In vitro and in vivo characterization of anthrax anti-protective antigen and anti-lethal factor monoclonal antibodies after passive transfer in a mouse lethal toxin challenge model to define correlates of immunity

Passive transfer of antibody may be useful for preexposure prophylaxis against biological agents used as weapons of terror, such as Bacillus anthracis. Studies were performed to evaluate the ability of anthrax antiprotective antigen (anti-PA) and antilethal factor (anti-LF) neutralizing monoclonal antibodies (mAbs) to protect against an anthrax lethal toxin (LeTx) challenge in a mouse model and to identify correlates of immunity to LeTx challenge. Despite having similar affinities for their respective antigens, anti-PA (3F11) and anti-LF (9A11), passive transfer of up to 1.5 mg of anti-PA 3F11 mAb did not provide significant protection when transferred to mice 24 h before LeTx challenge, while passive transfer of as low as 0.375 mg of anti-LF 9A11 did provide significant protection. Serum collected 24 h after passive transfer had LeTx-neutralizing activity when tested using a standard LeTx neutralization assay, but neutralization titers measured using this assay did not correlate with protection against LeTx challenge. However, measurement of LeTx-neutralizing serum responses with an LeTx neutralization assay in vitro employing the addition of LeTx to J774A.1 cells 15 min before the addition of the serum did result in neutralization titers that correlated with protection against LeTx challenge. Our results demonstrate that only the LeTx neutralization titers measured utilizing the addition of LeTx to J774A.1 cells 15 min before the addition of sample correlated with protection in vivo. Thus, this LeTx neutralization assay may be a more biologically relevant neutralization assay to predict the in vivo protective capacity of LeTx-neutralizing antibodies.     

Neutralizing monoclonal antibody against anthrax lethal factor inhibits intoxication in a mouse model

Anthrax toxin is the dominant virulence factor of Bacillus anthracis; drugs blocking its action could therefore have therapeutic benefit. We report here the production of a neutralizing monoclonal antibody (mAb) against anthrax lethal factor (LF) and the inhibition by the antibody of anthrax lethal toxin (LeTx) formation. The anti-LF monoclonal antibody LF8 neutralized the LeTx challenge both in vitro with macrophage J774A.1 cells and in vivo in nude mice. Our data suggested that LF8 binds LF at or near the PA binding domain. A set of dodecameric peptides was selected from a phage-displayed peptide library through their specific binding to anti-LF neutralizing mAb LF8. These small peptides compete with LF to bind LF8. Further investigation is undergoing to test the potential application of these peptides to the clinical treatment of anthrax infection by blocking LeTx formation.     

Enhancement of anthrax lethal toxin cytotoxicity: a subset of monoclonal antibodies against protective antigen increases lethal toxin-mediated killing of murine macrophages

We investigated the ability of using monoclonal antibodies (MAbs) against anthrax protective antigen (PA), an anthrax exotoxin component, to modulate exotoxin cytotoxic activity on target macrophage cell lines. Anthrax PA plays a critical role in the pathogenesis of Bacillus anthracis infection. PA is the cell-binding component of the two anthrax exotoxins: lethal toxin (LeTx) and edema toxin. Several MAbs that bind the PA component of LeTx are known to neutralize LeTx-mediated killing of target macrophages. Here we describe for the first time an overlooked population of anti-PA MAbs that, in contrast, function to increase the potency of LeTx against murine macrophage cell lines. The results support a possible mechanism of enhancement: binding of MAb to PA on the macrophage cell surface stabilizes the PA by interaction of MAb with macrophage Fcgamma receptors. This results in an increase in the amount of PA bound to the cell surface, which in turn leads to an enhancement in cell killing, most likely due to increased internalization of LF. Blocking of PA-receptor binding eliminates enhancement by MAb, demonstrating the importance of this step for the observed enhancement. The additional significance of these results is that, at least in mice, immunization with PA appears to elicit a poly-clonal response that has a significant prevalence of MAbs that enhance LeTx-mediated killing in macrophages.     

