Cross-Neutralization Activity of Single-Chain Variable Fragment (scFv) Derived from Anti-V3 Monoclonal Antibodies Mediated by Post-Attachment Binding

Abstract

SUMMARY:The V3 loop in the envelope (Env) of HIV-1 is one of the major targets of neutralizing antibodies. However, this antigen is hidden inside the Env trimer in most isolates and is fully exposed only during CD4-gp120 interaction. Thus, primary HIV-1 isolates are relatively resistant to anti-V3 antibodies because IgG is too large to access the V3 loop. To overcome this obstacle, we constructed singlechain variable fragments (scFvs) from anti-V3 monoclonal antibodies 0.5g, 5G2, and 16G6. Enhanced neutralization by 0.5g and 5G2 scFvs was observed in strains resistant to their IgG counterparts. Neutralization coverage by 0.5g scFv reached up to 90z of the tested viruses (tier 2 and 3 classes). The temperature-regulated neutralization assay revealed that extensive cross-neutralization of 0.5g scFv can be explained by post-attachment neutralization. Neutralization assay involving viruses carrying an intersubunit disulfide bond (SOS virus) showed that the neutralization-susceptible timeframe after attachment was 60 to 120 min. These results indicate that the scFvs efficiently access the V3 loop and subsequently neutralize HIV-1, even after virus attachment to the target cells. Based on its broad and potent neutralizing activity, further development of anti-V3 scFv for therapeutic and preventive strategies is warranted.

Unique binding modes for the broad neutralizing activity of single-chain variable fragments (scFv) targeting CD4-induced epitopes

“…These PCR products were combined by overlapping PCR using scFv167BL-F and scFvCH-R for 916B2, scFvKDIV-F and scFvCH-R for 4E9C, and scFvKDTT-F and scFvCH-R for 25C4b. The PCR fragments were inserted into a pETHiH vector, a modified pETCF [ 33 ] with a Stu I site, a His-tag and an HA-tag at the 3′ region of the Eco RI site, after digestion with Nde I and Stu I using GeneArt Seamless Cloning and Assembly kit (Invitrogen).…”

“…Anti-CD4i scFv proteins were produced in Escherichia coli Rosetta 2 (DE3) (Merck, Darmstadt, Germany) transformed by a scFv expression plasmid, as described previously [ 33 ]. Briefly, the transformed cells were cultured in Luria–Bertani (LB) broth containing 100 µg/ml carbenicillin, and production of scFv was induced with 2 mM isopropyl-b- d (−)-thiogalactopyranoside (IPTG, Wako Pure Chemical Industries).…”

“…The post-attachment neutralization activities of anti-CD4i Abs were measured using a temperature-regulated neutralization (TRN) assay as described previously [ 15 , 33 ]. Briefly, 100 μl of TZM-bl cells were plated at 2 × 10 4 cells per well in 96-well cell culture plates and incubated at 37 °C overnight for attachment.…”

Synergistic inhibition of cell-to-cell HIV-1 infection by combinations of single chain variable fragments and fusion inhibitors

“…The importance of size reduction for access to CD4i epitopes was demonstrated by improved neutralization using single chain variable fragments [scFvs] [~40 Å] constructed from their parent IgGs. The scFvs not only neutralized primary viruses resistant to the parental IgGs [20], but also showed broader inhibitory activity than the corresponding IgGs [21,22]. Post-binding neutralization played a crucial role in the improved inhibitory activity of the scFvs.…”

Exploration of broadly neutralizing antibody fragments produced in bacteria for the control of HIV

“…22,23 These fragments lack the constant region (Fc) of full-length Abs, but still bind to antigens through their complementarity determining regions (CDRs). ScFv constructs are able to mediate neutralization functions against viruses like HIV 24,25 and are a potential tool in the control of the pandemic.…”