Appl Microbiol Biotechnol
DOI 10.1007/s00253-016-7557-x
MINI-REVIEW
Lactic acid bacteria as mucosal delivery vehicles: a realistic
therapeutic option
Miao Wang 1 & Zeqian Gao 1 & Yongguang Zhang 1,2 & Li Pan 1,2
Received: 10 March 2016 / Revised: 12 April 2016 / Accepted: 14 April 2016
# Springer-Verlag Berlin Heidelberg 2016
Abstract Recombinant lactic acid bacteria (LAB), in partic-
ular lactococci and lactobacilli, have gained increasing interest
as mucosal delivery vehicles in recent years. With the devel-
opment of mucosal vaccines, studies on LAB expression sys-
tems have been mainly focused on the generation of genetic
tools for antigen expression in different locations.
Recombinant LAB show advantages in a wide range of as-
pects over other mucosal delivery systems and represent an
attractive candidate for the delivery of therapeutic and prophy-
lactic molecules in different applications. Here, we review the
recent data on the use of recombinant LAB as mucosal deliv-
ery vectors and the associated health benefits, including the
prevention and treatment of inflammatory bowel diseases
(IBDs), autoimmune disorders, and infections by pathogenic
microorganisms from mucosal surfaces. In addition, we dis-
cuss the use of LAB as vehicles to deliver DNA directly to
eukaryotic cells. Researches from the last 5 years demonstrate
that LAB as vectors for mucosal delivery of therapeutic mol-
ecules seem to be a realistic therapeutic option both in human
and animal diseases.
* Yongguang Zhang
yongguang.zhang@163.com
* Li Pan
panli@caas.cn
1
State Key Laboratory of Veterinary Etiological Biology, National
Foot-and-Mouth Disease Reference Laboratory, Lanzhou Veterinary
Research Institute, Chinese Academy of Agricultural Sciences, 1
Xujiaping, Yanchangbu, Lanzhou, Gansu 730046, China
2
Jiangsu Co-innovation Center for Prevention and Control of
Important Animal Infectious Diseases and Zoonoses,
Yangzhou, Jiangsu, China
Keywords Lactic acid bacteria . Mucosal immunity . Delivery
vehicles . Therapeutic molecules . Mucosal vaccines .
DNA vaccines
Introduction
Lactic acid bacteria (LAB), which include species of
Lactobacillus, Leuconostoc, Lactococcus, Pediococcus, and
Streptococcus, are a heterogeneous group of non-sporulating
and gram-positive bacteria of high relevance for the produc-
tion of fermented food and beverages and for human and
animal health (Gareau et al. 2010). LAB have been used for
centuries to produce and preserve food, such as cheese (spe-
cies of Lactococcus), pickled vegetables (species of
Lactobacillus and Lactococcus), and yoghurt (species of
Lactobacillus and Streptococcus); they are non-pathogenic
and are generally regarded as safe (GRAS) according to the
US Food and Drug Administration (FDA) (Bermudez-
Humaran et al. 2013; Wells and Mercenier 2008). LAB reside
in a diverse range of niches including milk, the surface of
plants, the oral cavity, gastrointestinal tract (GIT), and vagina
of humans and animals (Bermudez-Humaran et al. 2013).
Specific strains of LAB, Lactobacillus, Streptococcus, and
Bifidobacterium, are used as probiotics for human and animal
health, as they play a crucial role in maintaining gastrointesti-
nal homeostasis and influence other organs by producing bio-
active metabolites and by modulating immunological param-
eters and intestinal permeability (de Vrese and Schrezenmeir
2008; Tsai et al. 2012). In the past two decades, LAB strains,
in particular Lactococcus lactic and Lactobacillus plantarum
(Cano-Garrido et al. 2015), have generated increasing inter-
ests as vectors for the delivery of therapeutic and prophylactic
molecules via the intranasal, oral, or genital mucosal surfaces
(Dimitrijevic et al. 2014; Hiramatsu et al. 2014; Li et al.

2015a; Wang et al. 2014a; Xu et al. 2015a). Recombinant
LAB-mediated delivery of mucosal vaccines, particularly via
intranasal or oral immunization, is more applicable and con-
venient than the intravenous or intramuscular injection of tra-
ditional systemic vaccines, because mucosal vaccination can
save both time and labor, for instance, oral vaccine could be
made into powder and stirred into the feed, especially appli-
cable for scaled livestock and poultry, and meanwhile, muco-
sal immunization can reduce the pain compared to intravenous
or intramuscular injection. Moreover, mucosal delivery of
therapeutics and prophylactic molecules for chronic diseases
and infections associated with mucosal tissues may increase
their efficacy and specificity while minimizing the side effects
of systemic vaccine administration (LeBlanc et al. 2013; Pan
et al. 2014; Wang et al. 2011). Many LAB strains have the
capacity to survive transit through the GIT, and most of them
are present into the intestinal tract and eventually remain in the
lumen or are trapped in the mucus, which facilitates mucosal
targeting of therapeutic molecules (Daniel et al. 2011).
