Dual Immunization with SseB/Flagellin Provides Enhanced Protection against Salmonella Infection Mediated by Circulating Memory Cells

Seung-Joo Lee, Joseph Benoun, Brian S. Sheridan, Zachary Fogassy, Oanh Pham, Quynh-Mai Pham, Lynn Puddington and Stephen J. McSorley
J Immunol August 15, 2017, 199 (4) 1353-1361; DOI: https://doi.org/10.4049/jimmunol.1601357

Abstract

The development of a subunit Salmonella vaccine has been hindered by the absence of detailed information about antigenic targets of protective Salmonella-specific T and B cells. Recent studies have identified SseB as a modestly protective Ag in susceptible C57BL/6 mice, but the mechanism of protective immunity remains undefined. In this article, we report that simply combining Salmonella SseB with flagellin substantially enhances protective immunity, allowing immunized C57BL/6 mice to survive for up to 30 d following challenge with virulent bacteria. Surprisingly, the enhancing effect of flagellin did not require flagellin Ag targeting during secondary responses or recognition of flagellin by TLR5. Although coimmunization with flagellin did not affect SseB-specific Ab responses, it modestly boosted CD4 responses. In addition, protective immunity was effectively transferred in circulation to parabionts of immunized mice, demonstrating that tissue-resident memory is not required for vaccine-induced protection. Finally, protective immunity required host expression of IFN-γR but was independent of induced NO synthase expression. Taken together, these data indicate that Salmonella flagellin has unique adjuvant properties that improve SseB-mediated protective immunity provided by circulating memory.

Introduction

Salmonella enterica serovars infect humans, pets, livestock, and poultry, causing a variety of clinical diseases, depending on the serovar involved and the underlying susceptibility of the host (1, 2). In the United States, the economic impact of Salmonella infection is substantial, and periodic multistate outbreaks of gastroenteritis occur as the result of the ingestion of contaminated produce, meat, or processed foods (3). Public health measures have improved food handling and limited the dissemination of livestock waste, and vaccines have also been developed to reduce Salmonella carriage in livestock and poultry (4, 5). Despite these efforts, Salmonella infections cause >1 million illnesses in the United States every year (6).

The impact of Salmonella infection is considerably greater in low-income countries that lack access to clean water and basic sanitation. In this environment, human-restricted serovars S. enterica Typhi and Paratyphi can be transmitted, causing a life-threatening systemic disease known as typhoid or enteric fever (7). Recent estimates suggest that typhoid afflicts 21.65 million people and causes 433,000 deaths annually, with most cases localized to south and southeast Asia (8, 9). In contrast, nontyphoidal Salmonella serovars typically cause gastroenteritis but are also responsible for disseminated infections of young children or adults with compromised immunity, referred to as invasive nontyphoidal salmonellosis (iNTS) (10, 11). Importantly, iNTS strains are the most common bacterial isolates recovered from febrile presentations in adults and children in sub-Saharan Africa (12). In fact, estimates suggest that iNTS is responsible for 564,000 deaths annually, meaning that typhoidal and iNTS infections together cause almost 1 million deaths annually (9). Although these systemic bacterial infections can often be successfully treated with antimicrobials, Salmonella serovars are increasingly resistant to multiple antibiotics, and the development of an effective vaccine for vulnerable populations is required (8).

Vaccine development for typhoid has largely focused on improving the efficacy of live attenuated (Ty21a) and Vi polysaccharide (ViCPS) vaccines. These licensed vaccines are only moderately effective (50–60% over 3 y) and are poorly immunogenic in infants, and neither is widely used in endemic areas (8, 13–16). Ty21a is a live vaccine strain (LVS) of S. Typhi administered in four individual doses to patients older than 5 y of age. Although attempts have been made to improve the safety and immunogenicity of LVS of S. Typhi, this has proved unexpectedly difficult to achieve (13). Furthermore, there are natural impediments to administering live Salmonella vaccines to infants, the elderly, HIV-positive individuals, and immune-suppressed populations (17, 18). ViCPS is a purified capsule polysaccharide (CPS) that can effectively curtail a typhoid outbreak and provides protection to travelers visiting endemic areas (17, 18). The major limitation of this vaccine is that it induces short-term T cell–independent Ab responses and does not induce long-term immunological memory (19, 20). Next-generation ViCPS-conjugate vaccines are being studied in clinical trials, and one has already been licensed in India (16). Although these new Vi-conjugate vaccines should extend the duration of immunity provided by ViCPS, they will be unable to protect against iNTS or paratyphoid serovars because these bacteria lack expression of ViCPS (21). Thus, to develop new vaccines for typhoid and iNTS, viable alternatives to LVS and ViCPS vaccines need to be explored.

