The LVS strain of Ft was acquired from BEI (NR-646) and was grown in vitro in MHB, BHI, or BHI supplemented with casamino acids (BCA) as previously described (Hazlett et al., 2008; Hickey et al., 2011; Zarrella et al., 2011). LVS mutant strains used here have been described elsewhere (Su et al., 2007). Ft S4 was used within the CDC-certified A/BSL-3 facilities of Albany Medical College, the University of Pittsburgh, and the Trudeau Institute. Late-log phase Ft grown in MHB, BHI, or BCA were harvested by centrifugation (8,000 × g, 15 min, 20°C). Bacterial pellets were resuspended in the appropriate fresh growth media, transferred to pre-weighed Eppendorf tubes, pelleted as above after which the transfer supernatant was aspirated. Pellet wet weights were determined and used to estimate cell numbers based upon the estimate of 1 mg of wet weight = 5 × 10⁸ bacteria. Cells were resuspended to 2.5 × 10⁷/μl (higher bacterial concentrations promoted protein precipitation in subsequent steps) in 20 mM Tris, pH 8.0 containing 100 mM NaCl, 20 μl/ml protease inhibitor cocktail (Sigma, #P8849). After determining the protein concentrations (DC protein assay, BioRad, Hercules, CA), equilibrated bacterial suspensions were supplemented with EDTA to 5 mM, lysozyme to 100 μg/ml, Benzonase (1 μl/ml, Sigma), and Triton X-114 (Acros, NJ) to 1% from a 10% stock in PBS. Following a 1 h incubation at room temperature with periodic gentle agitation, a 1/20 vol. aliquot was saved as the whole-cell (WC) fraction; the WC and remaining sample were stored at −20°C overnight. The samples were thawed and incubated at RT until further viscosity reduction abated. Following 15 min incubation on ice, the samples were centrifuged (20 min, 16,500 × g, 4°C) to yield Triton X-114 soluble (TxS) and insoluble (TxI) fractions. The TxS fractions were transferred to fresh tubes and centrifuged again to remove any TxI carry-over; the TxI fractions were washed twice with cold PBS (1 vol). The TxS fractions were incubated in a 32°C water bath for ~10 min followed by centrifugation (7,000 × g, RT, 15 min) to effect phase partitioning (Bordier, 1981; Radolf et al., 1988; Sjostedt et al., 1991; Hazlett et al., 2001, 2005) into aqueous (top) and detergent (bottom) phases (any protein precipitation at this step rendered the samples useless, such experiments were discarded). Recovered phases were washed by supplementation (to 1% Triton X-114—A or 10 vol PBS—D) followed by phase separation; three washes per recovered phase were performed. The washed Triton insoluble (TxI) phases were resuspended in PBS containing 0.2% sarkosyl (Fisher Scientific, Fair Lawn, NJ) and incubated for 20 min with gentle agitation followed by centrifugation (30 min, 4°C, 16,500 × g). The sarkosyl-soluble (SS) fraction was recovered and centrifuged a second time to remove any carryover of insoluble material. The sarkosyl-insoluble (SI) material was washed once with PBS, 0.2% sarkosyl and dissolved in 10 mM Tris, pH = 8.0 containing 1% SDS. WC, A, D, SS, and SI fractions were resolved by SDS-PAGE. Occasionally the D, SS, and SI phases were pooled to represent the total membrane fraction.

