title: Attenuated poliovirus strain as a live vector: expression of regions of rotavirus outer capsid protein VP7 by using recombinant Sabin 3 viruses.
abstract: The ability to express heterologous antigens from attenuated poliovirus strains suggests the potential for use as live vectored vaccines. Full- or partial-length sequences of the gene encoding rotavirus major outer capsid protein VP7 were cloned into the open reading frame of a full-length cDNA copy of poliovirus Sabin type 3. They were inserted either at the 5' end or immediately after the capsid protein coding region, at the junction between precursors P1 and P2. A protease cleavage site for 3C protease was introduced 3' to the foreign sequences to enable proteolytic processing of the antigen from the poliovirus polyprotein. Infectious viruses were generated from several of the DNA constructs, and the presence of the foreign gene sequences was confirmed by reverse transcription of the viral RNA and PCR amplification. Viruses with inserts of about 300 bases maintained the foreign sequences during passage in Vero cells. Viruses carrying larger sequences were unstable, and deletions were generated within the foreign sequences. Expression of the VP7 polypeptides was demonstrated by immunoprecipitation with specific antiserum of labeled proteins from cells infected with Sabin 3 recombinant viruses. Comparative studies of RNA synthesis showed similar kinetics for Sabin 3 and the Sabin 3/VP7 recombinants. One-step growth curves showed that production of recombinant viruses was slower than that of Sabin 3 and that the final titers were 1 to 1.5 logs lower. Accumulation of VP7-containing precursors in infected cells suggests that slow cleavage at the engineered 3C protease site may be a limiting step in the growth of these recombinant Sabin polioviruses and may influence the permissible size of foreign sequence to be inserted.
authors.affiliation: Lederle-Praxis Biologicals, Pearl River, New York 10965.
authors.forename: N M
authors.initials: NM
authors.lastname: Mattion
authors.forename: P A
authors.initials: PA
authors.lastname: Reilly
authors.forename: S J
authors.initials: SJ
authors.lastname: DiMichele
authors.forename: J C
authors.initials: JC
authors.lastname: Crowley
authors.forename: C
authors.initials: C
authors.lastname: Weeks-Levy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000235:genetics; D001483:Base Sequence; D002213:Capsid / Q000235:genetics* / Q000276:immunology; D036022:Capsid Proteins; D004279:DNA, Viral / Q000235:genetics; D015870:Gene Expression; D005814:Genes, Viral; D005822:Genetic Vectors; D007700:Kinetics; D008969:Molecular Sequence Data; D017955:Poliovirus / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011055:Poliovirus Vaccine, Oral / Q000276:immunology; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D017384:Sequence Deletion; D014709:Vero Cells
chemical_list: D000956:Antigens, Viral; D036022:Capsid Proteins; D004279:DNA, Viral; D011055:Poliovirus Vaccine, Oral; D012367:RNA, Viral; C050779:VP7 protein, Rotavirus