HLA-Associated viral mutations are common in human immunodeficiency virus type 1 elite controllers

Elite controllers (EC) of human immunodeficiency virus type 1 (HTV-1) maintain viremia below the limit of detection without antiretroviral treatment. Virus-specific cytotoxic CD8+ T lymphocytes are believed to play a crucial role in viral containment, but the degree of immune imprinting and compensatory mutations in EC is unclear. We obtained plasma gag, pol, and nef sequences from HLA-diverse subjects and found that 30 to 40% of the predefined HLA-associated polymorphic sites show evidence of immune selection pressure in EC., compared to approximately 50% of the sites in chronic progressors. These data indicate ongoing viral replication and escape from cytotoxic T lymphocytes are present even in strictly controlled HTV-1 infection

Factors Associated With Viral Rebound in HIV-1-Infected Individuals Enrolled in a Therapeutic HIV-1 \u3ci\u3egag\u3c/i\u3e Vaccine Trial

Background. Human immunodeficiency virus type 1 (HIV-1) vaccines directed to the cell-mediated immune system could have a role in lowering the plasma HIV-1 RNA set point, which may reduce infectivity and delay disease progression. Methods. Randomized, placebo-controlled trial involving HIV-1-infected participants who received a recombinant adenovirus serotype 5 (rAd5) HIV-1 gag vaccine or placebo. Sequence-based HLA typing was performed for all 110 participants who initiated analytic treatment interruption (ATI) to assess the role of HLA types previously associated with HIV prognosis. Plasma HIV-1 gag and pol RNA sequences were obtained during the ATI. Virologic endpoints and HLA groups were compared between treatment arms using the 2-sample rank sum test. A linear regression model was fitted to derive independent correlates of ATI week 16 plasma viral load (w16 PVL). Results. Vaccinated participants with neutral HLA alleles had lower median w16 PVLs than did vaccinated participants with protective HLA alleles (P 5 .01) or placebo participants with neutral HLA alleles (P 5 .02). Factors independently associated with lower w16 PVL included lower pre-antiretroviral therapy PVL, greater Gag sequence divergence from the vaccine sequence, decreased proportion of HLA-associated polymorphisms in Gag, and randomization to the vaccine arm. Conclusions. Therapeutic vaccination with a rAd5-HIV gag vaccine was associated with lower ATI week 16 PVL even after controlling for viral and host genetic factors

Factors Associated With Viral Rebound in HIV-1-Infected Individuals Enrolled in a Therapeutic HIV-1 \u3ci\u3egag\u3c/i\u3e Vaccine Trial

Background. Human immunodeficiency virus type 1 (HIV-1) vaccines directed to the cell-mediated immune system could have a role in lowering the plasma HIV-1 RNA set point, which may reduce infectivity and delay disease progression. Methods. Randomized, placebo-controlled trial involving HIV-1-infected participants who received a recombinant adenovirus serotype 5 (rAd5) HIV-1 gag vaccine or placebo. Sequence-based HLA typing was performed for all 110 participants who initiated analytic treatment interruption (ATI) to assess the role of HLA types previously associated with HIV prognosis. Plasma HIV-1 gag and pol RNA sequences were obtained during the ATI. Virologic endpoints and HLA groups were compared between treatment arms using the 2-sample rank sum test. A linear regression model was fitted to derive independent correlates of ATI week 16 plasma viral load (w16 PVL). Results. Vaccinated participants with neutral HLA alleles had lower median w16 PVLs than did vaccinated participants with protective HLA alleles (P 5 .01) or placebo participants with neutral HLA alleles (P 5 .02). Factors independently associated with lower w16 PVL included lower pre-antiretroviral therapy PVL, greater Gag sequence divergence from the vaccine sequence, decreased proportion of HLA-associated polymorphisms in Gag, and randomization to the vaccine arm. Conclusions. Therapeutic vaccination with a rAd5-HIV gag vaccine was associated with lower ATI week 16 PVL even after controlling for viral and host genetic factors

Characteristics and Outcomes of Initial Virologic Suppressors during Analytic Treatment Interruption in a Therapeutic HIV-1 gag Vaccine Trial

Background: In the placebo-controlled trial ACTG A5197, a trend favoring viral suppression was seen in the HIV-1-infected subjects who received a recombinant Ad5 HIV-1 $gag$ vaccine. Objective: To identify individuals with initial viral suppression (plasma HIV-1 RNA set point <3.0 $log_{10}$ copies/ml) during the analytic treatment interruption (ATI) and evaluate the durability and correlates of virologic control and characteristics of HIV sequence evolution. Methods: HIV-1 $gag$ and $pol$ RNA were amplified and sequenced from plasma obtained during the ATI. Immune responses were measured by flow cytometric analysis and intracellular cytokine expression assays. Characteristics of those with and without initial viral suppression were compared using the Wilcoxon rank sum and Fisher's exact tests. Results: Eleven out of 104 participants (10.6%) were classified as initial virologic suppressors, nine of whom had received the vaccine. Initial virologic suppressors had significantly less CD4+ cell decline by ATI week 16 as compared to non-suppressors (median 7 CD4+ cell gain vs. 247 CD4+ cell loss, P = 0.04). However, of the ten initial virologic suppressors with a pVL at ATI week 49, only three maintained pVL <3.0 log10 copies/ml. HIV-1 Gag-specific CD4+ interferon-γ responses were not associated with initial virologic suppression and no evidence of vaccine-driven HIV sequence evolution was detected. Participants with initial virologic suppression were found to have a lower percentage of CD4+ CTLA-4+ cells prior to treatment interruption, but a greater proportion of HIV-1 Gag-reactive CD4+ TNF-α+ cells expressing either CTLA-4 or PD-1. Conclusions: Among individuals participating in a rAd5 therapeutic HIV-1 $gag$ vaccine trial, initial viral suppression was found in a subset of patients, but this response was not sustained. The association between CTLA-4 and PD-1 expression on CD4+ T cells and virologic outcome warrants further study in trials of other therapeutic vaccines in development. Trial Registration: ClinicalTrials.gov NCT0008010

Intersubtype differences in the effect of a rare p24 Gag mutation on HIV-1 replicative fitness.

