Enhanced Anti-HIV Functional Activity Associated with Gag-Specific CD8 T-Cell Responses

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.Studies aimed at correlating overall quantitative differences in breadth or magnitude of the gamma interferon (IFN-γ)-positive HIV-specific CD8 T-cell response and plasma HIV viral loads have failed to show an association with control of viremia (2, 18). However, multiple studies (10, 12-15, 18, 22, 29) have shown that broadly directed and/or dominant HIV-specific CD8 T-cell responses against the Gag protein, as measured by IFN-γ enzyme-linked immunospot (ELISPOT) assay, are associated with lower viremia in chronic HIV-1 infection. In contrast, non-Gag-specific T-cell responses, as shown in some studies, did not contribute to immune control. Indeed, more broadly directed CD8 T-cell responses directed to the Env protein have been associated with elevated viremia (15). The functional mechanism underlying enhanced viral control by Gag-specific CD8 T-cell responses has not been determined.One potential explanation for enhanced antiviral pressure by Gag-specific but not other virus-specific CD8 T-cell responses may be differences in the fitness cost associated with escape mutations within the highly conserved Gag protein compared to that of other viral proteins (5, 23, 27). Alternatively, the maturation phenotype and functional quality of HIV-specific CD8 T cells may be the more critical predictors of the effectiveness of a virus-specific response (1, 4, 7, 15, 20, 25). In addition to the secretion of IFN-γ, CD8 T cells exhibit a spectrum of additional antiviral functions, including cytolysis, cell proliferation, and production of cytokines and chemokines. The capacity of CD8 T cells to secrete multiple cytokines following stimulation with HIV peptides is also associated with long-term nonprogressive infection, although subsequent studies have argued that polyfunctionality may simply correlate with reduced antigen stimulation rather than being a direct mediator of viral control (4, 19, 28, 34). Increased expression of the negative immunoregulatory molecule PD-1 on HIV-specific CD8 T cells is associated with higher viral loads (8, 21, 30). Finally, high HIV-specific CD8 T-cell proliferative capacity is associated with lower HIV viral loads (9). However, a direct link between HIV-specific antiviral efficacy and any specific functional capacity has yet to be established.Following the resolution of acute HIV-1 infection, HIV-specific CD8 T-cell responses reduce viral replication to a set point, which varies dramatically among individuals but is a strong predictor of the rate of HIV disease progression (17). It is therefore plausible that more potent antiviral CD8 T-cell responses, at set point, that are able to contain viral replication more aggressively may provide enhanced control of disease progression. However, to date, the majority of studies aimed at defining differences in the viral suppressive properties of protective HIV-1-specific CD8 T-cell responses have focused narrowly either on single-peptide-specific cytotoxic T lymphocyte (CTL) clones or cell lines (7) or on specific subpopulations of study subjects such as “elite” controllers (25). Studies examining the relationship between in vitro inhibition of viral replication over a broad range of viral loads and antigen specificities have not been performed. Furthermore, little work has focused on defining the antiviral properties of HIV-specific CD8 T-cell responses in clade C infection (33).Thus, to address the potential role of antigen specificity in the antiviral properties of HIV-specific CD8 T-cell responses, we compared the phenotypic and functional characteristics of bulk CD8 T cells in a group of untreated chronically clade C-infected persons that broadly targeted Gag-specific responses (≥6 Gag-specific responses) to those of subjects that had very narrow or absent Gag-specific responses (≤1 Gag-specific response). Importantly, the two groups were selected such that total CD4 cell counts and total magnitude of HIV-specific CD8 T-cell responses by IFN-γ ELISPOT assay were matched. Our results confirm that, for the same level of CD4 cell count and overall magnitude of HIV-specific CD8 T-cell responses, subjects whose CD8 T-cell responses are dominantly and broadly directed against the Gag protein exhibit lower plasma viral loads than do subjects who target this protein less. Furthermore, we demonstrate that this enhanced viral control is associated with an enhanced ability of isolated CD8 T cells to inhibit replication of a heterologous HIV-1 strain in autologous CD4 cells in vitro, enhanced ability to proliferate in the presence of cognate antigen, and a more polyfunctional cytokine response, but not with a difference in the maturation status of HIV-specific CD8 T cells. These data indicate that the specificity of the CD8 T-cell response to HIV is important for viral control and that it is a distinct polyfunctional phenotype of CD8 T cells that is able to proliferate and secrete antiviral cytokines, which is indicative of effective antiviral CD8 T-cell function.     

Human immunodeficiency virus-specific gamma interferon enzyme-linked immunospot assay responses targeting specific regions of the proteome during primary subtype C infection are poor predictors of the course of viremia and set point

It is unknown whether patterns of human immunodeficiency virus (HIV)-specific T-cell responses during acute infection may influence the viral set point and the course of disease. We wished to establish whether the magnitude and breadth of HIV type 1 (HIV-1)-specific T-cell responses at 3 months postinfection were correlated with the viral-load set point at 12 months and hypothesized that the magnitude and breadth of HIV-specific T-cell responses during primary infection would predict the set point. Gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay responses across the complete proteome were measured in 47 subtype C HIV-1-infected participants at a median of 12 weeks postinfection. When corrected for amino acid length and individuals responding to each region, the order of recognition was as follows: Nef > Gag > Pol > Rev > Vpr > Env > Vpu > Vif > Tat. Nef responses were significantly (P < 0.05) dominant, targeted six epitopic regions, and were unrelated to the course of viremia. There was no significant difference in the magnitude and breadth of responses for each protein region with disease progression, although there was a trend of increased breadth (mean, four to seven pools) in rapid progressors. Correlation of the magnitude and breadth of IFN-γ responses with the viral set point at 12 months revealed almost zero association for each protein region. Taken together, these data demonstrate that the magnitude and breadth of IFN-γ ELISPOT assay responses at 3 months postinfection are unrelated to the course of disease in the first year of infection and are not associated with, and have low predictive power for, the viral set point at 12 months.     

