Zabrina L. Brumme

Adaptation of HIV-1 to human leukocyte antigen class I

The rapid and extensive spread of the human immunodeficiency virus (HIV) epidemic provides a rare opportunity to witness host–pathogen co-evolution involving humans. A focal point is the interaction between genes encoding human leukocyte antigen (HLA) and those encoding HIV proteins. HLA molecules present fragments (epitopes) of HIV proteins on the surface of infected cells to enable immune recognition and killing by CD8+ T cells; particular HLA molecules, such as HLA-B*57, HLA-B*27 and HLA-B*51, are more likely to mediate successful control of HIV infection. Mutation within these epitopes can allow viral escape from CD8+ T-cell recognition. Here we analysed viral sequences and HLA alleles from >2,800 subjects, drawn from 9 distinct study cohorts spanning 5 continents. Initial analysis of the HLA-B*51-restricted epitope, TAFTIPSI (reverse transcriptase residues 128–135), showed a strong correlation between the frequency of the escape mutation I135X and HLA-B*51 prevalence in the 9 study cohorts (P = 0.0001). Extending these analyses to incorporate other well-defined CD8+ T-cell epitopes, including those restricted by HLA-B*57 and HLA-B*27, showed that the frequency of these epitope variants (n = 14) was consistently correlated with the prevalence of the restricting HLA allele in the different cohorts (together, P < 0.0001), demonstrating strong evidence of HIV adaptation to HLA at a population level. This process of viral adaptation may dismantle the well-established HLA associations with control of HIV infection that are linked to the availability of key epitopes, and highlights the challenge for a vaccine to keep pace with the changing immunological landscape presented by HIV.

Predictors of HIV Drug-Resistance Mutations in a Large Antiretroviral-Naive Cohort Initiating Triple Antiretroviral Therapy

OBJECTIVE The objective of this study was to systematically characterize the incidence and determinants of antiretroviral resistance in the HOMER (Highly Active Antiretroviral Therapy [HAART] Observational Medical Evaluation and Research) cohort of 1191 human immunodeficiency virus-infected, antiretroviral-naive adults initiating HAART in British Columbia, Canada. METHODS All plasma samples with plasma virus loads (pVLs) >1000 copies/mL collected during the first 30 months of follow-up were genotyped for drug resistance. The primary outcome measure was time to the first detection of major drug-resistance mutation(s). Cox proportional hazard regression was used to identify factors significantly associated with the detection of drug-resistance mutations. RESULTS Drug-resistance mutations were detected in 298 subjects (25%). Factors significantly associated with detection of drug-resistance mutations included high baseline pVL (multivariate hazard ratio [HR], 1.59; P<.001) and adherence (estimated using prescription-refill data and/or untimed plasma drug-concentration measurements). When compared with subjects with low (0%-<20%) prescription-refill percentages, subjects at an elevated risk of harboring drug-resistance mutations were those with relatively high but imperfect prescription-refill percentages (80%-<90%; multivariate HR, 4.15; P<.001) and those with essentially perfect (>/=95%) refill percentages but with 2 plasma drug concentrations below the steady-state trough concentration minus 1 standard deviation (multivariate HR, 4.57; P<.001). Initial use of nonnucleoside reverse-transcriptase inhibitor-based HAART was significantly associated with multiclass drug resistance (multivariate HR, 1.84; P=.001). CONCLUSION High baseline pVLs and substantial but imperfect levels of adherence were major predictors of antiretroviral resistance.

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.

HLA-B57/B*5801 Human Immunodeficiency Virus Type 1 Elite Controllers Select for Rare Gag Variants Associated with Reduced Viral Replication Capacity and Strong Cytotoxic T-Lymphotye Recognition

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) elite controllers (EC) maintain viremia below the limit of commercial assay detection (<50 RNA copies/ml) in the absence of antiviral therapy, but the mechanisms of control remain unclear. HLA-B57 and the closely related allele B*5801 are particularly associated with enhanced control and recognize the same Gag240-249 TW10 epitope. The typical escape mutation (T242N) within this epitope diminishes viral replication capacity in chronically infected persons; however, little is known about TW10 epitope sequences in residual replicating viruses in B57/B*5801 EC and the extent to which mutations within this epitope may influence steady-state viremia. Here we analyzed TW10 in a total of 50 B57/B*5801-positive subjects (23 EC and 27 viremic subjects). Autologous plasma viral sequences from both EC and viremic subjects frequently harbored the typical cytotoxic T-lymphocyte (CTL)-selected mutation T242N (15/23 sequences [65.2%] versus 23/27 sequences [85.1%], respectively; P = 0.18). However, other unique mutants were identified in HIV controllers, both within and flanking TW10, that were associated with an even greater reduction in viral replication capacity in vitro. In addition, strong CTL responses to many of these unique TW10 variants were detected by gamma interferon-specific enzyme-linked immunospot assay. These data suggest a dual mechanism for durable control of HIV replication, consisting of viral fitness loss resulting from CTL escape mutations together with strong CD8 T-cell immune responses to the arising variant epitopes.

