Anthrax Vaccines Present Status

Published Date: 02 Nov 2017

Manpreet Kaur#1,2, Samer Singh#3 and Rakesh Bhatnagar*1,4

1: Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University, New Delhi - 110067, Delhi, India.

2: Present Address: Vaccine and Infectious Disease Research Center, Translational Health Science and Technology Institute, An autonomous institute of Department of Biotechnology, Govt. of India, Gurgaon- 122016, Haryana, India.

3: Special Centre for Nano Sciences, Jawaharlal Nehru University, New Delhi-110067, Delhi, India.

4: Kumaun University, Nainital-263001, Uttarakhand, India.

#: Both authors contributed equally to this work.

* Corresponding Author: Tel.: +91 11 26704079; Fax: +91 11 26742040.

E-mail addresses: [email protected], [email protected] (Prof. Rakesh Bhatnagar)

ABSTRACT

The management of anthrax remains a top priority among the biowarfare/bioterror agents. It was the B. anthracis spore attack through US mail system after Sept 11, 2001 terrorist attacks in USA that highlighted the potential of B. anthracis as a bioterrorism agent and the threat posed by its deliberate dissemination. These attacks invigorated the efforts towards understanding the anthrax pathogenesis and development of more comprehensive medical intervention strategies for its containment both in natural epidemic as well as manmade accidental or deliberate infection to non-suspecting population. Currently, efforts are directed towards development of more efficacious and safer vaccines as well as intervention tools for controlling the disease in advanced fulminant stage when toxemia has set in. This work presents the overview of current understanding of anthrax pathogenesis and recent advances made particularly after 2001 in the direction of successful management of anthrax and future perspective.

KEYWORDS

Anthrax, Bacillus anthracis, bioterrorism, in vivo challenge, lethal toxin, neutralizing antibodies, protection, protective antigen, therapeutics, vaccines.

ABBREVIATIONS

AT, anthrax toxins; AVA, anthrax vaccine adsorbed; AVP, Anthrax Vaccine Precipitated; EdTx, edema toxin; EF, edema factor; LeTx, lethal toxin; LF, lethal factor; MAPKKs, mitogen-activated protein kinase kinases; PA, protective antigen; TNA, toxin neutralizing antibody; γDPGA, poly-γ-D-glutamic acid.

INTRODUCTION

Anthrax is an epizootic disease which commonly affects hoofed animals such as sheep, cattle, and goats. Humans can be accidentally infected, upon contact with infected animals or their products [1]. In the past, people at risk for anthrax generally included farm workers, veterinarians, tannery and wool workers. Infection in humans most often involves the skin, gastrointestinal tract, or lungs, which may lead to fatal consequences. The etiological agent of anthrax is Bacillus anthracis, a Gram-positive rod-shaped, spore forming bacterium. The spores of B. anthracis are highly resilient and can survive extremes of temperature, low-nutrient environments or harsh chemical treatment over decades or centuries. The ease of spore production, weponization, transportation and dissemination to large population as aerosol makes B. anthracis a biological weapon of choice in any biowarfare or bioterror attack. Though, there have been few cases of intentional and unintentional releases of anthrax spores, their ability to cause panic and massive medical emergencies was aptly demonstrated by the 2001 postal attacks in the United States [2]. Even though the attacks resulted in 22 cases of anthrax (11 cutaneous and 11 inhalation), with five culminating in death; thousands of people were potentially exposed to anthrax spores [3, 4]. More than 30,000 individuals received prophylactic antibiotic therapy for 60 days, followed by another 40-day course of antibiotics and therapeutic vaccination with the anthrax vaccine adsorbed (AVA). Delivering antibiotics effectively following an anthrax attack is a tremendous public health challenge, as large number of people may be exposed and the brief time window available for employing effective antibiotic therapy to prevent illness and death; may be, limiting the scope. Therefore, it is extremely important to develop sensitive and efficient prophylactic as well as therapeutic methods for mass intervention in case of an anthrax-biowarfare attack. Consequently, tremendous efforts have been consolidated towards development of an effective anthrax vaccine [5] and developing therapeutic intervention strategies against advance stage anthrax. This article reviews the strategies currently being followed in this direction along with the promising strategies for the future.

BACKGROUND

Bacillus anthracis: the anthrax pathogen

Bacillus anthracis, the etiological agent of anthrax, is a Gram-positive, facultative anaerobic, spore forming, rod-shaped bacterium. It belongs to the Bacillus cereus group of bacteria which also includes B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis. B. mycoides, B. pseudomycoides and B. weinhenstephanensis [6, 7]. The genome of B. anthracis is extremely monomorphic, and despite high structural and physiological similarity with B. cereus and B. thuringiensis; it is the only obligate pathogen within the genus [8]. The diversity of phenotype within the B. cereus group is often attributed to plasmid encoded virulence factors. In accordance with this, plasmids pXO1 (182 kb) and pXO2 (96 kb) encoding major virulence factors, that is, anthrax toxins (AT) and the poly-γ-D-glutamic acid (γDPGA) capsule respectively have been established as key mediators of B. anthracis pathogenesis [9].

