TIBTEC 1321 No.

of Pages 13

Review
Food-Grade Organisms as
Vaccine Biofactories and Oral
Delivery Vehicles
Sergio Rosales-Mendoza,1,* Carlos Angulo,2,* and
Beatriz Meza2

The use of food-grade organisms as recombinant vaccine expression hosts and

Trends
delivery vehicles has been explored during the past 25 years, opening new
Recombinant food-grade organisms
avenues for vaccinology. Considering that oral immunization is a beneficial are being used for vaccine production
approach in terms of costs, patient comfort, and protection of mucosal tissues, and delivery as food-grade vaccines
(FGVs).
the use of food-grade organisms can lead to highly advantageous vaccines in
terms of costs, easy administration, and safety. The organisms currently used The concept of FGVs is highly advan-
for this purpose are bacteria (Lactobacillus and Bacillus), yeasts, algae, plants, tageous in terms of costs, administra-
tion, and safety.
and insect species. Herein, a comparative and updated scenario on the pro-
duction of oral vaccines in food-grade organisms is provided and placed in FGVs are currently produced in some
bacteria, yeast, algae, plants, and
perspective. The status of clinical evaluations and the adoption of this technol-
insect species.
ogy by the industry are highlighted.
FGVs with improved immunogenicity
Vaccine Production and Delivery Using Food-Grade Organisms are being successfully explored for
painless mucosal administration.
Vaccination has greatly reduced the burden of infectious diseases and has led to eradication of
smallpox, near eradication of polio, and the prevention of billions of deaths [1]. Despite the success Several FGVs are under clinical evalua-
of vaccination programs against formerly fearsome diseases, many efficacious vaccines are still tion, and the current adoption of this
technology by the industry indicates a
needed, especially in developing countries. While distinct platforms for subunit vaccine synthesis
potential to benefit global healthcare
are well established, there are some clear limitations to their applicability in global vaccination. systems.
Although Escherichia coli shows very high productivity, the antigens must be purified to eliminate
endotoxins before safe use in humans and this host often produces insoluble, incorrectly folded,
nonfunctional proteins [2]. Production systems using insect or animal cell lines require expensive
culture media, and have longer production times than those required for microbial systems [3].
Besides production costs, the vaccination programs should contemplate the cost for distribution
(cold chain) and administration (trained personnel) that hamper vaccination coverage [4]. In terms
1
of easy administration and immune protection at local mucosal tissues, oral vaccination is an Laboratorio de Biofarmacéuticos
Recombinantes, Facultad de Ciencias
attractive approach to fight against several diseases, particularly in poor countries where vaccines Químicas, Universidad Autónoma de
are more needed [5]. Thus far several oral vaccine formulations have been developed; however, San Luis Potosí, Avenida Dr. Manuel
these are composed of attenuated forms of disease-causing pathogens [6], and thus the risk of Nava 6, SLP, 78210, México
2
Grupo de Inmunología y Vacunología,
reversion into pathogenic forms exists [7]. In addition, the production of these vaccines usually Centro de Investigaciones Biológicas
requires high-level biosafety facilities that are not needed for producing subunit vaccines. Under del Noroeste, SC, Instituto Politécnico
this scenario, new approaches for the development of subunit vaccines that can surmount the Nacional 195, Playa Palo de Santa
Rita Sur, La Paz, Baja California Sur,
above-mentioned limitations are necessary. CP 23096, México

During the past two decades, food-grade organisms have attracted the attention in vaccinology
as both production hosts and delivery vehicles. Looking to minimize costs, the production of *Correspondence:
rosales.s@fcq.uaslp.mx
subunit vaccines becomes advantageous if an innocuous organism is used as the expression (S. Rosales-Mendoza) and
host for the recombinant vaccine and, moreover, if its biomass can support a straightforward eangulo@cibnor.mx (C. Angulo).

Trends in Biotechnology, Month Year, Vol. xx, No. yy http://dx.doi.org/10.1016/j.tibtech.2015.11.007 1

© 2015 Elsevier Ltd. All rights reserved.
 TIBTEC 1321 No. of Pages 13

Plants Microalgae Yeast Gram+ Insects Figure 1. Schematic Description of Glossary

