Low CCR5 expression protects HIV-specific CD4+ T cells of elite controllers from viral entry.

HIV elite controllers maintain a population of CD4 + T cells endowed with high avidity for Gag antigens and potent effector functions. How these HIV-specific cells avoid infection and depletion upon encounter with the virus remains incompletely understood. Ex vivo characterization of single Gag-specific CD4 + T cells reveals an advanced Th1 differentiation pattern in controllers, except for the CCR5 marker, which is downregulated compared to specific cells of treated patients. Accordingly, controller specific CD4 + T cells show decreased susceptibility to CCR5-dependent HIV entry. Two controllers carried biallelic mutations impairing CCR5 surface expression, indicating that in rare cases CCR5 downregulation can have a direct genetic cause. Increased expression of ß-chemokine ligands upon high-avidity antigen/TCR interactions contributes to autocrine CCR5 downregulation in controllers without CCR5 mutations. These findings suggest that genetic and functional regulation of the primary HIV coreceptor CCR5 play a key role in promoting natural HIV control.

Vivax infection alters peripheral B-cell profile and induces persistent serum IgM.

B cell-mediated humoral responses are essential for controlling malarial infection. Studies have addressed the effects of Plasmodium falciparum infection on peripheral B-cell subsets but not much is known for P. vivax infection. Furthermore, majority of the studies investigate changes during acute infection, but not after parasite clearance. In this prospective study, we analysed peripheral B-cell profiles and antibody responses during acute P. vivax infection and upon recovery (30 days post-treatment) in a low-transmission area in India. Dengue patients were included as febrile-condition controls. Both dengue and malaria patients showed a transient increase in atypical memory B cells during acute infection. However, transient B cell-activating factor (BAFF)-independent increase in the percentage of total and activated immature B cells was observed in malaria patients. Naïve B cells from malaria patients also showed increased TLR4 expression. Total IgM levels remained unchanged during acute infection but increased significantly at recovery. Serum antibody profiling showed a parasite-specific IgM response that persisted at recovery. A persistent IgM autoantibody response was also observed in malaria but not dengue patients. Our data suggest that in hypoendemic regions acute P. vivax infection skews peripheral B-cell subsets and results in a persistent parasite-specific and autoreactive IgM response.

DNA Vaccination by Electroporation Amplifies Broadly Cross-Restricted Public TCR Clonotypes Shared with HIV Controllers.

Rare patients who spontaneously control HIV replication provide a useful model to inform HIV vaccine development. HIV controllers develop particularly efficient antiviral CD4+ T cell responses mediated by shared high-affinity TCRs. To determine whether the candidate DNA vaccine ADVAX could induce similar responses, we analyzed Gag-specific primary CD4+ T cells from healthy volunteers who received ADVAX DNA by electroporation. Vaccinated volunteers had an immunodominant response to the Gag293 epitope with a functional avidity intermediate between that of controllers and treated patients. The TCR repertoire of Gag293-specific CD4+ T cells proved highly biased, with a predominant usage of the TCRß variable gene 2 (TRBV2) in vaccinees as well as controllers. TCRα variable gene (TRAV) gene usage was more diverse, with the dominance of TRAV29 over TRAV24 genes in vaccinees, whereas TRAV24 predominated in controllers. Sequence analysis revealed an unexpected degree of overlap between the specific repertoires of vaccinees and controllers, with the sharing of TRAV24 and TRBV2 public motifs (>30%) and of public clonotypes characteristic of high-affinity TCRs. MHC class II tetramer binding revealed a broad HLA-DR cross-restriction, explaining how Gag293-specific public clonotypes could be selected in individuals with diverse genetic backgrounds. TRAV29 clonotypes also proved cross-restricted, but conferred responses of lower functional avidity upon TCR transfer. In conclusion, DNA vaccination by electroporation primed for TCR clonotypes that were associated with HIV control, highlighting the potential of this vaccine delivery method. To our knowledge, this study provides the first proof-of-concept that clonotypic analysis may be used as a tool to monitor the quality of vaccine-induced responses and modulate these toward "controller-like" responses.

HbAHP-25, an In-Silico Designed Peptide, Inhibits HIV-1 Entry by Blocking gp120 Binding to CD4 Receptor.