Samples derived from 10 μg of Ft protein (~1 × 10⁸ cells) were mixed with Laemmli sample buffer and boiled for 10 min prior to resolution through 4–12% gradient SDS-PAGE pre-cast gels (Invitrogen). The running buffer was NuPAGE MES SDS buffer from Invitrogen; gels were variously run at 90–160 V. Resolved gels were stained with either Coomassie blue (BioRad) or transferred to nitrocellulose membranes. Coomassie-stained gels were scanned into Adobe Photoshop using an HP 2820 scanner. Membranes were blocked for 30 min with PBS, 0.05% Tween 20, 5% non-fat dry milk. Primary Abs were applied for overnight incubation at dilutions ranging from 1:1,000 to 1:60,000. Blots to be probed multiple times were first probed with mAb prior to stripping and reprobing with polyclonal sera. HRP-conjugated secondary Abs were used at dilutions ranging from 1:1,000 to 1:20,000. Occasionally the secondary conjugate was a biotinylated goat α-species-specific Ab followed by a tertiary conjugate of streptavidin-linked HRP. Development of the chemiluminescent substrate (SuperSignal West Pico, Pierce, Rockford, IL) was visualized using an Alpha Innotech imaging system in movie mode. Densitometric analysis of developed blots was performed on the same system.

S4-convalescent mouse serum. Six to eight week old C57BL/6 mice (Taconic, Germantown, NY) of either sex (n = 10) were used for raising antisera against Ft S4. Mice were anesthetized by intraperitoneal injection of a cocktail of Ketamine (5 mg/kg) (Fort Dodge Animal Health, Fort Dodge, IA) and Xylazine (4 mg/kg) (Phoenix Scientific, St. Joseph) and checked for the loss of toe-pinch reflex. Anesthetized mice were immunized intranasally (i.n.) on day zero with 2 × 10³ colony forming units (CFU) of live, MHB-grown Ft LVS in 20 μl (10 μl/nare) of sterile PBS. Identical booster immunizations were administered on d14 and d28. On d42, the immunized mice were challenged i.n. with 26 CFU of MHB-grown Ft S4. One hundred percent of these mice survived. The same mice were challenged again with 100 CFU of MHB-grown Ft S4 on d72; again all mice survived. On d100, mice were re-challenged with 500 CFU of MHB-grown Ft S4. On day 111, the surviving mice (100%) were bled by retro orbital venipuncture; sera were harvested and stored at −80°C until further use. The immunization and challenge doses were confirmed by plating serial dilutions on MHB chocolate agar plates. The plates were incubated at 37°C in the presence of 5% CO₂ for 48 h and the colonies were counted. S4-convalescent rabbit sera. Rabbit sera used here was previously generated (Reed et al., 2014) in New Zealand White rabbits by scarification on day zero with BHI-grown aroD Ft S4 followed by aerosol challenge on d28 with 50–500 LD₅₀ of BHI-grown WT Ft S4. Sera were harvested on d56. LVS-immunized, human sera. The USAMRIID Special Immunization Program supplied aliquots of previously-banked, pre- and post- LVS-immunization sera from 10 sero-positive, de-identified study participants. Provision and use of these human sera as described herein was approved by the Institutional Review Boards of USAMRIID and Albany Medical College. The Institutional Animal Care and Use Committees (IACUCs) of Albany Medical College, the University of Pittsburgh, and the Trudeau Institute approved animal protocols used herein.

Four independent challenge experiments were conducted. In the first two, conducted at Albany Medical College and the University of Pittsburgh respectively and shown in Figure 4, groups of female BALB/c mice (n = 6–8/group) were immunized three times on d0, d14, and d28 with PBS or 200 CFU of live Ft LVS. Mice were challenged on d42 with Ft S4 and monitored for 14d. For the AMC trial, mice (Taconic) were primed intradermally (i.d.) and boosted twice intranasally (i.n.) with PBS or live, MHB-Ft LVS prior to i.n. challenge with 25 CFU of MHB-Ft S4. For the University of Pittsburgh trial, mice (Charles River) were primed i.n. and boosted twice i.n. with PBS or BHI- Ft LVS prior to challenge with 34 CFU of BHI-Ft S4 delivered via aerosol. In the third experiment (Figure 5), two groups (n = 20/group) of C57/BL6 mice (Taconic; ½ male, ½ female) were primed i.d. and twice boosted i.n. on d11 and d25 at AMC with either PBS or 200 CFU of live, MHB-Ft LVS. Mice were challenged i.n. on d42 at the Trudeau Institute with 26 ± 2 CFU of either MHB- or BHI-Ft S4 and monitored for 21 d. In our final experiment (Figure 6), two groups (n = 22–24/group) of C57/BL6 mice (Taconic; ½ male, ½ female) were primed i.d. and boosted i.n. once on d21 at AMC with either PBS or 1,000 CFU of live, BHI-Ft LVS. On d42 the mice were challenged i.n. with 29.5 ± 2.5 CFU of Ft S4; half of each group was challenged at AMC with MHB-Ft S4; the other half was challenged at the Trudeau Institute with BHI-Ft S4. Survival was monitored for 21 d. The IACUCs of Albany Medical College, the University of Pittsburgh, and the Trudeau Institute approved animal protocols used herein. All challenge-survival data are presented as Kaplan–Meier survival curves with statistical analysis by the Log-rank (Mantel–Cox) test.

