Showing papers on "Anthrax vaccines published in 2010"
23 Jul 2010
TL;DR: The number of doses required to complete the pre-event and preexposure primary series is reduced from 6 doses to 5 doses, and the evidence regarding the effectiveness and efficacy, immunogenicity, and safety of AVA is summarized.
Abstract: These recommendations from the Advisory Committee on Immunization Practices (ACIP) update the previous recommendations for anthrax vaccine adsorbed (AVA) (CDC. Use of anthrax vaccine in the United States: Recommendations of the Advisory Committee on Immunization Practices [ACIP]. MMWR 2000;49:1-20; CDC. Use of anthrax vaccine in response to terrorism: supplemental recommendations of the Advisory Committee on Immunization Practices [ACIP]. MMWR 2002;51:1024-6) and reflect the status of anthrax vaccine supplies in the United States. This statement 1) provides updated information on anthrax epidemiology; 2) summarizes the evidence regarding the effectiveness and efficacy, immunogenicity, and safety of AVA; 3) provides recommendations for pre-event and preexposure use of AVA; and 4) provides recommendations for postexposure use of AVA. In certain instances, recommendations that did not change were clarified. No new licensed anthrax vaccines are presented. Substantial changes to these recommendations include the following: 1) reducing the number of doses required to complete the pre-event and preexposure primary series from 6 doses to 5 doses, 2) recommending intramuscular rather than subcutaneous AVA administration for preexposure use, 3) recommending AVA as a component of postexposure prophylaxis in pregnant women exposed to aerosolized Bacillus anthracis spores, 4) providing guidance regarding preexposure vaccination of emergency and other responder organizations under the direction of an occupational health program, and 5) recommending 60 days of antimicrobial prophylaxis in conjunction with 3 doses of AVA for optimal protection of previously unvaccinated persons after exposure to aerosolized B. anthracis spores.
123 citations
TL;DR: Basic characteristics of the bacterium and its products, key aspects of pathogenesis, and the pathologic changes commonly observed in each animal model species are focused on.
Abstract: Anthrax is a lethal disease caused by the bacterium Bacillus anthracis. There are three principal forms of the disease in humans-cutaneous, gastrointestinal, and inhalational-depending on the route of exposure. Of these, inhalational anthrax is the most dangerous; it is rapidly fatal; and it has been used as a deadly biological warfare agent in the last decade. Suitable animal models of inhalational anthrax have been utilized to study pathogenesis of disease, investigate bacterial characteristics such as virulence, and test effectiveness of vaccines and therapeutics. To date, mice, guinea pigs, rabbits, and nonhuman primates are the principal animal species used to study inhalational anthrax. Mice are valuable in studying early pathogenesis and bacterial characteristics. Few pathologic changes occur in the mouse models but may include marked bacteremia and lymphocyte destruction in the spleen and mediastinal lymph nodes. Rabbits and guinea pigs rapidly develop fulminate systemic disease, and pathologic findings often include necrotizing lymphadenitis; splenitis; pneumonia; vasculitis; and hemorrhage, congestion, and edema in multiple tissues. Nonhuman primates consistently develop the full range of classic lesions of human inhalational anthrax, including meningitis; lymphadenitis; splenitis; mediastinitis; pneumonia; vasculitis; and hemorrhage, congestion, and edema in multiple tissues. This review focuses on basic characteristics of the bacterium and its products, key aspects of pathogenesis, and the pathologic changes commonly observed in each animal model species.
78 citations
TL;DR: Results suggest that BslA functions as the surface adhesin of the anthrax pathogen B. anthracis strain Ames, and that few vegetative forms could be recovered from internal organs of animals infected with the bslA mutant.
Abstract: Microbial pathogens use adhesive surface proteins to bind to and interact with host tissues, events that are universal for the pathogenesis of infectious diseases. A surface adhesin of Bacillus anthracis, the causative agent of anthrax, required to mediate these steps has not been discovered. Previous work identified BslA, an S-layer protein, to be necessary and sufficient for adhesion of the anthrax vaccine strain, Bacillus anthracis Sterne, to host cells. Here we asked whether encapsulated bacilli require BslA for anthrax pathogenesis in guinea pigs. Compared with the highly virulent parent strain B. anthracis Ames, bslA mutants displayed a dramatic increase in the lethal dose and in mean time-to-death. Whereas all tissues of animals infected with B. anthracis Ames contained high numbers of bacilli, only few vegetative forms could be recovered from internal organs of animals infected with the bslA mutant. Surface display of BslA occurred at the poles of encapsulated bacilli and enabled the binding of vegetative forms to host cells. Together these results suggest that BslA functions as the surface adhesin of the anthrax pathogen B. anthracis strain Ames.
75 citations
TL;DR: Improvement of this improved expression vaccine strategy reduced the number of inoculations and length of the boosting period, leading to animal protection via efficacious bacterial adjuvanticity and safe oral delivery of this vaccine to mucosal immune cells, including dendritic cells.
