Study On Recombinant Human Antibodies And Affinity Maturation

Rabies is a zoonosis caused by the rabies virus, which mortality rate of almost100%after onset. The prevention of level III of the rabies post-exposure mainly adopts rabies vaccination combined with rabies immune globulin, According to the World Health Organization (WHO) recommendation. Human rabies immune globulin (HRIG) and Equine rabies immune globulin (ERIG) are two types RIG currently used, both of which are defective. The former is expensive, limited availability and potential pathogenic threat, and the latter inhibits antibody response to certain vaccines and side effects. Anti-rabies virus monoclonal antibodies (mAbs) neutralizes efficiently with high safety, low-cost, mass production, so it may be able to replace RIG for rabies post-exposure prophylaxis.Affinity maturation in vitro was to simulate the process of antibody maturation in vivo, A variety of strategies was adopted for antibody gene mutation and the mutated antibody library was Construct. According to the Affinity screening, the high affinity anti-rabies antibody could be selected. During the Antibody affinity maturation in vitro, mutation strategy can be divided into random mutation, substitution, and site-directed mutagenesis. In this study, high-affinity human-derived anti-Rabies virus neutralizing antibodies are screened in the technology platform of phage displaying, respectively, through random mutagenesis and chain shuffling methods, specific experiments have the following two parts: 1. Study on Construction and screening of Human Error-prone scFv antibody library anti-rabies virusHuman mAb RV08, as an anti-rabies virus neutralizing antibody, was determined against the conformational antigenic site II. We further chose scFv phage display technology to optimize RV08with random mutagenesis. At first, The light chains and heavy chains genes of RV08were cloned into phagmid vector pHAL14, random mutations were inserted by performing6-8rounds of error-prone PCR, and a phage antibody library was constructed. The library were panned and selected by using purified rabies virus antigen of CTN strain with phage displaying, scfv antibodies specific against the rabies virus glycoprotein were obtained by ELISA and IFA. Sequence analysis of all selected scfv clones with Vbase database revealed the presence of90unique clones.2. Expression and identification of IgG antibodiesOf these scFv antibodies, the light chains and heavy chains of90human scfv antibodies genes were cloned into IgG expression vector (the VHpress and VKepresss cassettes) and expressed in293T cells. Finally, five full human IgG antibodies were chosen to test by SDS-PAGE、ELISA and IFA. The affinity of the5IgG were up to10-9M through the identification of non-competitive ELISA. Especially, The affinity of HL2, HL37and HL46were1.56x10-9M,1.88×10-9M,1.30x10-9M, higher than the The affinity of RV08. Mutations were inserted into the CDRs of the light chains and the heavy chains except the CDR3of heavy chains. So the meaningful mutations in the CDR3of the heavy chains are not easy to generate.In summary, we generated90human scFv antibodies against rabies virus, using phage antibody library we have constructed. Further more,5of them were converted to IgG antibodies, which were highly neutralizing and affinity against antigenic site II of rabies virus glycoprotein. Study on human recombinant antibody has major implications for replacing RIG for rabies post-exposure prophylaxis.