An Adenovirus-Vectored Nasal Vaccine Confers Rapid and Sustained Protection against Anthrax in a Single-Dose Regimen

Bacillus anthracis is the causative agent of anthrax, and its spores have been developed into lethal bioweapons. To mitigate an onslaught from airborne anthrax spores that are maliciously disseminated, it is of paramount importance to develop a rapid-response anthrax vaccine that can be mass administered by nonmedical personnel during a crisis. We report here that intranasal instillation of a nonreplicating adenovirus vector encoding B. anthracis protective antigen could confer rapid and sustained protection against inhalation anthrax in mice in a single-dose regimen in the presence of preexisting adenovirus immunity. The potency of the vaccine was greatly enhanced when codons of the antigen gene were optimized to match the tRNA pool found in human cells. In addition, an adenovirus vector encoding lethal factor can confer partial protection against inhalation anthrax and might be coadministered with a protective antigen-based vaccine.     

Study of Immunization against Anthrax with the Purified Recombinant Protective Antigen of Bacillus anthracis

Protective antigen (PA) of anthrax toxin is the major component of human anthrax vaccine. Currently available human vaccines in the United States and Europe consist of alum-precipitated supernatant material from cultures of toxigenic, nonencapsulated strains of Bacillus anthracis. Immunization with these vaccines requires several boosters and occasionally causes local pain and edema. We previously described the biological activity of a nontoxic mutant of PA expressed in Bacillus subtilis. In the present study, we evaluated the efficacy of the purified mutant PA protein alone or in combination with the lethal factor and edema factor components of anthrax toxin to protect against anthrax. Both mutant and native PA preparations elicited high anti-PA titers in Hartley guinea pigs. Mutant PA alone and in combination with lethal factor and edema factor completely protected the guinea pigs from B. anthracis spore challenge. The results suggest that the mutant PA protein may be used to develop an effective recombinant vaccine against anthrax.     

Two-Photon Intravital Imaging of Lungs during Anthrax Infection Reveals Long-Lasting Macrophage-Dendritic Cell Contacts

The dynamics of the lung immune system at the microscopic level are largely unknown because of inefficient methods of restraining chest motion during image acquisition. In this study, we developed an improved intravital method for two-photon lung imaging uniquely based on a posteriori parenchymal tissue motion correction. We took advantage of the alveolar collagen pattern given by the second harmonic generation signal as a reference for frame registration. We describe here for the first time a detailed dynamic account of two major lung immune cell populations, alveolar macrophages and CD11b-positive dendritic cells, during homeostasis and infection by spores of Bacillus anthracis, the agent of anthrax. We show that after alveolar macrophages capture spores, CD11b-positive dendritic cells come in prolonged contact with infected macrophages. Dendritic cells are known to carry spores to the draining lymph nodes and elicit the immune response in pulmonary anthrax. The intimate and long-lasting contacts between these two lines of defense may therefore coordinate immune responses in the lung through an immunological synapse-like process.     

Toxin Inhibition of Antimicrobial Factors Induced by Bacillus anthracis Peptidoglycan in Human Blood

Here, we describe the capacity of Bacillus anthracis peptidoglycan (BaPGN) to trigger an antimicrobial response in human white blood cells (WBCs). Analysis of freshly isolated human blood cells found that monocytes and neutrophils, but not B and T cells, were highly responsive to BaPGN and produced a variety of cytokines and chemokines. This BaPGN-induced response was suppressed by anthrax lethal toxin (LT) and edema toxin (ET), with the most pronounced effect on human monocytes, and this corresponded with the higher levels of anthrax toxin receptor 1 (ANTXR1) in these cells than in neutrophils. The supernatant from BaPGN-treated cells altered the growth of B. anthracis Sterne, and this effect was blocked by LT, but not by ET. An FtsX mutant of B. anthracis known to be resistant to the antimicrobial effects of interferon-inducible Glu-Leu-Arg (ELR)-negative CXC chemokines was not affected by the BaPGN-induced antimicrobial effects. Collectively, these findings describe a system in which BaPGN triggers expression of antimicrobial factors in human WBCs and reveal a distinctive role, not shared with ET, in LT''s capacity to suppress this response.     

