The development of safe and effective mucosal vaccines are hampered by safety concerns associated with adjuvants or live attenuated microbes. We previously demonstrated that targeting antigens to the human-Fc-gamma-receptor-I (hFcγRI) eliminates the need for adjuvants, thereby mitigating safety concerns associated with the mucosal delivery of adjuvant formulated vaccines. Here we evaluated the role of the route of immunization in the mucosal immunity elicited by the hFcγRI-targeted vaccine approach. To enable Ag targeting, PspA from Streptococcus pneumoniae (Sp) was genetically fused with the hFcγRI-targeting antibody (α-hFcγRI) to generate PspA-FP. Intranasal (IN) immunization with the PspA-FP induced significantly higher IgA, IgG, and memory T cell response in the lung mucosa compared to that of the intramuscular (IM) route, while both routes exhibited similar increase in the systemic IgG response. The IN immunization elicited better resistance against nasal colonization (NC) of Sp compared to the IM immunization. Additionally, the resistance to NC with the IN administered PspA-FP was higher than the PspA-Alum formulation administered by the IM route. While the protection form lethal pulmonary Sp infection correlated with the systemic Ab response, the resistance from NC (of Sp) correlated with the mucosal immune response. Similar to the pneumococcal pneumoniae model, the hFcγRI-targeted vaccine (based on HA as Ag) was equally protective against pulmonary Influenza virus infection via both routes. However, the IN route promoted better protection compared to the IM route against a lethal pulmonary infection with Francisella tularensis (Ft). The enhanced protection against Ft correlated with the superior mucosal immune response elicited by the IN route compared to the IM route. These observations showed a differential requirement for mucosal delivery for protection depending on the type of pathogen. Moreover, this study revealed that the hFcγRI-targeted vaccine platform is broadly-effective as an adjuvant-free mucosal vaccine platform against respiratory pathogens.

Streptococcus pneumoniae (Spn) is a common pathogen causing a secondary bacterial infection following influenza, which leads to severe morbidity and mortality during seasonal and pandemic influenza. Therefore, there is an urgent need to develop bacterial vaccines that prevent severe post-influenza bacterial pneumonia. Here, an improved Yersinia pseudotuberculosis strain (designated as YptbS46) possessing an Asd+ plasmid pSMV92 could synthesize high amounts of the Spn pneumococcal surface protein A (PspA) antigen and monophosphoryl lipid A as an adjuvant. The recombinant strain produced outer membrane vesicles (OMVs) enclosing a high amount of PspA protein (designated as OMV-PspA). A prime-boost intramuscular immunization with OMV-PspA induced both memory adaptive and innate immune responses in vaccinated mice, reduced the viral and bacterial burden, and provided complete protection against influenza-mediated secondary Spn infection. Also, the OMV-PspA immunization afforded significant cross-protection against the secondary Spn A66.1 infection and long-term protection against the secondary Spn D39 challenge. Our study implies that an OMV vaccine delivering Spn antigens can be a new promising pneumococcal vaccine candidate.

The global rollout of COVID-19 vaccines has played a critical role in reducing pandemic spread, disease severity, hospitalizations, and deaths. However, the first-generation vaccines failed to block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and transmission, partially due to the limited induction of mucosal immunity, leading to the continuous emergence of variants of concern (VOC) and breakthrough infections. To meet the challenges from VOC, limited durability, and lack of mucosal immune response of first-generation vaccines, novel approaches are being investigated. Herein, we have discussed the current knowledge pertaining to natural and vaccine-induced immunity, and the role of the mucosal immune response in controlling SARS-CoV2 infection. We have also presented the current status of the novel approaches aimed at eliciting both mucosal and systemic immunity. Finally, we have presented a novel adjuvant-free approach to elicit effective mucosal immunity against SARS-CoV-2, which lacks the safety concerns associated with live-attenuated vaccine platforms.

