Evaluation of antiviral T cell responses and TSCM cells in volunteers enrolled in a phase I HIV-1 subtype C prophylactic vaccine trial in India.

T cells play an important role in controlling viral replication during HIV infection. An effective vaccine should, therefore, lead to the induction of a strong and early viral-specific CD8+ T cell response. While polyfunctional T cell responses are thought to be important contributors to the antiviral response, there is evidence to show that polyfunctional HIV- specific CD8+ T cells are just a small fraction of the total HIV-specific CD8+ T cells and may be absent in many individuals who control HIV replication, suggesting that other HIV-1 specific CD8+ effector T cell subsets may be key players in HIV control. Stem cell-like memory T cells (TSCM) are a subset of T cells with a long half-life and self-renewal capacity. They serve as key reservoirs for HIV and contribute a significant barrier to HIV eradication. The present study evaluated vaccine-induced antiviral responses and TSCM cells in volunteers vaccinated with a subtype C prophylactic HIV-1 vaccine candidate administered in a prime-boost regimen. We found that ADVAX DNA prime followed by MVA boost induced significantly more peripheral CD8+ TSCM cells and higher levels of CD8+ T cell-mediated inhibition of replication of different HIV-1 clades as compared to MVA alone and placebo. These findings are novel and provide encouraging evidence to demonstrate the induction of TSCM and cytotoxic immune responses by a subtype C HIV-1 prophylactic vaccine administered using a prime-boost strategy.

Association of circulatory Tfh-like cells with neutralizing antibody responses among chronic HIV-1 subtype C infected long-term nonprogressors and progressors.

HIV-1 vaccine functioning relies on successful induction of broadly neutralizing antibodies (bNAbs). CXCR3- circulatory T-follicular helper (cTfh) cells are necessary for inducing B-cells for generating bNAbs. Recent studies have suggested that CXCR3+ Tfh cells might also influence bNAb production. Plasma samples from 34 ART-Naïve HIV-1 infected individuals [long-term nonprogressors (LTNP)-19; Progressors-13] were tested against a heterologous virus panel (n = 11) from subtypes A, B, C, G, AC, BC and AE. Frequencies of CXCR3+ and CXCR3- cTfh-like cells in peripheral circulation were studied using flow cytometry. LTNP showed significantly lower CXCR3+ and higher CXCR3- cTfh-like cell frequencies, while neutralization breadth was observed to be broader in progressors. A positive correlation was observed between bNAb breadth and potency with CXCR3+PD-1+ cTfh-like cells in LTNP. Based on neutralization breadth, 9 HIV-1 infected individuals were classified as 'top neutralizers' and 23 as 'low neutralizers' and they did not show any correlations with CXCR3+ and CXCR3- cTfh-like cells. These preliminary data suggest that CXCR3+ similar to CXCR3- might possess significant functional properties for driving B-cells to produce bNAbs. Hence, an HIV vaccine which is capable of optimal induction of CXCR3+ cTfh cells at germinal centers might confer superior protection against HIV.

Induction and maintenance of bi-functional (IFN-γ + IL-2+ and IL-2+ TNF-α+) T cell responses by DNA prime MVA boosted subtype C prophylactic vaccine tested in a Phase I trial in India.

Effective vaccine design relies on accurate knowledge of protection against a pathogen, so as to be able to induce relevant and effective protective responses against it. An ideal Human Immunodeficiency virus (HIV) vaccine should induce humoral as well as cellular immune responses to prevent initial infection of host cells or limit early events of viral dissemination. A Phase I HIV-1 prophylactic vaccine trial sponsored by the International AIDS Vaccine Initiative (IAVI) was conducted in India in 2009.The trial tested a HIV-1 subtype C vaccine in a prime-boost regimen, comprising of a DNA prime (ADVAX) and Modified Vaccine Ankara (MVA) (TBC-M4) boost. The trial reported that the vaccine regimen was safe, well tolerated, and resulted in enhancement of HIV-specific immune responses. However, preliminary immunological studies were limited to vaccine-induced IFN-γ responses against the Env and Gag peptides. The present study is a retrospective study to characterize in detail the nature of the vaccine-induced cell mediated immune responses among volunteers, using Peripheral Blood Mononuclear Cells (PBMC) that were archived during the trial. ELISpot was used to measure IFN-γ responses and polyfunctional T cells were analyzed by intracellular multicolor flow cytometry. It was observed that DNA priming and MVA boosting induced Env and Gag specific bi-functional and multi-functional CD4+ and CD8+ T cells expressing IFN-γ, TNF-α and IL-2. The heterologous prime-boost regimen appeared to be slightly superior to the homologous prime-boost regimen in inducing favorable cell mediated immune responses. These results suggest that an in-depth analysis of vaccine-induced cellular immune response can aid in the identification of correlates of an effective immunogenic response, and inform future design of HIV vaccines.

Induction of circulating T follicular helper cells and regulatory T cells correlating with HIV-1 gp120 variable loop antibodies by a subtype C prophylactic vaccine tested in a Phase I trial in India.

