: HIV-1 sequence variations impact binding of inhibitory killer cell immunoglobulin-like receptors (KIRs) to human leukocyte antigen class I (HLA-I) molecules modulating natural killer cell function. HIV-1 strains encoding amino acids that mediate binding of inhibitory KIRs might therefore have a selective benefit in individuals expressing the respective KIR/HLA genotypes. Here, we demonstrate that HIV-1 clade C avoids a p24 Gag mutation that abolishes binding of KIR2DL2 to HLA-C03:04 and disinhibits natural killer cells in individual encoding for this genotype.

: HIV-1 sequence variations impact binding of inhibitory killer cell immunoglobulin-like receptors (KIRs) to human leucocyte class I (HLA-I) molecules modulating NK cell function. HIV-1 strains encoding amino acids that mediate binding of inhibitory KIRs might therefore have a selective benefit in individuals expressing the respective KIR/HLA genotypes. Here we demonstrate that HIV-1 clade C avoids a p24 Gag mutation that abolishes binding of KIR2DL2 to HLA-C03:04 and disinhibits NK cells in individual encoding for this genotype.

HIV-1 sequence variations impact binding of inhibitory killer cell immunoglobulin-like receptors (KIRs) to human leucocyte class I (HLA-I) molecules modulating NK cell function. HIV-1 strains encoding amino acids that mediate binding of inhibitory KIRs might therefore have a selective benefit in individuals expressing the respective KIR/HLA genotypes. Here we demonstrate that HIV-1 clade C avoids a p24 Gag mutation that abolishes binding of KIR2DL2 to HLA-C*03:04 and disinhibits NK cells in individual encoding for this genotype. Copyright © 2020 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Natural killer (NK) cells are an important component of the innate immune system, able to rapidly respond to target cells without prior sensitization. The effector function of NK cells to stimuli are determined by the interplay between activating and inhibitory receptors [1], including killer immunoglobulin receptors (KIRs). KIRs bind to human leukocyte antigen class I (HLA-I) molecules to monitor HLA-I expression levels on cells, and the affinity of these KIR/HLA-I interactions is dependent on the sequence of the HLA-I presented peptide [2, 3]. KIR/HLA-I compound genotypes have been associated with the speed of HIV-1 disease progression [4], and selection of viral sequence polymorphisms associated with specific KIR or KIR/HLA-I compound genotypes in HIV-1 infection have been reported [5-7], indicating KIR+ NK cell-mediated immune selection pressure. We previously showed selection of two minor HIV-1 sequence polymorphisms in a cohort of 392 clade C HIV-1 infected South Africans and demonstrated that these mutations within p24 Gag were associated with combined expression of KIR2DL3 and HLA-C*03:04 genotypes and mediated escape from NK cell responses through enhanced engagement of inhibitory KIR2DL3 [8]. Here we investigated another sequence polymorphism at position 84 of p24 Gag (TGag84V) associated with the combined HLA-C*03:04/KIR2DL2 genotype in this cohort. While 42 infected individuals expressing both KIR2DL2 and HLA-C*03:04 all harbored viruses encoding for the major variant threonine (T) at amino acid position 84 of HIV-1 Gag, the minor variant valine (V) at position 84 was not present in the KIR2DL2/HLA-C*03:04 individuals yet was present in 10% (35/350) of individuals not encoding for KIR2DL2 and HLA-C*03:04 (see section A of Supplementary Material; Fig 1A , http://links.lww.com/QAD/B855, P=0.02, Fischer’s Exact test). These data suggest a significant advantage for HIV-1 to encode for TGag84 in KIR2DL2/HLA-C*03:04 individuals. The TGag84V polymorphism is located within the C-terminal region of the HLA-C*03:04-restricted peptide RSLYNTVATL (RSTL10). The major variant (RSLYNTVATL (RSTL10)) and the minor variant (RSLYNTVAVL (RSVL10)) peptides equally stabilized HLA-C*03:04 expression (method described in section B of Supplementary Material , http://links.lww.com/QAD/B855) on transfected TAP-deficient 721.221 cells (221-TAPko-HLA-C*03:04) (T [mean 2.2 ± 0.20 SD] to V [mean 2.3± 0.26SD], P=0.8), and resulted in an up to 3-fold higher expression of HLA-C*03:04 compared to non-peptide-pulsed control cells (Fig 1B). These