Different distribution of HIV type 1 genetic variants in European patients with distinct risk practices.

The use of two genetic markers has permitted the analysis of the distribution of two different human immunodeficiency virus type 1 (HIV-1) variants in patients of the homosexual (HO) and intravenous drug user (IDU) groups in distinct European countries. In Germany, Holland, and Italy the variants circulating in each risk group of HO and IDU patients were genetically distinguishable according to the genetic markers used. In contrast, in France and Spain, the same variant has been recovered from patients with different risk practices. These data highlight the diversity of the HIV-1 epidemic in Europe and the different patterns of HIV-1 variant distribution in European countries.

Characterization of a highly replicative intergroup M/O human immunodeficiency virus type 1 recombinant isolated from a Cameroonian patient.

A Cameroonian patient with antibodies reacting simultaneously to human immunodeficiency virus type 1 (HIV-1) group O- and group M-specific V3-loop peptides was identified. In order to confirm that this patient was coinfected with both viruses, PCRs with O- and M-specific discriminating primers corresponding to different regions of the genome were carried out with both primary lymphocyte DNA and the corresponding viral strains isolated from three consecutive patient samples. The PCR data suggested that this patient is coinfected with a group M virus and a recombinant M/O virus. Indeed, only type M gag sequences could be amplified, while for the env region, both type M and O sequences were amplified, from plasma or from DNA extracted from primary lymphocytes. Sequence analysis of a complete recombinant genome isolated from the second sample (97CA-MP645 virus isolate) revealed two intergroup breakpoints, one in the vpr gene and the second in the long terminal repeat region around the TATA box. Comparison of the type M sequences shared by the group M and the recombinant M/O viruses showed that these sequences were closely related, with only 3% genetic distance, suggesting that the M virus was one of the parental viruses. In this report we describe for the first time a recombination event in vivo between viruses belonging to two different groups, leading to a replicative virus. Recombination between strains with such distant lineages (65% overall homology) may contribute substantially to the emergence of new HIV-1 variants. We documented that this virus replicates well and became predominant in vitro. At this time, group O viruses represent a minority of the strains responsible for the HIV-1 pandemic. If such recombinant intergroup viruses gained better fitness, inducing changes in their biological properties compared to the parental group O virus, the prevalences of group O sequences could increase rapidly. This will have important implications for diagnosis of HIV-1 infections by serological and molecular tests, as well as for antiviral treatment.

Serologic and genetic characterization of HIV type 1 subtypes on Reunion Island.

The aim of this study was to determine the HIV subtypes present on Reunion Island, a French island located in the Indian Ocean, where the first case of AIDS was diagnosed in 1987. Paired sera and blood samples were collected between September 1996 and September 1997 from 53 HIV-1-positive patients. Subtyping was performed by serotyping with a previously described subtype-specific enzyme immunoassay (SSEIA) and by genotyping with the heteroduplex mobility assay (HMA). When samples gave uninterpretable results with either of the methods, or discordant results, the V3 env region was sequenced and genetic subtypes were determined by phylogenetic analysis. Genetic subtyping showed that 48 of 53 patients were infected with HIV-1 subtype B (90.5%). This high prevalence of subtype B on Reunion Island is probably due to the regular exchanges with metropolitan France. The other five patients were infected with subtype A (9.5%); they had been directly linked to African populations. Of the 48 subtype B samples, 44 (91.7%) were correctly subtyped by SSEIA and 43 (89.6%) by HMA. However, the SSEIA did not allow the subtyping of A strains in three of five patients. Thus, the SSEIA could be an alternative routine technique for screening subtype B versus nonsubtype B HIV-1 strains.

Increase of HIV-1 subtype A in Central African Republic.