Abstract: Aim: Induction of protective immunity against pathogenic microbes, including Bacillus anthracis, requires efficient vaccines that potentiate antibody avidity and increase T-cell longevity. We recently reported that the delivery of targeted B. anthracis protective antigen (PA) genetically fused to a DC-binding peptide (DCpep) by Lactobacillus acidophilus induced mucosal and systemic immunity against B. anthracis challenge in mice. Materials & methods: Improvement of this oral vaccine strategy was attempted by use of the high copy and genetically stable q-replicating vector, pTRKH2, for expression of the targeted PA fusion protein in Lactobacillus gasseri, a common human commensal microbe, to vaccinate animals against anthrax Sterne infection. Results: Oral application of L. gasseri expressing the PA–DCpep fusion proteins elicited robust PA-neutralizing antibody and T-cell mediated immune responses against anthrax Sterne challenge, resulting in complete animal survival. Collectively, this improved expressio...
70 citations
TL;DR: It is demonstrated that electroporation mediated delivery enhanced the immunogenicity of DNA vaccines in nonhuman primates over 100-fold as compared to conventional intramuscular injection.
59 citations
TL;DR: This review summarizes the current scientific evidence about the relationship between squalene, anti-squalene antibodies and vaccination and concludes that neither the presence ofAnti-sQualene antibodies nor their titre is significantly increased by immunization with vaccines containingSqualene (or MF59) as an adjuvant.
54 citations
TL;DR: A single vaccine comprising protective regions from LF and PA would simplify production and confer a broader spectrum of protection than that seen with PA alone.
54 citations
TL;DR: CD4 T cell responses in patients from the Kayseri region of Turkey who were previously infected with cutaneous anthrax were investigated, finding some anthrax epitopes showed broad binding to several HLA class alleles, but others were more constrained in their HLA binding patterns.
Abstract: There has been a long history of defining T cell epitopes to track viral immunity and to design rational vaccines, yet few data of this type exist for bacterial infections. Bacillus anthracis, the causative agent of anthrax, is both an endemic pathogen in many regions and a potential biological warfare threat. T cell immunity in naturally infected anthrax patients has not previously been characterized, which is surprising given concern about the ability of anthrax toxins to subvert or ablate adaptive immunity. We investigated CD4 T cell responses in patients from the Kayseri region of Turkey who were previously infected with cutaneous anthrax. Responses to B. anthracis protective Ag and lethal factor (LF) were investigated at the protein, domain, and epitope level. Several years after antibiotic-treated anthrax infection, strong T cell memory was detectable, with no evidence of the expected impairment in specific immunity. Although serological responses to existing anthrax vaccines focus primarily on protective Ag, the major target of T cell immunity in infected individuals and anthrax-vaccinated donors was LF, notably domain IV. Some of these anthrax epitopes showed broad binding to several HLA class alleles, but others were more constrained in their HLA binding patterns. Of specific CD4 T cell epitopes targeted within LF domain IV, one is preferentially seen in the context of bacterial infection, as opposed to vaccination, suggesting that studies of this type will be important in understanding how the human immune system confronts serious bacterial infection.
52 citations
TL;DR: It is concluded that a synthetic peptide vaccine targeting the LND would be a potentially efficacious vaccine for anthrax.
Abstract: The current vaccines for anthrax in the United States and United Kingdom are efficacious in the two most accepted animal models of inhalation anthrax, nonhuman primates and rabbits, but require extensive immunization protocols. We previously demonstrated that a linear determinant in domain 2 of Bacillus anthracis protective Ag (PA) is a potentially important target for an epitope-specific vaccine for anthrax, as Abs specific for this site, referred to as the loop-neutralizing determinant (LND), neutralize lethal toxin in vitro, yet are virtually absent in PA-immunized rabbits. In this study, we evaluated the immunogenicity and protective efficacy in rabbits of multiple antigenic peptides (MAPs) consisting of aa 304-319 from the LND of PA colinearly synthesized at the C terminus (T-B MAP) or N terminus (B-T MAP) with a heterologous T cell epitope from Plasmodium falciparum. Immunogenicity studies demonstrated that both MAPs elicited toxin-neutralizing Ab in rabbits. To evaluate the MAPs as potential anthrax vaccines, we immunized groups of rabbits (n = 7) with each MAP in Freund's adjuvant and then exposed all rabbits to a 200-LD(50) challenge with aerosolized spores of B. anthracis Ames strain. All seven rabbits immunized with the B-T MAP and 89% (six of seven) of rabbits immunized with the T-B MAP survived the spore challenge. Corollary studies with reference sera from human vaccinees immunized with rPA or anthrax vaccine absorbed and nonhuman primates immunized with PA revealed no detectable Ab with specificity for the LND. We conclude that a synthetic peptide vaccine targeting the LND would be a potentially efficacious vaccine for anthrax.
