Protein G of respiratory syncytial virus (RSV) is an envelope glycoprotein that is structurally very different from its counterparts (haemagglutinin-neuraminidase and haemagglutinin) in other paramyxoviruses. In this study, we put forward a model for this unique viral envelope protein. We propose that protein G of RSV contains several independently folding regions, with the ectodomain consisting of a conserved central hydrophobic region located between two polymeric mucinlike regions. The central conserved region is probably the only relatively fixed and folded part of the ectodomain of RSV-G. This central conserved region contains four conserved cysteine residues which can form two disulphide bridges. Analysis of the proteolytic digestion products of a peptide corresponding to the central conserved region showed that one of the three theoretically possible combinations of disulphide connections could be eliminated. The final disulphide bridge assignment was established by affinity measurements with peptide variants in which different disulphide connections were formed. Additionally, peptide binding studies were used to map the binding site, at the amino acid level, of a monoclonal antibody directed against the central conserved region. These studies indicated the level of surface exposure of the amino acid side-chains. The surface exposure agreed with the structural model. The proteolytic digestion, the peptide binding studies and the affinity measurements with structural peptide variants support a structural model with disulphide connections that correspond to a structural motif called a cystine noose. This model provides a structural explanation for the location and molecular details of important antigenic sites.

To identify the principal neutralization determinant (PND) of simian immunodeficiency virus (SIV), antisera were generated using recombinant gpllO [

The attachment protein G of respiratory syncytial virus (RSV) has a modular architecture. The ectodomain of the protein comprises a small folded conserved region which is bounded by two mucin-like regions. In this study, a sequence and structural homology is described between this central conserved region of RSV-G and the fourth subdomain of the 55-kDa tumor necrosis factor receptor (TNFr). The three-dimensional structures of RSV-G and human TNFr were previously determined with NMR spectroscopy and X-ray crystallography, respectively. The C-terminal part of both subdomains fold into a cystine noose connected by two cystine bridges with the same spacing between cysteine residues and the same topology. Although a general structural similarity is observed, there are differences in secondary structure and other structural features. Molecular Dynamics calculations show that the BRSV-G NMR structure of the cystine noose is stable and that the TNFr crystal structure of the cystine noose drifts towards the BRSV-G NMR structure in the simulated solution environment. By homology modelling a model was built for the unresolved N-terminal part of the central conserved region of RSV-G. The functions for both protein domains are not known but the structural similarity of both protein domains suggests a similar function. Although the homology suggests that the cystine noose of RSV-G may interfere with the antiviral and apoptotic effect of TNF, the biological activity remains to be proven.

The three-dimensional solution structure of the immunodominant central conserved region of the attachment protein G (BRSV-G) of bovine respiratory syncytial virus has been determined by nuclear magnetic resonance (NMR) spectroscopy. In the 32-residue peptide studied, 19 residues form a small rigid core composed of two short helices, connected by a type I' turn, and linked by two disulfide bridges. This unique fold is among the smallest stable tertiary structures known and could therefore serve as an ideal building block for the design of de novo proteins and as a test case for modeling studies. A characteristic hydrophobic pocket, lined by conserved residues, lies at the surface of the peptide and may play a role in receptor binding. This work provides a structural basis for further peptide vaccine development against the severe diseases associated with the respiratory syncytial viruses in both cattle and man.

Peptides deduced from the central hydrophobic region (residues 158-189) of the G protein of bovine and ovine respiratory syncytial virus (RSV) and of human RSV subtypes A and B were synthesized. These peptides were used to develop ELISAs to measure specifically antibodies against these types and subtypes of RSV. We have evaluated the bovine RSV-G peptide in both an indirect ELISA and in a blocking ELISA. Specificity and sensitivity, relative to a routine diagnostic ELISA that detects antibodies against the RSV F-protein in bovine sera, were 98% and 92% respectively for the indirect peptide-based ELISA, and 98% and 98% for the blocking peptide-based ELISA. In paired serum samples, rises in antibody titer were detected more frequently with the indirect peptide-based ELISA than with the routine F-ELISA. Furthermore, the peptide-based G-ELISAs were able to differentiate between antibodies against BRSV and HRSV, and between those against BRSV and ORSV. In addition, the indirect peptide-based ELISA was selective for HRSV subtype A and B antibodies. This study shows that peptides, corresponding to the central hydrophobic region of the attachment protein G of several RSVs, can be used successfully as antigens in highly specific and sensitive immunoassays.

