ABSTRACT
Nontyphoidal Salmonella enterica serotypes (NTS) are the leading cause of hospitalization and death due to foodborne illnesses. NTS are the costliest of the foodborne pathogens and cause ∼$4 billion annually in health care costs. In Africa, new invasive NTS are the leading cause of bacteremia, especially in HIV-positive children and adults. Current vaccines against S. enterica are not broadly protective and most are directed at the typhoid-causing serotypes, not the NTS. All S. enterica strains require two type III secretion systems (T3SS) for virulence. The T3SS needle tip protein and the first translocator are localized to the T3SS needle tip and are required for pathogenesis of S. enterica. Collectively they are 95 to 98% conserved at the amino acid sequence level among all S. enterica strains. The Salmonella pathogenicity island 1 or 2 tip and first translocator proteins were genetically fused to produce the S1 and S2 fusion proteins, respectively, as potential vaccine candidates. S1 and S2 were then characterized using spectroscopic techniques to understand their structural and biophysical properties. Formulated at the proper pH, S1, S2, or S1 plus S2 (S1S2), admixed with adjuvant, was used to immunize mice followed by a lethal challenge with S. enterica serotype Typhimurium or S. enterica serotype Enteritidis. The S1S2 formulation provided the highest protective efficacy, thus demonstrating that an S1S2 subunit vaccine can provide broad, serotype-independent protection, possibly against all S. enterica serotypes. Such a finding would be transformative in improving human health.
INTRODUCTION
Salmonella enterica is comprised of over 2,500 serotypes that infect a wide variety of hosts and causes a broad range of diseases from enteric fever to gastroenteritis. While S. enterica serotype Typhi and S. enterica serotype Paratyphi are host restricted to humans and cause typhoid fever, other S. enterica serotypes, the nontyphoidal Salmonella enterica serotypes (NTS), cause gastroenteritis in humans and are the leading cause of hospitalization and death due to bacterial foodborne illness in the United States (1). Of the 1.2 million cases of NTS, 33% are due to contaminated beef, pork, and poultry products (2). In the developing world, children, especially those infected with HIV, are disproportionally affected by, and often die from, NTS-induced bacteremia (3). Prevention in industrial countries has been achieved with antibiotics, but emerging resistance has made their routine use in livestock unsustainable. According to recent reports, about 30% of the cattle processed harbor NTS isolates that are resistant to at least one antimicrobial agent (4). The best prevention against infectious diseases is vaccination. While licensed vaccines against typhoid fever are available, there are no vaccines that prevent infection by a broad spectrum of S. enterica serotypes.
Regardless of serotype, S. enterica uses two type III secretion systems (T3SS) to interact with host cells (5). The first TT3S is encoded on Salmonella pathogenicity island 1 (SPI-1) and is used to elicit uptake by host cells. The second is encoded on Salmonella pathogenicity island 2 (SPI-2) and is used to maintain the Salmonella-containing vacuole (6). Each T3SS uses an energized conduit, the T3S apparatus (T3SA), linking the bacterial cytoplasm to the host cell to facilitate the delivery of effector proteins that modulate host cell activities for the benefit of the bacterium (5). Each T3SA resembles a molecular syringe and needle, with a needle tip protein and the first translocator localizing to the distal end of the each T3SA needle (5). The tip and translocator proteins are required for pathogenesis of S. enterica and are 95 to 98% conserved among all S. enterica serotypes (7–9). For a potential broadly protective protein subunit vaccine, we genetically fused the SPI-1-encoded tip and first translocator proteins, SipD and SipB (7, 8), to give S1, while the SPI-2-encoded SseB (tip) and SseC (first translocator) (9) were genetically fused to give S2. In this study, the S1 and S2 proteins were recombinantly expressed and purified, and their biophysical characteristics were examined. After vaccination with S1, S2, and S1 plus S2 (S1S2), the immune response and resulting protective efficacy against challenge with S. enterica serotype Typhimurium and S. enterica serotype Enteritidis, two of the more important serotypes implicated in human and poultry disease, were assessed in mice.
