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Methods in
Molecular Biology 1143

Monica Rinaldi
Daniela Fioretti
Sandra Iurescia Editors

DNA Vaccines
Methods and Protocols
Third Edition
METHODS IN M O L E C U L A R B I O LO G Y

Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651
 DNA Vaccines

Methods and Protocols

Third Edition

Edited by

Monica Rinaldi
Section of Medical Biotechnology, Institute of Translational Pharmacology (IFT),
National Research Council (CNR), Rome, Italy

Daniela Fioretti
Section of Medical Biotechnology, Institute of Translational Pharmacology (IFT),
National Research Council (CNR), Rome, Italy

Sandra Iurescia
Section of Medical Biotechnology, Institute of Translational Pharmacology (IFT),
National Research Council (CNR), Rome, Italy
Editors
Monica Rinaldi Daniela Fioretti
Section of Medical Biotechnology Section of Medical Biotechnology
Institute of Translational Pharmacology (IFT) Institute of Translational Pharmacology (IFT)
National Research Council (CNR) National Research Council (CNR)
Rome, Italy Rome, Italy

Sandra Iurescia
Section of Medical Biotechnology
Institute of Translational Pharmacology (IFT)
National Research Council (CNR)
Rome, Italy

ISSN 1064-3745 ISSN 1940-6029 (electronic)

ISBN 978-1-4939-0409-9 ISBN 978-1-4939-0410-5 (eBook)
DOI 10.1007/978-1-4939-0410-5
Springer New York Heidelberg Dordrecht London

Library of Congress Control Number: 2014934311

© Springer Science+Business Media New York 2014

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this
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Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the
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for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution
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The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not
imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and
regulations and therefore free for general use.
While the advice and information in this book are believed to be true and accurate at the date of publication, neither
the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be
made. The publisher makes no warranty, express or implied, with respect to the material contained herein.

Printed on acid-free paper

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Springer is part of Springer Science+Business Media (www.springer.com)
Preface

Vaccination has had a profound positive effect on the quality of public health. Vaccines have
long been used to combat infectious disease; however, the last decade has witnessed a revo-
lution in the approach to vaccine design and development. Several groundbreaking studies
demonstrated that immunological responses could be generated against antigenic trans-
genes delivered via DNA vaccination. Since then new sophisticated technologies, advances
in molecular biology techniques, and new bioinformatics analysis tools to study and manip-
ulate the basic elements of an organism’s genome have been used for the rational design
and production of DNA vaccines.
Nowadays, DNA vaccination is the most important early application of nonviral gene
therapy, and it seems clear that the route of DNA vaccine and the methods of vaccine
preparation have strong effects on the immune response and the effectiveness of that
response in preventing or treating disease.
DNA Vaccines: Methods and Protocols, Third Edition reviews innovative approaches and
technologies used to design, deliver, and enhance the efficacy of DNA vaccines. In this
book, expert international authors critically review the current cutting-edge research in
DNA vaccine design and development. Topics also include methods of production and
purification. The book also has chapters on recent DNA vaccine applications which should
be of great value in moving vaccines from research to clinic. All of these chapters, as well as
the others presented in the previous DNA Vaccines editions, have the important role of
further documenting the potential of the DNA vaccination as a platform technology for
treatment and prevention of human diseases suitable also for developing nations. Several
peculiar features of DNA vaccines (i.e., preparation and purification, stability, cost-
effectiveness and non-requirement of cold chain) emphasize this prospect.
The current status of three gene vaccines licensed for veterinary use (i.e., the West Nile
virus DNA vaccine for horses, a fish DNA vaccine against the Infectious Haematopoietic
Necrosis virus, and the Canine Malignant Melanoma vaccine (ONCEPT™)) will pave the
way for future application in humans.
To date, no human DNA vaccine has been licensed; however, during recent years, more
than 100 clinical trials have been undertaken worldwide on DNA vaccines covering the full
range of prophylactic through to therapeutic vaccines against infections, cancers, and a range
of other disorders (details at: http://www.dnavaccine.com/; http://clinicaltrials.gov;
http://www.cancer.gov/clinicaltrials).
DNA-based vaccine technology has moved from pioneering animal studies to clinical
testing quite rapidly. However, more work is still required on design and delivery to lift the
immunogenicity of DNA vaccines to the levels required for human regulatory approval and
commercial exploitation.

v
vi Preface

Consistent with the approach of the Methods in Molecular Biology series, DNA Vaccines,
Third Edition contains detailed practical procedures on the latest DNA vaccine technology
and is recommended to microbiologists and vaccinologists, immunologists, infectious dis-
eases and public health physicians, and to the many scientists working on vaccine
development (e.g., biochemists, and molecular biologists).
In conclusion, we hope that this book will be a productive opportunity to further push
the recent improvements in DNA vaccine technology to their full clinical potential, moving
from the benchtop to the patient.

