Study On Recombinant Human Antibody Against Rabies Virus

Rabies is a zoonosis caused by the rabies virus, which mortality rate of almost 100% after onset. Rabies virus is enveloped Rhabdoviridae Lyssavirus and has 5 types of structural proteins, including the glycoprotein(G), which closely related to virulence and pathogenicity of the virus, is a ligand binding virus and host cell, can induce the production of neutralizing antibodies. 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. In this study, human-derived anti-Rabies virus neutralizing antibodies are screened in the technology platform of phage displaying, specific experiments have the following three parts:1. Construction, panning and screening of human recombinant Fab antibody library agaist rabies virusesAt first, the lymphocytes from the blood of vaccinated donors, in convalescent phase were isolated. The total RNA was extracted from the human isolated lymphocytes and the cDNAs was synthesized with using primer oligo-dT. The genes of Fd fragment and light chain of antibodies were amplified by a set of human IgG Fab antibody primers. The gel purified light chains and heavy chains were cloned into phagmid vector pComb3, and a phage antibody library with size 4.5×10 was constructed. Insertion rate of light chain and heavy chain inserts were 100%, fully meet the needs of our library screen.The library were panned and selected by using purified rabies virus antigen of aG or CTN strain with phage displaying. Fab antibodies specific against the rabies virus glycoprotein were obtained by ELISA、IFA. Sequence analysis of all selected Fab clones with Vbase database revealed the presence of 11 unique clones.2. Expression and identification of IgG antibodies in recombinant baculovirus/insect systemOf these Fab antibodies, the light chain and heavy chain of five human Fab antibodies genes were cloned into IgG expression vector (pAc-L-Fc).and expressed in recombinant baculovirus/insect system. The five full human IgG antibodies were tested by SDS-PAGE、ELISA and IFA. The affinity of the 5 IgG were up to 10-9M through the identification of non-competitive ELISA. And the result showed that the 5 IgG s neutralizing activity well, identified by the rapid fluorescent focus inhibition test (RFFIT).3. Study on the critical neutralizing epitopes on rabies recognized by neutralizing recombinant human antibodiesThe results showed that the relationship between 5 antibodies and their epitopes was overlapping through the identification using competitive ELISA.The further study on the identification of RVIgG57, which against antigenic site I of rabies virus glycoprotein, and the result demonstrated that the epitopes of 5 antibodies were not overlapping with RVIgG57 s. Then we analyzed the key antigenic sites of rabies virus glycoprotein,3 of them were designed for mutation using bioinformatics combined with other reports, and the results showed that the 5 antibodies we got were against antigenic site Ⅱ of rabies virus glycoprotein, while RVIgG57 against antigenic site I of rabies virus glycoprotein, as the literature’s description.In summary, we generated 11 human Fab antibodies against rabies virus, using phage antibody library we have constructed. Further more,5 of 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.