Fundisiwe Chonco

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

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

Extended high viremics: a substantial fraction of individuals maintain high plasma viral RNA levels after acute HIV-1 subtype C infection.

Objective:The present study addressed two questions: what fraction of individuals maintain a sustained high HIV-1 RNA load after the acute HIV-1C infection peak and how long is a high HIV-1 RNA load maintained after acute HIV-1C infection in this subpopulation? Design/methods:Plasma HIV-1 RNA dynamics were studied in 77 participants with primary HIV-1C infection from African cohorts in Gaborone, Botswana, and Durban, South Africa. HIV-infected individuals who maintained mean viral load of at least 100 000 (5.0 log10) copies/ml after 100 days postseroconversion (p/s) were termed extended high viremics. Individuals were followed longitudinally for a median [interquartile range (IQR)] of 573 (226–986) days p/s. Results:The proportion of extended high viremics was 34% [95% confidence interval (CI) 23–44%] during the period 100–300 days p/s and 19% (95% CI 9–29%) over the period of 200–400 days p/s. The median (IQR) duration of HIV-1 RNA load at least 100 000 copies/ml among extended high viremics was 271 (188–340) days p/s. For the subset with average viral load at least 100 000 copies/ml during 200–400 days p/s, the median (IQR) duration was 318 (282–459) days. The extended high viremics had a significantly shorter time to CD4 cell decline to 350 cells/&mgr;l (median: 88 vs. 691 days p/s for those not designated as extended high viremics; P < 0.0001, Gehan-Wilcoxon test). Conclusion:A high proportion of extended high viremics – individuals maintaining high plasma HIV-1 RNA load after acute infection – have been identified during primary HIV-1 subtype C infection. These extended high viremics likely contribute disproportionately to HIV-1 incidence.

Influence of Gag-Protease-Mediated Replication Capacity on Disease Progression in Individuals Recently Infected with HIV-1 Subtype C

ABSTRACT HLA class I-mediated selection of immune escape mutations in functionally important Gag epitopes may partly explain slower disease progression in HIV-1-infected individuals with protective HLA alleles. To investigate the impact of Gag function on disease progression, the replication capacities of viruses encoding Gag-protease from 60 individuals in early HIV-1 subtype C infection were assayed in an HIV-1-inducible green fluorescent protein reporter cell line and were correlated with subsequent disease progression. Replication capacities did not correlate with viral load set points (P = 0.37) but were significantly lower in individuals with below-median viral load set points (P = 0.03), and there was a trend of correlation between lower replication capacities and lower rates of CD4 decline (P = 0.09). Overall, the proportion of host HLA-specific Gag polymorphisms in or adjacent to epitopes was negatively associated with replication capacities (P = 0.04), but host HLA-B-specific polymorphisms were associated with higher viral load set points (P = 0.01). Further, polymorphisms associated with host-specific protective HLA alleles were linked with higher viral load set points (P = 0.03). These data suggest that transmission or early HLA-driven selection of Gag polymorphisms results in reduced early cytotoxic T-lymphocyte (CTL) responses and higher viral load set points. In support of the former, 46% of individuals with nonprotective alleles harbored a Gag polymorphism exclusively associated with a protective HLA allele, indicating a high rate of their transmission in sub-Saharan Africa. Overall, HIV disease progression is likely to be affected by the ability to mount effective Gag CTL responses as well as the replication capacity of the transmitted virus.

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

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

A Molecular Assay for Sensitive Detection of Pathogen- Specific T-Cells

Here we describe the development and validation of a highly sensitive assay of antigen-specific IFN-γ production using real time quantitative PCR (qPCR) for two reporters - monokine-induced by IFN-γ (MIG) and the IFN-γ inducible protein-10 (IP10). We developed and validated the assay and applied it to the detection of CMV, HIV and Mycobacterium tuberculosis (MTB) specific responses, in a cohort of HIV co-infected patients. We compared the sensitivity of this assay to that of the ex vivo RD1 (ESAT-6 and CFP-10)-specific IFN-γ Elispot assay. We observed a clear quantitative correlation between the two assays (P<0.001). Our assay proved to be a sensitive assay for the detection of MTB-specific T cells, could be performed on whole blood samples of fingerprick (50 uL) volumes, and was not affected by HIV-mediated immunosuppression. This assay platform is potentially of utility in diagnosis of infection in this and other clinical settings.

Impact of Select Immunologic and Virologic Biomarkers on CD4 Cell Count Decrease in Patients with Chronic HIV-1 Subtype C Infection: Results from Sinikithemba Cohort, Durban, South Africa

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.Background The extent to which immunologic and clinical biomarkers influence HIV outcomes remains incompletely characterized, particularly for non-B subtypes. Based on data supporting in vitro HIV 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 biomarkers, with rates of CD4 decline in subtype-C infection.

