Sharon Reddy

PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression

Functional impairment of T cells is characteristic of many chronic mouse and human viral infections. The inhibitory receptor programmed death 1 (PD-1; also known as PDCD1), a negative regulator of activated T cells, is markedly upregulated on the surface of exhausted virus-specific CD8 T cells in mice. Blockade of this pathway using antibodies against the PD ligand 1 (PD-L1, also known as CD274) restores CD8 T-cell function and reduces viral load. To investigate the role of PD-1 in a chronic human viral infection, we examined PD-1 expression on human immunodeficiency virus (HIV)-specific CD8 T cells in 71 clade-C-infected people who were naive to anti-HIV treatments, using ten major histocompatibility complex (MHC) class I tetramers specific for frequently targeted epitopes. Here we report that PD-1 is significantly upregulated on these cells, and expression correlates with impaired HIV-specific CD8 T-cell function as well as predictors of disease progression: positively with plasma viral load and inversely with CD4 T-cell count. PD-1 expression on CD4 T cells likewise showed a positive correlation with viral load and an inverse correlation with CD4 T-cell count, and blockade of the pathway augmented HIV-specific CD4 and CD8 T-cell function. These data indicate that the immunoregulatory PD-1/PD-L1 pathway is operative during a persistent viral infection in humans, and define a reversible defect in HIV-specific T-cell function. Moreover, this pathway of reversible T-cell impairment provides a potential target for enhancing the function of exhausted T cells in chronic HIV infection.

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.

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.

Enhanced anti-HIV functional activity associated with Gag-specific CD8 T-cell responses.

ABSTRACT Effective HIV-specific T-cell immunity requires the ability to inhibit virus replication in the infected host, but the functional characteristics of cells able to mediate this effect are not well defined. Since Gag-specific CD8 T cells have repeatedly been associated with lower viremia, we examined the influence of Gag specificity on the ability of unstimulated CD8 T cells from chronically infected persons to inhibit virus replication in autologous CD4 T cells. Persons with broad (≥6; n = 13) or narrow (≤1; n = 13) Gag-specific responses, as assessed by gamma interferon enzyme-linked immunospot assay, were selected from 288 highly active antiretroviral therapy (HAART)-naive HIV-1 clade C-infected South Africans, matching groups for total magnitude of HIV-specific CD8 T-cell responses and CD4 T-cell counts. CD8 T cells from high Gag responders suppressed in vitro replication of a heterologous HIV strain in autologous CD4 cells more potently than did those from low Gag responders (P < 0.003) and were associated with lower viral loads in vivo (P < 0.002). As previously shown in subjects with low viremia, CD8 T cells from high Gag responders exhibited a more polyfunctional cytokine profile and a stronger ability to proliferate in response to HIV stimulation than did low Gag responders, which mainly exhibited monofunctional CD8 T-cell responses. Furthermore, increased polyfunctionality was significantly correlated with greater inhibition of viral replication in vitro. These data indicate that enhanced suppression of HIV replication is associated with broader targeting of Gag. We conclude that it is not the overall magnitude but rather the breadth, magnitude, and functional capacity of CD8 T-cell responses to certain conserved proteins, like Gag, which predict effective antiviral HIV-specific CD8 T-cell function.

Targeting of a CD8 T cell env epitope presented by HLA-B*5802 is associated with markers of HIV disease progression and lack of selection pressure

In HIV-infected persons, certain HLA class I alleles are associated with effective control of viremia, while others are associated with rapid disease progression. Among the most divergent clinical outcomes are the relatively good prognosis in HLA-B*5801 expressing persons and poor prognosis with HLA-B*5802. These two alleles differ by only three amino acids in regions involved in HLA-peptide recognition. This study evaluated a cohort of over 1000 persons with chronic HIV clade C virus infection to determine whether clinical outcome differences associated with B*5801 (n = 93) and B*5802 ( n = 259) expression are associated with differences in HIV-1-specific CD8 (+) T cell responses. The overall breadth and magnitude of HIV-1-specific CD8(+) T cell responses were lower in persons expressing B*5802, and epitope presentation by B*5802 contributed significantly less to the overall response as compared to B*5801-restricted CD8 (+) T cells. Moreover, viral load in B*5802-positive persons was higher and CD4 cell counts lower when this allele contributed to the overall CD8 (+) T cell response, which was detected exclusively through a single epitope in Env. In addition, persons heterozygous for B*5802 compared to persons homozygous for other HLA-B alleles had significantly higher viral loads. Viral sequencing revealed strong selection pressure mediated through B*5801-restricted responses but not through B*5802. These data indicate that minor differences in HLA sequence can have a major impact on epitope recognition, and that selective targeting of Env through HLA-B*5802 is at least ineffectual if not actively adverse in the containment of viremia. These results provide experimental evidence that not all epitope-specific responses contribute to immune containment, a better understanding of which is essential to shed light on mechanisms involved in HIV disease progression.