Immunodominant HIV-Specific CD8+ T-Cell Responses Are Common to Blood and Gastrointestinal Mucosa,and Gag-Specific Responses Dominate in Rectal Mucosa of HIV Controllers

Previous studies have suggested that polyfunctional mucosal CD8⁺ T-cell responses may be a correlate of protection in HIV controllers. Mucosal T-cell breadth and/or specificity may also contribute to defining protective responses. In this study, rectal CD8⁺ T-cell responses to HIV Gag, Env, and Nef were mapped at the peptide level in four subject groups: elite controllers (n = 16; viral load [VL], <75 copies/ml), viremic controllers (n = 14; VL, 75 to 2,000 copies/ml), noncontrollers (n = 14; VL, >10,000 copies/ml), and antiretroviral-drug-treated subjects (n = 8; VL, <75 copies/ml). In all subject groups, immunodominant CD8⁺ T-cell responses were generally shared by blood and mucosa, although there were exceptions. In HIV controllers, responses to HLA-B27- and HLA-B57-restricted epitopes were common to both tissues, and their magnitude (in spot-forming cells [SFC] per million) was significantly greater than those of responses restricted by other alleles. Furthermore, peptides recognized by T cells in both blood and rectal mucosa, termed “concordant,” elicited higher median numbers of SFC than discordant responses. In magnitude as well as breadth, HIV Gag-specific responses, particularly those targeting p24 and p7, dominated in controllers. Responses in noncontrollers were more evenly distributed among epitopes in Gag, Env, and Nef. Viremic controllers showed significantly broader mucosal Gag-specific responses than other groups. Taken together, these findings demonstrate that (i) Gag-specific responses dominate in mucosal tissues of HIV controllers; (ii) there is extensive overlap between CD8⁺ T cells in blood and mucosal tissues, with responses to immunodominant epitopes generally shared by both sites; and (iii) mucosal T-cell response breadth alone cannot account for immune control.Despite more than two decades of intensive research, the immunologic correlates of protection from human immunodeficiency virus (HIV) infection and disease progression remain incompletely understood. To date, the majority of studies of HIV-specific T-cell responses have focused on the measurement of such responses in peripheral blood lymphocytes. Nevertheless, the majority of the body''s lymphocytes are housed in mucosal tissues, notably the gastrointestinal (GI) tract (18, 33, 40). The gastrointestinal mucosa also serves as a major target of HIV infection and CD4⁺ T-cell depletion (7, 25, 36), as well as an important site of transmission (18, 33, 40). Antigen-experienced T cells may preferentially traffic to tissue sites of infection (50), where they may also expand in an antigen-driven manner. Because of the unique role of the gastrointestinal mucosa in HIV pathogenesis, detailed studies of HIV-specific immune responses in this compartment may contribute important insights to our understanding of the disease process.An important question is the degree to which T-cell responses in mucosal tissues are “compartmentalized” and distinct in specificity and/or clonality from those found elsewhere in the body, including in peripheral blood. Because of the technical challenges associated with obtaining large numbers of viable lymphocytes from mucosal biopsy specimen tissue, comprehensive mapping of the fine specificity of mucosal HIV-specific T-cell responses has been difficult. Relying on a polyclonal expansion approach, Ibarrondo and colleagues successfully mapped HIV-specific CD8⁺ T-cell responses in blood and rectal mucosa of chronically infected persons to the level of peptide pools but not to individual epitopes (29). Their studies revealed a similar pattern of responses, and nearly identical immunodominance hierarchies, in the two tissue sites.We have focused our recent studies of mucosal immunity on a group of individuals who control HIV infection in the absence of antiretroviral therapy. These are often called “long-term nonprogressors” (LTNP) (14), referring to their ability to maintain normal CD4⁺ T-cell counts for more than 10 years without medication. LTNP are believed to account for 5 to 15% of the HIV-infected population. Several recent studies have used the term “HIV controllers,” defined as those who maintain undetectable plasma HIV RNA levels (“elite controllers”) and those who have persistently detectable but low plasma HIV RNA levels (“viremic controllers”). Elite controllers represent less than 1% of the HIV-infected population (14). In contrast, individuals with viral loads of >10,000 copies/ml in the absence of therapy are termed “noncontrollers.” Recently, we found that “polyfunctional” HIV-specific T cells, producing multiple antiviral factors, were significantly more abundant in gastrointestinal mucosa of HIV controllers than in those of noncontrollers or subjects on highly active antiretroviral therapy (HAART) (20). Furthermore, in many cases these strong, polyfunctional mucosal T-cell responses were not mirrored in peripheral blood, suggesting that HIV-specific T cells either preferentially traffic to or undergo expansion within mucosal tissues.Because of these findings, we undertook a follow-up study to determine the breadth and fine specificity, to the peptide level, of mucosal CD8⁺ T-cell responses to HIV Gag, Env, and Nef among HIV controllers, noncontrollers, and individuals on HAART. We hypothesized that controllers might harbor an unusually broad repertoire of HIV-specific CD8⁺ T cells in mucosal tissues. We found a similar response breadth in mucosal tissues of all three subject groups, arguing against a critical role for mucosal T-cell response breadth in determining the extent of HIV control. In contrast, we found that high-magnitude mucosal responses directed at well-conserved regions in Gag were a strong and consistent correlate of control. Finally, concordant responses, defined as those common to blood and mucosa, were generally stronger than discordant responses, underscoring the observation that T cells responding to immunodominant epitopes are broadly distributed throughout the body in both controllers and noncontrollers.     