Virulence factors and their mechanisms of action

Anthrax toxins: The plasmid pXO1 encodes a tripartite anthrax toxin (AT) complex comprising lethal factor (LF) - a metalloprotease, Edema Factor (EF) - a cyclic AMP modulator, and Protective Antigen (PA) - the non-toxic, cell binding component responsible for transporting LF and EF into the cell. These anthrax toxins primarily account for the anthrax pathogenicity while the γDPGA capsule contributes submissively. The anthrax toxins belong to the family of bacterial AB toxins, in which the A moiety (i.e. catalytic subunits LF and EF) acts within the cytosol of target cells and the B moiety (i.e. PA) binds to target cells and helps in translocating the A moiety into the cytosol. The binary combination of PA with LF is known as Lethal Toxin (LeTx; PA+LF) while that with EF is known as Edema Toxin (EdTx; PA+EF) [10]. Though the three proteins are secreted independently, they assemble at the mammalian cell surface into toxic complexes. PA binds to ubiquitously expressed cellular receptors, two of which Tumor Endothelial Marker and Capillary Morphogenesis Protein have been established as primary anthrax toxin receptors [11, 12]. Upon binding, PA undergoes proteolytic cleavage by cell-surface furin or furin-like proteases, resulting in release of PA20, a 20 kDa N-terminal fragment into the extracellular milieu [13]. The remaining carboxy-terminal 63 kDa receptor bound fragment PA63, then spontaneously oligomerizes into a heptamer that competitively binds EF and/or LF, to translocate them into the cytosol, where they exert their toxic effects [14, 15]. LeTx is the prime mediator of toxic shock and death [16]. The enzymatic moiety LF contains a thermolysin-like active site and zinc-binding consensus motif HExxH [17] that acts as a Zn2+ metalloprotease on a range of substrates, including peptide hormones and Mitogen-Activated Protein Kinases (MAPK). The MAPK cascade is essential for full induction of the oxidative burst and pro-inflammatory cytokine expression, its disruption by LeTx neutralizes macrophage activation and favors bacterial escape from lymph nodes during the initial phase of infection [18]. The other anthrax toxin, the EdTx exerts its effect through edema. The enzymatic moiety EF is a calcium- and calmodulin-dependent adenylate cyclase [19]. After translocation to the host cell cytoplasm, EF catalyzes the synthesis of cAMP in a calmodulin dependent manner [20], increasing the intracellular cAMP levels that leads to edema. The production of LeTx and EdTx soon after the spore germination impairs the immune system, allowing unimpeded replication of vegetative bacteria and its systemic dissemination resulting in massive bacteremia [21]. As the infection progresses, accumulation of toxins induces the development of cytokine independent shock, which is correlated with the direct injurious effects of LeTx on the endothelial cell function that ultimately contributes to host death [22]. Furthermore, LeTx and EdTx have been also shown to directly affect cardiovascular system along with toxin-specific alterations in blood counts in animal models. The LeTx associated toxicity is similar to that of acute cardiac failure, while that of EdTx can be correlated with direct vascular endothelial injury [23, 24, 25, 26]. These studies indicate that anthrax management should not be only cytokine or septic shock directed, rather therapies against effect of these toxins especially on the heart and blood vessels along with hemodynamic support, may prove beneficial [27].

The γDPGA capsule: Along with anthrax toxins, the capsule of B. anthracis serves as one of the principal virulence factors during the course of infection. By virtue of its negative charge, poly-D-glutamic acid (γDPGA) capsule inhibits phagocytosis and opsonisation of the bacilli [28]. Thus, in conjunction with LeTx and EdTx, the capsule allows virulent anthrax bacilli to grow virtually unimpeded in the infected host. The capsule synthesis genes capA, capB, capC and capE are present as an operon on the pXO2 plasmid [29]. These genes are necessary as well as sufficient for polyglutamate synthesis. An additional protein encoded by the dep gene located downstream from the cap region is a depolymerase that catalyzes the hydrolysis of γDPGA into lower molecular weight polyglutamates [30]. Although the biological functions of the dep gene are unknown, it is possible that the polyglutamates produced through its action may act as inhibitors to the host defense mechanisms. Additionally, capD is required for polyglutamate anchoring to the peptidoglycan layer [29]. Expression of cap genes and the capsule formation is regulated by serum, CO2 and temperature through products of the atxA, acpA and acpB genes which are also shown to regulate toxin expression, however, the details of the mechanism of regulation are not entirely understood [31, 32].

 

Spores: Central to the persistence of anthrax is the ability of B. anthracis to form long-lasting, highly resistant spores, which represent the dormant, metabolically inert form of the bacteria in nature [33]. B. anthracis spores invade the mammalian host through skin abrasions, inhalation or ingestion causing cutaneous, inhalational or gastrointestinal manifestations of the disease, respectively. In cutaneous infections, the secretion of the toxins following germination of spores results in the extensive edema and necrosis with characteristic eschar formation. Other symptoms include muscle aches and pain, headache, fever, nausea, and vomiting. Neutrophils, the first line of defense recruited to the site of cutaneous infection, are proficient in combating the bacilli, accounting for spontaneous healing of lesion in most of the cutaneous infections cases [34, 35]. Very rarely, the bacteria may enter the systemic circulation and replicate to high densities leading to large amount of toxin release causing shock, respiratory failure and death. In inhalation anthrax, alveolar macrophages and pulmonary dendritic cells engulf the inhaled spores and then transport them to central lymph nodes. Here spores germinate and usually spread systemically causing massive bacteremia and toxemia [36, 37]. Initial symptoms are subtle, mild and flu-like. Gradually, severe symptoms including enlarged lymph nodes, pulmonary edema with acute dyspnea (labored respiration) and cynosis (bluish discoloration of the skin caused by poor blood oxygenation) are observed. Subsequent, inactivation of the innate and adaptive immune systems, damage to endothelial cell function and induction of cytokine independent shock, eventually leads to death. Gastrointestinal anthrax is extremely rare and occurs mainly in Africa, the Middle East and Central and Southern Asia. This form of disease is infrequent in livestock and rarely occurs in humans from eating undercooked contaminated meat from anthrax-infected animals. The symptoms include nausea, loss of appetite, bloody diarrhea and fever followed by abdominal pain. The bacteria subsequently invade through the bowel wall and spread throughout the body via the bloodstream, resulting in deadly bacteremia and toxemia.

