Elsevier

Vaccine

Volume 25, Issue 2, 4 January 2007, Pages 346-355
Vaccine

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

https://doi.org/10.1016/j.vaccine.2006.07.037Get rights and content

Abstract

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.

Introduction

Anthrax caused by Bacillus anthracis is now a disease of considerable interest because of its use as an agent of bioterrorism. The disease typically appears in one of three forms: cutaneous, gastrointestinal and pulmonary [1] and two vaccines are currently licensed for human use. In the UK, an alum-precipitated filtrate of a B. anthracis Sterne strain culture that is administered by intra-muscular injection is used. In the USA, the AVA (anthrax vaccine adsorbed) is in use consisting mainly of protective antigen (PA) from cultures of the unencapsulated, toxin-producing B. anthracis V770-NP1-R strain adsorbed onto aluminum hydroxide and administered by the sub-cutaneous route. Both vaccines have a number of disadvantages including transient reactogenicity associated with the UK vaccine [2], [3], [4] and minor reactions at the injection site with the US vaccine [5], [6]. Finally, both require frequent boosting. Two virulence factors are produced by B. anthracis, the poly-d-glutamic acid capsule [7] and the tripartite toxin that is composed of PA together with edema factor or lethal factor [8]. PA is the essential factor to which protection is conferred in current vaccines [1]. This 83 kDa protein is secreted from the cell and binds to a host cell membrane receptor. Furin-mediated cleavage of a 20 kDa amino-fragment of PA (referred to as Domain 1a) at the cell surface produces the mature 63 kDa form of PA that carries 4 domains, 1b, 2, 3 and 4. Upon cleavage the 63 kDa cleaved form of PA next forms a heptameric pore to which either the edema factor or lethal factor can bind using domain 1b for interaction [9]. An essential attribute of an anthrax vaccine must therefore be neutralization of PA and prevention of its binding to the host cell surface. Domains 1b and 4 are both known to carry protective epitopes [10] and immunization with recombinant PA has been shown to induce protection against B. anthracis infection [10], [11].

Bacterial endospores have shown potential as vehicles for delivery of heterologous antigens with proof-of-principle studies demonstrating that orally delivered Bacillus subtilis spores expressing a Clostridium tetani antigen on the spore surface can protect mice against toxin challenge [12]. Other studies have shown that the B. subtilis spore can germinate in the murine gut and that this provides an additional route for antigen delivery [13], [14]. In this approach the antigen is expressed in the vegetative cell and relies upon germination of the spore in the gastrointestinal tract (GIT). Interestingly, it has also been shown that spores given orally can disseminate to the gut-associated lymphoid tissue (GALT) and enter the Peyer's Patches and mesenteric lymphoid tissues [12], [15]. Although this is likely to represent the fate of only a sub-population of ingested spores phagocytosed spores have been shown to germinate and persist within the phagosome before being destroyed [16]. Like B. anthracis, B. subtilis can cross the gut wall and spores can germinate in the phagosome. However, unlike B. anthracis, B. subtilis is unable to establish a productive infection or to cause disease. The heat-stability of spores coupled with a convenient route of delivery (e.g., oral) make spore vaccines promising candidates for use in developing countries and for military personnel.

We have considered that B. subtilis could provide a potential method for immunizing against anthrax. B. subtilis is a non-pathogen and is being used commercially as a probiotic [17]. Although the importance of cellular responses in anthrax immunity are currently poorly understood [6], [18] this feature could be important. Accordingly, we have engineered B. subtilis to express the B. anthracis protective antigen on the spore surface and/or from the germinating spore as a secreted protein. In this study we evaluate the systemic responses produced to these spores given to mice by a parenteral route and show that mice can be protected against a lethal dose of, B. anthracis, STI spores (100 median lethal dose [MLD]).

