Background: Analysis of human monoclonal antibodies (mAbs) developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein.The third variable region (V3) is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5) binding and presence of epitopes recognized by broadly neutralizing antibodies.Methods: Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females) within the age range of 20-57 years (median 033 years) were recruited in this study for mAb production.The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB) fusion protein.We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays.Results: We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals.The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while 904 exhibited cross reactivity also with subtype-B V3.Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown.The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively.Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite exposure of the epitopes recognized by these antibodies on the native viruses, as determined by intact virion binding assay with two representative subtype-C and B viruses (Du156.12and JRFL).Conclusion: Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses.Defining the epitope specificities of these mAbs and further experimental manipulations will be helpful in identification of epitopes, unique to clade C or shared with nonclade C viruses, for immunogen design.

Results Mice: Co-immunization with chemokines induced significant enhancement of HIV-1 specific CD8+ T cell IFNgamma secretion in the periphery and TNF-alpha, IL-2 and IFN-gamma by gut lymphocytes. Co-immunization with mucosal chemokines augmented HIV-1-specific sIgA in sera/fecal samples. Similar immunogenicity data was observed to Influenza A/PR8/34 hemagglutanin plasmid including responses that neutralized virus and protected mice from morbidity/mortality associated with lethal mucosal challenge. Macaque: In the periphery, we observed significant IFN-gamma in all groups (~6,000 SFU each). However intracellular cytokine staining on mucosal lymphocytes showed a trend toward an increase in CD8+T cells secreting TNF and IL-2 in CCL27 coimmunized macaques, levels greater than those observed in infected animals. Enhanced antigen-specific IgA was also detected in sera of chemokine-vaccinated macaques. A mucosal challenge is scheduled to determine if the functionality and phenotype of vaccine-induced immunity, either at the mucosa or periphery, is a driving determinant of protection.

Methods Ad-specific T cell responses were characterized in five seropositive and seronegative subjects from the Merck phase I 016 trial, the immediate STEP trial predecessor. Subjects received 3 × 1011 vector particles Merck Ad5 gag/pol/nef at weeks 0, 4 and 30. PBMC samples were obtained at weeks 0, 4, 8,18, 26, 30, 42, 52 and 78 relative to vaccination. T-cell responses to Ad were measured by stimulating PBMCs overnight with whole Ad vector before measuring functionality (IFN-γ, TNF-α, IL-2) memory phenotype (CD45RO, CCR7) and mucosal homing markers (α4, β7, CCR10, αE) by multicolor flow cytometry.