Identification of a Synthetic Peptide as Part of a Major Neutralization Epitope of Respiratory Syncytial Virus

Abstract

SUMMARYA 7000 Mr cleavage fragment of the F 1 subunit that carries the major neutralization epitope has been identified by chemical and enzymatic cleavage of the fusion protein of respiratory syncytial (RS) virus (Long strain) with an efficient RS virus-neutralizing monoclonal antibody. Based on the published mRNA-deduced sequence of the A2 strain, coupled to the hydropathicity profile and prediction of protein conformation, the neutralization epitope has tentatively been localized on the first third of the F1 protein N-terminal, probably in the region of amino acids 215Ser to 236Glu. Analysis of three peptides covering different portions of the 212Cys to 236Glu region of the F 1 fusion protein identified a peptide (Cys. 216Ash to 236Glu) that reacted strongly with the neutralizing monoclonal antibody and that was efficient in blocking neutralization and in plaque-reducing assays, confirming that the neutralization epitope was localized in that region. Further analysis with two other synthetic peptides (212Cys to 222Glu and Cys.221Ile to 236Glu) indicated that the dodecapeptide Ile-Glu-Phe-Gln-LysAsn-Asn-Arg-Leu-Leu-Glu mimicked either the whole or a major part of the neutralization epitope. This opens a promising avenue for the simple design of a synthetic peptide vaccine to control RS virus infection. INTRODUCTIONThe genus Pneumovirus of the Paramyxoviridae family consists of human and bovine strains of respiratory syncytial (RS) virus and pneumonia virus of mice. Human RS virus, an important pathogen in young children (Belshe et al., 1984), is thought to be a monotypic strain though minor differences in cross-neutralization assays have been noted. Using monoclonal antibodies (MAbs), Mufson et al. (1985) have been able to separate different isolates into subgroups A and B; however, studies by Prince et al. (1985) suggest that antigenic differences detected in vitro disappear when virus strains are compared in vivo. Recently, using a neutralizing MAb produced against the Long strain of RS virus, a major and highly conserved neutralization epitope was identified which was present on human, bovine and caprine strains of RS virus but not on pneumonia virus of mice. This epitope is localized on the F 1 fragment of the fusion glycoprotein (Trudel et al., 1986(Trudel et al., , 1987.The fusion glycoprotein is found as a 125K Mr dimer on the viral envelope and is involved in cell fusion leading to the formation of syncytia and cell penetration. It is composed of two disulphide-linked subunits: F1 with a reported Mr varying from 43K to 50K and F2 from 19K to 25K (Walsh et al., 1985, 1986;Trudel et al., 1986).Expression by recombinant vaccinia viruses of the major and fusion glycoproteins of RS virus (Olmsted et al., 1986) confirmed previous work carried out with MAbs and purified solubilized proteins showing the predominant role of the fusion glycoprotein in inducing immunity

Characterization of two antigenic sites recognized by neutralizing monoclonal antibodies directed against the fusion glycoprotein of human respiratory syncytial virus

“…The isolation and characterization of the MAbs used in this study have been documented (Taylor et al, 1984(Taylor et al, , 1992Trudel et al, 1987;Garcia-Barreno et al, 1989). Antibody AK 13A2, raised against the Long strain F protein, was a generous gift from Dr. P. Coppe (Centre d'Economie Rurale, Marloie, Belgium).…”

“…Monoclonal antibodies (MAbs) with the highest neutralization index recognize epitopes of the RSV F glycoprotein (Trudel et al, 1987;Anderson et al, 1988;Beeler & van Wyke Coelingh, 1989;. This protein is synthesized as an inactive precursor which is glycosylated and processed proteolytically during maturation to generate two subunits (F1 and F2) that remain linked by disulphide bridges (Gruber & Levine, 1983).…”

“…Several laboratories have located a few epitopes on the primary structure of the F protein by (i) using synthetic peptides to examine the binding of MAbs (Trudel et al, 1987;Bourgeois et al, 1991;Martin-Gallardo et al, 1991) or polyclonal antibodies (Scopes et al, 1990), or (ii) isolating viruses that escape neutralization by anti-F protein MAbs in order to identify amino acid residues essential for epitope integrity . Since both approaches have limitations (see Discussion), we have used the two complementary strategies to locate the epitopes recognized by several anti-F protein neutralizing MAbs on the primary structure of the protein.…”

Intracellular processing of the human respiratory syncytial virus fusion glycoprotein: amino acid substitutions affecting folding, transport and cleavage

“…MAbs 7C2 and L4, which define neutralization epitopes on the F protein at amino acid residues 221 to 232 and 283 to 315, respectively (Paradiso et al, 1989;Trudel et al, 1987), and MAbs C1, C2, C13, C14, C18, C19, C20 and C21, which recognize eight distinct epitopes from (0) and/or chase (1 or 3) periods, cell lysates were prepared and the samples were immunoprecipitated with anti-RSV serum. Half of each immunoprecipitated sample was treated with Endo H ( + ) to examine the state of maturation of the oligosaccharide residues.…”

Conformational constraints of conserved neutralizing epitopes from a major antigenic area of human respiratory syncytial virus fusion glycoprotein

“…The MAbs used throughout this study have been described previously (Taylor et al, 1984Samson et al, 1986;Trudel et al, 1987;Kennedy et al, 1988;GarciaBarreno et al, 1989). Antibodies AK13A2 (Dr P. Coppe, Centre D'Economie Rurale, Marloie, Belgium), IE3 (Samson et al, 1986) and 7C2 (Trudel et al, 1987) were kindly supplied by the respective laboratories. All MAbs were of murine origin, except MAbs B4, B5 and B 10, which were produced by heterohybridomas of bovine splenocytes and murine myeloma cells (Kennedy et al, 1988;Taylor et al, 1992).…”