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.     

Neutralizing activity of vaccine-induced antibodies to two Bacillus anthracis toxin components, lethal factor and edema factor

Anthrax vaccine adsorbed (AVA; BioThrax), the current FDA-licensed human anthrax vaccine, contains various amounts of the three anthrax toxin components, protective antigen (PA), lethal factor (LF), and edema factor (EF). While antibody to PA is sufficient to mediate protection against anthrax in animal models, it is not known if antibodies to LF or EF contribute to protection in humans. Toxin-neutralizing activity was evaluated in sera from AVA-vaccinated volunteers, all of whom had antibody responses to LF and EF, as well as PA. The contribution of antibodies to LF and EF was assessed using mouse macrophage J774A.1 cells by examining neutralization of LF-induced lysis using alamarBlue reduction and neutralization of EF-induced cyclic AMP increases by enzyme-linked immunosorbent assay. Antibody responses to LF and EF were low compared to those to PA, and the amount of LF or EF in the assay could exceed the amount of antibodies to LF or EF. Higher titers were seen for most individuals when the LF or EF concentration was limiting compared to when LF or EF was in excess, initially suggesting that antibody to LF or EF augmented protection. However, depletion of LF and EF antibodies in sera did not result in a significant decrease in toxin neutralization. Overall, this study suggests that AVA-induced LF and EF antibodies do not significantly contribute to anthrax toxin neutralization in humans and that antibodies to PA are sufficient to neutralize toxin activity.     

The high efficiency, human B cell immortalizing heteromyeloma CB-F7. Production of human monoclonal antibodies to human immunodeficiency virus

This paper describes the construction of a new heteromyeloma cell line designated CB-F7. The cell line was derived from xenogeneic somatic cell hybridization between normal human B lymphocytes and the murine HAT-sensitive P3X63Ag8/653 cell line. CB-F7 cells were characterized by rapid cell growth (doubling time about 16 h) and high cloning efficiencies in culture medium supplemented with 10% or 5% fetal calf serum, respectively. The karyotype of the cells consists of about 75-78 chromosomes as well as two chromosomal fragments. Fusions of the cells with human peripheral blood cells resulted in approximately 2-6 clones per 10(5) seeded lymphocytes. Furthermore, the cells are ouabain resistant and therefore suitable for fusions with EBV-transformed lymphoblastoid cell lines. Using CB-F7 as the parental cell line a number of specific human mAb producing hybrids were established. For the first time, we describe here the generation of hybrids secreting human monoclonal antibodies to human immunodeficiency virus (HIV). Two monoclonal antibodies of IgG type and one of IgM type reacted with the major core protein p25 and one IgG antibody reacted with the transmembrane protein gp41.     

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.     

Analysis of Defined Combinations of Monoclonal Antibodies in Anthrax Toxin Neutralization Assays and Their Synergistic Action

Antibodies against the protective antigen (PA) component of anthrax toxin play an important role in protection against disease caused by Bacillus anthracis. In this study, we examined defined combinations of PA-specific monoclonal antibodies for their ability to neutralize anthrax toxin in cell culture assays. We observed additive, synergistic, and antagonistic effects of the antibodies depending on the specific antibody combination examined and the specific assay used. Synergistic toxin-neutralizing antibody interactions were examined in more detail. We found that one mechanism that can lead to antibody synergy is the bridging of PA monomers by one antibody, with resultant bivalent binding of the second antibody. These results may aid in optimal design of new vaccines and antibody therapies against anthrax.     

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.     

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.     

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.     

Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen

Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim^®), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim^® attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim^® antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0.05) decreased relative to the amount of PA remained in the solution after passing through unmodified as well as protein A modified poly(AAm-AGE) cryogel columns, indicates efficient PA removal from spiked PBS over 60 min of circulation. The high adsorption capacity towards anthrax toxin PA of the cryogel adsorbents indicated potential application of these materials for treatment of Bacillus anthracis infection.     

