A Novel Mucosal Adjuvant System for Immunization against Avian Coronavirus Causing Infectious Bronchitis

According to 2017 U.S. agriculture statistics, the combined value of production and sales from broilers, eggs, turkeys, and chicks was $42.8 billion. Of this number, broiler sales comprised 67% of the industry value, with the production of >50 billion pounds of chicken meat. The economic success of the poultry industry in the United States hinges on the extensive use of vaccines to control infectious bronchitis virus (IBV) and other poultry pathogens. The majority of vaccines currently licensed for poultry health include both modified live vaccine and inactivated pathogens. Despite their proven efficacy, modified live vaccine constructs take time to produce and could revert to virulence, which limits their safety. The significance of our research stems from the development of a safer and potent alternative mucosal vaccine to replace live vaccines against IBV and other emerging coronaviruses.

ABSTRACT

Infectious bronchitis (IB) caused by infectious bronchitis virus (IBV) is currently a major threat to chicken health, with multiple outbreaks being reported in the United States over the past decade. Modified live virus (MLV) vaccines used in the field can persist and provide the genetic material needed for recombination and emergence of novel IBV serotypes. Inactivated and subunit vaccines overcome some of the limitations of MLV with no risk of virulence reversion and emergence of new virulent serotypes. However, these vaccines are weakly immunogenic and poorly protective. There is an urgent need to develop more effective vaccines that can elicit a robust, long-lasting immune response. In this study, we evaluate a novel adjuvant system developed from Quil-A and chitosan (QAC) for the intranasal delivery of nucleic acid immunogens to improve protective efficacy. The QAC adjuvant system forms nanocarriers (<100 nm) that efficiently encapsulate nucleic acid cargo, exhibit sustained release of payload, and can stably transfect cells. Encapsulation of plasmid DNA vaccine expressing IBV nucleocapsid (N) protein by the QAC adjuvant system (pQAC-N) enhanced immunogenicity, as evidenced by robust induction of adaptive humoral and cellular immune responses postvaccination and postchallenge. Birds immunized with pQAC-N showed reduced clinical severity and viral shedding postchallenge on par with protection observed with current commercial vaccines without the associated safety concerns. Presented results indicate that the QAC adjuvant system can offer a safer alternative to the use of live vaccines against avian and other emerging coronaviruses.

IMPORTANCE According to 2017 U.S. agriculture statistics, the combined value of production and sales from broilers, eggs, turkeys, and chicks was $42.8 billion. Of this number, broiler sales comprised 67% of the industry value, with the production of >50 billion pounds of chicken meat. The economic success of the poultry industry in the United States hinges on the extensive use of vaccines to control infectious bronchitis virus (IBV) and other poultry pathogens. The majority of vaccines currently licensed for poultry health include both modified live vaccine and inactivated pathogens. Despite their proven efficacy, modified live vaccine constructs take time to produce and could revert to virulence, which limits their safety. The significance of our research stems from the development of a safer and potent alternative mucosal vaccine to replace live vaccines against IBV and other emerging coronaviruses.

INTRODUCTION

Infectious bronchitis (IB) is an acute respiratory illness of domestic fowl caused by infectious bronchitis virus (IBV) (1). IBV is a member of the genus Gammacoronavirus, family Coronaviridae, order Nidovirales, with a 27.6-kb single-stranded positive-sense RNA genome encoding major structural proteins, spike glycoprotein (S), envelope (E), membrane (M), and nucleocapsid (N) (2). IB-associated clinical signs include tracheal rales, frequent sneezing with nasal exudate, lethargy, and labored breathing. Uncomplicated IB infections are not lethal, and virus with associated symptoms is cleared within 10 days (3). However, complications from other viral and bacterial infections usually lead to increased mortalities that can reach up to 60% of the flock (4). IBV control is of great economic importance, as IBV is related to increased mortalities, increased condemnation at the slaughterhouses for infected broilers, and a severe drop in egg quality and production for infected layers (5–7). Current commercial modified live virus (MLV) vaccines confer modest protection but fail to protect against newly emerging serotypes. Multiple serotypes cocirculate in birds, which complicates diagnosis and control of IBV. High antigenic variation in the spike glycoprotein (S) is a hallmark of different IBV serotypes (1, 7, 8). Sequence difference, even as little as 4% in the S1 subunit of S protein, can lead to vaccine failure (9, 10). Unfortunately, MLVs have been shown to persist and transmit in vaccinated birds, potentially mediating recombination with virulent serotypes, leading to the emergence of new serotypes (11–13). With the emergence of new serotypes, like GA98, linked to the excessive use of MLV (14), there is an urgent need to develop a safe and effective vaccine against IBV, the focus of the current study.

The use of plasmid DNA as potential immunogens was described almost 30 years ago (15); however, only 5 DNA-based vaccines have been licensed for veterinary use (16). Nucleic acid-based vaccines have significant advantages over MLV, as they have a superior safety profile, invoke robust cell-mediated immunity (CMI) with potent adjuvants, cost less to produce, and are thermostable, obviating the need for a cold chain (17). DNA-based vaccines encoding IBV S1, M, and N genes administered in ovo and intramuscularly (i.m.) have been studied with variable protection levels against IBV (18–26). The use of plasmid DNA vaccines in the field despite having practical advantages and being safe has been limited owing to poor immunogenicity and cellular availability. Nanocarriers increase the bioavailability of antigen cargo, generating an immediate uptake by immune cells; hence, they are potent adjuvants (27). Nanocarriers can protect plasmid DNA and antigen cargo from degradation in vivo, facilitating delivery in vaccine hostile mucosal surfaces (28, 29). To provide a safer alternative to current MLVs, we detail the development of nanocarriers composed of natural adjuvants, Quil-A and chitosan (QAC), for the delivery of IBV plasmid DNA immunogen. Chitosan is a biodegradable natural polysaccharide that is cationic in nature and can readily complex with negatively charged nucleic acids and proteins through electrostatic interactions (30). Chitosan forms stable DNA/protein complexes and has mucoadhesive properties, because of which they are widely applied for mucosal routes of administration (31). On the other hand, Quil-A is a potent adjuvant with mild surfactant properties (32) produced from the plant Quillaja saponaria, which can form nanoparticle compounds, like ISCOMs and those formed with chitosan (QAC), as we detail below.