41 citations
TL;DR: Results show that a small group of 'inducers' rapidly up-regulated a large network genes following CpG treatment of mice, and shed light on the mechanism underlying C pG-mediated immune activation and therapeutic activity.
37 citations
TL;DR: The vaccine was safe, well tolerated and stimulated a robust humoral and cellular response after two doses.
Abstract: Background: The fatal disease caused by Bacillus anthracis is preventable with a prophylactic vaccine. The currently available anthrax vaccine requires a lengthy immunization schedule, and simpler and more immunogenic options for protection against anthrax are a priority for development. In this report we describe a phase I clinical trial testing the safety and immunogenicity of an anthrax vaccine using recombinant Escherichia coli-derived, B. anthracis protective antigen (rPA). Methodology/Principal Findings: A total of 73 healthy adults ages 18–40 were enrolled and 67 received 2 injections separated by 4 weeks of either buffered saline placebo, or rPA formulated with or without 704 mg/ml AlhydrogelH adjuvant in increasing doses (5, 25, 50, 100 mg) of rPA. Participants were followed for one year and safety and immunologic data were assessed. Tenderness and warmth were the most common post-injection site reactions. No serious adverse events related to the vaccine were observed. The most robust humoral immune responses were observed in subjects receiving 50 mg of rPA formulated with AlhydrogelH with a geometric mean concentration of anti-rPA IgG antibodies of 283 mg/ml and a toxin neutralizing geometric 50% reciprocal geometric mean titer of 1061. The highest lymphoproliferative peak cellular response (median Lymphocyte Stimulation Index of 29) was observed in the group receiving 25 mg AlhydrogelH-formulated rPA. Conclusions/Significance: The vaccine was safe, well tolerated and stimulated a robust humoral and cellular response after two doses. Trial Registration: ClinicalTrials.gov NCT00057525
TL;DR: The results suggest that NKT-activating CD1d ligands could be exploited for the development of improved vaccines for Bacillus anthracis that increase not only neutralizing Ab titers but also the duration of the protection afforded by Ab.
Abstract: The current Bacillus anthracis vaccine consists largely of protective antigen (PA), the protein of anthrax toxin that mediates entry of edema factor (EF) or lethal factor (LF) into cells. PA induces protective antibody (Ab)-mediated immunity against Bacillus anthracis but has limited efficacy and duration. We previously demonstrated that activation of CD1d-restricted natural killer-like T cells (NKT) with a CD1d-binding glycolipid led to enhanced Ab titers specific for foreign antigen (Ag). We therefore tested the hypothesis that activation of NKT cells with the CD1d ligand (α-galactosylceramide [α-GC]) at the time of immunization improves PA-specific Ab responses. We observed that α-GC enhanced PA-specific Ab titers in C57BL/6 mice. In CD1d−/− mice deficient in type I and type II NKT cells the anti-PA Ab response was diminished. In Jα281−/− mice expressing CD1d but lacking type I α-GC-reactive NKT cells, α-GC did not enhance the Ab response. In vitro neutralization assays were performed and showed that the Ab titers correlated with protection of macrophages against anthrax lethal toxin (LT). The neutralization capacity of the Ab was further tested in lethal challenge studies, which revealed that NKT activation leads to enhanced in vivo protection against LT. Anti-PA Ab titers, neutralization, and protection were then measured over a period of several months, and this revealed that NKT activation leads to a sustained protective Ab response. These results suggest that NKT-activating CD1d ligands could be exploited for the development of improved vaccines for Bacillus anthracis that increase not only neutralizing Ab titers but also the duration of the protection afforded by Ab.
TL;DR: A single vaccine using the licensed Wyeth smallpox vaccine strain that is efficacious against both smallpox and anthrax due to the integration of immune-enhancing cytokine IL-15 and the protective antigen (PA) of B. anthracis into the Wyeth vaccinia virus is developed.
Abstract: Bioterrorism poses a daunting challenge to global security and public health in the 21st century. Variola major virus, the etiological agent of smallpox, and Bacillus anthracis, the bacterial pathogen responsible for anthrax, remain at the apex of potential pathogens that could be used in a bioterror attack to inflict mass casualties. Although licensed vaccines are available for both smallpox and anthrax, because of inadequacies associated with each of these vaccines, serious concerns remain as to the deployability of these vaccines, especially in the aftermath of a bioterror attack involving these pathogens. We have developed a single vaccine (Wyeth/IL-15/PA) using the licensed Wyeth smallpox vaccine strain that is efficacious against both smallpox and anthrax due to the integration of immune-enhancing cytokine IL-15 and the protective antigen (PA) of B. anthracis into the Wyeth vaccinia virus. Integration of IL-15 renders Wyeth vaccinia avirulent in immunodeficient mice and enhances anti-vaccinia immune responses. Wyeth/IL-15/PA conferred sterile protection against a lethal challenge of B. anthracis Ames strain spores in rabbits. A single dose of Wyeth/IL-15/PA protected 33% of the vaccinated A/J mice against a lethal spore challenge 72 h later whereas a single dose of licensed anthrax vaccine protected only 10%. Our dual vaccine Wyeth/IL-15/PA remedies the inadequacies associated with the licensed vaccines, and the inherent ability of Wyeth vaccinia virus to be lyophilized without loss of potency makes it cold-chain independent, thus simplifying the logistics of storage, stockpiling, and field delivery in the event of a bioterror attack involving smallpox or anthrax.
