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Africa remains significantly underrepresented in high-resolution Human Leukocyte Antigen (HLA) data, despite being one of the most genetically diverse regions in the world. This critical gap in genetic information poses a substantial barrier to HLA-based research on the continent. In this study, Class I HLA data from Eastern and Southern African populations were analysed to assess genetic diversity across the region. We examined allele and haplotype frequency distributions, deviations from Hardy-Weinberg Equilibrium (HWE), linkage disequilibrium (LD), and conducted neutrality tests of homozygosity across various populations. Additionally, the African HLA data were compared to those of Caucasian and African American populations using the Jaccard index and multidimensional scaling (MDS) methods. The study revealed that South African populations exhibited 50.4% more genetic diversity within the Class I HLA region compared to other African populations. Zambia showed an estimated 36.5% genetic diversity, with Kenya, Rwanda and Uganda showing 35.7%, 34.2%, and 31.1%, respectively. Furthermore, an analysis of in-country diversity among different tribes indicated an average Class I HLA diversity of 25.7% in Kenya, 17% in Rwanda, 2.8% in South Africa, 13.6% in Uganda, and 6.5% in Zambia. The study also highlighted the genetic distinctness of Caucasian and African American populations compared to African populations. Notably, the differential frequencies of disease-promoting and disease-preventing HLA alleles across these populations emphasize the urgent need to generate high-quality HLA data for all regions of Africa and its major ethnic groups. Such efforts will be crucial in enhancing healthcare outcomes across the continent., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

Introduction: Studying diseased human tissues offers better insights into the intricate interactions between pathogens and the human host. In conditions such as HIV and cancers, where diseases primarily manifest in tissues, peripheral blood studies are limited in providing a thorough understanding of disease processes and localized immune responses., Methods: We describe a study designed to obtain excisional lymph nodes from volunteers for HIV reservoir studies. Since study commencement in 2015, 181 lymph node excisions have been performed, resulting in collection of 138 lymph node tissues. Lymph nodes were surgically excised from study volunteers using a minimally invasive procedure, performed in a minor theater under local anesthesia., Results: The surgery takes less than 30 minutes to complete, minimizing risk and stress on the volunteer. The small incision made during the procedure typically heals within a week. The associated discomfort is generally manageable, and participants are often able to resume their regular activities within a day. Only 5.5% of the study participants experienced minor adverse events, such as swelling and prolonged wound healing, recovering within 2 weeks with no serious adverse events reported., Discussion: Our study demonstrates that when done with outmost care, obtaining excised lymph nodes for research is relatively safe and practical., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Khaba, Papadopoulos, Nkosi, Nxele, Ngubane, Jajbhay, Pansegrouw and Ndhlovu.)

Introduction: HIV reservoir quantification is essential for evaluation of HIV curative strategies and may provide valuable insights about reservoir dynamics during antiretroviral therapy. The Intact Proviral DNA Assay (IPDA) provides the unique opportunity to quantify the intact and defective reservoir. The current IPDA is optimized for HIV-1 subtype B, the dominant subtype in resource-rich settings. However, subtype C is dominant in Sub-Saharan Africa, jointly accounting for around 60% of the pandemic. We developed an assay capable of quantifying intact and defective proviral HIV-1 DNA of subtype B and C., Methods: Primer and probe sequences were strategically positioned at conserved regions in psi and env and adapted to subtype B&C. In silico analysis of 752 subtype B and 697 subtype C near-full length genome sequences (nFGS) was performed to predict  the specificity and sensitivity. Gblocks were used to determine the limit of blank (LoB), limit of detection (LoD), and different annealing temperatures were tested to address impact of sequence variability., Results: The in silico analysis showed that the HIV-1 B&C IPDA correctly identified 100% of the intact subtype B, and 86% of the subtype C sequences. In contrast, the original IPDA identified 86% and 12% of these subtype B and C sequences as intact. Furthermore, the HIV-1 B&C IPDA correctly identified hypermutated (87% and 88%) and other defective sequences (73% and 66%) for subtype B and C with comparable specificity as the original IPDA for subtype B (59% and 63%). Subtype B cis-acting sequences were more frequently identified as intact by the HIV-1 B&C IPDA compared to the original IPDA (39% and 2%). The LoB for intact proviral DNA copies was 0, and the LoD for intact proviral DNA copies was 6 (> 95% certainty) at 60 °C. Quantification of 2-6 copies can be performed with > 80% certainty. Lowering the annealing temperature to 55 °C slightly lowered the specificity but prevented exclusion of samples with single mutations in the primer/probe region., Conclusions: We developed a robust and sensitive assay for the quantification of intact and defective HIV-1 subtype B and C proviral DNA, making this a suitable tool to monitor the impact of (large-scale) curative interventions., (© 2024. The Author(s).)

