Dissemination bottleneck in a murine model of inhalational anthrax.

Inhalational anthrax is caused by the sporulating bacterium Bacillus anthracis. A current model for progression in mammalian hosts includes inhalation of bacterial spores, phagocytosis of spores in the nasal mucosa-associated lymphoid tissue (NALT) and lungs by macrophages and dendritic cells, trafficking of phagocytes to draining lymph nodes, germination of spores and multiplication of vegetative bacteria in the NALT and lymph nodes, and dissemination of bacteria via the bloodstream to multiple organs. In previous studies, the kinetics of infection varied greatly among mice, leading us to hypothesize the existence of a bottleneck past which very few spores (perhaps only one) progress to allow the infection to proceed. To test this hypothesis, we engineered three strains of B. anthracis Sterne, each marked with a different fluorescent protein, enabling visual differentiation of strains grown on plates. Mice were infected with a mixture of the three strains, the infection was allowed to proceed, and the strains colonizing the organs were identified. Although the inoculum consisted of approximately equal numbers of each of the three strains, the distal organs were consistently colonized by a majority of only one of the three strains, with the dominant strain varying among animals. Such dominance of one strain over the other two was also found at early time points in the cervical lymph nodes but not in the mediastinal lymph nodes. These results support the existence of a bottleneck in the infectious process.

Nonhuman primate model of pertussis.

Pertussis is a highly contagious, acute respiratory illness caused by the bacterial pathogen Bordetella pertussis. Despite nearly universal vaccine coverage, pertussis rates in the United States have been rising steadily over the last 20 years. Our failure to comprehend and counteract this important public health concern is due in large part to gaps in our knowledge of the disease and the mechanisms of vaccine-mediated protection. Important questions about pertussis pathogenesis and mechanisms of vaccine effectiveness remain unanswered due to the lack of an animal model that replicates the full spectrum of human disease. Because current animal models do not meet these needs, we set out to develop a nonhuman primate model of pertussis. We inoculated rhesus macaques and olive baboons with wild-type B. pertussis strains and evaluated animals for clinical disease. We found that only 25% of rhesus macaques developed pertussis. In contrast, 100% of inoculated baboons developed clinical pertussis. A strong anamnestic response was observed when convalescent baboons were infected 6 months following recovery from a primary infection. Our results demonstrate that the baboon provides an excellent model of clinical pertussis that will allow researchers to investigate pertussis pathogenesis and disease progression, evaluate currently licensed vaccines, and develop improved vaccines and therapeutics.

Development of a highly efficacious vaccinia-based dual vaccine against smallpox and anthrax, two important bioterror entities.

Bioterrorism poses a daunting challenge to global security and public health in the 21st century. Variola major virus, the etiological agent of smallpox, and Bacillus anthracis, the bacterial pathogen responsible for anthrax, remain at the apex of potential pathogens that could be used in a bioterror attack to inflict mass casualties. Although licensed vaccines are available for both smallpox and anthrax, because of inadequacies associated with each of these vaccines, serious concerns remain as to the deployability of these vaccines, especially in the aftermath of a bioterror attack involving these pathogens. We have developed a single vaccine (Wyeth/IL-15/PA) using the licensed Wyeth smallpox vaccine strain that is efficacious against both smallpox and anthrax due to the integration of immune-enhancing cytokine IL-15 and the protective antigen (PA) of B. anthracis into the Wyeth vaccinia virus. Integration of IL-15 renders Wyeth vaccinia avirulent in immunodeficient mice and enhances anti-vaccinia immune responses. Wyeth/IL-15/PA conferred sterile protection against a lethal challenge of B. anthracis Ames strain spores in rabbits. A single dose of Wyeth/IL-15/PA protected 33% of the vaccinated A/J mice against a lethal spore challenge 72 h later whereas a single dose of licensed anthrax vaccine protected only 10%. Our dual vaccine Wyeth/IL-15/PA remedies the inadequacies associated with the licensed vaccines, and the inherent ability of Wyeth vaccinia virus to be lyophilized without loss of potency makes it cold-chain independent, thus simplifying the logistics of storage, stockpiling, and field delivery in the event of a bioterror attack involving smallpox or anthrax.

Role of anthrax toxins in dissemination, disease progression, and induction of protective adaptive immunity in the mouse aerosol challenge model.

Anthrax toxins significantly contribute to anthrax disease pathogenesis, and mechanisms by which the toxins affect host cellular responses have been identified with purified toxins. However, the contribution of anthrax toxin proteins to dissemination, disease progression, and subsequent immunity after aerosol infection with spores has not been clearly elucidated. To better understand the role of anthrax toxins in pathogenesis in vivo and to investigate the contribution of antibody to toxin proteins in protection, we completed a series of in vivo experiments using a murine aerosol challenge model and a collection of in-frame deletion mutants lacking toxin components. Our data show that after aerosol exposure to Bacillus anthracis spores, anthrax lethal toxin was required for outgrowth of bacilli in the draining lymph nodes and subsequent progression of infection beyond the lymph nodes to establish disseminated disease. After pulmonary exposure to anthrax spores, toxin expression was required for the development of protective immunity to a subsequent lethal challenge. However, immunoglobulin (immunoglobulin G) titers to toxin proteins, prior to secondary challenge, did not correlate with the protection observed upon secondary challenge with wild-type spores. A correlation was observed between survival after secondary challenge and rapid anamnestic responses directed against toxin proteins. Taken together, these studies indicate that anthrax toxins are required for dissemination of bacteria beyond the draining lymphoid tissue, leading to full virulence in the mouse aerosol challenge model, and that primary and anamnestic immune responses to toxin proteins provide protection against subsequent lethal challenge. These results provide support for the utility of the mouse aerosol challenge model for the study of inhalational anthrax.

Anamnestic protective immunity to Bacillus anthracis is antibody mediated but independent of complement and Fc receptors.

The threat of bioterrorist use of Bacillus anthracis has focused urgent attention on the efficacy and mechanisms of protective immunity induced by available vaccines. However, the mechanisms of infection-induced immunity have been less well studied and defined. We used a combination of complement depletion along with immunodeficient mice and adoptive transfer approaches to determine the mechanisms of infection-induced protective immunity to B. anthracis. B- or T-cell-deficient mice lacked the complete anamnestic protection observed in immunocompetent mice. In addition, T-cell-deficient mice generated poor antibody titers but were protected by the adoptive transfer of serum from B. anthracis-challenged mice. Adoptively transferred sera were protective in mice lacking complement, Fc receptors, or both, suggesting that they operate independent of these effectors. Together, these results indicate that antibody-mediated neutralization provides significant protection in B. anthracis infection-induced immunity.