Rome, Italy Monica Rinaldi

Daniela Fioretti
Sandra Iurescia
Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

PART I DNA VACCINE DESIGN AND ENHANCEMENT

1 A Blueprint for DNA Vaccine Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Sandra Iurescia, Daniela Fioretti, and Monica Rinaldi
2 Enhancement of Plasmid-Mediated Transgene Expression . . . . . . . . . . . . . . . . 11
Daniela Fioretti, Sandra Iurescia, and Monica Rinaldi
3 Strategies for Improving DNA Vaccine Performance . . . . . . . . . . . . . . . . . . . . 21
Sandra Iurescia, Daniela Fioretti, and Monica Rinaldi
4 Enhancement of DNA Vaccine Efficacy
by Intracellular Targeting Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Elisabete Borges Freitas, Ana Margarida Henriques,
Miguel Fevereiro, Duarte Miguel Prazeres,
and Gabriel Amaro Monteiro
5 Progresses in DNA-Based Heterologous Prime-Boost
Immunization Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Ronald J. Jackson, David B. Boyle, and Charani Ranasinghe
6 Development of Antibiotic-Free Selection System
for Safer DNA Vaccination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Jeremy M. Luke, Aaron E. Carnes, and James A. Williams

PART II DELIVERY SYSTEM

7 Electroporation-Based DNA Delivery Technology:
Methods for Gene Electrotransfer to Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Anita Gothelf and Julie Gehl
8 DNA Vaccination in Skin Enhanced by Electroporation. . . . . . . . . . . . . . . . . . 123
Kate E. Broderick, Amir S. Khan, and Niranjan Y. Sardesai
9 Intradermal Vaccination by DNA Tattooing . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Joost H. van den Berg, Koen Oosterhuis, Ton N.M. Schumacher,
John B.A.G. Haanen, and Adriaan D. Bins
10 Microneedle Applications for DNA Vaccine Delivery to the Skin . . . . . . . . . . . 141
Hae-yong Seok, Hyemee Suh, Sunghyun Baek, and Yeu-Chun Kim
11 Multivalent DNA-Based Vectors for DNA Vaccine Delivery. . . . . . . . . . . . . . . 159
Young Hoon Roh, Kwang Lee, Jessica Jane Ye, and Dan Luo

vii
viii Contents

12 Superparamagnetic Nanoparticle Delivery of DNA Vaccine . . . . . . . . . . . . . . . 181

Fatin Nawwab Al-Deen, Cordelia Selomulya, Charles Ma,
and Ross L. Coppel

PART III PRODUCTION, PURIFICATION, AND QUALITY

13 Plasmid Fermentation Process for DNA Immunization Applications . . . . . . . . 197
Aaron E. Carnes and James A. Williams
14 Pharmaceutical Grade Large-Scale Plasmid DNA Manufacturing Process. . . . . 219
Marco Schmeer and Martin Schleef

PART IV NEW VACCINE APPLICATIONS

15 Protective and Therapeutic DNA Vaccination Against Allergic Diseases . . . . . . 243
Almedina Isakovic, Richard Weiss, Josef Thalhamer,
and Sandra Scheiblhofer
16 Immunotherapy for Alzheimer’s Disease:
DNA- and Protein-Based Epitope Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
Hayk Davtyan, Irina Petrushina, and Anahit Ghochikyan
17 Tetravalent DNA Vaccine Product as a Vaccine Candidate
Against Dengue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
Kevin R. Porter, Nimfa Teneza-Mora,
and Kanakatte Raviprakash
18 DNA Vaccination as a Treatment for Chronic Kidney Disease . . . . . . . . . . . . . 297
Yuan Min Wang and Stephen I. Alexander