Background: Understanding how HIV affects SARS-CoV-2 immunity is crucial for managing COVID-19 in sub-Saharan populations due to frequent coinfections. Our previous research showed that unsuppressed HIV is associated with weaker immune responses to SARS-CoV-2, but the underlying mechanisms are unclear. We investigated how pre-existing T cell immunity against an endemic human coronavirus HCoV-NL63 impacts SARS-CoV-2 T cell responses in people living with HIV (PLWH) compared to uninfected individuals, and how HIV-related T cell dysfunction influences responses to SARS-CoV-2 variants., Methods: We used flow cytometry to measure T cell responses following PBMC stimulation with peptide pools representing beta, delta, wild-type, and HCoV-NL63 spike proteins. Luminex bead assay was used to measure circulating plasma chemokine and cytokine levels. ELISA and MSD V-PLEX COVID-19 Serology and ACE2 Neutralization assays were used to measure humoral responses., Results: Regardless of HIV status, we found a strong positive correlation between responses to HCoV-NL63 and SARS-CoV-2. However, PLWH exhibited weaker CD4 + T cell responses to both HCoV-NL63 and SARS-CoV-2 than HIV-uninfected individuals. PLWH also had higher proportions of functionally exhausted (PD-1high) CD4 + T cells producing fewer proinflammatory cytokines (IFNγ and TNFα) and had elevated plasma IL-2 and IL-12(p70) levels compared to HIV-uninfected individuals. HIV status didn't significantly affect IgG antibody levels against SARS-CoV-2 antigens or ACE2 binding inhibition activity., Conclusion: Our results indicate that the decrease in SARS-CoV-2 specific T cell responses in PLWH may be attributable to reduced frequencies of pre-existing cross-reactive responses. However, HIV infection minimally affected the quality and magnitude of humoral responses, and this could explain why the risk of severe COVID-19 in PLWH is highly heterogeneous., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ng’uni, Musale, Nkosi, Mandolo, Mvula, Michelo, Karim, Moosa, Khan, Jambo, Hanekom, Sigal, Kilembe and Ndhlovu.)

Broadly neutralizing antibodies (bNAbs) for HIV-1 prevention or cure strategies must inhibit transmitted/founder and reservoir viruses. Establishing sensitivity of circulating viruses to bNAbs and genetic patterns affecting neutralization variability may guide rational bNAbs selection for clinical development. We analyzed 326 single env genomes from nine individuals followed longitudinally following acute HIV-1 infection, with samples collected at ~1 week after the first detection of plasma viremia; 300 to 1,709 days postinfection but prior to initiating antiretroviral therapy (ART) (median = 724 days); and ~1 year post ART initiation. Sequences were assessed for phylogenetic relatedness, potential N- and O-linked glycosylation, and variable loop lengths (V1 to V5). A total of 43 env amplicons (median = 3 per patient per time point) were cloned into an expression vector and the TZM-bl assay was used to assess the neutralization profiles of 15 bNAbs targeting the CD4 binding site, V1/V2 region, V3 supersite, MPER, gp120/gp41 interface, and fusion peptide. At 1 μg/mL, the neutralization breadths were as follows: VRC07-LS and N6.LS (100%), VRC01 (86%), PGT151 (81%), 10-1074 and PGT121 (80%), and less than 70% for 10E8, 3BNC117, CAP256.VRC26, 4E10, PGDM1400, and N123-VRC34.01. Features associated with low sensitivity to V1/V2 and V3 bNAbs were higher potential glycosylation sites and/or relatively longer V1 and V4 domains, including known "signature" mutations. The study shows significant variability in the breadth and potency of bNAbs against circulating HIV-1 subtype C envelopes. VRC07-LS, N6.LS, VRC01, PGT151, 10-1074, and PGT121 display broad activity against subtype C variants, and major determinants of sensitivity to most bNAbs were within the V1/V4 domains. IMPORTANCE Broadly neutralizing antibodies (bNAbs) have potential clinical utility in HIV-1 prevention and cure strategies. However, bNAbs target diverse epitopes on the HIV-1 envelope and the virus may evolve to evade immune responses. It is therefore important to identify antibodies with broad activity in high prevalence settings, as well as the genetic patterns that may lead to neutralization escape. We investigated 15 bNAbs with diverse biophysical properties that target six epitopes of the HIV-1 Env glycoprotein for their ability to inhibit viruses that initiated infection, viruses circulating in plasma at chronic infection before antiretroviral treatment (ART), or viruses that were archived in the reservoir during ART in subtype C infected individuals in South Africa, a high burden country. We identify the antibodies most likely to be effective for clinical use in this setting and describe mutational patterns associated with neutralization escape from these antibodies.

In some instances, unsuppressed HIV has been associated with severe COVID-19 disease, but the mechanisms underpinning this susceptibility are still unclear. Here, we assessed the impact of HIV infection on the quality and epitope specificity of SARS-CoV-2 T cell responses in the first wave and second wave of the COVID-19 epidemic in South Africa. Flow cytometry was used to measure T cell responses following peripheral blood mononuclear cell stimulation with SARS-CoV-2 peptide pools. Culture expansion was used to determine T cell immunodominance hierarchies and to assess potential SARS-CoV-2 escape from T cell recognition. HIV-seronegative individuals had significantly greater CD4 + T cell responses against the Spike protein compared to the viremic people living with HIV (PLWH). Absolute CD4 count correlated positively with SARS-CoV-2-specific CD4 + and CD8 + T cell responses (CD4 r=0.5, p=0.03; CD8 r=0.5, p=0.001), whereas T cell activation was negatively correlated with CD4 + T cell responses (CD4 r=-0.7, p=0.04). There was diminished T cell cross-recognition between the two waves, which was more pronounced in individuals with unsuppressed HIV infection. Importantly, we identify four mutations in the Beta variant that resulted in abrogation of T cell recognition. Taken together, we show that unsuppressed HIV infection markedly impairs T cell responses to SARS-Cov-2 infection and diminishes T cell cross-recognition. These findings may partly explain the increased susceptibility of PLWH to severe COVID-19 and also highlights their vulnerability to emerging SARS-CoV-2 variants of concern., Competing Interests: TN, CC, AP, TN, FK, MM, IG, KJ, WH, AS, ZN No competing interests declared, (© 2022, Nkosi, Chasara et al.)

