Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors

Abstract

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 = 33 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 mAb 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 the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL), suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition.

Conclusions: Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope specificities of these mAbs and further experimental manipulations will be helpful in identification of epitopes, unique to clade C or shared with non-clade C viruses, in context of V3 region.

Figure 1

Relative binding affinity of anti-V3 antibodies to HIV-1 derived peptides and proteins. The binding pattern of anti-V3 mAbs derived from Indian donors (red) and 447-52D (an anti-V3 Ab isolated from HIV-1 subtype-B infected American individual) (green) to consensus-C and B V3 peptides (1A), and subtype-C (Du156.12) and subtype-B (JRFL) derived envelope gp120 proteins (1B). The binding of anti-V3 mAbs was tested by ELISA using mAbs at a concentration ranging from 10 to 0.00003 μg/ml (12 dilutions). Human anti-parvovirus B19 mAb 1418 was used as negative control. Relative reactivity of anti-V3 plasma antibodies to consensus-B and C V3 peptides is shown in terms of 50% ELISA binding titers (Max50) for three patients from whom antibodies were generated (1C). Two plasma samples 277 and 904 showed cross clade reactive binding while 903 displayed subtype-C specific binding.

Figure 2

Binding of anti-V3 mAbs to native viruses. Binding of anti-V3 MAbs to native, intact HIV-1 virions, Du156.12 (subtype-C) and JRFL (subtype-B). The supernatants of the two viruses were used at a final p24 concentration of 25 ng/ml. Antibody 904 displayed binding to both viruses (Du156.12 and JRFL), while antibodies 277 and 903 showed subtype-C (Du156.12) specific binding (A). The unrelated mAb 1418 against parvovirus B19 served as a negative control.The anti-V3 mAb 447-52D (known to bind SF162, a clade-B virus) and SF162 were used to validate the experiment. The binding of 447-52D to intact virus was performed by using a fixed concentration of antibody (10 μg/ml) with two-fold dilution of SF162 virus starting with 50 ng/ml of virus (B). The virus capture is determined by measuring the level of p24 (picograms per milliliter) released when bound virus is lysed with detergent. The viruses (Du156.12 and JRFL) were chosen for intact virion binding assay on the basis of their resemblance to the consensus-B and C V3 loop sequences (C). The V3 sequence of these viruses is aligned with their corresponding consensus-C and B V3 loop sequence using Seqpublish program (http://hiv.lanl.gov).