The c3-v4 region is a major target of autologous neutralizing antibodies in human immunodeficiency virus type 1 subtype C infection.

The early autologous neutralizing antibody response in human immunodeficiency virus type 1 (HIV-1) subtype C infections is often characterized by high titers, but the response is type specific with little to no cross-neutralizing activity. The specificities of these early neutralizing antibodies are not known; however, the type specificity suggests that they may target the variable regions of the envelope. Here, we show that cross-reactive anti-V3 antibodies developed within 3 to 12 weeks in six individuals but did not mediate autologous neutralization. Using a series of chimeric viruses, we found that antibodies directed at the V1V2, V4, and V5 regions contributed to autologous neutralization in some individuals, with V1V2 playing a more substantial role. However, these antibodies did not account for the total neutralizing capacity of these sera against the early autologous virus. Antibodies directed against the C3-V4 region were involved in autologous neutralization in all four sera studied. In two sera, transfer of the C3-V4 region rendered the chimera as sensitive to antibody neutralization as the parental virus. Although the C3 region, which contains the highly variable alpha2-helix was not a direct target in most cases, it contributed to the formation of neutralization epitopes as substitution of this region resulted in neutralization resistance. These data suggest that the C3 and V4 regions combine to form important structural motifs and that epitopes in this region are major targets of the early autologous neutralizing response in HIV-1 subtype C infection.

Polymorphisms in Nef associated with different clinical outcomes in HIV type 1 subtype C-infected children.

The human immunodeficiency virus type 1 (HIV-1) negative factor, or Nef, has a variety of functions that are important in viral pathogenesis. Sequence analysis has identified nef mutations that are linked to the rate of disease progression in adults and children infected with HIV-1 subtype B. Here we have sequenced and analyzed HIV-1 subtype C nef sequences from 34 children with rapid (RP) or slow progressing (SP) disease and identified polymorphisms associated with disease stage including motifs involved in specific pathogenic functions. Unlike subtype B, insertions and deletions in the N-terminal variable region were observed exclusively in SP children (8 out of 25). Strong positive selection pressures were found in sites of known functional importance among SP sequences, whereas RP had strong negative selection across the gene. A lineage analysis of selection pressures indicated weaker pressure across the nef gene in SP sequences bearing a deletion in region 8-12, suggesting this deletion has functional importance in vivo. Together these results suggest a differential adaptation of certain Nef functions related to disease progression, some of which may be attributable to immune-imposed pressures. These data broadly reflect previous studies on subtype B, corroborate the decreased cytopathicity of SP viruses, but also highlight potential subtype differences that require further investigation.

Resistance mutational analysis of HIV type 1 subtype C among rural South African drug-naive patients prior to large-scale availability of antiretrovirals.

Baseline HIV-1 resistance data are important for resistance monitoring purposes especially in regions initiating large-scale antiretroviral treatment programs. We examined 40 protease and 35 reverse transcriptase amino acid sequences of HIV-1 subtype C from drug inexperienced patients from rural settings in South Africa for resistance mutations. Samples were collected between 2001 and 2004 prior to the availability of antiretrovirals through public health institutions. Ninety-five percent of patients had no major mutations in the protease gene, although substitutions M46L (2.5%) and G73S (2.5%), which according to the Stanford Genotypic Resistance Interpretation Algorithm are considered major mutations, were detected. In addition, a high prevalence of minor mutations was observed in the protease, with at least three minor resistance-associated mutations in 37% of the isolates. An isoleucine insertion at codon 37 was detected in one sequence. Most of the RT sequences were wild-type, although V118I (8.5%) and Y318F (5.7%) associated with resistance to lamivudine and nevirapine, respectively, were observed. Our data suggest that major resistance mutations among the drug-inexperienced population in South Africa may be rare, and routine resistance testing before the initiation of therapy in this initial stage of the treatment program may not be necessary.

