Human adenovirus type 7 was isolated and preserved in our laboratory. Female BALB/c mice aged 5 weeks old were purchased from the Experimental Animal Center of the Chinese Academy of Agricultural Sciences. The Institutional Animal Care and Use Committee (IACUC) of the Changchun University of Chinese Medicine approved the animal experimental protocols (Approval No. 2021079).

The hAd-7 hexon sequence was obtained from the Human adenovirus 7 strain BJ/CHN/2018 (GenBank: MH355567.1). The hexon gene was inserted into pFastBac HT A vector to obtain pFastBac-hexon recombinant plasmid. The recombinant plasmid was transformed into competent cells of DH10Bac™, coated on a blue-and-white spot screening plate containing tetracycline (10 mg/L), kanamycin sulfate (50 mg/L), gentamicin (7 mg/L) and IPTG/X-gal, and inverted at 37°C for 48 hours. White colonies were selected and added to SOC culture medium, and the recombinant baculocytes were extracted after shaking culture at 37°C for 3 hours. Then, 4 μg of recombinant baculovirus was mixed with 8 μL of Cellfectin II Reagent transfection reagent, and then added to 1.5 × 10⁶ viable Sf9 cells (Insect cell line)/ml. After standing at 27°C, the cell state was observed every day. When 60% of the cells showed CPE, the supernatant was collected as the first generation recombinant baculovirus, which was used to infect SF9 cells and spread to the third generation, and the recombinant baculovirus (rBV) containing the hAd-7 hexon gene was obtained. To produce the recombinant subunit vaccine, 2.5 × 10⁶ viable Sf9 cells (Insect cell line)/ml were infected with rBV at an MOI of 5 and incubated at 27°C for 72 hours. The expressed protein was purified by sucrose gradient ultracentrifugation and analyzed by western blot.

The mouse primary bone marrow cells were isolated according to the methods described in a previous study (Han et al., 2021), and then cultured in SF-900 medium containing 10% Fetal Bovine Serum, 20 ng/ml GM-CSF and 20 ng/ml IL-4 for 6 days. DCs were then collected and subcultured into 6-well plates at 5 × 10⁶ cells per well. After 24h, 1 μg LPS (1μg/ml) and 10 μg rBV-hexon (5μg/ml) were added and the cells were further incubated for 48 hours. The DCs cells were then harvested and stained with FITC dextran (1mg/ml) at 37 °C and 4 °C for 2h respectively. Flow cytometry was used to analyze the difference in the mean fluorescence intensity (MFI). In the DC maturation experiment, supernatants obtained from the culture of antigen treated DCs were collected and used to detect the presence of IL-6 (R&D, cat.DY406-05), IL-12p70 (R&D, cat.M1270), TNFα (R&D, cat. DY410-05) and IFN-γ (R&D, cat. DY485-05) according to the instructions of the ELISA kit.

After euthanasia, the spleen was taken out under sterile conditions and smashed with a 70μm cell strainer to prepare a single-cell suspension. The spleen lymphocytes were extracted with Mouse Spleen Lymphocyte Separation Kit (Solarbio, cat. P8860), and the red blood cells contained in the splenic lymphocytes were removed with Red Blood Cell Lysis Buffer (Solarbio, cat. R1010).

DC (2 × 10⁶ cells) and spleen lymphocytes (2 × 10⁶ cells) were incubated with APC-CD11c (BioLegend, cat.117310), PE-CD80 (BioLegend, cat.104707), PE-CD86 (BioLegend, cat.105007), PE-CD40 (BioLegend, cat.124609), FITC-I-A/I-E (BioLegend, cat.), PE-CD3 (BioLegend, cat.100205), PerCP-CD4 (BioLegend, cat.100431), and FITC-CD8 (BioLegend, cat.100705) antibodies and the proportion of positive cells was analyzed by flow cytometry.

DCs (5 × 10⁶ cells) were pretreated with 100 nM TAK-242 (TLR4 inhibitor, MedChemExpress, USA) or 20 mM PDTC (NF-κB inhibitor, MedChemExpress, USA) for 2 hours, and then incubated with 10 μg rBV-hexon for 48 hours at 37 °C. Cell surface markers were detected by flow cytometry and cytokines were detected by the ELISA kit.

