Human Exposure to Naturally Occurring Bacillus anthracis in the Kars Region of Eastern Türkiye.

Environmental contamination with Bacillus anthracis spores poses uncertain threats to human health. We undertook a study to determine whether inhabitants of the anthrax-endemic region of Kars in eastern Türkiye could develop immune responses to anthrax toxins without recognised clinical infection. We measured anti-PA and anti-LF IgG antibody concentrations by ELISA in serum from 279 volunteers, 105 of whom had previously diagnosed anthrax infection (100 cutaneous, 5 gastrointestinal). Of the 174 without history of infection, 72 had prior contact with anthrax-contaminated material. Individuals were classified according to demographic parameters, daily working environment, and residence type. All villages in this study had recorded previous animal or human anthrax cases. Stepwise regression analyses showed that prior clinical infection correlated strongly with concentrations at the upper end of the ranges observed for both antibodies. For anti-PA, being a butcher and duration of continuous exposure risk correlated with high concentrations, while being a veterinarian or shepherd, time since infection, and town residence correlated with low concentrations. For anti-LF, village residence correlated with high concentrations, while infection limited to fingers or thumbs correlated with low concentrations. Linear discriminant analysis identified antibody concentration profiles associated with known prior infection. Profiles least typical of prior infection were observed in urban dwellers with known previous infection and in veterinarians without history of infection. Four individuals without history of infection (two butchers, two rural dwellers) had profiles suggesting unrecognised prior infection. Healthy humans therefore appear able to tolerate low-level exposure to environmental B. anthracis spores without ill effect, but it remains to be determined whether this exposure is protective. These findings have implications for authorities tasked with reducing the risk posed to human health by spore-contaminated materials and environments.

Convolutional neural network-based quantitative structure-activity relationship and fingerprint analysis against inhibitors of anthrax lethal factor.

Aim: To develop a one-dimensional convolutional neural network-based quantitative structure-activity relationship (1D-CNN-QSAR) model to identify novel anthrax inhibitors and analyze chemical space. Methods: We developed a 1D-CNN-QSAR model to identify novel anthrax inhibitors. Results: The statistical results of the 1D-CNN-QSAR model showed a mean square error of 0.045 and a predicted correlation coefficient of 0.79 for the test set. Further, chemical space analysis showed more than 80% fragment pair similarity, with activity cliffs associated with carboxylic acid, 2-phenylfurans, N-phenyldihydropyrazole, N-phenylpyrrole, furan, 4-methylene-1H-pyrazol-5-one, phenylimidazole, phenylpyrrole and phenylpyrazolidine. Conclusion: These fragments may serve as the basis for developing potent novel drug candidates for anthrax. Finally, we concluded that our proposed 1D-CNN-QSAR model and fingerprint analysis might be used to discover potential anthrax drug candidates.

Ebola virus disease: In vivo protection provided by the PAMP restricted TLR3 agonist rintatolimod and its mechanism of action.

Ebola virus (EBOV) is a highly infectious and lethal pathogen responsible for sporadic self-limiting clusters of Ebola virus disease (EVD) in Central Africa capable of reaching epidemic status. 100% protection from lethal EBOV-Zaire in Balb/c mice was achieved by rintatolimod (Ampligen) at the well tolerated human clinical dose of 6 mg/kg. The data indicate that the mechanism of action is rintatolimod's dual ability to act as both a competitive decoy for the IID domain of VP35 blocking viral dsRNA sequestration and as a pathogen-associated molecular pattern (PAMP) restricted agonist for direct TLR3 activation but lacking RIG-1-like cytosolic helicase agonist properties. These data show promise for rintatolimod as a prophylactic therapy against human Ebola outbreaks.

Label-free high-precise nanopore detection of endopeptidase activity of anthrax lethal factor regulated by diverse conditions.

