Brucella lipopolysaccharide reinforced Salmonella delivering Brucella immunogens protects mice against virulent challenge

Accepted Manuscript Title: Brucella lipopolysaccharide reinforced Salmonella delivering Brucella immunogens protects mice against virulent challenge Authors: Jonathan Lalsiamthara, John Hwa Lee PII: DOI: Reference:

S0378-1135(17)30064-0 http://dx.doi.org/doi:10.1016/j.vetmic.2017.05.012 VETMIC 7642

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VETMIC

Received date: Revised date: Accepted date:

14-1-2017 14-5-2017 15-5-2017

Please cite this article as: Lalsiamthara, Jonathan, Lee, John Hwa, Brucella lipopolysaccharide reinforced Salmonella delivering Brucella immunogens protects mice against virulent challenge.Veterinary Microbiology http://dx.doi.org/10.1016/j.vetmic.2017.05.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Brucella lipopolysaccharide reinforced Salmonella delivering Brucella immunogens protects mice against virulent challenge

Jonathan Lalsiamthara, and John Hwa Lee*

College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Iksan Campus, 570-752, Iksan, Republic of Korea

*Corresponding author. E-mail address: [email protected] Highlights • • •

Salmonella delivering BLS, PrpA, SOD and Omp19 were evaluated as Brucella vaccine. Brucella abortus lipopolysaccharide was used to augment the immunogenicity of the vaccine cocktail. Highly conserved Brucella antigens used and can provide pan-Brucella vaccine. formulation provide platform for safer Brucella vaccine platform

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The

Abstract Intracellular pathogen Salmonella exhibits natural infection broadly analogous to Brucella, this phenomenon makes Salmonella a pragmatic choice for an anti-Brucella vaccine delivery platform. In this study we developed and formulated a combination of four attenuated Salmonella Typhimurium live vector strains secreting heterologous Brucella antigens (rBs), namely lumazine synthase, proline racemase subunit A, lipoprotein outer membrane protein19, and Cu-Zn superoxide dismutase. With an aim to develop a cross-protecting vaccine, Brucella pan-species conserved rBs were selected. The present study compared the efficacy of smooth and rough variants of Salmonella delivery vector and also evaluated the inclusion of purified Brucella lipopolysaccharide (LPS) in the formulation. Immunization of SPF-BALB/c mice with the vaccine combinations significantly (P≤0.05) reduced splenic wild-type Brucella abortus 544 colonization as compared to non-immunized mice as well as Salmonella only immunized mice. Increased induction of Brucella specific-IgG, sIgA production, and antigenspecific splenocyte proliferative responses were observed in the mice immunized with the formulations as compared to naïve or vector only immunized mice. Modulatory effects of rB and LPS on production of interleukin (IL)-4, IL-12, and interferon-γ were detected in splenocytes of mice immunized with the formulation. Rough Salmonella variant in combination with LPS could further enhance the efficacy of the delivery when applied intraperitoneally. Taken together, it is compelling that Brucella LPS-augmented Salmonella vector delivering immunogenic Brucella proteins may be more suitable than the current nonideal live Brucella abortus vaccine. The vaccine system also provides a basis for the development of crossprotecting vaccine capable of preventing multispecies brucellosis.

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Keywords: live bacterial vaccine vector; Brucella vaccine; protective efficacy; lipopolysaccharide; Salmonella delivery; brucellosis

1. Introduction Brucellosis is caused by gram-negative bacteria species of the genus Brucella and affects a wide range of hosts including marine mammals (Nymo et al., 2011; Pappas et al., 2005). The disease is sub-acute or chronic in cattle, sheep, goats, other ruminants, and pigs; the initial phase of the infection is often not apparent (Corbel, 2006). Four major Brucella species known to cause human brucellosis are B. melitensis, B. suis, B. abortus, and B. canis, in descending order of pathogenicity. Over the last century, human brucellosis has been controlled within domesticated animals by vaccination and culling (Corbel, 2006). Major hurdles in the development of ideal vaccines are lower protection, residual virulence, and abortion in some of the animal population. An alternative approach that avoids the residual virulence of Brucella live vaccines is the delivery of Brucella subunit components using live vaccine vectors. Live vaccine vectors in general provide lower protection as compared to live attenuated Brucella vaccine; however, when safety is a major concern, and especially for human deployment, it would be pragmatic to initiate anti-Brucella vaccine development based on live bacterial vaccine vectors. We hypothesized that the issue of lower protection might be resolved by using a mixture of live vectors each secreting a Brucella subunit antigen. In this study, with the aim of developing a cross-protecting multivalent Brucella vaccine, we selected highly conserved Brucella immunogens with approximately 100% amino acid sequence homology between B. melitensis, B. suis, B. abortus, and B. canis for vaccine construction. We developed a vaccine formulation composing purified Brucella LPS antigen and a cocktail of four live attenuated Salmonella vaccine vector strains each delivering 3

