An improved algorithm for determining HIV type 1 subtypes in a primary laboratory in Uganda.

A pilot study was undertaken with the objective of developing a simple, economical, and efficient algorithm through which to subtype HIV-1 in a large epidemiological cohort study in Uganda. A peptide enzyme immunoassay (PEIA) employing both V3 and gp41 regions and a heteroduplex mobility assay (HMA) were evaluated in comparison with DNA sequencing. Of 146 samples selected, 115 (79%) were successfully sequenced. Taking sequence data as the "gold standard," other assays were compared with these data. The HMA correctly identified 95 (83%) of the samples, and only 1 sample was wrongly identified. The V3 PEIA alone and in combination with gp41 peptides correctly identified 76 and 78% of the samples, respectively; however, the number of wrongly identified samples was four times less with the combination compared with V3 peptides alone (4 versus 16%). The sensitivity, specificity, and positive and negative predictive values for serotype A and D samples were greater for the combination than V3 peptides alone. We have described a new algorithm to segregate subtypes A and D. This algorithm uses the two peptide assays followed by HMA and then DNA sequencing for untypable samples, giving an accuracy of 95% at a cost of 37 and 21% for consumables compared with subtyping all the samples by HMA or DNA sequencing, respectively. This proposed approach is suitable for epidemiological studies in Uganda and other regions with a predominance of A and D subtypes.

Characterization of sera from subjects infected with HIV-1 subtypes B and E in Thailand by antibody binding and neutralization.

The range and specificity of the humoral immune response to HIV-1 subtypes B and E was investigated in Thai samples. Sera from HIV-1-positive subjects, consisting of subtypes B (n = 24) and E (n = 138), were characterized in relation to the neutralization of primary isolates and T-cell line-adapted (TCLA) strains and binding to monomeric gp120, the CD4/gp120 binding site (BS), and V3 peptides. A subtype-specific pattern of antibody binding was observed with the exception of the CD4/gp 120MN BS. Neutralization of TCLA strains (n = 4) was strongly type-specific (p = .002); however, neutralization of primary isolates (n = 8) was weak and group specific. Thus, the subtype specificity of B and E sera in the neutralization of TCLA strains, but not primary isolates, supports the dominance of the V3 region in TCLA virus neutralization but does not support the distinction of subtypes B and E as discrete neutralization serotypes in Thailand.

PIP: The range and specificity of the humoral immune response to HIV-1 subtypes B and E were investigated in sera and plasma collected from 168 infected patients from Thailand in 1990-94. Specifically, samples were examined for the presence of binding antibody to env regions within monomeric gp120, the CD4/gp120 binding site, and the V3 domain as well as neutralizing antibodies to T-cell line-adapted (TCLA) and primary HIV-1 isolates from subtypes B and E. A subtype-specific pattern of antibody binding was observed with the exception of the CD4/gp120MN binding site. Although neutralization of TCLA strains was highly type-specific, neutralization of primary isolates was weak and group-specific. This finding supports the dominance of the V3 region in TCLA virus neutralization but fails to confirm the distinction of subtypes B and E as discrete neutralization serotypes in Thailand.

Lack of correlation between V3-loop peptide enzyme immunoassay serologic subtyping and genetic sequencing.

