Open Access

Austin Journal of Vaccines &

Immunotherapeutics
Review Article

Future Clinical Applications of the Potential use of Lactic

Acid Bacteria as Vehicles to Deliver DNA Vaccines
Mancha-Agresti P1, Sousa CS1, Carmo FLR1,
Oliveira Junior AF1, Azevedo V1 and de Azevedo
MSP1*
Departamento de Biologia Geral, Universidade Federal de
Minas Gerais, Brazil
*Corresponding author: Marcela Santiago Pacheco de
Azevedo, Departamento de Biologia Geral, Laboratrio
de Gentica Celular e Molecular (LGCM), Instituto de
Cincias Biolgicas, Universidade Federal de Minas
Gerais, CP 486 CEP 31270-901, Belo Horizonte - MG,
Brazil
Received: June 16, 2015; Accepted: August 27, 2015;
Published: August 29, 2015

Abstract
The use of Lactic Acid Bacteria (LAB) as DNA delivery vehicles represents
an interesting strategy as they are regarded as safe. Within the group of LAB,
the Lactococcus lactis is deemed as a model microorganism, which is being
extensively used for antigen and cytokines production and delivery to the
mucosal level. Recently studies about these bacteria have focused on their
usage as vehicles for the delivery of genic vaccines. Wild type or recombinant
invasive L. lactis are able to trigger DNA expression by epithelial cells, both in
vitro and in vivo, important for effectiveness of the vaccine. For this, invasive
strains of L. lactis have been developed in order to increase the delivery
efficiency of these vaccines to host cells. DNA vaccines are plasmid structures
with genes that encode antigenic/therapeutic proteins or peptides capable of
triggering an immune response against a wide range of diseases. This review
summarizes the potential use of Lactic Acid Bacteria as vehicles to deliver DNA
vaccines.
Keywords: DNA vaccine; Delivery Vectors; Lactic Acid Bacteria;
Lactococcus Lactis

Introduction
The use of DNA as a strategy for vaccination has progressed very
quickly since the first publication, in 1992 [1]. DNA vaccines are the
third generation vaccine that contains the best-required elements of
standard vaccines to be used in humans. This vaccination strategy has
the ability to induce potent cellular immune responses, in addition to
antibodies and the elasticity to express multiple antigens or epitopes
using a single DNA vector [2]. Genetic immunization involves the
transfer of a gene encoding an antigenic protein cloned in expression
vectors to a eukaryotic cell from the host, leading to the induction
of an immune response against the expressed antigen [3]. Therefore
theses transfected mammalian cells are able to express in situ the
antigen (for vaccines) or the therapeutic protein (for gene therapy
applications) [4]. Furthermore, they do not have the inconvenient
of classical vaccines: they are safe, inexpensive, easy to produce, heat
stable and amenable to genetic manipulation [3]. The DNA vaccine
is composed of a plasmid backbone that contains a bacterial origin
of replication needed for the vectors maintenance and propagation
inside the bacteria, as well as a resistance marker, necessary to
permit a selective growth of the bacteria that carries the plasmid;
immunostimulatory sequences (ISS), for example, the CpG motifs
(cytosine-phosphate-guanineunmethylated). They are responsible
for increasing the magnitude of the immune response as they can
enhance T lymphocyte recruitment or expansion [58]. Moreover,
these ISS sequences can interact with Toll-like receptors (TLR),
such as TLR9, and add adjuvant activity [9]. Another component of
DNA vaccines is the transcriptional unit, necessary for eukaryotic
expression, which harbors a promoter/enhancer region, introns
with functional splicing donor and acceptor sites, as well as the ORF
(open reading frame) encoding the antigenic protein of interest, and
the polyadenylation sequence (poly A), signal required for efficient

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and correct transcription termination of the ORF and transfer of the

stable mRNA from the nucleus to the cytoplasm [3,10]. The polyA
sequence usually is derived from the bovine growth hormone, SV40,
or rabbit -globin gene [8]. The ORF encoding the protein of interest
contains a Kozak translation initiation sequence (ACCATGG)
harboring an initiation codon (ATG) for appropriate translation
[1113]. The insert also contains a termination codon (TAA, TGA,
or TAG) that signals a termination of translation. Major structures of
DNA vaccines are illustrated in (Figure 1).

Figure 1: Genetic element of a plasmid DNA vaccine. The vaccine

synthesis (eukaryotic expression region) harbors the enhancer/promoter,
introns, transgene of interest and a transcriptional terminators (poly A), which
together lead the protein synthesis. The Plasmid propagation in the microbial
host comprises a prokaryotic origin of replication (ColE1) and a selectable
marker and the immunostimulatory sequences (ISS).