Five-week-old female BALB/c mice were randomly divided into 6 groups (n=6 per group) and immunized with the rBV protein at 0 and 21 days. Blood was collected weekly to detect the level of specific antibody and neutralizing antibody levels were detected according to the methods in previous study (Li et al., 2016). The mice were euthanized on the fifth week and spleens were collected for pathological analysis. Spleen lymphocytes were extracted with the Mouse Spleen Lymphocyte Separation Kit (Solarbio, cat. P8860) and the number of cells was cell numbers were adjusted to 1 × 10⁶/ml with RPMI1640 culture medium. The lymphocyte proliferation assay was performed with inactivated hAd-7 as the specific stimulator and Con A as the positive stimulator, as previously described (Han et al., 2021).

Extracted The extracted mouse spleen lymphocytes cells (2 × 10⁶ cells) were stained with anti-CD3, CD4, and CD8, and then detected by flow cytometry. The cChanges in CD3⁺CD4⁺ and CD3⁺CD8⁺ counts in each group of mice were determined. The cCells were then stained with anti-IL-4 and IFN-γ and flow cytometry was used to detect changes in CD3⁺CD4⁺/IL-4/IFN-γ and CD3⁺CD8⁺/IL-4/IFN-γ in each group of mice.

Mice were observed daily for morphology, movement, respiration, and body weight changes throughout the experimental cycle, and a disease activity index (DAI) score was established (Blattner et al., 2016): (a) weight loss (0 point: no loss; 1-20 points: loss of 1-20%, 20 points: loss of over 20%; ); (b) Fur condition (0 point: shining; 2 points: matte; 4 points: ruffled); (c) Eyes condition (0 point: clear and clean; 3 points: unclean, closed); (d) Posture (0 point: normal; 10 points: hunched; 20 points: massively hunched); (e) Motility (0 point: normal; 1 point: spontaneous but reduced; 2 points: moderately reduced activity; 5 points: motility only after stimulation; 10 points: lethargy; 20 points: self-mutilation); (f) Respiratory condition (0 point: normal; 1 point: slightly changed; 10 points: accelerated breathing + 30% (tachypnoea); 20 points: strongly accelerated breathing + 50%); The daily DAI score is the sum of six items: A, B, C, D, E and F.

The histopathological scores of each tissue were as follows: extremely mild or no - 0 point, mild - 1 point, moderate - 2 points and severe - 3 points ( Table 1 ).

All data are presented as the mean ± SD. We used GraphPadPrism 6.0 to perform statistical analysis or analysis of variance (ANOVA) of unpaired double-tailed Student’s test. P < 0.05 is considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001.

Using the insect expression system, we constructed a recombinant baculovirus rBV expressing adenovirus type 7 hexon protein ( Figure 1A ) and PCR amplification confirmed the successful insertion of the hexon gene (2850 bp) ( Figure 1B ). Subsequently, the recombinant protein rBV-hexon was obtained by infecting SF-9 cells with rBV and subjected to western blot and indirect immunofluorescence experiments with His-labeled antibody. Successful expression of the hexon protein was observed ( Figures 1C, D ).

BMDCs were incubated with PBS, LPS or rBV-hexon and stained with FITC-Dextran for analysis of DC antigen uptake. The results showed that DCs cells in the PBS treatment group (△MFI=48933.64) had the highest antigen uptake, while DCs cells in the rBV-hexon-treated group had significantly reduced antigen uptake (△MFI=9139.25) (P <0.001), which was similar to that in the LPS group (△MFI=6340.29) ( Figure 2A ). Maturation begins when DCs cease taking up antigens, hence, the data suggests that rBV-hexon can could induce dendritic cell maturation.

3. The vaccine can promote DC maturation and induce secretion of pro-inflammatory cytokines

DCs need the assistance of MHC II and the co-stimulatory molecules CD80 and CD86 to participate in the antigen presentation process. In the previous section, we demonstrated that rBV-hexon could induce dendritic cell maturation. To further verify if rBV-hexon affects the expression of DC surface molecules, we first immunized the mice with rBV-hexon for three days and then analyzed the expression levels of CD80, CD86, CD40 and MHCII in CD11c+ spleen lymphocytes by flow cytometry ( Figure 2B ). After immunization with rBV-hexon, Compared with PBS group, the expression of CD80, CD86, CD40, and MHCII was significantly increased significantly after rBV-hexon immunization. compared to the PBS group. The results indicate that rBV-hexon promotes antigen presentation in vivo.

