Investigation of Anthrax Cases in North-East China, 2010-2014

We determined the genotypes of seven Bacillus anthracis strains that were recovered from nine anthrax outbreaks in North-East China from 2010 to 2014, and two approved vaccine strains that are currently in use in China. The causes of these cases were partly due to local farmers being unaware of the presence of anthrax, and butchers with open wounds having direct contact with anthrax-contaminated meat products. The genotype of five of the seven recovered strains was A.Br.001/002 sub-lineage, which was concordant with previously published research. The remaining two cases belongs to the A.Br.Ames sub-lineage. Both of these strains displayed an identical SNR pattern, which was the first time that this genotype was identified in North-East China. Strengthening education in remote villages of rural China is an important activity aimed at fostering attempts to prevent and control anthrax. The genotype of the vaccine strain Anthrax Spore Vaccine No.II was A.Br.008/009 and A.Br.001/002 for the vaccine strain Anthrax Spore Vaccine Non-capsulated. Further studies of their characteristics are clearly warranted.     

Special features of immune response to the lethal toxin of Bacillus anthracis

We and other authors have recently shown that the pattern of the immune response to components of anthrax, the Bacillus anthracis lethal toxin, is complex. In addition to the neutralizing antibodies, the antitoxin antibody pool contains antibodies enhancing the toxin lethal action. We mapped the epitopes in the protective antigen that are responsible for the induction of both antibody types. In this study, we obtained new data on the cytotoxicity of the B. anthracis lethal toxin toward the J774 A.1 cell line in the presence of monoclonal antibodies to various domains of the protective antigen and the lethal factor. The role of the Fc fragment of immunoglobulins in enhancing the lethal toxin action was shown. These results may serve as a basis for the development of a new generation vaccine for anthrax.     

A Single-Dose PLGA Encapsulated Protective Antigen Domain 4 Nanoformulation Protects Mice against Bacillus anthracis Spore Challenge

Bacillus anthracis, the etiological agent of anthrax, is a major bioterror agent. Vaccination is the most effective prophylactic measure available against anthrax. Currently available anthrax vaccines have issues of the multiple booster dose requirement, adjuvant-associated side effects and stability. Use of biocompatible and biodegradable nanoparticles to deliver the antigens to immune cells could solve the issues associated with anthrax vaccines. We hypothesized that the delivery of a stable immunogenic domain 4 of protective antigen (PAD4) of Bacillus anthracis encapsulated in a poly (lactide-co-glycolide) (PLGA) - an FDA approved biocompatible and biodegradable material, may alleviate the problems of booster dose, adjuvant toxicity and stability associated with anthrax vaccines. We made a PLGA based protective antigen domain 4 nanoparticle (PAD4-NP) formulation using water/oil/water solvent evaporation method. Nanoparticles were characterized for antigen content, morphology, size, polydispersity and zeta potential. The immune correlates and protective efficacy of the nanoparticle formulation was evaluated in Swiss Webster outbred mice. Mice were immunized with single dose of PAD4-NP or recombinant PAD4. The PAD4-NP elicited a robust IgG response with mixed IgG1 and IgG2a subtypes, whereas the control PAD4 immunized mice elicited low IgG response with predominant IgG1 subtype. The PAD4-NP generated mixed Th1/Th2 response, whereas PAD4 elicited predominantly Th2 response. When we compared the efficacy of this single-dose vaccine nanoformulation PAD4-NP with that of the recombinant PAD4 in providing protective immunity against a lethal challenge with Bacillus anthracis spores, the median survival of PAD4-NP immunized mice was 6 days as compared to 1 day for PAD4 immunized mice (p<0.001). Thus, we demonstrate, for the first time, the possibility of the development of a single-dose and adjuvant-free protective antigen based anthrax vaccine in the form of PAD4-NP. Further work in this direction may produce a better and safer candidate anthrax vaccine.     

Immunoproteomically identified GBAA_0345, alkyl hydroperoxide reductase subunit C is a potential target for multivalent anthrax vaccine