The concomitant presence of five distinct HIV-1 subtypes and of unclassified HIV-1 was reported in Bangui, Central African Republic (C.A.R.) between 1990 and 1991. This previous study was conducted in individuals belonging to the C.A.R. Armed Forces (FACA) Cohort and in patients from the University Hospital of Baugui. To follow the HIV-1 subtype distribution in Bangui over time, we conducted a cross-sectional surveillance of HIV-1 subtypes between 1987 and 1997 in three groups of individuals in Bangui: 47 men belonging to the FACA Cohort, 38 patients from the CNHUB hospital, and 51 individuals consulting the sexually transmitted diseases (STD) clinic. One hundred and ten HIV-1 were subtyped by heteroduplex mobility assay (HMA) and/or sequencing of env regions encompassing the V3 domain. By comparing the HIV-1 distribution in two time periods (1987-1991 and 1991-1996) in the FACA cohort, we observed a significant increase of subtype A from 43.7% to 83.9%. This subtype distribution does not seem specific to the FACA cohort, in that subtype A accounted for 46.7% of the HIV-1 infections in CNHUB patients in the first time period studied and for 69.6% in the second time period. In STD patients, subtype A infections were predominant in 1995 (72.7%) and 1997 (89.7%). Subtype E viruses could be identified in the second time period, but represented only between 6.5% and 21.8% of the infections in the three groups of individuals studied. Other subtypes (B, C, H) and non-classified HIV-1 in C2-V3 were detected with only a 3.2% to 9.1% frequency for each in the second time period. Phylogenetic analysis excluded infection by a single source for the individuals included in the study. Our data demonstrate an increase in the proportion of HIV-1 subtype A infections in Bangui that raises the question of a preferential transmissibility of specific HIV-1 variants.

High diversity of HIV-1 subtype F strains in Central Africa.

In this paper, we studied the variability of HIV-1 subtype F strains in Africa. For 11 viruses, mainly of Central African origin, different parts of the genome were genetically characterized. For all strains the V3-V5 region of the envelope gene was sequenced, and for 7 strains, the entire envelope gene was studied. For 10 strains, the p24 region of the gag gene was also sequenced. For each region studied, three subgroups in the F subtype were identified, F1, F2, and F3. These three subgroups were supported by high bootstrap values and the intra- and inter-subgroup F distances were comparable to those obtained for the known subtypes A, B, C, D, E, G, and H. In subgroup F1, some African strains clustered with previously described strains from Brazil and Romania, suggesting an African origin of the HIV-1 epidemic in these countries. A more detailed analysis of the gag and the envelope sequences allowed the identification of four recombinant viruses. Our data show a high diversity among subtype F strains, suggesting the presence of new subtypes in the regions studied. If biological differences exist among subtypes, it is important that these subtypes be well defined. The data from our study show that there is a need to clearly identify the different subgroups within the F subtype.

Genetic characterization of the nef gene from human immunodeficiency virus type 1 group M strains representing genetic subtypes A, B, C, E, F, G, and H.

Most efforts to characterize sequence variation of HIV isolates has been directed toward the structural envelope gene. Few studies have evaluated the sequence variability of auxiliary genes such as nef. In this study 41 new HIV-1 strains, representing the majority of the described envelope subtypes of HIV-1 (A to H), were genetically characterized in the nef region. Phylogenetic analysis showed that 34 strains could be classified in the same subtype in nef and env, and 7 (19%) of the 41 new viruses were recombinants. For two of the seven strains, recombination occurred upstream of the nef gene, whereas for five of the seven strains recombination occurred within the nef gene with a crossover close to the 5' end of the LTR (long terminal repeat). The low intersubtype distance between subtype B and D in the nef gene confirms previous observations in the pol, env, and gag genes, which suggest a common ancestor for these subtypes. The majority of all the previously described functional domains in the nef gene were relatively conserved among the different subtypes, with only minor differences being observed. The myristoylation signal among the different subtypes, with only minor differences being observed. The myristoylation signal was less conserved for subtype C, with one or more amino acid changes being observed at positions 3, 4, and 5. The highly conserved acidic region (positions 62 to 65), critical for the enhancement of viral synthesis with an increased virus growth rate, was less conserved among the subtype G strains from our study. At least three epitopic regions of the nef gene have been defined and each can be recognized by CTLs under a variety of HLA restrictions; all were also relatively well conserved between the different genetic subtypes. Despite the relatively important genetic variation in nef sequences obtained among the different genetic subtypes, functional domains and CTL epitopes were relatively well conserved. In vitro and/or in vivo studies are necessary to study the relevance of the observed differences.