The sequence similarity of IBV N protein between diverse serotypes is greater than 90%. For this reason, IBV N protein is an ideal immunogen candidate in an effort to develop an effective vaccine. Immunization with N protein elicited a robust cytotoxic T-lymphocyte (CTL) response, an important correlate of protection against IBV (20, 33, 34). Adoptively transferred IBV-reactive CD8⁺ T cells protect against IBV challenge in naive chickens (35, 36). In the present study, we evaluated the ability of a stable plasmid DNA construct expressing the IBV N protein complexed with the QAC adjuvant system (pQAC-N) given intranasally (i.n.) to protect immunized birds against challenge with a virulent strain of IBV. Our results indicate that pQAC-N vaccine elicits a CD8⁺ T-cell response that protects vaccinated birds against IBV challenge. Levels of protection in pQAC-N-vaccinated birds were higher than those of birds that were unadjuvanted or given a chitosan-only complexed plasmid vaccine. Our data demonstrate that intranasal immunization with pQAC-N induced a strong cell-mediated immune response that protected vaccinated birds with a significant reduction in clinical signs and viral load to levels on par with those of commercial MLV-vaccinated birds. Even better, when commercial birds were immunized, the pQAC-N construct lowered clinical signs of IB with no mortalities in that group compared to the MLV-vaccinated group.

RESULTS

Synthesis and characterization of pQAC-N nanovaccine.

The green fluorescent protein (GFP) gene (37) inserted into the pCAG plasmid (a gift from Connie Cepko; Addgene plasmid number 11150) was used as a DNA payload for nanoparticle (NP) characterization. Transmission electron microscopy (TEM) analysis of chitosan-plasmid DNA complexes indicated the presence of aggregated structures (Fig. 1A). However, when Quil-A was added to the chitosan-DNA complex, defined particles were formed with the disaggregation of chitosan-DNA complexes (Fig. 1B). Size estimations using TEM analysis indicated that QAC-DNA nanoparticles were <100 nm (Fig. 1B). Dynamic light scattering (DLS) was also used to measure the hydrodynamic size and zeta potential of QAC-pDNA particles. As expected, particles were 95 ± 25 nm in size (94%), with a net positive zeta potential of 15 ± 4.44 mV (Fig. 1C and D).

FIG 1.

Nanostructure of QAC adjuvant system. (A) Aggregates of chitosan-pCAG-GFP preparation (arrows) were seen with TEM. (B) Nanoparticles of QAC-pCAG-GFP preparation (arrows) with TEM. Scale bar, 100 nm. (C and D) Number-based DLS data (C) and Zeta potential (D) on QAC-pCAG-GFP nanoparticles at 25°C with Zetasizer software.

The ability of the QAC adjuvant system to deliver plasmid DNA payload was evaluated in vitro to examine its potential for immunization programs. The QAC nanoparticles encapsulating 5 μg pCAG-GFP construct were added to a suspension of Expi293F cells. At 72 h postaddition, the presence of fluorescent cells was observed using fluorescence microscopy (Fig. 2A), indicating the delivery and expression of the GFP from the construct. The release kinetics of the GFP from the pCAG-GFP construct was evaluated in phosphate-buffered saline (PBS) at pH 7.4 by quantifying the amount of starting and released plasmid DNA in buffer using spectrophotometry. The analysis showed that almost 42% of plasmid DNA within the nanoparticles was released within 15 days, the endpoint for our analysis. Overall, a biphasic release kinetics was observed with sustained release of DNA cargo for the first 10 days, followed by a plateau over the next 5 days (Fig. 2B). The encapsulation efficiency of DNA (percentage of encapsulated DNA relative to the starting DNA) in QAC nanoparticles ranged from 70 to 90%.

FIG 2.

QAC nanoparticle payload delivery and release. (A) GFP⁺ Expi293F cells postaddition of QAC-pCAG-GFP. (B) Sustained release kinetics observed in vitro.

Finally, following successful encapsulation and sustained release of plasmid DNA with QAC, we used the same nanocarrier (QAC) to encapsulate plasmid DNA encoding N protein from the IBV-Arkansas strain with a C-terminal 6×His tag, here referred to as pQAC-N. The expression of antigen was confirmed using Western blot analysis with an anti-6×His antibody (Fig. 3A and B).

FIG 3.

DNA vaccine construct. (A) Plasmid map of pCAG-IBV Ark N, with 6×His construct (pQAC-N) generated using Snapgene software. CMV, cytomegalovirus; SV40, simian virus 40. (B) Western blot analysis with anti-6×His-HRP antibody confirming expression of N6×His from pCAG-N6×His plasmid. Lanes: supernatant (lane 2) and pellet (lane 4) from Expi293F cells transfected with control pCAG plasmid, supernatant (lane 1) and pellet (lane 3) from Expi293F cells transfected with pCAG-N6×His plasmid, and purified N6×His protein (lane 5). (C) Western blot analysis with anti 6×His-HRP antibody confirming expression of S1 6×His from pCAG-S1 6×His plasmid used for S1 6×His purification. Lanes: supernatant (lane 1) from Expi293F cells transfected with control pCAG plasmid, supernatant (lane 2) from Expi29F3 cells transfected with pCAG-S1 6×His plasmid, and purified S1 6×His protein (lane 3).

QAC-based nanovaccine is well tolerated by chickens.

