Protein transfer-mediated modification of virus-like particles and plasma membrane vesicles: A means to adjuvant and deliver tumor antigens Public

Abstract

The optimal size and particulate nature of nano and microparticles make them ideal antigen delivery vehicles for immune activation. Encapsulation or surface attachment of antigen to these particles by chemical means has shown to enhance antigen-specific immune responses compared to administration of soluble antigen. However, chemical modifications can alter antigen immunogenicity and modify the particulate structure leading to decreased antigen-specific immunity. Herein, we propose a protein transfer method to modify lipid-based nano and microparticle surfaces with antigen and immunostimulatory molecules (ISMs). By protein transfer, glycosylphosphatidylinositol (GPI)-anchored forms of antigen and/or ISMs were incorporated stably onto influenza H5 virus-like particles (VLPs) or tumor-derived plasma membrane vesicles (PMVs) within 4 h. Multiple GPI-anchored proteins could be incorporated onto the particles simultaneously, and the incorporated proteins remained functional. Vaccination with GPI-GM-CSF-incorporated-VLPs led to enhanced VLP and virus-specific T helper 1 (Th1) and T helper 2 (Th2)-type IgG responses compared to unmodified VLPs as well as complete protection against a heterologous H5N1 viral challenge unlike vaccination with unmodified VLPs. Further, vaccination using influenza VLPs incorporated with a GPI-anchored form of tumor associated antigen, HER-2, led to enhanced protection against a HER-2-expressing murine breast cancer cell line compared to vaccination with corresponding levels of GPI-HER-2 alone. HER-2-specific antibody production characteristic of a Th1 and Th2-type immune response was also elevated in these mice. Similarly, vaccination with protein transferred-GPI-HER-2-PMVs also increased HER-2-specific Th1 and Th2-type immune responses and enhanced protection against live cell challenge of two murine breast cancers that express HER-2. Additionally, inclusion of GPI-IL-12 and GPI-B7-1 along with GPI-HER-2 on the PMVs by protein transfer led to complete protection against HER-2-expressing D2F2/E2 tumors in a prophylactic setting and delayed tumor growth with partial regression in a therapeutic setting. These studies suggest that protein transfer can be used to modify the surfaces of VLPs and PMVs to enhance the inherent particle immunogenicity, and to enhance immunogenicity against exogenously incorporated tumor antigens. These results have implications in enhancing the potency of particle-based vaccines against influenza viruses and the efficacy of protein-based vaccines against cancer.

Table of Contents

Chapter I: Introduction...1

Cell membranes...2

Modification of cell membranes...3

Protein transfer modification of cell membranes...4

Surface Engineering with GPI-anchored Proteins...5

Expression of proteins on the cell surface by protein transfer of GPI-APs...8

Characteristics of GPI-AP protein transfer...10

Advantages and limitations of GPI-AP protein transfer...13

Protein transfer of antigen-presenting cells with GPI-anchored MHC complexes...15

Protein transfer of GPI-APs in tumor immunotherapy...16

Nano and microparticles...18

References...22

Chapter II: Protein transfer-mediated surface engineering to adjuvantate virus-like nanoparticles for enhanced anti-viral immune responses...34

Chapter III: Influenza virus-like particles engineered by protein transfer with tumor-associated antigens induces protective antitumor immunity...84

Chapter IV: Plasma membrane vesicles engineered by protein transfer as an adjuvanted antigen delivery platform...123

Chapter V: Discussion...186

Use of enveloped VLPs to enhance immunogenicity of tumor antigens...188

Use of plasma membrane vesicles to enhance immunogenicity...191

Enhanced immunogenicity by GM-CSF...192

Enhanced tumor antigen-specific immune stimulation by IL-12 and B7-1...194

Advantages/disadvantages of protein transfer onto particles...198

Future studies using protein transfer technology onto lipid-based particles...199

Protein transfer for personalized cancer immunotherapy...201

Implications of protein transfer-modified VLPs and PMVs...202

References...203

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Health Sciences, Immunology Health Sciences, Oncology Biology, Virology
Mot-clé	membrane vesicles glycolipid anchor protein transfer virus-like particles
Committee Chair / Thesis Advisor	Selvaraj, Periasamy, Emory University
Committee Members	Spearman, Paul, Emory University Jacob, Joshy, Emory University Steinhauer, David, Emory University Kang, Sang-Moo, Georgia State University Compans, Richard W, Emory University

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