A glucan-particle based tularemia subunit vaccine induces T-cell immunity and affords partial protection in an inhalation rat infection model.

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 ß-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.

Layering vaccination with antibiotic therapy results in protection and clearance of Burkholderia pseudomallei in Balb/c mice.

Melioidosis is a disease that is difficult to treat due to the causative organism, Burkholderia pseudomallei being inherently antibiotic resistant and it having the ability to invade, survive, and replicate in an intracellular environment. Combination therapy approaches are routinely being evaluated in animal models with the aim of improving the level of protection and clearance of colonizing bacteria detected. In this study, a subunit vaccine layered with the antibiotic finafloxacin was evaluated in vivo against an inhalational infection with B. pseudomallei in Balb/c mice. Groups of mice vaccinated, infected, and euthanized at antibiotic initiation had a reduced bacterial load compared to those that had not been immunized. In addition, the subunit vaccine provided a synergistic effect when it was delivered with a CpG ODN and finafloxacin was initiated at 48 h post-challenge. Vaccination was also shown to improve the outcome, in a composite measure of survival and clearance. In summary, layering a subunit vaccine with the antibiotic finafloxacin is a promising therapeutic alternative for use in the treatment of B. pseudomallei infections.

Predatory bacteria can protect SKH-1 mice from a lethal plague challenge.

With the rise of antimicrobial resistance, novel ways to treat bacterial infections are required and the use of predatory bacteria may be one such approach. Bdellovibrio species have been shown in vitro to predate on a wide range of other Gram-negative bacteria, including CDC category A/B pathogens such as Yersinia pestis. The data reported here show that treatment of SKH-1 mice with Bdellovibrio bacteriovorus HD100 provided significant protection from a lethal challenge of Yersinia pestis CO92. This is the first report of protection conferred by predation in vivo against a systemic pathogen challenge. However, this protective effect was not observed in a preliminary study with Balb/c mice. Therefore the effects of the predatory bacteria are complex and may be dependent on immune status/genetics of the host. Overall, predatory bacteria may have utility as a therapeutic modality but further work is required to understand the predator-host interaction.

Protection induced by a Francisella tularensis subunit vaccine delivered by glucan particles.

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 ß-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.

A Bioluminescent Francisella tularensis SCHU S4 Strain Enables Noninvasive Tracking of Bacterial Dissemination and the Evaluation of Antibiotics in an Inhalational Mouse Model of Tularemia.

Bioluminescence imaging (BLI) enables real-time, noninvasive tracking of infection in vivo and longitudinal infection studies. In this study, a bioluminescent Francisella tularensis strain, SCHU S4-lux, was used to develop an inhalational infection model in BALB/c mice. Mice were infected intranasally, and the progression of infection was monitored in real time using BLI. A bioluminescent signal was detectable from 3 days postinfection (3 dpi), initially in the spleen and then in the liver and lymph nodes, before finally becoming systemic. The level of bioluminescent signal correlated with bacterial numbers in vivo, enabling noninvasive quantification of bacterial burdens in tissues. Treatment with levofloxacin (commencing at 4 dpi) significantly reduced the BLI signal. Furthermore, BLI was able to distinguish noninvasively between different levofloxacin treatment regimens and to identify sites of relapse following treatment cessation. These data demonstrate that BLI and SCHU S4-lux are suitable for the study of F. tularensis pathogenesis and the evaluation of therapeutics for tularemia.

Assessment of antimicrobial peptide LL-37 as a post-exposure therapy to protect against respiratory tularemia in mice.

Early activation of the innate immune response is important for protection against infection with Francisella tularensis live vaccine strain (LVS) in mice. The human cathelicidin antimicrobial peptide LL-37 is known to have immunomodulatory properties, and therefore exogenously administered LL-37 may be suitable as an early post-exposure therapy to protect against LVS infection. LL-37 has been evaluated for immunostimulatory activity in uninfected mice and for activity against LVS in macrophage assays and protective efficacy when administered post-challenge in a mouse model of respiratory tularemia. Increased levels of pro-inflammatory cytokine IL-6, chemokines monocyte chemoattractant protein 1 (MCP-1) and CXCL1 with increased neutrophil influx into the lungs were observed in uninfected mice after intranasal administration of LL-37. Following LVS challenge, LL-37 administration resulted in increased IL-6, IL-12 p70, IFNγ and MCP-1 production, a slowing of LVS growth in the lung, and a significant extension of mean time to death compared to control mice. However, protection was transient, with the LL-37 treated mice eventually succumbing to infection. As this short course of nasally delivered LL-37 was moderately effective at overcoming the immunosuppressive effects of LVS infection this suggests that a more sustained treatment regimen may be an effective therapy against this pathogen.