TL;DR: Differences in the magnitudes of the Fc receptor-mediated neutralization associated with the J774A.1 cell- and RAW 264.7 cell-based assays may account for some of the species dependence of the assays.
Abstract: Different types of anthrax toxin neutralization assays have been utilized to measure the antibody levels elicited by anthrax vaccines in both nonclinical and clinical studies. In the present study, we sought to determine whether three commonly used toxin neutralization assays—J774A.1 cell-, RAW 264.7 cell-, and CHO cell-based assays—yield comparable estimates of neutralization activities for sera obtained after vaccination with anthrax vaccines composed of recombinant protective antigen (rPA). In order to compare the assays, sera were assayed alongside a common reference serum sample and the neutralization titers were expressed relative to the titer for the reference sample in each assay. Analysis of sera from rabbits immunized with multiple doses of the rPA vaccine showed that for later bleeds, the quantitative agreement between the assays was good; however, for early bleeds, some heterogeneity in relative neutralization estimates was observed. Analysis of serum samples from rabbits, nonhuman primates, and humans immunized with the rPA vaccine showed that the relative neutralization estimates obtained in the different assays agreed to various extents, depending on the species of origin of the sera examined. We identified differences in the magnitudes of the Fc receptor-mediated neutralization associated with the J774A.1 cell- and RAW 264.7 cell-based assays, which may account for some of the species dependence of the assays. The differences in the relative neutralization estimates among the assays were relatively small and were always less than 2.5-fold. However, because toxin neutralization assays will likely be used to establish the efficacies of new anthrax vaccines, our findings should be considered when assay outputs are interpreted.
TL;DR: Heterologous prime/boost immunization regimens that involved an initial intranasal administration of a live influenza virus vector, followed by an intramuscular boost with either the killed RV vector or the VV vector were particularly effective, inducing antigen-specific antibodies at levels severalfold higher than homologous or alternative heterologous protocols.
Abstract: Viral vectors based on influenza virus, rabies virus (RV), and vaccinia virus (VV) were used to express large polypeptide segments derived from the Bacillus anthracis protective antigen (PA). For the infectious influenza virus vector and recombinant VV constructs, the receptor binding domain (RBD or domain 4) or the lethal and edema factor binding domain (LEF or domain 1′) were engineered into functional chimeric hemagglutinin (HA) glycoproteins. In the case of the RV vector, the viral glycoprotein (G) was used as a carrier for RBD in an inactivated form of the vector. These constructs were examined by using multiple homologous and heterologous prime/boost immunization regimens in order to optimize the induction of α-PA antibody responses. Several immunization combinations were shown to induce high titers of antibody recognizing the anthrax RBD and LEF domains, as well as the full-length PA protein in mice. The heterologous prime/boost immunization regimens that involved an initial intranasal administration of a live influenza virus vector, followed by an intramuscular boost with either the killed RV vector or the VV vector, were particularly effective, inducing antigen-specific antibodies at levels severalfold higher than homologous or alternative heterologous protocols. Furthermore, sera from several groups of the immunized mice demonstrated neutralization activity in an in vitro anthrax toxin neutralization assay. In some cases, such toxin-neutralizing activity was notably high, indicating that the mechanisms by which immunity is primed by live influenza virus vectors may have beneficial properties.
TL;DR: The immunobiology of major lung cell subsets and their cellular synapses are described to reveal the dynamics of B. anthracis infection and its effects on lung immune mechanisms and vaccines strategies that mobilize protective mucosal and systemic immunity against this deadly microbe are highlighted.
TL;DR: It is concluded that the relatively weaker anti‐PA immune response in the non-anaesthetized mice might be partially attributed to the reduced retention of the PA/LPD particles in the nasal cavity of the non‐anaesthedized mice.