RESULTS
EPDs show pH-dependent thermal stability and define an overlapping region of stability for S1 and S2.Since protein instability may lead to the failure of a subunit vaccine, we used various spectroscopic methods to examine the effects of accelerated thermal stress on S1 and S2 in buffers at pH 3 to 8. Three methods were used: far-UV circular dichroism (CD) spectroscopy (see Fig. S1 in the supplemental material) for monitoring changes in protein secondary structure, intrinsic tryptophan fluorescence peak position (PP) (see Fig. S2 in the supplemental material) for tertiary structure, and static light scattering (SLS) (Fig. S2) for quaternary structure and aggregation. A large data set was collected from the three spectroscopic methods under the combination of five pH values (pH 3 to 8) and increasing temperature of 10 to 90°C in 2.5°C increments. The data set was first normalized and then visualized as a single empirical phase diagram (EPD) (10). Briefly, normalized data from zero to one for each spectroscopic method were referred to as a structural index because they represent the relative amount of structural change measured under given conditions. Red-green-blue (RGB) colors were chosen to visualize changes in structural indices in two-dimensional spaces defined by pH and temperature variables. Red, green, and blue were assigned to secondary, tertiary, and quaternary structural indices, respectively (Fig. 1, right side of the EPD). For the CD index, the red faded to black, as the molar ellipticity at 222 nm decreased as the temperature increased. A decrease in molar ellipticity is indicative of a loss of secondary structure within the protein. Similarly, as the PP shifted to a higher wavelength due to increased temperature, the green fades to black. As the temperature increases, there is a reduction in the tertiary structure of the protein leading to a more hydrophilic environment surrounding the buried tryptophans, which increases the peak position wavelength. In contrast, black fades to blue as the SLS intensity increases concomitant with an increase in temperature. As the temperature increases, proteins begin to unfold, allowing hydrophobic regions to be exposed and promoting interaction with other regions within the protein or within other proteins. This leads to aggregation and increased light scattering. The three indices were combined to produce the three-index EPD with computer-generated clusters. In general, EPD colors close to yellow indicate a native state where both secondary and tertiary structure are intact and no aggregation is seen. Colors close to black indicate an unfolded state where there is minimal secondary and tertiary structure and no aggregation. Colors close to blue indicate minimal secondary and tertiary structure and maximal aggregation. Lastly, the colors close to brown indicate an intermediate state where there is some loss of secondary and tertiary structure.
S1 and S2 show pH-dependent thermal stability. Empirical phase diagrams for S1 (A) and S2 (B) are shown. The three-index empirical phase diagrams illustrate the thermal stability for each fusion protein as a function of pH 3 to 8. The red, green, and blue panels define individual component indices for secondary structure (CD), tertiary structure (fluorescence peak position), and aggregation behavior (SLS), respectively.
The three-index EPD for S1 revealed that the most thermally stable protein state occurs at pH 7 to 8 below a maximal temperature of 47.5°C, defined as region I (Fig. 1). Although region I extends through pH 3 to 6, SLS increased here, indicating some protein aggregation in these other sections of region I. Region II occurs at lower pH values and lower temperatures where there is an increase in SLS and a decrease in the secondary or tertiary structure of S1 based on CD or PP. The cluster designated region III includes the most unstable S1 conditions, as indicated by colors turning shades of blue at higher temperatures between pH 4 and 7. Similarly, at pH 3 and 8, the EPD again illustrates the instability of S1 with colors that are a darker mixture of blue, green, and oranges, indicating a loss of a mixture, to various degrees, of secondary, tertiary, and quaternary structures.
A similar thermal stability profile for S2 was visualized by an EPD which clustered in a unique manner. Region I extended over the entire range of pH 3 to 8, where pH 7 and 8 were less stable than pH 5 and 6, probably due to the aggregation of protein as indicated by SLS at pH 7 and 8. Region II is defined by a section ranging from pH 3 to 6 where there is a distinct boundary at ∼50°C at pH 3 and 30°C at pH 4. At pH 5 and 6, the region II cluster boundary marks a section where there is a more gradual transition between regions I and III. The colors of region III are darker or blue, indicating, as in the S1 EPD, a loss of a mixture, to various degrees, of secondary, tertiary, and quaternary structures.
