Christine Rousseau

CD8+ T-cell responses to different HIV proteins have discordant associations with viral load

Selection of T-cell vaccine antigens for chronic persistent viral infections has been largely empirical. To define the relationship, at the population level, between the specificity of the cellular immune response and viral control for a relevant human pathogen, we performed a comprehensive analysis of the 160 dominant CD8+ T-cell responses in 578 untreated HIV-infected individuals from KwaZulu-Natal, South Africa. Of the HIV proteins targeted, only Gag-specific responses were associated with lowering viremia. Env-specific and Accessory/Regulatory protein–specific responses were associated with higher viremia. Increasing breadth of Gag-specific responses was associated with decreasing viremia and increasing Env breadth with increasing viremia. Association of the specific CD8+ T-cell response with low viremia was independent of HLA type and unrelated to epitope sequence conservation. These population-based data, suggesting the existence of both effective immune responses and responses lacking demonstrable biological impact in chronic HIV infection, are of relevance to HIV vaccine design and evaluation.

Fitness Cost of Escape Mutations in p24 Gag in Association with Control of Human Immunodeficiency Virus Type 1

ABSTRACT Mutational escape by human immunodeficiency virus (HIV) from cytotoxic T-lymphocyte (CTL) recognition is a major challenge for vaccine design. However, recent studies suggest that CTL escape may carry a sufficient cost to viral replicative capacity to facilitate subsequent immune control of a now attenuated virus. In order to examine how limitations can be imposed on viral escape, the epitope TSTLQEQIGW (TW10 [Gag residues 240 to 249]), presented by two HLA alleles associated with effective control of HIV, HLA-B*57 and -B*5801, was investigated. The in vitro experiments described here demonstrate that the dominant TW10 escape mutation, T242N, reduces viral replicative capacity. Structural analysis reveals that T242 plays a critical role in defining the start point and in stabilizing helix 6 within p24 Gag, ensuring that escape occurs at a significant cost. A very similar role is played by Thr-180, which is also an escape residue, but within a second p24 Gag epitope associated with immune control. Analysis of HIV type 1 gag in 206 B*57/5801-positive subjects reveals three principle alternative TW10-associated variants, and each is strongly linked to concomitant additional variants within p24 Gag, suggesting that functional constraints operate against their occurrence alone. The extreme conservation of p24 Gag and the predictable nature of escape variation resulting from these tight functional constraints indicate that p24 Gag may be a critical immunogen in vaccine design and suggest novel vaccination strategies to limit viral escape options from such epitopes.

Founder Effects in the Assessment of HIV Polymorphisms and HLA Allele Associations

Escape from T cell–mediated immune responses affects the ongoing evolution of rapidly evolving viruses such as HIV. By applying statistical approaches that account for phylogenetic relationships among viral sequences, we show that viral lineage effects rather than immune escape often explain apparent human leukocyte antigen (HLA)–mediated immune-escape mutations defined by older analysis methods. Phylogenetically informed methods identified immune-susceptible locations with greatly improved accuracy, and the associations we identified with these methods were experimentally validated. This approach has practical implications for understanding the impact of host immunity on pathogen evolution and for defining relevant variants for inclusion in vaccine antigens.

Compensatory Mutation Partially Restores Fitness and Delays Reversion of Escape Mutation within the Immunodominant HLA-B*5703-Restricted Gag Epitope in Chronic Human Immunodeficiency Virus Type 1 Infection

ABSTRACT HLA-B*5703 is associated with effective immune control in human immunodeficiency virus type 1 (HIV-1) infection. Here we describe an escape mutation within the immunodominant HLA-B*5703-restricted epitope in chronic HIV-1 infection, KAFSPEVIPMF (Gag 162-172), and demonstrate that this mutation reduces viral replicative capacity. Reversion of this mutation following transmission to HLA-B*5703-negative recipients was delayed by the compensatory mutation S165N within the same epitope. These data may help explain the observed association between HLA-B*5703 and long-term control of viremia.

Central Role of Reverting Mutations in HLA Associations with Human Immunodeficiency Virus Set Point

ABSTRACT Much uncertainty still exists over what T-cell responses need to be induced by an effective human immunodeficiency virus (HIV) vaccine. Previous studies have hypothesized that the effective CD8+ T-cell responses are those driving the selection of escape mutations that reduce viral fitness and therefore revert posttransmission. In this study, we adopted a novel approach to define better the role of reverting escape mutations in immune control of HIV infection. This analysis of sequences from 710 study subjects with chronic C-clade HIV type 1 infection demonstrates the importance of mutations that impose a fitness cost in the control of viremia. Consistent with previous studies, the viral set points associated with each HLA-B allele are strongly correlated with the number of Gag-specific polymorphisms associated with the relevant HLA-B allele (r = −0.56, P = 0.0034). The viral set points associated with each HLA-C allele were also strongly correlated with the number of Pol-specific polymorphisms associated with the relevant HLA-C allele (r = −0.67, P = 0.0047). However, critically, both these correlations were dependent solely on the polymorphisms identified as reverting. Therefore, despite the inevitable evolution of viral escape, viremia can be controlled through the selection of mutations that are detrimental to viral fitness. The significance of these results is in highlighting the rationale for an HIV vaccine that can induce these broad responses.

