IVIAT™ and CMAT™
Background. IIVIAT™ and CMAT™ are two related platform technologies that enable the simple, fast identification of novel and potentially important gene targets associated with the natural onset and progression of infections, cancers and other diseases in humans and other living organisms, including plants. These technologies offer us the potential to generate and develop a number of product candidates for future out-licensing to corporate partners, particularly in the area of cancer and tuberculosis, as well as agricultural and other non-human uses.
The IVIAT™ technology platform was developed by Dr. Jeffrey Hillman and co-scientists. IVIAT™ stands for In Vivo Induced Antigen Technology. IVIAT™ can quickly and easily identify in vivo induced genes in human infections without the use of animal models, facilitating the discovery of new targets for the development of vaccines, antimicrobials and diagnostics. Dr. Hillman and his collaborators have further developed methods based on this approach to create CMAT™, which stands for Change Mediated Antigen Technology. CMAT™ can be used to identify gene targets associated with the onset and progression of cancerous processes and autoimmune diseases. It can also be used to identify novel genes in plant diseases, including genes expressed by the pathogen when it causes the disease and genes expressed by the plant in response to the disease. In April 2007, the National Cancer Institute notified Oragenics that it had been awareded a Phase 1 SBIR grant to help support the research for identifying colorectal cancer markers. CMAT can also be used to study a vast array of problems ranging from autoimmune disease to biofilm formation and biofouling to agriculturally important plant infections.
Microbial infections are complex, dynamic processes that evolve constantly within the host. In many instances, virulence gene expression is modulated in response to the changing environment encountered at the site of infection. It is unlikely that all regulated virulence determinants of a pathogen can be identified in vitro because it is technically impossible to determine and mimic all of the complex and changing environmental stimuli that occur at the site of an infection. Pioneering technologies such as in vivo expression technology were designed to identify genes specifically expressed in vivo, and thereby fill the gaps in our understanding of how microorganisms cause disease. All of these methods depend on a reasonable assumption: namely, those genes that are specifically induced during in vivo growth are likely to be important to the pathogenic process. Although remarkably powerful, all of these technologies have certain limitations. One of their major drawbacks is that they depend on the use of animal models of infection. Animal models are not available for many pathogens and, in those cases where animal models are available, they might not closely approximate the human condition. Consequently, a number of examples exist in the literature where the results of animal model testing have been misleading. IVIAT™ is a revolutionary approach for studying microbial pathogenesis. The major conceptual and technological breakthrough is that our method does not rely on animal models. Instead, it identifies genes expressed during an actual human infection.
Technical Background. IVIAT™ avoids the use of animal models by using serum from patients who have experienced disease caused by the pathogen of interest. By pooling sera from several different patients, we can be assured of finding the widest possible array of antigens produced during different stages of infection. The pooled serum is absorbed with whole cells and cellular extracts prepared from the pathogen grown in vitro. The resulting adsorbed serum contains the subpopulation of antibodies reactive against in vivo induced antigens. This adsorbed serum is used to probe an inducible expression library containing DNA from the pathogen of interest cloned into an appropriate host such as Escherichia coli. Reactive clones contain a DNA fragment from the pathogen that encodes an in vivo antigen. IVIAT™ has proven its ability to identify novel targets for a number of infectious agents including Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa. Oragenics is currently working with a collection of IVIAT™ identified genes of Mycobacterium tuberculosis that are specifically induced during active infection of human patients. These gene targets offer the promise of a new tuberculosis diagnostic test to meet a critical worldwide need, and could potentially serve as the basis for an effective new vaccine against tuberculosis infection.
The IVIAT™ process does not require known function to identify a gene target. Given the state of knowledge, a gene of unknown function that is active only during the process of disease may be the most valuable target of all, particularly from the point of view of developing an intellectual property position.
CMAT™ is more general than IVIAT™ and allows us to study situations where the host does not mount an antibody response. Proof of principle has been accomplished using Xanthomonas campestris infection of the common bean plant, where we have identified both novel virulence genes of the pathogen and novel resistance genes of the host. Essentially, plant tissue can be harvested and quick frozen or otherwise treated to preserve antigens of the pathogen that are present. At the same time, proteins made by the plant in response to the infection are also preserved. When this tissue is mixed with adjuvant and used to immunize an appropriate host, the immune serum that is obtained can be adsorbed with in vitro grown cells of the pathogen to create a probe for identifying virulence genes of the pathogen. It can also be adsorbed with healthy plant tissue to identify resistance genes of the host.
This method can also be used to study a vast array of problems ranging from autoimmune diseases to biofilms to cancer. In the last of these, for example, cancerous human tissue will be surgically excised and used to immunize an appropriate host. The immune serum will then be adsorbed with healthy tissue from the homologous human subject so that remaining antibodies will be directed at proteins made by the cancer.
Scientific Articles (Click links below to view articles if available)
Cheng, S., Nguyen, M.H., Zhang, Z., Jia, H., Handfield, M., Hillman, J.D., Progulske-Fox, A. and Clancy, C.J. Evaluating the roles of NOT5, NOT3, BUR2, and KEL1 in Candida albicans virulence using gene disruption strains that express URA3 from the native locus. J. Mol. Microbiol., submitted.
Harris, J.B., Baresch-Bernal, A., Rollins, S.M., Alam, A., Larocque, R.C., Bikowski, M., Peppercorn, A.F., Handfield, M., Hillman, J.D., Qadri, F., Calderwood, S.B., Hohmann, E., Breiman, R.F., Brooks, W.A., and Ryan, E.T. 2006. Identification of in vivo-induced bacterial protein antigens during human infection with Salmonella enterica serovar Typhi. Infect. Immun.74:5161-8.
Manohar, J., Kudva, I.T., Griffin, R.W., Dodson, A., McManus, B., Krastins, B., Sarracino, D., Progulske-Fox, A., Hillman, J.D., Handfield, M., Tarr, P.I. and Calderwood, S.B. Identification of Escherichia coli 0157:H7 proteins expressed specifically during human infection using in vivo-induced antigen technology (IVIAT). Mol. Microbiol., In press.
Kowthar, Y.S., Cvitkovitch, D.G., Chang, P., Blast, D.J., Handfield, M., Hillman, J.D. and de Azavedo, J.C.S. Identification of Group A Streptococcus antigenic determinants upregulated in vivo. Mol. Microbiol. In press.
Handfield, M., Progulske-Fox, and Hillman, J.D. 2005. In vivo induced genes in human diseases. Periodontol. 2000 38:123-134.
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