Human Immunodeficiency Virus (HIV-1) poses a serious threat to the developing world and sexual transmission continues to be the major source of new infections. Therefore, the development of molecules, which prevent new HIV-1 infections, is highly warranted. In the present study, a panel of human hemoglobin (Hb)-α subunit derived peptides and their analogues, with an ability to bind gp120, were designed in-silico and their anti-HIV-1 activity was evaluated. Of these peptides, HbAHP-25, an analogue of Hb-α derived peptide, demonstrated significant anti-HIV-1 activity. HbAHP-25 was found to be active against CCR5-tropic HIV-1 strains (ADA5 and BaL) and CXCR4-tropic HIV-1 strains (IIIB and NL4-3). Surface plasmon resonance (SPR) and ELISA revealed direct interaction between HbAHP-25 and HIV-1 envelope protein, gp120. The peptide prevented binding of CD4 to gp120 and blocked subsequent steps leading to entry and/or fusion or both. Anti-HIV activity of HbAHP-25 appeared to be specific as it failed to inhibit the entry of HIV-1 pseudotyped virus (HIV-1 VSV). Further, HbAHP-25 was found to be non-cytotoxic to TZM-bl cells, VK2/E6E7 cells, CEM-GFP cells and PBMCs, even at higher concentrations. Moreover, HbAHP-25 retained its anti-HIV activity in presence of seminal plasma and vaginal fluid. In brief, the study identified HbAHP-25, a novel anti-HIV peptide, which directly interacts with gp120 and thus has a potential to inhibit early stages of HIV-1 infection.

MicroRNA let-7f: a novel regulator of innate immune response in human endocervical cells.

PROBLEM: Endocervical epithelial cells express pattern recognition receptors (PRRs) that aid in innate immune responses. Mechanisms regulating signaling of PRRs are poorly understood. METHODS OF STUDY: Endocervical cells (End1/E6E7) were treated with ligands of TLR9 and RIG-I once or after pre-stimulation with same ligand. Cytokine responses were determined by ELISA. Differential gene expression was analyzed by microarray. Differentially expressed genes were validated by qPCR /Western blot. Role of let-7f was studied by inhibition and over-expression studies using commercial inhibitors and let-7f encoding plasmids, respectively. RESULTS: Single stimulation of cells with TLR9 ligand, but not RIG-I ligand, induced tolerance to subsequent challenge to the same ligand. Stimulation with TLR9 decreased let-7f and increased its target Blimp-1. Conversely, RIG-I stimulation increased let-7f and decreased Blimp-1 expression. Inhibition and over-expression revealed let-7f is involved in induction of immune tolerance. CONCLUSION: We identify let-7f as a novel regulator of PRR signaling in endocervical cells.

A rabbit vaginal cell-derived antimicrobial peptide, RVFHbαP, blocks lipopolysaccharide-mediated inflammation in human vaginal cells in vitro.

Antimicrobial peptides (AMPs) constitute a phylogenetically ancient form of innate immunity that provides host defense at various mucosal surfaces, including the vagina. Recently, we have identified one such AMP, rabbit vaginal fluid hemoglobin alpha peptide (RVFHbαP), from the vaginal lavage of rabbits (Oryctolagus cuniculus). The recent demonstration of a protective role of this peptide in erythrocytes and vaginal cells led us to investigate (i) the lipopolysaccharide (LPS) interactive domain in RVFHbαP and (ii) whether RVFHbαP of rabbit origin modulates the cellular immune responses of another species (humans) in vitro. HeLa-S3, a human vaginal epithelial cell line (hVEC), was exposed to LPS alone (10 µg/ml for 6 h), or LPS-induced cells were treated with RVFHbαP (70.45 µM for 1 h) and cultured for 24 h, and the results obtained were compared with the medium control. We show here that RVFHbαP exerts an anti-inflammatory activity in hVECs, as suggested by the prevention of LPS-induced production of extracellular (supernatant) and intracellular (lysate) levels of cytokines (interleukin 6 [IL-6] and IL-1α) and chemokines (IL-8 and monocyte chemoattractant protein 1 [MCP-1]). The demonstration of Toll-like receptor 4 (TLR4) and NF-κB expression in hVECs and the observations of RVFHbαP suppression of human ß-defensin-1 (hBD1) mRNA expression further support the hypothesis of a genomic activity of RVFHbαP. Confocal microscopy and flow cytometry results demonstrate that RVFHbαP inhibits LPS-induced phagocytosis of Escherichia coli by macrophages. The chemotaxis studies performed using the Boyden chamber Transwell method showed the increased migration of U937 cells when supernatants of LPS-induced hVECs were used, and this effect was inhibited by RVFHbαP. In conclusion, our study proposes a novel explanation for the protective role of RVFHbαP in inflammation-associated infections, which not only may provide the new cellular targets for the screening of RVFHbαP ligands acting in the vaginal tissue but also has the potential to develop RVFHbαP as a therapeutic agent for reproductive tract infections.