The present application is a competing renewal of a CCHI grant on the human immune response to Bacillus anthracis and the vaccine that protects the military, and that was first awarded in 2004. The goals of the original application were threefold: (a) To study the human immune response to a flawed vaccine, (b) To understand the mechanism for the high lethality of the inhalation form of the disease, (c) To understand the cellular basis of the host response to the pathogen. We have learned much about the human vaccine with our collection of nearly 3,000 samples, including samples of individuals who have naturally been infected with B. anthracis. Especially notable is our finding that fully 50% of vaccinees are unprotected. This is so, despite more than 6 vaccinations immunized against the pathogen’s toxins in an onerous vaccine schedule. We have evidence, in contrast to prevailing views, that the high rate of mortality is due to bacterial sepsis and not the anthrax toxins. We made the seminal discovery that immune complexes of peptidoglycan and pre-existing serum opsonins present in all humans may be the source of the massive inflammation and coagulopathy accompanying infection by B. anthracis. We have new evidence that the infection is accompanied by release of proinflammatory and procoagulant nucleosome material (DAMPs) and that the anthrax toxins can modulate the clearance of this material. In this renewal application, we will follow up on these exciting discoveries to determine: (a) In the early- and mid-stage of disease, how are DAMPS released by the host, how they are cleared by the host innate immune system and how does toxin affect these processes. (b) In the late stage of the disease, how does opsonized peptidoglycan influence the outcome of the disease. (c) Why the vaccine is imperfect in stimulating the maturation of germinal center B cells in adults. These studies are supported by 2 scientific cores: An animal core that applies an animal model we established in previous funding cycles and a flow cytometry core with state-of-the-art sorting and analyzing capacity. The studies in this renewal application are focused and thematically organized around the key roles of peptidoglycan and the anthrax toxins in the human innate and adaptive immune responses. They have great potential to identify novel means of interrupting the pathology caused by this model Gram-positive pathogen.
Our U19 Center consists of three Cores and three Projects:
ADMINISTRATIVE CORE
COGGESHALL, KENNETH MARK, Core Lead
The Administrative Core centralizes and coordinates all group activities. These include the monthly meetings, the annual report to the NIH, the annual CCHI meeting, and the selections of Opportunity Fund projects. These efforts involve the PI and a project coordinator.
FLOW CYTOMETRY CORE
THOMPSON, LINDA F., Core Lead
The Aim of the Flow Cytometry Core is to provide access to state-of-the art flow cytometry (sorting and analysis) for all the members of the Oklahoma Anthrax Group (U19). The Flow Cytometry Core has two high speed fluorescence activated cell sorters (Beckman-Coulter MoFlo XDP and BD FACSAria) and three analyzers (FACSCalibur, FACSCelesta and LSRII, all from BD). Dr. Linda Thompson, who is an expert in multicolor analysis and sorting of human lymphocytes, will serve as Core Director and technical assistance will be provided by Mr. Jacob Bass and Dr. Diana Hamilton. Mr. Bass and Dr. Hamilton will perform all the sorts. Funds for the purchase of monoclonal antibodies and user fees will be provided to all COBRE participants. All three research projects depend upon flow cytometry to accomplish their scientific goals. Thus, this Core will be heavily used and is critical for the success of the Oklahoma Anthrax Group.
