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Vaccine 31 (2012) 4048
Contents lists available at SciVerse ScienceDirect
Vaccine
j our nal home page: www. el sevi er . com/ l ocat e/ vacci ne
Review
Developing plant-based vaccines against neglected tropical diseases: Where are
we?
Sergio Rosales-Mendoza
a,
, Dania O. Govea-Alonso
a
, Elizabeth Monreal-Escalante
a
,
Gladis Fragoso
b
, Edda Sciutto
b
a
Laboratorio de biofarmacuticos recombinantes, Facultad de Ciencias Qumicas, Universidad Autnoma de San Luis Potos, Av. Dr. Manuel Nava 6, SLP, 78210, Mexico
b
Instituto de Investigaciones Biomdicas, Universidad Nacional Autnoma de Mxico, AP70228, Mexico DF 04510, Mexico
a r t i c l e i n f o
Article history:
Received 29 August 2012
Received in revised form9 October 2012
Accepted 25 October 2012
Available online 6 November 2012
Keywords:
Low-cost vaccination
Transgenic plant
Developing countries
Oral immunization
Vaccines
a b s t r a c t
Neglected tropical diseases (NTDs) impair the lives of 1 billion people worldwide, and threaten the health
of millions more. Although vaccine candidates have been proposed to prevent some NTDs, no vaccine
is available at the market yet. Vaccines against NTDs should be low-cost and needle-free to reduce the
logistic cost of their administration. Plant-based vaccines meet both requirements: plant systems allow
antigen production at low cost, and also yield an optimal delivery vehicle that prevents or delays digestive
hydrolysis of vaccine antigens. This review covers recent reports on the development of plant-based
vaccines against NTDs. Efforts conducted by a number of research groups to develop vaccines as a mean to
ght rabies, cysticercosis, dengue, and helminthiasis are emphasized. Future perspectives are identied,
such as the need to develop vaccination models for more than ten pathologies through a plant-based
biotechnological approach. Current limitations on the method are also noted, and molecular approaches
that might allow us to address such limitations are discussed.
2012 Elsevier Ltd. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2. Plant-based vaccines: an alternative for low cost massive immunization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.1. Cysticercosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.1.1. S3Pvac: a papaya-based vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
2.2. Human hydatidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.2.1. Approaches of plant-based hydatidosis vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.3. Helminthiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.3.1. Approaches of plant-based helminthiasis vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.4. Rabies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.4.1. Approaches of plant-based rabies vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.5. Dengue. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
2.5.1. Approaches of plant-based dengue vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
3. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
3.1. A great need of exploring plant-based production of well-known immunogens related to NTDs is observed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
3.2. Molecular strategies to improve vaccination models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
4. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Corresponding author at: Facultad de Ciencias Qumicas, Universidad Autnoma de San Luis Potos, Av. Dr. Manuel Nava 6, SLP, 78210, Mexico. Tel.: +52 444 826 2440;
fax: +52 444 826 2440.
E-mail address: rosales.s@fcq.uaslp.mx (S. Rosales-Mendoza).
0264-410X/$ see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.vaccine.2012.10.094
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S. Rosales-Mendoza et al. / Vaccine 31 (2012) 4048 41
1. Introduction
Tropical diseases (TDs) are dened as those diseases that mainly
occur in tropical or subtropical regions. This denition implies that
TDs prevail in hot, humid conditions. A special group among TDs,
designated as neglected tropical diseases (NTDs), includes 17 dis-
eases (Table 1), according to the rst WHO report on the subject. To
be regardedas a NTD, a disease must meet the following criteria: (1)
it prominently affects poor countries, (2) it affects low-income and
politically marginalized populations, (3) it does not spread widely
as its distribution is restricted by climate and the effects of cli-
mate on the distribution of vectors and reservoir hosts, (4) it causes
stigma and social discrimination, especially in women, (5) it has
a relevant impact on morbidity and mortality, (6) it is relatively
neglected by researchers, and (7) it can be controlled, prevented,
and possibly eliminated. NTDs impair the lives of 1 billion peo-
ple worldwide, and threaten the health of millions more [1]. They
debilitate poor populations, frustrate the achievement of universal
health in the Millennium Development Goals, and hamper global
development results (Table 2).
Strategic interventions to ght NTDs include preventive
chemotherapy, intensied case management, vector control, safe
water provision, sanitation and hygiene measures, and veterinary
public health. Therefore, signicant research is needed to develop
new diagnostic tests and medicines, and to make accessible for
the poor population those interventions aimed to prevent, cure,
and manage NTD complications. In addition, since putting in place
appropriate infrastructure to allow better sanitary conditions and
hygiene practices critically depends on economic and politic fac-
tors, it is frequently impossible to address this need in the short
term in extremely poor communities. In light of this situation,
vaccination is regarded as the best option to prevent infectious dis-
eases, as it may constitute an immediate intervention for disease
control. A crucial factor in a successful vaccination program is the
logistic cost involved. Therefore, developing efcient and low-cost
vaccines is fundamental in ghting NTDs. In particular, those vac-
cines suitable to be orally administered would constitute a realistic
and effective alternative, which potentially will provide long-term
NTD prevention, supporting successful control programs.
2. Plant-based vaccines: an alternative for lowcost massive
immunization
Many pathogens enter the human body through mucosal sur-
faces. Thus, mucosal immunization could be a promising strategy
to prevent such infections. Moreover, needle-free, oral vaccina-
tion is by far the most attractive method of vaccine delivery, for
it also avoids costly logistic problems, making it suitable for mass
immunization programs.
However, feworallyadministeredvaccines havebeenmarketed.
This could in part be due to the lack of effective oral delivery sys-
tems that delay digestive hydrolysis [2]. These limitations could be
overridden using plant cells for oral vaccine delivery [3,4]. Plant
systems enable us to produce antigens at low cost, avoiding the
expensive step of antigen purication and costly articial antigen
encapsulation technologies. Thus, they are very accessible and may
constitute anattractive approachfor massive immunizationinpoor
countries [5].