Visceral leishmaniasis (VL), caused by Leishmania donovani, is a major health concern in India. It represents T‐helper type 2 (Th2) bias of cytokines in active state and Th1 bias at cure. However, the role of the parasite in regulating Toll‐like receptor (TLR)‐mediated macrophage activation in VL patients remains elusive. In this report, we demonstrated that later stages of L. donovani infection rendered tolerance to macrophages, leading to incapability for the production of inflammatory cytokines like tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β in response to TLR stimulation. Overexpression of transforming growth factor (TGF)‐β1, but not IL‐10, resulted in suppressed lipopolysaccharide (LPS)‐induced production of TNF‐α and downregulation of TLR4 expression in L. donovani‐infected macrophages. Recombinant human (rh)TGF‐β1 markedly enhanced tyrosine phosphatase (Src homology region 2 domain‐containing phosphatase‐1) activity, but inhibited IL‐1 receptor‐activated kinase (IRAK)‐1 activation. Addition of neutralizing TGF‐β1 antibody reversed these effects, and thus suggesting the pivotal role of TGF‐β1 in promoting refractoriness for LPS in macrophages. Surprisingly, the use of a tyrosine phosphatase inhibitor (sodium orthovanadate, Na3VO4) promoted IRAK‐1 activation, confirming the negative inhibitory role of tyrosine phosphatase in macrophage activation. Furthermore, rhTGF‐β1 induced tolerance in infected macrophages by reducing inhibitory protein (IκBα) degradation in a time‐dependent manner. In addition, short interfering RNA studies proved that overexpression of A20 ubiquitin‐editing protein complex induced inhibitory activity of TGF‐β1 on LPS‐mediated nuclear factor‐κB activation. Thus, these findings suggest that TGF‐β1 promotes overexpression of A20 through tyrosine phosphatase activity that ensures transient activation of inflammatory signaling pathways in macrophages in active L. donovani infection.

Identifying correlates of protection (COPs) for vaccines against lethal human (Hu) pathogens, such as Francisella tularensis (Ft), is problematic, as clinical trials are currently untenable and the relevance of various animal models can be controversial. Previously, Hu trials with the live vaccine strain (LVS) demonstrated ~80% vaccine efficacy against low dose (~50 CFU) challenge; however, protection deteriorated with higher challenge doses (~2000 CFU of SchuS4) and no COPs were established. Here, we describe our efforts to develop clinically relevant, humoral COPs applicable to high-dose, aerosol challenge with S4. First, our serosurvey of LVS-vaccinated Hu and animals revealed that rabbits (Rbs), but not rodents, recapitulate the Hu O-Ag dependent Ab response to Ft. Next, we assayed Rbs immunized with distinct S4-based vaccine candidates (S4ΔclpB, S4ΔguaBA, and S4ΔaroD) and found that, across multiple vaccines, the %O-Ag dep Ab trended with vaccine efficacy. Among S4ΔguaBA-vaccinated Rbs, the %O-Ag dep Ab in pre-challenge plasma was significantly higher in survivors than in non-survivors; a cut-off of >70% O-Ag dep Ab predicted survival with high sensitivity and specificity. Finally, we found this COP in 80% of LVS-vaccinated Hu plasma samples as expected for a vaccine with 80% Hu efficacy. Collectively, the %O-Ag dep Ab response is a bona fide COP for S4ΔguaBA-vaccinated Rb and holds significant promise for guiding vaccine trials with higher animals.

Lack of safe and effective mucosal adjuvants has severely hampered the development of mucosal subunit vaccines. In this regard, we have previously shown that immunogenicity of vaccine antigens can be improved by targeting the antigens to the antigen-presenting cells. Specifically, groups of mice immunized intranasally with a fusion protein (Bivalent-FP) containing a fragment of pneumococcal-surface-protein-A (PspA) as antigen and a single-chain bivalent antibody raised against the anti-human Fc-gamma-receptor-I (hFcγRI) elicited protective immunity to pulmonary Streptococcus pneumoniae infection. In order to further enhance the immunogenicity, an additional hFcγRI-binding moiety of the single chain antibody was incorporated. The modified vaccine (Trivalent-FP) induced significantly improved protection against lethal pulmonary S. pneumoniae challenge compared to Bivalent-FP. In addition, the modified vaccine exhibited over 85% protection with only two immunizations. Trivalent-FP also induced S. pneumoniae-specific systemic and mucosal antibodies. Moreover, Trivalent-FP also induced IL-17- and IL-22-producing CD4+ T cells. Furthermore, it was found that the hFcγRI facilitated uptake and presentation of Trivalent-FP. In addition, Trivalent-FP also induced IL-1α, MIP-1α, and TNF-α; modulated recruitment of dendritic cells and macrophages; and induced CD80/86 and MHC-II expression on antigen presenting cells.

