Patricia Fellows

In vitro correlate of immunity in a rabbit model of inhalational anthrax

A serological correlate of vaccine-induced immunity was identified in the rabbit model of inhalational anthrax. Animals were inoculated intramuscularly at 0 and 4 weeks with varying doses of Anthrax Vaccine Adsorbed (AVA) ranging from a human dose to a 1:256 dilution in phosphate-buffered saline (PBS). At 6 and 10 weeks, both the quantitative anti-protective antigen (PA) IgG ELISA and the toxin-neutralizing antibody (TNA) assays were used to measure antibody levels to PA. Rabbits were aerosol-challenged at 10 weeks with a lethal dose (84-133 LD(50)) of Bacillus anthracis spores. All the rabbits that received the undiluted and 1:4 dilution of vaccine survived, whereas those receiving the higher dilutions of vaccine (1:16, 1:64 and 1:256) had deaths in their groups. Results showed that antibody levels to PA at both 6 and 10 weeks were significant (P<0.0001) predictors of survival.

Comparative efficacy of experimental anthrax vaccine candidates against inhalation anthrax in rhesus macaques

The authors examined the efficacy of Bacillus anthracis protective antigen (PA) combined with adjuvants as vaccines against an aerosol challenge of virulent anthrax spores in rhesus macaques. Adjuvants tested included i) aluminum hydroxide (Alhydrogel), ii) saponin QS-21 and iii) monophosphoryl lipid A (MPL) in squalene/lecithin/Tween 80 emulsion (SLT). Animals were immunized once with either 50 micrograms of recombinant PA plus adjuvant, or with Anthrax Vaccine Adsorbed (AVA), the licensed human anthrax vaccine. The serological response to PA was measured by enzyme linked immunosorbent assay. Lymphocyte proliferation and serum neutralization of in vitro lethal toxin cytotoxicity were also assayed. In all vaccine groups, anti-PA IgM and IgG titers peaked at 2 weeks and 4-5 weeks postimmunization, respectively. Five weeks postimmunization, animals in all vaccine groups demonstrated PA-specific lymphocyte proliferation and sera that neutralized in vitro cytotoxicity. Six weeks after immunization, the animals were challenged by aerosol with approximately 93 LD50 of virulent anthrax spores. Animals were bled daily for 1 week to monitor bacteremia, and deaths were recorded. Anti-PA ELISA titers in all groups of immunized animals were substantially increased 2 weeks after challenge. One dose of each vaccine provided significant protection (> 90%) against inhalation anthrax in the rhesus macaques.

The role of antibodies to Bacillus anthracis and anthrax toxin components in inhibiting the early stages of infection by anthrax spores.

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.

Efficacy of a human anthrax vaccine in guinea pigs, rabbits, and rhesus macaques against challenge by Bacillus anthracis isolates of diverse geographical origin

The efficacy of a licensed human anthrax vaccine (Anthrax Vaccine Adsorbed (AVA)) was tested in guinea pigs, rabbits, and rhesus macaques against spore challenge by Bacillus anthracis isolates of diverse geographical origin. Initially, groups of Hartley guinea pigs were vaccinated at 0 and 4 weeks with AVA, then challenged intramuscularly at 10 weeks with spores from 33 isolates of B. anthracis. Survival among the vaccinated groups varied from 6 to 100%, although there were no differences in mean time to death among the groups. There was no correlation between isolate virulence and variable number tandem repeat category or protective antigen genotype identified. New Zealand white rabbits were then vaccinated with AVA at 0 and 4 weeks, and challenged at 10 weeks by aerosol with spores from six of the isolates that were highly virulent in vaccinated guinea pigs. AVA completely protected the rabbits from four of the isolates, and protected 90% of the animals from the other two isolates. Subsequently, two of these six isolates were then used to challenge rhesus macaques, previously vaccinated with AVA at 0 and 4 weeks, and challenged at 10 weeks by aerosol. AVA protected 80 and 100% of the animals from these two isolates. These studies demonstrated that, although AVA confers variable protection against different B. anthracis isolates in guinea pigs, it is highly protective against these same isolates in both rabbits and rhesus macaques.

Experimental anthrax vaccines: efficacy of adjuvants combined with protective antigen against an aerosol Bacillus anthracis spore challenge in guinea pigs

The efficacy of several human anthrax vaccine candidates comprised of different adjuvants together with Bacillus anthracis protective antigen (PA) was evaluated in guinea pigs challenged by an aerosol of virulent B. anthracis spores. The most efficacious vaccines tested were formulated with PA plus monophosphoryl lipid A (MPL) in a squalene/lecithin/Tween 80 emulsion (SLT) and PA plus the saponin QS-21. The PA+MPL in SLT vaccine, which was lyophilized and then reconstituted before use, demonstrated strong protective immunogenicity, even after storage for 2 years at 4 degrees C. The MPL component was required for maximum efficacy of the vaccine. Eliminating lyophilization of the vaccine did not diminish its protective efficacy. No significant alteration in efficacy was observed when PA was dialyzed against different buffers before preparation of vaccine. PA+MPL in SLT proved superior in efficacy to the licensed United States human anthrax vaccine in the guinea pig model.

