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.
Highlights
The CD4-induced (CD4i) epitopes in gp120 includes the co-receptor binding site, which are formed and exposed after interaction with CD4
Immunoglobulin variable regions amplified from Epstein-Barr Virus-immortalized cell lines producing anti-CD4i Monoclonal antibodies (mAbs) were cloned into IgG expression vectors, and 916B2, 4E9C and 25C4b were used for the construction of Fab and scFv
The size of light chains was similar between all mAbs except 916B2, which had a smaller counterpart in addition to a light chain of normal size. 916B2, 4E9C and 25C4b scFvs had a molecular weight of approximately 30 kDa (Fig. 1b)
Summary
The CD4-induced (CD4i) epitopes in gp120 includes the co-receptor binding site, which are formed and exposed after interaction with CD4. Human immunodeficiency virus type 1 (HIV-1) entry into host cells is initiated by the interaction of CD4 of the host cell and gp120 of the viral envelope glycoprotein (Env) This interaction triggers conformational changes of gp120, exposing the co-receptor binding site on the surface of trimeric Env by the rearrangement of V1, V2 and V3 loops [1,2,3,4,5]. The bridging sheet consists of a four-stranded β-sheet structure composed of two double-strand β-sheet structures, Tanaka et al Retrovirology (2017) 14:44 hairpin 1 (H1 containing β2 and β3 in the stem of the V1/ V2 loops) and hairpin 2 (H2 containing β20 and β21 in the C4 region) [5, 10] These β-sheets are highly conserved among HIV-1 strains because the structure and amino acid sequences are critical for the interaction with the N-terminus of CCR5, suggesting it is the main component of the co-receptor binding site [11, 12]. The CD4-induced (CD4i) epitopes include the co-receptor binding site, which is formed and exposed after interacting with CD4
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