Novel candidate virulence factors

Though anthrax is toxinogenic disease, other factors may be required in the course of infection. Such virulence factors may also be feasible in understanding pathogenesis, immune mechanisms and directing therapies against the bacillus and/or toxins. pXO1 encodes for the gerX operon (germination operon) [38], and Cot43, a novel regulatory gene, which has been implicated in control of extracellular proteases [39, 40]. In addition to proteases, B. anthracis also secrete diverse lecithinases, hemolysins and Anthrolysin O (ALO), a cholesterol- dependent cytolysin CDC) toxin, responsible for some of the clinical manifestations of anthrax [41]. Likewise, pX02 plasmid encodes for the cap regulators, acpA and acpB, and the operon capBCAD involved in capsule biosynthesis and expression of general stress transcription factor gene sigB [42, 43, 44, 45]. A large number of chromosome products including acyl carrier protein, acyl-CoA synthase, AMP-binding protein, 2,3-dihydroxybenzoate-2,3-dehydrogenase, dTDP-glucose 4,6-dehydratase, enhancin, enterotoxin, hemolysin (hly), immune inhibitor A metalloprotease, internalin, iron compound ABC transporter, isochorismatase, preprotein translocase, protein export protein, phospholipase C (plC), siderophore biosynthesis protein, substrate-binding family protein, thiol-activated cytolysin, abrB and sasP have been shown to contribute towards pathogenicity and virulence of B. anthracis [301].

MANAGEMENT

The management of anthrax mainly relies on vaccination for pre-exposure cases while on antibiotics, anti-toxins and other supportive care for post-exposure cases.

Antibiotics: B. anthracis is sensitive to a number of antibiotics including penicillin, doxycycline, ciprofloxacin, amoxicillin, levofloxacin [46, 47]. However, due to the continuing action of toxins (PA, EF, and LF), antibiotic treatment is of limited value after the onset of toxemia symptoms, emphasizing the need of early administration of antibiotics [48, 49]. In case of cutaneous infection, eschar formation usually occurs despite timely treatment; however, it doesn’t lead to fatal consequences. In contrast, in gastrointestinal and inhalation anthrax the infection spreads systemically without overt symptoms and by the time it is diagnosed the toxemia has already set in making the antibiotic treatment largely ineffective. A therapeutic time window of only 40 to 48 hours from infection exists, to start an efficient antibiotic-mediated treatment [50]. Furthermore, as spores can persist longer in the system, a longer duration antibiotics treatment needs to be carried out. Current CDC recommendations for post exposure prophylaxis (PEP) following potential inhalation exposure to aerosolized B. anthracis spores are 60 days of oral antimicrobial therapy (using ciprofloxacin and doxycycline) in combination with a 3-doses of AVA (BioPort Corporation, Lansing, MI, USA) administered at time zero, two weeks, and four weeks [51, 52, 53]. 

Antitoxins: In view of the limited potential of antibiotic treatment, therapies directed against the action of anthrax toxins are going to be pivotal in the management of anthrax. Numerous strategies are being explored in this direction.

Antibodies: The antibodies (Abs) directed against anthrax virulence factors could be of immense significance in increasing the therapeutic window, decreasing the length of treatment, and overcoming toxemia from potential antibiotic resistant strains. Potential antibodies for therapy include anti-PA, anti-LF anti-EF antibodies that could interfere with PA and receptor interaction, PA cleavage by Furin, PA heptamerization, PA and LF/EF interactions, toxin endocytosis and translocation as well as those directed against Bacillus anthracis capsule. A number of therapeutic antibodies are under different stages of clinical development. Amongst these, ABthrax (raxibacumab), AnthimTM (ETI-204), MDX-1303 (ValortimTM) have successfully completed Phase 1 clinical trials [302].

Monoclonal antibodies directed against anthrax toxins could be of tremendous therapeutic value in a biowarfare event due to increased specificity. Monoclonals against PA [54,55, 302], LF [54, 302] or EF [302] are being developed in view of disrupting the action of toxins. Groen et al. showed that fully humanized monoclonal antibodies IQNPA-1 and 2 can effectively neutralize LeTx [201]. A bispecific monoclonal antibody (mAb, H10) with high affinity interaction for both EF and LF had also been generated [56]. H10 mAb not only neutralized the adenylate cyclase activity of EdTx but it also neutralized the cytotoxic activity of lethal toxin. Apart from anthrax toxins, other moieties like the anthrax toxin receptor and spore extract antigen had been also targeted [57, 3302]. To enhance the opsonophagocytosis of B. anthracis, ultimately leading to bacterial clearance, monoclonal antibodies directed to the capsule had been made. Mouse mAbs specific to γDPGA capsule successfully generated from mice immunized with γDPGA in combination with a CD40 agonist mAb protected mice against pulmonary anthrax [58, 59]. Recombinant lactobacilli expressing a neutralizing scFv (single-chain variable fragment) fragment against PA was found to provide protection against anthrax toxins both in vitro and in vivo [60]. The ability of lactobacilli to colonize the gastrointestinal tract may find its application in both prophylactics and therapeutics. For mass applications, Jeong and Rani optimized high cell density cultivations for preparative scale production of antibody M18. The purified antibody showed biological activity [61]. Such up-scaling strategy may be applicable to other monoclonals as well. Thus, these studies demonstrate the feasibility and potential of monoclonal antibodies for anthrax therapy.