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.     

Protective antigen-mediated antibody response against a heterologous protein produced in vivo by Bacillus anthracis

Bacillus anthracis secretes a lethal toxin composed of two proteins, the lethal factor (LF) and the protective antigen (PA), which interact within the host or in vitro at the surfaces of eukaryotic cells. Immunization with attenuated B. anthracis strains induces an antibody response against PA and LF. The LF-specific response is potentiated by the binding of LF to PA. In this study, we investigated the capacity of PA to increase the antibody response against a foreign antigen. We constructed a chimeric gene encoding the PA-binding part of LF (LF254) fused to the C fragment of tetanus toxin (ToxC). The construct was introduced by allelic exchange into the locus encoding LF. Two recombinant B. anthracis strains secreting the hybrid protein LF254-ToxC were generated, one in a PA-producing background and the other in a PA-deficient background. Mice were immunized with spores of the strains, and the humoral response and protection against tetanus toxin were assessed. The B. anthracis strain producing both PA and LF254-ToxC induced significantly higher antibody titers and provided better protection against a lethal challenge with tetanus toxin than did its PA-deficient counterpart. Thus, PA is able to potentiate protective immunity against a heterologous antigen, demonstrating the potential of B. anthracis recombinant strains for use as live vaccine vehicles.     

Frequency and Domain Specificity of Toxin-Neutralizing Paratopes in the Human Antibody Response to Anthrax Vaccine Adsorbed

Protective antigen (PA) is the cell surface recognition unit of the binary anthrax toxin system and the primary immunogenic component in both the current and proposed “next-generation” anthrax vaccines. Several studies utilizing animal models have indicated that PA-specific antibodies, acquired by either active or passive immunization, are sufficient to protect against infection with Bacillus anthracis. To investigate the human antibody response to anthrax immunization, we have established a large panel of human PA-specific monoclonal antibodies derived from multiple individuals vaccinated with the currently approved anthrax vaccine BioThrax. We have determined that although these antibodies bind PA in standard binding assays such as enzyme-linked immunosorbent assay, Western blotting, capture assays, and dot blots, less than 25% are capable of neutralizing lethal toxin (LT) in vitro. Nonneutralizing antibodies also fail to neutralize toxin when present in combination with other nonneutralizing paratopes. Although neutralizing antibodies recognize determinants throughout the PA monomer, they are significantly less common among those paratopes that bind to the immunodominant amino-terminal portion of the molecule. These findings demonstrate that PA binding alone is not sufficient to neutralize LT and suggest that for an antibody to effectively block PA-mediated toxicity, it must bind to PA such that one of the requisite toxin functions is disrupted. A vaccine design strategy that directed a higher percentage of the antibody response toward neutralizing epitopes may result in a more efficacious vaccine for the prevention of anthrax infection.The Bacillus anthracis binary toxin system contributes directly to anthrax pathogenicity in the host (3, 14). The cell surface recognition element of this toxin system is an 83-kDa protein known as protective antigen (PA₈₃). Antibodies that bind PA protect against infection (8, 12), and PA is the primary immunogenic component in the anthrax vaccine currently licensed for use in the United States (BioThrax, or anthrax vaccine adsorbed [AVA]; Emergent Biosystems). Ongoing attempts to develop a “next-generation” anthrax vaccine are relying on a recombinant form of PA as the sole immunogenic component. PA''s role as an important vaccine target has driven a significant amount of research into both the biology of this protein toxin and the immunobiology of its interaction with the immune system of the vaccinated or infected host.PA₈₃ binds to the cell surface receptors tumor endothelial marker 8 and the capillary morphogenesis gene 2 product (4, 20). Bound PA is cleaved by cell surface-associated furin proteases to release the 20-kDa amino-terminal portion of the molecule (PA₂₀), which has no further role in intoxication. Following proteolytic cleavage, cell-bound PA₆₃ self-assembles to form a heptameric prepore structure that can bind several molecules of the catalytic toxin components lethal factor (LF) and/or edema factor (EF). Receptor-mediated endocytosis results in the internalization of the complex, which inserts into the membrane of the endocytic vacuole. LF and/or EF is then actively translocated into the cytoplasm of the cell. The structure of PA, both as a monomer and as a heptamer, has been determined (15, 19), and the regions of the molecule (domains) involved in the various functions described above have been identified (1, 6, 15, 18, 19).The immunobiology of the immune response to PA in vaccinated humans has only recently been explored at the molecular level. PA elicits a polyclonal antibody response in vaccinated humans that utilizes a wide variety of immunoglobulin variable (V)-region genes. Preliminary studies have indicated that after vaccination, antibodies undergo the somatic hypermutation and class switch normally associated with affinity maturation (21). We have previously demonstrated the human antibody response to PA to be significantly biased toward epitopes associated with the amino-terminal domain of the PA protein (PA₂₀) and have postulated that these antibodies may be deficient in their ability to neutralize toxin (16).In this study, we determined the toxin neutralization potentials of a large panel of individual monoclonal antibodies isolated from seven individuals vaccinated with AVA vaccine, using a cell-based assay of LT-mediated cytotoxicity. We found that only 24% of the component antibodies that comprise the overall response are capable of neutralizing PA-mediated cytotoxicity in vitro. We found no direct correlation between the relative PA binding ability of the individual antibodies and their ability to neutralize anthrax toxin. We also determined that toxin-neutralizing paratopes occur less frequently among those antibodies that recognize the immunodominant epitopes associated with the amino-terminal domain of the PA monomer. These findings suggest that the efficacy of future PA-based vaccines might be improved by modifying the immunogen such that a greater proportion of the antibody response is directed toward those epitopes that lead to toxin neutralization.     