bacteria the Rationale of Food-Grade Vac-
Bacillus subtilis: a Gram-positive,
cines. This type of vaccine constitutes
catalase-positive bacterium which
a low-cost, easy to administer, and poten-
holds GRAS status; it can form a
tially efficacious vaccine. Mucosal and
Easy formulaon based in freeze-drying systemic immune responses are induced
tough, protective endospore that
process and encapsulaon allows it to tolerate extreme
as consequence of antigen (Ag) translo-
environmental conditions, features
cation by M (microfold) cells or dendritic
that are exploited in the development
cells (DC) which mediate antigen presen-
of convenient oral vaccines.
tation to lymphocytes for the induction of
Bombyx mori: a silkworm species
adaptive immune responses whose effec-
with GRAS status, currently used for
tor mechanisms involve IgA and IgG pro-
the production of FGVs with
Pain-free and duction and the recruitment of cytotoxic
promising prototypes.
safe oral lymphocytes.[2_TD$IF] Abbreviation: SIgA, secre-
administraon Chlamydomonas reinhartdii: a
tory IgA.
GRAS eukaryotic algae widely used
for the production of
Intesnal lumen biopharmaceuticals; in particular,
several vaccine prototypes based in
Oral Angen uptake
vaccine this species are under investigation.
Mucus ELELYSO® (taliglucerase alfa):
M recombinant glucocerebrosidase
Epithelial cells cell
Peyer’s Nave produced in carrot and indicated for
patch DC CD4+ DC
long-term enzyme replacement
therapy in patients with type 1
CD8+ Th1
cell
CD4+ Gaucher disease. The first plant-
Ag-loaded
DC based biopharmaceutical approved
Treg Th2 Mesenteric for human use.
cell cell
Plasma cell lymph node Food-grade vaccines (FGVs):
Mucosal SlgA CD4+
and CD8+ recombinant subunit vaccines
systemic
inmmunity
lgG Plasma cell B cell produced in safe organisms that may
also serve as the delivery vehicle.
Lactic acid bacteria (LAB): a group
of Gram-positive bacterial species
characterized by the production of
formulation of an oral vaccine. Thus, food-grade organisms can serve at the same time as lactic acid by glucose fermentation.
biofactory and oral delivery vehicle for subunit vaccines, avoiding costly purification processes. Plant-based vaccine: vaccine
We term this immunization approach food-grade vaccines (FGVs, see Glossary), which formulation produced in plants as a
convenient source of recombinant
represent an outstanding means to generate low-cost and easy to administer vaccines with
antigens.
the potential to facilitate universal coverage. Under this notion, several systems have been Saccharomyces cerevisiae: a
adopted for the production of FGVs, including bacterial species, yeasts, algae, plants, and GRAS yeast species that, in addition
insects (Figure 1). Each system possesses particular attributes and limitations that should be to serving for injectable vaccine
production, is being used for the
evaluated in selecting the most appropriate platform for vaccine development (Table 1).
production of FGVs based on whole
cells expressing a variety of target
Description of FGVs antigens.
Lactic Acid Bacteria-Based Vaccines
Lactic acid bacteria (LAB) are Gram-positive, non-[5_TD$IF]spore-forming cocci, coccobacilli or rods,
lacking catalase activity, which ferment glucose primarily to lactic acid or to lactic acid, CO2, and

Table 1. Comparison of Food-Grade Organisms Used in Oral Vaccine Development

Organism Diversity of Growth Post-Translational Glycoengineering Adjuvant Industrial
Genetic Rate Modification Tools Producer Production
Tools Capacity Experience

Plants ++++ + ++++ ++++ ++++ ++++

Yeast ++ +++ ++ ++++ +++ ++++

B. subtilis/lactic +++ ++++ + + ++ ++++

acid bacteria

Silkworm ++ ++ ++++ +++ ++ ++++

Algae ++ ++++ ++++ + ++++ ++++

2 Trends in Biotechnology, Month Year, Vol. xx, No. yy

 TIBTEC 1321 No. of Pages 13

ethanol [8]. Many LAB have served along the history of human beings as probiotics and have
been studied over the past two decades as delivery systems for recombinant proteins [9]. Most
of the sold probiotic LAB belong to the genera of Lactobacillus and Bifidobacterium that are very
attractive for vaccine production and delivery because of their ‘generally recognized as safe’
(GRAS) status, adjuvant properties, mucoadhesive ability, easy genetic manipulation, and the
availability of industrial production processes [10,11]. Although research in several LAB species
has been conducted, Lactococcus lactis is the main species used for FGV development [12].
Effective mucosal immunogenicity and protection after oral and nasal vaccination with Lacto-
bacillus strains expressing antigens has been achieved against several viral and bacterial
pathogens [13–18] (Table 2). It is encouraging that a clinical trial using the oral administration
of an E7-expressing L. casei-based vaccine, directed against human papillomavirus E7 for
the treatment of cervical intraepithelial neoplasia grade 3 (CIN3), demonstrated induction
of E7-specific mucosal immunity in the cervix of CIN3 patients [19]. After the completion of
clinical trials, this could become the first approved LAB-based therapy that will facilitate the
path for incoming applications including other vaccines. On the other hand, a less explored but
promising strategy is the delivery of DNA vaccines by LAB that offers fascinating opportunities
for oral vaccination [20].

