Table Of ContentCurrent Topics in
Microbiology
226 and Immunology
Editors
R.W. Compans, Atlanta/Georgia
M. Cooper, Birmingham/Alabama
J.M. Hogle, Boston/Massachusetts· Y. Ito, Kyoto
H. Koprowski, Philadelphia/Pennsylvania· F. Melchers, Basel
M. Oldstone, La Jolla/California· S. Olsnes, Oslo
M. Potter, Bethesda/Maryland· H. Saedler, Cologne
P.K. Vogt, La Jolla/California· H. Wagner, Munich
Springer
Berlin
Heidelberg
New York
Barcelona
Budapest
Hong Kong
London
Milan
Paris
Santa Clara
Singapore
Tokyo
DNA Vaccination/
Genetic Vaccination
Edited by H. Koprowski and D.B. Weiner
With 31 Figures and 14 Tables
, Springer
HILARY KOPROWSKI, M.D.
Professor
Thomas Jefferson University
Department of Microbiology and Immunology
Center for Neurobiology
Rm. M-85, Jefferson Alumni Hall
1020 Locust Street
Philadelphia, PA 19197-6799
USA
DAVID B. WEINER, Ph.D.
Associate Professor
University of Pennsylvania School of Medicine
Hospital of the University of Pennsylvania
Department of Pathology and Laboratory Medicine
500 Stellar-Chance Bldg.
422 Curie Boulevard
Philadelphia, P A 19104-6100
USA
Cover Illustration: Plasmid delivery to a host tissue results in tissue
specific protein production and specific activation of T cells, B cells
and antigen presenting cells generating specific immunity (by
courtesy of Bin Wang and Michael Chattergoon).
Cover Design: design & production GmbH, Heidelberg
ISBN-13: 978-3-642-80477-9 e-ISBN-13: 978-3-642-80475-5
DOl: 10.1007/978-3-642-80475-5
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Preface
Genetic / DNA immunization represents a novel approach to
vaccine and immune therapeutic development. The direct injec
tion of nucleic acid expression cassettes into a living host results
in a limited number of its cells becoming factories for production
of the introduced gene products. This host-inappropriate gene
expression has important immunological consequences, resulting
in the specific immune activation of the host against the gene
delivered antigen. The recent demonstration by a number of
laboratories that the induced immune responses are functional in
experimental models against both specific infectious diseases and
cancers is likely to have dramatic consequences for the develop
ment of a new generation of experimental vaccines and immune
therapies. This technology has the potential to enable the pro
duction of vaccines and immune-based therapies that are not only
effective immunologically but are accessible to the entire world
(rather than just to the most developed nations).
Vaccine Development
Vaccination against pathogenic microorganisms represents one of
the most important advances in the history of medicine. Vaccines,
including those against polio, measles, mumps, rubella, hepatitis
A, hepatitis B, pertussis and other diseases, have dramatically
improved and protected more human lives than any other avenue
of modern medicine. The vaccine against smallpox, for example,
has been so successful that it is now widely believed that this
malicious killer, responsible for more deaths in the twentieth
century than World Wars I and II combined, has been removed
from the face of the earth. Traditional vaccination has relied on
two specific types of microbiological preparations for producing
material suitable for immunization and the generation of a pro
tective immune response. These two broad categories involve
either live infectious material which has been manufactured in a
weakened or attenuated state, thus preventing the vaccine from
VI Preface
inducing disease, or alternatively, nonlive, inactivated, or subunit
preparations. Live attenuated vaccines, such as the polio and
smallpox vaccines, for example, stimulate protective immune re
sponses as they replicate in the host. However they are limited in
their ability to replicate in humans and, accordingly, do not
normally induce crippling or lethal disease in inoculated indi
viduals. Since these live preparations are actually produced in the
host as the attenuated virus replicates within the host cells, they
have several unique immunological properties associated with
this in vivo replication. The viral proteins produced within the
host are leaked or perhaps shed into the extracellular space sur
rounding the infected cells and are then picked up, internalized,
and digested by scavenger cells that patrol the body. These in
clude both macro phages and the several forms of dendritic cells,
as well as B cells; collectively they function to expand immune
response. These antigen presenting cells (APCs) then recirculate
small fragments of the antigen to their surface, attached to MHC
II or class II antigens. This complex of foreign peptide antigen
plus host MHC class II antigens forms part of the specific signal
with which APCs themselves, along with the MHC peptide
complex, trigger the action of a central population of immune
cells, the T helper lymphocytes cells. The second part of the ac
tivation signal comes from the APCs themselves: they display on
their cell surface, in addition to MHC-antigen complexes, co
stimulatory molecules. Together they can drive T cell expansion
and activation through interaction with their respective ligands,
the T cell receptor complex (TCR) and the costimulatory recep
tors CD28/CTLA4, present on the T cell surface. Activated T
helper cells secrete soluble molecules that serve as powerful ac
tivators of immune cells. In addition, as viral proteins are pro
duced within cells of the host, small fragments of viral proteins
are drawn to the cell surface, chaperoned by the host cell MHC I
antigens. These complexes are recognized by a second class of T
cells, the killer or cytotoxic T cells. This recognition, coupled with
secondary stimulation provided by professional APCs and helped
by cytokine production from the stimulated helper T cells, either
individually or collectively, is responsible for the development of
the mature effector T cytotoxic cells capable of destroying viral
factories, i.e., infected cells. In addition, in most instances live
infection induces an additional benefit, that of lifelong immunity.
