- Research article
- Open Access
Oral immunization with a novel attenuated Salmonella Gallinarum encoding infectious bronchitis virus spike protein induces protective immune responses against fowl typhoid and infectious bronchitis in chickens
© The Author(s) 2018
- Received: 8 July 2018
- Accepted: 3 September 2018
- Published: 12 September 2018
Fowl typhoid (FT), a septicemic disease caused by Salmonella Gallinarum (SG), and infectious bronchitis (IB) are two economically important avian diseases that affect poultry industry worldwide. Herein, we exploited a live attenuated SG mutant, JOL967, to deliver spike (S) protein 1 of IB virus (V) to elicit protective immunity against both FT and IB in chickens. The codon optimized S1 nucleotide sequence was cloned in-frame into a prokaryotic constitutive expression vector, pJHL65. Subsequently, empty pJHL65 or recombinant pJHL65-S1 plasmid was electroporated into JOL967 and the resultant clones were designated as JOL2068 and JOL2077, respectively. Our results demonstrated that the chickens vaccinated once orally with JOL2077 elicited significantly (p < 0.05) higher IBV-specific humoral and cell-mediated immunity compared to JOL2068 and PBS control groups. Consequently, on challenge with the virulent IBV strain at 28th day post-vaccination, JOL2077 vaccinated birds displayed significantly (p < 0.05) lower inflammatory lesions in virus-targeted tissues compared to control groups. Furthermore, 33.3% (2 of 6) of birds vaccinated with JOL2077 vaccine had shown virus recovery from tracheal tissues compared to 100% (6 of 6) recovery obtained in both the control groups. Against wild-type SG lethal challenge, both JOL2077 and JOL2068 vaccinated groups exhibited only 10% mortality compared to 80% mortality observed in PBS control group. In conclusion, we show that JOL2077 can induce efficient IBV- and carrier-specific protective immunity and can act as a bivalent vaccine against FT and IB. Further studies are warranted to investigate the potential of JOL2077 vaccine in broiler and young layer birds.
Infectious bronchitis virus (IBV), one of the prototype viruses of the Coronaviridae family, is an enveloped, single-stranded, positive-sense RNA virus that causes significant economic losses in poultry industry worldwide [1, 2]. In chickens, IBV causes an acute highly contagious disease which affects the respiratory tract, gut, kidneys, bursa and reproductive systems, and the disease is characterized by 50–100% morbidity and 0–25% mortality rates in affected flocks [3, 4]. The viral genome encodes four major structural proteins, namely membrane protein (M), nucleocapsid protein (N), spike protein (S) and envelope protein (E), that facilitate host entry and virus assembly . The S protein is post-translationally cleaved by host cell serine proteases to form two subunits, S1 and S2, and this cleavage plays an important role in viral infectivity . The S1 subunit binds to host cell receptors and is the main target for induction of serotype-specific neutralizing and hemagglutination-inhibiting antibodies [7, 8], and it is sufficient to induce protective immune responses . To prevent economic losses resulted by IB, live attenuated and inactivated oil adjuvanted IBV vaccines have been employed in the field ; however, these types of vaccines are associated with some limitations. Inactivated IBV vaccines are safe, but relatively costlier and less effective than live attenuated vaccines, while use of live attenuated vaccines may result in the emergence of novel variants of the virus [10, 11]. Furthermore, the existence of more than 20 IBV serotypes with little cross protective immunity among vaccine strains results in poor efficacy of the currently available vaccines. The introduction and development of an effective and safe vaccine against each IBV serotype is not an economically viable option for the poultry industry. Therefore, novel approaches should be devised that are not only egg independent and cost-effective, but easy to amplify and can provide efficient protection against the circulating IBV strains.
