The recombinant pseudorabies virus expressing porcine deltacoronavirus spike protein is safe and effective for mice

Background Porcine deltacoronavirus (PDCoV) is a new pathogenic porcine intestinal coronavirus, which has appeared in many countries since 2012. PDCoV disease caused acute diarrhea, vomiting, dehydration and death in piglets, resulted in significant economic loss to the pig industry. However, there is no commercially available vaccine for PDCoV. In this study, we constructed recombinant pseudorabies virus (rPRVXJ-delgE/gI/TK-S) expressing PDCoV spike (S) protein and evaluated its safety and immunogenicity in mice. Results The recombinant strain rPRVXJ-delgE/gI/TK-S obtained by CRISPR/Cas gE gene editing technology and homologous recombination technology has genetic stability in baby hamster syrian kidney-21 (BHK-21) cells and is safe to mice. After immunizing mice with rPRVXJ-delgE/gI/TK-S, the expression levels of IFN-γ and IL-4 in peripheral blood of mice were up-regulated, the proliferation of spleen-specific T lymphocytes and the percentage of CD4+ and CD8+ lymphocytes in mice spleen was increased. rPRVXJ-delgE/gI/TK-S showed good immunogenicity for mice. On the seventh day after booster immunity, PRV gB and PDCoV S specific antibodies were detected in mice, and the antibody level continued to increase, and the neutralizing antibody level reached the maximum at 28 days post- immunization (dpi). The recombinant strain can protect mice with 100% from the challenge of virulent strain (PRV XJ) and accelerate the detoxification of PDCoV in mice. Conclusion The recombinant rPRVXJ-delgE/gI/TK-S strain is safe and effective with strong immunogenicity and is expected to be a candidate vaccine against PDCoV and PRV.

Recombinant Pseudorabies Virus with TK/gE Gene Deletion and Flt3L Co-Expression Enhances the Innate and Adaptive Immune Response via Activating Dendritic Cells

Owing to viral evolution and recombination, emerging pseudorabies virus (PRV) strains have caused unprecedented outbreaks in swine farms even when the pigs were previously vaccinated, which might indicate that traditional vaccines were unable to provide effective protection. The development of safe and efficacious vaccines presents prospects to minimize the clinical signs and eventually eradicate the infection. In this study, we used an emerging PRV strain, HNX, as the parental strain to construct a recombinant PRV with TK/gE gene deletion and Fms-related tyrosine kinase 3 ligand (Flt3L) expression, named HNX-TK−/gE−-Flt3L. HNX-TK−/gE−-Flt3L enhanced the maturation of bone marrow derived dendritic cells (DCs) in vitro. Significantly more activated DCs were detected in HNX-TK−/gE−-Flt3L-immunized mice compared with those immunized with HNX-TK−/gE−. Subsequently, a remarkable increase of neutralizing antibodies, gB-specific IgG antibodies, and interferon-gamma (IFN-γ) was observed in mice vaccinated with HNX-TK−/gE−-Flt3L. In addition, a lower mortality and less histopathological damage were observed in HNX-TK−/gE−-Flt3L vaccinated mice with upon PRV lethal challenge infection. Taken together, our results revealed the potential of Flt3L as an ideal adjuvant that can activate DCs and enhance protective immune responses and support the further evaluation of HNX-TK−/gE−-Flt3L as a promising PRV vaccine candidate.

The recombinant pseudorabies virus expressing African swine fever virus CD2v protein is safe and effective in mice

Background African swine fever (ASF) leads to high mortality in domestic pigs and wild boar and is caused by the African swine fever virus (ASFV). Currently, no vaccine is commercially available for prevention, and the epidemic is still spreading. Here, we constructed a recombinant pseudorabies virus (PRV) (PRV-ΔgE/ΔgI/ΔTK-(CD2v)) that expresses the CD2v protein of ASFV and evaluated its effectiveness and safety as a vaccine candidate in mice. Methods A homologous recombination fragment containing ASFV CD2v was synthesized and co-transfected into HEK 293 T cells, a knockout vector targeting the PRV TK gene. The transfected cells were infected with PRV-ΔgE/ΔgI, and the recombinant strain (PRV-ΔgE/ΔgI/ΔTK-(CD2v)) was obtained by plaque purification in Vero cells. The expression of ASFV CD2v in the recombinant virus was confirmed by sequencing, Western blotting, and immunofluorescence analysis, and the genetic stability was tested in Vero cells over 20 passages. The virulence, immunogenicity and protective ability of the recombinant virus were further tested in a mouse model. Results The PRV-ΔgE/ΔgI/ΔTK-(CD2v) recombinant strain is stable in Vero cells, and the processing of CD2v does not depend on ASFV infection. The vaccination of PRV-ΔgE/ΔgI/ΔTK-(CD2v) causes neither pruritus, not a systemic infection and inflammation (with the high expression of interleukin-6 (IL6)). Besides, the virus vaccination can produce anti-CD2v specific antibody and activate a specific cellular immune response, and 100% protect mice from the challenge of the virulent strain (PRV-Fa). The detoxification occurs much earlier upon the recombinant virus vaccination and the amount of detoxification is much lower as well. Conclusions The PRV-ΔgE/ΔgI/ΔTK-(CD2v) recombinant strain has strong immunogenicity, is safe and effective, and maybe a potential vaccine candidate for the prevention of ASF and Pseudorabies.