HIV persistence in tissue sites despite ART is a major barrier to HIV cure. Detailed studies of HIV-infected cells and immune responses in native lymph node tissue environment is critical for gaining insight into immune mechanisms impacting HIV persistence and clearance in tissue sanctuary sites. We compared HIV persistence and HIV-specific T cell responses in lymph node biopsies obtained from 14 individuals who initiated therapy in Fiebig stages I/II, 5 persons treated in Fiebig stages III-V and 17 late treated individuals who initiated ART in Fiebig VI and beyond. Using multicolor immunofluorescence staining and in situ hybridization, we detect HIV RNA and/or protein in 12 of 14 Fiebig I/II treated persons on suppressive therapy for 1 to 55 months, and in late treated persons with persistent antigens. CXCR3 + T follicular helper cells harbor the greatest amounts of gag mRNA transcripts. Notably, HIV-specific CD8 + T cells responses are associated with lower HIV antigen burden, suggesting that these responses may contribute to HIV suppression in lymph nodes during therapy. These results reveal HIV persistence despite the initiation of ART in hyperacute infection and highlight the contribution of virus-specific responses to HIV suppression in tissue sanctuaries during suppressive ART., (© 2022. The Author(s).)

Background: HIV eradication efforts have been unsuccessful partly due to virus persistence in immune sanctuary sites such as germinal centres within lymph node (LN) tissues. Recent evidence suggests that LNs harbour a novel subset of regulatory T cells, termed follicular regulatory T cells (TFRs), but their role in HIV pathogenesis is not fully elucidated., Results: Paired excisional LN and peripheral blood samples obtained from 20 HIV-uninfected and 31 HIV-infected treated and 7 chronic untreated, were used to determine if and how HIV infection modulate frequencies, function and spatial localization of TFRs within LN tissues. Imaging studies showed that most TFRs are localized in extra-follicular regions. Co-culture assays showed TFRs suppression of TFH help to B cells. Importantly, epigenetic and transcriptional studies identified DPP4 and FCRL3 as novel phenotypic markers that define four functionally distinct TFR subpopulations in human LNs regardless of HIV status. Imaging studies confirmed the regulatory phenotype of DPP4 + TFRs., Conclusion: Together these studies describe TFRs dynamic changes during HIV infection and reveal previously underappreciated TFR heterogeneity within human LNs., (© 2022. The Author(s).)

CD8+ T cells play an important role in HIV control. However, in human lymph nodes (LNs), only a small subset of CD8+ T cells express CXCR5, the chemokine receptor required for cell migration into B-cell follicles, which are major sanctuaries for HIV persistence in individuals on therapy. Here, we investigate the impact of HIV infection on follicular CD8+ T cell (fCD8) frequencies, trafficking patterns, and CXCR5 regulation. We show that, although HIV infection results in a marginal increase in fCD8s in LNs, the majority of HIV-specific CD8+ T cells are CXCR5- (non-fCD8s) (P < .003). Mechanistic investigations using Assay for Transposase-Accessible Chromatin using sequencing showed that non-fCD8s have closed chromatin at the CXCR5 transcriptional start site (TSS). DNA bisulfite sequencing identified DNA hypermethylation at the CXCR5 TSS as the most probable cause of closed chromatin. Transcriptional factor footprint analysis revealed enrichment of transforming growth factors (TGFs) at the TSS of fCD8s. In vitro stimulation of non-fCD8s with recombinant TGF-β resulted in a significant increase in CXCR5 expression (fCD8s). Thus, this study identifies TGF-β signaling as a viable strategy for increasing fCD8 frequencies in follicular areas of the LN where they are needed to eliminate HIV-infected cells, with implications for HIV cure strategies., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The duodenum is a major site of HIV persistence during suppressive antiretroviral therapy despite harboring abundant tissue-resident memory (Trm) CD8+ T cells. The role of duodenal Trm CD8+ T cells in viral control is still not well defined. We examined the spatial localization, phenotype, and function of CD8+ T cells in the human duodenal tissue from people living with HIV (PLHIV) and healthy controls. We found that Trm (CD69+CD103hi) cells were the predominant CD8+ T cell population in the duodenum. Immunofluorescence imaging of the duodenal tissue revealed that CD103+CD8+ T cells were localized in the intraepithelial region, while CD103-CD8+ T cells and CD4+ T cells were mostly localized in the lamina propria (LP). Furthermore, HIV-specific CD8+ T cells were enriched in the CD69+CD103-/lo population. However, the duodenal HIV-specific CD8+ Trm cells rarely expressed canonical molecules for potent cytolytic function (perforin and granzyme B) but were more polyfunctional than those from peripheral blood. Taken together, our results show that duodenal CD8+ Trm cells possess limited perforin-mediated cytolytic potential and are spatially separated from HIV-susceptible LP CD4+ T cells. This could contribute to HIV persistence in the duodenum and provides critical information for the design of cure therapies.

Following the discovery of HIV as a causative agent of AIDS, the expectation was to rapidly develop a vaccine; but thirty years later, we still do not have a licensed vaccine. Progress has been hindered by the extensive genetic variability of HIV and our limited understanding of immune responses required to protect against HIV acquisition. Nonetheless, valuable knowledge accrued from numerous basic and translational science research studies and vaccine trials has provided insight into the structural biology of the virus, immunogen design and novel vaccine delivery systems that will likely constitute an effective vaccine. Furthermore, stakeholders now appreciate the daunting scientific challenges of developing an effective HIV vaccine, hence the increased advocacy for collaborative efforts among academic research scientists, governments, pharmaceutical industry, philanthropy, and regulatory entities. In this review, we highlight the history of HIV vaccine development efforts, highlighting major challenges and future directions., (Copyright © 2020 Ng’uni, Chasara and Ndhlovu.)

