- Research article
- Open Access
A live attenuated Salmonella Enteritidis secreting detoxified heat labile toxin enhances mucosal immunity and confers protection against wild-type challenge in chickens
- Jonathan Lalsiamthara1,
- Nitin Machindra Kamble1 and
- John Hwa Lee1Email author
- Received: 27 February 2016
- Accepted: 22 May 2016
- Published: 4 June 2016
Abstract
A live attenuated Salmonella Enteritidis (SE) capable of constitutively secreting detoxified double mutant Escherichia coli heat labile toxin (dmLT) was developed. The biologically adjuvanted strain was generated via transformation of a highly immunogenic SE JOL1087 with a plasmid encoding dmLT gene cassette; the resultant strain was designated JOL1641. A balanced-lethal host-vector system stably maintained the plasmid via auxotrophic host complementation with a plasmid encoded aspartate semialdehyde dehydrogenase (asd) gene. Characterization by western blot assay revealed the dmLT subunit proteins in culture supernatants of JOL1641. For the investigation of adjuvanticity and protective efficacy, chickens were immunized via oral or intramuscular routes with PBS, JOL1087 and JOL1641. Birds immunized with JOL1641 showed significant (P ≤ 0.05) increases in intestinal SIgA production at the 1st and 2nd weeks post-immunization via oral and intramuscular routes, respectively. Interestingly, while both strains showed significant splenic protection via intramuscular immunization, JOL1641 outperformed JOL1087 upon oral immunization. Oral immunization of birds with JOL1641 significantly reduced splenic bacterial counts. The reduction in bacterial counts may be correlated with an adjuvant effect of dmLT that increases SIgA secretion in the intestines of immunized birds. The inclusion of detoxified dmLT in the strain did not cause adverse reactions to birds, nor did it extend the period of bacterial fecal shedding. In conclusion, we report here that dmLT could be biologically incorporated in the secretion system of a live attenuated Salmonella-based vaccine, and that this construction is safe and could enhance mucosal immunity, and protect immunized birds against wild-type challenge.
Keywords
- Protective Efficacy
- Oral Immunization
- Intramuscular Route
- SIgA Level
- Buffer Peptone Water
Introduction
Salmonella enterica serovar Enteritidis is one of the most frequently isolated bacteria from human infections worldwide [1]. Foodborne salmonellosis is widespread in developing and developed countries, resulting in approximated 155 000 deaths every year [2]. Eggs, meat and meat products are the most common transmission vehicles of Salmonella infections [3]. Due to the ubiquitous presence and rapid spread of Salmonellae in poultry premises, enforcing control measures can be expensive and still does not ensure complete elimination of the organism. Control strategies, such as culling, antibiotic interventions, and Salmonella-free feed approaches, are being deployed with varying success to control the Salmonella transmission cycle [4]. In particular, poultry vaccination is the suggested ideal strategy for controlling Salmonella Enteritidis infections on poultry farms and thereby reducing food contamination [5, 6].
The heat-labile enterotoxin (LT) of E. coli is a potent oral adjuvant boosting both the humoral and cellular immune responses when co-administered with antigens. However, it also induces secretory diarrhea, even at low doses [7, 8]. It is composed of a monomeric A subunit and pentameric B subunits [9]. The usefulness of native LT as an adjuvant is eclipsed by its toxicity. Amino acid substitutions have been introduced into the native LT to generate active but non-toxic mutant protein adjuvants. The first such example was the mutant labile toxin, mLT or LT(R192G), in which the amino acid glycine was substituted for arginine in the A-subunit, thereby preventing enzymatic cleavage [10]. mLT showed reduced toxicity [11] and maintained adjuvanticity in vitro and in animal studies, inducing a balanced Th1/Th2 cytokine and antibody subclass profile equivalent to native LT [12–14]. However, at a higher dosage (100 µg of mLT), human subjects showed mild to moderate diarrhea [15]. To alleviate this problem, an additional mutation was added to further detoxify the toxin and thus generate the double mutant, LT (R192G/L211A), or dmLT [16]. Detoxified dmLT has reduced cyclic AMP activation and exhibited no enterotoxicity, but most importantly, it retained the ability to function as an oral mucosal adjuvant. LT and its variants can enhance immune responses to whole cell vaccines against enterotoxigenic E. coli, Streptococcus pneumonia and Helicobacter pylori in different mouse models [17, 18].