Sequential B-cell epitopes of Bacillus anthracis lethal factor bind lethal toxin-neutralizing antibodies

The bipartite anthrax lethal toxin (LeTx) consisting of protective antigen (PA) and lethal factor (LF) is a major virulence factor contributing to death from systemic Bacillus anthracis infection. The current vaccine elicits antibodies directed primarily to PA; however, in experimental settings serologic responses to LF can neutralize LeTx and contribute to protection against infection. The goals of the present study were to identify sequential B-cell epitopes of LF and to determine the capacity of these determinants to bind neutralizing antibodies. Sera of recombinant LF-immunized A/J mice exhibited high titers of immunoglobulin G anti-LF reactivity that neutralized LeTx in vitro 78 days after the final booster immunization and protected the mice from in vivo challenge with 3 50% lethal doses of LeTx. These sera bound multiple discontinuous epitopes, and there were major clusters of reactivity on native LF. Strikingly, all three neutralizing, LF-specific monoclonal antibodies tested bound specific peptide sequences that coincided with sequential epitopes identified in polyclonal antisera from recombinant LF-immunized mice. This study confirms that LF induces high-titer protective antibodies in vitro and in vivo. Moreover, the binding of short LF peptides by LF-specific neutralizing monoclonal antibodies suggests that generation of protective antibodies by peptide vaccination may be feasible for this antigen. This study paves the way for a more effective anthrax vaccine by identifying discontinuous peptide epitopes of LF.     

Prophylaxis and therapy of inhalational anthrax by a novel monoclonal antibody to protective antigen that mimics vaccine-induced immunity

The neutralizing antibody response to the protective antigen (PA) component of anthrax toxin elicited by approved anthrax vaccines is an accepted correlate for vaccine-mediated protection against anthrax. We reasoned that a human anti-PA monoclonal antibody (MAb) selected on the basis of superior toxin neutralization activity might provide potent protection against anthrax. The fully human MAb (also referred to as MDX-1303 or Valortim) was chosen from a large panel of anti-PA human MAbs generated using transgenic mice immunized with recombinant PA solely on the basis of in vitro anthrax toxin neutralization. This MAb was effective in prophylactic and postsymptomatic treatment of rabbits exposed to aerosolized anthrax spores, and a single intramuscular injection of 1 mg/kg of body weight fully protected cynomolgus monkeys challenged with aerosolized anthrax spores. Importantly, MAb 1303 defines a novel neutralizing epitope that requires Fc receptor engagement for maximal activity. F(ab′)2 fragments of MAb 1303, which retain equivalent affinity for PA, are 10- to 100-fold less potent in neutralizing anthrax toxin in vitro. Addition of Fc receptor-blocking antibodies also greatly reduced the activity of MAb 1303. Moreover, we found that the neutralizing activity of mouse, rabbit, and human antisera elicited by PA vaccines was effectively abrogated by blocking Fc receptors. Selection of an anti-PA MAb by using a functional assay that is a surrogate for protection has resulted in the identification of a fully human MAb with potent activity in vivo and uncovered a previously unrecognized mechanism of antibody-mediated toxin neutralization that is important for currently used anthrax vaccines.     

Role of toxin functional domains in anthrax pathogenesis

We investigated the role of the functional domains of anthrax toxins during infection. Three proteins produced by Bacillus anthracis, the protective antigen (PA), the lethal factor (LF), and the edema factor (EF), combine in pairs to produce the lethal (PA+LF) and edema (PA+EF) toxins. A genetic strategy was developed to introduce by allelic exchange specific point mutations or in-frame deletions into B. anthracis toxin genes, thereby impairing either LF metalloprotease or EF adenylate cyclase activity or PA functional domains. In vivo effects of toxin mutations were analyzed in an experimental infection of mice. A tight correlation was observed between the properties of anthrax toxins delivered in vivo and their in vitro activities. The synergic effects of the lethal and edema toxins resulted purely from their enzymatic activities, suggesting that in vivo these toxins may act together. The PA-dependent antibody response to LF induced by immunization with live B. anthracis was used to follow the in vivo interaction of LF and PA. We found that the binding of LF to PA in vivo was necessary and sufficient for a strong antibody response against LF, whereas neither LF activity nor binding of lethal toxin complex to the cell surface was required. Mutant PA proteins were cleaved in mice sera. Thus, our data provide evidence that, during anthrax infection, PA may interact with LF before binding to the cell receptor. Immunoprotection studies indicated that the strain producing detoxified LF and EF, isogenic to the current live vaccine Sterne strain, is a safe candidate for use as a vaccine against anthrax.     

