Development of a Nanoparticle Multiepitope DNA Vaccine against Virulent Infectious Bronchitis Virus Challenge

Infectious bronchitis virus (IBV), belonging to the genus Gammacoronavirus of the family Coronaviridae, is an enveloped, positive-sense, single-stranded RNA virus (1). The IBV genome is ∼27.6 kb in size and encodes 4 structural proteins, i.e., spike (S), membrane (M), small envelope (E), and nucleocapsid (N), and 15 nonstructural proteins (nsp2–16) (2). The S protein can be cleaved into S1 and S2 subunits by host cellular proteases on virus infection. The S1 subunit carries the receptor binding site and plays a critical role in the induction of neutralizing Ab (3–5). The S2 subunit anchors to the viral membrane and is responsible for membrane fusion (1). The IBV N protein is also a major immunogenic protein that induces protective immunity, and several T cell epitopes were identified in our previous study on N protein (6). Compared with S protein, the IBV N protein is more genetically conserved and suitable for the development of a novel cross-protective IBV vaccine (6, 7).

Vaccination is an efficacious approach to control infectious bronchitis (IB). Currently, both live attenuated and inactivated IBV vaccines are widely used in the field (8). However, live attenuated vaccines, produced by serial passage of pathogenic field isolates of IBV in embryonated specific-pathogen-free (SPF) eggs, have some limitations, such as their tendency to revert back to virulence after circulation in the field (9, 10) and their lack of genetic stability, through which new IBV variants are created via mutation and recombination events (11, 12). Inactive IBV vaccines generally elicit poor immune response in chickens and cannot protect chickens against challenge with virulent IBV strains when administrated alone (10). Currently, dozens of IBV variants have been identified in the field, causing significant economic losses to the poultry industry. For these reasons, there is an urgent need to develop a safer and more effective IBV vaccine.

DNA vaccination provides a new and valuable alternative in the development of IBV vaccines. DNA vaccines show significant advantages over live attenuated vaccines because they are safer and more cost effective. Furthermore, DNA vaccines can induce both humoral and cellular immune responses and can be used in the presence of maternal Abs (13). These advantages suggest that DNA vaccines hold great promise as future vaccines for inducing effective immune protection against IBV infection. Despite the advantages of plasmid DNA vaccines, their poor immunogenicity and cellular availability limit their practical use in the field. Chitosan represents a potential gene carrier for improving the effectiveness of DNA vaccines. Chitosan is a nontoxic, biocompatible, and biodegradable cationic polymer, and the positively charged properties of chitosan enable it to interact with negatively charged DNA macromolecules to form chitosan nanoparticles (NPs) of various sizes (14). These NPs not only protect the encapsulated DNA from nuclease degradation but also promote the entry of DNA into cells (15). Chitosan has been used for the delivery of drugs, genes, and vaccines previously (16–18).

In our previous study, we developed a recombinant viral vector-based IBV multiepitope vaccine (rLa Sota/SBNT) based on neutralizing and T cell epitopes. Our results showed that rLa Sota/SBNT could provide significant protection against homologous and heterologous IBV challenge (6). To further explore the potential application of this multiepitope vaccine in the control of IB, in this study, we designed a NP multiepitope DNA vaccine harboring the SBNT expression box gene and used chitosan as the gene carrier. The protective efficacy of the DNA vaccine against challenge with homologous IBV strains was evaluated in SPF chickens.

The QX-like IBV SZ230 and SD strains were stored in our laboratory (19). The 50% embryo infectious dose (EID₅₀) of both strains was determined by inoculating serial 10-fold dilutions of virus into 10-d-old embryonated SPF chicken eggs and was calculated by applying the Reed and Muench method (20). HEK293T cells were cultured in DMEM (Life Technologies, Grand Island, NY) with 10% FBS (Life Technologies). SPF embryonated eggs and SPF white leghorn chickens were purchased from Beijing Boehringer Ingelheim Vital Biotechnology Co. (Beijing, China). Our study was approved by the Animal Welfare and Ethical Censor Committee of China Agricultural University (CAU approval number: 2021–052).

