Pathology and Pathophysiology of Inhalational Anthrax in a Guinea Pig Model

ABSTRACTNonhuman primates (NHPs) and rabbits are the animal models most commonly used to evaluate the efficacy of medical countermeasures against anthrax in support of licensure under the FDA's “Animal Rule.” However, a need for an alternative animal model may arise in certain cases. The development of such an alternative model requires a thorough understanding of the course and manifestation of experimental anthrax disease induced under controlled conditions in the proposed animal species. The guinea pig, which has been used extensively for anthrax pathogenesis studies and anthrax vaccine potency testing, is a good candidate for such an alternative model. This study was aimed at determining the median lethal dose (LD50) of theBacillus anthracisAmes strain in guinea pigs and investigating the natural history, pathophysiology, and pathology of inhalational anthrax in this animal model following nose-only aerosol exposure. The inhaled LD50of aerosolized Ames strain spores in guinea pigs was determined to be 5.0 × 104spores. Aerosol challenge of guinea pigs resulted in inhalational anthrax with death occurring between 46 and 71 h postchallenge. The first clinical signs appeared as early as 36 h postchallenge. Cardiovascular function declined starting at 20 h postexposure. Hematogenous dissemination of bacteria was observed microscopically in multiple organs and tissues as early as 24 h postchallenge. Other histopathologic findings typical of disseminated anthrax included suppurative (heterophilic) inflammation, edema, fibrin, necrosis, and/or hemorrhage in the spleen, lungs, and regional lymph nodes and lymphocyte depletion and/or lymphocytolysis in the spleen and lymph nodes. This study demonstrated that the course of inhalational anthrax disease and the resulting pathology in guinea pigs are similar to those seen in rabbits and NHPs, as well as in humans.

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

ABSTRACTWe 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 withBacillus anthracisAmes 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 fromSchistosoma 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 theB. anthracisAmes 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.

Immunization of Mice with Formalin-Inactivated Spores from Avirulent Bacillus cereus Strains Provides Significant Protection from Challenge with Bacillus anthracis Ames

ABSTRACTBacillus anthracisspores are the infectious form of the organism for humans and animals. However, the approved human vaccine in the United States is derived from a vegetative culture filtrate of a toxigenic, nonencapsulatedB. anthracisstrain that primarily contains protective antigen (PA). Immunization of mice with purified spore proteins and formalin-inactivated spores (FIS) from a nonencapsulated, nontoxigenicB. anthracisstrain confers protection againstB. anthracischallenge when PA is also administered. To investigate the capacity of the spore particle to act as a vaccine without PA, we immunized mice subcutaneously with FIS from nontoxigenic, nonencapsulatedB. cereusstrain G9241 pBCXO1−/pBC210−(dcG9241), dcG9241 ΔbclA, or 569-UM20 or with exosporium isolated from dcG9241. FIS vaccination provided significant protection of mice from intraperitoneal or intranasal challenge with spores of the virulentB. anthracisAmes or Ames ΔbclAstrain. Immunization with dcG9241 ΔbclAFIS, which are devoid of the immunodominant spore protein BclA, provided greater protection from challenge with either Ames strain than did immunization with FIS from BclA-producing strains. In addition, we used prechallenge immune antisera to probe a panel of recombinantB. anthracisSterne spore proteins to identify novel immunogenic vaccine candidates. The antisera were variably reactive with BclA and with 10 other proteins, four of which were previously tested as vaccine candidates. Overall our data show that immunization with FIS from nontoxigenic, nonencapsulatedB. cereusstrains provides moderate to high levels of protection of mice fromB. anthracisAmes challenge and that neither PA nor BclA is required for this protection.

Germination and Amplification of Anthrax Spores by Soil-Dwelling Amoebas

ABSTRACTWhile 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 resilientB. anthracisspores. 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 asB. anthraciscompetes 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 ofB. anthracisspores by enabling germination and intracellular multiplication. Under simulated environmental conditions, we show thatB. anthracisgerminates and multiplies withinAcanthamoeba castellanii. The growth kinetics of a fully virulentB. anthracisAmes strain (containing both the pX01 and pX02 virulence plasmids) and vaccine strain Sterne (containing only pX01) inoculated as spores in coculture withA. castellaniishowed 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 withinA. 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 ofB. anthracisin natural environments.

