Attenuated Salmonella typhimurium delivering DNA vaccine encoding duck enteritis virus UL24 induced systemic and mucosal immune responses and conferred good protection against challenge
- Xia Yu†1, 2,
- Renyong Jia†1, 2, 3Email author,
- Juan Huang†1, 2,
- Bin Shu†1, 2,
- Dekang Zhu1, 2,
- Qing Liu3,
- Xinghong Gao1, 2,
- Meng Lin1, 2,
- Zhongqiong Yin3,
- Mingshu Wang1, 2, 3,
- Shun Chen1, 2, 3,
- Yin Wang3,
- Xiaoyue Chen1, 2 and
- Anchun Cheng1, 2, 3Email author
© Yu et al.; licensee BioMed Central Ltd. 2012
Received: 23 January 2012
Accepted: 6 July 2012
Published: 6 July 2012
Orally delivered DNA vaccines against duck enteritis virus (DEV) were developed using live attenuated Salmonella typhimurium (SL7207) as a carrier and Escherichia coli heat labile enterotoxin B subunit (LTB) as a mucosal adjuvant. DNA vaccine plasmids pVAX-UL24 and pVAX-LTB-UL24 were constructed and transformed into attenuated Salmonella typhimurium SL7207 resulting SL7207 (pVAX-UL24) and SL7207 (pVAX-LTB-UL24) respectively. After ducklings were orally inoculated with SL7207 (pVAX-UL24) or SL7207 (pVAX-LTB-UL24), the anti-DEV mucosal and systemic immune responses were recorded. To identify the optimum dose that confers maximum protection, we used different doses of the candidate vaccine SL7207 (pVAX-LTB-UL24) during oral immunization. The strongest mucosal and systemic immune responses developed in the SL7207 (pVAX-LTB-UL24) (1011 CFU) immunized group. Accordingly, oral immunization of ducklings with SL7207 (pVAX-LTB-UL24) showed superior efficacy of protection (60-80%) against a lethal DEV challenge (1000 LD50), compared with the limited survival rate (40%) of ducklings immunized with SL7207 (pVAX-UL24). Our study suggests that the SL7207 (pVAX-LTB-UL24) can be a candidate DEV vaccine.
Duck viral enteritis (DVE, also called duck plague), caused by Anatid herpesvirus 1 (Duck enteritis virus, DEV), is an acute, contagious viral disease of ducks, geese and swans, accounting for a high mortality rate in ducks and decreased egg production, leading to heavy economic losses [1–4]. The symptoms of this disease include vascular damage, eruptions at specific locations on the mucosal surface of the gastrointestinal tract, lesions of lymphoid organs and degenerative sequelae in parenchymatous organs .
Immunization of ducks is an efficient way to prevent DEV infection [6, 7]. The commonly used DEV attenuated live vaccine, provides a good protection against DEV infection . However, the production and supply of the vaccine is insufficient, considering the large number of domestic and wild ducks . Additionally, sometimes this vaccine fails to protect ducks after intramuscular or subcutaneous vaccination and, because it is grown in chick embryos, it may harbor other infectious agents such as H5N1 [6, 9]. Therefore, a novel and more effective vaccine to protect against DEV infection is urgently required. Recently, some enteropathogenic bacteria  have been used as effective carriers for DNA vaccine including attenuated strains of Listeria monocytogenes, Salmonella spp  and Shigella spp . These bacteria are attractive vectors to deliver DNA vaccines to immunological inductive sites at mucosal surfaces and antigen-presenting cells (APC), which can improve mucosal and systemic responses against pathogens [14, 15]. Amongst these bacteria, attenuated Salmonella has been extensively studied [15, 16]. However, the use of attenuated Salmonella typhimurium as a DNA vaccine carrier in DEV has not yet been reported. A few antigens derived from pathogenic microorganisms, such as Escherichia coli heat-labile enterotoxin (LT), can be used as adjuvants to improve systemic and mucosal responses . The nontoxic B subunit (LTB) is commonly used for this purpose [18, 19]. These strategies of DNA vaccine combined with adjuvant might provide new opportunities in the development of DEV vaccine.
