Comparative multilocus VNTR and SNP analysis of Bacillus anthracis vaccine strains

Comparative VNTR and SNP genotype analysis of four Bacillus anthracis strains (three vaccine and one virulent) was carried out using modern molecular-genetic typing methods. It is established that these strains formed four SNP patterns completely corresponding to the VNTR profiles. It was demonstrated none of the strains tested, except vaccine B. anthracis STI-1, could belong by SNP profile to three global genetic lines of the anthrax agent extended in the world.     

Does environmental replication contribute to Bacillus anthracis spore persistence and infectivity in soil?

Bacillus anthracis is the zoonotic causal agent of anthrax. Its infectious form is the spore, which can persist in soil. Herbivores usually acquire the disease from grazing in spore-contaminated sites. There are two schools of thought regarding B. anthracis activities in soil. One contends the bacteria are obligate animal parasites and soil-based spores remain inert until taken up by another animal host. Others contend that spores can germinate in soil and the bacteria replicate and re-sporulate to maintain and/or increase spore numbers. This review discusses whether soil replication of B. anthracis is an important part of its life cycle.     

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.     

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.     

Identification of differences in structure of methionine biosynthesis genes in Bacillus anthracis strains and phylogenetically related species of bacilli

Comparative analysis of nucleotide sequences in genes that encode synthesis of methionine metabolism enzymes in the Bacillus anthracis strains and closely related bacterial species was conducted. A 42-bp deletion in the hom2 gene, which determines homoserine dehydrogenase, was found in all tested strains of the anthrax microbe. No mutation in the hom2 gene blocking the biosynthesis of methionine and threonine was registered in the strains of other bacillary species. Single nucleotide polymorphism was detected in the asd1, metX, and metH genes, which allows the B. anthracis strains to be identified by means of sequence technology.     

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.     

A <Emphasis Type="Italic">Bacillus anthracis</Emphasis> system for acquisition of heme-bound iron

In Bacillus anthracis, an alternative to the use of chelating agents (siderophores) for acquiring iron consists in using a system of hemophores and transporter proteins for scavenging hemes from hemoglobin and delivering them into the bacterial cell. B. anthracis utilizes the siderophore-mediated mechanism of acquiring iron predominantly at the intracellular stage of its development, with the system of hemophores and transporter proteins being used at the extracellular stage of anthrax infection. The structure, genetic organization, and functions of the system for heme iron acquisition in B. anthracis, understanding of which may facilitate the development of new therapies for anthrax, are discussed.     

Bacillus anthracis Lethal Toxin Reduces Human Alveolar Epithelial Barrier Function

The lung is the site of entry for Bacillus anthracis in inhalation anthrax, the deadliest form of the disease. Bacillus anthracis produces virulence toxins required for disease. Alveolar macrophages were considered the primary target of the Bacillus anthracis virulence factor lethal toxin because lethal toxin inhibits mouse macrophages through cleavage of MEK signaling pathway components, but we have reported that human alveolar macrophages are not a target of lethal toxin. Our current results suggest that, unlike human alveolar macrophages, the cells lining the respiratory units of the lung, alveolar epithelial cells, are a target of lethal toxin in humans. Alveolar epithelial cells expressed lethal toxin receptor protein, bound the protective antigen component of lethal toxin, and were subject to lethal-toxin-induced cleavage of multiple MEKs. These findings suggest that human alveolar epithelial cells are a target of Bacillus anthracis lethal toxin. Further, no reduction in alveolar epithelial cell viability was observed, but lethal toxin caused actin rearrangement and impaired desmosome formation, consistent with impaired barrier function as well as reduced surfactant production. Therefore, by compromising epithelial barrier function, lethal toxin may play a role in the pathogenesis of inhalation anthrax by facilitating the dissemination of Bacillus anthracis from the lung in early disease and promoting edema in late stages of the illness.     

