Ashwini Shete

Safety and immunogenicity of DNA and MVA HIV-1 subtype C vaccine prime-boost regimens: a phase I randomised Trial in HIV-uninfected Indian volunteers.

Study Design A randomized, double-blind, placebo controlled phase I trial. Methods The trial was conducted in 32 HIV-uninfected healthy volunteers to assess the safety and immunogenicity of prime-boost vaccination regimens with either 2 doses of ADVAX, a DNA vaccine containing Chinese HIV-1 subtype C env gp160, gag, pol and nef/tat genes, as a prime and 2 doses of TBC-M4, a recombinant MVA encoding Indian HIV-1 subtype C env gp160, gag, RT, rev, tat, and nef genes, as a boost in Group A or 3 doses of TBC-M4 alone in Group B participants. Out of 16 participants in each group, 12 received vaccine candidates and 4 received placebos. Results Both vaccine regimens were found to be generally safe and well tolerated. The breadth of anti-HIV binding antibodies and the titres of anti-HIV neutralizing antibodies were significantly higher (p<0.05) in Group B volunteers at 14 days post last vaccination. Neutralizing antibodies were detected mainly against Tier-1 subtype B and C viruses. HIV-specific IFN-γ ELISPOT responses were directed mostly to Env and Gag proteins. Although the IFN-γ ELISPOT responses were infrequent after ADVAX vaccinations, the response rate was significantly higher in group A after 1st and 2nd MVA doses as compared to the responses in group B volunteers. However, the priming effect was short lasting leading to no difference in the frequency, breadth and magnitude of IFN-γELISPOT responses between the groups at 3, 6 and 9 months post-last vaccination. Conclusions Although DNA priming resulted in enhancement of immune responses after 1st MVA boosting, the overall DNA prime MVA boost was not found to be immunologically superior to homologous MVA boosting. Trial Registration Clinical Trial Registry CTRI/2009/091/000051

Is Prime Boost Strategy a Promising Approach in HIV Vaccine Development

Since the discovery of HIV vaccine three decades back, the quest for HIV vaccines has remained unquenched. There has been a transition of preferred approaches from candidates capable of inducing neutralizing antibody (Nab) or cytolytic T cell (CTL) response to vaccines that can induce broad spectrum responses. Heterologous prime boost strategy is believed to induce broad spectrum immunity of higher magnitude and breadth to effectively counter HIV diversity and hence is being studied extensively in the HIV vaccine field. It is important to understand factors affecting the immune responses generated by the prime-boost regimens to get leads for developing effective regimens. This review focuses on the results of completed clinical trials based on the three most frequently used prime-boost regimens, vector (ALVAC)/protein, DNA/vector (MVA) and DNA/vector (Ad5). It will also discuss probable protective immunological responses responsible for efficacy of the vaccine and role of prime boost strategy in eliciting them.

DIFFERENTIAL MODULATION OF PHENOTYPIC COMPOSITION OF HIV-INFECTED AND -UNINFECTED PBMCS DURING CRYOPRESERVATION

This article was designed to determine variations in phenotypic composition of fresh and frozen PBMCs for assessing utility of cryopreserved PBMCs for phenotypic assays. Relative percentages of effector memory cells increased significantly as against percentages of naïve cells which showed significant decrease after cryopreservation in HIV-uninfected samples. These differences were not significant in HIV-infected individuals. There was no significant difference in the expression of activation markers in fresh and frozen PBMCs except the HLA DR expression on CD8 cells in HIV-infected individuals, which was significantly decreased in frozen PBMCs. Thus, cryopreservation resulted in differential effect on phenotypic composition of PBMCs in HIV-infected and -uninfected individuals.

Indian Long-term Non-Progressors Show Broad ADCC Responses with Preferential Recognition of V3 Region of Envelope and a Region from Tat Protein

HIV-specific antibody-dependent cell cytotoxicity (ADCC) is likely to be important in governing protection from human immunodeficiency virus (HIV) and slowing disease progression. Little is known about the ADCC responses to HIV-1 subtype C. We characterized ADCC responses in HIV-1 subtype C-infected Indian subjects with slow disease progression and identified the dominant antigenic regions recognized by these antibodies. ADCC responses were measured in plasma from 34 long-term non-progressors (LTNPs), who were asymptomatic and maintained CD4 count above 500 cells/mm3 for the last 7 years in the absence of antiretroviral therapy (ART), and 58 ART naïve progressors with CD4 count <500 cells/mm3 against overlapping HIV-1 peptides using a flow cytometry-based antibody-dependent natural killer (NK) cell activation assay. The assay measured CD107a expression on NK cells as a marker of antibody-dependent NK cell activation and IFN-γ secretion by NK cells upon activation. The ADCC epitopes were mapped using the matrix of overlapping peptides. Indian LTNPs showed higher and broader ADCC responses compared to the progressors. The Env-C and Tat-specific ADCC responses were associated with lower plasma viral load, whereas the Env-C responses were also associated with higher CD4 counts. Five of 10 LTNP responders targeted epitopes in the V3 region (amino acids 288–330) of Env-C. Additionally, three Tat regions were targeted by ADCC antibodies from LTNPs. ADCC responses were associated with slow HIV progression in Indian subtype C-infected cohort. The frequently recognized peptides from the V3 loop of Env and the novel epitopes from Tat by the LTNPs warrants further study to understand the role of ADCC responses to these regions in control and prevention of HIV-1 infection.