A Toll/interleukin (IL)-1 receptor domain protein from Yersinia pestis interacts with mammalian IL-1/Toll-like receptor pathways but does not play a central role in the virulence of Y. pestis in a mouse model of bubonic plague.

The Toll/interleukin (IL)-1 receptor (TIR) domain is an essential component of eukaryotic innate immune signalling pathways. Interaction between TIR domains present in Toll-like receptors and associated adaptors initiates and propagates an immune signalling cascade. Proteins containing TIR domains have also been discovered in bacteria. Studies have subsequently shown that these proteins are able to modulate mammalian immune signalling pathways dependent on TIR interactions and that this may represent an evasion strategy for bacterial pathogens. Here, we investigate a TIR domain protein from the highly virulent bacterium Yersinia pestis, the causative agent of plague. When overexpressed in vitro this protein is able to downregulate IL-1ß- and LPS-dependent signalling to NFκB and to interact with the TIR adaptor protein MyD88. This interaction is dependent on a single proline residue. However, a Y. pestis knockout mutant lacking the TIR domain protein was not attenuated in virulence in a mouse model of bubonic plague. Minor alterations in the host cytokine response to the mutant were indicated, suggesting a potential subtle role in pathogenesis. The Y. pestis mutant also showed increased auto-aggregation and reduced survival in high-salinity conditions, phenotypes which may contribute to pathogenesis or survival.

An anthrax subunit vaccine candidate based on protective regions of Bacillus anthracis protective antigen and lethal factor.

Studies have confirmed the key role of Bacillus anthracis protective antigen (PA) in the US and UK human anthrax vaccines. However, given the tripartite nature of the toxin, other components, including lethal factor (LF), are also likely to contribute to protection. We examined the antibody and T cell responses to PA and LF in human volunteers immunized with the UK anthrax vaccine (AVP). Individual LF domains were assessed for immunogenicity in mice when given alone or with PA. Based on the results obtained, a novel fusion protein comprising D1 of LF and the host cell-binding domain of PA (D4) was assessed for protective efficacy. Murine protection studies demonstrated that both full-length LF and D1 of LF conferred complete protection against a lethal intraperitoneal challenge with B. anthracis STI spores. Subsequent studies with the LFD1-PAD4 fusion protein showed a similar level of protection. LF is immunogenic in humans and is likely to contribute to the protection stimulated by AVP. A single vaccine comprising protective regions from LF and PA would simplify production and confer a broader spectrum of protection than that seen with PA alone.

Small protective fragments of the Yersinia pestis V antigen.

Yersinia pestis is the causative agent of plague. Naturally occurring cases of the disease and the potential use of Y. pestis as a bioweapon fuel the need for efficacious vaccines. The most recent plague vaccine is a killed whole cell preparation that is expensive to manufacture and its side effects are common. The protective antigens F1 and V have been identified and are currently being developed as a combined subunit vaccine. Protective epitopes of the V antigen have previously been shown to reside in the central part of the protein. In order to identify the minimum protective fragment of the V antigen that can provide protection against plague, the structures of several small fragments of the antigen were modelled in silico and recombinant proteins were produced. These fragments were probed for the retention of a protective epitope using a protective monoclonal antibody and protection against Y. pestis in mice was determined. The smallest protective fragment of V antigen identified comprised amino acids 135-262. Finally the ability of this fragment to confer protection when given in the context of a DNA vaccine was confirmed.

Human monoclonal antibodies against anthrax lethal factor and protective antigen act independently to protect against Bacillus anthracis infection and enhance endogenous immunity to anthrax.

The unpredictable nature of bioterrorism and the absence of real-time detection systems have highlighted the need for an efficient postexposure therapy for Bacillus anthracis infection. One approach is passive immunization through the administration of antibodies that mitigate the biological action of anthrax toxin. We isolated and characterized two protective fully human monoclonal antibodies with specificity for protective antigen (PA) and lethal factor (LF). These antibodies, designated IQNPA (anti-PA) and IQNLF (anti-LF), were developed as hybridomas from individuals immunized with licensed anthrax vaccine. The effective concentration of IQNPA that neutralized 50% of the toxin in anthrax toxin neutralization assays was 0.3 nM, while 0.1 nM IQNLF neutralized the same amount of toxin. When combined, the antibodies had additive neutralization efficacy. IQNPA binds to domain IV of PA containing the host cell receptor binding site, while IQNLF recognizes domain I containing the PA binding region in LF. A single 180-mug dose of either antibody given to A/J mice 2.5 h before challenge conferred 100% protection against a lethal intraperitoneal spore challenge with 24 50% lethal doses [LD50s] of B. anthracis Sterne and against rechallenge on day 20 with a more aggressive challenge dose of 41 LD50s. Mice treated with either antibody and infected with B. anthracis Sterne developed detectable murine anti-PA and anti-LF immunoglobulin G antibody responses by day 17 that were dependent on which antibody the mice had received. Based on these results, IQNPA and IQNLF act independently during prophylactic anthrax treatment and do not interfere with the establishment of endogenous immunity.

