IMMUNODOMINANT EPITOPES DEFINED BY A YEAST-EXPRESSED LIBRARY OF RANDOM FRAGMENTS OF THE RABIES VIRUS GLYCOPROTEIN MAP OUTSIDE MAJOR ANTIGENIC SITES

Nineteen yeast colonies secreting rabies virus glycoprotein (G) peptid es immunoreactive with polyclonal anti-rabies virus sera were selected from a random expression library. The peptides, around 80 amino acids long, spanned amino acids 54-494 of the G protein. These peptides, to gether with two constructions including, respectively, immunodominant sites II and III, were analysed for their immunoreactivity with 40 ant i-G protein monoclonal antibodies (MAbs) composed of 12 MAbs that reac ted with SDS-treated protein in Western blot under reducing conditions (WB+) and 28 representative MAbs that did not react after denaturatio n (WB-). This last category represents 98% of anti-rabies virus G MAbs . None of the WB- MAbs bound peptides. Of the 12 WB+ MAbs, one bound t wo peptides situated before the transmembrane domain of the protein an d six bound peptides overlapping a region situated between amino acids 223 and 276. These six MAbs define a new antigenic region that would be considered 'immunodominant' if the peptide strategy had been used t o study the antigenicity of the protein; however, this region is only recognized by about 1% of our MAbs. Three of these WB+ MAbs had signif icant neutralizing activity; two were used for the selection of antige nic mutants (MAR mutants). Some mutants had a substitution within the region delimited by the peptides, confirming the pertinence of both th e peptide and escape mutant approaches. However, a few mutants had a s ubstitution outside the peptide-delimited region, suggesting that remo te mutation(s) could affect epitope accessibility in the native protei n.