Endopeptidase activity of anthrax lethal factor (aLF) prevents the destroy of anthracis spore intracellularly by host macrophages, meanwhile disables the signaling pathways extracellularly that leads to host lethality. Hence, inhibitory of this activity is expected to be an alternative option to cure anthrax infection. Herein, we fabricated a nanopore platform via transmembrane pore construction in vitro, which allows precise mimics, monitoring of intercellular proteinic transport and enables the quantitative detection of aLF endopeptidase activity towards MAPKK signaling protein at single molecule level. Next, we inhibited the aLF activity via screening approaches of protein-metal ion acquisition and other condition controlment (proton/hydroxide strength, adapted temperature, ionizing irradiation), which were identified by nanopore electrokinetic study. Upon the results, we found that Ca2+, Mg2+, Mn2+, Ni2+ collaborating with Zn2+ promote aLF activity efficiently. In contrary, Cd2+, Co2+, Cu2+ have great inhibitory effect. Result further revealed that, the speed of aLF endopeptidase activity with different ions functions as the nanopore signal frequency in linear manner, which enables evident distinction of those divalent ions using this proteinase assay. We also found the higher strength of the proton or hydroxide, the higher the inhibitory to aLF activity. Besides, adapted temperature and γ-ray also play integral roles in inhibiting this activity. Our results lay experimental basis for accurate detection of aLF activity, meanwhile provide new direction to screening novel stimuli-responsive inhibitors specific to aLF.

Role of site-directed mutagenesis and adjuvants in the stability and potency of anthrax protective antigen.

Anthrax is a zoonotic infection caused by the gram-positive, aerobic, spore-forming bacterium Bacillus anthracis. Depending on the origin of the infection, serious health problems or mortality is possible. The virulence of B. anthracis is reliant on three pathogenic factors, which are secreted upon infection: protective antigen (PA), lethal factor (LF), and edema factor (EF). Systemic illness results from LF and EF entering cells through the formation of a complex with the heptameric form of PA, bound to the membrane of infected cells through its receptor. The currently available anthrax vaccines have multiple drawbacks, and recombinant PA is considered a promising second-generation vaccine candidate. However, the inherent chemical instability of PA through Asn deamidation at multiple sites prevents its use after long-term storage owing to loss of potency. Moreover, there is a distinct possibility of B. anthracis being used as a bioweapon; thus, the developed vaccine should remain efficacious and stable over the long-term. Second-generation anthrax vaccines with appropriate adjuvant formulations for enhanced immunogenicity and safety are desired. In this article, using protein engineering approaches, we have reviewed the stabilization of anthrax vaccine candidates that are currently licensed or under preclinical and clinical trials. We have also proposed a formulation to enhance recombinant PA vaccine potency via adjuvant formulation.

Multiplexed, High-Sensitivity Measurements of Antibody Affinity Using Interferometric Reflectance Imaging Sensor.

Anthrax lethal factor (LF) is one of the enzymatic components of the anthrax toxin responsible for the pathogenic responses of the anthrax disease. The ability to screen multiplexed ligands against LF and subsequently estimate the effective kinetic rates (kon and koff) and complementary binding behavior provides critical information useful in diagnostic and therapeutic development for anthrax. Tools such as biolayer interferometry (BLI) and surface plasmon resonance imaging (SPRi) have been developed for this purpose; however, these tools suffer from limitations such as signal jumps when the solution in the chamber is switched or low sensitivity. Here, we present multiplexed antibody affinity measurements obtained by the interferometric reflectance imaging sensor (IRIS), a highly sensitive, label-free optical biosensor, whose stability, simplicity, and imaging modality overcomes many of the limitations of other multiplexed methods. We compare the multiplexed binding results obtained with the IRIS system using two ligands targeting the anthrax lethal factor (LF) against previously published results obtained with more traditional surface plasmon resonance (SPR), which showed consistent results, as well as kinetic information previously unattainable with SPR. Additional exemplary data demonstrating multiplexed binding and the corresponding complementary binding to sequentially injected ligands provides an additional layer of information immediately useful to the researcher.

