We have produced baculovirus recombinants expressing the human parainfluenza type 3 virus (PIV3) hemagglutinin-neuraminidase (HN) protein to extend our previous antigenic and functional characterization of the HN to include structural analysis. The HN protein expressed by initial baculovirus recombinants was shown to be glycosylated, biologically active, immunogenic and antigenically authentic, but was produced in relatively small quantities. We have now produced a second generation of recombinants which express HN as a fusion product. These new recombinants achieve a level of expression which is 20-fold higher than our initial recombinants. We have also generated recombinants which produce a truncated, secreted form of HN. Recombinant HN protein will be purified prior to structural analysis. To extend our antigenic and functional analysis of the PIV3 surfaced glycoproteins, we have produced monoclonal antibodies (MAbs) to the fusion (F) glycoprotein. These MAbs define 20 epitopes, 14 of which participate in neutralization. The neutralization epitopes are distributed among three non-overlapping antigenic sites (A,B, and C) and one bridge site (AB), and the six non-neutralization epitopes from four distinct antigenic sites. Analysis of the biological activities of MAbs indicated that antigenic sites AB,B, and C correspond to functional domains of the F protein. Antigenic variants have been selected using neutralizing and fusion-inhibiting MAbs,a nd sequence analysis of these variants and of naturally-occurring clinical PIV3 isolates is currently in progress. These studies will identify amino acid residues involved in neutralization by antibodies as well as residues important for fusion function.
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