Clone-specific high-frequency retrotransposition of a recombinant virus containing a VL30 promoter in SV40-transformed NIH3T3 cells.

A recombinant virus, containing the promoter of a VL30 LTR and tagged with the neomycin gene as a selection and indicator marker, was constructed to investigate transposition events in NIH3T3 cells after SV40 transformation. This retroviral construct was transfected into psi/CRE packaging cells, and pseudovirions were used to infect NIH3T3 cells. Clones resistant to G418 bearing single-copy integrations of the recombinant virus were isolated and transformed by SV40 virus. Transpositions were detected through RFLPs with a neomycin probe and 'retrotransposition' was further confirmed by inverse PCR and DNA sequencing of transposed and parental copies. We found that: (1) retrotransposition of this recombinant virus occurred with a high frequency in a parental clone transformed with SV40 virus suggesting that the frequency of retrotransposition depended on the initial site of provirus integration; (2) the transposition frequency was independent of the transcription level of the recombinant construct; and (3) analysis of transposition-positive transformants showed that the high transposition frequency appeared to be associated with the induction of endogenous reverse transcriptases.

Study of the V3 loop as a target epitope for antibodies involved in the neutralization of primary isolates versus T-cell-line-adapted strains of human immunodeficiency virus type 1.

Previous studies characterized the third variable (V3) loop of the envelope gp120 as the principal neutralizing determinant for laboratory T-cell-line-adapted (TCLA) strains of human immunodeficiency virus type 1 (HIV-1). However, primary viruses isolated from infected individuals are more refractory to neutralization than TCLA strains, suggesting that qualitatively different neutralizing antibodies may be involved. In this study, we investigated whether the V3 loop constitutes a linear target epitope for antibodies neutralizing primary isolates. By using peptides representative of the V3 regions of various primary isolates, an early, relatively specific and persistent antibody response was detected in sera from HIV-infected patients. To assess the relationship between these antibodies and neutralization, the same peptides were used in competition and depletion experiments. Addition of homologous V3 peptides led to a competitive inhibition in the neutralization of the TCLA strain HIVMN/MT-4 but had no effect on the neutralization of the autologous primary isolate. Similarly, the removal of antibodies that bind to linear V3 epitopes resulted in a loss of HIVMN/MT-4 neutralization, whereas no decrease in the autologous neutralization was measured. The different roles of V3-specific antibodies according to the virus considered were thereby brought to light. This confirmed the involvement of V3 antibodies in the neutralization of a TCLA strain but emphasized a more pronounced contribution of either conformational epitopes or epitopes outside the V3 loop as targets for antibodies neutralizing primary HIV-1 isolates. This result underlines the need to focus on new vaccinal immunogens with epitopes able to induce broadly reactive and efficient antibodies that neutralize a wide range of primary HIV-1 isolates.

Identification of a new human immunodeficiency virus type 1 distinct from group M and group O.

A highly divergent HIV-1 isolate, designated YBF 30, was obtained in 1995 from a 40-year-old Cameroonian woman with AIDS. Depending on the genes studied, phylogenetic analysis showed that YBF30 branched either with SIVcpz-gab or between SIVcpz-gab and HIV-1 group M. The structural genes and tat, vpr, and nef of YBF30 are approximately equidistant from those of HIV-1 group M and SIVcpz-gab. In contrast, vif and rev are closer to HIV-1 group M, and vpu is highly divergent. Using a YBF30 V3 loop peptide enzyme immunoassay, we screened 700 HIV-1-positive sera collected in Cameroon; three reacted strongly with the YBF30 peptides and one was confirmed as being related to YBF30 by genetic analysis of a pol fragment. YBF30 is as distinct from SIVcpz-gab as it is from HIV-1 group M and can thus be considered as the prototype strain of a new human immunodeficiency virus group.