Anthrax is caused by the spore‐forming bacterium Bacillus anthracis, which has been used as a weapon for bioterrorism. Although current vaccines are effective, they involve prolonged dose regimens and often cause adverse reactions. High rates of mortality associated with anthrax have made the development of an improved vaccine a top priority. To identify novel vaccine candidates, we applied an immunoproteomics approach. Using sera from convalescent guinea pigs or from human patients with anthrax, we identified 34 immunogenic proteins from the virulent B. anthracis H9401. To evaluate vaccine candidates, six were expressed as recombinant proteins and tested in vivo. Two proteins, rGBAA_0345 (alkyl hydroperoxide reductase subunit C) and rGBAA_3990 (malonyl CoA‐acyl carrier protein transacylase), have afforded guinea pigs partial protection from a subsequent virulent‐spore challenge. Moreover, combined vaccination with rGBAA_0345 and rPA (protective antigen) exhibited an enhanced ability to protect against anthrax mortality. Finally, we demonstrated that GBAA_0345 localizes to anthrax spores and bacilli. Our results indicate that rGBAA_0345 may be a potential component of a multivalent anthrax vaccine, as it enhances the efficacy of rPA vaccination. This is the first time that sera from patients with anthrax have been used to interrogate the proteome of virulent B. anthracis vegetative cells.     

Cloning,expression and purification of binding domains of lethal factor and protective antigen of Bacillus anthracis in Escherichia coli and evaluation of their related murine antibody

Anthrax is common disease between human and animals caused by Bacillus anthracis. The cell binding domain of protective antigen (PAD4) and the binding domain of lethal factor (LFD1) have high immunogenicity potential and always were considered as a vaccine candidate against anthrax. The aims of this study are cloning and expressing of PAD4 and LFD1 in Escherichia coli, purification of the recombinant proteins and determination of their immunogenicity through evaluating of the relative produced polyclonal antibodies in mice. PAD4 and LFD1 genes were cloned in pET28a(+) vector and expressed in E. coli Bl21(DE3)PlysS. Expression and purification of the two recombinant proteins were confirmed by SDS-PAGE and Western blotting techniques. The PAD4 and LFD1 were purified using Ni⁺-NTA affinity chromatography (95–98 %), yielding 37.5 and 45 mg/l of culture, respectively. The antigens were injected three times into mice and production of relative antibodies was evaluated by ELISA test. The results showed that both PAD4 and LFD1 are immunogenic, but LFD1 has higher potential to stimulate Murine immune system. With regard to the high level of LFD1 and PAD4 expression and also significant increment in produced polyclonal antibodies, these recombinant proteins can be considered as a recombinant vaccine candidate against anthrax.     

Enhanced Immune Response to DNA Vaccine Encoding Bacillus anthracis PA-D4 Protects Mice against Anthrax Spore Challenge

Anthrax has long been considered the most probable bioweapon-induced disease. The protective antigen (PA) of Bacillus anthracis plays a crucial role in the pathogenesis of anthrax. In the current study, we evaluated the efficiency of a genetic vaccination with the fourth domain (D4) of PA, which is responsible for initial binding of the anthrax toxin to the cellular receptor. The eukaryotic expression vector was designed with the immunoglobulin M (IgM) signal sequence encoding for PA-D4, which contains codon-optimized genes. The expression and secretion of recombinant protein was confirmed in vitro in 293T cells transfected with plasmid and detected by western blotting, confocal microscopy, and enzyme-linked immunosorbent assay (ELISA). The results revealed that PA-D4 protein can be efficiently expressed and secreted at high levels into the culture medium. When plasmid DNA was given intramuscularly to mice, a significant PA-D4-specific antibody response was induced. Importantly, high titers of antibodies were maintained for nearly 1 year. Furthermore, incorporation of the SV40 enhancer in the plasmid DNA resulted in approximately a 15-fold increase in serum antibody levels in comparison with the plasmid without enhancer. The antibodies produced were predominantly the immunoglobulin G2 (IgG2) type, indicating the predominance of the Th1 response. In addition, splenocytes collected from immunized mice produced PA-D4-specific interferon gamma (IFN-γ). The biodistribution study showed that plasmid DNA was detected in most organs and it rapidly cleared from the injection site. Finally, DNA vaccination with electroporation induced a significant increase in immunogenicity and successfully protected the mice against anthrax spore challenge. Our approach to enhancing the immune response contributes to the development of DNA vaccines against anthrax and other biothreats.     

Small molecule inhibitors of anthrax edema factor

Anthrax is a highly lethal disease caused by the Gram-(+) bacteria Bacillus anthracis. Edema toxin (ET) is a major contributor to the pathogenesis of disease in humans exposed to B. anthracis. ET is a bipartite toxin composed of two proteins secreted by the vegetative bacteria, edema factor (EF) and protective antigen (PA). Our work towards identifying a small molecule inhibitor of anthrax edema factor is the subject of this letter. First we demonstrate that the small molecule probe 5′-Fluorosulfonylbenzoyl 5′-adenosine (FSBA) reacts irreversibly with EF and blocks enzymatic activity. We then show that the adenosine portion of FSBA can be replaced to provide more drug-like molecules which are up to 1000-fold more potent against EF relative to FSBA, display low cross reactivity when tested against a panel of kinases, and are nanomolar inhibitors of EF in a cell-based assay of cAMP production.     

