Table Of ContentDNA Vaccines
M E T H O D S I N M O L E C U L A R M E D I C I N ETM
DNA Vaccines
Methods and Protocols
Edited by
Douglas B. Lowrie,
MD
Mycobacterial Research Laboratory, London, UK
and
Robert G. Whalen,
MD
Maxygen Inc., Redwood City, CA
Humana Press Totowa, New Jersey
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Methods in molecular medicine™.
DNA vaccines : methods and protocols / edited by Douglas B. Lowrie and Robert G. Whalen.
p. cm. — (Methods in molecular medicine™ ; 29)
Includes bibliographical references and index.
ISBN 0-89603-580-8 ( alk. paper).
1. DNA vaccines—Research—Methodology. I. Lowrie, Douglas B. II. Whalen, Robert G.
III. Series.
QR189.5.D53D63 1999
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Preface
The field of DNA vaccines has undergone explosive growth in the last
few years. As usual, some historical precursors of this approach can be dis-
cerned in the scientific literature of the last decades. However, the present
state of affairs appears to date from observations made discreetly in 1988 by
Wolff, Malone, Felgner, and colleagues, which were described in a 1989 patent
and published in 1990. Quite surprisingly, they showed that genes carried by
pure plasmid DNA and injected in a saline solution, hence the epithet “naked
DNA,” could be taken up and expressed by skeletal muscle cells with a low
but reproducible frequency. Such a simple methodology was sure to spawn
many applications. In a separate and important line of experimentation, Tang,
De Vit, and Johnston announced in 1992 that it was indeed possible to obtain
humoral immune responses against proteins encoded by DNA delivered to the
skin by a biolistic device, which has colloquially become known as the “gene
gun.”
The year 1993 saw the publication of further improvements in the meth-
ods of naked DNA delivery and, above all, the first demonstrations by several
groups of the induction of humoral and cytotoxic immune responses to viral
antigens expressed from injected plasmid DNA. In some cases, protection
against challenge with the pathogen was obtained. The latter result was un-
questionably the touchstone of a method of vaccination worthy of the name.
DNA or “genetic” vaccination (or, as the World Health Organization
suggested,“nucleic acid vaccination) was now really and truly delivered unto
the scientific world. In 1995 Bernard Dixon, Contributing Editor for Bio/Tech-
nology(nowNature Biotechnology), called this development the “Third Vac-
cine Revolution.” Some were less fervent believers, gravely reminding naïve
molecular biologists that it was assuredly not safe to inject DNA. They added
that DNA-based immunization was, après tout, only a technique, barren of
any intrinsic scientific interest. Such reactions are not unknown in the history
of science, although subsequent events appear to lend little support to this
idiosyncratic point of view.
Only a dozen or so publications on DNA vaccines appeared in 1993, and
less than 100 in all of 1995. The numbers have increased steadily and now
exceed 900 in total (June 1999). This rapid increase stems partly from the fact
v
vi Preface
that DNA-based immunization in experimental models is a relatively easy
methodology to implement. Indeed, the technical barriers to entry in this field
are so low that scientists in countries all over the world can readily begin to
study prototype vaccines to diseases that are of particular interest to their re-
gion of the globe.
This is a most salutary development since infectious agents know no
national boundaries. In contrast many diseases of the developing world often
receive scant notice from the large commercial entities in developed coun-
tries. A firm belief in the global utility of vaccination and the general useful-
ness of DNA vaccine methodology prompted one of us to create, in 1995, an
Internet site called The DNA Vaccine Web. This protean publication takes
advantage of the World Wide Web to promulgate information on new and
important developments.
In what might well be record time, DNA vaccines entered clinical trials
in mid-1995. Today, four years later, we have been assured of at least the
minimum level of safety for DNA vaccines when injected into humans, but
we are by and large still waiting for reports on the efficacy of the first trials. It
is still early days in the clinic. Doubtless, “naked” DNA vaccination will not
be the instantly successful panacea that one might have hoped for. Other for-
mulations of plasmid DNA will probably be required to ensure the reproduc-
ibility and efficacy of DNA-mediated vaccination in real-world settings.
DNA vaccination is a simple methodology. There are only so many ways
to explain how to inject a DNA-containing solution into muscle or skin. The
articles in this compendium, therefore, also provide detailed information on
the essentials of “upstream” and “downstream” analysis. The safety aspects,
quality control, and preparation of plasmid DNA will be of primary impor-
tance for those contemplating clinical trials for human or veterinary applica-
tions of DNA vaccines. Several authors discuss the use of classical adjuvants,
“genetic adjuvants” (involving coexpression of cytokines), and the
immunostimulatory properties of DNA or certain oligonucleotide sequences.
Details on formulations of DNA and some novel expression systems provide
extensions of the standard methodology that might prove highly beneficial in
the long run. With respect to DNA vaccines being “just” a technique, several
contributors provide strategic ideas on antigen engineering and describe some
of the particularly novel applications of DNA vaccine methodology that have
already been devised. The simplicity of experimental methods allows human
creativity to come to the fore in surprising ways.
Finally, we felt that it was essential to devote consideration to the topic
of dendritic cells, and antigen-presenting cells generally, which are the major
Preface vii
players in the aptitude of DNA vaccines to activate the immune system. As
Sigmund Freud said of the role of dream content in understanding the uncon-
scious, the injection of plasmid DNA might serendipitously provide us with
the “royal road” to greater comprehension of dendritic cell function. If so,
DNA vaccines hold great promise for influencing the immune response.
We are pleased to have secured for Humana Press an impressive list of
contributors whose expertise in various areas of DNA vaccines, immunology,
and related disciplines is without question. The Editors thank Prof. John Walker,
Series Editor, and Mr. Thomas Lanigan, President of Humana Press, for their
foresight, support, and assistance in the realization of this volume.
Douglas B. Lowrie, MD
Robert G. Whalen, MD
Contents
Preface .............................................................................................................v
Contributors...................................................................................................xiii
1 Purification of Supercoiled Plasmid
Anthony P. Green..................................................................................1
2 Production of Plasmid DNA in Industrial Quantities According to
cGMP Guidelines
Joachim Schorr, Peter Moritz, and Martin Schleef.........................11
3 Development and Characterization of Lyophilized DNA Vaccine
Formulations
Nancy L. Shen, Jukka Hartikka, Nancy A. Horn,
Marston Manthorpe, and Magda Marquet....................................23
4 Repeated Use of Qiagen Columns in Large-Scale Preparation
of Plasmid DNA
Derek Gregory, Ricardo E. Tascon, and Douglas B. Lowrie..........35
5 The Immunology of DNA Vaccines
Thomas Tüting, Jonathan Austyn, Walter J. Storkus, and
Louis D. Falo Jr..............................................................................37
6 Methodology Used in DNA-Based Prophylactic and Therapeutic
Immunization Against Hepatitis B Virus in Chimpanzees
Alfred M. Prince and Betsy Brotman.................................................65
7 Intramuscular and Intradermal Injection of DNA Vaccines
in Mice and Primates
Heather L. Davis...................................................................................71
8 Veterinary DNA Vaccines
Sylvia van Drunen Littel-van den Hurk, Ralph P. Braun,
and Lorne A. Babiuk.......................................................................79
9 DNA-Based Immunization of Neonatal Mice
Cynthia L. Brazolot Millan and Heather L. Davis.............................95
10 Intramuscular Injection of DNA Vaccines in Fish
Joël Heppell and Heather L. Davis....................................................99
11 Development of DNA Vaccines for Salmonid Fish
Eric D. Anderson and Jo-Ann C. Leong.........................................105
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