Directed Evolution Of Bacillus Anthracis Protective Antigen And Screening Of Anthrax Antitoxin And Vaccine Candidate

Anthrax is a zoonotic and fatal disease caused by Bacillus anthracis, a rod-shaped, gram-positive, spore-forming bacterium. The bacterium or its spores can invade the body through skin abrasions, ingestion, or inhalation, resulting in cutaneous, gastrointestinal, or inhalational anthrax. Of the three forms, inhalational anthrax is regarded as the most lethal, with a mortality rate approaching 80%. Although the naturally occurring inhalational form of anthrax is rare, malicious release of anthrax spores, particularly as weaponized anthrax spores, in biowarfare and bioterrorism not only kills civilians but also creates great panic. This has stimulated the search for effective methods for the therapy and prevention of anthrax.The principal virulence factors of B. anthracis consist of an anti-phagocytic capsule composed of poly-D-glutamic acid (PGA) and a secreted bacterial toxin. The anthrax toxin, which is predominantly responsible for the etiology of anthrax, belongs to the family of bacterial binary A-B type toxins, which consist of a receptor-binding B subunit. i.e., the protective antigen (PA), and two catalytic A subunits, i.e., the lethal factor (LF) and edema factor (EF). PA combines with either LF or EF to form the lethal toxin (LeTx) or edema toxin (EdTx), respectively. PA, functioning as a vehicle, mediates the entry of either LF or EF into the cytosol of eukaryotic target cells. LF is a zinc metalloprotease that inactivates mitogen-activated protein kinase kinase, thus inducing cell death, and EF is a calmoduline dependent adenylate cyclase impairing host defences. The combination of respective catalytic effects leads to the manifestation of the symptoms of anthrax in several ways and at different stages of infection.The threat of using B. anthracis as a a bioterror weapon has been the impetus for anthrax-related studies, including toxins structure-function study, exploitation of new detection methods, development of next-generation vaccines and antitoxic drugs for anthrax prophylaxis and therapy. In this study, we mainly aimed to screen new antitoxin agents and develop a highly effective anthrax vaccine candidate. The principal work and main findings were summarized as follows: 1. Expression and purification of biologically active EF in E.coliEF is a calmoduline dependent adenylate cyclase and combines PA to form edema toxin that can causes a rapid increase in the intracellular cAMP levels of some sensitive cells, like CHO-K1. In this study, EF coding gene cya was cloned into expression vector pGEX-6p-1, resulting in pGEX-EF. EF was expressed in solube form in E. coli BL21-CodonPlus (DE3)-RIL strain with 0.2 mM of IPTG inducation at 28?, and then purified to high homogeneity via a single step affinity chromatography. We also detected the biological activity of recombinant EF by cAMP ananlysis, and found that the intracellular cAMP concentration of CHO-K1 cells treated with 1 ug/mL of EF in the presence of 1?g/mL of PA was 3 times higer than that of untreated cells. This suggested that the recombinant EF was biologically active.2. Screening and identification of dominant-negative mutants of protective antigen by directed evolution.Certain mutations within PA, defined as dominant-negative (DN) mutants, has been proven to co-oligomerize with the wild type PA and block its function to transport LF or EF. In this study, we first constructed PA mutation library by error prone PCR-based random mutagenesis. Then, we screened this mutant library and obtained four new DN-PA:V377E, T380S, 1432C and N435C. The characteristics of the DN mutant were investigated by proteolytic cleavage assay, heptamer formation and SDS-resistance of the heptamer assays. The results indicated that these mutations impaired the prepore-pore transit, thereby blocking the pore formation and LF/EF translocation. DN inhibition assay in in vitro models showed all the DN muants had high antitoxin activity. Animal protection assay showed that the two strongest DN mutants, V377E and N435C, were able to protect BALB/c mice against the lethal toxin challenge. These new DN mutants may be useful for the structure-function study of PA and constructing new antitoxic therapeutics and vaccines against anthrax.3. A chimeric protein that functions both as an anthrax dual-target antitoxin and trivalent vaccineThe PA-binding domain of LF (LFn) or its derivatives is sufficient for binding to the PA63 formed prepore and can inhibit the anthrax toxin by competitively inhibiting the binding of LF to the prepore. Another powerful antitoxin is the dominant-negative mutant of PA (DPA), which can be proteolytically activated to form dominant-negative inhibitory PA63 (DPA63). DPA63 co-assembles with wild-type PA63 and blocks its ability to translate LF/EF. In this study, we first demonstrated that the chimeric protein LFn-PA, created by fusing PA-binding domain of lethal factor (LFn) to protective antigen (PA), retained functions of the respective molecules. Based on this observation, we attempted to develop an antitoxin that targets the binding of lethal factor (LF) and/or edema factor (EF) to PA and the transportation of LF/EF. Therefore, we replaced PA in LFn-PA with a dominant-negative inhibitory PA (DPA), i.e., PAF427D. In in vitro models of anthrax intoxication, the chimera LFn-DPA showed 3-fold and 2-fold higher potency than DPA in protecting sensitive cells against anthrax lethal toxin (LeTx) and edema toxin (EdTx), respectively. In animal models, LFn-DPA exhibited strong potency in rescuing mice from lethal challenge with LeTx. We also evaluated the immunogenicity and immunoprotective efficacy of LFn-DPA as an anthrax vaccine candidate. In comparison with the recombinant PA, LFn-DPA induced significantly higher levels of the anti-PA immune response. Moreover, LFn-DPA elicited an anti-LF antibody response that could cross-react with EF. Mice immunized with LFn-DPA tolerated a LeTx challenge that was 5 times its LD50 value. Thus, LFn-DPA represents a highly effective trivalent vaccine candidate for both pre-exposure and post-exposure vaccination. Overall, we have developed a novel and dually-functional reagent for the prophylaxis and treatment of anthrax.