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. Author manuscript; available in PMC: 2021 May 6.
Published in final edited form as: Vaccine. 2020 Apr 8;38(21):3720–3728. doi: 10.1016/j.vaccine.2020.03.055

Oral vaccination with live attenuated Yersinia pseudotuberculosis strains delivering a FliC180-LcrV fusion antigen confers protection against pulmonary Y. pestis infection

Amit K Singh 1, Xiuran Wang 1, Wei Sun 1,#
PMCID: PMC7285849  NIHMSID: NIHMS1581394  PMID: 32278523

Abstract

We incorporated the ΔPfur::TT araC PBAD fur deletion-insertion mutation on top of a previous Yersinia pseudotuberculosis mutant (Δasd ΔyopJ ΔyopK) to construct a new mutant designated as Yptb5, which manifests the arabinose-dependent regulated delayed fur (encoding ferric uptake regulator) shut-off. The Yptb5 strain was used to deliver an adjuvanted fusion protein, FliC180-LcrV. Levels of FliC180-LcrV synthesis were same in Yptb5 either harboring pSMV4, a p15A ori plasmid or pSMV8, a pSC101 ori plasmid containing the fliC180-lcrV fusion gene driven by Ptrc promoter. Tissue burdens of both Yptb5(pSMV4) and Yptb5(pSMV8) in mice had similar patterns. Mice vaccinated orally with 5×108 CFU of either Yptb5(pSMV4) or Yptb5(pSMV8) strain were primed high antibody titers with a balanced Th1/Th2 response, also developed potent T-cell responses with significant productions of IFN-γ, IL-17A and TNF-α. Immunization with each mutant strain conferred complete protection against pulmonary challenge with 5.5 × 103 CFU (55 LD50) of Y. pestis, but partial protection (50% survival) against 100 LD50 of Y. pestis. Our results demonstrate that arabinose-dependent regulated delayed fur shut-off is an effective strategy to develop live attenuated bacterial vaccines while retaining strong immunogenicity.

Keywords: Yersinia pseudotuberculosis, vaccine, plague, regulated delayed attenuation

1. Introduction

Plague is one of the most feared infectious diseases caused by Yersinia pestis. Three well-known plague epidemics (Justinian, Black death and Modern plague) caused a huge historical disaster and claimed more than 200 million human lives [1, 2]. Currently, no FDA-approved vaccine is available yet. Pneumonic plague caused by pulmonary infection of Y. pestis is the most lethal form [1]. So, development of a safe and effective vaccine for human application is a priority, particularly for people lives in plague endemic regions. Live vaccines established on Y. pseudotuberculosis, the Y. pestis progenitor [3], showed induction of potent antibody- and cell-mediated responses in mice, and provided significant protection against pulmonary challenge with virulent Y. pestis strains [4–7]. The basis built on previous studies can allow us seeking different means to optimize the live attenuated Y. pseudotuberculosis vaccine.

A regulated delayed attenuation system was developed in Salmonella [8] and Yersinia [6, 9], in which the recombinant strains display features of the wild-type virulent pathogen at the time of immunization to enable strain to effectively colonize lymphoid tissues and then become completely attenuated in vivo to preclude inducing disease symptoms. Studies demonstrated that antibodies against iron regulated outer membrane proteins (IROMPs), which block essential iron uptake of the bacterial pathogens, were capable of cross-reacting with IROMPs synthesized by other pathogens belonging to the same species [10]. In S. Typhimurium, deletion of fur encoding ferric uptake regulator enabled constitutive expression of IROMPs, attenuating infection of orally-challenged mice by 2–3 log fold [11]. To achieve a high constitutive level of synthesis of all components for iron acquisition including IROMPs, a S. Typhimurium strain with a ΔPfur::TT araC PBAD fur deletion-insertion mutation displayed that expression of the fur gene was solely dependent on the presence of arabinose, instead of iron concentration [8]. Growth of the S. Typhimurium mutant strain in media with a low level of arabinose resulted in very good colonization of lymphoid tissues, high attenuation and very high levels of induced protective immunity [8]. Yersinia iron acquisition systems are required for full virulence [12–16]. Both ybt and yfe loci in Y. pestis and Y. pseudotuberculosis have been found to be 97–100% identical. The Yersinia Fur protein represses transcription of genes located at ybt and yfe loci in the presence of excess iron [17, 18]. Yersinia grown in the iron deprived media results in enhanced synthesis of major IROMPs such as Psn, Irp1, Irp2, YbtA and so on [19–21]. Thus, a live attenuated Y. pseudotuberculosis strain with ΔPfur::TT araC PBAD fur deletion-insertion mutation would enhance synthesis and exposure of IROMPs in vivo, predictably priming robust immune responses and enhancing attenuation.