Identification and characterization of regional and most circulating serotypes plays an important role in the development of a successful vaccination program. Over the past decade, nephropathogenic IBV variants have been continuously evolved in South Korea via accumulated point mutations and by recombination with other existing strains . Thus, emphasizing the need for the development of efficient and effective vaccination strategies which respond to the circulating field strains in a very short notice. In accordance with this notion, the present study exploited Salmonella Gallinarum (SG) to deliver immunogenic S1 protein (20–263 amino acid residues) of most emerging and circulating nephropathogenic strain of IBV in South Korea. SG is an intracellular Gram-negative bacterium that causes fowl typhoid (FT) in domestic birds, primarily chickens, and rarely causes food poisoning in humans . In chickens, FT is characterized by severe inflammation and acute mortality that causes significant economic losses in poultry industry worldwide [13–15]. The disease has worldwide distribution and is endemic in many parts of the world. Besides farm hygiene and biosecurity measures, the control and eradication of FT can be effectively achieved by vaccination with live attenuated and inactivated killed vaccines. We previously have developed an attenuated SG vaccine strain (JOL916) that effectively controls FT infection in chickens . Exploiting live attenuated Salmonella system to deliver IBV S1 immunogenic protein is a highly economical vaccination strategy that allows for a quick response to deployment of vaccines, as it circumvents the need for a constant supply of specific-pathogen-free (SPF) embryonated eggs. Furthermore, Salmonella-based vaccination strategy induces efficient antigen-specific humoral and cell-mediated immunity [17–19], and these vaccines have already been approved for use in poultry . Herein, we show that attenuated SG mutant delivering immunogenic S1 protein elicited efficient IBV-specific humoral and cell-mediated immune (CMI) responses and conferred significant protection against a virulent IBV challenge. We also demonstrate that attenuated SG delivering S1 protein protected chickens against a lethal wild-type SG challenge.
Bacterial strains, plasmids and virus
Bacterial strains and plasmids used in this study
E. coli Δasd strain used for cloning of genes into asd+ plasmid
F−, ompT, hsdSB (r B − , m B − ), dcm, gal, λ (DE3), pLysS, Cmr
∆lon, ∆cpxR and ∆asd mutant of SG
∆lon and ∆cpxR mutant of SG
JOL967 with pJHL65 plasmid
JOL967 with pJHL65-S1 plasmid
BL21(DE3)pLysS with pET28a-S1 plasmid
An asd+ vector, pBR ori, β-lactamase signal sequence-based periplasmic secretion plasmid, 6xHis, high copy number
pJHL65 carrying IBV S1 gene sequence
IPTG-inducible expression vector; Kanr
pET28a plasmid carrying S1 gene
Construction of an attenuated SG mutant delivering IBV spike protein 1
The partial immunogenic S1 amino acid sequence20–263 of most emerging nephropathogenic IBV Korean strain (Accession No. ADQ01077.1) was chosen, in this study, based on the presence of major neutralizing B and T cell epitopes that are well-known to elicit complete protection against IBV infection in vaccinated chickens [22–24]. The S1 nucleotide sequence was codon optimized for efficient expression in SG. The optimized gene sequence was chemically synthesized (Bionee, Korea) and then built into the pJHL65 plasmid, an asd+ constitutive expression vector, and propagated in an asd mutated Escherichia coli strain as described previously . The S1 gene sequence was cloned in frame downstream to the beta-lactamase signal sequence (bla SS) of pJHL65 vector, so that the intended protein would get secreted out of the bacteria . The recombinant plasmid, pJHL65-S1, was subsequently transformed into an attenuated auxotrophic mutant of SG strain, JOL967, and the resultant clone was designated as JOL2077. The JOL967 strain was constructed by the deletion of the lon, cpxR, and asd genes from the wild-type SG strain, JOL394 isolate, using allelic exchange method as described and reported elsewhere , and used as the delivery vehicle for the S1 protein. To produce the coating antigen for determination of IBV-specific antibody responses, the S1 gene was cloned into pET28a (+) expression vector (Novagen, San Diego, USA) that was subsequently transformed into E. coli BL21 (DE3) pLysS strain (Novagen, USA) for protein expression. The expressed S1 protein in both S. typhimurium and E. coli was confirmed by Western blot analysis using polyclonal infectious bronchitis virus antibody (#ab31671, Abcam). The E. coli expressed S1 protein was purified by Ni–NTA affinity column chromatography and dialysed against PBS (three washes). Purified protein was quantified by a Bradford assay , filtered, and stored at −20 °C until further use.