Because of these positive features, LAB have emerged as an
attractive potential alternative to other mucosal delivery sys-
tems, such as microspheres, liposomes, nanoparticles,
immunostimulating complexes, and attenuated pathogens
(Daudel et al. 2007; Hu et al. 2001; Pabreja et al. 2014;
Wendorf et al. 2008). In this review, we summarize recent data
on recombinant LAB as mucosal delivery vehicles and discuss
their potential medical applications in animal studies and hu-
man clinical trials.
LAB delivery systems and advantages as mucosal
delivery vehicles
Mucosal vaccines have attracted significant attention in recent
years because of their remarkable properties and potential ap-
plications against a wide range of microbial pathogens. They
confer protection against infections at mucosal surfaces, pre-
vent antigen degradation, and promote uptake in the GIT, in
addition to activating adaptive immune responses. Despite
efforts to develop attenuated bacterial pathogens as mucosal
vaccines based on their ability to elicit host immune responses
against foreign antigens, there are limitations to the use of
attenuated live vaccines, such as the potential risk of reversion
to a virulent phenotype, side effects of varying severity, and
imbalances between immunogenicity and reactogenicity
(Roland et al. 2005; Wang et al. 2013). LAB can overcome
some of the limitations of attenuated vaccines as mucosal
delivery vehicles and fulfill the requirements of delivery sys-
tems for mucosal administration.
LAB are non-pathogenic microorganisms, and they are con-
sidered safer than some attenuated vectors. Recombinant LAB
strains have the capacity to express numerous heterologous
proteins, such as chimeric or non-chimeric antigens, cytokines,
Appl Microbiol Biotechnol
and enzymes, which could advance the development of multi-
valent vaccines (Lei et al. 2015a; Mobergslien et al. 2015;
Saraiva et al. 2015; Yang et al. 2015). Dietary LAB can survive
exposure to gastric acid and contact with bile, allowing uptake
into the inductive sites of the mucosal immune system such as
Peyer’s patches (Wang et al. 2014b). In addition, the mucosal
administration of LAB by oral, intranasal, or genital routes can
stimulate both mucosal and systemic immune responses. Their
ability to elicit the production of secretory IgA is one major
advantage of LAB as mucosal vaccines (Macpherson et al.
2015). LAB are not only used as live bacteria for mucosal
immunization, but certain killed recombinant strains have
shown efficacy as mucosal vehicles, mainly by intranasal vac-
cination (Almada et al. 2015). LAB expression systems (espe-
cially the NICE and the Self-Immobilizing Plasmid (pSIP) sys-
tems) enable the targeted expression of foreign antigens at spe-
cific locations (Fig. 1), such as the cytoplasm, cell wall, or the
medium, which prevents protein degradation under harsh con-
ditions associated with the administration route or through in-
teraction with the environment that may affect immunogenicity
(Nguyen et al. 2015; Zhou et al. 2015).
Prevention and treatment of inflammatory bowel
diseases
Inflammatory bowel disease (IBD) affects millions of people
worldwide and refers to two chronic diseases that cause inflam-
mation of the intestines: Crohn’s disease (CD) and ulcerative
colitis (UC). IBD symptoms include abdominal pain, cramps,
diarrhea, fatigue, fever, weight loss, and poor appetite
(Bermudez-Humaran et al. 2015). The first reported use of
recombinant LAB for the treatment of IBD was published
15 years ago, in a study that described the use of secreted IL-
10 in two mouse models of colitis (Steidler et al. 2000). This
was followed by several studies reporting on the use of recom-
binant LAB for human IL-10 delivery (Bermudez-Humaran
et al. 2015; Martin et al. 2014; Schotte et al. 2000; Steidler
et al. 2003). The effect of recombinant Lactococcus lactis
MG1363 secreting IL-10 generated using a stress-inducible
controlled expression (SICE) system was tested in a murine
model of chronic low inflammation in response to repeated
dinitrobenzene sulfonic acid challenge (Martin et al. 2014).