Four T cell epitope peptides (N_211–230: GTRITKAKADEMAHRRFCKR; N_261–280: EGIKDGRVTAMLNLTPSPHA; N_271–290: MLNLTPSPHACLFGSRVTPK; N_381–400: EERNNAQLEFDDEPKVINWG) identified in our previous study (6) were synthesized with high purity (>90%) by Shanghai Apeptide Co. (Shanghai, China). The peptides were dissolved in 70% DMSO (Solarbio, Beijing, China) to a concentration of 10 mM before being stored at −80°C.

The SBNT multiepitope gene expression box was designed as previously described (6). Briefly, four IBV-neutralizing epitope domains (S_24–61, S_132–149, S_291–398, and S_546–567) and four IBV T cell epitope peptides (N_211–230, N_261–280, N_271–290, and N_381–400) were joined together by proper linkers, namely, AAA, AAY, KK, and (G4S)3. The SBNT expression box amino acid residues were converted into corresponding nucleotide sequences, the start codon (ATG) and Kozak sequence (GCCACC) were placed at the N terminus, and the 6×His tag sequence, stop codon (TAA), and CpG motif were added to the C terminus (Fig. 1A, 1B). The SBNT expression box gene was synthesized by GenScript and inserted into a eukaryotic expression vector pcDNA3.1(+) (Invitrogen, Carlsbad, CA) using the HindIII and XhoI sites. In the constructed plasmid, designated pcDNA/SBNT, the sequence of the insert was validated by applying nucleotide sequence analysis.

The expression of SBNT multiepitope protein was detected by Western blotting and indirect immunofluorescence analysis (IFA) assays. For Western blotting, HEK293T cells were first transfected with the constructed plasmid pcDNA/SBNT or empty vector pcDNA3.1(+) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. At 24 h posttransfection, the cells were harvested and lysed with Pierce IP Lysis Buffer (Thermo Fisher Scientific, Waltham, MA). The treated protein samples were separated by 10% SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (Amersham Biosciences, Freiburg, Germany). The membranes were blocked with 5% (w/v) skim milk in TBST buffer (TBS with 0.1% Tween 20) for 2 h at room temperature. Mouse anti-6×His tag mAb (Abcam, Cambridge, MA) or mouse anti–β-actin mAb (Abcam) was used as the primary Ab to detect the multiepitope protein SBNT or cellular protein β-actin, respectively. After being washed three times with TBST buffer, the membranes were incubated with HRP-conjugated goat anti-mouse secondary Ab (Bioss Biotechnology, Beijing, China). Finally, the target proteins were visualized using a BeyoECL Plus kit (Beyotime Biotechnology, Shanghai, China).

For the IFA assays, HEK293T cells were first transfected with pcDNA/SBNT or pcDNA3.1(+) as described earlier. At 24 h posttransfection, the cells were fixed with methanol and then incubated with a mouse anti-6×His tag mAb at 37°C for 1 h. After five washes with PBS, the cells were incubated with FITC-conjugated goat anti-mouse IgG secondary Ab (Bioss Biotechnology) for 1 h at 37°C. Finally, the cells were further washed with PBS and then observed under a fluorescence microscope (Nikon, Tokyo, Japan).