Bacillus subtilis-Based Vaccines

Bacillus subtilis is a Gram-positive bacterium with GRAS status and is classified as a novel
food which is being used as a probiotic for both human and animal consumption. A singular
characteristic of this bacterial species is the formation of an endospore as part of its develop-
mental life cycle when starved of nutrients. Interestingly, the mature spore survives in a
metabolically dormant stage under extreme temperatures, desiccation, and exposure to sol-
vents and other noxious chemicals [21]. These characteristics make the spore an attractive
delivery vehicle for oral vaccination because spores act as heat-stable particulate carriers [22]
and can be produced at low cost through established industrial processes [23]. Currently, it is
possible to generate strains expressing the antigen in vegetative cells or spores that displaying
chimeric proteins in which the spore coat protein is fused to the antigen of interest, or to generate
strains based on adsorption of the recombinant antigen on wild-type spores [24].

Most of the studies have comprised immunoprotection assays (Table 2), where protection has
been observed in test animals against viral and bacterial pathogens. Because most soluble
antigen-based subunit vaccines are poor immunogens that require the presence of an adjuvant
[25], it deserves special attention that antibody- and cell-mediated immune responses have
been robustly induced by B. subtilis formulations delivered by nasal, sublingual, or oral routes.
Notably, B. subtilis-based vaccines have proved to induce effective immunoprotective
responses against several pathogens by mucosal administration without the need of adjuvants.
Overall, the ability of B. subtilis-based vaccines to induce strong immunoprotective responses
in several preclinical vaccination models is well proven.

Whole-Cell Yeast-Based Vaccines

Yeast species have been widely used for the expression of biopharmaceuticals for industrial
purposes [26–28]. The relevant attributes of these expression hosts include their GRAS status
and the capacity to perform post-translational modifications. Hyperglycosylation of the recom-
binant proteins is a drawback for these systems; however, this aspect has been partially
addressed by generating strains that are defective for N-glycosylation [29,30]. The use of whole
Saccharomyces cerevisiae cells expressing the vaccine antigen to implement innovative
immunization approaches can lead to low-cost vaccines because no purification is needed.
Whole-cell yeast vaccines have been applied to target non-communicable diseases [31,32].
From these, most candidates have been tested in parenteral administration schemes, and
only the vaccine candidate against muscular atrophy was orally administered, with successful

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 TIBTEC 1321 No. of Pages 13
Table 2. Representative FGVs Based on Bacterial and Yeast Species
Organism Target Disease Antigen Description
(Infectious Agent)
Bacteria Leishmania major Lactococcus lactis
expressing the protective
Leishmania antigen, LACK,
in the cytoplasm, secreted
or anchored to the bacterial
cell wall or co-expressing
mouse IL-12
White spot B. subtilis expressing Vp26
syndrome virus and Vp28
(WSSV)
Clonorchis sinensis B. subtilis expressing
enolase
Clostridium difficile B. subtilis expressing
carboxy-terminal repeat
domains of toxins A and B
Foot-and-mouth B. subtilis expressing
disease virus epitopes of foot-and-mouth
disease virus and cholera
toxin B subunit
Hepatitis E virus L. lactis expressing antigen
ORF2
Testicular cancer Lactobacillus plantarum
expressing testicular
cancer antigen NY-ESO-1
Peanut allergy L. lactis expressing peanut
allergen Ara h 2
Staphylococcus L. lactis expressing
aureus staphylococcal enterotoxin
B (SEB)
Helicobacter pylori B. subtilis expressing
urease B
Yeast Muscular atrophy S. cerevisiae expressing
myostatin
Infectious bursal Kluyveromyces lactis
disease virus (IBDV) expressing the VP2 protein
of IBDV
Red-spotted S. cerevisiae expressing the
grouper nervous capsid protein of RGNNV
necrosis virus
(RGNNV)
Actinobacillus S. cerevisiae expressing
pleuropneumoniae ApxIIA#5 antigen
4 Trends in Biotechnology, Month Year, Vol. xx, No. yy
Immunogenic Properties Ref.
Oral immunization using L. lactis [9]
secreting both LACK and IL-12
induced a LACK-specific mucosal
immune response and Th1 immune
response in splenocytes and
mesenteric lymph node cells, and
partially protected BALB/c mice
against L. major challenge.
Protects shrimp against WSSV [88]
challenge.
Induces humoral responses in rats [89]
and protects against challenge with
C. sinensis metacercariae (61.07%
worm reduction rate and 80.67%
egg reduction rate).
Induced in mice humoral responses [90]
at systemic and mucosal levels.
Protects hamster against challenge
with C. difficile spores.
Induced in mice humoral responses [91]
at systemic and mucosal levels and
protects against foot-and-mouth
disease virus challenge.
Induced in mice specific mucosal [92]
IgA and serum IgG and cellular
immunity.
Induced NY-ESO-1 specific [93]
antibodies and T cell responses in
mice.
Induced secretory IgA and [94]
regulatory T cells at the local level in
orally immunized mice.
Induced in mice cellular or systemic [95]
immune responses.
Induced in mice fecal IgA and serum [96]
IgG responses. A reduction of 84%
in the stomach bacterial load was
achieved in challenged mice.
Induced humoral responses in [31]
immunized mice. Induced enhanced
growth performance.
Induced IBDV-neutralizing [37]
antibodies in mice and chickens.
Immunized chickens were
protected against pathogen
challenge.
Induced strong humoral responses [39]
with neutralizing activity against
RGNNV in mice.
Induced humoral responses at [97]
systemic and local levels, and
systemic cellular responses in mice.
 TIBTEC 1321 No. of Pages 13