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

Phylogenetic Dependency Networks: Inferring Patterns of CTL Escape and Codon Covariation in HIV-1 Gag

HIV avoids elimination by cytotoxic T-lymphocytes (CTLs) through the evolution of escape mutations. Although there is mounting evidence that these escape pathways are broadly consistent among individuals with similar human leukocyte antigen (HLA) class I alleles, previous population-based studies have been limited by the inability to simultaneously account for HIV codon covariation, linkage disequilibrium among HLA alleles, and the confounding effects of HIV phylogeny when attempting to identify HLA-associated viral evolution. We have developed a statistical model of evolution, called a phylogenetic dependency network, that accounts for these three sources of confounding and identifies the primary sources of selection pressure acting on each HIV codon. Using synthetic data, we demonstrate the utility of this approach for identifying sites of HLA-mediated selection pressure and codon evolution as well as the deleterious effects of failing to account for all three sources of confounding. We then apply our approach to a large, clinically-derived dataset of Gag p17 and p24 sequences from a multicenter cohort of 1144 HIV-infected individuals from British Columbia, Canada (predominantly HIV-1 clade B) and Durban, South Africa (predominantly HIV-1 clade C). The resulting phylogenetic dependency network is dense, containing 149 associations between HLA alleles and HIV codons and 1386 associations among HIV codons. These associations include the complete reconstruction of several recently defined escape and compensatory mutation pathways and agree with emerging data on patterns of epitope targeting. The phylogenetic dependency network adds to the growing body of literature suggesting that sites of escape, order of escape, and compensatory mutations are largely consistent even across different clades, although we also identify several differences between clades. As recent case studies have demonstrated, understanding both the complexity and the consistency of immune escape has important implications for CTL-based vaccine design. Phylogenetic dependency networks represent a major step toward systematically expanding our understanding of CTL escape to diverse populations and whole viral genes

Whole Genome Deep Sequencing of HIV-1 Reveals the Impact of Early Minor Variants Upon Immune Recognition During Acute Infection

Deep sequencing technologies have the potential to transform the study of highly variable viral pathogens by providing a rapid and cost-effective approach to sensitively characterize rapidly evolving viral quasispecies. Here, we report on a high-throughput whole HIV-1 genome deep sequencing platform that combines 454 pyrosequencing with novel assembly and variant detection algorithms. In one subject we combined these genetic data with detailed immunological analyses to comprehensively evaluate viral evolution and immune escape during the acute phase of HIV-1 infection. The majority of early, low frequency mutations represented viral adaptation to host CD8+ T cell responses, evidence of strong immune selection pressure occurring during the early decline from peak viremia. CD8+ T cell responses capable of recognizing these low frequency escape variants coincided with the selection and evolution of more effective secondary HLA-anchor escape mutations. Frequent, and in some cases rapid, reversion of transmitted mutations was also observed across the viral genome. When located within restricted CD8 epitopes these low frequency reverting mutations were sufficient to prime de novo responses to these epitopes, again illustrating the capacity of the immune response to recognize and respond to low frequency variants. More importantly, rapid viral escape from the most immunodominant CD8+ T cell responses coincided with plateauing of the initial viral load decline in this subject, suggestive of a potential link between maintenance of effective, dominant CD8 responses and the degree of early viremia reduction. We conclude that the early control of HIV-1 replication by immunodominant CD8+ T cell responses may be substantially influenced by rapid, low frequency viral adaptations not detected by conventional sequencing approaches, which warrants further investigation. These data support the critical need for vaccine-induced CD8+ T cell responses to target more highly constrained regions of the virus in order to ensure the maintenance of immunodominant CD8 responses and the sustained decline of early viremia

Unique CRF01_AE Gag CTL Epitopes Associated with Lower HIV-Viral Load and Delayed Disease Progression in a Cohort of HIV-Infected Thais

Cytotoxic T Lymphocytes (CTLs) play a central role in controlling HIV-replication. Although numerous CTL epitopes have been described, most are in subtype B or C infection. Little is known about CTL responses in CRF01_AE infection. Gag CTL responses were investigated in a cohort of 137 treatment-naïve HIV-1 infected Thai patients with high CD4+ T cell counts, using gIFN Enzyme-Linked Immunospot (ELISpot) assays with 15-mer overlapping peptides (OLPs) derived from locally dominant CRF01_AE Gag sequences. 44 OLPs were recognized in 112 (81.8%) individuals. Both the breadth and magnitude of the CTL response, particularly against the p24 region, positively correlated with CD4+ T cell count and inversely correlated with HIV viral load. The breadth of OLP response was also associated with slower progression to antiretroviral therapy initiation. Statistical analysis and single peptide ELISpot assay identified at least 17 significant associations between reactive OLP and HLA in 12 OLP regions; 6 OLP-HLA associations (35.3%) were not compatible with previously reported CTL epitopes, suggesting that these contained new CTL Gag epitopes. A substantial proportion of CTL epitopes in CRF01_AE infection differ from subtype B or C. However, the pattern of protective CTL responses is similar; Gag CTL responses, particularly against p24, control viral replication and slow clinical progression