Spatial epidemiology of human anthrax in Son La province, Vietnam, 2003-2022.

AIMS: Anthrax is reported with frequency but poorly understood in Southeast Asian countries including Vietnam. In Vietnam, anthrax surveillance is national. However, case detection, prevention, and control are implemented locally at the provincial level. Here, we describe the epidemiological characteristics, identify spatial clusters of human anthrax, and compare the variation in livestock anthrax vaccine coverage to disease incidence in humans and livestock using historical data in Son La province, Vietnam (2003-2020). METHODS AND RESULTS: Most human cases occurred between April and September. Most of the patients were male, aged 15-54 years old. The human cases were mainly reported by public district hospitals. There was a delay between disease onset and hospitalization of ~5 days. We identified spatial clusters of high-high incidence communes in the northern communes of the province using the local Moran's I statistic. The vaccine coverage sharply decreased across the study period. The province reported sporadic human anthrax outbreaks, while animal cases were only reported in 2005 and 2022. CONCLUSIONS: These results suggest underreporting for human and livestock anthrax in the province. Intersectoral information sharing is needed to aid livestock vaccination planning, which currently relies on reported livestock cases. The spatial clusters identify areas for targeted surveillance and livestock vaccination, while the seasonal case data suggest prioritizing vaccination campaigns for February or early March ahead of the April peak. A regional approach for studying the role of livestock trading between Son La and neighbouring provinces in anthrax occurrence is recommended.

Characterization of the adaptive immune response of donors receiving live anthrax vaccine.

Live anthrax vaccine containing spores from attenuated strains STI-1 of Bacillus anthracis is used in Russia and former CIS (Commonwealth of Independent States) to prevent anthrax. In this paper we studied the duration of circulation of antibodies specific to spore antigens, the protective antigen (PA), the lethal factor (LF) and their domains (D) in donors' blood at different times after their immunization with live anthrax vaccine. The relationship between the toxin neutralization activity level and the level of antibodies to PA, LF and their domains was tested. The effect of age, gender and number of vaccinations on the level of adaptive post-vaccination immune response has been studied. It was shown that antibodies against PA-D1 circulate in the blood of donors for 1 year or more after immunization with live anthrax vaccine. Antibodies against all domains of LF and PA-D4 were detected in 11 months after vaccination. Antibodies against the spores were detected in 8 months after vaccination. A moderate positive correlation was found between the titers of antibodies to PA, LF, or their domains, and the TNA of the samples of blood serum from the donors.

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.

Evaluation of the AV7909 Anthrax Vaccine Toxicity in Sprague Dawley Rats Following Three Intramuscular Administrations.

AV7909 is a next-generation anthrax vaccine under development for post-exposure prophylaxis following suspected or confirmed Bacillus anthracis exposure, when administered in conjunction with the recommended antibacterial regimen. AV7909 consists of the FDA-approved BioThrax® vaccine (anthrax vaccine adsorbed) and an immunostimulatory Toll-like receptor 9 agonist oligodeoxynucleotide adjuvant, CPG 7909. The purpose of this study was to evaluate the potential systemic and local toxicity of AV7909 when administered via repeat intramuscular injection to the right thigh muscle (biceps femoris) to male and female Sprague Dawley rats. The vaccine was administered on Days 1, 15, and 29 and the animals were assessed for treatment-related effects followed by a 2-week recovery period to evaluate the persistence or reversibility of any toxic effects. The AV7909 vaccine produced no apparent systemic toxicity based on evaluation of clinical observations, body weights, body temperature, clinical pathology, and anatomic pathology. Necrosis and inflammation were observed at the injection sites as well as in regional lymph nodes and adjacent tissues and were consistent with immune stimulation. Antibodies against B. anthracis protective antigen (PA) were detected in rats treated with the AV7909 vaccine, confirming relevance of this animal model for the assessment of systemic toxicity of AV7909. In contrast, sera of rats that received saline or soluble CPG 7909 alone were negative for anti-PA antibodies. Overall, 3 intramuscular immunizations of Sprague Dawley rats with AV7909 were well tolerated, did not induce mortality or any systemic adverse effects, and did not result in any delayed toxicity.

