SBIR-STTR Award

Anthrax is considered a possible bioterrorist threat because of its lethality and its durability. Several nations have had bioweapons programs in the past that produced large quantities of anthrax. Numerous anthrax scares and the recent deaths from exposure to anthrax spores sent through the mail have heightened concerns about protecting the population against anthrax. No mass vaccination program is envisioned because the existing anthrax vaccine is in short supply. In the absence of a vaccine, passive immunization with intravenous immunoglobulin (IVIG) of people exposed to anthrax spores is a reasonable strategy. This proposal is to develop a large animal system for producing human polyclonal antibody against anthrax. It has been shown that transgenic mice carrying an artificial human chromosome (HAC) produce human antibody of all classes and with a broad repertoire when challenged with antigen. In the current work, a similar strategy would be applied to cattle, where the yield of antibody would be far greater than with mice. We have already shown that we can create cattle clones that have a HAC containing the human IG genes, and that the chromosome is stable and is expressed throughout fetal development into neonatal life. Work is proposed to characterize the immune response to anthrax vaccine and purified anthrax proteins in normal and in cloned calves. This novel and practical solution to the limited supply of human IVIG has several advantages. First, it would enable the production of large quanitites of human antibody at a reasonable cost. Second, it would provide greater flexibility in designing immunization strategies for producing high, titer, high specificity antibody beyond what is possible with human volunteers. Third, it would provide a new enabling technology for producing clinically important human antibody reagents against other bacterial and viral pathogens.

Thesaurus Terms:
Bacillus anthracis, antibacterial antibody, bioterrorism /chemical warfare, cow, immunologic substance development /preparation, transgenic animal anthrax, immunoglobulin gene, intravenous administration, neutralizing antibody biotechnology, enzyme linked immunosorbent assay

Anthrax is a high risk bioterrorist threat. Despite high dose antibiotic therapy, several deaths occurred following the 2001 mail attack. Passive immunization with anti-anthrax immunoglobulins, in combination with antibiotics, is a promising approach for post-exposure treatment. Hematech is developing a genetically modified line of cattle that will produce human instead of bovine immunoglobulin. These cattle may be hyperimmunized with anthrax antigens and produce highly efficacious polyclonal antibodies for protection against anthrax, and many other bioterrorist agents. In phase I, we introduced a human artificial chromosome containing the entire sequences of human immunoglobulin heavy and light chain genes into bovine fibroblasts and produced transchromosomic (Tc) cattle using somatic cell cloning technology. We showed that Tc calves retain the HAC and produce low levels of human IgG in blood (2-30 mg/L). We also developed an immunization scheme, in wild wildtype Holstein steers, using various antigen-adjuvant formulations and booster vaccinations to produce high titer antibodies to anthrax. Furthermore, we developed and optimized ELISA assays, which were used to detect IgG titers to anthrax protective antigen, edema factor and lethal factor antigens, and we refined an in-vitro toxin neutralization assay, to quantify and demonstrate the biological potency of the antibodies. Under Phase II support we will vaccinate and boost Tc cattle produced in Phase I, collect plasma and purify human and bovine antibodies separately. We will refine an in vivo mouse protection assay and evaluate the therapeutic potency of the purified human antibody as compared to purified bovine antibody. We will also produce Tc calves in which the bovine heavy chain genes have been knocked out in an effort to increase levels of human immunoglobulin production. The ultimate goal of phase II is to complete the development of a bovine system for production of human polyclonal antibodies with efficacy in the treatment of anthrax infections.

Thesaurus Terms:
Bacillus anthracis, antibacterial antibody, chimeric protein, immunologic substance development /preparation, protein purification anthrax, immunoglobulin gene, intravenous administration, neutralizing antibody biotechnology, bioterrorism /chemical warfare, cow, enzyme linked immunosorbent assay, genetically modified animal