Respiratory syncytial virus strains isolated from infants and children hospitalized with acute respiratory tract disease in Huntington, W.Va., from July 1978 through June 1988 were categorized by subgroup by using two panels of monoclonal antibodies specific for subgroup A and subgroup B. Of 405 strains tested, 319 (78.8%) were subgroup A, 73 (18.0%) were subgroup Bi, and 13 (3.2%) were subgroup B2. The subgroup B2 strains occurred as a single-season epidemic during the epidemiologic year July 1980 to June 1981, except for one strain isolated in the epidemiologic year 1978 to 1979. Subgroup B strains were associated with significantly fewer bronchiolitis illnesses than subgroup A strains. The severity of illnesses caused by subgroup BI and B2 strains was the same.
An antigenic site (represented by 15 amino acids, residues 174 to 188, designated peptide 12) of the large glycoprotein G of respiratory syncytial virus was demonstrated to be subgroup specific in peptide enzyme-linked immunosorbent assay tests with murine monoclonal antibodies and human postinfection sera. The role of individual amino acids in this subgroup-specific site was determined by use of single-amino-acid-deletion sets of peptides. When monoclonal antibodies were reacted with the deletion sets, a broad amino acid dependence of 11 or 12 residues, Cys-176 (Ile-175 in subgroup B) to Cys-186, was found. Human postinfection sera exhibited a narrower reaction profile (for subgroup A, Cys-182 to Trp-183; for subgroup B, Cys-176 to Lys-183). Reduction of peptides on microtiter plates by treatment with dithiothreitol completely destroyed their antigenic activity in tests with monoclonal antibodies and human postinfection sera of subgroup B. A variant of peptide 12 containing all four cysteines of the G protein (represented by 16 amino acids, residues 172 to 187, designated peptide 12var) also was subgroup specific. We concluded that the activity of the antigenic site in tests with monoclonal antibodies for subgroups A and B appears to depend on intrapeptide disulfide bonds. Reactions with postinfection sera of subgroup B also may depend on a disulfide bond. In contrast, postinfection sera of subgroup A appeared to have the capacity to identify a subgroup-specific site in a linear form of the selected 15-amino-acid-long peptide. Treatment of peptides with dithiothreitol had no effect on their antigenic activity in tests with human postinfection sera of subgroup A. These findings have relevance for molecular engineering of peptide antigens for use in respiratory syncytial virus subgroup-specific site-directed serology.
The location of the epitope recognized by monoclonal antibody (MAb) 63G on the primary structure of the human respiratory syncytial virus G glycoprotein was determined by testing the reactivity of synthetic peptides with the MAb. The role of individual amino acids in this epitope was determined by using a set of 13-mer peptides containing sigle residue deletions. Residues 204 to 209 were found to be essential for antibody binding. These results are in full agreement with previous sequence data for escape mutants selected with MAb 63G. Several peptides, free or bound to keyhole limpet haemocyanin (KLH), were used to raise antisera in rabbits. The antipeptide antibodies reacted with the G protein in Western blots. However, only peptide G1-KLH (residues 187 to 200 bound to KLH) induced antibodies that reacted with the intact G protein and inhibited infectivity. These findings are discussed in terms of the antigenic structure of the G glycoprotein and the molecular engineering of peptide antigens.
The SH protein of RSV, a small integrated hydrophobic membrane protein, consists of 64 amino acid residues in the polypeptide of subgroup A and 65 amino acid residues in the polypeptide of subgroup B. We synthesized five peptides, representing the SH protein of each RSV subgroup comprised of the following amino acid residues: 2-16, 12-26, 35-49, 45-60, and for subgroup A, 51-64 and for subgroup B, 51-65. Peptides 2-16 and 51-64/65 represented the N-terminal and C-terminal ends of the protein, respectively. In RIPA, under reducing conditions with mercaptoethanol, hyperimmune guinea pig (GP) serum against C-terminal peptide of the two subgroups precipitated the homologous 7.5 kDa and 21-30 kDa SH proteins. Under nonreducing conditions, the GP antipeptide sera precipitated all three SH proteins, suggesting that the 13-15 kDa protein exists as a dimer. The subgroup A 7.5 and 13-15 kDa proteins had apparent molecular weights about 1-2 kDa higher than the corresponding subgroup B proteins. The C-terminal peptides of subgroups A and B were used to characterize the immune response of 11 children, age 1 month to 1 year, with presumed primary RSV infection. Three of 4 children with subgroup A infection and 4 of 7 children with subgroup B infection developed homologous 4-fold rises in antibody to C-terminal peptide (aa 51-64/65) during convalescence. Except for one child with subgroup A and one child with subgroup B infection, the other 5 children developed heterologous rises also.(ABSTRACT TRUNCATED AT 250 WORDS)
Two synthetic peptides, designated peptides 12G(A) and 12G(B), representing amino acids 174-188 of the G glycoprotein of respiratory syncytial virus (RSV) subgroup A (strain A2) and subgroup B (strain CH18537) were evaluated for their properties as subgroup-specific antigens for enzyme immunoassay (ELISA). These peptides were used to characterize the immune response of children with naturally occurring RSV infection during six annual epidemics in the Huntington area, West Virginia, USA; viz. 1978-1979, 1979-1980, 1980-1981, 1983-1984, 1989-1990, and 1990-1991. The study group comprised 43 paired sera from 42 infants and children, who ranged in age between 1 month and 5.5 years of age (median age 16 months). The inclusion criteria were subgroup identification of RSV, respiratory tract illness requiring admission to hospital, and the availability of paired sera. Five of 30 children with subgroup A and 3 of 13 children with subgroup B infections developed homologous or dual fourfold or greater antibody responses to peptides 12G(A) and 12G(B) during convalescence; six of these eight children also developed antibody rises to whole virus antigens. Twenty children (14 subgroup A and 6 subgroup B) developed such responses in antibody only to whole virus (not to the peptides), and 15 children (11 subgroup A and 4 subgroup B) failed to develop a rise in antibody. Children who developed rises in antibody to the peptides were usually less than 9 months of age, suggesting that a response to peptides was more likely to occur during primary infection. Peptides 12G(A) and 12G(B) of RSV G protein lacked sufficient sensitivity and specificity to serve as antigens for ELISA for characterizing the subgroup-specific immune responses to RSV infection in infants and children.
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