People With Human Immunodeficiency Virus Receiving Suppressive Antiretroviral Therapy Show Typical Antibody Durability After Dual Coronavirus Disease 2019 Vaccination and Strong Third Dose Responses.

BACKGROUND: Longer-term humoral responses to 2-dose coronavirus disease 2019 (COVID-19) vaccines remain incompletely characterized in people living with human immunodeficiency virus (HIV) (PLWH), as do initial responses to a third dose. METHODS: We measured antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain, angiotensin-converting enzyme 2 (ACE2) displacement, and viral neutralization against wild-type and Omicron strains up to 6 months after 2-dose vaccination, and 1 month after the third dose, in 99 PLWH receiving suppressive antiretroviral therapy and 152 controls. RESULTS: Although humoral responses naturally decline after 2-dose vaccination, we found no evidence of lower antibody concentrations or faster rates of antibody decline in PLWH compared with controls after accounting for sociodemographic, health, and vaccine-related factors. We also found no evidence of poorer viral neutralization in PLWH after 2 doses, nor evidence that a low nadir CD4+ T-cell count compromised responses. Post-third-dose humoral responses substantially exceeded post-second-dose levels, though Omicron-specific responses were consistently weaker than responses against wild-type virus. Nevertheless, post-third-dose responses in PLWH were comparable to or higher than controls. An mRNA-1273 third dose was the strongest consistent correlate of higher post-third-dose responses. CONCLUSION: PLWH receiving suppressive antiretroviral therapy mount strong antibody responses after 2- and 3-dose COVID-19 vaccination. Results underscore the immune benefits of third doses in light of Omicron.

T-cell responses to sequentially emerging viral escape mutants shape long-term HIV-1 population dynamics.

HIV-1 strains harboring immune escape mutations can persist in circulation, but the impact of selection by multiple HLA alleles on population HIV-1 dynamics remains unclear. In Japan, HIV-1 Reverse Transcriptase codon 135 (RT135) is under strong immune pressure by HLA-B*51:01-restricted and HLA-B*52:01-restricted T cells that target a key epitope in this region (TI8; spanning RT codons 128-135). Major population-level shifts have occurred at HIV-1 RT135 during the Japanese epidemic, which first affected hemophiliacs (via imported contaminated blood products) and subsequently non-hemophiliacs (via domestic transmission). Specifically, threonine accumulated at RT135 (RT135T) in hemophiliac and non-hemophiliac HLA-B*51:01+ individuals diagnosed before 1997, but since then RT135T has markedly declined while RT135L has increased among non-hemophiliac individuals. We demonstrated that RT135V selection by HLA-B*52:01-restricted TI8-specific T-cells led to the creation of a new HLA-C*12:02-restricted epitope TN9-8V. We further showed that TN9-8V-specific HLA-C*12:02-restricted T cells selected RT135L while TN9-8T-specific HLA-C*12:02-restricted T cells suppressed replication of the RT135T variant. Thus, population-level accumulation of the RT135L mutation over time in Japan can be explained by initial targeting of the TI8 epitope by HLA-B*52:01-restricted T-cells, followed by targeting of the resulting escape mutant by HLA-C*12:02-restricted T-cells. We further demonstrate that this phenomenon is particular to Japan, where the HLA-B*52:01-C*12:02 haplotype is common: RT135L did not accumulate over a 15-year longitudinal analysis of HIV sequences in British Columbia, Canada, where this haplotype is rare. Together, our observations reveal that T-cell responses to sequentially emerging viral escape mutants can shape long-term HIV-1 population dynamics in a host population-specific manner.

Variation in HIV-1 Nef function within and among viral subtypes reveals genetically separable antagonism of SERINC3 and SERINC5.

