Differential Cultivation of Francisella tularensis Induces Changes in the Immune Response to and Protective Efficacy of Whole Cell-Based Inactivated Vaccines

Francisella tularensis (Ft) is a category A biothreat agent for which there is no Food and Drug Administration-approved vaccine. Ft can survive in a variety of habitats with a remarkable ability to adapt to changing environmental conditions. Furthermore, Ft expresses distinct sets of antigens (Ags) when inside of macrophages (its in vivo host) as compared to those grown in vitro with Mueller Hinton Broth (MHB). However, in contrast to MHB-grown Ft, Ft grown in Brain-Heart Infusion (BHI) more closely mimics the antigenic profile of macrophage-grown Ft. Thus, we anticipated that when used as a vaccine, BHI-grown Ft would provide better protection compared to MHB-grown Ft, primarily due to its greater antigenic similarity to Ft circulating inside the host (macrophages) during natural infection. Our investigation, however, revealed that inactivated Ft (iFt) grown in MHB (iFt-MHB) exhibited superior protective activity when used as a vaccine, as compared to iFt grown in BHI (iFt-BHI). The superior protection afforded by iFt-MHB compared to that of iFt-BHI was associated with significantly lower bacterial burden and inflammation in the lungs and spleens of vaccinated mice. Moreover, iFt-MHB also induced increased levels of Ft-specific IgG. Further evaluation of early immunological cues also revealed that iFt-MHB exhibits increased engagement of Ag-presenting cells including increased iFt binding to dendritic cells, increased expression of costimulatory markers, and increased secretion of pro-inflammatory cytokines. Importantly, these studies directly demonstrate that Ft growth conditions strongly impact Ft vaccine efficacy and that the growth medium used to produce whole cell vaccines to Ft must be a key consideration in the development of a tularemia vaccine.

directing the immune response toward an array of Ags that the host is likely to encounter during a natural infection maximizes the potential for a protective immune response to be generated upon vaccination. In this regard, the composition of the antigenic repertoire expressed by the constituent whole cell vaccine becomes very important, while PAMPs can provide molecular components required for the activation of Ag-presenting cells (APCs). Furthermore, activation of APCs is a key requirement for the generation of an optimal adaptive immune response against cognate Ags contained within such vaccines.
Importantly, the in vitro culture medium utilized to generate whole cell-based vaccines can significantly impact the expression of antigenic determinants and PAMPs on the infectious agent. Several microbes have been shown to differentially express Ags depending on the growth medium utilized, including Escherichia coli, Salmonella enterica (1), Listeria monocytogenes (2), Helicobacter pylori (3), and Mycobacterium bovis (BCG) (4,5). Direct evidence supporting an impact of growth medium on the efficacy of whole cell-based vaccines comes from a study involving BCG where its differential cultivation in Sauton versus Middlebrook medium significantly altered its protective efficacy (6). However, the mechanism responsible for this apparent difference has not been well established.
In regard to Francisella tularensis (Ft), it has been found that its antigenic repertoire can change depending on the growth medium employed for in vitro cultivation. Specifically, we previously demonstrated that the protein profile expressed by Brain-Heart Infusion (BHI)-grown Ft closely resembles that of in vivo circulating Ft, while Ft grown in Mueller Hinton Broth (MHB) exhibits a distinct protein profile (7,8). Interestingly, other investigations have revealed that BHI-and MHB-grown Ft also differ in their ability to interact with complement and antibodies (9). In addition, BHI-grown Ft exhibits a diminished ability to elicit pro-inflammatory cytokines by various APCs compared with inactivated Ft (iFt)-MHB (10,11). These features of BHI-versus MHB-grown Ft provide unique and necessary tools to investigate the dichotomy between the antigenic repertoire versus APCactivating components of whole cell-based Ft vaccines.
In this study, we examined the differences in protective efficacy of iFt vaccine prepared by growth in MHB or BHI and further evaluated if the difference in protective efficacy observed reflects changes in not only the immune response but also APC function. While we initially hypothesized that BHI-grown iFt, which more closely mimics the antigenic repertoire of in vivo macrophagegrown Ft, would provide the superior vaccine immunogen, we found that iFt-MHB generates superior protection compared to iFt-BHI. Other indicators of protection, such as bacterial burden, tissue damage, and production of pro-inflammatory cytokines, also correlated with the improved protective efficacy of iFt-MHB versus iFt-BHI. Furthermore, we found that iFt-MHB exhibited significantly higher binding to APCs, higher induction of dendritic cell (DC) maturation, and increased Ag presentation to Ft-specific T cells. Overall, this investigation suggests that APC activation components are important for protective efficacy of inactivated whole cell-based vaccines. Moreover, growth conditions employed to prepare these vaccines can significantly influence their immunological and protective efficacy.