Abstract: To better protect against inhalational anthrax infection, a nasal anthrax vaccine based on the protective antigen (PA) protein of Bacillus anthracis could be an attractive alternative to the current Anthrax-Vaccine-Adsorbed (AVA), which was licensed for cutaneous anthrax prevention. Previously, we have demonstrated that an anti-PA immune response comparable with that in mice subcutaneously immunized with PA protein adjuvanted with aluminium hydroxide was induced in both the systemic compartment and the mucosal secretions of the nose and lung of anaesthetized mice when they were nasally immunized with PA protein incorporated into previously reported LPD (Liposome-Protamine-DNA) particles. In this study, we evaluated the anti-PA immune response induced by the nasal PA/LPD particles in non-anaesthetized mice and compared it with that in anaesthetized mice. Our data showed that the anti-PA antibody response and the anthrax lethal toxin-neutralization activity induced by the nasal PA/LPD in non-anaesthetized mice was relatively weaker than that in anaesthetized mice. However, the splenocytes isolated from the nasally immunized mice, anaesthetized and non-anaesthetized, proliferated comparably after in-vitro re-stimulation. By evaluating the uptake of fluorescence-labelled LPD particles by phagocytes in the nasal and broncho-alveolar lavages of mice after the nasal administration, we concluded that the relatively weaker anti-PA immune response in the non-anaesthetized mice might be partially attributed to the reduced retention of the PA/LPD particles in the nasal cavity of the non-anaesthetized mice. Data collected in this study are expected to be useful for future anthrax nasal vaccine studies when mice are used as a model.
23 Jul 2010
TL;DR: In this paper, the authors provide a summary of the evidence regarding the effectiveness and efficacy, immunogenicity, and safety of AVA and provide recommendations for pre-event and preexposure use.
Abstract: : These recommendations from the Advisory Committee on Immunization Practices (ACIP) update the previous recommendations for anthrax vaccine adsorbed (AVA) (CDC. Use of anthrax vaccine in the United States: Recommendations of the Advisory Committee on Immunization Practices [ACIP]. MMWR 2000;49:1-20; CDC. Use of anthrax vaccine in response to terrorism: supplemental recommendations of the Advisory Committee on Immunization Practices [ACIP]. MMWR 2002;51:1024-6) and reflect the status of anthrax vaccine supplies in the United States. This statement 1) provides updated information on anthrax epidemiology; 2) summarizes the evidence regarding the effectiveness and efficacy, immunogenicity, and safety of AVA; 3) provides recommendations for pre-event and preexposure use of AVA; and 4) provides recommendations for postexposure use of AVA. In certain instances, recommendations that did not change were clarified. No new licensed anthrax vaccines are presented. Substantial changes to these recommendations include the following: 1) reducing the number of doses required to complete the pre-event and preexposure primary series from 6 doses to 5 doses, 2) recommending intramuscular rather than subcutaneous AVA administration for preexposure use, 3) recommending AVA as a component of postexposure prophylaxis in pregnant women exposed to aerosolized Bacillus anthracis spores, 4) providing guidance regarding preexposure vaccination of emergency and other responder organizations under the direction of an occupational health program, and 5) recommending 60 days of antimicrobial prophylaxis in conjunction with 3 doses of AVA for optimal protection of previously unvaccinated persons after exposure to aerosolized B. anthracis spores.
TL;DR: Mucosal priming with a safe and effective S. Typhi-based anthrax vaccine followed by PA-boost could serve as a practical and effective prophylactic approach to prevent anthrax early in life.
TL;DR: Data demonstrate that AVA stimulates a myriad of antibodies that recognize multiple neutralizing epitopes and confirm that change, loss, or occlusion of epitopes after PA is processed from PA83 to PA63 at the cell surface does not significantly affect in vitro neutralizing efficacy.
Abstract: Anthrax toxin (ATx) is composed of the binary exotoxins lethal toxin (LTx) and edema toxin (ETx). They have separate effector proteins (edema factor and lethal factor) but have the same binding protein, protective antigen (PA). PA is the primary immunogen in the current licensed vaccine anthrax vaccine adsorbed (AVA [BioThrax]). AVA confers protective immunity by stimulating production of ATx-neutralizing antibodies, which could block the intoxication process at several steps (binding of PA to the target cell surface, furin cleavage, toxin complex formation, and binding/translocation of ATx into the cell). To evaluate ATx neutralization by anti-AVA antibodies, we developed two low-temperature LTx neutralization activity (TNA) assays that distinguish antibody blocking before and after binding of PA to target cells (noncomplexed [NC] and receptor-bound [RB] TNA assays). These assays were used to investigate anti-PA antibody responses in AVA-vaccinated rhesus macaques (Macaca mulatta) that survived an aerosol challenge with Bacillus anthracis Ames spores. Results showed that macaque anti-AVA sera neutralized LTx in vitro, even when PA was prebound to cells. Neutralization titers in surviving versus nonsurviving animals and between prechallenge and postchallenge activities were highly correlated. These data demonstrate that AVA stimulates a myriad of antibodies that recognize multiple neutralizing epitopes and confirm that change, loss, or occlusion of epitopes after PA is processed from PA83 to PA63 at the cell surface does not significantly affect in vitro neutralizing efficacy. Furthermore, these data support the idea that the full-length PA83 monomer is an appropriate immunogen for inclusion in next-generation anthrax vaccines.