Previous findings have demonstrated a truncated FliC180 in Salmonella deletes the 180 amino acids containing the antigenically hypervariable domain of the flagellin antigen, but retains the conserved N- and C-terminal regions that interact with Toll like receptor-5 (TLR5) on immune cell surface to recruit/stimulate innate immune responses [22, 23]. Immunization with flagellin mixed or fused with Y. pestis antigen conferred clearly protection against plague challenge in mice and nonhuman primates [24–26].

Based on above information, we incorporated ΔPfur::TT araC PBAD fur deletion-insertion mutation into a live attenuated Y. pseudotuberculosis construction χ10069 (Δasd ΔyopJ ΔyopK) [4] to construct a novel vaccine strain, Yptb5 (Δasd ΔyopJ ΔyopK ΔPfur::TT araC PBAD fur). Furthermore, we fused FliC180 from S. Typhimurium with Y. pestis LcrV antigen and delivered this fusion antigen by Yptb5 strain to enhance immunogenicity. Mouse studies demonstrated oral immunization with Yptb5 delivering the FliC180-LcrV fusion antigen induced Y. pestis specific humoral and cell mediated immunity and conferred significant protection against pneumonic plague.

2. Materials and Methods

2.1. Bacterial cultures and media

Bacteria and plasmids used in this study were listed in Table 1. Escherichia coli χ7213 as a suicide plasmid donor strain was grown routinely at 37°C in Luria Bertani (LB) broth or on LB agar plates supplemented with diaminopimelic acid (50 μg/ml, DAP) or chloramphenicol (25 μg/ml, Cm) as necessary. The Y. pseudotuberculosis strain used in this study was routinely grown in LB medium at 28°C. LB plates containing 5% sucrose were used for sacB gene-based counter-selection in allelic exchange experiments for mutant constructions. Yersinia selective agar supplemented with Cefsulodin-Irgasan-Novobiocin (CIN) was applied for bacterial enumeration in different organs of mice administrated with Y. pseudotuberculosis. Y. pestis KIM6+(pCD1Ap) was used for challenge studies as previously reported [4] and grown routinely in heart-infusion broth (HIB) or HIB- Congo red agar plates at 28°C. HIB-Congo red agar plates were used to confirm the pigmentation (Pgm+) phenotype of virulent Y. pestis strain [27].

Table 1.

Strains and plasmids used in this study

Strain or Plasmid Genotype or relevant characteristics Source
Strains
E. coli χ6212 F– λ– θ80 Δ(lacZYA-argF) endA1 recA1 hsdR17 deoR thi-1 glnV44 gyrA96 relA1 ΔasdA4 [7]
E. coli χ7213 thi-1 thr-1 leuB6fhuA21 lacY1 glnV44 ΔasdA4 recA1 RP4 2-Tc::Mu [λpir]; Kmr [7]
Salmonella Typhimurium UK-1 mutant χ9206 fliC180 Dr. Roy Curtiss
Y. pseudotuberculosis PB1+ Serotype O:1b Dr. Roy Curtiss
Y. pseudotuberculosis χ10069 Δasd206 ΔyopJ315 ΔyopK108 [4]
Yptb-A5 Δasd206 ΔyopJ315 ΔyopK108 ΔPfur::TT araC PBAD fur This study
Y. pestis KIM6+(pCD1Ap) Pgm+, pMT1, pPCP1, pCD1Ap [27]
Plasmids
pRE112 Suicide vector, Cmr, mob− (RP4)R6K ori, sacB [28]
pYA3332 Asd+, p15A ori [28]
pYA3337 Asd+, pSC101 ori [44]
pYA4505 The fliC180 gene fragment under Ptrc promoter in an Asd+ plasmid (pBR ori) Dr. Roy Curtiss
pSMV4 The fliC180-lcrV DNA fragment was cloned into pYA3332 This study
pSMV7 The lcrV gene fragment was cloned into pYA3332 This study
pSMV8 The fliC180-lcrV DNA fragment was cloned into pYA3337 This study
pSMV31 The SD-fur and YPTS_1183’ fragments cloned into the XhoI/SacI sites and PstI/SphI sites of pYA3700 This study
pSMV32 The ΔPfur::TT araC PBAD fur DNA fragment was cloned into pRE112 This study
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2.2. Plasmid and strain constructions