Assessment of knowledge, attitudes and practices towards anthrax in Narok County, Southern Kenya.

INTRODUCTION: anthrax is endemic in some parts of Kenya causing mortalities in livestock and morbidity in humans. On January 20th, 2018, news media reported suspected anthrax in a remote southern Kenyan village after villagers became ill following consumption of meat from a dead cow that was confirmed, by microscopy, to have died of anthrax. We assessed community knowledge, attitude and practices (KAP) to identify intervention gaps for anthrax prevention. METHODS: we conducted a KAP survey in randomly selected households (HHs) in villages from selected wards. Using multi-stage sampling approach, we administered structured questionnaire to persons aged ≥15 years to collect KAP information from February 11th-21st, 2018. From a set of questions for KAP, we scored participants' response as "1" for a correct response and "0" for an incorrect response. Univariate analysis and Chi-square tests were performed to explore determinants of KAP. Concurrently, we gathered qualitative data using interview guides for thematic areas on anthrax KAP from key informant interviews and focus group discussions. Qualitative data were transcribed in Ms Word and analyzed along themes by content analysis. RESULTS: among 334 respondents: 187/334 (56%) were male; mean age, 40.7±13.6 years; 331/334 (99.1%) had heard of anthrax and 304/331 (91.8%) knew anthrax to be zoonotic. Transmission was considered to be through eating dead-carcasses by 273/331 (82.5%) and through contact with infected tissue by 213/331 (64.4%). About 59% (194/329) regularly vaccinated their livestock against anthrax, 53.0% (174/328) had slaughtered or skinned a dead-animal and 59.5% (195/328) practiced home slaughter while 52.9% (172/325) treated sick-animals by themselves. Sex (p≤0.001), age (p=0.007) and livestock-rearing years (p≤0.001) were significantly associated with knowledge and practice. CONCLUSION: there were differences in knowledge and practices towards anthrax by age-group and sex. Enhanced public health education and targeted interventions by relevant government agencies is recommended.

A putative exosporium lipoprotein GBAA0190 of Bacillus anthracis as a potential anthrax vaccine candidate.

BACKGROUND: Bacillus ancthracis causes cutaneous, pulmonary, or gastrointestinal forms of anthrax. B. anthracis is a pathogenic bacterium that is potentially to be used in bioterrorism because it can be produced in the form of spores. Currently, protective antigen (PA)-based vaccines are being used for the prevention of anthrax, but it is necessary to develop more safe and effective vaccines due to their prolonged immunization schedules and adverse reactions. METHODS: We selected the lipoprotein GBAA0190, a potent inducer of host immune response, present in anthrax spores as a novel potential vaccine candidate. Then, we evaluated its immune-stimulating activity in the bone marrow-derived macrophages (BMDMs) using enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Protective efficacy of GBAA0190 was evaluated in the guinea pig (GP) model. RESULTS: The recombinant GBAA0190 (r0190) protein induced the expression of various inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α) in the BMDMs. These immune responses were mediated through toll-like receptor 1/2 via activation of mitogen-activated protein (MAP) kinase and Nuclear factor-κB (NF-κB) pathways. We demonstrated that not only immunization of r0190 alone, but also combined immunization with r0190 and recombinant PA showed significant protective efficacy against B. anthracis spore challenges in the GP model. CONCLUSIONS: Our results suggest that r0190 may be a potential target for anthrax vaccine.

Anthrax toxin component, Protective Antigen, protects insects from bacterial infections.

