Result
1. Neutralizing ability of 12-2A12 against RABV street viruses
Here, we tested the neutralization effects of 12-2A12 by the
pseduovirus-based neutralization assays. First, we tested the
neutralization effects of the monoclonal antibody to a total of 59
street strains (including CVS-N2C) isolated from different regions
(Supplementary Table1). According to the neutralization result, the
neutralizing efficacy of the antibody targeting 49 of the 59 strains was
lower than 1000 ng/mL. The median
IC50 value of the antibody against the tested street viruses was only
12.37 ng/ml, with an IC50 of 20.85 ng/mL against CVS-N2C (Fig. 1a).
According to the isolation region, the 59 street strains were further
divided into four regions: Asia, America, Africa and Europe
(Supplementary Table1). The median IC50 values of the antibody against
the street strains isolated from Asia, America, Africa and Europe were
calculated to be 23.94 ng/mL, 11.47 ng/mL, 7.15 ng/mL and 50.46 ng/mL,
respectively. Targeting the four geographic isolates, there was no
significant statistical difference in terms of the neutralizing activity
of the antibody (P>0.05) (Fig. 1b).
Thus, these data demonstrated that 12-2A12 exhibited a strong
neutralization potency and a wide breadth against multiple street
viruses of RABV.
2. Binding affinity and kinetics of 12-2A12 to RABV G
The affinity of 12-2A12 binding to RABV G was that evaluated by surface
plasmon resonance (SPR). As expected, the antibody readily interacted
with the viral G protein, showing typical slow-on/slow-off binding
kinetics. The affinity KD of 12-2A12 bound to RABV G was determined to
be 1.26 × 10-9 M, with
Ka=6.23×105/Ms and Kd=7.84×10-4/s
(Table 1 and Supplementary Figure1).
3. Crystal structure of 12-2A12 bound to RABV G
To illustrate the neutralization basis of the antibody, we solved the
complex structure of 12-2A12 Fab bound to RABV G (Supplementary Table
2). Overall, the antibody mainly bound to the PHD domain of RABV G. The
region in PHD containing residues Y173-G203 has been proposed to
interact with nAChR, which is an important cellular receptor for rabies
virus entry [20, 21]. Peptides derived from this
region (well-known as RVG-29 or RVG-9R) have been widely used in
brain-targeting drug delivery[20, 22, 23].The
epitope in RABV G recognized by the 12-2A12 antibody is partially
overlapped with the nAChR recognition region. The antibody therefore
would interfere with the nAChR/G interaction to block the viral receptor
binding (Fig. 2a). In addition, 12-2A12 might also neutralize the viral
infection by shutting off the transition of the glycoprotein from its
neutral-pH conformation to the acidic-pH conformation. When the 12-2A12
antibody was aligned to a previously reported G structure in the acidic
state (PDB: 6LGW) [22], a clear steric clash was
observed, indicating that the antibody would otherwise prevent the viral
glycoprotein from changing into the acidic conformation (Fig. 2b). While
RABV enters cells via the endocytic pathway, it was believed that the
conformational change of the glycoprotein induced by the endosomal-pH is
a prerequisite for the G-mediated fusion between the viral envelope and
the host cell membrane [24-26]. Thus,the antibody
would further inhibit rabies virus infection by inhibiting
membrane-fusion after receptor engagement (Fig. 2b).