Mice
Six-to 8-week-old male and female C57Bl/6 mice were obtained from Taconic laboratories (Hudson, NY, USA). Mice were housed in the animal resource facility at Albany Medical Center. Food and water were provided ad libitum during the entirety of experiments. Animal studies were reviewed and approved by Institutional Animal Care and Use Committee of Albany Medical Center according to NIH guidelines.

Preparation of iFt
Francisella tularensis LVS was grown in MHB (Becton, Dickinson, Franklin Lakes, NJ, USA) or BHI (Becton, Dickinson) medium at 37°C to a density of ~0.5-1 × 10 9 colony-forming units (CFU)/ ml. To inactivate Ft LVS, 1 × 10 10 CFU/ml of live bacteria were incubated in 1 ml of sterile phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA) containing 2% paraformaldehyde (Sigma-Aldrich) for 2 h at room temperature with gentle shaking. Subsequently, fixed bacteria (iFt) were washed with sterile PBS three times. Inactivation was verified by culturing a 100 µl of sample (1 × 10 9 iFt organisms) on chocolate agar plates (Becton, Dickinson) for 7 days. Protein contents were quantified using a bicinchoninic acid assay (Sigma-Aldrich) as described (12). The iFt preparations were resuspended in PBS and stored in 1 ml aliquots at −20°C until use. Ft LVS for challenge was grown similarly and stored in liquid nitrogen until use.

immunization and challenge
Groups of six to eight mice with equal numbers of males and females were immunized on day 0 and 21 via the intranasal (i.n.) route. Each mouse was anesthetized by intraperitoneal injection of 20% ketamine plus 5% xylazine and subsequently administered 20 µl of PBS (vehicle), iFt-MHB, or iFt-BHI at a concentration of 37.5 ng, 75 ng, 150 ng, or 300 ng/20 μl. Immunized mice were then challenged on day 35 i.n. with 2× LD50 of Ft in 40 µl of PBS (single nostril) and subsequently monitored for survival for 21 days.

Quantification of Bacterial Burden
Following immunization and challenge, mice were euthanized at various time intervals, and lung and spleen were collected aseptically in PBS-containing protease inhibitor mixture (Roche Diagnostics, Indianapolis, IN, USA) and subjected to mechanical homogenization using Mini Bead Beater-8 (BioSpec Products, Bartlesville, OK, USA) using 1-mm zirconia/silica beads. Tissue homogenates were then diluted 10-fold in sterile saline, and 10 µl of each dilution was spotted onto chocolate agar plates in duplicate and incubated at 37°C for 48 to 72 h. The number of colonies on the plates were counted and expressed as CFU per milliliter for the respective tissue. Tissue homogenates were then centrifuged at 14,000 g for 20 min, and the clarified homogenates were stored at −20°C for subsequent cytokine analysis.
lactate Dehydrogenase (lDh) assay Serum LDH levels were quantified following the manufacturer's instructions (Sigma-Aldrich). Briefly, mice were immunized and challenged as described for quantification of bacterial burden. Sera were collected on days 1, 3, 5, and 7 postinfection. Prechallenge sera were collected 11 days after booster immunization. Serum samples were diluted 1:100 with LDH assay buffer. Fifty microliters of diluted serum samples, standard, and blanks was added to separate wells in a 96-well microtiter plate. Fifty microliters of LDH substrate was subsequently added to each well. LDH converts NAD to NADH, which can be read calorimetrically at 450 nm. The amount of NADH generated between Tinitial (absorbance at 450 nm immediately after the LDH substrate) and Tfinal (absorbance at 450 nm immediately after the LDH substrate) by the LDH activity is first calculated (B). The LDH activity of the sample is determined by the formula LDH activity = (B × sample dilution factor)/(reaction time × V), where V is the sample volume added to well. LDH activity is reported as milliunit per milliliter.