In contrast, killed or nonlive vaccines composed of whole or
even fragments of viruses, when inoculated as vaccines, are not
produced within host cells and upon administration mostly wind
up in the extracellular space and not within cells. Accordingly
they provide protection by directly generating T helper and hu-
Preface VII
moral immune responses against the pathogenic immunogen and
are therefore particularly useful in protecting against infection,
especially when antibodies are responsible for microbe inactiva
tion. In the absence of the cellular production of the foreign
antigen, these vaccines are usually devoid of the ability to induce
significant T cytotoxic responses. The lymphokine profiles of
these antigens are also often different from those induced by live
attenuated vaccines. In addition these vaccines are not actually
produced in the host and therefore are not customized by the
host. The immunity induced by these vaccines frequently wanes
during the lifespan of the inoculated hosts and may often require
repeated immunizations or boosting to achieve lifelong immuni
ty. However, nonlive vaccines offer some important advantages
over live vaccines. They are generally easier to manipulate and
usually easier to produce than live vaccines. Furthermore they
enjoy an inherent advantage: we can design them to contain only
the specific antigenic target of the pathogen that is involved in the
development of protective immunity and exclude all other viral
components. The latter, namely, can deflect the focus of the im
mune response and actually have unwanted immunological cross
reactivities that can cause problems for the vaccinated host.
Accordingly, live attenuated preparations are clearly the
vaccines of choice in terms of the diverse immune responses they
produce. However they pose risks of reversion to a more
pathogenic form: during their replication process they can
sometimes convert back into an actual disease-causing virus.
Furthermore they may retain the unwanted characteristic, even in
the attenuated state, of inducing disease in persons with weak or
compromised immune systems. This includes persons suffering
from genetic or drug induced immune deficiency, receiving cancer
chemotherapy, or suffering from a separate infection that weak
ens their immunity (AIDS patients, the elderly). An individual
vaccinated with a live attenuated virus can pass that attenuated
virus on to others, thus presenting risks of inadvertent spread to
an unknowing host (something health care workers need to keep
in mind). Mothers have become vaccinated with the live attenu
ated polio vaccine simply by changing the diapers of their re
cently vaccinated infants. In contrast, safely manufactured
nonlive vaccines that have specific immunity-inducing abilities
have no risk of inducing infection or disease due to a need to
maintain a replicating phenotype. They can be designed to ex
clude those portions of a pathogen that may induce unwanted
cross-reactive immune responses. Accordingly they have been the
vaccines of choice, based on both their safety and specificity and
certain manufacturing considerations.
VIII Preface
Recent work from a number of laboratories (including that
of the editors) has demonstrated that inoculation of a DNA
plasmid directly into a host with subsequent expression of the
encoded peptide sequences in vivo results in presentation of the
specific encoded protein(s) to the immune system. Since DNA
vaccines are nonreplicating and are produced within the host cells
they can be constructed to function with the safety advantages of
a subunit nonlive vaccine and yet to mimic immune potentiating
aspects of a live attenuated vaccine. This represents a distinct
advantage for initial vaccine and analysis as DNA vaccination
represents a rapid system for directly testing subunit vaccination
strategies without viral production or protein purification pro
cedures. The latter can significantly slow down the investigation
process. Furthermore, DNA vaccination facilitates a diverse ar
ray of immune analysis from a single immunization platform. It
may be particularly advantageous that such a system can deliver
an antigen which could be presented for development of both the
helper T cell and the cytotoxic T cell arms of the immune system
in the absence of live vector construction. Using direct DNA
immunization the genes cloned into the expression vectors can be
manipulated to present single proteins or an extended genome
with only the genes that might lead to pathogenesis removed.
Accordingly, this approach, if translatable into success in the
clinic, may present the best of both prior vaccine modalitieswith
the added benefit of decreased turnaround times for vaccine de
velopment.