Liposome-based nanocarriers loaded with anthrax lethal factor and armed with anti-CD19 VHH for effectively inhibiting MAPK pathway in B cells.

OBJECTIVE: One of the vital signaling pathways in cancer development and metastasis is mitogen-activated protein kinases (MAPKs). Bacillus anthracis Lethal Toxin (LT) is a potent MAPK signaling inhibitor. This toxin is comprised of two distinct domains, Lethal Factor (LF), MAPK inhibitor, and Protective Antigen (PA). To enter various cell lines, LF must be associated with the protective antigen (PA), which facilitates LF delivery. In the current study, to block MAPK signaling, LF was loaded into anti-CD19 immunoliposomes nanoparticle to deliver the cargo to Raji B cells. METHODS: The liposome nanoparticle was prepared using classical lipid film formation, then conjugated to anti-CD19 VHH. The binding efficiency was measured through flow cytometry. The targeted cytotoxicity of LF immunoliposome was confirmed by BrdU lymphoproliferation assay. This was followed by Real-Time PCR to assess the effect of formulation on pro-apoptotic genes. The inhibitory effect of LF on MAPK signaling was confirmed by western blot. RESULTS: Liposome nano-formulation was optimized to reach the maximum LF encapsulation and targeted delivery. Next, phosphorylation of MAPK pathway mediators like MEK1/2, P38 and JNK were inhibited following the treatment of Raji cells with LF-immunoliposome. The treatment also upregulated caspase genes, clearly illustrating cell death induced by LF through pyroptosis and caspase-dependent apoptosis. CONCLUSIONS: In conclusion, anti-CD19 VHH immunoliposome was loaded with LF, a potent MAPK inhibitor targeting B cells, which curbs proliferation and ushers B cells toward apoptosis. Thus, immunoliposome presents as a versatile nanoparticle for delivery of LF to block aberrant MAPK activation. To use LF as a therapy, it would be necessary to materialize LF without PA. In the current study, PA was substituted with anti-CD19 immunoliposome to make it targeted to CD19+ while keeping the normal cells intact.

Efficacy assessment of a triple anthrax chimeric antigen as a vaccine candidate in guinea pigs: challenge test with Bacillus anthracis 17 JB strain spores.

CONTEXT: Bacillus anthracis secretes a tripartite toxin comprising protective antigen (PA), edema factor (EF), and lethal factor (LF). The human anthrax vaccine is mainly composed of the anthrax protective antigen (PA). Considerable efforts are being directed towards improving the efficacy of vaccines because the use of commercial anthrax vaccines (human/veterinary) is associated with several limitations. OBJECTIVE: In this study, a triple chimeric antigen referred to as ELP (gene accession no: MT590758) comprising highly immunogenic domains of PA, LF, and EF was designed, constructed, and assessed for the immunization capacity against anthrax in a guinea pig model. MATERIALS AND METHODS: Immunization was carried out considering antigen titration and immunization protocol. The immunoprotective efficacy of the ELP was evaluated in guinea pigs and compared with the potency of veterinary anthrax vaccine using a challenge test with B. anthracis 17JB strain spores. RESULTS: The results demonstrated that the ELP antigen induced strong humoral responses. The T-cell response of the ELP was found to be similar to PA, and showed that the ELP could protect 100%, 100%, 100%, 80% and 60% of the animals from 50, 70, 90, 100 and 120 times the minimum lethal dose (MLD, equal 5 × 105 spore/ml), respectively, which killed control animals within 48 h. DISCUSSION AND CONCLUSIONS: It is concluded that the ELP antigen has the necessary requirement for proper immunization against anthrax and it can be used to develop an effective recombinant vaccine candidate against anthrax.

Development of a set of three real-time loop-mediated isothermal amplification (LAMP) assays for detection of Bacillus anthracis, the causative agent of anthrax.