OBJECTIVE: To compare the performance of V3-loop peptide enzyme immunoassay (PEIA) methodologies from four different laboratories for subtyping HIV-1, and to determine the causes for the lack of correlation between V3-loop PEIA serotyping and subtyping by sequencing. MATERIALS AND METHODS: Synthetic peptides derived from the amino-acid consensus sequences of the V3-loop of group M strains representing genetic subtypes A-F as well as reference strains were evaluated in PEIA by four different laboratories for their ability to accurately determine the subtype in a panel of 85 sera obtained from persons infected with known HIV-1 subtypes (28 subtype A, 34 subtype B, four subtype C, 10 subtype D, seven subtype F, one each of subtype H and G). Furthermore, the V3 loop of the corresponding virus was compared with the V3 loop of the peptides used in PEIA. RESULTS: The correlation between HIV-1 subtyping by sequencing and V3-loop PEIA from the different laboratories varied considerably for the different HIV-1 subtypes: subtype A (46-68%), B (38-85%), C (75-100%), D (29-50%), and F (17-57%). A 70% agreement between PEIA and sequencing subtypes was observed for samples with the concordant presence of the same octameric sequences in the V3 loop of the virus and the V3 loop of the peptide used in PEIA; however, only 42% of specimens with different V3-loop octameric viral and peptide sequences yielded concordant results in V3-loop serotyping and genetic subtyping. CONCLUSION: Our results indicate that V3-loop PEIA methodologies used in different laboratories correlate poorly with genetic subtyping, and that their accuracy to predict HIV-1 subtypes in sera of Belgian individuals infected with different HIV-1 subtypes (A, B, C, D, F, G and H) vary considerably. The poor correlation between serotyping and genetic subtyping was partly due to the simultaneous occurrence of subtype-specific octameric sequences at the tip of the V3 loop of viruses belonging to different genetic subtypes.

A sudden epidemic of HIV type 1 among injecting drug users in the former Soviet Union: identification of subtype A, subtype B, and novel gagA/envB recombinants.

The former Soviet Union republics have experienced an explosive human immunodeficiency virus type 1 (HIV-1) epidemic among injecting drug users (IDUs), consisting mainly of subtype A viruses originated from a point source (Bobkov et al.: AIDS Res Hum Retroviruses 1997;13:1195-1201). To determine whether new HIV-1 subtypes have entered the IDU population, 46 samples derived from IDUs in Russia (n = 39) and the Ukraine (n = 7) were genotyped by heteroduplex mobility assay (HMA). It was shown that 83% of IDU HIV-1 strains found in both countries belong to genetic subtype A. However, env subtype B was also found in 17% of cases. The sequence data showed a marked intrasubtype homogeneity of HIV-1 (the average means of interpatient genetic distance were 1.1 and 1.7% [in the gag gene] or 1.8 and 2.3% [in the env gene] for subtype A and subtype B, respectively), confirming the hypothesis of a point source of virus for each subtype variant. Moreover, recombinant gagA/envB variants originating from those two strains were also found in two samples collected in the Kaliningrad region of Russia. In conclusion, our results suggest that two strains of HIV-1 belonging to different genetic subtypes, A and B, as well as gagA/envB recombinants between genomes of these strains, are now circulating simultaneously among IDUs in the former Soviet Union.

Serotyping of HIV type 1 infections: definition, relationship to viral genetic subtypes, and assay evaluation. UNAIDS Network for HIV-1 Isolation and Characterization.

V3 serotyping refers to a system based on binding of antibody in patient sera to V3-loop peptides derived from HIV-1 env genetic subtypes. The V3x serotype represents reactivity of serum from an HIV-1-infected patient (regardless of viral genetic subtype), which reacts preferentially to a V3 peptide derived from the X subtype sequence. We have classified HIV-1 serotypes, determined the relationship between the HIV-1 V3 serotypes and viral genetic subtypes in a large study (n = 125), and evaluated the performance of three different V3 peptide-binding assays. Seven HIV-1 V3 serotypes were identified: A, B, B-Br, B-Th, C, D, and E. Serotypes B-Br and B-Th represent sera that react specifically to peptides derived from Brazilian B (B-Br, GWGR) and Thai B (B-Th, GPGQ) strains. The HIV-1 V3 B, C, and E serotypes correlated closely with their viral env genetic subtypes; 19-26 of 32 B sera (59-79%), 3-4 of 4 C sera (75-100%), and 19-22 of 23 E sera (83-96%) were identified as serotypes B, C, and E, respectively. In contrast, two major V3 serotypes were classified in A sera: A (14-18 of 36 [40-50%]) and C (12-19 of 36 [33-54%]). Similarly, two major V3 serotypes were classified in D sera: B (6-10 of 20 [30-50%]) and D (9-12 of 20 [45-60%]). Serotyping of subtype E sera showed the best concordance with genetic subtypes by all assays. Overall, HIV-1 V3 serotyping produced consistent results among three laboratories. However, HIV-1 V3 serotypes do not distinguish all HIV-1 genetic subtypes. The relative biological significance of the V3 serotypes remains to be elucidated.