recombinant Brucella abortus antigen (rB) namely lumazine synthase (BLS), proline racemase subunit A (PrpA), lipoprotein outer membrane protein-19 (Omp19), and Cu-Zn superoxide dismutase (SOD). BLS can elicit a mixed Th1-Th2 pathway response important for humoral and cell-mediated immune responses (Velikovsky et al., 2002). PrpA is a potent splenocyte and B-cell stimulant; the enzyme is also linked with chronicity of Brucella infections (Spera et al., 2006). PrpA immunization would not only have immunomodulatory effects but would also generate anti-PrpA antibodies in immunized animals, which in turn may reduce the chronicity of Brucella infection. Omp19 elicits adaptive interleukin (IL)-17 cytokine production that is important for mucosal immunity and also confers protection when used as a subunit or DNA vaccine platform (Pasquevich et al., 2011). SOD is a protective immunogen capable of inducing diverse immune responses (Onate et al., 1999). This study also investigates the effects of including purified Brucella lipopolysaccharide (LPS) in combination with the live vaccine system. Brucella abortus LPS is a highly immunogenic component of Brucella surface antigens and in combination with Omp19 can serve as a bacterial somatic antigen target for antibodies. In regard to the carrier vector strain, the efficacy of the O-antigen–deficient ST strain was also evaluated. Rough Salmonellae have been tested extensively as candidate vaccines (Dlabac et al., 1997; Nagy et al., 2004); however, to date very few rough strains have been evaluated as live vaccine vector. A Salmonella strain with O-Ag deficiency showed an increased rate of uptake by dendritic cells (DCs), altered intracellular processing, and increased degradation, and also boosted the activation of immune functions of DCs (Zenk et al., 2009). The present study evaluates the effectiveness of smooth (SrB) and rough (RSrB) Salmonella live vector strains for heterologous antigen delivery and protective efficacy against Brucella infection.

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This study reports a pilot trial of a novel Brucella-multispecies vaccine against Brucella abortus in a mouse model. We investigate the effects of immunization with regard to hostsafety issues, humoral and cellular immune responses, cytokine induction, relative cellular IFN-γ secretion, CD3+ CD4+/CD8+ FACS analysis, and protective efficacy compared with virulent wild-type challenge. To further evaluate the degree of protection, a live commercial Brucella abortus vaccine RB51 strain was used as a reference. 2. Materials and methods 2.1 Ethics and biosafety statement All animal experimental procedures were approved (CBNU2015-00085) by the Chonbuk National University Animal Ethics Committee in accordance with the guidelines of the Korean Council on Animal Care and Korean Animal Protection Law, 2007; Article 13 (Experiments with animals). All mice used in the study were housed and maintained humanely and were provided water and antibiotic-free food ad libitum. Biosafety level-3 organism Brucella abortus was handled with safety precautions and under the supervision of Ministry of Health & Welfare, South Korea. 2.2 Bacterial strains, plasmids, and primers The bacterial strains, plasmids, and primers used are listed in Table S1. All Salmonella strains and E. coli strains were grown in Luria-Bertani (LB) medium supplemented with 50 µg/mL diaminopimelic acid for growth of asd gene-deleted strains. Brucella abortus strains RB51 and 544 were grown routinely on BD Brucella medium (BB) in an aerobic or 5% CO2 atmosphere.

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2.3

Construction and validation of Salmonella Typhimurium strains secreting

Brucella immunogenic proteins The open reading frames of PrpA, Omp19, and SOD were amplified from the strain 544 genome and cloned into the delivery plasmid pJHL-65 via the incorporated EcoRI and HindIII restriction sites. The BLS gene cassette was synthesized in vitro because of restriction enzyme incompatibility (File S2). The constructed plasmid also contained the asd gene of ST, which assisted plasmid-host complementation. The gene of interest was fused with a bla secretion signal and a 6×His tag sequence and the fused cassette was constitutively expressed under the Ptrc promoter. The plasmid was used to electrotransformed the ST delivery strains, JOL912 and JOL1800 (Table S1). JOL912 is a ∆lon∆cpxRΔasd, smooth phenotype strain derived from JOL401. JOL1800 is a rough strain derived from JOL912. The LPS biosynthesis gene ΔrfaL encoding O-antigen ligase was knocked out using a lambda red engineering technique. Disruption of LPS synthesis in JOL1800 was confirmed using SDS-PAGE silver staining (unpublished data). For the purpose of comparative studies, a JOL1800 vector-only control was transformed with blank asd-positive pJHL65 lacking the Brucella gene cassette. The expression of Brucella antigen was confirmed from the culture supernatant of JOL strains using a previously described protocol with minor modifications (Lalsiamthara et al., 2016). Briefly, 200 mL of supernatant from broth cultures grown at 37ºC (OD600 0.8) was recovered after centrifugation at 4,000 g for 15 min. The supernatant containing the secreted protein was filtered 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 96ºC for 5 min, the proteins were separated using 15% SDS-PAGE gels. The separated proteins were blot transferred to polyvinylidene fluoride