A chitosan solution of 200 μg/ml was prepared by dissolving 0.01 g of chitosan (75–85% deacetylation; Sigma-Aldrich, St. Louis, MO) in 50 ml of 1.0% acetic acid solution (Sigma-Aldrich), and the pH was adjusted to 5.5 before use. The GFP expression plasmid pEGFP-N1 (Clontech, Mountain View, CA) was diluted to a concentration of 100 μg/ml with 10 mM Na₂SO₄ solution (Sigma-Aldrich). The chitosan/DNA NPs were prepared with a complex coacervation method as described previously with some modifications (21). Briefly, 500 μl of chitosan solution and an equal volume of plasmid DNA solution were heated in a 55°C water bath for 30 min separately, and the solutions were then mixed together in equal proportions, vortexed for 40 s, then incubated at room temperature for 1 h to form NPs. These NPs were designated pEGFP-N1-NPs. To evaluate the in vitro transfection efficiency of chitosan/DNA NPs, we seeded HEK293T cells into 24-well plates and grew them to a density of 80%. Then, the supernatant media were replaced with fresh DMEM with 2% FBS, and the pEGFP-N1-NP solutions (containing 2.5 μg of plasmid DNA) were added directly to the HEK293T cells and incubated at 37°C in 5% CO₂. The cells were observed under a fluorescence microscope every 24 h until 72 h to identify GFP⁺ cells. As a positive control, cells were transfected with plasmid pEGFP-N1 (2.5 μg) using Lipofectamine 2000. As a negative control (NC), cells were added to naked plasmid pEGFP-N1 (2.5 μg) or the blank chitosan particle control (the amount of chitosan equivalent to that in pEGFP-N1-NPs was added).

The chitosan NPs containing the constructed plasmid pcDNA/SBNT or empty plasmid pcDNA3.1(+) were prepared as described earlier and were designated pcDNA/SBNT-NPs and pcDNA-NPs, respectively. A gel retardation assay was applied for evaluating plasmid DNA condensation by loading mixtures onto a 1% agarose gel and running at 120 V for 30 min. To evaluate the ability of chitosan NPs to protect the plasmid DNA from DNase digestion, we incubated the naked plasmid pcDNA/SBNT (1 μg) and the pcDNA/SBNT-NP solutions (containing 1 μg of DNA) with 10 U of DNase I (Takara, Tokyo, Japan) at 37°C for 30 min. The ability of NPs to resist DNase degradation was determined by agarose gel electrophoresis. Spectrophotometry was performed to evaluate the encapsulation efficacy of the chitosan NP DNA vaccine. Briefly, the pcDNA/SBNT-NPs were centrifuged at 16,000 × g for 30 min at 4°C, and the amount of free plasmid DNA in the supernatant was measured with a spectrophotometer. The encapsulation efficacy was calculated as follows: Encapsulation efficacy = [(total amount of DNA) − (nonbound DNA)/total amount of DNA] × 100%.

The morphological and surface characteristics of the prepared NP multiepitope DNA vaccine were observed using a JEM-1230 transmission electron microscope (TEM) (JEOL, Tokyo, Japan). Briefly, the pcDNA/SBNT-NP and pcDNA-NP solutions were vortexed for 2 min; then 20 μl of the solution was transferred onto a carbon-coated copper grid, which was then air-dried at room temperature before viewing under a TEM. To analyze the size distribution and ζ potential of the pcDNA/SBNT-NPs and pcDNA-NPs, the samples were diluted with deionized water and measured using a laser particle size analyzer (Malvern Instruments, Worcestershire, U.K.) at 25°C.

Seven-day-old SPF chickens (n = 72) were randomly divided into four groups with 18 chickens in each group. Chickens in group 1 were vaccinated i.m. with pcDNA/SBNT-NPs (containing 150 μg of plasmid DNA), chickens in group 2 were vaccinated i.m. with pcDNA-NPs (containing 150 μg of plasmid DNA), chickens in group 3 were vaccinated i.m. with an equal volume of PBS, and chickens in group 4 were vaccinated intranasally with 10⁶ EID₅₀/bird of attenuated live vaccine SZ230 strain. All chickens were given a booster immunization after 14 d following the same process. After vaccination, all chickens were observed every day for any adverse reactions. To evaluate the humoral and cellular immune responses induced by the NP multiepitope DNA vaccine, 2 wk after the booster, we collected serum samples from three chickens per group and used them in a virus neutralization assay as described previously (6). In addition, splenocytes were isolated from two chickens per group and were stimulated with the four T cell epitope peptides (N_211–230, N_261–280, N_271–290, and N_381–400) for an ELISPOT assay as described previously (6). To increase any responses present, we stained splenocytes isolated earlier with 0.5 μM CFSE (Invitrogen) and then stimulated them with the four T cell epitope peptides for 5 d with the addition of 2 μg/ml recombinant chicken IL-2 (ChIL-2; Kingfisher Biotech, St. Paul, MN) for a CFSE staining assay as described previously (6).

