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

Abstract

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.

Differential Growth of Francisella tularensis, Which Alters Expression of Virulence Factors, Dominant Antigens, and Surface-Carbohydrate Synthases, Governs the Apparent Virulence of Ft SchuS4 to Immunized Animals

“…This pro-inflammatory nature which makes i Ft LVS a better immunogen (Kumar et al, 2016) could also make MHB- Ft S4 a worse pathogen. Previously, we stained early-log phase MHB- and BHI-grown Ft with a fluorescent vital dye mix and analyzed the stained bacteria by flow cytometry.…”

Differential In Vitro Cultivation of Francisella tularensis Influences Live Vaccine Protective Efficacy by Altering the Immune Response

“…The challenge pathogen Ft SchuS4 was cultured aerobically in MHM or BHIM broth (Becton Dickinson, Sparks, Maryland) and the active mid-log phase bacteria were harvested and used for mouse infection ( 8 , 9 ). Inactivated Ft (i Ft ) was generated, as previously described ( 5 ). For in vivo imaging studies, Ft LVS was transformed with the luminescence reporter plasmid (pXB173-lux), as described previously by Bina et al ( 20 ).…”

“…In addition to the antigen (Ag)/immunogen utilized, a number of other key factors influence vaccine efficacy, which include: bacterial strain ( 1 , 2 ), growth conditions of the attenuated or killed vaccine, challenge strain ( 3 – 5 ), as well as the genetic background ( 6 , 7 ), and gender of the host ( 8 – 10 ). Regarding bacterial growth conditions, immunogens used as attenuated or killed vaccines must first be grown in vitro .…”

“…Regarding bacterial growth conditions, immunogens used as attenuated or killed vaccines must first be grown in vitro . Studies have clearly demonstrated that the protective ability of such vaccines are dictated in part by the medium in which the vaccine candidate is generated with the choice of medium potentially altering the antigenic composition and thus efficacy of whole cell-based attenuated and killed vaccines ( 3 , 5 , 11 ). Specifically, the in vitro culture medium utilized to generate whole cell-based vaccines can impact the expression of antigenic determinants, pathogen-associated molecular patterns, and virulence factors expressed by the infectious agent.…”

“…Similarly, the influence of growth media on tularemia vaccination has also been demonstrated. Specifically, in the case of inactivated vaccine, we reported that the immune-stimulatory nature of MHM-grown inactivated Ft (i Ft ) generates better protection against Ft LVS challenge ( 5 ). In contrast, our preliminary studies using live Ft vaccination indicate BHIM- Ft LVS - immunized mice are better protected against Ft SchuS4 mucosal challenge than that of live Ft LVS grown in MHM ( 8 ).…”