antibody (ab) Titer Determination
Anti-Ft-Ab production in response to immunization with iFt-MHB versus iFt-BHI was measured by ELISA. Briefly, ELISA plates (Corning, Corning, NY, USA) were coated with 50 µl of Ft LVS (5 × 10 7 CFU/ml) in carbonate buffer [4.3 g/l sodium bicarbonate (Sigma-Aldrich) and 5.3 g/l sodium carbonate (Sigma-Aldrich) at pH 9.4] for 16 h at 4°C. The plates were then washed with washing buffer [PBS (Sigma-Aldrich) containing 0.5% bovine serum albumin (BSA) (Sigma-Aldrich)] and blocked for 2 h with 200 µl of PBS containing 5% BSA. Serial threefold dilutions of sera (starting with 1/10) were added to the plates (100 µl/well) and incubated for 2 h at 4°C. After three washes with washing buffer, alkaline phosphatase-conjugated anti-mouse Ab specific for IgG (Sigma-Aldrich) was added and incubated for 1 h at 4°C. Plates were then washed three times with washing buffer, and then 100 µl of BCIP/NBT alkaline phosphatase substrate (Sigma-Aldrich) was added. Plates were subsequently incubated for 2 h, and absorbance at 405 nm was measured using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
sodium Dodecyl sulfate Polyacrylamide gel electrophoresis (sDs-Page) and Western Blot analysis Samples derived from 10 µg (~1 × 10 8 cells) of Ft were mixed with Laemmli sample buffer (BioRad, Hercules, CA, USA) and boiled for 10 min prior to resolution through 4-12% gradient SDS-PAGE precast gels (Invitrogen, Carlsbad, CA, USA). The running buffer was NuPAGE MES SDS buffer (Invitrogen). Gels were run at 90-160 V. Resolved gels were stained with either Coomassie blue (Bio-Rad) or transferred to nitrocellulose membranes. Coomassie-stained gels were scanned into Adobe Photoshop. Membranes were then 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 1:500 to 1:60,000 in PBS-Tween. Following five washes with PBS-Tween, the membranes were incubated for 1 h with agitation with biotinylated goat anti-mouse IgG (H + L, Southern Biotech, Birmingham, AL, USA). Following an additional five washes with PBS-Tween, the membranes were incubated for 1 h with agitation with streptavidin-conjugated horseradish peroxidase (Southern Biotech). Development of the chemiluminescent substrate (SuperSignal West Pico, Pierce, Rockford, IL, USA) was visualized using an Alpha Innotech imaging system in movie mode. Densitometric analysis of developed blots was performed as previously described (7,9).
iFt Binding assay Binding of iFt was visualized by flow cytometry using GFPexpressing iFt, as previously described (13). GFP-expressing Ft LVS organisms were provided by Dr. Mats Forsman (Swedish Defense Research Agency, Stockholm) (14). Briefly, 1 × 10 6 BMDCs were incubated with 5, 10, 15, or 20 µg of GFP-expressing iFt-BHI or iFt-MHB in PBS. After 2 h incubation at 4°C, BMDCs were washed twice with PBS and stained with rat anti-mouse CD11c-Alexa 700 Ab and subsequently analyzed by flow cytometry (LSRII, BD, Franklin Lakes, NJ, USA). The percent of GFP-positive CD11c DCs (indicative of iFt bound to DCs) were analyzed in respective samples.