Two weeks after boost vaccination, all chickens in each group were challenged with 10⁶ EID₅₀/bird of IBV SD strain via the oculonasal route. The birds in each group were monitored daily for 14 d for clinical signs, scoring as described previously (6). Tracheal and oropharyngeal swab samples were collected from chickens of each group at 5 and 7 d postchallenge (dpc) and were used for virus shedding detection by real-time quantitative RT-PCR (qRT-PCR) as described previously (22). At 5 and 7 dpc, three chickens from each group were euthanized, and tracheal ciliary activity was evaluated as described previously (6). Trachea, lung, kidney, and bursa of Fabricius samples were collected and used for viral load detection by real-time qRT-PCR as described previously (22).

Statistical analyses were performed using GraphPad Prism version 6.0. One-way ANOVA, two-way ANOVA, and an unpaired t test were used to evaluate statistically significance differences between various groups. Data are expressed as the mean ± SD. Statistically significant differences are expressed as follows: *p < 0.05, **p < 0.01, and ***p < 0.001.

The SBNT expression box gene was successfully cloned into the eukaryotic expression vector pcDNA3.1(+), and the constructed recombinant plasmids pcDNA/SBNT were verified by sequencing analysis. The constructed plasmids pcDNA/SBNT and empty vector pcDNA3.1(+) were transfected into HEK293T cells, and the expression of multiepitope protein SBNT was confirmed by Western blotting and IFA. Multiepitope protein SBNT was successfully detected at a molecular mass of ∼56 kDa in the lysates of cells transfected with plasmid pcDNA/SBNT. However, in the lysates of mock- or empty vector pcDNA3.1(+)-transfected cells, no specific bands were detected using a mouse anti-6×His tag mAb (Fig. 1C). The results of the IFA further confirmed the expression of SBNT multiepitope protein in pcDNA/SBNT-transfected cells using a mouse anti-6×His tag mAb and a FITC-conjugated goat anti-mouse IgG secondary Ab; in contrast, no fluorescent signal was detected in mock- and pcDNA3.1(+)-transfected cells (Fig. 1D).

To evaluate the transfection efficiency of chitosan NPs, we used a GFP expression plasmid pEGFP-N1 for the preparation of chitosan/DNA NPs. The results revealed that, compared with liposome-mediated gene transfection, the chitosan/DNA NPs showed lower gene delivery efficiency and delayed protein expression in vitro. In positive control wells, a considerable number of cells were found to express GFP at 24 h posttransfection, whereas in pEGFP-N1-NP–transfected wells, only a few cells were found to express GFP at 24 h posttransfection. However, at 48 h posttransfection, around three to four times more fluorescent cells were detected than at 24 h. The number of fluorescent cells observed at 72 h was not significantly different from that observed at 48 h in pEGFP-N1-NP–transfected wells. Cells to which chitosan or plasmid DNA alone were added showed no green luminescence (Fig. 1E).

After production of the NP multiepitope DNA vaccine, gel retardation assays were performed to analyze the efficiency of NP complex formation. As shown in (Fig. 2A, the prepared pcDNA/SBNT-NPs remained within the wells and did not migrate through the gel, so no DNA band was observed. By contrast, an obvious DNA band was detected when naked plasmid pcDNA/SBNT samples were analyzed on a 1% agarose gel. The NP multiepitope DNA vaccine was also evaluated for its ability to protect DNA from DNase I degradation. As shown in (Fig. 2B, the naked plasmid DNA was completely degraded by DNase I, whereas the plasmid DNA encapsulated in chitosan NPs was protected throughout the 30-min incubation period as the brightness of the pcDNA/SBNT-NPs remained unchanged before and after DNase I digestion. A high encapsulation efficiency of 94.90 ± 1.35% was obtained during the preparation of the NP multiepitope DNA vaccine. These results indicated that plasmid DNA could be effectively encapsulated in chitosan to form a stable complex, and chitosan encapsulation protected the DNA from DNase digestion.

