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Although the role of glycans in shielding neutralizing epitopes has long been known (9-11), it has only recently become clear that many BCN responses directly target glycans, including the one [...], Neutralizing antibodies are likely to play a crucial part in a preventative HIV-1 vaccine. Although efforts to elicit broadly cross-neutralizing (BCN) antibodies by vaccination have been unsuccessful (1-3), a minority of individuals naturally develop these antibodies after many years of infection (4-7). How such antibodies arise, and the role of viral evolution in shaping these responses, is unknown. Here we show, in two HIV-1-infected individuals who developed BCN antibodies targeting the glycan at Asn332 on the gp120 envelope, that this glycan was absent on the initial infecting virus. However, this BCN epitope evolved within 6 months, through immune escape from earlier strain-specific antibodies that resulted in a shift of a glycan to position 332. Both viruses that lacked the glycan at amino acid 332 were resistant to the Asn332-dependent BCN monoclonal antibody PGT128 (ref. 8), whereas escaped variants that acquired this glycan were sensitive. Analysis of large sequence and neutralization data sets showed the 332 glycan to be significantly under-represented in transmitted subtype C viruses compared to chronic viruses, with the absence of this glycan corresponding with resistance to PGT128. These findings highlight the dynamic interplay between early antibodies and viral escape in driving the evolution of conserved BCN antibody epitopes.

Abstract Background The cytotoxic T-lymphocyte immune response is important in controlling HIV-1 replication in infected humans. In this immune pathway, viral peptides within infected cells are presented to T-lymphocytes by the polymorphic human leukocyte antigens (HLA). HLA alleles exert selective pressure on the peptide regions and immune escape mutations that occur at some of the targeted sites can enable the virus to adapt to the infected host. The pattern of ongoing immune escape and reversion associated with several human HLA alleles has been studied extensively. Such mutations revert upon transmission to a host without the HLA allele because the escape mutation incurs a fitness cost. However, to-date there has been little attempt to study permanent loss of CTL epitopes due to escape mutations without an effect on fitness. Results Here, we set out to determine the extent of adaptation of HIV-1 to three well-characterized HLA alleles during the initial exposure of the virus to the human cytotoxic immune responses following transmission from chimpanzee. We generated a chimpanzee consensus sequence to approximate the virus sequence that was initially transmitted to the human host and used a method based on peptide binding affinity to HLA crystal structures to predict peptides that were potentially targeted by the HLA alleles on this sequence. Next, we used codon-based phylogenetic models to quantify the average selective pressure that acted on these regions during the period immediately following the zoonosis event, corresponding to the branch of the phylogenetic tree leading to the common ancestor of all of the HIV-1 sequences. Evidence for adaptive evolution during this period was observed at regions recognised by HLA A*6801 and A*0201, both of which are common in African populations. No evidence of adaptive evolution was observed at sites targeted by HLA-B*2705, which is a rare allele in African populations. Conclusion Our results suggest that the ancestral HIV-1 virus experienced a period of positive selective pressure due to immune responses associated with HLA alleles that were common in the infected human population. We propose that this resulted in permanent escape from immune responses targeting unconstrained regions of the virus.

Abstract Background Positive selection pressure acting on protein-coding sequences is usually inferred when the rate of nonsynonymous substitution is greater than the synonymous rate. However, purifying selection acting directly on the nucleotide sequence can lower the synonymous substitution rate. This could result in false inference of positive selection because when synonymous changes at some sites are under purifying selection, the average synonymous rate is an underestimate of the neutral rate of evolution. Even though HIV-1 coding sequences contain a number of regions that function at the nucleotide level, and are thus likely to be affected by purifying selection, studies of positive selection assume that synonymous substitutions can be used to estimate the neutral rate of evolution. Results We modelled site-to-site variation in the synonymous substitution rate across coding regions of the HIV-1 genome. Synonymous substitution rates were found to vary significantly within and between genes. Surprisingly, regions of the genome that encode proteins in more than one frame had significantly higher synonymous substitution rates than regions coding in a single frame. We found evidence of strong purifying selection pressure affecting synonymous mutations in fourteen regions with known functions. These included an exonic splicing enhancer, the rev-responsive element, the poly-purine tract and a transcription factor binding site. A further five highly conserved regions were located within known functional domains. We also found four conserved regions located in env and vpu which have not been characterized previously. Conclusion We provide the coordinates of genomic regions with markedly lower synonymous substitution rates, which are putatively under the influence of strong purifying selection pressure at the nucleotide level as well as regions encoding proteins in more than one frame. These regions should be excluded from studies of positive selection acting on HIV-1 coding regions.