All primers used in this study were listed in Supplemental information Table S1. A primer set of FliC180-F/ FliC180-R was used for amplifying fliC180 DNA fragment encoding truncated FliC180 from S. Typhimurium UK-1 mutant strain, χ9206 (received from Dr. Roy Curtiss). Another primer set of LcrV-F/LcrV-R was used to amplify lcrV DNA fragment of Y. pestis. The chimeric DNA fragments (fliC180-lcrV) were fused by overlapping PCR using a primer set of FliC180-F/ LcrV-R. The fliC180-lcrV fragment digested by NcoI and HindIII was cloned into the same sites of two Asd+ plasmids, pYA3332 (p15A ori) and pYA3337 (pSC101 ori) to generate pSMV4 and pSMV8, respectively (Table 1 and Fig. 2A). Primer sets Pfur-1/Pfur-2 and Pfur-3/Pfur-4 were used for amplifying fur containing its original SD sequence (SD-fur), and YPTS_1183’ (−113 to −760 bp upstream of fur) fragments from wild-type Y. pseudotuberculosis PB1+ strain, respectively. The SD-fur and YPTS_1183’ fragments were cloned into the XhoI/SacI sites and PstI/SphI sites of pYA3700, respectively to form pSMV31. The ΔPfur::TT araC PBAD fur DNA fragment was digested from plasmid pSMV31 by SacI /SphI, blunted by T4 DNA polymerase and ligated into pRE112 to generate a suicide plasmid pSMV32. All constructed plasmids were confirmed by DNA sequencing.

Figure 2.

Figure 2.

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Kinetics of bacterial burden in mice administrated orally with 109 CFU of each Y. pseudotuberculosis PB1+, Yptb5(pSMV4), and Yptb5(pSMV8). Bacterial titers in (A) Peyer’s patches, (B) spleens, (C) livers and (D) lungs of infected mice at days 3, 6 and 9 post infection. Experiment was performed twice with equal numbers of animals (3 mice in each group); graphs represented pooled results. Statistical significance among groups were analyzed by two-way ANOVA multivariant applying Tukey’s post-hoc test. Mean value bars ± SD. *, P< 0.05; **, P< 0.01; ***, P< 0.001, ****, P< 0.0001.

The procedures for constructing the Y. pseudotuberculosis mutant were described as previous studies [4, 28]. Briefly, the suicide plasmid pSMV32 (ΔPfur::TT araC PBAD fur) was conjugationally transferred from E. coli χ7213 [29] to χ10069 (Δasd ΔyopJ ΔyopK) [4]. Single-crossover insertion strains were isolated on TBA agar plates containing Cm. Loss of the suicide vector after the second recombination between homologous regions (i.e., allelic exchange) was selected by using the sacB-based sucrose sensitivity counter-selection system. The colonies were screened for Cms (chloramphenicol sensitive) and verified by PCR using primers Pfur-1/ Pfur-4.

2.3. In-vitro assay of TLR5 activities

To determine stimulatory activity of FliC180-LcrV fusion via TLR5, HEK-Blue™-mTLR5 cells (InvivoGen, CA, USA) were maintained at 37 °C with 5% CO2 in DMEM (Gibco BRL, Grand Island, NY, USA) containing 10% FBS supplemented with 100 μg/ml penicillin, 100 μg/ml streptomycin and 100 μg/ml Normocin. Cells were seeded at a density of 5 × 104 cells per well in 96-well tissue culture plates (Costar, Washington, DC) and were stimulated with bacterial lysate from different strains (final concentration 20 μg/ml) for 6 hours. HEK-Blue™-Null cell without TLR5 was used as experimental control. Relative NF-κB activity was determined by measuring the embryonic alkaline phosphatase (SEAP) activity secreted into culture media according to the manufacturer’s instructions.

2.4. Mice and ethical statement

Animal care and experimental protocols were in accordance with the NIH “Guide for the Care and Use of the laboratory Animals” and were approved by the Institutional Animal Care and Use Committee at Albany Medical College (IACUC protocol# 20–01001). Six-week-old, male and female Swiss Webster mice were purchased from Charles River Laboratories (Wilmington, MA). Mice were acclimated for one week before experiments.

2.5. Bacterial inoculation and Immunization

The overnight grown cultures of Yptb5(pSMV4) and Yptb5(pSMV8) were re-inoculated in fresh LB broth with arabinose (0.05% v/v), respectively. Bacterial cultures were grown at 28 °C with 180 rpm constant agitation until exponential phase (O.D.600nm 0.8–0.9). Bacterial cells were pelleted at 4000 ×g for 12 min and resuspended in sterile phosphate buffered saline (PBS) pH 7.4 with required volume. Groups of mice (10 mice each group) were deprived of food and water for 6 hours to minimize intestinal contents and gavaged 0.2 ml bacterial suspension to each mouse with a single dose of 5.5 × 108 colony forming units (CFU) by using 20 gauge feeding needle. Equal number of sham mice were given 0.2 ml of sterile PBS [4, 7].