The targeting of immunogens/vaccines to specific immune cells is a promising approach for amplifying immune responses in the absence of exogenous adjuvants. However, the targeting approaches reported thus far require novel, labor-intensive reagents for each vaccine and have primarily been shown as proof-of-concept with isolated proteins and/or inactivated bacteria. We have engineered a plasmid-based, complement receptor-targeting platform that is readily applicable to live forms of multiple gram-negative bacteria, including, but not limited to, Escherichia coli, Klebsiella pneumoniae, and Francisella tularensis. Using F. tularensis as a model, we find that targeted bacteria show increased binding and uptake by macrophages, which coincides with increased p38 and p65 phosphorylation. Mice vaccinated with targeted bacteria produce higher titers of specific antibody that recognizes a greater diversity of bacterial antigens. Following challenge with homologous or heterologous isolates, these mice exhibited less weight loss and/or accelerated weight recovery as compared to counterparts vaccinated with non-targeted immunogens. Collectively, these findings provide proof-of-concept for plasmid-based, complement receptor-targeting of live gram-negative bacteria.

Adjuvants have been used in vaccines for over a century, however, the search for safe and effective vaccine adjuvants continues. In recent decades toll-like-receptor (TLR) agonists have been investigated as potential vaccine adjuvants. In this regard, the majority of the currently investigated TLR agonists are non-protein microbial components such as lipopolysaccharides, oligonucleotides, and lipopeptides. On the other hand, a growing number of studies reveal that TLR signaling and immune responses can be activated by numerous bacterial proteins. However, their potential roles as adjuvants have been somewhat overlooked. Herein, we discuss several such bacterial proteins which exhibit adjuvant properties, including the activation of TLR signaling, antigen presenting cell maturation, pro-inflammatory cytokine production and adaptive immune response. The protein nature of these TLR agonists presents several unique features not shared by non-protein TLR agonists. These properties include the amenability for modifying the structure and function as necessary for optimal immunogenicity and minimal toxicity. Protein adjuvants can be genetically fused to protein antigens which ensure the co-delivery of adjuvant-antigen not only into the same cell but also in the same endocytic cargo, leading to more effective activation of innate and adaptive immune response.

Protein-based subunit vaccines require adjuvants to increase their immunogenicity. However, there is lack of FDA-approved mucosal adjuvant. We have previously shown that targeting antigens to antigen presenting cells increases their immunogenicity. Specifically, Streptococcus pneumoniae (Sp) protective antigen (PspA) fused to a single-chain bivalent-anti human Fc-gamma-receptor-I (hFcgRI) antibody, when introduced into mice, generates protection against Sp. To further enhance the immunogenicity of the anti-hFcgRI-PspA fusion protein, we have incorporated an additional hFcgRI-binding moiety, with the resultant trivalent-anti-hFcgRI inducing better protection compared to the bivalent anti-hFcgRI-based vaccine. This improved vaccine requires fewer immunizations to confer over 90% protection, significantly higher than that of bivalent-anti-hFcgRI-PspA (less than 60%). The trivalent- anti-hFcgRI-PspA also induces significantly higher levels of Sp-specific antibodies. In addition, we observed elevated levels of mucosal IgA responses in trivalent-anti-hFcgRI-PspA-immunized mice compared to that of bivalent anti-hFcgRI-PspA. Both trivalent-anti-hFcgRI-PspA and bivalent anti-hFcgRI-PspA enhanced antigen presentation in FcgRI-dependent manner. We have further observed that trivalent-anti-hFcgRI-PspA induces recruitment of innate immune cells in the nasopharyngeal lymphoid tissue upon intranasal immunization, which is a hallmark of adjuvant activity. Thus, our study suggests that anti-hFcgRI-PspA compensates for the lack of adjuvant by inducing adjuvant effects, and can be utilized as a mucosal vaccine platform.