Section snippets

Bacterial strains and transformation

The laboratory strain of B. subtilis used in this work was strain PY79 a prototrophic, Spo+, derivative of the type strain 168 [19]. All recombinant strains described here are isogenic derivatives of PY79. Plasmid amplification for nucleotide sequencing, subcloning experiments and transformation of Escherichia coli competent cells were performed in the E. coli strain TG1 [20]. Methods for growth, sporulation and transformation of B. subtilis were detailed elsewhere previously [21]. B. anthracis

Expression of PA in vegetative cells and on spores of B. subtilis

The pagA gene, or gene fragments encoding domains of PA, were cloned in B. subtilis using vectors pDL242 or pDG364 (Section 2 and Fig. 1) to facilitate expression of heterologous genes within the vegetative cell or on the spore surface. For vegetative cell expression, DNA was cloned, in frame, with a short leader sequence of 23 codons (2.4 kDa) that was under the control of the B. subtilis PrrnO promoter and the sspA RBS to provide maximal levels of expression of cloned DNA during vegetative

Discussion

In this study, we have questioned whether the B. subtilis spore could be used to deliver PA either by display on the spore surface or by germination of the spore within the host animal and its subsequent expression within the germinating spore, i.e., the live vegetative cell. In previous work PA has been expressed in B. subtilis primarily to evaluate its use as a host expression system to produce rPA and thereby improve the safety of the existing anthrax vaccines [31], [32]. In these studies,

Acknowledgments

SMC was supported by the BBSRC (111/S19599). We gratefully acknowledge the technical assistance provided by Maggie Stokes and Tony Stagg.

References (40)

  • E.M.F. Mauriello et al.

    Display of heterologous antigens on the Bacillus subtilis spore coat using CotC as a fusion partner

    Vaccine

    (2004)
  • H.C. Flick-Smith et al.

    Mouse model characterisation for anthrax vaccine development: comparison of one inbred and one outbred mouse strain

    Microb Pathog

    (2005)
  • M. Isaka et al.

    Mucosal immunization against hepatitis B virus by intranasal co-administration of recombinant hepatitis B surface antigen and recombinant cholera toxin B subunit as an adjuvant

    Vaccine

    (2001)
  • C.K. Cote et al.

    The detection of protective antigen (PA) associated with spores of Bacillus anthracis and the effects of anti-PA antibodies on spore germination and macrophage interactions

    Microb Pathog

    (2005)
  • M. Mock et al.

    Anthrax

    Annu Rev Microbiol

    (2001)
  • L. Baillie

    The development of new vaccines against Bacillus anthracis

    J Appl Microbiol

    (2001)
  • S.H. Leppla et al.

    Development of an improved vaccine for anthrax

    J Clin Invest

    (2002)
  • B.D. Green et al.

    Demonstration of a capsule plasmid in Bacillus anthracis

    Infect Immun

    (1985)
  • K.R. Klimpel et al.

    Anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin

    Proc Natl Acad Sci USA

    (1992)
  • C. Petosa et al.

    Crystal structure of the anthrax toxin protective antigen

    Nature

    (1997)
  • Cited by (79)

    • Bacillus anthracis and other Bacillus species

      2023, Molecular Medical Microbiology, Third Edition
    • Boosting BCG with inert spores improves immunogenicity and induces specific IL-17 responses in a murine model of bovine tuberculosis

      2016, Tuberculosis
      Citation Excerpt :

      A number of heterologous boost vehicles for tuberculosis vaccines have been evaluated and (reviewed in [10]). Inert bioparticles, such as Bacillus subtilis spores, are effective delivery vehicles with immunomodulatory properties [11,12], capable of inducing protective immunity against disease [13,14], including showing promise via respiratory vaccination against TB [15]. These data, combined with the desirable properties of cost and scalability [16], suggest B. subtilis spores may represent an ideal vehicle candidate for boosting BCG in a vaccination strategy against bTB.

    View all citing articles on Scopus
    1

    These authors contributed equally to this paper.

    View full text