Evaluation of different V3 peptides in an enzyme immunoassay for specific HIV type 1 group O antibody detection.

Strategies to discriminate group O from group M infections need to be improved. We have developed and evaluated an HIV-1 group O V3 peptide-based enzyme immunoassay (PEIA) for specific HIV-1 group O antibody detection among HIV-1-infected patients. Synthetic peptides, derived from the amino acid sequences of the V3 loop of 15 different group O strains and 7 group O consensus sequences, were evaluated in a PEIA against a panel of genetically confirmed group O (n = 33), group M (n = 90), and HIV-1 antibody-negative sera (n = 17). The best-performing PEIA(s) were then used to screen 134 sera of European and 336 sera of Cameroonian origin for the presence of anti-HIV-1 group O antibodies. The reactivity of reference ("gold standard") sera to individual peptides in the PEIA resulted in the selection of five different peptides with sensitivities (sens), specificities (spec), and test efficiencies (TEs) in the range of 90 to 100%. Improvement of the PEIA was obtained with simultaneous reactivity of at least two different peptides in separate wells of an ELISA plate, together with stringent criteria for positivity. We were able to select seven peptide combinations each with a sens, spec, and TE of 96.9, 100, and 99.2%, respectively. None of the 134 European and 4 (1.2%) of the 336 Cameroonian samples sera were group O positive in the optimized HIV-1 group O PEIA; this was confirmed by the repeated presence of reactives, in agreement with the present knowledge of group O infection distribution. Finally, we were able to develop a strategy with a higher TE (99.2%) than the previously used ANT-70 (98.5%) and ANT-70/MVP5180 (95.7%). Our results show that optimal specificity rather than optimal sensitivity makes the V3 PEIA a sufficiently accurate epidemiological tool to be useful in estimating specifically group O infection among HIV-1-infected patients.

HIV type 1 diversity and the reliability of the heteroduplex mobility assay.

We investigated HIV-1 diversity by means of heteroduplex mobility assay (HMA) genotyping. We studied 199 samples from patients originating from 26 countries and living in France. The HMA successfully genotyped 182 (91%) of these samples, as follows: 77 (42%) subtype A, 57 (31%) subtype B, 5 (3%) subtype C, 5 (3%) subtype D, 8 (4%) subtype E, 22 (12%) subtype F, 5 (3%) subtype G, and 3 (2%) subtype H. We were not able to genotype 12 samples by means of the HMA. These latter strains were sequenced, and phylogenetic analyses revealed that they were highly divergent subtype A-, D-, or G-related strains. Eight (of 12) subtype D strains were indeterminate by HMA, owing to the broad intrasubtype diversity, suggesting that new reference subtype D plasmids are required, as previously proposed. Thirty-seven strains belonging to the different subtypes were sequenced, and the results showed perfect concordance with the HMA results. Interlaboratory quality controls confirmed the reliability of the HMA for HIV-1 subtyping, despite the extensive viral variability. However, plasmid selection must be continuously revised to cover viral diversification.

Cross-reactions between the cytotoxic T-lymphocyte responses of human immunodeficiency virus-infected African and European patients.

