Les Baillie

Murine Macrophages Kill the Vegetative Form of Bacillus anthracis

ABSTRACT Anti-protective antigen antibody was reported to enhance macrophage killing of ingested Bacillus anthracis spores, but it was unclear whether the antibody-mediated macrophage killing mechanism was directed against the spore itself or the vegetative form emerging from the ingested and germinating spore. To address this question, we compared the killing of germination-proficient (gp) and germination-deficient (ΔgerH) Sterne 34F2 strain spores by murine peritoneal macrophages. While macrophages similarly ingested both spores, only gp Sterne was killed at 5 h (0.37 log kill). Pretreatment of macrophages with gamma interferon (IFN-γ) or opsonization with immunoglobulin G (IgG) isolated from a subject immunized with an anthrax vaccine enhanced the killing of Sterne to 0.49 and 0.73 log, respectively, but the combination of IFN-γ and IgG was no better than either treatment alone. Under no condition was there killing of ΔgerH spores. To examine the ability of the exosporium to protect spores from macrophages, we compared the macrophage-mediated killing of nonsonicated (exosporium+) and sonicated (exosporium−) Sterne 34F2 spores. More sonicated spores than nonsonicated spores were killed at 5 h (0.98 versus 0.37 log kill, respectively). Pretreatment with IFN-γ increased the sonicated spore killing to 1.39 log. However, the opsonization with IgG was no better than no treatment or pretreatment with IFN-γ. We conclude that macrophages appear unable to kill the spore form of B. anthracis and that the exosporium may play a role in the protection of spores from macrophages.

Human Monoclonal Antibodies against Anthrax Lethal Factor and Protective Antigen Act Independently To Protect against Bacillus anthracis Infection and Enhance Endogenous Immunity to Anthrax

ABSTRACT The unpredictable nature of bioterrorism and the absence of real-time detection systems have highlighted the need for an efficient postexposure therapy for Bacillus anthracis infection. One approach is passive immunization through the administration of antibodies that mitigate the biological action of anthrax toxin. We isolated and characterized two protective fully human monoclonal antibodies with specificity for protective antigen (PA) and lethal factor (LF). These antibodies, designated IQNPA (anti-PA) and IQNLF (anti-LF), were developed as hybridomas from individuals immunized with licensed anthrax vaccine. The effective concentration of IQNPA that neutralized 50% of the toxin in anthrax toxin neutralization assays was 0.3 nM, while 0.1 nM IQNLF neutralized the same amount of toxin. When combined, the antibodies had additive neutralization efficacy. IQNPA binds to domain IV of PA containing the host cell receptor binding site, while IQNLF recognizes domain I containing the PA binding region in LF. A single 180-μg dose of either antibody given to A/J mice 2.5 h before challenge conferred 100% protection against a lethal intraperitoneal spore challenge with 24 50% lethal doses [LD50s] of B. anthracis Sterne and against rechallenge on day 20 with a more aggressive challenge dose of 41 LD50s. Mice treated with either antibody and infected with B. anthracis Sterne developed detectable murine anti-PA and anti-LF immunoglobulin G antibody responses by day 17 that were dependent on which antibody the mice had received. Based on these results, IQNPA and IQNLF act independently during prophylactic anthrax treatment and do not interfere with the establishment of endogenous immunity.

Importance of Nitric Oxide Synthase in the Control of Infection by Bacillus anthracis

ABSTRACT The spore-forming, gram-positive bacterium Bacillus anthracis, the causative agent of anthrax, has achieved notoriety due to its use as a bioterror agent. In the environment, B. anthracis exists as a dormant endospore. Upon infection, germination of endospores occurs during their internalization within the phagocyte, and the ability to survive exposure to antibacterial killing mechanisms, such as O2·−, NO·, and H2O2, is a key initial event in the infective process. Macrophages generate NO· from the oxidative metabolism of l-arginine, using an isoform of nitric oxide synthase (NOS 2). Exposure of murine macrophages (RAW264.7 cells) to B. anthracis endospores up-regulated the expression of NOS 2 12 h after exposure, and production of NO· was comparable to that achieved following other bacterial infections. Spore-killing assays demonstrated a NO·-dependent bactericidal response that was significantly decreased in the presence of the NOS 2 inhibitor l-N6-(1-iminoethyl)lysine and in l-arginine-depleted media. Interestingly, we also found that B. anthracis bacilli and endospores exhibited arginase activity, possibly competing with host NOS 2 for its substrate, l-arginine. As macrophage-generated NO· is an important pathway in microbial killing, the ability of endospores of B. anthracis to regulate production of this free radical has important implications in the control of B. anthracis-mediated infection.

Protective Role of Bacillus anthracis Exosporium in Macrophage-Mediated Killing by Nitric Oxide

ABSTRACT The ability of the endospore-forming, gram-positive bacterium Bacillus anthracis to survive in activated macrophages is key to its germination and survival. In a previous publication, we discovered that exposure of primary murine macrophages to B. anthracis endospores upregulated NOS 2 concomitant with an ·NO-dependent bactericidal response. Since NOS 2 also generates O2·−, experiments were designed to determine whether NOS 2 formed peroxynitrite (ONOO−) from the reaction of ·NO with O2·− and if so, was ONOO− microbicidal toward B. anthracis. Our findings suggest that ONOO− was formed upon macrophage infection by B. anthracis endospores; however, ONOO− does not appear to exhibit microbicidal activity toward this bacterium. In contrast, the exosporium of B. anthracis, which exhibits arginase activity, protected B. anthracis from macrophage-mediated killing by decreasing ·NO levels in the macrophage. Thus, the ability of B. anthracis to subvert ·NO production has important implications in the control of B. anthracis-induced infection.