In Vitro analysis of costimulatory Molecule expression and cytokine secretion
Expression of costimulatory molecules on BMDCs was analyzed by flow cytometry as previously described (13). Briefly, 2 × 10 5 BMDCs were incubated for 24 h with PBS or 10 µg of iFt-MHB or iFt-BHI in an equivalent volume of PBS. Cells were then harvested and stained with Abs to the following surface markers: rat anti-mouse CD40-PE, rat anti-mouse CD80-APC, and rat anti-mouse CD86-Alexa 700. Cells were subsequently analyzed via flow cytometry.
Cytokine secretion by BMDCs was assayed following previously described methods (13). Briefly, 2 × 10 5 BMDCs were incubated with PBS or with 10 µg iFt-MHB or iFt-BHI for 24 h at 37°C. Culture supernatants were then collected and analyzed for cytokines using the Bio-Plex assay system as described below. immunization studies retain the protein composition of their unfixed counter parts. Ten micrograms of live and fixed Ft LVS previously grown in Mueller Hinton Broth and Brain-Heart Infusion (BHI) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and stained with Coomassie blue (a) or transferred for western blot analysis (B). Asterisks in A mark bands, which are visibly more abundant in the western blot of BHI-grown Ft (B). The membrane was probed with a cocktail of monoclonal antibodies specific for IglB, IglC, and a FopA-specific polyclonal mouse serum. IglB and IglC have previously been shown to be more abundant in BHI-grown Ft (7,9). FopA is used here as a loading control. The ~18-kDa band visible under IglC is an unidentified, endogenously biotinylated Ft protein (likely AccB) detected by the streptavidin-horseradish peroxidase conjugate used during development of the western blots.

ag Presentation assays
Antigen presentation assays were done as previously described (13). Briefly, 2 × 10 5 BMDCs or PECs were incubated overnight at 37°C with PBS or 5 µg of iFt-MHB or iFt-BHI. A total of 1 × 10 5 Ft-specific T cells (FT25 6D10 hybridoma cells) (13) in 100 µl medium (RPMI 1640 supplemented with 10% FBS) were then added to each well. The plate was then incubated at 37°C for 24 h in CO2 incubator, and supernatants were collected. Supernatants were then assayed for interleukin (IL)-5 (secreted by these cells in response to Ag) using the Bio-Plex assay as described earlier.

statistics
The log-rank (Mantel-Cox) test was used for survival curves.
One-way analysis of variance or the unpaired Student's t-test was used for the remaining figures. Data analysis was performed using Graph-Pad Prizm 5 (Graph-Pad Software, San Diego, CA, USA).

resUlTs iFt exhibits Protein Profiles similar to its live counterpart
Before using iFt as immunogen, we first used SDS-PAGE and western blot analysis to examine the impact of our fixation procedure on the protein composition of Ft LVS. The overall protein profile of live and fixed Ft LVS was largely unchanged, although the fixed samples did show some expected evidence of crosslinked Ags ( Figure 1A). Consistent with previous reports (7,9), the BHI-derived samples contained elevated levels of ~23 and ~55 kDa proteins [ Figure 1A (asterisks)], which were apparent in both the live and fixed bacteria. Western blot analysis indicated that the live and fixed BHI-derived samples contained levels of IglB and IglC that were elevated relative to their MHB-grown counterparts ( Figure 1B). Densitometric analysis revealed that these increases were on the order of threefold to fivefold (Table  S1 in Supplementary Material), as reported previously (7,9). As our fixed iFt preparations appeared to be faithful representations of their live counterparts, we next examined the vaccine efficacy of these immunogens.