Neutralizing Monoclonal Antibody to Edema Toxin and Its Effect on Murine Anthrax

Edema factor (EF) is a component of an anthrax toxin that functions as an adenylate cyclase. Numerous monoclonal antibodies (MAbs) have been reported for the other Bacillus anthracis toxin components, but relatively few to EF have been studied. We report the generation of six murine hybridoma lines producing two IgM and four IgG1 MAbs to EF. Of the six MAbs, only one IgM neutralized EF, as assayed by an increase in cyclic AMP (cAMP) production by Chinese hamster ovary (CHO) cells. Analysis of the variable gene elements revealed that the single neutralizing MAb had a different binding site than the others. There was no competition between the neutralizing IgM and the nonneutralizing IgG MAbs indicative of different specificity. MAb-based capture enzyme-linked immunosorbent assay (ELISA) detected EF in liver lysates from mice infected with B. anthracis Sterne 34F2. Administration of the neutralizing IgM MAb to A/JCr mice lethally infected with B. anthracis strain Sterne had no significant effect on median time to death, but mice treated with the MAb were more likely to survive infection. Combining the neutralizing IgM to EF with a subprotective dose of a neutralizing MAb to protective antigen (PA) prolonged mean time to death of infected mice, suggesting that neutralization of EF and PA could produce synergistic beneficial effects. In summary, the results from our study and literature observations suggest that the majority of Abs to EF are nonneutralizing, but the toxin has some epitopes that can be targeted by the humoral response to generate useful Abs that may contribute to defense against anthrax.Bacillus anthracis is a Gram-positive, spore-forming bacterium and the causative agent of anthrax (4, 24). B. anthracis spores are naturally found in the soil, and the disease primarily affects animals such as sheep and cattle that ingest or inhale spores while grazing. Human anthrax is rare, and most cases occur in individuals who contract the disease via contact with farm animals or spore-infested animal hides. However, in recent years, B. anthracis has emerged as a potent biological weapon and there has been great interest in understanding its pathogenesis and developing new therapies (12). B. anthracis produces two large plasmids, pXO1 and pXO2, which encode the genes necessary for toxin production and formation of a poly-d-glutamic acid capsule, respectively. A major virulence factor, the toxins are made up of three protein components known as protective antigen (PA), lethal factor (LF), and edema factor (EF). PA interacts in a binary fashion with EF to produce edema toxin (EdTx) and LF to produce lethal toxin (LeTx) (4). PA interacts with EF and LF via a heptameric structure that facilitates their entry into the cell. EdTx acts as a calcium- and calmodulin-dependent adenylate cyclase that leads to cellular edema, whereas LeTx is a zinc metalloprotease, which cleaves mitogen-activated protein kinase kinases activating various cellular pathways and ultimately leading to cell death (4, 16). In considering the various treatment options for anthrax, the delivery of preformed antibodies (Abs) has some advantages over other measures of postexposure prophylaxis, such as antimicrobial agents (5). Due to their low side effect profile, high specificity, lack of selection for antimicrobial drug resistance, and ability to bind to preformed toxins, Abs could reduce damage during infection. Several monoclonal antibodies (MAbs) to PA were isolated and determined to show protective effects against LeTx in mice (1, 18, 31). Other studies have shown that LF MAbs are also able to protect against LeTx activity in rats (17) and mice (38); however, relatively few attempts have been made to isolate neutralizing MAbs to EF. Little et al. described 10 MAbs to EF; however, only 2 had a moderate effect in disrupting ¹²⁵I-EF binding to cell-bound PA in vitro, with just one of the two inhibiting the rounding of CHO cells, a feature known to result from exposure to EdTx (19). Recently, a neutralizing MAb to EF was described that mediated protection by interfering with the toxin interaction with calmodulin (3).Apart from their potential usefulness as therapeutic reagents, MAbs can also be used to define protective and nonprotective epitopes and for serological assays. Since there has been relatively little work done in the area of neutralizing MAbs against EF, there is an interest in trying to isolate additional MAbs to evaluate the efficacy of humoral immunity and for the design of toxin detection assays. This study describes the isolation of six MAbs against EF, one of which has potent neutralizing activity in vitro and modest protective abilities in vivo.     

Detoxified lethal toxin as a potential mucosal vaccine against anthrax

The nontoxic mutant lethal factor (mLF; which has the E687C substitution) and functional protective antigen (PA63) of Bacillus anthracis were evaluated for their use as mucosal vaccines against anthrax in A/J mice. Intranasal vaccination of three doses of 30 microg of mLF or 60 microg of PA63 elicited significant serum and mucosal antibody responses, with anthrax lethal toxin-neutralizing titers of 40 and 60 in immune sera, respectively. However, only 30% and 60% of the vaccinated animals in the two groups could survive a challenge with 100 times the 50% lethal dose of B. anthracis Sterne spores, respectively. In contrast, vaccination with three doses of the combination of 30 microg of mLF and 60 microg of PA63, the detoxified lethal toxin, elicited antibody responses against LF and PA significantly higher than those elicited after vaccination with mLF or PA63 individually by use of the same dose and schedule. Vaccination with the detoxified lethal toxin resulted in significantly higher lethal toxin-neutralizing antibody titers in sera (titer, 90). Animals vaccinated with three doses of the detoxified lethal toxin were completely protected against the spore challenge. The data suggest that mLF and PA63 have a mutual enhancement effect for evoking systemic and mucosal immune responses and that the detoxified lethal toxin can be used as an efficient mucosal vaccine against anthrax.     