ANIMAL MODEL CORE
LUPU, FLOREA, Core Lead
Our U19 program is focused on the investigation of the pathophysiology of B. anthracis infection and sepsis and potential novel therapies. The Animal Core will serve as an integral component of the U19 program, providing support to all three projects using a clinically relevant animal model of sepsis. The investigators will have a unique opportunity to answer critical questions by combining a genetically diverse, clinically relevant animal model, which has a proven history of mimicking human responses to sepsis with novel technologies being developed within our program. The Animal Core combines a unique set of facilities, management and technical expertise needed to achieve the Specific Aims of this program, as well as expert veterinary care, specialized reagent tools and strong expertise in advanced biochemical and microscopical analysis of tissue samples. The personnel involved in the Core have more than three decades of experience in infectious disease using this animal model. Optimal utilization of clinically relevant animal models for research on bioterrorism related pathogens such as B. anthracis requires a facility where animals are housed in appropriate biocontainment, research personnel are experienced in the methodologies required for infectious disease research, and development of novel therapeutics is required. Our Animal Core will use state-of-the-art facilities to develop animal models for studying disease pathogenesis and testing the efficacy and safety of novel therapeutics. Our facilities fully support biohazard research at ABSL-2 and meet all the NIH/CDC guidelines. Hence, this Core is a critical component in support of the proposals aimed at developing innovative therapeutics against B. anthracis, which we believe will be relevant to other Gram-positive pathogens.
IMMUNE CELL MECHANISMS OF BACILLUS ANTHRACIS SEPSIS
FARRIS, A DARISE, Project Lead
Susan Kovats, Project Co-Lead
Bacterial sepsis is a serious and difficult-to-treat condition with high mortality in part due to initial overactive innate immunity followed by immunosuppression that leaves the host vulnerable to new infections. Inhalational anthrax, a known and expected outcome of bioterrorism-related release of Bacillus anthracis (Ba) spores, inevitably leads to sepsis and high mortality without early intervention. Anti-inflammatory sepsis therapies developed in mice have failed in humans, indicating that improved understanding of molecular mechanisms of human immune cell involvement in Ba sepsis is needed. Prior work in our center showed that unchecked inflammation and sepsis pathology is worsened by elevated circulating nucleosomes released from dying cells. Clearance of apoptotic cells is mediated by macrophages via the process of efferocytosis. During sepsis, efferocytic function by tissue macrophages is likely crucial for clearance of apoptotic lymphocytes in secondary lymphoid organs. Our preliminary data show that Ba and its toxins impair human macrophage efferocytosis, although the mechanism is unclear. In Aim 1, we will test the hypothesis that Ba and its toxins inhibit human tissue macrophage efferocytosis by decreasing expression of efferocytic machinery and impairing efferocytic receptor signaling. Extensive lymphocyte apoptosis also contributes to immunosuppression, a major complication following sepsis survival. Long term immunosuppression correlates with the accumulation of hyporesponsive T cells bearing markers of exhaustion, suggesting the involvement of epigenetic changes. Our preliminary data show lymphopenia and T cells bearing exhaustion markers in an animal model of Ba induced sepsis. In Aim 2, we will test the hypothesis that live Ba and its toxins promote T lymphocyte apoptosis and exhaustion, leading to immunosuppressed T cell phenotypes enforced by epigenetic changes. Lipid-activated nuclear receptors respond to lipids of efferocytosed apoptotic cells by increasing transcription of efferocytic machinery. However, few studies have assessed the impact of such receptors on human tissue macrophage gene expression or lymphocyte apoptosis. In Aim 3, we will test the hypothesis that synthetic agonists of various lipid-activated nuclear receptors can mitigate negative impacts of Ba and its toxins on macrophages and T cells during sepsis. The results of these studies will lead to a better understanding of mechanisms of acute pathology and subsequent immunosuppression in sepsis and may accelerate the development of new therapeutic strategies for the treatment of septic patients.
