Bacillus anthracis, the causative agent of anthrax, has been the focus of much research and attention following the release of spores through the US mail system in 2001. 22 cases of infection resulted in 5 deaths, causing much concern regarding treatment, therapeutics and vaccine efficacy. Recently, the CDC discontinued the administration of the current anthrax vaccine (Anthrax Vaccine Adsorbed -AVA) due to adverse side effects observed in a large percentage of volunteers. This revocation of available vaccine has left healthcare workers, laboratory personnel and first responders with only limited means of protection following potential exposures to anthrax spores.
In humans, the anthracis bacilli can cause three types of infections: cutaneous via abrasions in the skin, gastrointestinal through ingestion of spores in contaminated meat and inhalation when spores less than 5 uM um are deposited into the lungs . The mortality rates vary between each form of the disease with cutaneous anthrax presenting as a self-limiting and treatable infection with only a 20% case fatality rate. When left untreated gastrointestinal infections can progress rapidly and have over 80% case fatality rates. Inhalation anthrax infections are rare but have a high case fatality rate (over 75%) even with antibiotic treatment.
Treatment options for patients presenting with symptoms of inhalational anthrax infections are limited and are generally ineffective at reducing mortality. Although antibiotic therapy is effective in the early stages of infection, it does not have any effect on the bipartite exotoxins, which are the major contributing factors to the mortality observed in acute anthrax infections . The current lack of an approved, available vaccine puts laboratory workers, military personnel and first responders at an increased risk of inhalational anthrax should another terrorist event, similar to the anthrax mailings in 2001, occur. Clearly there is a need for an effective vaccine as well as a well-tolerated, economical, post-exposure therapeutic for the treatment of human anthrax infections.
Passive immunotherapy is a non-chemical therapeutic providing immediate immunity to infectious agents and toxins. This treatment option has been shown to be effective against many diseases including anthrax [2–6] and other biothreat agents [7, 8]. Several approaches have been used previously for the production of immunotherapeutics specific for B. anthracis although they all have significant drawbacks. The pooling of immune serum from previously vaccinated volunteers yields highly protective anti-sera in very small quantities, limiting its use as a source of therapeutics for the Strategic National Stockpile or as a commercially available product. Monoclonal antibodies are highly specific, limiting their application to a single antigenic target and have a high cost associated with their development further limiting their feasibility for mass production and stockpiling. In the past animal vaccination has successfully been used to generate immunotherapeutic antiserum specific for infectious and toxic agents including snake venom, botulism toxin and Ebola virus [9–12] but limitations in quantity and safety have prevented their widespread use in the development of human therapeutics. Horses can provide large amounts of antiserum but are costly to maintain. Mice, rabbits and guinea pigs are inexpensive to maintain but yield limited volumes of anti-sera. Goats provide a renewable source of plasma and serum; however they have not been traditionally used in the generation of passive immunotherapeutics. We have plasmapheresed hyper immunized goats to successfully produce liters of GMP-grade antisera following a short immunization schedule (3 immunizations over 14 weeks), with minimal cost.
Bacillus anthracis produces two separate exotoxins, edema toxin (EdTx) and lethal toxin (LeTx). The two exotoxins utilize a common cell binding component termed protective antigen (PA83, 83 kDa) which binds to the ubiquitous anthrax toxin receptor (ATR) found on most cell surfaces. Once PA83 is bound to the host cell surface, a furin-like protease cleaves the full-length, inactive protein into the active form, PA63 (63 kDa), thereby exposing the binding sites for the catalytic components of the exotoxins (edema factor, EF or lethal factor, LF). A heptamer composed of PA63 + three LF/EF moieties [13, 14] forms on the cell surface and is internalized via receptor mediated endocytosis. The subsequent decrease in pH within the endosome causes conformational changes in PA63, so that it inserts into the endosomal membrane, forming a protease-stable pore; formation of this pore allows EF and LF to enter the cell and exert their toxic effects . LeTx is formed when PA63 is combined with LF, and is responsible for the most severe intoxicative effects of anthrax infection. EF is an adenylate cyclase capable of causing severe disregulation of cellular cAMP levels . LF has been shown to be a zinc-dependant metalloprotease with specificity for mitogen-activated protein kinase kinases (MAPKKs) capable of disrupting several cell signaling cascades; however, its specific mode of action is still unclear [17, 18]. Disruption of the binding of PA to ATR or LF would disrupt internalization of functional LeTx and would thereby prevent toxin-mediated death of the host following rapid multiplication of the bacilli.
Here we immunized goats with recombinant PA83, coupled to a novel non-toxic muramyl dipeptide derivative (NT-MDP) capable of inducing both innate and humoral immunity and does not induce clotting even when administered at high concentrations. The resulting polyclonal anti-sera conferred protection against in vitro and in vivo intoxication with the anthrax lethal toxin (LeTx) and in vivo intranasal challenge with virulent B. anthracis spores. Recently, we have shown that the passive transfer of goat-derived anti-HIV antibodies to failing therapy AIDS patients has been well tolerate, safe and effective [19–21].
In order to circumvent any hypersensitivity reactions associated with goat IgG, we have explored the use of F(ab')2 antibodies lacking the Fc region of the IgG molecule. The Fc region of the IgG is involved in the activation of complement, and patients with a pre-developed sensitivity to goat proteins may be at a higher risk of developing fatal allergic reactions following the administration of a goat-based antibody therapy. Removal of the Fc region allows for the retention of the dimeric antigen binding sites while increasing the safety of the immunotherapeutic without a significant loss in neutralizing capabilities.
Our data suggests that the administration of anti-PA83 goat IgG or F(ab')2 would provide an efficacious and well-tolerated passive immunotherapy for post-exposure treatment of acute human anthrax infections. Most notable is the rapidity with which the anti-sera were produced in goats and the volume of anti-sera generated from a single plasmapheresis. In addition, this data serves a proof of concept that a rapid, inexpensive, GMP-grade immunotherapeutic can be produced in a short enough timeframe for an emerging disease event like SARS-CoV.