Modeling gastrointestinal anthrax disease

Bacillus anthracis is a spore-forming microbe that persists in soil and causes anthrax disease. The most natural route of infection is ingestion by grazing animals. Gastrointestinal (GI) anthrax also occurs in their monogastric predators, including humans. Exposure of carcasses to oxygen triggers sporulation and contamination of the surrounding soil completing the unusual life cycle of this microbe. The pathogenesis of GI anthrax is poorly characterized. Here, we use B. anthracis carrying the virulence plasmids pXO1 and pXO2, to model gastrointestinal disease in Guinea pigs and mice. We find that spores germinate in the GI tract and precipitate disease in a dose-dependent manner. Inoculation of vegetative bacilli also results in GI anthrax. Virulence is impacted severely by the loss of capsule (pXO2-encoded) but only moderately in absence of toxins (pXO1-encoded). Nonetheless, the lack of toxins leads to reduced bacterial replication in infected hosts. B. cereus Elc4, a strain isolated from a fatal case of inhalational anthrax-like disease, was also found to cause GI anthrax. Because transmission to new hosts depends on the release of large numbers of spores in the environment, we propose that the acquisition of pXO1- and pXO2-like plasmids may promote the successful expansion of members of the Bacillus cereus sensu lato group able to cause anthrax-like disease.     

Molecular Confirmation of the Circulating Bacillus anthracis during Outbreak of Anthrax in Different Villages of Simdega District,Jharkhand

Aims and Objectives: Molecular confirmation of the circulating Bacillus anthracis during outbreak of anthrax in different villages of Simdega district, Jharkhand, India. Materials and Methods: Blood samples with swabs from skin lesions (eschar) were collected from the suspected cases of Anthrax from October 2014 to June 2016 from Simdega district, Jharkhand. All the swabs were inoculated on polymyxin lysozyme EDTA thallous acetate media, nutrient agar media as well as 5% sheep blood agar media. Gamma-phage lysis was done. DNA extraction was done using a QIAamp DNA Mini Kit (QIAGEN, Valencia, CA, USA) and subjected to polymerase chain reaction (PCR) using anthrax-specific primers. Results: On Gram and acid fast staining, purple rods and pink-coloured anthrax spores were detected. Capsular and M’Fadyean staining was done. Gamma-phage lysed B. anthracis culture. Of 39 suspected cases, 8 were culture and PCR positive and showed gamma-phage lysis. 3 deaths were reported. Discussion and Conclusion: The conventional and real-time PCR methods are suitable for both the clinical and the epidemiological practice.     

Anthrax Spore Detection by a Luminex Assay Based on Monoclonal Antibodies That Recognize Anthrose-Containing Oligosaccharides

The similarity of endospore surface antigens between bacteria of the Bacillus cereus group complicates the development of selective antibody-based anthrax detection systems. The surface of B. anthracis endospores exposes a tetrasaccharide containing the monosaccharide anthrose. Anti-tetrasaccharide monoclonal antibodies (MAbs) and anti-anthrose-rhamnose disaccharide MAbs were produced and tested for their fine specificities in a direct spore enzyme-linked immunosorbent assay (ELISA) with inactivated spores of a broad spectrum of B. anthracis strains and related species of the Bacillus genus. Although the two sets of MAbs had different fine specificities, all of them recognized the tested B. anthracis strains and showed only a limited cross-reactivity with two B. cereus strains. The MAbs were further tested for their ability to be implemented in a highly sensitive and specific bead-based Luminex assay. This assay detected spores from different B. anthracis strains and two cross-reactive B. cereus strains, correlating with the results obtained in direct spore ELISA. The Luminex assay (detection limit 10³ to 10⁴ spores per ml) was much more sensitive than the corresponding sandwich ELISA. Although not strictly specific for B. anthracis spores, the developed Luminex assay represents a useful first-line screening tool for the detection of B. anthracis spores.Anthrax is an acute zoonotic disease caused by the spore-forming bacterium Bacillus anthracis. It affects primarily herbivores in many countries of Southern Europe, South America, Asia, and Africa (24). Endospores are the infecting agent and remarkably resistant to extreme heat, dryness, chemicals, or irradiation, thus ensuring long-term survival. The principal virulence factors are a capsule and two exotoxins produced by the growing vegetative form. The major sources of human anthrax infection are direct or indirect contact with infected animals. A reliable identification of B. anthracis is challenging, due to the monomorphic nature of the B. cereus group, which comprises B. cereus, B. anthracis, B. thuringiensis, and B. mycoides (10). The similarity of spore cell surface antigens of the bacteria of this group makes it difficult to create selective, reliable, antibody-based detection systems. DNA-based assays and traditional phenotyping of bacteria are the most accurate detection systems but are also complex, expensive, or slow. The use of B. anthracis spores as a biological weapon has stressed the need to learn more about spore components that can be used for efficient vaccines and rapid detection systems.The B. anthracis endospore comprises a genome-containing core compartment and three protective layers called the cortex, coat, and exosporium (8). The glycoprotein Bacillus collagen-like protein of anthracis (BclA) is an immunodominant structural component of the exosporium that is extensively glycosylated with two O-linked carbohydrates, a 715-Da tetrasaccharide and a 324-Da disaccharide (6). The tetrasaccharide contains three rhamnose residues and an unusual terminal sugar, 2-O-methyl-4-(3-hydroxy-3-methylbutanamido)-4,6-dideoxy-d-glucopyranose, named anthrose. As anthrose was not found in spores of related strains of bacteria, it has been considered a potential biomarker for the detection of anthrax (6). In this study, the detection of B. anthracis spores was achieved by an assay based on the Luminex technology with monoclonal antibodies (MAbs) derived from mice immunized with anthrose-containing synthetic oligosaccharides.     

