Doctoral Dissertations

Fan Zhou

Date of Award

12-1992

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Leaf Huang

Committee Members

Steve J. Kennel, Robert N. Moore, Barry T. Rouse, Jeffrey M. Becker

Abstract

The major problem in inducing a CTL response in animals is that soluble antigens are processed in endocytic pathway for class Il-restricted presentation rather than entering the cytosolic pathway for the class I-restricted presentation. pH-sensitive liposomes, which can respond to pH changes and release spontaneously their entrapped contents, have been developed as vehicles for the cytosolic delivery of exogenous antigen from an acidic endocytic compartment. In previous studies, antigen presenting cells (APC) were sensitized with a soluble antigen delivered by pH-sensitive liposomes for CTL recognition. In order to optimize the delivery system for further mechanistic studies, we improved the method of loading antigen into pH-sensitive liposomes and characterized the liposomal delivery system in terms of cell uptake, catabolism, and presentation kinetics. In contrast to virus-infected cells which present constitutively endogenous viral antigen, antigen presenting cells sensitized by liposomal antigen exhibited transient kinetics of antigen presentation. By using immunoelectron microscopy, we confirmed that pH-sensitive liposomes delivered approximately 20 to 30% of ingested OVA to the cytosolic compartment. However, in vivo delivery of exogenous antigens for CTL induction did not require the acid sensitivity of the liposomes. pH-insensitive, conventional liposomes could also prime mice for specific CTL activity. Data obtained from a study on the CTL inductive formulations favor the conclusion that the membranous/lipidic structure of the antigen carrier is the critical component for a CTL inductive immunogen. Furthermore, CTL could not be induced by administering liposomal antigens after systemic depletion of macrophages with a specific macrophage killer, liposomes containing dichloromethylene diphosphonate. These data suggest that macrophages play an important role in processing and initiating the CTL response. Whether macrophages present the antigen directly to CTL precursors has yet to be demonstrated. Plain liposomes did not express optimal adjuvant activity for CTL induction. Incorporation of monophosphoryl lipid A (MPL), a potent immunomodulator, into the liposome formulation increased significantly the capacity to induce CTL. The MPL-containing liposomes also rendered the vaccine formulation capable of inducing CTL via other immunization routes (i.m., s.c., or i.p.) besides i.v. injection. Finally, we evaluated the role of CTL priming in antitumor immunity with a murine tumor which had been engineered to expresses a model tumor antigen, i.e. OVA. Immunization with liposomal OVA prolonged the mean survival time of mice when subsequently challenged with a lethal dose of OVA-expressing thymoma. The potential of liposomes in delivering soluble antigens for CTL immunity against tumors or other diseases was thus demonstrated.

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