Characterizing the role of CD4+ T cell immunoregulatory networks in peripheral blood and lymphoid tissue during HIV-1 clade C infection.

Masters Degree. University of KwaZulu-Natal, Medical School.HIV eradication efforts have been unsuccessful due to virus persistence in cellular and tissue reservoirs. Recent evidence suggests that germinal centers (GCs) within lymph nodes (LN) contain a novel subset of regulatory T cells (TREGs), termed follicular regulatory T (TFR) cells. These cells control the magnitude and specificity of the GC response and like TREGs are essential for the maintenance of self-tolerance and immune homeostasis. However, the exact role of TFR cells in HIV infection and their contribution to viral control is not completely understood, possibly due to their low frequency, heterogeneity and more so, the difficulty in accessing human lymphoid tissue samples to fully study them. Thus, we set out to comprehensively investigate TFR cells in LN and peripheral blood (PB) samples, using a multifaceted approach including flow cytometry, MHC class II tetramers, immunofluorescence microscopy (IF), ELISA, digital droplet PCR and singlecell RNA sequencing (SeqWell), in HIV-1 clade C infection. Furthermore, we aimed to determine the effect of very early treatment on the frequency and function of this cell subset. Overall, our studies contributed various notable findings to the field. Firstly, we were able to develop MHC class II tetramers, specific in our HIV-1 clade C setting, as a more sensitive method of identifying very low cell frequency antigen-specific CD4+ T cells without relying on function. Tetramers eliminate the bias associated with in vitro stimulation required for functional assays and the limitation associated with only detecting subsets of cells capable of secreting a cytokine. Notably, we used class II tetramers to demonstrate that HIV-specific CD4+ T cell responses restricted to DRB1*11-Gag41 are associated with immune control of HIV-1 infection. We next focused on understanding the role of CD4+ regulatory cells during HIV-1 infection. Firstly, we showed that TFR cell frequencies were significantly higher in LN compared to PB samples. Secondly, TFR are a phenotypically and transcriptionally distinct subset compared to regulatory T cells (TREGs) and T Follicular Helper cells (TFH). Thirdly, we were able to detect HIV-specific TFR using our newly synthesized MHC class II tetramers, and showed higher frequencies observed in LNs during untreated HIV infection. Fourthly, as measured by both flow cytometry and IF, most of TFR localized outside of the GC, with very early ART initiators displaying larger proportions of TFR within the GC. Lastly, TFR cells exhibited a potential suppressive functional capacity as they produced IL-10, which is a canonical suppressive cytokine and they were also positively associated with gp41 IgG antibodies titers. Overall, the data presented in this thesis highlights the advantage of MHC class II tetramers in evaluating HIV-specific CD4 + T cell responses in natural infections. More so, the results give important insights into regulatory cells within lymph nodes; their biology, function and their role in the setting of very ART initiation

Mapping immunodominant patterns and HLA class II restriction characteristics of HIV-specific CD4+ T cell responses in acute and chronic HIV-1 subtype C infection.

M. Med. Sc. University of KwaZulu-Natal, Durban 2014.Increasing evidence suggests that virus-specific CD4+ T cells contribute to immune-mediated control of HIV-1 infection. However, precise details of CD4+ T cell contribution to immune protection against HIV have not been adequately defined and most of the existing data was predominantly generated in clade B HIV-1 infection. Understanding the contribution of CD4+ T cell responses in clade C infection is important for developing vaccines that would be efficacious in sub-Saharan Africa which carries the highest burden of the HIV epidemic in the world. Therefore this study focused on the role of CD4+ T helper cells in the immune response to clade C HIV-1 infection. We tested the hypothesis that HIV-1-specific CD4+ T cell responses and protective class II HLA alleles are important determinants of effective immunological control of HIV-1 infection. Firstly, CD8 depleted PBMCs were used in an IFN-γ ELISPOT assay to conduct a comprehensive analysis of virus-specific CD4+ T cell responses in acute and chronic HIV-1 clade C infection. Thereafter the host genetic effects of class II HLA-DRB1 alleles on HIV viremia were assessed using the HLA-DRB1 restriction assay, where HLA class II-restriction characteristics of detectable responses were defined. Lastly, functional differences of HIV-specific CD4+ T cells were further characterized using flow cytometric analysis. In our study, Gag and Pol regions of the HIV proteome were found to be the most frequently targeted in acute HIV-1 infection (69% of total responses), with CD4+ T cell targeting across the proteome remaining relatively stable over time. In chronic HIV-1 clade C infection, dominant HIV-1-specific CD4+ T cell responses were detectable against a limited number of epitopes. Epitopes in the Gag region were the most targeted by CD4+ T cells (30/40 peptides), with OLP 41 in the Gag p24 region being the most dominant epitope targeted (15% of responses). There were no significant differences observed between total or Gag-specific CD4+ T cell responses and contemporaneous viral load. Interestingly, responses rarely targeted the envelope region in clade C infection, in contrast to multiple epitopes targeted in this protein in previous clade B studies. Functional analysis demonstrated that IFN-γ, IL-2 and TNFα were the most secreted cytokines by HIV-specific CD4+ T cells in 18/25 individuals, with IFN-γ being the most dominant response in individual subjects. The HLA class II DRB1 restriction in clade C HIV infected individuals showed epitope promiscuity, consistent with previous studies in clade B infection. The HLA-DRB1*13:01 allele variant was associated with the highest frequency of responders (22%) in our cohort and restricted the highest number of HIV-specific peptides (9/15). Together, our data identify immunodominant regions of HIV-specific CD4+ T cell responses and their association with viral control during clade C infection. Furthermore, our findings will inform studies aimed at elucidating the underlying mechanism by which CD4+ T cells modulate effective CD8+ T cell and B cell responses. Additionally, these data suggest that epitope promiscuity among class II HLA molecules should be taken into account for vaccines designed to induce CD4+ T cell responses. This information will be critical to vaccine efforts designed to induce these responses, as well as potential therapeutic manipulation of immunity in persons with acute and chronic HIV-1 infection