Receptor decoys: Inhibition of PA binding to its receptors, tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2) can effectively block anthrax intoxication. The potency of Receptor-like agonists such as the mammalian cell-expressed von Willebrand factor type A (vWA) domain of CMG2 (sCMG2), had been demonstrated against the anthrax toxin challenge [62]. Another moiety, L56A, a PA-binding-affinity-elevated mutant of sTEM8, was shown to inhibit anthrax toxin action as effectively as sCMG2 in Fisher 344 rats [63]. A Polyvalent liposome-based receptor-directed anthrax-toxin inhibitor that binds both TEM8 and CMG2 receptors was found to protect animals from toxin challenge [64]. Thus, receptor decoys based on TEM8 or CMG2 are promising anthrax toxin inhibitors in the advanced stage of anthrax infection, when considerable production and accumulation of toxins could render antibiotics treatment of limited use.

Dominant-negative inhibitors of translocation: A dominant negative inhibitor of translocation of PA, in which, 22-23 loop residues were replaced with amphipathic membrane-inserting loop of its homologue (iota-b toxin of Clostridium perfringens) was generated. The mutant was found to assemble into hetero-heptameric structures with wild-type PA. The mutant inhibited toxin activity both in vitro on J774A.1 cells and in vivo in Fischer 344 rats thereby exhibiting a dominant negative effect. Furthermore, it inhibited the channel-forming ability of wild-type PA resulting in the formation of mixed oligomers with defective functional activity. This indicated the significance of dominant negative inhibitor of translocation for anthrax therapy [65]. Interestingly, the immunization of rabbits with multiple antigenic peptide (MAP) vaccine displaying amino acids (a.a.) 304-319 from same 22-23 loop of PA, alone [66] or fused with p38/P4 helper T cell epitope from Schistosoma mansoni, protected them from aerosolized spore challenge with B. anthracis Ames strain [67].

Small-molecule inhibitors: Small molecules have also been investigated to disrupt the action of anthrax toxins [68]. Panchal et al. identified several small non-peptidic inhibitors of LF, which exhibited K i values in the micro-molar range and showed competitive inhibition [69]. Tonello et al. identified a highly potent hydroxamate based substrate analogue for LF, which conferred protection from LeTx-induced necrosis in macrophages [70]. Considering, possibility that hydroxamates may cross-react with other host metalloproteinases and cause metabolic instability; novel scaffolds which are not found in other metalloproteinases were also screened. Two compounds from the library protected LeTx-treated macrophages from cytolysis [70]. Peptide substrate for LF had also been shown to protect cultured macrophages from LF-mediated cytolysis [71]. Rubert et al. designed small molecular scaffolds which mimicked key residues of PA63 required for the intermolecular interactions that stabilize the heptamer. They demonstrated modest inhibition of PA activity in murine J774A.1 macrophage cells by hindering oligomerization of PA63 [72]. In another study, a series of compounds which inhibit EF-CaM (calmodulin) interaction and therefore the adenylyl cyclase activity of EF were tested. Nucleotide based inhibitors, similar to ATP, the natural substrate for EF activity, have been assessed for activity against EF [73]. However, these compounds had low activity and specificity for EF. Fluorenone-based inhibitors that inhibited the release of cAMP from cells treated with EF had shown promise for anthrax therapy. Molecules directed against EF - active site inhibited the adenylyl cyclase activity of EF in the micromolar range [74]. Hence, these molecules targeting the proven virulence factors with reasonable efficacy may form the basis of future designer anthrax toxins inhibitors for clinical applications to counteract anthrax toxemia and infection in vivo.

ANTHRAX VACCINES

Current Vaccines:

The threat of anthrax biowarfare has stimulated the development of improved medical countermeasures especially vaccines for mass scale immunization. The conventional Sterne’s live spore vaccine was derived from B. anthracis 34F2 strain, which had lost its ability to produce capsule due to differential subculturing [75]. Though, it was extensively used in the past and still finds application in Russia and China; it wasn’t received well for clinical application in the Western world, owing to its excessive toxicity as indicated by the animal experimentation. In the early 1950s, cell free filtrates were investigated for their ability to confer protection against anthrax. In US, aluminum hydroxide–adsorbed, formalin-treated, culture supernatant of a toxigenic, non-capsulated, non-proteolytic B. anthracis V770-NP1-R strain was employed [76, 77, 78]. The vaccine referred to as the anthrax vaccine adsorbed (AVA) has now been registered as BioThrax®. The UK version referred to as Anthrax Vaccine Precipitated (AVP) consists of aluminum-phosphate precipitated culture filtrate of the Sterne stain 34F2 [75]. Although there is no direct clinical data on the efficacy of AVA or AVP, several studies have strengthened confidence in their potential. Data from a 1950s trial of wool-sorters immunized with a similar vaccine, besides experience with AVP and AVA, have shown that a critical level of serum antibodies to PA confers immunity to anthrax [79, 80, 81]. Subsequently, British Ministry of Labour notified that regular immunization of staff of the Government Wool Disinfection Station in Liverpool protected them from anthrax infection despite high risk of exposure [82]. Efficacy of AVA was also shown in different animal models against cutaneous and inhalational challenge with B. anthracis [83, 84].

