Novel human monoclonal antibody combination effectively neutralizing natural rabies virus variants and individual in vitro escape mutants

Abstract

The need to replace rabies immune globulin (RIG) as an essential component of rabies postexposure prophylaxis is widely acknowledged. We set out to discover a unique combination of human monoclonal antibodies (MAbs) able to replace RIG. Stringent criteria concerning neutralizing potency, affinity, breadth of neutralization, and coverage of natural rabies virus (RV) isolates and in vitro escape mutants were set for each individual antibody, and the complementarities of the two MAbs were defined at the onset. First, we identified and characterized one human MAb (CR57) with high in vitro and in vivo neutralizing potency and a broad neutralization spectrum. The linear antibody binding site was mapped on the RV glycoprotein as antigenic site I by characterizing CR57 escape mutants. Secondly, we selected using phage display a complementing antibody (CR4098) that recognized a distinct, nonoverlapping epitope (antigenic site III), showed similar neutralizing potency and breadth as CR57, and neutralized CR57 escape mutants. Reciprocally, CR57 neutralized RV variants escaping CR4098. Analysis of glycoprotein sequences of natural RV isolates revealed that the majority of strains contain both intact epitopes, and the few remaining strains contain at least one of the two. In vitro exposure of RV to the combination of CR57 and CR4098 yielded no escape mutants. In conclusion, a novel combination of human MAbs was discovered suitable to replace RIG.

FIG. 1.

Lack of correlation between affinity and neutralizing potency of anti-RV antibodies. SPR binding analysis of anti-RV antibodies on glycoprotein from the Evelyn-Rokitnicky-Abelseth strain. Neutralizing potency as measured in a standard RFFIT is shown on the x axis in IU/mg. The reduced response after 12.5 min of subsequent dissociation is given on the y axis, calculated as a percentage of the association response. Antibodies with a relative high affinity are located in the upper part of the graph. Data points are labeled with the last three digits of the novel MAbs for reasons of clarity. The inset table shows the actual affinity K_D values for a subset of antibodies.

FIG. 2.

Neutralizing epitopes on RV glycoprotein. A schematic drawing of the RV glycoprotein is shown depicting the antigenic sites including the recently described CR57 epitope (19). The arrow indicates the location of the CR4098 epitope (antigenic site III). The signal peptide (19 aa) and transmembrane domain are indicated by black boxes. Disulfide bridges are indicated. Amino acid numbering is from the mature protein minus the signal peptide.

FIG. 3.

CR57 and CR4098 bind to different epitopes on RV glycoprotein. (A) SPR experiments were performed by injecting CR57 and CR4098 (1 μM) without regeneration between these injections. Time in seconds (x axis) is plotted against RU (y axis). RU levels are indicated for each injected antibody. Inset graph shows the reverse experiment. (B) CR57 and CR4098 were mixed in different ratios to obtain a 5-IU/ml antibody solution, which was then tested in a modified RFFIT for neutralizing activity. The activity (in IU/ml) of each combination is shown. Horizontal line indicates 5 IU/ml. The bars on the graph represent the following antibodies: 1, 100% CR4098; 2, 25% CR57 plus 75% CR4098; 3, 50% CR57 plus 50% CR4098; 4, 75% CR57 plus 25% CR4098; 5, 100% CR57.

FIG. 4.

Anti-RV MAbs protect Syrian hamsters from a lethal RV infection. Syrian hamsters were challenged with COSRV on day −1. Animals were vaccinated with rabies vaccine and treated with 10, 20, or 40 IU/kg of body weight of CR57, CR4098, or CR4144 on day 0. Control groups received either phosphate-buffered saline or 20 IU/kg HRIG. Animals were monitored twice daily and euthanized when clinical signs of rabies appeared. Kaplan-Meier survival curves are shown by plotting percent survival against time (in days).