A novel strategy for efficient production of anti-V3 human scFvs against HIV-1 clade C

Abstract

BackgroundProduction of human monoclonal antibodies that exhibit broadly neutralizing activity is needed for preventing HIV-1 infection, however only a few such antibodies have been generated till date. Isolation of antibodies by the hybridoma technology is a cumbersome process with fewer yields. Further, the loss of unstable or slowly growing clones which may have unique binding specificities often occurs during cloning and propagation and the strongly positive clones are often lost. This has been avoided by the process described in this paper, wherein, by combining the strategy of EBV transformation and recombinant DNA technology, we constructed human single chain variable fragments (scFvs) against the third variable region (V3) of the clade C HIV-1 envelope.ResultsAn antigen specific phage library of 7000 clones was constructed from the enriched V3- positive antibody secreting EBV transformed cells. By ligation of the digested scFv DNA into phagemid vector and bio panning against the HIV-1 consensus C and B V3 peptides followed by random selection of 40 clones, we identified 15 clones that showed V3 reactivity in phage ELISA. DNA fingerprinting analysis and sequencing showed that 13 out of the 15 clones were distinct. Expression of the positive clones was tested by SDS-PAGE and Western blot. All the 13 anti-V3 scFvs showed cross-reactivity against both the clade C and B V3 peptides and did not show any reactivity against other unrelated peptides in ELISA. Preliminary neutralization assays indicated varying degrees of neutralization of clade C and B viruses. EBV transformation, followed by antigen selection of lines to identify specific binders, enabled the selection of phage from un-cloned lines for scFv generation, thus avoiding the problems of hybridoma technology. Moreover, as the clones were pretested for antigen binding, a comparatively small library sufficed for the selection of a considerable number of unique antigen binding phage. After selection, the phage clones were propagated in a clonal manner.ConclusionsThis strategy can be efficiently used and is cost effective for the generation of diverse recombinant antibodies. This is the first study to generate anti-V3 scFvs against HIV-1 Clade C.

Cross-neutralizing anti-HIV-1 human single chain variable fragments(scFvs) against CD4 binding site and N332 glycan identified from a recombinant phage library

“…In order to determine the diversity of the library we randomly picked 57 clones from the unselected library. Amplification of these scFvs was done by PCR, followed by digestion with BstN142. DNA fingerprinting revealed that 45/57 (79%) clones were unique (Figure S3).…”

“…Earlier, we used a combined approach of EBV immortalization of V3 specific B cells selected from a single HIV-1 infected donor, followed by construction of antigen preselected human anti-V3 recombinant scFv library of limited diversity; the advantage of such antigen (V3) preselected library being the ease of isolating V3 directed scFv monoclonals42.…”

“…The procedure adopted for recombinant phage library construction was similar to that reported by us in our previous study42; with few modifications. Here, we did not EBV transform the cells or select based on reactivity to any HIV-1 antigen, but instead used the donor PBMCs directly for construction of a phage library.…”

“…Thirty combinations (6 forward primers and 5 reverse primers) were used to amplify kappa (κ) light chain, and 21 combinations (7 forward primers and 3 reverse primers) were used for lambda (λ) light chain amplification. Full scFvs were amplified through a pull through PCR reaction using Taq DNA polymerase and primers PTfw 5′ CCT TTC TAT GCG GCC CAG CCG GCC ATG GCC 3′ as forward primer and PAK Sfi 1 5′ TCA GCA T GG CCC CCG AGG CC G CAC GTT TRA T 3′ as reverse primer, using conditions described earlier42.…”

“…In order to determine the diversity of the library we randomly picked 57 clones from the unselected library. Amplification of these scFvs was done by pull through PCR (PTfw 5′ CCT TTC TAT GCG GCC CAG CCG GCC ATG GCC 3′ as forward primer and PAK Sfi 1 5′ TCA GCA T GG CCC CCG AGG CC G CAC GTT TRA T 3′ as reverse primer) as described above, followed by digestion with BstN14266. A phage library of 7.8 × 10 8 clones was successfully constructed.…”