HIV Nef counteracts cellular host restriction factors SERINC3 and SERINC5, but our understanding of how naturally occurring global Nef sequence diversity impacts these activities is limited. Here, we quantify SERINC3 and SERINC5 internalization function for 339 Nef clones, representing the major pandemic HIV-1 group M subtypes A, B, C and D. We describe distinct subtype-associated hierarchies for Nef-mediated internalization of SERINC5, for which subtype B clones display the highest activities on average, and of SERINC3, for which subtype B clones display the lowest activities on average. We further identify Nef polymorphisms that modulate its ability to counteract SERINC proteins, including substitutions in the N-terminal domain that selectively impair SERINC3 internalization. Our findings demonstrate that the SERINC antagonism activities of HIV Nef differ markedly among major viral subtypes and between individual isolates within a subtype, suggesting that variation in these functions may contribute to global differences in viral pathogenesis.

Are We Ready for NGS HIV Drug Resistance Testing? The Second "Winnipeg Consensus" Symposium.

HIV drug resistance is a major global challenge to successful and sustainable antiretroviral therapy. Next-generation sequencing (NGS)-based HIV drug resistance (HIVDR) assays enable more sensitive and quantitative detection of drug-resistance-associated mutations (DRMs) and outperform Sanger sequencing approaches in detecting lower abundance resistance mutations. While NGS is likely to become the new standard for routine HIVDR testing, many technical and knowledge gaps remain to be resolved before its generalized adoption in regular clinical care, public health, and research. Recognizing this, we conceived and launched an international symposium series on NGS HIVDR, to bring together leading experts in the field to address these issues through in-depth discussions and brainstorming. Following the first symposium in 2018 (Winnipeg, MB Canada, 21-22 February, 2018), a second "Winnipeg Consensus" symposium was held in September 2019 in Winnipeg, Canada, and was focused on external quality assurance strategies for NGS HIVDR assays. In this paper, we summarize this second symposium's goals and highlights.

HIV Subtype and Nef-Mediated Immune Evasion Function Correlate with Viral Reservoir Size in Early-Treated Individuals.

The HIV accessory protein Nef modulates key immune evasion and pathogenic functions, and its encoding gene region exhibits high sequence diversity. Given the recent identification of early HIV-specific adaptive immune responses as novel correlates of HIV reservoir size, we hypothesized that viral factors that facilitate the evasion of such responses-namely, Nef genetic and functional diversity-might also influence reservoir establishment and/or persistence. We isolated baseline plasma HIV RNA-derived nef clones from 30 acute/early-infected individuals who participated in a clinical trial of early combination antiretroviral therapy (cART) (<6 months following infection) and assessed each Nef clone's ability to downregulate CD4 and human leukocyte antigen (HLA) class I in vitro We then explored the relationships between baseline clinical, immunological, and virological characteristics and the HIV reservoir size measured 48 weeks following initiation of suppressive cART (where the reservoir size was quantified in terms of the proviral DNA loads as well as the levels of replication-competent HIV in CD4+ T cells). Maximal within-host Nef-mediated downregulation of HLA, but not CD4, correlated positively with post-cART proviral DNA levels (Spearman's R = 0.61, P = 0.0004) and replication-competent reservoir sizes (Spearman's R = 0.36, P = 0.056) in univariable analyses. Furthermore, the Nef-mediated HLA downregulation function was retained in final multivariable models adjusting for established clinical and immunological correlates of reservoir size. Finally, HIV subtype B-infected persons (n = 25) harbored significantly larger viral reservoirs than non-subtype B-infected persons (2 infected with subtype CRF01_AE and 3 infected with subtype G). Our results highlight a potentially important role of viral factors-in particular, HIV subtype and accessory protein function-in modulating viral reservoir establishment and persistence.IMPORTANCE While combination antiretroviral therapies (cART) have transformed HIV infection into a chronic manageable condition, they do not act upon the latent HIV reservoir and are therefore not curative. As HIV cure or remission should be more readily achievable in individuals with smaller HIV reservoirs, achieving a deeper understanding of the clinical, immunological, and virological determinants of reservoir size is critical to eradication efforts. We performed a post hoc analysis of 30 participants of a clinical trial of early cART who had previously been assessed in detail for their clinical, immunological, and reservoir size characteristics. We observed that the HIV subtype and autologous Nef-mediated HLA downregulation function correlated with the viral reservoir size measured approximately 1 year post-cART initiation. Our findings highlight virological characteristics-both genetic and functional-as possible novel determinants of HIV reservoir establishment and persistence.

