Identification of Multiple Protective Epitopes (Protectopes) in the Central Conserved Domain of a Prototype Human Respiratory Syncytial Virus G Protein

Abstract

A recombinant fusion protein (BBG2Na) comprising the central conserved domain of the respiratory syncytial virus subgroup A (RSV-A) (Long) G protein (residues 130 to 230) and an albumin binding domain of streptococcal protein G was shown previously to protect mouse upper (URT) and lower (LRT) respiratory tracts against intranasal RSV challenge (U. F. Power, H. Plotnicky-Gilquin, T. Huss, A. Robert, M. Trudel, S. Stahl, M. Uhlén, T. N. Nguyen, and H. Binz, Virology 230:155–166, 1997). Panels of monoclonal antibodies (MAbs) and synthetic peptides were generated to facilitate dissection of the structural elements of this domain implicated in protective efficacy. All MAbs recognized native RSV-A antigens, and five linear B-cell epitopes were identified; these mapped to residues 152 to 163, 165 to 172, 171 to 187 (two overlapping epitopes), and 196 to 204, thereby covering the highly conserved cysteine noose domain. Antibody passive-transfer and peptide immunization studies revealed that all epitopes were implicated in protection of the LRT, but not likely the URT, against RSV-A challenge. Pepscan analyses of anti-RSV-A and anti-BBG2Na murine polyclonal sera revealed lower-level epitope usage within the central conserved region in the former, suggesting diminished immunogenicity of the implicated epitopes in the context of the whole virus. However, Pepscan analyses of RSV-seropositive human sera revealed that all of the murine B-cell protective epitopes (protectopes) that mapped to the central conserved domain were recognized in man. Should these murine protectopes also be implicated in human LRT protection, their clustering around the highly conserved cysteine noose region will have important implications for the development of RSV vaccines.

Peptides as tools and drugs for immunotherapies

“…In contrast two doses of 20 µg of P40-Cys G144 -159 (N -terminal coupling) were poorly immunogenic and no lung protection was observed. Similar results were obtained when BB was used for conjugation, indicating that the results were independent of the carrier protein [28]. Using this panel of monoclonal antibodies and synthetic peptides, the structural elements of G2Na implicated in protective efficacy were mapped and five different B-cell epitopes (145-159, 164-176, 171-187, 172-187 and 190-204) were identified (Figure 4(B)), covering the highly conserved cystine nose domain [28,29].…”

“…Similar results were obtained when BB was used for conjugation, indicating that the results were independent of the carrier protein [28]. Using this panel of monoclonal antibodies and synthetic peptides, the structural elements of G2Na implicated in protective efficacy were mapped and five different B-cell epitopes (145-159, 164-176, 171-187, 172-187 and 190-204) were identified (Figure 4(B)), covering the highly conserved cystine nose domain [28,29]. Using the PEPSCAN technology, there was a noticeable absence of reactivity with the 12-mer peptides spanning the cysteine-rich region but the corresponding epitope was unambiguously identified using the correctly folded synthetic G4 hexadecapeptide.…”

“…Ninety overlapping 12-mer peptides spanning residues 130-230 (G2Na) of the human RSV-A G protein were synthesised on non-cleavable derived rods for B cell epitope mapping ( Figure 4(A)). The peptides were tested for their binding ability with Monoclonal Antibodies (MAbs) or animal and patient sera by ELISA [28,29]. Moreover, all three possible oxidised isomers of the G4a hexadecamer (PCSICSNNPTCWAICK) were obtained by solid-phase synthesis of the respective reduced derivative further treated under various oxidation conditions [27].…”

Current research on respiratory viral infections: Fifth International Symposium1

“…BBG2Na is a recombinant chimeric protein comprised of BB (the albumin binding domain of Streptococcal protein G) and G2Na (residues 130-230 of the RSV-A Long strain G protein) expressed in E. coli. In pre-clinical studies, we found that BBG2Na formulated in alum was immunogenic and protective against RSV-A and -B challenges , was immunogenic and protective in murine neonates, even in the presence of high anti-RSV maternal antibody titers (Brandt et al, 1997;Siegrist et al, 1999), did not induce evidence of enhanced pathology (Corvaia et al, 1997;Plotnicky-Gilquin et al, 1999b;Power et al, 2001a), and protected via antibodies and CD4 + T cells (Plotnicky-Gilquin et al, 1999aPower et al, 2001b).…”

Generation and epitope mapping of a sub‐group cross‐reactive anti‐respiratory syncytial virus G glycoprotein monoclonal antibody which is protective in vivo

“…In animals polyclonal antibodies generated either to virus infection or to wild type or recombinant G glycoprotein have little sub‐group cross reactivity [Stott et al, ; Johnson et al, ; Walsh et al, ; Murata and Catherman, ] and following primary infection of human infants, only lineage specific antibodies to the G glycoprotein were detected in the convalescent sera [McGill et al, ]. However, Plotnicky‐Gilquin et al [] have demonstrated that antibody responses to a synthetic peptide corresponding to the central conserved motif do occur both in hyperimmunized mice and in older human convalescents and Palomo et al [] have demonstrated antibodies which compete with monoclonal antibodies to this region in a number of human sera. Where present such responses may be beneficial as anti‐G MAbs, generated in several laboratories and whose epitopes overlap this motif, have been shown to be protective in both cotton rats and mice reducing virus replication and, independently, pulmonary inflammation [Walsh et al, ; Plotnicky‐Gilquin et al, ; Collarini et al, ; Haynes et al, ; Miao et al, ].…”