DEV was classified as a separate genus of the Alphaherpesvirinae subfamily in the Herpesviridae family [1, 2]. One gene UL24, is considered to be a core herpesvirus gene and is conserved among most of the herpes viruses [20, 21], and null mutations or mutations in the conversed regions of UL24 can confer a syncytial phenotype and result in decreased viral yields in cultured cells, indicating that UL24 is important for efficient viral replication [22, 23]. In addition, UL24 protein has the ability to elicit a specific antibody response .
In this study, we used LTB as an adjuvant fused to UL24 gene and the attenuated S. typhimurium aroA- strain SL7207 as a vector to deliver DEV DNA vaccines. The results indicate that oral immunization of the recombinant S. typhimurium could induce specific immune response against DEV.
Materials and methods
Bacterial strains, plasmids, experimental ducklings
Eukaryotic expression pVAX1 (Invitrogen, Carlsbad, California, USA), which contains the cytomegalovirus (CMV) promoter and bovine growth hormone (BGH) poly A signal, and enterotoxigenic E. coli K88ac were generously provided by Professor Sanjie Cao of Sichuan Agricultural University, China. The attenuated S. typhimurium aroA- strain SL7207 (S. typhimurium 2337–65 derivative hisG46, DEL407 [aroA::Tn10 (Tcs)]) was kindly provided by Professor Kai Schulze of Helmholtz Centre for Infection Research (Germany). 7-day-old Tianfu ducklings were purchased from commercial duck farms (Ya’an, China) and fed under standard conditions.
Construction of expression plasmids
Transient expression of the recombinant plasmids
When COS-7 cells growing in 6-well plates (Corning Life Sciences, Corning, New York, USA) were 60-90% confluent, the cells were transiently transfected with plasmids pVAX-UL24, pVAX-LTB-UL24 and pVAX1 using Lipofectamine 2000 (Invitrogen), respectively. Forty-eight hours after transfection, the cells were washed with phosphate-buffered-saline (PBS) and were fixed with 4% paraformaldehyde for 15 min at room temperature and washed again. After being permeabilized with 0.2% Triton X-100 (v/v in PBS) for 10 min, the cells were blocked in 5% BSA for 1 h at 37 °C. Then, the samples were incubated with diluted primary and secondary antibodies at 37 °C for 1 h, respectively. Primary antibodies used were purified rabbit anti-DEV UL24 polyclonal and secondary antibodies were fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Sigma, St. Louis, Missouri, USA). After the cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature, the coverslips were mounted onto glass slides with a drop of buffered glycerol and analyzed by fluorescence microscopy (Nikon, Tokyo, Japan).
Transformation of attenuated S. typhimurium strain SL7207 with DNA vaccine plasmids
The recombinant plasmids pVAX-UL24, pVAX-LTB-UL24 and control vector pVAX1 were transformed into attenuated S. typhimurium strain SL7207 by electroporation using the Micropulser Electroporator (Bio-Rad, Hercules, California, USA). The positive transformants were selected on LB agar containing kanamycin (50 μg/mL) and then were confirmed by sequencing and PCR. The constructs were named strain SL7207 (pVAX-UL24), strain SL7207 (pVAX-LTB-UL24) and strain SL7207 (pVAX1) respectively. The primers of UL24 gene were designed as follows: primer 3: 5'-ATGGCATCGAAGGTACAGAAA AAGC-3' (forward) and primer 4: 5'-CTCGAGCTAGTGTTTAGTGGTCTGAA-3' (reverse). The primers to amplify LTB-UL24 (about 1600 bp in length) were primer 1 (as aforementioned) and primer 4.
RT-PCR detection of transcripts in vivo
2-week-old ducklings were orally administered 1 × 1010 CFU of SL7207 (pVAX-UL24) or SL7207 (pVAX-LTB-UL24), and control ducklings were given the same dose of SL7207 (pVAX1). Three days after the immunization, ileums (from three ducklings immunized with SL7207 (pVAX-UL24), SL7207 (pVAX-LTB-UL24) or SL7207 (pVAX1)) were removed and pooled. The UL24 and LTB-UL24 transcripts (mRNA) were then analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR).