Neutralization of primary and T-cell line adapted isolates of human immunodeficiency virus type 1: role of V3-specific antibodies.

The role of the third variable domain (V3) of gp120 in the neutralization of primary and T-cell line adapted (TCLA) strains of human immunodeficiency virus type 1 (HIV-1) by serum from HIV-1-infected individuals was investigated. A primary virus isolate, M2424/4, when adapted to H9 cells, was more sensitive to neutralization on MT2 cells than the same stock passaged in PBMC. Neutralization of the PBMC-passaged stock by V3-specific MAbs was abrogated by addition of V3 (MN) peptide. However, exogenous V3 (MN) peptide failed to reduce the neutralization of this isolate on PBMC, or MT2 cells, by high titre anti-HIV-1 polyclonal human sera in contrast to the extensive reduction of neutralization by the same sera on MT2 cells using the prototype MN strain (4- to > or = 24-fold) and the TCLA M2424/H9 isolate (2- to 8-fold). These results indicate that the neutralization of primary virus isolates by serum from HIV-1-infected individuals is not significantly mediated by V3-specific antibodies.

HIV type 1 V3 serotyping of Tanzanian samples: probable reasons for mismatching with genetic subtyping.

HIV-1 V3 serotyping is used to classify immunodeficiency viruses on the basis of antibody binding to V3 peptides derived from env genetic subtypes. Although it shows a reasonable overlap, it has been reported to be distinct from viral genetic subtypes. The aim of this study is to determine the feasibility of HIV-1 serotyping to predict genetic subtypes in an East African setting, where multiple HIV-1 subtypes have coexisted for many years. HIV-1 genetic subtypes of 86 AIDS patients in Mbeya Town, southwest Tanzania, were determined, using env nucleic acid sequencing as the basis for comparison. Those data were compared with V3 serotyping results obtained by four different methodologies. Four HIV-1 genetic subtypes were identified, including A (25, 29%), C (47, 55%), D (13, 15%), and G (1, 1%). The sensitivity and specificity of those serotyping assays varied considerably: sensitivity for genetic subtype A (40-48%), C (52-96%), and D (9-31%); and specificity for genetic subtype A (77-95%), C (46-63%), and D (97-100%). We further tried to identify reasons for the discrepancies between serotyping results and genetic subtypes. By means of logistic regression analysis three amino acid residues within the V3 loop (positions 12, 13, and 19; V, H, and A for serotype A, I, R, and T for serotype C) were found to be most important for antibody binding; a deviation from the subtype-specific amino acids was highly related to mismatched results. In addition, we have shown that phenetic analysis of V3 amino acid sequence data could be used to predict the majority of V3 serotypes (93-94%). Our data demonstrated that for the majority of specimens HIV-1 V3 serotyping results closely match the subtype of the analyzed sample as revealed by the V3 loop amino acid sequence. However, our data demonstrate that HIV-1 serotyping is not sufficiently accurate to predict genetic subtypes in Tanzania, where subtypes A, C, D, and G are circulating. This was due to highly similar amino acid sequences throughout the prevalent genetic subtypes, which caused the inability of HIV-1 V3 serotyping to differentiate subtype A from C as well as D from C. Instead, the serotyping results reflect the frequency distribution of V3 serotypes. To investigate HIV-1 genetic subtypes in population-based studies in this African setting additional or modified algorithms are needed.

