in particular differential, diagnosis of the infection of an individual with a virus of the HIV-1 type using methods which are known to the skilled person.

The present invention encompasses all the peptides which are capable of being recognized by antibodies which are isolated from an infectious serum which is obtained after an infection with a non-M, non-O HIV-1 strain, and the peptides which are capable of being recognized by an antibody according to the invention.

The invention furthermore relates to a method for the in-vitro diagnosis of a non-M, non-O HIV-1 virus, which method is characterized in that it comprises bringing a biological sample, which has been taken from a patient, into contact with antibodies according to Claim 10, which may possibly be combined with anti-CPZGAB SIV antibodies, and detecting the immunological complexes which are formed between the HIV-1 antigens, which may possibly be present in the biological sample, and the said antibodies.

The invention also relates to a kit for diagnosing HIV-1, which kit is characterized in that it includes at least one reagent according to the invention.
 

Claim 1 of 10 Claims

1. An isolated nucleic acid, wherein the nucleic acid comprises SEQ ID NO: 5.
 

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