Implications of autophagy in anthrax pathogenicity

The etiological agent for anthrax is Bacillus anthracis, which produces lethal toxin (LT) that exerts a myriad of effects on many immune cells. In our previous study, it was demonstrated that LT and protective antigen (PA) induce autophagy in mammalian cells. Preliminary results suggest that autophagy may function as a cellular defense mechanism against LT-mediated toxemia. This degradation pathway may also be relevant to other aspects of the immune response in both innate and adaptive immunity. Understanding the role of autophagy in response to anthrax infection and the possibility of modulating this degradation pathway as potential countermeasures are subjects for further investigation.     

Responding to the threat of bioterrorism: a microbial ecology perspective – the case of anthrax

Anthrax is a disease of herbivores caused by the gram-positive bacterium Bacillus anthracis. It can affect cattle, sheep, swine, horses and various species of wildlife. The routes for the spread among wildlife are reviewed. There are three kinds of human anthrax – inhalation, cutaneous, and intestinal anthrax – which differ in their routes of infection and outcomes. In the United States, confirmation of cases is made by the isolation of B. anthracis and by biochemical tests. Vaccination is not recommended for the general public; civilians who should be vaccinated include those who, in their work places, come in contact with products potentially contaminated with B. anthracis spores, and people engaged in research or diagnostic activities. After September 11, 2001, there were bioterrorism anthrax attacks in the United States: anthrax-laced letters sent to multiple locations were the source of infectious B. anthracis. The US Postal Service issued recommendations to prevent the danger of hazardous exposure to the bacterium. B. anthracis spores can spread easily and persist for very long times, which makes decontamination of buildings very difficult. Early detection, rapid diagnosis, and well-coordinated public health response are the key to minimizing casualties. The US Government is seeking new ways to deter bioterrorism, including a tighter control of research on infectious agents, even though pathogens such as B. anthracis are widely spread in nature and easy to grow. It is necessary to define the boundary between defensive and offensive biological weapons research. Deterring bioterrorism should not restrict critical scientific research. Electronic Publication     

Anthrax Toxin Uptake by Primary Immune Cells as Determined with a Lethal Factor-β-Lactamase Fusion Protein

Background

To initiate infection, Bacillus anthracis needs to overcome the host innate immune system. Anthrax toxin, a major virulence factor of B. anthracis, impairs both the innate and adaptive immune systems and is important in the establishment of anthrax infections.

Methodology/Principal Findings

To measure the ability of anthrax toxin to target immune cells, studies were performed using a fusion of the anthrax toxin lethal factor (LF) N-terminal domain (LFn, aa 1–254) with β-lactamase (LFnBLA). This protein reports on the ability of the anthrax toxin protective antigen (PA) to mediate LF delivery into cells. Primary immune cells prepared from mouse spleens were used in conjunction with flow cytometry to assess cleavage and resulting FRET disruption of a fluorescent β-lactamase substrate, CCF2/AM. In spleen cell suspensions, the macrophages, dendritic cells, and B cells showed about 75% FRET disruption of CCF2/AM due to cleavage by the PA–delivered LFnBLA. LFnBLA delivery into CD4+ and CD8+ T cells was lower, with 40% FRET disruption. When the analyses were done on purified samples of individual cell types, similar results were obtained, with T cells again having lower LFnBLA delivery than macrophages, dendritic cells, and B cells. Relative expression levels of the toxin receptors CMG2 and TEM8 on these cells were determined by real-time PCR. Expression of CMG2 was about 1.5-fold higher in CD8+ cells than in CD4+ and B cells, and 2.5-fold higher than in macrophages.

Conclusions/Significance

Anthrax toxin entry and activity differs among immune cells. Macrophages, dendritic cells, and B cells displayed higher LFnBLA activity than CD4+ and CD8+ T cells in both spleen cell suspension and the purified samples of individual cell types. Expression of anthrax toxin receptor CMG2 is higher in CD4+ and CD8+ T cells, which is not correlated to the intracellular LFnBLA activity.     

