Studies on the immunochemistry of respiratory syncytial virus

Ward, Kevin Arnott (1983) Studies on the immunochemistry of respiratory syncytial virus. University of Southampton, Doctoral Thesis.

Abstract

Nine respiratory syncytial (RS) virus polypeptides, VP200, VGP95, VP68, VGP48, VPN41, VP35, VP27, VP23 and VGP20 were identified by comparing 35 S-cysteine/methionine labelled extracts of infected and uninfected HEp-2 cells, and by radioimmunoprecipitation using a hyperimmune human serum. Three of these polypeptides, VGP95, VGP48 and VGP20 were shown to be glycosylated by incorporation of 3H-glucosamine. VGP48, VP23 and VGP20 were shown to comprise a disulphide bond-linked protein as they were coprecipitated by a monoclonal antibody and resolved as a single band (Mr 75K) by polyacrylamide gel electrophoresis under non-reducing conditions. VPN41 was shown to be the major nucleoprotein by purification of the nucleocapsid. A rapid method for the partial purification of the nucleocapsid from infected cells was devised, and the conditions required to obtain detergent-soluble preparations of VPN41 were determined. Two-dimensional peptide mapping of VPN41 revealed considerable structural conservation among the nucleoproteins from strains of RS virus isolated in Southampton over five consecutive years, and from two laboratory strains. Bovine RS virus nucleoprotein was also shown to be closely related to nucleoproteins from the human strains by peptide mapping. Preliminary data obtained by an enzyme-linked immunoabsorbent assay showed cross-reactivity of monoclonal antibodies among nucleoproteins from all strains. Monoclonal antibodies were raised to the nucleoprotein and used to demonstrate that VPN41 was susceptible to cleavage into two major fragments, Mr 27K and 14K, during the preparation of nucleocapsids from infected Vero or bovine kidney cells, but not from HEp-2 cells. Similar cleavage of VPN41 was observed when nucleocapsids prepared from HEp-2 cells were treated with trypsin. Human serum antibody levels to the major nucleoprotein and to the two glycoproteins were measured by radioimmunoprecipitation using detergent soluble, radioiodinated antigens. Sera from a group of mothers whose babies escaped RS virus infection during a local epidemic showed increased antibody levels to VPN41 when compared to sera from mothers whose babies had become infected with RS virus within the first 6 months of life. In infants who remained uninfected with RS virus during the first 12 months of life the maternal gift of antibody decayed to about 50% at 3 months with traces of antibodies detected in a few sera at 12 months. The antibody levels detected in the sera of infants less than 3 months old convalescent from primary RS virus infection did not exceed the mean levels present in the serum of uninfected babies. Infants between the ages of 6 and 12 months were able to mount an IgG response to VPN41 and VGP48 but, unlike adults, a particularly striking finding was their failure to produce antibodies to VGP95.

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