Citation: Mancha-Agresti P, Sousa CS, Carmo FLR, Oliveira Junior AF, Azevedo V and de Azevedo MSP. Future
Clinical Applications of the Potential use of Lactic Acid Bacteria as Vehicles to Deliver DNA Vaccines. Austin J
Vaccines & Immunother. 2015; 2(1): 1006.

de Azevedo MSP

The promoter is the element used to drive the expression of the

transgene/antigen in eukaryotic cells. The most broadly used promoter
in traditional DNA formulations is the human cytomegalovirus
(hCMV), which induces strong and constitutive expression of a
protein in a variety of cell types [14,15]. An alternative to these viral
promoters is the use of both human polyubiquitin C (UbC) and the
elongation factor 1 (EF1 ) promoters, which has been shown to
persistent gene expression in mouse lung cells, leading to a fourfold increased protein expression level of an antigenic protein when
compared to viral promoters [16]. Another element found in DNA
vaccines localized after the promoter sequence and before the ORF
from the gene of interests are the introns. These genetic elements
were reported to increase promoter activity [6] and to avoid antigen
expression by the prokaryotic machinery from bacteria, turning
possible the Heterologous expression of the protein only by the
eukaryotic system [7].
Plasmid backbones commercially available approved for gene
therapy and vaccination include pVax1, pVAC, pDNAVACultra
and pcDNA and others. They contain an Escherichia coli origin
of replication such as pUC or pBR322, which allow plasmids to
replicate in bacterial cell generating many copies of the plasmid
in a short period of time [17]. The plasmids used in gene therapy
and vaccination preferentially have TN903 gene, coding for amino
glycoside enzyme that confers resistance to kanamycin, an antibiotic
that is not widely used in humans preventing the risk of allergic
responses when compared to others antibiotics [18]. DNA vaccines
have a broad range of features that offer them many advantages over
other vaccination platforms. The principal advantage concerning
the DNA vaccine refers to the fact that they are easy to handle and
rapid to construct. This is a fascinating attribute when considering
an emerging pandemic threat [4]. In addition to this property DNA
vaccines are (i) safe as plasmids do not replicate in human cells do
not have the potential to integrate into the human cellular DNA (ii)
No adverse effects have been reported neither tolerance to the antigen
nor autoimmunity [19]; (iii) They have been shown to stimulate
immunity through MHC I-mediated antigen presentation triggering
both proliferation and activation of T and B cells antigen-specific;
(iv) Vaccine manufacturing is simple and low cost as it requires
only cloning techniques in order to clone the protein of interest;
(v) they are stable at room temperature, easy to store and transport,
presents thermal stability and long life time [20,21]. To sum up, DNA
vaccines represents an attractive tool due to its property to induce all
three points of adaptive immunity: antibodies, helper T cells (TH)
and cytotoxic T lymphocytes (CTLs), as well as being capable of
stimulating innate immune responses [22].

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well as in humans for some reasons: the plasmid DNA administered

by this manner is inefficiently expressed, poorly distributed, and
rapidly degraded [23]. The low immunogenicity of DNA vaccines
observed in these studies with humans and primates have indicated
scientist to focus on other methods of administration where antigenpresenting cells (APCs) would be transfected in a higher efficiency
and stimulating stronger immune response [24,25]. Considering
the disadvantage of intramuscular injection, other possible routes of
administration have been studied, such as intradermal, subcutaneous,
intraperitoneal, sublingual, intrarectal, ocular, and application of the
DNA vaccines to mucosal surfaces (vaginal, nasal and oral) [26]. The
pathway of administration has been shown to influence the nature
and the power of immune responses. Mucosal immune responses are
most efficiently induced by administration of vaccines to the mucosa,
where as systemic immunization strategies rarely induce long lasting
or optimal mucosal immunity and are therefore less effective against
infection at the mucosal surfaces.
Course of plasmid inside the cell and development of
immunogenicity
Once the DNA vaccine is administrated, the DNA plasmid will
transfect different types of cells, for instance myocytes, keratinocytes,
resident Antigen Presenting Cells (APCs), such as dendritic cells
(DCs) and macrophages [4,27]. Inside the cells, the plasmids will
reach the nucleus surviving to the attack of endonuclease, using
a microtubule net [28]. Inside the nucleus, the plasmid DNA has
contact to the transcription machinery, which allows the transcription
of the gene of interest [4,26]. The host cell offers the necessary posttranslational modifications mimicking a real infection. The Bacterial
mediated transfer for plasmid DNA is illustrated in Figure 2.
This feature is one of the biggest advantages of the genetic
immunization [4,27]. The produced proteins are then presented to
the surface of the cells becoming a target to the immune system.
Antigenic proteins can be secreted as well generating both humoral
and cellular immune responses. The direct transfection of the
DNA plasmid to the APCs cells has a critical role in DNA vaccine
immunogenicity. DCs are probably the most important APCs
associated with the capture and processing of antigens via receptor-