We detected expression of 1,156 distinct Ft LVS proteins, 916 in the ^18/16O₂ labeled samples and an additional 240 by label-free detection. Our total detection rate of 67% of the hypothetical Ft proteome is identical to that reported for a related quantitative proteomics strategy recently used by Lenco et al. to interrogate the proteomes of Ft LVS and Ft S4 (Lenco et al., 2009). Of the 845 proteins for which we have high-quality quantitative data, 17% were more abundant [>1.5 FC that was statistically significant] in BHI-Ft whereas 9% were less abundant (Table S1).

As the quality of our Ft data appeared to be comparable to that of others, we next compared our BHI/MHB proteomics FCs to existing “omics” FC datasets in which Ft grown in mammalian cells and/or mice were compared to bacteria grown in broth (Twine et al., 2006a; Wehrly et al., 2009; Bent et al., 2013; Thomas-Charles et al., 2013). This comparison was possible as FC is a form of data expression common to both “omics” technics. Our goal was to very-broadly test the notion that BHI-Ft is more similar, than MHB-Ft, to what has been reported for host-adapted Ft. While comparing FCs across distinct “omics” platforms and between different laboratories presents some limitations, we perused this analysis with an eye toward broad trends (operons, regulons, and protein families) that were apparent in our data and within multiple reports for host-adapted Ft. As a control, we also included the proteomics data of Lenco et al. who analyzed Ft grown in vitro to stationary and log phase in CDM (Lenco et al., 2009). Our BHI/MHB FC dataset best fit the transcriptional datasets of Bent et al. (FCs derived from Ft L Daygrown in MΦs for 4 and 8 h vs. in BCA) (Bent et al., 2013) and Thomas-Charles et al. (FCs derived from Ft LVS grown in hepatocytes for 2 d vs. in MHB) (Thomas-Charles et al., 2013). A linear regression analysis of our data against the Bent et al. 4 h dataset revealed a very low correlation (R² = 0.04, Figure 1); the correlation between our data and that of Lenco was moderately increased (R² = 0.17). Our data showed the highest correlation with data derived from 8 and 48 h of intracellular growth (R² = 0.30 and 0.37 respectively); these latter two R²-values were not significantly different from each other (correlation significance analysis, http://vassarstats.net/). Collectively, these results suggest that on a broad scale, BHI-Ft more closely reflect what has been reported for bacteria that have grown in host cells for 8–48 h.

Identifying the virulence factors (VF) of Ft has been a significant area of interest for the tularemia field. Through the use of mutant Ft and various infection models, a collection of the bacterium's VFs is emerging (Baron and Nano, 1998; Santic et al., 2005, 2007; Brotcke et al., 2006; Tempel et al., 2006; Su et al., 2007; Bonquist et al., 2008; Sammons-Jackson et al., 2008; Barker et al., 2009; Buchan et al., 2009; Dean et al., 2009; Schmerk et al., 2009; Ahlund et al., 2010; McCaffrey et al., 2010; Broms et al., 2011, 2012a; Straskova et al., 2012; Robertson et al., 2013, 2014) and currently contains 190 Francisella proteins. In our proteomics analysis, we detected 92% of these proteins which is significantly higher (p < 0.0002; proportion analysis http://vassarstats.net/) than our overall Ft protein detection rate (67%). Among these VFs, 26% were up-regulated by 1.5-fold or more in BHI-Ft compared to MHB-Ft; 9% were down-regulated in BHI (Table S1). The percentage of BHI up-regulated VFs was significantly higher (p = 0.013; proportion analysis http://vassarstats.net/) than the percentage of BHI up-regulated proteins in the broader proteome (17%).