Roles of macrophages and neutrophils in the early host response to Bacillus anthracis spores in a mouse model of infection

The development of new approaches to combat anthrax requires that the pathogenesis and host response to Bacillus anthracis spores be better understood. We investigated the roles that macrophages and neutrophils play in the progression of infection by B. anthracis in a mouse model. Mice were treated with a macrophage depletion agent (liposome-encapsulated clodronate) or with a neutrophil depletion agent (cyclophosphamide or the rat anti-mouse granulocyte monoclonal antibody RB6-8C5), and the animals were then infected intraperitoneally or by aerosol challenge with fully virulent, ungerminated B. anthracis strain Ames spores. The macrophage-depleted mice were significantly more susceptible to the ensuing infection than the saline-pretreated mice, whereas the differences observed between the neutropenic mice and the saline-pretreated controls were generally not significant. We also found that augmenting peritoneal neutrophil populations before spore challenge did not increase resistance of the mice to infection. In addition, the bacterial load in macrophage-depleted mice was significantly greater and appeared significantly sooner than that observed with the saline-pretreated mice. However, the bacterial load in the neutropenic mice was comparable to that of the saline-pretreated mice. These data suggest that, in our model, neutrophils play a relatively minor role in the early host response to spores, whereas macrophages play a more dominant role in early host defenses against infection by B. anthracis spores.     

Advax-Adjuvanted Recombinant Protective Antigen Provides Protection against Inhalational Anthrax That Is Further Enhanced by Addition of Murabutide Adjuvant

Subunit vaccines against anthrax based on recombinant protective antigen (PA) potentially offer more consistent and less reactogenic anthrax vaccines but require adjuvants to achieve optimal immunogenicity. This study sought to determine in a murine model of pulmonary anthrax infection whether the polysaccharide adjuvant Advax or the innate immune adjuvant murabutide alone or together could enhance PA immunogenicity by comparison to an alum adjuvant. A single immunization with PA plus Advax adjuvant afforded significantly greater protection against aerosolized Bacillus anthracis Sterne strain 7702 than three immunizations with PA alone. Murabutide had a weaker adjuvant effect than Advax when used alone, but when murabutide was formulated together with Advax, an additive effect on immunogenicity and protection was observed, with complete protection after just two doses. The combined adjuvant formulation stimulated a robust, long-lasting B-cell memory response that protected mice against an aerosol challenge 18 months postimmunization with acceleration of the kinetics of the anamnestic IgG response to B. anthracis as reflected by ∼4-fold-higher anti-PA IgG titers by day 2 postchallenge versus mice that received PA with Alhydrogel. In addition, the combination of Advax plus murabutide induced approximately 3-fold-less inflammation than Alhydrogel as measured by in vivo imaging of cathepsin cleavage resulting from injection of ProSense 750. Thus, the combination of Advax and murabutide provided enhanced protection against inhalational anthrax with reduced localized inflammation, making this a promising next-generation anthrax vaccine adjuvanting strategy.     

Genotyping of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> vaccine strains by multilocus VNTR analysis

In the course of this investigation, we described the genotypic variations across 18 variable chromosomal loci of the five known anthrax bacillus vaccine strains from the collection of microbial cultures of the 48th Central Research Institute of the Ministry of Defense of the Russian Federation. The persistence of inheriting VNTR loci and their potential application in gene identification were estimated in closely related B. anthracis strains. The genetic profile of each of the studied vaccine strains has been determined for all 18 polymorphic loci. Sequencing of their amplification products was performed. We have confirmed that the variations in the electrophoretic mobility of the amplicons are due to the presence of repeats with different numbers of copies in their structure.     

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.     

