Abstract
BACKGROUND: Canine distemper virus (CDV) hemagglutinin protein (H), as one of the surface glycoproteins of the virus, helps attach the virus to the host cell through its interaction with cell receptors, makes hemagglutinin protein a key target for the development of neutralizing antibodies. METHODS: In this study, H protein sequences were analyzed by conservation analysis and prediction of multiple immune cell epitopes, three possible epitope sequences were identified. The epitopes were ligated to Ferritin vector with linkers to construct pET30a-Ferritin-Hemagglutinin-Canine distemper virus (FHCDV). pET30a-FHCDV was transfected into E.coli BL21, the expressed protein was extracted by nickel column affinity chromatography and dialysis concentration. Evaluate vaccine efficacy through nasal mucosal and muscular administration. RESULTS: The best expression conditions of FHCDV in E. coli transfected with pET30a-FHCDV were 25 °C and 1 mM Isopropyl β-D-1-Thiogalactopyranoside (IPTG). The results of Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis(SDS-PAGE) and Non-denaturing Polyacrylamide Gel Electrophoresis (Native-PAGE)showed that the purity of purified FHCDV was about 95% and had capable of self-assembly. The results of Dynamic Light Scattering (DLS) and Transmission Electron Microscope (TEM) characterization showed the recombinant protein FHCDV self-assembly into tetrameric caged nanoparticles with a diameter of 16.56 nm. Significant secreted IgA (sIgA) was generated in BALB/C female mice with nasal mucosal immunization. The titers of sIgA antibodies against FHCDV in nasal wash and bronchoalveolar lavage fluid were about 1865 ng/ml and 3943 ng/ml. FHCDV was used as an intramuscular vaccination to inject BALB/C female mice with aluminium adjuvant (Al), Oligodeoxynucleotide (CpG), and polyethyleneimine (PEI) respectively. All immunization groups produced distinct antibodies against the antigenic epitopes of FHCDV. The antibody titer of FHCDV group was 1.28 × 10(4), that of Ferritin-Hemagglutinin- Canine distemper virus + aluminium adjuvant (FHCDV + Al) group was 5.12 × 10(4), Ferritin-Hemagglutinin- Canine distemper virus + Oligodeoxynucleotide (FHCDV + CpG) group was 1.28 × 10(4), and Ferritin-Hemagglutinin- Canine distemper virus + polyethyleneimine (FHCDV + PEI) group was 2.56 × 10(4). The results of cytokine assays showed that FHCDV generated a Th1 immune response. Immunoaffinity protein serum exhibited varying degrees of neutralizing activity against Canine distemper virus-11(CDV-11); ID(50) of FHCDV groups was 39.8, that of FHCDV + Al groups was 106.7, FHCDV + CpG groups was 58.7, and FHCDV + PEI groups was 55.4. FHCDV did not cause damage to mouse organs. CONCLUSIONS: These results indicate that self-assembled multi-epitope FHCDV nano vaccines elicit effective immune responses with a favorable safety profile, providing a promising strategy for canine distemper virus vaccine development.