Despite its proven safety and efficacy in different studies, AVA wasn’t widely accepted. Firstly, the dosage of AVA was standardized according to the manufacturing process and potency assay involving protection of guinea pigs challenged intracutaneously with B. anthracis spores [84, 85]. There is lack of standards for vaccine-induced protective level of serum anti-PA antibody to evaluate efficacy antibodies in animals or humans vaccinated with AVA. Thus, it’s difficult to maintain consistency of AVA. Secondly, lack of well-characterized components raised concern over local and systemic adverse reactions [86]. Furthermore, the vaccine requires lengthy immunization schedule (six subcutaneous injections over an eighteen-month period along with annual boosters for those at risk) [87]. This not only adds to the cost of immunization; but also chances of failing to comply with multiple booster schedule required to induce a protective immunity.

Next generation anthrax vaccines (Experimental):

The concerns over present vaccination approach have prompted improvement in current vaccination strategies along with search for and development of additional immunogens, novel delivery methods and agents for a safer and more efficacious anthrax vaccine. These experimental anthrax vaccines that are at different stages of development or safety and immunogenicity evaluation are described below.

Capsule vaccines: The capsule is an essential virulence factor that enables protection to the bacterium from phagocytosis and evades generation of strong humoral immune response owing to predominantly inert nature [88]. Being a surface antigen, it appears to be a promising candidate for the development of anthrax vaccine; however, concerns arise owing to inherently weak capsule immunogenicity. Nevertheless, outcome of immune potential can be significantly enhanced through conjugation to a strongly immunogenic protein carrier. In this regard, B. anthracis γDPGA capsule covalently conjugated to the outer membrane protein complex of Neisseria meningitidis serotype B protected mice against parenteral anthrax challenge [89]. Robust anti-capsule antibody responses were mounted in rabbits and monkeys. Furthermore, monkeys, exhibited significant protection against a high aerosol spore challenge dose. In another study, γDPGA-peptidoglycan preparation (GluPG) elicited antibody response and partially protected mice against lethal challenges with a non-toxinogenic strain. Capsular preparations had also been investigated to improve the potential of other anthrax vaccines. The GluPG preparation, in conjunction with protective antigen had conferred complete protection against cutaneous B. anthracis challenge [90]. Garufi et al. developed sortase-conjugation method for improving the earlier used random chemical cross-linking. Sortase-conjugation was used to link the γDPGA capsule to the receptor binding domain (D4) of PA. This construct elicited robust antibody response against both the antigens. Moreover, it conferred complete protection against anthrax challenge with wild-type or pagA mutant B. anthracis Ames in guinea pigs [91]. Thus, addition of capsule components may broaden and enhance the protection afforded by protective antigen-based vaccines.

PA and its derivatives based Vaccines: Among anthrax toxin components, PA is central to host cell intoxication as it is the cell binding moiety that facilitates the entry of both toxic components EF and LF into the host cell [92, 93]. Various studies carried out with AVA and PA in different animal models of inhalation or cutaneous anthrax challenge indicate relevance of PA as the key component of the vaccine [94, 95, 96]. In a study, pX01- B. anthracis strain was shown to be avirulent and incapable of conferring immunity, due to its inability to express the anthrax toxins. It had been also demonstrated that the level of antibodies to PA, either induced through active immunization or passive administration, were the prime correlate of protection against anthrax [97]. Hence, PA has remained the prime target for various vaccination strategies. An ideal anthrax vaccine may comprise of safe, efficacious, single dose of PA.

In our laboratory, Escherichia coli based expression system was investigated for such a preparation of recombinant PA (rPA) [98]. The rPA was capable of serving as the receptor for anthrax toxin and it elicited toxin neutralizing antibodies that afforded protection in New Zealand White rabbits and Rhesus macaques against an aerosol challenge with Bacillus anthracis spores (IVRI strain, tox+cap+). It also induced a robust and rapid memory response. Furthermore, complete protection was observed for the group immunized with ≥25 g of rPA, following aerosol challenge with 1000 LD50. Thus, E. coli expressed rPA may be effectively used for vaccination against anthrax.

Several methods had been also employed to improve the immunogenecity of PA based vaccination. In a study, CIA05, a TLR4 agonist, was conjugated with rPA. The combination resulted in enhanced serum anti-PA IgG antibodies, including toxin-neutralizing antibody titers, along with increased IL-4 and GL7 levels. Thus, CIA05 was found capable of enhancing the efficacy of PA [99]. During anthrax infection, PA production occurs subsequent to spore germination. Accumulation of PA and exertion of its effects occur during later vegetative cell proliferation. Inclusion of spore-specific antigens with PA in vaccines seems reasonable modification for conferring better protective immunity againstthe disease. Using reverse vaccinology approach, a large number of novel spore antigens as well as candidate proteins from secretome had been identified as potential vaccine candidates [8]. Many of them are being explored for use as a vaccine candidate. Once such analysis is complete we will have a priority list of potential vaccine candidates which may be combined with PA to generate immunity that would be able to handle better the anthrax infection, including that from such B. anthracis strains that have been deliberately genetically modified to produce variant PA or other toxin components, not recognized by immune response generated by current PA based vaccines. Supplementation of PA with recombinant spore-surface antigens like BclA, ExsFA/BxpB and p5303 had been shown to augment the protection afforded in different animal models against challenge with spores derived from attenuated Sterne strain and fully virulent Ames strain [100]. Hence, these and probably other spore surface antigens could be used to develop future anthrax vaccine strategies.