Bioinformatic data processing pipelines in support of next-generation sequencing-based HIV drug resistance testing: the Winnipeg Consensus.

INTRODUCTION: Next-generation sequencing (NGS) has several advantages over conventional Sanger sequencing for HIV drug resistance (HIVDR) genotyping, including detection and quantitation of low-abundance variants bearing drug resistance mutations (DRMs). However, the high HIV genomic diversity, unprecedented large volume of data, complexity of analysis and potential for error pose significant challenges for data processing. Several NGS analysis pipelines have been developed and used in HIVDR research; however, the absence of uniformity in data processing strategies results in lack of consistency and comparability of outputs from different pipelines. To fill this gap, an international symposium on bioinformatic strategies for NGS-based HIVDR testing was held in February 2018 in Winnipeg, Canada, convening laboratory scientists, bioinformaticians and clinicians involved in four recently developed, publicly available NGS HIVDR pipelines. The goal of this symposium was to establish a consensus on effective bioinformatic strategies for NGS data management and its use for HIVDR reporting. DISCUSSION: Essential functionalities of an NGS HIVDR pipeline were divided into five analytic blocks: (1) NGS read quality control (QC)/quality assurance (QA); (2) NGS read alignment and reference mapping; (3) HIV variant calling and variant QC; (4) NGS HIVDR reporting; and (5) extended data applications and additional considerations for data management. The consensuses reached among the participants on all major aspects of these blocks are summarized here. They encompass not only recommended data management and analysis strategies, but also detailed bioinformatic approaches that help ensure accuracy of the derived HIVDR analysis outputs for both research and potential clinical use. CONCLUSIONS: While NGS is being adopted more broadly in HIVDR testing laboratories, data processing is often a bottleneck hindering its generalized application. The proposed standardization of NGS read QC/QA, read alignment and reference mapping, variant calling and QC, HIVDR reporting and relevant data management strategies in this "Winnipeg Consensus" may serve as a starting guideline for NGS HIVDR data processing that informs the refinement of existing pipelines and those yet to be developed. Moreover, the bioinformatic strategies presented here may apply more broadly to NGS data analysis of microbes harbouring significant genomic diversity.

Frequent cross-resistance to rilpivirine among subtype C HIV-1 from first-line antiretroviral therapy failures in South Africa.

Background Rilpivirine (TMC278LA) is a promising drug for pre-exposure prophylaxis of HIV-1 because of its sub-nanomolar potency and long-acting formulation; however, increasing transmission of non-nucleoside reverse transcriptase inhibitor-resistant HIV-1 with potential cross-resistance to rilpivirine could reduce its preventive efficacy. This study investigated rilpivirine cross-resistance among recombinant subtype C HIV-1 derived from 100 individuals failing on first-line non-nucleoside reverse transcriptase inhibitor-containing antiretroviral therapy in South Africa whose samples were sent for routine HIV-1 drug resistance testing to Lancet Laboratories (Johannesburg, South Africa). Methods Plasma samples were selected from individuals with HIV-1 RNA > 10,000 copies/ml and ≥1 non-nucleoside reverse transcriptase inhibitor-resistance mutation in reverse transcriptase. Recombinant HIV-1LAI-containing bulk-cloned full-length reverse transcriptase sequences from plasma were assayed for susceptibility to nevirapine (NVP), efavirenz (EFV) and rilpivirine in TZM-bl cells. Fold-change (FC) decreases in drug susceptibility were calculated against a mean IC50 from 12 subtype C HIV-1 samples from treatment-naïve individuals in South Africa. Cross-resistance was evaluated based on biological cutoffs established for rilpivirine (2.5-FC) and the effect of mutation combinations on rilpivirine phenotype. Results Of the 100 samples from individuals on failing antiretroviral therapy, 69 had 2.5- to 75-fold decreased susceptibility to rilpivirine and 11 had >75-fold resistance. Rilpivirine resistance was strongly associated with K103N especially in combination with other rilpivirine-associated mutations. Conclusion The frequently observed cross-resistance of HIV-1 suggests that the preventive efficacy of TMC278LA pre-exposure prophylaxis could be compromised by transmission of HIV-1 from individuals with failure of first-line non-nucleoside reverse transcriptase inhibitor-containing antiretroviral therapy.