Complement protein C3 binding to Bacillus anthracis spores enhances phagocytosis by human macrophages

Alveolar macrophages are thought to play a central role in the pathogenesis of inhalational anthrax. Receptors present on macrophages that mediate phagocytosis of Bacillus anthracis spores have yet to be completely defined. To begin to determine if soluble factors that are present in the lung such as immunoglobulin and complement are involved, we characterized the binding of human IgG and C3 to the surface of B. anthracis spores at different concentrations of nonimmune human serum. Furthermore we investigated the uptake of B. anthracis spores by human monocyte-derived macrophages in the presence of nonimmune human serum. Here we show that C3b is bound to B. anthracis spores and is activated through the classical pathway by IgG bound to the spore surface. Furthermore, we show that C3 serves as an opsonin for B. anthracis spores resulting in enhanced phagocytosis by human macrophages. These studies provide evidence that nonimmune serum contains IgG which binds to B. anthracis spores but is not sufficient to initiate phagocytosis. However, surface-bound IgG does initiate the classical pathway of complement activation, which is active in the lung, resulting in deposition of the opsonin C3b on the spore surface.     

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.     

Recombinant GroEL enhances protective antigen-mediated protection against Bacillus anthracis spore challenge

The fatal inhalation infection caused by Bacillus anthracis results from a complex pathogenic cycle involving release of toxins by bacteria that germinate from spores. Currently available vaccines against anthrax consist of protective antigen (PA), one of the anthrax toxin components. However, these PA-based vaccines are only partially protective against spore challenge in mice. This shows that exclusive elicitation of high anti-PA titer does not directly correlate with protection. Here, we demonstrate that inclusion of GroEL of B. anthracis with PA elicits enhanced protection against anthrax spore challenge in mice. GroEL was included as it has been reported to be present both on the exosporium and in the secretome in addition to the cell surface of B. anthracis. It has also been found protective against other pathogens. In the present study, immunization with GroEL alone was also potent enough to induce high humoral and cell-mediated response and significantly prolonged the mean time to death in spore-challenged mice. As a surface antigen, opsonization of spores with anti-GroEL IgG showed increased uptake of treated spores and therefore accelerated rate of spore destruction by phagocytic cells leading to the protection of mice. We found that GroEL was able to enhance nitric oxide release from lymphocytes and also reduce bacterial load from the organs, probably through the activation of macrophages and over-expression of certain innate immunity receptors. Therefore, the present study emphasizes that GroEL is an effective immunomodulator against B. anthracis infection.     

Novel Role for the yceGH Tellurite Resistance Genes in the Pathogenesis of Bacillus anthracis

Bacillus anthracis, the causative agent of anthrax, relies on multiple virulence factors to subvert the host immune defense. Using Caenorhabditis elegans as an infection model, we screened approximately 5,000 transposon mutants of B. anthracis Sterne for decreased virulence. One of the attenuated mutants resulted in loss of expression of yceG and yceH, the last two genes in a six-gene cluster of tellurite resistance genes. We generated an analogous insertional mutant to confirm the phenotype and characterize the role of yceGH in resistance to host defenses. Loss of yceGH rendered the mutants more sensitive to tellurite toxicity as well as to host defenses such as reactive oxygen species and the cathelicidin family of antimicrobial peptides. Additionally, we see decreased survival in mammalian models of infection, including human whole blood and in mice. We identify a novel role for the yceGH genes in B. anthracis Sterne virulence and suggest that C. elegans is a useful infection model to study anthrax pathogenesis.     