iFt-MhB generates superior Protection against Ft lVs-induced Mortality versus iFt-Bhi
First, we evaluated whether the differential expression of antigenic repertoire and PAMPs affected by cultivation in MHB versus BHI has any impact on the protective efficacy of iFt. We used iFt generated in MHB or BHI to immunize mice. Following a lethal challenge with Ft LVS, we observed that iFt-MHBimmunized mice were better protected than iFt-BHI-immunized mice (Figure 2). Of the four doses used, the 150-and 300-ng doses exhibited statistically significant differences in the protection afforded by iFt-MHB versus iFt-BHI (Figures 2C,D). Importantly, in Figures 2A-D mice were challenged with MHB grown Ft LVS. Thus, to evaluate any potential bias of growth medium used to culture the challenge organism (Ft LVS) on the outcome of survival, we similarly immunized mice with PBS or 150 ng of iFt-MHB or iFt-BHI, but challenged with BHI-grown Ft LVS Once again, we observed that iFt-MHB provided better protection compared to iFt-BHI ( Figure 2E).    Following the above observation of differential protection afforded by iFt-MHB versus iFt-BHI, we investigated the ability of the two iFt immunogens to protect against bacterial replication and tissue damage exerted by Ft LVS infection. Following immunization and Ft LVS challenge, we found significantly lower bacterial burdens in the lungs (Days 5 and 7) ( Figure 3A) and spleen (Day 7) (Figure 3B) of mice immunized with iFt-MHB as compared to that of iFt-BHI. Francisella tularensis LVS infection inflicts systemic tissue damage in infected mice, and serum LDH levels are commonly used as a biomarker of tissue damage (15). Thus, we evaluated serum LDH levels at early and late stages of Ft LVS infection in mice immunized with iFt-MHB versus iFt-BHI. At days 5 and 7 after Ft LVS infection, significantly lower levels of LDH activity were found in iFt-MHB-immunized mice versus that of iFt-BHIimmunized mice (Figure 4).

Differential Protection against Ft lVs-induced inflammation by iFt-MhB versus iFt-Bhi
Systemic inflammation and/or sepsis are hallmarks of the tularemia in advanced stages of infection. We previously observed that mice protected by immunization exhibit lower levels of proinflammatory cytokines in the lungs and spleens following Ft infection, compared to those of unprotected mice (16,17). In this case, analogous to our previous studies, we observed that mice immunized with iFt-MHB, which are better protected, also exhibited lower levels of IL-6 ( Figure 5A), interferon (IFN)-γ (Figure 5C), and monocyte chemoattractant protein (MCP)-1 ( Figure 5D) in the lungs in the latter stage of disease (day 7 postinfection). MCP-1 levels were also lower at day 3 postinfection in the lungs of iFt-MHB immunized mice compared to that   of iFt-BHI immunized mice. MCP-1 is a chemoattractant for inflammatory monocytes, which can induce to tissue damage. Therefore, regulation of MCP-1 levels is important for protection against systemic tissue damage and subsequent mortality inflicted by Ft LVS infection. Interestingly, higher levels of IL-17 were also found at day 3 postinfection in iFt-MHB immunized mice compared to that of iFt-BHI immunized (Figure 5B). IL-17 has been found to be protective against mucosal infections with bacteria including Ft (18,19). Thus, higher IL-17 levels at early stages of infection correlate with superior protection afforded by iFt-MHB compared to iFt-BHI. Similarly, in the spleens, significantly lower levels of tumor necrosis factor (TNF)-α (at day 7 postinfection) ( Figure 5F), IFN-γ (day 5 postinfection) ( Figure 5G), and MCP-1 (day 7 postinfection) ( Figure 5H) were observed in mice immunized with iFt-MHB compared to that of iFt-BHI. However, level of IL6 (Figure 5E) in the spleens were comparable among mice immunized with iFt-MHB and iFt-BHI.