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
Contributors

FATIN NAWWAB AL-DEEN • Department of Chemical Engineering, Monash University,

Clayton, VIC, Australia
STEPHEN I. ALEXANDER • Centre for Kidney Research, Children’s Hospital at Westmead,
The University of Sydney, Westmead, NSW, Australia
SUNGHYUN BAEK • Department of Chemical and Biomolecular Engineering,
Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea
JOOST H. VAN DEN BERG • Division of Immunology, The Netherlands Cancer Institute,
Amsterdam, The Netherlands
ADRIAAN D. BINS • Division of Immunology, The Netherlands Cancer Institute,
Amsterdam, The Netherlands
DAVID B. BOYLE • Australian Animal Health Laboratory, CSIRO Animal, Food,
and Health Sciences, Geelong, Victoria, Australia
KATE E. BRODERICK • Inovio Pharmaceuticals, Blue Bell, PA, USA
AARON E. CARNES • Nature Technology Corporation, Lincoln, NE, USA
ROSS L. COPPEL • Department of Microbiology, Monash University, Clayton, VIC, Australia
HAYK DAVTYAN • Department of Molecular Immunology, Institute for Molecular Medicine,
Huntington Beach, CA, USA
MIGUEL FEVEREIRO • INIAV, Instituto Nacional de Investigação Agrária e Veterinária,
Lisboa, Portugal
DANIELA FIORETTI • Section of Medical Biotechnology, Institute of Translational
Pharmacology (IFT), National Research Council (CNR), Rome, Italy
ELISABETE BORGES FREITAS • Institute of Biotechnology and Bioengineering, Centre for
Chemical and Biological Engineering, Instituto Superior Técnico, Lisboa, Portugal
JULIE GEHL • Center for Experimental Drug and Gene Electrotransfer (C*EDGE),
Department of Oncology, Copenhagen University Hospital Herlev, Herlev, Denmark
ANAHIT GHOCHIKYAN • Department of Molecular Immunology, Institute for Molecular
Medicine, Huntington Beach, CA, USA
ANITA GOTHELF • Center for Experimental Drug and Gene Electrotransfer (C*EDGE),
Department of Oncology, Copenhagen University Hospital Herlev, Herlev, Denmark
JOHN B.A.G. HAANEN • Division of Immunology, The Netherlands Cancer Institute,
Amsterdam, The Netherlands
ANA MARGARIDA HENRIQUES • INIAV, Instituto Nacional de Investigação Agrária e
Veterinária, Lisboa, Portugal
ALMEDINA ISAKOVIC • Division of Allergy and Immunology, Department of Molecular
Biology, University of Salzburg, Salzburg, Austria
SANDRA IURESCIA • Section of Medical Biotechnology, Institute of Translational
Pharmacology (IFT), National Research Council (CNR), Rome, Italy
RONALD J. JACKSON • Molecular Mucosal Vaccine Immunology Group, Department of
Immunology, The John Curtin School of Medical Research, The Australian National
University, Canberra, ACT, Australia

ix
x Contributors

AMIR S. KHAN • Inovio Pharmaceuticals, Blue Bell, PA, USA

YEU-CHUN KIM • Department of Chemical and Biomolecular Engineering, Korea Advanced
Institute of Science and Technology (KAIST), Daejeon, Korea
KWANG LEE • Department of Biological and Environmental Engineering, Cornell University,
Ithaca, NY, USA
JEREMY M. LUKE • Nature Technology Corporation, Lincoln, NE, USA
DAN LUO • Department of Biological and Environmental Engineering, Cornell University,
Ithaca, NY, USA
CHARLES MA • Department of Microbiology, Monash University, Clayton, VIC, Australia
GABRIEL AMARO MONTEIRO • Institute of Biotechnology and Bioengineering, Centre for
Chemical and Biological Engineering, Instituto Superior Técnico, Lisboa, Portugal;
Department of Bioengineering, Instituto Superior Técnico, University of Lisbon, Lisboa,
Portugal
KOEN OOSTERHUIS • Division of Immunology, The Netherlands Cancer Institute,
Amsterdam, The Netherlands
IRINA PETRUSHINA • Institute for Memory Impairments and Neurological Disorders,
University of California, Irvine, CA, USA
KEVIN R. PORTER • Infectious Diseases Directorate, Naval Medical Research Center,
Silver Spring, MD, USA
DUARTE MIGUEL PRAZERES • Institute of Biotechnology and Bioengineering, Centre for
Chemical and Biological Engineering, Instituto Superior Técnico, Lisboa, Portugal;
Department of Bioengineering, Instituto Superior Técnico, University of Lisbon, Lisboa,
Portugal
CHARANI RANASINGHE • Molecular Mucosal Vaccine Immunology Group, Department of
Immunology, The John Curtin School of Medical Research, The Australian National
University, Canberra, ACT, Australia
KANAKATTE RAVIPRAKASH • Viral and Rickettsial Diseases Department, Infectious Disease
Directorate, Naval Medical Research Center, Silver Spring, MD, USA
MONICA RINALDI • Section of Medical Biotechnology, Institute of Translational
Pharmacology (IFT), National Research Council (CNR), Rome, Italy
YOUNG HOON ROH • David H. Koch Institute for Integrative Cancer Research,
Massachusetts Institute of Technology, Cambridge, MA, USA
NIRANJAN Y. SARDESAI • Inovio Pharmaceuticals, Blue Bell, PA, USA
SANDRA SCHEIBLHOFER • Division of Allergy and Immunology, Department of Molecular
Biology, University of Salzburg, Salzburg, Austria
MARTIN SCHLEEF • PlasmidFactory GmbH & Co. KG, Bielefeld, Germany
MARCO SCHMEER • PlasmidFactory GmbH & Co. KG, Bielefeld, Germany
TON N.M. SCHUMACHER • Division of Immunology, The Netherlands Cancer Institute,
Amsterdam, The Netherlands
CORDELIA SELOMULYA • Department of Chemical Engineering, Monash University,
Clayton, VIC, Australia
HAE-YONG SEOK • Department of Chemical and Biomolecular Engineering, Korea Advanced
Institute of Science and Technology (KAIST), Daejeon, Korea
HYEMEE SUH • Department of Chemical and Biomolecular Engineering, Korea Advanced
Institute of Science and Technology (KAIST), Daejeon, Korea
NIMFA TENEZA-MORA • Infectious Diseases Directorate, Naval Medical Research Center,
Silver Spring, MD, USA
 Contributors xi