In vitro correlate of immunity in an animal model of inhalational anthrax

The incidence of anthrax in humans is extremely low. Human vaccine efficacy studies for inhalational anthrax cannot be conducted. The identification of a correlate of protection that predicts vaccine efficacy is crucial for determining the immune status of immunized humans. This surrogate marker of immunity can only be established by using an appropriate animal model. Numerous studies showed that protective antigen (PA) is the principle protective antigen in naturally‐ or vaccine‐induced immunity. However, attempts to correlate the quantity of anti‐PA antibodies with protective immunity in the guinea pig model for anthrax and various vaccine formulations have failed. In these studies, we used the licensed anthrax vaccine adsorbed (AVA) in rabbits.

Efficacy of a standard human anthrax vaccine against Bacillus anthracis spore challenge in guinea-pigs

The efficacy of an anthrax vaccine licensed for human use, MDPH-PA, was tested in guinea-pigs intramuscularly challenged with 10, 100 or 1000 LD50 of spores from two virulent strains of Bacillus anthracis, Vollum 1B and Ames. As demonstrated in other investigations, immunization with MDPH-PA provided better protection against challenge with the Vollum 1B strain than with the Ames strain, although vaccine efficacy against the Ames strain was better than previously reported. Enzyme-linked immunosorbent assay of serum antibody titres to B. anthracis protective antigen showed that there was no significant correlation between survival and antibody titres.

Protection in mice passively immunized with serum from cynomolgus macaques and humans vaccinated with recombinant plague vaccine (rF1V)

Passive transfer models were developed to evaluate the ability of antibodies generated in cynomolgus macaques and humans vaccinated with a recombinant plague vaccine (rF1V) to protect naïve Swiss Webster mice against pneumonic plague. Development of the passive transfer model is intended to support clinical and nonclinical development of the rF1V vaccine. To evaluate protection, unfractionated serum collected from rF1V vaccinated cynomolgus macaques and human volunteers with known antibody titers to rF1, rV and rF1V was transferred into naïve Swiss Webster mice via the intraperitoneal route. Results of these studies demonstrated that passive immunization protected mice from challenge or extended mean survival time and that the passive transfer assay can be used to evaluate the functional role of antibodies induced by rF1V vaccination in protection against aerosol exposure.

Anthrax vaccine efficacy in golden Syrian hamsters

The efficacy of a licensed human anthrax vaccine (anthrax vaccine adsorbed, AVA) was tested in golden Syrian hamsters against a virulent Bacillus anthracis spore challenge. Groups of golden Syrian hamsters were vaccinated at either 0 and 4 weeks or 0, 4 and 8 weeks, then challenged subcutaneously (s.c.) at 10 weeks with spores of various B. anthracis isolates. Although ELISA and toxin neutralization assays demonstrated high titers, none of the AVA-vaccinated hamsters were protected from challenge or demonstrated a significantly extended time to death compared to that of control animals. The results of the study demonstrate that the golden Syrian hamster is not an appropriate model for investigating human anthrax vaccine efficacy.

Establishment of a Swiss Webster Mouse Model of Pneumonic Plague To Meet Essential Data Elements under the Animal Rule

ABSTRACT A recombinant vaccine (rF1V) is being developed for protection against pneumonic plague. This study was performed to address essential data elements to establish a well-characterized Swiss Webster mouse model for licensing the rF1V vaccine using the FDAs Animal Rule. These elements include the documentation of challenge material characteristics, aerosol exposure parameters, details of the onset and severity of clinical signs, pathophysiological response to disease, and relevance to human disease. Prior to animal exposures, an evaluation of the aerosol system was performed to determine and understand the variability of the aerosol exposure system. Standardized procedures for the preparation of Yersinia pestis challenge material also were developed. The 50% lethal dose (LD50) was estimated to be 1,966 CFU using Probit analysis. Following the LD50 determination, pathology was evaluated by exposing mice to a target LD99 (42,890 CFU). Mice were euthanized at 12, 24, 36, 48, 60, and 72 h postexposure. At each time point, samples were collected for clinical pathology, detection of bacteria in blood and tissues, and pathology evaluations. A general increase in incidence and severity of microscopic findings was observed in the lung, lymph nodes, spleen, and liver from 36 to 72 h postchallenge. Similarly, the incidence and severity of pneumonia increased throughout the study; however, some mice died in the absence of pneumonia, suggesting that disease progression does not require the development of pneumonia. Disease pathology in the Swiss Webster mouse is similar to that observed in humans, demonstrating the utility of this pneumonic plague model that can be used by researchers investigating plague countermeasures.