Plant-based orally administered vaccines are formulated with
biomass from transgenic plant cells or tissues expressing anti-
genic immunoprotective heterologous proteins. Plant cell walls can
protect antigens from further degradation in the digestive tract,
enabling them to reach the gut-associated lymphoid tissue. This
approach has proved to be an effective and accessible strategy for
immunization. The companies currently developing this type of
vaccines and the number of products that are close to be marketed
constitute a good evidence of the viability and current advances of
this technology [6]. Currently, Protalix BioTherapeutics has on the
market a plant-derived product named ELELYSO, a recombinant
hydrolytic lysosomal glucocerebroside-specic enzyme indicated
for long-term enzyme replacement therapy (ERT) for adults with
conrmed diagnosis of Type 1 Gaucher disease. This example
reects the high potential of plant-derived biopharmaceuticals to
be marketed in the near future.
As NTDs are characterized by having a negative impact on
poor populations, it would be expected that research efforts on
plant-based vaccines were directed to these diseases at higher pri-
orities. A revision of the current scientic literature indicates that
plant-based vaccine developments are under way for the following
NTDs: soil-transmitted taeniasis/cysticercosis, human echinococ-
cosis, several helminthiases, dengue, and rabies. The following
sections describe the research efforts accomplished by various
research groups all over the world to develop plant-based vac-
cines against these NTDs. Current obstacles and possible solutions
to provide a roadmap for future approaches are also discussed.
2.1. Cysticercosis
Neurocysticercosis (NC) is caused by the Taenia soliummetaces-
tode when located in the human brain. The great majority of NC
cases occur in developing countries [7,8], but it is being consid-
ered as an emerging disease in the developed world due to human
immigration [9]. NC is one of the most frequent parasitic diseases
of the central nervous system (CNS) and the main cause of adult
epilepsy in Mexico. Cysticercosis outside the central nervous sys-
tem (CNS) is frequently reported in Asia and Africa, but less so in
America [10]. More than 80% of the worlds 50 million people who
are affected by epilepsy live in endemic developing countries. Cys-
ticercosis mainly affects the health and livelihoods of subsistence
farmers in developing countries of Africa, Asia and Latin America,
as it leads to epilepsy and even death in humans and also reduces
the market value of pigs as it makes pork unsafe to eat [11]. Cur-
rently, no exact data on the world prevalence of cysticercosis are
available. WHO estimates about two and half million persons liv-
ing with intestinal taeniosis globally [12]. NC is an endemic disease
linked to poverty and ignorance. Then, hygiene and sanitation con-
jointly with direct public guidance on the traits of the disease may
also contribute to its elimination.
Although theoretically susceptible to control and declared erad-
icable by the International Task Force for Disease Eradication in
1993, cysticercosis remains a neglected disease [12]. This is mainly
due to a lack of (i) information about its burden and transmission,
(ii) diagnostic tools for use on the eld, and (iii) validation of simple
intervention packages used as part of integrated helminth control
strategies.
Therefore, its eradication remains a major challenge, partic-
ularly in non-developed countries where cestode life-cycles are
rmly established in the social, cultural, and economic context.
Intervention programs aimed to improve sanitary conditions, like
health education, detection and treatment of tapeworm carri-
ers, and pig vaccination have been proposed and successfully
tested locally [13,14]. However, these limited actions have not
been enough to achieve wide and long-term control due to the
complex economic and social networks underlying cestode trans-
mission [15]. Increased resistance to the parasite establishment
by improving the specic host immune status through vaccina-
tion is a realistic strategy to impact on transmission; furthermore,
it does not require improving the development level of marginal-
ized countries as a prerequisite. By improving the mucosal specic
immunity, oral vaccination of humans against taeniasis would
be the most effective mean to prevent cysticercosis, attacking
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42 S. Rosales-Mendoza et al. / Vaccine 31 (2012) 4048
Table 1
The seventeen diseases grouped as neglected tropical diseases by the WHO (2010). Diseases for which plant-based vaccine models have been reported are specied.
Disease Causal agent Plant-based vaccination models
Dracunculiasis Dracunculus medinesis Not reported
Lymphatic lariasis Wuchereria bancrofti and Brugia spp. Not reported
Onchocerciasis Onchocerca volvulus Not reported
Schistosomiasis Schistosoma spp. Not reported
Soil-transmitted helminthiases Ascaris lumbricoides, Trichuris trichiura and the hookworms Rice
Taeniasis/cysticercosis Taenia saginata and T. solium Papaya
Human echinococcosis Echinococcus granulosus and E. multilocularis Alfalfa
Blinding trachoma Chlamydia trachomatis Not reported
Fascioliasis Fasciola spp. Not reported
Yaws Treponema pertenue Not reported
Dengue Flavivirus Lettuce and tobacco
Rabies Rhabdovirus Spinach and tobacco
Cutaneous and mucocutaneous
leishmaniasis/visceral leishmaniasis
Leishmania sp Not reported
Leprosy Mycobacteriumleprae and Mycobacteriumlepromatosis Not reported
Buruli ulcer Mycobacteriumulcerans Not reported
Chagas disease Trypanosoma cruzi Not reported
Human African trypanosomiasis Trypanosoma brucei gambiense Not reported
this central focus in the transmission. Considering the difcul-
ties of developing human vaccines, a transmission disrupt through
pig vaccination has been proposed. Indeed, the essential role of
pigs as obligate intermediate hosts in the parasite life allows us
to effectively interrupt the transmission by pig vaccination. This
is a feasible strategy, better accepted by pig owners since it is
not conscatory. Vaccination is also economy-conscious, as the
pigs will resist infection even though rustically bred, and the
meat will reach higher prices if it meets inspection standards in
abattoirs.
