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

Mimotopes: realization of an unlikely concept

Theoretically it seems highly unlikely that relatively small peptides could mimic functionally discontinuous epitopes of antigens. Nevertheless various recent reports show this to be the case. Peptide mimics of protein-, polysaccharide- and DNA-epitopes have been shown to be able to replace the native epitope. Moreover, some of them are able to induce, when used in a vaccine, antibodies with the same activity as that of the antibody used as a template. These mimics, called mimotopes, can be used in vaccines and diagnostics and can be developed more or less systematically using solely antibodies and random, semi-random and dedicated peptide arrays or libraries. Furthermore, the mimotope concept which seems to have proven itself for antibody and antigen interaction can be applied equally well to many receptor ligand interactions and thus may form a new generic approach to the development of drugs. Copyright (C) 2000 John Wiley and Sons, Ltd

In vitro inhibition of the bioactivity of follicle-stimulating hormone by antisera against a peptide representing part of the FSH-receptor

The aim of the present work was to define an FSH receptor (FSHR) peptide that can induce antibodies that will inhibit the bioactivity of FSH. Therefore, the hFSHR sequence was aligned with that of all other known G-protein coupled receptors. An area with increased sequence homology was identified between the FSH-, LH-, TSH receptors, the C5a receptor and the IL8 receptor. The similarity consists of a richness in acidic (D and E) and hydrophobic (Y and F) residues. In hFSHR the sequence is EDNESSYSRGFDMTYTEFDYDLCNEVVD (amino acid 299–326). Research on both the C5a- and IL8-receptor has indicated that this part is responsible for hormone binding but not for signal transduction. Protamine, an antagonist for both the C5a- and IL8 receptor also inhibited the bioactivities of FSH and LH when tested in a bioassay. This suggests that in the hFSHR this region might also be involved in hormone binding. Specificity of this region towards the diverse ligands all binding to the C5a or to the IL8 receptor might be attributed to differences in the profile of alternating basic and hydrophobic residues. Therefore, the hypothesis was tested as to whether antisera raised against peptides of this FSHR-domain would inhibit FSH-bioactivity but not LH-bioactivity. Indeed antisera were found (anti-hFSHR 309–322) that inhibited the biological activity of FSH in a bioassay. These antisera proved to be specific since they did not inhibit the bioactivity of LH. These data suggest that the core sequence (hFSHR 309-322) of the aligned domain of the hFSHR, in analogy to the IL8- and C5a receptors, is involved in hormone binding and ligand specificity. This domain therefore forms a valuable tool in FSH- and FSHR research for scientific and medical purposes