Weaker HLA Footprints on HIV in the Unique and Highly Genetically Admixed Host Population of Mexico.

HIV circumvents HLA class I-restricted CD8+ T-cell responses through selection of escape mutations that leave characteristic mutational "footprints," also known as HLA-associated polymorphisms (HAPs), on HIV sequences at the population level. While many HLA footprints are universal across HIV subtypes and human populations, others can be region specific as a result of the unique immunogenetic background of each host population. Using a published probabilistic phylogenetically informed model, we compared HAPs in HIV Gag and Pol (PR-RT) in 1,612 subtype B-infected, antiretroviral treatment-naive individuals from Mexico and 1,641 individuals from Canada/United States. A total of 252 HLA class I allele subtypes were represented, including 140 observed in both cohorts, 67 unique to Mexico, and 45 unique to Canada/United States. At the predefined statistical threshold of a q value of <0.2, 358 HAPs (201 in Gag, 157 in PR-RT) were identified in Mexico, while 905 (534 in Gag and 371 in PR-RT) were identified in Canada/United States. HAPs identified in Mexico included both canonical HLA-associated escape pathways and novel associations, in particular with HLA alleles enriched in Amerindian and mestizo populations. Remarkably, HLA footprints on HIV in Mexico were not only fewer but also, on average, significantly weaker than those in Canada/United States, although some exceptions were noted. Moreover, exploratory analyses suggested that the weaker HLA footprint on HIV in Mexico may be due, at least in part, to weaker and/or less reproducible HLA-mediated immune pressures on HIV in this population. The implications of these differences for natural and vaccine-induced anti-HIV immunity merit further investigation.IMPORTANCE HLA footprints on HIV identify viral regions under intense and consistent pressure by HLA-restricted immune responses and the common mutational pathways that HIV uses to evade them. In particular, HLA footprints can identify novel immunogenic regions and/or epitopes targeted by understudied HLA alleles; moreover, comparative analyses across immunogenetically distinct populations can illuminate the extent to which HIV immunogenic regions and escape pathways are shared versus population-specific pathways, information which can in turn inform the design of universal or geographically tailored HIV vaccines. We compared HLA-associated footprints on HIV in two immunogenetically distinct North American populations, those of Mexico and Canada/United States. We identify both shared and population-specific pathways of HIV adaptation but also make the surprising observation that HLA footprints on HIV in Mexico overall are fewer and weaker than those in Canada/United States, raising the possibility that HLA-restricted antiviral immune responses in Mexico are weaker, and/or escape pathways somewhat less consistent, than those in other populations.

Promises and pitfalls of Illumina sequencing for HIV resistance genotyping.

Genetic sequencing ("genotyping") plays a critical role in the modern clinical management of HIV infection. This virus evolves rapidly within patients because of its error-prone reverse transcriptase and short generation time. Consequently, HIV variants with mutations that confer resistance to one or more antiretroviral drugs can emerge during sub-optimal treatment. There are now multiple HIV drug resistance interpretation algorithms that take the region of the HIV genome encoding the major drug targets as inputs; expert use of these algorithms can significantly improve to clinical outcomes in HIV treatment. Next-generation sequencing has the potential to revolutionize HIV resistance genotyping by lowering the threshold that rare but clinically significant HIV variants can be detected reproducibly, and by conferring improved cost-effectiveness in high-throughput scenarios. In this review, we discuss the relative merits and challenges of deploying the Illumina MiSeq instrument for clinical HIV genotyping.