Immunization and challenge studies
All animal experimentation work was approved by the Chonbuk National University Animal Ethics Committee (CBNU2015-00085) and the chicken experiment was carried out according to the guidelines of the Korean Council on Animal Care. One-day-old female layer chickens (Corporation of Join hatchery, Republic of Korea) were maintained under standard conditions and provided antibiotic-free food and water ad libitum. Four weeks later, the chickens were randomly divided into three groups (n = 15) and vaccinated once orally with sterile phosphate buffered saline (PBS, 100 µL in each bird), JOL2068 (SG carrying an empty pJHL65 vector) or JOL2077 (SG-pJHL65-S1). The 109 colony forming units (CFU) of JOL2068 or JOL2077 were used for inoculation in each bird. Blood was drawn from jugular vein of six randomly vaccinated chickens on day 28 post-vaccination and serum samples were subsequently isolated for IgG analysis. To assess mucosal secretory (s) IgA responses, five chickens on day 28 post-vaccination were sacrificed from each group and intestinal wash samples were collected in PBS (pH 7.2) containing antibiotics and protease inhibitor cocktail (Sigma Aldrich).
Four weeks post-vaccination, all the vaccinated chickens were intranasally challenged with a virulent dose of IBV (104 EID50) and 6 days later post-challenge, the tracheal tissues (n = 6) were aseptically processed and inoculated into 15–16 day old chicken embryos to isolate the challenged virus as described previously . The clinical protection against IBV was assessed based on the death and/or stunting of embryos. For histopathological analysis of trachea, lungs, kidneys and bursa, each tissue from sacrificed birds (n = 6) was aseptically collected on day 6 post-challenge and fixed with 10% formalin, embedded in paraffin and cut into 5 μm thin sections, which were subsequently processed as previously described . The hematoxylin and eosin (H&E) stained sections were examined by light microscopy (Axio Imager 2, Zeiss, Germany) and digital imaging software (Axio Vision, Zeiss, Germany). We also separately maintained three groups (n = 10) vaccinated once orally with JOL2068, JOL2077 or sterile PBS, and 4 weeks later, all the birds were challenged orally with 100 µL of a suspension containing 1.6 × 106 CFU of a wild-type SG (JOL394 strain) as described previously . The protective efficacy of the SG-S1 based vaccine (JOL2077) against JOL394 was evaluated in the context of the mortality of the challenged birds, which was monitored daily for 12 consecutive days. At the end of the observation period post-challenge, the surviving birds were sacrificed to determine the bacterial load in liver and spleen as previously described .
Systemic IgG and mucosal IgA antigen-specific humoral responses
The systemic IBV and SG-ompA specific IgG and mucosal IgA responses were analysed in vaccinated sera and in intestinal wash samples, respectively, by an indirect ELISA . Purified S1 protein and ompA antigen (each 250 ng/well) were used as coating antigens in an indirect ELISA to determine the IBV and the SG specific humoral responses, respectively.
IBV-specific cellular immune responses
The IBV-specific cell-mediated immunity elicited by vaccination was evaluated by a lymphocyte proliferation test. Four weeks post-vaccination, in vitro proliferative capacity of vaccinated peripheral blood mononuclear cells (PBMCs) (1 × 106, n = 6) in response to the recall S1 protein was determined by a MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] based assay . The PBMCs were isolated using Histopaque density gradient as previously described . The proliferative capacity of vaccinated PBMCs in response to recall S1 antigen was further analyzed by flow cytometry. To this end, PBMCs (1 × 106, n = 6) isolated from vaccinated birds on day 28 post-vaccination were stimulated with S1 antigen (10 µg/mL) for 48 h and then S1-specific CD4+ and CD8+ T cell responses were analysed by flow cytometry as previously described .
For analysis of Th1 (IFN-γ) and Th2 (IL-4 and IL-10) cytokine responses, PBMCs (1 × 106, n = 6) were isolated from vaccinated and control birds and restimulated with S1 protein (10 µg/mL) in vitro for 24 h incubation at 37 °C in 5% CO2. Then total RNA isolated from stimulated cells was analysed for IFN-γ, IL-4 and IL-10 mRNA transcription levels by qRT-PCR assay as previously described .
All the obtained data was analysed using GraphPad prism 6.00 program (San Diego, CA, USA). Statistical significance was determined by one-way ANOVA (with Tukey’s multiple comparisons tests). A non-parametric Chi square test was used to analyze significant differences in mortality of birds following SG challenge. Data are represented as mean ± standard deviation. p values of < 0.05 were considered statistically significant.