Taken together, the three-index EPDs for S2 and S1 indicate that each protein structure is sensitive to changes in temperature and pH conditions. The shared region showing the greatest stability for both fusion proteins is defined by pH 7 to 8 and temperatures below about 45°C. Alternatively, for future excipient screening, pH 6 at 45°C could be used because it is at a shared boundary where stability studies can be carried out at an accelerated rate. In two clusters, the most dramatic change in structure is located at pH 6 and 50°C. For the results presented here, we chose pH 7.0 with an ionic strength of 0.15 for subsequent vaccination experiments. Because S1 and S2 were to be adsorbed onto Alhydrogel (AH), no phosphate buffers could be used. Previously, we used a histidine buffer, which we again used here.
Intramuscular administration of S1, S2, or S1S2 elicits high serum IgG responses but no fecal IgA.S1 and S2 were adsorbed onto Alhydrogel. Monophosphoryl lipid A (MPL) liposomes were added to produce S1, S2, or S1S2 with combination adjuvants. Mice (n = 15) were immunized intramuscularly (i.m.) on days 0, 14, and 28 with adjuvanted S1, S2, or S1S2. Two groups were vaccinated orally on day 28, with one group receiving S. Typhimurium ΔSPI-1/2 and one group receiving S. Enteritidis ΔSPI-1/2. These two S. enterica strains, used as live-attenuated vaccine strains, are null for inv and ssaV, whose gene products are required for functional SPI-1 and SPI-2 T3SS, respectively. Blood and fecal samples were collected from all immunized mice on days −1, 13, 27, 42, and 55.
Serum samples were evaluated for anti-SipB, -SipD, -SseB, and -SseC IgG titers (Fig. 2). As expected, mice vaccinated with S1 elicited strong anti-SipB and -SipD IgG responses, while mice vaccinated with S2 generated strong anti-SseB and -SseC IgG titers. Mice immunized with S1S2 elicited high serum IgG titers against all four individual protein antigens. No serum IgG titers to any of the four proteins were detected in the groups vaccinated with phosphate-buffered saline (PBS), S. Typhimurium ΔSPI-1/2, or S. Enteritidis ΔSPI-1/2 (results for S. Typhimurium ΔSPI-1/2 samples are shown). Fecal samples were tested for protein-specific IgA titers, but no IgA was detected in any of the groups (data not shown).
Immunization elicits an antigen-specific serum IgG response. Mice (n = 15) were immunized intramuscularly (i.m.) on days 0, 14, and 28 with adjuvanted S1, S2, or S1S2. Two groups were vaccinated orally on day 28, with one group receiving S. Typhimurium ΔSPI-1/2 and one group receiving S. Enteritidis ΔSPI-1/2. Blood and fecal samples were collected at days −1, 13, 27, 41, and 55. Serum IgG antibodies specific for SipD (A), SipB (B), SseB (C), and SseC (D) were analyzed by enzyme-linked immunosorbent assay (ELISA). The individual titers for serum IgG are represented as endotoxin units (EU) ml−1. Titers under 100 EU ml−1 were adjusted to baseline titer. Data are represented as mean ± SEM for 10 mice per group. The serum IgG response was tested for levels of significance using an unpaired t test (**, P < 0.001 [S1 versus S1S2], #, P < 0.05 [S2 versus S1S2] using a two-way ANOVA). No antigen-specific IgA was detected in any of the fecal samples.
S1, S2, or S1S2 immunization generates IgG-secreting cells in spleens and bone marrow.The frequency of antibody-secreting cells (ASCs) in cell suspensions from spleens and bone marrow of mice at day 56 were quantified by enzyme-linked immunosorbent spot (ELISpot) assay (Fig. 3). The frequencies of IgG ASCs within the spleen or bone marrow against SipB and SipD were comparable regardless of whether the vaccine was S1 or S1S2. Similarly, the frequencies of IgG ASCs were comparable in spleen and bone marrow against SseC for mice vaccinated with S2 or S1S2. In contrast, there was a significant increase in frequency of IgG ASCs against SseB from the spleen and bone marrow for the S1S2 group compared to the S2 group. The importance of the higher frequency of anti-SseB IgG ASCs remains to be determined, since the IgG titers were higher in the S2 group. No antigen-specific IgG ASCs were observed in spleen or bone marrow cells from mice in the groups vaccinated with PBS, S. Typhimurium ΔSPI-1/2, or S. Enteritidis ΔSPI-1/2 (results for S. Typhimurium ΔSPI-1/2 samples are shown). Similarly, there were no antigen-specific IgA ASCs detected in spleen or bone marrow in any groups, consistent with the lack of detectable fecal IgA.