PIP: HIV-1 V3 serotyping is used to classify immunodeficiency viruses on the basis of antibody binding to V3 peptides derived from env genetic subtypes. Findings are reported from a study conducted to determine whether HIV-1 serotyping could be effectively used to predict genetic subtypes in an East African setting, where multiple HIV-1 subtypes have coexisted for many years. The HIV-1 genetic subtypes of 86 people with AIDS in Mbeya Town, southwest Tanzania, were determined, using env nucleic acid sequencing as the basis for comparison. Those data were then compared with V3 serotyping results obtained by analysis with tests manufactured by Behring and the Pettenkofer Institute, tests conducted by St. Mary's Hospital Medical School, tests conducted by Georg-Speyer-Haus, and tests conducted by Universite Francois Rabelais. The following HIV-1 genetic subtypes were identified: 25 cases of A (29%), 47 of C (55%), 13 of D (15%), and 1 of G (1%). The sensitivity and specificity of the serotyping assays varied considerably. These data indicate that HIV-1 serotyping is not accurate enough to predict genetic subtypes in Tanzania. This conclusion was reached based upon the highly similar amino acid sequences throughout the prevalent genetic subtypes, which caused the inability of HIV-1 V3 serotyping to differentiate subtype A from C as well as D from C. The serotyping results instead reflect the frequency distribution of V3 serotypes.

An HIV type 1 epidemic among injecting drug users in the former Soviet Union caused by a homogeneous subtype A strain.

Epidemiological data have demonstrated rapid growth of HIV-1 infections among injecting drug users (IDUs) in the Ukraine and Russia, during 1996. Here we describe the results of genetic analysis of isolates derived from 12 HIV-1-infected IDUs in different sites of Russia and the Ukraine. The blood samples were taken within a 1- to 2-month period after the first HIV-1-positive test. The results of the heteroduplex mobility assay as well as gag/env phylogenetic analysis reveal that all sequences belong to gag/env genetic subtype A. Moreover, interpatient genetic distances between the nucleotide sequences encompassing the C2-V3, the V4-V5, and p17-encoding regions within this group were low (the average means were 0.9, 1.3, and 0.4%, respectively). These data show a marked homogeneity of HIV-1, probably spreading during primary infection. It is possible that the current epidemic of subtype A HIV-1 among IDUs in the former Soviet Union is caused by a point source exposure.

Sequence analysis of an HIV type 1 env subtype E isolate from Thailand with discordant V3-loop serotyping.

PIP: HIV-1 genetic subtypes B and E are currently circulating in Thailand. Subtype E accounts for more than 90% of heterosexual transmission nationwide, while subtype B is transmitted mainly among IV drug users. This paper reports an HIV-1 Thai E isolate which yielded discordant results in serotyping and genotyping assays. An HIV-1-infected mother enrolled in Bangkok in a perinatal transmission study was identified independently as subtype B by V3 serotype by St. Mary's and HIV/AIDS Collaboration laboratories using different in-house assays. Her plasma was also screened for other HIV-1-specific immune responses, yielding a pattern of antibody reactivity similar to other subtype E sera. The genetic subtype of the isolate was identified by heteroduplex mobility assay (HMA) to further characterize it. Analysis results suggest that the woman was infected with HIV-1 subtype E. The env region encoding C2-V3 was subsequently sequenced to clarify the discordant results between the V3 serotype B and the genetic subtype E.^ieng

[Genotyping and phylogenetic analysis of HIV-1 isolates circulating in Russia]. / Genotipirovanie i filogeneticheskii analiz izoliatov VICh-1, tsirkuliruiushchikh v Rossii.

HIV-1 genetic subtypes were analyzed by the heteroduplex mobility assay (HMA) in 125 samples derived in 1995-1996 from residents of the European part of Russia. The results indicate the prevalence of six subtypes (A, B, C, D, G, and H) in the Russian Federation, with four types (A, B, C, and G) predominating (95%). The viruses belonging to subtypes A, B, and C spread via heterosexual contacts, subtype B mainly through homosexual intercourse. The majority of subtype G isolates are epidemiologically linked with previously reported nosocomial infection of children and their mothers with HIV-1 in Southern Russia. Nucleotide sequence analysis of env encoded region confirmed the results of HMA.

A pilot phase II study of the safety and immunogenicity of HIV p17/p24:VLP (p24-VLP) in asymptomatic HIV seropositive subjects.

