The national blueprint for biodefense concluded that the United States is underprepared for biological threats. The licensed anthrax vaccine absorbed vaccine, BioThrax, requires administration of at least 3-5 intramuscular doses. The anthrax vaccine absorbed vaccine consists of complex cell-free culture filtrates of a toxigenic Bacillus anthracis strain and causes tenderness at the injection site and significant adverse events. We integrated a codon-optimized, protective antigen gene of B. anthracis (plus extracellular secretion machinery), into the chromosome of the licensed, oral, live-attenuated typhoid fever vaccineTy21a to form Ty21a-PA-01 and demonstrated excellent expression of the gene encoding protective antigen. We produced the vaccine in a 10-L fermenter; foam-dried and vialed it, and characterized the dried product. The vaccine retained ~50% viability for 20 months at ambient temperature. Sera from animals immunized by the intraperitoneal route had high levels of anti-protective antigen antibodies by enzyme-linked immunosorbent assay and anthrax lethal toxin-neutralizing activity. Immunized mice were fully protected against intranasal challenge with ~5 LD50 of B. anthracis Sterne spores, and 70% (7/10) of vaccinated rabbits were protected against aerosol challenge with 200 LD50 of B. anthracis Ames spores. There was a significant correlation between protection and antibody levels determined by enzyme-linked immunosorbent assay and toxin-neutralizing activity. These data provide the foundation for achievement of our ultimate goal, which is to develop an oral anthrax vaccine that is stable at ambient temperatures and induces the rapid onset of durable, high-level protection after a 1-week immunization regimen.

National Institute of Allergy and Infectious Diseases has classified all the emerging infectious diseases agents under three categories. Among Category A priority pathogens comes Bacillus anthracis -the causative agent of Anthrax. It is a gram positive spore bearing bacteria, and the disease is typically associated with grazing animals, and affects the people as a zoonosis. The disease can be classically transmitted by three routes namely: cutaneous, gastrointestinal and pulmonary, with a fourth route recently identified as "injection anthrax", seen in intravenous drug abusers. Cutaneous anthrax is the commonest form in humans, accounting for 95% of all the cases. There are two main virulence factors of this bacteria, a capsule and an exotoxin, each carried by a separate toxin. Two models have been used for explaining the pathogenesis of this infection. The earlier one or "Trojan horse" model is now replaced with "jail-break" model. Centers for disease control (CDC) has issued updated guidelines for diagnosis, post-exposure prophylaxis and treatment. For immunization, anthrax vaccine absorbed is available.

Second generation anthrax vaccines focus on the use of recombinant protective antigen (rPA) to elicit a strong, toxin neutralizing antibody responses in immunized subjects. The main difference between the rPA vaccines compared to the current licensed vaccine, anthrax vaccine absorbed (AVA), is the rPA vaccines are highly purified preparations of only rPA. These second generation rPA vaccines strive to elicit strong immune responses with substantially fewer doses than AVA while provoking less side effects. Many of the rPA candidates have shown to be effective in pre-clinical studies, but most of the second generation molecules have stability issues which reduce their efficacy over time. These stability issues are evident even under refrigerated conditions and thus emphasis has been directed to stabilizing the rPA molecule and determining an optimized final formulation. Stabilization of vaccines for long-term storage is a major challenge in the product development life cycle. The effort required to identify suitable formulations can be slow and expensive. The ideal storage for stockpiled vaccines would allow the candidate to withstand years of storage at ambient temperatures. The Fraunhofer Center for Molecular Biotechnology is developing a plant-produced rPA vaccine candidate that shows instability when stored under refrigerated conditions in a solution, as is typical for rPA vaccines. Increased stability of our plant-produced rPA vaccine candidate was achieved in a spray dried powder formulation that could eliminate the need for conventional cold chain allowing greater confidence to stockpile vaccine for civilian and military biodefense.

Bacillus anthracis is a sporulating Gram-positive bacterium that is the causative agent of anthrax and a potential weapon of bioterrorism. The U.S.-licensed anthrax vaccine is made from an incompletely characterized culture supernatant of a nonencapsulated, toxigenic strain (anthrax vaccine absorbed [AVA]) whose primary protective component is thought to be protective antigen (PA). AVA is effective in protecting animals and elicits toxin-neutralizing antibodies in humans, but enthusiasm is dampened by its undefined composition, multishot regimen, recommended boosters, and potential for adverse reactions. Improving next-generation anthrax vaccines is important to safeguard citizens and the military. Here, we report that vaccination with recombinant forms of a conserved domain (near-iron transporter [NEAT]), common in Gram-positive pathogens, elicits protection in a murine model of B. anthracis infection. Protection was observed with both Freund's and alum adjuvants, given subcutaneously and intramuscularly, respectively, with a mixed composite of NEATs. Protection correlated with an antibody response against the NEAT domains and a decrease in the numbers of bacteria in major organs. Anti-NEAT antibodies promote opsonophagocytosis of bacilli by alveolar macrophages. To guide the development of inactive and safe NEAT antigens, we also report the crystal structure of one of the NEAT domains (Hal) and identify critical residues mediating its heme-binding and acquisition activity. These results indicate that we should consider NEAT proteins in the development of an improved antianthrax vaccine.