TL;DR: LFn-DPA represents a highly effective trivalent vaccine candidate for both preexposure and postexposure vaccination and has developed a novel and dually functional reagent for the prophylaxis and treatment of anthrax.
Abstract: Inhalational anthrax, caused by inhalation of the adversity-resistant spores, is a fatal disease, with a mortality rate approaching 80% (30). Although the naturally occurring inhalational form of anthrax is rare, malicious release of anthrax spores, particularly as weaponized anthrax spores, in a bioterrorism event kills civilians as well as creates great panic. This has stimulated the search for effective methods for the therapy and prevention of anthrax.
The principal virulence factors of Bacillus anthracis consist of an antiphagocytic capsule composed of poly-d-glutamic acid (PGA) and a secreted bacterial toxin. The former is encoded by genes located on plasmid pXO1, and the latter is encoded by plasmid pXO2 (47). The anthrax toxin, which is predominantly responsible for the etiology of anthrax, belongs to the family of bacterial binary AB-type toxins, which consist of a receptor-binding B subunit known as the protective antigen (PA) and two catalytic A subunits, i.e., the lethal factor (LF) and edema factor (EF). PA combines with either LF or EF to form the lethal toxin (LeTx) and edema toxin (EdTx), respectively (47). Currently, the standard approach for anthrax therapy is to kill the germinating bacilli by administering aggressive antibiotics. However, antibiotic therapy is ineffective once systematic anthrax symptoms appear because by that time, fatal concentrations of the anthrax toxin have accumulated in the patient's body (41). Moreover, the emergence of antibiotic-resistant strains as a result of natural evolution or intentional modification by genetic engineering also poses a new challenge to traditional antibiotic treatment (13, 14). Therefore, the development of an antitoxin for combined use with antibiotic therapy is of high priority.
At present, the process by which anthrax toxins enter cells and act is relatively well understood. Initially, the B subunit, i.e., the 83-kDa PA (PA83), binds to specific cell surface receptors through its C-terminal binding domain, and this is then proteolytically cleaved by furin or furin-like protease into a 20-kDa N-terminal fragment (PA20) and an active 63-kDa C-terminal fragment (PA63) (5, 15, 19, 28). After dissociation of PA20, cell-bound PA63 self-assembles into a ring-shaped homo-oligomer (heptamer or octamer) termed a prepore (18, 52). Simultaneously, the prepore competitively binds up to three molecules of LF and/or EF to form toxin complexes (9, 23, 33). These complexes are then internalized into the cells by receptor-mediated endocytosis and delivered to an endosome, where the acidic pH triggers the conformational transition of the prepore to generate the pore (31). Ultimately, LF and EF are translocated through the pore into the cytosol, where they exert their respective catalytic effects, leading to the manifestation of the anthrax symptoms (32).
The elucidation of the molecular mechanism of anthrax toxin action has provided us with new strategies for developing antitoxins for anthrax treatment. To date, several potential antitoxins that target different steps of anthrax toxin intoxication are under development (37). The PA-binding domain of LF (LFn) or LFn-based fusion proteins is sufficient for binding to the PA63 formed prepore and can inhibit the anthrax toxin by competitively inhibiting the binding of LF to the prepore (1, 3, 20, 34). Another powerful antitoxin is the dominant-negative mutant of PA (DPA), which can be proteolytically activated to form dominant-negative inhibitory PA63 (DPA63). DPA63 coassembles with wild-type PA63 and blocks its ability to translate LF and EF (42, 46). On the basis of these findings, we decided to combine the competitive inhibitory activity of LFn and the dominant-negative inhibitory activity of DPA into a single-component reagent. Toward this end, we first constructed the chimera LFn-PA and demonstrated that this chimera could be proteolytically activated to produce an active PA63 moiety and a functional LFn-PA20 part. We then further replaced the PA moiety with a dominant-negative mutation, PAF427D, which has been described in sufficient detail elsewhere, to generate LFn-DPA (6, 21, 46). We next investigated the antitoxin potency of LFn-DPA in vitro and in vivo.
Vaccination with the anthrax vaccine is the best strategy for preexposure prevention and postexposure prophylaxis (45). Now the only licensed anthrax vaccine for human use in the United States is the anthrax vaccine absorbed (AVA), also known as BioThrax. AVA has several disadvantages, such as batch-to-batch variation, an ill-defined composition, side effects, and a lengthy vaccination schedule (47). A second generation of vaccines based on highly purified recombinant PA (rPA) is under development. Although well-defined and homogeneous, rPA-based vaccines have immunogenicity and potency similar to those of AVA (17). This means that vaccination with rPA-based vaccines may be burdensome, just like vaccination with AVA. This underscores the need for developing new and more effective anthrax vaccines. Therefore, we evaluated the potential of LFn-DPA in anthrax prophylaxis.
