Vaccines are for dinnerDavid W. Pascual*Department of Veterinary
Molecular Biology, Montana State University, Bozeman, MT
59717-3610
Transgenic plants have beensought not only as bioreactorsbut
also as potential scaffoldsfor oral vaccines. Tobacco was
initially exploited for the successful ex-pression of
Streptococcus mutans surfaceprotein A as a potential dental
cariesvaccine (1) and for hepatitis B surfaceantigen (2) as a
vaccine bioreactor alter-native for viral hepatitis B. Today,
anumber of edible plants (Table 1), in-cluding potatoes, tomatoes,
maize, andsoybeans, have been genetically modi-fied to express a
variety of vaccinetargets, including hepatitis B surfaceantigen
(2), Norwalk virus particles (3,4), heat-labile enterotoxin B
subunit (5,6), and others (711) that can benefitboth humans (68)
and livestock (911).The advantage of edible vaccines is thatoften
the plant products, whether leaves,fruit, or seed, can be readily
consumedwith limited or no processing. Viableoral platforms such as
edible productssuitable for human use are in demandto deliver
vaccines (12). Obviously, thefact that food products are
consumedobviates many of the health concernsthat arise in the oral
vaccination of hu-mans (as reviewed in ref. 13). Ediblevaccines can
also have the advantage ofcircumventing cold-storage issues
(thecold chain), because plant tissues canbe dried or, as when the
seeds are tar-geted, have low moisture content. Like-wise, water-
or oil-based plant extractscan provide additional storage
conve-nience. In this issue of PNAS, Nochi etal. (14) describe a
rice-based oral vac-cine that potentially addresses many ofthese
topics. At hand is the need forvaccine development and strategies
toaid underserved nations with the abilityto produce vaccines
locally in a cost-effective manner. Because rice is pro-duced in
many such areas, this currentwork shows the feasibility of
propagat-ing rice-based vaccines that are trulyedible vaccines,
unlike in the earlierwork with tobacco that ultimately pro-vided
the mechanisms for edible vaccinedevelopment (1, 2). In addition,
this ap-proach breaks the cold-chain barrierthat for many
conventional vaccinesdrives up cost and creates storage prob-lems.
In fact, it is estimated that re-moval of this barrier could give
anadded benefit of as much as $300 mil-lion per year, which could
providevaccines for an additional 10 millionchildren (15). In this
regard, the workby Nochi et al. shows that their trans-
genic rice was stable for 18 months atroom temperature or at 4C.
Becausethese vaccines are needle-free (16), theyhave the added
advantage of eliminatingthe associated waste and potential
fordissemination of bloodborne infections.The propagation of
transgenic rice alsohas a lesser impact on the environmentbecause
of rices limited pollen scatter-ing, unlike with maize or wheat
(13).Thus, Nochi et al. have addressed manyof the issues that
prevent vaccines fromcoming to market, including cost, cold-chain,
needle-associated, and regulatoryconcerns.The authors examination
of the type
of vaccine produced is also a significantachievement. A major
obstacle to fur-thering the field of edible vaccines is theneed to
produce effective immunity.Typically, vaccines applied to
mucosalsurfaces in the absence of adjuvant failto stimulate an
immunogenic responseand resort to the default pathway, ortolerance
(as reviewed in ref. 12). Obvi-ously, we need to be tolerant of
food;the genetic modification of foods maybe potentially
problematic and couldresult in food allergies, thus
jeopardizingfuture consumption of the unmodifiedfood in the diet.
For rice, a majorworldwide staple, this would be espe-cially
problematic. Nochi et al. (14)nicely show that oral immunization
withthe transgenic rice encoding the choleratoxin B subunit (CTB)
does not stimu-late a serum IgG response against ricestorage
protein or, presumably, a secre-tory IgA response. This will be a
keyelement in the success of transgenicrice. Moreover, they show
that the en-coded CTB is resistant to the gut envi-ronment because
of its expression inendosperm that normally is resistant
togastrointestinal digestion. In overcoming
these obstacles, Nochi et al. show mod-est fecal IgA and serum
anti-CTB anti-body responses after the mice wereorally immunized
with the equivalent of75150 g of rice-generated CTB perdose, given
six times over a 10-week vac-cination schedule. When compared
withCTB rice-immunized mice, the miceorally dosed with recombinant
CTBshowed equivalent serum IgG antibodyresponses but weak fecal IgA
responses.This latter finding is surprising, but theauthors did
show protection againstnative cholera toxin challenge, as
evi-denced by reduced intestinal water con-tent (diarrhea). This
level of protectionwas equivalent to that in mice given therice
CTB, whereas mice given nativerice or PBS were not protected.The
selection of CTB as a candidate
vaccine for testing in rice is appropriatebecause of its
adjuvant properties (17).Nochi et al. (14) were able to show
thatthe rice CTB could enter via the gut sam-pling cells or M cells
(18), which are re-sponsible for continually sampling the
gutcontents for aberrant antigens. A numberof pathogens can also
enter via these cellsto cause infection, thus subverting
thisdefensive mechanism. A major obstaclefor successful oral
transgenic plant vac-cines is the potential outcome of
toleriza-tion rather than immunity, but this is truewith any oral
vaccine given in the absenceof mucosal adjuvant. Tolerance, or lack
ofresponsiveness, occurs when a particularantigen or vaccine is fed
without costimu-lation of the innate immune system. Con-
Author contributions: D.W.P. wrote the paper.