Anthrax is a major zoonotic disease of wildlife, and in places like West Africa, it can be caused by Bacillus anthracis in arid nonsylvatic savannahs, and by B. cereus biovar anthracis (Bcbva) in sylvatic rainforests. Bcbva-caused anthrax has been implicated in as much as 38% of mortality in rainforest ecosystems, where insects can enhance the transmission of anthrax-causing bacteria. While anthrax is well-characterized in mammals, its transmission by insects points to an unidentified anthrax-resistance mechanism in its vectors. In mammals, a secreted anthrax toxin component, 83 kDa Protective Antigen (PA83), binds to cell-surface receptors and is cleaved by furin into an evolutionary-conserved PA20 and a pore-forming PA63 subunits. We show that PA20 increases the resistance of Drosophila flies and Culex mosquitoes to bacterial challenges, without directly affecting the bacterial growth. We further show that the PA83 loop known to be cleaved by furin to release PA20 from PA63 is, in part, responsible for the PA20-mediated protection. We found that PA20 binds directly to the Toll activating peptidoglycan-recognition protein-SA (PGRP-SA) and that the Toll/NF-κB pathway is necessary for the PA20-mediated protection of infected flies. This effect of PA20 on innate immunity may also exist in mammals: we show that PA20 binds to human PGRP-SA ortholog. Moreover, the constitutive activity of Imd/NF-κB pathway in MAPKK Dsor1 mutant flies is sufficient to confer the protection from bacterial infections in a manner that is independent of PA20 treatment. Lastly, Clostridium septicum alpha toxin protects flies from anthrax-causing bacteria, showing that other pathogens may help insects resist anthrax. The mechanism of anthrax resistance in insects has direct implications on insect-mediated anthrax transmission for wildlife management, and with potential for applications, such as reducing the sensitivity of pollinating insects to bacterial pathogens.

Bacillus subtilis spore vaccines displaying protective antigen induce functional antibodies and protective potency.

BACKGROUND: Bacillus anthracis is the causative agent of anthrax, a disease of both humans and various animal species, and can be used as a bioterror agent. Effective vaccines are available, but those could benefit from improvements, including increasing the immunity duration, reducing the shot frequency and adverse reactions. In addition, more sophisticated antigen delivery and potentiation systems are urgently required. The protective antigen (PA), one of three major virulence factors associated with anthrax was displayed on the surface of Bacillus subtilis spores, which is a vaccine production host and delivery vector with several advantages such as a low production cost, straightforward administration as it is safe for human consumption and the particulate adjuvanticity. Mice were immunized orally (PO), intranasally (IN), sublingually (SL) or intraperitoneally (IP) with the PA displaying probiotic spore vaccine. Clinical observation, serological analysis and challenge experiment were conducted to investigate the safety and efficacy of the vaccine. RESULTS: A/J mice immunized with the PA spore vaccine via PO, IN, SL, and IP were observed to have increased levels of active antibody titer, isotype profiles and toxin neutralizing antibody in sera, and IgA in saliva. The immunized mice were demonstrated to raise protective immunity against the challenge with lethal B. anthracis spores. CONCLUSIONS: In this study, we developed a B. subtilis spore vaccine that displays the PA on its surface and showed that the PA-displaying spore vaccine was able to confer active immunity to a murine model based on the results of antibody isotype titration, mucosal antibody identification, and a lethal challenge experiment.

Characterization of Bacillus anthracis Spore Proteins Using a Nanoscaffold Vaccine Platform.

Subunit vaccines are theoretically safe and easy to manufacture but require effective adjuvants and delivery systems to yield protective immunity, particularly at critical mucosal sites such as the lung. We investigated nanolipoprotein particles (NLPs) containing the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) as a platform for intranasal vaccination against Bacillus anthracis. Modified lipids enabled attachment of disparate spore and toxin protein antigens. Intranasal vaccination of mice with B. anthracis antigen-MPLA-NLP constructs induced robust IgG and IgA responses in serum and in bronchoalveolar and nasal lavage. Typically, a single dose sufficed to induce sustained antibody titers over time. When multiple immunizations were required for sustained titers, specific antibodies were detected earlier in the boost schedule with MPLA-NLP-mediated delivery than with free MPLA. Administering combinations of constructs induced responses to multiple antigens, indicating potential for a multivalent vaccine preparation. No off-target responses to the NLP scaffold protein were detected. In summary, the NLP platform enhances humoral and mucosal responses to intranasal immunization, indicating promise for NLPs as a flexible, robust vaccine platform against B. anthracis and potentially other inhalational pathogens.

Progress towards the Development of a NEAT Vaccine for Anthrax II: Immunogen Specificity and Alum Effectiveness in an Inhalational Model.

