Anatomy of plasmid vectors used in genetic immunization
Brazilian Journal of Medical and Biological Research (1999) 32: 147-153
Main features of DNA-based immunization vectors
Departamentos de 1Biologia Geral, 2Microbiologia, and3
Bioquímica e Imunologia, Instituto de Ciências Biológicas,
Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil4Instituto de Investigaciones en Ingenieria Genetica y Biologia Molecular(INGEBI-CONICET-UBA), Buenos Aires, Argentina
Abstract Correspondence
DNA-based immunization has initiated a new era of vaccine research. Key words
One of the main goals of gene vaccine development is the control of
the levels of expression in vivo for efficient immunization. Modifying
the vector to modulate expression or immunogenicity is of critical
importance for the improvement of DNA vaccines. The most fre-
quently used vectors for genetic immunization are plasmids. In this
article, we review some of the main elements relevant to their design
such as strong promoter/enhancer region, introns, genes encoding
antigens of interest from the pathogen (how to choose and modify
them), polyadenylation termination sequence, origin of replication for
plasmid production in Escherichia coli, antibiotic resistance gene as
selectable marker, convenient cloning sites, and the presence of
immunostimulatory sequences (ISS) that can be added to the plasmid
to enhance adjuvanticity and to activate the immune system. In this
review, the specific modifications that can increase overall expression
as well as the potential of DNA-based vaccination are also discussed. Introduction
therefore they are of limited interest for the
purpose of immunization. In contrast, DNA
plasmids encoding antigen(s) are more fre-
quently used because they do not have the
involves transfer of a gene encoding an anti-
inconvenience of classical vaccines: they are
genic protein cloned in expression vectors to
safe, inexpensive, easy to produce, heat stable
a host, leading to the induction of an immune
response. During the last two decades, dif-
this review, we will deal with some of the
main elements relevant to the design of vec-
have been developed as well as new methods
tors used for genetic vaccination. These vec-
for direct gene transfer. Several reviews on
tors are Escherichia coli-derived plasmids
the subject have been published (1-3). Direct
capable of expressing foreign genes in eu-
gene transfer may be undertaken using either
karyotic cells. Conceptually, their struc-
viral vectors or recombinant plasmid DNA.
ture can be divided into two distinct units: i)
Viral vectors have the disadvantages of be-
a transcription complex unit that drives anti-
ing derived from pathogens like traditional
gen synthesis and that contains a promoter/
vaccines based on attenuated viruses, and
enhancer region, introns with functional splic-
ing donor and acceptor sites, sequences en-
coding an antigenic protein, and signals re-
quired for efficient polyadenylation of the
transcript. ii) Prokaryotic elements such as
lian promoters, have also been used. These
replication origin, multiple cloning sites and
promoter elements are particularly interest-
a selectable marker to facilitate the construc-
ing for vaccine development since they may
tion, propagation and amplification of re-
combinant vectors in bacteria. This plasmid
pathogenic or tumor-causing viruses for ap-
backbone can also carry immunostimulatory
plications involving humans or animals.
sequences (ISS) with adjuvant activity (Fig-
Gurunathan et al. (14) reported that the bo-
vine promoter/enhancer from the major his-
Basic structure of plasmid DNA
gene gave significantly better protection than
vaccines
that obtained with hCMV-derived plasmids. The transcription complex unit
including those from the beta-actin, muscle-
specific heavy chain of myosin and muscle
Enhancer/promoter regions. Almost all
creatine kinase genes (mck) (7,15,16).
carry the immediate early promoter/enhancer
exogenous enhancer elements of aB crystal-
from pathogenic viruses. Although these pro-
line gene (cryB) or mck can be incorporated
moter elements are from pathogenic viruses,
upstream in relation to the enhancer/pro-
moter region. Hartikka et al. (17) and Dai et
therapy and genetic immunization thanks to
al. (18) reported that these entities prolong
their high transcription initiation ability in
and increase expression by the CMV promo-
most mammalian tissues (4). The most com-
tor/enhancer. In fact, the main function of
monly used promoter is the one from human
these elements is to control the gene initia-
tion transcription rate. In spite of the exist-
strong and constitutive expression in a vari-
ence of various gene delivery systems, the
ety of cell types (5-7). The use of alternative
main question to be addressed is if the initia-
tion of the transferred gene transcription is
cussed, including Rous sarcoma virus (RSV),
efficient and how long it lasts. The modula-
tion of this initiation rate can be essential to
obtain a positive genetic immunization ef-
Figure 1 - Plasmid DNA patternfor genetic immunization. i) The
repeat (LTR) promoter (8-10). Among them,
It is sometimes advisable to introduce an
the hCMV promoter gave the highest levels
inducible promoter to control in vivo expres-
of expression of the reported genes tested
sion. This was done by Dhawan et al. (19)
and Liang et al. (20) who modified plasmids
to make tetracycline (Tc)-dependent tran-
scriptions. When tetracycline is administered
to transfected mice it can act in a repressive
or activating way depending on the position
of the tetracycline-operator (tetO) control
sequence within these Tc-controlled plas-
livers adjuvant and mitogenicactivity via immunostimulatory
mids. The inducible systems have the advan-
tage of overcoming potential immunological
Anatomy of plasmid vectors used in genetic immunization
tolerance that may exist in genetic immuni-
cording to the different transcriptional gene
terminators. Moreover, as the rate of tran-
Introns. Intervening sequences (or in-
scriptional initiation is increased by the use
trons) have a beneficial effect on antigen
of strong promoter/enhancer, the process of
expression. This can be attributed to an en-
transcriptional termination may become rate-
hanced rate of RNA polyadenylation and/or
limiting (18). Although transcriptional ter-
minators are not widely recognized as gene
(21), but can also indicate the presence of
regulatory elements, Hartikka et al. (17)
transcriptional enhancers within the introns
showed that modifications in transcriptional
terminator sequences such as their replace-
ment by other types of terminator sequences
nation also include intron A from hCMV.