2.1.1. S3Pvac: a papaya-based vaccine
An injectable vaccine against pig cysticercosis named S3Pvac,
based on three peptides (KETc7, KETc1, KETc12) expressed by
another cestode (Taenia crassiceps), was developed. S3Pvac syn-
thetically and recombinantly expressed reduced in about 50% the
Table 2
Summary of reports addressing plant-based vaccination against specic neglected tropical diseases.
Pathogen Plant species Target antigen Expression strategy Immunogenic properties Reference
Taenia solium Carica papaya L. KETc1, KETc12,
KETc7
Biolistic, stable
expression
Plant-derived antigens are immunogenic
by s.c. route and protects against T.
crassiceps, T. solium, and T. pisiformis
[18,19]
Dengue virus Nicotiana benthamiana D2EIII Viral infection,
transient expression
Plant-derived antigen retains antigenicity
and immunogenicity when i.m.
administered, inducing neutralizing
antibodies in mice
[59]
Nicotiana tabacum CTB-EIII Agrobacterium
tumefaciens, stable
expression
Plant-derived antigen displays antigenic
determinants on ELISA
[61]
Nicotiana benthamiana Et, CMEt,
HBcore-DV2d3
Magnifection, transient
expression
Plant-derived antigen displays antigenic
determinants in Western blots
[62]
Lactuca sativa DENV3prM/E Biolistic, stable
expression
Plant-derived antigen displays antigenic
determinants in Western blot
[63]
Rabies virus Nicotiana tabacum CTB-G Agrobacterium
tumefaciens, stable
expression
Plant-derived antigen displays antigenic
determinants on ELISA
[56]
Nicotiana tabacum CPDrg24 CPMNV3 Viral infection,
transient expression
Plant-derived antigens are immunogenic
in mice when i.p. administered and induce
neutralizing antibodies to CVS-11 rabies
virus
[51]
Nicotiana benthamiana and Spinacia oleracea Drg24 Viral infection,
transient expression
Plant-derived antigen induces local and
systemic immune response when i.p.
administered and protects against rabies
virus challenge
[52]
Nicotiana tabacum G protein Agrobacterium
tumefaciens, stable
transformation
Plant-derived antigen induces complete
protective immunity in mice when i.p.
administered
[54]
Zea mays G protein Biolistics, stable
expression
Plant-derived antigen induces neutralizing
antibodies in mice when orally
administered and protects against rabies
virus challenge
[55]
Ascaris suum Oryza sativa As16-CTB Agrobacterium
tumefaciens, stable
transformation
Plant-derived antigen is immunogenic in
mice when orally administered along with
CT
[43]
Echinococcus
granulosus
Medicago sativa Eg95-EgA31 Stable transformation Plant-derived antigen induces Th1
response when i.g. and i.n. administered
and protects mice against E. granulosus
challenge
[33,34]
s.c., subcutaneous; i.m., intramuscular; i.p., intraperitoneal; i.g., intragastrical; i.n., intranasal; p.o., oral; ELISA, enzyme-linked immuno sorbent assay.
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S. Rosales-Mendoza et al. / Vaccine 31 (2012) 4048 43
number of infected pigs, and in 8090% the number of established
cysticerci under natural conditions of transmission [16,17]. In an
effort to develop an oral version of the vaccine, KETc1, KETc12, and
KETc7-were expressed in three independent papaya embryogenic
cell lines, obtained by bioballistics [18]. The vaccine, composed by
the three clones, was designated as S3Pvac-papaya. The expression
of the respective peptide in each clone was conrmed at the trans-
criptional level by RT-PCR. Soluble extracts from the transgenic
papaya clones were found to be immunogenic when administered
to mice by subcutaneous route. Indeed, all three clones express-
ing the vaccine peptides induced high level of protection against
murine cysticercosis when injected to mice. These achievements
highlight the great potential of this technology to render a highly
effective and affordable vaccine against cysticercosis.
Recently, S3Pvac-papaya also demonstrated a high protective
capacity against T. pisiformis cysticercosis [19] when orally admin-
istered to rabbits. Furthermore, oral S3Pvac-papaya induced a
protection level as high as the injectable synthetic version of the
vaccine, and even higher than the recombinant S3Pvac version
expressed in lamentous phages. S3Pvac-papaya also demon-
strated to be immunogenic in pigs when orally administered [18].
Currently, this vaccine is being evaluated in pigs under oral immu-
nization schemes (Edda Sciutto, personal communication).
To further explore the potential of an oral multi-epitope vac-
cine, the addition of antigens that have shown to elicit protective
immunity was considered. In this respect, the most promising
candidate is TSOL18/HP6. This antigen has been proved capable
of protecting pigs against T. solium experimental infection [20].
More recently, its protective effect was evaluated when coadmin-
istered with oxfendazole on a eld trial study at rural communities
in Cameroon with very promising results [21]. Later on, it was
further evaluated on the eld [22] combined with another protec-
tive recombinant antigen (TSOL16) [23]. Although the protective
capacity of these antigens when orally administered has not been
evaluated, it is possible that if expressed in an adequate delivery
systemthey could be immunogenic, and considered to be included
as additional epitopes in the plant-derived S3Pvac vaccine.
2.2. Human hydatidosis
Hydatid disease, caused by Echinococcus granulosus or
Echinococcus multilocularis, is another important cestodiasis
affecting human and veterinary health [24]. It is also considered
a NTD. Cystic and alveolar echinococcosis are caused in humans
by ingesting eggs of E. granulosus or E. multilocularis, respectively,
shed in the feces of dogs harboring adult stages of these tape-
worms. Echinococcosis has a global distribution and causes serious
morbidity and even death if left untreated. Interestingly, various
vaccination candidates against hydatidosis have been reported.
