Mary van der Stok

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.

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.

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.

Detection of Polyfunctional Mycobacterium tuberculosis-Specific T Cells and Association with Viral Load in HIV-1-Infected Persons

BACKGROUND The human immunodeficiency virus type 1 (HIV-1) epidemic is associated with a significant increase in the incidence of tuberculosis (TB); however, little is known about the quality of Mycobacterium tuberculosis (MTB)-specific cellular immune responses in coinfected individuals. METHODS A total of 137 HIV-1-positive individuals in Durban, South Africa, were screened with the use of overlapping peptides spanning Ag85A, culture filtrate protein 10 (CFP-10), early secretory antigen target 6 (ESAT-6), and TB10.4, in an interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) assay. Intracellular cytokine staining for MTB-specific production of IFN-gamma, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-2 was performed, as was ex vivo phenotyping of memory markers on MTB-specific T cells. RESULTS A total of 41% of subjects responded to ESAT-6 and/or CFP-10, indicating the presence of latent MTB infection. The proportion of MTB-specific IFN-gamma(+)/TNF-alpha(+) CD4(+) cells was significantly higher than the proportion of IFN-gamma(+)/IL-2(+) CD4(+) cells (P = .0220), and the proportion of MTB-specific IL-2-secreting CD4 cells was inversely correlated with the HIV-1 load (P = .0098). MTB-specific CD8 T cells were predominately IFN-gamma(+)/TNF-alpha(+)/IL-2(-). Ex vivo memory phenotyping of MTB-specific CD4 and CD8 T cells indicated an early to intermediate differentiated phenotype for the population of effector memory cells. CONCLUSIONS Polyfunctional MTB-specific CD4 and CD8 T cell responses are maintained in the peripheral blood of HIV-1-positive individuals, in the absence of active disease, and the functional capacity of these responses is affected by HIV-1 disease status.

Proliferative capacity of epitope-specific CD8 T-cell responses is inversely related to viral load in chronic human immunodeficiency virus type 1 infection

ABSTRACT The relationship between the function of human immunodeficiency virus (HIV)-specific CD8 T-cell responses and viral load has not been defined. In this study, we used a panel of major histocompatibility complex class I tetramers to examine responses to frequently targeted CD8 T-cell epitopes in a large cohort of antiretroviral-therapy-naïve HIV type 1 clade C virus-infected persons in KwaZulu Natal, South Africa. In terms of effector functions of proliferation, cytokine production, and degranulation, only proliferation showed a significant correlation with viral load. This robust inverse relationship provides an important functional correlate of viral control relevant to both vaccine design and evaluation.

Gag-Protease-Mediated Replication Capacity in HIV-1 Subtype C Chronic Infection: Associations with HLA Type and Clinical Parameters

ABSTRACT The mechanisms underlying HIV-1 control by protective HLA class I alleles are not fully understood and could involve selection of escape mutations in functionally important Gag epitopes resulting in fitness costs. This study was undertaken to investigate, at the population level, the impact of HLA-mediated immune pressure in Gag on viral fitness and its influence on HIV-1 pathogenesis. Replication capacities of 406 recombinant viruses encoding plasma-derived Gag-protease from patients chronically infected with HIV-1 subtype C were assayed in an HIV-1-inducible green fluorescent protein reporter cell line. Viral replication capacities varied significantly with respect to the specific HLA-B alleles expressed by the patient, and protective HLA-B alleles, most notably HLA-B*81, were associated with lower replication capacities. HLA-associated mutations at low-entropy sites, especially the HLA-B*81-associated 186S mutation in the TL9 epitope, were associated with lower replication capacities. Most mutations linked to alterations in replication capacity in the conserved p24 region decreased replication capacity, while most in the highly variable p17 region increased replication capacity. Replication capacity also correlated positively with baseline viral load and negatively with baseline CD4 count but did not correlate with the subsequent rate of CD4 decline. In conclusion, there is evidence that protective HLA alleles, in particular HLA-B*81, significantly influence Gag-protease function by driving sequence changes in Gag and that conserved regions of Gag should be included in a vaccine aiming to drive HIV-1 toward a less fit state. However, the long-term clinical benefit of immune-driven fitness costs is uncertain given the lack of correlation with longitudinal markers of disease progression.

Immunodominant HIV-1 Cd4+ T Cell Epitopes in Chronic Untreated Clade C HIV-1 Infection

Background A dominance of Gag-specific CD8+ T cell responses is significantly associated with a lower viral load in individuals with chronic, untreated clade C human immunodeficiency virus type 1 (HIV-1) infection. This association has not been investigated in terms of Gag-specific CD4+ T cell responses, nor have clade C HIV-1–specific CD4+ T cell epitopes, likely a vital component of an effective global HIV-1 vaccine, been identified. Methodology/Principal Findings Intracellular cytokine staining was conducted on 373 subjects with chronic, untreated clade C infection to assess interferon-gamma (IFN-γ) responses by CD4+ T cells to pooled Gag peptides and to determine their association with viral load and CD4 count. Gag-specific IFN-γ–producing CD4+ T cell responses were detected in 261/373 (70%) subjects, with the Gag responders having a significantly lower viral load and higher CD4 count than those with no detectable Gag response (p<0.0001 for both parameters). To identify individual peptides targeted by HIV-1–specific CD4+ T cells, separate ELISPOT screening was conducted on CD8-depleted PBMCs from 32 chronically infected untreated subjects, using pools of overlapping peptides that spanned the entire HIV-1 clade C consensus sequence, and reconfirmed by flow cytometry to be CD4+ mediated. The ELISPOT screening identified 33 CD4+ peptides targeted by 18/32 patients (56%), with 27 of the 33 peptides located in the Gag region. Although the breadth of the CD4+ responses correlated inversely with viral load (p = 0.015), the magnitude of the response was not significantly associated with viral load. Conclusions/Significance These data indicate that in chronic untreated clade C HIV-1 infection, IFN-γ–secreting Gag-specific CD4+ T cell responses are immunodominant, directed at multiple distinct epitopes, and associated with viral control.