HLA-C*03:04-stabilization data demonstrated that the TGag84V polymorphism identified within HIV-1 p24 Gag did not impact the ability of the respective peptides to bind to HLA-C*03:04. Next, the RSTL10 and RSVL10 peptides were assessed for their ability to mediate KIR2DL2-binding to HLA-C*03:04, using a KIR2DL2-Fc fusion construct, as previously described [8], (see also section C of Supplementary Material , http://links.lww.com/QAD/B855). The HLA-C*03:04-presented GAVDPLLAL (GAL) peptide derived from the human importin-α-1 subunit (residues 204-212) [9], is known to bind KIR2DL2 [9] and was used as positive control, whereas the variant GAVDPLLKL (GKL) peptide which stabilizes HLA-C*03:04 at similar levels but does not bind KIR2DL2 [9] was Copyright © 2020 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. used as a negative control. Results from 5 independent KIR2DL2-Fc-binding assays consistently demonstrated weaker binding of KIR2DL2-Fc to HLA-C*03:04 presenting the minor variant peptide RSVL10, while the major variant peptide RSTL10 mediated significantly higher binding of KIR2DL2-Fc to HLA-C*03:04 (Fig 1C, P=0.008, Mann Whitney test). Higher KIR2DL2-Fc-binding furthermore had significant functional consequences for KIR2DL2 NK cells, as demonstrated by increased inhibition of CD107a expression as a read-out for NK cell activation [10], (Supplementary Figure 1 , http://links.lww.com/QAD/B855), of KIR2DL2 primary NK cells following co-incubation of 221-TAPko-HLA-C*03:04 cells pulsed with the major variant RSTL10 peptide compared to the minor variant RSVL10 peptide (method described in section D of Supplementary Material , http://links.lww.com/QAD/B855) (Fig 1D, P=0.007, Mann Whitney test, N = 8). In summary, the results from these KIR2DL2/HLA-C*03:04 binding assays and functional assays using KIR2DL2 primary NK cells demonstrate that the minor VGag84 HIV-1 variant that was circulating in KIR2DL2/HLA-C*03:04 negative individuals, but not detected in individuals encoding for KIR2DL2/HLA-C*03:04, mediated significantly reduced binding of KIR2DL2 to HLA-C*03:04, resulting in stronger activation of primary KIR2DL2 NK cells. Previous studies have reported minor HIV-1 sequence polymorphisms selected in individuals expressing specific KIRs or KIR/HLA-I combinations that enhanced binding of inhibitory KIRs and inhibit KIR NK cells, consistent with KIR NK cell-mediated selection of viral escape variants [3, 6-8]. Here we report a sequence polymorphism within HIV-1 Gag (TGag84V) for which the major variant (TGag84) was encoded in all individuals expressing KIR2DL2/HLA-C*03:04, while the minor variant (VGag84) was detected in some individuals not encoding for this KIR/HLA compound genotype. The combined KIR2DL2/HLA-C*03:04 genotype is frequent in the studied population of HIV-1 clade C infected individuals from South Africa (42 of 392 individuals, 10.7%), and these observations are consistent with the selection and potential accumulation of the TGag84 variant over time, similar to what has been described for HLA-B*51restricted epitope TAFTIPSI in different populations [11]. The current study was a cross sectional analysis in chronically HIV-1 clade C infected individuals in which the timing of HIV-1 infection was not known. It was therefore not possible to determine whether the TGag84 polymorphism was indeed selected in KIR2DL2/HLA-C*03:04 individuals infected with the minor VGag84 variant; and the fact that less than 10% of circulating viruses in these 392 individuals encoded for VGag84 suggests that most individuals were initially infected with the TGag84 polymorphism. Longitudinal studies with accurate timing of primary infection will be required to determine whether the TGag84 variant is indeed selected in KIR2DL2/HLA-C*03:04 individuals infected with the minor VGag84 variant, leading to the accumulation of the TGag84 variant over time. In summary, these data suggest potential accumulation of sequence polymorphisms that reduce recognition by KIR NK cells over time, eventually representing the major variant in the circulating HIV-1 strains, similar to the enrichment of HIV-1 variants selected in response to CD8T-cell-mediated immune pressure [11]. Copyright © 2020 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.