The antigen candidate most thoroughly evaluated against
hydatidosis is named Eg95 [2527]. This injectable recombinant
vaccine was proved to be efcient in an extensive eld trial per-
formed in sheep, goats and cattle [2830]. Eg95 has been registered
for use in China and Argentina, and it has been produced commer-
cially for large scale use in control programs, assuming that its high
cost will be heavily subsidized by health authorities. However, cost
could be an important limitation for its extensive use along with
the proposed schedule, which required two immunizations and a
booster toattaina highimmune response. Moreover, the parenteral
administration is not suitable for large scale application [31]. Thus,
the use of Vaccinia as a delivery system to improve Eg95 immu-
nity and reduce production costs is under development [32]. Other
approaches are also under experimentation. Among them gure
EgTrp-tropomyosin and EgA31-tropomyosin, antigens expressed
inthe larval andthe adult stages of the parasite whichelicit promis-
ing protective responses in experimental trials [31]. While these
approaches seemfeasible, the use of plant cells as delivery vectors
for an orally administered vaccine would yield convenient vaccines
as well, at lower costs.
2.2.1. Approaches of plant-based hydatidosis vaccines
Several plant-basedvaccinecandidates against hydatidosis have
been reported. Both EgA31 by itself and combined with Eg95 were
used to produce transgenic alfalfa plants by nuclear transforma-
tion. The immunogenic properties of these cells were evaluated in
the hydatidosis mouse model. Oral and intranasal immunization
with transgenic alfalfa signicantly reduced larval size and elicited
a pro-inammatory response, probably involved in this protective
capacity [33,34].
It has also interest to mention that the S3Pvac vaccine can pro-
tect not only against cysticercosis but also against hydatidosis,
as shown in a eld trial test performed 187 vaccinated and 204
control pigs. Macroscopic and histological evidences showed that
protective efcacy of S3Pvac-phage vaccination against porcine
cysticercosis and hydatidosis are of 61.7% and 56.1%, respectively
[35]. Then, S3Pvac expressed in papaya, orally administered may
possible prevent also both infections.
2.3. Helminthiasis
Soil-transmitted helminths cause most infections affecting the
poorest populations. As these pathogens enter the human body
through mucosal surfaces, mucosal immunization could be a
promising strategy to prevent such infections. The main causal
agents of these pathologies are Ascaris lumbricoides, Trichuris
trichiura, Ancylostoma duodenale, and Necator americanus. Accord-
ing to recent estimates, A. lumbricoides infects over 1 billion people,
T. trichiura 795 million, and hookworms 740 million. The great
majority of soil-transmitted helminth infections occur in sub-
Saharan Africa, the Americas, China, and East Asia. Infection is
caused by ingestion of eggs fromcontaminated soil (A. lumbricoides
and T. trichiura) or by active skin penetration by larvae in the soil
(A. duodenale and N. americanus). Soil-transmitted helminths pro-
duce a wide range of symptoms, includingintestinal manifestations
(diarrhea and abdominal pain), general malaise and weakness,
which may affect working and learning ability and impair physi-
cal growth. In especial, hookworms cause chronic intestinal blood
loss leading to anemia [36].
Currently, global distribution of anthelmintic drugs to control
this health issue is at a historical peak due to numerous coordina-
tion initiatives with donors, governments and local communities.
However, in spite of these efforts a much larger and rapidly grow-
ing childhood population in these regions remains untreated and
harboring more than one parasite. Another limitation is rapid rein-
fection rates, which demand periodic administrationof these drugs
[37]. A feasible alternative could be the conjoint development
of effective vaccines and new technologies to produce affordable
formulations. Since most helminthes induce immune-mediated
resistance against further challenge infections, it is likely that vac-
cination could offer a viable alternative for wormcontrol.
2.3.1. Approaches of plant-based helminthiasis vaccines
Very fewstudies on plant cells expressing heminthiasis-related
antigens have been performed. Only a couple of reports on Ascaris
suum antigen production have been published. These reports
are relevant since this pathogen is used in the swine model
for helminthiasis. The extensive similarities in natural history,
pathology, and antigenic composition between A. suum and A.
lumbricoides suggest that protective vaccine antigens against one
species could be used against the other.
A. suuminfection begins when embryonated eggs encapsulating
third-stage larvae are ingested by host animals. Parasites hatch in
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44 S. Rosales-Mendoza et al. / Vaccine 31 (2012) 4048
the hosts small intestine, migrate to the liver and lungs via the por-
tal vein, and nally reach the cecumand/or proximal colon, where
theydevelopintoadult worms. Recent studies showingthat A. suum
can also infect humans have stressed its relevance as a zoonotic
parasite [38,39]. Previous studies showed that animals can be pro-
tected from A. suum infection by immunization with L3 or with a
cuticle component [40]. This evidence led to describe a protective
16-kDa antigen (As16), which is expressed in the intestine, hypo-
dermis, and cuticles of larva and adult A. suum [41]. It has been
proved that antibodies elicited by immunization with As16 kills A.
suum L3 [41,42]. Then, As16 is a viable vaccine antigen candidate
against this parasite.
In 2009, Matsumoto et al. reported the evaluation of rice
plants for As16 production as a chimeric protein fused to the
cholera toxin B subunit (CTB). Expression levels up to 50g/g seed
[43] were observed. Feeding mice with the transgenic rice seeds
elicited an As16-specic serum antibody response when admin-
istered conjointly with cholera toxin (CT) as a mucosal adjuvant.
Even though no response was observed when no adjuvant was
used, subcutaneous booster immunizationwithbacteria-expressed
As16 successfully induced antibody responses; therefore, a prim-
ing effect by the transgenic rice is postulated. Notably, mice orally
immunized with transgenic rice/CT showed lower lung wormbur-
dens after challenge with A. suum eggs. This suggests a good
potential for the rice-derived antigen as a low-cost vaccine can-
didate to control A. suum infection in animals, but it could also
serve as a model for developinghumanvaccines against heminthia-
sis. Cross-protective immunity against A. lumbricoides, for example,
shouldbeexploredas it couldleadtoanother successful application
for this approach.