The results showed that oral administration of L. lactis secret-
ing active IL-10 (LL-IL10) decreased gut hyperpermeability,
improved dysfunctional parameters, and enhanced immune ac-
tivation in immunized mice. Lactococci strains containing the
recombinant plasmid and the empty plasmid both
counterbalanced cytokine levels and LL-IL10 T cell levels,
confirming previous data in which the immunomodulatory ca-
pacities of wild-type L. lactis were observed in in vitro and
in vivo assays (Smelt et al. 2012). The protective effects of
IL-10 delivered by recombinant L. lactis MG1363 were further
Appl Microbiol Biotechnol
Fig. 1 Four approaches based on the use of lactic acid bacteria (LAB) as
delivery vehicles for protein antigens or DNA in the gastrointestinal tract.
Cell wall-anchored proteins produced by LAB interact with cells in the
intestinal tract (a). Cytoplasmic proteins expressed by LAB are released
into the lumen after lysis of LAB (b). LAB directly secrete protein
tested using two expression systems, the SICE system and a
complementary DNA (cDNA) delivery system named pValac,
in a murine colitis model mimicking the relapse nature of IBD
(del Carmen et al. 2014). The beneficial effects of non-invasive
recombinant L. lactis strains through two different ways be-
tween DNA and protein delivery were compared, which
showed that both approaches had a similar anti-inflammatory
effect in a chronic 2,4,6-trinitrobenzenesulfonic acid-induced
colitis model, underscoring the relevance of L. lactis delivered
IL-10 for the treatment of IBD. The efficacy of LAB-mediated
delivery to the gut lumen was tested using other anti-
inflammatory molecules. A recent study compared the efficacy
of recombinant L. lactis, MG1363 and NZ9000, secreting two
types of anti-inflammatory molecules, namely cytokines and
serine protease inhibitors, to identify the best strategy for the
treatment of IBD in a dextran sulfate sodium-induced colitis
mouse model (Bermudez-Humaran et al. 2015). The results
showed that treatment with recombinant L. lactis strains deliv-
ering the serine protease inhibitors elafin and secretory leuko-
cyte protease inhibitor (SLPI) was more efficient than treatment
with anti-inflammatory cytokines (IL-10 and TGF-β1) for al-
leviating the symptoms of colitis. Elafin and SLPI released in
the lumen by LAB attenuated the effects of proteases produced
by inflammatory cells, downregulated pro-inflammatory tran-
scription factors, restored barrier functions, and promoted anti-
microbial activity (Butler et al. 2006; Henriksen et al. 2004;
Motta et al. 2012), indicating that they exerted anti-
inflammatory effects more efficiently than cytokines. These
antigens into the lumen (c). The strains harboring a eukaryotic
expression system transfer DNA directly to eukaryotes and transduce
host cells to express and release proteins into the lumen (d). DC
dendritic cell, M cell microfold cell, LAB lactic acid bacteria
results highlight the importance of other anti-inflammatory
molecules (especially serine protease inhibitors) expressed by
recombinant LAB for the treatment of IBD in addition to the
L. lactis IL-10 recombinant approach (de LeBlanc et al. 2015).
Taken together, these studies indicate that the use of recom-
binant LAB for mucosal therapy could be valuable for the
treatment of IBD in humans in the future.
Preventing infections by pathogenic microorganisms
and parasites at mucosal surfaces
Over the past 10 years, several anti-infective bioshield strate-
gies with recombinant LAB have been applied for preventing
infection at the mucosal surface (Table 1). The first report on
the use of live L. lactis for the delivery of a vaccinal antigen
was published in 1993 (Wells et al. 1993). In that study, the
protective role of recombinant L. lactis expressing the tetanus
fragment C (TTFC) was initially tested via subcutaneous in-
jection in mice after tetanus toxin challenge and later via oral
and nasal administration using live L. lactis producing TTFC
(Norton et al. 1997; Robinson et al. 1997; Wells et al. 1993).
Although the protective efficacy was the same, nasal immu-
nization elicited higher levels of serum IgG and mucosal IgA
than oral delivery. Since then, numerous studies have reported
on the use of recombinant LAB as mucosal vaccines express-
ing antigens derived from viral, bacterial, fungal, or parasitic
pathogens.