Several studies have utilized conjugation of purified recombinant dmLT protein with candidate antigens for immunological studies, dose optimizations, and vaccine development [10, 19, 20]. However, based on our previous studies with adjuvanted vaccine strains, we perceived that the use of strains inherently secreting [21] or displaying [22] adjuvant molecules would be more practical for bulk production and may be more convenient for field deployments. In this study, to improve the vaccine-biological adjuvant system and to surpass the multistep processing of dmLT-conjugations, we investigated the use of a highly immunogenic live attenuated SE strain that secretes dmLT adjuvant molecules constitutively as a vaccine candidate. The protective efficacy against virulent challenge, immune responses, and safety as a vaccine candidate, were assessed in a chicken model.
Materials and methods
Experimental birds, bacterial strains and plasmids
Bacterial strains and plasmids used in this study
Strain/plasmid | Description | Reference |
---|---|---|
Salmonella Enteritidis | ||
JOL1182 | Wild type isolate from chicken, challenge strain | Lab stock |
JOL860 | Wild type isolate from chicken for antigen preparations | Lab stock |
JOL1087 | ΔlonΔcpxRΔasd, used as base vaccine strain | [21] |
JOL1641 | JOL1087 containing pJHL65-dmLT | This study |
Plasmids | ||
pJHL65 | asd + vector, pBR ori, b-lactamase signal sequence-based periplasmic secretion plasmid, 6xHis, high copy number | [34] |
pJHL65-dmLT | pJHL65 containing dmLT constitutively express under Ptrc promoter, secreted under bla secretory system | This study |
Construction of plasmids harboring dmLT
Components of pJHL65-dmLT. The SE asd + plasmid with ori pBR (pJHL65) harboring a constitutive expression system under which the dmLT cassette was expressed along with a fused bla secretion signal sequence. Lower case DNA sequence (inside the box) depicts codon substitution and corresponding non-synonymous amino acid substitutions, R192G and L211A.
Immunoblot confirmation of dmLT expression in culture supernatants
dmLT subunit expression was confirmed from the culture supernatant of JOL1641 using a previously described protocol with minor modifications [21]. Briefly, 200-mL supernatants from broth cultures grown at 37 °C (OD600 0.8) were recovered following centrifugation at 3380 g for 15 min. The supernatant containing the secreted protein was filtered through a 0.22-µm pore-size filter and precipitated with chilled 20% trichloroacetic acid overnight. The precipitated proteins were pelleted at 30 000 g for 20 min, washed with acetone and resuspended in PBS. After heating at 96 °C for 5 min, the proteins were separated using 15 and 18% SDS-PAGE gels for LTA and LTB, respectively. The separated proteins were blot transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA) and overnight blocking was performed in 5% skim milk. The LTA subunit was detected via the incorporated histidine tags, using primary mouse IgG1 anti-His-tag (Penta-His™, Life Technologies, Eugene, OR, USA) and secondary anti-mouse IgG1 antibody-HRPO conjugate (Sigma-Aldrich, USA) antibodies. The LTB subunit was detected using a lab-generated hyperimmune serum primary antibody (rabbit anti-LTB) and a secondary anti-rabbit IgG antibody-HRPO conjugate (Sigma-Aldrich). Reactive bands were developed using the West-One™ Western Blot Detection System (iNTRON, KOR) and bands were visualized using a KODAK Image Station (Kodak, New Haven, CT, USA).
GM1 ganglioside binding assay
GM1 based functional assay was conducted to validate the presence of dmLT in culture supernatants. GM1-based sandwich ELISA was performed based on a previously reported protocol with minor modifications [22]. The modifications were application of dmLT protein and use of anti-LTA antibody. Briefly, 5 µg/mL of purified ganglioside GM1 from bovine brain (Santa Cruz Biotechnology, USA) was used for coating the ELISA plate, and the remaining binding sites were blocked by incubating the plates with 200 μL of 5% skimmed milk. After washing three times with PBST, precipitated supernatants of JOL1640 was added and incubated at 37 °C for 2 h. The unbound sites were blocked with 200 μL of 5% skimmed milk. A 1:5000 dilution of anti-LTA rabbit serum was added to the wells and then incubated at 37 °C for 1 h. After washing five times with PBST, the plates were incubated with a 1:10 000 dilution of HRP-conjugated goat anti-rabbit IgG secondary antibody. The activity of bound HRP was measured using OPD (Sigma-Aldrich, St. Louis, MO, USA). A negative binding control involving all the steps was also carried out in parallel, except that GM1 coating was replaced with 5% skimmed milk.