The typical morphology of the pcDNA/SBNT-NPs (Fig. 2C) and pcDNA-NPs (Fig. 2D) was spherical, with a smooth surface and good dispersion, as revealed under a TEM. The pcDNA/SBNT-NPs and pcDNA-NPs had average particle sizes of 140.9 ± 73.2 and 134.2 ± 57.1 nm and mean ζ potentials of +16.8 ± 4.3 and +16.3 ± 4.8 mV, respectively.

Two weeks after boost vaccination, serum samples and splenocytes were isolated from each group and used for the evaluation of humoral and cellular immune responses. The results showed that, compared with the PBS-inoculated chickens, the serum samples from pcDNA/SBNT-NP–vaccinated chickens have a higher level of mean neutralizing Ab titer, but no significant difference was obtained between them (p > 0.05). The chickens vaccinated with attenuated live vaccine SZ230 strain have a significantly higher level of mean neutralizing Ab titer than those of chickens inoculated with PBS (p < 0.001), whereas no neutralizing Abs could be detected in the serum from pcDNA-NP–vaccinated chickens (Fig. 3).

An ELISPOT assay was initially used to evaluate the cellular immune response induced by the NP multiepitope DNA vaccine. As shown in (Fig. 4A, compared with the PBS-inoculated chickens, the splenocytes from pcDNA/SBNT-NP–vaccinated chickens produced significantly higher levels of chicken IFN-γ (ChIFN-γ) when stimulated with the two T cell epitope peptides, N_261–280 and N_381–400 (p < 0.001), whereas no significant ChIFN-γ was released when stimulated with the other two T cell epitope peptides, N_211–230 and N_271–290 (p > 0.05). As expected, the splenocytes from attenuated live vaccine SZ230-vaccinated chickens produced significantly higher levels of ChIFN-γ when stimulated with the four T cell epitope peptides (p < 0.001), whereas no significant ChIFN-γ secretion was detected in splenocytes of pcDNA-NP–vaccinated chickens. To increase any responses present, we further performed CFSE staining assays to evaluate the proliferation of CD4⁺ and CD8⁺ T cells after their stimulation with the T cell epitope peptides for 5 d with the addition of recombinant ChIL-2. The results showed that, compared with the splenocytes from PBS-inoculated chickens, a significantly higher rate of CD8⁺ T cell proliferation was obtained in splenocytes from pcDNA/SBNT-NP–vaccinated chickens after their simulation with peptides N_261–280 and N_271–290 (p < 0.001); peptide N_261–280 also induced a significantly higher rate of CD4⁺ T cell proliferation in splenocytes from pcDNA/SBNT-NP–vaccinated chickens, suggesting that peptide N_261–280 composed in the pcDNA/SBNT-NPs construct can be recognized by both CD4⁺ and CD8⁺ T cells in vivo. At the same time, compared with the splenocytes from PBS-inoculated chickens, the splenocytes from pcDNA/SBNT-NP–vaccinated chickens seemed to have a higher rate of CD8⁺ T cell proliferation when stimulated with peptide N_211–230, but no significant difference in values was obtained between them (p > 0.05). Consistent with our previous study (6), peptide N_381–400 did not stimulate a significant T cell proliferation in CFSE staining assays, even when it has been found by ELISPOT assay to induce ChIFN-γ release in splenocytes from pcDNA/SBNT-NP–vaccinated chickens. As expected, the splenocytes from attenuated live vaccine SZ230-vaccinated chickens produced a significantly higher rate of CD8⁺ T cell proliferation when stimulated with the three T cell epitope peptides (N_211–230, N_261–280, and N_271–290) (p < 0.001) and produced a significantly higher rate of CD4⁺ T cell proliferation when stimulated with the peptide N_261–280 (p < 0.001), whereas no significant CD8⁺ or CD4⁺ T cell proliferation was detected in splenocytes of pcDNA-NP–vaccinated chickens (Fig. 4B, 4C) when stimulated with the four T cell epitope peptides. These results suggested that the NP multiepitope DNA vaccine can induce an IBV-specific immune response in vaccinated chickens.