Cellular immunity is critical for controlling intracellular pathogens, but individual cellular dynamics and cell-cell cooperativity in evolving human immune responses remain poorly understood. Single-cell RNA-sequencing (scRNA-seq) represents a powerful tool for dissecting complex multicellular behaviors in health and disease 1,2 and nominating testable therapeutic targets 3 . Its application to longitudinal samples could afford an opportunity to uncover cellular factors associated with the evolution of disease progression without potentially confounding inter-individual variability 4 . Here, we present an experimental and computational methodology that uses scRNA-seq to characterize dynamic cellular programs and their molecular drivers, and apply it to HIV infection. By performing scRNA-seq on peripheral blood mononuclear cells from four untreated individuals before and longitudinally during acute infection 5 , we were powered within each to discover gene response modules that vary by time and cell subset. Beyond previously unappreciated individual- and cell-type-specific interferon-stimulated gene upregulation, we describe temporally aligned gene expression responses obscured in bulk analyses, including those involved in proinflammatory T cell differentiation, prolonged monocyte major histocompatibility complex II upregulation and persistent natural killer (NK) cell cytolytic killing. We further identify response features arising in the first weeks of infection, for example proliferating natural killer cells, which potentially may associate with future viral control. Overall, our approach provides a unified framework for characterizing multiple dynamic cellular responses and their coordination.

Introduction: Immunological damage in acute HIV infection (AHI) may predispose to detrimental clinical sequela. However, studies on the earliest HIV-induced immunological changes are limited, particularly in sub-Saharan Africa. We assessed the plasma cytokines kinetics, and their associations with virological and immunological parameters, in a well-characterized AHI cohort where participants were diagnosed before peak viremia., Methods: Blood cytokine levels were measured using Luminex and ELISA assays pre-infection, during the hyperacute infection phase (before or at peak viremia, 1-11 days after the first detection of viremia), after peak viremia (24-32 days), and during the early chronic phase (77-263 days). Gag-protease-driven replicative capacities of the transmitted/founder viruses were determined using a green fluorescent reporter T cell assay. Complete blood counts were determined before and immediately following AHI detection before ART initiation., Results: Untreated AHI was associated with a cytokine storm of 12 out of the 33 cytokines analyzed. Initiation of ART during Fiebig stages I-II abrogated the cytokine storm. In untreated AHI, virus replicative capacity correlated positively with IP-10 (rho = 0.84, P < 0.001) and IFN-alpha (rho = 0.59, P = 0.045) and inversely with nadir CD4 + T cell counts (rho = - 0.58, P = 0.048). Hyperacute HIV infection before the initiation of ART was associated with a transient increase in monocytes (P < 0.001), decreased lymphocytes (P = 0.011) and eosinophils (P = 0.003) at Fiebig stages I-II, and decreased eosinophils (P < 0.001) and basophils (P = 0.007) at Fiebig stages III-V. Levels of CXCL13 during the untreated hyperacute phase correlated inversely with blood eosinophils (rho = - 0.89, P < 0.001), basophils (rho = - 0.87, P = 0.001) and lymphocytes (rho = - 0.81, P = 0.005), suggesting their trafficking into tissues. In early treated individuals, time to viral load suppression correlated positively with plasma CXCL13 at the early chronic phase (rho = 0.83, P = 0.042)., Conclusion: While commencement of ART during Fiebig stages I-II of AHI abrogated the HIV-induced cytokine storm, significant depletions of eosinophils, basophils, and lymphocytes, as well as transient expansions of monocytes, were still observed in these individuals in the hyperacute phase before the initiation of ART, suggesting that even ART initiated during the onset of viremia does not abrogate all HIV-induced immune changes.

Sustained viremia after acute HIV infection is associated with profound CD4 + T cell loss and exhaustion of HIV-specific CD8 + T cell responses. To determine the impact of combination antiretroviral therapy (cART) on these processes, we examined the evolution of immune responses in acutely infected individuals initiating treatment before peak viremia. Immediate treatment of Fiebig stages I and II infection led to a rapid decline in viral load and diminished magnitude of HIV-specific (tetramer + ) CD8 + T cell responses compared to untreated donors. There was a strong positive correlation between cumulative viral antigen exposure before full cART-induced suppression and immune responses measured by MHC class I tetramers, IFN-γ ELISPOT, and CD8 + T cell activation. HIV-specific CD8 + T responses of early treated individuals were characterized by increased CD127 and BCL-2 expression, greater in vitro IFN-γ secretion, and enhanced differentiation into effector memory (T em ) cells. Transcriptional analysis of tetramer + CD8 + T cells from treated persons revealed reduced expression of genes associated with activation and apoptosis, with concurrent up-regulation of prosurvival genes including BCL-2 , AXL , and SRC Early treatment also resulted in robust HIV-specific CD4 + T cell responses compared to untreated HIV-infected individuals. Our data show that limiting acute viremia results in enhanced functionality of HIV-specific CD4 + and CD8 + T cells, preserving key antiviral properties of these cells., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY).)

Some closely related human leukocyte antigen (HLA) alleles are associated with variable clinical outcomes following HIV-1 infection despite presenting the same viral epitopes. Mechanisms underlying these differences remain unclear but may be due to intrinsic characteristics of the HLA alleles or responding T cell repertoires. Here we examine CD8 + T cell responses against the immunodominant HIV-1 Gag epitope TL9 (TPQDLNTML 180-188 ) in the context of the protective allele B*81:01 and the less protective allele B*42:01. We observe a population of dual-reactive T cells that recognize TL9 presented by both B*81:01 and B*42:01 in individuals lacking one allele. The presence of dual-reactive T cells is associated with lower plasma viremia, suggesting a clinical benefit. In B*42:01 expressing individuals, the dual-reactive phenotype defines public T cell receptor (TCR) clones that recognize a wider range of TL9 escape variants, consistent with enhanced control of viral infection through containment of HIV-1 sequence adaptation.

