The terrorism events of September 11, 2001, and the subsequent B. anthracis spore attacks through the U.S. mail system have demonstrated their feasibility as a bioterrorism weapon. Moreover, they have shown that the West needs to be prepared for an increasing number of terrorist attacks, which may include the use of biological warfare. In the absence of specific intelligence and integrated real-time detection systems, the unpredictable nature of bioterrorism events calls for the development of medical countermeasures, which will enable the authorities to treat the exposed individuals. 1,2 Following a biological anthrax attack, vaccination in conjunction with antibiotic administration appears to be the most effective and economical form of mass protection. However, such a countermeasure is not as effective as it may seem because the current vaccines have drawbacks and the interval between initial exposure and the onset of treatment can be lengthy 3 as well. For these reasons, therapeutic strategies that emphasize toxin inactivation are seen to be worth pursuing. At present, physicians have antibiotic options at their disposal to eliminate an anthrax infection, but they have no therapeutic options to combat the lethal factor (LF a ) mediated toxemia and tissue destruction or the residual toxemia that persists even after the bacteria have been eliminated by antibiotics. 4 New details regarding anthrax pathogenesis and host responses have emerged since the terrorist attack in 2001, which demonstrate that not only a better understanding of the biology of virulence factors but also their interaction with the host during infection could lead to the development of improved options against anthrax. 5,6 Research efforts for the development of improved treatment modalities have increased dramatically, leading to a huge expansion in the existing literature, and it is precisely this that makes reviewing such a topic so difficult. The present study focuses on the most important medical countermeasures against inhalational anthrax that have been developed mainly since 2001 and also highlights current problems and the possible development of improved human anti-infective strategies.

Current and Future Medical Approaches To Combat the Anthrax Threat

The terrorist attacks of 2001 involving anthrax underscore the imperative that safe and effective medical countermeasures should be readily available. Vaccination appears to be the most effective form of mass protection against a biological attack, but the current vaccines have drawbacks that justify the enormous amount of effort currently being put into developing more effective vaccines and other treatment modalities. After providing a comprehensive overview of the organism Bacillus anthracis as a biological weapon and its pathogenicity, this review briefly summarizes the current knowledge vital to the management of anthrax disease. This knowledge has been acquired since 2001 as a result of the progress on anthrax research and focuses on the possible development of improved human anti-infective strategies targeting B. anthracis spore components, as well as strategies based on host–pathogen interactions.

Potential biological targets of Bacillus anthracis in anti-infective approaches against the threat of bioterrorism.

Chimeric hepatitis B virus core particles carrying an epitope of anthrax protective antigen induce protective immunity against Bacillus anthracis

Anthrax lethal and edema toxins (LeTx and EdTx, respectively) form by binding of lethal factor (LF) or edema factor (EF) to the pore-forming moiety protective antigen (PA). Immunity to LF and EF protects animals from anthrax spore challenge and neutralizes anthrax toxins. The goal of the present study is to identify linear B-cell epitopes of EF and to determine the relative contributions of cross-reactive antibodies of EF and LF to LeTx and EdTx neutralization. A/J mice were immunized with recombinant LF (rLF) or rEF. Pools of LF or EF immune sera were tested for reactivity to rLF or rEF by enzyme-linked immunosorbent assays, in vitro neutralization of LeTx and EdTx, and binding to solid-phase LF and EF decapeptides. Cross-reactive antibodies were isolated by column absorption of EF-binding antibodies from LF immune sera and by column absorption of LF-binding antibodies from EF immune sera. The resulting fractions were subjected to the same assays. Major cross-reactive epitopes were identified as EF amino acids (aa) 257 to 268 and LF aa 265 to 274. Whole LF and EF immune sera neutralized LeTx and EdTx, respectively. However, LF sera did not neutralize EdTx, nor did EF sera neutralize LeTx. Purified cross-reactive immunoglobulin G also failed to cross-neutralize. Cross-reactive B-cell epitopes in the PA-binding domains of whole rLF and rEF occur and have been identified; however, the major anthrax toxin-neutralizing humoral responses to these antigens are constituted by non-cross-reactive epitopes. This work increases understanding of the immunogenicity of EF and LF and offers perspective for the development of new strategies for vaccination against anthrax.

https://iai.asm.org/content/iai/77/11/4714.full.pdf

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

Single-step purification of recombinant anthrax lethal factor from periplasm of Escherichia coli.