Expression of either Lethal Toxin or Edema Toxin by Bacillus anthracis Is Sufficient for Virulence in a Rabbit Model of Inhalational Anthrax

ABSTRACTThe development of therapeutics against biothreats requires that we understand the pathogenesis of the disease in relevant animal models. The rabbit model of inhalational anthrax is an important tool in the assessment of potential therapeutics againstBacillus anthracis. We investigated the roles ofB. anthraciscapsule and toxins in the pathogenesis of inhalational anthrax in rabbits by comparing infection with the Ames strain versus isogenic mutants with deletions of the genes for the capsule operon (capBCADE), lethal factor (lef), edema factor (cya), or protective antigen (pagA). The absence of capsule or protective antigen (PA) resulted in complete avirulence, while the presence of either edema toxin or lethal toxin plus capsule resulted in lethality. The absence of toxin did not influence the ability ofB. anthracisto traffic to draining lymph nodes, but systemic dissemination required the presence of at least one of the toxins. Histopathology studies demonstrated minimal differences among lethal wild-type and single toxin mutant strains. When rabbits were coinfected with the Ames strain and the PA− mutant strain, the toxin produced by the Ames strain was not able to promote dissemination of the PA− mutant, suggesting that toxigenic action occurs in close proximity to secreting bacteria. Taken together, these findings suggest that a major role for toxins in the pathogenesis of anthrax is to enable the organism to overcome innate host effector mechanisms locally and that much of the damage during the later stages of infection is due to the interactions of the host with the massive bacterial burden.

Anthrax Vaccine Antigen-Adjuvant Formulations Completely Protect New Zealand White Rabbits against Challenge with Bacillus anthracis Ames Strain Spores

ABSTRACTIn an effort to develop an improved anthrax vaccine that shows high potency, five different anthrax protective antigen (PA)-adjuvant vaccine formulations that were previously found to be efficacious in a nonhuman primate model were evaluated for their efficacy in a rabbit pulmonary challenge model usingBacillus anthracisAmes strain spores. The vaccine formulations include PA adsorbed to Alhydrogel, PA encapsulated in liposomes containing monophosphoryl lipid A, stable liposomal PA oil-in-water emulsion, PA displayed on bacteriophage T4 by the intramuscular route, and PA mixed withEscherichia coliheat-labile enterotoxin administered by the needle-free transcutaneous route. Three of the vaccine formulations administered by the intramuscular or the transcutaneous route as a three-dose regimen induced 100% protection in the rabbit model. One of the formulations, liposomal PA, also induced significantly higher lethal toxin neutralizing antibodies than PA-Alhydrogel. Even 5 months after the second immunization of a two-dose regimen, rabbits vaccinated with liposomal PA were 100% protected from lethal challenge with Ames strain spores. In summary, the needle-free skin delivery and liposomal formulation that were found to be effective in two different animal model systems appear to be promising candidates for next-generation anthrax vaccine development.

Role of Purine Biosynthesis inBacillus anthracisPathogenesis and Virulence

ABSTRACTBacillus anthracis, the etiological agent of anthrax, is a spore-forming, Gram-positive bacterium and a category A biothreat agent. Screening of a library of transposon-mutagenizedB. anthracisspores identified a mutant displaying an altered phenotype that harbored a mutated gene encoding the purine biosynthetic enzyme PurH. PurH is a bifunctional protein that catalyzes the final steps in the biosynthesis of the purine IMP. We constructed and characterized definedpurHmutants of the virulentB. anthracisAmes strain. The virulence of thepurHmutants was assessed in guinea pigs, mice, and rabbits. The spores of thepurHmutants were as virulent as wild-type spores in mouse intranasal and rabbit subcutaneous infection models but were partially attenuated in a mouse intraperitoneal model. In contrast, thepurHmutant spores were highly attenuated in guinea pigs regardless of the administration route. The reduced virulence in guinea pigs was not due solely to a germination defect, since both bacilli and toxins were detectedin vivo, suggesting that the significant attenuation was associated with a growth defectin vivo. We hypothesize that an intact purine biosynthetic pathway is required for the virulence ofB. anthracisin guinea pigs.