Despite decades of focused research, the field has yet to develop a prophylactic vaccine for HIV-1 infection. In the RV144 vaccine trial, nonneutralizing antibody responses were identified as a correlate for prevention of HIV acquisition. However, factors that predict the development of such antibodies are not fully elucidated. We sought to define the contribution of circulating T follicular helper (cTfh) subsets to the development of nonneutralizing antibodies in HIV-1 clade C infection. Study participants were recruited from an acute HIV-1 clade C infection cohort. Plasma anti-gp41, -gp120, -p24, and -p17 antibodies were screened using a customized multivariate Luminex assay. Phenotypic and functional characterizations of cTfh cells were performed using HLA class II tetramers and intracellular cytokine staining. In this study, we found that acute HIV-1 clade C infection skewed the differentiation of functional cTfh subsets toward increased Tfh1 ( P = 0.02) and Tfh2 ( P < 0.0001) subsets, with a concomitant decrease in overall Tfh1-17 (which shares both Tfh1 and Tfh17 properties) ( P = 0.01) and Tfh17 ( P < 0.0001) subsets, compared to the subsets found in HIV-negative subjects. Interestingly, the frequencies of Tfh1 cells during acute infection (5.0 to 8.0 weeks postinfection) correlated negatively with the set point viral load ( P = 0.03, Spearman rho [ r ] = -60) and were predictive of p24-specific plasma IgG titers at 1 year of infection ( P = 0.003, r = 0.85). Taken together, our results suggest that the circulating Tfh1 subset plays an important role in the development of anti-HIV antibody responses and contributes to HIV suppression during acute HIV-1 infection. These results have implications for vaccine studies aimed at inducing long-lasting anti-HIV antibody responses. IMPORTANCE The HIV epidemic in southern Africa accounts for almost half of the global HIV burden, with HIV-1 clade C being the predominant strain. It is therefore important to define immune correlates of clade C HIV control that might have implications for vaccine design in this region. T follicular helper (Tfh) cells are critical for the development of HIV-specific antibody responses and could play a role in viral control. Here we showed that the early induction of circulating Tfh1 cells during acute infection correlated positively with the magnitude of p24-specific IgG and was associated with a lower set point viral load. This study highlights a key Tfh cell subset that could limit HIV replication by enhancing antibody generation. This study underscores the importance of circulating Tfh cells in promoting nonneutralizing antibodies during HIV-1 infection., Competing Interests: We declare no conflict of interest., (Copyright © 2018 American Society for Microbiology.)

Background: HIV incidence among young women in sub-Saharan Africa remains high and their inclusion in vaccine and cure efforts is crucial. We aimed to establish a cohort of young women detected during Fiebig stage I acute HIV infection in whom treatment was initiated immediately after diagnosis to advance research in this high-risk group., Methods: 945 women aged 18-23 years in KwaZulu-Natal, South Africa, who were HIV uninfected and sexually active consented to HIV-1 RNA testing twice a week and biological sampling and risk assessment every 3 months during participation in a 48-96 week life-skills and job-readiness programme. We analysed the effect of immediate combination antiretroviral therapy (ART) on viraemia and immune responses, sexual risk behaviour, and the effect of the socioeconomic intervention., Findings: 42 women were diagnosed with acute HIV infection between Dec 1, 2012, and June 30, 2016, (incidence 8·2 per 100 person-years, 95% CI 5·9-11·1), of whom 36 (86%) were diagnosed in Fiebig stage I infection with a median initial viral load of 2·97 log 10 copies per mL (IQR 2·42-3·85). 23 of these 36 women started ART at a median of 1 day (1-1) after detection, which limited the median peak viral load to 4·22 log 10 copies per mL (3·27-4·83) and the CD4 nadir to 685 cells per μL (561-802). ART also suppressed viral load (to <20 copies per mL) within a median of 16 days (12-26) and, in 20 (87%) of 23 women, prevented seroconversion, as shown with western blotting. 385 women completed the 48 week socioeconomic intervention, of whom 231 were followed up for 1 year. 202 (87%) of these 231 women were placed in jobs, returned to school, or started a business., Interpretation: Frequent HIV screening combined with a socioeconomic intervention facilitated sampling and risk assessment before and after infection. In addition to detection of acute infection and immediate treatment, we established a cohort optimised for prevention and cure research., Funding: Bill & Melinda Gates Foundation, National Institute of Allergy and Infectious Diseases, International AIDS Vaccine Initiative, Wellcome Trust, Howard Hughes Medical Institute., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Immune control of viral infections is heavily dependent on helper CD4 + T cell function. However, the understanding of the contribution of HIV-specific CD4 + T cell responses to immune protection against HIV-1, particularly in clade C infection, remains incomplete. Recently, major histocompatibility complex (MHC) class II tetramers have emerged as a powerful tool for interrogating antigen-specific CD4 + T cells without relying on effector functions. Here, we defined the MHC class II alleles for immunodominant Gag CD4 + T cell epitopes in clade C virus infection, constructed MHC class II tetramers, and then used these to define the magnitude, function, and relation to the viral load of HIV-specific CD4 + T cell responses in a cohort of untreated HIV clade C-infected persons. We observed significantly higher frequencies of MHC class II tetramer-positive CD4 + T cells in HIV controllers than progressors ( P = 0.0001), and these expanded Gag-specific CD4 + T cells in HIV controllers showed higher levels of expression of the cytolytic proteins granzymes A and B. Importantly, targeting of the immunodominant Gag41 peptide in the context of HLA class II DRB1*1101 was associated with HIV control ( r = -0.5, P = 0.02). These data identify an association between HIV-specific CD4 + T cell targeting of immunodominant Gag epitopes and immune control, particularly the contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infection. Furthermore, these results highlight the advantage of the use of class II tetramers in evaluating HIV-specific CD4 + T cell responses in natural infections. IMPORTANCE Increasing evidence suggests that virus-specific CD4 + T cells contribute to the immune-mediated control of clade B HIV-1 infection, yet there remains a relative paucity of data regarding the role of HIV-specific CD4 + T cells in shaping adaptive immune responses in individuals infected with clade C, which is responsible for the majority of HIV infections worldwide. Understanding the contribution of HIV-specific CD4 + T cell responses in clade C infection is particularly important for developing vaccines that would be efficacious in sub-Saharan Africa, where clade C infection is dominant. Here, we employed MHC class II tetramers designed to immunodominant Gag epitopes and used them to characterize CD4 + T cell responses in HIV-1 clade C infection. Our results demonstrate an association between the frequency of HIV-specific CD4 + T cell responses targeting an immunodominant DRB1*11-Gag41 complex and HIV control, highlighting the important contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infections., (Copyright © 2017 American Society for Microbiology.)

