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.  PIVIAT is a revolutionary approach for studying microbial pathogenesis.  The major conceptual and technological breakthrough is that this method does not rely on animal models.  Instead, it identifies genes expressed during an actual human infection.

Technical Background.  PIVIAT 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, one 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.  PIVIAT has proven its ability to identify novel targets for a number of infectious agents including Vibrio cholera, Escherichia coli and Pseudomonas aeruginosa.  Oragenics is currently working with a collection of PIVIAT identified genes of Mycobacterium tuberculosis that are specifically induced during active infection of human patients.  These genes, and expressed proteins, are being validated by preeminent tuberculosis experts to determine their potential usefulness in new diagnostic and vaccine applications.
            The PIVIAT 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.
            PCMAT is more general than PIVIAT and allows the study of 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 Oragenics’ scientists 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 the 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 can be surgically excised and used to immunize an appropriate host.  The immune serum can then be adsorbed with healthy tissue from the homologous human subject so that remaining antibodies are directed at proteins made by the cancer.  With PCMAT, the proof-of-concept for a human disease was demonstrated for colorectal cancer, CRC.  Partial funding for this project was provided through an SBIR grant from NCI.  Initial work was focused on Stages 1,2 and 4 of this disease and over 60 proteins were identified from each stage.  Bioinformatics analysis showed that approximately 50% of the targets were previously identified, but that approximately half from each stage of CRC were relevant, and these markers were the subject of a patent application filed in 2008.  Oragenics and its collaborator on this project, a large international diagnostics firm, have culled the markers for stages 1, 2 and 4 down to thirteen candidates, which will be further reduced to the most promising 2-3 markers using immunocytochemistry approaches for ultimate incorporation into a diagnostic assay for CRC.

Scientific Articles (Click links below to view articles if available)

Hillman, J.D. 1978. Lactate dehydrogenase mutants of Streptococcus mutans: isolation and preliminary characterization. Infect Immun 21: 206-212.

Johnson, C.P., Gross, S.M. and Hillman, J.D. 1980. Cariogenic potential in vitro in man and in vivo in the rat of lactate dehydrogenase mutants of Streptococcus mutans. Arch Oral Biol 25: 707-713.

Socransky, S.S., Tanner, A.C.R., Haffajee, A.D., Hillman, J.D., and Goodson, J.M. 1982. Present status of studies on the microbial etiology of periodontal disease. In: Host-Bacterial Interactions in Periodontal Disease (R.J. Genco and P. Mergenhagen, eds.), A.S.M. Publications, Washington D.C., pp. 1-12.

Hillman, J.D. and Socransky, S.S. 1982.  Bacterial interference in the oral ecology of Actinobacillus actinomycetemcomitans and its relationship to human periodontosis. Arch Oral Biol 27: 75-77.

Hillman, J.D., Socransky, S.S., and Shivers, M. 1985. The relationships between streptococcal species and periodontopathic bacteria in human dental plaque. Arch Oral Biol 30: 791-795.

Hillman, J.D., Yaphe, B.I., and Johnson, K.P. 1985. Colonization of the human oral cavity by a strain of Streptococcus mutans. J Dent Res 64: 1272-1274.

Socransky, S.S., Haffajee, A.D., Dzink, J.L., and Hillman, J.D. 1988. Associations between microbial species in subgingival plaque samples. Oral Microbiol Immunol 3: 1-7.

Hillman, J.D. and Shivers, M. 1988. Interaction between wild-type, mutant and revertant forms of the bacterium Streptococcus sanguis and the bacterium Actinobacillus actinomycetemcomitans in vitro and in the gnotobiotic rat. Arch Oral Biol 33: 395-401.

Hillman, J.D., McDonell, E., Hillman, C.H., and Zahradnik, R.T.  Safety assessment of ProBiora3™, a probiotic mouthwash: subchronic toxicity study in rats.
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Zahradnik, R.T., Magnusson, I., Walker, C., McDonell, E., Hillman, C.H., and Hillman, J.D.  Preliminary assessment of safety and effectiveness in humans of ProBiora3™, a probiotic mouthwash. J Applied Micro. In Press.