Anthrax lethal toxin induces human endothelial cell apoptosis

Because of its ease of dispersal and high lethality, Bacillus anthracis is one of the most feared biowarfare agents. A better understanding of anthrax pathogenesis is urgently needed to develop new therapies for systemic disease that is relatively unresponsive to antibiotics. Although experimental evidence has implicated a role for macrophages in anthrax pathogenesis, clinical and pathological observations suggest that a direct insult to the host vasculature may also be important. Two bacterial toxins, lethal toxin and edema toxin, are believed to mediate the clinical sequelae of anthrax. Here, I examined whether these toxins are directly toxic to endothelial cells, the cell type that lines the interior of blood vessels. I show for the first time that lethal toxin but not edema toxin reduces the viability of cultured human endothelial cells and induces caspase-dependent endothelial apoptosis. In addition, this toxicity affects both microvascular and large vessel endothelial cells as well as endothelial cells that have differentiated into tubules within a type I collagen extracellular matrix. Finally, lethal toxin induces cleavage of mitogen-activated protein kinase kinases in endothelial cells and inhibits phosphorylation of ERK, p38, and JNK p46. Based on the contributions of these pathways to endothelial survival, I propose that lethal toxin-mediated cytotoxicity/apoptosis results primarily through inhibition of the ERK pathway. I also hypothesize that the observed endothelial toxicity contributes to vascular pathology and hemorrhage during systemic anthrax.     

Comprehensive Analysis and Selection of Anthrax Vaccine Adsorbed Immune Correlates of Protection in Rhesus Macaques

Humoral and cell-mediated immune correlates of protection (COP) for inhalation anthrax in a rhesus macaque (Macaca mulatta) model were determined. The immunological and survival data were from 114 vaccinated and 23 control animals exposed to Bacillus anthracis spores at 12, 30, or 52 months after the first vaccination. The vaccinated animals received a 3-dose intramuscular priming series (3-i.m.) of anthrax vaccine adsorbed (AVA) (BioThrax) at 0, 1, and 6 months. The immune responses were modulated by administering a range of vaccine dilutions. Together with the vaccine dilution dose and interval between the first vaccination and challenge, each of 80 immune response variables to anthrax toxin protective antigen (PA) at every available study time point was analyzed as a potential COP by logistic regression penalized by least absolute shrinkage and selection operator (LASSO) or elastic net. The anti-PA IgG level at the last available time point before challenge (last) and lymphocyte stimulation index (SI) at months 2 and 6 were identified consistently as a COP. Anti-PA IgG levels and lethal toxin neutralization activity (TNA) at months 6 and 7 (peak) and the frequency of gamma interferon (IFN-γ)-secreting cells at month 6 also had statistically significant positive correlations with survival. The ratio of interleukin 4 (IL-4) mRNA to IFN-γ mRNA at month 6 also had a statistically significant negative correlation with survival. TNA had lower accuracy as a COP than did anti-PA IgG response. Following the 3-i.m. priming with AVA, the anti-PA IgG responses at the time of exposure or at month 7 were practicable and accurate metrics for correlating vaccine-induced immunity with protection against inhalation anthrax.     

Toxin-deficient mutants of Bacillus anthracis are lethal in a murine model for pulmonary anthrax

Bacillus anthracis, the etiologic agent of anthrax, produces at least three primary virulence factors: lethal toxin, edema toxin, and a capsule. The capsule is absolutely required for dissemination and lethality in a murine model of inhalation anthrax, yet the roles for the toxins during infection are ill-defined. We show in a murine model that when spores of specific toxin-null mutants are introduced into the lung, dissemination and lethality are comparable to those of the parent strain. Mutants lacking one or more of the structural genes for the toxin proteins, i.e., protective antigen, lethal factor, and edema factor, disseminated from the lung to the spleen at rates similar to that of the virulent parental strain. The 50% lethal dose (LD50) and mean time to death (MTD) of the mutants did not differ significantly from those of the parent. The LD50s or MTDs were also unaffected relative to those of the parent strain when mice were inoculated intravenously with vegetative cells. Nonetheless, histopathological examination of tissues revealed subtle but distinct differences in infections by the parent compared to some toxin mutants, suggesting that the host response is affected by toxin proteins synthesized during infection.     

