A Triple Gene-Deleted Pseudorabies Virus-Vectored Subunit PCV2b and CSFV Vaccine Protect Pigs against a Virulent CSFV Challenge

Abstract

Classical swine fever (CSF) remains one of the most economically significant viral diseases affecting domestic pigs and wild boars worldwide. To develop a safe and effective vaccine against CSF, we have constructed a triple gene-deleted pseudorabies virus (PRVtmv)-vectored bivalent subunit vaccine against porcine circovirus type 2b (PCV2b) and CSFV (PRVtmv+). In this study, we determined the protective efficacy of the PRVtmv+ against virulent CSFV challenge in pigs. The results revealed that the sham-vaccinated control group pigs developed severe CSFV-specific clinical signs characterized by pyrexia and diarrhea, and became moribund on or before the seventh day post challenge (dpc). However, the PRVtmv+-vaccinated pigs survived until the day of euthanasia at 21 dpc. A few vaccinated pigs showed transient diarrhea but recovered within a day or two. One pig had a low-grade fever for a day but recovered. The sham-vaccinated control group pigs had a high level of viremia, severe lymphocytopenia, and thrombocytopenia. In contrast, the vaccinated pigs had a low-moderate degree of lymphocytopenia and thrombocytopenia on four dpc, but recovered by seven dpc. Based on the gross pathology, none of the vaccinated pigs had any CSFV-specific lesions. Therefore, our results demonstrated that the PRVtmv+ vaccinated pigs are protected against virulent CSFV challenge.

Keywords: CSFV challenge; PCV2 capsid; PRV trivalent vaccine; classical swine fever virus (CSFV); glycoproteins E2 and Erns; granulocytic monocyte-colony stimulating factor (GM-CSF); pig; pseudorabies virus; subunit vaccine; triple mutant; vaccine efficacy; vectored vaccine.

Figure 1

PRVtmv+ immunization in pigs. (A) Schematic showing the PRVtmv+ vaccination, sample collection, and CSFV challenge scheme for the animal experiment. (B) Pigs (n = 5 in each group) were immunized with PRVtmv+ vaccine (for each pig, intranasally—8 × 10⁷ plaque forming units (PFU), and subcutaneously—4 × 10⁷ PFU; 0 days post vaccination (dpv)), or sham-vaccinated and sera samples were collected on 0 and 28 dpv to determine the classical swine fever virus (CSFV)-specific neutralizing antibody titer [33]. The dot plot graph shows each animal’s individual CSFV titer with the mean values of the group (n = 5). *** p < 0.001.

Figure 2

Clinical assessment, survival curve, and classical swine fever virus (CSFV)-associated viremia in sham-vaccinated control and PRVtmv+ vaccinated pigs following CSFV challenge. (A) Pigs were challenged with CSFV at 28 days post vaccination (0 days post challenge (dpc)), and rectal temperature was recorded until 21 dpc. Mean rectal temperatures with standard deviation (SD) are given (n = 5). All pigs in the CSFV control group were euthanized on days 6 and 7 dpc. (B) A survival rate is given in terms of percentage in each group (n = 5). (C) Blood samples were collected from pigs on days 0, 4, 7, 14, and 21 post challenge, and the CSFV titer was determined in cell culture. The mean CSFV titer in the blood of each animal from both groups is shown. The dot plot graph represents mean + individual values in each group (n = 5). TCID50—50% tissue culture infectious dose; * p < 0.05 and *** p < 0.001.

Figure 3

Percent changes in leukocyte, lymphocyte, and platelet count following the classical swine fever virus (CSFV) Brescia strain (BICv) challenge in both groups. Whole blood was collected from pigs on 28 dpv/0 dpc and 49 dpv/21 dpc, (A) leukocyte, (B) lymphocyte, and (C) platelet counts were determined, and their percent changes were calculated. The normal range in pigs: (i) leukocytes—11–22 × 10³/μL; (ii) lymphocytes—4.6–12 × 10³/μL; (iii) platelets—200–500 × 10³/μL [34].

Figure 4

Photograph of spleen, kidney, and tonsils of PRVtmv+ vaccinated pigs (#84, #87, #88, #91, and #92) euthanized at 21 days post challenge. Spleen (A–E), kidney (F–J), and tonsils (K–O). The spleen, kidney, and tonsils lack CSFV-specific lesions. The focal areas marked with circles in D most likely represent areas of blood pooling/incomplete extrusion of blood; this is sometimes seen in the spleens of various species during postmortems after euthanasia using barbiturate compounds. Splenic infarctions, even red-type infarcts, are usually sharply demarcated from the rest of the parenchyma.