 Appl Microbiol Biotechnol

Table 1 Current anti-infective applications of lactic acid bacteria (LAB) delivery

Application Vehicle Recombinant protein Model Route References

Bacillus Lactobacillus paracasei scFv antibody Mouse Oral Andersen et al. (2011)
anthracis
Candida albicans Lactobacillus casei Enolase 1 protein Mouse Oral Shibasaki et al. (2014)
Clostridium Lactococcus lactis Cytotoxins Mouse Oral Guo et al. (2015)
difficile
Cryptosporidium L. casei Surface adhesion protein Mouse Oral Geriletu et al. (2011)
parvum P23
CSFV Lactobacillus plantarum E2 protein with Pig Oral Xu et al. (2015b)
thymosin -1
EHEC L. lactis EspB Mouse Oral Ahmed et al. (2014)
Eimeria tenella L. lactis 3-1E protein Chicken Oral Ma et al. (2013)
ETEC L. casei F41 Mouse Oral and Liu et al. (2014b)
intranasal
Helicobacter Lactobacillus acidophilus, Adhesin Hp0410, CTB- Mouse Oral Hongying et al. (2014), Li et al. (2014a),
pylori L. lactis UE, UreB with IL-2 Zhang et al. (2014)
Hepatitis E virus L. lactis ORF2 protein Mouse Oral Gao et al. (2015)
HPAI L. lactis NA, HA with LTB Chicken, Oral and Lei et al. (2015b, c, d)
ferret Intranasal
HPV L. lactis, L. casei E7 protein, E7 protein Mouse, Intranasal Kawana et al. (2014), Li et al. (2014b),
with IL-12 human and oral Ribelles et al. (2013)
IBV L. lactis EpiC peptide with SPA Chicken Oral and Cao et al. (2013)
Intranasal
Influenza viruses L. lactis, L. casei, NA, HA, NA with CTB, Mouse, Oral and Chowdhury et al. (2014), Lei et al. (2015a, e),
Lactobacillus rhamnosus, sM2 with CTA1, sM2 human intranasal Li et al. (2015b), Shi et al. (2014), Song
L. plantarum, and HA2 with CTA1 et al. (2014), Waki et al. (2014)
Lactobacillus brevis
PRRSV L. lactis ORF6 Mouse Intranasal Wang et al. (2014c)
Rotavirus L. rhamnosus, L. acidophilus Protein G, ARP1, oral Mouse, Oral Alvarez et al. (2015), Gunaydin et al. (2014),
attenuated human pig Liu et al. (2014a)
rotavirus
Salmonella L. acidophilus HMLABs Caco-2, Oral Chen et al. (2013)
mouse
Staphylococcus L. lactis SEB Mouse Oral Asensi et al. (2013)
aureus
Streptococcus Lactobacillus zeae scFv antibody Mouse Oral Andersen et al. (2011)
mutans
TGEV L. casei Muramyl dipeptide and Mouse Oral Jiang et al. (2014)
tuftsin fusion protein

scFv antibody single-chain fragment variable antibody, CSFV classical swine fever virus, EHEC entero-hemorrhagic E. coli, EspB E. coli secreted
protein B, ETEC entero-toxigenic Escherichia coli, CTB-UE cholera toxin B subunit and urease subunits, UreB urease B, HPAI highly pathogenic H5N1
avian influenza virus, NA neuraminidase, HA hemagglutinin, LTB heat-labile toxin B subunit, HPV human papillomavirus, IL interleukin, IBV infectious
bronchitis virus, SPA Staphylococcus aureus protein A, CTA1 cholera toxin subunit A1, sM2 consensus matrix protein-2, PRRSV porcine reproductive
and respiratory syndrome virus, ARP1 anti-rotavirus protein 1, HMLABs the heat-killed multispecies combination of LAB, TGEV transmissible
gastroenteritis virus, SEB Staphylococcal enterotoxin B

The health benefits of recombinant LAB in the prevention
of human immunodeficiency virus (HIV)-1 infection at the
mucosa have been clearly demonstrated; however, in recent
years, research focus has shifted toward their effects on influ-
enza virus, rotavirus, and human papillomavirus infections
(Lei et al. 2015a, c, d; Li et al. 2014b, 2015b; Wells and
Mercenier 2008). Two recombinant Lactobacillus strains
(LA4356-pH and DLD17-pH) expressing the highly patho-
genic avian influenza (HPAI) virus protein hemagglutinin
(HA)-1 were successfully generated, and the mucosal and
systemic responses stimulated by these two strains were eval-
uated following oral administration in mice (Wang et al.