To investigate the protective efficacy of the DNA vaccine, we challenged all immunized chickens with virulent IBV SD strain 2 wk after the booster vaccination. The clinical signs of each group were monitored and scored daily for 14 dpc. The chickens in the pcDNA/SBNT-NPs-SD group showed significantly milder signs than chickens in the PBS-SD group, especially at 8–14 dpc. No significant difference was detected in the clinical signs score between the pcDNA-NPs-SD and PBS-SD groups throughout the observation period. The chickens in the SZ230-SD group were relatively asymptomatic, with a significantly lower clinical sign score at 5–14 dpc compared with the PBS-SD group (Fig. 5A). The mortality rate for the pcDNA/SBNT-NPs-SD group was 10%, with one mortality at 9 dpc. By contrast, mortality rates of 60% and 40% were obtained in the PBS-SD and pcDNA-NPs-SD groups, respectively (Fig. 5B).

Tracheal ciliary activity (Fig. 5C) and virus shedding (Fig. 5D) were evaluated at 5 and 7 dpc for the different groups. The results showed that, compared with the PBS-SD group, chickens in the SZ230-SD group had significantly lower ciliostasis scores and virus shedding at both 5 and 7 dpc (p < 0.001). However, our results did not show a significant difference in ciliostasis scores and virus shedding between the pcDNA/SBNT-NPs-SD, pcDNA-NPs-SD, and PBS-SD groups at 5 and 7 dpc (p > 0.05).

The viral loads in various organs of the different groups were detected by real-time qRT-PCR. Similar to the results of virus shedding, the viral loads in the trachea of the SZ230-SD group were significantly lower than those of the PBS-SD group at 5 and 7 dpc (p < 0.05), whereas no significant difference was detected between the pcDNA/SBNT-NPs-SD, pcDNA-NPs-SD, and PBS-SD groups at 5 and 7 dpc (p > 0.05) (Fig. 6A). The viral loads in the lungs of the pcDNA/SBNT-NPs-SD group were lower than those in the PBS-SD group at 5 and 7 dpc, but this difference was not statistically significant (p > 0.05). Compared with the PBS-SD group, the viral loads in the lungs of the SZ230-SD group were significantly reduced only at 7 dpc (p < 0.05) (Fig. 6B). Furthermore, the viral loads in the kidney (Fig. 6C) and bursa of Fabricius (Fig. 6D) in the pcDNA/SBNT-NPs-SD and SZ230-SD groups were lower than those in the PBS-SD group at 5 dpc, but this difference was not statistically significant (p > 0.05), whereas at 7 dpc, a significantly reduced viral load was detected in the kidney and bursa of Fabricius compared with the PBS/SD group (p < 0.05).

IB is a major viral disease that causes huge economic losses to the poultry industry worldwide. Live attenuated and inactivated IBV vaccines are widely used to control this disease (8). However, it has been reported that live attenuated IBV vaccines can revert back to virulence or recombine with field strains, leading to the emergence of new IBV variants (23, 24). Furthermore, inactivated IBV vaccines alone usually induce poor immune responses (10). Therefore, a more effective and safe vaccine is needed.

DNA vaccines as new generation vaccines have great potential as future alternatives to the traditional IBV vaccine. Although several DNA vaccines have been licensed for veterinary use against viral infections (13), IB DNA vaccines have not been licensed to date. In our previous study, a multiepitope gene expression box SBNT comprising four neutralizing epitope domains and four T cell epitope peptides was designed and inserted into the genome of Newcastle disease virus strain La Sota between the P and M genes. The constructed recombinant viral vector IBV multiepitope vaccine candidate rLa Sota/SBNT provided significant protection against homologous and heterologous IBV challenge (6). In this study, to investigate the application potential of IBV multiepitope DNA vaccine, we constructed a recombinant plasmid pcDNA/SBNT by inserting the SBNT expression box gene into a eukaryotic expression vector pcDNA3.1(+). CpG DNA, a potent Th-1 like adjuvant, has proven immunomodulatory effects (25). Thus, in this study, chicken-specific CpG motifs (26) were also incorporated into the recombinant plasmid pcDNA/SBNT to enhance the immune response.