Immunological Features of DNA Vaccine

Routes of administration
The therapeutic delivery of nucleic acid has recently been
recognized as a promising tool for the treatment of several infectious
and genetic diseases. In order to exert their protective effects, DNA
vaccines needs a suitable delivery technology to produce a desired
immune response.
Intramuscular injection is the most broadly used method to
administrate DNA vaccine. Nevertheless, it has been shown that this
method is inefficient to induce immune response in large animals as
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Figure 2: Schematic representation of bacterial mediated transfer

of plasmid DNA into mammalian cells. First, the entrance of bacteria
in mammalian cells. Second, the phagolysosome engulfs the bacteria to
cleaves it. Third, the DNA plasmid escapes from the vesicle and reaches
the nucleus of the mammalian cells. Fourth, in the nucleus the transgene
is transcribed and the protein synthesis is realized by host cell. Finally, the
protein is exposed the immune system.

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mediated endocytosis as it presents antigens trough MHC class I or II

directly to T nave cells, leading to TCD4+ and TCD8+ lymphocytes
activation. The stimulation of this immunological repertoire raises
both humoral and cellular protection against the antigen inside the
host [29,30].
Clinical advance in DNA vaccine platform
FDA still did not approve the use of DNA vaccines in humans.
Phase I clinical studies have been reported for the prevention and/
or treatment of HIV, malaria, hepatitis B, SARS and many other
infectious agents [31]. In order to strongly trigger the innate immune
mechanisms and to go guarantee an optimal expression of the antigen,
it is essential to efficiently deliver the DNA into the cells ensuring their
transfer to the nuclei [32]. Actually, the inefficient uptake/transfer of
the plasmids into mammalian cells is one of the principal hurdles in
DNA vaccinology [21]. The lack of reproducibility of many results
obtained in mice after the applications in larger animals, as well as
the failure of DNA vaccines to induce potent immune responses in
humans have not yet been elucidated [33]. For this reason, novel
strategies to improve transfection efficiency are emerging. Among
them, can be highlighted the delivery methods employed to introduce
the DNA vaccine in the organism [33]. These methods include physical
and chemical approaches or the biological strategies, such as the use
of viral and bacterial vectors. The physical methods of delivering
include: gene gun, tattooing, ultrasound (US), electroporation
(EP), laser and dermal patches. The chemical vectors most studied
are lipid and polymer complexes. The principal advantages of these
vectors are that they can tightly compact and protect the DNA and
be recognized by specific cell-surface receptors expressed in DCs or
macrophages. Moreover, they can disrupt the endosomal membrane
to deliver DNA plasmids allowing their transfer to the nucleus when
the pH of the medium is reduced to below to six [33]. Even though
physical and chemical vectors have shown to be interesting tools to
delivery DNA, other methods are being explored as well, such as
viruses and bacteria. Regarding the use of viruses as delivery vehicles,
a severe adverse effect has occurred during in a gene therapy trial
raising serious safety concerns. Attenuated viruses have been studied
for gene therapy/antigen delivery, however it was shown that their
genome could integrate the host cellular chromatin (oncoretrovirus
and lentivirus), which may cause genetic diseases or favor the
development of cancerous cells [34].
Regarding the use of biological vehicles, bacterial vectors have
been shown to be an excellent choice, as they can transfer the genetic
material into mammalian cells such as viruses and does not present
the same problems associated with their use. Both attenuated bacterial
pathogens or live recombinant bacteria are considered good models
for DNA delivery due to their ability to protect the DNA vaccine
from enzymatic digestion, to stimulate the immune system as they
can target inductive sites of the body generating effective adaptive
immunity [35].
Bacterial as delivery vectors for DNA vaccines
The innate tropism of some pathogenic bacterial strains have for
specific tissues of the host directed the attention of researchers to
study them as a vehicle to deliver DNA vaccine, as this characteristic
is indispensable for the elicitation of immune response. Several
advantages can be highlighted: they can keep the plasmid in a high