Differential Clade-Specific HLA-B*3501 Association with HIV-1 Disease Outcome Is Linked to Immunogenicity of a Single Gag Epitope

The strongest genetic influence on immune control in HIV-1 infection is the HLA class I genotype. Rapid disease progression in B-clade infection has been linked to HLA-B*35 expression, in particular to the less common HLA-B*3502 and HLA-B*3503 subtypes but also to the most prevalent subtype, HLA-B*3501. In these studies we first demonstrated that whereas HLA-B*3501 is associated with a high viral set point in two further B-clade-infected cohorts, in Japan and Mexico, this association does not hold in two large C-clade-infected African cohorts. We tested the hypothesis that clade-specific differences in HLA associations with disease outcomes may be related to distinct targeting of critical CD8⁺ T-cell epitopes. We observed that only one epitope was significantly targeted differentially, namely, the Gag-specific epitope NPPIPVGDIY (NY10, Gag positions 253 to 262) (P = 2 × 10⁻⁵). In common with two other HLA-B*3501-restricted epitopes, in Gag and Nef, that were not targeted differentially, a response toward NY10 was associated with a significantly lower viral set point. Nonimmunogenicity of NY10 in B-clade-infected subjects derives from the Gag-D260E polymorphism present in ∼90% of B-clade sequences, which critically reduces recognition of the Gag NY10 epitope. These data suggest that in spite of any inherent HLA-linked T-cell receptor repertoire differences that may exist, maximizing the breadth of the Gag-specific CD8⁺ T-cell response, by the addition of even a single epitope, may be of overriding importance in achieving immune control of HIV infection. This distinction is of direct relevance to development of vaccines designed to optimize the anti-HIV CD8⁺ T-cell response in all individuals, irrespective of HLA type.     

Human immunodeficiency virus-specific CD8+ T-cell activity is detectable from birth in the majority of in utero-infected infants

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.     

Non-Immunogenicity of Overlapping Gag Peptides Pulsed on Autologous Cells after Vaccination of HIV Infected Individuals

Background

HIV Gag-specific CD4+ and CD8+ T-cell responses are important for HIV immune control. Pulsing overlapping Gag peptides on autologous lymphocytes (OPAL) has proven immunogenic and effective in reducing viral loads in multiple pigtail macaque studies, warranting clinical evaluation.

Methodology

We performed a phase I, single centre, placebo-controlled, double-blinded and dose-escalating study to evaluate the safety and preliminary immunogenicity of a novel therapeutic vaccine approach ‘OPAL-HIV-Gag(c)’. This vaccine is comprised of 120 15mer peptides, overlapping by 11 amino acids, spanning the HIV Gag C clade sequence proteome, pulsed on white blood cells enriched from whole blood using a closed system, followed by intravenous reinfusion. Patients with undetectable HIV viral loads (<50 copies/ml plasma) on HAART received four administrations at week 0, 4, 8 and 12, and were followed up for 12 weeks post-treatment. Twenty-three people were enrolled in four groups: 12 mg (n = 6), 24 mg (n = 7), 48 mg (n = 2) or matching placebo (n = 8) with 18 immunologically evaluable. T-cell immunogenicity was assessed by IFNγ ELIspot and intracellular cytokine staining (ICS).

Results

The OPAL-HIV-Gag(c) peptides were antigenic in vitro in 17/17 subjects. After vaccination with OPAL-HIV-Gag(c), 1/6 subjects at 12 mg and 1/6 subjects at 24 mg dose groups had a 2- and 3-fold increase in ELIspot magnitudes from baseline, respectively, of Gag-specific CD8+ T-cells at week 14, compared to 0/6 subjects in the placebo group. No Gag-specific CD4+ T-cell responses or overall change in Rev, Nef, Tat and CMV specific responses were detected. Marked, transient and self-limiting lymphopenia was observed immediately post-vaccination (4 hours) in OPAL-HIV-Gag(c) but not in placebo recipients, with median fall from 1.72 to 0.67 million lymphocytes/mL for active groups (P<0.001), compared to post-placebo from 1.70 to 1.56 lymphocytes/ml (P = 0.16).

Conclusion/Significance

Despite strong immunogenicity observed in several Macaca nemestrina studies using this approach, OPAL-HIV-Gag(c) was not significantly immunogenic in humans and improved methods of generating high-frequency Gag-specific T-cell responses are required.

Name of Registry

ClinicalTrials.gov, Registry number: {"type":"clinical-trial","attrs":{"text":"NCT01123915","term_id":"NCT01123915"}}NCT01123915, URL trial registry database: http://www.clinicaltrials.gov/ct2/results?term=OPAL-HIV-1001&Search=Search     

Central role of reverting mutations in HLA associations with human immunodeficiency virus set point

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.     