The most studied VFs of Ft include the 17 Francisella Pathogenecity Island (FPI) proteins, which likely form a type-6 secretion system for the delivery of Ft effector proteins into target cells (de Bruin et al., 2007; Ludu et al., 2008; Broms et al., 2009, 2012a,b; Clemens et al., 2015). As shown in Table 1 (and Table S1), we detected all FPI proteins with the exception of the 135 kDa protein PdpD (FTT1715)—whose gene is truncated in Ft LVS (Lenco et al., 2009). The average FC for the FPI proteins (excluding IglF [high level of variation]) was 5.3 in BHI-Ft relative to MHB-Ft. In general, our FPI data compares favorably with that of Bent et al. and Thomas-Charles et al. particularly for the later time points of intracellular (IC) growth (Table 1 and Table S1). Similarly, our findings generally mirror those derived from mouse and MΦ models with Ft S4. Twine et al. purified Ft S4 from murine spleens 4 d post-infection and used proteomics to compare these to MHB-Ft (Twine et al., 2006a); Wehrley et al. used transcriptional analysis to compare Ft S4 grown within cultured MΦs (for 1–24 h) to Ft S4 grown in CDM (Wehrly et al., 2009). Based on the available Ft S4 data, the FPI expression we observed with Ft LVS in BHI appears to echo S4 FPI induction reported in Ft from mouse spleens (Twine et al., 2006a) and in MΦs at ~12–16 h (Wehrly et al., 2009) (Table S1). To confirm key findings of our proteomics data, we performed SDS-PAGE and western blot analysis of MHB-, BHI-, and BCA-grown Ft LVS along with BHI-grown Ft S4. As shown in Figure S1, BHI-grown Ft contained elevated levels of VgrG (FTL0123/FTT1702), IglC (FTL0113/FTT1712), and IglB (FTL0112/FTT1713). IglC and IglB levels were comparable in BHI-grown LVS and S4 while VgrG levels were slightly higher in S4 than LVS—as previously reported (Lenco et al., 2009).

Regulation of Ft virulence is also an active area of investigation with ~15 Ft proteins identified as having various degrees of influence on VF/FPI gene expression. The most well-characterized of these are MglA (FTL1185) and SspA (FTL1606) (homologs of the E. coli stringent starvation protein A—SspA), and the DNA-binding protein FevR (FTL0449, also called PigR). In the presence of ppGpp, FevR binds both to DNA up-stream of VF promoters and to a protein complex including MglA, SspA, and RNA polymerase to affect transcription of the MglA/SspA/FevR regulon which contains many Ft VF genes—including fevR and most of the (Brotcke et al., 2006; Charity et al., 2007, 2009; Brotcke and Monack, 2008; Rohlfing and Dove, 2014; Ramsey et al., 2015). We observed a 5.0 FC for FevR in BHI-Ft relative to MHB-Ft (Table 1 and Table S1)—values that closely track the average FPI FC. Among the remaining FPI regulators, we observed that PmrA (FTL0552) levels were slightly higher (FC = 2.0, Table 1 and Table S1) in BHI-Ft as we previously reported (Hazlett et al., 2008). Levels of SspA, OxyR (FTL1014), QscC (FTL1762), TrmE (FTL1177), SpoT (FTL1413), Fur (FTL1831), and Hfq (FTL0898) were unchanged, and levels of MglA, MglB (FTL1184), MigR (FTL1542), and CphA (FTL0831) trended lower in BHI-Ft (Table 1 and Table S1). The changes we observed for the FPI regulators (particularly for FevR) in BHI-Ft appear to mirror those reported for bacteria grown in host cells (Wehrly et al., 2009; Bent et al., 2013) and are greater than that observed for Ft grown to stationary phase in CDM (Lenco et al., 2009).