Immunization triggers production of antibody-secreting cells. Antibody-secreting cells (ASCs) were quantified in bone marrow (A) and spleen (B). Mice (n = 5 per group concomitant with mice for Fig. 2) were vaccinated on days 0, 14, and 28 and euthanized on day 55 to collect spleen and bone marrow for assessment of antibody-secreting cells. Single-cell suspensions obtained from spleen and bone marrow were incubated in SipD-, SipB-, SseB-, or SseC-precoated plates. IgG-secreting cells were detected by ELISpot assay, counted, and plotted as specific ASCs/106 cells ± standard deviation (SD). *, P < 0.05 (t test). No IgA-secreting cells were detected.
The S1S2 fusion protein confers protection from S. enterica lethal challenge.The protective efficacy of each vaccine formulation was assessed by orogastric challenge for each group of mice with 2 × 108 CFU of the homologous wild-type S. Typhimurium SL1344 (Fig. 4A) or 5 × 107 CFU of the heterologous wild-type S. Enteritidis 125109 (Fig. 4B). All mice vaccinated with PBS died after challenge. Similarly, the mice vaccinated with S1 or S2 demonstrated poor to no survival. Rapid decreases in overall health and weight were also observed in these mice (data not shown). In contrast, immunization with S1S2 elicited an immune response that resulted in 60% survival regardless of the challenge serotype. This survival was comparable (P < 0.05) to the one provided by vaccination with attenuated S. Typhimurium ΔSPI-1/2 and S. Enteritidis ΔSPI-1/2 serotypes, which exhibited protective efficacies of 80% and 70%, respectively.
Protective efficacy of the vaccine formulations. Mice (n = 10 per group used for Fig. 2) were vaccinated on days 0, 14, and 28. On day 56, 2 × 108 CFU of wild-type S. Typhimurium SL1344 (A) and 5 × 107 CFU of wild-type S. Enteritidis 125109 (B) were administered by gavage. Survival was monitored for 20 days postchallenge. *, P < 0.05 compared to survival of mice vaccinated with PBS using the log rank test; n.s., not significant when survival of the S1S2-immunized group was compared with those of the S. Typhimurium ΔSPI-1/2- and S. Enteritidis ΔSPI-1/2-vaccinated groups (P > 0.05 using log rank test).
The immune response elicited by immunization with S1S2 reduces cecal inflammation.Streptomycin treatment of mice induced a colitis marked by cecal inflammation, which was scored by levels of submucosal edema, polymorphonuclear leukocyte (PMN) infiltration, a loss of goblet cells, and a loss of epithelial integrity. Thus, mice vaccinated at day 0, 14, or 28 with PBS, S1, S2, S1S2, or S. Typhimurium ΔSPI-1/2 were treated orally with 20 mg streptomycin by gavage 24 h prior to challenge to eliminate the gut microbiota and allow colonization of the gut (11). Mice were then challenged orally with 200 CFU in 100 μl of the wild-type S. Typhimurium SL1344. At 4 days postchallenge, the mice were euthanized and the cecum extracted to assess cecal inflammation by histochemical staining (Fig. 5). As expected, the mice vaccinated with PBS and infected with wild-type S. Typhimurium SL1344 exhibited extensive submucosal edema, PMN infiltration, a loss of goblet cells, and a loss of epithelial integrity (Fig. 5E). S1-vaccinated mice displayed pathology similar to that of the PBS-vaccinated mice (Fig. 5A). S2-vaccinated mice, however, developed pathology scores significantly lower than that of S1- or PBS-vaccinated mice (Fig. 5B). Consistent with the challenge data in Fig. 4, the pathology of the samples from the S1S2-vaccinated mice showed a substantial decrease in overall cecal inflammation as indicated by the suppressed submucosal edema and a reduction in the number of PMNs in the lamina propria region (Fig. 5C). These observations are comparable to the pathology of the mice immunized with S. Typhimurium ΔSPI-1/2. Tissue sections from S1S2-immunized mice illustrated minimal loss of the integrity of the mucosal epithelial cells as well as a minimal loss of goblet cells. Collectively, these features contributed to lowering the overall pathoscores for cecal tissues from the S1S2-immunized mice relative to the other protein-immunized groups (Fig. 5F). Pathoscores for the S1-, S2-, and PBS-immunized mouse groups were significantly higher (P < 0.05) than those of the combination S1S2-immunized group and the mice immunized with S. Typhimurium ΔSPI-1/2.