Control of Human Immunodeficiency Virus Type 1 Is Associated with HLA-B*13 and Targeting of Multiple Gag-Specific CD8+ T-Cell Epitopes

ABSTRACT To better understand relationships between CD8+ T-cell specificity and the immune control of human immunodeficiency virus type 1 (HIV-1), we analyzed the role of HLA-B*13, an allele associated with low viremia, in a cohort of 578 C clade-infected individuals in Durban, South Africa. Six novel B*13-restricted cytotoxic T lymphocyte epitopes were defined from analyses of 37 B*13-positive subjects, including three Gag epitopes. These B*13-restricted epitopes contribute to a broad Gag-specific CD8+ response that is associated with the control of viremia. These data are consistent with data from studies of other HLA-class I alleles associated with HIV control that have shown that the targeting of multiple Gag epitopes is associated with relative suppression of viremia.

Broad and Gag-Biased HIV-1 Epitope Repertoires Are Associated with Lower Viral Loads

Background HLA class-I alleles differ in their ability to control HIV replication through cell-mediated immune responses. No consistent associations have been found between the breadth of Cytotoxic T Lymphocytes (CTL) responses and the control of HIV-1, and it is unknown whether the size or distribution of the viral proteome-wide epitope repertoire, i.e., the intrinsic ability to present fewer, more or specific viral epitopes, could affect clinical markers of disease progression. Methodology/Principal Findings We used an epitope prediction model to identify all epitope motifs in a set of 302 HIV-1 full-length proteomes according to each individuals HLA (Human Leukocyte Antigen) genotype. The epitope repertoire, i.e., the number of predicted epitopes per HIV-1 proteome, varied considerably between HLA alleles and thus among individual proteomes. In a subgroup of 270 chronically infected individuals, we found that lower viral loads and higher CD4 counts were associated with a larger predicted epitope repertoire. Additionally, in Gag and Rev only, more epitopes were restricted by alleles associated with low viral loads than by alleles associated with higher viral loads. Conclusions/Significance This comprehensive analysis puts forth the epitope repertoire as a mechanistic component of the multi-faceted HIV-specific CTL response. The favorable impact on markers of disease status of the propensity to present more HLA binding peptides and specific proteins gives impetus to vaccine design strategies that seek to elicit responses to a broad array of HIV-1 epitopes, and suggest a particular focus on Gag.

HLA Class I-Driven Evolution of Human Immunodeficiency Virus Type 1 Subtype C Proteome: Immune Escape and Viral Load

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) mutations that confer escape from cytotoxic T-lymphocyte (CTL) recognition can sometimes result in lower viral fitness. These mutations can then revert upon transmission to a new host in the absence of CTL-mediated immune selection pressure restricted by the HLA alleles of the prior host. To identify these potentially critical recognition points on the virus, we assessed HLA-driven viral evolution using three phylogenetic correction methods across full HIV-1 subtype C proteomes from a cohort of 261 South Africans and identified amino acids conferring either susceptibility or resistance to CTLs. A total of 558 CTL-susceptible and -resistant HLA-amino acid associations were identified and organized into 310 immunological sets (groups of individual associations related to a single HLA/epitope combination). Mutations away from seven susceptible residues, including four in Gag, were associated with lower plasma viral-RNA loads (q < 0.2 [where q is the expected false-discovery rate]) in individuals with the corresponding HLA alleles. The ratio of susceptible to resistant residues among those without the corresponding HLA alleles varied in the order Vpr > Gag > Rev > Pol > Nef > Vif > Tat > Env > Vpu (Fishers exact test; P ≤ 0.0009 for each comparison), suggesting the same ranking of fitness costs by genes associated with CTL escape. Significantly more HLA-B (χ2; P = 3.59 × 10−5) and HLA-C (χ2; P = 4.71 × 10−6) alleles were associated with amino acid changes than HLA-A, highlighting their importance in driving viral evolution. In conclusion, specific HIV-1 residues (enriched in Vpr, Gag, and Rev) and HLA alleles (particularly B and C) confer susceptibility to the CTL response and are likely to be important in the development of vaccines targeted to decrease the viral load.

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.

Extensive Intrasubtype Recombination in South African Human Immunodeficiency Virus Type 1 Subtype C Infections

ABSTRACT Recombinant human immunodeficiency virus type 1 (HIV-1) strains containing sequences from different viral genetic subtypes (intersubtype) and different lineages from within the same subtype (intrasubtype) have been observed. A consequence of recombination can be the distortion of the phylogenetic signal. Several intersubtype recombinants have been identified; however, less is known about the frequency of intrasubtype recombination. For this study, near-full-length HIV-1 subtype C genomes from 270 individuals were evaluated for the presence of intrasubtype recombination. A sliding window schema (window, 2 kb; step, 385 bp) was used to partition the aligned sequences. The Shimodaira-Hasegawa test detected significant topological incongruence in 99.6% of the comparisons of the maximum-likelihood trees generated from each sequence partition, a result that could be explained by recombination. Using RECOMBINE, we detected significant levels of recombination using five random subsets of the sequences. With a set of 23 topologically consistent sequences used as references, bootscanning followed by the interactive informative site test defined recombination breakpoints. Using two multiple-comparison correction methods, 47% of the sequences showed significant evidence of recombination in both analyses. Estimated evolutionary rates were revised from 0.51%/year (95% confidence interval [CI], 0.39 to 0.53%) with all sequences to 0.46%/year (95% CI, 0.38 to 0.48%) with the putative recombinants removed. The timing of the subtype C epidemic origin was revised from 1961 (95% CI, 1947 to 1962) with all sequences to 1958 (95% CI, 1949 to 1960) with the putative recombinants removed. Thus, intrasubtype recombinants are common within the subtype C epidemic and these impact analyses of HIV-1 evolution.