Host HLA B*Allele-Associated Multi-Clade Gag T-Cell Recognition Correlates with Slow HIV-1 Disease Progression in Antiretroviral Therapy-Na?ve Ugandans

Background

Some HIV infected individuals remain asymptomatic for protracted periods of time in the absence of antiretroviral therapy (ART). Virological control, CD4 T cell loss and HIV-specific responses are some of the key interrelated determinants of HIV-1 disease progression. In this study, possible interactions between viral load, CD4 T cell slopes, host genetics and HIV-specific IFN-γ responses were evaluated in chronically HIV-1-infected adults.

Methodolology/Principal Findings

Multilevel regression modeling was used to stratify clade A or D HIV-infected individuals into disease progression groups based on CD4 T cell slopes. ELISpot assays were used to quantify the frequency and magnitude of HIV-induced IFN-γ responses in 7 defined rapid progressors (RPs) and 14 defined slow progressors (SPs) at a single time point. HLA typing was performed using reference strand conformational analysis (RSCA). Although neither the breadth nor the magnitude of the proteome-wide HIV-specific IFN-γ response correlated with viral load, slow disease progression was associated with over-representation of host immunogenetic protective HLA B* alleles (10 of 14 SPs compared to 0 of 7; p = 0.004, Fisher''s Exact) especially B*57 and B*5801, multiclade Gag T-cell targeting (71%, 10 of 14 SPs compared to 14%, 1 of 7 RPs); p = 0.029, Fisher''s Exact test and evident virological control (3.65 compared to 5.46 log10 copies/mL in SPs and RPs respectively); p<0.001, unpaired student''s t-test

Conclusions

These data are consistent with others that associated protection from HIV disease with inherent host HLA B allele-mediated ability to induce broader Gag T-cell targeting coupled with apparent virological control. These immunogenetic features of Gag-specific immune response which could influence disease progression may provide useful insight in future HIV vaccine design.     

A Correlate of HIV-1 Control Consisting of Both Innate and Adaptive Immune Parameters Best Predicts Viral Load by Multivariable Analysis in HIV-1 Infected Viremic Controllers and Chronically-Infected Non-Controllers

HIV-1 infected viremic controllers maintain durable viral suppression below 2000 copies viral RNA/ml without anti-retroviral therapy (ART), and the immunological factor(s) associated with host control in presence of low but detectable viral replication are of considerable interest. Here, we utilized a multivariable analysis to identify which innate and adaptive immune parameters best correlated with viral control utilizing a cohort of viremic controllers (median 704 viral RNA/ml) and non-controllers (median 21,932 viral RNA/ml) that were matched for similar CD4⁺ T cell counts in the absence of ART. We observed that HIV-1 Gag-specific CD8⁺ T cell responses were preferentially targeted over Pol-specific responses in viremic controllers (p = 0.0137), while Pol-specific responses were positively associated with viral load (rho = 0.7753, p = 0.0001, n = 23). Viremic controllers exhibited significantly higher NK and plasmacytoid dendritic cells (pDC) frequency as well as retained expression of the NK CD16 receptor and strong target cell-induced NK cell IFN-gamma production compared to non-controllers (p<0.05). Despite differences in innate and adaptive immune function however, both viremic controllers (p<0.05) and non-controller subjects (p<0.001) exhibited significantly increased CD8⁺ T cell activation and spontaneous NK cell degranulation compared to uninfected donors. Overall, we identified that a combination of innate (pDC frequency) and adaptive (Pol-specific CD8⁺ T cell responses) immune parameters best predicted viral load (R² = 0.5864, p = 0.0021, n = 17) by a multivariable analysis. Together, this data indicates that preferential Gag-specific over Pol-specific CD8⁺ T cell responses along with a retention of functional innate subsets best predict host control over viral replication in HIV-1 infected viremic controllers compared to chronically-infected non-controllers.     

Increased Sequence Coverage through Combined Targeting of Variant and Conserved Epitopes Correlates with Control of HIV Replication

A major challenge in the development of an HIV vaccine is that of contending with the extensive sequence variability found in circulating viruses. Induction of HIV-specific T-cell responses targeting conserved regions and induction of HIV-specific T-cell responses recognizing a high number of epitope variants have both been proposed as strategies to overcome this challenge. We addressed the ability of cytotoxic T lymphocytes from 30 untreated HIV-infected subjects with and without control of virus replication to recognize all clade B Gag sequence variants encoded by at least 5% of the sequences in the Los Alamos National Laboratory HIV database (1,300 peptides) using gamma interferon and interleukin-2 (IFN-γ/IL-2) FluoroSpot analysis. While targeting of conserved regions was similar in the two groups (P = 0.47), we found that subjects with control of virus replication demonstrated marginally lower recognition of Gag epitope variants than subjects with normal progression (P = 0.05). In viremic controllers and progressors, we found variant recognition to be associated with viral load (r = 0.62, P = 0.001). Interestingly, we show that increased overall sequence coverage, defined as the overall proportion of HIV database sequences targeted through the Gag-specific repertoire, is inversely associated with viral load (r = −0.38, P = 0.03). Furthermore, we found that sequence coverage, but not variant recognition, correlated with increased recognition of a panel of clade B HIV founder viruses (r = 0.50, P = 0.004). We propose sequence coverage by HIV Gag-specific immune responses as a possible correlate of protection that may contribute to control of virus replication. Additionally, sequence coverage serves as a valuable measure by which to evaluate the protective potential of future vaccination strategies.     