Recombinant Single-Chain Variable Fragment Antibodies Directed against Clostridium difficile Toxin B Produced by Use of an Optimized Phage Display System

Recombinant antibody cloning and phage display technologies were used to produce single-chain antibodies (scFv) against Clostridium difficile toxin B. The starting material was the mouse B cell hybridoma line 5A8, which generates a monoclonal antibody against the toxin. The integrated cloning, screening, and phage display system of Krebber et al. (J. Immunol. Methods 201:35-55, 1997) allowed us to rapidly obtain toxin B-binding scFv sequences derived from the hybridoma cell line. The best candidate scFv sequences, based on preliminary enzyme-linked immunosorbent assay (ELISA) screening data were then subcloned into the compatible expression vector. Recombinant single-chain antibodies were expressed in Escherichia coli. A 29-kDa band was observed on polyacrylamide gel electrophoresis as predicted. The expressed product was characterized by immunoblotting and detection with an anti-FLAG antibody. The toxin B-binding function of the single-chain antibody was shown by a sandwich ELISA. The antibody was highly specific for toxin B and did not cross-react with material isolated from a toxin B-negative C. difficile strain. The sensitivity of the soluble single-chain antibody is significantly higher than the original monoclonal antibody based on ELISA data and could detect a minimum of 10 ng of toxin B/well. Competitive ELISAs established that the affinity of the 5A8 parent antibody and the best representative (clone 10) of the single-chain antibodies were similar and in the range of 10⁻⁸ M. We propose that recombinant antibody technology is a rapid and effective approach to the development of the next generation of immunodiagnostic reagents.     

Anthrax Toxin

Anthrax is primarily a disease of herbivores caused by Gram-positive, aerobic, spore-forming Bacillus anthracis. Humans are accidental hosts through the food of animal origin and animal products. Anthrax is prevelant in most parts of the globe, and cases of anthrax have been reported from almost every country. Three forms of the disease have been recognized: cutaneous (through skin), gastrointestinal (through alimentary tract), and pulmonary (by inhalation of spores).

