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.

16‐17 July 2010, International AIDS Society’s Workshop “Towards a Cure”: HIV Reservoirs and Strategies to Control Them, Vienna, Austria

The enumeration of antigen-specific T cell responses has been greatly facilitated in recent years by the development of methods based on the detection of cytokines. In particular, the enzyme-linked immunospot (ELISPOT) and cytokine flow cytometry (CFC) assays have become popular. Since both assays are likely to continue to be in widespread use, it is important to evaluate whether their results are comparable. In the current study, we compared the results obtained in the ELISPOT and CFC assays using peptide pools corresponding to CMV and HIV-1 proteins in chronically HIV-1-infected individuals. Analysis of T cell responses to peptide pools indicated that the CMV pp65 and HIV-1 Gag CFC and ELISPOT-derived results were statistically correlated. However, the results obtained with each assay differed in important ways: the magnitude of the response was consistently higher in the CFC assay while the CFC assay was less likely than the ELISPOT assay to detect low-level responses. Furthermore, there was a lack of numeric agreement between ELISPOT and CFC results. For studies that require the detection of low-level responses, or definition of responses as positive or negative, the ELISPOT assay may be preferable. In contrast, the CFC has a greater dynamic range and allows for phenotypic discrimination of responding cells, making it the assay of choice for most other applications.