Early Gag Immunodominance of the HIV-Specific T-Cell Response during Acute/Early Infection Is Associated with Higher CD8+ T-Cell Antiviral Activity and Correlates with Preservation of the CD4+ T-Cell Compartment

The important role of the CD8⁺ T-cell response on HIV control is well established. Moreover, the acute phase of infection represents a proper scenario to delineate the antiviral cellular functions that best correlate with control. Here, multiple functional aspects (specificity, ex vivo viral inhibitory activity [VIA] and polyfunctionality) of the HIV-specific CD8⁺ T-cell subset arising early after infection, and their association with disease progression markers, were examined. Blood samples from 44 subjects recruited within 6 months from infection (primary HIV infection [PHI] group), 16 chronically infected subjects, 11 elite controllers (EC), and 10 healthy donors were obtained. Results indicated that, although Nef dominated the anti-HIV response during acute/early infection, a higher proportion of early anti-Gag T cells correlated with delayed progression. Polyfunctional HIV-specific CD8⁺ T cells were detected at early time points but did not associate with virus control. Conversely, higher CD4⁺ T-cell set points were observed in PHI subjects with higher HIV-specific CD8⁺ T-cell VIA at baseline. Importantly, VIA levels correlated with the magnitude of the anti-Gag cellular response. The advantage of Gag-specific cells may result from their enhanced ability to mediate lysis of infected cells (evidenced by a higher capacity to degranulate and to mediate VIA) and to simultaneously produce IFN-γ. Finally, Gag immunodominance was associated with elevated plasma levels of interleukin 2 (IL-2) and macrophage inflammatory protein 1β (MIP-1β). All together, this study underscores the importance of CD8⁺ T-cell specificity in the improved control of disease progression, which was related to the capacity of Gag-specific cells to mediate both lytic and nonlytic antiviral mechanisms at early time points postinfection.     

Gag-Specific CD4 and CD8 T-Cell Proliferation in Adolescents and Young Adults with Perinatally Acquired HIV-1 Infection Is Associated with Ethnicity — The ANRS-EP38-IMMIP Study

The ANRS-EP38-IMMIP study aimed to provide a detailed assessment of the immune status of perinatally infected youths living in France. We studied Gag-specific CD4 and CD8 T-cell proliferation and the association between the proliferation of these cells, demographic factors and HIV disease history. We included 93 youths aged between 15 and 24 years who had been perinatally infected with HIV. Sixty-nine had undergone valid CFSE-based T-cell proliferation assays. Gag-specific proliferation of CD4 and CD8 T cells was detected in 12 (16%) and 30 (38%) patients, respectively. The Gag-specific proliferation of CD4 and CD8 T cells was more frequently observed in black patients than in patients from other ethnic groups (CD4: 32% vs. 4%, P = 0.001; CD8: 55% vs. 26%, P = 0.02). Among aviremic patients, the duration of viral suppression was shorter in CD8 responders than in CD8 nonresponders (medians: 54 vs. 20 months, P = 0.04). Among viremic patients, CD8 responders had significantly lower plasma HIV RNA levels than CD8 nonresponders (2.7 vs. 3.7 log₁₀ HIV-RNA copies/ml, P = 0.02). In multivariate analyses including sex and HIV-1 subtype as covariables, Gag-specific CD4 T-cell proliferation was associated only with ethnicity, whereas Gag-specific CD8 T-cell proliferation was associated with both ethnicity and the duration of viral suppression. Both CD4 and CD8 responders reached their nadir CD4 T-cell percentages at younger ages than their nonresponder counterparts (6 vs. 8 years, P = 0.04 for both CD4 and CD8 T-cell proliferation). However, these associations were not significant in multivariate analysis. In conclusion, after at least 15 years of HIV infection, Gag-specific T-cell proliferation was found to be more frequent in black youths than in patients of other ethnic groups, despite all the patients being born in the same country, with similar access to care.     

Role of human immunodeficiency virus (HIV)-specific T-cell immunity in control of dual HIV-1 and HIV-2 infection

Progressive immune dysfunction and AIDS develop in most cases of human immunodeficiency virus type 1 (HIV-1) infection but in only 25 to 30% of persons with HIV-2 infection. However, the natural history and immunologic responses of individuals with dual HIV-1 and HIV-2 infection are largely undefined. Based on our previous findings, we hypothesized that among patients with dual infection the control of HIV-1 is associated with the ability to respond to HIV-2 Gag epitopes and to maintain HIV-specific CD4⁺ T-cell responses. To test this, we compared the HIV-specific ex vivo IFN-γ enzyme-linked immunospot (ELISPOT) assay responses of 19 dually infected individuals to those of persons infected with HIV-1 or HIV-2 only. Further, we assessed the functional profile of HIV Gag-specific CD4⁺ and CD8⁺ T cells from nine HIV dually infected patients by using a multicolor intracellular cytokine staining assay. As determined by ELISPOT assay, the magnitude and frequency of IFN-γ-secreting T-cell responses to gene products of HIV-1 were higher than those to gene products of HIV-2 (2.64 versus 1.53 log₁₀ IFN-γ spot-forming cells/10⁶ cells [90% versus 63%, respectively].) Further, HIV-1 Env-, Gag-, and Nef- and HIV-2 Gag-specific responses were common; HIV-2 Nef-specific responses were rare. HIV-specific CD4⁺ T helper responses were detected in nine of nine dually infected subjects, with the majority of these T cells producing gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) and, to a lesser extent, interleukin-2. The HIV-1 plasma viral load was inversely correlated with HIV-2 Gag-specific IFN-γ-/TNF-α-secreting CD4⁺ and HIV-2 Gag-specific IFN-γ-secreting CD8⁺ T cells. In conclusion, the T-cell memory responses associated with containment of single HIV-1 and HIV-2 infection play a similar significant role in the immune control of dual HIV-1 and HIV-2 infection.     