Rational vaccine development can be informed by detailed knowledge of Ag targeting in immune individuals (22). Indeed, understanding of Ag targeting in human and murine Salmonella infection has recently improved, and subunit vaccine development for Salmonella has received greater attention (23–28). It has long been known that common bacterial products, like LPS and flagellin, are targeted by host Ab responses during Salmonella infection, making these serological responses incredibly useful for diagnostic purposes (29). Studies in a mouse model of Salmonella infection have confirmed that flagellin is a major target Ag of Ab and CD4 T cell responses and that flagellin can be modestly protective when used to immunize susceptible mice (30–32). Many outer membrane proteins of Salmonella have also been identified as targets of the adaptive immune response, and several of these proteins have similar modest protective efficacy to flagellin (33–35). Large proteomic studies have provided a more comprehensive understanding of Salmonella protein targeting by the adaptive immune system, and several Ag targets show limited protective efficacy when examined in mice (25, 26, 28). One surprise to emerge from these studies is that components of the Salmonella type III secretion system are common targets of the adaptive immune response and can also provide limited protective immunity (23, 25, 36–39). Thus, many experiments using individual Ags have shown modest protective efficacy as part of a subunit vaccine formulation; however, it is not clear whether a combination of multiple Salmonella Ags could match the much greater protective efficacy provided by an LVS Salmonella immunization.

In this article, we report that immunizing mice with two Salmonella proteins, SseB and flagellin, vastly improves the overall protection against bacterial challenge. This enhanced protection required an adjuvant effect of flagellin but did not require host TLR5 expression. The enhanced protection mediated by SseB/flagellin immunization also correlated with a modestly increased CD4 T cell response to SseB and was transferrable to naive mice via circulation. Finally, this protective response required host expression of IFN-γR but did not require inducible NO synthase (iNOS) expression.

Materials and Methods

Mouse strains

C57BL/6 mice were purchased from the National Cancer Institute or the Jackson Laboratory and were used at 8–16 wk of age. IFN-γR–deficient and iNOS-deficient mice were purchased from the Jackson Laboratory. TLR5-deficient mice were bred at the University of California Davis from a line developed in the Akira laboratory (40); these mice do not suffer from basal inflammatory or metabolic defects (41). All mice were cared for in accordance with University of California Davis Animal guidelines.

Bacterial strains and infection

Attenuated Salmonella typhimurium strain BRD509 (AroA−) was provided by Dr. D. Xu, (University of Glasgow, Glasgow, U.K.) (42). Flagellin-deficient S. typhimurium strain BC490 was a gift from Dr. B. Cookson (University of Washington, Seattle, WA) (31). LPS-deficient S. typhimurium χ4700 was a kind gift from Dr. R. Curtiss (Arizona State University, Tempe, AZ). Salmonella were cultured overnight in Luria–Bertani broth without shaking and diluted in PBS after an estimation of bacterial concentration using a spectrophotometer. For immunization experiments, 5 × 105 BRD509 were administered i.v. in the lateral tail vein. Immunized mice were challenged with 1000 wild-type (WT) S. typhimurium SL1344 i.v. or 1 × 106 SL1344 administered orally by gavage. The administered dose of bacteria was confirmed by plating serial dilutions onto MacConkey agar plates and counting colonies after overnight culture at 37°C. Infected mice were monitored daily, and mice were euthanized if they developed a moribund state (unresponsive to gentle prodding). To assess bacterial colonization, spleens and livers from infected mice were homogenized in PBS, and serial dilutions were plated onto MacConkey agar plates. After overnight incubation at 37°C, bacterial colonies were counted, and bacterial burdens were calculated for each individual organ.