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(PVDF) membranes (Millipore, USA) and blocked in 5% skim milk. Expressed protein was detected via the incorporated histidine tags using primary mouse IgG1 anti-His-tag antibody (Anti-HIS6, AprilBio, South Korea) and secondary anti-mouse IgG1 antibody-HRPO conjugate (Sigma-Aldrich, USA) antibodies, diluted at 1:2500 (1% BSA-PBS) and 1:5000 (PBS) respectively. Reactive bands were developed using DAB substrate conversion or the West-OneTM Western Blot Detection System (iNTRON biotechnology, South Korea) and bands were visualized using a KODAK Image Station (Kodak, New Haven, CT, USA). Purified Brucella LPS was extracted from smooth Brucella abortus strain 544 using a phenol-based commercial LPS extraction kit (iNtRON biotechnology, South Korea). Purified LPS was electrophoresed on a 15% PAGE gel, which was fixed and stained using a silver staining kit (Silver Stain Plus, Bio-Rad, US). An LPS oxidation step using 200 µL of periodic acid (20% w/v) was included in the staining process. Protein and nucleic acid contamination was reduced by the addition of proteinase K and nucleases respectively. The level of protein contamination was determined using Bradford assay. Following vacuum evaporation, the concentration of LPS was determined by dry weight- measurement. For vaccine formulation, purified LPS resuspended in 10mM Tris-HCl buffer (pH 8.0) was directly added to prespecified Salmonella-PBS suspension, at 10 µg/dose concentration (Table 1). BLS gene was cloned in pET28 (+) plasmid and expressed in a BL21 E. coli expression system (Table S1). BLS recombinant protein was purified and used as antigen for indirect ELISA-based detection of delivered antigen-antibody response. 2.4 Immunization, challenge, and protective efficacy A total of 128 5-week-old specific pathogen free (SPF) female BALB/c mice were divided equally into 8 groups (n = 16/group), fasted for 20 h, and were immunized according to the 7

scheme presented in Table 1. The mice were then simultaneously used for vaccine-related safety assessment (i.e., observation for any immunization-induced morbidity or mortality), immunological profiling, and protective efficacy studies. Six mice were euthanized at day 21 post-immunization and harvested splenocyte samples were subjected to FACS analysis, invitro stimulation and cytokine quantification, ELISPOT assay, and lymphocyte proliferation analysis. All mouse groups were challenged at 30 days post-immunization with 2 × 105 CFU/200 µL IP using a mouse-passaged virulent B. abortus strain 544 (Table 1). At day 15 post-challenge, ten mice were euthanized and 544 splenic bacterial load was determined as previously described (Lalsiamthara et al., 2015). Briefly, the spleen was excised aseptically and homogenized in a 15-mL tube containing 2 mL PBS. After complete homogenization with a sterile wooden applicator, 200 µL of the inoculum was seeded and spread on Brucella agar plate. Colonies were counted after incubation for 7 days at 37ºC in a 5% CO2 atmosphere. Brucella cell counts of each group were mathematically transformed to conform to statistical fitting, using the formula: y = log(x/log x) as described previously (Bosseray and Plommet, 1990). Protective index (PI) was calculated as PI = y value of test vaccine subtracted from y value of PBS. 2.5 Humoral immunity against whole cell Brucella lysate and specific BLS antigen To determine the level of anti-Brucella antibodies generated, humoral responses of control mice and mice immunized with cocktails of delivered Brucella antigens and LPS were investigated using an indirect ELISA format. Serum and intestinal lavage samples were collected at weekly intervals for 4 weeks post-immunization. Secretory IgA levels were measured from intestinal lavage samples collected as described previously (Lalsiamthara and Lee, 2016) with minor modifications suitable for mice. Briefly, mice were intra-gastrically