Attenuated SG mutant efficiently expressed and secreted IBV S1 protein
To construct the SG-based IB vaccine, we predicted highly antigenic epitopes of IBV S1 protein using BepiPred 1.0 Server program based on the protein characteristics such as structural domains, hydrophilicity residues, antigenicity and surface probability (Additional file 1) . To direct the expressed protein to the periplasmic space, the S1 gene was cloned in frame downstream to the bla SS of the pJHL65 vector. The insertion of S1 into pJHL65 vector was confirmed by digestion of the positive clones with EcoR1 and HindIII to release a fragment of 738 bp. Subsequently, the pJHL65-S1 gene construct was electroporated into JOL967 strain and the resultant clone was designated as JOL2077. Western blot analysis revealed that JOL2077 strain efficiently expressed and secreted S1 protein in the culture supernatant of cultured JOL2077 bacteria (Additional file 2). The S1 protein was biologically active as evidenced by the reactivity with polyclonal IBV-specific antibody and by the induction of the antigen-specific immune responses in vaccinated chickens as observed in this study.
Orally administered JOL2077 vaccine induces efficient S1-specific systemic and mucosal antibody responses
Orally administered JOL2077 vaccine elicits efficient IBV-specific cellular immunity
Orally administered JOL2077 provides protection against virulent IBV challenge
Protective efficacy of orally administered SG-based S1 vaccine (JOL2077) in brown nick layer chickens
Recovery of virus from trachea
No. positive/no. tested
JOL2077 vaccine induces SG-specific protective immune responses
Mortality and bacterial load in the chickens post-challenge
This study was aimed to investigate whether attenuated SG delivering S1 protein could induce protective immune responses against both IB and FT in chickens. Our strategy of employing Salmonella system as a delivery system for S1 protein has certain advantages over live attenuated or inactivated IB vaccines. These types of vaccines are easy to prepare, stable, convenient, economical, and easy to administer in chickens. Moreover, these types of vaccines does not require exogenous adjuvants as Salmonella bacteria provide an appropriate danger signals to the immune system, acting as natural adjuvant, thereby promoting efficient maturation and activation of dendritic cells (DCs) [25, 30], which is prerequisite for the induction of potent adaptive immune responses. Also SG-based IB vaccination can differentiate vaccinated birds from infected ones as only structural and immunogenic antigens are exploited for the vaccine development. Previously, Salmonella-based system has been exploited to deliver DNA encoded foreign antigens to induce protective immune responses against a variety of pathogens, especially those invading via the mucosal route [19, 31, 32]. However, a potential limitation associated with the Salmonella delivered DNA vaccines is the inability to stably maintain the plasmid DNA inside the bacterium. Moreover, the gene expressions of heterologous antigens encoded by DNA is inefficient, resulting in poor immunogenicity of the vaccine , thus, requiring multiple immunizations to elicit some level of significant protection in vaccinated animals . In accordance with this notion, we hypothesized that SG delivering S1 antigen in protein format could induce efficient IBV-specific protective immunity in chickens. We show that single oral inoculation of SG-based S1 vaccine induced efficient protective immune responses against IBV in chickens. We also show that SG delivering S1 protein does not affect the induction of SG-specific humoral and cell-mediated protective immune responses.