Generation of recombinant and purified proteins

Salmonella Ag, SseB, was cloned from Salmonella genomic DNA, inserted into the His-tag pRSET vector, and overexpressed in Escherichia coli BL21 Star (DE3) cells (Thermo Fisher Scientific). To generate an SseB protein expressing the 2W1S epitope (EAWGALANWAVDSA; SseB-2W), the 2W1S sequence was cloned in-frame onto the C terminus of SseB and expressed in the same E. coli expression system. Additional Salmonella Ags, CirA, IroN, and SlyB, were similarly cloned and expressed in E. coli. Recombinant E. coli strains were cultured in Luria–Bertani broth and harvested after 14 h of 1 mM IPTG induction. The bacteria pellet was resuspended with BugBuster (EMD Millipore), and inclusion bodies were resuspended with 8 M urea buffer. Recombinant proteins were purified using ProBond (Thermo Fisher Scientific) or a Ni-NTA His∙Bind Resin (EMD Millipore), according to the manufacturer’s protocol, before dialysis against 1× PBS to remove traces of urea. Samples were subsequently concentrated using Amicon centrifugal instruments (EMD Millipore), and protein concentration were determined using the BCA method (Thermo Fisher Scientific). Because the methodology required the use of E. coli LPS as an adjuvant for immunization, residual LPS was not specifically removed from recombinant proteins prior to use.

Flagellin purification

LPS-deficient S. typhimurium χ4700 was used to purify flagellin with a modified acid-shock protocol (43, 44). Briefly, an overnight bacteria culture was spun down, washed, and resuspended in HCl/PBS (pH 2) for 30 min at room temperature. Supernatants were collected, and flagellin was harvested by ultracentrifugation and ammonium sulfate precipitation. Monomeric flagellin was prepared by depolymerizing samples at 70°C for 1 h, and LPS was removed using Detoxi-Gel columns.

Immunization

LPS was purchased from Axxora, MPLA was purchased from InvivoGen, Alum was purchased from Thermo Fisher Scientific, and IFA and CFA were purchased from Sigma-Aldrich. Mice were immunized via the lateral tail vein at 4-wk intervals with 100 μg recombinant protein (SseB or SseB-2W1S) and/or 100 μg purified flagellin mixed with 10 μg LPS or other adjuvants. In some experiments, mice were immunized subcutaneously with SseB, flagellin, IroN, CirA, or SlyB combined with CFA during the primary immunization and combined with IFA during boosting, 4 wk later.

Ab ELISA

IgG2c responses to SseB immunization were measured using an ELISA method. Briefly, 96-well microtiter plates were coated with 10 μg/ml recombinant SseB, and serum samples were added in serial dilution in 10% FBS/PBS. After incubation for 2 h at 37°C, plates were washed four times with 0.05% Tween 20/PBS before the addition of biotin-conjugated Ab specific for the anti-mouse IgG2c (BD Biosciences). After an additional incubation for 1 h at 37°C, plates were washed six times and incubated for 1 h at 37°C with HRP-conjugated streptavidin Ab (Extravidin; Sigma-Aldrich) diluted in 10% FBS/PBS. Plates were then washed eight times, and an HRP substrate (O-phenylenediamine dihydrochloride; Sigma-Aldrich) was used to develop the plates. After sufficient color change was observed, the reaction was stopped by adding 50 μl of 2 N H2SO4, and plates were analyzed using a spectrophotometer (SpectraMax M2; Molecular Devices).

Tetramer staining of 2W1S-specific CD4 T cells

PE-conjugated 2W1S::I-Ab tetramer was generated in-house from 2W1S::I-Ab monomers produced in insect cells, as previously described (45, 46). Spleen and livers were harvested and stained with PE-conjugated 2W1S::I-Ab tetramer in the presence of Fc block (culture supernatant from the 2.4G2 hybridoma, 2% mouse serum, 2% rat serum) at room temperature for 1 h. After washing with 2% FBS/PBS, cells were stained with fluorochrome-conjugated Abs specific for CD3, CD4, CD8, and CD44 (Affymetrix). Cells were then analyzed by flow cytometry using a BD LSR Fortessa (BD Biosciences). All data sets were analyzed using FlowJo software (TreeStar).

Parabiosis experiments

Mice were immunized and boosted with SseB and flagellin mixed with MPLA, and 60 d later, mice were surgically joined to generate a shared circulation. Parabionts were housed together for 28 d, allowing for the formation of an anastomosis and adequate transfer of vascularized lymphocytes between animals. Mice were then separated and allowed 2 wk to recover from separation surgery. Postrecovery, mice were challenged with 1000 virulent Salmonella (SL1344) i.v.; all groups of mice were euthanized 5 d later, and bacterial burdens were measured in tissues, as described above.

Statistical analyses

Statistical differences between groups of normally distributed data were examined using Prism (GraphPad). Experimental groups were compared using an unpaired t test and were considered significantly different with a p value < 0.05.