increased ab responses are induced by iFt-MhB versus iFt-Bhi
Next we investigated whether iFt generated in MHB versus BHI medium also has a differential impact on the humoral immune response against Ft. We quantified Ft-specific serum-IgG by ELISA as a measure of humoral immune response to Ft. Live Ft LVS grown in BHI and MHB medium were coated onto ELISA plates and reacted with sera obtained from iFt-MHB versus iFt-BHI-immunized mice. IgG induced by iFt-MHB compared to iFt-BHI reacted more strongly to Ft surface Ags expressed on both MHB-or BHI-grown Ft LVS, as evidenced by their relative titers (Figures 6A,B). This is consistent with superior protection afforded by iFt-MHB compared to that of iFt-BHI. The choice of Ags employed [Ft-MHB ( Figure 6A) versus Ft-BHI ( Figure 6B)] to detect Ab titers did not alter the relative pattern of Ab production observed in iFt-MHB or iFt-BHI-immunized mice.
To further characterize these Ab responses, we also used the above sera to probe by western blot lysates of MHB-and BHI-grown Ft LVS along with a BHI-grown mutant Ft (WbtA), which is deficient for O-Ag. As shown in Figure 7, the anti-iFt-MHB sera recognized a more diverse array of Ags than did the anti-iFt-BHI sera. One of the Ags preferentially recognized by the anti-iFt-MHB sera appeared to have a ladder pattern typical of LPS, and consistent with this, this ladder pattern was markedly reduced/absent in the WbtA mutant Ft. In addition, the Ag specificity of the anti-iF-BHI sera appeared to be more focused on a ~43 kDa protein with reduced O-Ag-dependent reactivity. Collectively, the data in Figures 6 and 7, as well as in data not shown, indicate that immunization with iFt-MHB induces higher titers of anti-Ft Ab and that a larger proportion of this Ab is dependent on O-Ag for binding.

iFt-MhB and iFt-Bhi Differentially Bind to BMDcs and Differentially induce expression of costimulatory Molecules
Having observed differential protective efficacy between iFt generated in MHB versus BHI, we sought to further identify the immune mechanisms underlying the differences in protective efficacy of iFt-MHB versus iFt-BHI. In our previous studies (7,9) and Figure 1, we demonstrated that iFt-BHI exhibits an antigenic profile more similar to that of in vivo circulating Ft, as apposed to that of Ft-MHB. This is in contrast to our expectation that by virtue of exposing the host to iFt-BHI, which is more similar antigenically to in vivo replicating Ft, a better protective immune response would be achieved. This led us to further investigate the immune mechanisms responsible for this unexpected observation. By using a GFP-expressing Ft LVS, we investigated whether iFt generated in MHB versus BHI have different abilities to bind to and activate APCs. In fact, we observed significantly higher binding of iFt-MHB-(GFP) to BMDCs compared to iFt-BHI-(GFP) (Figure 8). Increasing the concentration of the iFt-MHB-(GFP) successively increased the number of BMDCs harboring iFt-MHB. In contrast, iFt-BHI-(GFP) exhibited relatively poor binding to BMDCs at all concentrations used in this assay. Furthermore, costimulatory molecules are essential components for T cell activation. As demonstrated in Figure 9, iFt-MHB induced significantly higher expression of the costimulatory molecules CD80 (Figures 9A,B) and CD86 (Figures 9C,D), as compared to iFt-BHI, although CD40 (Figures 9E,F) expression was stimulated to a similar degree by both immunogens.

iFt-MhB Mediates higher Pro-inflammatory cytokine Production and increased ag Presentation to Ft-specific T cells
In addition to Ag binding and expression of costimulatory molecules on APCs, secretion of pro-inflammatory cytokines is also critical for DC-mediated orchestration of the adaptive immune response. We observed that iFt-MHB induces higher levels of the pro-inflammatory cytokines [IL-1β (Figure 10A), IL-6 ( Figure 10C), and TNF-α ( Figure 10D)], as compared to iFt-BHI, while both immunogens induced similar levels of IL-10. Finally, we investigated the presentation of iFt-MHB versus iFt-BHI using an Ft-specific T cell hybridoma (FT25 6D10), which is specific to an epitope corresponding to Ft 50 s-ribosomal protein (20) and secretes IL-5 on engagement with cognate major histocompatibility complex (MHC)peptide complex (13). Co-cultures of FT25 6D10 and APCs (PECs or BMDCs) exposed to iFt-MHB exhibited higher levels of IL-5 compared to that of iFt-BHI (Figure 11). Together, data presented in Figures 8-10 suggest that iFt-MHB harbors a superior ability to stimulate APC function as compared to iFt-BHI.

