title: Development and in vitro characterization of recombinant vaccinia viruses expressing bovine leukemia virus gp51 in combination with bovine IL4 or IL12.
abstract: Type 1 and type 2 immune responses are modulated by IL12 or IL4, respectively, at the time of lymphocyte priming. Importantly, type 1 responses have been associated with resistance to retroviral infection in mice, humans, and ruminants. Specifically, vaccination of sheep with vaccinia virus expressing bovine leukemia virus (BLV) gp51 resulted in protective immunity with the characteristics of a type 1 response, whereas vaccination of cattle resulted in a non-protective type 2 response. In order to test the hypothesis that cattle inoculated with BLV gp51 and IL12 will respond with a type 1 response, a recombinant vaccinia virus expressing BLV gp51 together with bovine IL12 was developed and characterized in vitro. For induction of type 2 responses a recombinant vaccinia virus expressing gp51 with bovine IL4 was similarly constructed and characterized. In this study recombinant cassettes were developed containing either the BLVenv gene alone or in combination with bovine IL4 or the two genes, p35 and p40, encoding bovine IL12. Correct alignment with p7.5 or p11 vaccinia promoters and orientation was confirmed by complete sequencing. Recombinant vaccinia viruses were generated by homologous recombination, selected based on large plaque formation due to reconstitution of the vp37 gene, and structurally confirmed by Southern blotting. Transcription of recombinant BLVenv, bovine IL4, p35 and p40 was demonstrated by RT-PCR. Expression of BLVenv gp51 protein and bovine IL4 was shown by immunofluorescence and immunoblotting. Biologically active bovine IL4 expressed by vaccinia virus stimulated lymphoblast proliferation, B lymphocyte proliferation in the presence of CD40L, and inhibited IFN gamma secretion from PHA activated PBMC in a dose dependent fashion. Finally, bovine IL12 expression and biological function was confirmed by dose dependent induction of IFN gamma secretion by PHA activated PBMC and the moderate enhancement of lymphoblast proliferation. In conclusion, bovine IL12 and IL4 expressed by recombinant vaccinia virus in vitro clearly exhibited type 1-type 2 modulating properties.
authors.affiliation: Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman 99164-7040, USA. bvbeust@vetmed.wsu.edu
authors.forename: B R
authors.initials: BR
authors.lastname: Von Beust
authors.forename: W C
authors.initials: WC
authors.lastname: Brown
authors.forename: D M
authors.initials: DM
authors.lastname: Estes
authors.forename: D S
authors.initials: DS
authors.lastname: Zarlenga
authors.forename: T F
authors.initials: TF
authors.lastname: McElwain
authors.forename: G H
authors.initials: GH
authors.lastname: Palmer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D018664:Interleukin-12 / Q000096:biosynthesis / Q000235:genetics*; D015847:Interleukin-4 / Q000096:biosynthesis / Q000235:genetics*; D001909:Leukemia Virus, Bovine / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics*