Discussion
Although RABV infection has a mortality rate of ~100%, it can be prevented by PEP. In recognition of the disadvantages for RIG derived from the plasma of vaccinated people or horses, monoclonal antibody is widely accepted as the most promising substitute. While the G protein of RABV is the most important antigen that can stimulate the body to produce antibodies, it is therefore a feasible way to use anti-G monoclonal antibody (MAb) cocktails to replace RIG. The WHO Collaborating Centre for Rabies (WHO RCC) has identified five mouse Mabs (E559, M727-5-1, M777-16-3 and 1112-1 can recognize antigenic site II of G protein, 62-71-3 can recognize antigenic site I) and combined them into three MAb cocktails (62-71-3/E559, 62-71-3/M727-5-1 and 62-71-3/M777-16-3), which exhibit comparable protective effect as HRIG in animals[27-30]. The study reminds us that monoclonal neutralizing antibodies targeting RABV G could represent a potential better biological alternative for RIG. Therefore, in this study, we used the mouse hybridoma technology to screen for monoclonal neutralizing antibodies and identified MAb m12-2A12. In order to reduce the immunogenicity of m12-2A12, we humanized m12-2A12 by replacing its constant region with the human IgG1 antibody sequences, leading to the human-mouse chimeric antibody 12-2A12, which was successfully expressed using the mammalian cell expression system.
Previous studies showed that the distribution of RABV isolates has regional characteristics[31-34]. It is therefore necessary to determine the neutralizing efficacy of 12-2A12 against street viruses isolated from different regions. According to the study of Cai et al.[35], a total of 59 street virus strains are classified into four geographic regions, including Asia, America, Africa and Europe. The neutralizing capacity of 12-2A12 against these street strains were measured by the pseudovirus neutralization system. The result showed that there was no statistical difference in the neutralizing ability against the viral strains in the four regions, demonstrating that 12-2A12 could effectively neutralize RABV street viruses isolated from different regions.
As an important receptor for RABV G, nAChR plays an important role in the viral infection. Via the high resolution complex structure, we find that the epitope recognized by 12-2A12 partially overlaps with the nAChR recognition region. This structural observation indicates that the antibody may interfere with the binding of G to nAChR, preventing viral particles from adhering to cells. In the next step, when RABV enters cells through endocytosis, the acidic pH of the endosome would induce the conformational change of the G trimer to trigger the fusion between the viral envelope and the cell membrane. Our structure also showed that 12-2A12 should be able to prevent the viral glycoprotein from changing into its acidic conformation. Such observation means that 12-2A12 could further prevent the membrane fusion mediated by G. In light of its dual-mechanism for neutralization, we believe 12-2A12 has the potential to be used in human rabies PEP.