In general, the direct delivery of naked plasmid DNA into cells in vivo is unrealistic due to the inability of negatively charged DNA molecules to enter negatively charged cellular membranes (27). Encapsulation of naked plasmid DNA with a carrier is considered a possible strategy to improve the efficacy of DNA vaccines. Cationic lipids and polymers have been extensively studied as carriers for the delivery of genes and drugs. Cationic lipids usually have a high transfection efficacy in vitro, but their potential toxicity and short life span in vivo need to be addressed before clinical trials (28). In recent years, cationic polymers such as chitosan have gained increasing interest as safe and effective gene delivery carriers. Chitosan is a natural polymer that has several advantages, such as low immunogenicity, low toxicity, and high biocompatibility, and it can form a complex with negatively charged plasmid DNA to form NPs and regulate gene delivery (14). A transfection assay confirmed that plasmid pEGFP-N1 could be successfully delivered into cells by chitosan NPs. However, the chitosan/DNA complexes delivered by gene transfection showed delayed protein expression in cells, which may be associated with the slow-release properties of chitosan NPs (29), a phenomenon reported in a previous study (30). The slow-release nature of chitosan NPs may extend the residence time of the plasmid DNA on the target site, thereby inducing a long-lasting immune response.

Encapsulation efficiency is an important index for evaluating the quality of chitosan/DNA NPs. In our study, a high encapsulation efficiency of 94.90 ± 1.35% was obtained during the preparation of NP multiepitope DNA vaccines, indicating that the plasmid DNA was wrapped tightly by chitosan molecules (31). This phenomenon was also verified by a gel retardation assay that indicated that the chitosan/DNA complexes remained in the wells, with no DNA molecules migrating through the 1% agarose gel. This observation indicated that the complex coacervation method used to prepare the NP multiepitope DNA vaccines in our study allowed the chitosan to capture all of the DNA plasmid molecules. The plasmid DNA is susceptible to degradation by nucleases in vivo, and this presents another challenge for the application of DNA vaccines (32). The pcDNA/SBNT-NPs prepared in this study provided full protection of plasmid DNA from DNase degradation, which is important for the effectiveness of DNA vaccines on administration to the host.

The efficacy of NP uptake by cells is strongly dependent on the physicochemical properties of NPs, in particular shape, size, and surface charge (33). Chithrani et al. (34) reported that the spherical NPs can be taken up by cells more efficiently than their rod-shaped counterparts. In our study, the pcDNA/SBNT-NPs were spherical in shape and showed good dispersion, properties that are favorable for cellular transfection. Moreover, the size of these NPs was similar to that of pathogens (20–200 nm), making them easily recognizable by APCs (35). However, the particle size determined under a TEM was smaller than that measured by the laser particle size analyzer, which may be because of the NPs in the prepared solution becoming swollen and then shrunken after drying, a phenomenon that has been reported previously (36). In general, negatively charged cellular membranes preferentially interact with positively charged particles (37). The NPs prepared in our study had positive ζ potentials, which would be expected to promote the interaction of pSBNT-CS-NPs with cell membranes.