History of DNA Inoculation in Vivo
The ability of genetic material to deliver genes for therapeutic
purposes has been appreciated for some time. Some of the earliest
experiments describing the transfer of DNA into the cells of a
living animal were reported by Stasmey (1950), Parchkis (1955),
and Ito (1958). These reports described the ability of chromatin
preparations, crude preparations of DNA isolated from tumors,
to induce formation of tumors after injection in laboratory ro
dents. Later experiments further purified the genetic material and
confirmed that direct DNA gene injection in the absence of viral
vectors can result in the expression of the inoculated genes in the
host. If genes encoding cancer antigens were injected, cellular
transformation and cancer, with a reproducible frequency, would
result in the inoculated animals. Studies using purified DNA and
RNA derived from viruses were also reported. Most importantly,
Atanasia and colleagues (1962) demonstrated that the subcuta-
Preface IX
neous administration of purified polyoma virus DNA trans
forming sequences as part of the viral genome to newborn
hamsters resulted in the generation of anti-polyoma antibodies to
the virus and the induction of tumors from the transforming
genes. In a similar study, Orth and coworkers (1964) injected
either newborn or 40-day-old hamsters with DNA isolated from
polyoma virus grown in tissue culture. Thirty days following
subcutaneous injection of purified DNA, 34 of the 35 newborn
hamsters had developed antibodies and 26 developed tumors.
The results described by these studies are explained by the uptake
of the injected DNA by cells of the inoculated host followed by
translation of the injected DNA sequences into messenger RNA.
The host translation system then drives expression of viral en
coded genes and their functions, which in the case of the ad
ministration of transforming gene sequences, is cellular
transformation. Crucial for this discussion however is the out
come of this delivery of viral genes: it resulted in the presentation
of these genes to the immune system, leading to the production of
an immune response against the virus, as determined by the in
vivo antibody production observed in these studies.
Additional experiments extend these findings to recombinant
DNA molecules. Israel and colleagues (1979) reported that re
combinant clones of purified DNA containing head to tail dimers
of the polyoma virus resulted in seroconversion of inoculated
animals after injection of such DNA vectors into weanling mice.
Similar experiments using plasmids containing hepatitis B genetic
material were reported by Will (1984). The in vivo gene expres
sion of plasmid material was further studied in regard to suc
cessful insulin delivery as a gene therapy approach in living
animals (Dobensky 1985). Elegant studies from the laboratory of
Jon Wolff (University of Wisconsin) detailed the long-term ex
pression of injected plasmids in vivo (1990). Together, these
studies and others served to support the idea that purified nucleic
acids could be directly delivered into a host and proteins would
be produced. Such proteins could be immunogenic in some set
tings. However, challenge studies in these systems have not yet
been widely reported.
In 1992 Tang and Johnston reported that the delivery of
human growth hormone gene in an expression cassette in vivo
resulted in protein expression in the inoculated animals. These
authors utilized a genetic gun to shoot gold particles coated with
DNA through the skin layers of mice. The inoculated animals
produced detectable levels of human growth hormone. Interest
ingly they reported that antibodies developed against the human
growth hormone produced in the mice. These authors termed this
X Preface
immunization procedure genetic immunization, describing the
ability of inoculated genes to be individual immunogens. These
studies elegantly brought together and focused the earlier work in
this area.
Anti-Pathogen Immune Responses Induced
By DNA Immunization In Vivo
Almost simultaneously with the publication by Tang and John
ston the annual vaccines meeting at Cold Spring Harbor in the
fall of 1992 brought together several independent groups that
collectively reported on the use of DNA immunization technol
ogy for the generation of protective immune responses in vivo.
These presentations, which used unique approaches and were
directed at different viral targets, were not the first public pre
sentations of any of the laboratories involved. However, the
collective success of these independent investigators and the large
amount of data they presented from diverse systems could not be
ignored by the vaccine community. Specifically, Margaret Liu of
Merck and her collaborators at Vical reported on the induction
of immune responses to influenza A virus following intramus
cular inoculation of mice with highly purified plasmid DNA en
coding influenza A genes. Following three inoculations over 6
weeks using lOOg doses of plasmid DNA, the resulting immune
responses to the influenza proteins included antibodies, cellular
immune responses, and evidence of protection from viral chal
lenge. Harriet Robinson and colleagues (University of Mass
achussetts) utilized the gene gun to deliver influenza genes in
plasmids in vivo; they observed that DNA doses on the nano
gram level were able to induce both antibodies and cellular im
mune responses. These doses were also able to protect mice from
challenge in a similar influenza challenge model. One of us
(Weiner) reported that direct injection of plasmids encoding core
HIV genes, rev and the envelope genes induced humoral and
cellular immunity against HIV-l, the virus which causes AIDS.
The immune responses induced were able to prevent HIV infec
tion in in vitro assays.
There have been many important studies, which, together,
form the basis of this emerging field. Some of the seminal pio
neers in this arena have contributed chapters to this volume.
Specifically, using a variety of delivery methods and DNA con
structs, DNA immunization in animals has been reported to
generate host immunity against herpes simplex virus (B. Rouse