As the PA based vaccination may not elicit any direct response against the bacilli, inclusion of capsular components in the vaccine preparation may facilitate immune response generation both against the bacilli and toxin. In this direction, Aulinger et al. developed a dual vaccine by conjugating the -DPGA capsule with PA, which elicited antibodies against both the bacilli and toxins. The construct was further improved by replacing native PA with dominant-negative inhibitory (DNI) mutant of PA. The DNI--DPGA construct elicited enhanced antibody response, in comparison to the native conjugates [101]. Higher immune-potential of the epitopes or better processing might be responsible for improved immune response. The potential of DNI to inhibit anthrax toxin was established in post-exposure incidents. These constructs would be suited for both prophylactic and therapeutic application. Intranasal immunization of mice with PA along with PGA-BSA protein conjugate had elicited immune response against both PA and PGA. The anti-PA antibodies were capable of LeTx neutralization while anti-PGA Abs could activate complement and kill PGA-producing bacteria [102]. Recombinant adeno-associated virus type 1 (rAAV1) vectors encoding activated PA (PA63) or LF had also been investigated as anthrax vaccine. The monovalent vaccines (PA63/LF) and bivalent vaccine (PA63+LF) elicited significant antibody response against respective antigens, in rabbits [103]. Furthermore, single shot of vaccine was capable of inducing robust neutralizing antibody response demonstrating potential of rAAV1-based vectors for development of future anthrax vaccines.

The threat of Bacillus anthracis spore attacks have accelerated in the modern society. Apart from Bacillus anthracis- the causative agent of anthrax, Variola major virus- the etiological agent of smallpox, poses immense potential to inflict mass casualties.

Though vaccines are available for both them, their potency in a combined attack to inflict loses remains a big question. In view of this, Merkel et al developed a dual vaccine, by integrating anthrax PA and immune-enhancing cytokine IL-15 into the Wyeth vaccinia virus (smallpox vaccine strain). The resultant, Wyeth/IL-15/PA, had proven its efficacy against both smallpox and anthrax [94]. Furthermore, the dual vaccine was cold-chain independent making it amenable to storage and transport in an episode of a bioterror attack.

Though, these studies showed the feasibility of PA based vaccines, the extent of immune response elicited and protection conferred by them in different experimental models was variable. Despite the advancement in PA based vaccines (i.e. recombinant, purified, modified) over AVA and other traditional vaccines, they still fail to confer complete protection and induce memory response similar to AVA. In spite of induction of toxin neutralizing antibodies, their repertoire may vary. Additionally, cellular immune response may be essential for limiting the anthrax infection and conferring complete protection. This calls for investigation of other immunogens, refinement in immunizing strategies, and a greater understanding of the immune mechanisms. These understandings may lead to development of single-shot, safe, efficacious, well characterized, standardized "ideal" anthrax vaccine in future.

Epitope vaccines: There are instances when the immune response induced by the natural immunogens is suboptimal. In such cases isolation and optimization of specific components, i.e. the epitope, could enhance the immune response as it could provide the platform for investigating type and breadth of adaptive immunity [105]. A highly conserved epitope could be of tremendous application for targeting multiple pathogens or variants. In contrast, use of multiple-epitopes may broaden the immune response, reducing the chance of a pathogen to escape immune response from a single epitope [106]. Immunization of rabbits with a multiple antigenic peptide (MAP) displaying a.a. 305 to 319 from the 2β2-2β3 loop in domain 2 of PA was capable of eliciting antibody specific to a linear determinant in the 2β2-2β3 loop, which mediated high-titer neutralization of LeTx in vitro [107]. Furthermore, T-B MAPs (comprising a.a. 305 to 319 co-linearly synthesized with the P30 helper epitope of tetanus toxin) elicited high-titer, durable antibody responses in rabbits which exhibited potent neutralization of LeTx in vitro [108]. We had also demonstrated the robust immunogenicity of ID-II epitope (a.a. 626–676 of PA). It afforded protection at par with whole PA establishing the feasibility of epitopes for anthrax prophylaxis [109]. Using solid-phase epitope mapping and confirmatory assays, several neutralization-associated humoral epitopes were also identified [110]. Additionally, passively transferred antibodies directed against these epitopes provided protection in an in vivo lethal toxin mouse model. The epitope mapping of PA, LF and EF could further provide candidate immunodominant peptides for a better anthrax vaccine.

Subunit vaccines: The better understanding of molecular pathogenesis and immune mechanisms; [111] combined with elucidation of PA structure had hurled the era of evaluation of PA and its domain for subunit vaccines. Antibodies directed against PA-D4 were shown to protect mice from infection with B. anthracis [112]. It is postulated that addition of other anthrax proteins or protein domains can enhance the effectiveness of a PA-D4 based vaccine. The inclusion of LF and EF in engineered strains expressing PA and EF/LF had resulted in an heightened antibody response which was more effective in protecting animals against a spore challenge than strains expressing PA alone [113]. A study employing display of fusion products of PA, LF and EF on bacteriophage T4 capsid, either individually or in combinations demonstrated induction of strong antigen-specific as well as LeTx neutralizing antibodies when PA, LF and EF were displayed together as compared to the phage displaying PA alone [114]. Baillie et al. investigated several subunit combinations, and indicated the potency of a novel fusion protein comprising D1 of LF with D4 of PA. The fusion conferred robust broader spectrum protection level in comparison to PA alone [115].