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administered 600 µL of lavage solution (0.2 M Na2SO4, 0.2 M NaHCO3, 0.1 M KCl, 0.25 M NaCl, 1× serine trypsin protease inhibitor cocktail, 50 mM EDTA, 16.25% polyethylene glycol in distilled water) and enclosed separately in disinfected inverted glass beakers with a provision for mesh flooring and a petri dish for sample collection. Ten minutes postadministration, the mice were injected intramuscularly with 100 μL of 5% pilocarpine solution. Approximately 200- to 400-µL samples of mucinous droppings were collected in microcentrifuge tubes. For preservation, 5 μL each of 5% bovine serum albumin, 10% sodium azide, and 1% PMSF (phenylmethylsulfonyl fluoride) was added and the samples were stored at -20°C until analysis. Indirect ELISA was conducted using 544 whole cell lysate as well as purified BLS recombinant protein antigen. Briefly, 500 ng of lysate/well or 350 ng of BLS/well was used to coat ELISA plates (Maxisorp, NUNC). Primary mouse serum and lavage samples were diluted 1:100 and 1:5 respectively with PBS, and secondary horseradish peroxidase (HRP)-conjugated anti-mice IgG and sIgA antibodies were used at 1:8,000 and 1:5,000 dilutions, respectively. Colorimetric changes resulting from the action of HRP on OPD (Sigma–Aldrich, US) were measured (TECAN, Austria) at 492 nm, 10 and 15 minutes post-development for IgG and sIgA samples, respectively. The values for binding of IgG and sIgA to respective antigens were expressed as the mean OD value ± standard error (SE). 2.6 FACS-based enumeration of antigen- induced splenocyte proliferation The lymphocyte proliferation assay (LPA) was performed at 21 days post-immunization. The proliferative capability of splenocytes was evaluated based on a previously described protocol with minor modifications. Splenocytes were harvested aseptically and separated using 1077 histopaque density gradient centrifugation. Viable cells were seeded in triplicate in 96-well plates at 1 × 105 cells/well. Cells were treated with 4 µg/mL of ST purified OMP, Brucella

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abortus bacterial lysate suspension, 10 µg/mL concanavalin A, or RPMI alone and incubated at 40°C in a humidified 5% CO2 atmosphere for 72 h. The cellular proliferative response against a specific antigen was measured using DAPI stained-direct FACS cell counting and gating out of cell debris, and the mean stimulation index (SI) was determined. 2.7 qPCR quantification of relative IL-4 and IL-12 levels Quantitative analysis of cytokines was performed based on a protocol described previously with minor modifications (Jawale and Lee, 2016). The assay was performed on splenocytes of the immunized mice at 21 days post-immunization. Viable cells were seeded at a density of 2 × 105 cells/well. After 24 h incubation at 42ºC in a 5% CO2 incubator, the splenocytes were stimulated with sonicated Brucella 544 antigens at 5 µg/well, followed by incubation for 16 h. Total RNA was extracted from the culture cells and complementary DNA was synthesized. Real-time polymerase chain reaction (PCR) quantification was performed using qPCR Master Mix (QuantiTect®, Qiagen, Germany) to measure the levels of IL-4 and IL-12 cytokines. The reference gene β-actin was used as an endogenous control. DataAssistTM (ThermoScientific Inc.) was used to analyze the cycle threshold (CT) values. Fold changes for the cytokines were calculated as the log2 mean value of 2-ΔΔCT of a stimulated sample divided by the log2 mean value of 2-ΔΔCT of an unstimulated sample (Livak and Schmittgen, 2001).

2.8 INF-γ cytokine quantification by ELISPOT assay Antigen-specific induction of INF-γ was measured at the cellular level using mouse IFN-γ ELISpotPLUS (Mabtech AB, Sweden). The assay was performed at 21 days post-immunization using splenocytes of the immunized mice. A total of 2 × 105 cells/wells were seeded in triplicate and incubated with RPMI only, 500 ng of ST JOL401 outer membrane antigen, and 544 sonicated whole-cell lysate. After 30 h incubation in a 37ºC humidified 5% CO2 10

atmosphere, INF-γ spots were detected using biotin-conjugated anti-INF-γ primary antibody. Streptavidin-HRP (1:1,000) was used to bind the primary antibodies and TMB substrate solution was used to develop insoluble spots. Distinct spots were counted manually and total mean count was determined. 2.9 FACS analysis of CD3+CD4+ and CD3+CD8+ T cells Flow cytometric analyses of T-cell populations were carried out on splenocytes of mice at 21 days post-immunization. Briefly, 5 × 105 viable cells were exposed to ST 401 OMP, Brucella whole-cell sonicated antigen, or RPMI media alone for 48 h and then the cells were harvested and stained with phycoerythrin (PE)-labeled CD3e (17A2), PerCP-Vio700-labeled anti-CD4, and fluorescein isothiocyanate (FITC)-labeled anti-CD8a monoclonal antibodies (Miltenyi Biotec, Germany) for 30 min in the dark at 4°C. The cells were washed with 1 mL FACS running buffer twice and data were acquired using MACSQuant® Analyzer (Miltenyi Biotec, Germany) and analyzed using FlowJo software (Tree Star, Inc., OR, USA). The CD3+ cell population and T-cell subsets of CD3+CD4+/CD3+CD8+ lymphocytes were determined.

2.10 Statistical analysis Statistical analyses were used where applicable. One-way analysis of variance (ANOVA) and Student’s t-tests were used to determine statistically significant differences, with a P value ≤0.05 considered significant. Tukey’s test was applied for post hoc analysis. Analyses were performed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). Data represent two independent set of experiments unless indicated.

