Identification Of Env-specific Monoclonal Antibodies From Chinese HIV-1 Infected Person's Memory B Cell

In vitro human B cell activation and single cell RT-PCR techniques developed in recent years greatly improved the efficiency of screening broadly neutralizing antibodies.With this technology,Laura M.Walker isolated two potent neutralizing antibodies PG9 and PG16 from a subtype C HIV-1 infected individual in 2009.Xueling Wu isolated one neutralizing antibody which can neutralize more than 90%strains in 2010.However,it's reported that HIV-1 has become more resistant to antibody neutralization during the circulation.Virus variants isolated from individuals between 2003 and 2006 were significantly more resistant to neutralization by VRC01 and PG16 than viruses from individuals between 1985 and 1989.Despite this increased neutralization resistance,all recently transmitted viruses were sensitive to one other broadly neutralizing antibodies.These studies suggest that it is possible to prevent HIV-1 infection given more and more potent broadly neutralizing antibodies were found and used with combination.Except the neutralizing antibodies in HIV-1 infected individual,some antibodies have weak or no neutralizing activities,they can also kill HIV-1 infected cells by antibody-dependent cell-mediated cytotoxicity(ADCC).The results of recent Thailand RV144 vaccine trial showed that the vaccine was protective to participants and ADCC may play an important role.Other study on long-term nonprogressors also found the ADCC activities correlated with HIV suppression.HIV-1 epidemic strains in China include three major genetic groups:subtype B',CRF01_AE and subtype C/CRF07_BC/CRF08_BC/B'C.Many viruses are resistant to one or more bnmAb,including those known to have high potency against diverse viruses from outside China.Therefore,it is necessary to screening protective antibodies with neutralizing or ADCC activity from Chinese HIV-1 infected individuls.In this study,we adapted a technology for isolating antigen specific monoclonal antibody from human memory B cells through in vitro B cell activation coupled with RT-PCT and expression cloning.In brief,human B cells were purified by negative selection from PBMCs of HIV-1 infected individuals and memory B cells were further enriched using anti-CD27 microbeads.Two hundred memory B cells per well were cultured in 96-well round-bottom plates with feeder cells in medium containing EBV and CpG.Env-specific antibodies in supernatants were screened by ELISA after 1?2 weeks'culture.Cells from positive wells of Env-specific antibody were harvested and total RNA was isolated.Human VH and V? or V? genes were amplified by RT-PCR and cloned into IgG1 and ? or ? expressing vectors.Functional VH and V? or V? were identified by cotransfecting 293T cells with individual heavy chain and light chain clones followed by analysis of culture supernatants by ELISA for Env-specific antibodies.Finally,corresponding mAb was produced by transient transfection of 293T cells with the identified VH and V?/? pair and purified by protein A affinity chromatography.Purified monoclonal antibodies were evaluated by HIV-1 specific ADCC and neutralizing activity assay.With these methods,eight monoclonal Env-specific antibodies were isolated from three Chinese HIV-1 infected individuals.Four of them showed neutralizing activity and three showed strong ADCC activity against HIV-1.Further studies on their application in therapeutic or prophylactic vaccines against HIV-1 should be evaluated.