Immunization against anthrax using Bacillus subtilis spores expressing the anthrax protective antigen.

Protective immunity to anthrax can be achieved by antibodies raised against the secreted protective antigen (PA) and this forms the basis of the current acellular vaccines for human use. Bacillus subtilis spores have previously been used for delivery of heterologous antigens by the oral and nasal routes and their intrinsic heat-stability make them attractive vaccine vehicles. In this study we have expressed PA, or segments of PA, in B. subtilis using two strategies. First, display on the spore coat, and second, in the germinated spore (or vegetative cell). Using parenteral delivery we show that recombinant spores can be used to confer protective immunity in a murine model using an in vitro toxin neutralization assay and a challenge experiment with the latter showing protection to 100 median lethal dose of B. anthracis spores. PA must be secreted from the live bacterium or alternatively displayed on the spore surface to confer protective immunity. Intracellular expression of PA failed to confer protective immunity. The highest levels of protective immunity were achieved when PA was displayed on the spore surface as well as in the germinating spore.

Evaluation of the VP22 protein for enhancement of a DNA vaccine against anthrax.

BACKGROUND: Previously, antigens expressed from DNA vaccines have been fused to the VP22 protein from Herpes Simplex Virus type I in order to improve efficacy. However, the immune enhancing mechanism of VP22 is poorly understood and initial suggestions that VP22 can mediate intercellular spread have been questioned. Despite this, fusion of VP22 to antigens expressed from DNA vaccines has improved immune responses, particularly to non-secreted antigens. METHODS: In this study, we fused the gene for the VP22 protein to the gene for Protective Antigen (PA) from Bacillus anthracis, the causative agent of anthrax. Protective immunity against infection with B. anthracis is almost entirely based on a response to PA and we have generated two constructs, where VP22 is fused to either the N- or the C-terminus of the 63 kDa protease-cleaved fragment of PA (PA63). RESULTS: Following gene gun immunisation of A/J mice with these constructs, we observed no improvement in the anti-PA antibody response generated. Following an intraperitoneal challenge with 70 50% lethal doses of B. anthracis strain STI spores, no difference in protection was evident in groups immunised with the DNA vaccine expressing PA63 and the DNA vaccines expressing fusion proteins of PA63 with VP22. CONCLUSION: VP22 fusion does not improve the protection of A/J mice against live spore challenge following immunisation of DNA vaccines expressing PA63.

Mucosal or parenteral administration of microsphere-associated Bacillus anthracis protective antigen protects against anthrax infection in mice.

Existing licensed anthrax vaccines are administered parenterally and require multiple doses to induce protective immunity. This requires trained personnel and is not the optimum route for stimulating a mucosal immune response. Microencapsulation of vaccine antigens offers a number of advantages over traditional vaccine formulations, including stability without refrigeration and the potential for utilizing less invasive routes of administration. Recombinant protective antigen (rPA), the dominant antigen for protection against anthrax infection, was encapsulated in poly-L-lactide 100-kDa microspheres. Alternatively, rPA was loosely attached to the surfaces of microspheres by lyophilization. All of the microspheric formulations were administered to A/J mice with a two-dose schedule by either the intramuscular route, the intranasal route, or a combination of these two routes, and immunogenicity and protective efficacy were assessed. An intramuscular priming immunization followed by either an intramuscular or intranasal boost gave optimum anti-rPA immunoglobulin G titers. Despite differences in rPA-specific antibody titers, all immunized mice survived an injected challenge consisting of 10(3) median lethal doses of Bacillus anthracis STI spores. Immunization with microencapsulated and microsphere-associated formulations of rPA also protected against aerosol challenge with 30 median lethal doses of STI spores. These results show that rPA can be encapsulated and surface bound to polymeric microspheres without impairing its immunogenicity and also that mucosal or parenteral administration of microspheric formulations of rPA efficiently protects mice against both injected and aerosol challenges with B. anthracis spores. Microspheric formulations of rPA could represent the next generation of anthrax vaccines, which could require fewer doses because they are more potent, are less reactogenic than currently available human anthrax vaccines, and could be self-administered without injection.

A recombinant carboxy-terminal domain of the protective antigen of Bacillus anthracis protects mice against anthrax infection.

The immunogenicity and protective efficacy of overlapping regions of the protective antigen (PA) polypeptide, cloned and expressed as glutathione S-transferase fusion proteins, have been assessed. Results show that protection can be attributed to individual domains and imply that it is domain 4 which contains the dominant protective epitopes of PA.