Generation of ppGpp to activate the FevR regulon is dependent on RelA (FTL-0285) and/or SpoT activity (Charity et al., 2009) and is inducible through amino acid starvation experimentally-triggered by serine hydroxamate (Dean et al., 2009; Faron et al., 2013). Under these conditions, ppGpp levels rise by 2- to 3-fold and correspond with a 2- to 3-fold increase in iglA promoter activity (Faron et al., 2013). By comparing the proteomes of Ft grown in BHI and BCA [BHI supplemented with casamino acids (the amino acid source in MHB)] we were able to explore the extent to which amino acid starvation contributes the differences in MHB- and BHI-grown Ft. Consistent with the reported impact of serine hydroxamate, we found the absence of casamino acids induced an average FPI protein induction of 2.2-fold and a 2.6 induction of FevR (Table 1). These values are similar to those reported for Ft grown in MΦ for 4 h (ave FPI-2.2, FevR 3.2) but lower than the full FPI induction reported for Ft grown in MΦ for 8 h (ave-FPI 4.6, FevR 6.9) and what we observed between BHI- and MHB- grown Ft (ave FPI-5.3, FevR 5.0). These data suggest that while amino acid limitation alone can induce the FevR regulon 2- to 3-fold, additional, possibly nutritional, signals contribute to full FPI induction. Classically, nutrient starvation and induction of the stringent starvation response (mediated by RelA/SpoT and ppGpp) results in decreased abundance of ribosomes. Of the 47 ribosomal proteins for which we had robust FC data, 24 were statistically less abundant in BHI-Ft compared to MHB-Ft (Table 1 and Table S2) Among these 47 proteins, 8 were reduced in BHI compared to BCA; 12 and 32 are reportedly reduced after 4 and 8 h respectively of growth in MΦ. Thus in our data, as well as that of Bent for LVS (Bent et al., 2013) and Lenco for LVS and S4 (Lenco et al., 2009), FPI induction appears to correlate with ribosomal protein reduction.

Collectively, the protein fingerprint of log-phase BHI-Ft (compared to MHB Ft) is one of nutrient-depleted bacteria that—through induction of a stringent starvation-like response—have induced the FevR regulon for high-level expression of the bacterium's VFs.

Another category of Ft proteins that contribute significantly to virulence are the ~65 enzymes that synthesize/modify/incorporate carbohydrates into the bacterium's surface-coating of polysaccharides. Principal among these polysaccharides is the four sugar, O-antigen moiety synthesized by the Wbt enzymes and incorporated into polymers found in both the bacterium's LPS and the discrete O-Ag capsule. Previously, we demonstrated that BHI-Ft have higher molecular weight capsular polysaccharides and longer polymers of LPS O-Ag compared to MHB-Ft although the mechanistic basis for this observation was unknown (Zarrella et al., 2011). In our proteomics data (Table 1 and Table S1) for BHI-Ft, we observe moderately increased levels of several Wbt enzymes (M, H, E, and D) as well as KdsB and ManB—enzymes that contribute to synthesis of the KDO core which links O-Ag to Lipid A. Among the remaining ~40 LPS/Capsule related proteins (Lpx, Waa, Lpc, Lpt, Flm, and Nax members) for which we have robust data, no significant differences were noted between MHB-Ft and BHI-Ft (Table S1). As with many Ft LPS/capsule synthases, perturbations of WbtM, WbtE, WbtD, and ManB reduce the bacterium's polysaccharide coat rendering the pathogen highly susceptible to host defenses (Thomas et al., 2007; Lai et al., 2010; Lindemann et al., 2011; Twine et al., 2012b; Okan et al., 2013; Rasmussen et al., 2015). Currently, it is unclear if the increased abundance of the Wbt, Kds, and Man enzymes reported here for BHI-Ft is sufficient to account for the increased surface polysaccharides we previously described. Another potential factor, increased retention of capsular material, has also been postulated for BHI-Ft (Faith et al., 2012) and a mechanism of such retention, lipid-modification of O-Ag capsule, has recently been reported for BHI-Ft (Barker et al., 2016).