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copy number, they are easy to manufacture, they are less laborious
and has low cost as there is no need to amplify and purify the plasmid
before [7,36], large-size plasmid are able to be housed inside the
bacteria, permitting the insertion of multiple genes of interest, and the
bacterial cell can protect the DNA against endonuclease degradation
[37,38].
Additionally to these features of bacterial vectors is the possibility
to use them for oral administration, important feature to stimulate
both mucosal and systemic immune responses [39]. Considering the
increased numbers of vaccines administered all over the word, the
fact that they can be inoculated without the use of a needle turns them
a cheap and safe method [40].
DNA delivery from bacterial to mucosal surfaces:
immunological features
Bacteria carrying a DNA vaccine are able to cross the intestinal
barrier, mainly via M cell (specialized epithelial cells named Micro
fold cells) overlying Peyers patches (PPs). The PPs are isolated
lymphoid follicles in draining gut mesenteric lymph nodes, considered
more accessible to antigens and bacteria present in the luminal
compartment. Another manner by which bacteria may have access
to the body is through immature dendritic cells (DC) that reside in
PPs. They are capable to open tight junctions between epithelial cells,
extend their dendrites outside the epithelium and directly sample
bacteria, thereby monitoring the contents of the intestinal lumen
[41]. Moreover, bacterial vectors are able to enter inside the host body
by invading intestinal epithelial cells (IECs) lining mucosal surfaces
through the expression of some proteins named invasins. This
characteristic refers to the capacity of attenuated pathogenic vectors
to deliver DNA vaccines as they naturally produce invasins.
Regarding the vectors based on attenuated pathogenic species,
once inside the cells they have the ability to escape from the
phagolysosome vesicles by the secretion of a variety of phospholipases
and pore-forming cytolysins and enter the cytoplasm of the host
cells [36,37]. The plasmids can then reach the nucleus through
the microtubules net; once in the nucleus using the host cells
transcription machinery the protein of interest carried by the plasmid
can be encoded, translated, and secreted afterwards [36,42]. The
antigenic proteins may be secreted outside the cell or be presented
on the surface of epithelial cell or DCs. The Major Histocompatibility
Complex (MHC) class-II, from APCs, presents the exogenous
proteins, turning nave T cells activated into T helper cells (CD4+
T-cells). Furthermore, the exogenous protein may also be processed
into small peptides, which are then presented on the surface of MHC
class-I molecules to cytotoxic T-cells (CD8+ T-cells), stimulating
them [43].
Other important components of immunity are the pattern
recognition receptors (Toll-like and Nod-like receptors) expressed
by IECs, B-lymphocytes and DCs that are located in the sub
epithelial lamina propria. These receptors are able to recognize
some bacterial components known as microbe-associated molecular
patterns (MAMPs), triggering intracellular signaling pathways
that lead to cytokine secretion and immune cell activation [44,45].
The production of a merged immune response encompassing the
induction of humoral and cell mediated immunity (CMI) effectors
like CD8+ and CD4+ T cells after DNA vaccination using bacterial
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vectors is a well established event known as cross-priming [46,47].

The bacterial recognition by the immune system modulates the
innate immune response, therefore, supporting a vigorous and lasting
adaptive response [37].
Another important characteristic is the communication between
activated immune cells localized in the mucosal surface with the
systemic blood where they can travel around the body via the lymph
[48]. The production and secretion of antigen-specific secretory
immunoglobulin A (SIgA) responses by plasmocytes is another
important advantage to be considered when using bacteria as mucosal
delivery vehicles for DNA vaccines.
Commensal bacteria can interact with IECs and deliver
tolerogenic signals that are transmitted to the underlying cells of
the immune system [49]; consequently, they are not ignored by
the intestinal immune system. The gastrointestinal mucosa offers
immunological tolerance against nonpathogenic bacteria and
antigens; this phenomenon avoids reactions against proteins and
commensal bacteria. Thus, mucosal tolerance protects the mucosa
from detrimental inflammatory immune responses [50]. Actually,
allergic disease development and cancer, especially colon cancer, has
been associated with alterations in the intestinal micro biota [51].
Bacteria as a delivery vector for gene transfer
Salmonella typhi, Listeria monocytogenes, Shigella Flexner, Yesinia
enterocolitica and Escherichia coli are the principal enteropathogenic
species most widely used as bacterial delivery systems into mammalian
cells [36] due to their natural tropism for DCs and macrophages in
the lymphoid tissue of the intestinal mucosal surface [7]. There are
several reports showing the use of these strains as bacterial vectors.
For example: Salmonella typhimurium is probably the most broadly
used bacteria for antigen delivery applications, such as encoding duck
enteritis virus UL24 [52], encoding HIV gp140 [53], among others;
Yesinia enterocolitica encoding Brucella abortus antigens [54]; Listeria
monocytogenes as a gene delivery vector for targeting cancer cells [55]
as it could effectively target tumors expressing surface bound antigens
(Her2/neu), intracellular antigens (HPV-16 E7) or secreted antigens
(PSA) [56].
The use of human enteric bacterial strains, as a bacterial carrier,
is being considered an advantage because of their capacity to infect
human colonic mucosa after oral administration. However, for
this proposal enteropathogenic species needs to be attenuated or
inactivated. Nonetheless, attenuated strains has a restrict use as they
present the risk to revert to the virulent phenotype compromising
its safety, as reported for the oral polio vaccine (e.g., Sabin 3)
[57]. Therefore, World and Health Organization (WHO) does
not recommend their use in children and immunocompromised
individuals. Thus, to counteract this severe problem, it has been
investigated the use of non-pathogenic bacteria, such as LAB as
vectors for genetic immunization [58].
Lactic acid bacteria
Lactic acid bacteria (LAB) are distributed in diverse ecological
niches; for example in plants, fermented foods, as well as in the
gastrointestinal tract of many animals, including humans, where
some species can live as commensal microorganisms [59]. LAB
comprises mainly species of Lactobacillus, Lactococcus, Leuconostoc,