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3. Results 3.1 Construction and validation of Salmonella secreting Brucella immunogenic proteins The pJHL-rB plasmids were successfully transformed into the ST delivery strains JOL912 and JOL1800. Electro-transformation of the plasmid into host strains was confirmed using specific colony PCR (Fig. S3). Expression and secretion of Brucella antigens by the vaccine vector was validated using precipitated secretory protein harvested from cultures. Proteins were separated by SDS-PAGE and subjected to western blot analysis using anti-His tag antibody to detect the recombinant proteins (Fig. S4). The expected reactive bands were detected on sample lanes and corresponded approximately to protein markers at 23.48 kDa, 39.76 kDa, 20.68 kDa, and 21 kDa for BLS, PrpA, Omp19, and SOD, respectively of the in silico translated protein molecular weights. The molecular weight of each protein was increased by 3 kDa due to fusion with the leader 6×His tags. 3.2

SrBL immunization induced humoral response against Brucella whole-cell lysate

and delivered antigens The systemic and mucosal humoral immune responses elicited by the vaccine strains were investigated. Mice groupings and immunization scheme is given in table 1. All immunized mice developed antibody production against the Brucella whole-cell lysate except for mice immunized with JOL1800 vector control. Anti-Brucella IgG production peaked at the 3rd week post immunization among the mice groups that were immunized orally with SrBL, the increment was significantly higher compared to the control group (Fig. 1A). Mice immunized with SrBL via IP showed a pattern of increased IgG production beyond the 4th week postimmunization. Mice immunized with live RB51 vaccine showed a significant increase in IgG compared with SrB or SrBL vaccinated groups (P≤0.05). Mouse groups immunized via 12

oral or IP routes showed similar patterns for IgG levels during the course of the study; relative IgG levels were higher than in the control group irrespective of the administration route (Fig. 1A). To evaluate the role of vaccine strains in inducing mucosal immunity we measured the levels of intestinal sIgA. At the second week, the IgA levels of orally immunized mice were higher than those of corresponding IP or control non-immunized mice (P≤0.05), including group G and H. At the third week post-immunization, the IgA levels were highest in group F mice, which showed significant differences compared with other groups excluding group H (Fig. 1B). The combined data of IgG and sIgA revealed that RB51 also induced humoral responses significantly different from controls; however, the difference was not significant from that of Salmonella delivered groups. The degree of humoral response induced by the delivered antigens was also determined. For this purpose, purified recombinant BLS with high humoral immunogenicity was used. We observed increased levels of IgG and sIgA among immunized mice in response to the delivered antigens (Fig. 1C, 1D). Significant increases (P≤0.05) in humoral responses were observed in immunized mice compared with group A non-immunized mice. Overall, levels of anti-BLS antibodies were higher for Salmonella delivery compared with RB51 immunized mice. 3.3 Delivered Brucella rB immunogens enhance splenocyte proliferation The cell proliferative responses to Salmonella and Brucella antigens among the mice groups were investigated by FACS-based LPA. The splenocytes of groups B, C, D, E, and F showed a significantly higher proliferative index than the control group upon Salmonella antigen stimulation (Fig. 2). It was evident that inclusion of rBs in the vaccine preparation augmented

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cellular responses in groups B, C, D, E, and F, whereas group G mice that were immunized with vector alone showed a comparatively lower degree of proliferation. Group E and H showed a significant proliferative response upon Brucella antigenic stimulation compared with group A control (Fig. 2). Overall, we observed that Salmonella stimulated stronger proliferative responses than Brucella antigens among the mouse groups. 3.4 SrBL immunization induced significantly increased IL-4 and IL-12 production We next investigated the magnitude of the Th1 and Th2 immune response through cytokine induction in response to the vaccine formulations. The levels of IL-4 and IL-12 cytokines, which are important for Th2 and Th1 cell differentiation respectively, were measured from RNA isolated from in vitro stimulated splenocytes of mice. The quantitative PCR data revealed that all mouse groups showed alterations in cytokine profiles upon stimulation with Brucella antigen. Significant increases in IL-4 were observed in groups B, D, E, and H (P≤0.05), while mice in groups C, F, and G did not show significant changes (Fig. 3A). Group D mice showed remarkably elevated IL-4 production compared with other groups. SrBL vaccines induced significant IL-4 production with both oral and IP route. IL-12 secretion was highest in group C, whereas significant increases were observed in groups B, C, D and H (Fig. 3B). 3.5 SrBL exhibited an immunomodulatory effect on cellular IFN-γ secretion Antigen-specific induction of Brucella-protective cytokine INF-γ was determined at the cellular level using ELISPOT assay. Mean spot counts were enumerated from unstimulated and Salmonella and Brucella antigen stimulated samples. Upon Salmonella antigenic stimulation, remarkably high INF-γ production was observed in group D mice. Significant increases in INF-γ production were also observed in groups B to F (Fig. 4). Upon Brucella 14