Immunization and serum sampling
Ducklings, randomly divided into 6 groups (40 per group), were allowed to adapt to the new environment for 7 days, deprived of food and water for 4 h prior to immunization, and immunized three times at 7-day intervals. For oral immunization, each duckling received 100 μL of 10% NaHCO3 intragastrically 30 min before immunization to neutralize gastric acids. Ducklings of group A, B and C were intragastrically inoculated with SL7207 (pVAX-LTB-UL24) at doses of 1011, 1010 and 109 CFU per duckling respectively. Ducklings in groups D and E were inoculated intragastrically with SL7207 (pVAX-UL24) and the control strain SL7207 (pVAX1), respectively, at 1010 CFU per duckling. Group F ducklings received PBS and were used as a negative control.
At weeks 1, 2, 3, 4, 5, 6 and 8 after the first immunization, three ducklings of each group were sacrificed for sera, bile and duodenum. To prepare duodenal fluid, its connective and fat tissues in serosa were removed in PBS and were opened to expose their lumens. After a 1 cm section of this intestinal tract was placed in 1 mL of PBS containing 100 μg/mL of trypsin inhibitor (Sigma), the mixture was centrifuged and the supernatant was collected. Accordingly, the bile obtained was centrifuged and the supernatant was collected. All of these samples were stored at −20 °C prior to analysis.
Measurement of antibody levels
Enzyme-linked immunosorbent assay (ELISA) was used to analyze DEV-specific antibodies in serum, duodenum and bile. In brief, 96-well polystyrene microtitre plates were coated with 100 μL 0.25 μg/mL purified DEV-antigen and incubated overnight at 4 °C. The plates were washed three times with PBS containing 0.05% Tween 20 (PBST) and blocked by incubation with 100 μL of blocking solution (1% BSA in PBST) for 1 h at 37 °C. After washing, a 100-μL volume of diluted duckling serum was added to each well, and incubated for 1 h at 37 °C. Rabbit anti-duck IgG or IgA-horseradish peroxidase conjugate (Sigma) was used as the secondary antibody at 1:2000 dilutions and incubated for 1 h at 37 °C, then washed again. The substrate, 3,3’,5,5’-tetramethy1 benzidine (TMB), added and incubated for 30 min at 37 °C. 50 μL of 2 mol/L H2SO4 was added to stop the reaction, and then the optical density (OD) at 450 nm was measured using an ELISA reader.
Measurement of neutralizing antibody responses
Serum samples were inactivated at 56 °C for 30 min and diluted in serial twofold dilutions in MEM (GIBCO, Grand Island, New York, USA). Each sample was mixed with an equal volume containing 200 TCID50 of DEV and incubated for 1 h at 37 °C. One-hundred microliters of the above serum-virus mixture was transferred to duck embryo fibroblast monolayers on 96-well culture plates (Corning Life Sciences) and incubated at 37 °C for 1 h. The mixture in each well was then replaced with MEM containing 2% FCS (GIBCO). Cytopathic effects were observed for 4 days and the end-point dilution of each serum sample was calculated by the Reed-Muench formula .
Detection of CD4+ and CD8+ T cells by flow cytometry
Peripheral blood lymphocytes (PBL) were isolated and indirect staining of the cells was carried out as described elsewhere . Briefly, PBL were isolated from heparinized blood samples, washed twice by PBS, and adjusted to a final concentration of 5 × 105 cells per mL. Then 1:200 diluted anti-duck CD4 monoclonal antibody and 1:1000 diluted anti-duck CD8 monoclonal antibody (AbD Serotec Ltd, Oxford, UK) were added into the cells. After incubation for 30 min at 4 °C in the dark, FITC-labeled goat anti-mouse IgG (AbD Serotec Ltd) was added. Subsequently, the cells were washed with PBS and resuspended in 500 μL PBS, followed by flow cytometric analysis. Viable lymphocytes were gated on the basis of forward and side scatter characteristics, and 10 000 events were analyzed for positive staining with FITC. Data analysis was carried out using BD FACSAria software.