Vaccines save millions of lives each year from various life-threatening infectious diseases, and there are more than 20 vaccines currently licensed for human use worldwide. Moreover, in recent decades immunotherapy has become the mainstream therapy, which highlights the tremendous potential of immune response mediators, including vaccines for prevention and treatment of various forms of cancer. However, despite the tremendous advances in microbiology and immunology, there are several vaccine preventable diseases which still lack effective vaccines. Classically, weakened forms (attenuated) of pathogenic microbes were used as vaccines. Although the attenuated microbes induce effective immune response, a significant risk of reversion to pathogenic forms remains. While in the twenty-first century, with the advent of genetic engineering, microbes can be tailored with desired properties. In this review, I have focused on the use of genetically modified bacteria for the delivery of vaccine antigens. More specifically, the live-attenuated bacteria, derived from pathogenic bacteria, possess many features that make them highly suitable vectors for the delivery of vaccine antigens. Bacteria can theoretically express any heterologous gene or can deliver mammalian expression vectors harboring vaccine antigens (DNA vaccines). These properties of live-attenuated microbes are being harnessed to make vaccines against several infectious and noninfectious diseases. In this regard, I have described the desired features of live-attenuated bacterial vectors and the mechanisms of immune responses manifested by live-attenuated bacterial vectors. Interestingly anaerobic bacteria are naturally attracted to tumors, which make them suitable vehicles to deliver tumor-associated antigens thus I have discussed important studies investigating the role of bacterial vectors in immunotherapy. Finally, I have provided important discussion on novel approaches for improvement and tailoring of live-attenuated bacterial vectors for the generation of desired immune responses.

Francisella tularensis (Ft) is a biothreat agent for which there is no FDA-approved human vaccine. Currently, there are substantial efforts underway to develop both vaccines and the tools to assess these vaccines. Tularemia laboratory research has historically relied primarily upon a small number of inbred mouse strains, but the utility of such findings to outbred animals may be limited. Specifically, C57BL/6 mice are more susceptible than BALB/c mice to Ft infection and less easily protected against challenge with highly virulent type A Ft. Thus, depending on the inbred mouse strain used, one could be misled as to which immunogen(s)/vaccine will ultimately be effective in an outbred human population. Accordingly, we evaluated an outbred Swiss Webster (SW) mouse model in direct comparison to a well-established, inbred C57BL/6 mouse model. Mucosal vaccination with the live, attenuated Ft LVS superoxide dismutase (sodB) mutant demonstrated significantly higher protection in outbred SW mice compared to inbred C57BL/6 mice against Ft SchuS4 respiratory challenge. The protection observed in vaccinated outbred mice correlated with lower bacterial density, reduced tissue inflammation, and reduced levels of pro-inflammatory cytokine production. This protection was CD4+ and CD8+ T cell-dependent and characterized by lower titers of serum antibody (Ab) that qualitatively differed from vaccinated inbred mice. Enhanced protection of vaccinated outbred mice correlated with early and robust production of IFN-γ and IL-17A. Neutralizing Ab administered at the time of challenge revealed that IFN-γ was central to this protection, while IL-17A neutralization did not alter bacterial burden or survival. The present study demonstrates the utility of the outbred mouse as an alternative vaccination model for testing tularemia vaccines. Given the limited MHC repertoire in inbred mice, this outbred model is more analogous to the human in terms of immunological diversity.

