title: Assembly and analysis of a functional vaccinia virus "amplicon" containing the C-repeat region from the M protein of Streptococcus pyogenes.
abstract: Previous studies have shown that when inoculated intranasally into mice, vaccinia virus (VV) recombinants expressing the carboxyl half of the Streptococcus pyogenes M protein [which contains the C-repeat region (CRR)] could elicit a protective immune response against subsequent challenge by both homologous and heterologous serotypes of pathogenic group A streptococci. In the present study, an insertion plasmid was constructed that contained three tandem in-frame repeats of a 310-base-pair DNA sequence encoding the CRR from streptococcal M6 protein under control of a constitutive viral promoter. The plasmid was used to introduce the bacterial sequences into the VV genome by homologous recombination. Surprisingly, the recombinant VV:CRR3X virus that was isolated appeared to represent not an individual recombinant virus but a complex mixture of variants that contained from 1 to greater than 20 tandem copies of the CRR region at the insertion site. This genomic complexity was mirrored at the transcriptional level in that a nested set of coterminal transcripts was detected in VV:CRR3X-infected cells, which increased in size from 1400 to 6600 bases by increments of approximately 300 bases. All transcripts containing two or more CRR inserts appeared functional, as Western (immuno) blot analyses of VV:CRR3X-infected cell extracts revealed a family of CRR-related proteins with apparent molecular masses that increased from 30 kDa upward in increments of 10 kDa. All data are consistent with the hypothesis that variation in the VV:CRR3X recombinants is from random crossover events that occur within the CRR region during viral DNA replication. These results suggest that the genomic diversity generated by the "recombinogenic" properties of vaccinia recombinants containing tandem foreign inserts could be used to facilitate induction of a broadly protective immune response against antigenically diverse pathogenic agents.
authors.affiliation: Department of Microbiology, Oregon State University, Corvallis 97331-3804.
authors.forename: D E
authors.initials: DE
authors.lastname: Hruby
authors.forename: O
authors.initials: O
authors.lastname: Schneewind
authors.forename: E M
authors.initials: EM
authors.lastname: Wilson
authors.forename: V A
authors.initials: VA
authors.lastname: Fischetti
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002352:Carrier Proteins; D004252:DNA Mutational Analysis; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D014616:Vaccinia virus / Q000235:genetics*
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D009841:Oligonucleotides; D012333:RNA, Messenger; C014689:streptococcal M protein