or the construction of chimeric termination
Indeed, some studies have revealed that ex-
sequences with more efficient polyadenyla-
pression and stimulation of an immune re-
tion signal or the lack of 3' untranslated
sponse were enhanced by the addition of an
region (UTR) sequences led to increases in
intron sequence upstream of the coding re-
expression which can reflect the differences
gion (9,23). This location prevents the utili-
in transcriptional termination efficiency.
zation of possible cryptic 5' donor splicing
sites within DNA sequences (21). These cryp-
carried by DNA vaccine constructs usually
tic sites hamper expression due to aberrant
sites of different genes, and the branchpoint
site can be optimized to match the consensus
late SV40-derived sequence terminators. The
sequence leading to an increased expression
efficient and increases the steady-state level
of RNA approximately 5-fold more than the
improved transcriptional efficiency 10- to
early SV40 polyadenylation signal (27).
served little effect on gene expression when
multiple cloning sites. This location facili-
compared to cells transfected in culture.
tates efficient processing of cloned genes
Various interpretations are possible but the
which may not have an efficient polyadeny-
most plausible explanation is that introns
contain DNA sequences that are recognized
Sequences encoding an antigenic pro-tein. Recombinant DNA technology has en-
not required after transfection, in established
cell lines. In conclusion, the studies that
with an optimized structure for genetic im-
examine the role of introns in expression
munization. To be optimized, synthetic genes
plasmid vectors show that the increase in in
require elimination of large hairpin struc-
vivo expression depends not only on the
tures in the 5'-end UTR mRNAs. It has been
presence or absence of introns, but also on
shown that these structures reduce the level
the position of introns within the transcrip-
of in vitro and in vivo translation in higher
eukaryotes (28,29). To initiate translation,
Polyadenylation signal. Polyadenylation
an ATG must be present in the inserted gene.
This ATG must also be the first one in the 5'
and translation (26) which in turn vary ac-
region and in the translational start site. Out-
of-frame ATGs can reduce the rate of gene
produces a transcription unit capable of mod-
translation (29). Kozak (29) has proposed a
ulating genetic expression. The exact modu-
lation of the expression level elicits the de-
located around the start site (-9 GCCGCCA/
GCCAUGG+4). She also pointed out that an Discovery of new antigenic proteins.
efficient translation is obtained in the -3
position containing a purine base. In the
opportunity to quickly discover new anti-
absence of a purine base, the efficiency of
gens and to handle the antigenicity of the
translation can be maintained with a guanine
protein at the sequence level with the advan-
tage of not requiring protein production and
organisms and some eukaryotic genes do not
purification. Since it is easy and rapid to
have this consensus sequence. Its insertion
clone and modify genes in plasmid expres-
into the 5'-region of these genes might in-
produced and tested in a short period of time
In contrast to prokaryotic sequences, limi-
such as entire expression libraries which can
tations of size and availability often dictate
be cloned and injected in a shot-gun fashion
that the eukaryotic sequences to be expressed
very interesting application of DNA vacci-
struct a synthetic gene it may be necessary to
nation has been developed to identify new
protective antigens. This strategy (termed
excise them during processing of the pri-
mary transcript. The presence of introns dur-
ing processing can sometimes increase the
levels of cytoplasmic messenger RNA (30).
