Recombinant Hexon Protein as a New Bovine Adenovirus Type 3 Subunit Vaccine Candidate.

Introduction: Bovine adenovirus (BAdV) type 3 causes respiratory and gastroenteric diseases of varying severity in cattle, particularly newborn calves. Trials have been conducted of a vaccination against the diseases caused by BAdV using both modified live-virus and inactivated-virus preparations in cattle, but no commercial BAdV-3 vaccine has yet reached the market. Therefore, there is an urgent need to develop new, safe, and effective vaccines against BAdV-3. Material and Methods: Recombinant hexon protein (rhexon) of BAdV-3 was expressed in the E. coli system to evaluate immune response in mice and goats. Antibody responses and cytokine levels were analysed and the effects of administrations of different amounts of recombinant protein compared. Long-term antibody production was evaluated by indirect ELISA, and the total immunoglobulin G secreted by goats and mice immunised with the purified rhexon protein was determined. Results: The immunised mice had a stronger antibody response than the control group at eight weeks post vaccination. The immunised groups also showed significantly higher (P ˂ 0.05) expression of interferon-γ, interleukin 2 (in mice), and interleukin 21 (in goats) at four weeks. Furthermore, vaccination with rhexon was able to induce long-term antibody production for at least 16 weeks in mice and goats. Conclusion: The rhexon protein induced immune responses, especially long-term antibody production and T helper 1 cell cytokine production in mice and goats. The immunogenic properties of this protein make it a promising subunit vaccine antigen.

Immunogenicity of a secreted, C-terminally truncated, form of bovine viral diarrhea virus E2 glycoprotein as a potential candidate in subunit vaccine development.

Both current live, attenuated, and killed virus vaccines for bovine viral diarrhea virus (BVDV) have their limitations. Here, we report the development of a BVDV subunit vaccine by (i) the expression of a secreted form of a recombinant E2 glycoprotein using BHK21 cells and (ii) determination of the immune responses in mice. The E2 glycoprotein was modified by deletion of the C-terminal transmembrane anchor domain and fusion to a V5 epitope tag. This allowed detection using anti-V5 monoclonal antibodies together with simple purification of the expressed, secreted, form of E2 from the cell media. Furthermore, we genetically fused green fluorescent protein (GFP) linked to E2 via a Thosea asigna virus 2A (T2A) ribosome skipping sequence thereby creating a self-processing polyprotein [GFP-T2A-BVDV-E2trunk-V5], producing discrete [GFP-T2A] and [E2trunk-V5] translation products: GFP fluorescence acts, therefore, as a surrogate marker of E2 expression, BALB/c mice were inoculated with [E2trunk-V5] purified from cell media and both humoral and cellular immune responses were observed. Our antigen expression system provides, therefore, both (i) a simple antigen purification protocol together with (ii) a feasible strategy for further, large-scale, production of vaccines.

Cloning and characterization of a 7 transmembrane receptor from the adherent cells of chicken peripheral blood mononuclear cells.

A cDNA encoding a 7 transmembrane (7TM) receptor gene from the adherent cells of chicken peripheral blood mononuclear cells (PBMC) was cloned and characterized. The open reading frame of the chicken-7TM (Ch-7TM) receptor gene was 1008 nucleotides long, encoding a protein of 335 amino acid residues with a molecular mass of approximately 37.1 kDa. Hydrophobic stretches indicated the presence of 7 TM domains. Moreover, the complete nucleotide sequences encoding 7TM of duck (Du-7TM) and goose (Go-7TM), corresponding to the open reading frame of Ch-7TM, were determined. Each of the Du- and Go-7TM encoding regions comprised 990 nucleotides, representing an 18-nucleotide deletion in alignment with the Ch-7TM encoding region, resulting in a 6-amino-acid deletion at the 3'-end. No signal peptides were predicted. Six phosphorylation sites were predicted and conserved for all three 7TMs. The proteins of the three 7TMs were similar, with 11 conserved cysteine residues. No glycosylation sites could be predicted. The results of the pairwise comparisons indicated that the Ch-7TM encoding region and Ch-7TM protein were the least similar to those of Du- and Go-7TMs. These results were in accordance with those of the phylogenetic analysis, which indicated that the Du- and Go-7TM encoding regions clustered, but were separated from the Ch-7TM encoding region. Monoclonal antibody B28D5 was prepared from spleens of mice immunized with the bacterially expressed N-terminal (55 amino acid residues) region of the Ch-7TM protein for further use. Double staining with B28D5 and KUL01 suggested that Ch-7TM was expressed in subsets of the adherent cells, among which a subset that was recognized with both antibodies was likely of monocyte and macrophage lineage. However, the fluorescence intensities of B28D5 and, particularly, KUL01 decreased after the adherent cells were incubated for additional 48 h.