Francisella tularensis (Ft) is a biothreat agent for which there is no FDA-approved human vaccine. Currently, there are substantial efforts underway to develop both vaccines and improved tools to assess these vaccines. Ft expresses distinct sets of antigens (Ags) in vivo as compared to those expressed in vitro. Importantly, Ft grown in brain-heart infusion medium (BHIM) more closely mimics the antigenic profile of macrophage-grown Ft when compared to Mueller-Hinton medium (MHM)-grown Ft. Thus, we predicted that when used as a live vaccine BHIM-grown Ft (BHIM-Ft) would provide better protection, as compared to MHM-Ft. We first determined if there was a difference in growth kinetics between BHIM and MHM-Ft. We found that BHIM-Ft exhibited an initial growth advantage ex vivo that manifests as slightly hastened intracellular replication as compared to MHM-Ft. We also observed that BHIM-Ft exhibited an initial growth advantage in vivo represented by rapid bacterial expansion and systemic dissemination associated with a slightly shorter mean survival time of naive animals. Next, using two distinct strains of Ft LVS (WT and sodB), we observed that mice vaccinated with live BHIM-Ft LVS exhibited significantly better protection against Ft SchuS4 respiratory challenge compared to MHM-Ft-immunized mice. This enhanced protection correlated with lower bacterial burden, reduced tissue inflammation, and reduced pro-inflammatory cytokine production late in infection. Splenocytes from BHIM-Ft sodB-immunized mice contained more CD4+, effector, memory T-cells, and were more effective at limiting intracellular replication of Ft LVS in vitro. Concurrent with enhanced killing of Ft LVS, BHIM-Ft sodB-immune splenocytes produced significantly higher levels of IFN-γ and IL-17A cytokines than their MHM-Ft sodB-immunized counterparts indicating development of a more effective T cell memory response when immunizing mice with BHIM-Ft.

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.

The gram-negative bacterium Francisella tularensis (Ft) is both a potential biological weapon and a naturally occurring microbe that survives in arthropods, fresh water amoeba, and mammals with distinct phenotypes in various environments. Previously, we used a number of measurements to characterize Ft grown in Brain-Heart Infusion (BHI) broth as (1) more similar to infection-derived bacteria, and (2) slightly more virulent in naïve animals, compared to Ft grown in Mueller Hinton Broth (MHB). In these studies we observed that the free amino acids in MHB repress expression of select Ft virulence factors by an unknown mechanism. Here, we tested the hypotheses that Ft grown in BHI (BHI-Ft) accurately displays a full protein composition more similar to that reported for infection-derived Ft and that this similarity would make BHI-Ft more susceptible to pre-existing, vaccine-induced immunity than MHB-Ft. We performed comprehensive proteomic analysis of Ft grown in MHB, BHI, and BHI supplemented with casamino acids (BCA) and compared our findings to published “omics” data derived from Ft grown in vivo. Based on the abundance of ~1,000 proteins, the fingerprint of BHI-Ft is one of nutrient-deprived bacteria that—through induction of a stringent-starvation-like response—have induced the FevR regulon for expression of the bacterium's virulence factors, immuno-dominant antigens, and surface-carbohydrate synthases. To test the notion that increased abundance of dominant antigens expressed by BHI-Ft would render these bacteria more susceptible to pre-existing, vaccine-induced immunity, we employed a battery of LVS-vaccination and S4-challenge protocols using MHB- and BHI-grown Ft S4. Contrary to our hypothesis, these experiments reveal that LVS-immunization provides a barrier to infection that is significantly more effective against an MHB-S4 challenge than a BHI-S4 challenge. The differences in apparent virulence to immunized mice are profoundly greater than those observed with primary infection of naïve mice. Our findings suggest that tularemia vaccination studies should be critically evaluated in regard to the growth conditions of the challenge agent.

Numerous studies have demonstrated that administration of antigen (Ag)-pulsed dendritic cells (DCs) is an effective strategy for enhancing immunity to tumors and infectious disease organisms. However, the generation and/or isolation of DCs can require substantial time and expense. Therefore, using inactivated F. tularensis (iFt) Ag as a model immunogen, we first sought to determine if DCs could be replaced with peripheral blood mononuclear cells (PBMCs) during the ex-vivo pulse phase and still provide protection against Ft infection. Follow up studies were then conducted using the S. pneumoniae (Sp) vaccine Prevnar ®13 as the Ag in the pulse phase followed by immunization and Sp challenge. In both cases, we demonstrate that PBMCs can be used in place of DCs when pulsing with iFt and/or Prevnar ®13 ex vivo and re-administering the Ag-pulsed PBMCs as a vaccine. In addition, utilization of the i.n. route for Ag-pulsed PBMC administration is superior to use of the i.v. route in the case of Sp immunization, as well as when compared to direct injection of Prevnar ®13 vaccine i.m. or i.n. Furthermore, this PBMC-based vaccine strategy provides a more marked and enduring protective immune response and is also capable of serving as a multi-organism vaccine platform. The potential for this ex-vivo vaccine strategy to provide a simpler, less time consuming, and less expensive approach to DC-based vaccines and vaccination in general is also discussed.