Lack of Duffy antigen receptor for chemokines: no influence on HIV disease progression in an African treatment-naive population.

Dear Editors: He et al recently reported an association between a single-nucleotide polymorphism (SNP, rs2814778, -46T→C) in the erythroid specific promoter region of the Duffy antigen receptor for chemokine (DARC) and HIV-1 acquisition and disease progression in African Americans (He et al., 2008). The authors described a higher frequency of the null homozygous genotype (-46C/C) in HIV-infected (70%) versus non-infected individuals (60%). Based on these findings they argued that the null allele confers susceptibility to infection with HIV-1 and suggested that up to 11% of the HIV-1 burden in sub-Saharan Africa could be attributed to homozygosity for the null allele. Oddly, however, these authors reported that the null genotype associated with better outcomes amongst those who became infected, including longer survival and more particularly decelerated loss of CD4+ T-lymphocytes. Another report showed an association between the -46C/C genotype and significantly reduced total white blood cells (WBC) in African Americans explaining ∼20% of population variation in WBC (Nalls et al., 2008). The -46C/C genotype is nearly fixed in those sub-Saharan African populations where Plasmodium vivax is endemic, apparently because it confers resistance against P. vivax malaria, but -46C is virtually absent in individuals of European descent (Tournamille et al., 1995; Hamblin and Di Rienzo, 2000). In those regions of sub-Saharan Africa that are not endemic for P. vivax, the -46C allele is not fixed, however, and the wildtype (-46T) allele is frequently observed (Rosenberg et al., 2007). Considering the potential importance of DARC on HIV-1 disease progression in Africans, we studied the effect of the DARC -46 genotype on outcomes to HIV infection in a cohort of untreated black South Africans. This region of Africa is not endemic for P. vivax and lies outside of the area of 96% mean fixation for -46C; thus, the null allele of DARC is not fixed in our study population and the allele frequency of -46T (the wildtype allele) is about 20%. We assessed the effects of the null (C/C), heterozygous (T/C), and wildtype (T/T) genotypes on rates of CD4 decline and mean HIV-1 viral loads in a group of 381 untreated black South Africans. Since the -46C/C genotype has previously been associated significantly with reduced total WBC (Nalls et al., 2008), we also determined WBC in our cohort in order to verify that our cohort is sufficiently powered to detect an effect of the -46 genotype, where -46C/C is expected to associate with low WBC as previously demonstrated.

Limited Immunogenicity of HIV CD8+ T-Cell Epitopes in Acute Clade C Virus Infection

BACKGROUND Human immunodeficiency virus type 1 (HIV-1)-specific CD8(+) responses contribute to the decline in acute peak viremia following infection. However, data on the relative immunogenicity of CD8(+) T-cell epitopes during and after acute viremia are lacking. METHODS We characterized CD8(+) T-cell responses in 20 acutely infected, antiretroviral-naive individuals with HIV-1 subtype C infection using the interferon-γ enzyme-linked immunosorbent spot assay. Eleven of these had not fully seroconverted at the time of analysis. Viruses from plasma were sequenced within defined cytotoxic T-lymphocyte (CTL) cell epitopes for selected subjects. RESULTS At approximately 28 days after estimated initial infection, CD8(+) T-cell responses were directed against an average of 3 of the 410 peptides tested (range, 0-6); 2 individuals had no detectable responses at this time. At 18 weeks, the average number of peptides targeted had increased to 5 (range 0-11). Of the 56 optimal Gag CTL epitopes sequenced, 31 were wild-type in the infecting viruses, but only 11 of 31 elicited measurable CD8(+) T-cell responses. CONCLUSIONS These data demonstrate that the majority of CD8(+) responses are not elicited during acute HIV infection despite the presence of the cognate epitope in the infecting strain. There is a need to define factors that influence lack of induction of effective immune responses and the parameters that dictate immunodominance in acute infection.

Broad and persistent Gag-specific CD8+ T-cell responses are associated with viral control but rarely drive viral escape during primary HIV-1 infection.

Objective:We characterized protein-specific CD8+ T-cell immunodominance patterns during the first year of HIV-1 infection, and their impact on viral evolution and immune control. Methods:We analyzed CD8+ T-cell responses to the full HIV-1 proteome during the first year of infection in 18 antiretroviral-naïve individuals with acute HIV-1 subtype C infection, all identified prior to seroconversion. Ex-vivo and cultured interferon-&ggr; ELISPOT assays were performed and viruses from plasma were sequenced within defined CTL Gag epitopes. Results:Nef-specific CD8+ T-cell responses were dominant during the first 4 weeks after infection and made up 40% of the total responses at this time; yet, by 1 year, responses against this region had declined and Gag responses made up to 47% of all T-cell responses measured. An inverse correlation between the breadth of Gag-specific responses and viral load set point was evident at 26 weeks after infection (P = 0.0081, r = −0.60) and beyond. An inverse correlation between the number of persistent responses targeting Gag and viral set point was also identified (P = 0.01, r = −0.58). Gag-specific responses detectable by the cultured ELISPOT assay correlated negatively with viral load set point (P = 0.0013, r = −0.91). Sequence evolution in targeted and nontargeted Gag epitopes in this cohort was infrequent. Conclusions:These data underscore the importance of HIV-specific CD8+ T-cell responses, particularly to the Gag protein, in the maintenance of low viral load levels during primary infection, and show that these responses are initially poorly elicited by natural infection. These data have implications for vaccine design strategies.