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

ABSTRACT: More than 50% of HIV-1 infection globally is caused by subtype_C viruses. Majority of the broadly neutralizing antibodies (bnAbs) targeting HIV-1 have been isolated from non-subtype_C infected donors. Mapping the epitope specificities of bnAbs provides useful information for vaccine design. Recombinant antibody technology enables generation of a large repertoire of monoclonals with diverse specificities. We constructed a phage recombinant single chain variable fragment (scFv) library with a diversity of 7.8 × 10⁸ clones, using a novel strategy of pooling peripheral blood mononuclear cells (PBMCs) of six select HIV-1 chronically infected Indian donors whose plasma antibodies exhibited potent cross neutralization efficiency. The library was panned and screened by phage ELISA using trimeric recombinant proteins to identify viral envelope specific clones. Three scFv monoclonals D11, C11 and 1F6 selected from the library cross neutralized subtypes A, B and C viruses at concentrations ranging from 0.09 μg/mL to 100 μg/mL. The D11 and 1F6 scFvs competed with mAbs b12 and VRC01 demonstrating CD4bs specificity, while C11 demonstrated N332 specificity. This is the first study to identify cross neutralizing scFv monoclonals with CD4bs and N332 glycan specificities from India. Cross neutralizing anti-HIV-1 human scFv monoclonals can be potential candidates for passive immunotherapy and for guiding immunogen design.

SUBMITTER: Khan L

PROVIDER: S-EPMC5362912 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The 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.We 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 ?V3 truncation. In contrast, binding of 916B2 was dependent on the H1 region, which is composed of ?2 and ?3 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.Identification 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.

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

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