Vaccines and Therapeutics for Anthrax

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

Anthrax toxin, a major virulence factor of Bacillus anthracis, consists of the cellular binding moiety protective antigen (PA) and the enzymatic moieties lethal factor (LF) and edema factor (EF). To intoxicate host organisms, PA binds to its cellular receptors TEM8 and CMG2 and is proteolytically activated by the ubiquitously expressed cell surface furin protease, resulting in the formation of active PA heptamer, which in turn translocates LF and EF into the cytosol of cells. PA and LF form lethal toxin (LT), and PA and EF form edema toxin (ET). LF cleaves several MEKs, thereby inactivating the ERK, p38, and JUNK MAPK pathways. EF is an adenylate cyclase that generates abnormally high concentrations of cAMP. To study the roles of TEM8 and CMG2 in anthrax pathogenesis, we previously generated TEM8 and CMG2 knockout mice and found that CMG2 is the major anthrax toxin receptor in vivo. To further investigate how B. anthracis overcomes host innate immune responses to initiate infection, in the year of 2011 we generated myeloid-lineage specific CMG2-deficient mice to examine the roles of macrophages, neutrophils, and other myeloid cells in anthrax pathogenesis. Macrophages and neutrophils isolated from these mice were resistant to anthrax toxin. However, the myeloid-specific CMG2-deficient mice remained fully sensitive to both anthrax lethal and edema toxins, demonstrating that targeting of myeloid cells is not responsible for anthrax toxin-induced lethality. Surprisingly, the myeloid-specific CMG2-deficient mice were completely resistant to B. anthracis infection. This work demonstrates that anthrax toxin uptake through CMG2 and the resulting impairment of myeloid cells are essential to anthrax infection. This work indicates that antitoxin therapeutics including antibodies against PA, LF, and EF would be effective in particular when used in early infection to protect the innate immune cells and thus allow these cells to clear B. anthracis. In addition to anthrax toxin, another essential virulence factor of B. anthracis is the DPGA capsule, the most outer layer structure of the bacterium. Therefore, development of antibodies against DPGA could be useful in anthrax therapy. In the year of 2011, we are extending collaborative work with Drs. Purcell and Zhao (LID, NIAID) to develop chimpanzee/human monoclonal antibodies (mAbs) to DPGA. Five DPGA-specific antibody antigen-binding fragments (Fabs) were generated from immunized chimpanzees. The two selected for further study, Fabs 11D and 4C, were both converted into full-length IgG1 and IgG3 mAbs having human IgG1 or IgG3 constant regions. These two mAbs had similar binding affinities, in vitro opsonophagocytic activities, and in vivo efficacies, with the IgG1 and IgG3 subclasses reacting similarly. A single 30 microgram dose of either mAb given to BALB/c mice 18 h before challenge conferred about 50% protection against a lethal intratracheal spore challenge by the virulent B. anthracis Ames strain. More importantly, either mAb given 8 h or 20 h after challenge provided significant protection against lethal infection. Thus, these anti-DPGA mAbs should be useful, alone or in combination with antitoxin mAbs, for achieving a safe and efficacious postexposure therapy for anthrax. Our previous collaborative work with Dr. Prosper N. Boyaka (Ohio State University) showed that intranasal coapplication of anthrax PA together with an EF mutant having reduced adenylate cyclase activity (i.e., EF-S414N) not only enhances anti-PA responses, but also acts as a mucosal adjuvant for coadministered unrelated antigens. To elucidate the role of ET components in its adjuvanticity, in the year of 2011, we continued the collaborative work with Dr. Boyaka to examined how a PA mutant lacking the ability to bind EF (PA-U7) or another mutant that allows the cellular uptake of EF, but fails to efficiently mediate its translocation into the cytosol (PA-dFF), would affect ET-induced adaptive immunity. Native ET promotes costimulatory molecule expression by macrophages and B lymphocytes and induces a broad spectrum of cytokine responses by cervical lymph node cells in vitro. These effects were reduced or abrogated when cells were treated with EF plus PA-dFF, or PA-U7 instead of PA. We also intranasally immunized groups of mice with a recombinant fusion protein of Yersinia pestis F1 and LcrV Ags (F1-V) together with ET variants consisting of wild-type or mutant PA and EF. Analysis of serum and mucosal antibody responses against F1-V or ET components revealed that a fully functional PA and a minimum of adenylate cyclase activity are needed for ET to act as a mucosal adjuvant. One area of work in this project seeks to use modified anthrax toxins to target cancer. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Although considerable progress has been made in elucidating the etiology of the disease, the prognosis for individuals diagnosed with HNSCC remains poor, underscoring the need for development of additional treatment modalities. HNSCC is characterized by the upregulation of a large number of proteolytic enzymes, including urokinase plasminogen activator (uPA) and an assortment of matrix metalloproteinases (MMPs) that may be expressed by tumor cells, by tumor-supporting stromal cells or by both. We previously found that the toxicity of anthrax toxin can be redirected to cancers by changing PA's furin specificity to cancer-selective protease specificities. Therefore, we constructed uPA and MMP activated anthrax lethal toxins. In 2011, collaborating with Dr. Bugge (NIDCR), we explored the use of an intercomplementing anthrax toxin that requires combined cell surface uPA and MMP activities for cellular intoxication and specifically targets the ERK/MAPK pathway for the treatment of HNSCC. We found that this toxin displayed strong systemic anti-tumor activity towards a variety of xenografted human HNSCC cell lines by inducing apoptotic and necrotic tumor cell death, and by impairing tumor cell proliferation and angiogenesis. Interestingly, the human HNSCC cell lines were insensitive to the intercomplementing toxin when cultured ex vivo, suggesting that either the toxin targets the tumor-supporting stromal cell compartment or that the tumor cell requirement for ERK/MAPK signaling differs in vivo and ex vivo. This intercomplementing toxin warrants further investigation as an anti-HNSCC agent.