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Oenococcus, Pediococcus and Streptococcus [60,61]. They are widely

used in the industry for the production and the preservation of foods
due to their ability to acidify the medium (pH 3.5 to 4.5). Therefore,
LAB are ingested diary by humans in different types of foods such as
cheese, wine, yogurt, fermented milks, pickles, kefir, butter, among
others. It have been reported the importance of enteric lactic bacteria
during their life into the human gastrointestinal tract, as they have
an important impact on host metabolism, participating in microbialmammalian co-metabolism [62]. Therefore, FDA has granted some
strains of LAB group as Generally Recognized as Safe (GRAS)
for human consumption [63]. Another important characteristics
of LAB are their capacity to restore the normal intestinal flora,
eliminate intestinal pathogens, reinforce the intestinal barrier
capacity to foreign antigens, stimulate nonspecific immunity such as
phagocytosis, stimulation of humoral immunity and production of
anti-inflammatory products [64,65]. Due to all these positive effects,
some strains are considered as probiotics because when consumed at
adequate levels they can positively influence the human health. Thus,
scientific is extensively exploring these probiotics as an alternative
treatment for some diseases, and to serve as a tool for genetic
immunization [66].
L. lactis is the best-characterized member within the LAB group
being considered the model organism. They are facultative anaerobic,
catalase negative, and do not form endospores. Lactococcus lactis
ssp. lactis was originally found as a milk-souring isolate, but it is also
associated with plants. Lactococcus lactis ssp. cremoris is used as a
starter culture for the manufacture of Cheddar cheese, in which it
contributes with highly prized flavor [60]. L. lactis does not colonize
the digestive tract of men and animals. Besides to this economic
significance of L. lactis, other important features have been described:
(i) it has a completely sequenced genome [67]; (ii) they are GRAS,
(iii) it is genetically easy to manipulate; and (iv) many genetic tools
have already been developed for this species [68], and (v) does not
contain Lip polysaccharide (LPS) avoiding endotoxin shock after
being administered to humans [69].
Lactococcus lactis: heterologous protein delivery
Besides its traditional and safe use in the food industry, L. lactis
is presenting to be a very an interesting tool to be used as a cell
factory for the high-level protein production. This new role assigned
for L. lactis is due to the fact that this bacterium does not produce
endotoxins or other toxic metabolic products [70]; few proteins are
secreted in L. lactis; Usp45 (Unknown Secreted Protein of 45 kDa) is
the only one on e secreted in quantities large enough to be detected in
Coomassie-stained protein gels [71]. This feature is very important, as
it simplifies purification step after bacterial growth in fomenters [72].
Transformation protocols, cloning or screening-vectors,
mutagenesis systems, protein expression and targeting-systems are
examples of the availability of genetic tools that have been developed
for L. lactis. These tools have been used to engineer L. lactis to produce
intra- or extra-cellular recombinant proteins of viral, bacterial or
eukaryotic origins [72]. These tools also allow the expression of
these proteins in a controlled manner. To this end, various vectors
containing constitutive or inducible promoters PlacA [73]; PnisA
[74]; PT7 [75]; P170 [76]; P59 [77]; PxylT [78]; Pzn [79] have been
developed and represent the basis of all expression systems in L. lactis
and other LAB [80].
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leprae [90]; S-layer protein (SlpA) of Lactobacillus brevis [91], among

others.
Lactococcus lactis as live mucosal delivery vectors for
vaccine

Figure 3: Schematic representation of the NICE system development

in L. lactis. The expression of genes in response to nisin stimulus involves
a membrane-located sensor protein (NisK) and a cytoplasmic response
regulator (NisR) that controls transcriptional activation from the misA
promoter (PnisA). The auto-induction mechanism of nisin is used as a
controlled heterologous gene expression system in L. Lactic.

