1 Instituto de Virología, CICV, INTA-Castelar, CC77, Morón 1708, Buenos Aires, Argentina
2 Consejo Nacional e Investigaciones Científicas y Técnicas (CONICET), Argentina
3 Instituto de Genética E. A. Favret, CICA, INTA-Castelar, Buenos Aires, Argentina
4 Departamento de Biotecnología and Centro de Investigación en Sanidad Animal, INIA, Valdeolmos, 28140 Madrid, Spain
5 University of Saskatchewan, VIDO, Saskatoon, SK, Canada, S7N 5E3
Correspondence
Manuel V. Borca
mborca{at}piadc.ars.usda.gov
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ABSTRACT |
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These authors contributed equally to the results presented in this article.
Present address: Plum Island Animal Disease Center, ARS, USDA, Greenport, NY 11944, USA.
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INTRODUCTION |
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In addition to their use as bioreactors for the production of vaccine antigens, plants also provide an adequate system for oral delivery of recombinant immunogens by including them in the diet. A number of viral and bacterial antigens produced in plants have already been shown to be immunogenic when orally administered: among them are Norwalk virus capsid protein (Mason et al., 1996), Escherichia coli heat-labile enterotoxin (Haq et al., 1995
), cholera toxin B subunit (Arakawa et al., 1998
), gS glycoprotein of transmissible gastroenteritis virus (Gómez et al., 2000
), VP1 of foot-and-mouth disease virus (Wigdorovitz et al., 1999
) and the enterotoxigenic E. coli fimbrial and rotavirus enterotoxin (Yu & Langridge, 2001
). This approach is especially relevant when used for enteric pathogens since oral immunization may be capable of eliciting appropriate immune mechanisms for the induction of protective responses.
Rotaviruses are the principal aetiological agents of severe acute gastroenteritis in numerous mammalian species throughout the world (Kapikian & Chanock, 1996). Newborn calves are susceptible to rotavirus infection during the first weeks of life, thus making it difficult to actively immunize the animals before exposure to the virulent pathogen. However, colostral antibodies produced in rotavirus-vaccinated mothers have been reported to confer passive protection to the newborn, as shown in an experimental murine model (Offit & Clark, 1985
) as well as in the natural host (Fernandez et al., 1996
; Kim et al., 2002
; Saif et al., 1983
).
Rotavirus particles possess six structural proteins that are organized in an inner core, and a surrounding viral capsid composed of two protein layers. One of the outer capsid proteins, VP4, forms spikes composed of homodimers of the protein that emerge from the virion surface layer (Shaw et al., 1993). The amino acid sequence involved in the adsorption of bovine rotavirus (BRV) to epithelial cells has been mapped to a VP4 region made up of 24 aa (eBRV4) (Ijaz et al., 1995
), which also included at least one neutralizing epitope. In vivo experiments performed in mice demonstrated that peptides representing the eBRV4 sequence were able to mediate protection by induction of neutralizing antibodies, as well as by competing with the virus in the infection of epithelial cells (Ijaz et al., 1998
).
In this study, we present the development of an experimental edible immunogen based on the use of recombinant eBRV4 peptide expressed in transgenic alfalfa plants. The eBRV4 peptide was expressed fused to the enzyme -glucuronidase (
GUS) in order to reduce the proteolysis of the peptide within plant cells, and also to facilitate the screening of a large number of individuals to select those plants expressing the highest levels of the recombinant antigen (Dus Santos et al., 2002
; Gil et al., 2001
). Transgenic alfalfa plants expressing significant levels of the fusion protein eBRV4
GUS were used to demonstrate the ability of the chimeric protein to induce protective lactogenic immunity against a virulent rotavirus in suckling mice born from immunized adult female mice.
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METHODS |
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Detection of the eBRV4GUS gene in transgenic plants.
The presence of the recombinant genes in transgenic alfalfa plants was detected by PCR (Carrillo et al., 1998). Total nucleic acids were extracted from samples of approximately 50 mg of leaves using DNAzol (Gibco) following the manufacturer's specifications. The primers 5'-TCGACGGATCCATGTGTAAC-3' (forward) and 5'-CGATACCCGGGATAGCGATA-3' (reverse) were used to specifically amplify a 96 bp DNA fragment from the eBRV4
GUS recombinant gene.
Analysis of eBRV4GUS expression in transgenic plants
Expression of the eBRV4GUS fusion product in transgenic plants was initially studied by measuring
GUS enzymic activity and detecting expression of the eBRV4 epitope by Western blot analysis.