Combinations of Monoclonal Antibodies to Anthrax Toxin Manifest New Properties in Neutralization Assays

Monoclonal antibodies (MAbs) are potential therapeutic agents against Bacillus anthracis toxins, since there is no current treatment to counteract the detrimental effects of toxemia. In hopes of isolating new protective MAbs to the toxin component lethal factor (LF), we used a strain of mice (C57BL/6) that had not been used in previous studies, generating MAbs to LF. Six LF-binding MAbs were obtained, representing 3 IgG isotypes and one IgM. One MAb (20C1) provided protection from lethal toxin (LeTx) in an in vitro mouse macrophage system but did not provide significant protection in vivo. However, the combination of two MAbs to LF (17F1 and 20C1) provided synergistic increases in protection both in vitro and in vivo. In addition, when these MAbs were mixed with MAbs to protective antigen (PA) previously generated in our laboratory, these MAb combinations produced synergistic toxin neutralization in vitro. But when 17F1 was combined with another MAb to LF, 19C9, the combination resulted in enhanced lethal toxicity. While no single MAb to LF provided significant toxin neutralization, LF-immunized mice were completely protected from infection with B. anthracis strain Sterne, which suggested that a polyclonal response is required for effective toxin neutralization. In total, these studies show that while a single MAb against LeTx may not be effective, combinations of multiple MAbs may provide the most effective form of passive immunotherapy, with the caveat that these may demonstrate emergent properties with regard to protective efficacy.     

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.     

Novel Chimpanzee/Human Monoclonal Antibodies That Neutralize Anthrax Lethal Factor,and Evidence for Possible Synergy with Anti-Protective Antigen Antibody

Three chimpanzee Fabs reactive with lethal factor (LF) of anthrax toxin were isolated and converted into complete monoclonal antibodies (MAbs) with human γ1 heavy-chain constant regions. In a macrophage toxicity assay, two of the MAbs, LF10E and LF11H, neutralized lethal toxin (LT), a complex of LF and anthrax protective antigen (PA). LF10E has the highest reported affinity for a neutralizing MAb against LF (dissociation constant of 0.69 nM). This antibody also efficiently neutralized LT in vitro, with a 50% effective concentration (EC₅₀) of 0.1 nM, and provided 100% protection of rats against toxin challenge with a 0.5 submolar ratio relative to LT. LF11H, on the other hand, had a slightly lower binding affinity to LF (dissociation constant of 7.4 nM) and poor neutralization of LT in vitro (EC₅₀ of 400 nM) and offered complete protection in vivo only at an equimolar or higher ratio to toxin. Despite this, LF11H, but not LF10E, provided robust synergistic protection when combined with MAb W1, which neutralizes PA. Epitope mapping and binding assays indicated that both LF10E and LF11H recognize domain I of LF (amino acids 1 to 254). Although domain I is responsible for binding to PA, neither MAb prevented LF from binding to activated PA. Although two unique MAbs could protect against anthrax when used alone, even more efficient and broader protection should be gained by combining them with anti-PA MAbs.Anthrax is a highly lethal infectious disease caused by the spore-forming bacterium Bacillus anthracis. The deliberate distribution of anthrax spores through the U.S. mail system in 2001 resulted in five deaths among the 11 individuals who contracted inhalational anthrax (18). This incident highlighted the great threat posed by the potential use of anthrax in terrorism and warfare. The lethality of inhalational anthrax is primarily due to the action of anthrax toxins. The bacterium produces three toxin components; these are protective antigen (PA) (83 kDa), lethal factor (LF) (85 kDa), and edema factor (EF) (89 kDa) (13, 32). PA binds to host cell anthrax toxin receptors and is cleaved by cell surface furin to produce a 63-kDa peptide, PA63 (activated PA). Anthrax toxin receptor-bound PA63 oligomerizes to a heptamer and translocates up to three molecules of LF or EF from the cell surface via endosomes to the cytosol. Therefore, PA functions as a vehicle to mediate the cellular uptake of LF and EF (for a review, see reference 44). PA with LF forms lethal toxin (LT), and PA with EF forms edema toxin (ET). LF is a zinc-dependent endopeptidase that cleaves mitogen-activated protein kinase kinases and disrupts intracellular signaling (8, 30, 40). LT can replicate symptoms of anthrax disease when injected into animals (27). EF is a calcium-calmodulin-dependent adenylate cyclase that transforms ATP to cyclic AMP, and ET has a range of toxic effects in the host (12, 20). These toxins are the dominant virulence factors for anthrax disease, and vaccination against their common component, PA, is sufficient for protection against anthrax disease.Currently antibiotics are the only choice for clinical treatment of anthrax disease. Although effective, antibiotics have limitations. Exposure to the bacterium followed by bacterial division leads to production of large quantities of the anthrax toxins. Thus, unless exposure is diagnosed early enough for antibiotic treatment to prevent significant replication, patients will succumb to disease even after the killing of all bacteria. The current PA-based vaccine approved by the U.S. Food and Drug Administration is also not effective postexposure in protecting newly infected individuals, as it requires repeated administration and at least 4 weeks for development of anti-PA protective titers. Thus, in the absence of any small-molecule toxin inhibitors, monoclonal antibodies (MAbs) against toxin components are the only viable candidates for immediate neutralization of the effects of toxin. Although PA has been the primary target for passive protection (5, 25, 31, 35, 41, 43), it has been suggested that immunity to LF and EF can also play an important role in protection (14, 33, 34), and thus these proteins may represent alternative targets for antibody therapy against anthrax. In a previous study, the protective effects of anti-PA and anti-LF antibodies were greatly synergized by their combination (3). Furthermore, concerns that PA may be mutated within currently recognized neutralization epitopes such that anti-PA therapies would no longer be effective against this toxin warrant the further development of antibodies targeting the other toxin components. A cocktail of more than one MAb that could recognize distinct epitopes on multiple toxin proteins (PA, LF, and EF) could certainly broaden the spectrum of protection against anthrax. In recent years, several anti-LF neutralizing MAbs have been reported (1, 21, 24, 37, 46). However, only one of them was a human antibody; the others were rodent MAbs that would need further manipulation before use in humans.Chimpanzee immunoglobulins (Igs) are virtually identical to human Igs and may have clinically useful applications (9). As part of a larger study (5), we recovered chimpanzee MAbs specific for LF from a combinatorial cDNA library of antibody genes developed from chimpanzees that had been immunized with anthrax toxins. In this work we describe the detailed characterization of these anti-LF antibodies.     