The present study demonstrated that orally administered JOL2077 vaccine elicited efficient circulating IgG responses in vaccinated chickens. The role of circulating antibody titers in conferring protection against IBV remains controversial. Some studies have demonstrated that circulating antibodies did not correlate well with the protection from IBV infection [33, 34], while others have shown the importance of humoral immunity in disease recovery and virus clearance [35, 36]. Nevertheless, JOL2077 vaccination had conferred significant protection against IBV challenge and had efficiently contained IBV replication in virus-targeted tissues. Furthermore, JOL2077 vaccinated chickens displayed insignificant inflammatory lesions in IBV-targeted tissues. The role of IgA antibodies in providing resistance to IBV infection is well-documented [35, 37]. The elicitation of IgA antibodies at mucosal surfaces plays an important role in limiting spread of pathogens that primarily enter into systemic circulation via mucosal route. This has direct implication on the pathogenesis and the clinical outcome of the disease. We found significantly higher IgA responses in JOL2077 vaccinated chickens compared to the JOL2068 control group. This finding might explain why JOL2077 vaccinated chickens displayed insignificant inflammatory lesions in targeted tissues and achieved higher level of protection than control groups. Consistent with the notion that cellular immunity is protective against IBV infection , we next investigated cytokine and T cell recall responses elicited by our JOL2077 vaccine. After antigenic stimulation, CD4+ T cells secrete anti-viral cytokines, which generate potent antibody formation and CD8+ cytotoxic T lymphocytes, thus playing a critical role in controlling IBV infection [39, 40]. The present study demonstrated that chickens vaccinated with JOL2077 vaccine displayed higher CD4+ and CD8+ T cell responses compared to the control chickens. Consequently, the higher protection rate was observed in chickens vaccinated with JOL2077 vaccine. The nature of the cytokines produced after antigenic stimulation in vitro is an important parameter to define the type of immunity elicited . Our data showed that JOL2077 vaccine induced efficient IBV-specific both Th1 (IFN-γ) and Th2 (IL-4 and IL-10) cytokine responses, which is indicative of the mixed-type of immunity. IFN-γ has potent anti-viral activity through the promotion of natural-killer cells and macrophage activation that are likely to contribute to the containment of IBV replication and spread within the host. Further, IFN-γ upregulates MHC-I and MHC-II molecules and induces IL-12, nitric oxide and superoxide production in macrophages, all of which are important in the elimination of intracellular pathogens . These findings might further explain the higher protection rate observed in JOL2077 vaccinated chickens. The IL-4 and IL-10 have a clear role in promoting humoral immune responses [43, 44]. Previous studies have reported that IL-10 augment the proliferation and plasma differentiation of the B cells that have switched to IgA production, suggesting that IL-10 plays an important role in development of mucosal immunity. Considering that both humoral and cell-mediated immunity are important in containment of IBV replication and clinical disease, our study clearly shows that JOL2077 vaccine is capable of eliciting efficient humoral and CMI responses, and can, thus, offer significant protection against IBV in chickens.
The induction of SG-specific IgG responses following vaccination strongly correlates with the recovery from FT and protection from subsequent challenge infections in chickens [16, 28]. The systemic IgG mediate protection during the early stages of infection when SG circulates extracellularly before it penetrates the target cells . The present study demonstrated that systemic IgG responses were highest in chickens vaccinated with either JOL2068 or JOL2077 strain compared to the PBS control group. Consequently, the mortality rate was lowest in both JOL2068 and JOL2077 vaccinated chickens compared to the chickens that received PBS only. The induction of immune responses at mucosal surfaces serves as the first line defense against pathogens that enter into systemic circulation through mucosal surfaces. Importantly, the presence of antigen-specific secretory (s) IgA antibodies in the intestinal mucus acts as an immunological barrier, thereby preventing the adherence of Salmonella bacteria to the intestinal lining and subsequent penetration into deeper tissues . In the present study, we observed that the sIgA levels were highest in JOL2068 and JOL2077 vaccinated chickens. Consequently, the lower bacterial load was observed in spleen and liver of chickens vaccinated with either JOL2068 or JOL2077 strain. These findings clearly suggest that SG can be exploited to deliver viral antigens to elicit carrier and virus-specific humoral and cell mediated protective immune responses.
In conclusion, we show that SG delivering IBV-specific S1 protein can elicit efficient both SG and S1-specific humoral and cell-mediated immunity and can offer dual protection against FT and IB in chickens. Other studies have shown that efficacious live attenuated IB vaccines have protected chickens against clinical signs and mortality and increased significant resistance in birds to infection . Our results clearly show that vaccination with SG-based IB vaccine induced efficient protection against IBV virulent challenge as evidenced by insignificant inflammation in trachea, lungs, kidneys and bursa and by efficient containment of viral replication in tracheal tissues. Further studies are warranted to investigate the protective efficacy of JOL2077 vaccine in both broiler and young layer chickens.
The authors declare that they have no competing interests.
The work was designed by JHL and IAH. Experiments and sampling were done by IAH and JK. Manuscript draft preparation and statistical analysis was done by IAH. All authors read and approved the final manuscript.
This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) through Agri-Bio industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (Grant Number. 118055-03).
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