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.Lethal rabies is prevented by postexposure prophylaxis (PEP) through the combined administration of a rabies vaccine and rabies immune globulin (RIG). Two types of RIG are used: human RIG (HRIG) and equine RIG, both derived from pooled sera of human donors or horses vaccinated against rabies, respectively. The need to replace these hyperimmune serum preparations is widely recognized (29), and monoclonal antibodies (MAbs) that neutralize rabies virus (RV) offer the opportunity to do so.Mouse MAbs, as well as human MAbs, have been shown to protect rodents from a lethal RV challenge (6,9,12,14,20,22,24). One of the most potent human MAbs, SO57, neutralizing a variety of RV strains, was described by Dietzschold et al. (6). A cocktail of three human MAbs including SO57 and SOJA and SOJB showed effective protection of mice from a lethal dose of RV (22). We reformatted these three MAbs (renamed CR57, CRJA, and CRJB) into our own expression system for production in PER.C6 cells (19). However, we showed that the CRJA and CRJB MAbs were not suitable in combination with CR57 for use in PEP (19) because of overlapping epitope recognition, lack of neutralizing potency, and shared escape mutants. Novel anti-RV MAbs were generated using phage display technology and were characterized with special emphasis on CR57 complementarity.We considered several criteria to be of crucial importance for the inclusion of human MAbs into a cocktail aimed at effectively blocking an RV infection in humans. First, the MAbs should target distinct, nonoverlapping epitopes and should not compete for binding to RV glyco...

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