of the pathogen. It was initially developed by
Barry et al. (34) who demonstrated that im-
sequences by addition or deletion of secre-
munization with partial expression libraries
tory control signals have also been evalu-
made from genomic DNA of Mycoplasma
ated, and surprisingly they either had no
pulmonis protects mice challenged with the
pathogen. Similarly, Alberti et al. (35) in-
(23,31). However, Hoffman et al. (32) re-
jected mice with an expression genomic li-
ported that in-frame gene fusions with the
brary of Trypanosoma cruzi. Although the
sequence encoding the leader peptide of hu-
protection was not assayed, it was possible
to detect expression of T. cruzi antigens in
the muscle and the specific IgG antibodies
to a DNA vaccine, Boyle et al. (33) directed
The identification of antigens, their com-
the antigen to sites of immune-response in-
binations and forms is the most effective
way to raise protective responses and is one
antigen-ligand fusion proteins. These two
ligands bind to receptors that are present on
endothelial venule cells of lymph nodes or
Plasmid backbone unit
that both the humoral and cellular immune
Multiple cloning region, replication ori-
enhanced using either antigen-targeting strat-
gin and prokaryotic selectable marker. The
egy. The construction of optimized synthetic
backbone of plasmid DNA vectors carries a
multiple cloning site (MCS) that can be con-
Anatomy of plasmid vectors used in genetic immunization
sidered to be additional elements of the tran-
cassette is possible but does not ease public
scription unit. This MCS is especially de-
concern over its potential clinical use. The
signed to avoid formation of hairpin struc-
use of auxotrophic markers can also be pos-
tures in the 5' end of the transcribed RNA
sible but there is a high contamination risk
since hairpin structures can interfere with
with the essential component that is not nec-
host. It should be remembered that applica-
tion of direct gene transfer for genetic immu-
multicopy plasmid, ColE1. It is the best char-
nization requires the availability of plasmid
acterized copy number and incompatibility
DNA that is free of all contaminants, partic-
system and allows the maintenance at steady
ularly toxic or immunogenic substances. This
level of more than 20 copies per E. coli cell,
resulting in a high DNA plasmid yield, im-
purification technology combined with se-
portant in gene vaccine production. The pres-
lected E. coli strains and growth optimiza-
ervation of a high number of copies depends
tion is used for each plasmid construction
on the mechanisms which initiate replica-
tion and is controlled by the ColE1 origin
Sequences as immunostimulatory ele-
(36). DNA vectors can also contain a viral
ments. It has been shown that certain DNA
origin of replication. In this regard, SV40
sequences can induce cytokine secretion and
parently, the inclusion or deletion of such
stimulatory, whereas similar sequences from
other species are not. These observations
levels of the foreign peptides (37). These
suggest that manipulation of DNA vaccines
findings, together with the necessity of satis-
to contain or to avoid these sequences may
fying concerns related to their potential clini-
affect the immunogenicity of the antigens
cal use, indicate that replication sequences
expressed by the vector. In this regard, Sato
of viral origin should be deleted from DNA
containing a bacterial ampR produced a stron-
ger immune response than a similar expres-
select for growth only in those cells which
sion vector containing the kanamycin resis-
contain a vector. Such markers are of two
tance gene (kanR). In vitro transfection ex-
types: drug resistant and auxotrophic. Drug
periments revealed that the ability to stimu-
resistant markers enable cells to detoxify an
late the immune response was not due to the
different amounts of antigen expressed by
wise kill the cell. Auxotrophic markers al-
low cells to synthesize an essential compo-
ISS was identified within the kanR gene.
prokaryotic selectable marker is the beta-
Klinman et al. (41) demonstrated that the
lactamase ampicillin resistance gene (ampR)
elimination of CpG motifs from the plasmid
which confers resistance to penicillin-based
antibiotics. Penicillin can induce anaphylac-
immunogenicity, and that this effect could
tic shock in sensitized individuals. Trace
be reversed by co-administering exogenous
amounts of this antibiotic may be present as
beta-lactamase gene with another antibiotic
equaled the effect of incorporating these
to test various regulatory modules, to clone
ISS, to remove unwanted or safetywise un-
findings indicate that the backbone of the
acceptable sequences, to identify and modify
immunoprotective epitopes, are already avail-
genic activity via ISS, suggesting that the
able. In addition, obtaining high-level gene
expression is not a difficult task. However,
important consideration in designing a DNA
there is some controversy over the role of
high gene expression levels in genetic im-
munization. The key to the problem depends
vaccines induces B-cell proliferation, im-
on the antigen used as well as the type of
immune response expected, i.e., cellular,
cretion, and promotes the generation of a
humoral, or both. The exact modulation of
the expression level is necessary for each
newly tested antigen to elicit the desired
immune responses without modifying or shut-
used in DNA vaccines. Optimizing the num-
ber and the exact sequences of ISS can greatly
negative effects similar to those of tradi-
enhance the potency of DNA-based vaccina-
have been identified, efforts must be con-
Concluding remarks
centrated on testing their different combina-
tions and evaluating their immunogenic po-
tential. The study of potential biosafety risks
of DNA vaccines such as chromosomal inte-
deal of modularity. It is therefore possible to
efficient combinations of promoter, enhancer,
Acknowledgments
introns, gene from a pathogen and signal of
polyadenylation to undertake specific tasks
in the genetic immunization protocol. The
adjuvanticity of CpG motifs turns them into
Cultura Inglesa, Belo Horizonte, Brazil for
helpful comments and for reviewing the En-
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