induction of therapeutic immune responses [31]. In regard to vaccines against infectious

diseases, 10 whole-yeast vaccine prototypes have been reported targeting distinct pathogens;
most were viral pathogens, and only one candidate was designed against the parasite Plas-
modium [33,34]. Remarkably, most candidate vaccines against infectious agents have resulted
in immunoprotection in preclinical studies. Interestingly, the whole-yeast vaccine against hepa-
titis C virus was evaluated in a Phase I clinical trial with promising results [35]. In addition to the
conventional S. cerevisiae, several yeast species are being adopted in this area (Table 2). The
subunit vaccine candidates against hepatitis B virus (HBV) and Plasmodium have been pro-
duced in Pichia pastoris [34,36]. A vaccine candidate against infectious bursal disease virus was
expressed in Kluyveromyces lactis [37], and another candidate against HBV was developed in a
Hansenula polymorpha strain [38]. Encouragingly, the vaccines against infectious bursal disease
virus [37] and the red-spotted grouper necrosis virus [39] conferred protective immunity
following oral administration, indicating a promising potential for the formulation of oral vaccines
using yeasts. The positive outcomes obtained with these vaccines could be associated to the
adjuvant activity of yeast cell compounds because these interact closely with the immune
system [40].

Algae-Based Vaccines
The algae-based vaccine concept began with the first report by Sun et al. [41], and algae are
a promising platform owing to particular advantages including rapid transformation, no
need for growth regulators, and the ability to properly fold complex proteins [42]. Antigens
from various viral pathogens have been produced in microalgae species such as
Chlamydomonas reinhardtii (freshwater), Dunaliella salina (marine), Schizochytrium spp.
(marine), and Phaeodactylum tricornutum (marine) [43–46]. Interestingly, C. reinhardtii has
GRAS status, as have omega-3 fatty acids (DHA) from Schizochytrium, b-carotene from
D. salina, and fucoxanthin from P. tricornutum, for which industrial processes have been
developed. Promising C. reinhardtii-based vaccine candidates against Plasmodium have
been shown to be immunogenic by the oral route [47] and were even able to induce
immunoprotection against Plasmodium or Staphylococcus aureus in a mouse infection
model [44,48] (Table 3).

Plant-Based Vaccines
After 25 years of exploring the concept of plant-based vaccines, several edible plants have been
used to develop plant-based vaccine candidates. The main attribute is that edible plant
tissues are very economical to produce in a process that can be easily scaled-up. Plant cells can
be genetically engineered to transiently or stably express antigens in the nucleus or in the
chloroplast. Nuclear expression offers high biosynthetic capacity because complex post-trans-
lational modifications take place. A myriad of proof-of-concept have been published. Several
vaccine prototypes were analyzed in detail at the preclinical level targeting virus [49], bacteria
[50–54], and parasite pathogens as well as autoimmune diseases [55]. Many other preclinical
studies against several diseases are ongoing with a view to entering clinical trials or trials in
definitive animal hosts for veterinary species (e.g., [54,56,57]). Remarkably, several clinical trials
have been conducted to assess the safety and immunogenicity of oral plant-based vaccines
including antigens from HBV, delivered in potato [58]; enterotoxigenic E. coli, delivered in maize
[59]; Newcastle disease virus, delivered in potato [60]; and rabies virus, delivered in spinach [49].
The findings reflect a potential to induce specific humoral responses in humans.

The first plant-produced biopharmaceutical approved for human use (taliglucerase alfa for
Gaucher disease treatment, named ELELYSO®[4_TD$IF]) was produced in carrot, and this species
provides an excellent platform because of the experience gained in terms of production at the
industrial level and, moreover, the establishment of proper conditions that meet the regulations
governing biopharmaceuticals for human use (www.ifpma.org/innovation/biotherapeutics.html).

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 TIBTEC 1321 No. of Pages 13

Table 3. Representative FGVs Based on Algae and Plant Species

Organism Target Disease Antigen Description Immunogenic Properties Ref.
(Infectious Agent)

Algae Malaria Chimeric protein comprising Mucosal IgA response to [44]

(Plasmodium the P. falciparum surface both CTB and Pfs25, and
falciparum) protein Pfs25 antigen fused to systemic IgG response to
cholera toxin B subunit CTB in mice.
expressed in C. reinhardtii
chloroplast genome. A yield up
to 0.09% of total soluble
protein (TSP) was obtained.

