title: L1R, A27L, A33R and B5R vaccinia virus genes expressed by fowlpox recombinants as putative novel orthopoxvirus vaccines.
abstract: BACKGROUND
  The traditional smallpox vaccine, administered by scarification, was discontinued in the general population from 1980, because of the absence of new smallpox cases. However, the development of an effective prophylactic vaccine against smallpox is still necessary, to protect from the threat of deliberate release of the variola virus for bioterrorism and from new zoonotic infections, and to improve the safety of the traditional vaccine. Preventive vaccination still remains the most effective control and new vectors have been developed to generate recombinant vaccines against smallpox that induce the same immunogenicity as the traditional one. As protective antibodies are mainly directed against the surface proteins of the two infectious forms of vaccinia, the intracellular mature virions and the extracellular virions, combined proteins from these viral forms can be used to better elicit a complete and protective immunity.
  
  
  METHODS
  Four novel viral recombinants were constructed based on the fowlpox genetic background, which independently express the vaccinia virus L1 and A27 proteins present on the mature virions, and the A33 and B5 proteins present on the extracellular virions. The correct expression of the transgenes was determined by RT-PCR, Western blotting, and immunofluorescence.
  
  
  CONCLUSIONS
  Using immunoprecipitation and Western blotting, the ability of the proteins expressed by the four novel FPL1R, FPA27L, FPA33R and FPB5R recombinants to be recognized by VV-specific hyperimmune mouse sera was demonstrated. By neutralisation assays, recombinant virus particles released by infected chick embryo fibroblasts were shown not be recognised by hyperimmune sera. This thus demonstrates that the L1R, A27L, A33R and B5R gene products are not inserted into the new viral progeny. Fowlpox virus replicates only in avian species, but it is permissive for entry and transgene expression in mammalian cells, while being immunologically non-cross-reactive with vaccinia virus. These recombinants might therefore represent safer and more promising immunogens that can circumvent neutralisation by vector-generated immunity in smallpox-vaccine-experienced humans.
authors.affiliation: Department of Medical Biotechnologies and Translational Medicine, University of Milan, via Vanvitelli 32, 20129 Milan, Italy.
authors.forename: Sole Maria
authors.initials: SM
authors.lastname: Pacchioni
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Bissa
authors.forename: Carlo
authors.initials: C
authors.lastname: Zanotto
authors.forename: Carlo De Giuli
authors.initials: Cde G
authors.lastname: Morghen
authors.forename: Elena
authors.initials: E
authors.lastname: Illiano
authors.forename: Antonia
authors.initials: A
authors.lastname: Radaelli
mesh_terms: D000818:Animals; D002642:Chick Embryo; D002522:Chlorocebus aethiops; D005347:Fibroblasts / Q000378:metabolism; D005587:Fowlpox virus / Q000235:genetics*; D005814:Genes, Viral; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D009500:Neutralization Tests; D012900:Smallpox Vaccine / Q000235:genetics* / Q000276:immunology; D019076:Transgenes; D014614:Vaccines, Synthetic / Q000235:genetics* / Q000276:immunology; D014616:Vaccinia virus / Q000235:genetics*; D014709:Vero Cells; D014765:Viral Vaccines / Q000235:genetics*
chemical_list: D012900:Smallpox Vaccine; D014614:Vaccines, Synthetic; D014765:Viral Vaccines