Manjeetha Jaggernath

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

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

Prevalence and Characteristics of Hepatitis B Virus (HBV) Coinfection among HIV-Positive Women in South Africa and Botswana

There is progressive concern about the evolving burden of morbidity and mortality caused by coinfection with HIV-1 and hepatitis B virus (HBV) in sub-Saharan Africa, but the epidemiology and impact of this problem are not well defined. We therefore set out to assimilate more information about the nature of HBV/HIV coinfection in this region by undertaking a retrospective observational study of southern African adult women. We used samples from previously recruited HIV-1 positive women attending antenatal clinics in three settings in South Africa and Botswana (n = 950) and added a small cohort of HIV-negative antenatal South African women for comparison (n = 72). We tested for HBsAg and followed up HBsAg-positive samples by testing for HBeAg, HBV DNA, HBV genotype, presence of drug-resistance associated mutations (RAMs) and HDV. We identified HBsAg in 72 individuals (7% of the whole cohort), of whom 27% were HBeAg-positive, and the majority HBV genotypes A1 and A2. We did not detect any HDV coinfection. HBV prevalence was significantly different between geographically distinct cohorts, but did not differ according to HIV status. Among adults from South Africa, HBV/HIV coinfected patients had lower CD4+ T cell counts compared to those with HIV-monoinfection (p = 0.02), but this finding was not replicated in the cohort from Botswana. Overall, these data provide a snapshot of the coinfection problem at the heart of the HIV/HBV co-epidemic, and are important to inform public health policy, resource allocation, education, surveillance and clinical care.

Evaluation of the NucliSens EasyQ v2.0 Assay in Comparison with the Roche Amplicor v1.5 and the Roche CAP/CTM HIV-1 Test v2.0 in Quantification of C-Clade HIV-1 in Plasma

Human immunodeficiency virus type 1 (HIV-1) genetic diversity poses a challenge to reliable viral load monitoring. Discrepancies between different testing platforms have been observed, especially for non-clade-B virus. Therefore we compare, in antiretroviral therapy (ART)-naïve South African subjects predominantly infected with HIV-1 clade-C, three commercially available assays: the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test version 2.0 by Roche (CAP/CTM v2.0), the BioMérieux NucliSens Version 2.0 Easy Q/Easy Mag (NucliSens v2.0) and the Roche COBAS Amplicor HIV-1 Monitor Test Version 1.5 (Amplicor v1.5). Strong linear correlation was observed and Bland-Altman analyses showed overall good agreement between the assays with mean viral load differences of 0.078 log cp/ml (NucliSens v2.0 – Amplicor v1.5), 0.260 log cp/ml (CAP/CTM v2.0 – Amplicor v1.5) and 0.164 log cp/ml (CAP/CTM v2.0 – NucliSens v2.0), indicating lower mean viral load results for the Amplicor v1.5 and higher mean readings for the CAP/CTM v2.0. Consistent with observations following previous comparisons of CAP/CTM v2.0 versus Amplicor v1.5, the CAP/CTM v2.0 assay detected low-level viremia (median 65 cp/ml) in more than one-third of those in whom viremia had been undetectable (<20 cp/ml) in assays using the NucliSens platform. These levels of viremia are of uncertain clinical significance but may be of importance in early detection of ART resistance in those on treatment. Overall the three assays showed good comparability of results but with consistent, albeit relatively small, discrepancies for HIV-1 clade-C samples, especially in the low-viremic range that should be taken into account when interpreting viral load data.

Prospective Monitoring Reveals Dynamic Levels of T Cell Immunity to Mycobacterium Tuberculosis in HIV Infected Individuals

Monitoring of latent Mycobacterium tuberculosis infection may prevent disease. We tested an ESAT-6 and CFP-10-specific IFN-γ Elispot assay (RD1-Elispot) on 163 HIV-infected individuals living in a TB-endemic setting. An RD1-Elispot was performed every 3 months for a period of 3–21 months. 62% of RD1-Elispot negative individuals were positive by cultured Elispot. Fluctuations in T cell response were observed with rates of change ranging from −150 to +153 spot-forming cells (SFC)/200,000 PBMC in a 3-month period. To validate these responses we used an RD1-specific real time quantitative PCR assay for monokine-induced by IFN-γ (MIG) and IFN-γ inducible protein-10 (IP10) (MIG: r = 0.6527, p = 0.0114; IP-10: r = 0.6967, p = 0.0056; IP-10+MIG: r = 0.7055, p = 0.0048). During follow-up 30 individuals were placed on ARVs and 4 progressed to active TB. Fluctuations in SFC did not correlate with CD4 count, viral load, treatment initiation, or progression to active TB. The RD1-Elispot appears to have limited value in this setting.

High Frequency of Transmitted HIV-1 Gag HLA Class I-Driven Immune Escape Variants but Minimal Immune Selection over the First Year of Clade C Infection

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.

Diagnostic Accuracy of the HemoCue Hb 301, STAT-Site MHgb and URIT-12 Point-of-Care Hemoglobin Meters in a Central Laboratory and a Community Based Clinic in Durban, South Africa.

In South Africa, various point-of-care hemoglobin meters are used. However, the regulatory framework for approval, implementation and oversight of use of point-of-care hemoglobin meters is suboptimal. We assessed the diagnostic accuracy of the HemoCue Hb 301, STAT-Site MHgb and URIT-12 point-of-care hemoglobin meters, compared to a central laboratory based reference assay, in a central laboratory and a community based clinic in Durban, South Africa. Differences in performance of the point-of-care assays, compared to the reference assay, were more pronounced in the community based clinic. Results were reasonable for the HemoCue Hb 301, but poor for the STAT-Site MHgb and the URIT-12. Poor test performance of point-of-care hemoglobin meters, and inadequate evaluations and oversight in South Africa, leads to suboptimal clinical care and clinical research, and increased costs. There is a need for proper evaluation and quality assurance of point-of-care tests, the results of which should be made widely available to key stakeholders.

Reduced Expression of Siglec-7, NKG2A and CD57 on Terminally Differentiated CD56-CD16+ NK Cell Subset is associated with NK Cell Dysfunction in Chronic HIV-1 clade C Infection

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.

Association of interleukin-10 promoter genetic variants with T-cell and B-cell activation in HIV-1 infection

Background Interleukin-10 (IL-10) is a potent immunoregulatory cytokine, with promoter polymorphisms that have previously been associated with HIV-1 susceptibility and pathogenesis. Association of IL-10 SNPs with markers of CD4, CD8 and B cell activation has not previously been investigated. Methods

KIR-HLA footprints and NK cell-mediated recognition of HIV-1

Background Increasing data suggest an important role of KIR+ NK cells in the control of HIV-1, however the precise mechanisms on how NK cells recognize HIV-1-infected cells remain poorly understood. KIRs can bind to HLA class I molecules, but the binding affinity of these interactions is dependent on the HLA class I presented epitope. Recent reports have suggested that sequence variations within HIV-1 epitopes presented by HLA class I can affect the binding of inhibitory KIRs expressed on NK cells, potentially modulating NK cell responses to infected cells. Here we investigated whether HIV-1 might adapt to the combined KIR/HLA genotypes on a population level to identify areas within HIV-1 that might be targeted by KIR+ NK cells.

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.