Impact of mucosal inflammation on cervical human immunodeficiency virus (HIV-1)-specific CD8 T-cell responses in the female genital tract during chronic HIV infection

The female genital tract is the major route of heterosexual human immunodeficiency virus (HIV) acquisition and transmission. Here, we investigated whether HIV-specific CD8 T-cell-mediated immune responses could be detected in the genital mucosa of chronically HIV-infected women and whether these were associated with either local mucosal HIV shedding or local immune factors. We found that CD8⁺ T-cell gamma interferon responses to Gag were detectable at the cervix of HIV-infected women but that the magnitude of genital responses did not correlate with those similarly detected in blood. This indicates that ex vivo HIV responses in one compartment may not be predictive of those in the other. We found that increased genital tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) levels correlated significantly with levels of Gag-specific CD8⁺ T cells at the cervix. Women who were detectably shedding virus in the genital tract had significantly increased cervical levels of TNF-α, IL-1β, IL-6, and IL-8 compared to women who were not detectably shedding virus. We were, however, unable to detect any association between the magnitude of cervical HIV-specific responses and mucosal HIV shedding. Our results support the hypothesis that proinflammatory cytokines in the female genital tract may promote HIV replication and shedding. In addition, we further show that inflammatory cytokines are associated with increased levels of HIV-specific CD8 effector cells at the genital mucosa but that these were not able to control genital HIV shedding.     

Broadening of Virus-Specific CD8+ T-Cell Responses Is Indicative of Residual Viral Replication in Aviremic SIV Controllers

Control of HIV replication is a rare immunological event, providing clues to understand the viral control mechanism. CD8⁺ T-cell responses are crucial for virus control, but it is unclear whether lasting HIV containment can be achieved after establishment of infection. Here, we describe lasting SIV containment in a macaque AIDS model. Analysis of ten rhesus macaques that controlled viremia for 2 years post-infection found accumulation of proviral gag and nef CD8⁺ T-cell escape mutations in four of them. These four controllers mounted CD8⁺ T cells targeting Gag, Nef, and other viral proteins at 4 months, suggesting that broadening of CD8⁺ T-cell targets can be an indicator of the beginning of viral control failure. The remaining six aviremic SIV controllers, however, harbored proviruses without mutations and showed no or little broadening of their CD8⁺ T-cell responses in the chronic phase. Indeed, three of the latter six exhibiting no change in CD8⁺ T-cell targets showed gradual decreases in SIV-specific CD8⁺ T-cell frequencies, implying a concomitant reduction in viral replication. Thus, stability of the breadth of virus-specific CD8⁺ T-cell responses may represent a status of lasting HIV containment by CD8⁺ T cells.     

Efficacious Early Antiviral Activity of HIV Gag- and Pol-Specific HLA-B*2705-Restricted CD8+ T Cells