CD1d-Dependent B-Cell Help by NK-Like T Cells Leads to Enhanced and Sustained Production of Bacillus anthracis Lethal Toxin-Neutralizing Antibodies

The current Bacillus anthracis vaccine consists largely of protective antigen (PA), the protein of anthrax toxin that mediates entry of edema factor (EF) or lethal factor (LF) into cells. PA induces protective antibody (Ab)-mediated immunity against Bacillus anthracis but has limited efficacy and duration. We previously demonstrated that activation of CD1d-restricted natural killer-like T cells (NKT) with a CD1d-binding glycolipid led to enhanced Ab titers specific for foreign antigen (Ag). We therefore tested the hypothesis that activation of NKT cells with the CD1d ligand (α-galactosylceramide [α-GC]) at the time of immunization improves PA-specific Ab responses. We observed that α-GC enhanced PA-specific Ab titers in C57BL/6 mice. In CD1d^−/− mice deficient in type I and type II NKT cells the anti-PA Ab response was diminished. In Jα281^−/− mice expressing CD1d but lacking type I α-GC-reactive NKT cells, α-GC did not enhance the Ab response. In vitro neutralization assays were performed and showed that the Ab titers correlated with protection of macrophages against anthrax lethal toxin (LT). The neutralization capacity of the Ab was further tested in lethal challenge studies, which revealed that NKT activation leads to enhanced in vivo protection against LT. Anti-PA Ab titers, neutralization, and protection were then measured over a period of several months, and this revealed that NKT activation leads to a sustained protective Ab response. These results suggest that NKT-activating CD1d ligands could be exploited for the development of improved vaccines for Bacillus anthracis that increase not only neutralizing Ab titers but also the duration of the protection afforded by Ab.The current AVA anthrax vaccine administered to U.S. military personnel consists of the Bacillus anthracis protective antigen (PA) and induces PA-specific antibody (Ab) titers sufficient to neutralize anthrax toxin (28, 29). However, the anti-PA Ab titers are not sustained, and individuals require administration of multiple booster vaccines to maintain toxin-neutralizing Ab titers (http://www.anthrax.osd.mil/vaccine/schedule.asp#). Thus, there is a need for improving the efficacy of the current anthrax vaccine.PA is an 83-kDa protein that forms heptameric pores on the surface of target cells expressing anthrax toxin receptors (capillary morphogenesis protein 2 [CMG-2] and tumor endothelial marker 8 [TEM-8]) (5, 6). PA heptamers interact with lethal factor (LF) and edema factor (EF) to form lethal toxin (LT) and edema toxin (ET), which together are referred to as anthrax toxin (6). The PA heptamer facilitates entry of EF and LF into the target cell. Following cell entry, EF generates supraphysiological levels of cyclic AMP via the protein''s calmodulin-dependent adenylate cyclase activity (20). Within the intoxicated cell LF functions as a zinc-dependent metalloprotease and cleaves mitogen-activated protein kinase kinases and has recently been found to disrupt the inflammasome (10). Both toxins are lethal in animal models and cause a broad range of defects in target cells, including altered cell cycle, cell growth, and survival and attenuated inflammatory responses (24). Collectively these activities of anthrax toxin cripple the host immune system and allow B. anthracis to grow to high numbers in the bloodstream (6). Hence, immune neutralization of PA prevents all of the detrimental effects of anthrax edema toxin and lethal toxin, providing a crucial advantage to the host during early stages of disease.PA-specific Abs effectively neutralize LT and ET in vitro and protect immunized animals in vivo following a lethal challenge with the toxins (1, 4, 25, 26, 30, 33). There is a good correlation between PA-specific Ab titers and toxin neutralization by sera from patients who have survived B. anthracis infection (29). Consequently, there is considerable interest in development of efficacious vaccines which incorporate PA as the immunogen but involve fewer immunizations, boost immunological memory, and prolong neutralizing Ab production while stimulating a minimal inflammatory response (28, 29).Recent work by our laboratory and others has shown that activation of a specialized subset of T cells known as natural killer-like T cells (NKT cells) improves Ab responses against model and pathogen-derived antigens (3, 8, 13, 16, 17). More recently, we and others have also demonstrated that B-cell memory responses and the duration of plasma cell responses are enhanced as a result of administration of an NKT-activating glycolipid known as α-galactosylceramide (α-GC) (8, 11). The α-GC ligand consists of a galactose headgroup with an α-anomeric linkage to hydrophobic sphingosine and acyl chains. The lipid moiety is loaded into hydrophobic pockets in the CD1d molecule expressed by professional antigen-presenting cells, orienting the galactose headgroup for recognition by NKT cells (14, 32). The predominant NKT subset is represented by the type I NKT cells, which express an invariant Vα14, Jα18 T-cell antigen receptor (TCR) in mice and a Vα24, Jα28 TCR in humans (14, 32). Type II NKT cells have variable Vα usage, and while CD1d restricted they are thought to be stimulated by a variety of glycolipids but are not stimulated by α-GC (14, 32).In this study we therefore tested the hypothesis that coadministration of α-GC and PA leads to enhanced anti-PA Ab titers and that the Ab produced neutralizes LT in vitro and protects in vivo against a lethal challenge. Our results show that NKT cells are required for generation of PA-specific LT-neutralizing Abs and that activation of NKT cells with exogenous CD1d ligand at the time of immunization boosts the toxin-neutralizing Ab titer and leads to enhanced and sustained in vivo protection against a lethal challenge with anthrax toxin.     