2012). Both recombinant strains improved the production of
specific anti-HA1 IgA antibody in the mucosa and IgG in
serum, as well as splenic T lymphocyte proliferative re-
sponses; however, DLD17-pH was more effective in inducing
mucosal and systemic immune responses than LA4356-pH,
promoting higher anti-HA1-specific IgA and IgG levels. In a
trial testing oral vaccines against avian influenza virus (AIV),
a recombinant L. plantarum NC8 (CCUG 61730) strain
Appl Microbiol Biotechnol
expressing the HA gene of H9N2 AIV via the pSIP system
was orally administered in mice (Shi et al. 2014). The recom-
binant NC8 strain induced mucosal and systemic immune
responses, eliciting the production of sIgA, IgG, and hemag-
glutination inhibition antibodies and conferring complete pro-
tection against challenge with mouse-adapted H9N2 virus. In
a different approach to develop a safer and more effective
mucosal vaccine against AIV, a Lactobacillus casei strain
L525 was designed to express a conserved matrix protein with
the cholera toxin subunit (pgsA-CTA1-sM2/L. casei) on the
surface and the sM2 protein without CTA1 as a control
(Chowdhury et al. 2014). After verification of the surface
localization, the effects of the vaccine were tested in mice in
response to oral or nasal inoculation. The recombinant L. casei
expressing CTA1-conjugated sM2 conferred better protection
against divergent influenza virus subtypes than pgsA-sM2/
L. casei but less potent mucosal and systemic immune re-
sponses. A further study aimed at improving the efficacy of
this system led to the induction of strong mucosal immune
responses with CTA1 using L. casei as a delivery vehicle (Li
et al. 2015b). In this latter strategy, the HA2 gene from H5N1
strain was used as an additional component of the vaccine as
well as conserved sM2. Both oral and nasal administrations of
pgs-CTAsM2HA2/L. casei in mice generated high levels of
serum IgG (IgG1 and IgG2a) and mucosal IgA and enhanced
sM2- or HA2-specific cell-mediated immunity, with high
levels of IFN-γ and IL-4. HA2 is one of the highly conserved
subunit of HA which was investigated and found to be effec-
tive for cross-protective antibodies against several subtypes of
viruses in mice, and therefore, HA2-based recombinant vac-
cines demonstrated better effects against AIV. Furthermore,
the recombinant pgs-CTAsM2HA2/L. casei showed protec-
tive effects and elicited long-lasting immunity against diver-
gent influenza subtypes. Intranasal administration induced
stronger immune responses and better protection than oral
administration despite shorter immunization times and lower
dosages, supporting the greater efficacy of intranasal inocula-
tion for this mucosal vaccine. Although the HA protein is
the target of conventional influenza vaccines, neuramini-
dase (NA), the other major glycoprotein on the surface
of the influenza virus, is attracting increasing attention.
Recombinant L. lactis (NZ3000)/pNZ2103-NA adminis-
tered orally in the absence of adjuvant to evaluate the
protective immunity against homologous H5N1 virus chal-
lenge elicited significant humoral and mucosal immune
responses in a chicken model (Lei et al. 2015c). In anoth-
er approach, the immunogenicity of an L. lactis (NZ9000)
vectored vaccine expressing nucleoprotein (NP) was en-
hanced using the cholera toxin B (CTB) subunit as a
mucosal adjuvant (Lei et al. 2015a). Oral administration
of L. lactis/pNZ8008-NP with CTB in mice induced ef-
fective humoral and mucosal responses, providing good
protection against divergent influenza viruses.
Nowadays, it is feasible to express antibody fragments by
LAB against infectious agents. The single-chain fragment var-
iable (scFv) antibodies can stop pathogen from reaching the
epithelial cells by preventing pathogen adherence or neutral-
izing bacteria toxin. Based on those characteristics, scFv se-
creted by L. casei was used to block cell-associated HIV-1
transmission across a cervical epithelial monolayer in vitro
(Chancey et al. 2006). For in vivo immunotherapy, a recom-
binant scFv antibody fragment was constructed and expressed
by Lactobacillus zeae, and it recognized streptococcal antigen
I/II. The transformed lactobacilli exerted the therapeutic action
on experimental animals by decreasing the quantity of
Streptococcus mutans bacteria and reducing the development
of caries (Kruger et al. 2002). The recombinant LAB were
also used to deliver antibody fragments against surface epi-
topes of many pathogens, such as Bacillus anthracis,
Porphyromonas gingivalis, and rotavirus (Andersen et al.
2011; Marcotte et al. 2006; Pant et al. 2006). This strategy
could be considered for immunotherapy or prophylaxis and
will overcome the need for daily administration of purified
antibodies or antibody fragments.