antigen stimulation, splenocytes cultures of group C and H showed production of IFN-γ which higher than Salmonella stimulated cells. Overall, Brucella antigen induced weaker INF-γ cytokine secretion than Salmonella antigen. 3.6 FACS analysis of T-cell subpopulations The cell proliferative responses to Salmonella and Brucella antigens among the mouse groups were investigated by FACS analysis. The profiles of CD3+CD4+ and CD3+CD8+ T-cell subset populations were assessed at day 21 post-immunization. The flow cytometry data revealed overall increases in both T-cell populations upon live bacterial immunization. Determination of CD4+ and CD8+ T-cell fractions among the mouse groups (Table S5) showed the highest CD3+ T-cell percentage in group H mice. The ratio of CD4+:CD8+ was also determined. Lymphocyte cell populations were gated from the other splenocyte populations, followed by the PE-labeled CD3+ population, PerCP-Vio700-labeled CD4+ and FITC-labeled CD8 subsets of gated CD3+ cells. CD4+ cells were plotted at the first quadrant and CD8+ cells were plotted at the third quadrant. Upon immunization by delivery of Salmonella, a shift toward CD3+CD8+ T cells was observed in groups B, C, D, E, and F. Control non-immunized group A and the RB51 immunized group showed an increase in CD3+CD4+ T cells (Table S5).

3.7

Rough phenotype SrBL immunization confers the highest protection against

wildtype challenge The protective efficacy of immunization with the Salmonella delivery vaccines was investigated in the mouse model. Four-week-old SPF-BALB/c female mice were immunized with vaccine candidate strains/formulations and then challenged at 30 days post-immunization with virulent Brucella abortus 544. Mice were euthanized at 15 days post-challenge. The magnitude of challenge-bacterial load recovered from the spleen reflected the efficacy of 15

immunization. In comparison with PBS and vector controls, SrBL-oral, RSrBL-oral, SrBL-IP, RSrBL-IP, RSrB-IP, and RB51-IP immunized mice shown significantly higher protections, further, SrBL-IP, RSrBL-IP, and RB51-IP immunized mice group formed different subset (Fig. 5; S6). 4. Discussion Using Salmonella-based vector, Brucella protective immunogens can be delivered to critical immunological sites comparable to Brucella infection.

In this study we evaluated the

immunogenicity and protective efficacy of four highly conserved Brucella antigens: BLS, PrpA, Omp19, and SOD. Purified Brucella LPS was also added to the formulation since several experiments have shown the importance of LPS in protective immunity against wildtype challenge (Jacques et al., 1992; Montaraz and Winter, 1986). Rough mutants that are deficient in LPS have lower protective efficacy (González et al., 2008; Monreal et al., 2003). It was also reported that LPS alone as a subunit vaccine candidate could confer some level of protection in mice (Bhattacharjee et al., 2006; Monreal et al., 2003). In contrast, there were also reports describing that non-canonical Brucella LPS played a role in immunosuppression, stealth infections, and virulency of intact Brucella bacterium (Barquero-Calvo et al., 2007; Forestier et al., 2000; Martirosyan et al., 2011); although the immuno-modulatory effects of the LPS were in part dose-dependent (Forestier et al., 2000). The present study did not show any conspicuous down regulation of immunity among the mice inoculated with LPS formulations. The key advantage of our vaccine formulation is that the Brucella antigens selected are highly conserved across Brucella species; there is 100% amino acid sequence homology between B. suis, B. canis, and B. melitensis for BLS, SOD, and Omp19 proteins and 99%

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homology in the case of PrpA. It can be speculated that a similar level of cross-protection would be achievable against these species. Further, considering human brucellosis, the B. abortus LPS component of the current formulation can also be replaced with LPS derived from B. melitensis. Additionally, the concentration of the LPS incorporated in the vaccine can be optimized to the minimal level required to achieve adequate protective immune responses, while the induction of anti-LPS IgG is low enough not to interfere with LPS-based Brucella serodiagnosis. Hence, balanced optimization would provide an option for vaccinedifferentiating infected and vaccinated animals (DIVA) capability. Although Brucella is an intracellular pathogen it has been reported that during a secondary recall response both Brucella-specific humoral response and Th1 cells played an important role in conferring protective immunity to B. melitensis infection in the spleen (Vitry et al., 2014). Complement killing and opsonization could be an appropriate defense mechanism before the organism becomes intracellular (Corbeil et al., 1988). The present study determined the humoral responses against whole-cell lysate of a Brucella wild-type strain (Vitry et al., 2014). We presumed that mice inoculated with RB51 would show the highest IgG response against the lysate antigen, followed by the group immunized intraperitoneally with RSrBL. However, humoral response assessment based on purified BLS protein-ELISA revealed that the SrB and RSrB vaccines performed better. This observation may be related to the quantity of BLS proteins produced by RB51 or Salmonella vector. Remarkably, mice in groups D (RSrBL) and F (RSrB) that were immunized via a non-mucosal route, i.e. the IP route, showed a significant increase in anti-BLS SIgA antibodies. It is tempting to infer that Salmonella administered via the IP route may reach mucosal sites of the intestines via systemic invasion.