Genetic polymorphisms and susceptibility to lung disease

Susceptibility to infection by bacterium such as Bacillus anthracis has a genetic basis in mice and may also have a genetic basis in humans. In the limited human cases of inhalation anthrax, studies suggest that not all individuals exposed to anthrax spores were infected, but rather, individuals with underlying lung disease, particularly asthma, sarcoidosis and tuberculosis, might be more susceptible. In this study, we determined if polymorphisms in genes important in innate immunity are associated with increased susceptibility to infectious and non-infectious lung diseases, particularly tuberculosis and sarcoidosis, respectively, and therefore might be a risk factor for inhalation anthrax. Examination of 45 non-synonymous polymorphisms in ten genes: p47phox (NCF1), p67phox (NCF2), p40phox (NCF4), p22phox (CYBA), gp91phox (CYBB), DUOX1, DUOX2, TLR2, TLR9 and alpha 1-antitrypsin (AAT) in a cohort of 95 lung disease individuals and 95 control individuals did not show an association of these polymorphisms with increased susceptibility to lung disease.     

Direct inhibition of T-lymphocyte activation by anthrax toxins in vivo

The causative agent of anthrax, Bacillus anthracis, produces two toxins that contribute in part to its virulence. Lethal toxin is a metalloprotease that cleaves upstream mitogen-activated protein kinase kinases. Edema toxin is a calmodulin-dependent adenylate cyclase. Previous studies demonstrated that the anthrax toxins are important immunomodulators that promote immune evasion of the bacterium by suppressing activation of macrophages and dendritic cells. Here we showed that injection of sublethal doses of either lethal or edema toxin into mice directly inhibited the subsequent activation of T lymphocytes by T-cell receptor-mediated stimulation. Lymphocytes were isolated from toxin-injected mice after 1 or 4 days and stimulated with antibodies against CD3 and CD28. Treatment with either toxin inhibited the proliferation of T cells. Injection of lethal toxin also potently inhibited cytokine secretion by stimulated T cells. The effects of edema toxin on cytokine secretion were more complex and were dependent on the length of time between the injection of edema toxin and the isolation of lymphocytes. Treatment with lethal toxin blocked multiple kinase signaling pathways important for T-cell receptor-mediated activation of T cells. Phosphorylation of the extracellular signal-regulated kinase and the stress-activated kinase p38 was significantly decreased. In addition, phosphorylation of the serine/threonine kinase AKT and of glycogen synthase kinase 3 was inhibited in T cells from lethal toxin-injected mice. Thus, anthrax toxins directly act on T lymphocytes in a mouse model. These findings are important for future anthrax vaccine development and treatment.     