To date, 81 Francisella proteins have been reported one or more times to be immunogenic in an immunization or sub-lethal infection model (Twine et al., 2006b, 2010, 2012a; Janovska et al., 2007; Sundaresh et al., 2007; Savitt et al., 2009; Fulton et al., 2011; Nuti et al., 2011; Golovliov et al., 2013; Chu et al., 2014). We detected 100% of these proteins, which is significantly (p < 0.0002) higher than our overall Ft protein detection rate (67%). Among the immunogenic proteins, 18% were more abundant in BHI-Ft while 13% were more abundant in MHB-Ft. In the course of our analysis we made an intriguing observation—the proteins most frequently reported to be immunogenic were among those that we measured as the most abundant. For example, in our label-free, spectral count data, GroEL (FTL1714) and DnaK (FTL1194) were the 1st and 3rd most abundant proteins in Ft (Table 1) and these proteins have been described as immunogenic by nearly every group that has performed Ft immunoproteomics. To further explore the relationship between protein abundance and reports of immunogenicity, we plotted the spectral counting abundance (normalized for protein size as larger proteins generate more peptides) for >1,100 proteins against the number of published reports of immunogenicity for each protein. As shown in Figure 2 there is a strong linear trend between a protein's abundance and the likelihood that the protein will have been reported as immunogenic. This trend was similar whether we used data from BHI- or MHB-grown Ft. The mean normalized abundance for the proteins in each category (1 report up to >3 reports) was significantly higher than the abundance of proteins that had not been reported as immunogenic.

As several immunogenic proteins are also differentially expressed to become highly abundant in BHI-Ft (Table 1), we hypothesized that sera from S4-surviving subjects might react differently with Ft that had been differentially cultivated. Inspired by recent reports from the Bosio group (Griffin et al., 2015; Roberts et al., 2016) indicating that C57BL/6 mice can be successfully immunized against challenge with MHB-Ft S4, we generated S4-convalescent mouse sera using live LVS in a prime-boost-boost strategy followed by an increasing challenge-dose regimen (26-100-500 CFU) with MHB-Ft S4. As a source of outbred S4-convalescent Ab, we used rabbits that had been primed with Ft S4 ΔaroD and challenged with BHI-Ft S4 prior to serum collection (Reed et al., 2014). For comparison, we also used sera from another outbred population, LVS-immunized humans. With these 3 sera, we probed by western blot, whole cell (WC), soluble, and membrane fractions of Ft LVS that had been grown in MHB, BHI, or BCA. As shown in Figure 3, the outbred rabbit and human sera strongly recognized the typical “ladder pattern” indicative of reactivity with the LPS O-antigen (Ag). The ladder pattern was present in the WC and membrane fractions of WT Ft LVS (Figure 3) and absent in preparations from O-Ag mutant Ft (data not shown). In contrast, our hyper-immune Mo sera displayed markedly reduced LPS-reactivity and instead showed prominent reactivity with a discrete number of soluble and membrane proteins (Figure 3); this pattern was unchanged when we probed preparations from O-Ag deficient Ft (data not shown). A similar discord between human and mouse immune serum reactivity with Ft LPS has been noted previously by Huntley et al. (2007).

