An Improved Tobacco Mosaic Virus (TMV)-Conjugated Multiantigen Subunit Vaccine Against Respiratory Tularemia.

Francisella tularensis, the causative agent of the fatal human disease known as tularemia is classified as a Category A Select Agent by the Centers for Disease Control. No licensed vaccine is currently available for prevention of tularemia in the United States. Previously, we published that a tri-antigen tobacco mosaic virus (TMV) vaccine confers 50% protection in immunized mice against respiratory tularemia caused by F. tularensis. In this study, we refined the TMV-vaccine formulation to improve the level of protection in immunized C57BL/6 mice against respiratory tularemia. We developed a tetra-antigen vaccine by conjugating OmpA, DnaK, Tul4, and SucB proteins of Francisella to TMV. CpG was also included in the vaccine formulation as an adjuvant. Primary intranasal (i.n.) immunization followed by two booster immunizations with the tetra-antigen TMV vaccine protected 100% mice against i.n. 10LD100 challenges dose of F. tularensis live vaccine strain (LVS). Mice receiving three immunization doses of tetra-antigen TMV vaccine showed only transient body weight loss, cleared the infection rapidly, and showed minimal histopathological lesions in lungs, liver, and spleen following a lethal respiratory challenge with F. tularensis LVS. Mice immunized with the tetra-antigen TMV vaccine also induced strong ex vivo recall responses and were protected against a lethal challenge as late as 163 days post-primary immunization. Three immunization with the tetra-antigen TMV vaccine also induced a stronger humoral immune response predominated by IgG1, IgG2b, and IgG2c antibodies than mice receiving only a single or two immunizations. Remarkably, a single dose protected 40% of mice, while two doses protected 80% of mice from lethal pathogen challenge. Immunization of Interferon-gamma (IFN-γ)-deficient mice with the tetra-antigen TMV vaccine demonstrated an absolute requirement of IFN-γ for the generation of protective immune response against a lethal respiratory challenge with F. tularensis LVS. Collectively, this study further demonstrates the feasibility of TMV as an efficient platform for the delivery of multiple F. tularensis antigens and that tetra-antigen TMV vaccine formulation provides complete protection, and induces long-lasting protective and memory immune responses against respiratory tularemia caused by F. tularensis LVS.

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.

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.

Temperature-Dependent Gentamicin Resistance of Francisella tularensis is Mediated by Uptake Modulation.

Gentamicin (Gm) is an aminoglycoside commonly used to treat bacterial infections such as tularemia - the disease caused by Francisella tularensis. In addition to being pathogenic, F. tularensis is found in environmental niches such as soil where this bacterium likely encounters Gm producers (Micromonospora sp.). Here we show that F. tularensis exhibits increased resistance to Gm at ambient temperature (26°C) compared to mammalian body temperature (37°C). To evaluate whether F. tularensis was less permeable to Gm at 26°C, a fluorescent marker [Texas Red (Tr)] was conjugated with Gm, yielding Tr-Gm. Bacteria incubated at 26°C showed reduced fluorescence compared to those at 37°C when exposed to Tr-Gm suggesting that uptake of Gm was reduced at 26°C. Unconjugated Gm competitively inhibited uptake of Tr-Gm, demonstrating that this fluorescent compound was taken up similarly to unconjugated Gm. Lysates of F. tularensis bacteria incubated with Gm at 37°C inhibited the growth of Escherichia coli significantly more than lysates from bacteria incubated at 26°C, further indicating reduced uptake at this lower temperature. Other facultative pathogens (Listeria monocytogenes and Klebsiella pneumoniae) exhibited increased resistance to Gm at 26°C suggesting that the results generated using F. tularensis may be generalizable to diverse bacteria. Regulation of the uptake of antibiotics provides a mechanism by which facultative pathogens survive alongside antibiotic-producing microbes in nature.

Naturally occurring hypothermia is more advantageous than fever in severe forms of lipopolysaccharide- and Escherichia coli-induced systemic inflammation.

The natural switch from fever to hypothermia observed in the most severe cases of systemic inflammation is a phenomenon that continues to puzzle clinicians and scientists. The present study was the first to evaluate in direct experiments how the development of hypothermia vs. fever during severe forms of systemic inflammation impacts the pathophysiology of this malady and mortality rates in rats. Following administration of bacterial lipopolysaccharide (LPS; 5 or 18 mg/kg) or of a clinical Escherichia coli isolate (5 × 10(9) or 1 × 10(10) CFU/kg), hypothermia developed in rats exposed to a mildly cool environment, but not in rats exposed to a warm environment; only fever was revealed in the warm environment. Development of hypothermia instead of fever suppressed endotoxemia in E. coli-infected rats, but not in LPS-injected rats. The infiltration of the lungs by neutrophils was similarly suppressed in E. coli-infected rats of the hypothermic group. These potentially beneficial effects came with costs, as hypothermia increased bacterial burden in the liver. Furthermore, the hypotensive responses to LPS or E. coli were exaggerated in rats of the hypothermic group. This exaggeration, however, occurred independently of changes in inflammatory cytokines and prostaglandins. Despite possible costs, development of hypothermia lessened abdominal organ dysfunction and reduced overall mortality rates in both the E. coli and LPS models. By demonstrating that naturally occurring hypothermia is more advantageous than fever in severe forms of aseptic (LPS-induced) or septic (E. coli-induced) systemic inflammation, this study provides new grounds for the management of this deadly condition.

Humoral and cell-mediated immunity to the intracellular pathogen Francisella tularensis.

SUMMARY: Francisella tularensis can cause fatal respiratory tularemia in humans and animals and is increasingly being isolated in the United States and several European countries. The correlates of protective immunity against this intracellular bacterium are not known, and currently there are no licensed vaccines available for human use. Cell-mediated immunity has long been believed to be critical for protection, and the importance of humoral immunity is also now recognized. Furthermore, synergy between antibodies, T cell-derived cytokines, and phagocytes appears to be critical to achieve sterilizing immunity against F. tularensis. Thus, novel vaccine approaches should be designed to induce robust antibody and cell-mediated immune responses to this pathogen.