Results

Dual SseB and flagellin immunization enhances protection against Salmonella infection

IgG responses to 117 Salmonella target Ags were previously identified by probing a proteomic array with sera from Salmonella-infected mouse strains and humans with iNTS (25). However, when examined individually, many of these immune-dominant Ags provided little or no protection to susceptible C57BL/6 mice after Salmonella challenge (data not shown). This would suggest that immunogenicity is a poor predictor of any target Ag’s protective efficacy in Salmonella infection. Indeed, a recent study examining another large protein set for protective efficacy in BALB/c mice has reached similar conclusions (26). Indeed, it seems possible that Salmonella specifically direct host immune responses onto a set of largely irrelevant nonprotective Ags (47), further complicating subunit vaccine development.

Several studies report that immunization with flagellin or SseB provides susceptible mice with a modest degree of protection against Salmonella infection (23, 25, 31, 39). However, these two protein Ags have never been directly compared or combined together for protection studies. As a positive control, prior immunization with an LVS of Salmonella reduced bacterial burdens by four orders of magnitude and allowed the majority of mice to survive subsequent infection with virulent Salmonella (Fig. 1, LVS Salmonella). In contrast, C57BL/6 mice immunized and boosted with SseB and E. coli LPS as an adjuvant had significantly reduced bacterial burdens in the spleen and liver (Fig. 1A, 1B, SseB) but displayed only minimal prolonged survival. Similarly, mice immunized with flagellin plus LPS had lower bacterial burdens than unimmunized mice, although this was only statistically significant in the liver (Fig. 1A, 1B, flagellin). As found with SseB alone, flagellin immunization had only a minor effect on survival following Salmonella infection (Fig. 1E). Thus, immunization of susceptible C57BL/6 mice with individual Salmonella Ags provides very limited protective immunity that compares poorly with the robust protective efficacy observed with LVS immunization.

FIGURE 1.
FIGURE 1.

Dual SseB and flagellin immunization enhances protection against Salmonella infection. C57BL/6 mice were immunized i.v. twice at 4-wk intervals with 100 μg of SseB, flagellin, or a combination of SseB and flagellin (SseB/flagellin) mixed with 10 μg of LPS. As a positive control, groups of mice were vaccinated i.v. with 5 × 105 an LVS of S. typhimurium. Four weeks after the second immunization, all groups of mice were infected i.v. with 1000 virulent WT Salmonella SL1344 (A and B) or were orally infected with 5 × 107 SL1344 (C and D). Bacterial loads were determined in spleens (A and C) or livers (B and D) at 4 d postinfection. Horizontal lines show the mean bacterial burden per group with individual mice shown as scatter plots. The p values show the statistical significance between unimmunized (naive) mice and each immunized group. (E) Four weeks after boosting, mice were infected orally with 1 × 106 SL1344 and were monitored for the development of a moribund state. Data show the percentage of surviving mice in each group and are representative of 8–12 mice per group.

Given the modest individual efficacy of SseB or flagellin, we explored whether immunization with both Ags would enhance protective immunity. Surprisingly, dual immunization with SseB/flagellin was highly effective, especially in the liver, where bacterial burdens in SseB/flagellin-immunized mice approached levels observed in LVS-immunized mice (Fig. 1A, 1B, SseB/flagellin). Importantly, dual immunization with SseB/flagellin was also effective against mucosal challenge with Salmonella, reducing bacterial burdens to levels close to LVS-immunized mice (Fig. 1C, 1D). In agreement with the much larger reduction in bacterial counts observed with dual immunization, SseB/flagellin-immunized mice survived for an extended period following challenge with virulent Salmonella (Fig. 1E). Indeed, SseB/flagellin-immunized mice had similar survival rates to LVS-immunized mice for 1 mo after oral challenge (Fig. 1E). Interestingly, between 1 and 3 mo postinfection, many SseB/flagellin-immunized mice slowly succumbed to Salmonella infection (Fig. 1E). A recent report has identified several Salmonella Ags that were modestly protective in BALB/c mice after s.c. injection with CFA (26). We confirmed that some of these Ags were protective in C57BL/6 mice, but addition of these new Ags did not enhance the protective efficacy of SseB/flagellin after subcutaneous immunization (Supplemental Fig. 1). Taken together, these data demonstrate that dual immunization with SseB/flagellin strikingly elevates protection against systemic or mucosal infection with highly virulent Salmonella.