Other recent anti-infective strategies are based on LAB
prototype vaccines against different viral infections including
human papillomavirus (Kawana et al. 2014; Li et al. 2014a),
rotavirus (Alvarez et al. 2015; Liu et al. 2014a), and hepatitis
E virus (Gao et al. 2015); bacterial infections including
Staphylococcus aureus (Veloso et al. 2015), Candida albicans
(Shibasaki et al. 2014), and Clostridium difficile (Guo et al.
2015); and parasitic infections including Babesia bovis
(Bautista-Garfias et al. 2015), Eimeria tenella (Ma et al.
2013), and Cryptosporidium parvum (Geriletu et al. 2011).
Despite the promising features of mucosal vaccination as an
anti-infective approach against numerous antigens, most of
these vaccines are still in the experimental stage and only a
handful are currently approved for human and animal use
(Holmgren and Czerkinsky 2005; Wang et al. 2015).
Prevention and treatment of autoimmune disorders
Recombinant LAB have attracted attention for their potential
in the treatment of autoimmune disorders, such as allergy,
diabetes, and obesity (Walters et al. 2014). The potential of
LAB as an oral allergy vaccine has been researched extensive-
ly because of their safety as food microorganisms and their
potent adjuvant activity in triggering anti-allergic immune re-
sponses (Cano-Garrido et al. 2015). In a recent study, the
prophylactic effect of recombinant L. plantarum NCL21 ex-
pressing a common Japanese cedar pollen allergen, Cry j 1
(Cry j 1-LAB), was evaluated in vivo (Ohkouchi et al. 2012).
Oral vaccination with Cry j 1-LAB ameliorated cedar
pollinosis-like clinical symptoms and allergen-specific IgE
responses in a murine model of cedar pollinosis, providing a

new platform of safe and effective oral immunotherapy
against Japanese cedar pollinosis. Another engineered
L. plantarum WCFS1 (CCUG 61730) was generated by co-
valent attachment of the Fes p1 allergen (from Festuca
pratensis) to the bacterial cell wall and consequent surface
display (Minic et al. 2015). Assessment of its effects in a
mouse model showed that oral administration of recombinant
and non-recombinant live L. plantarum reduced peripheral
blood eosinophilia and increased serum IgG2A levels, which
was attributed to L. plantarum itself, whereas mice treated
with the recombinant LAB construct showed increased spe-
cific serum IgA levels, indicating that cell surface expression
of the Fes p1 allergen potentiated the beneficial therapeutic
effect of orally administered L. plantarum in an allergen-
specific manner. Another important anti-allergic application
of antigen-producing LAB is in the treatment of celiac disease
(CD), an often-undiagnosed autoimmune disease caused by
gluten in wheat-containing foods. Prolyl endopeptidases
(PEPs) have the capacity to hydrolyze the peptide bond on
the carboxyl side of an internal proline residue and can de-
grade immunotoxic peptides responsible for CD, such as a 33-
residue gluten peptide (33-mer). Modified LAB provide an
effective approach to the local delivery of PEP, which catalyze
the cleavage of the gliadin fragment leading to the detoxifica-
tion of dietary gluten. Oral administration of PEP is therefore a
potential therapeutic approach for the treatment of CD
(Alvarez-Sieiro et al. 2014). Two food-grade L. casei strains
(L. casei IPLA1503 and L. casei IPLA1506) were engineered
to deliver PEP in an in vitro model mimicking the environ-
ment of the small intestine: One strain was designed to secrete
PEP into the extracellular medium and the other one for
retaining the enzyme in the intracellular environment. The
results showed that the former was more effective at degrading
the immunotoxic 33-mer peptide than the latter and showed
resistance against simulated gastrointestinal stress while main-
taining the ability to secrete PEP, suggesting its potential for
the delivery of PEP to the duodenum of celiac patients.
Type 1 diabetes (T1D) is a prominent example of a chronic
autoimmune disease characterized by the selective disruption
of insulin-producing pancreatic ß-cells by pathogenic self-
reactive T cells. Although antibody-based immunotherapies
and broad immunosuppressive drugs have been developed
to control this disease, these treatments are accompanied by
short- and long-term toxic effects and failure of discrimination
between autoreactive and non-autoimmune effectors (Luo
et al. 2010; Petrovsky 2010; Van Belle et al. 2011). Antigen-
based immunotherapies are therefore becoming the focus of
research because of their ability to selectively target disease-
relevant T cells while preserving the function of the immune
system. Mucosal delivery of modified L. lactis MG1363 se-
creting the proinsulin autoantigen or glutamic acid decarbox-
ylase (GAD) along with IL-10 resulted in the stabilization of
insulitis, preserved functional β-cell mass, and restored
Appl Microbiol Biotechnol
normoglycemia in non-obese diabetic (NOD) mice when used
in combination with low-dose systemic anti-CD3 (Robert
et al. 2014; Takiishi et al. 2012). L. lactis-mediated delivery
of T1D autoantigens, such as GAD65 and tyrosine
phosphatase-like protein ICA512, in combination with human
IL-10 has been successfully used to treat T1D (Robert et al.
