Novel Rabies Virus-Neutralizing Epitope Recognized by Human Monoclonal Antibody: Fine Mapping and Escape Mutant Analysis

Abstract

Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We produced two previously described potent rabies virus-neutralizing human MAbs, CR57 and CRJB, in human PER.C6 cells. The two MAbs competed for binding to rabies virus glycoprotein. Using CR57 and a set of 15-mer overlapping peptides covering the glycoprotein ectodomain, a neutralization domain was identified between amino acids (aa) 218 and 240. The minimal binding region was identified as KLCGVL (aa 226 to 231), with key residues K-CGV-identified by alanine replacement scanning. The critical binding region of this novel nonconformational rabies virus epitope is highly conserved within rabies viruses of genotype 1. Subsequently, we generated six rabies virus variants escaping neutralization by CR57 and six variants escaping CRJB. The CR57 escape mutants were only partially covered by CRJB, and all CRJB-resistant variants completely escaped neutralization by CR57. Without exception, the CR57-resistant variants showed a mutation at key residues within the defined minimal binding region, while the CRJB escape viruses showed a single mutation distant from the CR57 epitope (N182D) combined with mutations in the CR57 epitope. The competition between CR57 and CRJB, the in vitro escape profile, and the apparent overlap between the recognized epitopes argues against including both CR57 and CRJB in a MAb cocktail aimed at replacing classical immunoglobulin preparations.

The human antibody repertoire specific for rabies virus glycoprotein as selected from immune libraries

“…9). Several of the IgG 1 displayed affinities in the same range as the previously described [17] antibodies CR57 and CRJA.…”

“…8 summarizes the data, showing relative binding of each IgG 1 compared to CR57 as reference. CR57 is a wellcharacterized, potent RV-neutralizing IgG 1 isolated from a vaccinated human donor using B cell immortalization technology [9,16], which recognizes an epitope within antigenic site I [17]. To analyze the affinity of a subset of the antibodies, surface plasmon resonance (SPR) analysis was performed.…”

“…Sequencing of the gp gene of the resulting CR4098 escape virus E98-4 revealed a single point mutation localized in the welldefined antigenic site III [18], strongly suggesting that antibody CR4098 is directed against antigenic site III [19]. Six previously described CR57 escape viruses containing mutations in antigenic site I [17], as well as the CR4098 escape virus E98-4, were used in a modified RFFIT for analysis of the panel of IgG 1 . As summarized in Fig.…”

“…The vectors were transfected into PER.C6 cells and cultured in the presence of Geneticin (Gibco) to generate stable cell lines. Expression of gp was analyzed using FCM and mAb CR57 [17] and VI7 [30] specific for RV and VSV gp, respectively.…”

First administration to humans of a monoclonal antibody cocktail against rabies virus: Safety, tolerability, and neutralizing activity

“…These mAbs, produced on the PER.C6 ® human cell line, are directed against distinct, non-overlapping rabies virus epitopes and do not compete for binding to rabies glycoprotein [9,10]. CL184 has demonstrated broad We have performed two phase I studies, one in the USA (RAB-M-A001) and one in India (RAB-M-A002), with the primary objective of investigating the safety and tolerability of CL184 in healthy adult subjects.…”