A Phase I HIV-1 vaccine trial sponsored by the International AIDS Vaccine Initiative (IAVI) was conducted in India in 2009 to test a subtype C prophylactic vaccine in a prime-boost regimen comprising of a DNA prime (ADVAX) and MVA (TBC-M4) boost. The trial demonstrated that the regimen was safe and well tolerated and resulted in enhancement of HIV-specific immune responses. Preliminary observations on vaccine-induced immune responses were limited to analysis of neutralizing antibodies and IFN-γ ELISPOT response. The present study involves a more detailed analysis of the nature of the vaccine-induced humoral immune response using specimens that were archived from the volunteers at the time of the trial. Interestingly, we found vaccine induced production of V1/V2 and V3 region-specific antibodies in a significant proportion of vaccinees. Variable region antibody levels correlated directly with the frequency of circulating T follicular helper cells (Tfh) and regulatory T cells (Treg). Our findings provide encouraging evidence to demonstrate the immunogenicity of the tested vaccine. Better insights into vaccine-induced immune responses can aid in informing future design of a successfulHIV-1 vaccine.

Broad neutralization response in a subset of HIV-1 subtype C-infected viraemic non-progressors from southern India.

Broadly neutralizing antibodies (bnAbs) have been considered to be potent therapeutic tools and potential vaccine candidates to enable protection against various clades of human immunodeficiency virus (HIV). The generation of bnAbs has been associated with enhanced exposure to antigen, high viral load and low CD4+ T cell counts, among other factors. However, only limited data are available on the generation of bnAbs in viraemic non-progressors that demonstrate moderate to high viraemia. Further, since HIV-1 subtype C viruses account for more than 50 % of global HIV infections, the identification of bnAbs with novel specificities is crucial to enable the development of potent tools to aid in HIV therapy and prevention. In the present study, we analysed and compared the neutralization potential of responses in 70 plasma samples isolated from ART-naïve HIV-1 subtype C-infected individuals with various disease progression profiles against a panel of 30 pseudoviruses. Among the seven samples that exhibited a neutralization breadth of ≥70 %, four were identified as 'elite neutralizers', and three of these were from viraemic non-progressors while the fourth was from a typical progressor. Analysis of the neutralization specificities revealed that none of the four elite neutralizers were reactive to epitopes in the membrane proximal external region (MPER), CD4-binding site and V1V2 or V3 glycan. However, two of the four elite neutralizers exhibited enhanced sensitivity towards viruses lacking N332 glycan, indicating high neutralization potency. Overall, our findings indicate that the identification of potent neutralization responses with distinct epitope specificities is possible from the as yet unexplored Indian population, which has a high prevalence of HIV-1 subtype C infection.

A semisynthetic Eph receptor tyrosine kinase provides insight into ligand-induced kinase activation.

We have developed a methodology for generating milligram amounts of functional Eph tyrosine kinase receptor using the protein engineering approach of expressed protein ligation. Stimulation with ligand induces efficient autophosphorylation of the semisynthetic Eph construct. The in vitro phosphorylation of key Eph tyrosine residues upon ligand-induced activation was monitored via time-resolved, quantitative phosphoproteomics, suggesting a precise and unique order of phosphorylation of the Eph tyrosines in the kinase activation process. To our knowledge, this work represents the first reported semisynthesis of a receptor tyrosine kinase and provides a potentially general method for producing single-pass membrane proteins for structural and biochemical characterization.

Crystal structure of the ligand-binding domain of the promiscuous EphA4 receptor reveals two distinct conformations.

Eph receptors and their ephrin ligands are important mediators of cell-cell communication. They are divided in two subclasses based on their affinities for each other and on sequence conservation. Receptor-ligand binding within each subclass is fairly promiscuous, while binding cross the subclasses happens rarely. EphA4 is an exception to this general rule, since it has long been known to bind both A- and B-class ephrin ligands but the reason for this exceptional behavior has not been worked out at molecular level. Recent structural and biochemical studies on EphA4 ligand-binding domain alone and in complex with its ligands have addressed this question. However, the published structures of EphA4/ephrin complexes differ considerably from each other and strikingly different explanations for the exceptional promiscuity of EphA4 were proposed. To address these contradictory findings, we have determined a crystal structure of the EphA4 ligand-binding domain at 2.3A resolution and show that the receptor has an unprecedented ability to exist in two very different, well-ordered conformations even in the unbound state. Our results suggest that the ligand promiscuity of the Ephs is directly correlated with the structural flexibility of the ligand-binding surface of the receptor.

Toward the semisynthesis of multidomain transmembrane receptors: modification of Eph tyrosine kinases.

Expressed protein ligation (EPL) is a protein engineering approach that allows the modification or assembly of a target protein from multiple recombinant and synthetic polypeptides. EPL has been previously used to modify intracellular proteins and small integral membrane proteins for structural and functional studies. Here we describe the semisynthetic site-specific modification of the complete, multidomain extracellular regions of both A and B classes of Eph receptor tyrosine kinases. We show that the ectodomains of these receptors can be ligated to different peptides under carefully established experimental conditions, while their biological activity is retained. This work extends the boundaries of the EPL technique for semisynthesis of multidomain, extracellular, disulfide-bonded, and glycosylated proteins and highlights its potential application for reconstituting entire single-pass transmembrane proteins.