T-cell epitopes identified by BALB/c mice immunized with vaccinia expressing HIV-1 gag lie within immunodominant regions recognized by HIV-infected Indian patients

Background: Human immunodeficiency virus (HIV) antigens from transmitted strains of HIV would prove crucial in vaccine designing for prevention of HIV infection. Immune response generated by Vaccinia construct expressing the HIV-1 gag gene from transmitted Indian HIV-1 subtype C strain (Vgag) in BALB/c mice is reported in the present study along with the identification of epitopes responsible for induction of the immune response. Aims: The aim of this study was to determine immune response generated by the constructs in a mouse model and to understand the epitope specificities of the response. Settings and Design: This was an observational study carried out in BALB/c mice. Materials and Methods: The immunogenecity of Vgag construct was evaluated in BALB/c mice after multiple immunizations. T-cell response was monitored by the interferon-γ ELISPOT assay using HIV-1 C Gag overlapping peptides and anti-P24 antibodies were estimated by ELISA. Statistical Analysis Used: Graphpad prism software was used for statistical analysis and for plotting graphs. Results: IFN-γ-secreting T cells and antibodies were detected against HIV Gag in mice after immunization. Although after repeated immunizations, antibody-mediated immune response increased or remained sustained, the magnitude of IFN-γ-secreting T cell was found to be decreased over time. The Gag peptides recognized by mice were mainly confined to the P24 region and had a considerable overlap with earlier reported immunodominant regions recognized by HIV-infected Indian patients. Conclusion: Vaccinia construct with a gag gene from transmitted HIV-1 virus was found to be immunogenic. The Gag regions identified by mice could have important implications in terms of future HIV vaccine designing.

Development of IFN-γ secretory ELISPOT based assay for screening of ADCC responses

Antibody dependent cell mediated cytotoxicity has been established as one of the important protective immune mechanisms against HIV making it essential to evaluate it while testing immunogenicity of emerging vaccine candidates. IFN-γ secretory ELISPOT assay, widely used for evaluation of CTL response in HIV vaccine trials, was adapted for measuring ADCC responses and the results were compared with the standard ICS based assays. IFN-γ responses elicited by plasma samples of 23 HIV infected individuals against Env and Gag peptides using granulocytes as antigen presenting cells were assessed by both the methods. Supernatants of the activated cells in ELISPOT assay were also assessed for cytokine/chemokine estimation. ELISPOT assays detected significantly more ADCC responders against HIV-Env and Gag peptide pools than ICS assay. The magnitude of IFN-γ response in both the assay correlated significantly (p=0.002). NK cells were found to be the predominant cell type secreting IFN-γ in the assay. Although IFN-γ and IL-6 levels were significantly higher in supernatants of Env peptides stimulated cells, IP-10 and MCP-1α levels were found to be more against Gag peptides. Thus, IFN-γ secretory ELISPOT assay was found to be more sensitive in detecting ADCC responders than ICS assay making it a valuable tool for screening of ADCC responses in future vaccine trials. Differences in cytokine pattern of Env versus Gag stimulated cells warrants a need for investigating their role in protection against HIV infection.

Effect of combination antiretroviral therapy on HIV-1-specific antibody-dependent cellular cytotoxicity responses in subtype B- and subtype C-infected cohorts.

Background: There is growing interest in immune therapies to clear the latent HIV-1 after combination antiretroviral therapy (cART). There is limited information on the effect of cART on antibody-dependent cellular cytotoxicity (ADCC), and no studies have directly compared ADCC in HIV-1 subtype B- and subtype C-infected subjects. The effect of improving immunocompetence on ADCC to influenza also remains unexplored. Methods: The effect of cART on HIV-1- and influenza-specific ADCC was analyzed in 2 cohorts (39 subtype B- and 47 subtype C-infected subjects) before and after 2 years of cART. ADCC analyses included an enzyme-linked immunosorbent assay–based dimeric recombinant soluble (rs) Fc&ggr;RIIIa-binding assay, antibody-dependent natural killer cell activation assay, and ADCC-mediated killing assays. Results: HIV-1 subtype B and C Env-specific antibody binding to dimeric rsFc&ggr;RIIIa were reduced in subtypes B- and C-infected cohorts after 2 years of cART (both P < 0.05). Reduced ADCC-mediated killing of target cells expressing subtype B Env in the subtype B-infected cohort (P = 0.003) was observed after 96 weeks of cART, but not of subtype C Env in the subtype C-infected cohort. A greater reduction in ADCC was detected in subjects with baseline CD4 counts >300 cells/&mgr;L (P < 0.05). The resolving immunodeficiency after 96 weeks of cART resulted in improved HA-specific ADCC to 6 strains of influenza (all P < 0.01). Conclusions: cART results in HIV-1 antigen loss and reductions in HIV-1 Env-specific antibodies with Fc functionality in both subtype B- and C-infected subjects, particularly in immunocompetent subjects. Simultaneously, cART improves ADCC to diverse strains of influenza, suggesting reduction in influenza disease after cART.