Human Immunodeficiency Virus-Specific CD8+ T-Cell Activity Is Detectable from Birth in the Majority of In Utero-Infected Infants

ABSTRACT Human immunodeficiency virus (HIV)-infected infants in sub-Saharan Africa typically progress to AIDS or death by 2 years of life in the absence of antiretroviral therapy. This rapid progression to HIV disease has been related to immaturity of the adaptive immune response in infants. We screened 740 infants born to HIV-infected mothers and tracked development and specificity of HIV-specific CD8+ T-cell responses in 63 HIV-infected infants identified using gamma interferon enzyme-linked immunospot assays and intracellular cytokine staining. Forty-four in utero-infected and 19 intrapartum-infected infants were compared to 45 chronically infected children >2 years of age. Seventy percent (14 of 20) in utero-infected infants tested within the first week of life demonstrated HIV-specific CD8+ T-cell responses. Gag, Pol, and Nef were the principally targeted regions in chronic pediatric infection. However, Env dominated the overall response in one-third (12/36) of the acutely infected infants, compared to only 2/45 (4%) of chronically infected children (P = 0.00083). Gag-specific CD4+ T-cell responses were minimal to undetectable in the first 6 months of pediatric infection. These data indicate that failure to control HIV replication in in utero-infected infants is not due to an inability to induce responses but instead suggest secondary failure of adaptive immunity in containing this infection. Moreover, the detection of virus-specific CD8+ T-cell responses in the first days of life in most in utero-infected infants is encouraging for HIV vaccine interventions in infants.

Impact of HLA in Mother and Child on Disease Progression of Pediatric Human Immunodeficiency Virus Type 1 Infection

ABSTRACT A broad Gag-specific CD8+ T-cell response is associated with effective control of adult human immunodeficiency virus (HIV) infection. The association of certain HLA class I molecules, such as HLA-B*57, -B*5801, and -B*8101, with immune control is linked to mutations within Gag epitopes presented by these alleles that allow HIV to evade the immune response but that also reduce viral replicative capacity. Transmission of such viruses containing mutations within Gag epitopes results in lower viral loads in adult recipients. In this study of pediatric infection, we tested the hypothesis that children may tend to progress relatively slowly if either they themselves possess one of the protective HLA-B alleles or the mother possesses one of these alleles, thereby transmitting a low-fitness virus to the child. We analyzed HLA type, CD8+ T-cell responses, and viral sequence changes for 61 mother-child pairs from Durban, South Africa, who were monitored from birth. Slow progression was significantly associated with the mother or child possessing one of the protective HLA-B alleles, and more significantly so when the protective allele was not shared by mother and child (P = 0.007). Slow progressors tended to make CD8+ T-cell responses to Gag epitopes presented by the protective HLA-B alleles, in contrast to progressors expressing the same alleles (P = 0.07; Fishers exact test). Mothers expressing the protective alleles were significantly more likely to transmit escape variants within the Gag epitopes presented by those alleles than mothers not expressing those alleles (75% versus 21%; P = 0.001). Reversion of transmitted escape mutations was observed in all slow-progressing children whose mothers possessed protective HLA-B alleles. These data show that HLA class I alleles influence disease progression in pediatric as well as adult infection, both as a result of the CD8+ T-cell responses generated in the child and through the transmission of low-fitness viruses by the mother.

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.

Alterations in Natural Killer Cell Receptor Profiles During HIV Type 1 Disease Progression Among Chronically Infected South African Adults

Recent studies suggest that innate immune responses by natural killer (NK) cells play a significant role in restricting human immunodeficiency virus type-1 (HIV-1) pathogenesis. Our aim was to characterize changes in NK cells associated with HIV-1 clade C disease progression. Here we used multiparametric flow cytometry (LSRII) to quantify phenotype and function of NK cells in a cross-sectional analysis of cryopreserved blood samples from a cohort of 41 chronically HIV-1-infected, treatment-naive adult South Africans. These individuals ranged in disease severity from early (CD4 count >500) to advanced HIV-1 disease (CD4 count <50). We found that the frequency of NK cells expressing KIR2DL1, an inhibitory receptor, and/or KIR2DS1, an activating receptor, tended to decrease with increasing HIV-1 viral load. We also discovered a significant increase (p < 0.05) in overall NK cell degranulation with disease progression. We found that acutely activated NK cells (CD69(pos)) were deficient in NKp46 expression ex vivo. In conclusion, we observed that with viremia and advanced HIV-1 disease, activated NK cells lack NKp46 expression, and KIR2DS1(pos) and/ or KIR2DL1(pos) NK cells are reduced in frequency. These findings suggest that modulation of receptor expression on NK cells may play a role in HIV-1 pathogenesis, and provide new insights on immunological changes in advanced HIV-1 disease.

Detection of HIV Type 1 Gag-Specific CD4+ T Cell Responses in Acutely Infected Infants

Multiple HIV-1-specific cytokine and proliferative responses by CD4(+) T cells have not been studied in acutely infected infants. Using an intracellular cytokine staining assay, 34 untreated clade C HIV-1-infected infants (2-102 days old) were assessed for IFN-gamma, 28/34 for IL-2, and 26/34 for TNF-alpha responses to all HIV-1 proteins. Responses were detected in 29%, 36%, and 15% of infants, respectively. Twelve of the original 34 infants were then studied longitudinally for 14 months to determine the effect of viral load on IFN-gamma Gag-specific responses: seven infants were treated for 1 year, stopped treatment, and resumed when CD4% was < 20 and five infants were treated only when the CD4% was <20. Following treatment cessation, there was an immediate increase in viral load followed by an increase in the magnitude of CD4(+) Gag-specific responses. Despite this, the majority of infants (54%) had to restart treatment by 24 months of age, indicating that the immune responses were antigen driven but not associated with protection. Among untreated infants HIV-specific CD4(+) responses were detected sporadically indicating a dysfunctional immune response in the face of constant exposure to high levels of viremia.