Plant vaccines: Plants offer a convenient alternate source of anthrax vaccine. Plant based vaccines would be particularly attractive for being potentially economical, least challenging for manufacture/process development, practically devoid of any animal pathogens; with added benefit of being able to induce both humoral and cell mediated immunity- a prerequisite for efficacious anthrax vaccine. The suitability of PA for expression in transgenic chloroplasts had been shown by us and others [116, 117, 118]. The transgenic chloroplast produced PA had been shown to induce generation of neutralization antibodies and being amenable to economical purification. It is estimated that one acre of such transgenic tobacco plantation could yield more than 360 million doses of PA based anthrax vaccine [117]. The recombinant PA expressed in tomato- an edible crop [118], had effectively circumvented the need of PA purification as well as storage and stability issues associated with conventional anthrax vaccines. Recently, work from our laboratory demonstrated that PA domain 4 when expressed in transgenic chloroplasts could make up to 5.3% of total soluble protein (TSP) and protect orally immunized mice against B. anthracis spore challenge [119]. Similarly, plant based subunit or multiple antigen anthrax vaccines are also a good proposition. A subunit vaccine comprising domain 4 of PA (PAD4) and domain 1 of lethal factor (LFD1) as fusions to lichenase (LicKM), a thermostable enzyme from Clostridium thermocellum, expressed in Nicotiana benthamiana was shown to generate LeTx neutralizing antibodies [120]. Thus, plant based vaccines present an attractive alternative to conventional anthrax vaccine in terms of being economical and suitable for mass immunization.

DNA vaccines: These vaccines would offer advantage over conventional vaccine in terms of ease of construction and manufacture with concomitant elimination of protein expression and antigen purification steps from vaccine making. Furthermore a number of antigens can be combined together. A cationic lipid based bivalent DNA vaccine encoding genetically detoxified protective antigen (PA) and lethal factor (LF) proteins that was tested against anthrax spore challenge in mice models [121] had shown promise in nonhuman primates as well as passed the clinical trial evaluating immunogenicity and safety parameters [122]. There is evidence that the immunogenicity and type of immune response generated by DNA vaccines can be further modified/tweaked and enhanced by targeting the antigen to different cellular location. Work from our laboratory had demonstrated that immunogenicity, immune response and protection to anthrax spore challenge depended on targeting of expressed PA antigen to specific subcellular location [123, 124].

Live bacterial vaccines: Towards development of PA based oral vaccines a number of live attenuated strains of bacteria (e.g. B. anthracis, B. subtilis, Salmonella, Lactobacillus, vaccinia virus) engineered to deliver PA in vivo had been evaluated for their potency [125]. The generation of toxin neutralizing antibodies and protective immunity following immunization with these vaccine candidates was found promising [125]. The major disadvantage of these live systems had been to culture and store the organism prior to use [125]. Generation of lyophilized bacterial stocks that are stable at room temperature could be the answer to these drawbacks of live vaccine systems. The potential of live system was further demonstrated by successfully conferring protective immunity to animal models against aerosolized B. anthracis spores challenge by oral immunization with a vaccine strain of Salmonella [125, 126]. The recombinant live bacterial vectors could be further refined and developed to combine multiple antigens including other biowarfare agents or common infectious agents- providing a comprehensive protection against such threats.

Virus-like particle (VLP) vaccines: Virus-like particles (VLPs) are multiprotein structures that mimic the organization and conformation of native viruses but lack the viral genome [127]. Therefore, they are non-infectious and provide a safer and cost effective platform for delivery cum presentation of vaccine candidate antigens including that of anthrax as opposed to live virus based such as recombinant adeno associated virus type 1 (rAAV1) [103] or T4 phage [114] based. Parvovirus B19 was used to develop VLP expressing the domain 4 of PA (PA-D4). The recombinant VLP elicited robust neutralizing anti-PA antibody [128]. Manayani et al. used the Flock House virus as a platform to display 180 copies of the high affinity, PA-binding von Willebrand A domain of the ANTXR2 cellular receptor [129]. The chimeric VLP inhibited lethal toxin action.

FIVE YEAR VIEW:

Anthrax as a biowarfare or bioterror agent, poses its own unique set of issues in terms of its successful and acceptable management. In case of inhalational anthrax, the initial symptoms are flu-like (prodrome stage; 1-5 days from infection) followed by rapid progression to fulminant stage of inhalation anthrax that is marked by sudden onset of high fever, sweating, and shortness of breath culminating into death. Though starting aggressive antibiotic treatment in predrome stage could prevent death in 60% cases in case of anthrax terror attack, the antibiotics would become largely useless in fulminant stage due to extensive toxemia [130]. Effective management of anthrax both in case of natural epidemic or biological warfare warrants development of better health care system, development of diagnostic tools for its faster detection and measures to manage fulminant anthrax. Next 5 years will see rapid progress towards development and testing of portable rapid response diagnostic kits that can be used in fields in any eventuality of a bioterror attack. Furthermore, technological advancement in future would make testing/diagnosis of bioterror agents including anthrax a routine. Next human anthrax vaccine will most probably be comprised of aluminum hydroxide precipitated dominant negative PA mutant/ individual immunogenic PA domains combined with PA binding amino-terminal domains of LF/EF. Later, this would be replaced by recombinant vaccines containing additional spore and capsule immunogens (e.g. SLH domain proteins, γ-DPGA), providing more comprehensive protection against anthrax challenge. The inclusion of dominant negative PA variants would make future vaccines safer as it could both be used as immunogen as well as anti-toxin because of their virtue to interfere with LeTx and EdTx action. The management of post-exposure /fulminant stage anthrax would require development of aggressive anti-toxin therapy along with the standard supportive care that is currently recommended. There had been a lot of push in identifying, developing, and characterization of polyclonal and monoclonal antibodies against PA as well as other major virulence factors that can be used as antitoxins to mitigate the toxemia when antibiotic therapy becomes ineffective. Next 5 years will see characterization and standardization of these antibodies to treat anthrax toxemia with additional more effective antitoxin antibodies including humanized single chain antibodies being discovered as well as designed with the aid of molecular modeling and bioinformatics tools available. New and more efficient small molecule inhibitors interfering with anthrax pathogenesis, particularly interaction of PA to EF/LF and cellular receptors; LF to cellular targets; γ-DPGA to host cells will be designed and discovered that will supplement the treatment modalities available and would eventually replace the antitoxin/antibody therapy practiced in post-exposure treatment modality. These small molecule inhibitors combined with antibiotics are going to replace the anthrax therapy in future.

