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

Abstract

BackgroundThe CD4-induced (CD4i) epitopes in gp120 includes the co-receptor binding site, which are formed and exposed after interaction with CD4. Monoclonal antibodies (mAbs) to the CD4i epitopes exhibit limited neutralizing activity because of restricted access to their epitopes. However, small fragment counterparts such as single-chain variable fragments (scFvs) have been reported to neutralize a broad range of viruses compared with the full-size IgG molecule. To identify the CD4i epitope site responsible for this broad neutralization we constructed three scFvs of anti-CD4i mAbs from a human immunodeficiency virus type 1 (HIV-1)-infected elite controller, and investigated the neutralization coverage and precise binding site in the CD4i epitopes.ResultsWe constructed scFvs from the anti-CD4i mAbs, 916B2, 4E9C, and 25C4b and tested their neutralization activity against a panel of 66 viruses of multi-subtype. Coverage of neutralization by the scFvs against this panel of pseudoviruses was 89% (59/66) for 4E9C, 95% (63/66) for 25C4b and 100% (66/66) for 916B2. Analysis using a series of envelope glycoprotein mutants revealed that individual anti-CD4i mAbs showed various dependencies on the hairpin 1 (H1) and V3 base. The binding profiles of 25C4b were similar to those of 17b, and 25C4b bound the region spanning multiple domains of H1 and hairpin 2 (H2) of the bridging sheet and V3 base. For 4E9C, the V3-base dependent binding was apparent from no binding to mutants containing the ΔV3 truncation. In contrast, binding of 916B2 was dependent on the H1 region, which is composed of β2 and β3 strands, because mutants containing the H1 truncation did not show any reactivity to 916B2. Although the H1 region structure is affected by CD4 engagement, the results indicate the unique nature of the 916B2 epitope, which may be structurally conserved before and after conformational changes of gp120.ConclusionsIdentification of a unique structure of the H1 region that can be targeted by 916B2 may have an important implication in the development of small molecules to inhibit infection by a broad range of HIV-1 for the purpose of HIV treatment and prevention.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-017-0369-y) contains supplementary material, which is available to authorized users.

Neutralization Breadth and Potency of Single-Chain Variable Fragments Derived from Broadly Neutralizing Antibodies Targeting Multiple Epitopes on the HIV-1 Envelope

“…scFv may have better access to their epitopes than IgG due to their smaller size, as has been reported using other antibody constructs. For example, antigen-binding fragments (Fab) of antibodies targeting the CD4-induced (CD4i) epitope showed significant improvement in potency over the IgG versions (48)(49)(50). Moreover, scFv isolated from phage display libraries demonstrated good breadth and potency (25,51).…”

CD4-Binding Site Directed Cross-Neutralizing scFv Monoclonals from HIV-1 Subtype C Infected Indian Children

“…One of the targets of bnAbs, the CD4 induced (CD4i) site, is only transiently exposed on conformational changes following binding of the viral envelope gp120 to the CD4 receptor on immune cells. Recently and in the previous studies, the scFvs, targeting the CD4i site, exhibited broad neutralization potential with 80–100 percent breadth against the HIV-1 viruses tested from different subtypes globally ( 62 – 64 ).…”

Functional analysis of a monoclonal antibody reactive against anti-cluster A epitope obtained from a patient infected with HIV-1 CRF02_AG

“…The 293T, 293A and TZM-bl cells were maintained in Dulbecco's modi ed Eagle's medium (DMEM; Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Mediatech Inc., Corning, Manassas, VA, USA). Mab 4E9C (45,46) and CD4mc YIR-821 (42) have previously been reported. Soluble CD4 was purchased commercially (sCD4, R&D systems, Inc., Minneapolis, MN, USA).…”

“…4). The addition of CD4mc (YIR-821) and an anti-CoRBS antibody (17b or 4E9C) (45,46) markedly enhanced the binding activity of 1E5. This enhancement required both CD4mc and anti-CoRBS antibody, and the addition of either CD4mc or anti-CoRBS antibody alone did not affect 1E5 binding signi cantly.…”