Lawrence K. Silbart, Ph.D.
Associate Professor

Abbreviated C.V.

Project 1: Mucosal vaccines to protect livestock from primary infection with foot-and-mouth disease virus (FMDV). We have an ongoing USDA NRI grant to develop peptide vaccines to protect livestock from FMDV. This work involves the development of peptide vaccines, mucosal adjuvants, and novel delivery systems. We are currently using a porcine model, and perform vaccination and a variety of immunological assays to assess correlates of immune protection (ELISA, ELISPOT, flow cytometry and immunohistochemistry). More recent work focuses on designing vaccines that are structurally similar to secondary structures found in the viral VP1 G-H loop. We are also working collaboratively with Dr. Roberto Gaxiola and Dr. Jane Knapp to express FMDV epitopes fused to mucosal delivery molecules, in transgenic plants to induce mucosal immunity through feeding. Dr. John Zinckgraf, Roger Barrett, Rupa Challa and Debra Rood are actively involved in these projects.

Project 2: Determining the correlates of immune protection of a live attenuated Mycoplasma gallisepticum vaccine for poultry. In collaboration with Dr. Steven Geary's laboratory, we have participated in the development of a live-attenuated M. gallisepticum vaccine that confers protection to chickens. This protection appears to be antibody-mediated, yet several paradoxical observations emerged during our assessments of immune protection.  The first unanticipated observation was that only very moderate amounts of IgG are produced in respiratory secretions following vaccination with the attenuated vaccine strain, and little or no secretory IgA was detected. We are currently trying to determine if the IgG was produced by resident plasma cells within the respiratory mucosa, or simply transudated from serum. The second paradox was observed in sham immunized birds challenged with pathogenic M. gal. These birds produced copious amounts of secretory IgA anti-M. gal., yet were unable to clear the organism. In addition, a vigorous inflammatory response resulted in mucosal thickening, desquamation, accompanied by a massive lymphocytic/heterophilic infiltration of the mucosa. This "futile" IgA response and inflammation is clearly maladaptive, and is undoubtedly driven by immunomodulatory mechanisms that have evolved in Mycoplasma. We hope to characterize the mediators responsible for this response (cytokine, chemokine etc.) and identify the mechanisms responsible for this aberrant response. We are currently pursuing the hypothesis that Mycoplasmal phage variation, in which one of 38 different genes encoding surface variable lipoproteins are expressed on the surface of the organism, is responsible for the slow clearance of the organism. We are using techniques such as ELISA, ELISPOT, flow cytometry, immunohistochemistry and electron microscopy to address these questions. Mohammed Javed and Debra Rood are actively involved in this project, as well as several students from Dr. Geary and Dr. Frasca's lab (PVS).

Project 3: Development of more effective DNA vaccines. One of the problems plaguing DNA vaccines is their relatively low efficiency, especially in large, agriculturally important species such as livestock. One of the reasons for this inefficiency is due to the low level of expression of DNA constructs upon cell transfection. Almost all DNA vaccines use strong viral promoters (such as the cytomegalovirus immediate early promoter) to maximize expression, but very little thought is given to downstream regulatory elements at the 3'-end of the gene. Traditionally, the 3' regulatory elements that are used in DNA vaccine consist of the bovine growth hormone polyadenylation region. This region does not contain any enhancer elements, and provides little more than mRNA stability to DNA vaccines through poly-adenylation. We have recently substituted three different 3'-polyadenylation sequences into otherwise identical DNA vaccines and have found that enhancer regions derived from the Hepatitis B virus can dramatically enhance expression. When animals were vaccinated with such constructs, we observed stronger antibody responses, higher seroconversion rates, and alterations in cytokine levels. A manuscript describing these results has recently been accepted in the journal "Vaccine." Since submission of the manuscript, we have found that these vaccines also provide a much better prime/boost response when protein is used in the booster vaccination. We intend to examine a number of regulatory sequences within the 3'-untranslated region to further improve responses to DNA vaccines. We also intend to examine alterations in the immune response when low-efficiency plasmids are used to determine if a state of immunological tolerance or "ignorance" can be achieved. This project will require students with a strong interest in genetic engineering approaches.

Project 4. We are working to develop a transgenic tobacco plant expressing a recombinant single chain antibody that will bind to an important tobacco specific nitrosamine that is, in part, responsible for initiating cancer in the oral cavity. We hope to test the hypothesis that by intercepting the carcinogen prior to its interaction with the oral epithelial surface, the bioactivation and subsequent DNA damage in epithelial cells can be reduced or prevented. An undergraduate student, Dan Zapata is working on this project, along with Ms.Debra Rood. Dr. John Zinckgraf is overseeing parts of this project (primarily the molecular biology). This project will be performed collaboratively with Dr. Jan Knapp once the constructs have been created.

Recent publications and manuscripts submitted:

• Immune Response to a Mucosally Administered Aflatoxin B₁ Vaccine (.pdf)
• Modulating gene expression using DNA vaccines with different 3´-UTRs influences antibody titer, seroconversion and cytokine profiles. (.pdf)
• Intranasal immunization of guinea pigs with an immunodominant FMDV Peptide Conjugate induces mucosal and humoral antibodies and protection against challenge. (.pdf)
• A modified live Mycoplasma gallisepticum vaccine to protect chickens from respiratory disease. (.pdf)