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The present study investigated the effects of vaccination on splenocyte proliferation and T-cell differentiation. Salmonella lysate antigens induced strong proliferations among the immunized mice due to their potent mitogenic activity (Fig. 2). The mice groups immunized with SrBL, RSrB, and RSrBL by either oral or IP routes showed increased splenocyte proliferation upon antigenic recall. However, group G mice immunized intraperitoneally with vector-only JOL1800 did not show a significant increase in splenocyte proliferation compared with administration of RSrB or RSrBL by the same route. It may be inferred that Brucella antigens and LPS acted synergistically as adjuvants and further enhanced the cellular responses. Several researchers have reported that protective immunity against B. abortus is mediated in part by T cells of the CD8+ phenotype (Oliveira and Splitter, 1995; Yingst and Hoover, 2003). We assessed the dynamics of T-cell populations upon antigenic stimulation at day 21 post-immunization. Immunized mice showed an increase in the percentage of CD3+ lymphocytes among the splenocyte population (Table S5). We also determined the ratio of CD3+CD4+ and CD3+CD8+ cells and showed a significant shift toward CD8+ lymphocyte proliferation among mice immunized with SrB, RSrBL, and RSrB. It has been reported that IL-12 production is strongly related to suppression of Brucella infection in mice. Spleen cells from IL-12–depleted mice show a reduced ability to produce nitrite (Zhan and Cheers, 1995), and increased production of IL-12 indirectly enhances levels of INF-γ, an important protective cytokine. IFN-γ is a critical cytokine for host control of Brucella infection (Murphy et al., 2001). The present study determined the levels of IL-4, IL12, and cellular level INF-γ production in splenocytes of mice upon antigenic recall (Fig. 3, Fig. 4). The immunomodulatory effect of Brucella antigens was evident from mice

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immunized with SrBL and RSrBL. Significant increases in INF-γ secretion were observed upon Brucella antigen exposure in mice from groups B, C, D, and E that were inoculated with SrBL and RSrBL. This finding correlated with the IL-12 cytokine levels and also with the protective indices observed in the present study. For potency determination and routine vaccine-batch testing, Brucella vaccines are generally assessed using the mice challenge strain-splenic recovery model (Grilló et al., 2000; Lalsiamthara et al., 2015). Splenic bacterial load revealed that the most efficient clearance and reduction of 544 occurred in mice group that were immunized with RSrBL via the IP route (Fig. 5). It is also noteworthy that mice immunized via the oral route in the present study exhibited some degree of immunologic response yet showed a remarkably low level of protection (Fig. 5). Although the reason for this is not completely clear, it may be presumed that oral immunization is hindered by factors such as interfering digestive contents, degrading enzymes, and gastric acids. In future, this issue could be remediated by incorporating shielding enteric-coated formulations suitable for oral administration. We also observed that the vector only immunized mice showed a considerable degree of protection, this phenomenon may correlate with Mackaness effect (Mackaness, 1964). Although the present study was not set up to investigate the probable explanation for why delivery of rough Salmonella confers enhanced protection, it is tempting to presume that a rough Salmonella strain with more exposed bacterial surface proteins such as fliC would have a stronger capability to induce diverse cytokines like TNF-α (Ciacci-Woolwine et al., 1998). It has been reported that TNF-α is an important cytokine for Brucella clearance (Caron et al., 1994). It has also been reported that the uptake of Salmonella by macrophages and bone marrow-derived dendritic cells is higher in rough type than wild type, and that rough Salmonella Typhimurium increases the expression of CD86, MHCII, TLR4, and myD88 molecules (Zenk et al., 2009). Furthermore, 19

JOL1800 is an O-antigen deficient strain, in which the lipid A moiety of the outer membrane may be readily exposed. Lipid A is a strong activator of monocytes to release immune stimulators such as the pro-inflammatory cytokines TNF-γ and IL-6. These properties might have concerted effects to enhance protection, as revealed by delivery of the rough phenotype. 5. Conclusion In conclusion, the present study provides compelling evidence that Salmonella delivering selected Brucella immunogens in combination with Brucella LPS antigen work synergistically and enhances protection in a mouse challenge model. We also demonstrated that delivered Brucella antigen acts as an immunomodulator by influencing both the humoral and cellmediated response. The increased production of IL-12 cytokine among immunized mice strongly correlated with increased INF-γ production and cell proliferation. All of these attributes are necessary for efficient protection against wild-type infection. Taken together, these findings revealed that SrBL and RSrBL have potential as substitutes for the non-ideal anti-Brucella vaccines in human and animal health sectors. Conflict of interest The authors declare that they have no competing interests. Funding This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea [grant number: HI16C2130]

