Current prophylaxis for anthrax
There are two licensed anthrax vaccines available (Little, 2005; Wang and Roehrl, 2005). The US anthrax vaccine adsorbed (AVA; Emergent BioDefense Corporation; also known as BioThrax®, Emergent Biosolutions Incorporated, Rockville, MD, USA) is extracted from a cell-free culture filtrate of an unencapsulated, toxin-producing strain of Bacillus anthracis (V770-NP1R). The UK vaccine (Health Protection Agency) is prepared from a similar strain called Sterne 34F2. Both vaccines contain the protective antigen (PA) adsorbed to aluminium hydroxide and contain small amounts of lethal factor (LF) and oedema factor (EF). The vaccines are both effective against anthrax infection when administered prophylactically, although the vaccination protocols differ (Little, 2005; Wang and Roehrl, 2005; Scorpio et al., 2006). The US vaccine is administered in a six-dose primary series at 0, 2 and 4 weeks and 6, 12 and 18 months with an annual booster, while the UK vaccine requires four single injections: three injections 3 weeks apart, followed by a 6 month dose, with an annual booster.
For post-exposure prophylaxis against inhalation anthrax the Centre for Disease Control and Prevention (CDC) recommends that the vaccine AVA be used at 0, 2 and 4 weeks in combination with selected oral antibiotics. The combined use of AVA and antibiotics has been shown to prevent inhalation anthrax (Schneemann and Manchester, 2009) and may also shorten the required period of antibiotic therapy (Bossi et al., 2004a). However, this regime has not been approved by the United States Food and Drug Administration (FDA). Caution should be taken with children as the PA component of the vaccine may associate directly with the toxin components produced by the invading bacterium thereby potentially augmenting intoxication (Aulinger et al., 2005).
Although current human anthrax vaccines are effective against anthrax, they still suffer from batch-to-batch variation in composition, require multiple doses and yearly booster injections and have been associated with occasional adverse reactions (reactogenicity) (Pittman et al., 2001; Pittman et al., 2004). These limitations have prompted the development of novel vaccines that are less reactogenic, but equally efficacious with fewer doses. Research efforts focus on: (i) development of subunit vaccines targeting PA (and to a lesser extent EF and LF); (ii) evaluation of alternative vaccine delivery routes (e.g. i.m. and mucosal administration); and (iii) identification of new vaccine targets (e.g. spore and capsule antigens). Excellent reviews have been published on a number of the major achievements (Brey, 2005; Little, 2005; Wang and Roehrl, 2005; Scorpio et al., 2006).
Mucosal vaccination has proven to be a practical, non-invasive and efficacious method for the induction of both mucosal and systemic immune responses. Recently, a mucosal anthrax vaccine, based on a non-toxic mucosal adjuvant (NE) and a recombinant protective antigen (rPA), was developed (Bielinska et al., 2007). Guinea pigs immunized intra-nasally (i.n.) with the vaccine were protected from an intra-dermal (i.d.) challenge (1000 × LD50) of B. anthracis Ames spores. Another mucosal anthrax vaccine composed of rPA, MPL (a toll-like 4 receptor agonist) and ChiSys (a chitosan mucoadhesive agent) is available in the form of a dry powder (Klas et al., 2008). The vaccine protects rabbits from lethal aerosol spore challenge up to 9 weeks after a single i.n. immunization.
An anthrax vaccine based on live attenuated Salmonella vaccine strain (Ty21a) has also been reported (Stokes et al., 2007). Administration of Ty21a (p.o.) expressing the full-length rPA conferred significant protection against lethal exposure to aerosolized B. anthracis spores in mice. Further modification of rPA by its fusion to two distinct transport proteins (HlyA and ClyA) (Baillie et al., 2008) resulted in significant PA-specific immune responses when mice were immunized with Ty21a expressing the ClyA-PA fusion protein and then boosted with either rPA or AVA. CpG (unmethylated sequences of DNA) oligodeoxynucleotides (ODN) have also been evaluated as an adjuvant for AVA (Klinman et al., 2007). Mice immunized i.p. or i.n. with AVA + CpG ODN showed significantly increased host immunity to infection via aerosolized anthrax spores, in contrast to animals immunized with AVA alone. Interestingly the enhanced immunity correlates with the induction of strong systemic rather than mucosal immune responses (Klinman et al., 2007).