In ovo and spray vaccinations are two strategies used in the field for mass vaccinations of poultry flocks (38, 39). The safety of pQAC-N in chicken hosts was evaluated through two routes of administration, in ovo and intranasal, a proxy for field spray vaccinations. Embryo development and hatch rate of pQAC-N (100 μg)-inoculated specific-pathogen-free (SPF) embryonated chicken eggs (ECE) was similar to that of ECEs inoculated with PBS (100%) (Fig. 4A). In addition, 1-day-old SPF chicks were immunized with pQAC-N construct intranasally and monitored for general or respiratory distress, depression, or inappetence and weight gain over the course of 30 days postvaccination. No signs for respiratory distress were observed in chicks immunized at 1 day of age, and weight gain over 30 days was not statistically different from that of chicks inoculated with PBS (Fig. 4B). Overall, our analysis in chickens and chicken embryos indicated that pQAC-N is well tolerated.

FIG 4.

Safety of pQAC-N. (A) Hatch rate percent in ECEs inoculated with pQAC-N vaccine. (B) Weight gain of chicks immunized with pQAC-N construct at 30 days postvaccination (DPV). Data shown are means ± standard deviations (SD). Significance (*, P < 0.05) or nonsignificance (ns) was determined by one-way ANOVA with multiple comparisons.

Immunization with pQAC-N induces a robust immune response.

Harderian glands play a critical role in the control of IBV infection in the upper respiratory tract by secreting IBV-specific IgA antibodies into the lachrymal fluid (tears) (34). Accordingly, we examined the ability of pQAC-N vaccine to elicit IBV-specific immune responses in birds following intranasal delivery. Lachrymal fluid samples collected at different time points, 10 and 20 days postvaccination (DPV; prechallenge) and 3 days postchallenge (DPC), were examined using enzyme-linked immunosorbent assay (ELISA) plates coated with IBV Arkansas S1 and N proteins. Purified proteins were used for detection to reduce ELISA background, which was observed when whole inactivated virus was used. IBV-specific IgA titers were detectable in pQAC-N immunized birds at 20 DPV at levels higher than those seen in chitosan pCAG-N-immunized birds (Fig. 5A). Albeit detectable, IgA levels were not significantly different from levels seen in the other groups, naked (unadjuvanted) and chitosan-complexed pCAG-N. IgA levels were 10-fold higher in birds immunized with commercial MLV than in the other experimental vaccine groups (Fig. 5A), most likely because of the IgA against S1, which was not included in the pQAC-N but present in MLV.

FIG 5.

pQAC-N vaccine immunogenicity. Groups of white leghorn SPF chicks were either unvaccinated (PBS) or immunized with MLV (day 1), naked unadjuvanted pCAG-N, chitosan-complexed pCAG-N, or pQAC-N vaccine (100 μg pQAC-N) at day 1 and day 14. (A) IBV-specific IgA in tears. Significance (*, P < 0.05) was determined by two-way ANOVA. (B) Lymphocyte proliferation assay on PBMCs harvested at day 20 postvaccination. Significance (*, P < 0.05; **, P < 0.01) or nonsignificance (ns) was determined by one-way ANOVA with multiple comparisons. Data show means ± SD.

To assess the effect of IBV-specific cellular immune responses induced by pQAC-N, we measured the ability of peripheral blood mononuclear cells (PBMCs) from immunized chickens to respond to IBV antigen stimulation. PBMCs were harvested from vaccinated birds at 20 DPV (prechallenge time point) and processed for antigen-specific cell proliferation assay (MTT assay). The stimulation of PBMCs from chickens vaccinated with pQAC-N resulted in significantly higher proliferation (P < 0.05) than that of PBMCs from other groups, including MLV (Fig. 5B), suggesting a better cellular immunity following pQAC-N immunization.

Reduced clinical severity and viral burden in immunized birds.

To investigate the protective efficacy of pQAC-N vaccine, all immunized birds were challenged with virulent IBV Arkansas DPI serotype at 3 weeks after the first vaccination (21 DPV), and the clinical severity of bird groups was scored up to 8 DPC (Fig. 6A). Vaccination with naked pCAG-N (unadjuvanted) and chitosan-complexed pCAG-N conferred partial protection against clinical signs associated with IBV. On the other hand, pQAC-N and commercial MLV-immunized bird groups were relatively asymptomatic, with a significant reduction in clinical severity compared to that of unvaccinated birds (Fig. 6A).

FIG 6.

Protective efficacy of pQAC-N vaccine. Groups of white leghorn SPF chicks were either unvaccinated (PBS) or immunized with MLV (day 1), naked pCAG-N, Chitosan pCAG-N, or pQAC-N vaccine (100 μg) at day 1 and day 14. (A) Clinical sign severity, represented as average score/bird over 8 days postchallenge in each group. (B) IBV log viral load/10 μl lachrymal fluid at 6 days postchallenge. Significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001) or nonsignificance (ns) was determined by one-way ANOVA with multiple comparisons. Data show means ± SD.

In addition, we used quantitative reverse transcription-PCR (qRT-PCR) to assess the level of viral RNA in lachrymal fluid of all birds at 6 DPC. A significant reduction in viral load (∼2.5 logs) was found in the pQAC-N-immunized birds compared to control birds at levels comparable to those of commercial MLV-vaccinated birds (Fig. 6B). A partial reduction in viral load was also observed in naked pCAG-N- and chitosan pCAG-N-administered birds (Fig. 6B). Overall, reduction in viral load was higher in the pQAC-N than other DNA-based vaccine constructs, suggesting an important role played by Quil-A in the induced immunity in chickens.

Localized IBV-specific cellular responses in immunized birds.

Induction of robust T-cell responses has been identified as a relevant correlate of protection against IBV infection in previous studies (34). An antigen-specific T-cell proliferation assay based on CellTrace violet cell dye staining of lung cells to trace proliferating T cells was developed. Different T-cell subsets responding to antigen stimulation were identified using flow cytometry-assisted T-cell assay. Twenty days after the first vaccination, the IBV Ark N protein-specific proliferation was measured. The stimulation index (SI), which is the fold increase in stimulated to unstimulated cells, was calculated. Lung cells from pQAC-N-vaccinated birds responded well to antigen stimulation, which was higher than that of negative and MLV control groups (Fig. 7A). An increase in the stimulation of proliferating CD8⁺ and TCRγδ⁺ T cells was observed in pQAC-N-vaccinated birds compared to control birds (Fig. 7B and D), suggesting a role for CD8⁺ and TCRγδ⁺ cells in pQAC-N immunity. On the other hand, the CD4⁺ T-cell proliferation was higher in MLV-vaccinated birds (Fig. 7C).

