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STABILIZATION OF AN OLIGOMERIC PROTEIN ANTIGEN ENHANCES IMMUNOGENICITY BY ALTERNATIVE ROUTES OF IMMUNIZATION

Weldon, William Clyde (2010)
Dissertation (134 pages)
Committee Chair / Thesis Adviser: Compans, Richard W
Committee Members: Jacob, Joshy ; Katz, Jacqueline M ; Steinhauer, David
Research Fields: Health Sciences, Immunology; Biology, Virology
Keywords: influenza HA trimer; influenza subunit vaccine; microneedle vaccination; immune response
Program: Laney Graduate School, Biological and Biomedical Sciences (Immunology)
Permanent url: http://pid.emory.edu/ark:/25593/8kcfc

Abstract

STABILIZATION OF AN OLIGOMERIC PROTEIN ANTIGEN ENHANCES IMMUNOGENICITY BY ALTERNATIVE ROUTES OF IMMUNIZATION By William C. Weldon Structural studies of recombinant sHA indicate that the oligomeric status varies with HA subtype and immunogenicity is associated with trimeric proteins. To stabilize the oligomeric structure, we modified the C-terminus of the sHA derived from the H3N2 influenza virus A/Aichi/2/68 was modified with the GCN4pII trimerization repeat. This modification was found to stabilize the H3 sHA trimers while unmodified H3 sHA was a mixture of trimers, dimers and monomers. Conformation-specific monoclonal antibodies indicate that the trimeric sHA presented native epitopes while the unmodified sHA presented low-pH conformation epitopes. In vaccination studies, we observed enhanced immune responses with the stabilized trimeric sHA resulting in 100% protection against lethal challenge. Our results suggest that stabilization of trimeric H3 sHA enhances immunogenicity and confers protective immune responses by preserving epitopes present in the native trimeric structure of HA. We also investigated the efficacy of skin-based vaccination using microneedles coated with recombinant H3 sHA. Our data indicate that the modified trimeric sHA induces improved immune responses resulting in 100% protection against lethal challenge, and improved clearance of influenza virus from lungs of vaccinated mice after lethal challenge compared to subcutaneous vaccination. Coated-microneedle vaccination with the trimeric sHA induced increased serum and mucosal IgA compared to the unmodified sHA. In addition, we observed an increased Th1 helper T cell phenotype following microneedle vaccination with trimeric sHA. Our results demonstrate the efficacy of skin- based vaccination with a recombinant HA subunit vaccine. We extended our studies of stabilized H3 sHA trimers to investigate the effect of trimer stabilization of the H1 sHA derived from the swine-origin influenza virus A/California/04/2009. Modification of the H1 sHA with GCN4pII generated stabilized trimers while unmodified sHA formed monomers. However, vaccination studies indicate that the trimeric sHA was less immunogenic than the monomeric sHA, with neither recombinant H1 sHA conferring complete protection from lethal challenge. These data indicate that a different strategy for antigen design is required for the H1 sHA

Table of Contents

TABLE OF CONTENTS -- ABSTRACT -- ACKNOWLEDGEMENTS -- LIST OF TABLES -- LIST OF FIGURES -- INTRODUCTION -- 1 -- CHAPTER 1 - ENHANCED IMMUNOGENICITY OF STABILIZED TRIMERIC -- SOLUBLE INFLUENZA HEMAGGLUTININ -- 36 -- CHAPTER 2 - MICRONEEDLE VACCINATION WITH STABILIZED -- RECOMBINANT INFLUENZA HEMAGGLUTININ INDUCES -- IMPROVED PROTECTIVE IMMUNITY -- 63 -- CHAPTER 3 - RECOMBINANT TRIMERIC PANDEMIC SWINE-ORIGIN H1N1 -- INFLUENZA HEMAGGLUTININ -- 94 -- DISCUSSION -- 113

Files

application/pdf Dissertation/Thesis 134 pages (3.9 MB) [Access copy of Dissertation/Thesis]
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