Bacillus anthracis is the causative agent of anthrax disease, presents with high mortality, and has been at the center of bioweapon efforts. The only currently U.S. FDA-approved vaccine to prevent anthrax in humans is anthrax vaccine adsorbed (AVA), which is protective in several animal models and induces neutralizing antibodies against protective antigen (PA), the cell-binding component of anthrax toxin. However, AVA requires a five-course regimen to induce immunity, along with an annual booster, and is composed of undefined culture supernatants from a PA-secreting strain. In addition, it appears to be ineffective against strains that lack anthrax toxin. Here, we investigated a vaccine formulation consisting of recombinant proteins from a surface-localized heme transport system containing near-iron transporter (NEAT) domains and its efficacy as a vaccine for anthrax disease. The cocktail of five NEAT domains was protective against a lethal challenge of inhaled bacillus spores at 3 and 28 weeks after vaccination. The reduction of the formulation to three NEATs (IsdX1, IsdX2, and Bslk) was as effective as a five-NEAT domain cocktail. The adjuvant alum, approved for use in humans, was as protective as Freund's Adjuvant, and protective vaccination correlated with increased anti-NEAT antibody reactivity and reduced bacterial levels in organs. Finally, the passive transfer of anti-NEAT antisera reduced mortality and disease severity, suggesting the protective component is comprised of antibodies. Collectively, these results provide evidence that a vaccine based upon recombinant NEAT proteins should be considered in the development of a next-generation anthrax vaccine.

Immunogenicity and Biodistribution of Anthrax DNA Vaccine Delivered by Intradermal Electroporation.

PURPOSE: Anthrax is a lethal bacterial disease caused by gram-positive bacterium Bacillus anthracis and vaccination is a desirable method to prevent anthrax infections. In the present study, DNA vaccine encoding a protective antigen of Bacillus anthracis was prepared and we investigated the influence of DNA electrotransfer in the skin on the induced immune response and biodistribution. METHODS AND RESULTS: The tdTomato reporter gene for the whole animal in vivo imaging was used to assess gene transfer efficiency into the skin as a function of electrical parameters. Compared to that with 25 V, the transgene expression of red fluorescent protein increased significantly when a voltage of 90 V was used. Delivery of DNA vaccines expressing Bacillus anthracis protective antigen domain 4 (PAD4) with an applied voltage of 90 V induced robust PA-D4-specific antibody responses. In addition, the in vivo fate of anthrax DNA vaccine was studied after intradermal administration into the mouse. DNA plasmids remained at the skin injection site for an appropriate period of time after immunization. Intradermal administration of DNA vaccine resulted in detection in various organs (viz., lung, heart, kidney, spleen, brain, and liver), although the levels were significantly reduced. CONCLUSION: Our results offer important insights into how anthrax DNA vaccine delivery by intradermal electroporation affects the immune response and biodistribution of DNA vaccine. Therefore, it may provide valuable information for the development of effective DNA vaccines against anthrax infection.

Effect of raxibacumab on immunogenicity of Anthrax Vaccine Adsorbed: a phase 4, open-label, parallel-group, randomised non-inferiority study.