Functional analysis of T-cell responses in HIV-infected individuals has indicated that virus-specific CD8 + T cells with superior antiviral efficacy are well represented in HIV-1 controllers but are rare or absent in HIV-1 progressors. To define the role of individual T-cell receptor (TCR) clonotypes in differential antiviral CD8 + T-cell function, we performed detailed functional and mass cytometric cluster analysis of multiple CD8 + T-cell clones recognizing the identical HLA-B*2705-restricted HIV-1 epitope KK10 (KRWIILGLNK). Effective and ineffective CD8 + T-cell clones segregated based on responses to HIV-1-infected and peptide-loaded target cells. Following cognate peptide stimulation, effective HIV-specific clones displayed significantly more rapid TCR signal propagation, more efficient initial lytic granule release, and more sustained nonlytic cytokine and chemokine secretion than ineffective clones. To evaluate the TCR clonotype contribution to CD8 + T-cell function, we cloned the TCR α and β chain genes from one effective and two ineffective CD8 + T-cell clones from an elite controller into TCR-expressing lentivectors. We show that Jurkat/MA cells and primary CD8 + T cells transduced with lentivirus expressing TCR from one of the ineffective clones exhibited a level of activation by cognate peptide and inhibition of in vitro HIV-1 infection, respectively, that were comparable to those of the effective clonotype. Taken together, these data suggest that the potent antiviral capacity of some HIV-specific CD8 + T cells is a consequence of factors in addition to TCR sequence that modulate functionality and contribute to the increased antiviral capacity of HIV-specific CD8 + T cells in elite controllers to inhibit HIV infection. IMPORTANCE The greater ex vivo antiviral inhibitory activity of CD8 + T cells from elite controllers than from HIV-1 progressors supports the crucial role of effective HIV-specific CD8 + T cells in controlling HIV-1 replication. The contribution of TCR clonotype to inhibitory potency was investigated by delineating the responsiveness of effective and ineffective CD8 + T-cell clones recognizing the identical HLA-B*2705-restricted HIV-1 Gag-derived peptide, KK10 (KRWIILGLNK). KK10-stimulated "effective" CD8 + T-cell clones displayed significantly more rapid TCR signal propagation, more efficient initial lytic granule release, and more sustained cytokine and chemokine secretion than "ineffective" CD8 + T-cell clones. However, TCRs cloned from an effective and one of two ineffective clones conferred upon primary CD8 + T cells the equivalent potent capacity to inhibit HIV-1 infection. Taken together, these data suggest that other factors aside from intrinsic TCR-peptide-major histocompatibility complex (TCR-peptide-MHC) reactivity can contribute to the potent antiviral capacity of some HIV-specific CD8 + T-cell clones., (Copyright © 2017 American Society for Microbiology.)

Unlabelled: 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., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Previous studies have shown that elite controllers with minimal effector T cell responses harbor a low-frequency, readily expandable, highly functional, and broadly directed memory population. Here, we interrogated the in vivo relevance of this cell population by investigating whether the breadth of expandable memory responses is associated with the magnitude of residual viremia in individuals achieving durable suppression of HIV infection. HIV-specific memory CD8(+) T cells were expanded by using autologous epitopic and variant peptides. Viral load was measured by an ultrasensitive single-copy PCR assay. Following expansion, controllers showed a greater increase in the overall breadth of Gag responses than did untreated progressors (P = 0.01) as well as treated progressors (P = 0.0003). Nef- and Env-specific memory cells expanded poorly for all groups, and their expanded breadths were indistinguishable among groups (P = 0.9 for Nef as determined by a Kruskal-Wallis test; P = 0.6 for Env as determined by a Kruskal-Wallis test). More importantly, we show that the breadth of expandable, previously undetectable Gag-specific responses was inversely correlated with residual viral load (r = -0.6; P = 0.009). Together, these data reveal a direct link between the abundance of Gag-specific expandable memory responses and prolonged maintenance of low-level viremia. Our studies highlight a CD8(+) T cell feature that would be desirable in a vaccine-induced T cell response., Importance: Many studies have shown that the rare ability of some individuals to control HIV infection in the absence of antiretroviral therapy appears to be heavily dependent upon special HIV-specific killer T lymphocytes that are able to inhibit viral replication. The identification of key features of these immune cells has the potential to inform rational HIV vaccine design. This study shows that a special subset of killer lymphocytes, known as central memory CD8(+) T lymphocytes, is at least partially involved in the durable control of HIV replication. HIV controllers maintain a large proportion of Gag-specific expandable memory CD8(+) T cells involved in ongoing viral suppression. These data suggest that induction of this cell subset by future HIV vaccines may be important for narrowing possible routes of rapid escape from vaccine-induced CD8(+) T cell responses., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