Production of Functionally Active and Immunogenic Non-Glycosylated Protective Antigen from Bacillus anthracis in Nicotiana benthamiana by Co-Expression with Peptide-N-Glycosidase F (PNGase F) of Flavobacterium meningosepticum

Bacillus anthracis has long been considered a potential biological warfare agent, and therefore, there is a need for a safe, low-cost and highly efficient anthrax vaccine with demonstrated long-term stability for mass vaccination in case of an emergency. Many efforts have been made towards developing an anthrax vaccine based on recombinant protective antigen (rPA) of B. anthracis, a key component of the anthrax toxin, produced using different expression systems. Plants represent a promising recombinant protein production platform due to their relatively low cost, rapid scalability and favorable safety profile. Previous studies have shown that full-length rPA produced in Nicotiana benthamiana (pp-PA83) is immunogenic and can provide full protection against lethal spore challenge; however, further improvement in the potency and stability of the vaccine candidate is necessary. PA of B. anthracis is not a glycoprotein in its native host; however, this protein contains potential N-linked glycosylation sites, which can be aberrantly glycosylated during expression in eukaryotic systems including plants. This glycosylation could affect the availability of certain key epitopes either due to masking or misfolding of the protein. Therefore, a non-glycosylated form of pp-PA83 was engineered and produced in N. benthamiana using an in vivo deglycosylation approach based on co-expression of peptide-N-glycosidase F (PNGase F) from Flavobacterium meningosepticum. For comparison, versions of pp-PA83 containing point mutations in six potential N-glycosylation sites were also engineered and expressed in N. benthamiana. The in vivo deglycosylated pp-PA83 (pp-dPA83) was shown to have in vitro activity, in contrast to glycosylated pp-PA83, and to induce significantly higher levels of toxin-neutralizing antibody responses in mice compared with glycosylated pp-PA83, in vitro deglycosylated pp-PA83 or the mutated versions of pp-PA83. These results suggest that pp-dPA83 may offer advantages in terms of dose sparing and enhanced immunogenicity as a promising candidate for a safe, effective and low-cost subunit vaccine against anthrax.     

Poly-γ-d-glutamic acid and protective antigen conjugate vaccines induce functional antibodies against the protective antigen and capsule of Bacillus anthracis in guinea-pigs and rabbits

Anthrax is a lethal infectious disease caused by the spore-forming Bacillus anthracis . The two major virulence factors of B. anthracis are exotoxin and the poly-γ- d -glutamic acid (PGA) capsule. The three components of the exotoxin, protective antigen (PA), lethal factor and edema factor act in a binary combination, which results in massive edema and organ failure in the progress of anthrax disease. The antiphagocytic PGA capsule disguises the bacilli from immune surveillance and allows unimpeded growth of bacilli in the host. Because PA can elicit a protective immune response, it has been a target of the anthrax vaccine. In addition to PA, efforts have been made to include PGA as a component of the anthrax vaccine. In this study, we report that PA–PGA conjugates induce expressions of anti-PA, anti-PGA and toxin-neutralizing antibodies in guinea-pigs and completely protect guinea-pigs against a 50 × LD₅₀ challenge with fully virulent B. anthracis spores. Polyclonal rabbit antisera produced against either PA or ovalbumin conjugated to a PGA-15mer offer a partial passive protection to guinea-pigs against B. anthracis infection, indicating that anti-PGA antibodies play a protective role. Our results demonstrate that PA–PGA conjugate vaccines are effective in the guinea-pig model, in addition to the previously reported mouse model.     

Improvement of a Potential Anthrax Therapeutic by Computational Protein Design

Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 °C, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis.     

Bacillus anthracis Lethal Toxin Disrupts TCR Signaling in CD1d-Restricted NKT Cells Leading to Functional Anergy

Exogenous CD1d-binding glycolipid (α-Galactosylceramide, α-GC) stimulates TCR signaling and activation of type-1 natural killer–like T (NKT) cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT) on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA)-mediated intracellular delivery of lethal factor (LF), a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8) and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis–derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen.     

Erythrocytic Mobilization Enhanced by the Granulocyte Colony-Stimulating Factor Is Associated with Reduced Anthrax-Lethal-Toxin-Induced Mortality in Mice

Anthrax lethal toxin (LT), one of the primary virulence factors of Bacillus anthracis, causes anthrax-like symptoms and death in animals. Experiments have indicated that levels of erythrocytopenia and hypoxic stress are associated with disease severity after administering LT. In this study, the granulocyte colony-stimulating factor (G-CSF) was used as a therapeutic agent to ameliorate anthrax-LT- and spore-induced mortality in C57BL/6J mice. We demonstrated that G-CSF promoted the mobilization of mature erythrocytes to peripheral blood, resulting in a significantly faster recovery from erythrocytopenia. In addition, combined treatment using G-CSF and erythropoietin tended to ameliorate B. anthracis-spore-elicited mortality in mice. Although specific treatments against LT-mediated pathogenesis remain elusive, these results may be useful in developing feasible strategies to treat anthrax.     

