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

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

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 Biology, Virology
Palavra-chave	microneedle vaccination influenza subunit vaccine influenza HA trimer immune response
Committee Chair / Thesis Advisor	Compans, Richard W, Emory University
Committee Members	Jacob, Joshy, Emory University Steinhauer, David, Emory University Katz, Jacqueline M, Emory University

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