Abstract Francisella tularensis (Ft) is a Category A biothreat agent for which there currently is no FDA-approved vaccine. Thus, there is a substantial effort underway to develop an effective tularemia vaccine. While it is well established that gender can significantly impact susceptibility to primary infection, the impact of gender on vaccine efficacy is not well established. Thus, development of a successful vaccine against tularemia will require an understanding of this impact. In this study, a role for gender in vaccine-induced protection against tularemia is identified for the first time. Mucosal vaccination with inactivated Ft (iFt) LVS elicited gender-based protection in C57BL/6 mice against respiratory challenge with Ft LVS. Specifically, vaccinated male mice were more susceptible to subsequent Ft LVS challenge. This increased susceptibility in male mice correlated with increased bacterial burden, tissue inflammation, and elevated proinflammatory cytokine production late in infection. In contrast, improved survival of iFt-vaccinated female mice correlated with reduced bacterial burden and enhanced levels of Ft-specific Abs in serum and broncho-alveolar lavage (BAL) fluid. Furthermore, vaccination with a live attenuated vaccine consisting of an Ft LVS super oxide dismutase (SodB) mutant demonstrated a similar gender bias in protection following Ft SchuS4 challenge. Of particular significance is the fact that these are the first studies to demonstrate that gender differences impact disease outcome in the case of lethal tularemia following mucosal vaccination. In addition, these studies further emphasize the fact that gender differences must be a serious consideration in any future tularemia vaccine development studies

Francisella tularensis (Ft) is a gram-negative intercellular pathogen and category A biothreat agent. However, despite 15 years of strong government investment and intense research focused on the development of a US Food and Drug Administration-approved vaccine against Ft, the primary goal remains elusive. This article reviews research efforts focused on developing an Ft vaccine, as well as a number of important factors, some only recently recognized as such, which can significantly impact the development and evaluation of Ft vaccine efficacy. Finally, an assessment is provided as to whether a US Food and Drug Administration-approved Ft vaccine is likely to be forthcoming and the potential means by which this might be achieved.

Francisella tularensis (Ft) can survive in a variety of habitats with a remarkable ability to adapt to changing environmental conditions. For example, Ft expresses a distinct set of antigens (Ags) in the macrophage, which are not expressed in Ft grown in vitro with Mueler Hinton Broth (MHB) medium. In contrast, Ft grown in Brain Heart Infusion (BHI) medium exhibits an antigenic profile more closely resembling that of in vivo growth conditions. In addition, studies also suggest an alteration in immunological properties of Ft when grown in BHI versus MHB medium. Thus, we investigated the protective efficacy of BHI and MHB-grown Ft, when inactivated and used as an immunogen to protect against Ft LVS challenge. Inactivated Ft (iFt) generated in MHB (iFt-MHB) was found to have superior protective efficacy as compared to that of the iFt generated in BHI (iFt-BHI). The enhanced protection generated by iFt- MHB-immunized mice was also associated with lower bacterial burden and sepsis-like conditions in the lungs and spleens of iFt-MHB versus iFt-BHI-immunized mice. Further evaluation of early immunological events revealed, in the case of iFt-MHB immunogen, superior Ag binding to Ag-presenting dendritic cells (DCs), increased MHC class II expression by DCs, and enhanced Ag presentation by DCs, as apposed to the use of iFt-BHI. Also, iFt-MHB induced increased levels of Ft-specific IgG as well as IFNg and IL17 production versus that of iFt-BHI. These studies thus demonstrate the critical importance of considering bacterial growth medium in Ft vaccine development.