2.6. Colonization of Y. pseudotuberculosis mutant

Groups of mice were gavaged with 5.5 ×109 CFU of Y. pseudotuberculosis to assess the kinetics of bacterial burden in different vital organs including intestine, spleen, liver and lung. At day 3, 6 and 9 post infection, 3 mice from each group were humanly euthanized and perfused with 5 ml sterile PBS before extracting lung, liver, spleen and Peyer’s patch, aseptically. Tissue samples were homogenized by bullet blender (Bullet Blender Blue; Averill Park, NY, USA) and serially diluted homogenates were spread on Yersinia selective agar plates (CIN agar) in duplicates. After 48 h incubation at 28 °C, CFU were counted and calculated according to the initial weight (mg) or/and volume (ml) of the organ [4, 7].

2.7. Antibody responses

On day 14 and 28 post immunization, blood was collected from immunized and sham mice by sub-mandibular vein puncture for antibody analysis. For measuring secreted IgA in bronchoalveolar lavage fluid (BALF), 4 representative mice from each group were euthanized at 42nd day post immunization and BALF was collected as previous description [4]. An enzyme-linked immunosorbent assay (ELISA) was used to measure antibodies in serum and BALF to YpL (Y. pestis whole-cell lysate) or LcrV [4].

2.8. Antigen specific T-cell responses

Single cells obtained from spleens and collagenase treated lungs were seeded (2 × 106) in 12-well cell culture plates. Cells were in-vitro stimulated with 20 μg/ml YPL or LcrV protein for 72 h at 37 °C and 5% CO2. Four hours before collection, cell culture media in each well were supplemented with brefeldin-A and monensin cocktail (1:1 ratio) to plug Golgi-mediated cytokine secretion. For flowcytometric analysis of T-cell population and their corresponding cytokines, induced cells were harvested and re-suspended in FACS staining buffer containing CD16/32 antibodies (1:200) for 10 min on ice. T-cell specific markers were stained using anti-mouse CD3 (FITC), CD4 (PE) and CD8 (APC) antibodies followed by intracellular cytokines (IFN-γ, Percp Cy5.5; TNF-α, HV510; IL-2, PECy7; IL17A, APCCy7) staining by using BioLegend Perm-fix solution and buffer as per manufacturer’s protocol. All antibodies were purchased from BioLegend. The entire staining process was performed on ice with 1 h incubation at each step. Events were acquired on BD flow cytometers (LSRII) with FACS Diva software and analyzed by using FlowJo v.10.

2. 9. Animal survival study

At 42nd day of immunization, immunized and sham mice were shifted to ABSL-3 to perform pneumonic plague challenge. Liquid cultures of Y. pestis KIM6+(pCD1Ap) were grown in HIB supplemented with Xylose (0.2% w/v) and ampicillin (100 μg/ml) at 26°C for overnight. The bacterial cultures were then diluted to an OD600 of 0.1 in 10 ml fresh HIB with Xylose (0.2% w/v), ampicillin (100 μg/ml), and CaCl2 (2.5 mM) and grown at 37°C with constant shaking until 0.6–0.8 of OD600. Bacterial cells were harvested by 4000 rpm centrifugation for 12 min and pellet resuspended in 1 ml of sterile PBS. Then, the resuspended bacterial solution was diluted to an appropriate concertation for animal challenge. Ketamine (xylazine/ketamine 1:5 ratio) anesthetized mice were infected with 40 μl Y. pestis suspension via nostril [4, 7]. Mortality and morbidity of infected mice were observed daily for the next 15 days.

2. 10. Statistical analysis

All experimental data were presented in mean value ± standard deviation (SD) obtained from minimum 3 biological replicated and repeated 2 times in similar condition. Data were analyzed using GraphPad PRISM 8.0 software. Statistical analyses of data were evaluated by Two-way ANOVA using Tukey’s post hoc tests. The log-rank test was used for analysis of the survival curves. Data represented significance at * P< 0.05, ** P< 0.01, *** P< 0.001, **** P<0.0001.

3. Results

3. 1. Construction of a regulated delayed fur expression strain, and determination of FliC180-LcrV synthesis and TLR5 activation

The ΔPfur::TT araC PBAD fur deletion-insertion mutation was added on top of χ10069 (Δasd ΔyopJ ΔyopK) to construct a new mutant strain designated as Yptb5 (Fig. 1A and Table 1). In Yptb5 mutant strain, fur expression is dependent on the presence of arabinose. No Fur was detected in Yptb5 grown in the absence of arabinose, while Yptb5 synthesized roughly the same amount of Fur as wild-type Y. pseudotuberculosis (Fig. 1B). Yptb5 grown in the absence of arabinose or iron-deprived media significantly increased levels of Psn (pesticin receptor) (Fig. 1B), an Y. pestis protective antigen [30, 31]. Strain Yptb5 without arabinose supplementation grew slowly at initial and log phases but could reach the same final OD600 at stationary phase as Yptb5 with arabinose and its patent strain χ10069 at 28°C in HIB medium (Fig. 1C). Then, we individually introduced low-copy-number Asd+ plasmids, pSMV4 (p15A ori) and pSMV8 (pSC101 ori) (Fig. 1D) into Yptb5 strain to determine fliC180-lcrV expression. Results showed that the amount of FliC180-LcrV fusion was similar in both Yptb5(pSMV4) and Yptb5(pSMV8) strains (Fig. 1E).