Nisin Controlled Gene Expression (NICE) is the most used

amongst all expression systems developed for use in LAB. It offers
significant potential for regulated gene expression. NisR and NisK
genes encode a two-component system (NisRK), which controls the
expression of the nisA gene via signal transduction. NisK functions
as a membrane sensor that detects extracellular nisin. The signal is
transferred to NisR through a phosphorylation process turning this
gene capable of activating gene transcription, which is controlled by
the PnisA promoter [74].
Therefore, in the presence of Nisin the promoter can induce the
transcription of the molecule of interest [81] (Figure 3). NisK and
NisR genes were isolated from the nisin gene cluster and inserted
into the chromosome of L. lactis subsp. cremoris MG1363 (nisinnegative), creating NZ9000 strain (nisin positive) [82,83]. Depending
on the presence or absence of the corresponding targeting signals,
the protein is expressed into the cytoplasm, anchored to the cell wall
or secreted to the extracellular medium. Many exogenous proteins
have been expressed in L. lactis through this system [74,84,85]. NICE
system has been tested in other LAB, such as Leuconostoc lactis,
Lactobacillus Helveticas, Streptococcus sp., Bacillus sp., Enterococcus
sp. [86] and Lactobacillus Plant arum [87], proving its versatility.
Another expression and targeting system that allows different
cellular locations of the gene of interest for use in L. lactis is the
xylose-inducible expression system (XIES), which is based on the
use of a xylose inducible lactococcal promoter, PxylT from L. lactis
NCDO2118 strain. In the presence of sugars (glucose, fructose and/
or mannose) PxylT is tightly repressed. However, in the presence of
xylose, PxylT is transcription ally activated [88,89]. Staphylococcus
aurous nuclease genes (nuc) fused or not to the lactococci Usp45
signal peptide was adopted to test the capacity of this system to express
the cytoplasm or secreted form of nuc protein. Xylose-inducible nuc
expression was found to be tightly controlled resulting in high-level,
long-term protein production, and correct targeting either to the
cytoplasm or to the extracellular medium. This expression system is
versatile and can be easily switched on or off by adding either xylose
or glucose, respectively [78]. XIES system has been employed in the
biotechnology field for production of different heterologous protein,
for example: 65-kDa heat shock proteins (HSPs) of Mycobacterium
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Regarding L. lactis as a vehicle to deliver DNA vaccines, many

interesting features can be highlighted: (1) it was proved in different
laboratories all over the world that they can carry recombinant
plasmids and express antigens and therapeutic molecules at different
cellular localizations [73,92]; (2) it was successfully demonstrated that
L. lactis can deliver DNA into eukaryotic cells and in vivo to mice IECs
[9395]; (3) they can induce both systemic and mucosal immunity
when administrated at mucosa surfaces [96,97]; (4) they can resist
to the acid environment of the stomach, being able to survive into
the gastrointestinal tract, ensuring recombinant protein or plasmid
delivery [20]. Because L. lactis is not very immunogenic, it can be
orally administrated multiple times [98], regarding its extraordinary
safety profile [99]. Furthermore, L. lactis has the ability to stimulate
the phagocytic system of the host [100]. All this characteristics
turns it a good option for being used in immunization programs
[101]. In accordance with the benefits of L. lactis, several researchers
have developed different recombinant strains to be used for genetic
immunization. Table 1 summarizes some studies that were performed
in different laboratories around the world corroborating the ability of
L. lactis in inducing long-lasting immune responses.
L. lactis expressing invasions for DNA delivering
Studies demonstrated both in vitro [98] and in vivo [93] that wildtype (wt) L. lactis could be used as a vector for genetic immunization.
However, the percentage of gene transferred observed was low, as well
as a low and transitory Th1-type immune response after immunization
trials [93]. For this reason, with the aim to increase the capacity of
lactococci to deliver DNA, some strains of L. lactis expressing invasins
have been developed. The first engineered L. lactis to express invasins
was reported in 2005 by Guimares and co-workers; InlA gene from
L. monocytogenes was cloned and expressed under transcriptional
control of the native promoter. This work showed that recombinant
lactococci could efficiently express the cell wall anchored form of
InlA, and the invasion rates of LL-InlA+ strain in Caco-2 cells was
approximately 100-fold higher than the wt lactococci. Moreover, after
oral inoculation in guinea pigs, this recombinant strain was capable
to invade intestinal cells [94]. However, the use of LL-InlA+ strain
showed to be inconvenient because InlA cannot bind to its receptor
in mice, murine E-cadherin, thus limiting the in vivo studies as the
effect of LL-InlA+ strain was only possible to be explored in guinea
pigs or transgenic mice, which may be laborious and/or expensive
[102]. To improve this strategy another invasive strain of L. lactis
was constructed by cloning the gene encoding fibronectin-binding
protein A (FnBPA), from Staphylococcus aureus (LL-FnBPA+) [103].
FnBPA production at the surface of L. lactis increases invasiveness
of the cells 1000 fold and increased plasmid transfer 30-fold in vitro
(Innocent in et al., 2009). L. lactis InlA+ and L. lactis FnBPA+, showed
comparable internalization rates in Caco-2 cells [93]. Therefore,
another recombinant invasive lactococci was developed producing a
mutated form of Internal in A (mInlA), appropriate to be used in a
murine model [90].
In order to evaluate recombinant L. lactis expressing invasins a
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Table 1: Antigens expressed by L. lactis, inducing long-lasting immune responses.