BACKGROUND: Raxibacumab is a monoclonal antibody against protective antigen, which is the cell-binding part of Bacillus anthracis toxin, and is approved for treatment and postexposure prophylaxis of inhalational anthrax. Anthrax Vaccine Adsorbed (AVA), for anthrax prophylaxis, consists primarily of adsorbed protective antigen. We did a postapproval study to assess the effect of raxibacumab on immunogenicity of AVA. METHODS: We did an open-label, parallel-group, randomised non-inferiority study at three centres in the USA. We enrolled healthy volunteers (aged 18-65 years) with no evidence of exposure to protective antigen. Participants were randomly allocated (1:1) according to a pregenerated balanced independent randomisation schedule to either subcutaneous 0·5 mL AVA on days 1, 15, and 29 or raxibacumab intravenous infusion (40 mg/kg) immediately before AVA on day 1, followed by AVA only on days 15 and 29. It was an open-label study to investigators and participants; however, the sponsor remained blinded during the study. The primary outcome was the ratio of geometric mean concentrations (GMCs) of anti-protective antigen antibodies (attributable to the immune response to AVA) between AVA and AVA plus raxibacumab 4 weeks after the first AVA dose in the per-protocol population. The per-protocol population comprised all individuals who received the allocated treatment within the protocol-specified visit window and completed the primary study outcome assessment, without a protocol deviation requiring exclusion. The non-inferiority margin for the ratio of GMCs was predefined (upper limit of 90% CI <1·5). This trial is registered with ClinicalTrials.gov, NCT02339155. FINDINGS: Between Feb 24, 2015, and June 6, 2017, 873 participants were screened for eligibility, of whom 300 were excluded. 573 were randomly allocated either AVA (n=287) or AVA plus raxibacumab (n=286). The per-protocol population comprised 276 individuals assigned AVA and 269 allocated AVA plus raxibacumab. At week 4, the GMC of anti-protective antigen antibodies in participants allocated AVA was 26·5 µg/mL (95% CI 23·6-29·8) compared with 22·5 µg/mL (20·1-25·1) among individuals allocated AVA plus raxibacumab. The ratio between groups was 1·18 (90% CI 1·03-1·35; p=0·0019), which met the predefined non-inferiority margin. Adverse events in the safety population were similar across groups (87 [30%] of 286 in the AVA group vs 80 [29%] of 280 in the AVA plus raxibacumab group) and no treatment-related serious adverse events were reported. INTERPRETATION: Co-administration of raxibacumab with AVA does not negatively affect AVA immunogenicity. This finding suggests that combining raxibacumab with AVA might provide added benefit in postexposure prophylaxis against inhalational anthrax. FUNDING: US Biomedical Advanced Research and Development Authority, and GlaxoSmithKline.

BA3338, a surface layer homology domain possessing protein augments immune response and protection efficacy of protective antigen against Bacillus anthracis in mouse model.

AIM: Category A classified Bacillus anthracis is highly fatal pathogen that causes anthrax and creates challenges for global security and public health. In this study, development of a safe and ideal next-generation subunit anthrax vaccine has been evaluated in mouse model. METHOD AND RESULTS: Protective antigen (PA) and BA3338, a surface layer homology (SLH) domain possessing protein were cloned, expressed in heterologous system and purified by IMAC. Recombinant PA and BA3338 with alum were administered in mouse alone or in combination. The humoral and cell-mediated immune response was measured by ELISA and vaccinated animals were challenged with B. anthracis spores via intraperitoneal route. The circulating IgG antibody titre of anti-PA and anti-BA3338 was found significantly high in the first and second booster sera. A significant enhanced level of IL-4, IFN-γ and IL-12 was observed in antigens stimulated supernatant of splenocytes of PA + BA3338 vaccinated animals. A combination of PA and BA3338 provided 80% protection against 20 LD50 lethal dose of B. anthracis spores. CONCLUSION: Both antigens induced admirable humoral and cellular immune response as well as protective efficacy against B. anthracis spores. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has been evaluated for the first time using BA3338 as a vaccine candidate alone or in combination with well-known anthrax vaccine candidate PA. The findings of this study demonstrated that BA3338 could be a co-vaccine candidate for development of dual subunit vaccine against anthrax.

Development of a guinea pig inhalational anthrax model for evaluation of post-exposure prophylaxis efficacy of anthrax vaccines.

A next-generation anthrax vaccine candidate, AV7909, is being developed for post-exposure prophylaxis (PEP) of inhalational anthrax in combination with the recommended course of antimicrobial therapy. Clinical efficacy studies of anthrax countermeasures in humans are not ethical or feasible, therefore, licensure of AV7909 for PEP is being pursued under the US Food and Drug Administration (FDA) Animal Rule, which requires that evidence of effectiveness be demonstrated in an animal model of anthrax, where results of studies in such a model can establish reasonable likelihood of AV7909 to produce clinical benefit in humans. Initial development of a PEP model for inhalational anthrax included evaluation of post-exposure ciprofloxacin pharmacokinetics (PK), tolerability and survival in guinea pigs treated with various ciprofloxacin dosing regimens. Three times per day (TID) intraperitoneal (IP) dosing with 7.5 mg/kg of ciprofloxacin initiated 1 day following inhalational anthrax challenge and continued for 14 days was identified as a well tolerated partially curative ciprofloxacin treatment regimen. The added benefit of AV7909 vaccination was evaluated in guinea pigs given the partially curative ciprofloxacin treatment regimen. Groups of ciprofloxacin-treated guinea pigs were vaccinated. 1 and 8 days post-challenge with serial dilutions of AV7909, a 1:16 dilution of AVA, or normal saline. A group of untreated guinea pigs was included as a positive control to confirm lethal B. anthracis exposure. Post-exposure vaccination with the AV7909 anthrax vaccine candidate administered in combination with the partially curative ciprofloxacin treatment significantly increased survival of guinea pigs compared to ciprofloxacin treatment alone. These results suggest that the developed model can be useful in demonstrating added value of the vaccine for PEP.

