Anthrax is a well-characterized infectious disease caused by the sporulating bacteria Bacillus anthracis. The disease is historically associated with animal infections, especially herbivores such as cows, sheep, and goats, and is not typically found in humans. However, humans working with animal products where infection occurs are at risk of contracting anthrax. Some regions of the Middle East and sub-Saharan Africa are hyperendemic for anthrax, though the organism can often be found in many areas of the world. The disease manifests in three different ways: cutaneous, gastrointestinal and inhalation anthrax result from exposure of an open wound to spores, ingesting spores in contaminated meat products, or inhaling spores, respectively. While cutaneous anthrax has a fatality rate of up to 25 percent, gastrointestinal or inhalation anthrax results in nearly 100 percent fatalities. Definitive diagnosis of anthrax infection often comes too late to provide resuscitative care.
The principal virulence factor of B. anthracis is a multi-component toxin secreted by the organism. The toxin consists of three proteins designated protective antigen (PA), lethal factor (LF) and edema factor (EF), which are encoded by the genes pag, lef, and cya, respectively. PA is a 735 amino acid protein of molecular weight 83 kDa. It binds to the anthrax toxin receptor (ATR) on a mammalian cell surface, and subsequently undergoes a furin-mediated cleavage to yield a 63 kDa receptor-bound product. The 63 kDa PA fragment forms a heptameric complex on the cell surface which is capable of interacting with either LF or EF, and this complex is subsequently internalized. LF is a zinc metalloprotease that cleaves several isoforms of MAP kinase, thereby disrupting signal transduction events within a cell, eventually leading to cell death. LF is considered responsible for the lethal outcome of anthrax infection. EF is a calmodulin-dependent adenylate cyclase that causes deregulation of cellular physiology, leading to clinical manifestations that include edema. PA and LF together are referred to as lethal toxin.
The CDC lists anthrax as a category A disease agent and estimates the cost of an anthrax attack to exceed $26 billion per 100,000 persons exposed. Presently, the only vaccine licensed for human use in the U.S., Biothrax (formerly Anthrax vaccine adsorbed, or AVA), is an aluminum hydroxide-adsorbed, formalin-treated subunit vaccine based on protective antigen, PA. It is delivered by subcutaneous injection and induces immunity against lethal toxin secreted by the bacillus. The vaccine is produced from the filtered culture supernatant fraction of the V770-NP1-R strain of B. anthracis. The production process is complex, there is variation from batch-to-batch in vaccine preparation lots, and the precise composition of the vaccine is undetermined. Furthermore, since alum is included as an adjuvant with the current vaccine, a cold chain must be maintained during vaccine storage and distribution, adding inconvenience and cost. The vaccine is administered by injection, which can complicate the logistics of mass treatments. Thus, it would be desirable to have additional reagents capable of countering the infectious potential of an anthrax outbreak or attack.
Monoclonal antibodies are of increasing importance for a variety of therapeutic as well as diagnostic, industrial, and research purposes. For example, several animal studies have demonstrated anthrax toxin-specific antibodies from vaccinated animals can passively protect recipients from lethal effects of infection. However, animal-derived sera has obvious drawbacks which prevent widespread use as therapeutics. Monoclonal antibodies produced by hybridomas must be harvested from medium in which the hybridomas are cultured or harvested from mouse ascites fluid. Unfortunately, these production systems are expensive, labor-intensive, and have other significant disadvantages. For these reasons and others it would be desirable to be able to utilize alternative production systems for monoclonal antibodies such as production systems involving recombinant DNA technology.
Concerns regarding sufficient access and limited supply of reagents, product cost, and reagent purity underscore the urgent need for improved products and reagents. Thus, there exists a clear need and urgency for improved approaches to counter potential anthrax infection, as well as for improved methods of diagnostic detection, and research tools useful in examination of anthrax infection mechanism. Furthermore, it is desirable to provide production methods that allow for mass-production of products useful in such applications at reasonable cost.
USPTO Backgrounds
USPTO Backgrounds