Figure 1.

Figure 1.

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The features of Y. pseudotuberculosis construction. (A) Schematic chromosome structure of Y. pseudotuberculosis mutants, χ10069 (Δasd ΔyopJ ΔyopK), and Yptb5 (Δasd ΔyopJ ΔyopK ΔPfur::TT araC PBAD fur). (B) The fur expression in χ10069 and Yptb5 cultured in LB media with or without 0.05% arabinose at 28°C. The psn expression in χ10069 and Yptb5 cultured in LB media with or without 0.05% arabinose and/or 100μM 2,2’-dipyridyl at 28°C. (C) The growth curve of χ10069 and Yptb5 cultured in LB media with or without 0.05% arabinose at 28°C. (D) Physic maps of plasmids pSMV4 (p15A ori) and pSMV8 (pSC101 ori) harboring the fliC180-lcrV fusion gene under Ptrc promoter. (E) Determine FliC180-LcrV synthesis in Yptb5(pSMV4) and Yptb5(pSMV8). (F) Comparison of the secreted embryonic alkaline phosphatase (SEAP) activities in HEK-Blue™ cells with or without murine toll-like receptor 5 (TLR5). HEK-Blue™ mTLR5 (InvivoGen) cells were co-cultured with 20μg/ml whole-cell lysate from Yptb5(pYA3332), Yptb5(pAY4505, fliC180), Yptb5(pSMV4, fliC180-lcrV), Yptb5(pSMV7, lcrV) and Yptb5(pSMV8, fliC180-lcrV) strains for 6 hours, respectively. HEK-Blue™ Null1-v cells or PBS were used as negative controls. Each symbol represented data obtained from individual well, with mean value bars ± SD. ****, P< 0.0001 was obtained by two-way ANOVA with Tukey’s post-hoc tests.

To determine TLR5-activating abilities of FliC180-LcrV fusion protein, HEK-blue mTLR5 reported cells containing an NF-κB-inducible SEAP reporter gene were incubated with 20μg/ml of soluble protein fraction obtained from whole-cell lysates of each strain for 6 h. Protein fractions from both Yptb5(pSMV4) and Yptb5(pSMV8) strains synthesizing FliC180-LcrV strongly activated TLR5 signaling and showed significant increase of SEAP activity (4–5 fold), although the SEAP activity was lower than that induced by protein fraction from Yptb5(pYA4505) synthesizing FliC180 alone as a positive control (Fig. 1F). In contrast, protein fractions from Yptb5(pSMV7) expressing LcrV alone and Yptb5(pYA3332) harboring an empty plasmid did not show any induction. HEK-blue null cells without TLR5 incubated with each protein fraction also did not show any TLR5 stimulation (Fig. 1F). Thus, results indicated that FliC180-LcrV fusion antigen still retained strong activation of TLR5.

3. 3. Kinetics of Y. pseudotuberculosis mutant strains after oral administration

In order to investigate colonization of mutant strains, Swiss Webster mice (n=3 per time point) were gavaged with 200 μl (5.5 × 109 CFU) of Yptb5(pSMV4), Yptb5(pSMV8) and wild-type Y. pseudotuberculosis PB1+. At day 3 post infection, the wild-type PB1+ infected mice were found with overwhelming infection in Peyer’s patches and obvious micro-damage in intestinal epithelium, and bacterial load increased progressively at day 6 and 9 post infection (Fig. 2A). However, the numbers of Yptb5(pSMV4) and Yptb5(pSMV8) strains in Peyer’s patches gradually decreased and were substantially lower than that of wild-type PB1+ strain at day 3, 6 and 9 post-infection and the bacterial titers of Yptb5(pSMV8) reduced further than those of Yptb5(pSMV4) at day 6 and 9 post administration (Fig. 2A). The PB1+ strain also effectively colonized in livers and spleens at 3 and steadily heightened, at 6 days and 9 days post-infection (Fig. 2B and C). Interestingly, Yptb5(pSMV8) rapidly disseminated into spleens and livers at 3 days post-infection compared with Yptb5(pSMV4) and WT PB1+. Then, titers of both Yptb5(pSMV4) and Yptb5(pSMV8) dramatically decreased at day 6 and 9 post infection (Fig. 2B and C). Significantly reduced burdens of Yptb5(pSMV4) and Yptb5(pSMV8) in Peyer’s patches, spleens and livers clearly manifested their attenuation properties. The lung burden profiles of Yptb5(pSMV4) and Yptb5(pSMV8) were also very intriguing. Yptb5(pSMV4) rapidly disseminated to lung in majority of mice at early stages of infection (day 3), while Yptb5(pSMV8) could reach to lung at day 6 post infection, and both strains were persistent until day 9 post administration. However, wild-type PB1+ strain was able to reach lungs of half mice only by day 9 post infection (Fig. 2D).