Evaluation of the protective efficacy of recombinant protective antigen vaccine (GC1109)-immunized human sera using passive immunization in a mouse model.

The protective efficacy of human sera from vaccinated individuals with a new recombinant protective antigen anthrax vaccine (GC1109) against lethal spore challenge was evaluated in a mouse model. Eighteen human sera were selected from the vaccinated individuals based on their toxin neutralizing assay (TNA) titer (ED50 of 55 to 668). The selected sera were diluted and passively transferred to A/J mice and the mice were subsequently challenged with 100 × LD50 of Bacillus anthracis Sterne spores. The correlation between the survival rate of passively immunized mice and the TNA ED50 of transferred sera was presented (r = 0.873, P-value < 0.001). The estimated TNA titer for 50% survival rate against lethal challenge was 197 (95% confidence interval of 149 and 260). The result suggest that GC1109 is protective against exposure to B. anthracis and the TNA titer of vaccinated serum can be an indicator for protective efficacy.

A fucoidan-quaternary chitosan nanoparticle adjuvant for anthrax vaccine as an alternative to CpG oligodeoxynucleotides.

We examined the efficacy of fucoidan-N-(2-hydroxy-3-trimethylammonium)propylchitosan nanoparticles (FUC-HTCC NPs) as adjuvants for anthrax vaccine adsorbed (AVA). Positively and negatively surface-charged FUC-HTCC NPs were prepared via polyelectrolyte complexation by varying the mass ratio of FUC and HTCC. When cultured with L929 cells or JAWS II dendritic cells, both charged NPs showed high cell viability and low cytotoxicity, observed via MTT assay and lactate dehydrogenase release assay, respectively. In addition, we have monitored excellent NPs uptake efficacy by dendritic cells and observed that combining FUC-HTCC NPs with AVA significantly increases the magnitude of IgG-anti-protective antigen titers in A/J mice compared to that by CpG oligodeoxynucleotides plus AVA or AVA alone, and PA-specific IgG1 and IgG2a analysis confirmed that FUC-HTCC NPs strongly stimulated humoral immunity. Furthermore, FUC-HTCC NPs plus AVA provided a superior survival rate (100%) of A/J mice compared to CpG oligodeoxynucleotides plus AVA (75%) or AVA alone (50%) following anthrax lethal toxin challenge. The findings support FUC-HTCC NPs as a potential adjuvant of AVA for rapid induction of protective immunity.

Protective antigen domain 4 of Bacillus anthracis as a candidate for use as vaccine for anthrax.

Existing research for using the protective antigen (PA) of Bacillus anthracis as a vaccine component shows that protection against anthrax may be obtained using fragments of this protein. The aim of the research is to check whether the selected protein fragment of the protective antigen (domain 4) encoded by an appropriate nucleotide sequence of gene pag of B. anthracis, was expressed in the bacterial system of E. coli. In order to examine the selected sequence of the pag gene, a PCR reaction and a highly effective TOPO cloning strategy were used, followed by purification of the recombinant proteins and their detection by a western-blot method. In the planning of the PA4 antigen expression a higher level of effectiveness in production of small protein - domain 4 - was anticipated. As a result, the 139 amino acids protein fragment of B. anthracis PA (domain 4) was isolated. The research may have found the basis for in vivo research aimed at finding potential anthrax vaccine components.

Vaccines against anthrax based on recombinant protective antigen: problems and solutions.