Immune Response
Antigens Expressed by L. lactis
Observed
EDIII antigen from dengue virus type 2

Neutralization of the virus in vitro

Envelope protein of the human

immunodeficiency virus 1 (HIV-1)
Antigenic protein of
Proteus mirabilis (HBPM)
PspA antigen derived
from Streptococcus pneumoniae
Antigen lcrV from Yersinia
pseudotuberculosis
EspB antigen from the type III secretion
system (T3SS) of E. coli serotype
O157:H7
Leishmania antigen LACK and the
proinflammatory cytokine IL-12

High levels of IgG and IgA antibodies

against the antigen observed
Protection to the animals against
challenge with P.mirabilis virulent strain
Better protected
against challenge with the virulent strain
Humoral and cellular immune responses,
conferring protection against challenge
Significant levels of
specific serum Ig and
faecal IgA
Protection against
challenge

Mature murine IFNgamma

--

Antigen envolved with LPS transport

(wzm) from Vibrio cholerae O1 strain

Increase of systemic and mucosal

immunity
Inhibition of chemically induced
colon cancer

Catalase-producing L. lactis
IL-10

Anti-inflammatory effects controlling

intestinal inflammation

new vector have been developed resulted from the co-integration of

two replicons: one from E. coli and the other from L. lactis, named
pValac (Vaccination using Lactic acid bacteria). The pValac is
formed by the fusion of (i) cytomegalovirus promoter (CMV), that
allows the expression of the antigen of interest in eukaryotic cells,
(ii) polyadenylation sequences from the bovine Growth Hormone
(BGH), essential to stabilize the RNA transcript, (iii) origins of
replication that allow its propagation in both E. coli and L. lactis
hosts, and (iv) a chloramphenicol resistance gene for selection of
strains harboring the plasmid. The functionality of pValac was
observed after transfecting plasmids harboring the gfp ORF into
Pk15 cells. The vector demonstrated to be functional as PK15 cells
were able to express GFP protein. Moreover, invasiveness assays of
L. lactis inlA+ carrying pValac: gfp into Caco-2 cells showed that this
strain could deliver the vector to epithelial cells, in vitro [104]. This
assay demonstrated that L. lactis expressing invasins and harboring
functional plasmids can serve as tools for genetic immunization [93].
Besides the effort to construct invasive L. lactis strains and
plasmids to be used for genetic immunization, other works have
attempted to test L. lactis in the vaccination field.
De Azevedo and co-workers used L. lactis expressing a mutated
form of L. monocytogenes Internal in A (LL-mInlA) carrying pValac:
BLG. BLG is the bovine lacto globulin, a major cows milk allergen.
They were able to show the production of mInlA enhanced invasivity
and allowed plasmid transfer in a higher efficiency in in vitro
experiments. Besides than, were done in vivo experiments showing
slightly increased plasmid transfer after oral administration [90].
An elegant work has been done with recombinants L. lactis
FnBPA, L. lactis mInlA and wt strains (L. lactis non invasive), all
of them carrying pValac: BLG. It was showed that the intranasal
immunization of L. lactis non invasive strain carrying pValac: BLG
elicited a TH1 immune response. However, when immune response
elicited by L. lactis FnBPA and L. lactis mInlA were evaluated, both
strains carrying pValac: BLG, it was observed the secretion of IL-4
and IL-5 in medium of BLG reactivated splenocytes, after both oral
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Application