The impact of clinical, demographic and risk factors on rates of HIV transmission: a population-based phylogenetic analysis in British Columbia, Canada.

BACKGROUND: The diversification of human immunodeficiency virus (HIV) is shaped by its transmission history. We therefore used a population based province wide HIV drug resistance database in British Columbia (BC), Canada, to evaluate the impact of clinical, demographic, and behavioral factors on rates of HIV transmission. METHODS: We reconstructed molecular phylogenies from 27,296 anonymized bulk HIV pol sequences representing 7747 individuals in BC-about half the estimated HIV prevalence in BC. Infections were grouped into clusters based on phylogenetic distances, as a proxy for variation in transmission rates. Rates of cluster expansion were reconstructed from estimated dates of HIV seroconversion. RESULTS: Our criteria grouped 4431 individuals into 744 clusters largely separated with respect to risk factors, including large established clusters predominated by injection drug users and more-recently emerging clusters comprising men who have sex with men. The mean log10 viral load of an individual's phylogenetic neighborhood (composed of 5 other individuals with shortest phylogenetic distances) increased their odds of appearing in a cluster by >2-fold per log10 viruses per milliliter. CONCLUSIONS: Hotspots of ongoing HIV transmission can be characterized in near real time by the secondary analysis of HIV resistance genotypes, providing an important potential resource for targeting public health initiatives for HIV prevention.

Theoretical and experimental assessment of degenerate primer tagging in ultra-deep applications of next-generation sequencing.

Primer IDs (pIDs) are random oligonucleotide tags used in next-generation sequencing to identify sequences that originate from the same template. These tags are produced by degenerate primers during the reverse transcription of RNA molecules into cDNA. The use of pIDs helps to track the number of RNA molecules carried through amplification and sequencing, and allows resolution of inconsistencies between reads sharing a pID. Three potential issues complicate the above applications. First, multiple cDNAs may share a pID by chance; we found that while preventing any cDNAs from sharing a pID may be unfeasible, it is still practical to limit the number of these collisions. Secondly, a pID must be observed in at least three sequences to allow error correction; as such, pIDs observed only one or two times must be rejected. If the sequencing product contains copies from a high number of RT templates but produces few reads, our findings indicate that rejecting such pIDs will discard a great deal of data. Thirdly, the use of pIDs could influence amplification and sequencing. We examined the effects of several intrinsic and extrinsic factors on sequencing reads at both the individual and ensemble level.

Trends in plasma HIV-RNA suppression and antiretroviral resistance in British Columbia, 1997-2010.

OBJECTIVES: To examine temporal trends in plasma viral load (pVL) suppression and antiretroviral resistance from 1997 to 2010 in British Columbia (BC), Canada, and determine characteristics, pVL ranges, and resistance profiles of HIV-positive individuals with unsuppressed pVL in 2010. METHODS: HIV-positive individuals ≥19 years old in the provincial database at the BC Centre for Excellence in HIV/AIDS were included. Virological suppression was defined as 2 consecutive pVL <500 copies per milliliter within each calendar year. Temporal trends were evaluated using the Cochran-Armitage test. Persons with suppressed vs. unsuppressed pVL in 2010 were compared using the Pearson χ² or Fisher exact test (categorical variables) and the Wilcoxon rank-sum test (quantitative variables), including unsuppressed individuals only if they were on antiretroviral therapy (ART) in 2010 or their baseline CD4 count was <350 cells per cubic millimeter or <500 cells per cubic millimeter, in separate analyses. RESULTS: The proportion of individuals with suppressed pVL increased from 24% to 80% (P < 0.001). In comparative analyses, individuals with unsuppressed pVL (877 of 6142) were more likely to be female (30% vs. 16%), younger (median, 43 vs. 48 years), have injection drug use history (38% vs. 30%), report Aboriginal ancestry (30% vs. 16%), and have hepatitis C coinfection (57% vs. 34%) (all P < 0.001). Similar patterns were observed using the <500 cells per cubic millimeter CD4 cutoff. The median pVL of all unsuppressed individuals in 2010 was 12,896 copies per milliliter (interquartile range, 1495-47,763). CONCLUSIONS: The proportion of individuals achieving pVL suppression in BC has increased markedly since 1997; however, further efforts are needed to maximize the individual and societal benefits of modern antiretroviral therapy.