Anthrax toxin consists of three separate proteins, protective antigen (PA), lethal factor (LF) and edema factor (EF). EF is bacterial adenylate cyclase which, upon activation by its eukaryotic cofactor, calmodulin, causes a rapid increase in the intracellular cAMP level of host cells. EF can reduce the protective ability of host animal. In order to further research the mechanism of anthrax toxin, the expression plasmid was constructed and the structural gene for anthrax edema factor (EF) was expressed in Escherichia coli. Recombinant EF (rEF) was purified to homogeneity by a three-step procedure involving metal chelating affinity chromatography, cation-exchange chromatography and gel chromatography. From 1 liter of culture, 5mg of biologically active EF was easily purified. Rabbits were immuned with rEF, anti-EF antibodies were prepared and can neutralize rEF in cells. Tests in vitro proved rEF have good biological activity. rEF can competed the binding regions of PA with rLF in J774A.1 and CHO cells. rEF and rLF can restrain each other by competition. The successful expression of rEF has placed a solid foundation for the research on toxicity mechanism of EF, and screening for inhibitors against EF.

Expression and analysis of biological activity of the recombination anthrax edema factor

The gene encoding anthrax lehtal factor (LF) was cloned into a secretory expression plasmid and then expressed in periplasmic space of E. coli. The recombinant LF (rLF) expressed was about 4% of the total proteins in E. coli. About 3 mg electrophoresis purity rLF could be obtained after the purification of 1 liter culure using ion exchange chromatography and gel filtration. The result of sequencing assay showed that the N-terminal amino acid sequence of rLF was identical to the N-terminal sequence of natural LF. In vitro toxicity analysis also shows that rLF has an excellent biological activity. The successful expression of rLF has placed a solid foundation for the research on toxicity mechanism of LF, developing new anthrax vaccines, and screening for inhibitors against anthrax toxin.

Expression and characterization of the recombination anthrax lethal factor

Anthrax toxin from Bacillus anthracis is a three-component toxin consisting of lethal factor (LF), edema factor (EF), and protective antigen (PA) PA binds to target cells and transports LF or EF into the cell cytosol where they carry out their enzymatic functions PA can induce protective immunity to the infection of the bacterium and is the major component in the only anthrax vaccine approved by FDA of USA Mouse hybridoma clones specifically secreting anti-PA monoclonal antibodies (MAbs) were generated by cell fusion technique and their ability to neutralize anthrax lethal toxin activities was screened in vitro on a toxin-sensitive cell line Nine toxin-neutralizing MAbs obtained were then characterized for the domains of PA they recognize, and the epitope regions they bind were analyzed by competitive binding ELISA It was found that these MAbs bind four potential neutralizing epitope regions in three different domains of PA Four MAbs bind to two non-overlapping epitope regions in domain 4 of PA and may prevent the binding of PA to its cell receptor Four MAbs bind to domain 2, a domain involved in membrane insertion One MAb binds to domain 3, a region involved in the oligomerization of PA The results provided supporting evidence that PA has several neutralizing epitopes, and offered potential immunotherapeutic agents for the treatment of anthrax

[Toxin-neutralizing monoclonal antibodies to the different domains of anthrax protective antigen].

Anthrax, as a fulminating infectious disease, threatens human' s health seriously. Bacillus anthracis, the agent of anthrax, was classified into the second kinds of pathogenic microorganisms (one kind of the highly pathogenic microorganism) in the List of Human Pathogenic Microorganisms issued by the Chinese government. The spores formed by B. anthracis are potential material for biological warfare agent and biological terror. Therefore, it is very important and pressing to develop sensitive, efficient detection methods for the bacteria. For detection methods of B. anthracis, there are four types of targets : spores, vegetative cells, genes and anthrax toxin proteins. Among them, detection methods targeting spores and vegetative cells are developed. However, owing to disadvantages in specificity and clinical practicality, these methods are far from satisfaction. Detection methods targeting genes of B. anthracis are satisfactory in specificity and sensitivity, while it is short in clinical diagnosis. At the same time, the development of detection methods targeting anthrax toxin makes it possible to acquire information about main causative agent directly, which brings about great help in clinical diagnosis as well as epidemiology research. Herein, we summarized briefly detection methods of B. anthracis developed currently, investigated their application ranges and detection capacity, and discussed the development trend of related research, expecting favoring the profession developing detection methods of B. anthracis.

Wei Chen

Papers

Expression and analysis of biological activity of the recombination anthrax edema factor

Expression and characterization of the recombination anthrax lethal factor

[Toxin-neutralizing monoclonal antibodies to the different domains of anthrax protective antigen].

[Present status and prospects for the detection of Bacillus anthracis--a review].