Construction and Evaluation of Quadruple‐Gene‐Deleted Pseudorabies Virus Platforms for ASFV Antigen Delivery

Figures

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  Figure 1. Immunogenicity of rPRV-p54+p72 and rPRV-CD2v+pp62 in mice. (A) Schematic representation of the immunization protocol. Mice were immunized with rPRV-p54+p72, rPRV-CD2v+pp62, or DMEM at indicated time points. (B) Survival rates of mice after immunization with rPRV-p54+p72 and rPRV-CD2v+pp62. (C) PRVgB specific antibody levels in sera of immunized mice detected by ELISA. (D) p72-specific antibody levels in sera of immunized mice detected by commercial blocking ELISA. (E) Specific antibody levels against p54, p72, CD2v, and pp62 in sera of immunized mice detected by established ELISA methods. Serum samples were collected at different time points post-immunization.

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  Figure 2. Evaluation of immunogenicity and safety of rPRV-p54+p72 and rPRV-CD2v+pp62 immunization in piglets. (A) Schematic diagram of the prime-boost vaccination schedule in pigs. (B) Survival rate of pigs. (C) Body temperature curve of piglets. (D) Changes in serum IFN-γ levels in piglets after immunization with recombinant virus strains. (E) Effects of p54, p72, CD2v, and pp62 antigen stimulation on PBMCs proliferation in piglets. (F) No histopathological lesions observed in various organs of piglets following immunization with rPRV-p54+p72 and rPRV-CD2v+pp62. H&E, hematoxylin and eosin.

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  Figure 3. Construction of recombinant virus strains rPRV-p54+p72 and rPRV-CD2v+pp62. (A) Verification of the optimized ASFV p54, p72, CD2v, and pp62 gene expression in BHK-21 cells at 24 h by IFA. (B) and (C) Schematic diagram of the construction and purification of recombinant virus strains rPRV-p54+p72 and rPRV-CD2v+pp62. (D) PCR identification of UL24/TK and gI/gE deletions, along with ASFV p54, p72, CD2v and pp62 insertions in recombinant viruses: rPRV-p54+p72 and rPRV-CD2v+pp62.

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  Figure 4. Characterization of rPRV-p54+p72 and rPRV-CD2v+pp62. (A) Assessment of antigen expression in rPRV-ASFV by immunocytochemistry. (B) Analysis of antigen expression in HEK-293T cells infected with rPRV-p54+p72 and rPRV-CD2v+pp62 by western blot. (C) PCR verification of UL24/TK and gI/gE gene deletions and the stable integration of ASFV p54, p72, CD2v and pp62 genes in rPRV-p54+p72 and rPRV-CD2v+pp62 after 20 serial passages. (D) Growth curves of the parental strain PRV SX-10, rPRV-p54+p72 and rPRV-CD2v+pp62. (E) and (F) Measurement of plaque size and morphology of rPRV-p54+p72, rPRV-CD2v+pp62 and PRV SX-10. The plaque areas were counted and calculated using ImageJ software. ⁣∗∗∗p < 0.001, ns: not significant.

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  Figure 5. Immunohistochemical detection of epitope-tagged ASFV antigens in mouse spleen. Representative images showing positive immunostaining (brown) of ASFV antigens in spleen tissues from mice infected with recombinant PRVs. Flag antibody was used to detect p54 and CD2v expression, while HA antibody was used to detect p72 and pp62 expression in the recombinant virus strains.

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  Figure 6. Effects of p54, p72, CD2v, and pp62 antigen immunization on immune responses in mice. (A) Detection of IL-2, IL-4, and IFN-γ levels secreted by splenocytes from mice immunized with rPRV-p54+p72 and rPRV-CD2v+pp62 using ELISA. (B) Effects of p54, p72, CD2v, and pp62 antigen stimulation on mouse splenocyte proliferation. (C, D) Flow cytometric analysis of CD3+, CD4+, and CD8+ T cell percentages in spleens of mice immunized with different antigens. ⁣∗p < 0.05, ⁣∗∗p < 0.01, ⁣∗∗∗p < 0.001. ns: not significant.

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  Figure 7. rPRV-p54+p72 and rPRV-CD2v+pp62 protected mice against challenge with PRV SX-10 infection. (A) Survival rates of mice after challenge with PRV SX-10 strain. (B) Histopathological lesions in various organs of mice following PRV challenge. Tissue samples were collected as indicated for histopathological examination. (C) Cytokine expression levels of TNF-α, IL-6, and IL-10 in mouse sera were detected at 3 dpc using ELISA. ⁣∗p < 0.05, ⁣∗∗p < 0.01, ⁣∗∗∗p < 0.001, ⁣∗∗∗∗p < 0.0001, and ns as not significant.

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  Figure 8. Changes in specific antibody levels in sera of piglets after immunization with rPRV-p54+p72 and rPRV-CD2v+pp62. (A) Anti-PRVgB antibody titers in piglet sera at different time points after immunization. (B) Detection of p54 and p72 antibody titers in piglet sera using commercial ELISA kits. (C) Detection of p54, p72, CD2v, and pp62 antibody titers in piglet sera using in-house developed ELISA methods.