Anthrax vaccine adsorbed (AVA, BioThrax) was recently approved by the Food and Drug Administration (FDA) for a post-exposure prophylaxis (PEP) indication in adults 18-65years of age. The schedule is three doses administered subcutaneous (SC) at 2-week intervals (0, 2, and 4weeks), in conjunction with a 60-day course of antimicrobials. The Public Health Emergency Medical Countermeasures Enterprise (PHEMCE) developed an animal model to support assessment of a shortened antimicrobial PEP duration following Bacillus anthracis exposure. A nonhuman primate (NHP) study was completed to evaluate the efficacy of a two dose anthrax vaccine absorbed (AVA) schedule (0, 2weeks) aerosol challenged with high levels of B. anthracis spores at week4- the time point at which humans would receive the third vaccination of the approved PEP schedule. Here we use logistic regression models to combine the survival data from the NHP study along with serum anthrax lethal toxin neutralizing activity (TNA) and anti-PA IgG measured by enzyme linked immunosorbent assay (ELISA) data to perform a cross-species analysis to estimate survival probabilities in vaccinated human populations at this time interval (week4 of the PEP schedule). The bridging analysis demonstrated that high levels of NHP protection also yield high predicted probability of human survival just 2weeks after the second dose of vaccine with the full or half antigen dose regimen. The absolute difference in probability of human survival between the full and half antigen dose was estimated to be at most approximately 20%, indicating that more investigation of the half-antigen dose for vaccine dose sparing strategies may be warranted.

AdVAV is a replication-deficient adenovirus type 5-vectored vaccine expressing the 83-kDa protective antigen (PA83) from Bacillus anthracis that is being developed for the prevention of disease caused by inhalation of aerosolized B. anthracis spores. A noninferiority study comparing the efficacy of AdVAV to the currently licensed Anthrax Vaccine Absorbed (AVA; BioThrax) was performed in New Zealand White rabbits using postchallenge survival as the study endpoint (20% noninferiority margin for survival). Three groups of 32 rabbits were vaccinated with a single intranasal dose of AdVAV (7.5 × 10(7), 1.5 × 10(9), or 3.5 × 10(10) viral particles). Three additional groups of 32 animals received two doses of either intranasal AdVAV (3.5 × 10(10) viral particles) or intramuscular AVA (diluted 1:16 or 1:64) 28 days apart. The placebo group of 16 rabbits received a single intranasal dose of AdVAV formulation buffer. All animals were challenged via the inhalation route with a targeted dose of 200 times the 50% lethal dose (LD50) of aerosolized B. anthracis Ames spores 70 days after the initial vaccination and were followed for 3 weeks. PA83 immunogenicity was evaluated by validated toxin neutralizing antibody and serum anti-PA83 IgG enzyme-linked immunosorbent assays (ELISAs). All animals in the placebo cohort died from the challenge. Three of the four AdVAV dose cohorts tested, including two single-dose cohorts, achieved statistical noninferiority relative to the AVA comparator group, with survival rates between 97% and 100%. Vaccination with AdVAV also produced antibody titers with earlier onset and greater persistence than vaccination with AVA.

Bacillus anthracis, the causative agent of anthrax, produces a tripartite toxin composed of two enzymatically active subunits, lethal factor (LF) and edema factor (EF), which, when associated with a cell-binding component, protective antigen (PA), form lethal toxin and edema toxin, respectively. In this preliminary study, we characterized the toxin-specific antibody responses observed in 17 individuals infected with cutaneous anthrax. The majority of the toxin-specific antibody responses observed following infection were directed against LF, with immunoglobulin G (IgG) detected as early as 4 days after the onset of symptoms in contrast to the later and lower EF- and PA-specific IgG responses. Unlike the case with infection, the predominant toxin-specific antibody response of those immunized with the US anthrax vaccine absorbed and UK anthrax vaccine precipitated licensed anthrax vaccines was directed against PA. We observed that the LF-specific human antibodies were, like anti-PA antibodies, able to neutralize toxin activity, suggesting the possibility that they may contribute to protection. We conclude that an antibody response to LF might be a more sensitive diagnostic marker of anthrax than to PA. The ability of human LF-specific antibodies to neutralize toxin activity supports the possible inclusion of LF in future anthrax vaccines.

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.