Immunization and challenge of chickens
Immunization of hens with the Salmonella Enteritidis strains and challenge
Group (n = 10) | Constructed strain | Route | Dosagea | Challengeb |
---|---|---|---|---|
A | PBS control | Oral | 200 µL sterile PBS | |
JOL1182 1 × 109 cells/200 µL PBS | ||||
B | JOL1087 | Oral | 1 × 108 | |
Intramuscular | 1 × 107 | |||
C | JOL1641 | Oral | 1 × 108 | |
Intramuscular | 1 × 107 |
Safety and fecal shedding of vaccine strains
The safety qualities of the strains were investigated through regular monitoring of the birds until euthanization. The investigated parameters included anorexia, depression, and diarrhea. The presence of the vaccine strains in fecal samples was monitored 3, 7, 14 and 21 days post-immunization. Fecal samples were processed using a protocol described in detail elsewhere [21]. Birds were kept in clean disinfected buckets prior to fecal sampling. The fecal samples were collected and resuspended at a ratio of 1:10 in buffered peptone water. After brief pelleting, various dilutions of the supernatant were plated directly on BGA plates. Simultaneously, 1 mL of the supernatant was also enriched in 4 mL RV broth and incubated 48 h at 42 °C. A loop of the enrichment broth was streaked onto BGA and incubated at 37 °C for 16 h. Salmonella-like colonies that appeared after plating or streaking on BGA were further confirmed using SE-specific [23] and candidate strains specific primers.
Protective efficacy and bacterial recovery from infected organs
To evaluate the protective efficacy of the SE-dmLT strains, immunized birds were challenged with the SE virulent strain JOL1182 as described above. The protective efficacy of the dmLT-secreting strain JOL1641 was compared with those of the non-immunized control and the parental strain JOL1087. The birds were classified into Groups A, B and C corresponding to inoculation with PBS only and strains JOL1087 and JOL1641 respectively. The challenge experiment was carried out according to the protocol described previously with minor modifications [21]. At days 7 and 14 post-challenge, the birds were euthanized, and organ bacterial recovery and post-mortem examination were performed. To determine bacterial loads, samples of the liver, spleen, and cecum were weighed and then homogenized in 2 mL buffered peptone water. Hundred µL of the homogenate sample was plated on BGA for direct culture. The resulting colonies were counted after incubation at 37 °C for 16 h. In parallel, the remaining 1 mL homogenate was enriched in 4 mL of RV broth and incubated 48 h at 42 °C. A loop of the enrichment broth was streaked onto BGA and incubated at 37 °C for 16 h. Salmonella-like colonies were further confirmed using specific PCR primers. The number of bacterial colonies obtained via direct culturing were determined and expressed as the mean log10 CFU/g of samples. For the purposes of statistical analysis, a sample that was positive only after enrichment was rated as log10 = 1.0. A sample that was negative after enrichment was assigned as log10 = 0 [24].
Assessment of systemic and mucosal humoral immune responses
To measure anti-SE-specific antibodies, blood, and intestinal lavage samples were collected at weekly intervals for 5 weeks post-immunization. Heparinized peripheral blood was used to recover plasma samples to determine systemic immunoglobulin G (IgG) concentrations. Intestinal lavage samples were collected to determine secretory IgA (SIgA) concentrations, as per the protocol described elsewhere [25]. Indirect ELISA was performed for IgG and SIgA with an outer membrane protein (OMP) fraction extracted from wild-type JOL860, following a previously described protocol with minor modifications [26]. Changes included 40 min centrifugation at 20 000 g of sonicated pellet and final harvesting at 130 000 g for 1 h at 4 °C. IgG and SIgA samples were diluted with PBS at ratios of 1:100 and 1:10, respectively. Each sample was tested in duplicate, and the mean OD492 was calculated for each time point and compared to that of control samples.
Statistical analysis
Statistical analyses were applied wherever applicable. Analysis of variance (ANOVA) was used to analyze the differences among the group means; Tukey’s HSD post hoc analysis was further used to differentiate within groups. Differences were considered statistically significant at a P value of ≤0.05. In order to achieve a better statistical fit, bacterial enumeration log counts “x” at day 14 post-challenge (orally immunized groups) were mathematically transformed using the equation y = (x + 1).
