Newcastle Disease Virus-Vectored African Swine Fever Virus Antigen Cocktail Delays the Onset of ASFV-SY18 but Is Not Protective

Figures

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  Figure 1. Structure of the plasmid pcDNA3.1-rNDV-ASFV. The full-length cDNA of the Newcastle Disease Virus (NDV) vaccine strain La Sota was assembled in pcDNA3.1-HH, a modified plasmid featuring a CMV promoter, a hammerhead ribozyme (HamRz), a hepatitis delta virus ribozyme (HDVRz), and a BGH polyA signal. A gene-start and gene-end sequence (GS-GE), comprising the Kozak sequence and MluI site, was incorporated into the intergenic region between the P and M genes of NDV cDNA. The open reading frame (ORF) of the African Swine Fever Virus (ASFV) genome was introduced at the MluI site to construct the plasmid pcDNA3.1-rNDV-ASFV.

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  Figure 2. The presence of ASFV genes in rNDV-ASFV was confirmed using RT-PCR with primers targeting the ASFV gene. M indicates the DNA marker, followed by the names of the ASFV genes.

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  Figure 3. Reaction of rNDV-ASFV-expressed antigens with serum from a convalescent pig infected with ASFV-SY18. M indicates the protein marker, followed by the names of the ASFV proteins expressed by rNdV-ASFVs and their corresponding gene names in parentheses. Positive bands are marked with dark-red arrows.

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  Figure 4. Immune response in mice after injection with rNDV-ASFVs expressing immunodominant antigens. (A) Virus neutralization (VN) is expressed as the percentage of positive serum relative to negative PBS serum, with the latter indicating a neutralization value of 0. Values above the baseline indicate a neutralizing effect against the virus, while values below the baseline may suggest enhanced viral proliferation. (B) ASFV antigen-specific T-cell proliferation of mice splenic lymphocytes was tested by a Cell Counting Kit-8 assay. Cells were stimulated with rAdV-ASFVs expressing the same ASFV proteins as rNDV-ASFVs, while ConA stimulation served as a control. The data are presented as the mean with SD. Comparisons were made using the parametric unpaired t-test. * p < 0.05; ** p < 0.01.

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  Figure 5. Clinical and virological parameters in the pigs post-challenge. Rectal temperature (A) and the clinical score (B) were measured for each pig. The dotted line represents the normal rectal temperature for pigs (40.0 °C); a temperature exceeding 40.0 °C is classified as fever. (C) Survival rates were evaluated by a Kaplan–Meier curve. Different tissues from autopsies (D) as well as blood (E), oral swabs (F), and anal swabs (G) collected at different time points were tested for ASFV viral load. The green lines in (A,B,E–G) represent the control group, while the red lines represent the rNDV-ASFV group.

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  Figure 6. Immune responses post-inoculation and post-challenge in pigs. (A) Antibodies against NDV in porcine serum samples were detected by an HI assay. (B) Antibodies to the ASFV p72 were assessed by a blocking ELISA, with samples showing a percent of inhibition (PI) of < 40% classified as negative. (C) ASFV-specific IFN-γ-secreting PBMC responses were detected by ELISPOT. The data are presented as the mean with SD. Comparisons were made using the parametric unpaired t-test. * p < 0.05; ** p < 0.01.

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  Figure 7. The kinetics of serum cytokines. The concentrations of the pro-inflammatory cytokines TNF-α (A–C), IL-1β (D–F), and IL-12 (G–I) as well as the anti-inflammatory cytokine IL-10 (J–L) were quantified using ELISA. Panels (B,E,H,K) show the levels of TNF-α, IL-1β, IL-12, and IL-10 before inoculation and 14 days post-prime. Panels (C,F,I,L) show the levels before the challenge and 14 days post-challenge. The data are presented as the mean with SD. Comparisons were made using the parametric unpaired t-test. * p < 0.05; ** p < 0.01.