HIV-1 viremia has been shown to induce several phenotypic and functional abnormalities in natural killer (NK) cells. To assess immune defects associated with HIV viremia, we examined NK cell function, differentiation status, and phenotypic alterations based on expression of inhibitory and activating receptors on NK cells in HIV-1 subtype C chronically infected participants from Durban, South Africa. NK cell phenotypic profiles were characterized by assessing sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7), NKG2A, and NKG2C markers on frozen peripheral blood mononuclear cells from viremic, antiretroviral therapy (ART)-naive HIV-1 chronically infected participants (n = 23), HIV-1 chronically infected participants who had been on combination antiretroviral therapy (cART) for at least 12 months (n = 23) compared with healthy donors (n = 23). NK cell differentiation was assessed by measurement of killer immunoglobulin receptor (KIR) and NKG2A expression; CD57 and CD107a measurements were carried out in HIV viremic and healthy donors. All phenotypic and functional assessments were analyzed by using multicolor flow cytometry. HIV-1-infected participants displayed greater frequencies of the CD56-CD16+ (CD56negative) NK cell subset compared with healthy donors (p < .0001). Downregulation of Siglec-7 and NKG2A and upregulation of NKG2C were more pronounced in the CD56negative NK cell subset of viremic participants. The CD56negative subset demonstrated a differentiated (KIR+NKG2A-) phenotype with reduced CD57 expression and lower degranulation capacity in HIV-1-infected participants compared with healthy donors. HIV-1 infection induces the expansion of the CD56negative NK cell subset marked by altered receptor expression profiles that are indicative of impaired function and may explain the overall NK cell dysfunction observed in chronic HIV-1 infection.

ABSTRACT The mechanisms of viral control and loss of viral control in chronically infected individuals with or without protective HLA class I alleles are not fully understood. We therefore characterized longitudinally the immunological and virological features that may explain divergence in disease outcome in 70 HIV-1 C-clade-infected antiretroviral therapy (ART)-naive South African adults, 35 of whom possessed protective HLA class I alleles. We demonstrate that, over 5 years of longitudinal study, 35% of individuals with protective HLA class I alleles lost viral control compared to none of the individuals without protective HLA class I alleles ( P = 0.06). Sustained HIV-1 control in patients with protective HLA class I alleles was characteristically related to the breadth of HIV-1 CD8 + T cell responses against Gag and enhanced ability of CD8 + T cells to suppress viral replication ex vivo . In some cases, loss of virological control was associated with reduction in the total breadth of CD8 + T cell responses in the absence of differences in HIV-1-specific CD8 + T cell polyfunctionality or proliferation. In contrast, viremic controllers without protective HLA class I alleles possessed reduced breadth of HIV-1-specific CD8 + T cell responses characterized by reduced ability to suppress viral replication ex vivo . These data suggest that the control of HIV-1 in individuals with protective HLA class I alleles may be driven by broad CD8 + T cell responses with potent viral inhibitory capacity while control among individuals without protective HLA class I alleles may be more durable and mediated by CD8 + T cell-independent mechanisms. IMPORTANCE Host mechanisms of natural HIV-1 control are not fully understood. In a longitudinal study of antiretroviral therapy (ART)-naive individuals, we show that those with protective HLA class I alleles subsequently experienced virologic failure compared to those without protective alleles. Among individuals with protective HLA class I alleles, viremic control was associated with broad CD8 + T cells that targeted the Gag protein, and CD8 + T cells from these individuals exhibited superior virus inhibition capacity. In individuals without protective HLA class I alleles, HIV-1-specific CD8 + T cell responses were narrow and poorly inhibited virus replication. These results suggest that broad, highly functional cytotoxic T cells (cytotoxic T lymphocytes [CTLs]) against the HIV-1 Gag protein are associated with control among those with protective HLA class I alleles and that loss of these responses eventually leads to viremia. A subset of individuals appears to have alternative, non-CTL mechanisms of viral control. These controllers may hold the key to an effective HIV vaccine.