It seemed likely that the increased efficacy of dual SseB/flagellin immunization was due to the initiation of two adaptive immune responses against two target Ags. Indeed, we reported previously that flagellin and another type III secretion system protein, SseJ, induce anatomically and functionally distinct CD4 T cell responses (36) that could work cooperatively. The alternative explanation was that the intrinsic adjuvant properties of flagellin (48) might have simply enhanced the immune response to SseB. To discriminate between these two possibilities, we examined whether dual SseB/flagellin immunization could protect against flagellin-deficient Salmonella (BC490). Unimmunized C57BL/6 mice displayed a significantly higher bacterial burden after challenge with flagellin-deficient Salmonella (Fig. 2A, 2B), but there was no difference in survival time after oral challenge with WT or flagellin-deficient bacteria (Fig. 2C). Thus, the absence of flagellin modestly enhanced bacterial virulence, agreeing with previous data that forced expression of flagellin hindered bacterial replication in C57BL/6 mice (44). Interestingly, C57BL/6 mice vaccinated with LVS Salmonella or SseB/flagellin displayed robust protective immunity against challenge with WT and flagellin-deficient bacteria (Fig. 2). Thus, the protective immunity mediated by SseB/flagellin immunization did not require a flagellin-specific adaptive immune response for effective Salmonella clearance and suggested, instead, that flagellin was functioning as an adjuvant.

FIGURE 2.
FIGURE 2.

Protection by dual SseB/flagellin immunization does not require a flagellin-specific adaptive immune response. Groups of C57BL/6 mice were immunized i.v. twice with a combination of SseB and flagellin and LPS at 4-wk intervals. As a positive control, mice were vaccinated i.v. with 5 × 105 LVS Salmonella. Four weeks after boosting, groups of mice were infected i.v. with 1000 WT Salmonella (SL1344) or flagellin-deficient Salmonella (BC490), and bacterial burdens were determined in spleens (A) and livers (B) 4 d later. Horizontal lines show the mean bacterial burden per group with individual mice shown as scatter plots. The p values indicate statistical significance between unimmunized (naive) mice and each immunized group. (C) Four weeks after the second immunization, groups of mice were infected orally with 1 × 106 WT or flagellin-deficient bacteria and were monitored for the development of a moribund state. Data show the percentage of surviving mice in each group and are representative of three to five mice per group.

Bacterial flagellins are recognized by the innate immune system via the surface receptor TLR5, cytosolic sensors NAIP5/NLRC4, and a poorly defined mechanism distinct from both known pathways (49–51). We next examined whether the efficacy of dual SseB/flagellin immunization required host expression of TLR5. Immunization with LVS Salmonella was effective at lowering bacterial burdens in the spleen and liver of WT and TLR5-deficient mice (Fig. 3A, 3B, LVS Salmonella). Furthermore, almost all WT and TLR5-deficient mice immunized with LVS Salmonella survived challenge infection with virulent bacteria (Fig. 3C). Similarly, dual SseB/flagellin immunization lowered bacterial burdens in the spleen and liver of WT and TLR5-deficient mice (Fig. 3A, 3B). As noted above, SseB/flagellin immunization allowed C57BL/6 mice to survive for an extended period after challenge infection, and this long-term survival was comparable in immunized TLR5-deficient mice (Fig. 3C). Thus, although flagellin appears to function as an effective adjuvant for SseB, this protective effect does not require host expression of TLR5.

FIGURE 3.
FIGURE 3.

Protection by SseB/flagellin immunization does not require host expression of TLR5. Groups of C57BL/6 (WT) and TLR5-deficient (TLR5) mice were immunized i.v. twice at 4-wk intervals with SseB and flagellin mixed with additional LPS. As a positive control, WT and TLR5-deficient mice were vaccinated i.v. with 5 × 105 LVS Salmonella. Four weeks after boosting, groups of mice were infected i.v. with 1000 virulent Salmonella, and bacterial burdens were determined in the spleen (A) and liver (B) 4 d later. Horizontal lines represent the mean bacterial burden per group with individual mice shown as scatter plots. The p values indicate statistical significance between unimmunized (naive) mice and each immunized group. (C) Immunized mice were infected orally with 1 × 106 SL1344 and examined for the development of a moribund state. Data show the percentage of surviving mice in each group and are representative of two to seven mice per group.