Cecal inflammation during lethal challenges with S. Typhimurium. Immunized mice (n = 5; as described in Materials and Methods) were treated on day 55 with streptomycin to clear gut flora. On day 56, all mice were challenged with 200 CFU of wild-type S. Typhimurium SL1344. The cecum of each mouse was collected at day 4 postchallenge (day 60) and fixed in OCT. Thin cryosections (4 μm) of cecum were stained with hematoxylin and eosin. Sections were visualized and photographed at a magnification of ×10 (A to E), whereas the pathoscore was evaluated at a magnification of ×400 (F). The stained sections were evaluated independently on the basis of pathological changes that included submucosal edema (score, 0 to 3), PMN infiltration (score, 0 to 4), loss of goblet cells (score, 0 to 3), and epithelial integrity (score, 0 to 3), with a final pathoscore of 0 to 12, reflecting the overall degree of inflammation. The pathoscores were determined by averaging the scores. The combined scores ranged from 0 to 12 arbitrary units covering the inflammation levels as follows: intact intestine (pathoscore, 0); minimal inflammation (pathoscore, 1 or 2), which is commonly found in the ceca of mice; slight inflammation (pathoscore, 3 or 4); moderate inflammation (pathoscore, 5 to 8); and significant inflammation (pathoscore, 9 to 12) (11). Pathoscores are represented graphically as mean ± SEM. All the sections were imaged at a magnification of ×10 with a Nikon Eclipse 80i microscope, and the best representative image from each group is presented. Representative sections are presented to show differences in the cecal inflammation with respect to the individual vaccine formulation. SE, submucosal edema; Lu, cecal lumen; LP, lamina propria. Scale bar, 100 μm. *, P < 0.05 using ANOVA; **, P < 0.01; n.s. not significant.
DISCUSSION
NTS usually cause self-limiting gastroenteritis but can cause fatal systemic disease, particularly in young children or those who are immunocompromised (3). It is becoming increasingly difficult to treat NTS with antibiotics due to mounting resistance to fluoroquinolones and cephalosporins, and new antimicrobials are not economically feasible for low-income countries (12). Therefore, there is an urgent medical need for vaccines with broadly protective, serotype-independent coverage against all S. enterica strains. Subunit vaccines offer excellent safety profiles, but the key challenge in developing such vaccines is identification of suitable antigens that can be properly formulated into a protective human vaccine. We have previously reported that the T3SA tip (IpaD) and first translocator (IpaB) proteins of Shigella flexneri can be used as an efficient subunit vaccine (13). Furthermore, DBF, the fusion of IpaD and IpaB, provided better immunogenic potential and conferred excellent protection against homologous and heterologous Shigella sp. infections (14). Thus, the tip and first translocator proteins from each of the two Salmonella pathogenicity islands were fused to assess their protection of mice from challenges with S. Typhimurium and S. Enteritidis.