White spot Dunaliella salina expressing the D. salina-VP28-vaccinated [46]

syndrome virus VP28 protein of WSSV. crayfish had significantly
(WSSV) higher survival rates (41%)
than controls.

Malaria Chimeric protein comprising Systemic IgG production [47]

(Plasmodium the P. falciparum MSP1
falciparum, P. antigen, P. berghei MSP1, and
berghei) P. berghei AMA1, C-terminal
domains only, fused to
granule-bound starch
synthase. The antigens were
expressed in C. reinhardtii
nuclear genome, targeted to
the chloroplast starch
granules. A yield of 0.2 to
1.0 mg of protein per mg
purified starch was obtained.
and protection against P.
berghei challenge in a
mouse model. Immune
sera and purified IgG
specific to starch-bound
PfMSP1-19 antigen
blocked red blood cell entry
by P. falciparum in vitro.

Staphylococcosis Chimeric protein comprising Mucosal IgA and systemic [48]

(Staphylococcus the D2 fibronectin-binding
aureus) domain of S. aureus fused to
cholera toxin B subunit,
expressed in C. reinhardtii
chloroplast genome. A yield up
to 0.7% of TSP was obtained.
IgG responses, and
protection (80% survival)
against lethal S. aureus
challenge in mice.

Plants Vibrio cholerae Rice-made B subunit from Induced IgG and mucosal [54]
cholera toxin IgA antibodies with toxin-
neutralizing activity in mice
and macaques.

HIV-1 Carrot-made p24 Induced IgG in sera from [56]

mice primed with the
carrot-made p24.

HBV Maize-made HBsAg Induced long-term memory [57]

(sustained fecal IgA, and
serum IgA, IgG) in mice
primed by the parental
route and boosted with the
rice-based vaccine orally.

HIV-1 Lettuce-made multi-epitope Induced broad serum IgG [87]

C4(V3)6 protein and Th responses in mice.

Silkworm-Based Vaccines
The silkworm Bombyx mori has been used for silk production for centuries; however, this
species is also a source of high-quality proteins and lipids [61]. The use of silkworm pupae as
food has been approved as a ‘new food raw material originated from traditional food’ by the
Ministry of Health of the People's Republic of China [62], and silk protein food powder holds
GRAS status. As a result, silkworm pupae are consumed in several countries including Korea,
China, Assam, Vietnam, among others. Since Maeda et al. [63] first reported the production of

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Table 4. Representative Oral Vaccine Prototypes Based on Genetically Engineered Silkworm

Target Disease [3_TD$IF](Infectious Agent) Antigen Description Immunogenic Properties Ref.

Diabetes Fusion protein composed Immunological tolerance [66]

of cholera toxin B subunit against T cell-mediated
(CTB) and insulin produced autoimmune diabetes by
in silkworm. regulatory T cell induction was
elicited in mouse model.

Gastric ulcer and cancer (H. pylori) Urease B subunit (UreB) Strong IgG antibody response [98]
and heat shock protein A was induced in mice.
subunit of H. pylori
produced in silkworm.

Grass carp reovirus VP6 protein of grass carp Specific antibody response to [99]
reovirus produced in VP6 protein was generated in
silkworm. Grass carp model.

Canine parvovirus VP2 protein of canine Systemic immune response [100]

parvovirus produced in and long-lasting immunity were
silkworm. induced in a dog model.

Alzheimer Fusion protein composed Specific anti-Ab42 antibodies [101]

of CTB and 42-amino acid were induced in[1_TD$IF] mice, leading
isoform of the amyloid-b to a decreased Ab deposition
peptide (Ab42) produced in in the brain of immunized mice.
silkworm.

human /-interferon in silkworm, the production of many other proteins has been achieved.
TORAY Industries (Tokyo, Japan) has used silkworms to produce two recombinant proteins for
veterinary use (www.toray.com) and Immuno-Biological Laboratories (Japan) are developing
several vaccines using transgenic silkworms (www.ibl-japan.co.jp/en/search). The protein
expression in silkworm offers several advantages such as high protein expression levels
(50–1000-fold higher than those for insect cell lines [64]), low cost, and the capacity to produce
large and multiple proteins in addition to performing co-translational and post-translational
modifications that are present in mammalian cell-based systems [65]. In addition, the proteins
created for oral consumption are stable in the stomach and intestines because of the presence
of protease inhibitors and biocapsule-like fat in silkworms [66]. The first report regarding
silkworm pupae as a feasible delivery system for an oral subunit vaccine was by Gong et al.
[66]. Currently, antigens from various pathogens including Helicobacter pylori, foot and mouth
disease virus, canine parvovirus, white spot syndrome virus, and others have been successfully
expressed in this platform (Table 4). These reports have demonstrated the ability of silkworm-
based oral vaccines to induce strong immunoprotective responses in several vaccination
approaches at the preclinical level. Collectively, these features highlight the use of silkworms
as a promising FGV.