Proteomic analyses of murine macrophages treated with Bacillus anthracis lethal toxin

Bacillus anthracis is the etiological agent of anthrax and the bacterium produces a tripartite anthrax toxin composed of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA represents the binding domain of the toxin and acts in concert with either LF, a metalloprotease, or EF, an adenylate cyclase, to form lethal toxin (LeTx) or edema toxin (EdTx), respectively. We analyzed the proteomics response of two murine macrophage cell lines (J774.1A and RAW264.7) following B. anthracis LeTx treatment to detect unique host proteins involved in anthrax infection using difference in-gel electrophoresis (DIGE) followed by nanoLC-MS for identification of the proteins. The comparative proteomics approach identified a set of proteins in each cell line that was consistently upregulated when the two macrophage cell lines were treated with LeTx. The upregulated proteins include those involved in energy metabolism, cytoskeleton structure and stress response. A subset of five proteins (ATP synthase beta subunit, beta-actin, Hsp70, vimentin, and Hsp60 homolog) was identified that were commonly upregulated in both cell lines. The proteomic data suggest the involvement of reactive oxygen species (ROS) in cell lysis as seen by the upregulation of proteins that lead to the production of ROS in both the cell lines used in our study. However, proteins that afford protection against ROS may play an important role in the survival of the macrophage to LeTx infection as shown by the differences in proteomic responses of the two cell lines to the action of LeTx. These identified proteins may have the potential to be used as biomarkers for diagnostics and therapeutics.     

Synthetic Peptide Vaccine Targeting a Cryptic Neutralizing Epitope in Domain 2 of Bacillus anthracis Protective Antigen