Antigen Load and Viral Sequence Diversification Determine the Functional Profile of HIV-1–Specific CD8+ T Cells

Background Virus-specific CD8+ T lymphocytes play a key role in the initial reduction of peak viremia during acute viral infections, but display signs of increasing dysfunction and exhaustion under conditions of chronic antigen persistence. It has been suggested that virus-specific CD8+ T cells with a “polyfunctional” profile, defined by the capacity to secrete multiple cytokines or chemokines, are most competent in controlling viral replication in chronic HIV-1 infection. We used HIV-1 infection as a model of chronic persistent viral infection to investigate the process of exhaustion and dysfunction of virus-specific CD8+ T cell responses on the single-epitope level over time, starting in primary HIV-1 infection. Methods and Findings We longitudinally analyzed the polyfunctional epitope-specific CD8+ T cell responses of 18 patients during primary HIV-1 infection before and after therapy initiation or sequence variation in the targeted epitope. Epitope-specific CD8+ T cells responded with multiple effector functions to antigenic stimulation during primary HIV-1 infection, but lost their polyfunctional capacity in response to antigen and up-regulated programmed death 1 (PD-1) expression with persistent viremic infection. This exhausted phenotype significantly decreased upon removal of stimulation by antigen, either in response to antiretroviral therapy or by reduction of epitope-specific antigen load in the presence of ongoing viral replication, as a consequence of in vivo selection of cytotoxic T lymphocyte escape mutations in the respective epitopes. Monofunctionality increased in CD8+ T cell responses directed against conserved epitopes from 49% (95% confidence interval 27%–72%) to 76% (56%–95%) (standard deviation [SD] of the effect size 0.71), while monofunctionality remained stable or slightly decreased for responses directed against escaped epitopes from 61% (47%–75%) to 56% (42%–70%) (SD of the effect size 0.18) (p < 0.05). Conclusion These data suggest that persistence of antigen can be the cause, rather than the consequence, of the functional impairment of virus-specific T cell responses observed during chronic HIV-1 infection, and underscore the importance of evaluating autologous viral sequences in studies aimed at investigating the relationship between virus-specific immunity and associated pathogenesis.

Influence of HLA-C expression level on HIV control

Thwarting HIV Multiple genome-wide association studies have revealed that human leukocyte antigen (HLA) genes of the major histocompatibility complex locus have the strongest impact on HIV. In particular, a single-nucleotide polymorphism 35 base pairs upstream of HLA-C shows significant association with viral load and protection against HIV. How HLA-C mediates these effects is unknown. Apps et al. (p. 87) now demonstrate that increasing surface expression of HLA-C is associated with reduced viral load and reduced rate of progression to low CD4+ T cell counts in African and European Americans. High HLA-C expression likely promoted improved HIV control through a more effective cytotoxic CD8+ T cell response. In contrast to HIV infection, high HLA-C expression was associated with a higher risk of the inflammatory bowel disease, Crohns disease. Increased levels of human leukocyte antigen C are associated with control of HIV infection but increased susceptibility to Crohn’s disease. 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.

Marked Epitope- and Allele-Specific Differences in Rates of Mutation in Human Immunodeficiency Type 1 (HIV-1) Gag, Pol, and Nef Cytotoxic T-Lymphocyte Epitopes in Acute/Early HIV-1 Infection