Immunodominant HIV-1-specific HLA-B- and HLA-C-restricted CD8+ T cells do not differ in polyfunctionality

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.

Immunodominance and viral ditness in Gag may contribute to differential viral control in HLA-B*7 supertype individuals acutely infected with HIV-1C

Background HLA-B*7 supertype alleles are common among people of African descent and are associated with viral control. In particular, HLA-B*81 has been previously associated with reduced viral fitness. We analyzed the immunodominance of CD8+ T cell responses targeted by the B*7 supertype alleles, viral evolution and fitness dynamics over 1yr in acutely infected patients. Methods Six HLA-B*7 supertype participants [HLA-B*81 (n=2), HLA-B*4201 (n=3) and HLA-B*4202 (n=1)] identified with acute HIV-1 infection (antibody negative, vRNA positive) in KwaZulu-Natal, South Africa were studied. CD8+ T cell responses were measured by the IFN-g ELIspot assay. Replication capacities of viruses encoding Gag-protease were measured. Full-length HIV-1 Gag clonal sequencing of plasma was performed at ~14 days post infection and 1yr later. Results The average viral set point of the 4 HLA-B*42 individuals was higher than the 2 HLA-B*81, 4.89 vs 4.16 respectively. Approximately 28 days after viral infection, CD8+ T cell responses were directed to an average of 2/5 (range 2-4) HLA-B*42 Gag-specific epitopes, median magnitude of 490 (range 170–2,480 SFC/million PBMCs). None of these 4 individuals had selected for escape mutations in the immunodominant TL9 epitope at 1yr post-infection. Interestingly, CD8+ T cell responses were only against the TL9 epitope for the 2 HLA-B*81 patients with a median magnitude of 950 (range 300–1780 SFC/million PBMCs). One patient had a single wild type epitope in the transmitted virus, compared to 4/5 wild type epitopes in the second patient. However, CD8+ T cell responses were only elicited at the TL9 epitope with a low magnitude against T186S in the 1 patient with a much lower viral fitness.

Longitudinal assessment of HIV-1-specific T-cell responses generated during acute subtype C HIV-1 infection and associations with viral set point

Background HIV-1-specific CD8+ T-cell responses generated during acute infection play a key role in determining the course of disease. However, these early responses may disappear as the infection progresses. Tracking the phenotype, functional ability and fate of these early responses may help elucidate features of CD8+ T-cells that contribute to viral control. Methods We characterized the magnitude and breadth of HIV-1specific CD8+T-cell responses using the overnight IFN-g ELISPOT assay in 20 HIV-1 subtype C acutely infected, antiretroviral naive individuals as early as 28 days post initial exposure and up to 12 months post-infection. Cultured IFN-g ELISPOT assays were used to assess early epitope-specific CD8+ T-cell responses that were below the limit of detection by overnight ELISPOT assays at 12 months post-infection. Results The initial CD8+ T-cell responses detected during the decline in acute phase peak viremia were narrowly directed: T-cell responses were directed against an average of three (range 0-6) of the 410 peptides tested. At 12 months post-infection, immune responses had broadened with an average of seven peptides targeted (range 2-11). The majority of persistent T-cell responses targeted epitopes within Gag and Pol (50% and 30%, respectively). The breadth of persistent CD8+T-cell responses correlated negatively with viral set point (P=0.02). An average of 65% of earlier CD8+ T-cell responses which where not detected by the overnight ELISPOT at later time points, were detected by the cultured ELISPOT assay. There was no correlation between the magnitude or breadth of responses measured using the cultured ELISPOT assay and viral set point. Conclusion These data suggest that persistent CD8+ T-cell responses generated during the earliest stages of HIV-1C infection may play a role in viral control. Furthermore, effector T-cell responses that disappear following acute infection are maintained as memory T-cell responses and our preliminary data suggest that they have no significant impact on viral control.

P07-02. Cytotoxic T lymphocyte-mediated immune responses in HIV-1 clade C infected mother-child pairs

Address: 1Paediatrics and child Health, HIV Pathogenesis Programme, Doris Duke Medical Research Institute, UKZN, Durban, South Africa, 2HIV Pathogenesis Programme, Doris Duke Medical Research Institute, Durban, South Africa, 3Institute of Massachusetts General Hospital, Massachusetts, Massachusetts, MA USA and 4Department of Pediatrics, University of Oxford, England, Oxford, UK * Corresponding author