Mechanisms of innate and specific resistance during inhalational anthrax infections

Bacillus anthracis is the etiologic agent of inhalation, cutaneous, and gastrointestinal anthrax. Biothrax (AVA) is the only licensed vaccine for human anthrax in the United States, and the predominant antigen component is protective antigen (PA). The lengthy and complex dosing schedule, along with adverse injection site reactions, forms the basis of ongoing research to develop more efficient and effective vaccines and therapeutic treatments. In the first part of this study, we sought to identify whether passive immunization of guinea pigs with human monoclonal anti-PA (AVP-21D9) would significantly alter the adaptive immune response to active immunization with rPA or AVA. We treated guinea pigs with two s.c. doses of AVA (300il) or rPA (25mug) every two weeks with or without a single dose of AVP-21D9 (10 mg/kg) at time 0. After 6 weeks, the animals were challenged by aerosol inoculation with 41-53 LD50 of B. anthracis Ames spores. PA-ELISA and MTT neutralization assays were performed on all sera collected during immunization. The serological results suggested that there was no significant difference in the adaptive immune response in guinea pigs immunized with a combination of AVA and AVP-21D9 compared to immunization with AVA alone. Similarly, immunization with rPA in combination with AVP-21D9 also did not show a significant difference compared to guinea pigs immunized with rPA alone. Statistically significant protection after aerosol challenge was limited to 4-6 days in animals immunized with both AVP-21D9 and AVA (or rPA). The results suggest the intriguing possibility that individuals exposed to anthrax spores could be protected with a single dose of AVP-21D9, as opposed to 60 days of antibiotic therapy, and concomitantly, could be effectively vaccinated with AVA or rPA with little or no antigenic interference.;The second part of this work focused on Natural Killer cells and B. anthracis. We hypothesized that NK cells are activated to have bactericidal activity in response to B. anthracis and play an important immunological role following infection. To determine the potential for NK cells to play a role in host defense to B. anthracis, we assessed the in vitro antibacterial activity of primary human NK cells against B. anthracis (Ames strain) bacilli and spores and determined the outcome of B. anthracis infection in a mouse model following in vivo depletion of murine NK cells. Our results demonstrated that human NK cells kill both extracellular and intracellular B. anthracis bacilli but do not have activity against spores. Using electron microscopy, transwell separation, and chemical blockade, we further demonstrated that NK cells make intimate contact with B. anthracis bacilli and that antibacterial activity is both contact- and granule-dependent. We also observed that the functional activity of NK cells is not sensitive to immunosuppression by B. anthracis lethal or edema toxin as has been demonstrated for other leukocytes. Murine NK cells have similar antibacterial activity to that of human NK cells when exposed to B. anthracis in our studies. In vivo depletion of murine NK cells using anti-asialo GM1 antibody does not alter animal survival following intranasal infection with B. anthracis in our studies, but significantly increases the bacterial load in the blood of infected animals. Our studies demonstrated that NK cells might have an important antibacterial role that contributes to host defense against B. anthracis. Immunomodulation to augment NK cell function in the early stages of anthrax should be explored further in animal models as a clinical intervention strategy to complement antibiotic therapy.