CD8(+) T cells contribute to the control of HIV, but it is not clear whether initial immune responses modulate the viral set point. We screened high-risk uninfected women twice a week for plasma HIV RNA and identified 12 hyperacute infections. Onset of viremia elicited a massive HIV-specific CD8(+) T cell response, with limited bystander activation of non-HIV memory CD8(+) T cells. HIV-specific CD8(+) T cells secreted little interferon-γ, underwent rapid apoptosis, and failed to upregulate the interleukin-7 receptor, known to be important for T cell survival. The rapidity to peak CD8(+) T cell activation and the absolute magnitude of activation induced by the exponential rise in viremia were inversely correlated with set point viremia. These data indicate that rapid, high magnitude HIV-induced CD8(+) T cell responses are crucial for subsequent immune control of acute infection, which has important implications for HIV vaccine design., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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-γ 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.

Ag-specific CD8 T cells play a critical role in controlling HIV infection but eventually lose antiviral functions in part because of expression and signaling through the inhibitory programmed death-1 (PD-1) receptor. To better understand the impact of prolonged TCR ligation on regulation of PD-1 expression in HIV-specific CD8 T cells, we investigated the capacity of virus-specific CD8 T cells to modify the PD-1 epigenetic program after reduction in viral load. We observed that the transcriptional regulatory region was unmethylated in the PD-1(hi) HIV-specific CD8 T cells, whereas it remained methylated in donor-matched naive cells at acute and chronic stages of infection. Surprisingly, the PD-1 promoter remained unmethylated in HIV-specific CD8 T cells from subjects with a viral load controlled by antiviral therapy for >2 y or from elite controllers. Together, these data demonstrate that the epigenetic program at the PD-1 locus becomes fixed after prolonged exposure to HIV virus.

Unlabelled: The development of immunomonitoring models to determine HIV-1 vaccine efficacy is a major challenge. Studies suggest that HIV-1–specific CD8 T cells play a critical role in subjects achieving spontaneous viral control (HIV-1 controllers) and that they will be important in immune interventions. However, no single CD8 T-cell function is uniquely associated with controller status and the heterogeneity of responses targeting different epitopes further complicates the discovery of determinants of protective immunity. In the present study, we describe immunomonitoring models integrating multiple functions of epitope-specific CD8 T cells that distinguish controllers from subjects with treated or untreated progressive infection. Models integrating higher numbers of variables and trained with the least absolute shrinkage and selection operator (LASSO) variant of logistic regression and 10-fold cross-validation produce “diagnostic tests” that display an excellent capacity to delineate subject categories. The test accuracy reaches 75% area under the receiving operating characteristic curve in cohorts matched for prevalence of protective alleles. Linear mixed-effects model analyses show that the proliferative capacity, cytokine production, and kinetics of cytokine secretion are associated with HIV-1 control. Although proliferative capacity is the strongest single discriminant, integrated modeling of different dimensions of data leverages individual associations. This strategy may have important applications in predictive model development and immune monitoring of HIV-1 vaccine trials., Key Points: Immune monitoring models integrating multiple functions of HIV-1-specific CD8 T cells distinguish controllers from subjects with progressive HIV-1 infection. This strategy may have important applications in predictive model development and immune monitoring of HIV-1 vaccine trials.

The human leukocyte antigens HLA-B27 and HLA-B57 are associated with protection against progression of disease that results from infection with human immunodeficiency virus type 1 (HIV-1), yet most people with alleles encoding HLA-B27 and HLA-B57 are unable to control HIV-1. Here we found that HLA-B27-restricted CD8(+) T cells in people able to control infection with HIV-1 (controllers) and those who progress to disease after infection with HIV-1 (progressors) differed in their ability to inhibit viral replication through targeting of the immunodominant epitope of group-associated antigen (Gag) of HIV-1. This was associated with distinct T cell antigen receptor (TCR) clonotypes, characterized by superior control of HIV-1 replication in vitro, greater cross-reactivity to epitope variants and enhanced loading and delivery of perforin. We also observed clonotype-specific differences in antiviral efficacy for an immunodominant HLA-B57-restricted response in controllers and progressors. Thus, the efficacy of such so-called 'protective alleles' is modulated by specific TCR clonotypes selected during natural infection, which provides a functional explanation for divergent HIV-1 outcomes.

Analyses of the breadth and specificity of virus-specific CD8(+) T cell responses associated with control of HIV have largely relied on measurement of cytokine secretion by effector T cells. These have resulted in the identification of HIV elite controllers with low or absent responses in which non-T-cell mechanisms of control have been suggested. However, successful control of HIV infection may be associated with central memory T cells, which have not been consistently examined in these individuals. Gag-specific T cells were characterized using a peptide-based cultured enzyme-linked immunosorbent spot assay (ELISpot). Peripheral blood mononuclear cells from HIV elite controllers (n = 10), progressors (n = 12), and antiretroviral-treated individuals (n = 9) were cultured with overlapping peptides for 12 days. Specificity was assessed by tetramer staining, functional features of expanded cells were assessed by cytokine secretion, and virus inhibition and phenotypic characteristics were assessed by cell sorting and coculture assays. After peptide stimulation, elite controllers showed a greater number of previously undetectable (new) responses compared to progressors (P = 0.0008). These responses were highly polyfunctional, with 64.5% of responses having 3 to 5 functions. Expandable epitope-specific CD8(+) T cells from elite controllers had strong virus inhibitory capacity and predominantly displayed a central memory phenotype. These data indicate that elite controllers with minimal T cell responses harbor a highly functional, broadly directed central memory T cell population that is capable of suppressing HIV in vitro. Comprehensive examination of this cell population could provide insight into the immune responses associated with successful containment of viremia.