The association between HLA-B*2705 and the immune control of human immunodeficiency virus type 1 (HIV-1) has previously been linked to the targeting of the HLA-B*2705-restricted Gag epitope KRWIILGLNK (KK10) by CD8⁺ T cells. In order to better define the mechanisms of the HLA-B*2705 immune control of HIV, we first characterized the CD8⁺ T-cell responses of nine highly active antiretroviral therapy (HAART)-naïve B*2705-positive subjects. Unexpectedly, we observed a strong response to an HLA-B*2705-restricted Pol epitope, KRKGGIGGY (KY9), in 8/9 subjects. The magnitude of the KY9 response was only marginally lower than that of the KK10-specific response (median, 695 versus 867 spot-forming cells [SFC]/million peripheral blood mononuclear cells [PBMCs]; not significant [NS]), and viral escape mutants were observed in both KY9 and KK10, resulting from selection pressure driven by the respective CD8⁺ T-cell response. By comparing inhibitions of viral replication by CD8⁺ T cells specific for the Gag KK10, Pol KY9, and Vpr VL9 HLA-B*2705-restricted epitopes, we observed a consistent hierarchy of antiviral efficacy (Gag KK10 > Pol KY9 > Vpr VL9). This hierarchy was associated with early recognition of HIV-1-infected cells, within 6 h of infection, by KK10- and KY9-specific CD8⁺ T cells but not until 18 h postinfection by VL9-specific CD8⁺ T cells. There was no association between antiviral efficacy and proliferative capacity, cytotoxicity, polyfunctionality, or T-cell receptor (TCR) avidity. These data are consistent with previous studies indicating an important role for the B*2705-Gag KK10 response in the control of HIV but also suggest a previously unrecognized role played by the subdominant Pol-specific KY9 response in HLA-B*2705-mediated control of HIV and that the recognition of HIV-infected cells by CD8⁺ T cells early in the viral life cycle may be important for viral containment in HIV-infected individuals.Current human immunodeficiency virus (HIV) vaccine strategies are focused on emulating the protective effect observed for HIV-infected individuals carrying alleles such as B*2705 by inducing the virus-specific CD8⁺ T-cell responses that are thought to be responsible for delaying or preventing disease progression. Understanding why such alleles confer protection facilitates a rational approach to vaccine design. It has been hypothesized that the slow progression to AIDS exhibited by HLA-B*2705-positive (HLA-B*2705⁺) HIV-infected individuals is due to the immunodominant B*27-restricted CD8⁺ T-cell response toward the p24 Gag epitope KRWIILGLNK (KK10) (Gag residues 263 to 272). Escape from this epitope typically occurs late in infection and is associated with rapid progression to AIDS (14, 16). The commonly selected mutation R264K abrogates CD8⁺ T-cell recognition but also confers a substantial fitness cost to the virus, and the selection of compensatory mutations is required to restore viral replicative capacity (19, 29, 30). This has prompted the hypothesis that CD8⁺ T-cell responses that can drive escape mutations that reduce viral fitness are a contributing factor in the immune control of HIV, either by promoting the outgrowth of a viral quasispecies with a lower replicative capacity or by delaying the selection of escape mutations, both of which may slow the onset of AIDS (11, 21, 25).To better understand how CD8⁺ T cells can be most effective against HIV, recent studies have directly assessed the antiviral activity of CD8⁺ T cells via the viral suppression of HIV-infected CD4⁺ T cells during coculture. Such studies indicated that Gag-specific CD8⁺ T cells have a higher potency for viral suppression than Env-specific CD8⁺ T cells (10), supporting previous data indicating that broad CD8⁺ T-cell targeting of Gag epitopes was associated a with lower viral set point and, hence, slower progression to AIDS (20). A recent study of simian immunodeficiency virus (SIV) suggested that the protective effect of Gag-specific CD8⁺ T cells is mediated by the early presentation of Gag epitopes, processed from the viral Gag protein from incoming virions during infection, which can sensitize target cells for lysis by Gag-specific CD8⁺ T cells within 6 h of infection (26, 27). In addition, it was proposed previously that the ability of CD8⁺ T cells to secrete multiple cytokines may also be an important correlate of immune protection (6), and a further recent study demonstrated a more polyfunctional cytokine profile of Gag-specific B*2705-KK10 CD8⁺ T-cell responses than those of other HIV-specific CD8⁺ T-cell responses (1). The ability of CD8⁺ T cells to proliferate in response to the cognate epitope peptide has also been associated with immune control (1, 12). Other studies demonstrated the importance of lytic granule loading of CD8⁺ T cells for the effective elimination of HIV-infected cells (6, 22). However, the induction of a Gag KK10-specific CD8⁺ T-cell vaccine response in a B*2705-positive vaccinee did not protect against rapid progression following subsequent HIV-1 infection (5). This anecdotal case suggests the possibility that HLA-B*2705-associated immune control of HIV-1 may not be dependent on the Gag KK10-specific CD8⁺ T-cell response alone.Since current vaccine strategies hope to induce a protective effect, such as that observed for HLA-B*2705⁺ HIV-infected individuals, the study of the functional and phenotypic characteristics of B*2705-specific CD8⁺ T cells provides an opportunity to redefine the proposed correlates of immune protection essential for rational vaccine design. In this study we analyze three different specificities of HLA-B*2705-restricted CD8⁺ T cells from chronically HIV-infected individuals in order to directly compare antiviral activity with potential correlates of immune protection, including the kinetics of viral inhibition, cytokine profile, granzyme production, proliferative capacity, and cytotoxicity.     

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

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; Fisher''s 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.Human immunodeficiency virus (HIV)-specific CD8⁺ T cells play a central role in controlling viral replication (12). It is the specificity of the CD8⁺ T-cell response, particularly the response to Gag, that is associated with low viral loads in HIV infection (7, 17, 34). Although immune control is undermined by the selection of viral mutations that prevent recognition by the CD8⁺ T cells, evasion of Gag-specific responses mediated by protective class I HLA-B alleles typically brings a reduction in viral replicative capacity, facilitating subsequent immune control of HIV (2, 20, 21). The same principle has been demonstrated in studies of simian immunodeficiency virus infection (18, 22).Recent studies showed that the class I HLA-B alleles that protect against disease progression present more Gag-specific CD8⁺ T-cell epitopes and drive the selection of more Gag-specific escape mutations than those alleles that are associated with high viral loads (23). These protective HLA-B alleles not only are beneficial to infected individuals expressing those alleles but also benefit a recipient following transmission, since the transmitted virus carrying multiple Gag escape mutations may have substantially reduced fitness (3, 4, 8). However, there is no benefit to the recipient if he or she shares the same protective allele as the donor because the transmitted virus carries escape mutations in the Gag epitopes that would otherwise be expected to mediate successful immune control in the recipient (8, 11).The sharing of HLA alleles between donor and recipient occurs frequently in mother-to-child transmission (MTCT). The risk of MTCT is related to viral load in the mother, and a high viral load is associated with nonprotective alleles, such as HLA-B*18 and -B*5802. This may contribute in two distinct ways to the more rapid progression observed in pediatric HIV infection (24, 26, 27). First, because infected children share 50% or more of their HLA alleles with the transmitting mother, they are less likely than adults to carry protective HLA alleles (16). Thus, infected children as a group carry fewer protective HLA alleles and more nonprotective HLA alleles. Second, even when the child has a protective allele, such as HLA-B*27, this allele does not offer protection if the maternally transmitted virus carries escape mutations within the key Gag epitopes that are presented by the protective allele (11, 19).However, it is clear that infected children who possess protective alleles, such as HLA-B*27 or HLA-B*57, can achieve durable immune control of HIV infection if the virus transmitted from the mother is not preadapted to those alleles (6, 10). HIV-specific CD8⁺ T-cell responses are detectable from birth in infected infants (32). Furthermore, as in adult infection (3, 8), HIV-infected children have the potential to benefit from transmission of low-fitness viruses in the situation where the mother possesses protective HLA alleles and the child does not share those protective alleles. MTCT of low-fitness viruses carrying CD8⁺ T-cell escape mutations was recently documented (28; J. Prado et al., unpublished data).In this study, undertaken in Durban, South Africa, we set out to test the hypothesis that HIV-infected children are less likely to progress rapidly to disease if either the infected child or the transmitting mother possesses a protective HLA allele that is not shared. The HLA alleles most strongly associated with low viral loads and high CD4 counts in a cohort of >1,200 HIV-infected adults in Durban are HLA-B*57 (-B*5702 and -B*5703), HLA-B*5801, and HLA-B*8101 (16; A. Leslie et al., unpublished data). These four alleles all present Gag-specific CD8⁺ T-cell epitopes, and in each case the escape mutations selected in these epitopes reduce viral replicative capacity (2-4, 8, 21, 23).Analyzing a previously described cohort of 61 HIV-infected children in Durban (24, 26, 32), South Africa, who were all monitored from birth, we first addressed the question of whether possession of any of these four alleles by either mother or child is associated with slower disease progression in the child and then determined whether sharing of protective alleles by mother and child affects the ability of the child to make the Gag-specific CD8⁺ T-cell responses restricted by the shared allele.     