FIG 7.

pQAC-N induces a robust T-cell response. (A) Lung cell proliferative capacity measured by CellTrace violet dye dilution in unvaccinated and MLV- and pQAC-N-vaccinated chickens. Proliferation was measured in total lung cells (A) and CD8α⁺ (B), CD4⁺ (C), and TCRγδ⁺ (D) lung T cells after 4 days in culture after antigen stimulation.

Protective efficacy of pQAC-N in the presence of MDA.

Maternally derived antibodies (MDA), mainly IgY, are transferred from vaccinated hens to progeny via the yolk. The presence of IBV-specific MDA was shown to protect against IBV challenge in chickens (40–42). Although effective against IBV infections, MDA can interfere with MLV vaccination and dampen the development of active immunity (42). In one experiment, we investigated the ability of pQAC-N to mediate protection in the presence of interfering MDA. Commercial white leghorn chicks with high levels of circulating IBV-specific IgY, which persisted until about day 24 of age, was used (data not shown). As seen with the SPF chicks, pQAC-N-immunized commercial birds were also protected against IBV Arkansas DPI challenge, with a significant reduction in lachrymal fluid viral load and clinical severity scores compared to those of unvaccinated birds.

In a second experiment, we evaluated the ability of pQAC-N to reduce viral shedding in trachea compared to that in MLV-vaccinated birds. Similar to the previous trial, white leghorn chicks with high levels of IBV-specific circulating IgY, as seen in the negative-control PBS group, were used (Fig. 8A). IBV-specific IgY in serum (Fig. 8A) and IgA in tears (Fig. 8B) was significantly higher in MLV-vaccinated than in pQAC-N-vaccinated commercial birds, as observed before. The pQAC-N-vaccinated birds had significantly reduced viral shedding in tracheal swabs and clinical severity postchallenge on par with MLV vaccinated birds (Fig. 8C and D), suggesting the ability of the pQAC-N to protect birds without interference by MDA. Interestingly, MLV-vaccinated birds showed signs of respiratory distress as late as 15 DPV, with one bird dying at 10 DPV, which was not observed in the other groups and with SPF birds in the previous trial. Both necropsy and diagnostic bacteriology and virology revealed the presence of Staphylococcus aureus and Escherichia coli superinfection in the dead MLV-vaccinated bird, which was not detected in a euthanized control bird. Severe locally extensive pulmonary edema was reported in the lungs of the MLV-vaccinated bird, which was a result of bacteremia/sepsis, the likely cause of death. MLV-vaccinated birds were significantly lighter in weight and had reduced weight gain between 14 and 28 DPV (data not shown), a potential consequence of bacterial superinfection.

FIG 8.

Reduced tracheal viral shedding in pQAC-N-vaccinated commercial birds. Groups of commercial white leghorn chicks were either unvaccinated (PBS) or immunized with MLV (day 1) or pQAC N vaccine (100 μg) at day 1 and day 14. IBV-specific IgY in serum (A) and IgA in lachrymal fluid (B) significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001) was determined by two-way ANOVA. (C) Clinical sign severity, represented as average score/bird over 8 days postchallenge in each group. (D) IBV viral load in tracheal swabs at 6 days postchallenge. Significance (***, P < 0.001; ****, P < 0.0001) or nonsignificance (ns) was determined by one-way ANOVA with multiple comparisons. Data show means ± SD.

DISCUSSION

Many experimental subunit and plasmid DNA vaccines against IBV have been developed and their efficacies detailed in previous studies. Intramuscularly (i.m.) and in ovo-administered experimental vaccines without adjuvants or complemented with natural and molecular adjuvants, like interleukin-2 and granulocyte-macrophage colony-stimulating factor, have been shown to reduce viral shedding and clinical signs and improve protection rates against IBV (18–26). Alternative routes, such as in ovo administration of S1 plasmid DNA vaccine, have also been shown to elicit a modest immune response (19). Compared to S1 protein, higher protection rates against IBV challenge and increased total T cells were shown with i.m. immunization with plasmid encoding N protein (20, 23, 25). In this study, we detail the development of a safe mucosal vaccine adjuvant (QAC) for intranasal immunization, a highly desired feature of mass vaccinations under field conditions. The safety of QAC nanocarrier was examined by two common routes of field immunization, intranasal and in ovo. Birds intranasally immunized by a single dose or double doses (at 1 and 14 days of age) did not show any signs of respiratory distress, and weight gain was the same as that observed with control birds. The in ovo immunization also did not affect embryo development and egg hatch rates. Previously, both Quil-A and chitosan were shown to be biocompatible, with no adverse effects reported when administered to animals (30–32, 43). Similarly, our results indicate that the QAC adjuvant is safe, tolerable, and biocompatible in chicken hosts.