COMPLEMENT AND COAGULATION IN B. ANTHRACIS PEPTIDOGLYCAN-INDUCED SEPSIS
COGGESHALL, KENNETH MARK, Project Lead
Narcis Popescu, Project Co-Lead
Our work demonstrates that peptidoglycan (PGN), a major component of the cell wall of all Gram-positive bacteria, promotes inflammation and coagulation. Animals responded to in vivo PGN challenge with features of systemic inflammation and disseminated intravascular coagulopathy (DIC) similar to those seen in patients with inhalation anthrax. We found evidence of activation of both the intrinsic and extrinsic coagulation pathways. In vitro, we found PGN stimulated robust cytokine production in human innate immune cells and prothrombinase activity in human platelets exposed to highly purified polymeric PGN derived from B. anthracis or Staphylococcus aureus. Innate immune cell responses required PGN recognition by surface Fcγ receptors, phagocytosis, digestion in lysosomes, and stimulation of cytoplasmic NOD sensors. In the last iteration of this grant, we showed that these processes were dependent on human serum opsonins, IgG and serum amyloid P. We also showed that PGN-stimulated human monocytes expressed tissue factor (TF), an initiator of the extrinsic coagulation pathway, in part by virtue of the proinflammatory cytokines. This project will provide mechanistic insight into PGN-stimulated pathologies. First, we will test whether and how the anthrax toxins affect the immune system activation events we have documented to date and listed above. Second, we will identify the pathways that regulate PGN-induced activation of the intrinsic coagulation pathway in vivo. Lastly, we will apply existing biological inhibitors to each pathway (intrinsic, extrinsic coagulation and complement) to ask which of these contributes most to the pathology, organ failure and death in PGN-challenged animals. The results will greatly inform treatment options for patients with Gram-positive bacteremia.
MECHANISMS OF HUMORAL IMMUNE PROTECTION INDUCED BY ANTHRAX VACCINE ADSORBED
JAMES, JUDITH A, Project Lead
To mitigate the ongoing threat of anthrax infection due to bioterrorism, active military members are vaccinated with Anthrax Vaccine Adsorbed (AVA). However, some AVA recipients may remain unprotected against anthrax infection and a better understanding of mechanisms leading to impaired vaccine response and rapidly waning immunity are needed. In the largest real-world cohort of AVA vaccinees (>2,900 individuals), less than 50% of AVA vaccinees showed significant in vitro lethal toxin neutralization, and both antibody levels and neutralization capacity waned quickly after vaccination. Therefore, the goal of this project is to identify mechanisms of poor neutralization after AVA vaccination. The primary antigen in AVA is protective antigen (PA). Our previous studies have identified common sequential epitopes recognized by serum anti-PA, and have shown differential epitope binding of neutralizing vs. non-neutralizing responses. Poorly neutralizing responses have also been associated with impaired avidity of anti-PA and enhanced IgG4 production. In addition, suboptimal AVA responses are more common in African American vs. European American vaccinees. Preliminary data suggest that African American individuals have enriched responses against non-neutralizing epitopes after AVA vaccination and have marked differences in immune cell subsets and immune pathways compared to European American individuals. Critical questions remain in understanding the mechanisms of rapidly waning or impaired adult vaccination responses, such as those against AVA. This project addresses mechanisms of impaired protection after AVA immunization by comparing anti-PA antibodies from high and low neutralizers in the AVA cohort described above as well as new recruits for domain specificity, anti-PA avidity, and anti-PA IgG4 responses, all of which may inhibit protective human Bacillus anthracis immunity (Aim 1). Additional mechanisms of protective responses will be dissected using neutralizing PA-specific human monoclonal antibodies previously generated by our lab. In addition, although anti-PA domain specificity is related to neutralizing capacity, the conformational epitopes of PA bound by the serum of neutralizers have not been elucidated. Therefore, this project uses novel hydrogen-deuterium exchange mass spectrometry tech- niques to map anti-PA binding sites on PA using sera from high and low neutralizers (Aim 2). Finally, mech- anisms of impaired AVA responses will be evaluated in African American vs. European American vaccinees (Aim 3). Antibodies, immune cell profiles, and regulatory pathways will be compared by mass cytometry (CyTOF), intracellular cytokine production, flow cytometry and ELISpot at various times after AVA vaccination. Epitope specificity and genetic predisposition to impaired anti-PA responses will be tested. These studies will provide new insights to optimize future anthrax vaccines and other adult vaccinations across racial groups.