JOSEF THALHAMER • Division of Allergy and Immunology, Department of Molecular

Biology, University of Salzburg, Salzburg, Austria
YUAN MIN WANG • Centre for Kidney Research, Children’s Hospital at Westmead,
The University of Sydney, Westmead, NSW, Australia
RICHARD WEISS • Division of Allergy and Immunology, Department of Molecular Biology,
University of Salzburg, Salzburg, Austria
JAMES A. WILLIAMS • Nature Technology Corporation, Lincoln, NE, USA
JESSICA JANE YE • School of Medicine, Yale University, New Haven, CT, USA
 Part I

DNA Vaccine Design and Enhancement

 Chapter 1

A Blueprint for DNA Vaccine Design

Sandra Iurescia, Daniela Fioretti, and Monica Rinaldi

Abstract
Although safety concerns have been overcome, lower immunogenicity profiles of DNA vaccines have
­hindered their progress in humans. DNA vaccines need to make up for this limitation by altering plasmid
construction through vector design innovations intended for enhancement of transgene expression and
immunogenicity. The next-generation vectors also address safety issues such as selection markers. This
chapter discusses (a) plasmid backbone design, (b) enhancement of antigenic protein expression and
immunogenicity, and (c) vector modification to increase innate immunity. Modifications of the basic
design, when combined with improved delivery devices and/or prime/boost regimens, may enhance
DNA vaccine performance and clinical outcomes.

Key words DNA vaccines, Plasmid design, Regulatory elements, Antigen, Immunogenicity,
Transgene expression, Marker selection, DNA uptake, Innate immunity

1 Introduction

DNA vaccine efficacy is hampered by low level of transgene

­expression. Basically, a DNA vaccine consists in a self-replicating,
circular double-stranded DNA molecule (the plasmid) joining
­bacterial regions necessary for selection and replication in E. coli
host with eukaryotic sequences that regulate expression of the
encoded antigen in the target tissue.
Ongoing efforts are focused to optimize the DNA vaccine
platform to improve antigen (Ag) expression and immunogenicity
as increased antigen expression correlates with improved immuno-
genicity and augmented levels of immune response in humans and
large animal models [1].
This blueprint presents an overview of the issues facing the
development of a platform technology for the production of
improved DNA vaccines. Specifically, this chapter deals with strate-
gies relevant to (a) engineering the plasmid backbone to obtain a

Sandra Iurescia and Daniela Fioretti contributed equally to this work.

Monica Rinaldi et al. (eds.), DNA Vaccines: Methods and Protocols, Methods in Molecular Biology, vol. 1143,
DOI 10.1007/978-1-4939-0410-5_1, © Springer Science+Business Media New York 2014

3
4 Sandra Iurescia et al.

more efficient expression plasmid, (b) enhancement of antigenic

protein expression and immunogenicity, and (c) complementary
plasmid design modifications to increase innate immunity. All
modifications outlined herein are made at the level of molecular
cloning (see Note 1).
The aspects next described depict potential design strategies
and technologies necessary to carry out the development of
improved DNA vaccines. A selection of relevant patents employed
to improve DNA vaccine immunogenicity through several strate-
gies such as the use of tissue-specific transcriptional elements,
nuclear localization signaling, and codon optimization are reported
in Fioretti et al. [2].