Higher homologous and lower cross-reactive Gag-specific T-cell responses in human immunodeficiency virus type 2 (HIV-2) than in HIV-1 infection

Human immunodeficiency virus type 2 (HIV-2) infection results in slower CD4⁺ T-cell decline, lower plasma viral load levels, and hence slower progression of the disease than does HIV-1 infection. Although the reasons for this are not clear, it is possible that HIV-2 replication is more effectively controlled by host responses. We used aligned pools of overlapping HIV-1 and HIV-2 Gag peptides in an enhanced gamma interferon enzyme-linked immunospot assay to compare the levels of homologous and cross-reactive Gag-specific T-cell responses between HIV-1- and HIV-2-infected patients. HIV-2-infected patients showed broader and stronger homologous Gag-specific T-cell responses than HIV-1-infected patients. In contrast, the cross-reactive T-cell responses in HIV-2-infected patients were both narrower and weaker than those in HIV-1-infected patients, in line with overall weaker correlations between homologous and heterologous T-cell responses among HIV-2-infected patients than among HIV-1-infected patients. Cross-reactive responses in HIV-2-infected patients tended to correlate directly with HIV-1/HIV-2 Gag sequence similarities; this was not found in HIV-1-infected patients. The CD4⁺ T-cell counts of HIV-2-infected patients correlated directly with homologous responses and inversely with cross-reactive responses; this was not found in HIV-1-infected patients. Our data support a model whereby high-level HIV-2-specific T-cell responses control the replication of HIV-2, thus limiting viral diversification and priming of HIV-1 cross-reactive T-cell responses over time. However, we cannot exclude the possibility that HIV-2 replication is controlled by other host factors and that HIV-2-specific T-cell responses are better maintained in the context of slow viral divergence and a less damaged immune system. Understanding the nature of immune control of HIV-2 infection could be crucial for HIV vaccine design.     

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.     

The Use of Directed Evolution to Create a Stable and Immunogenic Recombinant BCG Expressing a Modified HIV-1 Gag Antigen

Numerous features make Mycobacterium bovis BCG an attractive vaccine vector for HIV. It has a good safety profile, it elicits long-lasting cellular immune responses and in addition manufacturing costs are affordable. Despite these advantages it is often difficult to express viral antigens in BCG, which results in genetic instability and low immunogenicity. The aim of this study was to generate stable recombinant BCG (rBCG) that express high levels of HIV antigens, by modification of the HIV genes. A directed evolution process was applied to recombinant mycobacteria that expressed HIV-1 Gag fused to the green fluorescent protein (GFP). Higher growth rates and increased GFP expression were selected for. Through this process a modified Gag antigen was selected. Recombinant BCG that expressed the modified Gag (BCG[pWB106] and BCG[pWB206]) were more stable, produced higher levels of antigen and grew faster than those that expressed the unmodified Gag (BCG[pWB105]). The recombinant BCG that expressed the modified HIV-1 Gag induced 2 to 3 fold higher levels of Gag-specific CD4 T cells than those expressing the unmodified Gag (BCG[pWB105]). Mice primed with 10⁷ CFU BCG[pWB206] and then boosted with MVA-Gag developed Gag-specific CD8 T cells with a frequency of 1343±17 SFU/10⁶ splenocytes, 16 fold greater than the response induced with MVA-Gag alone. Levels of Gag-specific CD4 T cells were approximately 5 fold higher in mice primed with BCG[pWB206] and boosted with MVA-Gag than in those receiving the MVA-Gag boost alone. In addition mice vaccinated with BCG[pWB206] were protected from a surrogate vaccinia virus challenge.