Among the protein Ags recognized by these sera were several differentially abundant species. In particular, all three sera reacted with a soluble ~ 23 kDa moiety present in BHI-Ft at apparently higher levels than in MHB-Ft or BCA-Ft (Figures 3B–D, soluble, asterisk). This pattern of Ab-reactivity was mirrored by the abundance of a ~23 kDa soluble protein visible on coomassie-stained SDS-PAGE gels (Figure 3A) and by the reactivity with mAb against IglC (Figure S2). Within the membrane fractions, the only visible, differentially abundant Ag was a ~120 kDa band of reactivity in the outbred Rb and Hu sera (Figures 3C,D, membrane, asterisks). The abundant O-Ag reactivity of these sera may obscure additional differentially abundant membrane species. Collectively, these results with sera from multiple models and immunization schedules indicate that vaccine-induced immunity recognizes several differentially-expressed Ft Ags.

As several Ag differences in MHB-Ft and BHI-Ft were recognized by immune/convalescent sera, we hypothesized that immunized hosts might be differentially susceptible to challenge with these two forms of the bacteria. Previously, we observed that naïve mice infected with BHI-Ft (LVS or S4) showed a slightly shorter median survival time (MST) compared to that of mice challenged with MHB-Ft (Hazlett et al., 2008; Zarrella et al., 2011). Here, we envisioned two distinct scenarios for immunized mice challenged with Ft S4 previously grown in MHB or BHI. The elevated reactivity of the differentially-expressed, dominant Ags could render a challenge inoculum of BHI-Ft S4 more susceptible to pre-existing immunity (which could result in increased host survival). Alternatively, the thicker carbohydrate coating, coupled to the immediate benefit of pre-formed VFs, could make the BHI-Ft S4 challenge inoculum less susceptible to pre-existing immunity (which should result in decreased host survival).

Our first indication that the latter might be true came inadvertently from independent vaccination trials. For both trials, BALB/c mice were immunized using a prime-boost-boost strategy in which live Ft LVS served as the positive control for protection against challenge with Ft S4. In one case, the Ft strains were grown in MHB; in the second case both Ft strains were cultured in BHI. While LVS immunization provided significant protection (p ≤ 0.0001) against S4 challenge in both cases, protection for the MHB-based trial was absolute (100%) but only partial (38% survival, 12.5 d MST) for the BHI-based trial (Figure 4). The MST for control mice challenged with MHB-Ft S4 was 6.5 vs. 5 d for those challenged with BHI-Ft S4. While these results were intriguing, differences between the independent trails necessitated further experimentation.

To assess more rigorously the impact of differential cultivation on Ft vaccination, we set up a large-scale trial in which mice vaccinated with live MHB-Ft LVS in a prime-boost-boost strategy were challenged with either MHB- or BHI-grown Ft S4. For these experiments, we used C57BL/6 mice, which are harder to protect against challenge with Ft S4 (Twine et al., 2012a), as a highly stringent test of immunization efficacy. For LVS-immunized mice, we again observed complete (100%) protection against challenge with MHB-Ft S4 (Figure 5), which was significantly (p = 0.007) higher than the survival observed for mice challenged with BHI-Ft S4 (44% survival, 13 d MST). Survival of control mice was not significantly different between the MHB-Ft S4 group (8.3 d MST) and the BHI-Ft S4 challenged group (7.5 d MST). In the former experiment, the Ft LVS used for vaccination had been grown in MHB raising the specter that survival among the mice challenged with MHB-Ft S4 might have been the result of a more specifically tailored, vaccine-induced immunity. To test this notion, we assessed the ability of BHI-Ft LVS to protect against challenge with MHB- and BHI-grown Ft S4. C57BL/6 mice were primed and boosted a single time with live BHI-Ft LVS. Contrary to the notion of immunogen-tailoring, we again observed that immunized mice survived challenge with MHB-Ft S4 (70% survival, undefinable MST) significantly better (p = 0.038) than those challenged with BHI-Ft S4 (33% survival, 13.75 d MST) (Figure 6). Survival of PBS-immunized control mice was not significantly different (p = 0.398) between the groups challenged with MHB-Ft S4 (6 d MST) and those challenged with BHI-Ft S4 (5.8 d MST).

Collectively, our vaccination studies demonstrate that challenge with BHI-Ft S4 provides a more stringent test of vaccine efficacy than challenge with MHB-Ft S4.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer VP and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.