2015; Vandenbroucke et al. 2004). Heat shock proteins (Hsps)
as autoantigens are delivered by LAB-mediated mucosal ad-
ministration, and oral administration of L. lactis expressing
Hsp shows potential for enhancing immune tolerance in
T1D. Nasal administration of the fusion protein Hsp65 with
tandem repeats of P227 (Hsp65-6P277) decreases the inci-
dence of T1D in NOD mice (Jin et al. 2010). Recombinant
LAB producing Hsp65-6P277 are used to deliver large
amounts of antigen and enhance long-term immune tolerance.
Oral administration of recombinant L. lactis NZ9000 express-
ing the fusion protein prevents hyperglycemia, increases glu-
cose tolerance, and reduces insulitis (Ma et al. 2014).
In summary, the use of recombinant LAB strains is a po-
tentially effective strategy for the treatment of autoimmune
disorders, and novel therapeutic approaches for mucosally
induced immunological tolerance are urgently needed.
DNA vaccines
Among recent strategies based on genetically modified LAB
as live delivery vectors, the direct delivery of DNA into eu-
karyotic cells has several advantages, including the induction
of potent cellular and humoral immune responses and the
potential to express multiple antigens or epitopes in a single
DNA vector. LAB are more attractive than other vectors such
as Salmonella spp., Listeria monocytogenes, and Escherichia
coli for the delivery of DNA to eukaryotic cells because LAB
are GRAS and considered as transiting and non-invasive bac-
teria (Daudel et al. 2007; Grillot-Courvalin et al. 1998).
A new invasive L. lactis strain (NZ9000) was engineered to
produce a mutant form of internalin A (mInlA) of
L. monocytogenes (LL-InlA+) that allows the binding of
mInlA to murine E-cadherin (de Azevedo et al. 2012). The
mInlA protein was successfully expressed on the surface of
L. lactis, as validated by fluorescence microscopy. The LL-
mInlA+ strain attached to Caco-2 cells with an intracellular
localization and increased the invasiveness of the cell by
1000-fold and the plasmid containing the cDNA of bovine
ß-lactoglobulin (BLG) transfer 10 times in vitro, whereas in
vivo, the proportion of mice producing BLG increased only
slightly. One possible interpretation of this result is that a
reduction in the direct interaction of L. lactis with the mouse
intestinal epithelium inhibited the binding of the recombinant
mInlA to the corresponding receptors. Another hypothesis is
the release of the plasmid induced by lysed Lactococcus under
the physicochemical conditions of the gut lumen. Considering
Appl Microbiol Biotechnol
these factors, Lactobacillus could be a better option because of
its longer persistence in the gut. The third probable reason is
that epithelial cells saturate with bacteria, even in the presence
of invasin (Zelmer et al. 2005). To test the roles of dendritic
cells (DCs) in the process of LAB DNA vaccination, mouse
bone marrow-derived DCs (BMDCs) were incubated with
invasive L. lactis strains expressing LL-mIlA+ and S. aureus
fibronectin-binding protein A (LL-FnBPA+), both carrying a
plasmid DNA vaccine (pValac) encoding for BLG (de
Azevedo et al. 2015). In this experiment, both recombinant
invasive strains were able to transfer DNA directly to
BMDCs, resulting in the secretion of IL-12 by the cells, inde-
pendent of the invasive status. The recombinant strains were
approximately 100 times more effective at invading the Caco-
2 monolayer than the wild-type strains. Furthermore, data
from a co-culture model indicated that pValac:BLG could be
transferred to DCs across a monolayer of epithelial cells by the
LL-mInlA strain, suggesting that DNA vaccines can cross the
epithelial barrier and express antigens in DCs.
In a previous study, the Lactobacillus acidophilus SW1
(LASW1) strain derived from swine was shown to act as an
immunoadjuvant when used as a live vector for DNA vacci-
nation against foot-and-mouth disease virus (FMDV) (Li et al.
