Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: Fine mapping and escape mutant analysis

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

Cleavage of polypeptide chain initiation factor eIF4GI during apoptosis in lymphoma cells: characterisation of an internal fragment generated by caspase-3-mediated cleavage

Polypeptide chain initiation factor eIF4GI undergoes caspase-mediated degradation during apoptosis to give characteristic fragments. The most prominent of these has an estimated mass of approximately 76 kDa (Middle-Fragment of Apoptotic cleavage of eIF4G; M-FAG). Subcellular fractionation of the BJAB lymphoma cell line after induction of apoptosis indicates that M-FAG occurs in both ribosome-bound and soluble forms. Affinity chromatography on m7GTP-Sepharose shows that M-FAG retains the ability of eIF4GI to associate with both the mRNA cap-binding protein eIF4E and initiation factor eIF4A and that the ribosome-bound form of M-FAG is also present as a complex with eIF4E and eIF4A. These data suggest that the binding sites for eIF4E, eIF4A and eIF3 on eIF4GI are retained in the caspase-generated fragment. M-FAG is also a substrate for cleavage by the Foot-and-Mouth-Disease Virus-encoded L protease. These properties, together with the pattern of recognition by a panel of antibodies, define the origin of the apoptotic cleavage fragment. N-terminal sequencing of the products of caspase-3-mediated eIF4GI cleavage has identified the major cleavage sites. The pattern of eIF4GI degradation and the possible roles of the individual cleavage products in cells undergoing apoptosis are discussed

First Administration to Humans of a Monoclonal Antibody Cocktail Against Rabies Virus: Safety, Tolerability, and Neutralizing Activity

Immediate passive immune prophylaxis as part of rabies post-exposure prophylaxis (PEP) often cannot be provided due to limited availability of human or equine rabies immunoglobulin (HRIG and ERIG, respectively). We report first clinical data from two phase I studies evaluating a monoclonal antibody cocktail CL184 against rabies. The studies included healthy adult subjects in the USA and India and involved two parts. First, subjects received a single intramuscular dose of CL184 or placebo in a double blind, randomized, dose-escalation trial. Second, open-label CL184 (20 IU/kg)was co-administered with rabies vaccine. Safety was the primary objective and rabies virus neutralizing activity (RVNA) was investigated as efficacy parameter. Pain at the CL184 injection site was reported by less than 40% of subjects; no fever or local induration, redness or swelling was observed. RVNA was detectable from day 1 to day 21 after a single dose of CL184 20 or 40 IU/kg. All subjects had adequate (\u3e0.5 IU/mL) RVNA levels from day 14 onwards when combined with rabies vaccine. CL184 appears promising as an alternative to RIG in PEP