In chronic HIV infection, CD8+ T cell responses to Gag are associated with lower viral loads, but longitudinal studies of HLA-restricted CD8+ T cell-driven selection pressure in Gag from the time of acute infection are limited. In this study we examined Gag sequence evolution over the first year of infection in 22 patients identified prior to seroconversion. A total of 310 and 337 full-length Gag sequences from the earliest available samples (median = 14 days after infection [Fiebig stage I/II]) and at one-year post infection respectively were generated. Six of 22 (27%) individuals were infected with multiple variants. There was a trend towards early intra-patient viral sequence diversity correlating with viral load set point (p = 0.07, r = 0.39). At 14 days post infection, 59.7% of Gag CTL epitopes contained non-consensus polymorphisms and over half of these (35.3%) comprised of previously described CTL escape variants. Consensus and variant CTL epitope proportions were equally distributed irrespective of the selecting host HLA allele and most epitopes remained unchanged over 12 months post infection. These data suggest that intrapatient diversity during acute infection is an indicator of disease outcome. In this setting, there is a high rate of transmitted CTL escape variants and limited immune selection in Gag during the first year of infection. These data have relevance for vaccine strategies designed to elicit effective CD8+ T cell immune responses.

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-naive individuals with acute HIV-1 subtype C infection, all identified prior to seroconversion. Ex-vivo and cultured interferon-gamma 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. (C) 2014 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins

AIDS Research and Human RetrovirusesVol. 30, No. S1 Innate ImmunityPhenotypic and Functional Characteristics of Siglec-7, NKG2A and NKG2C Expressing NK Cells in Chronic HIV-1 Clade C InfectionMichael Z. Zulu, Kewreshini Naidoo, Zenele Mncube, Manjeetha Jaggernath, Philip Goulder, Thumbi Ndung'u, Marcus Altfeld, and Christina ThobakgaleMichael Z. ZuluDoris Duke Medical Research Institute, University of KwaZulu-Natal, HIV Pathogenesis Programme, Durban, South AfricaSearch for more papers by this author, Kewreshini NaidooDoris Duke Medical Research Institute, University of KwaZulu-Natal, HIV Pathogenesis Programme, Durban, South AfricaSearch for more papers by this author, Zenele MncubeDoris Duke Medical Research Institute, University of KwaZulu-Natal, HIV Pathogenesis Programme, Durban, South AfricaSearch for more papers by this author, Manjeetha JaggernathDoris Duke Medical Research Institute, University of KwaZulu-Natal, HIV Pathogenesis Programme, Durban, South AfricaSearch for more papers by this author, Philip GoulderDoris Duke Medical Research Institute, University of KwaZulu-Natal, HIV Pathogenesis Programme, Durban, South AfricaPeter Medawar Building for Pathogen Research, University of Oxford, Department of Paediatrics, London, United KingdomSearch for more papers by this author, Thumbi Ndung'uDoris Duke Medical Research Institute, University of KwaZulu-Natal, HIV Pathogenesis Programme, Durban, South AfricaRagon Institute of MIT, MGH and Harvard, Boston, MA, United StatesSearch for more papers by this author, Marcus AltfeldDoris Duke Medical Research Institute, University of KwaZulu-Natal, HIV Pathogenesis Programme, Durban, South AfricaRagon Institute of MIT, MGH and Harvard, Boston, MA, United StatesHeinrich-Pette Institute, Department of Viral Immunology, Hamburg, GermanySearch for more papers by this author, and Christina ThobakgaleDoris Duke Medical Research Institute, University of KwaZulu-Natal, HIV Pathogenesis Programme, Durban, South AfricaSearch for more papers by this authorPublished Online:30 Oct 2014https://doi.org/10.1089/aid.2014.5256.abstractAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Phenotypic and Functional Characteristics of Siglec-7, NKG2A and NKG2C Expressing NK Cells in Chronic HIV-1 Clade C Infection." AIDS Research and Human Retroviruses, 30(S1), p. A130FiguresReferencesRelatedDetails Volume 30Issue S1Oct 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Michael Z. Zulu, Kewreshini Naidoo, Zenele Mncube, Manjeetha Jaggernath, Philip Goulder, Thumbi Ndung'u, Marcus Altfeld, and Christina Thobakgale.Phenotypic and Functional Characteristics of Siglec-7, NKG2A and NKG2C Expressing NK Cells in Chronic HIV-1 Clade C Infection.AIDS Research and Human Retroviruses.Oct 2014.A130-A130.http://doi.org/10.1089/aid.2014.5256.abstractPublished in Volume: 30 Issue S1: October 30, 2014PDF download

