Monoclonal antibodies recognize at least five epitopes on the SIV Nef protein and identify an in vitro-induced...

Eleven monoclonal antibodies (MAbs) to SIV Nef were produced and characterized. Five antibody-binding sites on SIV Nef were identified on the basis of the reactivity of the antibodies with recombinant proteins. Two of the five epitopes were defined using overlapping peptides. A further three epitopes could not be defined with peptides but all antibodies reacted in Western blot, suggesting that the epitopes were at least partially conformation dependent. Antibodies in two of the five epitope groups were further differentiated by competition analysis. The panel of MAbs described is able to distinguish between a number of recombinant Nef proteins currently under investigation in vivo in macaques. Two of the MAbs described are able to distinguish between the Nef protein from pathogenic (J5) and attenuated (C8) strains of SIV, thus providing useful tools for studying the relevance of the Nef protein in the pathogenesis of SIV infection. In FACScan analysis two of the MAbs, KK70 and KK75, were used to identify an in vitro-induced mutation in J5 Nef grown in C8166 cells. Sequence analysis of the phenotypic variants identified a mutation of the tryptophan (TGG) at amino acid 214 to a stop codon (TGA), thus truncating the Nef protein. The functional significance of this observation remains unclear but highlights the need to interpret data with caution if virus has been cultured in vitro even for a short period of time.