Bacillus anthracis, the causative agent of anthrax is a Gram-positive, non-motile, spore forming bacterium. Its spores can persist in soil and water for years and can also be aerosolized. A rapid, sensitive and specific method to detect B. anthracis is important for clinical management and preventing spread of anthrax. Loop-mediated isothermal amplification (LAMP) assay is a rapid technique that amplifies target DNA in isothermal conditions with high sensitivity and specificity. In this study, a LAMP assay set targeting a chromosomal and two plasmid markers was developed. The individual assays of the LAMP set targeting pXO1 plasmid (lef), pXO2 plasmid (capB), and chromosome (BA5345) sequences could detect 10, 250, and 100 fg of genomic DNA and 10, 100, and 50 copies of the DNA targets harboured in recombinant plasmids, respectively. The lef and capB LAMP assays could detect ≥ 1 × 103 CFU per mL of bacteria in spiked human blood samples, while BA5345 LAMP assay could detect ≥ 1 × 104 CFU of bacteria per mL of spiked blood. The amplification was monitored in real-time by turbidimeter, and visual detection was also accomplished under normal and UV light after adding SYBR Green 1 dye on completion of the reaction. The assay set was found to be highly sensitive and did not cross-react with the closely related Bacillus spp. and other bacterial strains used in the study.

Mechanism of Action of Monoclonal Antibodies That Block the Activity of the Lethal Toxin of Bacillus Anthracis.

Neutralization of the lethal toxin of Bacillus anthracis is an important topic of both fundamental medicine and practical health care, regarding the fight against highly dangerous infections. We have generated a neutralizing monoclonal antibody 1E10 against the lethal toxin of Bacillus anthracis and described the stages of receptor interaction between the protective antigen (PA) and the surface of eukaryotic cells, the formation of PA oligomers, assembly of the lethal toxin (LT), and its translocation by endocytosis into the eukaryotic cell, followed by the formation of a true pore and the release of LT into the cell cytosol. The antibody was shown to act selectively at the stage of interaction between Bacillus anthracis and the eukaryotic cell, and the mechanism of toxin-neutralizing activity of the 1E10 antibody was revealed. The interaction between the 1E10 monoclonal antibody and PA was found to lead to inhibition of the enzymatic activity of the lethal factor (LF), most likely due to a disruption of true pore formation by PA, which blocks the release of LF into the cytosol.

Production of recombinant lethal factor of Bacillus anthracis in Bacillus subtilis.

Cancer is considered as a disease with high rates of mortality and morbidity. The limitations and side effects of common treatments have prompted the need for innovative cancer therapies. Furthermore, selectivity and targeting of cancer cells are crucial factors to successful treatment of cancer. One of these methods is the use of bacterial toxins including Bacillus anthracis toxin to aid cancer therapy. This toxin is composed of three polypeptides: protective factor (PA), lethal factor (LF), and edema factor (EF). PA can bind to various surface receptors of all types of human cells and it internalizes the lethal factor and edema factor subunits of the toxin in the cytosol. In the present study, we cloned and expressed the lef gene of B. anthracis as the lethal part of the toxin in Bacillus subtilis WB600 by a shuttle expression vector PHT4. The rLF made in B. subtilis is efficiently secreted by the host into the culture medium which facilitates downstream processing. The rLF can be used to study cancer treatment. Abbreviations: EF: edema factor; LF: lethal factor; PA: protective factor; rLF: recombinant lethal factor; rPAm: recombinant protective factor mutants; uPA: urokinase-type plasminogen activator; uPAR: urokinase-type plasminogen activator receptor.

Clindamycin Protects Nonhuman Primates Against Inhalational Anthrax But Does Not Enhance Reduction of Circulating Toxin Levels When Combined With Ciprofloxacin.

