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).