DiscUssiOn
In the present investigation, we observed that iFt propagated in MHB versus BHI medium exhibits a differential ability to stimulate an immune response to and protection against Ft challenge. Specifically, iFt-MHB immunogen induced improved protection against lethal Ft LVS challenge as compared to iFt-BHI. Consistent with the superior protective activity of iFt-MHB immunogen, bacterial burden and tissue damage were also reduced. Furthermore, immunization with iFt-MHB leads to reduced inflammation in the lungs and spleens. Importantly, it has been demonstrated that mice protected against Ft LVS challenge via immunization exhibit lower pro-inflammatory cytokines in their lungs and spleen late in infection (16,17), which we also observed in the case of iFt-MHB immunization. Notably, in contrast to the above, however, we observed elevated levels of IL-17 in lungs of mice immunized with iFt-MHB on day 3 postinfection with Ft LVS IL-17 has been implicated in protection against mucosal infections including Ft LVS (18,19). Immunization with iFt-MHB also induced higher levels of Ftspecific IgG, which have been shown to play a role in protection against Ft LVS (21). Apart from quantitative differences in IgG titers, Abs produced in response to iFt-MHB and iFt-BHI also differed qualitatively. While the dominant reactivity of anti-iFt-BHI was to a 43 kDa protein, the anti-iFt-MHB response was more broadly distributed among a range of Ags including Ft LPS (O-Ag). Ft LPS has been found to be an important Ag for protection against Ft infection (22). Although, at present, we do not know the significance of the differences in the Ab responses elicited by iFt-MHB versus iFt-BHI, further studies in this regard may reveal their impact on protection.
In regard to the mechanisms(s) by which iFt-MHB is a better immunogen, APCs are the gateway to adaptive immune response. APCs present Ags to T cells in association with MHC, which provides the primary signal for T cell activation. Other signals required for T cell activation include costimulatory molecules, e.g., CD80 and CD86 (23)(24)(25). T cells that receive signals only via TCR by engagement with MHC-peptide complex, without receiving the second signal from costimulatory ligands, are destined to undergo anergy (26). Moreover, the local cytokine niche determines the type of T cell response generated. For example, IL-12 and IFN-γ favor a T helper 1 immune response, while IL-10 and transforming growth factor-β favor a regulatory T cell immune response (27,28). Furthermore, APCs can be activated to express MHC II, costimulatory molecules, and pro-inflammatory cytokines by exposing them to various pathogen-associated molecular patterns (23). Therefore, it is important that APCs are exposed to vaccine Ags in the context of appropriate APC activation signals (29). In this investigation, iFts derived from growth in MHB medium exhibited a superior ability to activate APCs versus iFt-BHI. Specifically, MHB-derived iFt exhibited an increased ability to induce CD80 and CD86 on BMDCs. iFt-MHB also induced higher pro-inflammatory cytokine production in BMDCs. Moreover, we also observed higher levels of Ag presentation/T cell activation induced by BMDCs exposed to iFt-MHB versus to iFt-BHI. Notably, iFt-MHB also exhibited comparatively greater binding to BMDCs than iFt-BHI. The increased binding may explain the higher levels of pro-inflammatory cytokine stimulation and Ag presentation by iFt-MHB (Figure 7). Observations from other investigators are consistent with our observations (10, 11) that iFt-MHB can induce higher pro-inflammatory cytokines compared to iFt-BHI. Thus, collectively, it is clear that iFt-MHB exhibits a superior ability to impact the nature and magnitude of adaptive immune response, thereby enhancing its protective efficacy. The apparent differences in APC engagement between iFt-MHB and iFt-BHI may be a reflection of their structural differences. In this regard, we have demonstrated that BHI-grown Ft LVS expresses additional layers of capsular polysaccharides, which have been implicated in hindering Fts' ability to bind with complement and Abs (9). Capsular polysaccharides have also been implicated in masking TLR2 and Tul4 ligands on the surface of Ft (9). Furthermore, Singh et al demonstrated that Ft grown