Many studies have shown that protective immunity against Salmonella infection is mediated by the contribution of Salmonella-specific CD4 T cells and B cells (52). Thus, we examined whether coadministration of flagellin altered T or B cell responses to SseB. In C57BL/6 mice, the Salmonella-specific Ab response is dominated by the IgG2c isotype (53); thus, we focused attention on SseB-specific IgG2c responses. After immunization and boosting, serum SseB-specific IgG2c responses were similar in mice immunized with SseB or SseB/flagellin (Fig. 4A). Thus, modification of specific IgG2c did not correlate with the observed enhanced protection. Although epitope mapping has identified an immunodominant epitope of SseB in humans (37), peptide library screening of SseB-specific T cells in our laboratory has so far failed to uncover the immunodominant epitope in mice (S.J. Lee, unpublished observations). Therefore, to examine the effect of flagellin on CD4 T cell responses to SseB, we fused the 2W1S epitope to the C terminus of SseB and used 2W1S MHC class II tetramers to examine SseB-2W–specific CD4 T cells. A population of tetramer-specific CD4 T cells could be detected in the spleen and liver of mice immunized with SseB-2W, with and without flagellin coadministration (Fig. 4B). The addition of flagellin significantly increased expansion of 2W1S-specific CD4 T cells in Sse-B2W–immunized mice (Fig. 4C), indicating that flagellin has a modest enhancing effect on CD4 T cell expansion in response to SseB immunization.

FIGURE 4.
FIGURE 4.

Dual SseB/flagellin immunization modestly enhances Salmonella-specific CD4 T expansion. C57BL/6 mice were immunized twice i.v. with 100 μg of SseB-2W1S (SseB) or a combination of SseB-2W1S and flagellin (SseB/flagellin) mixed with 10 μg of LPS. (A) Four weeks after the second immunization, sera were collected and examined for the presence of SseB-specific IgG2c by ELISA. Data show OD405 of SseB-specific IgG2c in SseB- or SseB/flagellin-immunized mice. Data represent the mean ± SEM of three to eight mice per group. (B) Four weeks after boosting, mice were reimmunized and 7 d later, spleens and livers were harvested and stained with 2W1S::I-Ab tetramer, CD4, and CD44 to detect 2W1S-specific CD4 T cells. Data are representative FACS plots showing tetramer-positive CD4 T cells. (C) Mice were immunized with LVS Salmonella-2W, 100 μg of SseB-2W1S (SseB), or 100 μg of SseB-2W1S and 10 μg flagellin (SseB/flagellin) and 10 μg of LPS. Seven days later, spleens and livers were harvested and stained with 2W1S::I-Ab tetramer, CD4, and CD44 to detect 2W1S-specific CD4 T cells. Bar graph shows absolute number of 2W1S-specific CD4 T cells in the spleen of immunized mice. These data combined two independent experiments each with two or three mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

To gather more information on the protective immunity mediated by dual SseB/flagellin immunization, we next examined whether the protection was mediated by resident or circulating memory responses. T central memory cells and T effector memory cells circulate in peripheral blood, whereas tissue-resident memory (TRM) cells remain localized to tissues (54–56). The role of circulating versus resident T memory cells has been examined in many infectious diseases but has not yet been examined in Salmonella infection. We used parabiosis surgery of immunized congenic mice to examine whether the protective effect of SseB/flagellin immunization could be conferred to naive mice via the circulation. Unpaired C57BL/6 mice and C57BL/6 mice that had only been surgically paired had equivalent bacterial burdens in the spleen and liver after Salmonella infection (Fig. 5, naive and surgery control). Thus, parabiosis surgery itself does affect Salmonella-specific immunity. As expected, unpaired and paired mice that had been directly immunized with SseB/flagellin had significantly lower bacterial counts in the spleen and liver compared with naive controls (Fig. 5, SseB/flagellin). Importantly, unimmunized mice that shared a circulation with immunized mice also had significantly lower bacterial burdens in the spleen and liver (Fig. 5). Thus, circulating memory mediates protective immunity conferred by SseB/flagellin immunization.

FIGURE 5.
FIGURE 5.

Protective immunity mediated by SseB/flagellin immunization is transferred in the circulation. C57BL/6 mice were immunized twice i.v. with 100 μg of SseB-2W1S (SseB) or a combination of SseB-2W1S and flagellin (SseB/flagellin) mixed with 10 μg of MPLA. Four weeks after the second immunization, mice were surgically joined for 28 d. Postseparation surgery, mice were rested for 2 wk before all groups were infected i.v. with 1000 virulent WT Salmonella SL1344. Bacterial loads were determined in spleens or livers at 5 d postinfection. Bars show the mean bacterial burden per group with individual mice shown as scatter plots. ***p < 0.001, ****p < 0.0001.