3. 4. Antigen-specific antibody titers in serum and BALF

Sera and BALF obtained from Yptb5(pSMV4) and Yptb5(pSMV8) immunized mice were evaluated for antibody titers and isotypes by ELISA using YPL (Y. pestis whole-cell lysate) and LcrV as target antigens. Mice immunized with Yptb5(pSMV4) or Yptb5(pSMV8) mounted high anti-YPL IgG titers with similar log10 mean values in sera at 14th day post immunization and maintained high IgG titers without substantial increase at day 28 (Fig. 3A). Analysis of IgG subclasses (IgG1, IgG2a and IgG2b) to YpL showed that ratios of IgG2a/IgG1 and IgG2b/IgG1 in mice immunized with either Yptb5(pSMV4) or Yptb5(pSMV8) were close to 1, respectively (Fig. 3B and C). Also, mice immunized with Yptb5(pSMV4) or Yptb5(pSMV8) were primed high anti-LcrV IgG titers with similar log10 mean values in sera at 14th day post immunization (Fig. 3D). At day 28 post immunization, anti-LcrV IgG titers in the Yptb5(pSMV4)-immunized group were substantial increase, while in the Yptb5(pSMV8)-immunized group were maintained (Fig. 3D). Ratios of IgG2a/IgG1 and IgG2b/IgG1 to LcrV in mice immunized with either Yptb5(pSMV4) or Yptb5(pSMV8) were also close to 1 at day 28 post immunization, respectively (Fig. 3E and F), indicating that mixed Th1/Th2 responses were stimulated by oral immunization with both vaccine candidates.

Figure 3.

Figure 3.

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Antibody responses in sera of mice immunized orally with Yptb5(pSMV4), and Yptb5(pSMV8) at day 14 and 28 post immunization, respectively. Y. pestis whole-cell lysate (YPL) and recombinant LcrV were used as the coating antigens. (A) Serum anti-YPL IgG titers in immunized mice. (B) Ratio of serum anti-YPL IgG2a and IgG1 titers in mice. (C) Ratio of serum anti-YPL IgG2b and IgG1 titers in mice. (D) Serum anti-LcrV IgG titers in immunized mice. (B) Ratio of serum anti-LcrV IgG2a and IgG1 titers in mice. (C) Ratio of serum anti-LcrV IgG2b and IgG1 titers in mice. Statistical significance among groups were analyzed by two-way ANOVA multivariant applying Tukey’s post-hoc test. *, P< 0.05; ****, P< 0.0001. Mean value bars ± SD.

Further analysis of IgM and IgA indicated that both immunized groups were primed a moderate induction of anti-YPL and -LcrV serum IgM and IgA titers at day 14 and maintained until day 28 (Fig. S1A, B, C and D). The mucosal immune response at lung was characterized by assessing secretory IgA (sIgA) in BALF obtained at the 42nd day post immunization. Mice immunized with Yptb5(pSMV4) or Yptb5(pSMV8) produced high levels of YPL-specific sIgA titers with similar log10 values, while immunization with Yptb5(pSMV8) generated significantly higher titers of anti-LcrV sIgA in lung than that with Yptb5(pSMV4) (Fig. S1E). No conspicuous antibody titers or isotypes were observed in sera or BALF from PBS inoculated mice.

3. 5. T-cell mediated immune responses

In addition to induction of antibody-mediated humoral immunity, an increasing body of evidence has demonstrated that an ideal pneumonic plague vaccine is required to induce cellular immunity [32, 33]. Therefore, lung and splenic T-cell responses and their corresponding cytokine productions were analyzed to verify the development of cell-mediated protective immunity at mucosal and systemic sites. Lung cells from both Yptb5(pSMV4)- and Yptb5(pSMV8)- immunized mice in vitro stimulated with YPL showed a substantial induction of (CD3+) T-cell populations compared with lung cells from sham mice (Fig. 5A). Higher proliferation of lung CD4+ and CD8+ T cells in the Yptb5(pSMV4)-immunized group (CD4, 5.5× 105) than that in the Yptb5(pSMV8)-immunized group. In vitro induction by YPL resulted in higher production of IFN-γ in both CD4+ and CD8+ T cells from Yptb5(pSMV4)-immunized mice than that from Yptb5(pSMV8)-immunized mice (Fig. 5A). Compared with lung lymphocytes from sham mice, incubation of lung lymphocytes with YPL led to substantial induction of IL-2, IL-17A and TNF-α in CD4+ T-cell subsets of Yptb5(pSMV4)- and Yptb5(pSMV8)-immunized mice, rather in CD8+ T-cell subsets (Fig. 5A).