2007). Immune responses to the DNA vaccine were only test-
ed after injection; however, mucosal immunization could
stimulate a specific immune response. Therefore, further in-
vestigation should be aimed at making LAB effective for mu-
cosal delivery as DNA vaccines and at demonstrating their
immune efficacy in animal models. Another current study
was published that LASW1 was used as an oral adjuvant for
a DNA vaccine (pRC/CMV-vp1) harboring the FMDV VP1
gene (Su et al. 2014). Unlike the former study, the strain was
orally administered rather than as a DNA vaccine by intramus-
cular routes, avoiding the unstable replication of recombinant
DNA plasmid in LASW1. The results showed that oral ad-
ministration of this strain significantly enhanced FMDV-
specific humoral and cellular responses and improved the pro-
duction of IFN rather than IL-4 and IL-10. These results sug-
gested that LASW1 is a promising immune adjuvant for DNA
vaccination via oral administration and may become a candi-
date DNA delivery vector.
To increase the efficiency of LAB DNA vaccination, efforts
should focus on improving the transfer efficacy of the plasmid
from the cytoplasm to the nucleus; furthermore, in vivo ex-
periments are necessary to test the effects of bacterial physi-
ology and select LAB species that persist for long periods in
the gut along with invasin inserted via adaptive plasmids.
Conclusion
The development of LAB expression systems has stimulated
studies aimed at determining the cellular localization of
expressed antigens (cytoplasmic, secreted, or cell wall-
anchored) and the effect of localization on their immunogenic
capacity. LAB expressing antigens on the surface may show
more pronounced immunological effects than those express-
ing cytoplasmic antigens, since cell surface antigens have the
ability to directly interact with the environment and avoid
proteolytic degradation (Nouaille et al. 2003; Pontes et al.
2011). However, in some cases, intracellular antigens are more
immunogenic than those expressed in other locations because
intracellular proteins avoid exposure to harsh environmental
conditions (Shaw et al. 2000; Wells and Mercenier 2008). On
the other hand, biologically active molecules, such as hor-
mones, enzymes, and cytokines, require an extracellular local-
ization to produce a functional protein. In addition, different
mucosal administration routes elicit different immune effects.
Intranasal administration is more effective than the intragastric
route for inducing systemic responses, and killed recombinant
LAB are more suitable for intranasal vaccination (Almada
et al. 2015; Hanniffy et al. 2007; Vintini and Medina 2011).
However, intragastric immunization is more feasible, econom-
ic, and applicable for the animal feed business. Despite broad
prospects in application of recombinant LAB, there are still
some issues existing which need to be solved, such as safety,
stability, and persistence. The foreign genes have a potential to
affect safety status of LAB and even have unsuspected harm-
ful effects on the host because of producing poisonous metab-
olizing substances. To address this issue, lots of animal exper-
iments and clinical trials need to be tested frequently and
repeatedly. Although many protection studies have been per-
formed in small animals, determining doses and side effects in
humans requires vaccination studies in larger animals. In most
experiments, plasmids are used as vectors to express foreign
genes, but the transgenes could potentially be transferred to
other individuals. A strategy to solve the above problem is the
genome recombination of LAB, which has been adopted in
the first human trial with recombinant L. lactis producing IL-
10 to treat CD (Braat et al. 2006). The thymidylate synthase
gene of genetically modified L. lactis (LL-Thy12) was re-
placed with synthetic mature human IL-10, which is biologi-
cally contained and avoids systemic side effects (Steidler et al.
2003). Meanwhile, many emerging technologies can increase
the efficiency of genome recombination, such as CRIPR/
Cas9. Most LAB cannot persist in animals or humans for a
long time, but Lactobacillus has longer persistence in the gut
than other genera, exerting therapeutic effects for a few days
after administration (Wells and Mercenier 2008). To improve
the design of therapies using LAB as vehicles, the following
issues should be addressed: (i) co-expression of different ther-
apeutic molecules in recombinant LAB; (ii) selection of per-
sistent LAB species, especially in the genus Lactobacillus;
(iii) development of appropriate mucosal delivery systems,
including ideal antigen delivery systems and specific adju-
vants; and (iv) design of highly efficient expression systems.

Additional studies and clinical trials will facilitate the use of
recombinant LAB as a novel therapeutic approach to the treat-
ment of a wide variety of human diseases in the near future.
Acknowledgments This study was financially supported by the
Chinese B863^ National Programs for High Technology Research and
Development (grant no 2011AA10A211), the National Pig Industrial
System (CARS-36-06B), and the Special Fund for Agro-scientific
Research in the Public Interest (201203039).
Compliance with ethical standards
Conflict of interest The authors declare no conflicts of interests.
Ethical statement This article does not contain any studies with human
participants or animals performed by any of the authors.
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