Surface display of the 20-kDa N-terminal fragment of anthrax protective antigen based on attenuated recombinant <Emphasis Type="Italic">Bacillus anthracis</Emphasis>

Extracellular antigen 1 (EA1), a major component of the Bacillus anthracis surface layer (S-layer), was used as a fusion partner for the expression of heterologous antigen. A recombinant B. anthracis strain was constructed by integrating a translational fusion harboring the DNA fragments encoding the cell wall–targeting domain of the S-layer protein EA1 and the 20-kDa N-terminal fragment of anthrax protective antigen (PA20) into the chromosome. A thermosensitive plasmid expressing Cre recombinase was introduced at a permissive temperature to remove the antibiotic marker. Cre recombinase action at the loxP sites excised the spectinomycin resistance cassette. The final derivative strains were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Western blot analysis, and immunofluorescence analysis. PA20 was successfully expressed on the S-layer of the recombinant antibiotic marker-free strain. Guinea pigs were immunized with the attenuated recombinant B. anthracis strain, and the bacilli elicited a humoral response to PA20. This antibiotic marker-free strain and the correlative experiment method may have potential applications for the generation of a live attenuated anthrax vaccine.     

The Genome of a Bacillus Isolate Causing Anthrax in Chimpanzees Combines Chromosomal Properties of B. cereus with B. anthracis Virulence Plasmids

Anthrax is a fatal disease caused by strains of Bacillus anthracis. Members of this monophyletic species are non motile and are all characterized by the presence of four prophages and a nonsense mutation in the plcR regulator gene. Here we report the complete genome sequence of a Bacillus strain isolated from a chimpanzee that had died with clinical symptoms of anthrax. Unlike classic B. anthracis, this strain was motile and lacked the four prohages and the nonsense mutation. Four replicons were identified, a chromosome and three plasmids. Comparative genome analysis revealed that the chromosome resembles those of non-B. anthracis members of the Bacillus cereus group, whereas two plasmids were identical to the anthrax virulence plasmids pXO1 and pXO2. The function of the newly discovered third plasmid with a length of 14 kbp is unknown. A detailed comparison of genomic loci encoding key features confirmed a higher similarity to B. thuringiensis serovar konkukian strain 97-27 and B. cereus E33L than to B. anthracis strains. For the first time we describe the sequence of an anthrax causing bacterium possessing both anthrax plasmids that apparently does not belong to the monophyletic group of all so far known B. anthracis strains and that differs in important diagnostic features. The data suggest that this bacterium has evolved from a B. cereus strain independently from the classic B. anthracis strains and established a B. anthracis lifestyle. Therefore we suggest to designate this isolate as “B. cereus variety (var.) anthracis”.     

Comparison of Growth and Toxin Production in Two Vaccine Strains of Bacillus anthracis

Two vaccine strains of Bacillus anthracis were monitored in a 10-liter fermentor to compare growth patterns and toxin production. Under identical conditions, the Sterne strain produced all three components of anthrax toxin, whereas strain V770 produced only the protective antigen.     

Germination and Amplification of Anthrax Spores by Soil-Dwelling Amoebas

While anthrax is typically associated with bioterrorism, in many parts of the world the anthrax bacillus (Bacillus anthracis) is endemic in soils, where it causes sporadic disease in livestock. These soils are typically rich in organic matter and calcium that promote survival of resilient B. anthracis spores. Outbreaks of anthrax tend to occur in warm weather following rains that are believed to concentrate spores in low-lying areas where runoff collects. It has been concluded that elevated spore concentrations are not the result of vegetative growth as B. anthracis competes poorly against indigenous bacteria. Here, we test an alternative hypothesis in which amoebas, common in moist soils and pools of standing water, serve as amplifiers of B. anthracis spores by enabling germination and intracellular multiplication. Under simulated environmental conditions, we show that B. anthracis germinates and multiplies within Acanthamoeba castellanii. The growth kinetics of a fully virulent B. anthracis Ames strain (containing both the pX01 and pX02 virulence plasmids) and vaccine strain Sterne (containing only pX01) inoculated as spores in coculture with A. castellanii showed a nearly 50-fold increase in spore numbers after 72 h. In contrast, the plasmidless strain 9131 showed little growth, demonstrating that plasmid pX01 is essential for growth within A. castellanii. Electron and time-lapse fluorescence microscopy revealed that spores germinate within amoebal phagosomes, vegetative bacilli undergo multiplication, and, following demise of the amoebas, bacilli sporulate in the extracellular milieu. This analysis supports our hypothesis that amoebas contribute to the persistence and amplification of B. anthracis in natural environments.