, the etiological agent of anthrax, is a gram-positive, aerobic, spore-forming bacterium (25
). Dormant spores are highly resistant to adverse environmental conditions and are able to reestablish vegetative growth in the presence of favorable environmental conditions (29
). Anthrax is initiated by the entry of spores into the host through the skin, the gastrointestinal tract, or the respiratory epithelium after inhalation of airborne spores (10
). The inhalational form of anthrax is the most severe and is associated with rapid progression of disease and death (3
). The best described virulence determinants of B. anthracis
are encoded on two large plasmids (pXO1 [185 kb] and pXO2 [97 kb]) (20
). The three genes that encode the proteins that combine to form the B. anthracis
, and pag
) are found on the pXO1 plasmid (16
). The combination of the pag
-encoded protective antigen (PA) and the cya
-encoded edema factor (edema toxin) causes edema when injected subcutaneously, and the combination of PA and the lef
-encoded lethal factor (lethal toxin) causes death when injected intravenously (36
). Capsule, composed of poly-d
-glutamic acid, is encoded on the pXO2 plasmid. The capsule is believed to protect vegetative cells from microbicidal activity and serum proteins (14
). Although the recent interest in B. anthracis
pathogenesis is rooted in its potential as a bioterrorist weapon, it should be remembered that B. anthracis
remains endemic throughout the world, and many people die yearly from anthrax due to environmental exposure. In many parts of the world anthrax outbreaks occur regularly in herds of wild and domestic animals (5
). These outbreaks have environmental, as well as economic impact, on the affected regions and provide a source of infection for the human population. In contrast to many pathogens that appear to be host limited, B. anthracis
is able to efficiently infect and overwhelm the immune response of a remarkably wide range of hosts. Some aspects of its complex interactions with the host immune response have been partially illuminated by recent efforts to develop more effective vaccines.
Efforts to develop improved vaccines have focused on specific bacterial components. Since PA was shown to be the principle immunogen of the licensed vaccine (41
), it has been studied extensively as the primary component of numerous recombinant vaccine formulations. Antibodies to PA protect animals against lethal disease, although other antigens may also contribute to protective immunity (4
). Fab fragments recognizing PA have been shown to be protective, suggesting that antibody neutralization of PA is sufficient to protect against lethal disease (26
). In addition to understanding the host response to vaccination, there is significant value in increasing our understanding of the biology of the anthrax organism, including its complex interactions with the host immune response. In particular, identifying mechanisms involved in protective immunity following infection, which may be different from those induced by current vaccination approaches, could have important applications.
Antibodies can function by three main mechanisms: complement activation, opsonization for FcR-mediated phagocytosis, or neutralization, which refers to antibodies’ ability to interfere with pathogen functions simply by binding. Antibody-mediated clearance of bacterial pathogens can require any one, or combinations, of these activities. For example, bacteria in the lungs can be unaffected by antibodies in the absence of complement components or FcRs, indicating that a complex combination of Fc-associated effector functions is required for bacterial clearance (22
). Although neutralization is likely to be the mechanism by which PA-based vaccines work, it is not clear that B. anthracis
infection-induced immunity provides subsequent protection by the generation of anti-PA antibodies. Also, it is not clear whether anti-PA antibodies contribute to a reduction in bacterial numbers during an infection. Therefore, the mechanisms of protection elicited by PA vaccine-induced immunity, which protects against toxin-mediated pathology, are likely to differ from those that are induced by infection with viable spores.
toxins can interfere with innate, inflammatory, and adaptive immune responses at various levels. Lethal toxins can kill or inactivate immune cells such as monocytes, macrophages, and neutrophils (2
). Edema toxin can hinder lipopolysaccharide-induced cytokine production by macrophages (19
). By suppressing activation of macrophages or dendritic cells, B. anthracis
toxins may interfere with antigen presentation pathways involved in the generation of adaptive immunity (1
). Furthermore, anthrax toxins have been shown to act directly on adaptive immune cells, blocking multiple kinase signaling pathways involved in T-cell activation (6
). Treating mice with toxins alone has been shown to inhibit the ability of T cells to proliferate and secrete cytokines. Thus, B. anthracis
can manipulate host immunity at various levels, some of which appear to be dependent on complexities of local concentrations of bacteria, toxins, and various immune cells. These complex interactions between host and bacterial components cannot be simulated in vitro or with purified bacterial components and/or toxins in vivo but are best studied in the context of infection.
Here we explore the immunological mechanisms involved in the generation of B. anthracis induced immunity after aerosol exposure to spores. We have taken the approach of experimentally infecting immunodeficient mice to determine which immune factors are required for the generation of protective anamnestic immunity. Our results indicate that both B and T cells were required, which is probably attributable to their respective roles in the induction of antibody production. T-cell-deficient mice failed to produce significant levels of immunoglobulin G (IgG) antibody to PA, and the adoptive transfer of anti-B. anthracis serum was sufficient for protection against challenge. Adoptively transferred antibodies were protective in mice lacking both complement and FcRs. Together, these data indicate that protective immunity induced by toxigenic, nonencapsulated B. anthracis infection acts via an antibody-dependent mechanism that does not require antibody Fc effector functions.