Identification Of Neutralizing Monoclonal Antibodies Against HIV-1 And Study On Monovalent Antibodies

Broad-spectrum neutralizing antibodies are able to neutralize many different strains of the virus. They play an important role in the prevention and treatment of viral infections, becoming the first line of defense against HIV. Researchers will focus in the search for inducing broadly neutralizing antibodies to develop therapeutic vaccines. In recent years, with the breakthrough of B cell culture technology, the quantity of broad-spectrum neutralizing antibody has been greatly improved from HIV infected persons. For example, VRC01, PT and PGT series of antibodies can neutralize the most world’s epidemic strains, especially VRC01 can neutralize more than 90% of the world’s epidemic strains. We adapted a technique for isolating memory B cells from peripheral blood. B cells were cultured with irradiated 3T3-h CD40 L feeder cells in medium containing cytokines IL-2 and IL-21. The memory B cells expressing the Env-specific antibody were screened by micro-neutralizing assay. By RT-PCR, Human VH and Vκ or Vλ genes were cloned into eukaryotic expression vectors and transfected into 293 T cells. The binding activity of antibodies to Env were tested by ELISA. The neutralizing activity of antibodies to Env were tested by micro-neutralization assay. In addition, we tested the affinity between the antibody and gp120. In a result, three Env-specific antibodies(8E7,19GF3 and 55B3) were identified from HIV-1 infected individual. Two of them could bind with gp120, the other one binds gp120 and FLSC with similar binding activity. 8E7 which has large variation of gene has neutralizing activity against with three subtypes(A 、 B 、 C)of the virus. 55B3 only has neutralizing activity against with SF162. 19GF3 has no neutralizing activity. The affinity of these three monoclonal antibodies are very strong. 8E7, 19GF3, 55B3 to Gp120 protein affinity reached 6.45×10-10 mol/L、1.12×10-10 mol/L、2.76×10-10 mol/L.The distribution of spikes in the envelope of HIV on the surface of the very sparse, and the two antigen binding epitopes of neutralizing antibody were difficult to bind to two spikes on a virus particle. That is to say, only one of the two epitopes can play a role in neutralization. In order to better overcome the steric hindrance, all Fabs can play a role, our laboratory constructed IgG half molecule(IgG-HM) in the early study. Preliminary results showed that the neutralizing activity of IgG-HM was improved compared with the parental IgG antibody. In this study, we have made a more systematic examination of the neutralizing activity of IgG-HM, and detected the activity of ADCC and the half-life of the antibody in vivo.The results showed that the neutralizing activity of IgG-HM was significantly improved compared with the IgG, especially VRC01-HM was increased 3.74 times.In addition, to different pseudotyped virus in vitro neutralization test, pairwise combination of HM antibodies were stronger than IgG antibodies, mainly the IC80 of combinations of HM are generally lower than IgG. For the THRO.c/2626 virus, which is difficult to neutralize, the combination of NIH45-46-HM and PG9-HM, and the combination of NIH45-46-HM and VRC01-HM have obvious neutralizing activity, which corresponds to the combination of IgG were not detected to neutralizing activity. The IC80 of combination of three HM antibodies was significantly lower than the combination of two antibodies. Also, using 2G12 antibody as a model, we compared the cytotoxic activity of NK cells in antibodies mediated by IgG or HM that kill the CEM-NK ’-CCR5 cells loaded with Gp120 protein. Results show that IgG antibody has significant ADCC activity, but HM antibody has no ADCC activity. But comparing to half-life comparison in mice in vivo, there was no significant change between IgG and HM antibodies. Although the HM antibody has smaller molecules, but not soon it is cleared. The characteristic of small Fab and scFv in vivo which were cleared have great differences. It has more advantages in the practical the application.On this basis, we constructed HM antibodies of the Env HIV-1 antigen binding activity of monoclonal antibodies(8E7, 55B3 and 19GF3). In addition, we researched the binding activity and neutralizing activity of them. There was no significant difference in binding activity between the constructed monoclonal antibody and the parent monoclonal antibody in vitro. In the neutralization activity, we carried out neutralization test in vitro to test IC50. The results showed that 8E7 can neutralize the virus, HM(half molecule IgG, IgG-HM) can also be neutralized, which shows that HM antibody basically maintained the neutralizing activity of Ig G antibody.Briefly, we successfully stimulated B cell to secrete IgG efficiently in vitro in 96 well plates. With the HIV pseudovirion neutralization test and RT-PCR technology, we can screen broadly neutralizing antibodies. This method is not only applied to the screening of HIV neutralizing antibodies, but also has a good application prospect in the screening of monoclonal antibodies against other infectious diseases.Besides,Our results suggest that in the combination of two or more antibodies, IgG-HM was significantly improved compared with the IgG. This lays the foundation for the further studies on their application in therapeutic or prophylactic vaccines against HIV-1.