A 14-kDa protective surface antigen fromA. suumL3 larvae has
been produced in rice by the same research group. The antigen was
fused with the mucosal carrier cholera toxin B subunit (CTB), and
the expression was driven by the endosperm-specic glutelin-B
promoter. This strategy allowed for the production of the expected
recombinant proteins at levels of 1.5g per seed. This study repre-
sents an advance in the eld that justies further functional studies
[44]. Further advances on this eld require assessing the immuno-
genic properties of these transgenic plants.
The dramatic rise in drug-resistant helminths of veterinary
importanceraises concerns over thelong-termuseof current drugs.
Drug discovery programs, based on the screening of chemical com-
pounds against parasitic or non-parasitic helminths, will require
to be expanded. The study of immune-mediated expulsion of adult
worms could potentially benet this process through the identi-
cation of novel anthelminthic molecules. A novel immunotherapy
approachis the administrationof IL-4/IL-13topromote adult worm
expulsion. In particular, an increase in the intestine RELMb level
results in an inhospitable environment for the wormand interferes
with essential wormfunctions such as feeding [45]. As a number of
cytokines retaining their functional properties have been produced
in plants, the use of this platformto accomplish cytokine-mediated
treatment of heminthiasis is also a possibility [46,47].
2.4. Rabies
Rabies is a viral zoonotic infection of the central nervous system
caused by a lyssavirus. Rabies virus causes the highest number of
deaths among all human pathogenic viruses. In 2005, World Health
organization (WHO) pointed out that 50% of the yearly-reported
victims were 15 years-old or younger. Of these cases, 95% occur in
Asia andAfrica, and99%of themare transmittedbydogs. Ingeneral,
this transmission occurs in exposed humans through a bite by a
rabies virus-infectedanimal or throughmucosal contact withvirus-
contaminateduids. Therefore, rabies is consideredone of the most
NTDs indeveloping countries, withthe greatest burdenput onpoor
rural communities, and disproportionately on children. The efforts
by WHO to reduce rabies burden and to eradicate the disease in
humans involve coordinated efforts to procure and deliver safe and
efcacious rabies vaccines in those countries where they are most
needed, to achieve preventive immunization in animals and pre-
exposure and post-exposure prophylaxis in humans [37].
Rabies virus has a single-stranded RNA genome encoding for
ve structural proteins: nucleoprotein (N), phosphoprotein (P),
matrix protein (M), glycoprotein (G), and RNA-dependent RNA
polymerase (L). Among these proteins, the surface glycoprotein (G)
is the most important in viral pathogenesis, and it may function
as a protective antigen, since it is the only target for neutraliz-
ing antibodies (NAs), which provide full protection against virus
challenge. Several research groups have produced this glycopro-
tein in recombinant yeast or insect cells, or in transgenic plants.
The immunogenicity of the recombinant protein has been found
different in the diverse tested systems; this is probably due to
the structural complexity of the rabies virus glycoprotein, which
carries two N-linked oligosaccharide branches [48]. Most rabies
virus NAs bind to conformation-dependent epitopes on the native
glycoprotein that seem to be expressed preferentially as multi-
meric proteins rather than as the monomeric forms of cleaved
glycoprotein secreted by infected cells or commonly produced in
recombinant expression systems [49].
Although rabies is a vaccine-preventable disease, effective pre-
vention in humans with category III bites requires the combined
administration of rabies immunoglobulin (RIG) and rabies vaccine.
Cell culture-based rabies vaccines have become widely avail-
able in developing countries, virtually replacing the inferior and
unsafe nerve tissue-based vaccines. Limitations inherent to the
conventional RIG of either equine or human origin have prompted
scientists to look for monoclonal antibody-based human RIG as an
alternative. This approach is attractive but has a severe drawback,
as theproductionof suchvaccines is costly, makingthemless acces-
sible to the general population. Different alternative approaches
expressing potential antigenic proteins have been developed for
reliable andaccessible vaccines. Amongthem, plant-basedvaccines
are the most promising candidates.
2.4.1. Approaches of plant-based rabies vaccines
Several approaches of plant-based vaccines against rabies have
been reported. In a pioneering study, tomato plants were engi-
neered at the nuclear level to express the G-protein, which was
successfully immunoprecipitated and detected by Western blot
from leaves and fruit. Electron microscopy of leaf tissue using
immunogold-labeling and antisera specic for rabies G-protein
showed that G-protein located at Golgi bodies, vesicles, plas-
malemma, and cell walls of vascular parenchyma cells, suggesting
that tomato can serve as a functional expression platform for this
vaccine [50]. Later, Yusibov et al. reported the expression of the
antigenic protein designated CPDrg24, comprised by the G5-24 B-
cell epitope fromrabies glycoprotein and a 31D T cell epitope from
rabies nucleoprotein, fused with alfalfa mosaic virus (AIMV) coat
protein (CP). This chimeric protein was cloned in the 30BRz vec-
tor, which allowed its expression in infected plants, yielding viral
particles that were successfully puried frominfected plant tissue
and used to immunize mice intraperitoneally (i.p.). Fourteen days
after the last CPDrg24 immunization, high serum titers of rabies-
specic antibodies were detected by ELISA in mice immunized
either with or without complete Freunds adjuvant. Thus, plant-
produced rabies virus antigen is capable of inducing an immune
response in mice in an adjuvant-free system. Moreover, in an
in vitro assay, the authors proved that these antibodies are capable
of neutralizing CVS-11 strain rabies virus [51].
