Establishment of a Bovine Herpesvirus 4 based vector expressing a secreted form of the Bovine Viral Diarrhoea Virus structural glycoprotein E2 for immunization purposes

A recombinant bovine herpesvirus 4 (BoHV-4CMV-IgKE2-14ΔTK) expressing an enhanced secreted form of the bovine viral diarrhea virus (BVDV) structural glycoprotein E2 (gE2-14), obtained by the removal of the putative transmembrane domain and addition of a 14 amino acids peptide at its carboxyl terminal and an immunoglobulin K signal peptide to the amino terminal, was successfully constructed using a Recombineering (recombination -mediated genetic engineering) approach on BoHV-4 cloned as bacterial artificial chromosome. The galactokinase – based recombineering system was modified by the introduction of a kanamycin expression cassette and a kanamycin selection step that allowed a significant reduction of the untargeted background clones. BoHV-4CMV-IgKE2-14ΔTK infected cell lines highly expressed gE2-14, which maintained native antigenic properties in a serum neutralization inhibition test. When rabbits and sheep were immunized with BoHV-4CMV-IgKE2-14ΔTK, high levels of serum neutralized antibodies against BVDV were generated.This work highlights the engineerization of BoHV-4 genome as a vector for vaccine purposes and may provide the basis for BVDV vaccination exploiting the BoHV-4- based vector that delivers an improved secreted version of the BVDV structural glycoprotein E2.Bovine herpesvirus 4 (BoHV-4) has been isolated from a variety of samples and cells from healthy cattle and from cattle that have experienced abortion or affected by metritis, pneumonia, diarrhoea, respiratory infection, and mammary pustular dermatitis [1]. The virus was first isolated in Europe from cattle with respiratory and ocular diseases by Bartha et al. [2] and later in the United States by Mohanty et al. [3]. Subsequently, distinct BoHV-4 isolates were obtained both in Europe and in the United States [1,4-6]. However, the pathogenic role of BoHV-4 remains unclear and despite its tropism for bovine endometrial cells [7], experimental disease has been reproduced by only a limited number of i