Current evidence suggests that protective antigen (PA)-based anthrax vaccines may elicit a narrow neutralizing antibody repertoire, and this may represent a vulnerability with PA-based vaccines. In an effort to identify neutralizing specificities which may complement those prevalent in PA antiserum, we evaluated whether sequences within the 2β2-2β3 loop of PA, which are apparent in the crystal structure of heptameric but not monomeric PA, might represent a target for an epitope-specific vaccine for anthrax and, further, whether antibodies to these sequences are induced in rabbits immunized with monomeric PA. We evaluated the immunogenicity in rabbits of a multiple antigenic peptide (MAP) displaying copies of amino acids (aa) 305 to 319 of this region. Overall, four out of six rabbits vaccinated with the MAP peptide in Freund''s adjuvant developed high-titer, high-avidity antibody responses which cross-reacted with the immobilized peptide sequence comprising aa 305 to 319 and with PA, as determined by an enzyme-linked immunosorbent assay, and which displayed potent and durable neutralization of lethal toxin (LeTx) in vitro, with peak titers which were 452%, 100%, 67%, and 41% of the peak neutralization titers observed in positive-control rabbits immunized with PA. Importantly, analysis of sera from multiple cohorts of rabbits with high-titer immunity to PA demonstrated a virtual absence of this potent antibody specificity, and work by others suggests that this specificity may be present at only low levels in primate PA antiserum. These results highlight the potential importance of this immunologically cryptic neutralizing epitope from PA as a target for alternative and adjunctive vaccines for anthrax.Bacillus anthracis has a long and storied history as the causative agent of anthrax in wildlife, livestock, and human hosts. More recently, the easy distribution and extreme toxicity associated with inhalation of its endospores have positioned B. anthracis as an accessible yet formidable bioweapon for use in warfare and terrorism. The morbidity and mortality associated with inhalation of anthrax spores among humans is largely a direct result of the elaboration of lethal toxin (LeTx) during vegetative growth of virulent strains of B. anthracis. The anthrax toxins LeTx and edema toxin are classic A-B toxins, where lethal factor (LF) and edema factor (EF) represent the active moieties and protective antigen (PA) the binding moiety. PA binds the anthrax toxin receptor (ATR), either CMG2 or TEM8, and forms a heptameric prepore which binds EF or LF. Under the acidic conditions of the late endosome, EF and LF are transported into the cell cytosol, where they exert their enzymatic activities as edema toxin and LeTx, respectively (9, 11, 24).PA-specific humoral immunity has been demonstrated to protect animals from experimental challenge with anthrax even in the absence of LF and EF immunity (25, 37, 58). Animal model studies have shown that anthrax vaccine adsorbed (AVA), the currently licensed anthrax vaccine in the United States, provides protection by stimulating antibodies against PA (38, 57), and AVA has been shown to confer a high degree of protection from an inhalation spore challenge in rabbits and primates (14, 20, 38). However, the multiple injections and yearly boosts required for establishment and maintenance of immunity, and the reactogenicity of and potential adverse reactions to AVA, have raised broad concern and have motivated commitment to the development of next-generation anthrax vaccines (10, 21, 39, 52).Most of the efforts to develop new vaccines for anthrax have focused on the elicitation of immunity to PA. Ongoing research is also focused on the design and testing of vaccines targeting antigens other than PA in an effort to broaden the breadth of immunity induced through vaccination (8, 15, 30, 40). This was motivated in part by the realization, informed primarily by analysis of PA-specific monoclonal antibodies (MAbs) in mice and humans, that the antibody specificities responsible for LeTx neutralization may be limited to only a few dominant specificities (1, 7, 28, 29, 44). These are focused primarily on domains 1′ and 4 of PA63, which are involved in binding to LF and EF and to the anthrax toxin receptors, respectively. The limited breadth of the neutralizing repertoire induced through PA immunization could leave vaccinees vulnerable to possible maliciously altered or selected B. anthracis strains resistant to the neutralizing specificities, a contingency for which proof of principle has now been demonstrated (2, 48).The solution of the 1TZN crystal structure revealed the PA heptamer bound to the CMG2 cell receptor, including sequences within the 2β2-2β3 loop of PA which were unresolved in previous crystal structures (23). The 2β2-2β3 loop had previously been identified as containing the chymotrypsin cleavage site and was shown to be critical for LeTx function, specifically for translocation of EF and LF into the cytosol (33, 34, 54, 55). The surface-exposed nature of this sequence, as deduced through protein-structure algorithms and through experimental demonstration that the site is accessible to protease cleavage, led us to believe that it might represent an effective target for an epitope-specific vaccine for anthrax.To date, efforts to develop vaccines targeting specific epitopes within PA or LF have been limited, and there are no published accounts of efficacious peptide vaccines targeting PA. Our hypothesis that this site in domain 2 of PA might represent a neutralizing determinant was confirmed when two groups independently reported mouse MAbs specific for the 2β2-2β3 loop region, which possessed LeTx-neutralizing activity (16, 61). Here, we demonstrate that a multiple antigenic peptide (MAP) consisting of four copies per molecule of amino acids (aa) 305 to 319 of PA can elicit humoral immunity in rabbits that is specific for the 2β2-2β3 loop neutralizing determinant (LND) and which demonstrates potent neutralization of LeTx in vitro. We further show that antibody specific for the LND is not induced in rabbits immunized with full-length PA.     