In immune protection assays, the chickens vaccinated with pcDNA/SBNT-NPs produced slightly higher levels of neutralizing Ab titer than those of chickens inoculated with pcDNA-NPs or PBS, in which no neutralizing Abs could be detected. Two of the four T cell epitope peptides (N_261–280 and N_381–400) were recognized by the splenocytes from pcDNA/SBNT-NP–vaccinated chickens and produced a high level of ChIFN-γ in ELISPOT assays. Unexpectedly, no significant ChIFN-γ release was observed from splenocytes when stimulated with the other two T cell epitope peptides (N_211–230 and N_271–290). However, in CFSE staining assays, peptides N_261–280 and N_271–290 could be recognized by the splenocytes from pcDNA/SBNT-NP–vaccinated chickens and stimulated a high rate of CD8⁺ T cell proliferation; peptide N_261–280 also stimulated a high rate of CD4⁺ T cell proliferation, indicating that peptide N_261–280 can be recognized by both CD8⁺ and CD4⁺ T cells in pcDNA/SBNT-NP–vaccinated chickens. We can speculate on the reasons for the differing results between ELISPOT and CFSE staining assays. First, compared with N_261–280 and N_381–400 epitope-specific T cells, the frequencies of T cells responding to peptides N_211–230 and N_271–290 in splenocytes from pcDNA/SBNT-NP–vaccinated chickens are extremely low, below the detection threshold of the ELISPOT assay. However, for CFSE staining assays, several rounds of clonal expansion may increase the numbers of such low frequencies of responsive T cells so that they become detectable by flow cytometry. Second, in some situations, some T cells do not produce IFN-γ before proliferation, and in other situations, some memory and effector T cells respond to their specific Ags with an effector response (e.g., cytokine release or cytotoxicity) and undergo activation-induced apoptosis before possible proliferative responses (38, 39). However, these reasons are only speculation, and the specific mechanism needs to be studied further.

After being challenged with virulent IBV SD strain, the chickens immunized with pcDNA/SBNT-NPs showed milder clinical symptoms and a lower mortality rate compared with those in the PBS-SD group, suggesting that the NP multiepitope DNA vaccine prepared in our study conferred partial protection against virulent IBV challenge. Vaccination with pcDNA/SBNT-NPs did not appear to protect the respiratory tract of chickens as the ciliostasis scores, virus shedding, and viral loads in the trachea and lung did not differ significantly from those of the PBS-SD group. However, the viral loads in the kidney and bursa of Fabricius in the pcDNA/SBNT-NPs-SD group were lower than those of the PBS-SD group, especially at 7 dpc, when a statistically significant difference was detected. The QX-like IBV strains initially replicated in the tracheal mucosa, resulting in damage to tracheal ciliary activity. This was followed by secondary replication in many nonrespiratory tissues, such as the kidney, bursa of Fabricius, and oviduct, resulting in a high mortality rate (40). In general, mucosal and systemic immunity are considered to be independently regulated and play different roles in different disease processes (41). It has been proved that vaccination via the mucosal route can induce both local mucosal and systemic immunity, whereas systemic vaccination usually fails to elicit strong mucosal immunity (42, 43). Kotani et al. (44) showed that CTLs infiltrating the tracheal mucosa are critical for clearance of IBV in the early stage of infection. However, the development of local mucosal immunity requires the direct interaction between local mucosal epithelial cells and pathogens. Therefore, chickens vaccinated with pcDNA/SBNT-NPs via the i.m. route may develop systemic immunity rather than mucosal immunity, thereby not protecting the respiratory tract of chickens but preventing secondary replication of IBV to some extent in nonrespiratory tissues, which may manifest as a reduction in the viral load in the kidneys and bursa of Fabricius. Indeed, nephritis is the major cause of mortality in nephropathogenic IBV (1). Compared with the PBS-SD group, the lower mortality rates in the pcDNA/SBNT-NPs-SD group may be related to the reduced viral load in the kidneys. Whether the NP multiepitope DNA vaccine pcDNA/SBNT-NPs potentially protect against infection with other heterologous IBV strains requires further investigation.

In summary, our study developed a novel IBV multiepitope DNA vaccine using chitosan NPs as the gene carrier. The NP multiepitope DNA vaccine confers partial protection against QX-like IBV infection. We postulate that the addition of other immunoadjuvants, such as cytokines and pattern-recognition receptor ligands, to the pcDNA/SBNT-NPs construct, or a combination with other cationic polymers, such as polylactic-coglycolic acid, during the preparation of the chitosan NPs, may be a useful strategy to improve the effectiveness of the NP multiepitope DNA vaccine.

We thank Liwen Bianji (Edanz China, Beijing, China) for editing the language of a draft of this manuscript.

The authors have no financial conflicts of interest.