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.

Impaired cytotoxic T lymphocyte recognition due to genetic variations in the main immunogenic region of the human immunodeficiency virus 1 NEF protein.

Human immunodeficiency virus (HIV) induces strong responses from human histocompatibility leukocyte antigen (HLA) class I-restricted cytotoxic T lymphocytes (CTL). In a previous report we identified an immunodominant region (amino acids 73-144) in the NEF protein that was recognized by CD8+ class I-restricted CTL of most asymptomatic individuals. Analysis of the 73-144 region by peptide sensitization, experiments using overlapping peptides corresponding to the LAI isolate identified the peptide sequences located between residues 73 and 82 or 84 and 92 and the peptide sequence between residues 134 and 144 as cognate peptides for HLA-A11- and HLA-B18-restricted epitopes, respectively. This report describes the variable demonstrable reactivities of CTL obtained from HLA-A11 or HLA-B18 seropositive, asymptomatic patients who all had a response to the virus NEF protein, but who did not always recognize appropriate cognate peptides. The high mutation rate of HIV probably facilitates the selection of mutants that can avoid the cellular immune response. We therefore analyzed the variability of these epitopes restricted by HLA-A11 and HLA-B18. We sequenced several viral isolates from HLA-A11 and HLA-B18 donors who recognized certain HLA-peptide complexes and from those who did not. A CTL sensitization assay was used to show that some mutations led to a great reduction in CTL activity in vitro. This might be due to failure of the mutated epitope to bind major histocompatibility complex class I molecule. A simple assay was used to detect peptides that promoted the assembly of class I molecules. Some of these mutations at major anchor positions prevented HLA-A11/peptide binding, and consequently impaired recognition of the HLA-peptide complex by the T cell receptor.

In vivo activation by ultraviolet rays of the human immunodeficiency virus type 1 long terminal repeat.

It has been previously shown in vitro that the human immunodeficiency virus type 1 long terminal repeat (LTR) is activated by ultraviolet irradiation. In order to analyze if a similar effect could occur in vivo, transgenic mice carrying the lacZ gene under the control of the viral LTR were irradiated at 280-300 and 254 nm. These mice spontaneously expressed the transgene in the epidermis and the lens of both adults and embryos. Irradiations caused a significant increase in skin beta-galactosidase activity. This phenomenon might be involved in viral activation and could be of interest in regard to the skin pathology observed during an HIV infection.

Isolation of cellular genes differentially expressed in mouse NIH 3T3 cells and a simian virus 40-transformed derivative: growth-specific expression of VL30 genes.

We constructed and screened a cDNA library made from simian virus 40 (SV40)-transformed NIH 3T3 cells, and we isolated cDNAs representing genes that are differentially expressed between the parental cell and its SV40-transformed derivative. We found only a small number of cDNAs representing such genes. Two isolated cDNA clones represented RNAs expressed at elevated levels in the transformed cell line in a manner relatively independent of growth conditions. The expression of two other cDNAs was growth specific because transformed cells and nonconfluent parental cells contained higher levels of the homologous RNAs than did confluent, contact-inhibited parental cells. Another cDNA was well expressed in confluent parental and confluent transformed cells, but not in nonconfluent cells. The expression of some of these cDNAs varied strikingly in different mouse cell lines. Thus the genotype or histories of different cell lines can also affect the expression of certain genes. Interestingly, the only cDNA isolated that was expressed exclusively in the transformed cell was from an SV40 message. We focused on a growth-specific cDNA which we show is derived from a mouse endogenous retrovirus-like family called VL30. We sequenced the 3' long terminal repeat (LTR) of this transcriptionally active VL30 gene. This LTR has good homology with other VL30 LTR sequences, but differences occur, particularly upstream of the VL30 promoter. We found that VL30 gene expression varied in different mouse cell lines such that C3H cell lines had very low levels of VL30 transcripts relative to NIH 3T3 cell lines. However, Southern analysis showed that both cell lines had about the same number of VL30 genes homologous to our probe and that the position of the majority of these genes was conserved. We discuss possible explanations for this difference in VL30 expression.

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.

Diversity of the V3 region of HIV in Paris, France.

