Listeria monocytogenes as a biologic vaccine vector against feline immunodeficiency virus (FIV)

by Stevens, Rosemary Greer.

Stevens, Rosemary. Listeria monocytogenes as a Biologic Vaccine Vector Against Feline Immunodeficiency Virus (FIV). (Under the direction of Dr. Gregg A. Dean) Listeria monocytogenes is attractive as a vaccine vector against human immunodeficiency virus (HIV) for multiple reasons. It induces strong cell-mediated immunity, can be delivered mucosally, is easily manipulated to express foreign antigens, is amenable to transport plasmids, is inexpensive to produce and is responsive to antibiotic treatment. Recent murine studies with orally delivered recombinant L. monocytogenes expressing HIV Gag resulted in strong CTL responses to Gag in the spleen and gut-associated lymphoid tissues. As the gut is the site of early immune defects caused by HIV infection, a successful vaccine will need to target this vulnerable site. Using the feline immunodeficiency virus (FIV) model of HIV we evaluated a recombinant L. monocytogenes in an FIV challenge system. In the first study, five cats were immunized with recombinant L. monocytogenes that expresses FIV Gag and delivers an FIV Env-expressing DNA vaccine (LMgag/pND14-Lc-env). Control cats were either sham immunized or immunized with wild-type L. monocytogenes (10403S, LM-wt). All cats were challenged vaginally three months post immunization with FIV strain NCSU1. One year post challenge all animals were sacrificed and tissues were harvested. Provirus was not detected by real-time PCR in any tissue evaluated from cats immunized with the recombinant bacteria. These cats also maintained normal CD4:CD8 ratios in mesenteric lymph nodes while control cats had inverted ratios. Recombinant L. monocytogenes vaccinated cats also maintained normal percentages of CD4 and CD8 T cells in intestinal epithelium while control cats showed depletion of both populations. Vaginal FIV-specific immunoglobulin A was present in three immunized cats before challenge and in all five at one-year post challenge. This study demonstrates that while a single, low-dose immunization with LMgag/pND14-Lc-env did not prevent FIV infection, it did reduce viral loads and enabled immunized cats to control viral replication. The second study addresses two questions, 1) vaccine efficacy in the face of preexisting vector immunity, and 2) evaluation of peripheral blood as an indicator of vaccine-induced immune responses. Pre-existing immunity is a critical consideration in light of diminished effectiveness of adenovirus and recombinant vaccinia virus upon repeated exposure. We examined the immunogenicity of LMgag/pND14-Lc-env in cats previously infected with LM-wt. Eight subcutaneously infected cats were divided into two groups of four. One group received 5x106 cfu LM-wt orally followed two months later with 1x108 cfu oral LMgag/pND14-Lc-env (SQ/Oral). The other group received only 1x108 cfu oral LMgag/pND14-Lc-env. After a single oral dose of LMgag/pND14- Lc-env, cats with existing anti-L. monocytogenes immune responses developed anti-FIV Gag IgA titers in vaginal secretions, saliva, and feces. Similarly, FIV Gag and Env specific IFN-? ELISPOT responses were measurable in spleen and lymph node, but at a statistically higher frequency in cats exposed to a single subcutaneous dose of wild-type L. monocytogenes versus those cats exposed both subcutaneously and orally. These two groups of cats were also used to answer the question of appropriateness of peripheral blood as a measurement of systemic and mucosal vaccine-specific immune responses. Our data show that extended culture was required to measure low-frequency antigen-specific immune responses in peripheral blood. Our results also show that there is no relationship between response measured in PBMC and that observed in splenic or lamina propria lymphocytes. Together, these studies show the ability of the oral vaccine LMgag/pND14-Lc-env to control viral load upon vaginal challenge and to generate anti-FIV antibodies and CD8 T cell response in the face of existing anti-listerial immunity. Listeria monocytogenes as a Biologic Vaccine Vector against Feline Immunodeficiency Virus (FIV) By Rosemary Stevens A dissertation submitted to the Graduate faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Immunology Raleigh 2004 Approved by Dr. Frederick J. Fuller Dr. Edward A. Havell Dr. Mary Jo Burkhard Dr. Gregg A. Dean Chair of Advisory Committee