BACKGROUND: Inhalational anthrax is rare and clinical experience limited. Expert guidelines recommend treatment with combination antibiotics including protein synthesis-inhibitors to decrease toxin production and increase survival, although evidence is lacking. METHODS: Rhesus macaques exposed to an aerosol of Bacillus anthracis spores were treated with ciprofloxacin, clindamycin, or ciprofloxacin + clindamycin after becoming bacteremic. Circulating anthrax lethal factor and protective antigen were quantitated pretreatment and 1.5 and 12 hours after beginning antibiotics. RESULTS: In the clindamycin group, 8 of 11 (73%) survived demonstrating its efficacy for the first time in inhalational anthrax, compared to 9 of 9 (100%) with ciprofloxacin, and 8 of 11 (73%) with ciprofloxacin + clindamycin. These differences were not statistically significant. There were no significant differences between groups in lethal factor or protective antigen levels from pretreatment to 12 hours after starting antibiotics. Animals that died after clindamycin had a greater incidence of meningitis compared to those given ciprofloxacin or ciprofloxacin + clindamycin, but numbers of animals were very low and no definitive conclusion could be reached. CONCLUSION: Treatment of inhalational anthrax with clindamycin was as effective as ciprofloxacin in the nonhuman primate. Addition of clindamycin to ciprofloxacin did not enhance reduction of circulating toxin levels.

Effect of pH and denaturants on the fold and metal status of anthrax lethal factor.

Anthrax lethal factor (LF) is a critical component of the anthrax toxin, and functions intracellularly as a zinc-dependent endopeptidase targeting proteins involved in maintaining critical host signaling pathways. To reach the cytoplasm, LF requires to be unfolded and guided through the narrow protective antigen pore in a pH-dependent process. The current study sought to address the question as to whether LF is capable of retaining its metal ion when exposed to a low-pH environment (similar to that found in late endosomes) and an unfolding stress (induced by urea). Using a combination of tryptophan fluorescence spectroscopy and chelation studies, we show that a decrease in the pH value (from 7.0 to 5.0) leads to a pronounced shift in the onset of structural alterations in LF to lower urea concentrations. More importantly, the enzyme was found to retain its Zn2+ ion beyond the unfolding transitions monitored by Trp fluorescence, a finding indicative of tight metal binding to LF in a non-native state. In addition, an analysis of red-edge excitation shift (REES) spectra suggests the protein to maintain residual structure (a feature necessary for metal binding) even at very high denaturant concentrations. Furthermore, studies using the chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR) revealed LF's Zn2+ ion to become accessible to complexation at urea concentrations in between those required to cause structural changes and metal dissociation. This phenomenon likely originates from the conversion of a PAR-inaccessible (closed) to a PAR-accessible (open) state of LF at intermediate denaturant concentrations.

Development of a receptor-based inhibitory penta-unit-conjugated peptide to enhance anthrax toxin neutralization.

Anthrax toxin is a key virulence factor for Bacillus anthracis. The cell-binding component of anthrax toxin, protective antigen (PA), mediates the entry of the toxin into cells by first binding to the extracellular von Willebrand factor A (VWA) domain of the cellular anthrax toxin receptor (ATR). Herein, we targeted the VWA domain of the cellular receptor to develop a more effective antitoxin agent for neutralization of anthrax toxin. We selected ATR-binding peptides by using a phage display: among these, we identified two novel peptides binding to the ATR with high affinity and specificity, and that neutralized anthrax toxicity in cells. Furthermore, to enhance the functional efficiency of the probes, the peptides were modified and conjugated to three polyvalent probe backbones: a 17 amino-acid-based cyclic form penta-unit, poly-d-lysine (PDL), or the M13 bacteriophage. One of the functionally modified polyvalent peptide probes, the penta-unit-conjugated probe (PUCP) produced the most potent neutralization of anthrax toxin, with half-maximal inhibitory concentration (IC50) of 20 nM. The PUCP disrupted anthrax toxin binding to its receptor and reduced endocytosis of anthrax toxin. This peptide-based approach may, therefore, represent a promising strategy to combat anthrax toxicosis and other bacterial diseases and may be efficient for disease treatment.

