Ethics statement
This study was approved by the Animal Care and Use Committee of the Jiaxing University with protocol number JUMC2019007. All animal experiments were performed in accordance with the guidelines of the Jiaxing University Animal Care and Use Committee. Immunization and sampling were performed under anesthesia.
Cell lines and virus gene
Sf9 insect cells were maintained in SF900II (Life Technologies, San Diego, CA, USA) at 27 °C in cell culture, and CDV H gene was obtained from giant panda/SX/2014. A recombinant Onderstepoort strain expressing green fluorescent protein used for virus neutralization was constructed and generated using a reverse genetic system based on RNA polymerase II for CDV by our laboratory.
Purification and characterization of recombinant CDV hemagglutinin proteins
We constructed recombinant plasmid-GCN4 sequence-stabilized tetrameric H (tH), consisting of a signal peptide-encoding sequence from honeybee melittin to facilitate protein expression in sf9 cells, full-length CDV H gene, foreign tetramerization motif GCN4, and His tag gene at the C-terminal (Fig. 1a). The full CDV H fusion gene was cloned into pFastbacI, and recombinant plasmid was transformed into DH10 Bac Escherichia coli (Life Technologies, San Diego, CA, USA) to obtain recombinant bacmid. The bacmid was transfected into sf9 to produce recombinant baculovirus (rBV) after bacmid was identified. rBV expression was generated using a Bac-to-Bac system (Invitrogen, Grand Island, NY, USA). Recombinant H protein was purified by infecting sf9 cells with rBVs at a MOI of 1 and incubated at 27 °C for 48 h. Supernatants were collected, and recombinant CDV H protein was purified using a His tag purification kit (Beyotime, Beijing, China). The sample of infected sf9 cells and pure recombinant CDV H protein was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blot analysis. Flagellin expression was determined using the Bac-to-Bac system, purified through his tag, and stored at − 80 ℃ for further use.
Preparation of nanoparticles
Exactly 1 μg of recombinant CDV H pure protein was incubated at room temperature in the presence of Bis [sulfosuccinimidyl] (BS3) at final concentrations of 6 mM for 30 min. Then, 1 M of Tris–HCl (pH 8.0) was added to reach a final concentration of 50 mM and stop the crosslinking reaction. Then, the sample was separated via SDS-PAGE followed by Western blot analysis using an anti-his tag antibody (Beyotime, Beijing, China) to identify hemagglutinin tetramers (tetrameric hemagglutinin [tH]).
Immunization and sampling
Female 6-week-old BALB/c mice were randomly divided into three groups of 15 mice each group. The mice were intranasally (i.n.) and intramuscularly (i.m.) immunized with 10 μg of soluble recombinant H protein (group 1, G1), 10 μg of nanoparticle tH (group 2, G2), or 10 μg of nanoparticle tH + 1 μg of flagellin (group 3, G3) at weeks 0, 4, and 8, respectively (Fig. 1b). Based on our previous reports, flagellin is an effective mucosal adjuvants [19]. Sera and nasal samples were collected 2 weeks after each immunization (we tested the samples collected previously). Lymphocytes from the spleen and lymph node were collected after the last sample was collected and used for ELISPOT testing. Inguinal lymph nodes were collected after primary immunization for flow cytometry assays.
Neutralization assay and ELISA
CDV H-specific antibody titers, IgG, IgG1, IgG2a, and IgA in immune samples were detected by ELISA using purified recombinant CDV H protein as coating antigens at 1 μg/mL. The diluted samples were added to each well and incubated. After washing, the plates were incubated with HRP-conjugated goat anti-mouse IgG, IgG1, IgG2a, and IgA antibodies (Southern Biotechnology Associates, Birmingham, AL, USA). TMB was used to develop the color, and an ELISA reader was used to read the OD value at 450 nm.
Neutralizing antibody was assessed by incubating the double dilution of serum with 100 TCID50 CDV for 1 h and adding it into vero cells at 105 cells/well in 96-well plates. The NA titers were calculated using the method of Reed and Muench.
Cytokine ELISpot
Interferon gamma (INF-γ) and interleukin 4 (IL-4) secretions from immunized mouse splenocytes and lymph node cells were evaluated using ELISpot kits (eBioscience, San Diego, CA) in accordance with the manufacturer’s instructions.
Flow cytometry assays for DCs
The lymph nodes were collected at 3, 6, and 9 days after primary immunization. Single-cell suspensions (1 × 106 cells/mL) were prepared in PBS containing 2% FBS and stained with anti-mouse CD11c, CD80, and CD86 antibodies (BD Biosciences, Franklin, TN, USA) for 30 min at 4 °C. After staining, the labeled cells were washed two times with PBS containing 2% FBS and analyzed using a flow cytometer.
Statistical analysis
The analyses were performed by using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA). P-values less than 0.05 (*P < 0.05) were considered to be statistically significant: **P < 0.01; ***P < 0.001; n.s., P > 0.05.