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.

Development of a novel adjuvanted nasal vaccine: C48/80 associated with chitosan nanoparticles as a path to enhance mucosal immunity.

In a time in which mucosal vaccines development has been delayed by the lack of safe and effective mucosal adjuvants, the combination of adjuvants has started to be explored as a strategy to obtain potent vaccine formulations. This study describes a novel adjuvant combination as an effective approach for a nasal vaccine - the association of the mast cell activator compound 48/80 with chitosan based nanoparticles. It was hypothesized that mucoadhesive nanoparticles would promote the cellular uptake and prolong the antigen residence time on nasal cavity. Simultaneously, mast cell activation would promote a local microenvironment favorable to the development of an immune response. To test this hypothesis, two different C48/80 loaded nanoparticles (NPs) were prepared: Chitosan-C48/80 NP (Chi-C48/80 NP) and Chitosan/Alginate-C48/80 NP (Chi/Alg-C48/80 NP). The potential as a vaccine adjuvant of the two delivery systems was evaluated and directly compared. Both formulations had a mean size near 500nm and a positive charge; however, Chi-C48/80 NP was a more effective adjuvant delivery system when compared with Chi/Alg-C48/80 NP or C48/80 alone. Chi-C48/80 NP activated mast cells at a greater extent, were better internalized by antigen presenting cells than Chi/Alg-C48/80 NP and successfully enhanced the nasal residence time of a model antigen. Superiority of Chi-C48/80 NP as adjuvant was also observed in vivo. Therefore, nasal immunization of mice with Bacillus anthracis protective antigen (PA) adsorbed on Chi-C48/80 NP elicited high levels of serum anti-PA neutralizing antibodies and a more balanced Th1/Th2 profile than C48/80 in solution or Chi/Alg-C48/80 NP. The incorporation of C48/80 within Chi NP also promoted a mucosal immunity greater than all the other adjuvanted groups tested, showing that the combination of a mast cell activator and chitosan NP could be a promising strategy for nasal immunization.

Evaluation of mucoadhesive carrier adjuvant: toward an oral anthrax vaccine.

The aim of present study was to evaluate the potential of mucoadhesive alginate-coated chitosan microparticles (A-CHMp) for oral vaccine against anthrax. The zeta potential of A-CHMp was -29.7 mV, and alginate coating could prevent the burst release of antigen in simulated gastric fluid. The results indicated that A-CHMp was mucoadhesive in nature and transported it to the peyer's patch upon oral delivery. The immunization studies indicated that A-CHMp resulted in the induction of potent systemic and mucosal immune responses, whereas alum-adjuvanted rPA could induce only systemic immune response. Thus, A-CHMp represents a promising acid carrier adjuvant for oral immunization against anthrax.

Increasing the potency of an alhydrogel-formulated anthrax vaccine by minimizing antigen-adjuvant interactions.

Aluminum salts are the most widely used vaccine adjuvants, and phosphate is known to modulate antigen-adjuvant interactions. Here we report an unexpected role for phosphate buffer in an anthrax vaccine (SparVax) containing recombinant protective antigen (rPA) and aluminum oxyhydroxide (AlOH) adjuvant (Alhydrogel). Phosphate ions bind to AlOH to produce an aluminum phosphate surface with a reduced rPA adsorption coefficient and binding capacity. However, these effects continued to increase as the free phosphate concentration increased, and the binding of rPA changed from endothermic to exothermic. Crucially, phosphate restored the thermostability of bound rPA so that it resembled the soluble form, even though it remained tightly bound to the surface. Batches of vaccine with either 0.25 mM (subsaturated) or 4 mM (saturated) phosphate were tested in a disease model at batch release, which showed that the latter was significantly more potent. Both formulations retained their potency for 3 years. The strongest aluminum adjuvant effects are thus likely to be via weakly attached or easily released native-state antigen proteins.

Aluminum hydroxide injections lead to motor deficits and motor neuron degeneration.