The great variability of protein sequences from human immunodeficiency virus (HIV) type 1 (HIV-1) isolates represents a major obstacle to the development of an effective vaccine against this virus. The surface protein (Env), which is the predominant target of neutralizing antibodies, is particularly variable. Here we examine the impact of variability among different HIV-1 subtypes (clades) on cytotoxic T-lymphocyte (CTL) activities, the other major component of the antiviral immune response. CTLs are produced not only against Env but also against other structural proteins, as well as some regulatory proteins. The genetic subtypes of HIV-1 were determined for Env and Gag from several patients infected either in France or in Africa. The cross-reactivities of the CTLs were tested with target cells expressing selected proteins from HIV-1 isolates of clade A or B or from HIV type 2 isolates. All African patients were infected with viruses belonging to clade A for Env and for Gag, except for one patient who was infected with a clade A Env-clade G Gag recombinant virus. All patients infected in France were infected with clade B viruses. The CTL responses obtained from all the African and all the French individuals tested showed frequent cross-reactions with proteins of the heterologous clade. Epitopes conserved between the viruses of clades A and B appeared especially frequent in Gag p24, Gag p18, integrase, and the central region of Nef. Cross-reactivity also existed among Gag epitopes of clades A, B, and G, as shown by the results for the patient infected with the clade A Env-clade G Gag recombinant virus. These results show that CTLs raised against viral antigens from different clades are able to cross-react, emphasizing the possibility of obtaining cross-immunizations for this part of the immune response in vaccinated individuals.

First case of mother-to-infant HIV type 1 group O transmission and evolution of C2V3 sequences in the infected child. French HIV Pediatric Cohort Study Group.

We report the first case of mother-to-infant transmission and follow-up for an HIV-1 group O virus from Cameroon. Isolates were obtained from the mother at delivery and from the child at birth and when 16 and 30 months old. We analyzed the viral evolution within mother and child by examining 51 sequences spanning C2V3 regions of the viral envelope gene. The mother carried two genotypes, v1 and v2. The genotype v1 was dominant in the child at birth, and persisted as a minor genotype at age 30 months. The genotype v2 was absent in the child sequences. The variability of the nucleotide sequences of the isolates from the child increased with age from 0.8 to 6%, and a novel genotype (v3) appeared at age 30 months. The nonsynonymous-to-synonymous mutation ratio increased with the age of the child, from 0.75 at birth to 1.86 at 30 months, indicating a high rate of fixation of amino acid changes in the child. The overall pattern was similar to that reported by Ganeshan et al. (J Virol 1997;71:663-677) for group M viruses infecting child with a slow-developing form of the disease.

Susceptibility of human immunodeficiency virus type 1 group O isolates to antiretroviral agents: in vitro phenotypic and genotypic analyses.

We investigated the phenotypic and genotypic susceptibility of 11 human immunodeficiency virus type 1 (HIV-1) group O strains to nucleoside and nonnucleoside reverse transcriptase (RT) inhibitors and protease inhibitors in vitro. Phenotypic susceptibility was determined by using a standardized in vitro assay of RT inhibition, taking into account the replication kinetics of each strain. HIV-1 group M and HIV-2 isolates were used as references. DNA from cocultured peripheral blood mononuclear cells was amplified by using pol-specific group O primers and cloned for sequencing. Group O isolates were highly sensitive to nucleoside inhibitors, but six isolates were naturally highly resistant to all of the nonnucleoside RT inhibitors tested. Phylogenetic analysis of the pol gene showed that these isolates formed a separate cluster within group O, and genotypic analysis revealed a tyrosine-to-cysteine substitution at residue 181. Differences in susceptibility to saquinavir and ritonavir (RTV) were not significant between group O and group M isolates, although the 50% inhibitory concentration of RTV for group O isolates was higher than that for the HIV-1 subtype B strains. The study of HIV-1 group O susceptibility to antiretroviral drugs revealed that the viruses tested had specific phenotypic characteristics contrasting with the group M phenotypic expression.

Diversity of the immunodominant epitope of gp41 of HIV-1 subtype O and its validity for antibody detection.