Anthrax Toxin Detection: From in Vivo Studies to Diagnostic Applications.

Anthrax toxins are produced by Bacillus anthracis throughout infection and shape the physiopathogenesis of the disease. They are produced in low quantities but are highly efficient. They have thus been long ignored, but recent biochemical methods have improved our knowledge in animal models. This article reviews the various methods that have been used and how they could be applied to clinical diagnosis.

Atomic Structures of Anthrax Prechannel Bound with Full-Length Lethal and Edema Factors.

Pathogenesis of anthrax disease involves two cytotoxic enzymes-edema factor (EF) and lethal factor (LF)-which are individually recruited by the protective antigen heptamer (PA7) or octamer (PA8) prechannel and subsequently translocated across channels formed on the endosomal membrane upon exposure to low pH. Here, we report the atomic structures of PA8 prechannel-bound full-length EF and LF. In this pretranslocation state, the N-terminal segment of both factors refolds into an α helix engaged in the α clamp of the prechannel. Recruitment to the PA prechannel exposes an originally buried ß strand of both toxins and enables domain organization of EF. Many interactions occur on domain interfaces in both PA prechannel-bound EF and LF, leading to toxin compaction prior to translocation. Our results provide key insights into the molecular mechanisms of translocation-coupled protein unfolding and translocation.

Rapid and Sensitive Multiplex Assay for the Detection of B. anthracis Spores from Environmental Samples.

: Prompt and accurate detection of Bacillus anthracis spores is crucial in the event of intentional spore dissemination in order to reduce the number of expected casualties. Specific identification of these spores from environmental samples is both challenging and time-consuming. This is due to the high homology with other Bacillus species as well as the complex composition of environmental samples, which further impedes assay sensitivity. Previously, we showed that a short incubation of B.anthracis spores in a defined growth medium results in rapid germination, bacterial growth, and secretion of toxins, including protective antigen. In this work, we tested whether coupling the incubation process to a newly developed immune-assay will enable the detection of secreted toxins as markers for the presence of spores in environmental samples. The new immune assay is a flow cytometry-based multiplex that simultaneously detects a protective antigen, lethal factor, and edema factor. Our combined assay detects 1 × 103-1 × 104/mL spores after a 2 h incubation followed by the ~80 min immune-multiplex detection. Extending the incubation step to 5 h increased assay sensitivity to 1 × 102/mL spore. The protocol was validated in various environmental samples using attenuated or fully virulent B. anthracis spores. There was no substantial influence of contaminants derived from real environmental samples on the performance of the assay compared to clean samples, which allow the unequivocal detection of 3 × 103/mL and 3 × 102/mL spores following 2 and 5 hour's incubation, respectively. Overall, we propose this method as a rapid, sensitive, and specific procedure for the identification of B. anthracis spores in environmental samples.

Immunogenicity and protective efficacy of Burkholderia pseudomallei BLF1-N and BLF1-C terminal domains against BLF1 toxin.