ABSTRACT During acute human immunodeficiency virus type 1 (HIV-1) infection, early host cellular immune responses drive viral evolution. The rates and extent of these mutations, however, remain incompletely characterized. In a cohort of 98 individuals newly infected with HIV-1 subtype B, we longitudinally characterized the rates and extent of HLA-mediated escape and reversion in Gag, Pol, and Nef using a rational definition of HLA-attributable mutation based on the analysis of a large independent subtype B data set. We demonstrate rapid and dramatic HIV evolution in response to immune pressures that in general reflect established cytotoxic T-lymphocyte (CTL) response hierarchies in early infection. On a population level, HLA-driven evolution was observed in ∼80% of published CTL epitopes. Five of the 10 most rapidly evolving epitopes were restricted by protective HLA alleles (HLA-B*13/B*51/B*57/B*5801; P = 0.01), supporting the importance of a strong early CTL response in HIV control. Consistent with known fitness costs of escape, B*57-associated mutations in Gag were among the most rapidly reverting positions upon transmission to non-B*57-expressing individuals, whereas many other HLA-associated polymorphisms displayed slow or negligible reversion. Overall, an estimated minimum of 30% of observed substitutions in Gag/Pol and 60% in Nef were attributable to HLA-associated escape and reversion events. Results underscore the dominant role of immune pressures in driving early within-host HIV evolution. Dramatic differences in escape and reversion rates across codons, genes, and HLA restrictions are observed, highlighting the complexity of viral adaptation to the host immune response.

Evidence of Differential HLA Class I-Mediated Viral Evolution in Functional and Accessory/Regulatory Genes of HIV-1

Despite the formidable mutational capacity and sequence diversity of HIV-1, evidence suggests that viral evolution in response to specific selective pressures follows generally predictable mutational pathways. Population-based analyses of clinically derived HIV sequences may be used to identify immune escape mutations in viral genes; however, prior attempts to identify such mutations have been complicated by the inability to discriminate active immune selection from virus founder effects. Furthermore, the association between mutations arising under in vivo immune selection and disease progression for highly variable pathogens such as HIV-1 remains incompletely understood. We applied a viral lineage-corrected analytical method to investigate HLA class I-associated sequence imprinting in HIV protease, reverse transcriptase (RT), Vpr, and Nef in a large cohort of chronically infected, antiretrovirally naïve individuals. A total of 478 unique HLA-associated polymorphisms were observed and organized into a series of “escape maps,” which identify known and putative cytotoxic T lymphocyte (CTL) epitopes under selection pressure in vivo. Our data indicate that pathways to immune escape are predictable based on host HLA class I profile, and that epitope anchor residues are not the preferred sites of CTL escape. Results reveal differential contributions of immune imprinting to viral gene diversity, with Nef exhibiting far greater evidence for HLA class I-mediated selection compared to other genes. Moreover, these data reveal a significant, dose-dependent inverse correlation between HLA-associated polymorphisms and HIV disease stage as estimated by CD4+ T cell count. Identification of specific sites and patterns of HLA-associated polymorphisms across HIV protease, RT, Vpr, and Nef illuminates regions of the genes encoding these products under active immune selection pressure in vivo. The high density of HLA-associated polymorphisms in Nef compared to other genes investigated indicates differential HLA class I-driven evolution in different viral genes. The relationship between HLA class I-associated polymorphisms and lower CD4+ cell count suggests that immune escape correlates with disease status, supporting an essential role of maintenance of effective CTL responses in immune control of HIV-1. The design of preventative and therapeutic CTL-based vaccine approaches could incorporate information on predictable escape pathways.

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≤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 ∼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≤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.

Genetic Characterization of Human Immunodeficiency Virus Type 1 in Elite Controllers: Lack of Gross Genetic Defects or Common Amino Acid Changes

ABSTRACT Despite reports of viral genetic defects in persons who control human immunodeficiency virus type 1 (HIV-1) in the absence of antiviral therapy, the extent to which such defects contribute to the long-term containment of viremia is not known. Most previous studies examining for such defects have involved small numbers of subjects, primarily focused on subjects expressing HLA-B57, or have examined single viral genes, and they have focused on cellular proviral DNA rather than plasma viral RNA sequences. Here, we attempted viral sequencing from 95 HIV-1 elite controllers (EC) who maintained plasma viral loads of <50 RNA copies/ml in the absence of therapy, the majority of whom did not express HLA-B57. HIV-1 gene fragments were obtained from 94% (89/95) of the EC, and plasma viral sequences were obtained from 78% (61/78), the latter indicating the presence of replicating virus in the majority of EC. Of 63 persons for whom nef was sequenced, only three cases of nef deletions were identified, and gross genetic defects were rarely observed in other HIV-1 coding genes. In a codon-by-codon comparison between EC and persons with progressive infection, correcting for HLA bias and coevolving secondary mutations, a significant difference was observed at only three codons in Gag, all three of which represented the historic population consensus amino acid at the time of infection. These results indicate that the spontaneous control of HIV replication is not attributable to shared viral genetic defects or shared viral polymorphisms.