Background: Women in the CAPRISA 004 trial assigned to use 1% tenofovir (TFV) microbicide gel, who became HIV-1 infected, had higher viral load set-point and slower antibody avidity maturation compared with placebo participants. We investigated whether TFV gel was selected for viruses with altered genetic characteristics. Setting: The participants of the CAPRISA 004 trial (n = 28 TFV and 43 placebo) were from KwaZulu-Natal Province, South Africa and were infected with HIV-1 subtype C. After HIV-1 diagnosis, they were recruited into the CAPRISA 002 cohort. Methods: We analyzed gag sequences from the earliest time point post infection (within 3 months of estimated time of infection). Transmission index was measured using a model which predicts the likelihood of an amino acid to be transmitted. Phylogenetic distance from a regional consensus sequence was calculated from a maximum likelihood phylogenetic tree. Results: Transmission index and distance from the most common (consensus) sequence have been shown to be markers of transmission fitness. We found that viruses infecting TFV gel recipients were closer to the consensus sequence of regional strains (P = 0.003) and had higher transmission index (P = 0.01). The transmission index was weakly correlated with concomitant viral load (Spearman r = 0.22, P = 0.06). Conclusion: Decreased acquisition risk may have increased the barrier to infection therefore selecting for fitter, more consensus-like viruses. Such virus fitness effects will need to be considered for future pre-exposure prophylaxis and vaccine trials. [ABSTRACT FROM AUTHOR]

Background Identification of the epitopes targeted by antibodies that can neutralize diverse HIV-1 strains can provide important clues for the design of a preventative vaccine. Methods We have developed a computational approach that can identify key amino acids within the HIV-1 envelope glycoprotein that influence sensitivity to broadly cross-neutralizing antibodies. Given a sequence alignment and neutralization titers for a panel of viruses, the method works by fitting a phylogenetic model that allows the amino acid frequencies at each site to depend on neutralization sensitivities. Sites at which viral evolution influences neutralization sensitivity were identified using Bayes factors (BFs) to compare the fit of this model to that of a null model in which sequences evolved independently of antibody sensitivity. Conformational epitopes were identified with a Metropolis algorithm that searched for a cluster of sites with large Bayes factors on the tertiary structure of the viral envelope. Results We applied our method to ID50 neutralization data generated from seven HIV-1 subtype C serum samples with neutralization breadth that had been tested against a multi-clade panel of 225 pseudoviruses for which envelope sequences were also available. For each sample, between two and four sites were identified that were strongly associated with neutralization sensitivity (2ln(BF) > 6), a subset of which were experimentally confirmed using sitedirected mutagenesis. Conclusions Our results provide strong support for the use of evolutionary models applied to crosssectional viral neutralization data to identify the epitopes of serum antibodies that confer neutralization breadth. [ABSTRACT FROM AUTHOR]

Certain immune-driven mutations in HIV-1, such as those arising in p24Gag, decrease viral replicative capacity. However, the intersubtype differences in the replicative consequences of such mutations have not been explored. In HIV-1 subtype B, the p24Gag M250I mutation is a rare variant (0.6%) that is enriched among elite controllers (7.2%) (P = 0.0005) and appears to be a rare escape variant selected by HLA-B58 supertype alleles (P < 0.01). In contrast, in subtype C, it is a relatively common minor polymorphic variant (10 to 15%) whose appearance is not associated with a particular HLA allele. Using site-directed mutant viruses, we demonstrate that M250I reduces in vitro viral replicative capacity in both subtype B and subtype C sequences. However, whereas in subtype C downstream compensatory mutations at p24Gag codons 252 and 260 reduce the adverse effects of M250I, fitness costs in subtype B appear difficult to restore. Indeed, patient-derived subtype B sequences harboring M250I exhibited in vitro replicative defects, while those from subtype C did not. The structural implications of M250I were predicted by protein modeling to be greater in subtype B versus C, providing a potential explanation for its lower frequency and enhanced replicative defects in subtype B. In addition to accounting for genetic differences between HIV-1 subtypes, the design of cytotoxic- T-lymphocyte-based vaccines may need to account for differential effects of host-driven viral evolution on viral fitness. [ABSTRACT FROM AUTHOR]

Cytotoxic T lymphocyte (CTL) responses are thought to be essential for the control of HIV-1 replication in vivo and immunogens that elicit CTL responses are currently a major focus of HIV vaccine research. Here we investigated two aspects of the CTL response to HIV-1 subtype C that are important for vaccine design and efficacy monitoring. First, we assessed the relationship between the CTL response and sequence diversity, using a robust statistical method. While peptides that were most frequently recognized by the CTL response in Nef and p24 tended to be conserved, this was not the case for p17 where epitope recognition coincided with highly variable regions. Second, we investigated the relationship between observed and predicted CTL responses, given the HLA genotype of infected individuals. Only 52% of the Nef peptides and 64% of the Gag peptides that elicited a CTL response contained sequence motifs thought to be required for binding by the HLA-A or -B alleles found in the corresponding patient. In a comparable subtype B dataset a much higher proportion of the peptides that elicited a CTL response were consistent with the patient HLA genotype (96% and 83% for Nef and Gag, respectively). We demonstrate that this difference between subtypes C and B is likely to result from a combination of a tendency for HLA alleles common in Southern African populations to be poorly characterized, as well as a tendency for sequence motifs associated with HLA recognition to be overspecified for sequence variation found in the B clade. Our results suggest that knowledge of HLA binding motifs is likely to be biased toward certain populations and subtypes. This can have important implications for understanding immune escape and predicting vaccine efficacy in the context of populations primarily infected with non-B subtype HIV-1.