Gulf War Syndrome is a multi-system disorder afflicting many veterans of Western armies in the 1990-1991 Gulf War. A number of those afflicted may show neurological deficits including various cognitive dysfunctions and motor neuron disease, the latter expression virtually indistinguishable from classical amyotrophic lateral sclerosis (ALS) except for the age of onset. This ALS "cluster" represents the second such ALS cluster described in the literature to date. Possible causes of GWS include several of the adjuvants in the anthrax vaccine and others. The most likely culprit appears to be aluminum hydroxide. In an initial series of experiments, we examined the potential toxicity of aluminum hydroxide in male, outbred CD-1 mice injected subcutaneously in two equivalent-to-human doses. After sacrifice, spinal cord and motor cortex samples were examined by immunohistochemistry. Aluminum-treated mice showed significantly increased apoptosis of motor neurons and increases in reactive astrocytes and microglial proliferation within the spinal cord and cortex. Morin stain detected the presence of aluminum in the cytoplasm of motor neurons with some neurons also testing positive for the presence of hyper-phosphorylated tau protein, a pathological hallmark of various neurological diseases, including Alzheimer's disease and frontotemporal dementia. A second series of experiments was conducted on mice injected with six doses of aluminum hydroxide. Behavioural analyses in these mice revealed significant impairments in a number of motor functions as well as diminished spatial memory capacity. The demonstrated neurotoxicity of aluminum hydroxide and its relative ubiquity as an adjuvant suggest that greater scrutiny by the scientific community is warranted.

Evaluation of a plasmid DNA-based anthrax vaccine in rabbits, nonhuman primates and healthy adults.

VCL-AB01, a cationic lipid-formulated plasmid DNA (pDNA)-based vaccine that contains genes encoding genetically detoxified Bacillus anthracis protective antigen (PA) and lethal factor (LF), was assessed in a Phase 1, dose-escalating clinical trial in healthy adults for safety and immunogenicity, and in nonhuman primates for immunogenicity and efficacy against challenge with a lethal dose of B. anthracis spores. Healthy 18-45 year old subjects were randomly assigned to receive either the investigational vaccine containing 0.2 mg, 0.6 mg, or 2 mg of total pDNA per dose, or saline placebo, administered at 0, 1 and 2 months. The 0.2 mg and 0.6 mg dose levels were generally well tolerated; however, dose-limiting reactogenicity was observed among subjects given the first 2 mg dose and the remaining two injections in the 2 mg group were reduced to 0.6 mg. Dose-related increases in seroconversion frequencies were observed. Overall, 10%, 33.3% and 80% of subjects in the 0.2, 0.6 and 2 mg groups, respectively, developed antibodies to PA and/or LF as measured by ELISA; however, antibodies with toxin neutralizing activity (TNA) were detected in only one subject. In monkeys that received a 0.6 mg dose three times at 2 week intervals, low levels of antibodies were detected by ELISA but not by the TNA assay in all animals just prior to challenge. Despite the absence of TNA, 75% animals survived the lethal challenge. In summary, VCL-AB01 was generally well tolerated in humans at a dose that provided immunity in monkeys despite the lack of robust TNA titers in either species.

Protective antigen composite nanofibers as a transdermal anthrax vaccine.

Anthrax, a disease caused by the gram positive bacteria Bacillus anthracis, has become an increasing threat to public health in the last several years, due to its use as an agent of biological warfare. The currently utilized human anthrax vaccine, which confers immunity through the host antibody recognition of protective antigen (PA), requires a three dose regimen and annual booster shots after the initial vaccination to maintain its efficacy. The long term goal of this project is to produce an anthrax vaccine that is capable of delivering protective antigen through human skin. The novel method for transdermal vaccine delivery that we propose utilizes the high surface area to volume ratio offered by protein-containing nanofiber membranes, prepared by the electrospinning technique. Research has already been undertaken to study the effect the main virulent agent of anthrax, lethal toxin (LT), has on a human monocytic cell line, Monomac 6 cells (MM6). Lethal toxin is said to comprise of a Zn2+ -dependent metalloprotease known as lethal factor (LF), and a binding protein known as protective antigen. The successful encapsulation of the protective antigen within the nanofibrous membrane was analyzed with the use of an in vitro MM6 assay. The assay was designed to ensure the functionality of PA through the harsh environment of the electrospinning process. Quantitative analysis of IL-6 cytokine production by lipopolysaccharide (LPS) stimulated MM6 cells in the presence of LF and PA provided proof that PA retained its biological activity through the process of electrospinning. This finding provides an innovative platform for the development of a transdermal anthrax vaccine.