TL;DR: Competitive assays suggested that humans may have a more significant secondary population of IgG antibodies that bind to partially unfolded or incorrectly folded PA, which will be useful when linking animal protection data to humans via an antibody bridge to establish efficacy of new anthrax vaccines.
Abstract: The licensed anthrax vaccine and many of the new anthrax vaccines being developed are based on protective antigen (PA), a nontoxic component of anthrax toxin. For this reason, an understanding of the immune response to PA vaccination is important. In this study, we examined the antibody response elicited by PA-based vaccines and identified the domains of PA that contribute to that response in humans as well as nonhuman primates (NHPs) and rabbits, animal species that will be used to generate efficacy data to support approval of new anthrax vaccines. To this end, we developed a competitive enzyme-linked immunosorbent assay (ELISA), using purified recombinant forms of intact PA and its individual domains. We found that PA-based vaccines elicited IgG antibodies to each of the four PA domains in all three species. We also developed a competitive toxin neutralization assay, which showed that rabbits, NHPs, and humans all have functional antibody populations that bind to domains 1, 3, and 4. While the domain specificities of the antibody responses elicited by PA-based vaccines were similar in humans, NHPs, and rabbits, competitive assays suggested that humans may have a more significant secondary population of IgG antibodies that bind to partially unfolded or incorrectly folded PA. These findings provide information that will be useful when linking animal protection data to humans via an antibody bridge to establish efficacy of new anthrax vaccines.
TL;DR: Oscherwitz et al. as mentioned in this paper showed that humans exposed to Bacillus anthracis and whole PA recognize and generate memory to PA 305-319, suggesting that it is immunodominant rather than cryptic.
Abstract: In a recent article, Oscherwitz et al. endeavor to enhance the immunogenicity of a multiple antigenic peptide (MAP) vaccine that targets a loop-neutralizing determinant (LND) of Bacillus anthracis protective antigen (PA) (1). They showed in a previous study (2) that this MAP, consisting of four copies of amino acids 305 to 319 of PA (PA 305-319) extending from a lysine core, can elicit humoral immunity in rabbits that is specific and strongly neutralizing for the 2β2-2β3 loop in domain 2 of PA. However, LND-specific antibodies were not detected in rabbits immunized with whole PA, and it was concluded that PA 305-319 may be a cryptic epitope. T cell assays in PA 305-319-immunized mice showed that this peptide appeared to lack activity as a T helper cell epitope.
We have good evidence that humans exposed to PA, either following cutaneous anthrax infection or through vaccination, generate long-term, T cell memory to both whole PA and PA 305-319. In a study including samples from naturally exposed patients recovered from cutaneous anthrax, from vaccine-hyperimmunized AVP (anthrax vaccine precipitated) donors, and from recombinant PA-vaccinated donors, positive T cell gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay responses were detected across all groups on restimulation of peripheral blood mononuclear cells (PBMC) with both whole PA and PA 305-319 (Table 1; unpublished data). This shows that humans exposed to B. anthracis and whole PA recognize and generate memory to PA 305-319, suggesting that it is immunodominant rather than cryptic. Donors expressed a range of HLA haplotypes, with a majority being HLA-DR4 positive. While many important discoveries on the immunology of infection are made in experimental models, the human T cell response to PA in this respect differs from that of both rabbits and mice.
In this light, the epitope used by Oscherwitz and colleagues may indeed be more relevant to eliciting appropriate human immunity than previously envisaged.
TL;DR: Assay quality control data establish the functionality of the quality control system and demonstrates the reliability of the serological data generated using these assays.
Journal Article•
TL;DR: Based on history of sudden death, oozing of anthrax in humans is correlated with industrial unclotted blood at natural orifices and blood smear activities such as handling potentially contaminated examination it was diagnosed as anthrax outbreak and materials like hides, fur, wool, hair, meat, or bone meal control measures were undertaken immediately.