Results
Construction and validation of dmLT
Western blot validation of dmLT from JOL1641. Lane M, prestained protein marker; lane 1, supernatant from the blank vector control; lane 2, JOL1641. A Immunoblot analysis of the LTA subunit, black arrow head poinitng the ~32-kDa reactive protein band. B Immunoblot analysis of the LTB subunit, white arrow head indicating the ~12-kDa reactive protein band.
Safety and fecal bacterial shedding
Faecal shedding of constructed strains post immunization
Group | Constructed strain | Route | Day | |||
---|---|---|---|---|---|---|
3 | 7 | 14 | 21 | |||
A | PBS control | Oral | 0/6a | 0/6 | 0/6 | 0/6 |
B | JOL1087 | Oral | 4/6 | 3/6 | 0/6 | 0/6 |
Intramuscular | 2/6 | 0/6 | 0/6 | 0/6 | ||
C | JOL1641 | Oral | 4/6 | 2/6 | 0/6 | 0/6 |
Intramuscular | 0/6 | 0/6 | 0/6 | 0/6 |
Systemic and mucosal humoral immune responses
Relative plasma IgG levels in immunized birds. IgG antibodies produced against the SE outer membrane protein antigens were measured using indirect ELISA. Bird groups were inoculated with: A, PBS control; B, JOL1087 and C, JOL1641. A Groups inoculated via the oral route. Immunized birds showed significant differences in their IgG levels as compared to the control group. This significant difference was observed at the 2nd week post-immunization. B Groups inoculated via the intramuscular route. Birds in Group C showed prominent increase in the IgG levels that declined gradually. As compared to the non-immunized group, the immunized groups showed significant differences at all sampling time points, except during the first week. *Significant difference compared to the control group (P ≤ 0.05), **Significant difference between immunized groups (P ≤ 0.05).
Relative intestinal SIgA levels in immunized birds. SIgA antibodies produced against the SE outer membrane protein antigens were measured using indirect ELISA. Bird groups were inoculated with: A, PBS control; B, JOL1087 and C, JOL1641. A Bird groups immunized via oral route. At the second week post-immunization, Group C birds showed significant rises in SIgA levels as compared to the other groups. B Bird groups immunized via the intramuscular route. At the first week post-immunization, Group C birds showed significant increases in SIgA. Birds in Group C showed consistent increases in SIgA levels 2, 3 and 4 weeks post-immunization. *Significant difference compared to the other groups (P ≤ 0.05).
Challenge and protection study
Recovery of challenge strain from internal organs of chickens. Enumeration of wild type bacterial load was performed in liver, spleen, and cecum of the bird after virulent wild type challenge. (A) Log10 organ bacterial counts of bird groups immunized orally, at the 7th day post-challenge. Spleens from Group C showed significant reduction in challenge bacterial load as compared to Group A and B (P ≤ 0.05) birds. (B) Log10 organ bacterial counts of bird groups immunized intramuscularly, at the 7th day post-challenge. The splenic bacterial loads were significantly lower in Group B and C as compared to Group A (P ≤ 0.05). The caecal bacterial load was significantly lower in Group C, birds immunized with adjuvanted strain (P ≤ 0.05). (C) Log10 organ bacterial counts of bird groups immunized orally, at the 14th day post-challenge. Spleens and ceca of Group B and C showed significant decreased in bacterial loads as compared to the control group A (P ≤ 0.05). (D) Log10 organ bacterial counts of bird groups immunized intramuscularly, at the 14th day post-challenge. In Group B and C, the number of birds determined to be completely negative of the challenge strain were 4 of 10.
Discussion
Upon infection via the oral route, Salmonella spreads rapidly, crossing the intestinal mucosa and invading the spleen and liver. The ability of Salmonella to evade killing by phagocytic cells and its pan tropism towards a variety of non-phagocytic cells leads to massive bacterial replication, resulting in high bacterial loads and systemic Salmonella infection [27]. It would be pragmatic to halt the invading Salmonella organisms before they penetrate their replication niche. Essentially, SIgA antibodies present in the intestinal mucus act as an important immunological barrier, preventing adherence and subsequent invasion of the intestinal lining by Salmonella. The present study investigated the effectiveness of the complete molecule of detoxified E. coli labile toxin dmLT in enhancing immunity against wild type Salmonella Enteritidis infection. The double mutant labile toxin gene was fused with the bla secretion signal sequence and was expressed under the constitutive promoter Ptrc. Furthermore, the stability of the plasmid was maintained by host-plasmid complementation using the asd gene. Salmonella asd gene knock-out strains transformed with this plasmid were stable and constitutively produced dmLT proteins, which are directed to the bacterial periplasmic space and then secreted. In order to achieve a strong protective ability and immunogenic properties, the dmLT adjuvanted strain JOL1641 was developed based on the highly immunogenic lon and cprxR gene-deleted SE strain (JOL1087). Deletion of lon and cprxR renders the organism attenuated by impairing intracellular replication while increasing its immunogenicity by up-regulating adhesion and invasion [28–30]. These properties of JOL1087 added additional suitable qualities to the vaccine candidate as evidenced by plasma IgG levels (Figure 3), intestinal lavage SIgA antibody levels (Figure 4) and protection (Figure 5).