For vaccine efficacy, our experimental vaccine (pQAC-N) was compared to the current commercial MLV in a challenge model adopted based on recommended guidelines from the Food and Drug Administration, with modifications (FDA-9CFR). The superiority of the QAC adjuvant system was shown in a challenge model where birds were challenged immediately at 7 days following vaccine boost with a relatively high dose of IBV Ark DPI. Specifically, we demonstrated the ability of the QAC adjuvant system to enhance immunogenicity and protective efficacy of pCAG-N plasmid vaccine, which otherwise conferred partial protection when administered independently (naked pCAG-N). It is possible that N protein uptake and processing by antigen-presenting cells (APCs) is a key step in the downstream activation of B and T cells for developing the robust memory immune response (44) that was shown in the pQAC-N-immunized birds. Factors that influence APC uptake of antigens include particle surface charge, size, hydrophobicity, and others (45). Soluble viral immunogens by themselves, owing to their small size (<20 nm), are poorly taken up by APCs. Particulate adjuvant systems with a size range of 20 to 200 nm have been shown to promote APC uptake by endocytosis (45). Our findings have shown that QAC-based formulations formed spherical disaggregated particles of optimal size (<100 nm) for efficient APC uptake and processing, as expected when the Quil-A surfactant is added. Delivery systems with net positive surface charge, like QAC (positive zeta potential, 15 ± 4.4 mV), temporarily disrupt membrane of cells, causing membrane flipping and/or fusion at cell surfaces mediating payload delivery, or enter cells via the clathrin-mediated endocytic pathway, a potential pathway for effective antigen uptake (46). Chitosan DNA particles promote the slow release of packaged DNA, and, similarly, we observed the sustained release of DNA payload complexed with QAC in vitro (47). Our in vitro analysis indicated that QAC could promote the targeted delivery of payload into cells and/or act as antigen depots, maintaining a sustained release of payload-priming immune cells continually. Overall, the encapsulation of plasmid DNA by QAC mediated the slow release of immunogen, which could help in continuous priming of antigen-presenting cells and overcome the need for multiple immunizations.

The pQAC-N-vaccinated SPF birds had a significantly lower viral burden than unvaccinated birds. Chitosan by itself did not reduce viral shedding, as observed with the naked unadjuvanted pCAG-N construct, suggesting a key role of the nanocarrier size and composition used here. Interestingly, a strong correlation between reduced clinical severity and reduction in viral load was observed, as suggested before (39). The protective efficacy of pQAC-N was comparable to that of MLV, with similar levels of reduced clinical severity and nonsignificant reduction in viral load observed between the groups. To our knowledge, with the exception of one study, most experimental IBV DNA vaccines have been tested against serotypes not endemic to the United States, via the i.m. route, without a comparable commercial live virus group (20–26). Here, we observed that the pQAC-N vaccine, when administered i.n., was able to protect vaccinated birds against a field-relevant IBV Arkansas DPI, most likely because of the induced localized immunity, as suggested before (34, 48). PBMCs harvested from pQAC-N-vaccinated birds responded to antigen stimulation ex vivo with significantly higher proliferation than that seen with control and other plasmid DNA vaccine groups. Moreover, the analysis of different immunological parameters indicated that pQAC-N induces strong CMI responses, in contrast to MLV, which induced potent antibody responses. The induction of CMI responses could be a hallmark for the pQAC-N nanovaccine. IBV N protein is a highly immunogenic antigen with mapped CTL epitopes in the C terminus that mediate potent CTL memory responses. IBV-specific memory CD8⁺ T-cell responses restrict IBV replication efficiently and are strong correlates of protection for IBV control (34). It is noteworthy here that birds vaccinated with experimental IBV N-based vaccines generally have a higher percentage and proliferation of CD3⁺ CD8⁺ T cells, albeit not specific to IBV, a limitation of assaying total T-cell numbers (20, 23, 25).

To decipher QAC vaccine-mediated IBV-specific immunity, we used a flow cytometry-assisted lymphocyte proliferation assay to identify and quantify subsets of T cells responding to IBV antigen. Similar to results with the MTT assay, we noticed lung cells from pQAC-N-vaccinated birds had higher stimulation ex vivo than the control groups. More reactive CD8⁺ and TCRγδ⁺ T cells were present in pQAC-N-vaccinated birds, albeit at nonsignificant levels. Large variations in recall proliferation within the pQAC-N-vaccinated group were observed, a phenomenon that has been reported in other published studies investigating chicken immune responses (49). Presented results suggest that vaccination with pQAC-N confers protection against IBV challenge to levels similar to those of MLV vaccination and that protection is attributed to an induction of CD8⁺ and TCRγδ⁺ memory T-cell responses. Although we did not observe the induction of cellular responses in PBMCs of MLV-vaccinated birds with the MTT assay, we noticed robust induction of CD4⁺ T cells in the lungs, as measured by the more sensitive flow cytometry-based assay. Further studies to elucidate the exact mechanism of pQAC-N-mediated immunity are needed.

Most commercial breeders are immunized with IBV vaccines and transfer IBV-specific IgY to their progeny via egg yolk, which can interfere with vaccine efficacy. Prior evidence suggests that DNA vaccination could overcome limitations of early vaccinations by priming the immune system even in the presence of interfering MDA (50–52). Our results indicate that pQAC-N can mediate protection in the presence of MDA, reducing viral shedding in lachrymal fluid and in the trachea. As observed in the trial with SPF chicks, IBV-specific IgA and IgY were detectable in pQAC N-vaccinated birds at 3 DPC, albeit at significantly lower levels than those in MLV-vaccinated birds. The pQAC-N-induced protective immunity in commercial birds was similar to that of the SPF birds, even though there was no IBV-specific IgA or IgY prechallenge. It is possible that pQAC-N induced IBV-specific protective T-cell responses in commercial birds, as established with pQAC-N-vaccinated SPF birds. Mortality associated with IBV outbreaks in the field is usually low unless compounded by secondary bacterial infections (4). Interestingly, in the latest trial conducted, MLV-vaccinated commercial birds had active bacterial superinfection, leading to reduced weight gain, the presence of respiratory clinical signs in all the birds, and mortality in one bird (∼8%), which was not seen in pQAC-N-vaccinated birds. This observation underscores the inferior safety profile of commercial MLV vaccines.

Although protective, the addition of other inexpensive biocompatible adjuvants to generate a complementing humoral response could be used in the current pQAC-N construct. In summary, we detailed the development of a safe plasmid DNA vaccine complemented by a mucosal adjuvant system (QAC) that protects SPF and commercial birds against IBV challenge by eliciting a strong T-cell immune response. We postulate that the QAC nanoadjuvant system can be used as a vaccine adjuvant for the delivery of plasmid DNA and protein immunogens against other respiratory viruses and intracellular pathogens for poultry and other animals.

MATERIALS AND METHODS

Ethics statement.

All the animals used in this study were cared for in accordance with established guidelines, and the experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Wisconsin at Madison.

