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. 2022 Aug 4:13:943707.
doi: 10.3389/fmicb.2022.943707. eCollection 2022.

The Inactivated gE/ TK Gene - Deleted Vaccine Against Pseudorabies Virus Type II Confers Effective Protection in Mice and Pigs

Yu-Lan Jin  1   2 , Di Yin  1 , Gang Xing  1 , Yan-Ming Huang  1 , Chun-Mei Fan  1 , Cheng-Fei Fan  1 , Xiao-Huo Qiu  1 , Wei-Ren Dong  1 , Yan Yan  1 , Jin-Yan Gu  1 , Ji-Yong Zhou  1   3
Affiliations

The Inactivated gE/ TK Gene - Deleted Vaccine Against Pseudorabies Virus Type II Confers Effective Protection in Mice and Pigs

Yu-Lan Jin et al. Front Microbiol. .

Abstract

The highly virulent and antigenic variant of Pseudorabies virus (PRV) that emerged from classical Bartha-K61-vaccinated pig herds has caused substantial economic losses to the swine industry in China since 2011. A safe and more effective vaccine is most desirable. In this study, a gE/TK gene-deficient PRV, namely, HD/c, was constructed based on a PRV type II DX strain isolated from a commercial vaccine-immunized farm and the HD/c-based inactivated vaccine was formulated and evaluated for its safety, immunogenicity, and protective efficacy in mice and piglets. The resulting PRV HD/c strain has a similar growth curve to the parental DX strain. After vaccination, the inactivated HD/c vaccine did not cause any visible gross pathological or histopathological changes in the tissues of mice and piglets and provided rapid and potent protection against the challenge of the classical and variant PRVs at day 21 post-vaccination in mice. A single immunization of 108.5TCID50 inactivated PRV HD/c strain-elicited robust immunity with high titer of neutralizing antibody and provided complete protection from the lethal challenge of PRV DX strain in piglets. These results indicated that the inactivated PRV HD/c vaccine with the deletion of gE/TK genes was a safe and effective PRV vaccine candidate for the control of PRV.

Keywords: Pseudorabies virus type II; gE/TK deletion; inactivated vaccine; protection; safe.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Schematic diagrams of recombinant plasmids.
Figure 2
Figure 2
Identification of recombinant PRV. (A) Red fluorescence on Vero cells after the cotransfection of pUC18-gE-loxp-Cherry and PRV DX viral DNA. (B) Vero cells infected with PRV DX gE−/Cherry+. (C) Green fluorescence on Vero cells after the cotransfection of PRV DX gE-viral DNA and pUC18-TK-loxp-EGFP. (D) Green fluorescence on Vero cells infected with PRV DX gE−/TK−/EGFP+. (E) PCR analysis of recombinant PRV (p30) with primers gE332; Lane 1: PRV DX; Lane 2: PRV HD/c; M: DNA ladder 3000. (F) PCR analysis of recombinant PRV (p30) with primers TK623; M: DNA ladder 3000; Lane 1: PRV DX; Lane 2: PRV HD/c. (G) One-step growth curves of the PRV DX and PRV HD/c. Vero cells at high confluence (>90%) were infected (MOI = 10) with the PRV DX or PRV HD/c. The cell cultures were harvested at the indicated time points (4, 8, 12, 18, 24, 30, 36, 48, 60, and 72 h), and titers were determined on the Vero cells. The mean values with standard error of mean are shown.
Figure 3
Figure 3
Immunologic safety assay. (A,B) Pathological changes of injection site of double-dose (0.4 ml) inactivated PRV HD/c and DMEM immunized piglets at 14 dpi, respectively. (C,D) Histopathological observation of inactivated PRV HD/c and DMEM immunized piglets, respectively. Sections were stained with hematoxylin and eosin (HE) and photographed at 100 × magnification. (E,F) Pathological changes of mice immunized with double-dose inactivated PRV HD/c and untreated group at 7 dpi, respectively.
Figure 4
Figure 4
Protective efficacy of inactivated PRV HD/c immunization in mouse model. Survival rates of mice (n = 10) immunized with different dose inactivated PRV HD/c (106.3, 106.8, 107.3, and 107.8 TCID50) challenge with wide type PRV DX strain (105.6TCID50) at 21 dpi (A), 28 dpi (B), and 35 dpi (C). (D) Survival rates of inoculated mice (n = 10) challenged with PRV DX or SC (105.6 TCID50) at 21 dpi.
Figure 5
Figure 5
Protective efficacy of inactivated PRV HD/c immunization in pigs. (A) Survival percentage of piglets after challenge with DX strain. (B) The titers of neutralizing antibody against PRV DX or SC, ***p < 0.0001. (C) PRV gE-specific antibodies were detected with PRV gE Test Kit (IDEXX, USA) based on blocking ELISA. If S/N ratio < 0.6, the sample is positive for gE antibodies. (D) PRV gB-specific antibodies were detected with PRV gB Test Kit (IDEXX, USA) based on blocking ELISA. If S/N ratio < 0.6, the sample is positive for gB antibodies. Standard deviations were shown as error bars.
Figure 6
Figure 6
Pathological changes of inactivated piglets challenged with the PRV strain DX. Groups of pigs (n = 5 per group) were vaccinated with inactivated PRV HD/c (108.5TCID50) or DMEM, and challenged with the PRV strain DX (105.4TCID50) at 21 dpi. The brain, tonsil, lung, and liver of inactivated piglets were collected and subjected to pathological examinations.
Figure 7
Figure 7
Histopathological observation of vaccinated piglets challenged with the PRV strain DX. Groups of pigs (n = 5 per group) were vaccinated with inactivated PRV HD/c (108.5TCID50) or DMEM, and challenged with the strain DX (105.4TCID50) at 21 dpi. Histopathological changes were observed in brain, tonsil, lung and liver of piglets. Sections were stained with hematoxylin and eosin (HE) and photographed at 200 × magnification.
Figure 8
Figure 8
Protective efficacy of maternal antibodies. (A) Survival percentage of piglets after challenge with PRV strain DX. (B) PRV gB-specific antibodies were detected with PRV gB Test Kit (IDEXX, USA) at 0 dpc. If S/N ratio < 0.6, the sample is positive for gB antibodies. (C) PRV gE-specific antibodies were detected with PDRV gE Test Kit (IDEXX, USA) at 0 dpc. If S/N ratio < 0.6, the sample is positive for gE antibodies. Standard deviations were shown as error bars.
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