Figure 5.

Figure 5.

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Survival of mice challenged by virulent Y. pestis KIM6+(pCD1Ap). Groups of Swiss-Webster mice (n=10, 5 males and 5 females) were orally vaccinated with a dose of 5×108 CFU of Yptb5(pSMV4), Yptb5(pSMV8), PBS as a negative control. (A) Schematic strategy of the entire animal experiment. (B) Swiss-Webster mice were intranasally challenged with a medium dose of Y. pestis (5.5 × 103 CFU). (c) Swiss-Webster mice were intranasally challenged with a high dose of Y. pestis (1.04 × 104 CFU). Mortality and morbidity were recorded in surviving mice for 15 days post infection. The log-rank test was used for analysis of the survival curves. (***, P< 0.001; ****, P< 0.0001).

Also, substantial T cells (CD3+), and CD4+ and CD8+ T-cell subsets were proliferated in splenocytes from both Yptb5(pSMV4)- and Yptb5(pSMV8)-immunized mice in vitro stimulated with YPL compared with cells from sham mice (Fig. 5B), implying development of Y. pestis specific cell-mediated systemic immunity. CD4+ T-cell subsets in splenocytes from both Yptb5(pSMV4)- and Yptb5(pSMV8)-immunized mice produced higher levels of IFN-γ, IL-2, IL-17A and TNF-α in response to re-stimulation with YPL than cells from sham mice (Fig. 5B). Compared with sham mice, CD8+ T-cell subsets in splenocytes from both Ypt5(pSMV4)- and Ypt5(pSMV8)-immunized mice produced substantial levels of IFN-γ, but displayed limited production of IL-2, IL-17A and TNF-α (Fig. 5B).

3. 6. Protection against pneumonic plague challenge

Groups of mice (10 mice per group, 5 males and 5 females) were orally immunized with a single dose of each mutant strain (5 × 108 CFU), with PBS as a negative control. At 42nd day post immunization (Fig. 5A), mice immunized with Yptb5(pSMV4) or Yptb5(pSMV8) were afforded 100% protection against intranasal challenge with 55 LD50 (5.5 × 103 CFU) of virulent Y. pestis (Fig. 5B), while ~50% survival against intranasal challenge with high dose (100 LD50) of Y. pestis (Fig. 5C). At the 14th day post infection, all surviving animals were found to have healthy conditions without any external signs of disease. No bacteria were detected in lungs, livers and spleens of surviving animals, thus demonstrating induction of sterilizing immunity. All sham mice administered with PBS succumbed to infection within 3–5 days. Male and female immunized mice were found unbiased for protection against Y. pestis infection.

4. Discussion

Our previously designed an Y. pseudotuberculosis vaccine candidate, χ10069 (Δasd ΔyopJ ΔyopK) harboring plasmid pYA5199 for delivering YopEnt138-LcrV by Type three secretion system, was shown substantial protection against pneumonic plague [4]. However, histopathological analysis of lung showed moderate sign of infection in χ10069 immunized mice, have raised certain safety concerns. Since χ10069 strain was shown high attenuation in mice [4], adding any essential gene deletions on top of χ10069 leaded to hyper-attenuation and subsequent loss of immunogenicity during screening Y. pseudotuberculosis vaccine candidates (unpublished data). Thus, Yptb5 incorporated with the arabinose regulated expression of fur gene to enable strain to colonize lymphoid tissues as its parent strain χ10069 and then become more attenuation than χ10069 in vivo to preclude inducing disease symptoms, which would retain immunogenicity but further reduce pathologic changes in tissues.

Flagellin, being a potent activator of innate and adaptive immunity, was utilized in previous plague vaccine studies [26, 34]. Hence, incorporation of an adjuvant to enhance immune responses would be a reasonable approach to maximize protection against Y. pestis infection. We initially tried to clone the fliC180-lcrV fusion gene fragment into an Asd+ plasmid with high copy number, pYA3342 (pBR ori) [35]. However, significantly slower growth of Yptb5 harboring this plasmid was observed, which may be due to overexpression of fliC180-lcrV causing a metabolic burden in host strain. Thus, a shift to a low copy plasmid pSMV4 (p15A ori) or pSMV8 (pSC101 ori) was performed in this study to eliminate this problem. Yptb5 strain harboring either pSMV4 or pSMV8 showed similar growth rate as Yptb5 harboring an empty plasmid pYA3332 (unpublished data).