Detection of anthrax toxin in the serum of animals infected with Bacillus anthracis by using engineered immunoassays

Several strategies that target anthrax toxin are being developed as therapies for infection by Bacillus anthracis. Although the action of the tripartite anthrax toxin has been extensively studied in vitro, relatively little is known about the presence of toxins during an infection in vivo. We developed a series of sensitive sandwich enzyme-linked immunosorbent assays (ELISAs) for detection of both the protective antigen (PA) and lethal factor (LF) components of the anthrax exotoxin in serum. The assays utilize as capture agents an engineered high-affinity antibody to PA, a soluble form of the extracellular domain of the anthrax toxin receptor (ANTXR2/CMG2), or PA itself. Sandwich immunoassays were used to detect and quantify PA and LF in animals infected with the Ames or Vollum strains of anthrax spores. PA and LF were detected before and after signs of toxemia were observed, with increasing levels reported in the late stages of the infection. These results represent the detection of free PA and LF by ELISA in the systemic circulation of two animal models exposed to either of the two fully virulent strains of anthrax. Simple anthrax toxin detection ELISAs could prove useful in the evaluation of potential therapies and possibly as a clinical diagnostic to complement other strategies for the rapid identification of B. anthracis infection.     

Protection against anthrax lethal toxin challenge by genetic immunization with a plasmid encoding the lethal factor protein

The ability of genetic vaccination to protect against a lethal challenge of anthrax toxin was evaluated. BALB/c mice were immunized via gene gun inoculation with eucaryotic expression vector plasmids encoding either a fragment of the protective antigen (PA) or a fragment of lethal factor (LF). Plasmid pCLF4 contains the N-terminal region (amino acids [aa] 10 to 254) of Bacillus anthracis LF cloned into the pCI expression plasmid. Plasmid pCPA contains a biologically active portion (aa 175 to 764) of B. anthracis PA cloned into the pCI expression vector. One-micrometer-diameter gold particles were coated with plasmid pCLF4 or pCPA or a 1:1 mixture of both and injected into mice via gene gun (1 microg of plasmid DNA/injection) three times at 2-week intervals. Sera were collected and analyzed for antibody titer as well as antibody isotype. Significantly, titers of antibody to both PA and LF from mice immunized with the combination of pCPA and pCLF4 were four to five times greater than titers from mice immunized with either gene alone. Two weeks following the third and final plasmid DNA boost, all mice were challenged with 5 50% lethal doses of lethal toxin (PA plus LF) injected intravenously into the tail vein. All mice immunized with pCLF4, pCPA, or the combination of both survived the challenge, whereas all unimmunized mice did not survive. These results demonstrate that DNA-based immunization alone can provide protection against a lethal toxin challenge and that DNA immunization against the LF antigen alone provides complete protection.     

Functional analysis of Bacillus anthracis protective antigen by using neutralizing monoclonal antibodies

Protective antigen (PA) is central to the action of the lethal and edema toxins produced by Bacillus anthracis. It is the common cell-binding component, mediating the translocation of the enzymatic moieties (lethal factor [LF] and edema factor) into the cytoplasm of the host cell. Monoclonal antibodies (MAbs) against PA, able to neutralize the activities of the toxins in vitro and in vivo, were screened. Two such MAbs, named 7.5 and 48.3, were purified and further characterized. MAb 7.5 binds to domain 4 of PA and prevents the binding of PA to its cell receptor. MAb 48.3 binds to domain 2 and blocks the cleavage of PA into PA63, a step necessary for the subsequent interaction with the enzymatic moieties. The epitope recognized by this antibody is in a region involved in the oligomerization of PA63; thus, MAb 48.3 does not recognize the oligomer form. MAbs 7.5 and 48.3 neutralize the activities of anthrax toxins produced by B. anthracis in mice. Also, there is an additive effect between the two MAbs against PA and a MAb against LF, in protecting mice against a lethal challenge by the Sterne strain. This work contributes to the functional analysis of PA and offers immunotherapeutic perspectives for the treatment of anthrax disease.     

A new triple chimeric protein as a high immunogenic antigen against anthrax toxins: theoretical and experimental analyses

Background: Anthrax is a zoonotic disease caused by Bacillus anthracis and it can be deadly in 6 days. Considerable efforts have been conducted toward developing more effective veterinary and human anthrax vaccines because these common vaccines have several limitations. B. anthracis secretes a tripartite toxin, comprising protective antigen (PA), edema factor (EF), and lethal factor (LF). Several studies have shown important role of PA in protection of anthrax. LF and EF induce production of toxin neutralizing antibodies too. PA in fusion form with LF/EF has synergistic effects as a potential subunit vaccine.