Anthrax has been a major cause of death in grazing animals and an occasional cause of death in humans for thousands of years. Since the late 1800s there has been an exceptional international history of anthrax vaccine development. Due to animal vaccinations, the rate of infection has dropped dramatically. Anthrax vaccines have progressed from uncharacterized whole-cell vaccines in 1881, to pXO2-negative spores in the 1930s, to culture filtrates absorbed to aluminum hydroxide in 1970, and likely to recombinant protective antigen in the near future. Each of these refinements has increased safety without significant loss of efficacy. The threat of genetically engineered, antibiotic and vaccine resistant strains of Bacillus anthracis is fueling hypothesis-driven research and global techniques--including genomics, proteomics and transposon site hybridization--to facilitate the discovery of novel vaccine targets. This review highlights historical achievements and new developments in anthrax vaccine research.

The transcriptional responses in recombinant protective antigen (PA)-stimulated peripheral blood mononuclear cells (PBMCs) from Anthrax Vaccine Absorbed (AVA)-vaccinated rhesus macaques were evaluated using Affymetrix HGU133 Plus 2.0 GeneChips. PBMCs from animals vaccinated at 0, 4, and 26 weeks were harvested at week 30, stimulated with PA, and RNA isolated. The expression of 295 unigenes was significantly increased in PA-stimulated compared to non-stimulated PBMCs; no significant decrease in gene expression was observed. These upregulated transcripts encoded for proteins functioning in both innate and adaptive immunity. Results were corroborated for several genes by real-time RT-PCR. This study provides information on the potential underlying transcriptional mechanisms in the immune response to PA in AVA-vaccinated rhesus macaques.

A validation of the performance characteristics of a toxin neutralization assay is presented. This in vitro assay measures the functional ability of antisera, containing antibodies to anthrax lethal toxin, to specifically protect J774A.1 cells against Bacillus anthracis lethal toxin cytotoxicity. This colormetric assay is based upon the reduction of MTT by living cells. Human and rabbit antisera produced against anthrax vaccine absorbed (AVA) were used to validate the assay. Results showed a high level of repeatability and reproducibility, particularly for a bio-assay. Inter-assay variability in absorbance values was the most prominent negative finding however, an acceptable level was demonstrated with a ratio [neutralization ratio (NR)] of the test serum 50% effective dose (ED(50)) to the reference standard ED(50). Accuracy was maintained, even in samples with minimal neutralizing capacity, and linearity was noted when sample dilutions resulted in accurate prediction of the Y(max)and Y(min). Specificity tests demonstrated that normal sera did not have an observable effect on the ability of the reference standard to neutralize toxin. The assay remained stable against time, temperature, and freeze/thaw effects on the reference standards, but not on the toxin. The assay also remained stable against media and solution storage effects. Cell passage number and cell plating density were two critical parameters identified during the robustness studies that may be responsible for inter-assay variability in absorbance values. The work was performed in accordance with the FDA's Bioanalytical Method Validation Guidance for Industry and the FDA's Good Laboratory Practice for Nonclinical Laboratory Studies (21 CFR Part 58).

Vaccines which are efficacious against anthrax, such as the human vaccine, Anthrax Vaccine Absorbed (AVA), contain the protective antigen (PA) component of the anthrax toxins as the major protective immunogen. Although AVA protects against inhalational anthrax, the immune responses to and role in protection of PA and possibly other antigens have yet to be fully elucidated. Sera from animals immunized with a toxin-producing, unencapsulated live vaccine strain of Bacillus anthracis have been reported to have anti-spore activities associated with the antitoxin humoral response. The authors performed studies to determine whether anti-PA antibody (Ab)-containing preparations stimulated spore uptake by phagocytes and suppressed the germination of spores in vitro. AVA- and PA-immune sera from several species enhanced the phagocytosis by murine peritoneal macrophages of spores of the virulent Ames and the Sterne vaccine strains. Antitoxin Abs appeared to contribute significantly, although not solely, to the enhanced uptake. Rabbit antisera to PA purified from either Sterne or a PA-producing pX01-cured recombinant, affinity-purified anti-PA IgG, and monkey antisera to AVA were used to assess the role of anti-PA ABS: Rabbit anti-PA Abs promoted the uptake of spores of the PA-producing strains Sterne, Ames and RP42, a mutant of Sterne producing only PA, but not of the pX01-Sterne-1 strain, Ames strain, or RP4, a mutant of Sterne with deletions in the loci encoding PA and the oedema factor (EF) toxin component and producing only the lethal factor toxin component. Rabbit anti-PA and monkey anti-AVA Abs also significantly inhibited spore germination in vitro compared to preimmune serum or medium. Spore-associated proteins recognized by anti-PA Abs were detected by electron microscopy and confirmed by immunoblotting of spore coat extracts. Thus, the anti-PA Ab-specific immunity induced by AVA has anti-spore activity and might have a role in impeding the early stages of infection with B. anthracis spores.