Polyvalent mosaic HIV immunogens offer a potential solution for generating vaccines that can elicit immune responses against genetically diverse viruses. However, it is unclear whether key T cell epitopes can be processed and presented from these synthetic Ags and recognized by epitope-specific human T cells. In this study, we tested the ability of mosaic HIV immunogens expressed by recombinant, replication-incompetent adenovirus serotype 26 vectors to process and present major HIV clade B and clade C CD8 T cell epitopes in human cells. A bivalent mosaic vaccine expressing HIV Gag sequences was used to transduce PBMCs from 12 HIV-1-infected individuals from the United States and 10 HIV-1-infected individuals from South Africa; intracellular cytokine staining, together with tetramer staining, was used to assess the ability of mosaic Gag Ags to stimulate pre-existing memory responses compared with natural clade B and C vectors. Mosaic Gag Ags expressed all eight clade B epitopes tested in 12 United States subjects and all 5 clade C epitopes tested in 10 South African subjects. Overall, the magnitude of cytokine production induced by stimulation with mosaic Ags was comparable to clade B and clade C Ags tested, but the mosaic Ags elicited greater cross-clade recognition. Additionally, mosaic Ags induced HIV-specific CD4 T cell responses. Our studies demonstrate that mosaic Ags express major clade B and clade C viral T cell epitopes in human cells, as well as support the evaluation of mosaic HIV-1 vaccines in humans.

The functional capacities of CD8(+) T cells important for virus clearance are influenced by interactions with antigen presenting cells (APCs) and CD4(+) T cells during initial selection, subsequent expansion, and development of memory. Recently, investigators have shown that polyfunctional T cells correlate best with long-term protection, however, it is still unknown how to stimulate T cells to achieve these responses. To study this, we examined the phenotypes and functions of CD8(+) T cells specific for two different virus antigens stimulated ex vivo using either autologous monocyte-derived dendritic cells (moDCs) or HLA-A2-Ig-based artificial APCs (aAPCs). Although similar numbers of influenza virus and measles virus tetramer-positive cells were generated by stimulation with peptide-loaded moDCs and aAPCs, T cell function, assessed by expression of IL-2, IFN-gamma, TNF-alpha, MIP1beta, and CD107a, showed that aAPC-generated CD8(+) T cells were multifunctional, whereas moDC-generated cells were mostly monofunctional. aAPC-generated cells also produced more of each cytokine per cell than CD8(+) T cells generated with moDCs. These phenotypes were not fixed, as changing the culture conditions of expanding T cells from aAPCs to moDCs, and moDCs to aAPCs, reversed the phenotypes. We conclude that CD8(+) T cells are heterogeneous in their functionality and that this is dependent, in a dynamic way, on the stimulating APC. These studies will lead to understanding the factors that influence induction of optimal CD8(+) T cell function.

Background: Endemic transmission of measles continues in many countries that have a high human immunodeficiency virus (HIV) burden. The effects that HIV infection has on immune responses to measles and to measles vaccine can impact measles elimination efforts. Assays to measure antibody include the enzyme immunoassay (EIA), which measures immunoglobulin G (IgG) to all measles virus (MV) proteins, and the plaque reduction neutralization (PRN) assay, which measures antibody to the hemagglutinin and correlates with protection. Antibody avidity may affect neutralizing capacity., Methods: HIV-infected and HIV-uninfected Zambian children were studied after measles vaccination (n=44) or MV infection (n=57). Laboratory or wild-type MV strains were used to infect Vero or Vero/signaling lymphocyte-activation molecule (SLAM) cells in PRN assays. IgG to MV was measured by EIA, and avidity was determined by ammonium thiocyanate dissociation., Results: HIV infection impaired EIA IgG responses after vaccination and measles but not PRN responses measured using laboratory-adapted MV. Avidity was lower among HIV-infected children 3 months after vaccination and 1 and 3 months after measles. Neutralization of wild-type MV infection of Vero/SLAM cells correlated with IgG avidity., Conclusion: Lower antibody quality and quantity in HIV-infected children after measles vaccination raise challenges for assuring the long-term protection of these children. Antibody quality in children receiving antiretroviral therapy requires assessment.

Evaluation of the immune responses induced by childhood vaccines requires measurement of T-cell, as well as antibody, responses. However, cellular immune responses are often not analyzed because of technical hurdles and the volume of blood required. Therefore, a sensitive and specific assay for antigen-specific T cells that utilizes a small volume of blood would facilitate new vaccine evaluation. We developed a novel assay for quantifying virus-specific CD8(+) T cells that combines the use of HLA-A2 immunoglobulin-based artificial antigen-presenting cells (aAPCs) for stimulation of antigen-specific CD8(+) T cells in whole blood with quantitative real-time reverse transcription-PCR (qRT-PCR) to detect gamma interferon (IFN-gamma) mRNA. This assay was optimized using a well-established cytomegalovirus (CMV) CD8(+) T-cell system. The aAPC-qRT-PCR assay had comparable sensitivity to intracellular cytokine staining (ICS) in detecting CMV-specific CD8(+) T cells with a detection limit of less than 0.004%. The assay was applied to the detection of low-frequency measles virus (MV)-specific CD8(+) T cells by stimulating blood from five MV-immune HLA-A*0201 donors with four different MV-specific peptides (MV peptide aAPCs). Stimulation with three of the MV peptide aAPCs resulted in significant increases in IFN-gamma mRNA ranging from 3.3- to 13.5-fold. Our results show that the aAPC-qRT-PCR assay is highly sensitive and specific and can be standardized for screening MV-specific CD8(+) T cells in vaccine trials. The technology should be transferable to analysis of CD8(+) T-cell responses to other antigens.