Research On Antiviral Activity And Mechanism Of HIV-1 Monoclonal Neutralizing Antibody Y498

In order to achieve effective antibody-based vaccine and passive immunotherapy, research on HIV-1 neutralizing antibody identified from peripheral blood mononuclear lymphocyte (PBMC) of donors infected with HIV-1 appeared unprecedented prosperity in recent years. Characterization and isolation of human monoclonal antibodies were providing important insights into the specificities that underlie broad neutralization of HIV-1.So far,7 clades including A, B’,B, C, CRF07_BC/CRF08_BC, CRF01_AE, CRF02_AG of HIV-1 have found in China. We identified monoclonal antibody Fab Y498 isolated from phage antibody library constructed from a Chinese popular subtype B/C patient, firstly we expressed and purified antibody Fab and IgG of Y498, then confirmed that Y498 had neutralizing activity, found that Y498 had cross reactivity to various subtypes of HIV-1, SPR experiments confirmed that antibody Y498IgG (Y498) had a very high affinity to JRFL-gp120, KD of Y498 (0.86 nM). The affinity of Y498 were higher than that of b12 (1.11 nM) and VRC01 (5.35 nM), and have a higher affinity to CN54-gp120, KD of Y498 (0.46 nM) higher than b12 (13.28 nM) and VRC01 (25.29 nM). And 81 strains of virus detected were effectively neutralized up to 26% among which capacity of neutralization towards the subtype B/C reached 32%, and neutralization capacity of Y498 was better than that of b12 and VRC01 for some pseudoviruses. We concluded that Y498 antibodies had a strong cross reactivity and a somewhat broad spectrum of neutralizing ability to different pseudovirus.In addition, we proved Y498 antibody recognized conformational epitope of gp120 which mimicked that of b12 and VRC01 antibody. Then we predicted a total of 15 amino acids both overlapping bioinformatic and 12 peptide phage display. Using pepitope service software we forecasted 24 amino acid residues related to recognization epitope, and then found that there were four important amino acid sites affecting activity of the antibody. These 4 amino acids (F277, D279, D368 and E370) were located at CD4 binding zone of the gp120. Once mutated D368 and E370, neutralizing ability of Y498 completely lost. PyMOL software found that the four amino acids, mainly in loop D (F277, D279), CD4 binding loop (D368, E370).As a somewhat broad-spectrum antibody, Y498 recognizes CD4 binding site, by blocking action of CD4 receptor to prevent HIV-1. The isolation and identification of the antibody tends to understand the molecular mechanism of neutralizing antibody immune response among donors infected with HIV-1. Therefore may thus be a complement to current monoclonal antibody-based approaches to immunotherapies, prophylaxis and vaccine design.