2 Materials

1. Plasmid DNA backbone.

2. DNA coding for the transgene.
3. Oligonucleotide primers to amplify target transgene from the
DNA template by polymerase chain reaction (PCR) (see Note 2).
4. Regulatory elements addressing antigen expression and immu-
nogenicity augmentation.

3 Methods

3.1 Plasmid DNA DNA vaccine design is relatively simple. Basically, the construction
Backbone of a DNA vaccine requires a plasmid backbone such as
pcDNA™3.1 or pVAX1© (Invitrogen, Life Technologies
Corporation). An important component of the plasmid backbone
is the promoter that drives expression of the transgene of interest.
For most expression plasmids, the human cytomegalovirus
(CMV) promoter (reviewed in ref. 3) is the usual choice as it pro-
motes high-level constitutive expression in a wide range of mam-
malian cells. Some early investigations reported that CMV
immediate/early enhancer/promoter activity was consistently the
highest among several constructs tested in mammalian somatic tis-
sues [4, 5]. Otherwise, the use of host tissue-specific promoters pre-
vents antigen expression in inappropriate tissues yet leading to
sufficient stimulation of immune responses [6]. A Kozak sequence
conforming to the consensus gccgccRccAUGG (R = G or A, AUG
start codon underlined, critical residues in caps) is included immedi-
ately prior to the ATG start codon ensuring efficient translation.
Approaches to increase transcription and translation thereby improve
DNA vaccine immunogenicity. This can be achieved by optimiza-
tion of regulatory elements in the plasmid backbone [2]. The inclu-
sion of an intron (i.e., the intronA of the CMV immediate–early
 A blueprint for DNA vaccine design 5

gene) in the vector backbone downstream of the promoter can

enhance the stability of mRNA and may improve gene expression.
Furthermore, the presence of the polyadenylation (polyA) signal
site, which contains accessory sequences upstream and downstream
of the polyA tail, ensures proper termination of transcription,
increased mRNA levels, and export of the mRNA from the nucleus
resulting in improved transgene expression. Both enhancer elements
and transcriptional transactivators may increase promoter efficiency.
For example, incorporation of the human T-cell leukemia virus type
I R 5′ untranslated region (UTR) (HTLV-I R-U5) downstream of
the CMV promoter increased mRNA translation efficiency [7] and
immunogenicity of DNA vaccines encoding multiple antigens in
small animals and in nonhuman primates [8].
The transcribed 3′ and 5′ UTRs should not contain cryptic
open reading frames (ORFs) since unforeseen immunogenic epit-
opes could be generated able to elicit a cytotoxic T lymphocyte
(CTL) response [9].
A critical step of the DNA vaccine design is a careful selection
and assembly of bacterial regions that provide both replication ori-
gin and selection marker necessary for propagation in different
E. coli host strains. The compositions and orientations of the so-
called relaxed origins of replication (i.e., pUC origin) can interfere
with the transgene expression, manufacturing yields, and plasmid
quality. Reduced expression may in part be due to the presence of
TATA-containing cryptic promoter within the replication origin or
the selectable marker generating spurious transcripts that triggers
protein kinase R (PKR)-mediated selective translational shutdown
or RNA interference (see Williams J.A. for a review) [10].
The use of antibiotic-resistance selection markers in DNA vac-
cines has safety issues and represents a key aspect for high-scale
plasmid production. The European Medicines Agency (EMA)
stated that “neomycin and kanamycin are of importance for veteri-
nary and human use and that their current and potential future use
cannot be classified as of no or only minor therapeutic relevance”
due to current use in critical clinical settings [11]. To address these
regulatory concerns, alternative non-antibiotic selection methods
are being developed [10, 12]. In Chapter 6 of this book, Williams
and colleagues describe the development of RNA-based antibiotic-­
free selection system for safer DNA vaccination.
In vector development, an important objective is plasmid
backbone shortening and removal of bacterial elements. Plasmid
size reduction improves pDNA structural stability and enhances
the transfection efficiency, leading to increased duration of antigen
expression [10, 12]. Overall, smaller plasmid size is therefore ben-
eficial for gene delivery efficiency. Minicircle DNA technology was
developed to obtain a plasmid backbone nearly devoid of any pro-
karyotic sequence by using site-specific recombination. The result-
ing miniplasmid contains almost exclusively the gene of interest
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