アクティブボード・２０１７年９月
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研究発表を行った学会；
　　　9th IAS Conference on HIV Science (IAS 2017)　
　　　2017年 7月23日〜26日（パリ、フランス）
タイトル；Structures within the Coreceptor Binding Site Account for the Broad Neutralizing Activity of Single Chain Variable Fragments (scFv) Targeting CD4-induced Epitopes.
発表者；田中　和樹　氏
　　　（熊本大学　エイズ学研究センター　松下プロジェクト研究室）
要旨；
[Background]
A CD4-induced (CD4i) epitope, which overlaps with the coreceptor binding site, is highly conserved, and is a favorable target for neutralizing monoclonal antibodies (nMAbs). However, the CD4i epitope is hidden inside trimeric Env, before CD4 binds gp120. Moreover, the close physical proximity of gp120 and the cellular membrane blocks the access of anti-CD4i Abs to this CD4i epitope after the CD4-gp120 interaction. In order to overcome this problem, single-chain variable fragments (scFv) of anti-CD4i nMAbs were tested for neutralization to multi-clade HIV-1 strains.
[Methods]
Single-chain variable fragments (scFv) from three anti-CD4i nMAbs, 16B2, 4E9C and 25C4b, were produced in E. coli, and their neutralization activities were compared with their counterparts. The binding properties of anti-CD4i nMAbs were determined by ELISA using gp120BaL mutants.
[Results]
The full length IgGs corresponding to the scFvs in this study were unable to neutralize Clade B HIV-1. In contrast, the anti-CD4i scFvs neutralized the tier 2 and tier 3 viruses of clade B. Futhermore, the neutralizing activities of scFvs were significantly higher than those of the corresponding Fabs. Moreover, neutralization assays using 66 pseudoviruses belonging to 7 subtypes revealed broad neutralization coverage by scFvs, 16B2 (100 %), 4E9C (83 %), 25C4b (92 %). Post-attachment neutralization assay, which regulated a fusion process by temperature, showed that the scFvs neutralized viruses resistant to the corresponding IgGs after the virus-cell attachment. Binding profiles of 25C4b against the series of gp120 mutants were comparable to 17b which interacts with the hairpin 1 (H1), hairpin 2 (H1) and requires the V3-base. On the other hand, binding of 4E9C was found to be dependent of V3-base region. Finally, 16B2 was found to recognize a CD4-bound structure composed of H1 and H2.
[Conclusions]
Anti-CD4i scFvs can effectively neutralize multi-clade HIV-1 strains after the CD4-gp120 interaction. The gp120 structure induced by CD4-binding targeted by the neutralizing scFvs is associated with conserved regions including hairpins H1 and H2.