Reference

Dengue virus
control strategy

Sim et al., 2008 (107)

HIV vaccine

Xin et al., 2003 (108)

Control of urinary
tract infections
Vaccine against
pneumonia
Vaccine against Far
East scarlet-like fever

Hanniffy et al., 2007 (110)

New strategie to fight against

enterohemorrhagic E. coli

Ahmed et al., 2012 (112)

Vaccine to combat
Leishmaniosis
Adjuvant tool
Vaccine against cholera
Cancer therapy
Treatment of allergy
and inflammatory
bowel diseases (IBD)

Scavone et al., 2007 (109)

Daniel et al., 2009 (111)

Hugentobler et al.,
2012 (113)
Bermdez-Humarn
et al., 2008 (114)
Zamri et al., 2012 (115)
De Moreno de
LeBlanc et al., 2008 (116)
Cortes-Perez et al., 2007 (117); Braat
et al. 2006(118) ; Marinho et al., 2010
(119)

and intranasal administration. It was concluded that non invasive

lactococci elicits a Th1 immune profile while the immunization with
the recombinant invasive strains elicited a Th2 immune response
[105].
Another work using L. lactis as DNA delivery vehicle involved the
construction and evaluation of a DNA vaccine against Tuberculoses.
In this work BALB/C mice were orally administered with L. lactis
expressing FnBPA carrying pValac coding for the 6-kDa early secreted
antigenic target (ESAT-6) gene of Mycobacterium tuberculosis. This
report showed significant increase of interferon gamma (IFN-)
production in spleen cells, as well as a significant increase of specific
secretory immunoglobulin A (SIgA) production in colon tissue and
fecal extracts [20].
The administration of L. lactis FnBPA, has been used for the
administration of other therapeutic molecule, such as IL10, for the
treatment of Inflammatory Bowel Disease (IBD), an inflammatory
condition of the TGI being presented in two forms: Ulcerative
colitis and Crohns disease. To this end, the therapeutic effect of L.
lactis expressing FnBPA carrying pValac coding for Interleukin-10
from Mus musculus was evaluated using a model of acute
trinitrobenzenesulfonic acid (TNBS)-induced colitis in mouse. It
was observed a decrease in the severity of the inflammation with
lower macroscopic and microscopic inflammatory scores in the large
intestine, decrease of microbial translocation to the liver and less body
weight loss. Furthermore, they were able to shown a decrease in antiinflammatory cytokine, IL-17. This work suggested that recombinant
L .lactis expressing FnBPA invasins (pValac:il-10) is a suitable option
to maintain an anti-inflammatory status in the GIT, especially for
chronic Crohns disease patients [106].

Conclusion
Food-grade bacteria, such as LAB, have recently been proposed as
a vehicle to express recombinant antigens and therapeutic molecules,
as well as to deliver DNA vaccines. One of the LAB models, L. lactis,
has been shown to act as DNA delivery vehicles and to deliver many

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de Azevedo MSP

different proteins with biomedical and biotechnological interest at the

mucosal level. In spite of the necessity of other studies, the approach of
using wild type or recombinant L. lactis as a tool to deliver therapeutic
plasmids can be considered a promising future. An important point
regarding the use of L. lactis as a DNA delivery vector is the strategy
to use L. lactis expressing invasins. As recombinant invasive strains
improved the delivery of DNA, especially in vitro, scientists explored
new horizons testing them for many applications, for instance in
the vaccination area. To date, there are no DNA vaccines available
to be administered in humans. The only licensed DNA vaccine is
for veterinary purposes. Being L. lactis safe for use in humans and
capable to efficiently deliver DNA vaccines, it Fs use as immunization
vehicles is an excellent choice for clinical applications in near future.
Especially because this bacterium is easy to handle, GRAS and has a
large number of genetic tools already developed.
Recombinant strains, as well as new vectors have attracted
researchers attention and a great number of studies are in progress
with the aim to develop systems using LAB to deliver DNA vaccine.
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Citation: Mancha-Agresti P, Sousa CS, Carmo FLR, Oliveira Junior AF, Azevedo V and de Azevedo MSP. Future
Clinical Applications of the Potential use of Lactic Acid Bacteria as Vehicles to Deliver DNA Vaccines. Austin J
Vaccines & Immunother. 2015; 2(1): 1006.

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