The aim of this phase II study was to evaluate the safety, immunogenicity and tolerability of the yeast-derived virus-like particle immunogen, Ty.p24.VLP (p24-VLP), in HIV-antibody-positive asymptomatic volunteers. Fifteen informed and consented volunteers, with p24 Antibody titres >1/100, p24 Antigen <20 pg/l, and CD4>350 x 10(9)/l were enrolled. Five were immunized with aluminium hydroxide placebo, five with 25 microg, and five with 100 microg p24-VLP in Alum adjuvant at weeks 0 and 4 by the intramuscular route. Patients were followed for 16 weeks post vaccination and the main outcome assessments were CD4 and CD8 lymphocyte counts, p24 antigen and antibody, Ty antibody and quantitative viral cultures. No serious adverse events were observed in any of the groups. There were increases in CD4 counts in the treated groups but not in the controls, although these changes were not statistically significant. There were no significant intrasubject or intergroup changes in the other parameters, such as p24 antigen and antibody. No pattern of change in plasma viraemia was detected, and most cultures were negative. Therefore we conclude that p24-VLP immunizations of 25 microg and 100 microg are well tolerated, and the CD4 changes are encouraging, but higher doses and larger numbers are required to see if there are significant humoral or cellular responses, and extended phase II studies are now in progress.

Genetic heterogeneity of HIV type 1 in Russia: identification of H variants and relationship with epidemiological data.

HIV-genetic subtypes were analyzed in 130 subjects from the Russian Federation, by the HMA technique. Six subtypes were identified in heterosexuals, including A, B, C, D, G, and H; however, homosexual men were infected predominantly with the B subtype (33 of 35). The subtype A isolates were found in population of intravenous drug users. HMA successfully identifies 128/130 DNA samples; the phylogenetic analysis of the V1/V5 gp120 encoding region derived from another two samples demonstrated that these isolate belong to subtype H.

No evidence of antibody to human foamy virus in widespread human populations.

The first human foamy virus (HFV) to be described was isolated from nasopharyngeal carcinoma tissue from a Kenyan patient. Early seroepidemiology concluded that there was a significant infection rate, particularly among Africans. Awareness of foamy viruses as potential vectors has stimulated interest in the natural seroprevalence of HFV infection. We, therefore, investigated the prevalence of HFV infection in more than 5000 human sera collected from diverse populations. To maximize the chances of including the major antigenic epitopes, recombinant proteins derived from the HFV gag and env genes divided into three (the 5' amino terminal, the 3' carboxy terminal, and an internal overlapping region) were used as antigens in an ELISA. In contrast to most other seroepidemiological investigations of HFV infection, highly reactive sera identified by ELISA were subjected to further analysis by additional serological assays and, where PBMCs were available, PCR. None of the serum samples were confirmed as positive. It is worth noting that with our ELISA, the highest level of serum reactivity to HFV was found in subjects from Pacific islands (17%), and in Central Africa (34% in Malawi), areas previously cited as having a high level of HFV infection. Taken together with sequence analysis endorsing the phylogenetic closeness of HFV to SFV-6/7, these data strongly suggest that HFV is not naturally found in the human population.

Sequence analysis of the glycoprotein 120 coding region of a new human immunodeficiency virus type 1 subtype G strain from Russia.

PIP: This paper reports the sequencing of HIV-1 subtype G from the blood sample of a 39-year-old Russian male taken in November 1992. The heterosexual, White male and his wife living in Uzbekistan found in 1991 that they were infected with HIV-1. The man reported living in Mozambique during 1984-85 where he had no sexual relations with African residents, yet was admitted to the hospital on several occasions. However, in 1986-87, shortly after returning to the Soviet Union, he reported having a sexual relationship with a woman other than his wife. That woman and her husband were also found to be HIV-positive in 1991. The subject died in November 1993, and his wife in March 1995. His peripheral blood mononuclear cells were separated and kept at -70 degrees Celsius until used. When the blood sample was obtained, the man had a CD4 count of 10 cells/mcl, had lost more than 10% of his weight, and developed candidal esophagitis and chronic diarrhea associated with Cryptosporidia.^ieng