in MHB exhibited increased engagement of cytosolic DNA sensors, possibly due to structural defects resulting in an increased release of DNA into cytosol after uptake by APCs, thereby inducing AIM 2-dependent cytokine production (11). The LPS of Ft LVS has a peculiar structure that minimizes its interaction with TLR4. Also, recently it has been shown that genetic engineering of microbes that express E. coli LPS significantly enhances their TLR4 engagement with bone marrowderived macrophage (30). Thus, in future, one potential means to improve Fts' ability to engage and activate APCs may be to genetically engineer Ft to express modified TLR agonists such as E. coli LPS.
It is also important to note that environmental conditions such as temperature, metal ions present, pH, and other media ingredients used for in vitro growth can significantly influence antigenic expression by bacterial pathogens (8). Various investigations have demonstrated the significance of growth conditions on bacterial recognition by Abs (1)(2)(3). In this investigation, we demonstrate that the two growth media employed for preparation of iFt LVS whole cell vaccines significantly altered the vaccine-induced immune response and protection against cognate pathogen. Recently, BCG vaccine exhibited differential protective efficacy based on the presence or absence of detergents in the growth medium. The latter was associated with differential expression of capsular polysaccharides, the presence of capsule specific Ab, splenic IL-17 and IFN-γ production, and multifunctional CD4+ T cell responses (31). We have also observed differential protective efficacy of BHI-and MHB-grown live Ft LVS vaccine, where BHI-grown Ft LVS exhibited better protection against lethal infection with Ft SchuS4 (32). This contrasting observation may be due to the distinct immunological requirements for protection against Ft LVS and Ft SchuS4 or live versus inactivated vaccine. Thus, we are further investigating this distinction in our laboratory. Nevertheless, these studies strongly emphasize the critical importance of growth conditions when developing whole cell vaccines.
cOnclUsiOn When we started this investigation, the goal was to improve the efficacy of Ft LVS-based whole cell vaccines. Our assumption was that by exposing the host to an antigenic repertoire more similar to that of the infecting agent in vivo (iFt-BHI), a superior protective immune response would be generated as compared to iFt-MHB (7). However, as depicted in Figure 12, we observed that iFt-MHB elicited a superior ability to stimulate the immune response and protection, as compared to iFt-BHI. In conclusion, our observations indicate that to endow hosts with protective immune response, vaccine formulations should simultaneously deliver a variety of protective Ags as well as APC activation components. This investigation also emphasizes that in vitro growth conditions, which influence Ag expression as well as APC activation capacity, are critical variables that must be considered when developing whole cell-based vaccines.

eThics sTaTeMenT
This study was carried out in accordance with the recommendations of the Institutional Animal Care and Use Committee (IACUC) of Albany Medical Center, Albany, NY, USA. The protocol was approved by the IACUC of Albany Medical Center, Albany, NY, USA.
aUThOr cOnTriBUTiOns EG, KH, and SK conceptualized and designed the study. SK, RS, GP, BF, and SR performed the experiments and acquired and analyzed the data. SK drafted the manuscript. EG and KH critically revised the manuscript. All the authors approved the publication of the manuscript and agreed to be accountable for all aspects of the work.

acKnOWleDgMenTs
The authors thank Yili Lin and Li Barry, Manager, Immunology core facility, Department of Immunology and Microbial Diseases, Albany Medical College, for their assistance in acquiring flow cytometry data.

FUnDing
This work was supported by grants from the National Institutes of Health (R01AI100138-KH & EG; RO1AI123129-KROH; P01AI056320-EG and KH). The funders have no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

sUPPleMenTarY MaTerial
The Supplementary Material for this article can be found online at http://journal.frontiersin.org/article/10.3389/fimmu. 2016.00677/full#supplementary-material.