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United States Department of Agriculture

Agricultural Research Service

Title: Development and in Vitro Characterization of Recombinant Vaccinia Viruses Expressing Blv Gp51 in Combination with Bovine Il4 Or Il12

Authors
item Von Beust, Barbara - WASHINGTON STATE UNIV
item Brown, Wendy - WASHINGTON STATE UNIV
item Estes, D - UNIV OF MISSOURI
item Zarlenga, Dante
item Mcelwain, Terry - WASHINGTON STATE UNIV
item Palmer, Guy - WASHINGTON STATE UNIV

Submitted to: Journal of Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 23, 1998
Publication Date: N/A

Interpretive Summary: Interleukin 12 is a heterodimeric host derived protein that functions as a potent immune modulator in attenuating infectious diseases in a many hosts. However, the mechanism of action of this protein is still not well understood. The purpose for this work was to produce recombinant proteins of two counteractive immune modulators, IL4 and IL12, each simultaneously with a third protein from bovine leukemia virus which has been documented to protect the host against infection. The new gene constructs were then tested for there ability to produce all the proteins necessary to elicit protective type cellular responses. The results from this study indicated that both recombinant IL4 and IL12, when produced in conjunction with a third protein from bovine leukemia virus, functioned as modulators to enhance the activity of the virus protein. These results are significant in that they demonstrate the importance of presenting both the correct protein and immune modulators to the host to elicit protection and will lay the foundation for synthesizing a new class of recombinant vaccine molecules for use in protecting animals against infectious diseases.

Technical Abstract: 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. 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 immunoblotting and immunofluorescence. Biologically active 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 and type 2 modulating properties.

Last Modified: 4/20/2014
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