OBJECTIVE: To carry out, within France, a large-scale molecular epidemiological investigation on the principal neutralizing determinant of HIV-1, located in the third variable region (V3) of the envelope protein. Such investigations are of the utmost importance in the identification and monitoring of the distribution and spread of different viral strains internationally. DESIGN: Using polymerase chain reaction (PCR), we examined the genetic variation of the V3 region sequences of 28 HIV-infected patients from Paris, France. RESULTS: Comparison of the Parisian V3 loop sequences with other published data indicates that the range of diversity in France is included within that of a large group that contains sequences from North America, the rest of Europe, Japan, India and Africa. Variability appears to be lower in the V3 loop than in its flanking regions. Five out of the six putative N-linked glycosylation sites show preferential alterations to charged amino acids. We report two motifs at the tip of the loop that have not been described previously. CONCLUSIONS: The structural homogeneity and the wide geographic representation of the major V3 group suggests that a common strategy could be applied to a large proportion of isolates in the development of a broad-spectrum HIV vaccine.

Lack of screening test sensitivity during HIV-1 non-subtype B seroconversions.

OBJECTIVE: To evaluate the serological consequences of HIV-1 group M diversity we studied the ability of screening tests to detect anti-HIV antibodies in early seroconverters infected by different HIV subtypes. SETTING: Virology Department, Bichat-Claude Bernard Hospital, Paris, France. DESIGN AND METHODS: Symptomatic patients with serial samples and with infective strains characterized by heteroduplex mobility assay. In each case, two sera were selected. The first (pre-seroconversion sample) was the last p24 antigen-positive/Western blot-non-reactive sample. The second (seroconversion sample) was the first Western blot-reactive sample. One second-generation enzyme immunoassay (EIA; Abbott) based on anti-human immunoglobulin (Ig) G-conjugate and three third generation EIA (Abbott; Enzygnost; Genscreen) based on the double antigen sandwich principle, detecting IgM and IgG, were used. RESULTS: Ten patients had subtype B strains and nine had non-B strains (seven were A, one E and one G). The Abbott third-generation test was more sensitive than the second generation test for pre-seroconversion subtype B samples (nine versus four out of 10; P < 0.05), but not for non-B subtypes; only two of the nine non-B sera tested were positive by both EIA. Positivity rates and optical densities differed (P < 0.05) between B and non-B subtypes in all third-generation EIA. There was no significant difference between the subtype B and non-B groups with regard to the interval between the pre-seroconversion sample and the seroconversion sample (subtype B, 6.7 +/- 2.6 days; non-B, 5.2 +/- 1.7 days). No significant difference in positivity rates and optical densities were found between B and non-B subtypes in these seroconversion samples. CONCLUSION: The shorter time since HIV infection required for sera to become reactive in third-generation EIA screening tests is due to better sensitivity for subtype B strains only. These results stress the importance of strict donor selection, the need to test screening kits against large panels of all subtypes, and the place of p24 antigen testing in closing the window of seroconversion.

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.

A peptide substitution in HIV-1 gp120 first hypervariable domain enhances its immunogenicity in mice.

The envelope (Env) protein from HIV-1 is the focus of several vaccine trials in humans. It could be considered for the optimization of Env vaccinal preparations to add within the molecule defined epitopes, for instance epitopes conserved among viral isolates from HIV-1, such as from Gag or Nef proteins. As a first step to this approach, we have constructed by in vitro mutagenesis HIV-1(LAI) Env gp120 molecules in which a 12-amino acid sequence in the first (V1) or the third (V3) hypervariable region was substituted by the hemagglutinin (HA) 307-318 peptide from the influenza virus, a dominant T helper cell epitope in humans. The proteins were produced by recombinant vaccinia viruses. They had kept their structural properties in terms of serological recognition and binding to CD4. Of note, we observed that the gp120 protein substituted in the V1 domain elicited a stronger serological immune response in mice compared to native or V3 substituted gp120. This indicates that gp120 can accommodate large substitutions without major structural perturbations and that, on the contrary, some of them could prove beneficial in terms of immunogenicity.

HIV type 1 diversity in northern Paris, France.

During a 6-month period, we studied the diversity of HIV-1 subtypes in 392 adult patients seen in Bichat-Claude Bernard Hospital, northern Paris, France. All the samples were serotyped and a subset was genotyped by means of HMA. Serotyping was performed with a new peptide subtype-specific EIA (SSEIA), based on in vitro competition for antibody binding between the representative V3 peptides of the different clades (A to E). HMA with plasmids from clades A to H gave unambiguous results on 105 of the 116 samples tested. The agreement between SSEIA and HMA was 36/41 for subtype B, 2/2 for subtype D, and 4/5 for subtype E. We found a discrepancy in the results between clade A and C: the patients with sera reacting to peptide C were confirmed by HMA as being infected by clade A strains. Three patients reactive with peptide A were infected by a subtype F. These results indicate that peptide cross-reactivity, even in the SSEIA format, hinders serotyping. In 11 samples, all from African patients, the subtype remained indeterminate because PCR or HMA failed. Caucasian patients (n = 223) were mainly infected by subtype B. HMA and/or SSEIA revealed non-subtype B infection in 14 Caucasians, who were infected by the sexual route overseas or in France. Patients originating from other countries (mainly in Africa) exhibited a broad strain diversity, with most of the different subtypes so far described being represented. This study confirms the frequency of subtype B strains in Caucasians living in France, but emphasizes the emergence of the different HIV-1 subtypes in Paris, together with the extent of strain trafficking. Discordances between serotype and genotype assays confirm that both tests require additional development.

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.

V3 loop sequence analysis of seven HIV type 1 group O isolates phenotyped in peripheral blood mononuclear cells and MT-2 cells.

HIV-1-infected individuals from which syncytium-inducing (SI) viruses are isolated most often progress more rapidly to AIDS than individuals carrying only non-syncytium-inducing (NSI) viruses. The syncytium-inducing capacity of virus isolates is commonly determined in conjunction to replication in MT-2 cells. Comparison of HIV-1 env sequences and a site-directed mutagenesis study have indicated that the presence of a positively charged amino acid at position 11 or 25 in the V3 loop is minimally required for the SI capacity of HIV-1 subtype B viruses. Studies have also shown a similar correlation between positively charged signature amino acids in the V3 loop and syncytium formation in MT-2 cells for HIV-1 subtypes A, D, and E. In the present study virus phenotype was determined and compared to the V3 loop sequence of seven HIV-1 group O isolates. Three of the HIV-1 group O isolates showed the NSI/non-MT-2 tropic phenotype and two showed the SI/MT-2 tropic phenotype, whereas two isolates presented an uncommon NSI/MT-2 tropic phenotype. The V3 loop of the two SI/MT-2 tropic isolates had a high net positive charge and contained a positively charged amino acid at position 11 or 25. The V3 loop of the two NSI/MT-2 tropic isolates had a low net positive charge and contained a single positively charged amino acid at position 37.

Neutralization of primary human immunodeficiency virus type 1 isolates: a study of parameters implicated in neutralization in vitro.

Various studies have reported that primary human immunodeficiency viruses seem to be more refractory to neutralization by HIV-positive sera than T cell line-adapted strains. In this study we also show that adaptation of the HIV-1SF-2 strain, produced in PBMCs, to the cell line CEM-SS renders this isolate sensitive to neutralization by almost all the sera tested. Further neutralization studies should thus focus on the development of an assay involving primary isolates in order to detect antibodies having a neutralizing activity in vivo. Neutralization protocols currently use either an antibody end-point dilution assay, which combines a fixed inoculum of virus with serial dilutions of antibody, or an infectivity reduction assay, which uses serial dilutions of virus with a single dilution of antibody. We have developed an assay designed for studying the neutralization of primary isolates that combines these two approaches. Performing the assay on PBMCs allows all primary isolates to be analyzed, not just those multiplying in T cell lines. The neutralizing titer measured on PBMCs for human HIV-positive sera is low, but reproducible and independent of the virus titer in a given experiment. It can be increased about five-fold by changing the temperature and duration of virus-serum interaction (overnight at 4 degrees C instead of 1 hr at 37 degrees C). These results emphasize the need for a relevant neutralization assay involving primary isolates and primary cells for a better understanding of the role of humoral response in HIV infection.

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.

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.

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.

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.

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.

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.