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P1.035 Expression, Purification and Characterization of Anti-V3 Human scFvs Against HIV-1 Clade C
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P1.035 Expression, Purification and Characterization of Anti-V3 Human scFvs Against HIV-1 Clade C
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P1.035 Expression, Purification and Characterization of Anti-V3 Human scFvs Against HIV-1 Clade C
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Journal Article
P1.035 Expression, Purification and Characterization of Anti-V3 Human scFvs Against HIV-1 Clade C
2013
Overview
Background Production of human monoclonal antibodies that shows broadly neutralising activity is needed for the prevention of HIV-1. Here we devised a novel approach and produced 11 different human scFvs against the V3 region of HIV-1 envelope. Method: The peripheral blood mononuclear cells (PBMCs) were isolated from an HIV-1 clade C infected drug naïve Indian patient whose plasma exhibited neutralising antibodies against a panel of viruses. PBMCs were EBV transformed in 96 well plate and then expanded into 24 well stage followed by 6 well and finally to flask stage. The transformed cells (wells) were screened against HIV-1 consensus C V3 peptide at each stage and only the positive cells (wells) were carried onto the next stage. Total RNA from these enriched positive antibody secreting cells at the flask stage was isolated and cDNA was synthesised. Results: By EBV transformation and preselection of V3 specific clones we successfully constructed a small phage library. One round of bio panning was done against HIV-1 consensus C and B V3 peptides. Randomly, 40 clones were selected and checked for their binding. Out of 40 clones, 13 showed positivity in phage ELISA. DNA fingerprinting analysis using BstN1 followed by sequencing showed that 11 out of 13 clones were distinct. Expression of positive clones was tested by SDS-PAGE and Western blot. All the 11 anti -V3 scFvs showed cross-reactivity against both the V3 peptides and did not show any reactivity against other unrelated peptides. The scFvs showed varying degrees of neutralisation against tier 1 and tier 2 viruses and cross neutralising activity against clade A, B and C viruses. Conclusion This is the first study to generate anti-V3 scFvs against HIV-1 Clade C. Further assessment of the neutralisation efficiency of these scFvs would reveal their potential for passive immunotherapy.