Structure of TCR and antigen complexes at an immunodominant CTL epitope in HIV-1 infection.

We investigated the crystal structure of an HLA-A*2402-restricted CTL epitope in the HIV-1 nef gene (Nef134-10) before (pHLA) or after TCR docking. The wild type epitope and two escape mutants were included in the study. Y135F was an early-appearing major mutation, while F139L was a late-appearing mutation which was selected in the patients without Y135F. F139 was an eminent feature of the Nef134-10 epitope. Wild type-specific TCR was less fit to F139L mutant suggesting that F139L is an escape from the CTL against the wild type epitope. Although Y135F mutation disrupted the hydrogen bond to HLA-A*2402 His70, newly formed hydrogen bond between T138 and His70 kept the conformation of the epitope in the reconstituted pMHC. TCR from Y135F- or dually-specific CTL had unique mode of binding to the mutant epitope. Y135F has been reported as a processing mutant but CTL carrying structurally adequate TCR can be found in the patients.

Influence of HLA-C expression level on HIV control.

A variant upstream of human leukocyte antigen C (HLA-C) shows the most significant genome-wide effect on HIV control in European Americans and is also associated with the level of HLA-C expression. We characterized the differential cell surface expression levels of all common HLA-C allotypes and tested directly for effects of HLA-C expression on outcomes of HIV infection in 5243 individuals. Increasing HLA-C expression was associated with protection against multiple outcomes independently of individual HLA allelic effects in both African and European Americans, regardless of their distinct HLA-C frequencies and linkage relationships with HLA-B and HLA-A. Higher HLA-C expression was correlated with increased likelihood of cytotoxic T lymphocyte responses and frequency of viral escape mutation. In contrast, high HLA-C expression had a deleterious effect in Crohn's disease, suggesting a broader influence of HLA expression levels in human disease.

Definition of the viral targets of protective HIV-1-specific T cell responses.

BACKGROUND: The efficacy of the CTL component of a future HIV-1 vaccine will depend on the induction of responses with the most potent antiviral activity and broad HLA class I restriction. However, current HIV vaccine designs are largely based on viral sequence alignments only, not incorporating experimental data on T cell function and specificity. METHODS: Here, 950 untreated HIV-1 clade B or -C infected individuals were tested for responses to sets of 410 overlapping peptides (OLP) spanning the entire HIV-1 proteome. For each OLP, a "protective ratio" (PR) was calculated as the ratio of median viral loads (VL) between OLP non-responders and responders. RESULTS: For both clades, there was a negative relationship between the PR and the entropy of the OLP sequence. There was also a significant additive effect of multiple responses to beneficial OLP. Responses to beneficial OLP were of significantly higher functional avidity than responses to non-beneficial OLP. They also had superior in-vitro antiviral activities and, importantly, were at least as predictive of individuals' viral loads than their HLA class I genotypes. CONCLUSIONS: The data thus identify immunogen sequence candidates for HIV and provide an approach for T cell immunogen design applicable to other viral infections.

Factors associated with viral rebound in HIV-1-infected individuals enrolled in a therapeutic HIV-1 gag 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 = .01) or placebo participants with neutral HLA alleles (P = .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. CLINICAL TRIALS REGISTRATION: NCT00080106.

Viral adaptation to immune selection pressure by HLA class I-restricted CTL responses targeting epitopes in HIV frameshift sequences.