Cells and viruses.

Expi293F cells obtained from ThermoFisher Scientific were used for confirming the expression of IBV Ark N6×His protein from the vaccine construct. The cells were cultured in Expi293 medium at 37°C, 125 rpm, 8% CO₂ atmosphere in plastic flasks with ventilated caps. The virulent IBV Arkansas DPI strain (a kind gift from B. S. Ladman and J. Gelb) was propagated in 9-day-old SPF ECEs and allantoic fluid harvested 4 days after infection. The stock virus titer was determined using qRT-PCR (see below) and also titrated and expressed as 50% embryo infectious dose (EID₅₀) (53). The IBV S1 gene sequence of the Ark DPI challenge isolate is GenBank accession no. AF006624.

Preparation of plasmids.

Arkansas nucleocapsid (N6×His) and S1 gene (S1 6×His) were amplified from reverse-transcribed cDNA synthesized from IBV Arkansas 99 (ATCC VR-841) with a C-terminal 6×His tag. The forward primer (5′-ATCACTGAATTCACCATGGCAAGCGGTAAAGCAG-3′) and reverse primer (5′-ATCACTGCGGCCGCTTAGTGGTGATGGTGATGATGACCTCCTCCAAGTTCATTCTCTCCTAGAGCTGC-3′) were employed for amplifying N6×His. The forward primer (5′-ATCACTGAATTCACCATGTTGGTGAAGTCACTGTTTCTAGTG-3′) and reverse primer (5′-ATCACTGCGGCCGCTCAGTGGTGATGGTGATGATGCCCTCCGCCGGAGGATCCAGTTCCATTAGTGATCTTAATGTAAAACTGGTTTTC-3′) were employed for amplifying S1 6×His. Amplified gene fragments were cloned into EcoRI and NotI restriction sites of pCAG-GFP plasmid, a gift from Connie Cepko (Addgene plasmid number 11150) (Fig. 3A). To confirm the insertion of genes in the correct orientation, DNA sequencing was performed at the UW-Madison Biotechnology Center with an ABI Prism 3730XL DNA analyzer using BigDye terminators (Applied Biosystems, CA). To confirm the expression of N6×His and S1 6×His protein, Expi293F cells seeded in 6-well format were transfected with an optimized ratio of DNA (4 μg) to TransIT PRO transfection reagent (2 μl) according to the manufacturer’s instructions (Mirus Bio, WI, USA). Three days posttransfection, cells were harvested for Western blot analysis. Cell fractions were boiled in Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) and resolved on a 4 to 20% SDS-PAGE gel by electrophoresis using a Mini-PROTEAN 3 system (Bio-Rad, CA). Polyacrylamide gels were electroblotted onto nitrocellulose membranes using a Turboblot system. Membranes were blocked in 5% (wt/vol) skim milk and probed with polyclonal anti-6×His horseradish peroxidase (HRP) antibody (MA1-21315-HRP; ThermoFisher Scientific). Membranes were developed using a solid-phase 3,30, 5,50-tetramethylbenzidine (TMB) substrate system.

Characterization of nanoparticles.

Quil-A (VET-SAP, Desert King) stock solution of 0.2% was made in nuclease-free water. Chitosan stock solution (≥75% deacetylated; Sigma) of 0.4% in 1% glacial acetic acid was prepared and diluted to 0.04% in 5 mM sodium acetate buffer, pH 5.5. Plasmid DNA was diluted to 100 μg/ml in 50 mM sodium sulfate buffer, and Quil-A was added to a final concentration of 0.002%. Size distribution and zeta potential of QAC-NPs in aqueous dispersion was measured by dynamic light scattering (DLS) on a Malvern Zetasizer instrument at 25°C. For size distribution, 50 μl of QAC-NPs in solution was placed in a low-volume cuvette and analyzed directly. For zeta potential measurement, approximately 1 ml of the QAC-NPs in solution was placed in a disposable capillary zeta potential cell, available from the Zetasizer Nano series. TEM experiments were performed at the Medical School Electron Microscopy Facility of the University of Wisconsin-Madison using a Philips CM120 transmission electron microscope (FEI, Eindhoven, the Netherlands) at 80 kV. The size and morphology of vaccine preparations were reexamined via negative staining using the drop method. QAC NPs loaded with 1 mg total DNA were resuspended in 600 μl of 0.05 M phosphate-buffered saline (PBS, pH 7.4) at 37°C. At each time point, suspensions were removed and centrifuged at 14,000 relative centrifugal force for 20 min. The supernatant was removed and replaced with PBS and returned to incubation. Supernatant samples were quantified for released DNA from the QAC using a NanoDrop spectrophotometer and compared to the total DNA used. QAC-GFP DNA (5 μg) was added to 3 × 10⁷ Expi293F cells seeded into a 6-well plate. Seventy-two hours after addition, the presence of GFP⁺ cells was identified using an upright fluorescence microscope. Background fluorescence was normalized using GFP⁻ Expi293F control cells.

Vaccine safety study.

In this study, the tolerability and biocompatibility of pQAC-N was evaluated in 1-day-old white leghorn SPF chicks and ECEs. A total of 30 chicks were divided into 3 groups of 10 each. Chicks from the first group were inoculated with PBS (negative control). Chicks from the other groups were inoculated with either a single dose (100 μg) or 2× the dose (200 μg) of pQAC-N at day 1 via the intranasal route. Chicks were monitored for general or respiratory distress, depression, or inappetence and weight gain over the course of 30 days postinoculation. In another experiment, 6 embryonated chicken eggs (ECEs) were divided into 2 groups of 3 each. At 18.5 days postincubation, ECEs were inoculated with PBS (negative control) or with 100 μg of pQAC-N construct into the allantoic cavity. Embryo development and hatch rate of inoculated ECEs were monitored.

Vaccine efficacy studies.