Detection of GUS activity.
Expression of GUS in plant tissues was quantified by fluorimetry as described by Dus Santos et al. (2002)
. Briefly, 100 mg of fresh plant tissue was frozen using liquid nitrogen and ground, and the resulting powder resuspended in GUS extraction buffer (50 mM sodium phosphate pH 7·0, 10 mM
-mercaptoethanol, 10 mM Na2EDTA pH 8·0, 0·1 % L-laurylsarcosine, 0·1 % Triton X-100). Samples were clarified at 12 000 g for 10 s and total soluble protein (TPS) was quantified by the Bradford assay (Bio-Rad). Five micrograms of TPS was dissolved in 400 µl of GUS reaction buffer, containing 1 mM 4-methylumbelliferyl
-D-glucuronide, and incubated at 37 °C. The reaction was stopped at different times by the addition of 0·2 M Na2CO3 and read by fluorimetry against a standard concentration curve obtained with different concentrations of 4-methylumbelliferone (MU). The results were expressed as nmol MU produced min1 (mg TPS)1 in the leaf extracts of the transgenic plants. The assay was repeated independently, at least twice, with each of the tested plants.
Western blot.
Fresh plant tissue (100 mg) was ground as described above and resuspended in 0·1 ml of extraction buffer containing 1 mM PMSF in PBS.
Immunoprecipitation was done according to Al-Yousif et al. (2000), using sera from mice immunized with semi-purified C486 BRV particles, and protein GSepharose 4 Fast Flow (Pharmacia Biotech). Samples were mixed with an equal volume of sample buffer (50 mM Tris/HCl pH 7·5, 1 mM PMSF, 8 M urea, 1 % SDS, 2 mM DTT and 2 %
-mercaptoethanol), boiled for 10 min, separated by a 12·5 % SDS-PAGE and blotted to Immobilon P (Millipore) membranes. Blots were blocked overnight with PBS containing 0·05 % Tween 20 (PBST)/3 % skimmed milk (all subsequent steps were performed using this buffer) and incubated with a bovine serum raised against BRV C486. The membrane was then washed with PBST, and incubated with a goat anti-bovine IgG peroxidase-labelled serum (KPL, MD, USA) for 1 h at 37 °C. After washing three times, the reaction was developed by addition of chemiluminescence reagent (NEN).
Animal immunization
BALB/c female mice (6090 days old) were used for all immunization experiments.
Intraperitoneal vaccination.
Mice utilized for serum antibody determinations received six doses, every 3 weeks, with 0·3 ml of an oil vaccine formulated with incomplete Freund's adjuvant containing 150 µl of crude extract obtained from 15 to 20 mg (containing approximately 0·5 µg of the eBRV4 peptide) of fresh leaf tissue from eBRV4GUS-expressing plants or non-transformed plants. Those dams used for passive protection experiments received three doses following the same protocol as described above.
Oral immunization.
Mice were placed in individual cages and 4 h before treatment were deprived of food and then fed with 0·3 g (containing approximately 6 µg of the eBRV4 peptide) of freshly harvested transgenic or control leaves. This treatment was carried out once a week over a 2 month period.
Analysis of antibody response to plant-expressed eBRVP4 polypeptide
Sera from immunized mice were studied for the presence of BRV-specific antibodies by ELISA and Western blot analysis.
ELISA.
A synthetic peptide (p232254) comprising the amino acid sequence of the eBRVP4 epitope was directly adsorbed onto a microtitre plate at a concentration of 15 µg ml1 in carbonate/bicarbonate buffer pH 9·6. Plates were then incubated with PBS/0·05 % PBST/3 % normal horse serum (blocking buffer) for 1 h at 37 °C. Blocking buffer was discarded and mouse serum samples were added to the plates, diluted 1 : 20 in the same blocking buffer, and incubated for 1 h at 37 °C. Plates were washed with PBST and were incubated as described above with horseradish peroxidase-conjugated goat anti-mouse IgG (KPL). After extensive washing with PBST, the reaction was developed with o-phenylenediamine (OPD)/H2O2 diluted in citrate buffer pH 5·0. Colour development was stopped by addition of an equal volume of 6 M H2SO4 and absorbance was measured 3 min later at 490 nm in an NR 500 Microplate Reader (Dynatech).
Western blot.