Comparative Analysis of Hosts for FGV Production: Positive Features and

Bottlenecks
Biosynthetic Capacity
Although bacterial systems offer fast development and short production times, the production of
complex antigens such as those requiring post-translational modifications (e.g., glycosylation
and multimer assembly) is a difficult task and in some cases unviable. In the case of bacteria-
based live-vector vaccines, another disadvantage is the risk of releasing genetically modified
organisms into the environment from the host after immunization. However, non-recombinant
strategies can be also pursued in Bacillus subtilis-based vaccines, but production costs are
increased owing to the requirement for pure antigens. Yeast is an attractive system in view of fast
growth, high biomass yield, simple handling, and industrial production knowledge. However,

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 TIBTEC 1321 No. of Pages 13

hyperglycosylation (excess of mannose residues) is a factor that should be evaluated case by

case because it may lead to undesired immunogenic effects. Genetic engineering approaches to
modify glycosylation machinery are ongoing.

In the case of algae, biopharmaceuticals are currently being produced in chloroplasts with
success. However the use of nucleus-based expression, which offers the production of complex
proteins, has yet to be optimized to reach high yields.

Although in its infancy, currently available reports indicate that silkworms constitute a promising
platform for vaccine production and oral delivery. Now that the industry has adopted this system
important advances in vaccine development including preclinical and clinical trials can be
expected in the following years. Notably, the impact of glycosylation on antigen structure
and functionality should be also analyzed in this system.

The plant cell possesses high biosynthetic capacity that will allow the production of complex
biopharmaceuticals. Differential glycosylation respect to the native antigen should be taken into
consideration [67], and glycoengineering approaches to avoid plant glycosylation or add human
glycosylation can be used when this factor is crucial for antigen functionality or safety [68]. Clear
advantages of plants over microbial systems include the possibility to implement production
processes that do not require expensive reactors for biomass production and the possibility to
scale-up the process in greenhouses. In addition, the plant system is attractive because
bioencapsulation of antigens in distinct plant cell compartments within different plant tissues
or organs can maintain vaccine stability and efficacy over weeks or months at environmental
temperature.

Optimization of Oral Immunization

Several aspects are involved in the development of an optimal oral immunization scheme in
which stability, bioavailability, and overcoming tolerance are key aspects. In general, FGVs
possess the advantage of being particulate antigen-containing systems that provide acceptable
stability in freeze-dried formulations and which can achieve sufficient bioavailability to elicit
immune responses when orally administered.

Among the FGV production platforms, plant-based oral vaccines are very attractive but most of
the vaccines near to commercialization are produced in plants that are not appropriate for oral
delivery, thus requiring purification. Therefore, an important goal in this area is to reinforce the
development of oral vaccines that require minimum processing for production.

Although immune tolerance is important for preventing allergic and autoimmune diseases or
allograft rejection, it also represents a challenge in the development of oral vaccines against
pathogens [69]. The main reason is that factors inducing tolerance (i.e., dose and frequency of
administration) are also crucial for immunization. Therefore, overcoming the balance between
immunogenicity/tolerance is a key challenge for FGVs. Notably, the development of oral
tolerogenic plant-based vaccines is currently being explored to modulate the immune response
in allergic and autoimmune diseases [70,71], with the potential of being applied in the induction
of tolerance to avoid allograft rejection.

To evoke antigen-specific mucosal immune responses, the ingested antigens that reach the
mucosal surfaces must be transported across the epithelium into the mucosa-associated
lymphoid tissue, mainly by the specialized antigen-sampling microfold (M) cells. Thereafter,
the oral tolerance is mediated by two main mechanisms depending on the dose and frequency
of antigen administration. High doses of antigen may induce clonal deletion and/or anergy of
lymphocyte T cells associated with suppression of type 1 T helper (Th1) and Th2 responses, and

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 TIBTEC 1321 No. of Pages 13

repeated administration of low doses of antigen may induce active suppression characterized by
the generation of regulatory T cells and a shift toward a Th2/Th3 response [72]. For the latter,
three main regulatory cell populations are involved: IL-10-producing CD4+ CD25 FOXP3 cells
(IL-10, an anti-inflammatory cytokine) known as Tr1 cells; TGF-b-producing CD4+ CD25+
FOXP3+ cells (TGF-b, an immune suppressive cytokine involved in the induction of tolerance)
known as Th3 cells; and CD4+ CD25+ FOXP3+ cells (regulatory T cells, Tregs), mediating
suppression through cytokines such as TGF-b and IL-10 [73]. Although these regulatory T cells
are implicated in the induction of oral tolerance, the contributions of the respective subpopu-
lations are not yet fully understood.