The protective efficacy of the pQAC-N construct was evaluated in 1-day-old white leghorn SPF chicks (Charles River Laboratories). A total of 50 chicks was divided equally into 5 groups (n = 10 each) and used for the efficacy study; the first 2 groups were inoculated with PBS (negative control) or commercial Arkansas MLV (positive control; Mildvac-Ark; Merck Animal Health USA) via direct intranasal instillations (dose according to the manufacturer’s instructions). The other groups were vaccinated naked (no adjuvant) or with chitosan-complexed or pQAC-N at day 1, followed by a booster dose at day 14 via the i.n. route. A vaccine dose (100 ng/bird) was administered at each vaccination time point. At 20 DPV, PBMCs were harvested from blood collected using previously described protocols for the proliferation assay (see below). For all the vaccine experiments, birds were challenged with a dose of 6.5E9 genome copy numbers or 10^6.5 EID₅₀/bird of virulent IBV Arkansas DPI strain via direct intranasal instillations. The challenge dose was determined in an independent infection experiment, wherein the challenge dose resulted in discernible clinical signs as early as 3 DPC and peak viral load replication was observed at 6 DPC. Lachrymal fluid (tears) samples were harvested for ELISA at 10 and 20 DPV and 3 DPC and at 6 DPC for viral load estimation (see below). Lachrymation was induced by placing sodium chloride (salt) crystals on the eyes, and tears were collected using micropipettes (54). Clinical severity was noted every day postchallenge for 8 days, as described before (39). The severity scores of clinical signs of IBV were the following: 0, normal; 1, infrequent sneezing (single event during observation); 2, frequent sneezing (more than one event during observation); 1, mild rales; 2, severe rales; 2, presence of nasal exudate. The severity scores of IBV clinical signs, described in the figure legends, were recorded once a day for each chicken for 8 days after challenge. Lachrymal fluid harvested at 6 DPC was analyzed for viral RNA using IBV N gene-specific qRT-PCR.

To test vaccine performance in commercial chicks, pQAC-N was used to immunize 1-day-old commercial white leghorn chicks (Cackle Hatchery, MO, USA). A total of 10 chicks were divided into 2 groups. Chicks from the first group (n = 4) were inoculated with PBS (negative control), while the second group (n = 6) was immunized with pQAC-N construct at day 3, followed by a booster dose at day 14 via the i.n. route. Blood was collected from birds in the negative-control group at days 10, 20, and 24 in age to quantitate MDA IgY. All birds were sampled, challenged, and monitored for clinical scores and virus titers as described above for the SPF chicks. In another experiment, pQAC-N was used to immunize 3-day-old commercial white leghorn chicks. A total of 35 chicks were divided into 3 groups. Chicks were inoculated with PBS (negative control, n = 11), commercial Arkansas MLV (positive control, n = 12; Mildvac-Ark; Merck Animal Health USA), or pQAC-N construct at day 3, followed by a booster dose at day 17 (14 DPV) via the i.n. route as described above. Blood was collected from all the birds at 0, 10, 20, and 24 DPV to quantitate MDA IgY and lachrymal fluid from all the birds at 10, 20, and 24 DPV to quantitate IBV-specific IgA. All birds were sampled, challenged, and monitored for clinical scores, as described above for the SPF chicks. Tracheal swabs were taken at 6 DPC and analyzed for viral RNA using IBV N gene-specific qRT-PCR. One bird in the MLV group died at 10 DPV, and one bird from MLV and PBS control groups each were euthanized at 12 DPV for necropsy and diagnosis by the Wisconsin Veterinary Diagnostic Laboratory (WVDL), Madison, WI, USA.

Recombinant protein purification.

To improve ELISA sensitivity, the pCAG constructs were used to produce purified S1 and N proteins. The pCAG-S1 and pCAG-N plasmids with 6×His tags were transfected into Expi293F cells as described above using TransIT PRO transfection reagent according to the manufacturer’s instructions (Mirus Bio, WI, USA). For S1 purification, supernatant was harvested, while cells were harvested for N purification at 3 days after transfection. The samples were purified using Thermo Scientific HisPur Ni-NTA resin according to the manufacturer’s instructions (batch method). The protein was eluted with elution buffer (50 mM sodium phosphate, 0.3 M sodium chloride, 250 mM imidazole, pH 8.0). The eluate was concentrated using a polyethylene glycol (PEG) 20 solution (800 mg/ml) hygroscopically and dialyzed using Thermo Scientific SnakeSkin dialysis tubing (7,000 molecular weight cutoff) against PEG-free PBS. Protein concentration was determined by a Thermo Scientific Pierce bicinchoninic acid protein assay kit.

IBV-specific ELISA.

Sera and lachrymal fluid from different time points were screened for humoral response against the IBV Arkansas serotype. To measure IgY and IgA antibody levels in plasma and tears of chicken, respectively, an IBV-specific enzyme-linked immunosorbent assay (ELISA) was developed as described previously, with modifications (55). Briefly, ELISA plates were coated with inactivated IBV Arkansas (100 ng/well, IgY) or IBV Arkansas S1 and N6×His protein (50 ng total/well, IgA) diluted in carbonate-bicarbonate buffer, pH 9.6, and incubated overnight at 4°C, followed by blocking with 5% skim milk to reduce background. A volume of 50 μl of diluted serum (1/200) or tears (1/50), harvested at different time points from immunized chickens, was added to the wells and incubated at 37°C for 1 h. After washing (PBS‐Triton X-100, 0.1%), either HRP-conjugated anti-chicken IgY (NBP1-74778; NOVUS Bio) or anti-chicken IgA (NB7284; NOVUS Bio) at a dilution of 1/1,000 was added to the wells and incubated at 37°C for 1 h. After washing, 50 μl of TMB substrate solution was added and incubated for 20 min or until color developed. The reaction was stopped by the addition of 1 M sulfuric acid, and plates were read at 450 nm. To generate standard curves, sera and tears from severely IBV-infected chickens from previous experiments were used. Twofold serial dilutions were assigned arbitrary values and used for analysis.

Assessment of IBV-specific lymphocyte proliferation assay.

