There is a requirement for an efficacious vaccine to protect people against infection fromFrancisella tularensis, the etiological agent of tularemia. The lipopolysaccharide (LPS) ofF. tularensisis suboptimally protective against a parenteral lethal challenge in mice. To develop a more efficacious subunit vaccine, we have used a novel biosynthetic technique of protein glycan coupling technology (PGCT) that exploits bacterial N-linked glycosylation to recombinantly conjugateF. tularensisO-antigen glycans to the immunogenic carrier proteinPseudomonas aeruginosaexoprotein A (ExoA). Previously, we demonstrated that an ExoA glycoconjugate with two glycosylation sequons was capable of providing significant protection to mice against a challenge with a low-virulence strain ofF. tularensis. Here, we have generated a more heavily glycosylated conjugate vaccine and evaluated its efficacy in a Fischer 344 rat model of tularemia. We demonstrate that this glycoconjugate vaccine protected rats against disease and the lethality of an inhalational challenge withF. tularensisSchu S4. Our data highlights the potential of this biosynthetic approach for the creation of next-generation tularemia subunit vaccines.

Francisella tularensis is an intracellular pathogen causing the disease tularemia, and an organism of concern to biodefence. There is no licensed vaccine available. Subunit approaches have failed to induce protection, which requires both humoral and cellular immune memory responses, and have been hampered by a lack of understanding as to which antigens are immunoprotective. We undertook a preliminary in silico analysis to identify candidate protein antigens. These antigens were then recombinantly expressed and encapsulated into glucan particles (GPs), purified Saccharomyces cerevisiae cell walls composed primarily of β-1,3-glucans. Immunological profiling in the mouse was used to down-selection to seven lead antigens: FTT1043 (Mip), IglC, FTT0814, FTT0438, FTT0071 (GltA), FTT0289, FTT0890 (PilA) prior to transitioning their evaluation to a Fischer 344 rat model for efficacy evaluation. F344 rats were vaccinated with the GP protein antigens co-delivered with GP-loaded with Francisella LPS. Measurement of cell mediated immune responses and computational epitope analysis allowed down-selection to three promising candidates: FTT0438, FTT1043 and FTT0814. Of these, a GP vaccine delivering Francisella LPS and the FTT0814 protein was able to induce protection in rats against an aerosol challenge of F. tularensis SchuS4, and reduced organ colonisation and clinical signs below that which immunisation with a GP-LPS alone vaccine provided. This is the first report of a protein supplementing protection induced by LPS in a Francisella vaccine. This paves the way for developing an effective, safe subunit vaccine for the prevention of inhalational tularemia, and validates the GP platform for vaccine delivery where complex immune responses are required for prevention of infections by intracellular pathogens.

The immune response against mycobacterial infections is dependant upon a complex interaction between T lymphocytes and macrophages in the context of the granuloma. For this study, we performed the analysis of 18 stage I or II, and 13 stage III or IV granulomas found in lymph nodes from 8 experimentally and 2 naturally infected cattle. T-cell subpopulations (CD3⁺, CD4⁺, CD8⁺, WC1⁺, CD25⁺) were investigated by immunohistochemistry. In the majority of stage I/II lesions, CD8⁺ and CD25⁺ cells were predominantly found in the lymphocytic outer region of the granuloma, suggesting a possible role for activated CD8⁺ cells in the initial attempt to restrain the granuloma growth. CD4⁺ T cells appeared equally distributed in the lymphocytic mantle and in the internal areas of the granulomas. WC1⁺ cells appeared interspersed among the macrophages. We speculated that this could indicate a role for these 2 subsets in the maintenance and the maturation of the granuloma. In stage III/IV lesions, all of the T-cell subsets investigated appeared interspersed among the mononuclear component of the granulomas. In general terms, there was a higher density of CD8⁺ cells compared with CD4⁺ cells. However, there was no sense of rimming effect for any of the investigated cell populations.

Tularemia is a zoonotic disease caused by the facultative intracellular gram-negative bacterium Francisella tularensis. F. tularensis has a very low infection dose by the aerosol route which can result in an acute, and potentially lethal, infection in humans. Consequently, it is classified as a Category A bioterrorism agent by the US Centers for Disease Control (CDC) and is a pathogen of concern for the International Biodefence community. There are currently no licenced tularemia vaccines. In this study we report on the continued assessment of a tularemia subunit vaccine utilising β-glucan particles (GPs) as a vaccine delivery platform for immunogenic F. tularensis antigens. Using a Fischer 344 rat infection model, we demonstrate that a GP based vaccine comprising the F. tularensis lipopolysaccharide antigen together with the protein antigen FTT0814 provided partial protection of F344 rats against an aerosol challenge with a high virulence strain of F. tularensis, SCHU S4. Inclusion of imiquimod as an adjuvant failed to enhance protective efficacy. Moreover, the level of protection afforded was dependant on the challenge dose. Immunological characterisation of this vaccine demonstrated that it induced strong antibody immunoglobulin responses to both polysaccharide and protein antigens. Furthermore, we demonstrate that the FTT0814 component of the GP vaccine primed CD4⁺ and CD8⁺ T-cells from immunised F344 rats to express interferon-γ, and CD4⁺ cells to express interleukin-17, in an antigen specific manner. These data demonstrate the development potential of this tularemia subunit vaccine and builds on a body of work highlighting GPs as a promising vaccine platform for difficult to treat pathogens including those of concern to the bio-defence community.

Antimicrobial resistance is a global issue, and the investigation of alternative therapies that are not traditional antibiotics are warranted. Novel bacterial type II topoisomerase inhibitors (NBTIs) have recently emerged as a novel class of antibiotics with reduced potential for cross-resistance to fluoroquinolones due to their novel mechanism of action. This study investigated the in vitro activity of a series of cyclohexyl–oxazolidinone bacterial topoisomerase inhibitors against type strains of Francisella tularensis and Burkholderia pseudomallei. Broth microdilution, time-kill, and cell infection assays were performed to determine activity against these biothreat pathogens. Two candidates were identified that demonstrated in vitro activity in multiple assays that in some instances was equivalent to ciprofloxacin and doxycycline. These data warrant the further evaluation of these novel NBTIs and future iterations in vitro and in vivo.

The timing of the initiation of antibiotic treatment has been shown to impact the clinical outcome of many bacterial infections, including Q fever. Delayed, suboptimal or incorrect antibiotic treatment has been shown to result in poor prognosis, resulting in the progression of acute disease to long-term chronic sequalae. Therefore, there is a requirement to identify an optimal, effective therapeutic regimen to treat acute Q fever. In the study, the efficacies of different doxycycline monohydrate regimens (pre-exposure prophylaxis, post-exposure prophylaxis or treatment at symptom onset or resolution) were evaluated in an inhalational murine model of Q fever. Different treatment lengths (7 or 14 days) were also evaluated. Clinical signs and weight loss were monitored during infection and mice were euthanized at different time points to characterize bacterial colonization in the lungs and the dissemination of bacteria to other tissues including the spleen, brain, testes, bone marrow and adipose. Post-exposure prophylaxis or doxycycline treatment starting at symptoms onset reduced clinical signs, and also delayed the systemic clearance of viable bacteria from key tissues. Effective clearance was dependent on the development of an adaptive immune response, but also driven by sufficient bacterial activity to maintain an active immune response. Pre-exposure prophylaxis or post-exposure treatment at the resolution of clinical signs did not improve outcomes. These are the first studies to experimentally evaluate different doxycycline treatment regimens for Q fever and illustrate the need to explore the efficacy of other novel antibiotics.