Background: HLA class I genotype is a major determinant of the outcome of HIV infection, and the impact of certain alleles on HIV disease outcome is well studied. Recent studies have demonstrated that certain HLA class I alleles that are in linkage disequilibrium, such as HLA-A*74 and HLA-B*57, appear to function co-operatively to result in greater immune control of HIV than mediated by either single allele alone. We here investigate the extent to which HLA alleles - irrespective of linkage disequilibrium - function co-operatively.Methodology/Principal Findings: We here refined a computational approach to the analysis of >2000 subjects infected with C-clade HIV first to discern the individual effect of each allele on disease control, and second to identify pairs of alleles that mediate 'co-operative additive' effects, either to improve disease suppression or to contribute to immunological failure. We identified six pairs of HLA class I alleles that have a co-operative additive effect in mediating HIV disease control and four hazardous pairs of alleles that, occurring together, are predictive of worse disease outcomes (q<0.05 in each case). We developed a novel 'sharing score' to quantify the breadth of CD8+ T cell responses made by pairs of HLA alleles across the HIV proteome, and used this to demonstrate that successful viraemic suppression correlates with breadth of unique CD8+ T cell responses (p = 0.03).Conclusions/Significance: These results identify co-operative effects between HLA Class I alleles in the control of HIV-1 in an extended Southern African cohort, and underline complementarity and breadth of the CD8+ T cell targeting as one potential mechanism for this effect.

BACKGROUND:The HLA class II molecules play a central role in the generation of human immunodeficiency virus (HIV)-specific CD4(+) T-helper cells, which are critical for the induction of cytotoxic CD8(+) T cell responses. However, little is known about the impact of HLA class II alleles on HIV disease progression.METHODS:In this study we investigated the effect of HLA class II alleles on HIV disease outcome and HIV-specific T cell responses in a cohort of 426 antiretroviral therapy-naive, HIV-1 clade C-infected, predominantly female black South Africans.RESULTS:The HLA class II allele DRB1*1303 was independently associated with lower plasma viral loads in this population (P = .02), an association that was confirmed in a second cohort of 1436 untreated, HIV-1 clade B-infected, male European Americans, suggesting that DRB1*1303-mediated protection is independent of ethnicity, sex, and viral clade. Interestingly, DRB1*1303 carriage was not associated with an increased frequency of interferon (IFN) γ-positive HIV-specific CD4(+) T cell responses.CONCLUSIONS:These data demonstrate the independent effect of an HLA class II allele, DRB1*1303, on HIV disease progression, in the absence of increased IFN-γ-positive HIV-specific CD4(+) T cell frequencies, suggesting that the protective activity of DRB1*1303 may be mediated via an alternative mechanism.

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.

Pediatric HIV-1 infection is characterized by rapid disease progression and without antiretroviral therapy (ART), more than 50% of infected children die by the age of 2 years. However, a small subset of infected children progresses slowly to disease in the absence of ART. This study aimed to identify functional characteristics of HIV-1-specific T cell responses that distinguish children with rapid and slow disease progression. Fifteen perinatally HIV-infected children (eight rapid and seven slow progressors) were longitudinally studied to monitor T cell polyfunctionality. HIV-1-specific interferon (IFN)-γ(+) CD8(+) T cell responses gradually increased over time but did not differ between slow and rapid progressors. However, polyfunctional HIV-1-specific CD8(+) T cell responses, as assessed by the expression of four functions (IFN-γ, CD107a, TNF-α, MIP-1β), were higher in slow compared to rapid progressors (p=0.05) early in infection, and was associated with slower subsequent disease progression. These data suggest that the quality of the HIV-specific CD8(+) T cell response is associated with the control of disease in children as has been shown in adult infection.

ABSTRACT It is unknown whether favorable HLA class II alleles may attenuate HIV-1 through selection pressure in a manner similar to that of protective HLA class I alleles. We investigated the relationship between HLA class II alleles and in vitro replication capacities of recombinant viruses encoding HIV-1 subtype C Gag-protease from chronically infected individuals. No associations were found between individual alleles and lower replication capacity, suggesting no significant HIV-1 attenuation by HLA class II-restricted Gag-specific CD4 + T cell immune pressure.