Activity of Dalbavancin against Bacillus anthracis In Vitro and in a Mouse Inhalation Anthrax Model

ABSTRACT Bacillus anthracis, the causative agent of anthrax, can produce fatal disease when it is inhaled or ingested by humans. Dalbavancin, a novel, semisynthetic lipoglycopeptide, has potent activity, greater than that of vancomycin, against Gram-positive bacteria and a half-life in humans that supports once-weekly dosing. Dalbavancin demonstrated potent in vitro activity against B. anthracis (MIC range, ≤0.03 to 0.5 mg/liter; MIC50 and MIC90, 0.06 and 0.25 mg/liter, respectively), which led us to test its efficacy in a murine inhalation anthrax model. The peak concentrations of dalbavancin in mouse plasma after the administration of single intraperitoneal doses of 5 and 20 mg/kg of body weight were 15 and 71 mg/kg, respectively. At 20 mg/kg, the dalbavancin activity was detectable for 6 days after administration (terminal half-life, 53 h), indicating that long intervals between doses were feasible. The mice were challenged with 50 to 100 times the median lethal dose of the Ames strain of B. anthracis, an inoculum that kills untreated animals within 4 days. The efficacy of dalbavancin was 80 to 100%, as determined by the rate of survival at 42 days, when treatment was initiated 24 h postchallenge with regimens of 15 to 120 mg/kg every 36 h (q36h) or 30 to 240 mg/kg every 72 h (q72h). A regimen of ciprofloxacin known to protect 100% of animals was tested in parallel. Delayed dalbavancin treatment (beginning 36 or 48 h postchallenge) with 60 mg/kg q36h or 120 mg/kg q72h still provided 70 to 100% survival. The low MICs and long duration of efficacy in vivo suggest that dalbavancin may have potential as an alternative treatment or for the prophylaxis of B. anthracis infections.

Processing, assembly and localization of a Bacillus anthracis spore protein

All Bacillus spores are encased in macromolecular shells. One of these is a proteinacious shell called the coat that, in Bacillus subtilis, provides critical protective functions. The Bacillus anthracis spore is the infectious particle for the disease anthrax. Therefore, the coat is of particular interest because it may provide essential protective functions required for the appearance of anthrax. Here, we analyse a protein component of the spore outer layers that was previously designated BxpA. Our data indicate that a significant amount of BxpA is located below the spore coat and associated with the cortex. By SDS-PAGE, BxpA migrates as a 9 kDa species when extracted from Sterne strain spores, and as 11 and 14 kDa species from Ames strain spores, even though it has predicted masses of 27 and 29 kDa, respectively, in these two strains. We investigated the possibility that BxpA is subject to post-translational processing as previously suggested. In B. subtilis, a subset of coat proteins is proteolysed or cross-linked by the spore proteins YabG or Tgl, respectively. To investigate the possibility that similar processing occurs in B. anthracis, we generated mutations in the yabG or tgl genes in the Sterne and Ames strains and analysed the consequences for BxpA assembly by SDS-PAGE. We found that in a tgl mutant of B. anthracis, the apparent mass of BxpA increased. This is consistent with the possibility that Tgl directs the cross-linking of BxpA into a form that normally does not enter the gel. Unexpectedly, the apparent mass of BxpA also increased in a yabG mutant, suggesting a relatively complex role for proteolysis in spore protein maturation in B. anthracis. These data reveal a previously unobserved event in spore protein maturation in B. anthracis. We speculate that proteolysis and cross-linking are ubiquitous spore assembly mechanisms throughout the genus Bacillus.

Recombinant Sindbis Virus Vectors Designed To Express Protective Antigen of Bacillus anthracis Protect Animals from Anthrax and Display Synergy with Ciprofloxacin

ABSTRACT Recombinant Sindbis viruses were engineered to express alternative forms of the protective antigen (PA) of Bacillus anthracis. The recombinant viruses induced PA-specific immunoglobulin G and neutralizing antibodies in Swiss Webster mice. Vaccination with the recombinant viruses induced immunity that offered some protection from a lethal Ames strain spore challenge and synergized the protective effects of ciprofloxacin.