Our study showed that the titers of Yptb5(pSMV4) and Yptb5(pSMV8) were significantly lower, but persistence in Peyer’s patches compared with WT PB1+ strain (Fig. 2A), which was in accordance with a previous report that the highly attenuated yopK mutant Y. pseudotuberculosis was able to colonize in Peyer’s patches and mesenteric lymph nodes but failed to cause systemic infection in mice [36]. Somehow, Yptb5(pSMV4) or Yptb5(pSMV4) could reach spleen and liver more rapidly than WT PB1+ strain at day 3 post administration, but titers of mutant strains significantly decreased at day 6 and 9 post inoculation (Fig. 2B and C), which might be related with the arabinose-dependent regulated fur expression in Yptb5(pSMV4) or Yptb5(pSMV8). In those mutant strains, Fur might be completely shut off at day 6 and 9 post inoculation causing further attenuation. Intriguingly, both Yptb5(pSMV4) and Yptb5(pSMV8) could persist higher bacterial titers than WT PB1+ strain in lungs by day 9 post infection (Fig. 2D). Regarding previous reports, pathogenic bacteria loss of Fur had dramatic attenuation by oral administration and lower dissemination to second lymphoid or non-lymphoid organs [8, 37–39]. So, this discrepancy might be due to alteration of Y. pseudotuberculosis invasion by fur mutant or different mouse lines used between this study and other studies, but the exact mechanisms behind those observations need to be investigated further.

Studies suggested that a plague vaccine candidate primarily stimulating antibody-mediated humoral immunity was not enough [25, 40, 41], but induction of humoral response was able to mirror cellular response to certain extend. Antibody analysis showed that Yptb5(pSMV4)- or Yptb5(pSMV8)-immunization induced a balanced Th1/Th2 response (Fig. 3), which implies concurrent induction of T-cell responses with significant productions of IFN-γ, IL-17A and TNF-α in both lungs and spleens (Fig. 4) and is consistent to previous studies [41, 42]. Both cellular and humoral immunity were primed by Yptb5(pSMV4) or Yptb5(pSMV8) strain afforded substantial protection against pulmonary Y. pestis infection (Fig. 5). In comparison to protective efficacy of χ10069(pYA5199) [4], there was no significant difference observed in Yptb5(pSMV4)- or Yptb5(pSMV8)-immunized mice (Fig. 5). Possible explanation is that a single-dose immunization of live Y. pseudotuberculosis vaccine candidates may not go beyond the maximal cap of protection against pneumonic plague. Another possible reason may be that lack of F1 antigen in above vaccine candidates can’t reach comprehensive protection. Recently, a phase I human clinical trial showed that the Flagellin-F1/V vaccine exhibited a dose dependent increase in immunogenicity and induced both humoral and cellular immune responses [34, 43]. So, we are going to incorporate another important Y. pestis antigen F1 into this new Y. pseudotuberculosis construction, as well as alter immunization regime using homologous or heterologous prime-boost strategy to further enhance protection.

Figure 4.

Figure 4.

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Analysis of antigen-specific T-cell responses from lungs and spleens. After the 42nd day of immunization, lymphocytes were aseptically isolated from lungs and spleens of mice and in vitro stimulated with Y. pestis whole cell lysate (YPL) at 20 μg/ml for 72 h to assess specific CD4+ and CD8+ T cells producing IFN-γ, IL-2, IL-17and TNF-α. The cells from sham mice were considered as controls. (A) Lung total T cell numbers, numbers of CD4+ and CD8+ T-cell subsets, numbers of CD4+ IFN-γ+, IL-2+, IL-17+ or TNF-α+ positive cells, and numbers of CD8+ IFN-γ+, IL-2+, IL-17+ or TNF-α+ positive cells. (B) Spleen total T cell numbers, numbers of CD4+ and CD8+ T-cell subsets, numbers of CD4+ IFN-γ+, IL-2+, IL-17+ or TNF-α+ positive cells, and numbers of CD8+ IFN-γ+, IL-2+, IL-17+ or TNF-α+ positive cells. Each symbol represented data obtained from individual mice, with horizontal mean value bars ± SD. *, P< 0.05; **, P< 0.01; ***, P< 0.001; ****, P< 0.0001 were obtained by two-way ANOVA with Tukey’s post-hoc tests.

Supplementary Material

1

Acknowledgement

We thank Drs. Roy Curtiss III and Shifeng Wang at the University of Florida for sharing different strains and pYA plasmids with us. This work was supported by the National Institutes of Health grant R01AI125623 and R21AI139703 to WS and the Albany Medical College start-up fund.

Footnotes

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Conflict of interest. All authors declare that they have no conflicts of interest.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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