Binding of neutralizing monoclonal antibodies to regions of the fusion protein of respiratory syncytial virus expressed in Escherichia coli

Biological activity, binding site and affinity of monoclonal antibodies to the fusion protein of respiratory syncytial virus

“…It may be that the full epitope recognized by all of these antibodies involves other elements, possibly distant in primary structure, and that recognition of these additional sites, not amino acids 253 to 289, determines the level of functional activity of the antibody. This would be in agreement with our previous observation that F protein fragments containing the 253 to 289 amino acid sequence fail to induce antibodies capable of neutralizing the virus (Lounsbach et al, 1993). A major conformation-dependent element in the epitope of these antibodies may also explain their apparent low avidity as conformational changes may occur on binding of proteins to plastic adversely affecting antibody binding to non-linear epitopes (Darst et al, 1988).…”

“…The F1-574 chimera was expressed from the full coding sequence of the F protein gene and contains all 574 amino acids encoded by the open reading frame of the F protein gene. The binding sites of MAbs RS348 (Lounsbach et aL, 1993) and 19 are indicated.…”

“…Other authors have found patterns similar to these. Although the binding sites of a number of neutralizing and fusion-inhibiting antibodies have been mapped to two regions of the protein, involving or close to the amino acids 265 to 272 (Arbiza et al, 1992;Lounsbach et al, 1993;Martin-Gallardo et al, 1991) and amino acids 422 to 438 (Lounsbach et aL, 1993). Each fragment is labelled according to the amino acids of the F protein sequence expressed in the chimera.…”

“…MAbs (Table 1) were derived from Balb/c mice immunized intranasally with 104 p.f.u, of live RS virus (strain A2) under ether anaesthesia. In subsequent immunizations the mice were boosted with different antigens; immunoaffinity purified A2 strain F protein in Freund's adjuvant (Routledge et al, 1988) was used to generate MAbs 5A8, 1C1 and 2D7; a live recombinant vaccinia virus (v-F) expressing F from RS virus strain A2 (kindly provided by Dr Gail Wertz et al, 1987) was used to generate MAb 5E2 and a fl-galactosidase chimeric protein containing A2-strain F protein residues 253 to 389 (Lounsbach et al, 1993) in Freund's adjuvant was used to generate MAbs 5A6 and 10D4. At 2 weeks and at 3 days prior to hybridoma production all mice were inoculated subcutaneously and intravenously, respectively with sucrose gradient-purified A2 strain virus (Ueba, 1978).…”

“…The binding of the MAb panel was mapped by dotblotting on overlapping fragments of the F protein polypeptide expressed as fl-galactosidase chimeras in Escherichia coli (Lounsbach et al, 1993) (Fig. 1).…”