Protection against anthrax via current anthrax vaccines is mediated largely by antibody (humoral) responses to the protective antigen (PA); however, cellular immunity has been shown to also play an important role (Glomski et al., 2007). Mice immunized with formaldehyde-inactivated spores (FIS) of a non-encapsulated B. anthracis strain were then challenged with an encapsulated non-toxinogenic B. anthracis strain. Sera, splenocytes and CD4 T lymphocytes were isolated from the FIS-induced mice and administered to naïve mice. The mice were then challenged with the encapsulated non-toxinogenic B. anthracis with results indicating that only interferon (IFN)-γ-producing CD4 T lymphocytes provide significant protection against anthrax infection. This study provides the first evidence of protective cellular immunity against encapsulated B. anthracis.
A plasmid DNA-based approach has been applied successfully to anthrax vaccine development to boost cellular immunity (Zhang et al., 2008). Vaccination of mice with plasmid constructs expressing either PA or EA1 (an S-layer antigen) produced both Th1 and Th2 cellular responses demonstrating that this approach may be used to generate durable immune responses against anthrax. This method has also been used in conjunction with a replication-defective adenovirus vector in a prime-boost vaccination strategy (McConnell et al., 2007). Mice primed and boosted with plasmid DNA and adenovirus DNA, respectively, were fully protected from anthrax spore challenge. Interestingly the adenovirus-based prime-boost immunization produced 10-fold the anti-PA antibodies than AVA after a single injection.
The toxin components PA and LF are composed of four domains, of which the PA domain 4 interacts with the host cell receptor, while the LF domain 1 binds to PA63 (the active form of PA). Antibodies raised against the PA domain 4 were protective against anthrax infection when tested in mice (Flick-Smith et al., 2002). The PA domain 4 and the LF domain 1 were fused to a thermostable lichenase from the bacterium Clostridium thermocellum and then expressed in the plant Nicotiana benthamiana (Chichester et al., 2007). Immunization of mice with the fusion protein resulted in high titres of antibodies capable of neutralizing the lethal toxin in vitro.
A novel vaccine with the combined function of vaccine and antitoxin has been reported (Manayani et al., 2007). In this vaccine multiple copies of the PA-binding domain VWA of the anthrax toxin receptor ANTXR2 were expressed and displayed on the surface of an insect virus. The resultant chimeric virus particles protected rats from anthrax intoxication, and when loaded with PA, induced a potent immune response against lethal toxin challenge in a single dose without adjuvant.
Previous studies have shown that whole spore-based vaccines are more effective against virulent strains of B. anthracis than the current PA-based vaccines (Little and Knudson, 1986; Welkos and Friedlander, 1988; Brossier et al., 2002). However, these vaccines are unlikely to be used in humans because of safety concerns. Mice primed with suboptimal amounts of PA followed by the spore surface antigen BclA were protected from lethal anthrax spore challenge (Brahmbhatt et al., 2007). BclA promotes opsonophagocytosis of spores by macrophages thereby inhibiting intra-macrophage spore germination. More recently, spore surface antigens p5307 and BxpB were identified (Cybulski et al., 2008). Mice immunized with suboptimal amounts of anthrax PA followed by p5307 and BxpB had enhanced protection against lethal anthrax spore challenge compared with animals immunized with PA alone. Although antibodies raised against either antigen reduced the rate of spore germination in vitro, both produced enhanced phagocytic uptake and phagocyte-mediated spore destruction in the mice. Holistically, these results demonstrate that spore surface antigens are potential immuno-enhancers to PA-based vaccines.
Catalytic mutants of LF (LFE687A) and EF (EFH351A) have been evaluated in combination with PA for prophylactic use. Studies in mice demonstrated the ability of LFE687A and EFH351A, co-administered with PA, to reduce lethality following lethal anthrax spore challenge (Gupta et al., 2007).
The advances in vaccine development for anthrax over the last 2 years, as outlined above, have been undertaken in preclinical animal studies and more definitive outcomes in human clinical trials are required.