Recombinant Vaccine Displaying the Loop-Neutralizing Determinant from Protective Antigen Completely Protects Rabbits from Experimental Inhalation Anthrax

We previously showed that a multiple antigenic peptide (MAP) vaccine displaying amino acids (aa) 304 to 319 from the 2β2-2β3 loop of protective antigen was capable of protecting rabbits from an aerosolized spore challenge with Bacillus anthracis Ames strain. Antibodies to this sequence, referred to as the loop-neutralizing determinant (LND), are highly potent at neutralizing lethal toxin yet are virtually absent in rabbit and human protective antigen (PA) antiserum. While the MAP vaccine was protective against anthrax, it contains a single heterologous helper T cell epitope which may be suboptimal for stimulating an outbred human population. We therefore engineered a recombinant vaccine (Rec-LND) containing two tandemly repeated copies of the LND fused to maltose binding protein, with enhanced immunogenicity resulting from the p38/P4 helper T cell epitope from Schistosoma mansoni. Rec-LND was found to be highly immunogenic in four major histocompatibility complex (MHC)-diverse strains of mice. All (7/7) rabbits immunized with Rec-LND developed high-titer antibody, 6 out of 7 developed neutralizing antibody, and all rabbits were protected from an aerosolized spore challenge of 193 50% lethal doses (LD₅₀) of the B. anthracis Ames strain. Survivor serum from Rec-LND-immunized rabbits revealed significantly increased neutralization titers and specific activity compared to prechallenge levels yet lacked PA or lethal factor (LF) antigenemia. Control rabbits immunized with PA, which were also completely protected, appeared sterilely immune, exhibiting significant declines in neutralization titer and specific activity compared to prechallenge levels. We conclude that Rec-LND may represent a prototype anthrax vaccine for use alone or potentially combined with PA-containing vaccines.     

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.     

Evaluation of Immunogenicity and Efficacy of Anthrax Vaccine Adsorbed for Postexposure Prophylaxis

Antimicrobials administered postexposure can reduce the incidence or progression of anthrax disease, but they do not protect against the disease resulting from the germination of spores that may remain in the body after cessation of the antimicrobial regimen. Such additional protection may be achieved by postexposure vaccination; however, no anthrax vaccine is licensed for postexposure prophylaxis (PEP). In a rabbit PEP study, animals were subjected to lethal challenge with aerosolized Bacillus anthracis spores and then were treated with levofloxacin with or without concomitant intramuscular (i.m.) vaccination with anthrax vaccine adsorbed (AVA) (BioThrax; Emergent BioDefense Operations Lansing LLC, Lansing, MI), administered twice, 1 week apart. A significant increase in survival rates was observed among vaccinated animals compared to those treated with antibiotic alone. In preexposure prophylaxis studies in rabbits and nonhuman primates (NHPs), animals received two i.m. vaccinations 1 month apart and were challenged with aerosolized anthrax spores at day 70. Prechallenge toxin-neutralizing antibody (TNA) titers correlated with animal survival postchallenge and provided the means for deriving an antibody titer associated with a specific probability of survival in animals. In a clinical immunogenicity study, 82% of the subjects met or exceeded the prechallenge TNA value that was associated with a 70% probability of survival in rabbits and 88% probability of survival in NHPs, which was estimated based on the results of animal preexposure prophylaxis studies. The animal data provide initial information on protective antibody levels for anthrax, as well as support previous findings regarding the ability of AVA to provide added protection to B. anthracis-infected animals compared to antimicrobial treatment alone.     

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.     

Immunogenicity of recombinant <Emphasis Type="Italic">Bacillus</Emphasis> strains with a cloned gene for synthesis of the protective antigen of <Emphasis Type="Italic">Bacillus anthracis</Emphasis>

A hybrid plasmid pUB110PA-1, which functions stably in cells of Bacillus strains and contains the gene for synthesis of the protective antigen of the anthrax microbe, Bacillus anthracis, has been constructed. Recombinant strains were obtained that surpass anthrax Bacillus cultures in the secretory synthesis of the protective antigen. Their immunologic effectiveness was assessed. A single immunization of guinea pigs with the recombinant strains in a dose of 5 × 10⁷ spores provides effective protection from infection by the anthrax pathogen. The immune response is characterized by high values of the immunity indices and titers of antibodies against the protective antigen. The recombinant strains do not possess residual virulence for guinea pigs and BALB/mice, in contrast to anthrax vaccine preparations.