The major virulence factors of Bacillus anthracis are a poly-D glutamic acid capsule and a three-component protein exotoxin. The genes coding for the toxin and the enzymes responsible for capsule production are carried on plasmid pXO1 and pXO2, respectively. The three proteins of the exotoxin are protective antigen (PA, 83?kDa), lethal factor (LF, 90?kDa), and edema factor (EF, 89?kDa). The toxins follow the A-B model with PA being the B moeity and LF/EF, the alternative A moeities. LF and EF are individually nontoxic, but in combination with PA form two toxins causing different pathogenic responses in animals and cultured cells. PA + LF forms the lethal toxin and PA + EF forms the edema toxin. During the process of intoxication, PA binds to the cell surface receptor and is cleaved at the sequence RKKR (167) by cell surface proteases such as furin generating a cell-bound, C-terminal 63?kDa protein (PA63). PA63 possesses a binding site to which LF or EF bind with high affinity. The complex is then internalized by receptor-mediated endocytosis. Acidification of the vesicle leads to instertion of PA63 into the endosomal membrane and translocation of LF/EF across the bilayer into the cytosol where they exert their toxic effects. EF has a calcium- and calmodulin-dependent adenylate cyclase activity. Recent reports indicate that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and 2), and this cleavage inactivates MAPKK1 and thus inhibits the mitogen-activated protein kinase signal transduction pathway. We describe in detail the studies so far done on unraveling the molecular mechanisms of pathogenesis of Bacillus anthracis.     

The effect of deletion of the edema factor on Bacillus anthracis pathogenicity in guinea pigs and rabbits

Bacillus anthracis secretes three major components, which assemble into two bipartite toxins: lethal toxin (LT), composed of lethal factor (LF) and protective antigen (PA) and edema toxin (ET), composed of edema factor (EF) and PA. EF is a potent calmodulin-dependent adenylate cyclase, which is internalized into the target cell following PA binding. Once inside the cell, EF elevates cAMP levels, interrupting intracellular signaling. Effects of ET were demonstrated on monocytes, neutrophils and T-cells. In an earlier work we demonstrated that a deletion of LF in a fully virulent strain had no effect in guinea pigs and a significant, but not major, effect in the rabbit model. These results suggested that EF might play an important role in the development of infection and mortality following exposure to B. anthracis spores. To evaluate the role of EF in B. anthracis pathogenicity we deleted the cya gene, which encodes the EF protein, in the fully virulent Vollum strain. The Δcya mutant was fully virulent in the guinea pig model as determined by LD₅₀ experiments. In the rabbit model, when infected subcutaneously, the absence of EF had no effect on the virulence of the mutant. However an increase of two orders of magnitude in the LD₅₀ was demonstrated when the rabbits were infected by intranasal instillation accompanied with partial mortality and increased mean time to death.These results argue that in the guinea pig model the presence of one of the toxins, ET or LT is sufficient for the development of the infection. In the rabbit model ET plays a role in respiratory infection, most probably mediating the early steps of host colonization.     

Development of specific IgG-producing human hybridomas by fusions of lymphocytes from actively immunized persons

More than 13,500 initial hybridoma lines derived from fusions of lymphocytes from non-boosted persons were tested for IgG production against Tetanus Toxoid. However, only 2 were found to produce IgG monoclonal antibodies of the desired specificity. Peripheral blood lymphocytes were then taken from actively immunized donors 3, 7, 14 or 60 days after boosting and fused to the HAT-sensitive heteromyeloma cell line CB-F7. A strong enhancement of both IgG-producing hybridomas and specific IgG-producers was detected in fusions of lymphocyte material derived 7 days after booster injection (239 of 731 IgG-producing lines showed anti-TT-specificity). Two months after boosting no more specific IgG-producing hybridomas could be established from the peripheral blood of the donors. Data were discussed regarding an optimized human monoclonal antibody production against bacterial antigens.     