To assess the protection of DNA vaccines against DEV in immunized ducklings, 10 ducklings from each group were orally challenged with 1000 lethal doses, 50% (LD50) of DEV at 6 weeks after primary immunization. These ducklings were monitored daily for survival for 10 days after challenge.
Construction and transient expression of pVAX-UL24 and pVAX-LTB-UL24 in COS-7 cells
Transcripts of UL24 and LTB-UL24 genes in vivo
Humoral immune responses of ducklings
Mucosal antibody responses of ducklings
Induction of DEV neutralizing antibodies
Neutralizing antibody titers in ducklings immunized with DEV DNA vaccines
Analysis of T lymphocytes in PBL
The strongest induction of CD4+ and CD8+ T cells was observed in the 1 × 1011 CFU SL7207 (pVAX-LTB-UL24) vaccinated group at all time points. The CD4+ population of this group was significantly higher than those in other oral groups at 6 weeks post immunization (P < 0.05). Compared with the vaccinated groups, the number of CD8+ in 1 × 1011 CFU SL7207 (pVAX-LTB-UL24) group was slightly higher but showed no difference in significance (P > 0.05) during the entire experiment. The number of CD4+ and CD8+ T cells generated in group B was greater than that in group D, but no significant difference was observed (P > 0.05).
Protection of ducklings against DEV challenge
DEV has the ability to establish latent infections and an asymptomatic carrier state in waterfowl, which further reinforces the difficulties in the control and prevention of the transmission of DEV [7, 27], and may explain why conventional DEV vaccines (inactivated and attenuated DEV preparations) sometimes fail to protect ducks. Therefore, it is urgent to develop a novel or more effective vaccine to control DEV. A DNA vaccine is considered a good choice as it has several advantages, including the simplicity of manufacture, biological stability, cost effectiveness, safety, ease of transport in lyophilized form and the ability to act in the presence of maternal immunity . More importantly, DNA vaccines elicit both humoral and cell-mediated immunity (CMI) stimulated by the presentation of peptide fragments. They have the potential to immunize against a broad range of pathogens, since eukaryotic expression can improve protein folding and therefore enable surface-exposed epitopes to be correctly presented and enable the introduction of post-translational modification [29–32]. These immune responses can be improved by other strategies such as improvements in DNA delivery and the inclusion of adjuvant (either as a gene or co-administered agent) . The evidence supporting this hypothesis comes from this study where we used LTB as a molecular adjuvant and developed a new Salmonella-based DNA vaccine delivery system. Ultimately, we demonstrated that DNA vaccines delivered by attenuated Salmonella expressing UL24 gene have a great ability to elicit systemic and mucosal immune responses and LTB from E. coli is a very useful adjuvant for DEV DNA vaccines.
As expression of antigens in antigen-presenting cells (APC) by DNA vaccine is a potential means of priming immune responses, targeting DNA to APC is likely to increase DNA vaccine potency [34–36]. In this study, we detected the transcripts of UL24 and LTB-UL24 genes by RT-PCR in vivo, indicating that both SL7207 (pVAX-UL24) and SL7207 (pVAX-LTB-UL24) can be expressed by APC (Figure 2). Because of the low number of APC present in muscle, delivery of DNA vaccine through traditional administration (intramuscularly or subcutaneously) would result in poor immune responses [33, 37]; while a large fraction of bacteria carrying DNA vaccine are taken up by APC which express and present the antigen after crossing the intestinal mucosal barrier . Therefore we used the oral route to immunize the ducklings. In addition, some component of gram-negative bacteria such as lipopolysaccharide (LPS) has been shown to act as a potent adjuvant which can enhance immune responses . Moreover, attenuated strains of Salmonella are attractive candidates for mucosal vaccine vectors, because they elicit both mucosal and systemic immune responses against carried antigens [39, 40]. Therefore, attenuated S. typhimurium is not only a promising vaccine carrier, but may also act as an adjuvant to stimulate immune responses . Accordingly, the results in this work were consistent with the aforementioned findings. Oral delivery of DEV DNA vaccines was an efficient way of inoculation, achieving high neutralizing antibody titers and ELISA antibodies (Table 1, Figures 4 and 5). Cell-mediated immune responses were significantly stimulated by DNA vaccines delivered by attenuated S. typhimurium (Figure 6).
