アクティブボード・２０１５年　２月
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研究発表を行った学会；
・HIV Research for Prevention (HIV R4P) 2014 AIDS Vaccine, Microbicide and ARV-based Prevention Science
　２０１４年１０月２８日〜３１日（Cape Town, South Africa）
タイトル；Post-attachment Neutralization by Single-chain Variable Fragment (scFv) from an Anti-V3 Monoclonal Antibody with Cross-reactivity.
発表者；丸田　泰広　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）
Abstract；
Background:
The neutralization antibody response against HIV-1 is crucial for controlling HIV-1 infection. Such is the case of the epitope in the V3 loop that is exposed after binding of gp120 to CD4. However, it is difficult for anti-V3 IgGs to bind their epitope following CD4-gp120 interaction because IgG is too large to access the close physical proximity of gp120 and the cellular membrane.

Methods
We constructed scFv from 1C10, an anti-V3 monoclonal antibody with cross-reactivity. The 1C10 scFv was produced in E.coli, purified and refolded by "on column refolding" process. In addition to the usual single cycle neutralization assay, we employed temperature regulated neutralization assay (TRN assay) and neutralization assay using SOSJR-FL virus (SOS assay) to evaluate post-attachment neutralization using TZM-bl cells as a target. TRN assay and SOS assay allows determining whether scFv can access efficiently to the narrow space between HIV-1 and the V3 epitope of the envelope after attachment of the virus to CD4.

Results
Neutralization activity of 1C10 scFv was greater than that of IgG counterpart against JR-FL, YU2 and 89.6 strains of HIV-1 (IC50 values of scFv were about 5, 3 and 3 times better than values with IgG, respectively). In addition, 1C10 scFv neutralized viruses resistant for neutralization by 1C10 IgG, such as SVPB8 (IC50 of IgG; >50 μg/ml, scFv; 5.23 μg/ml). TRN assay results showed that neutralization capacity of IgG was considerably reduced after the virus bound to CD4. On the other hand, neutralization activity of scFv at TRN assay was equivalent to that at usual single cycle neutralization assay. In SOS assay, 1C10 scFv neutralized SOSJR-FL, but 1C10 IgG did not.

Conclusion
These results suggest that the anti-V3 scFv can neutralize HIV-1 even after attachment of the virus to the target cells. For this reason, the use of scFv may be one of the promising strategies to overcome neutralization resistance of HIV-1.