Anthrax infection poses a continued, significant bioterrorism threat; however, this threat could be minimized by therapeutic strategies that either improve the adaptive immune responses and/or target the mortality-promoting interactions between Bacillus anthracis virulence factors and host immunity. The major virulence factors of 6. anthracis include lethal toxin (LT), which is composed of protective antigen (PA) and lethal factor (LF), and edema toxin (ET), which is composed of PA and edema factor (EF). Active and passive vaccination strategies are important for protection against anthrax infection. As such, over the last 9 years, our group has assembled and analyzed samples from a broad array of individuals exposed to anthrax or anthrax vaccines, including US military personnel, UK-Anthrax Vaccine Precipitated (AVP)-vaccinated individuals and cutaneous anthrax survivors from Turkey, providing critical resources for this renewal application. Our collaboration with Walter Reed military sites has led to the largest, real-world collection of AVA-vaccinated individuals (n=2,735 and still recruiting). Surprisingly, over 30% fail to produce serum LT neutralization activity better than unvaccinated controls, even while mounting anti-PA responses. Even more vulnerable than vaccinated military personnel is the general civilian population. Indeed, a near 50% mortality rate was observed during the 2001 B. anthracis spore attacks even with admittance into an intensive care hospital unit and antibiotic therapy. Despite their importance in anthrax infection, the mechanisms by which toxins support virulence late in infection have remained elusive, likely because of a reliance on rodent models challenged with isolated toxins. Such approaches ignore the lethal septic responses promoted by vegetative B. anthracis, a condition that cannot be adequately studied in rodents and one that our team has shown to be responsible for high anthrax mortality. Pioneering work from our institution has shown that nucleosomal components promote lethal septic responses. LT promotes myeloid cell death, and ET causes high levels of intracellular cAMP, which inhibits efferocytosis, or the removal of apoptotic or necrotic cells by phagocytes. We propose that these actions of LT and ET promote high levels of circulating nucleosomes in the lethal, bacteremic stage of anthrax infection. Our unique, extensive and well-characterized collection of human plasma and anti-PA mAbs, combined with our experienced, integrated team of investigators and authoritative local understanding of sepsis, place us in a unique position to unravel the mechanism of mortality-promoting host-pathogen interactions late in anthrax infection. The results of these studies are likely to lead to improved therapeutic approaches to reducing mortality not only in anthrax but also in other, more common septic bacterial infections such as those caused by Pseudomonas aeruginosa, a serious nosocomial pathogen that also produces cAMP-elevating toxins. We therefore hypothesize that individuals with anti-PA responses, but inadequate toxin neutralization, have lessened antibody avidity, altered subclass usage, and/or favor non-neutralizing humoral epitopes and cannot mount an effective protective response. Additionally, we hypothesize that anthrax toxins enhance anthrax sepsis lethality by inhibiting the timely clearance of sepsis-promoting nucleosomal material by efferocytic and phagocytic leukocytes and that antibody-mediated neutralization of toxic histones and nucleosomes, in conjunction with effective antibody-mediated toxin neutralization, will reduce the mortality of anthrax septicemia. Aim 1: Identify the characteristics that distinguish highly protective vs. poorly-protective humoral immunity elicited by AVA by: a) testing if high avidity anti-PA has higher LT neutralization activity, b) assessing the effects of antibody subclass usage on LT neutralization, c) identifying the common sequential and domain-specific antigenic responses in anti-PA positive vaccinated individuals with and without neutralizing responses, and d) evaluating association of plasma cytokine production with impaired toxin neutralization. Aim 2: Determine the roles of LT and ET in promoting elevated nucleosome levels through inhibition of efferocytosis by: a) quantifying the capacity of human monocyte-derived and tissue (liver, spleen and lung) macrophages (MO) to efferocytose LT-killed human monocytes and apoptotic neutrophils (PMNs) in the presence of LT, ET or tripartite toxin; b) quantifying the capacity of human monocyte-derived and tissue MOs to efferocytose and kill B. anthracis upon co-culture with bacilli-loaded, apoptotic human monocytes and PMNs in the presence of LT, ET or tripartite toxin; and c) determining whether and by what mechanism human PMNs NET upon exposure to B. anthracis bacilli in the presence of LT, ET or tripartite toxin. Aim 3: Use a baboon model of anthrax septicemia to test whether anthrax toxins increase circulating nucleosomes and evaluate the protective capacity of histone-specific mAbs alone or in combination with protective PA-specific human mAbs.