Identification of a surrogate marker for infection in the African green monkey model of inhalation anthrax

In 2001, a bioterrorism attack involving Bacillus anthracis spore-laced letters resulted in 22 cases of inhalation anthrax, with five fatalities. This incident identified gaps in our health care system and precipitated a renewed interest in identifying both therapeutics and rapid diagnostic assays. To address those gaps, well-characterized animal models that resemble the human disease are needed. In addition, a rapid assay for a reliable diagnostic marker is key to the success of these efforts. In this study, we exposed African green monkeys to B. anthracis spores; examined clinical signs and physiological parameters, including fever, heart rate, complete blood count, and bacteremia; and evaluated the PCR assay and electrochemiluminescence (ECL) immunoassay for the biomarkers protective antigen and capsule. The results demonstrated that although there were neither objective clinical nor physiological signs that consistently identified either infection or the onset of clinical anthrax disease, the African green monkey is a suitable animal model exhibiting a disease course similar to that observed in the rhesus model and humans. We also demonstrated that detection of the biomarkers protective antigen and capsule correlated with bacterial loads in the blood of these nonhuman primates. The ECL immunoassay described here is simple and sensitive enough to provide results in one to two hours, making this assay a viable option for use in the diagnosis of anthrax, leading to timely initiation of treatment, which is a key component of B. anthracis therapeutic development.     

Detection of anthrax toxin genetic sequences by the solid phase oligo-probes

Purpose: There is an urgent need to detect a rapid field-based test to detect anthrax. We have developed a rapid, highly sensitive DNA-based method to detect the anthrax toxin lethal factor gene located in pXO1, which is necessary for the pathogenicity of Bacillus anthracis. Materials and Methods: We have adopted the enzyme-linked immunosorbent assay (ELISA) so that instead of capturing antibodies we capture the DNA of the target sequence by a rapid oligo-based hybridization and then detect the captured DNA with another oligoprobe that binds to a different motif of the captured DNA sequences at a dissimilar location. We chose anthrax lethal factor endopeptidase sequences located in pXO1 and used complementary oligoprobe, conjugated with biotin, to detect the captured anthrax specific sequence by the streptavidin-peroxidase-based colorimetric assay. Result: Our system can detect picomoles (pMoles) of anthrax (approximately 33 spores of anthrax) and is >1000 times more sensitive than the current ELISA, which has a detection range of 0.1 to 1.0 ng/mL. False positive results can be minimized when various parameters and the colour development steps are optimized. Conclusion: Our results suggest that this assay can be adapted for the rapid detection of minuscule amounts of the anthrax spores that are aerosolized in the case of a bioterrorism attack. This detection system does not require polymerase chain reaction (PCR) step and can be more specific than the antibody method. This method can also detect genetically engineered anthrax. Since, the antibody method is so specific to the protein epitope that bioengineered versions of anthrax may not be detected.     

Lethal Factor Active-Site Mutations Affect Catalytic Activity In Vitro

The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093–1100, 1994). LF is hypothesized to act as a Zn²⁺ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase.     

Bacillus anthracis Capsule Activates Caspase-1 and Induces Interleukin-1β Release from Differentiated THP-1 and Human Monocyte-Derived Dendritic Cells