However, it is important to note that overcoming tolerance is feasible, as has been proven in a
myriad of studies where FGVs induced mucosal immunity [74,75]. An ideal FGV system should
be based in organisms with safe immunogenic compounds that can be used as vaccine delivery
vehicles [76].

Remarkably, acid lactic bacteria-, Bacillus subtilis-, and yeast-based FGVs seem to exert more
adjuvanticity than the other systems as a result of particular immunostimulant compounds. For
example, the use of probiotic Lactobacillus has been demonstrated to possess mucosal
immunogenic activity [77]. Similarly, b-glucans of yeast have immunoprotective properties
against infectious diseases [78]. Moreover, Bacillus subtilis spores exert adjuvant activity,
and this is of special importance given the poor immunogenicity shown by most of the orally
administered antigens [17]. Spores are also able to germinate in the mouse gut, a fact that has
been associated with the ability to induce strong mucosal immune responses that are thought to
reflect intracellular presentation of antigens taking place when spores germinate inside antigen-
presenting cells (APCs), which are killed in a short period of time [18–20]. However, it should be
considered that plant-based vaccines can also exert adjuvant effects due to the presence of
metabolites, or by the use of protein bodies or oil bodies which enhance antigen uptake [79].

The case of plant-based vaccines against HBV, based on the expression of the HBV surface
antigens, is a highly representative example of the field. Initial attempts had several limitations,
including low expression levels and suboptimal immunization schemes [80]. However, recent
developments have generated a promising outlook. The formulation of oral vaccines, based on
the HBV small surface antigen (S-HBsAg), in a standardized process consisting on freeze-drying
lettuce and the use of an excipient (sucrose) protocol, has significant promise for developing a
low-cost oral vaccine retaining immunogenicity and acceptable stability [81]. In addition, a recent
report on a maize-based vaccine, which was able to induce long-lasting immune responses at
the systemic and mucosal levels following oral administration in mice, supports the potential of
oral vaccines to fight HBV [57].

In addition, it should be considered that the use of safe adjuvants and appropriate delivery
vehicles can also be applied to overcome tolerance and to improve the immunoprotective
efficacy of oral vaccines. One trend with promising results in mucosal vaccinology is the use of
Toll-like receptor (TLR) agonists in vaccine formulations; these include CpG oligodeoxynucleo-
tides (targeting TLR9), polyinosinic:polycytidylic acid (poly:IC, TLR3), monophosphoryl lipid A
(MPL, TLR4), and flagellin (TLR5). The latter was fused to the M2e antigen (flagellin-M2e) from
influenza A virus and produced in Nicotiana benthamiana, generating a chimeric protein that
conferred protection in mice following intranasal immunization [82]. Several of the mentioned
adjuvants are currently in clinical trials and may be incorporated into FGVs. Similarly, new
advances in the era of nanotechnology have prompted the development of safe nano-delivery
vehicles for drugs and vaccines, not only as virus-like particles produced in plants [83] but
also gold-, chitosan-, and lipid (among others)-based nanoparticles with adjuvant properties
that can be used in FGV formulations to improve vaccine efficacy [84,85].

Trends in Biotechnology, Month Year, Vol. xx, No. yy 9

 TIBTEC 1321 No. of Pages 13

Industry Adoption
Interestingly, several companies are investing in the implementation of FGV production platforms.
One company is producing B. subtilis-based vaccines (SporeGen®, www.sporegen.com), and at
least one company has adopted the production of whole yeast-based vaccines to offer new
therapies in the short term (GlobeImmune, www.globeimmune.com). It is envisaged that the
adoption of new yeast species for producing this type of vaccine as well as for expanding the
range of targeted diseases will positively impact on opportunities to develop low-cost vaccines.

LAB-based technology to produce biopharmaceuticals has been developed by Bioneer (www.

bioneer.dk). Similarly, ActoGeniX (www.actogenix.com) developed ActoBioticsTM that provides
L. lactis-based technology for the production and delivery of biopharmaceuticals such as anti-
inflammatory interleukins (for inflammatory bowel disease and celiac disease), toxin-neutralizing
antibodies (for Clostridium difficile-associated enteropathy), antigens (for celiac disease and type
1 diabetes), allergens (for allergic asthma), and antibody– and peptide–toxin conjugates (target-
ing microbiome modulation).

In the case of algae, Triton Health and Nutrition (www.tritonhn.com/) have developed the
PhycoLogixTM Platform based on C. reinhardtii that enables oral delivery of proteins to improve
animal and human health. Triton's first PhycoShieldTM commercialized product is the mammary
associated amyloid protein that stimulates the production of mucus coating in the digestive tract
to prevent colonization by pathogenic microorganisms. Another example is of ALGENICS (www.
algenicss.fr), founded in 2008, which is using marine diatom P. tricornutum-based GRAS
technology to produce recombinant therapeutics for animal and human healthcare. Of special
interest are the microalgal cell lines engineered to produce glycosylated therapeutics such as
monoclonal antibodies and viral envelope antigens. Therefore it is envisaged that new compa-
nies will focus on microalgae-based technology for vaccines in the near future because many
genetic approaches and industrial processes have already been developed.