Expression of dmLT was confirmed by immunoblot assay, which revealed the presence of the LTA and LTB proteins in the secretions of JOL1641 (Figure 2). As reported earlier, the double mutations in the gene for E. coli labile toxin eLT resulted in inhibition of proteolytic cleavage of LT-A into A1 and A2. It exhibited reduced enzymatic activity and no detectable toxicity either in vitro or in vivo [16]. Our observations confirmed that the incorporation of dmLT in the strain did not cause any additional undesirable effects, like general illness, diarrhea or prolonged fecal shedding (Table 3). In addition, no immunization-induced pathological lesions were observed in the internal organs upon postmortem examination.
Native toxins such as cholera and labile toxins and their variant toxoids can enhance both humoral and cell-mediated immunity [17, 31, 32]. Humoral immunity plays an important role in the early stages of infection, during which extracellular Salmonella Enteritidis is opsonized by antibodies, thereby preventing cell penetration [33]. Systemic antibodies opsonize Salmonella Enteritidis and thus enhance receptor-mediated uptake by macrophages. Our data revealed that the candidate strain was highly immunogenic when administered either orally or intramuscularly. The strain was capable of inducing strong anti-Salmonella specific systemic IgG, and the levels were significantly different from those of control birds. We observed a significant induction effect of mucosal adjuvant on intestinal SIgA production by the 2nd week post-immunization upon oral administration among Group C birds (Figure 4A). Interestingly, SIgA production in the intestinal lavages was significantly higher during the 1st week post-immunization via the intramuscular route among Group C birds; this significant rise was observed 1 week earlier compared to the oral route of immunization. Overall, our data indicated that oral and intramuscular administration of SE-dmLT resulted in a significant elevation of SIgA production, compared to the non-adjuvanted SE strain.
Isolation of challenge strain from internal organs of birds
Group | Constructed strain | Route | Day post challenge | Organa | ||
---|---|---|---|---|---|---|
Liver | Spleen | Caecum | ||||
A | PBS control | PBS | 7 | 10/10 | 10/10 | 10/10 |
B | JOL1087 | Oral | 7 | 10/10 | 10/10 | 10/10 |
Intramuscular | 7 | 8/10 | 8/10 | 10/10 | ||
C | JOL1641 | Oral | 7 | 8/10 | 8/10 | 10/10 |
Intramuscular | 7 | 5/10 | 5/10 | 6/10 | ||
B | JOL1087 | Oral | 14 | 3/10 | 5/10 | 5/10 |
Intramuscular | 14 | 6/10 | 6/10 | 6/10 | ||
C | JOL1641 | Oral | 14 | 5/10 | 5/10 | 5/10 |
Intramuscular | 14 | 6/10 | 4/10 | 6/10 |
Our data demonstrated that JOL1641 could significantly protect immunized birds from virulent wild-type challenge. The inclusion of adjuvant dmLT in the secretions of JOL1641 strain increased SIgA production in the intestines of immunized birds. These increased SIgA levels may correlate with better splenic protection during early infection. In conclusion, this study reports a unique vaccine development strategy involving a live attenuated vaccine and an intrinsically incorporated mucosal adjuvant. We also provided compelling data that this novel strain could be a new tool for poultry anti-Salmonella Enteritidis vaccination, especially as a means to boost the humoral immune response required for neutralization during the early phases of invading Salmonellae infection.
Declarations
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JHL conceived and designed the study. NMK carried out cloning and construction of dmLT plasmid. JL and NMK carried out animal experimentations, sample collection, and processing. JL performed indirect ELISA, bacterial counts, statistical analyses and interpretation of the data. JL and JHL wrote the manuscript. All authors read and approved the final manuscript.
Acknowledgements
This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korea government (MISP) [No. 2015R1A2A1A14001011].
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Authors’ Affiliations
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