The Major Neutralizing Antibody Responses to Recombinant Anthrax Lethal and Edema Factors Are Directed to Non-Cross-Reactive Epitopes

Anthrax lethal and edema toxins (LeTx and EdTx, respectively) form by binding of lethal factor (LF) or edema factor (EF) to the pore-forming moiety protective antigen (PA). Immunity to LF and EF protects animals from anthrax spore challenge and neutralizes anthrax toxins. The goal of the present study is to identify linear B-cell epitopes of EF and to determine the relative contributions of cross-reactive antibodies of EF and LF to LeTx and EdTx neutralization. A/J mice were immunized with recombinant LF (rLF) or rEF. Pools of LF or EF immune sera were tested for reactivity to rLF or rEF by enzyme-linked immunosorbent assays, in vitro neutralization of LeTx and EdTx, and binding to solid-phase LF and EF decapeptides. Cross-reactive antibodies were isolated by column absorption of EF-binding antibodies from LF immune sera and by column absorption of LF-binding antibodies from EF immune sera. The resulting fractions were subjected to the same assays. Major cross-reactive epitopes were identified as EF amino acids (aa) 257 to 268 and LF aa 265 to 274. Whole LF and EF immune sera neutralized LeTx and EdTx, respectively. However, LF sera did not neutralize EdTx, nor did EF sera neutralize LeTx. Purified cross-reactive immunoglobulin G also failed to cross-neutralize. Cross-reactive B-cell epitopes in the PA-binding domains of whole rLF and rEF occur and have been identified; however, the major anthrax toxin-neutralizing humoral responses to these antigens are constituted by non-cross-reactive epitopes. This work increases understanding of the immunogenicity of EF and LF and offers perspective for the development of new strategies for vaccination against anthrax.Infectious agents with biological-weapon potential have become the focus of intense interest since the malicious release of anthrax spores through the U.S. postal system in 2001. Bacillus anthracis, the etiological agent of anthrax infection, is a gram-positive, rod-shaped, spore-forming bacterium that is commonly found in soil (37). The use of this agent as a bioterror weapon has highlighted the importance of developing improved vaccination strategies.The virulence of B. anthracis is attributable to a tripartite protein complex consisting of the receptor binding component protective antigen (PA) and two catalytic components, lethal factor (LF) and edema factor (EF). Combination of PA and LF forms lethal toxin (LeTx), and combination of PA and EF forms edema toxin (EdTx) (26). Interestingly, the PA-binding domains of both EF and LF, corresponding to the N-terminal regions, have been shown to share large regions of structural and amino acid similarities that have been implicated in binding to PA (6, 9, 17, 20). The simultaneous addition of an excess of LF to cells treated with EF plus PA (EdTx) prevented an increase of cyclic AMP (cAMP) in vitro (21). Monoclonal antibodies have also been shown to inhibit the binding of EF to PA, and these antibodies also recognize epitopes within the PA-binding domain of EF (22). In addition, binding of LF-neutralizing antibodies to EF by enzyme-linked immunosorbent assays (ELISAs) suggests that host immune responses against these domains may prevent toxin components from entering target cells (23). While studies have shown that even when aggressive, early antibiotic therapy eradicates bacterial load within 72 h, anthrax toxins are still present in concentrations sufficient to cause death (8, 16). Since death can result even with bacterial clearance, vaccine- or toxin-directed immunotherapeutic development is essential to prevent or stop infection at an early stage.The human vaccine currently available in the United States, anthrax vaccine absorbed (AVA), contains mainly PA as the protective component. AVA has many disadvantages, including a complicated dosing schedule (five intramuscular injections with yearly boosters), batch-to-batch variation of the protective bacterial components, limited duration of protection, requirement for containment facilities for production, and transient reactogenicity in many vaccinees (14, 15, 30, 37). Second-generation vaccines based on recombinant PA are currently in development; however, these vaccines will not elicit antibodies to LF and EF (2). Although PA has been shown to be the main protective component in the currently licensed vaccine, studies in which mice were immunized with strains of mutant B. anthracis revealed the significant individual contributions of antibodies to EF and LF toward immunoprotection (27, 29). Further studies have shown that immunization with a DNA construct encoding the N-terminal fragment of EF elicited protective immunity against a subcutaneous challenge of A/J mice with the B. anthracis Sterne strain (38). Moreover, our studies have demonstrated that immunization with recombinant LF (rLF) can induce high-titer protective antibodies in vivo and in vitro (28).Despite significant achievements toward understanding the contribution of EF and LF antibodies to protection, considerable gaps remain in understanding the fine specificity of the protective responses to these components of the tripartite toxin. The purposes of this study were to identify sequential B-cell epitopes within EF and to determine the relative contributions of cross-reactive antibodies in the conserved PA-binding domains of EF and LF to LeTx and EdTx neutralization. Host immune responses against these cross-reactive domains may prevent both EF and LF from gaining access into cells. We hypothesized that the protective host immune response following EF and LF vaccination would include antibodies directed to cross-reactive epitopes that prevent binding to PA and thus entry into target cells. We found that immunization of A/J mice with recombinant EF (rEF) generated high-antibody titers capable of neutralizing EdTx in vitro and in vivo. Furthermore, cross-reactive B-cell epitopes in the PA-binding domains of whole rLF and rEF have been identified. However, there was no evidence for contribution of these epitopes to toxin neutralization.     