EXPERT COMMENTARY

With the anthrax biowarfare or bioterrorist threat looming large, the development of safer and better vaccines along with medical interventions required to treat anthrax patient in post-exposure/fulminant stage remains a priority. Though PA based vaccine had been a success in terms of providing satisfactory immunity so far, the drawbacks of variable protective immunogenicity (batch to batch variation), reactogenicity, requirement of multiple boosters, etc. needs to be addressed. Immediate safety and efficacy concerns of the current anthrax vaccine can be alleviated by employing purified recombinant PA, preferably the dominant negative or non-functional variants. Further improvements could include presentation of PA in host by recombinant bacteria, virus, transgenic plant, DNA vaccines etc.; delivery through nasal, gastrointestinal routes; amenability to long term storage at room temperature without loss in immunogenicity; and decrease in number of doses required and induction of long-term protective immunity.

We would also need to consider the possibility of future anthrax spore attack using a genetically engineered B. anthracis or worse an engineered organism expressing altered PA and LF not recognized by antibodies generated through vaccination by conventional human or animal anthrax vaccines. To be able to successfully handle such eventualities, we would need to focus on designing/developing reagents that could interfere/inhibit essential steps/interactions among toxin components themselves or with host cell components. For now, we may rely on dominant negative PA variants [101] and 22-23 loop multiple antigenic peptides [67] for prophylactic vaccination but we would need to look into better possibilities and make ourselves ready for such eventuality. Humanized single chain antibodies or monoclonal antibodies generated against such interacting surfaces among toxin components which are essential for activity; use of peptides masking furin site in PA, or interfering with its interaction with LF or cellular receptors; peptides mimicking surface of the PA cellular receptors that interacts with PA are some of the promising leads. Additionally, small molecules offer a rapid course to design and develop such inhibitor compounds that would interfere with essential steps of toxin intoxication and thus, could effectively protect from B. anthracis attacks including that from genetically engineered ones.

EXECUTIVE SUMMARY

Anthrax: Primarily a disease of herbivores caused by Bacillus anthracis (Gram positive, spore forming bacteria) with accidental human infection while working with infected animal or their products; major biowarfare/bioterror agent.

Types: Cutaneous (least lethal; black eschar formation), Gastrointestinal, Inhalational (most lethal)

Symptoms: Cutaneous anthrax: Characterized by edema and necrosis followed by black

eschar formation at the site of infection that most of the time gets healed spontaneously

Gasterointestinal/Inhalational: Initially subtle flu like (Prodrome) followed by rapid progression to enlarged lymph nodes, pulmonary edema with labored respiration and cynosis (fulminant). Subsequent inactivation of the innate and adaptive immune systems, damage to endothelial cell function and induction of cytokine independent shock leads to death.

Major Virulence Factors:

Lethal Toxin [Protective Antigen (PA) + Lethal Factor (Zn metalloprotease)] cleaves MAPKK and causes cell death

Edema Toxin [Protective Antigen (PA) + Edema Factor (Calmodulin dependent adenylate cyclase)] increases cAMP level causes edema

poly-γ-D-glutamic acid (γDPGA) Capsule: Negatively charged, helps in evading phagocytosis and opsonisation of the bacilli

Management: Following suspected B. anthracis exposure, 60 days of oral antimicrobial

therapy (using ciprofloxacin and doxycycline) in combination with a 3-dose series of AVA at 0, 2, 4 weeks is prescribed.

Antibiotics: Sensitive to number of antibiotics (e.g. penicillin, amoxicillin, levofloxacin). Ciprofloxacin and doxycycline are antibiotics of choice.

Antitoxins: Antibodies (mAb and scAb) against virulence factors (e.g.PA, LF, EF, γDPGA, SLH domain proteins).

PA Receptor Decoys based on CMG2 and TEM8 (e.g. vWA domain of CMG2; L56A a mutant sTEM8)

Dominant Negative inhibitors of translocation

Vaccines: Current human vaccines AVA or AVP are PA based. They provide adequate prophylactic short term immunity but have very limited use in post exposure fulminant stage anthrax treatment or protection against a genetically modified B. anthracis producing an engineered PA molecule.

Next Generation Vaccines could be based on Spore and Capsule immunogens along with dominant negative PA ± amino-terminal PA binding domain of LF/EF delivered employing novel methods and adjuvants to alleviate problems associated with current vaccines.

Future Perspective: Design and development of small molecule inhibitors that could

interfere/inhibit essential interactions of toxin components/virulence factor required for the pathogenesis would form the backbone of any futuristic anthrax therapy. These molecules would also be able to effectively manage pathogenesis of such genetically modified strains against which currently available or next generation experimental vaccines would be ineffective.

FINANCIAL DISCLOSURE/ACKNOWLEDGEMENTS

The authors acknowledge support from Department of Biotechnology, Department of Science and Technology, Council of Scientific and Industrial Research, Indian Council of Medical Research and NATP-World Bank. SS is a Ramalingaswami Fellow supported by DBT, India. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from that disclosed. No writing assistance was utilized in production of this manuscript.