Methods: In this study, for the first time, a triple chimeric protein called ELP was modeled by fusing three different domains of anthrax toxic antigens, the N-terminal domains of EF and LF, and the C-terminal domain of PA as a high immunogenic antigen using Modeller 9.19 software. Immunogenicity of the ELP was assessed in guinea pigs using enzyme-linked immunosorbent assay (ELISA) test and MTT assay.

Results: Theoretical studies and molecular dynamics (MD) simulation results suggest that the ELP model had acceptable quality and stability. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified ELP, its domains, and PA were matched with their molecular size and confirmed by western blotting analysis. In the immune guinea pigs, antibody was produced against all of the ELP domains. It was observed that ELP induced strong humoral response and could protect murine macrophage cell line (RAW 264.7 cells) against anthrax lethal toxin (LeTx).

Conclusions: ELP chimeric antigen could be considered as a high immunogenic antigen.     

Immunogenicity of recombinant protective antigen and efficacy against aerosol challenge with anthrax

Immunization with a recombinant form of the protective antigen (rPA) from Bacillus anthracis has been carried out with rhesus macaques. Rhesus macaques immunized with 25 mug or more of B. subtilis-expressed rPA bound to alhydrogel had a significantly increased immunoglobulin G (IgG) response to rPA compared with macaques receiving the existing licensed vaccine from the United Kingdom (anthrax vaccine precipitated [AVP]), although the isotype profile was unchanged, with bias towards the IgG1 and IgG2 subclasses. Immune macaque sera from all immunized groups contained toxin-neutralizing antibody and recognized all the domains of PA. While the recognition of the N terminus of PA (domains 1 to 3) was predominant in macaques immunized with the existing vaccines (AVP and the U.S. vaccine anthrax vaccine adsorbed), macaques immunized with rPA recognized the N- and C-terminal domains of PA. Antiserum derived from immunized macaques protected macrophages in vitro against the cytotoxic effects of lethal toxin. Passive transfer of IgG purified from immune macaque serum into naive A/J mice conferred protection against challenge with B. anthracis in a dose-related manner. The protection conferred by passive transfer of 500 mug macaque IgG correlated significantly (P = 0.003; r = 0.4) with the titers of neutralizing antibody in donor macaques. Subsequently, a separate group of rhesus macaques immunized with 50 mug of Escherichia coli-derived rPA adsorbed to alhydrogel was fully protected against a target dose of 200 50% lethal doses of aerosolized B. anthracis. These data provide some preliminary evidence for the existence of immune correlates of protection against anthrax infection in rhesus macaques immunized with rPA.     

Characterization of neutralizing antibodies to West Nile virus

We produced nine monoclonal antibodies (MAbs) directed against the West Nile virus E glycoprotein using three different immunization strategies: inactivated virus, naked DNA, and recombinant protein. Most of the MAbs bound to conformation dependent epitopes in domain III of the E protein. Four of the MAbs neutralized WNV infection and bound to the same region of domain III with high affinity. The neutralizing MAbs were obtained from mice immunized with inactivated virus alone or in combination with a DNA plasmid. In contrast, MAbs obtained by immunization with a soluble version of the E glycoprotein did not exhibit neutralizing activity. These non-neutralizing antibodies were cross-reactive with several other flaviviruses, including Saint Louis encephalitis, Japanese encephalitis, Yellow Fever and Powassan viruses. Interestingly, some non-neutralizing MAbs bound with high affinity to domains I or III, indicating that both affinity and the precise epitope recognized by an antibody are important determinants of WNV neutralization.     

Neutralizing antibodies and persistence of immunity following anthrax vaccination

Anthrax toxin consists of protective antigen (PA) and two toxic components, lethal factor (LF) and edema factor (EF). PA binds to mammalian cellular receptors and delivers the toxic components to the cytoplasm. PA is the primary antigenic component of the current anthrax vaccine. Immunity is due to the generation of antibodies that prevent the PA-mediated internalization of LF and EF. In this study, we characterized sera obtained from vaccinated military personnel. Anthrax vaccine is administered in a series of six injections at 0, 2, and 4 weeks and 6, 12, and 18 months, followed by annual boosters. The vaccination histories of the subjects were highly varied; many subjects had not completed the entire series, and several had not received annual boosters. We developed a simple colorimetric assay using alamarBlue dye to assess the antibody-mediated neutralization of LF-mediated toxicity to the J774A.1 murine macrophage cell line. Recently vaccinated individuals had high antibody levels and neutralizing activity. One individual who had not been boosted for 5 years had low immunoglobulin G antibody levels but a detectable neutralization activity, suggesting that this individual produced low levels of very active antibodies.