Subsequently, the expression of chimeric Drg24 rabies virus
peptideinvirus-infectedspinachleaves was reported, showingthat
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S. Rosales-Mendoza et al. / Vaccine 31 (2012) 4048 45
either i.p. or orally (p.o.) immunized mice developed local and sys-
temic immune responses. Both groups were subjected to challenge
experiments, and40%of thei.p. immunizedanimals wereprotected
against lethal challenge. On the other hand, oral administration of
the antigen not only stimulated serum IgG and IgA synthesis, but
also ameliorated the clinical signs caused by intranasal infection
with an attenuated rabies virus strain [52].
In another approach, Yusibov et al. [53] reported the production
of a chimeric peptide carrying antigenic determinants of the Gpro-
tein (amino acids 253275) and nucleoprotein (N protein) (amino
acids 404418), whichwas fused to the alfalfa mosaic virus (A1MV)
coat protein (CP). This protein was expressed in transgenic Nico-
tiana tabacumcv. Samsun NN plants, providing in trans replicative
functions for full-length infectious RNA3 from A1MV (NF1-g24).
In addition, Nicotiana benthamiana and spinach (Spinacia oleracea)
plants were used as expression systems using autonomously repli-
cating tobacco mosaic virus (TMV) lacking native CP (Av/A4-g24).
The recombinant virus was puried from N. tabacum cv. Samsun
NN cells, and it retained its capacity to elicit humoral responses
in mice when parenterally administered. Moreover, recombinant
virus-containing unprocessed raw spinach leaves conferred pro-
tection to mice against oral challenge. Based on these results, this
research group assessed its efcacy in a pilot study on human
volunteers. Three of 5 volunteers previously immunized with con-
ventional vaccine, specically responded against the peptide after
ingesting spinach leaves inoculated with the recombinant virus,
while 5 of 9 non-immune individuals (fed with the same mate-
rial) exhibited signicant antibody responses to either rabies virus
or AIMV. After a single dose of conventional rabies virus vaccine,
three of these unvaccinated individuals showed detectable levels
of neutralizing antibodies against rabies virus. These ndings show
the potential for the plant virus-based expression systems as sup-
plementary oral booster in rabies immunization schemes [53].
Some research groups have attempted to engineer virus coat
proteins (CPs) to use them as carriers for genetically fused rabies
specic antigen. Such carrier proteins could have the potential
to self-assembly and form recombinant virus particles, which are
oftenhighlyimmunogenic anddisplaythedesiredepitopes ontheir
surfaces. In the scope of this revision, the viability of this approach
as a development strategy for highly efcient vaccines is noted.
Ashraf et al. [54] have reported a detailed approach to achieve
high G protein expression levels. The gene was optimized in codon
usage, and the native signal peptide of the pathogenesis-related
proteinPR-S, as well as the endoplasmic reticulumretentionsignal,
was included. Tobaccoplants transformedwiththis construct at the
nuclear level were able to express the protein at levels up to 0.38%
of TSP. According to the study, mice i.p. immunized with the plant-
derived G protein puried from tobacco leaf microsomal fraction
showed signicant immune responses, as high as that induced by
the commercial inactivated virus-vaccine. More importantly, this
immunizationschemeinducedcompleteprotectionagainst alethal
intracerebral rabies virus challenge. This constituted a signicant
step toward the development of a feasible and accessible vaccine
against rabies [54].
On the other hand, transgenic maize expressing the Vnukovo
strain rabies virus glycoprotein (G) has been developed by Loza-
Rubio et al. [55]. Interestingly, the expression levels reached up
to 1% of TSP. This research group evaluated the immunogenicity
of the heterologous protein when orally administered. Adult mice
received a single oral dose of kernels containing 50g of G pro-
tein, and 90 days post-vaccination they were challenged with a
lethal dose of a vampire bat rabies virus. In a relevant result, a 100%
protection was recorded in this experiment. It is concluded that G
protein fromVnukovo strain provides rabies cross-protection and
that these cornlines are promising candidates to formulate a highly
effective rabies vaccine [55].
Rabies surface glycoprotein (G protein) has also been expressed
in tobacco plants as a fused protein with the B subunit of cholera
toxin (CTB), in an effort to use this protein as an immunogenic
carrier. The expected recombinant protein was accumulated at lev-
els up to 0.4% of TSP in leaves and it was functionally active in
the GM1 binding assay, having a higher afnity for GM1 than the
native bacterial CTB; these results have interesting implications
since this binding activity is associated with a higher immuno-
genic potential. The pentameric fusion was immunoreactive both
with anti-cholera toxin and anti-rabies antibodies, suggesting that
the antigenic determinants of both components were preserved.
Nonetheless, the immunoprotective ability against rabies is a pend-
ing objective [56]. Immunogenicity evaluation for these proteins
and their capacity to neutralize the virus in humans remain as a
high-priority perspective.
All these attempts to express virus antigenic proteins in differ-
ent plant systems point to its future use in applied vaccinations
alternatives both for animals and humans.
2.5. Dengue
Dengue is caused by a virus of the family aviviridae (DENV).
Four dengue virus serotypes, designated DENV-1 to 4, have been
foundconcomitantly indifferent worldregions. This virus is related
totheyellowfever virus (YFV), hepatitis Cvirus (HCV), andtheWest
Nile (WNV), Japanese (JEV), and St. Louis encephalitis viruses. Each
virioncontains a single positive-strandRNAcoding for dengue viral
proteins in a long open reading frame comprising capsid (C), pre-
membrane (prM), envelope (E), and nonstructural (NS) 15 genes.
Human infection with DENV results in either an asymptomatic or
a symptomatic disease, ranging fromclassical dengue fever (DF) to
more severe cases of dengue hemorrhagic fever (DHF) and dengue
shock syndrome (DSS) [57].