The author declares no conflict of interest.
See companion article on page 10986.
*E-mail: [email protected].
2007 by The National Academy of Sciences of the USA
Table 1. Edible transgenic plant vaccines
VaccineEdibleplant Ref.
Norwalk virus particle Potato 3Tomato 4
Heat-labile enterotoxin B subunit Potato 5Maize 6Soybean 20
Cholera toxin B subunit Rice 14Potato 21
Enterotoxigenic Escherichia coli fimbrial subunit Soybean 11
Japanese cedar pollen peptide Rice 19
www.pnas.orgcgidoi10.1073pnas.0704516104 PNAS June 26, 2007 vol.
104 no. 26 1075710758
COMMENTARY
sequently, instead of being immunized,the host becomes actively
unresponsive,and subsequent challenge with the antigenleaves the
host unresponsive. Taking ad-vantage of this known behavior, Takagi
etal. (19) produced transgenic rice that en-coded a fusion protein
between soybeanseed-storage protein glycinin AlaB1b andknown
allergen peptides from Japanesecedar pollen for targeted expression
in therice seed endosperm. When mice were fedwith this transgenic
rice expressing thepollen peptides known to be reactive forT cells,
they were resistant to Japanesecedar pollen challenge, showing
reducedserum histamine release, reduced allergicIgE antibodies, and
reduced Th2 cells thatsupport the allergic response. The
authorsalso showed that cooking the transgenicrice did not affect
its ability to tolerize thehost (19). Although this outcome wouldbe
expected because T cell peptides arerequired for tolerization, the
investigatorsshow that oral feeding with transgenic ricecan
potentially treat autoimmune diseases(19), which is an advantage of
unadju-vanted oral vaccines (12). CTB also hasbeen used to induce
oral tolerance (re-viewed in ref. 17). Although immunity
ortolerance can be driven by CTB, presum-ably by interaction with
host M cells onPeyers patches, a major limitation of anyedible
vaccine will be the requiredcoadministration of a mucosal
adjuvant,unless mucosal adjuvants can be success-fully coexpressed
with the desired ediblevaccine. Being able to demonstrate immu-nity
using their transgenic rice representsa significant accomplishment
for Nochiet al.The greater protein content of rice is
an advantage over some of the starch-based edible vaccines
described previ-ously (Table 1) and for heat-labileenterotoxin B
subunit (LTB; ref. 5).
LTB is very similar to CTB in exhibitingadjuvant activity. When
maize-derivedLTB was fed in three 1.0-mg doses tohuman volunteers,
seven of nine volun-teers produced a serum IgG response,whereas
only four individuals showeda fecal IgA response (6).
Protein-basedseeds such as soybeans have the uniqueadvantage of a
high protein content(3540%), as opposed to rice and maize
that have 810% protein. When LTBwas expressed in soybeans, as
much as2.4% of the total seed protein was LTB(20). Mice fed or
parenterally immu-nized with soybean-derived LTB showedIgG and IgA
anti-LTB antibody re-sponses that could protect against diar-rhea.
Thus, these collective studiesdemonstrate, regardless of the
plant-derived vaccine used, the importanceof developing coexpressed
mucosal adju-vants with the edible vaccine. AlthoughNochi et al.
(14) show no anti-rice stor-age protein response, neither the
ex-pressed CTB nor the recombinant CTBhave the adjuvant potency of
nativecholera toxin. However, when mice werefed wild-type rice in
conjunction withnative cholera toxin, an anti-rice storageprotein
antibody response was elicited(14), suggesting that the expression
ofhighly potent mucosal adjuvants may beproblematic if these were
to stimulateimmune responses to the food product,
be it rice, maize, wheat, or soybeans.Perhaps plant-derived
alternative adju-vants need to be sought, or alternativeadjuvants
need to be added exogenouslyto the prepared edible vaccine.The
future of edible vaccines will de-
pend on the feasibility of producingsufficient quantities of
immunogenicvaccines. The edible vaccines that in-nately possess
immunogenicity and donot require additional adjuvant willprobably
be the first successful vaccinesfor human or livestock use. The
selec-tion of the transgenic plant platformwill largely depend on
the vaccine andthe region where it will be propagated.The
grain-based vaccines, as describedby Nochi et al. (14), will be the
mostlikely candidates because pollen disper-sion or potential
contamination of nor-mal food supplies with transgenic pollencan be
controlled. The future use ofthese vaccines also will depend on
thedevelopment of stable transgenic linesthat effectively maintain
the vaccine ex-pression for subsequent plant genera-tions. In
addition, edible vaccines mayhave to withstand food processing
andpossibly cooking. Nevertheless, a de-mand for the development of
edible vac-cines persists because they can eliminatemany of the
problems associated withconventional vaccines, including
storageissues, injection risks and associatedwaste, high production
costs, and easeof distribution in underserved areas.Who knows? It
may nice to have a littlevaccine with supper tonight!
This work was supported by Public HealthService Grants AI-41123,
AI-55563, and AI-56286 and in part by Montana AgriculturalStation
and U.S. Department of AgricultureFormula funds.
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The future of ediblevaccines will depend
on producingsufficient quantities.
10758 www.pnas.orgcgidoi10.1073pnas.0704516104 Pascual