Magnitude and Kinetics of CD8+ T Cell Activation during Hyperacute HIV Infection Impact Viral Set Point

CD8[superscript +] 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[superscript +] T cell response, with limited bystander activation of non-HIV memory CD8[superscript +] T cells. HIV-specific CD8[superscript +] 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[superscript +] 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[superscript +] T cell responses are crucial for subsequent immune control of acute infection, which has important implications for HIV vaccine design.Bill & Melinda Gates FoundationCollaboration for AIDS Vaccine DiscoveryWitten Family FoundationDan and Marjorie SullivanUrsula BrunnerGary and Loren CohenMark and Lisa Schwartz Foundation,International AIDS Vaccine Initiative (UKZNRSA1001)National Institute of Allergy and Infectious Diseases (U.S.) (R37AI067073)Center for AIDS Research (P30 AI060354

Continued Follow-Up of Phambili Phase 2b Randomized HIV-1 Vaccine Trial Participants Supports Increased HIV-1 Acquisition among Vaccinated Men.

CAPRISA, 2015.Abstract available in pdf

Magnitude and Kinetics of CD8+ T Cell Activation during Hyperacute HIV Infection Impact Viral Set Point.

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

Magnitude and Kinetics of CD8+ T Cell Activation during Hyperacute HIV Infection Impact Viral Set Point

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

Safety and immunogenicity of a subtype C ALVAC-HIV (vCP2438) vaccine prime plus bivalent subtype C gp120 vaccine boost adjuvanted with MF59 or alum in healthy adults without HIV (HVTN 107): A phase 1/2a randomized trial.

BackgroundAdjuvants are widely used to enhance and/or direct vaccine-induced immune responses yet rarely evaluated head-to-head. Our trial directly compared immune responses elicited by MF59 versus alum adjuvants in the RV144-like HIV vaccine regimen modified for the Southern African region. The RV144 trial of a recombinant canarypox vaccine vector expressing HIV env subtype B (ALVAC-HIV) prime followed by ALVAC-HIV plus a bivalent gp120 protein vaccine boost adjuvanted with alum is the only trial to have shown modest HIV vaccine efficacy. Data generated after RV144 suggested that use of MF59 adjuvant might allow lower protein doses to be used while maintaining robust immune responses. We evaluated safety and immunogenicity of an HIV recombinant canarypox vaccine vector expressing HIV env subtype C (ALVAC-HIV) prime followed by ALVAC-HIV plus a bivalent gp120 protein vaccine boost (gp120) adjuvanted with alum (ALVAC-HIV+gp120/alum) or MF59 (ALVAC-HIV+gp120/MF59) or unadjuvanted (ALVAC-HIV+gp120/no-adjuvant) and a regimen where ALVAC-HIV+gp120 adjuvanted with MF59 was used for the prime and boost (ALVAC-HIV+gp120/MF59 coadministration).Methods and findingsBetween June 19, 2017 and June 14, 2018, 132 healthy adults without HIV in South Africa, Zimbabwe, and Mozambique were randomized to receive intramuscularly: (1) 2 priming doses of ALVAC-HIV (months 0 and 1) followed by 3 booster doses of ALVAC-HIV+gp120/MF59 (months 3, 6, and 12), n = 36; (2) 2 priming doses of ALVAC-HIV (months 0 and 1) followed by 3 booster doses of ALVAC-HIV+gp120/alum (months 3, 6, and 12), n = 36; (3) 4 doses of ALVAC-HIV+gp120/MF59 coadministered (months 0, 1, 6, and 12), n = 36; or (4) 2 priming doses of ALVAC-HIV (months 0 and 1) followed by 3 booster doses of ALVAC-HIV+gp120/no adjuvant (months 3, 6, and 12), n = 24. Primary outcomes were safety and occurrence and mean fluorescence intensity (MFI) of vaccine-induced gp120-specific IgG and IgA binding antibodies at month 6.5. All vaccinations were safe and well-tolerated; increased alanine aminotransferase was the most frequent related adverse event, occurring in 2 (1.5%) participants (1 severe, 1 mild). At month 6.5, vaccine-specific gp120 IgG binding antibodies were detected in 100% of vaccinees for all 4 vaccine groups. No significant differences were seen in the occurrence and net MFI of vaccine-specific IgA responses between the ALVAC-HIV+gp120/MF59-prime-boost and ALVAC-HIV+gp120/alum-prime-boost groups or between the ALVAC-HIV+gp120/MF59-prime-boost and ALVAC-HIV+gp120/MF59 coadministration groups. Limitations were the relatively small sample size per group and lack of evaluation of higher gp120 doses.ConclusionsAlthough MF59 was expected to enhance immune responses, alum induced similar responses to MF59, suggesting that the choice between these adjuvants may not be critical for the ALVAC+gp120 regimen.Trial registrationHVTN 107 was registered with the South African National Clinical Trials Registry (DOH-27-0715-4894) and ClinicalTrials.gov (NCT03284710)