Th1 cell production of IFN-γ causes macrophage production of NO and eventual resolution of Salmonella infection (1). Thus, mice that are genetically deficient in T-bet or IFN-γ display increased susceptibility to Salmonella infection (57, 58). To examine the mechanism of protection after SseB/flagellin immunization, we immunized and boosted WT, IFN-γR–, and iNOS-deficient mice before challenge with virulent Salmonella. As expected, unimmunized IFN-γR–deficient mice had higher bacterial burdens than unimmunized WT mice (Fig. 6A, 6B, naive), demonstrating the critical role for IFN-γ in primary resolution of infection. Notably, in this set of experiments, protection mediated by SseB/flagellin immunization was lower than previously observed but was still statistically significant (Fig. 6A, 6B, SseB/flagellin). However, bacterial burdens in the spleen and liver of SseB/flagellin-immunized IFN-γR–deficient mice were still higher than in WT mice (Fig. 6A, 6B), indicating that IFN-γR is essential for protective immunity. In contrast, SseB/flagellin-immunized iNOS-deficient mice had bacterial loads that were similar to SseB/flagellin-immunized WT mice, suggesting that iNOS is dispensable for the protective immunity induced by SseB/flagellin immunization.

FIGURE 6.
FIGURE 6.

IFN-γ signaling is essential for protective immunity by SseB/flagellin immunization. Groups of C57BL/6 (WT) and IFN-γR–deficient mice (IFN-γR-KO) (A and B) or WT and iNOS-deficient mice (iNOS-KO) (C and D) were immunized i.v. twice with a combination of SseB and flagellin mixed with LPS. Four weeks after the second immunization, mice were infected i.v. with 1000 virulent Salmonella. Bacterial burdens were determined in spleens (A and C) and livers (B and D) 4 d later. Horizontal lines show the mean bacterial burden per group with individual mice shown as scatter plots. Numbers represent the p values and indicate statistical significance between groups of mice.

Discussion

A large genome-wide association study of enteric fever in Asia identified an MHC class II association with resistance to human typhoid (59), confirming prior data in a mouse model of typhoid (60). In addition, humans and mice with natural or acquired deficiencies in CD4 T cells display increased sensitivity to invasive Salmonella infections (61–64). Taken together, these data point to the importance of CD4 T cells in protective immunity to Salmonella infection, as expected for an intramacrophage pathogen (11). However, it should also be noted that many experiments also support a protective role for Salmonella-specific B cells, via Ab-dependent and Ab-independent mechanisms (53, 65–69).

Given the requirement for cellular immunity in protection against Salmonella infection, it is understandable that vaccine development has focused on LVSs that induce strong mucosal T cell responses (13). Typically, studies that have explored a subunit vaccine approach using purified or recombinant bacterial Ags have detected protection that is several orders of magnitude lower than LVS Salmonella (23, 25, 26, 28, 31–34), especially when using highly susceptible mouse strains. Our data with dual SseB/flagellin immunization demonstrate that unexpected gains in protective immunity can be achieved by simply combining two well-documented Salmonella Ags. Indeed, highly susceptible C57BL/6 mice immunized with SseB/flagellin survived for more than a month following challenge with bacteria that normally cause death within a week to 10 d. This finding provides some confidence that further modulation of subunit vaccine formulation or delivery strategy could result in a subunit Salmonella vaccine with efficacy approaching LVS Salmonella. Unfortunately, the addition of IroN, CirA, or SlyB (26) did not increase protection, but it seems likely that there are protective Ags that could improve the efficacy of SseB/flagellin immunization. Indeed, several new protective Ags were identified by Ferreira et al. (28) that could be explored in this regard. Although the i.v. immunization approach used in our study is impractical for human vaccine delivery, the purpose was to maximally stimulate systemic immunity to determine the limits of protection without using a live vaccine. Furthermore, it is interesting that i.v. priming with SseB/flagellin was effective against systemic and mucosal challenge with Salmonella. This fits well with previous data demonstrating that IgA or pIgR is not required for immunity against Salmonella (25) and that a focus on initiating robust systemic immunity, rather than mucosal protection, is the most effective approach to limiting Salmonella replication.