CD8+ cytotoxic T lymphocyte (CTL)-mediated immune responses to HIV contribute to viral control in vivo. Epitopes encoded by alternative reading frame (ARF) peptides may be targeted by CTLs as well, but their frequency and in vivo relevance are unknown. Using host genetic (human leukocyte antigen [HLA]) and plasma viral sequence information from 765 HIV-infected subjects, we identified 64 statistically significant (q<0.2) associations between specific HLA alleles and sequence polymorphisms in alternate reading frames of gag, pol, and nef that did not affect the regular frame protein sequence. Peptides spanning the top 20 HLA-associated imprints were used to test for ex vivo immune responses in 85 HIV-infected subjects and showed responses to 10 of these ARF peptides. The most frequent response recognized an HLA-A*03-restricted +2 frame-encoded epitope containing a unique A*03-associated polymorphism at position 6. Epitope-specific CTLs efficiently inhibited viral replication in vitro when viruses containing the wild-type sequence but not the observed polymorphism were tested. Mutating alternative internal start codons abrogated the CTL-mediated inhibition of viral replication. These data indicate that responses to ARF-encoded HIV epitopes are induced during natural infection, can contribute to viral control in vivo, and drive viral evolution on a population level.

HLA-associated immune escape pathways in HIV-1 subtype B Gag, Pol and Nef proteins.

BACKGROUND: Despite the extensive genetic diversity of HIV-1, viral evolution in response to immune selective pressures follows broadly predictable mutational patterns. Sites and pathways of Human Leukocyte-Antigen (HLA)-associated polymorphisms in HIV-1 have been identified through the analysis of population-level data, but the full extent of immune escape pathways remains incompletely characterized. Here, in the largest analysis of HIV-1 subtype B sequences undertaken to date, we identify HLA-associated polymorphisms in the three HIV-1 proteins most commonly considered in cellular-based vaccine strategies. Results are organized into protein-wide escape maps illustrating the sites and pathways of HLA-driven viral evolution. METHODOLOGY/PRINCIPAL FINDINGS: HLA-associated polymorphisms were identified in HIV-1 Gag, Pol and Nef in a multicenter cohort of >1500 chronically subtype-B infected, treatment-naïve individuals from established cohorts in Canada, the USA and Western Australia. At q< or =0.05, 282 codons commonly mutating under HLA-associated immune pressures were identified in these three proteins. The greatest density of associations was observed in Nef (where close to 40% of codons exhibited a significant HLA association), followed by Gag then Pol (where approximately 15-20% of codons exhibited HLA associations), confirming the extensive impact of immune selection on HIV evolution and diversity. Analysis of HIV codon covariation patterns identified over 2000 codon-codon interactions at q< or =0.05, illustrating the dense and complex networks of linked escape and secondary/compensatory mutations. CONCLUSIONS/SIGNIFICANCE: The immune escape maps and associated data are intended to serve as a user-friendly guide to the locations of common escape mutations and covarying codons in HIV-1 subtype B, and as a resource facilitating the systematic identification and classification of immune escape mutations. These resources should facilitate research in HIV epitope discovery and host-pathogen co-evolution, and are relevant to the continued search for an effective CTL-based AIDS vaccine.

Transmission and long-term stability of compensated CD8 escape mutations.

Human immunodeficiency virus effectively evades CD8(+) T-cell responses through the development of CD8 escape mutations. Recent reports documenting reversion of transmitted mutations and the impact of specific escape mutations upon viral replication suggest that complex forces limit the accumulation of CD8 escape mutations at the population level. However, the presence of compensatory mutations capable of alleviating the impact of CD8 escape mutations on replication capacity may enable their persistence in an HLA-mismatched host. Herein, we illustrate the long-term stability of stereotypic escape mutations in the immunodominant HLA-B27-restricted epitope KK10 in p24/Gag following transmission when accompanied by a specific compensatory mutation.

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.