Extraction and detection of DNA from Bacillus anthracis spores and the vegetative cells within 1 min

The use of a combination of low-cost technologies to both extract and detect anthrax DNA from spores and vegetative cells in two steps within 1 min is described. In a cavity, microwave energy is highly focused using thin-film aluminum bow-tie structures, to extract DNA from whole spores within 20 s. The detection of the released DNA, from less than 1000 vegetative cells, without additional preprocessing steps is accomplished in an additional 30 s by employing the microwave-accelerated metal-enhanced fluorescence technique. The new platform technology presented here is a highly attractive alternative method for DNA extraction and the fast detection of gram-positive bacteria and potentially other pathogenic species and cells as well.

Bacillus anthracis spores and lethal toxin induce IL‐1β via functionally distinct signaling pathways

Previous reports suggested that lethal toxin (LT)‐induced caspase‐1 activity and/or IL‐1β accounted for Bacillus anthracis (BA) infection lethality. In contrast, we now report that caspase‐1‐mediated IL‐1β expression in response to BA spores is required for anti‐BA host defenses. Caspase‐1–/– and IL‐1β–/– mice are more susceptible than wild‐type (WT) mice to lethal BA infection, are less able to kill BA both in vivo and in vitro, and addition of rIL‐1β to macrophages from these mice restored killing in vitro. Non‐germinating BA spores induced caspase‐1 activity, IL‐1β and nitric oxide, by which BA are killed in WT but not in caspase‐1–/– mice, suggesting that the spore itself stimulated inflammatory responses. While spores induced IL‐1β in LT‐susceptible and ‐resistant macrophages, LT induced IL‐1β only in LT‐susceptible macrophages. Cooperation between MyD88‐dependent and ‐independent signaling pathways was required for spore‐induced, but not LT‐induced, IL‐1β. While both spores and LT induced caspase‐1 activity and IL‐1β, LT did not induce IL‐1β mRNA, and spores did not induce cell death. Thus different components of the same bacterium each induce IL‐1β by distinct signaling pathways. Whereas the spore‐induced IL‐1β limits BA infection, LT‐induced IL‐1β enables BA to escape host defenses.

Role of Bacillus anthracis Spore Structures in Macrophage Cytokine Responses

ABSTRACT The innate immune response of macrophages (Mφ) to spores, the environmentally acquired form of Bacillus anthracis, is poorly characterized. We therefore examined the early Mφ cytokine response to B. anthracis spores, before germination. Mφ were exposed to bacilli and spores of Sterne strain 34F2 and its congenic nongerminating mutant (ΔgerH), and cytokine expression was measured by real-time PCR and an enzyme-linked immunosorbent assay. The exosporium spore layer was retained (exo+) or removed by sonication (exo−). Spores consistently induced a strong cytokine response, with the exo− spores eliciting a two- to threefold-higher response than exo+ spores. The threshold for interleukin-1β (IL-1β) production by wild-type Mφ was significantly lower than that required for tumor necrosis factor alpha expression. Cytokine production was largely dependent on MyD88, suggesting Toll-like receptor involvement; however, the expression of beta interferon in MyD88−/− Mφ suggests involvement of a MyD88-independent pathway. We conclude that (i) the B. anthracis spore is not immunologically inert, (ii) the exosporium masks epitopes recognized by the Mφ, (iii) the Mφ cytokine response to B. anthracis involves multiple pattern recognition receptors and signaling pathways, and (iv) compared to other cytokines, IL-1β is expressed at a lower spore concentration.

A non-glycosylated, plant-produced human monoclonal antibody against anthrax protective antigen protects mice and non-human primates from B. anthracis spore challenge

The health and economic burden of infectious diseases in general and bioterrorism in particular necessitate the development of medical countermeasures. One proven approach to reduce the disease burden and spread of pathogen is treatment with monoclonal antibodies (mAb). mAbs can prevent or reduce severity of the disease by variety of mechanisms, including neutralizing pathogen growth, limiting its spread from infected to adjacent cells, or by inhibiting biological activity of toxins, such as anthrax lethal toxin. Here, we report the production of glycosylated (pp-mAbPA) and non-glycosylated (pp-mAbPANG) versions of a plant-derived mAb directed against protective antigen (PA) of Bacillus anthracis in Nicotiana benthamiana plants using agroinfiltration. Both forms of the antibody were able to neutralize anthrax lethal toxin activity in vitro and protect mice against an intraperitoneal challenge with spores of B. anthracis Sterne strain. A single 180 µg intraperitoneal dose of pp-mAbPA or pp-mAbPANG provided 90% and 100% survival, respectively. When tested in non-human primates, pp-mAbPANG was demonstrated to be superior to pp-mAbPA in that it had a significantly longer terminal half-life and conferred 100% protection against a lethal dose of aerosolized anthrax spore challenge after a single 5 mg/kg intravenous dose compared to a 40% survival rate conferred by pp-mAbPA. This study demonstrates the potential of a plant-produced non-glycosylated antibody as a useful tool for the treatment of inhalation anthrax.