The poly-γ-d-glutamic acid (PGA) capsule is one of the major virulence factors of Bacillus anthracis, which causes a highly lethal infection. The antiphagocytic PGA capsule disguises the bacilli from immune surveillance and allows unimpeded growth of bacilli in the host. Recently, efforts have been made to include PGA as a component of anthrax vaccine; however, the innate immune response of PGA itself has been poorly investigated. In this study, we characterized the innate immune response elicited by PGA in the human monocytic cell line THP-1, which was differentiated into macrophages with phorbol 12-myristate 13-acetate (PMA) and human monocyte-derived dendritic cells (hMoDCs). PGA capsules were isolated from the culture supernatant of either the pXO1-cured strain of B. anthracis H9401 or B. licheniformis ATCC 9945a. PGA treatment of differentiated THP-1 cells and hMoDCs led to the specific extracellular release of interleukin-1β (IL-1β) in a dose-dependent manner. Evaluation of IL-1β processing by Western blotting revealed that cleaved IL-1β increased in THP-1 cells and hMoDCs after PGA treatment. Enhanced processing of IL-1β directly correlated with increased activation of its upstream regulator, caspase-1, also known as IL-1β-converting enzyme (ICE). The extracellular release of IL-1β in response to PGA was ICE dependent, since the administration of an ICE inhibitor prior to PGA treatment blocked induction of IL-1β. These results demonstrate that B. anthracis PGA elicits IL-1β production through activation of ICE in PMA-differentiated THP-1 cells and hMoDCs, suggesting the potential for PGA as a therapeutic target for anthrax.Anthrax is a highly lethal infectious disease caused by the spore-forming bacterium Bacillus anthracis (24). After entering the host, anthrax spores are rapidly exposed and phagocytosed by antigen-presenting cells (APCs), such as macrophages and dendritic cells (DCs), and carried to regional lymph nodes (25, 27). Normally, at this stage, APCs act as the first line of defense against microbial pathogens by engulfing and killing infectious agents. In addition to macrophages, immature DCs have then reported to capture anthrax spores by phagocytosis and migrate to lymphoid organs, and the lymph nodes, where they liaise with and activate antigen-specific T cells (2). Macrophages and immature DCs both produce numerous modulators of the inflammatory response to recruit and activate additional cells of the immune system, which link to the adaptive immune response (5). During this process, however, anthrax spores are able to survive, germinate into vegetative bacilli, multiply, and escape the control of the innate immune system (25, 27). The vegetative form of B. anthracis then penetrates into the circulatory system by disrupting these cells, secretes high levels of exotoxin, and spreads systemically, reaching 10⁸ organisms per ml of blood (20, 25, 27, 39). Because APCs are used to bypass the host immune system in B. anthracis infection, it is important to define the interactions between APCs and spore as well as the virulence factors of B. anthracis.The major virulence factor of B. anthracis, exotoxin, is composed of three distinct proteins—protective antigen (PA), edema factor (EF), and lethal factor (LF)—which are secreted separately as nontoxic monomers (24). The binding of LF or EF to PA results in the formation of active lethal toxin (LT) or edema toxin, respectively (24). LF is a metalloprotease that cleaves most isoforms of mitogen-activated protein kinase kinase (MAPKK) (15), and EF is a calmodulin-dependent adenylate cyclase that causes a prolonged increase of cytosolic cyclic AMP (22, 31). B. anthracis contains another virulence factor, the capsule, which is composed of poly-γ-d-glutamic acid (PGA) (24). The weakly immunogenic and antiphagocytic PGA capsule disguises the bacilli from immune surveillance in a similar manner to that of the capsular polysaccharides that protect pathogens such as pneumococci and meningococci (12, 23). Like other T-cell-independent polysaccharide antigens, the immunogenicity of PGA is enhanced when it is conjugated with other proteins such as PA (4, 35, 38). Recently, oral administration of high-molecular-weight PGA to C57BL/6 mice revealed natural killer (NK) cell-mediated antitumor activity and gamma interferon (IFN-γ) secretion (18), which indicates a role for PGA in the immune response. However, the immunogenic effect of PGA on APCs, including downstream effectors such as cytokines, remains to be elucidated.In the present study, we have characterized the innate immune response to PGA in the human monocytic cell line THP-1, which was differentiated into macrophages by phorbol 12-myristate 13-acetate (PMA) and ex vivo using human monocyte-derived dendritic cells (hMoDCs). PGA capsules were isolated from the culture supernatant of either the pXO1-cured strain of B. anthracis H9401 (37) or B. licheniformis ATCC 9945a (35). PGA treatment of differentiated THP-1 cells and hMoDCs induced significant amount of the proinflammatory cytokine IL-1β in a dose-dependent manner through the activation of caspase-1, also known as IL-1β-converting enzyme (ICE). The production of other proinflammatory cytokines such as interleukin-4 (IL-4), IL-6, and IL-18 was not affected by PGA treatment. These results indicate that PGA, as well as exotoxin, involves the regulation of the innate immune response by macrophages and DCs. The present study is the first to examine the immunological effects of B. anthracis PGA both in vitro using human macrophage cell line and ex vivo with hMoDCs.