S1 and S2 were subjected to biophysical characterization to assess their basic stabilities since they were to be used together in a vaccine formulation. A unique structural feature was exhibited by S1 and to a certain extent by S2. As illustrated by the thermal melting curves obtained by CD at pH 7 and 8, S1 did not completely unfold at 90°C and refolded almost entirely after cooling (see Fig. S1 in the supplemental material). S2 showed less refolding. This phenomenon appears to be attributable to the thermal resilience imparted by the zwitterionic detergent N,N-dimethyldodecylamine N-oxide (LDAO) on the T3SA tip and first translocator proteins. We have examined many other T3SA tip proteins and both translocator proteins formulated with LDAO and have found that they maintain some degree of folding at higher temperatures. Once cooled, these proteins completely or nearly completely refolded, as illustrated by a CD spectrum that is nearly identical to the one obtained prior to thermal melting (15, 16). When model proteins such as bovine serum albumin (BSA) or lysozyme, as well as other T3SS proteins such as the translocator chaperone, were heated in the presence of LDAO, no such thermal resilience was exhibited, with a complete loss of secondary structure at 90°C (15). While thermal stability of vaccines is important in relation to low-income countries, it is unclear whether LDAO would be in the final formulation.
The three-index EPDs identified the structurally stable and unstable conditions for each protein. Identification of stable regions at the same pH is important for the development of a final formulation. For example, at temperatures below ∼47°C at pH 7 and 8, region I is quite similar in S1 and S2, yet they are not the same since S2 exhibits some aggregation that is detected by SLS. This is reflected in the pink color rather than the orange of S1 in this same region I. Thus, excipients will now be screened to prevent the aggregation of S2 at pH 7 and 8 as well as to increase the overall stability of both S1 and S2. For the experiments described here, pH 7 was chosen, which appeared to produce somewhat less aggregation for S2. While citrate-phosphate buffer was used to provide broad pH, phosphate binds to aluminum hydroxide (Alhydrogel) to form aluminum hydroxyphosphate. For these studies, Alhydrogel was preferred due to the positive results obtained with IpaD plus IpaB in preventing Shigella infections (17). Thus, histidine was used to replace citrate-phosphate.
Immunizations with S1, S2, and S1S2 elicited efficient serum IgG titers with the magnitudes of antibody titers being comparable across the three groups, demonstrating that no interference occurred from any one of the proteins. On day 56, the anti-SseB titer of the S2 group was significantly higher (P = 0.05) than the titer in the S1S2 group. In contrast, there was a significant increase in the anti-SseB IgG ASCs in the S1S2 group in comparison to the S2 group in both the bone marrow and spleen cells. In the typhoid-like model, S1S2 elicited partial protection (60%) against homologous and heterologous challenges, while neither of the individual proteins provided protection (10%). In a second challenge model, a streptomycin-pretreated mouse colitis model was used, in which successful colonization by Salmonella induces cecal inflammation (11). As in the typhoid model, S1S2 immunization supported the overall survival data by preventing cecal inflammation upon Salmonella challenge, which resulted in keeping the gut in a nearly normal physiological state. In contrast, moderate to severe cecal inflammation in S1- and S2-immunized mice resulted in poor survival of the host after lethal challenge.
Many publications refer to new vaccines to prevent S. enterica infections. The fact that proteins secreted from the S. enterica T3SSs, including SseB, were used to provide protection has been demonstrated in these publications (18–22). Based on the Shigella DBF vaccine, this study was designed to develop a potential vaccine by combining two recombinant fusions, S1 and S2, formulating with an approved adjuvant system (MPL-AH), and delivering via an approved vaccination route to protect against two S. enterica serovars, S. Typhimurium and S. Enteritidis. Further work to understand the basic immunological targets and correlates of protection was beyond the scope of this study.
Taken together, this initial study demonstrates that the fusions resulting from the T3SA tip and translocator proteins are viable vaccine candidates. Further research must be performed to increase the protective efficacy. It is unlikely that a stand-alone subunit or a combination of two subunits with adjuvant will be protective in humans. While a particle composed of MPL-AH and proteins was used for this study, either a better formulation or a unique particle may be required to maintain the antigen in lymph nodes to elicit memory (23, 24). Nevertheless, the most important finding is that the vaccine formulation of S1S2 provided protection not only against the homologous S. Typhimurium but also against the heterologous S. Enteritidis. Although it would be difficult to test all of the >2,500 S. enterica serotypes, these two common serotypes provide prima facie evidence that this subunit vaccine could provide broadly protective, serotype-independent protection against this costly and deadly pathogen.
