Construction Of HIV-4 CRF07＿BC Subtype Gp140 Probe And Identification Of The Isolated Monoclonal Neutralizing Antibody

Background AIDS is an infectious disease that seriously threatens human health.Currently there are no effective therapies and reliable vaccines to cure AIDS.Highly active anti-retroviral therapy has played an important role in controlling HIV-1 infection,but failed to achieve the goal of ending AIDS/HIV.Vaccines that can induce broadly neutralizing antibodies are the most effective tools to prevent AIDS/HIV transmission.In recent years,studies have found that HIV-1 broadly neutralizing antibodies can prevent infection,reduce the plasma viral load of infected people and slow the disease progress.Isolation and identification of HIV-1 broadly neutralizing antibodies has become a research hotspot in HIV immunology.The isolated HIV-1 broadly neutralizing antibodies can not only be directly used in the prevention and treatment of AIDS,but also help us solve key scientific issues such as the mechanism and evolutionary path of these antibodies,and provide scientific basis for the design of immunogens in vaccine research.Objective To isolate envelope-specific memory B cells from PBMCs of HIV-1 infected individuals in China,obtain monoclonal antibodies and identify their biological characteristics by constructing the HIV-1 CRF07＿BC subtype gp140 probe（CN54 gp140-Avi）.Methods Using the Pfuse-CN54 gp140 plasmid stored in the laboratory,a biotinylated specific molecular probe（CN54 gp140-Avi）was constructed by PCR.Envelope-specific memory B cells were sorted from PBMCs of the HIV-1 clade BC infected individual by the biotinylated molecular probe.The variable region genes of heavy and light chain of Ig G were amplified by reverse transcription polymerase chain reaction（RT-PCR）separately.The pairable light and heavy chain variable region genes of the antibodies were found through the IMGT database and then were cloned into expression vectors.The pairable light and heavy chain plasmids were co-transfected into 293F cells to generate monoclonal antibodies.The binding activity of the produced antibodies was measured by enzyme-linked immunosorbent assay（ELISA）.The neutralizing activity of monoclonal antibodies was tested using the TZM-bl cell-pseudovirus system.The antibody-mediated cellular cytotoxic effects were performed by flow cytometry.Results The biotinylated molecular probe（CN54 gp140-Avi）was successfully constructed.The probe was used to sort single B cells from PBMCs of HIV-1 clade BC infected individual and a total of 29 B cells were obtained.Two B cells（B3,B4）that the Ig G light and heavy chain gene sequences can be paired were collected in a 96-well plate.We amplified the heavy chain and light chain variable region genes of Ig G by RT-PCR separately and analyzed their gene family by the IMGT database.Finally we isolated a monoclonal antibody CBJB363-B4.The heavy chain of CBJB363-B4 originates from IGHV1-3*01F family,and its light chain is Lambda chain which belongs to IGLV1-40*01F family;both the heavy and light chain have lower somatic mutation rates.The antibody of CBJB363-B4 could bind CN54 gp140 and RSC3protein well.It could neutralize MW965,X2278 and 398-F1 strain with 50%inhibitory concentrations(IC₅₀)of 7.2μg/m L,3.83μg/m L and 17.08μg/m L separately.B4 antibody could mediate antibody-dependent cellular cytotoxic effects efficiently and the inhibition rate can reach 65.1%when the concentration of antibody reaches 50μg/m L.Conclusion In this study,CN54 gp140-Avi probe was successfully generated and a monoclonal antibody with neutralizing activity was obtained.Using the probe,we expect to obtain more neutralizing antibodies in the future,and provide new tools and ideas for the development of antibody-based drugs and basic vaccine research against AIDS/HIV.