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Tables Table 1. Immunization and challenge scheme Groups N=128 n=16

Strains & Immunogen

Route

Formulation & Dose

PBS

A

Non-immunized Control

IP

B

SrBLa

Oral

C

RSrBLb

Oral

D

SrBL

IP

E

RSrBL

IP

F

RSrBc

IP

2 X 107 each (4 strains) + strain 544 LPS (10µg) 2 X 107 each (4 strains) + strain 544 LPS (10µg) 2 X 107 each (4 strains) + strain 544 LPS (5µg) 2 X 107 each (4 strains) + strain 544 LPS (5µg) 2 X 107 each (4 strains)

G

Vector control

IP

8x107

H

RB51

IP

107

Wild type Challenge

30 days post immunization (2 X 105 CFU, IP)

Splenic Brucella recovery

15 days post challenge

IP- intraperitoneal. a

SrBL formulation, four smooth strains JOL912 Salmonella live vectors-JOL1874, JOL1875, JOL1876, and JOL1877, each constitutively expressing Brucella immunogenic protein i.e. SOD, BLS, PrpA, and Omp19; combined with purified LPS isolated from strain 544.

b

RSrBL formulation, four rough strains JOL1800 Salmonella live vectors- JOL1878, JOL1879, JOL1880, and JOL1881, each constitutively expressing Brucella immunogenic protein i.e. SOD, BLS, PrpA, and Omp19; combined with purified LPS isolated from strain 544.

c

RSrB formulation, four rough strains JOL1800 Salmonella live vectors- JOL1878, JOL1879, JOL1880, and JOL1881, each constitutively expressing Brucella immunogenic protein i.e. SOD, BLS, PrpA and Omp19; LPS excluded.

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Figure legends Fig. 1. Humoral immune responses. The systemic and mucosal humoral immune responses elicited by the vaccine strains were investigated by measuring the relative levels of plasma IgG and intestinal lavage SIgA from samples obtained from immunized and non-immunized mice. (A) Systemic IgG response against Brucella lysate among mice groups. All immunized mice groups except vector control group, showed a significant difference (P≤0.05) at the 3rd week of immunization. (B) Intestinal secretory IgA response against Brucella lysate among mice groups. (C) Systemic IgG response against recombinant BLS antigen among immunized mice. A significant difference (P≤0.05) was observed between immunized and vector control only immunized mice group. (D) Intestinal IgA response against recombinant BLS antigen among immunized mice groups. A significant difference (P≤0.05) was observed between immunized and vector control only immunized mice group. Antibody levels are expressed as means OD492 ± standard errors of the mean. The asterisks indicate significant differences between the antibody titers of the immunized and vector control only immunized mice group. (P≤0.05). 1, 2, 3, 4, denotes sampling weeks and applies for each group. Fig. 2. Stimulation Index. Lymphocyte proliferative responses against Salmonella outer membrane or Brucella sonicated lysate antigen in immunized and non-immunized mice at 21 days post-immunization. The antigen-specific lymphocyte proliferative response is expressed as the mean stimulation index ± standard error of mean. The asterisks indicate significant differences between cell proliferative index of the immunized and non-immunized groups (P≤0.05). SS- Salmonella antigen stimulated, BS- Brucella antigen stimulated.

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Fig. 3. Cytokine profile of mice groups. Fold change in expression of splenocyte cytokines upon in vitro Brucella sonicated lysate antigen stimulation was determined by qPCR using the comparative Ct method. Values represent relative expression of the cytokine normalized to βactin endogenous control. (A) Relative expression of IL-4 cytokine. (B) Relative expression of IL-12 cytokine. Relative cytokine levels are expressed as log2 means ± standard errors of the mean. The asterisks indicate significant differences between cytokine expression of the immunized and non-immunized groups (P≤0.05). Fig. 4. ELISPOT-based quantification of splenocyte IFN-γ level. In vitro antigen-induced production of INF-γ cytokine production was determined for splenocytes of mice groups at 21 days post-immunization. Mean spot counts ± standard errors of mean were determined from un-stimulated and Salmonella outer membrane– or Brucella lysate antigen–stimulated cells. The asterisks indicate significant differences between immunized and non-immunized groups, * indicates P≤0.05, ** indicates P≤0.01. SS- Salmonella antigen stimulated; BS- Brucella antigen stimulated. Fig. 5. Protective efficacy of Salmonella delivery. Mice were challenged with virulent strain 544 at 30 days post-immunization and euthanized at 15 days post-challenge. The degree of challenge-bacterial load recovered from the spleen reflected the potency of immunization. Protective index (PI) was derived by subtracting the log value of test vaccine from log value of control non-immunized group. PI is expressed as mean PI value ± SE. The asterisks indicate significant differences (P≤0.05) in log count compared to vector control, ‘a’ indicates different subset.

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