MATERIALS AND METHODS
Preparation of Salmonella fusion proteins.Salmonella Typhimurium SL1344 T3SS-1 sipD and sipB were cloned in pET28a (NdeI-SalI-XhoI) to construct the plasmid s1/pET28a for expression of the SipD-SipB fusion protein, S1. T3SS-2 sseB and sseC were cloned in pET15b (NdeI-SacI-BamHI) to construct the plasmid s2/pET15b for expression of the SseB-SseC fusion protein, S2. For expression of S1, Escherichia coli NovaBlue(DE3) was cotransformed with s1/pET28a and sicA-pACYC Duet-1, which encodes the SipB cognate chaperone. Similarly, for expression of S2, E. coli NovaBlue(DE3) was cotransformed with s2/pET215b and sseA-pACYC Duet-1, which encodes the SseC cognate chaperone. S1 and S2 were expressed in E. coli Tuner(DE3) grown in autoinduction medium and Terrific broth, respectively. Harvested cells were lysed, and proteins were purified using standard denaturing immobilized metal affinity chromatography (IMAC) procedures. The purified proteins were refolded using a stepwise dialysis method and subjected to a second IMAC to remove any remaining chaperone. Proteins were further dialyzed into PBS containing 0.05% (wt/vol) LDAO, filtered using 0.22-μm filters and stored at −80°C (see Fig. S3 in the supplemental material).
For biophysical analyses, proteins were dialyzed into 20 mM citrate-phosphate buffer containing 0.05% (wt/vol) LDAO at each pH from 3.0 to 8.0, their ionic strength was adjusted to 0.150 using NaCl, and they were filtered using 0.22-μm filters. For vaccine studies, proteins were dialyzed in 10 mM histidine (pH 7.0), 150 mM sodium chloride, and 0.05% LDAO, filtered using 0.22-μm filters, and stored at −80°C as needed. Protein concentrations was determined by measuring absorbance at 280 nm (25).
Spectroscopic analysis of the fusion proteins and production of multi-index EPDs.Far-UV circular dichroism (CD) spectra, intrinsic tryptophan fluorescence spectra, and static light-scattering measurements were collected as previously described (10, 15). Three-index empirical phase diagrams (EPDs) were prepared as previously described (10, 15).
Attenuated Salmonella enterica strains (ΔSPI-1/2).S. Typhimurium SL1344 and S. Enteritidis P125109 double mutants (ΔinvC ssaV::aphT) were developed by phage transduction (26). Briefly, ΔinvC mutants for both S. Typhimurium and S. Enteritidis were kindly provided by M. Suar (KIIT University, India). A P22 phage lysate of ssaV::aphT was transduced into the recipient ΔinvC mutant strain to obtain stable ΔinvC ssaV::aphT double mutants of both serotypes. These mutants are abbreviated S. Typhimurium ΔSPI-1/2 or S. Enteritidis ΔSPI-1/2 since they have deletions in SPI-1 and SPI-2. S. Typhimurium ΔSPI-1/2 and S. Enteritidis ΔSPI-1/2 colonies were purified three times on LB agar supplemented with kanamycin (50 μg/ml). S. Typhimurium ΔSPI-1/2 and S. Enteritidis ΔSPI-1/2 were grown at 37°C in LB medium for 12 h, diluted 1:20 in fresh LB medium, and subcultured for another 4 h at 37°C until the desired optical density was achieved. The bacteria were washed, and 1 × 108 CFU was suspended in 100 μl cold PBS for immunization in each mice.
Mice and immunization.Six- to 8-week-old female BALB/c mice (Charles River Laboratories, Wilmington, MA) were used for all experiments, where the group size is n = 10 in all cases except for ASC frequency, where n = 5. These mice, however, were part of the same experiment, so the IgG titers and protection are associated with the ASC results. Animals were housed and handled in agreement with the guidelines of the University of Kansas Institutional Animal Care and Use Committee (IACUC). For intramuscular administration, S1 and S2 (20 μg/dose) and the combination were admixed with 50 μg Alhydrogel (AH) (Brenntag, Denmark) for 45 min at room temperature, at which point 50 μg monophosphoryl lipid A (MPL) (Sigma, USA) was added as previously described (14), with the final volume being 100 μl. PBS alone was included as a negative control. Vaccines were administered at days 0, 14, and 28 of the study. S. Typhimurium ΔSPI-1/2 and S. Enteritidis ΔSPI-1/2 were used as the positive controls and were administered by gavage (1 × 108 CFU) at day 28 of the study.