Burkholderia lethal factor 1 (BLF1), a glutamine deamidase, is a key virulence factor that plays significant role in B. pseudomallei pathogenesis. To elucidate the BLF1 immunological responses, two truncated BLF1 structural units, BLF1-C (90-211 amino acids) with structural similarity to T. maritima Chemoreceptor glutamine deamidase (CheD) protein, and BLF1-N (1-89 amino acids) disparate to CheD were identified from the 23 kDa BLF1 protein. Both the components were devoid of toxicity in mice and elicited an antibody titer of 1:16,000 that reacted with the respective truncated proteins and BLF1. A549 cell lines supplemented with anti BLF1-N and BLF1-C antibodies exhibited 73.47% and 83.24% survival when treated with BLF1 toxin. Passive i.p. transfer with antibodies elicited by BLF1-C that contained LSGC active site resulted in 80% protection while anti BLF1-N (devoid of LSGC) antibodies provided 51.4% protection, establishing the role of BLF1-N terminal also in deamidase action. The truncated proteins also elicited cell mediated immune responses through proliferation of CD4+ T cells, IFN-γ and IL-4 cytokines but with bias towards Th2 subsets. BLF1-C and BLF1-N immunization resulted in 80% and 60% active protection when challenged with BLF1 toxin while the sham immunized mice exhibited severe histopathological changes like necrosis in liver, lung, spleen and kidney similar to that observed in melioidosis and were killed within 7 days post challenge. The higher level of active and passive protection by BLF1-C protein could be attributed to the comparatively higher level of immune responses and inclusion of LSGC residues.

QSAR and molecular docking studies of lethal factor protease inhibitors against Bacillus anthracis.

Bacillus anthracis is considered as a biological warfare agent because it is the causative agent of the serious infectious anthrax disease. Delay in treatment leads to lethal factor-mediated toxaemia which is very critical due to lack of therapeutic options. Consequently, attempts have been made to discover potent lethal factor (LF) protease inhibitors such as small-molecule synthetic 2-thio-1,3-thiazolidine-4-one (rhodanine) compounds. But computed descriptor-based quantitative structure-activity relationship (QSAR) and drug design studies on such aspect are poorly represented. Therefore, an attempt was made for developing QSAR models using structural descriptors for 1,3-thiazolidine-4-one compounds. The models were developed on a series of 49 LF protease inhibitors using the combination of constitutional, functional group, atom-centred fragment and molecular property descriptors. The best QSAR model included four variables, namely, C-040, nR05, GVWAI-80 and ALOGP that correlated well with the anti-LF protease activity with a good correlation coefficient (r = 0.870) of good statistical significance (F4, 29 = 14.09 (α = 0.001) F4, 29 = 6.19). This model was also validated and explained 58.1% of variances of the Bacillus anthracis inhibitory activities of the studied compounds with r2pred = 0.710 which denotes external predictability. Finally, molecular docking was carried out to predict the mode of binding of some highly active congeneric compounds. It was shown that VAL 1403 is an important residue for phenyl ring. TYR 1456 and HIS 1418 are responsible for interaction with the rhodanine nucleus. Therefore, these residues are considered responsible for the inhibition of LF protease anthrax and can predict significant dimension of essential structural features of these inhibitors to evaluate, screen and help priorities of the synthesis of the candidates against anthrax bioterrorism.

Development of a novel chimeric PA-LF antigen of Bacillus anthracis, its immunological characterization and evaluation as a future vaccine candidate in mouse model.

Tremendous efforts are being made to develop an anthrax vaccine with long term protection. The main component of traditional anthrax vaccine is protective antigen (PA) with the trace amount of other proteins and bacterial components. In this study, we developed a recombinant PA-LF chimera antigen of Bacillus anthracis by fusing the PA domain 2-4 with lethal factor (LF) domain 1 and evaluated its protective potential against B. anthracis in mouse model. The anti-PA-LF chimera serum reacted with both PA and LF antigen, individually. The chimera elicited a strong antibody titer in mice with predominance of IgG1 isotype followed by IgG2b, IgG2a and IgG3. Cytokines were assessed in splenocytes of immunized mice and a significant up-regulation in the expression of IL-4, IL-10, IFN-γ and TNF-α was observed. The PA-LF chimera immunized mice exhibited 80% survival after challenge with virulent spores of B. anthracis. Pathological studies showed normal architecture in vital organs (spleen, lung, liver and kidney) of recovered immunized mice on 20 DPI after spore challenge. These findings suggested that PA-LF chimera of B. anthracis elicited good humoral as well as cell mediated immune response in mice, and thus, can be a potent vaccine candidate against anthrax.