Effects of Canine IL-12 on the Immune Response Against the Canine Parvovirus VP2 Protein

Abstract

Background: Canine parvovirus (CPV) is a highly pathogenic virus that predominantly affects puppies, with mortality rates exceeding 70%. Although commercial multivalent live attenuated vaccines (MLV) are widely employed, their efficacy is often compromised by maternal antibody interference. Consequently, the development of novel vaccines remains imperative for effective CPV control. Methods: Recombinant CPV VP2 protein (rVP2) and canine interlukine 12 protein (rcIL-12) were expressed using the Bac-to-Bac baculovirus expression system and the biological activity of these proteins was assessed through hemagglutination, Cell Counting Kit-8 (CCK8) and IFN-γ induction assays. The combined immunoenhancement effect of rVP2 and rcIL-12 protein was evaluated in puppies. Results: Both rVP2 and rcIL-12 were successfully expressed and purified, exhibiting confirmed antigenicity, immunogenicity, and bioactivity. Co-administration of rVP2 with rcIL-12 elicited higher neutralizing antibody titer (6-7 times higher), complete challenge protection efficiency (no clinical symptoms and tissue and organ lesions), fewer viral shedding (decreasing significantly 8-day post challenge) and superior viral blockade (lower viral load in the organism) compared to rVP2 alone. Conclusions: Our findings demonstrate that rVP2 co-administered with rcIL-12 induces robust protective immunity in puppies and significantly mitigated the inhibitory effects of maternal antibodies. This represents a promising strategy for enabling earlier vaccination in puppies and rational design of CPV subunit vaccines.

Keywords: IL-12; VP2; baculovirus expression system; canine parvovirus.

Figure 1

SDS-PAGE and Western blot analysis (A) WB probed with anti-CPV antibody. The Arrow marks the 69.5 kDa target band. (B) WB probed with canine IL-12 antibody. The Arrow marks the 65.9 kDa target band. (C) Coomassie blue-stained SDS-PAGE gel of rVP2. (D) Coomassie blue-stained SDS-PAGE gel of rcIL-12.

Figure 2

Bioactivity analysis of recombinant. (A) Hemagglutination assay. rVP2 protein were incubated with 0.1% guinea pig RBCs. PBS served as a control. (B) Cell proliferation assay. Canine PBMCs were treated with rcIL-12 and canine IL-2. Proliferation was measured by CCK-8 absorbance (450 nm). (C) IFN-γ induction. Canine PBMCs were stimulated with diluted rcIL-12. IFN-γ transcript level in PBMC was measured using RT-PCR. (D) Supernatants of (C) in 4-fold diluted were quantified by ELISA kit. Data represents mean ± S.D. (n = 5). ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001 and ns indicates no significance.

Figure 3

Evaluation of rVP2-IL-12 vaccine efficacy in challenged beagles in rVP2 (red), rVP2+IL-12 (blue) and control (black) groups. (A) Clinical scores (0–10 points). (B) Survival curves (14-day observation). Mortality: rVP2 (3/3), rVP2+IL-12 (0/3), control (3/3). (C) Neutralizing antibody titers at day 7 post-vaccination interval. (D) Virus shedding (qPCR, copies/μL) days 1–8 post-challenge. Data represents mean ± S.D. (n = 3). * indicates p < 0.05, ** indicates p < 0.01, ns indicates no significance, *** indicates p < 0.001, and **** indicates p < 0.0001.

Figure 4

Histopathological and immunohistochemical analysis. (A) HE staining. Representative images show, e.g., inflammatory infiltration, necrosis, or tissue structure. Scale bar: 200 µm. (B) IHC staining for CPV. Red-brown HRP signals indicate CPV positioning propagation. Scale bar: 200 µm. (C) Quantitative analysis of IHC signals. CPV positive cells were counted in random fields per sample and analyzed. Data represents mean ± S.D. (n ≥ 5). * indicates p < 0.05, ** indicates p < 0.01 and **** indicates p < 0.0001.

Figure 4

Histopathological and immunohistochemical analysis. (A) HE staining. Representative images show, e.g., inflammatory infiltration, necrosis, or tissue structure. Scale bar: 200 µm. (B) IHC staining for CPV. Red-brown HRP signals indicate CPV positioning propagation. Scale bar: 200 µm. (C) Quantitative analysis of IHC signals. CPV positive cells were counted in random fields per sample and analyzed. Data represents mean ± S.D. (n ≥ 5). * indicates p < 0.05, ** indicates p < 0.01 and **** indicates p < 0.0001.