Mannose-rich glycosylation patterns on HIV-1 subtype C gp120 and sensitivity to the lectins, Griffithsin, Cyanovirin-N and Scytovirin.

Griffithsin (GRFT), Cyanovirin-N (CV-N) and Scytovirin (SVN) are lectins that inhibit HIV-1 infection by binding to multiple mannose-rich glycans on the HIV-1 envelope glycoproteins (Env). Here we show that these lectins neutralize subtype C primary virus isolates in addition to Env-pseudotyped viruses obtained from plasma and cervical vaginal lavages. Among 15 subtype C pseudoviruses, the median IC(50) values were 0.4, 1.8 and 20.1nM for GRFT, CV-N and SVN, respectively, similar to what was found for subtype B and A. Analysis of Env sequences suggested that concomitant lack of glycans at positions 234 and 295 resulted in natural resistance to these compounds, which was confirmed by site-directed mutagenesis. Furthermore, the binding sites for these lectins overlapped that of the 2G12 monoclonal antibody epitope, which is generally absent on subtype C Env. This data support further research on these lectins as potential microbicides in the context of HIV-1 subtype C infection.

Genetic characteristics of HIV-1 subtype C envelopes inducing cross-neutralizing antibodies.

This study aimed to characterize genetic features of HIV-1 subtype C envelope glycoproteins capable of eliciting cross-reactive neutralizing antibodies during natural infections. The gp160 sequences were determined for 36 HIV-1 subtype C isolates (donor viruses) from infected individuals residing in Malawi, Zimbabwe, Zambia and South Africa, whose sera displayed a range of cross-neutralizing activities against a panel of 5 subtype C and 5 subtype B viruses (panel viruses). Hierarchical clustering analysis of neutralization data of the panel viruses predicted phylogenetic relationships between subtype B and C panel viruses, suggesting some subtype-specific neutralization determinants. A similar comparison of subtype C donor viruses showed no significant correlation; however of three donor sequence pairs resolvable by phylogenetic analysis, two were also associated within the neutralization clustering dendrogram, suggesting that closely related viruses may elicit antibodies targeting common neutralization determinants. Significantly, viruses that had shorter V1-V4 loops induced antibodies that showed more neutralization breadth against the subtype C panel viruses (p=0.0135). This study indicates that that some structural features of envelope, such as shorter variable loops, may facilitate the elicitation of cross-reactive neutralizing antibodies in natural infections. Collectively these data provide some insights into design features of an envelope immunogen aimed at inducing neutralizing antibodies.

High specificity of V3 serotyping among human immunodeficiency virus type-1 subtype C infected patients with varying disease status and viral phenotype.

V3 serotyping is a technique for determining HIV-1 genetic subtype based on the binding of antibodies from patient sera or plasma to synthetic V3 peptides derived from subtype consensus sequences. Variation in the performance of this assay has been attributed to V3 sequence heterogeneity, the degree of which varies with patient disease progression, virus co-receptor usage, and genetic subtype. This study assessed the performance of a competitive peptide enzyme immunoassay (cPEIA) in samples from HIV-1 subtype C infected patients with varying disease profiles, including those with syncytium (SI) and non-syncytium-inducing (NSI) viruses. Out of 90 sera tested, 94.4% reacted strongly against the subtype C peptide. There was no significant difference in assay sensitivity among samples from advanced AIDS patients in which humoral immune response may be lower, nor among SI viruses which carry changes in the V3 sequence. Four samples were found to be cross-reactive with other subtypes and one acutely infected patient sample was non-reactive due to low anti-gp120 antibody titers. A significantly higher number of samples showed secondary reactivity to subtype A, compared to other subtypes (P < 0.005). In conclusion, the assay was able to identify HIV-1 subtype C infection with a high level of sensitivity (94%) irrespective of the stage of disease and therefore provides a valuable resource for the large-scale epidemiological monitoring of the spread of HIV-1 subtypes in South Africa.