Evaluation of the immune response induced by a nasal anthrax vaccine based on the protective antigen protein in anaesthetized and non-anaesthetized mice.

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.

II. Cationic lipid-formulated plasmid DNA-based Bacillus anthracis vaccine: evaluation of plasmid DNA persistence and integration potential.

Several formulated plasmid DNA (pDNA)-based vaccines are being evaluated for safety and efficacy in healthy human subjects. A safety concern for any vaccine that contains genetic material, be it whole organism, live-attenuated, or gene-based, is the potential for integration into genomic DNA (gDNA). To address this concern, a preclinical pDNA persistence/integration study was conducted in rabbits to determine the level of pDNA in muscle 2, 28, and 64 days after intramuscular injection of DMRIE:DOPE-formulated pDNAs encoding Bacillus anthracis detoxified LF and PA proteins (VCL-AB01 vaccine). Total DNA was extracted from day 64 muscle tissue and fractionated by column agarose gel electrophoresis (CAGE). Plasmid copy number (PCN) in muscle 64 days after injection (geometric mean, 2808 PCN/microg of total DNA or 150,000 diploid genomes) was determined by quantitative polymerase chain reaction. Analysis of total DNA from five VCLAB01- injected rabbits revealed that two of five samples had no detectable PCN in the high molecular weight fraction after one round of CAGE, two samples had PCN under the lower limit of quantitation, and the remaining sample had 123 PCN/microg. All PCN in the latter sample cleared after an additional round of CAGE. It appears, therefore, that persisting PCN fractionate as low molecular weight material and are most likely not integrated into gDNA. Even if the worst-case assumption is made that the highest PCN found associated with gDNA represented covalently integrated pDNA inserts, the frequency of mutation would still be 500-fold lower than the autosomal spontaneous mutation rate.

An overview of adverse events reported by participants in CDC's anthrax vaccine and antimicrobial availability program.

PURPOSE: The CDC's Anthrax Vaccine and Antibiotic Availability Program was implemented under an Investigational New Drug (IND) application to provide additional post-exposure prophylaxis for individuals potentially exposed to Bacillus anthracis in the fall of 2001. Participants were provided with two options: (1) 40 additional days of antimicrobial prophylaxis (i.e., ciprofloxacin, doxycycline, or amoxicillin); or (2) 40 additional days of antimicrobial prophylaxis plus three doses of anthrax vaccine adsorbed (AVA). METHODS: Participants were monitored for adverse events (AEs). Participants were asked to complete 2-week AE diaries for 6 weeks post-enrollment, and approximately 2 months after enrollment, active surveillance was conducted through telephone interviews with 1113 (64%) participants. RESULTS: A total of 1727 of approximately 10 000 previously prophylaxed persons enrolled to receive 40 additional days of antibiotics. Of these, 199 opted at enrollment to receive three doses of AVA in addition to the additional 40 days of antibiotic. Overall, 28% of participants reported at least one AE on their diaries. Results varied by surveillance mechanism, the diary data indicated differences in the proportion reporting AEs between participants receiving antibiotic only and participants receiving antibiotic and AVA. However, during the active 2-month telephone follow-up, the rates of AEs reported for both the antibiotic only and antibiotic plus AVA treatment regimens were similar. Additionally, ciprofloxacin and doxycycline had similar AE profiles, with only rigors reported significantly more often among ciprofloxacin recipients. CONCLUSIONS: Overall, the rates of AEs experienced by all participants were acceptable given the seriousness of potential B. anthracis exposure.