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.