HSV-specific CD4+ and CD8+ T cells are protective in animal models during HSV infection and the antigen-specific CD4+ T cells support later expression of antigen-specific CD8+ T cells [41, 42]. In our tests, the number of CD4+ and CD8+ T cells showed an obvious rise in the early stage after immunization with DNA vaccines. A dramatic increase was seen in the CD4+ population, while CD8+ rose to a lesser degree. The most significant induction in CD4+ and CD8+ T cells was observed in 1 × 1011 CFU SL7207 (pVAX-LTB-UL24) immunized group at all time points, indicating that SL7207 (pVAX-LTB-UL24) can induce strong cellular immune responses. In addition, CD4+ T cells play important roles in modulating immune responses, up-regulating the costimulatory molecules on APC cells and enhancing their ability to process and present antigen . Therefore the CD4+ T cell response is a prerequisite when using a potential vaccine. Our work also demonstrated that DEV vaccines could provoke both specific CD4+ and CD8+ T cell responses to prevent or control infection.
The cholera toxin (CT) and heat-labile toxin (LT) from E. coli are the most commonly used mucosal adjuvants for immunization. Co-administration of CT or LT with antigen can result in substantial enhancement of antigen-specific secretory and systemic antibody responses [19, 44, 45]. LTB is widely used as an effective adjuvant to induce protective mucosal and systemic immune responses since LTB exhibits low toxicity, which was also demonstrated by our work (Table 1, Figures 45 and 6). Amongst the oral groups, SL7207 (pVAX-LTB-UL24) induced obviously better mucosal and systemic immune responses, compared with SL7207 (pVAX-UL24). When comparing the three different doses of SL7207 (pVAX-LTB-UL24), we found that immunogenicity of the DEV DNA vaccine was dose-dependent, with higher doses of SL7207 (pVAX-LTB-UL24) (e.g. 1 × 1010 CFU or 1 × 1011 CFU) inducing better immune responses. Delivery of the DNA vaccine at a low dose (109 CFU) induced systemic and mucosal immune responses, which were enhanced by increasing the dose of attenuated Salmonella. Moreover, ducklings immunized with 1011 CFU of SL7207 (pVAX-LTB-UL24) showed the highest anti-DEV mucosal and systemic immune response throughout the experiment.
Challenge studies revealed that higher mucosal and systemic immune responses were more protective against DEV infection. These findings suggest that the DEV DNA vaccine with the LTB gene fused to UL24 and delivered by attenuated S. typhimurium induced protective prophylactic responses against DEV challenge and this DNA vaccine at a high dose (1011 CFU) showed a superior protection, demonstrating that LTB is a potential and effective adjuvant of the DEV DNA vaccine.
Taken together, our results indicate that oral immunization with attenuated S. typhimurium as a DNA vaccine carrier induces efficient systemic and mucosal immune responses. We report here the first attempt to develop oral DNA vaccines against DEV infection and have shown that orally administered attenuated S. typhimurium co-expressing UL24 and LTB was able to protect ducklings against DEV infection. Consequently, a DNA vaccine encoding the UL24 gene of DEV and LTB gene of E. coli delivered by attenuated S. typhimurium may be a promising DEV vaccine.
We thank Dr Kenneth Roland from the Center for Infectious Disease and Vaccinology at Arizona State University for editing the manuscript. The research was supported by Changjiang Scholars and Innovative Research Team in University (PCSIRT0848), China Agricultural Research System (CARS-43-8), the Ministry of Science and Technology Support Program (2009GJF00034), Sichuan Province Research Program (2009JY0070/09ZX005/11ZA084) and China 973 program (2011CB111606).
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