HIV-1 specific HLA-B-restricted CD8+ T cell responses differ from HLA-C-restricted responses in antiviral effectiveness. To investigate possible reasons for these differences, we characterized the frequency and polyfunctionality of immmunodominant HLA-B*57/B5801- and HLA-Cw*07-restricted CD8+ T cells occurring concurrently in nine study subjects assessing IFN-γ, TNF-α, IL-2, MIP-1β, and CD107a by flow cytometry and analyzed sequence variation in targeted epitopes. HLA-B*57/5801 and HLA-Cw*07 restricted CD8+ T cells did not differ significantly in polyfunctionality (p=0.84). Possession of three or more functions correlated positively with CD4+ T cell counts (r=0.85; p=0.006) and monofunctional CD8+ T cells inversely correlated with CD4 cell counts (r=−0.79; p=0.05). There were no differences in polyfunctionality of CD8+ T cells specific to wildtype versus mutated epitopes. These results suggest that loss of polyfunctionality and increase in monofunctional HIV-1-specific CD8+ T cells are associated with disease progression independent of restricting HLA allele. Furthermore, sequence variation does not appear to significantly impact CD8+ T cell polyfunctionality in chronic HIV-1 infection.

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.

Recent studies suggest that natural killer T (NKT) cells play a role in early antiviral pathogenesis and are rapidly depleted in chronic human immunodeficiency virus type 1 (HIV-1) clade B infection. We aimed to characterize the phenotypic and functional characteristics of NKT cells in HIV-1 clade C-infected Africans at different stages of HIV-1 disease. NKT cell frequencies, subsets, and ex vivo effector functions were assessed using multiparametric flow cytometry in a cross-sectional analysis of cryopreserved peripheral blood mononuclear cells from a cohort of 53 HIV-1 clade C chronically infected South African adults with CD4 T cell counts ranging from 94 to 839 cells/μl. We observed a significant decline of NKT cell numbers in advanced HIV-1 disease as well as activation and functional impairment of NKT cells in individuals with low CD4 T cell counts. The loss of NKT cells was largely driven by a reduction in the CD4(+) and CD4(-)CD8(-) NKT cell subsets in advanced disease. These findings demonstrate significant impairment of the NKT cell compartment in progressive HIV-1 clade C disease that might play an important role in the modulation of immune function in HIV-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.

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-gamma) responses by CD4+ T cells to pooled Gag peptides and to determine their association with viral load and CD4 count. Gag-specific IFN-gamma-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-gamma-secreting Gag-specific CD4+ T cell responses are immunodominant, directed at multiple distinct epitopes, and associated with viral control.

BACKGROUND:The extent to which immunologic and clinical biomarkers influence human immunodeficiency virus type 1 (HIV-1) infection outcomes remains incompletely characterized, particularly for non-B subtypes. On the basis of data supporting in vitro HIV-1 protein-specific CD8 T lymphocyte responses as correlates of immune control in cross-sectional studies, we assessed the relationship of these responses, along with established HIV-1 biomarkers, with rates of CD4 cell count decrease in individuals infected with HIV-1 subtype C. METHODS:Bivariate and multivariate mixed-effects models were used to assess the relationship of baseline CD4 cell count, plasma viral load, human leukocyte antigen (HLA) class I alleles, and HIV-1 protein-specific CD8 T cell responses with the rate of CD4 cell count decrease in a longitudinal population-based cohort of 300 therapy-naive, chronically infected adults with baseline CD4 cell counts >200 cells/mm(3) and plasma viral loads >500 copies/mL over a median of 25 months of follow-up. RESULTS:In bivariate analyses, baseline CD4 cell count, plasma viral load, and possession of a protective HLA allele correlated significantly with the rate of CD4 cell count decrease. No relationship was observed between HIV-1 protein-specific CD8 T cell responses and CD4 cell count decrease. Results from multivariate models incorporating baseline CD4 cell counts (201-350 vs >350 cells/mm(3)), plasma viral load (< or =100,000 vs >100,000 copies/mL), and HLA (protective vs not protective) yielded the ability to discriminate CD4 cell count decreases over a 10-fold range. The fastest decrease was observed among individuals with CD4 cell counts >350 cells/mm(3) and plasma viral loads >100,000 copies/mL with no protective HLA alleles (-59 cells/mm(3) per year), whereas the slowest decrease was observed among individuals with CD4 cell counts 201-350 cells/mm(3), plasma viral loads < or =100,000 copies/mL, and a protective HLA allele (-6 cells/mm(3) per year). CONCLUSIONS:The combination of plasma viral load and HLA class I type, but not in vitro HIV-1 protein-specific CD8 T cell responses, differentiates rates of CD4 cell count decrease in patients with chronic subtype-C infection better than either marker alone.