Although dual immunization with SseB and flagellin markedly improved protective immunity compared with either Ag alone, all immunized mice eventually died. Therefore, although it is encouraging that there was a strong protective effect with this subunit approach, it is clearly less effective than LVS Salmonella. However, because there are many populations that cannot be administered a live attenuated vaccine strain, it would still be of interest to develop a safe subunit vaccine that could provide partial protection. In addition, it might be possible to enhance the moderate efficacy of LVS-Salmonella or Vi-CPS vaccines by complementation with a partially protective subunit vaccine. It is also interesting that SseB/flagellin-immunized mice appear healthy for several weeks and only succumb at late time points, with some mice becoming moribund around 90 d after challenge. High bacterial CFU were cultured from the spleen at these late time points, confirming that death is due to relapsing Salmonella infection. It is perplexing that apparently healthy mice can maintain bacteria for several months without developing effective immunity. This pattern of delayed bacterial outgrowth is reminiscent of an enrofloxacin treatment model in which primary infection can be temporarily curtailed but slowly relapses (70). In this relapse model, this correlates with incomplete Th1 immunity due to inappropriate T cell priming (70, 71). Both models suggest that if small numbers of Salmonella can evade CD4 T cell killing for a prolonged period, the adaptive immune response is eventually exhausted and overcome. Future studies will be needed to examine why the immune response fails at late time points in the SseB/flagellin-immunization model.

A surprising feature of our data is the efficacy of flagellin as an adjuvant, in combination with SseB. It has been known for several years that flagellin increases dendritic cell expression of CD80/86, induces inflammatory cytokines, and, thus, enhances B cell and T cell responses to coadministered Ags (49, 72). These inflammatory processes can be initiated when flagellin is detected at the cell surface or in the cytosol by membrane and cytosol sensors, TLR5 and NAIP5/NLRC4. Interestingly, there is also a third mechanism for flagellin recognition based on the activity of flagellin in TLR5- and inflammasome-deficient mice (51). It has been noted that flagellin functions as a relatively weak adjuvant compared with LPS or other TLR ligands (44, 49, 73, 74). However, our experiments demonstrate that flagellin has adjuvant capabilities distinct from LPS or other adjuvants. Single immunization with SseB is markedly less effective than SseB/flagellin, despite the fact that LPS is added to both formulations. Therefore, greater understanding of the role of flagellin as a vaccine adjuvant is required, particularly with respect to TLR5- and inflammasome-independent activity in vivo.

It is broadly accepted that Salmonella-specific CD4 T cells are required for protective immunity (64, 75, 76), but the relative role of circulating T central memory cells/T effector memory cells and TRM cells has not been examined. Our data show that the protection mediated by SseB/flagellin immunization can be transferred effectively in the circulation, suggesting that circulating memory populations are likely critical for Salmonella immunity. Given the large bacterial burden in highly vascularized tissues, this may not be entirely unexpected; however, it is possible that the superior protective immunity elicited by LVS Salmonella is due to the capacity of a live vaccine to elicit TRM CD4 T cells. Parabiosis experiments to examine this possibility are underway in our laboratory. Although flagellin modestly enhanced the CD4 T cell response to SseB-2W1S, this enhancement was less striking than the combined effect on protective immunity. Therefore, it remains possible that other non-CD4 cells contribute to the heightened protection offered by dual SseB/flagellin immunization.

Overall, our data show that combining two previously known antigenic targets of Salmonella-specific immunity markedly improves protective efficacy against challenge infection. Surprisingly, this enhancement is largely due to an adjuvant effect of flagellin and correlates with a modestly increased frequency of vaccine-specific CD4 T cells. The protection mediated by SseB/flagellin immunization was transferrable in the circulation and required expression of IFN-γR, suggesting that circulating effector Th1 cells are responsible for enhanced protection. Together, these data increase confidence in the possibility of developing a simple subunit vaccine to protect against systemic salmonellosis and support continued investigation to explore alternatives to live attenuated Salmonella vaccines.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by grants from the National Institutes of Health (NIH) to S.J.M. (AI055743, AI076278, and AI056172). Z.F. was supported by an NIH T32 training grant (AI60555). O.P. was supported by a Vietnam Education Foundation fellowship.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    CPS
    capsule polysaccharide
    iNOS
    inducible NO synthase
    iNTS
    invasive nontyphoidal salmonellosis
    LVS
    live vaccine strain
    TRM
    tissue-resident memory
    WT
    wild-type.

  • Received August 4, 2016.
  • Accepted June 19, 2017.

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