Abstract: Anthrax, an acute to per acute infectious disease Diagnosis, treatment and control measures of all domestic animals and human beings, is caused All the blood smears examined were positive for by Bacillus anthracis and characterized by septicemia Bacillus anthracis organisms. Smears showed and sudden death with the exudation of dark tarry characteristic McFadyean reaction characterized by colored and unclotted blood from the natural orifices. purple capsule around the gram positive bacilli which Human beings can get anthrax from contact with were seen in short chains with truncated or rounded infected animals, wool, meat, or hides. Occurrence of ends. Based on history of sudden death, oozing of anthrax in humans is correlated with industrial unclotted blood at natural orifices and blood smear activities such as handling potentially contaminated examination it was diagnosed as anthrax outbreak and materials like hides, fur, wool, hair, meat, or bone meal control measures were undertaken immediately. Ailing (Ammann and Brandl, 2007). The spores are very animals were isolated in a separate shed and treated resistant to unfavorable environmental extremes of for the disease as follows. They were administered with heat, cold, desiccation, chemicals and irradiation. The intramuscular injections of Streptopenicillin@1 LD incidence of anthrax varies with the type of the soil and twice daily, 10 ml of Melonex® once in a day, Chloril® climate. It is many times restricted to a particular area 10 ml once in a day and Tribivet® 10 ml once in a day for where it is endemic and such areas are known as 7 days. All the 7 animals clinically recovered after 7 “Anthrax belts”. Cattle and sheep are very susceptible days. Blood smear examination of these animals after to anthrax and dogs and cats are quite resistant. There 7 days of treatment revealed no Bacillus anthracis are only few reports of anthrax outbreak in domestic organisms. The carcass of the dead animals, feed, animals in India. Venkatesha, et al., (2006) reported 2 Manure, bedding materials of dead and ailing animals anthrax outbreaks in Hassan and Kolar districts of were burnt. The healthy animals were vaccinated with Karnataka State. anthrax spore vaccine (IAH & VB, Bangalore), which Hisotry and Clinical Examination contains a suspension of live spores of Sterne’s strain There was a history of sudden death of seven of Bacillus anthracis in 50% glycerine saline. The cattle cows and two bullocks in Jaligere village of were injected with 1 ml of anthrax vaccine and sheep Doddaballapur taluk, Bangalore rural district during the were given with a dose of 0.2 ml subcutaneously. The month of June, 2008. Upon external examination of the left over vaccines were disposed as per the standard carcass, it was found that there was unclotted blood procedures. From then, till date no further anthrax near the nostril, at anal orifice and on the floor, where outbreaks were noticed in this region. the animals were lying dead. There were around seven Discussion (7) ailing animals showing the clinical signs like high
Journal Article•
TL;DR: In this article, the authors determined the wavelength and linear range for spectrophotometry of anthrax vaccine, based on which the stability of test samples and repro-ducibility of the method were analyzed.
Abstract: Objective To determine the concentration of anthrax vaccine by spectrophotometry. Methods The wavelength and linear range for spectrophotometry of anthrax vaccine were determined, based on which the stability of test samples and repro - ducibility of the method were analyzed. Results The wavelength for spectrophotometry of anthrax vaccine was determined as 600 nm. The A600 value showed a good relationship(r = 0. 999 1, df = 3, P 0. 01)to anthrax vaccine concentration at a range of(0. 25 ～ 1. 25)× 108 bacteria / ml. The stability of test samples increased after treatment with formaldehyde. The method showed high reproducibility. Conclusion Spectrophotometry may be used for determination of anthrax vaccine concentration instead of turbidimetry or as a method for confirmation.
01 Aug 2010
TL;DR: Results showed that C48/80 is an effective mucosal adjuvant in rabbits and SFD powder formulations freshly prepared or stored for over two years at room temperature were able to elicit a significant serum anti-body and functional titer comparable to IM injection.
Abstract: Sheena Hailin Wang: Novel Delivery Systems for Nasal Administration of Anthrax Vaccine (Under the direction of Dr. Anthony J. Hickey, Ph.D., D.Sc.) There is current biodefense interest in protection against Anthrax. The inhaled form of anthrax is difficult to diagnose and can be fatal without prompt antibiotic intervention. Prophylactic vaccination has become a critical measure for protection of individuals at risk of exposure. This thesis makes the efforts to develop a new generation of stable, effective and affordable anthrax vaccine. This project studies the immune response elicited by rPA with a mast cell activator, Compound 48/80 as adjuvant. The vaccine formulation was prepared in a dry powder form by spray-freeze-drying (SFD) under optimized conditions to produce particles with a target size of 25μm median diameter, suitable for rabbit nasal delivery. Physicochemical properties of the powder vaccines were characterized in order to assess the powder delivery and storage potentials. Structural stability of PA after the SFD process and storage were confirmed by two spectroscopic techniques (CD and ATR-FTIR), while functional stability of both the antigen and adjuvant were monitored via cell-based assays. Animal studies were performed to evaluate the in vivo efficacy of the powder vaccines in rabbits using a Unitdose powder device. Results showed that C48/80 is an effective mucosal adjuvant in rabbits. SFD powder formulations freshly prepared or stored for over two years at room temperature were able to elicit a significant serum anti-body and functional titer comparable to IM injection.
TL;DR: After purification and immunization studies, the purified r PA may be used in the development of the human recombinant anthrax vaccine and also in diagnosis of anthrax disease.
Dissertation•
19 Mar 2010
14 May 2010
TL;DR: This document provides a summary of Frequently Asked Questions (FAQs) on the treatment of anthrax disease caused by a wide-area release of Bacillus anthracis spores as an act bioterrorism.
Abstract: This document provides a summary of Frequently Asked Questions (FAQs) on the treatment of anthrax disease caused by a wide-area release of Bacillus anthracis spores as an act bioterrorism These FAQs are intended to provide the public health and medical community, as well as others, with guidance and communications to support the response and long-term recovery from an anthrax event