A cDNA clone representing the mRNA coding sequence of the fusion glycoprotein (F) gene of human respiratory syncytial virus (RSV) was constructed and inserted into the thymidine kinase gene of vaccinia virus (WR strain) under the control of a vaccinia virus promoter. The resulting recombinant vaccinia virus, vaccinia F, expressed the F1 and F2 cleavage products (48 and 20 kDa, respectively) of the F glycoprotein in cell culture. F1 and F2 were indistinguishable from their authentic RSV counterparts with respect to glycosylation, disulfide linkage, electrophoretic mobility, cellsurface expression, and antigenic specificity. Cotton rats infected intradermally with vaccinia F developed a high titer of serum F-specific antibodies, which neutralized infectivity of RSV. This neutralizing antibody response exceeded that induced by infection of the respiratory tract with RSV and was 6-fold higher than that induced by vaccinia G, a recombinant vaccinia virus that expressed the RSV G glycoprotein gene. Immunization with vaccinia F stimulated almost complete resistance to replication of RSV in the lower respiratory tract as well as significant resistance in the upper respiratory tract.
Amino acid sequence of human respiratory syncytial virus envelope glycoprotein (G) was deduced from the DNA sequence of a recombinant plasmid and confirmed by limited amino acid microsequencing of purified 90K G protein. The calculated molecular mass of the protein encoded by the only long open reading frame of 298 amino acids was 32,588 daltons and was somewhat smaller than the 36K polypeptide translated in vitro from mRNA selected by this plasmid. Inspection of the sequence revealed a single hydrophobic domain of 23 amino acids capable of membrane insertion at 41 residues from the N-terminus. There was no N-terminal signal sequence and the hydrophilic N-terminal 20 residues probably represent the cytoplasmic tail of the protein. The N-terminally oriented membrane insertion was somewhat analogous to paramyxovirus hemagglutinin-neuraminidase (HN) and influenza neuraminidase (NA). The protein was moderately hydrophilic and rich in hydroxy-amino acids. It was both N- and O-glycosylated with the latter contributing significantly to the net molecular mass 90K.
A cDNA copy of the G glycoprotein gene of human respiratory syncytial virus (RSV) was placed under control of a vaccinia virus promoter and inserted into the thymidine kinase locus of the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and expressed a 93-kDa protein that migrated with the authentic RSV G glycoprotein upon polyacrylamide gel electrophoresis. Glycosylation of the expressed protein and transport to the cell surface were demonstrated in the absence of other RSV proteins. Cotton rats that were inoculated intradermally with the infectious recombinant virus produced serum antibody to the G glycoprotein that neutralized RSV in vitro. Furthermore, the vaccinated animals were resistant to lower respiratory tract infection upon intranasal inoculation with RSV and had reduced titers of RSV in the nose, (4,5). The larger one, designated G, has a molecular mass of about 90 kDa and is thought to be needed for virus adsorption. The smaller one, designated F because of its probable role in membrane fusion (6), consists of a 70-kDa protein that is proteolytically processed into 8). Passive immunization of cotton rats (9) and mice (10) with monoclonal antibodies to either glycoprotein provides protection against lower respiratory tract infection with RSV. Additional studies to delineate the roles of the glycoproteins in protective immunization have been hampered by the difficulty in obtaining sufficient amounts of purified materials. The recent cloning of cDNA copies of the mRNAs for both glycoproteins (11_14), however, opens the possibility of using expression vectors to produce the 90-and 70-kDa proteins.Vaccinia virus provides a novel eukaryotic vector system (15, 16) that should be particularly suitable for studies with RSV. Surface antigen genes from a variety of have been expressed by recombinant vaccinia viruses. When properly engineered, the proteins were synthesized, processed, and transported to the plasma membrane. In most cases, experimental animals were shown to produce neutralizing antibodies and were protected against a challenge with the corresponding virus. Evidence for priming of a cytotoxic T-cell response also has been obtained with recombinant vaccinia viruses that express influenza virus genes (25,26). We now describe a recombinant vaccinia virus that expresses the RSV G glycoprotein and protectively immunizes cotton rats against lower respiratory tract infection with RSV.
MATERIALS AND METHODSViruses and Cells. Vaccinia virus (WR strain) was grown in HeLa (human) cells and purified from cytoplasmic extracts by sucrose gradient centrifugation. RSV (strain A2) was propagated on HEp-2 (human) cell monolayers.Isolation of DNA. DNA was isolated from purified vaccinia virus as described (27). Plasmid DNA was prepared by using a modified alkaline sodium dodecyl sulfate (NaDodSO4) procedure (28). Lysozyme was omitted from the lysis step and the DNA was purified from RNA by passage through a Sepharose-4B column. DNA fragments were' purified from agarose gels by e...
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