Introduction: Anthrax is a dangerous bio-terror agent because Bacillus anthracis spores are highly resilient and can be easily aerosolized and disseminated. There is a threat of deliberate use of anthrax spores aerosol that could lead to serious fatal diseases outbreaks. Existing control measures against inhalation form of the disease are limited. All of this has provided an impetus to the development of new generation vaccines. Areas сovered: This review is devoted to challenges and achievements in the design of vaccines based on the anthrax recombinant protective antigen (rPA). Scientific databases have been searched, focusing on causes of PA instability and solutions to this problem, including new approaches of rPA expression, novel rPA-based vaccines formulations as well as the simultaneous usage of PA with other anthrax antigens. Expert opinion: PA is a central anthrax toxin component, playing a key role in the defense against encapsulated and unencapsulated strains. Subunit rPA-based vaccines have a good safety and protective profile. However, there are problems of PA instability that are greatly enhanced when using aluminum adjuvants. New adjuvant compositions, dry formulations and resistant to proteolysis and deamidation mutant PA forms can help to handle this issue. Devising a modern anthrax vaccine requires huge efforts.

Development of a novel multiepitope chimeric vaccine against anthrax.

Bacillus anthracis (BA), the etiological agent of anthrax, secretes protective antigen (PA), lethal factor (LF), and edema factor (EF) as major virulence mediators. Amongst these, PA-based vaccines are most effective for providing immunity against BA, but their low shelf life limits their usage. Previous studies showed that B-cell epitopes, ID II and ID III present in PA domain IV possess higher toxin neutralization activity and elicit higher antibody titer than ID I. Moreover, N-terminal region of both LF and EF harbors PA-binding sites which share 100% identity with each other. Here, in this study, we have developed an epitope-based chimeric vaccine (ID-LFn) comprising ID II-ID III region of PA and N-terminal region of LF. We have also evaluated its protective efficacy as well as stability and found it to be more stable than PA-based vaccine. Binding reactivities of ID-LFn with anti-PA/LF/EF antibodies were determined by ELISA. The stability of chimeric vaccine was assessed using circular dichroism spectroscopy. ID-LFn response was characterized by toxin neutralization, lymphocyte proliferation isotyping and cytokine profiling. The protective efficacy was analyzed by challenging ID-LFn-immunized mice with B. anthracis (pXO1+ and pXO2+). ID-LFn was found to be significantly stable as compared to PA. Anti-ID-LFn antibodies recognized PA, LF as well as EF. The T-cell response and the protective efficacy of ID-LFn were found to be almost similar to PA. ID-LFn exhibits equal protective efficacy in mice and possesses more stability as compared to PA along with the capability of recognizing PA, LF and EF at the same time. Thus, it can be considered as an improved vaccine against anthrax with better shelf life. ID-LFn, a novel multiepitope chimeric anthrax vaccine: ID-LFn comprises of immunodominant epitopes of domain 4 of PA and N-terminal homologous stretch of LF and EF. The administration of this protein as a vaccine provides protection against anthrax.

Immunogenicity of anthrax recombinant peptides and killed spores in goats and protective efficacy of immune sera in A/J mouse model.

Anthrax is primarily recognized as an affliction of herbivores with incubation period ranging from three to five days post-infection. Currently, the Sterne live-spore vaccine is the only vaccine approved for control of the disease in susceptible animals. While largely effective, the Sterne vaccine has several problems including adverse reactions in sensitive species, ineffectiveness in active outbreaks and incompatibility with antibiotics. These can be surmounted with the advent of recombinant peptides (non-living) next generation vaccines. The candidate vaccine antigens comprised of recombinant protective antigen (PA), spore-specific antigen (bacillus collagen-like protein of anthracis, BclA) and formaldehyde inactivated spores (FIS). Presently, little information exists on the protectivity of these novel vaccine candidates in susceptible ruminants. Thus, this study sought to assess the immunogenicity of these vaccine candidates in goats and evaluate their protectivity using an in vivo mouse model. Goats receiving a combination of PA, BclA and FIS yielded the highest antibody and toxin neutralizing titres compared to recombinant peptides alone. This was also reflected in the passive immunization experiment whereby mice receiving immune sera from goats vaccinated with the antigen combination had higher survival post-challenge. In conclusion, the current data indicate promising potential for further development of non-living anthrax vaccines in ruminants.