In terms of the algal species used for FGV production, there is a need to explore in more detail
additional species such as Chlorella, Dunaliella, and Schizochytrium.

Until now, plants seem to be the most promising platform for FGV development because over
two decades the technology has been widely accepted. This technology has been adopted by
several companies with numerous vaccine candidates in clinical trials (i.e., Medicago Inc., www.
medicago.com; iBio Inc., www.ibioinc.com). These vaccines are based on parenteral formu-
lations, and their production system alleviates biosafety concerns related to undesirable gene
flow because production under full containment was implemented (e.g., production in green-
houses or bioreactors). However, oral vaccines that avoid the need for purification are the ideal
goal. Fortunately, several research groups are focusing on advancing the development of
vaccines constituted by freeze-dried biomass from edible plants. Among these, several vaccine
prototypes with immunoprotective activity in preclinical evaluations have been developed
(Table 3). A key example is the tolerogenic vaccine targeting coagulation factor IX that is
produced at the industrial scale in a process consisting of freeze-drying the plant biomass,
resulting in a functional and highly stable oral vaccine formulation [86].

This outlook reflects the promising potential of plant-based vaccines. However, some chal-
lenges remain: attaining enhanced expression levels, developing effective oral vaccines, per-
forming more-extensive safety evaluations, and finding/establishing a proper regulatory
framework to achieve approval for human use [87].

There is also evidence of industrial application of vaccine production in silkworms (Immuno-

Biological Laboratories Company; www.ibl-japan.co.jp).

10 Trends in Biotechnology, Month Year, Vol. xx, No. yy

 TIBTEC 1321 No. of Pages 13

Concluding Remarks and Future Perspectives Outstanding Questions

It has been demonstrated that food-grade organisms are capable of expressing a myriad of Can FGVs provide a global solution for
target antigens, and even whole viral particles, that induce immunoprotective mucosal large-scale vaccination through easy
and low-cost oral administration with-
responses. Although several FGVs are currently in clinical trials with promising perspectives, out the need for expensive purification,
several challenges need to be overcome in the field to enhance their impact on global health. cold chain, and trained personnel
These include: (i) expanding the availability of genetic engineering tools to optimize the expres- requirements?
sion of immunogens, especially in algae; (ii) optimizing oral vaccine formulations (e.g., use of new
Can FGVs overcome the valley of death
adjuvants); ([7_TD$IF]iii) implementing strategies to avoid loss of viability/bioactivity when stored at and become a real alternative in the
ambient temperature; ([8_TD$IF]iv) detailed characterization of the adjuvant activity of the food-grade fight against high-impact epidemio-
expression hosts; ([9_TD$IF]v) overcoming the balance between immunogenicity/tolerance that usually logic diseases and neglected diseases
in the poorest populations?
occurs when an antigen is orally administered; ([10_TD$IF]vi) optimizing industrial production processes;
([1_TD$IF]vii) assessing immunoprotection at the preclinical level because most candidates have only Can efficient oral immunization
been characterized in terms of induction of humoral responses, but without pathogen challenge approaches be implemented using
studies; ([12_TD$IF]viii) studying the long-lasting immune responses induced by FGVs; ([13_TD$IF]ix) achieving the food-grade organisms?

implementation/maturation of regulatory systems for the approval of this type of vaccines; ([14_TD$IF]x)
enhancing research on biosafety aspects, for [15_TD$IF]examples, gene marker removal and developing
full-containment production systems.

The financial interest surrounding global immunization programs can be alleviated by technolo-
gies that achieve a substantial reduction in vaccine production costs. The current outlook
indicates that plants, algae, yeasts, silkworms, L. lactis, and B. subtilis constitute promising
platforms to address this goal. Most of the candidates are still in preclinical trials, and thus
substantial research and funding efforts must be continued to advance towards the completion
of preclinical evaluations and starting clinical trials. The next generation of platforms based on
food-grade organisms allowing subunit vaccine production, ambient temperature storage, and
mucosal delivery may be translated into low-cost and easy to administer vaccines. A central
observation is the growth of new spin-off companies that underscore the open opportunity to
commercialize biopharmaceuticals produced in food-grade organisms.

Acknowledgments
Current investigations from the group are supported by the Consejo Nacional de Ciencia y Tecnologia (CONACYT)[16_TD$IF], México,
and UASLP, México (grants INFR-2014-01-225843 and FAI/UASLP[17_TD$IF]/2015 to S.R.M.; grants CB-2010-01-151818, INFR-
2014-01-225924, and PDCPN2014-01-248033 to C.A.).

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