Dengue is an acute febrile, mosquito-borne, viral disease. In
recent years, transmission rate has increased predominantly in
urban and semi-urban areas in tropical and sub-tropical regions.
The incidence of this infection has grown dramatically around the
world in recent decades. Over 2.2 million cases were reported in
2010 in the Americas, South-East Asia, and Western Pacic [58].
At the present, no vaccines against dengue are available. However,
various strategies havebeenattemptedas efforts todevelopdengue
vaccine candidates, including models of attenuated, recombinant,
subunit, chimeric, and DNA vaccines [59].
The viral proteins prM, E, and NS1 are considered relevant to
provide immunity, since passively transferred antibodies against
each of these proteins have been reported to protect mice from
lethal challenge. Therefore, prM, E, and NS1 genes have been used
to develop dengue subunit vaccines [57]. Most of these strategies
have been focused on the dengue virus E protein. This 495-aa pro-
tein consists of three structurally distinct domains, labeled I, II, and
III [60]. This component constitutes the major structural protein
exposed on the mature virion surface, having a major role in host
cell attachment and viral entry. Thus, it has been regarded as the
primary antigen to attain protective immunity [59].
Despite signicant efforts exerted in many countries, no com-
mercially viable dengue vaccine is available. Currently, attention is
focused on developing either live attenuated vaccines or live atten-
uated chimeric vaccines using a variety of backbones. Alternate
vaccine approaches, such as whole inactivated virus and subunit
vaccines, are in early development stages, and each poses differ-
ent problems. Subunit vaccines offer the advantage of providing
a well-dened antigen, without adding the risk of introducing
other extraneous genetic material intothesubject beinginoculated.
Preliminary trials of subunit vaccines (using dengue E protein)
in rhesus monkeys have shown promising results. However, the
primary disadvantages of dengue subunit vaccines are the low
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46 S. Rosales-Mendoza et al. / Vaccine 31 (2012) 4048
expression levels of dengue proteins in mammalian or insect cells,
as well as the added unknown risks of antigens from mammalian
cells containing other potential contaminants.
2.5.1. Approaches of plant-based dengue vaccines
In a pioneering approach, domain III of dengue 2-envelope
protein (D2EIII, 298400-aa) was successfully expressed in
Nicotinana benthamiana cells using a tobacco mosaic virus (TMV)-
based transient expression system. The recombinant protein was
immunoreactive to both anti-D2EIII polyclonal and anti-His tag
antibodies. Mice i.m. immunized with the plant-derived vaccine
elicited anti-dengue virus humoral responses. Moreover, neutral-
izing activity against type-2 dengue virus by sera fromimmunized
animals was proven by the plaque reduction neutralization test
using the S16803 type-2 dengue virus strain and monkey kidney
cells (LLC-MK2), where the neutralizing antibody titer is dened as
the highest serumdilution that reduced the number of input virus
plaques by 90%. This result indicates that the TMV expression sys-
tem produces a dengue virus-derived antigen in plant cells that
exhibits appropriate antigenicity and immunogenicity [59]. The
observed response, however, was induced only when an adjuvant
was co-administered with the antigen. Therefore, planning alter-
native strategies to achieve a highly immunogenic formulation is
a mandatory step. For example, adjuvant co-expression or yet an
antigen-adjuvant genetic fusion would allow for inducing higher
responses even by oral immunization. It is well known that the
cholera toxin B subunit the heat-labile E. coli enterotoxin B subunit
can serve as advantageous carrier proteins with adjuvant proper-
ties, and in addition they have been expressed in a number of plant
species and retained their antigenic and immunogenic properties.
This approach has been explored by other research groups. CTB
was used as a carrier for the poorly immunogenic EIII domain
(297394-aa). The protein, calledCTB-EIII, was producedintobacco
plants following the standard Agrobacterium-mediated transfor-
mation procedure. This systemallowed for producing the CTB-EIII
protein at levels near to 0.019% of TSP. Interestingly, this plant-
derived antigen retained the ability to bind GM1, which is critical
for its biologic activity. Immunogenicity in mice would be the next
logical stepinthis project. It is alsoof interest toexpress this protein
at the chloroplast level, in search for better yields [61].
In another adjuvant-based approach, an E-protein truncated
version was expressed in Nicotiana benthamiana using decon-
structed viral modules [62]. The following congurations were
tested: (1) a truncated version of E (Et), lacking the membrane
anchor domain; (2) the co-expression of Et with DV struc-
tural proteins C and prM (CMEt); and (3) the fusion of HBcore
with DV serotype 2 domain III of the envelope protein (DV2d3)
(HBcore-DV2d3). These proteins were proved antigenic, since they
reactedwiththe correspondingantibodies (anti-Eandanti-HBcAg).
Immunogenicity studies, therefore, are also in the near future for
these efforts (Fig. 1).
A transplastomic approach was recently assessed through
the expression of dengue-3 serotype polyprotein (prM/E) con-
sisting of partial capsid, complete premembrane (prM) and
truncated envelope (E) proteins [63]. This system was success-
fully attained in lettuce chloroplasts. The rationale of this approach
is to produce an antigen capable of assembling into VLPs, and
therefore assuming a highly immunogenic form. Using Western
blot analyses, the authors proved that prM/E polyprotein was
expressed in different forms: as monomers (65kDa) or possi-
bly heterodimers (130kDa), or multimers. Virus-like particles
of 20nm diameter in chloroplast extracts from transplastomic
prM/E protein-expressing lettuce cells were detected. Immuno-
genicity in tests animals, however, still requires to be analyzed. A
comparison of the immune responses attained by this antigen with
monomeric approaches would be interesting, to ascertain whether
Fig. 1. Comparative number of reports on plant-based vaccine experimentation at
NCBI (2012). Number of reports addressing global spread diseases is compared to
those related to neglected tropical diseases (NTDs). As plant based-vaccines show
singular advantages to be useful in poor and developing countries, a great necessity
of addressing plant-based vaccines against NTDs is identied.
or not multimeric forms possess enhanced immunogenic proper-
ties.
