Induction of protective immunity against lethal anthrax challenge with a patch

MAJOR ARTICLE Induction of Protective Immunity against Lethal Anthrax Challenge with a Patch Richard T. Kenney,1 Jianmei Yu,1 Mimi Guebre-Xabier,1 Sarah A. Frech,1 Adam Lambert,1 Barbara A. Heller,1 Larry R. Ellingsworth,1 James E. Eyles,2 E. Diane Williamson,2 and Gregory M. Glenn1 1 IOMAI, Gaithersburg, Maryland; 2Defence Science and Technology Laboratory, Porton Down, Salisbury, United Kingdom Anthrax has been recognized as a disease for centuries, but it recently has become a more serious threat to public health as a tool of bioterrorism. Bacillus anthracis can form endospores that are easily transported and highly resistant to inactivation. The intentional contamination of the US mail in late 2001 attests to the potential havoc that can result from a small-scale bioterrorism effort, which, in itself, was limited, compared with the potential morbidity and mortality that would result from airborne release [1]. Postexposure antibiotic treatment can be used but has many limitations, not the least of which is the difficulty of compliance with a 60-day course of medication [2]. Vaccination is a Received 26 November 2003; accepted 5 March 2004; electronically published 13 July 2004. Financial support: National Institutes of Health (grant AI052963). Potential conflict of interest: All authors have a financial interest in the development of the anthrax vaccine patch. Reprints or correspondence: Dr. Richard T. Kenney, IOMAI, 20 Firstfield Rd., Ste. 250, Gaithersburg, MD 20878 (). The Journal of Infectious Diseases 2004; 190:774–82  2004 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2004/19004-0016$15.00 774 • JID 2004:190 (15 August) • Kenney et al. more ideal prophylaxis for large numbers of people who are at risk of contracting anthrax via inhalation. The only US-licensed vaccine, anthrax vaccine adsorbed (AVA), which is in short supply and currently is limited to the military and recently exposed individuals, has not been optimized clinically and is not well accepted, given the severity of local adverse events [3]. AVA is a culture supernatant–derived vaccine that has protective antigen (PA) protein as its principal active component and is adjuvanted with alum [4, 5]. Second-generation vaccines composed of recombinant PA (rPA) also adjuvanted with alum [6] are just entering early clinical testing. Although they are immunogenic and probably are effective, one disadvantage of parenteral formulations incorporating rPA is the logistics of administration, which requires trained personnel to inject the vaccine. Epidemic exposure to anthrax dictates broad and immediate availability of vaccine. An easily administered formulation incorporating rPA could overcome these disadvantages and facilitate widespread vaccination by minimally trained personnel. There is an urgent need for an effective alternative approach to prophylaxis against this deadly infection. Background. Transcutaneous immunization (TCI) is a needle-free technique that delivers antigens and adjuvants to potent epidermal immune cells. To address critical unmet needs in biodefense against anthrax, we have designed a novel vaccine delivery system using a dry adhesive patch that simplifies administration and improves tolerability of a subunit anthrax vaccine. Methods. Mice and rabbits were vaccinated with recombinant protective antigen of Bacillus anthracis and the heat-labile toxin of Escherichia coli. Serologic changes, levels of toxin-neutralizing antibodies (TNAs), and pulmonary and nodal responses were monitored in the mice. A lethal aerosolized B. anthracis challenge model was used in A/J mice, to demonstrate efficacy. Results. The level of systemic immunity and protection induced by TCI was comparable to that induced by intramuscular vaccination, and peak immunity could be achieved with only 2 doses. The addition of adjuvant in the patch induced superior TNA levels, compared with injected vaccination. Conclusions. Anthrax vaccine patches stimulated robust and functional immune responses that protected against lethal challenge. Demonstration of responses in the lung suggests that a mechanism exists for protection against challenge with aerosolized anthrax spores. A formulated, pressure-sensitive, dry adhesive patch, which is stable and can be manufactured in large scale, elicited comparable immunoglobulin G and TNA responses, suggesting that an anthrax vaccine patch is feasible and should advance into clinical evaluation. form the membrane attack complex to kill bacteria [24], are exposed to a live, nonencapsulated, toxin-producing anthrax strain as an aerosolized spore challenge, to more closely approximate human pathogenesis [25]. MATERIALS AND METHODS Animals. A/J mice were obtained from Jackson Laboratories and Harlan UK. Mice (6–10 weeks old) were maintained in pathogen-free conditions and fed rodent chow and water ad libitum. New Zealand White rabbits were obtained from Covance Research Products and studied under good laboratory practices (GLP) conditions. Challenge studies were conducted at the Defence Science and Technology Laboratory (Porton Down, UK) under biosafety level 3 (BL3) conditions. All studies were approved by the animal care and use committees in both the United Kingdom and Maryland. Antigens, adjuvants, and bacteria. B. anthracis rPA was produced from E. coli and was purified to remove endotoxin. LT was purchased from Berna Biologics. The STI vaccine strain of B. anthracis, an avirulent isolate (pX01+; pX02⫺) that was used extensively as an attenuated live spore vaccine in the former Soviet Union, was used during inhalation challenge experiments [25]. Vaccination. Mice were immunized by TCI, as described elsewhere [11]. In brief, mice were shaved on the dorsum and rested for 48 h. Mice were anesthetized with ketamine/xylazine during the procedure to prevent self-grooming. The skin was hydrated with saline-drenched gauze for 5–10 min, pretreated with 10 strokes using emery paper (GE Medical Systems), rehydrated, and blotted dry. Twenty-five microliters of immunizing solution was placed on a 1-cm2 gauze patch that was covered and left on the area for 24 h. The mice were then extensively washed (tails down under running tap water for ∼30 s), patted dry, and washed again. Control mice were immunized by intramuscular (im) injection with 5 mg of rPA adjuvanted with 25 mg of alum (Rehydrogel; Reheis) mixed in PBS and given in a split dose—that is, 12.5 mL in each upper thigh. Immunization was done similarly using a dry adhesive patch instead of an immunizing solution with a gauze patch. Mice received 1-cm2 patches left in place for 24 h, and rabbits received 15-cm2 patches left in place for 8 h. The immune response was evaluated 2 weeks after each immunization. ELISA. Antibody levels against LT and rPA were determined by use of ELISA, essentially as described elsewhere [26]. Immulon-2 polystyrene plates (Dynex Laboratories) were coated with 2 mg/mL antige (...truncated)