PBMCs were prepared from harvested blood as described previously (49). PBMCs were adjusted to 10⁷ cells/ml in RPMI 1640 (Invitrogen) supplemented with 10% inactivated fetal calf serum, and 100 μl cells per well was transferred into flat-bottomed 96-well plates. Equal volumes of medium containing stimulant (IBV Ark DPI live virus; multiplicity of infection of 1) was added in triplicate, and cultures were incubated for 2 days at 41°C, 5% CO₂. Negative controls received 100 μl RPMI 1640 medium only. After incubation, to each well, 15 μl of MTT reagent (CellTiter 96 non-radioactive cell proliferation assay; Promega) and cells incubated for a further 4 h at 41°C, 5% CO₂, until development of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide] formazan was observed. Postincubation, dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals and incubated for an hour at 41°C, 5% CO₂. The absorbance of the purple color was taken at 570 nm in an ELISA plate reader. PBMCs from 4 chicks/group were used to assess proliferative capacity. The output stimulation index (SI) is the ratio of absorbance poststimulation to the absorbance in unstimulated conditions.

Viral load measurement.

RNA was extracted from lachrymal fluid (10 μl) or tracheal swabs (100 μl) collected from chickens using a Zymo Direct-Zol RNA mini prep kit (Zymo Research, CA, USA) according to the manufacturer’s instructions. qRT-PCR was conducted in two steps: cDNA synthesis (Invitrogen SuperScript III first-strand synthesis system) and qPCRs. cDNA synthesis was performed with 0.5 μl (50 ng/μl) random hexamers, 0.5 μl of 10 mM deoxynucleoside triphosphates, and 4 μl RNA, heated at 65°C for 5 min, and chilled on ice, followed by addition of 1 μl of 10× RT buffer, 1 μl of 0.1 M dithiothreitol, 1 μl of 25 mM MgCl₂, 0.5 μl of RNaseOUT, and 0.5 μl of SuperScript III enzyme in a final volume of 10 μl. The reaction conditions included 25°C for 5 min, 50°C for 60 min, and 70°C for 15 min. SYBR green qRT-PCR was performed using an IBV N gene-specific primer pair set (forward primer, 5′-ATGCTCAACCTAGTCCCTAGCA-3′; reverse primer, 5′-TCAAACTGCGGATCATCACGT-3′) amplifying 128 nucleotides of the N gene of IBV Arkansas DPI. PCRs were performed using a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA) under the following conditions: one cycle at 95°C for 2 min, followed by 40 cycles of 95°C for 3 s and 60°C for 30 s. Each 20-μl reaction was carried out using 1 μl of diluted cDNA (1/10), 10 μl of GoTaq qPCR master mix (Promega), 2 μl of forward and reverse primers, and 7 μl of nuclease-free water. A serial 10-fold dilution of pCAG-IBV Ark N6×His plasmid was used to establish the standard curve. Temperature melt curve analysis was used to confirm the specificity of the product. The challenge dose, as estimated with the above-described method, was 6.5E9 genome copy numbers, which roughly translated to 10^6.5 EID₅₀.

Flow cytometric assessment of IBV specific proliferation.

Subgroups of additional chicks (N = 4 each) from each vaccine group in the SPF chick vaccine efficacy study were used for flow cytometric assessment. All chicks were euthanized at 20 DPV, and single-cell suspensions from lungs were prepared using standard techniques and used for T-cell proliferation assay. Briefly, lungs were excised and placed in a gentleMACS dissociator M tube (130-093-236; Miltenyi) with 5 ml collagenase B (2 mg/ml; Roche). Lung tissue was processed using the gentleMACS dissociator, followed by incubation for 30 min at 37°C. Single-cell lung suspensions were prepared by gently squeezing through a 70-mm cell strainer (Falcon) after lysing red blood cells using 1× BD Biosciences BD Pharm Lyse. A total of 10⁷ cells/ml were stained with CellTrace violet cell proliferation dye (C34557; Thermo Scientific), according to the manufacturer’s instructions, and 100 μl of cells plated/well in RPMI 1640 with 10% chicken immune serum. After overnight incubation at 41°C, 5% CO₂, cells were stimulated with 130 ng of IBV Arkansas N6×His protein complexed with chitosan per well in 100 μl of RPMI 1640 with 10% chicken immune serum. Four days poststimulation, cells were stained for surface markers CD4-Alexa Fluor 647 (AF647) (clone CT-4) and CD8α-fluorescein isothiocyanate (FITC) (clone 3-298) together and TCRγδ-FITC (clone TCR-1) independently for flow cytometry analysis. All antibodies were purchased from SouthernBiotech (Birmingham, AL, USA). All samples were acquired on a BD LSR Fortessa flow cytometer. Data were analyzed with FlowJo software (BD Biosciences). The strategy for gating on proliferating CD4⁺ and CD8a⁺ T cells was debris exclusion on the forward scatter (FSC)-side scatter (SSC) dot plot, followed by exclusion of dead cells by fixable viability dye eFluor 780 (number 65-0865-14; Invitrogen) staining. Out of the live cells, total proliferated cells were gated positive using a histogram plot with ef450 on the x axis (for CellTrace violet). Finally, CD4 cells were gated positive at the AF647 axis and CD8a cells were gated positive at the FITC axis in an FITC-AF647 dot plot. A similar approach was used for identifying proliferating TCRγδ⁺ T cells. The output, SI, is the ratio of percent proliferating cells poststimulation to the percent proliferating cells under the unstimulated condition.

Statistical analysis.

Statistical analyses were performed using GraphPad software (La Jolla, CA). Weight gain, cellular immune assays, clinical severity scoring, and viral loads were compared using an ordinary one-way analysis of variance (ANOVA) test with multiple comparisons, where P values of <0.05 (*), <0.01 (**), <0.001 (***), and <0.0001 (****) were considered significantly different among groups. Antibody titers and absolute weight of birds were compared using a two-way ANOVA test, where P values of <0.05 (*), <0.01 (**), <0.001 (***), and <0.0001 (****) were considered significantly different among groups.