The immunodominant regions of the gp41 from 13 HIV-1 subtype O strains from Cameroon, 11 from France and one from Germany were sequenced. The amino acid sequences were compared to those of the 3 published HIV-1 subtype O isolates, ANT70, MVP-5180 and VAU. All HIV-1 subtype O isolates had a very conserved amino acid sequence in this region and showed a subtype O specific structure. Within the cysteine loop there was a positive charge of two basic amino acids, arginine and lysine. Only two strains (CM.6778 and CM.8161) showed an acidic amino acid in this loop. None of the isolates showed the same amino acid sequence in this immunodominant region. A 25 residue peptide from the immunodominant domain of gp41 of the MVP-5180 strain was synthesized, cycled to form the cysteine-loop and coated to microtiter plates. Antibody binding was detected by indirect ELISA using an enzyme labeled anti-human IgG. Out of 111 anti-HIV-1 positive specimens, collected mainly from Cameroonian HIV infected patients, only 10 were not reactive in this assay. The 42 anti-HIV-1 subtype O positive specimens gave all a reaction above cut off. Despite the diversity found in the amino acid sequences within the 25 isolates a peptide-based indirect ELISA representing the immunodominant epitope of the strain MVP-5180 successfully detected all the anti-HIV-O sera so far tested, pointing to the importance of adding such a peptide for correct identification of HIV-1 subtype O infected patients, while some assays without HIV-O specific antigens partially fail to detect all anti-HIV-O specimens.

Characterization and titration of an HIV type 1 subtype E chimpanzee challenge stock.

A subtype E human immunodeficiency virus type 1 (HIV-1) isolate from the Central African Republic (E/90CR402) was adapted to growth on chimpanzee peripheral blood mononuclear cells (PBMCs) by cocultivation of irradiated, infected human PBMCs with chimpanzee PBMCs. The resulting virus was passaged in chimpanzee PBMCs to generate a stock of chimpanzee-adapted virus. Although its V3 region sequence was identical to that of the parental isolate, the chimpanzee-adapted virus had a syncytium-inducing phenotype as opposed to the non-syncytium-inducing phenotype of the parental virus. After demonstrating in one animal each that the passaged virus could infect chimpanzees following intravenous (i.v.) or cervical inoculation, the i.v. infectious titer of the stock was determined. Exposure of three chimpanzees to different doses of the virus indicated that the titer was between 2 and 5 TCID50. Thus, the HIV-1 E/90CR402 chimpanzee challenge stock established persistent infections in chimpanzees by both the i.v. and genital routes and should be valuable for future HIV-1 vaccine studies to evaluate cross-protection between HIV-1 subtypes.

Serological and virological characterization of HIV-1 group O infection in Cameroon.

OBJECTIVES: To study the presence of HIV-1 group O infection among HIV-infected people in Cameroon and to further characterize the HIV-1 group O infections. DESIGN AND METHODS: During a 2-year survey (1994-1995), all samples tested positive in screening methods in the National Reference and Public Health Laboratory, Centre Pasteur, Yaoundé, Cameroon were identified as HIV-1 group M, HIV-1 group O or HIV-2 by using a serological algorithm. HIV-1 group M and HIV-1 group O were distinguished on the basis of competitive enzyme-linked immunosorbent assay (ELISA) reactivity against gp41 group M recombinant protein. HIV-1 group O infections were confirmed by using group O-specific V3 synthetic peptides. HIV-1 group O strains were isolated by lymphocyte cocultures, proviral DNA was amplified with specific primers, and sequencing was performed on the C2V3 and gag regions. RESULTS: Of the 8,331 screened samples, 3,193 were HIV-reactive, 2,376 (74%) of which were considered to belong to group M. The 817 (26%) that had reacted poorly or not at all against group M gp41 were further characterized: 10 were confirmed as HIV-2 and 82 as HIV-1 group O, the others being indeterminate (n = 285) or negative (n = 440). The frequency of group O relative to group M ranged from 1% in Far North province to 6.3% in the capital. There was no difference in sex, age or frequency of clinical manifestations between group M and group O infections. Group O infection was confirmed in a subset of cases by polymerase chain reaction (n = 14), with perfect concordance. Sequencing and phylogenetic analyses confirmed the high variability inside group O. CONCLUSIONS: Group O and group M epidemiological patterns are known to be similar so the reason for the lower prevalence of group O remains to be found. The wide distribution of group O infection in all Cameroonian provinces underlines the importance of further characterizing the epidemic spread and diffusion of this group.