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.

Streptococcus suis ( S. suis ) and Pasteurella multocida ( P. multocida ) are pathogens that can cause zoonotic diseases. P . multocida toxin (PMT) is an important virulence factor that causes atrophic rhinitis in pigs. Suilysin (Sly) is an extracellular protein of S. suis and has been shown to be a potential adjuvant. Previous studies have indicated that subunit vaccines containing several fragments of PMT as antigens are safer than traditional inactivated or live-attenuated vaccines. However, protein-based vaccines need strong adjuvants to enhance their immunogenicity. In this study, recombinant PMT-NC (rPMT-NC) protein antigen was formulated with either recombinant Sly (rSly) or CpG oligodeoxynucleotides (CpG) as the adjuvant. The immune responses elicited by these vaccines and the protective efficacy after challenge with live P. multocida were evaluated in piglets. In the dose-dependent test, piglets immunized with the low dose (100 µg) of rSly had increased antigen-specific total IgG, interferon (IFN)-γ gene expression, and CD4 + and CD8 + T-cell populations. Compared to piglets in the commercial (Al-gel) adjuvant and the control groups ( p < 0.05), piglets in the biological adjuvant groups showed significantly reduced turbinate atrophy, nasal distortion, and lung lesion scores after challenge with P. multocida serotype A. Vaccines containing rSly or CpG adjuvant enhanced humoral and cellular immune responses and protection against P. multocida . This combination of a protein-based antigen formulated with a biological adjuvant showed synergistic and protective effects against atrophic rhinitis and has potential to be developed as part of a bivalent vaccine.

Background Cyclic peptide nanotubes (cPNTs) formed from the spontaneous beta-sheet stacking of peptide rings may serve as a safe and effective oral delivery vehicle/adjuvant for DNA vaccines. Aim In this study, we sought to determine if a DNA vaccine expressing the VP2 protein of goose parvovirus, adjuvanted with cPNTs, may elicit virus-specific antibody response through oral vaccination. Material and methods Forty 20-day-old Muscovy ducks were randomly assigned to two groups of 20 ducks each and vaccinated. Ducks were orally vaccinated (Day 0) and boosted (Day 1 and Day 2) or were mock-vaccinated with saline as the negative control. For immunohistochemical staining, the primary antibody used comprised a rabbit anti-GPV antibody, and the secondary antibody was a goat anti-rabbit antibody. Goat-anti-mouse-IgG was used as a tertiary antibody. IgG and IgA antibody titers in serum were analyzed by the GPV virus-coated ELISA. For IgA antibody analysis, intestine lavage was harvested too. Results A DNA vaccine, coated with cPNTs, can induce a significant antibody response in ducklings. Immunohistochemical staining of tissues from vaccinated ducklings showed that VP2 proteins can be detected in the intestines and livers for up to six weeks, confirming the antigen expression by the DNA vaccine. Antibody analysis found that this vaccine formulation was very efficient at inducing IgA antibodies in the serum and the intestinal tract. Conclusion A DNA vaccine adjuvanted with cPNTs can effectively express the antigen and can significantly induce an antibody response against goose parvovirus through oral vaccination.

Aims Riemerella anatipestiferinfections of goslings and ducklings can result in high mortality. Since there are at least 21 serotypes ofR. anatipestifer, cross-protection is an important goal for vaccine development. Methods and Results In this study, we evaluated the immunostimulatory effect of different immunization regimens - the traditional inactivated vaccinevsprime-boost regimens using DNA and protein subunit vaccines (DNA+subunit, subunit+subunit, subunit+inactivated and DNA+DNA). Results showed that, when compared to the inactivated vaccine, prime-boost regimens induced higher and up to 16-week longer lasting levels of antibody responses, significantly elevated the percentage of the cytotoxic CD8(+)T cell and higher expression levels of IFN-gamma, IL-6 and IL-12 mRNAs. Furthermore, as an indication of cross-protection, sera from prime-boost regimens were able to recognize lysates ofR. anatipestiferserotypes 1, 2 and 6. Conclusions Prime-boost regimens especially DNA-prime and protein-boost, induce strong long-term immune response and may prove protective for breeder ducks requiring long-term protection. Significance and Impact of the Study It is worth mentioning that the subunit+inactivated regimen group also elicited strong immune response. The cost of this regimen may only be half of the other prime-boost regimens, making this subunit + inactivated combination an attractive option.