This panoramashows that plant-basedvaccines candidates have
thepotential of elicitingspecic immuneresponses against dengue.
However, is it necessary to evaluate whether the elicited anti-
bodies are capable of preventing the virus from entering the host
cell. An effort to obtain an effective plant-based vaccine may also
be directed to attain the simultaneous expression of prM, E and
NS1 proteins with the goal of inducing a broad specic immune
response trough a single formulation.
3. Perspectives
3.1. A great need of exploring plant-based production of
well-known immunogens related to NTDs is observed
Even though many conventional vaccines against NTDs are in
advanced stages of development, most of these NTDs have not
been assessed in models of plant-based vaccines. Surprisingly, no
report exists ondevelopingplant-basedvaccinationmodels against
dracunculiasis, lymphatic lariasis, onchocerciasis, schistosomia-
sis, blinding trachoma, fascioliasis, yaws, leishmaniasis, leprosy,
Buruli ulcer, Chagas disease and human African trypanosomiasis.
This group for twelve diseases shows the need of performing more
substantial research efforts on the eld.
Of special importance is toconsider the frequent obstacles inthe
development of plant-based vaccines, which involve low expres-
sion levels and difculties to produce more than one immunogen
at the same time and at similar levels trough a single plant trans-
formation event. The following subheadings identify key molecular
strategies with the potential to impact positively on these issues in
the short term.
3.2. Molecular strategies to improve vaccination models
A frequent need when developing subunit vaccines is the pres-
ence of various antigens to constitute a polyvalent vaccine. This
objective is especially important when several target antigens are
associated with high vaccination efcacy. In the light of this need,
it is of interest that plant modication in the chloroplast genome
offers the possibility of expressing more thanone ORF inthe formof
multigene operons. This approach has been successfully employed
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to achieve the expression of three different ORFs; for example,
tobacco plants were engineered to express three enzymes involved
in the PHB biosynthetic pathway by plastid transformation. The
transplastomic plants accumulated the biodegradable polyester
polyhydroxybutyrate. Although phenotypic alterations were
observed, which are believed to be a consequence of PHA toxicity
in plant cells, the study proved the concept of a metabolic engi-
neering approach in higher plants by means of the introduction
of a bacteria-derived polycistron into a plastid genome [64]. This
kind of approach has been used in other cases where simultaneous
expression of several ORFs must be accomplished. For example,
Quesada-Vargas et al. have reported that heterologous operons
expressed via the tobacco (Nicotiana tabacum) chloroplast genome
led to large accumulation of the recombinant proteins, denoting
abundant translation rates [65]. Therefore, multigene expression
in plastid is both time- and cost-effective and provides a source
of well-regulated expression, which is not typically affected by
co-suppression caused by gene silencing [66]. However, it is
important to consider that glycosylation does not take place in
plastids, and therefore this technology is not a choice for those
systems where complex post-translational modications are
critical to obtain functional heterologous proteins retaining the
desired immunogenic properties.
Interestingly, while these approaches have not been applied to
vaccine development, they augur a great potential on the devel-
opment of multicomponent vaccines, with the additional implied
advantages, such as high productivity and maternal inheritance
[66]. In the vaccine development context, these multigene oper-
ons will allow for targeting a simultaneous expression of several
antigens trough a single transformation event, which obviously
reduce the technical work involved in generating and characteriz-
ing the transformed lines, and will permit to assess newcandidates
for highly efcient vaccines. Vaccines based on the immunization
with more than one antigen face the challenge of achieving multi-
component vaccine formulations in a straightforward manner. This
strategy opens a new avenue for multigene transformation, since
the traditional approachto this goal is a multistep, time-consuming
and costly procedure. It is therefore expected that these transplas-
tomic approaches will profoundly impact on the development of
multicomponent vaccines in the years to come.
On the other hand, designing chimeric proteins is also a viable
approach. However, the main disadvantage of this strategy lies
in our limited capacity to predict the functionality of each anti-
genic determinant in the chimera. Different parameters such as
peptide order, the number of multimers to be included, as well
as the linkers, play an important role on determining the overall
immunogenicity [67].
4. Concluding remarks
It is clear that a number of research groups over the world are
currently making efforts to develop vaccines against NTDs. How-
ever, many challenges remain to be addressed. It is expected that
in the following years the available models can yield enough data
to encourage clinical trials for these vaccines. In this situation,
plant-based vaccines are likely to have a profound impact on NTD
prevention. There is, however, a signicant number of NTDs not
considered yet for plant-based vaccine prevention. This demon-
strates that the development of plant-based vaccines against NTDs
is still in its infancy, and shows the necessity to expand the appli-
cation of this biotechnological tool to other immunoprotective
antigens, toassess newexperimental models, andtoyielddata sup-
porting the approach in other cases of interest. Since only a modest
attention has been paid to NTDs in the eld of plant-based vacci-
nation, there is a clear necessity of addressing projects where this
biotechnological approachcanbe appliedto low-cost vaccine mod-
els. Although several edible crops can be efciently transformed,
few groups have reported the use of edible crops to assess plant-
based vaccination models. This aspect is also of special importance
since it will constitute a step forward on the development of prod-
ucts suitable for evaluation in clinical trials. The discovery of new
immunogens along with an increased knowledge on the immuno-
logical mechanisms mediating immunoprotection may lead in the
near future to new developments that will facilitate the exploita-
tion of plants as biofactories and delivery systems for low-cost
vaccines, with the potential to improve the life quality of people
affected by NTDs.
Acknowledgements
Projects from the group are funded by grants from CONA-
CYT (102109/56980), PROMEP-2010 to Bioprocess CA. Francisco
Rodrguez corrected the English version of the manuscript.
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