Due to the advantageous properties of synthetic molecules compared to biological ones biological molecules in diagnostic tests are replaced increasingly by synthetic ones, usually synthetic peptides or related molecules. The replacement of biological antigens by synthetic peptides is most advanced at present, as well as the use of site-specific antibodies induced with synthetic peptides. Moreover recent results indicate that synthetic molecules may also replace antibodies. Ultimately this will lead to diagnostic assays built of synthetic molecules only.

The cellular immune response to respiratory syncytial virus (RSV) is important in both protection and immunopathogenesis. In contrast to HLA class I, HLA class II-restricted RSV-specific T-cell epitopes have not been identified. Here, we describe the generation and characterization of two human RSV-specific CD4؉ -T-cell clones (TCCs) associated with type 0-like cytokine profiles. TCC 1 was specific for the matrix protein and restricted over HLA-DPB1*1601, while TCC 2 was specific for the attachment protein G and restricted over either HLA-DPB1*0401 or -0402. Interestingly, the latter epitope is conserved in both RSV type A and B viruses. Given the high allele frequencies of HLA-DPB1*0401 and -0402 worldwide, this epitope could be widely recognized and boosted by recurrent RSV infections. Indeed, peptide stimulation of peripheral blood mononuclear cells from healthy adults resulted in the detection of specific responses in 8 of 13 donors. Additional G-specific TCCs were generated from three of these cultures, which recognized the identical (n ‫؍‬ 2) or almost identical (n ‫؍‬ 1) HLA-DP4-restricted epitope as TCC 2. No significant differences were found between the capacities of cell lines obtained from infants with severe (n ‫؍‬ 41) or mild (n ‫؍‬ 46) RSV lower respiratory tract infections to function as antigen-presenting cells to the G-specific TCCs, suggesting that the severity of RSV disease is not linked to the allelic frequency of HLA-DP4. In conclusion, we have identified an RSV G-specific human T helper cell epitope restricted by the widely expressed HLA class II alleles DPB1*0401 and -0402. Its putative role in protection and/or immunopathogenesis remains to be determined. Respiratory syncytial virus (RSV), a member of the genusPneumovirus of the family Paramyxoviridae, is a major cause of severe lower respiratory tract disease in infants, immunocompromised individuals, and the elderly (7, 29). RSV infections cause yearly epidemics in the winter season of moderate climate zones and are most often associated with relatively mild upper respiratory tract infections (29). In general, specific immunity is insufficient for protection, and RSV infections continue to occur throughout life.At present, no licensed RSV vaccine is available. During vaccine trials in the 1960s, vaccination with a formalin-inactivated whole-virus preparation (FI-RSV) was found to predispose for enhanced clinical disease following natural infection with RSV (17). Although the exact mechanism of this apparently immunopathological phenomenon remains unclear, studies of both rodent and nonhuman primate models have suggested that a skewed RSV-specific T helper type 2 (Th2) response was a key factor in this process (11,23). Several studies have suggested that primary infections in young infants resulting in severe RSV bronchiolitis are also associated with Th2 responses (24, 28). However, in two other cohort studies of infants with either severe RSV bronchiolitis or relatively mild RSV upper respiratory tract infection, this observation was...

Epitopes were resolved at the amino acid level for nine monoclonal antibodies (MAbs) directed against the central conserved region of protein G of bovine respiratory syncytial virus (BRSV-G). Peptide binding studies showed which amino acids in the epitope contributed to antibody binding. The details of the epitopes were compared with the high-resolution structure of a synthetic peptide corresponding to the central conserved region of BRSV-G, and this indicated which face of the central conserved region is the antigenic structure. The major linear epitope of the central conserved region of BRSV-G is located at the tip of the loop, overlapping a relatively flat surface formed by a double disulfide-bonded cystine noose. At least one, but possibly two sulfur atoms of a disulfide bridge that line the conserved pocket at the center of the flat surface, is a major contributor to antibody binding. Some of the residue positions in the epitope have mutated during the evolution of RSV-G, which suggests that the virus escaped antibody recognition with these mutations. Mutations that occur at positions 177 and 180 may have only a local effect on the antigenic surface, without influencing the structure of the backbone, whereas mutations at positions 183 and 184 will probably have major structural consequences. The study explains the antigenic, structural, and functional importance of each residue in the cystine noose which provides information for peptide vaccine design. Additionally, analysis of the epitopes demonstrated that two point mutations at positions 180 and 205 define the preliminary classification of BRSV subgroups.