Subsequently, we analyzed the effect of rBV-hexon on the expression of molecules on DCs surface in vitro. After incubating BMDCs with PBS, LPS and rBV-hexon, we analyzed the expression of MHC II, CD80, CD86 and CD40.We noted that DCs in the rBV-hexon group showed significant surface marker expression similar to that in the LPS group ( Figure 2C ). In addition, DC antigen presentation is known to release large quantities of pro-inflammatory cytokines. Therefore, we analyzed the release of TNFα, IFNγ, IL-6, and IL-12p70, and found that rBV-hexon significantly increased the secretion of these four cytokines ( Figure 2D ). These results indicated that rBV-hexon could activate DC maturation and induce the release of a large number of pro-inflammatory cytokines, which in turn could promote the proliferation and differentiation of other immune cells.

Studies have shown that the LPS receptor (TLR4) can recognize viral glycoprotein (Zhang et al., 2003), and that LPS-induced cytokines are mainly associated with NF-κB downstream of the TRL4 pathway (Kawai and Akira, 2011). In this study, we found that the expression levels of rBV-hexon-activated DCs surface markers were similar to those of LPS-activated DCs. Therefore, we hypothesized that rBV-hexon might activate DCs via the TLR4/NFκB pathway. In this study, we used inhibitors to inhibit TLR4 and NF-κB and established that the expression level of DCs surface markers was significantly decreased after PDTC (NF-κB inhibitor) treatment, while only MHC II and CD86 positive cells were significantly decreased after TAK242 (TLR4 inhibitor) treatment ( Figures 3A–D ). In terms of cytokine production ( Figures 3E–H ), both inhibitors inhibited the secretion of TNFα, IFNγ and IL-6, while TAK242 had a minor effect on IL-12p70. Then we analyzed the expression of TRAF6, IKKα/β, IκBα and NF-κB proteins ( Figure 3I ). The result shows that rBV-hexon can activate TRAF6 downstream of TLR4, and then activate IKK. The phosphorylation of IK will activate NF-κB signal transduction pathway. In conclusion, rBV-hexon activates DCs maturation via the TLR4/NF-κB pathway.

Weekly tail vein blood collection and testing for specific antibodies towards adenovirus type 7 were performed over the mice immunization period ( Figure 4A ). During the immune period of mice, blood was collected from the tail vein every week and specific antibodies against adenovirus type 7 were tested ( Figure 4A ). The results showed that the mouse specific antibody titers of mice in the vaccine group was were significantly higher (P <0.05) than that in those of the control group and the adjuvant group. From the second week onwards, the effect of the high dose vaccine group was superior to that of the low dose vaccine group, and the adjuvant (Al(OH)₃) was able to improve the specific antibody levels induced by the vaccine ( Figure 4B ). We then tested for neutralizing antibodies at two weeks after the initial vaccine injection and found that the titer of neutralizing antibodies was 1:50 after the first injection and improved to 1:150 after the second immunization. The adjuvant was able to increase the level of the neutralizing antibodies produced by the vaccine ( Figure 4C ). The above results showed that the vaccine was able to produce high levels of specific and neutralizing antibodies, demonstrating a high level of immunogenicity.

Spleen lymphocytes were isolated from immunized mice and stimulated to induce lymphocyte proliferation. Compared to the adjuvant group and control group, when treated with the adenovirus type 7 antigen, the vaccine group could significantly stimulate the specific proliferation of lymphocytes (P<0.01), but there was no significant difference between the adjuvant and control groups (P>0.05) ( Figure 4D ).

Lymphocyte typing showed that the proportion of CD4 and CD8 positive cells in the vaccine group was significantly higher (P <0.05) than that of the control group ( Figures 5A, B ; S1 ). Furthermore, the levels of CD4⁺IFN-γ and CD4⁺IL-4, CD8⁺IFN-γ and CD8⁺IL-4 in the vaccine group were higher than those in the control group ( Figures 5C–F ; S2-3 ). We also noted that the effectiveness of the vaccine improved in the presence of adjuvant and taken together, these results show that the vaccine is highly immunogenic in vivo. Pathological examination of important organs and muscles at the injection site of the immunized mice revealed no significant pathological changes in the tissues of immunized mice compared to the control group ( Figures 5G, H ). In addition, we can also find from the DAI score of mice that the immunized vaccine did not cause abnormal clinical symptoms in mice ( Figures 5I, J ). These findings imply that the rBV-hexon vaccine is safe for administration.