Assessment of immune response.Serum IgG and fecal IgA titers as well as frequency of antibody-secreting cells (ASCs) were determined as previously described (13, 14, 17).
S. Typhimurium and S. Enteritidis lethal challenge.Wild-type S. Typhimurium SL1344 and S. Enteritidis 125109 were grown at 37°C in LB medium for 12 h, diluted 1:20 in fresh LB medium, and subcultured for another 4 h at 37°C until the desired optical density was achieved. The bacteria were washed and were suspended in 100 μl PBS. On day 56, mice were orally challenged with 100 μl of S. Typhimurium (2 × 108 CFU) or S. Enteritidis (5 × 107 CFU). Serial dilutions of bacterial suspension were plated to confirm the challenge dose. Health score and weight loss were monitored for 20 days. Mice that became moribund or remained under 80% of their starting weight for more than 48 h were humanely euthanized. Survival was plotted, and a log rank test was used to evaluate the differences in overall survival.
S. Typhimurium infection of streptomycin-treated mice with histopathological evaluation.Mice (n = 5) were vaccinated as described above (days 0, 14, and 28) with S1, S2, and the combination adsorbed to AH, and then MPL was added. S. Typhimurium ΔSPI-1/2 was administered on day 28. On day 55, mice were treated orally with 20 mg of streptomycin by gavage at 24 h prior to challenge to eliminate the gut microbiota and allow colonization of the gut (11). Mice were challenged orally on day 56 with 200 CFU of wild-type S. Typhimurium SL1344 in 100 μl. At 4 days postchallenge, the mice were euthanized, and the cecum was extracted to assess cecal inflammation and fixed in OCT (11). Thin cryosections (4 μm) of cecum were stained with hematoxylin and eosin. Sections were visualized and photographed (Fig. 5A to E) at a magnification of ×10, whereas the pathoscore (Fig. 5F) was evaluated at a magnification of ×400 with a Nikon Eclipse 80i microscope (the best representative image from each group is presented). The stained sections were evaluated independently on the basis of pathological changes that included submucosal edema (score, 0 to 3), PMN infiltration (score, 0 to 4), loss of goblet cells (score, 0 to 3), and epithelial integrity (score, 0 to 3), with a final pathoscore of 0 to 12, reflecting the overall degree of inflammation. The pathoscores were determined by averaging the scores. The combined scores ranged from 0 to 12 arbitrary units, covering the inflammation levels as follows: intact intestine (pathoscore, 0); minimal inflammation (pathoscore, 1 or 2), which is commonly found in the ceca of mice; slight inflammation (pathoscore, 3 or 4); moderate inflammation (pathoscore, 5 to 8); and significant inflammation (pathoscore, 9 to 12) (11). Pathoscores are represented graphically as mean ± standard error of the mean (SEM).
Statistical analysis for animal analysis.All graphics and comparisons were done using a t test and log rank (Fig. 4) and analysis of variance (ANOVA) (Fig. 2) tests using GraphPad Prism software (Prism 5 version 5.04; GraphPad Software, La Jolla, CA, USA). A P value of <0.05 was considered statistically significant in all determinations.
ACKNOWLEDGMENTS
We thank the members of the Picking lab for excellent technical assistance in sample processing, protein production, and reviewing the manuscript.
This work was supported by funds from the University of Kansas to W.D.P. and W.L.P.
The University of Kansas had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
FOOTNOTES
- Received 6 July 2017.
- Returned for modification 30 August 2017.
- Accepted 17 December 2017.
- Accepted manuscript posted online 8 January 2018.
Supplemental material for this article may be found at https://doi.org/10.1128/IAI.00473-17.
- Copyright © 2018 American Society for Microbiology.