Production of human monoclonal antibodies to myeloperoxidase.

Two mouse-human heterohybridomas secreting human antibodies to myeloperoxidase (MPO) were derived from the peripheral blood of a patient who developed microscopic polyarteritis as the result of long-term treatment with hydralazine. Forty-five immunoglobulin-secreting lines were obtained from the fusion of patient lymphocytes with the CB-F7 heteromyeloma cell line. Of these, two antibodies, one IgG and one IgM, bound to myeloperoxidase in solid phase ELISA and gave a perinuclear staining pattern on ethanol-fixed human neutrophil cytospin preparations. The staining patterns were similar to those seen with serum from the patient. Antigen-inhibition studies revealed that the affinity of the IgG monoclonal antibody was 28 times higher (k = 1.4 x 10(-7)) than the IgM antibody (k = 5 x 10(-5)). Cross-inhibition studies further suggested that the two monoclonal antibodies recognized the same epitope on MPO. Of the other secreting cell lines, none produced antibody which reacted with the panel of autoantigens used for testing. Neither mononuclear antibody reacted with this panel indicating that they were not simply polyreactive natural autoantibodies. These are the first human monoclonal antibodies to native myeloperoxidase to be reported.     

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.     

Protective immunity induced by Bacillus anthracis toxin-deficient strains.

The two toxins secreted by Bacillus anthracis are composed of binary combinations of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). Six mutant strains that are deficient in the production of one or two of these toxin components have been previously constructed and characterized (C. Pezard, E. Duflot, and M. Mock, J. Gen. Microbiol. 139:2459-2463, 1993). In this work, we examined the antibody response to the in vivo production of PA, LF, and EF in mice immunized with spores of strains producing these proteins. High titers of antibody to PA were observed after immunization with all strains producing PA, while titers of antibodies to EF and LF were weak in animals immunized with strains producing only EF or LF. In contrast, immunization with strains producing either PA and EF or PA and LF resulted in an increased antibody response to EF or LF, respectively. The differing levels of protection from a lethal anthrax challenge afforded to mice immunized with spores of the mutant strains not only confirm the role of PA as the major protective antigen in the humoral response but also indicate a significant contribution of LF and EF to immunoprotection. We observed, however, that PA-deficient strains were also able to provide some protection, thereby suggesting that immune mechanisms other than the humoral response may be involved in immunity to anthrax. Finally, a control strain lacking the toxin-encoding plasmid was unable to provide protection or elicit an antibody response against bacterial antigens, indicating a possible role for pXO1 in the survival of B. anthracis in a host.