HIV-infected CD4+ T Cells Use T-bet-dependent Pathway for Production of IL-10 Upon Antigen Recognition.

Interleukin (IL)‐10 has been implicated in persistence of pathogens in a number of chronic infections. Infected CD4+ cells upon reactivation with HIV antigens were also shown to produce IL‐10, which might contribute to their persistence. Hence, it is crucial to determine mechanisms regulating IL‐10 production after activation with HIV antigens for devising effective blocking strategies. In this study, ERK‐, T‐bet‐ and FoxP3‐dependent pathways were evaluated for their possible roles in IL‐10 production by infected CD4+ cells after reactivation with HIV Env. Intracellular and secreted IL‐10 levels were determined by flow cytometry and Bioplex assay after treating PBMCs with PD98059, tipifarnib and cyclosporin A for blocking of ERK‐, T‐bet‐and FoxP3‐dependent pathways, respectively. Baseline levels of T‐bet, pERK were higher in P24+ CD4+ cells as compared to uninfected CD4+ cells, which increased further after activation with Env. Inhibition of T‐bet resulted in 2.3‐fold reduction of IL‐10 expression whereas ERK and FoxP3 inhibition failed to cause suppression of IL‐10 expression. Conversely, IL‐10 secreted by PBMCs was inhibited maximally after ERK inhibition suggesting its role in regulation of cytokine secretory pathway. IFN‐γ was found to be suppressed after treatment with inhibitors of all these pathways. Thus, the study highlighted need for IL‐10 blockade along with the use of antigens for therapeutic vaccinations or latency reversal and identified the T‐bet‐dependent pathway as an important pathway regulating IL‐10 production by infected CD4+ cells. However, simultaneous blockade of IFN‐γ precludes use of inhibitor of this pathway as an IL‐10 blocking strategy.

In Vitro Sensitization of T Cells with DC-Associated/Delivered HIV Constructs Can Induce a Polyfunctional CTL Response, Memory T-Cell Response, and Virus Suppression

The absence of a suitable animal model for HIV infection is one of the major obstacles to the development of a preventive HIV vaccine. Vaccines showing good response in animal studies may fail in human efficacy trials. We have demonstrated DC-mediated in vitro sensitization of autologous T cells against three HIV constructs. The in vitro sensitized T cells were able to demonstrate a polyfunctional T-cell response, as well as central and effector memory T cells, and virus lysis in a virus inhibition assay, three potentially protective responses. However, none of the constructs could induce all three responses. Also there were variations from volunteer to volunteer. These may be due to genetic and other factors. This study provides evidence of an in vitro system that can be used to assess the immune response against a candidate vaccine, and may also provide the opportunity to modify vaccine constructs to achieve the goal of developing an ideal vaccine.

Higher expression of human telomerase reverse transcriptase in productively-infected CD4 cells possibly indicates a mechanism for persistence of the virus in HIV infection: Persistence in productive HIV infection

Mechanisms involved in survival of productively‐infected memory CD4+cells after initial antigenic stimulation and their subsequent reversion to the resting state are critical for the development of a predominant replication‐competent HIV reservoir. These mechanisms may also counter their elimination after HIV reactivation through latency‐reversing agents (LRA). Thus, their evaluation is critical when using an appropriate HIV latency model that recapitulates the predominant replication‐competent HIV reservoir to develop strategies for HIV eradication. The model for evaluating the possible survival mechanisms after T cell receptor (TCR) stimulation was developed by infecting memory CD4+cells with an HIV‐1C primary isolate and cytokine secretion and gene expression patterns determined. Infected cells showed compromised functionality as evident from 6.8‐fold lower secretion of IL‐2 than from uninfected control cells. After TCR stimulation, the infected cells showed significantly higher fold increases in CD27 and CCR5 and smaller increases in CD5 mRNA over baseline values. Because CD27 expression may influence telomerase activity through AKT phosphorylation, CD27, human telomerase reverse transcriptase (hTERT) and pAKT expression in productively‐infected cells from HIV‐infected patients was evaluated by flow cytometry. HIV harbored in memory CD4+ cells was reactivated by HIV‐1 envelope peptides, which have been shown to act as effective LRA. P24+CD4+cell showed significantly higher expression of CD27, hTERT and pAKT than P24−CD4+cells. These findings indicate compromised functionality of HIV‐infected cells after TCR stimulation, which may interfere with their elimination by the immune system. They also indicate that pAKT and hTERT induction are possible survival mechanisms of productively‐infected CD4+cells.