Linear antigenic and immunogenic regions of the respiratory syncytial virus P protein

Abstract

Three linear antigenic regions on the P protein from human respiratory syncytial virus (RSV) subgroup A (strain A2) were represented by peptides that reacted with monoclonal antibodies and with sera from humans with recent or previous RSV infection. The determinants were localized within three hydrophilic regions of the P protein: Prog~ to AspH0, Ser16~ to LySls 0 and Glu221 to Phe241. The role of individual amino acids in the epitopes defined by monoclonal antibodies was determined. Two monoclonal antibodies reacting with the same antigenic site were found to detect epitopes that had different amino acid dependencies. Rabbit hyperimmune sera raised against selected peptides specifically precipitated different forms of the P protein from RSV-infected 35S-labelled cell extracts in a radioimmune precipitation assay. These findings have implications for forthcoming structural-functional studies of RSV capsid component interactions and also for serological diagnosis of RSV infection.

Linear antigenic and immunogenic regions of human respiratory syncytial virus N protein

“…Recently we reported similar studies of the RSVP protein (Leonov et al, 1994). In the present study, we identified three immunodominant linear antigenic regions by use of synthetic overlapping peptides representing the RSV N protein.…”

“…ELISA with purified RSV antigen and peptides was performed as previously described (Akerlind-Stopner et al, 1990;Leonov et al, 1994). The results were expressed either as A492 (mean of three determinations) or, for substituted peptide analogues, as the percentage of peptides binding.…”

“…The peptide competition ELISA was performed as described previously (Leonov et al, 1994). Briefly, different concentrations (from 0I-20 gg/ml) of the competitive peptide together with a standardized dilution of MAb (giving 50 % of the maximum A49 ~) in ELISA dilution buffer were mixed, added to the wells pre-adsorbed with either homologous or heterologous peptide and allowed to react for 3 h at 37 °C.…”

“…The preparations of RIPA RSV antigen (from subgroup A, strain A2-and subgroup B, strain CH 1 g537-infected Vero cells) and the RIPA procedure were slightly modified from previously described methods (Leonov et al, 1994 …”

“…After centrifugation (90 min, 12 °C, 260000 g) the 'free' (FF) protein pool fraction from the top of the 20% sucrose and the 'nucleocapsid-associated' (NCF) protein pool fraction from the 20-60 % sucrose interface were sequentially collected by gentle aspiration and dissolved in lysis buffer (Leonov et al, 1994); after final centrifugation (13000 g) for 5 min the supernatant was used as RIPA antigen. A mixture ofprotease inhibitors (Protease inhibitors set), prepared according to the manufacturer (Boehringer Mannheim) was added to the samples at each step of RIPA antigen preparation.…”