Concentrated semi-purified C486 BRV was produced according to Yuan et al. (2001). BRV antigen was resuspended in SDS-PAGE sample buffer. Electrophoresis and blotting, as well as blocking and washing steps, were performed as already described for detection of the eBRVP4 epitope in the plant tissues. Blots were subsequently incubated for 2 h at 37 °C with mouse serum samples diluted 1/30, washed with PBST and incubated with an alkaline phosphatase-labelled anti-mouse Ig goat antiserum (KPL) for 1 h at 37 °C. After extensive washing the reaction was developed by addition of NBT/BCIP substrate (Bio-Rad).
Challenge experiment.
Three weeks before the third immunization, female mice were mated with rotavirus seronegative BALB/c males at a 1 : 1 male-to-female ratio. Challenge experiments with pups born to orally or intraperitoneally immunized dams were performed independently. Newborn mice were left to freely suckle their dams and 5 days after birth were orally challenged with 104 fluorescence focus units (f.f.u.) of virulent C486 rotavirus per mouse. This infective virus dose produced diarrhoea in 100 % of control naive animals in mock-treated control groups in our work, similar to that reported by P. A. Offit (Offit & Clark, 1985; Offit et al., 1986
). Infectivity controls of the virulent BRV were repeated for each individual challenge experiment and such results were reproduced consistently. Virus preparations were administered by intubation of the stomach with a soft flexible plastic feeding tube and pups were inspected for diarrhoea for 24 h after challenge by gentle palpation of the abdomen (Ijaz et al., 1987
).
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RESULTS |
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Analysis of GUS activity and its correlation with eBRV4 epitope expression in eBRV4
GUS transgenic alfalfa plants
GUS activity was assessed in 62 transgenic individuals harbouring the eBRV4
GUS gene. As expected, recombinant plants presented variable levels of
GUS activity (Fig. 2
a), but approximately 4 % of the individuals tested had a significantly high level of expression of the
GUS protein. Some plants presenting significantly high
GUS activity (plants 4, 10, 17 and 46; Fig. 2a
) were analysed by Western blotting for expression of the eBRV4 epitope fused to
GUS protein. As shown in Fig. 2(b)
, expression of the BRV epitope was effectively associated with
GUS activity in the tested lines. Transgenic eBRV4 peptide reacted against a specific anti-VP4 antiserum in Western blot analysis giving a unique distinctive band of approximately 67 kDa, the molecular mass expected for the eBRV4
GUS fusion protein (Fig. 2b
). The approximate concentration of the recombinant eBRV4
GUS expressed in transgenic lines 17 and 46 was estimated at between 0·4 and 0·9 mg (g TPS)1 using a methodology based on the
GUS enzymic activity obtained with a known control, p135-160-
GUS (Dus Santos et al., 2002
), and that obtained with eBRVP4 (data not shown).
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Also of interest was the observation that the BRV antibody response elicited in a second group of dams, immunized by either the intraperitoneal or oral routes, was also passively transferred to their offspring. Pups born from intraperitoneally or orally immunized dams showed levels of anti-BRV specific antibodies that were consistently positive, and comparable to those of their respective mothers (Fig. 3c and e, respectively).
Virus challenge of pups born to dams immunized with plant-produced eBRV4GUS fusion protein
Since dams transferred significant passive antibody to their offspring, we determined whether the pups would be protected against rotavirus challenge. Pups were orally inoculated using 104 f.f.u. of BRV isolate C486 and then observed over a 24 h period for the presence of diarrhoea. Challenge experiments demonstrated that, regardless of the route of immunization administered to the dams, all groups showed a significant degree of protection when compared with animals immunized with extracts from plants transformed with a non-related gene, as determined by Fisher's exact test (Table 1).
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DISCUSSION |
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The demonstration that some of these antigens were immunogenic when orally administered (Arakawa et al., 1998; Gómez et al., 2000
; Haq et al., 1995
; Mason et al., 1996
; Gil et al., 2001
; Wigdorovitz et al., 1999
) encouraged the study of other proteins and peptides expressed in plants in order to develop edible vaccines, especially for those pathogens which invade the host organism via the oral route. In addition, in diseases affecting newborn animals, passive protection conferred by colostrum and milk is the main protective mechanism against these infections.
In the present work, we describe the production of transgenic alfalfa plants expressing the eBRV4 epitope from structural protein VP4 of rotavirus fused to GUS protein, and their use in conferring passive protection in an experimental host.
Fusion of the eBRV4 peptide to the reporter protein allowed us to easily detect the highest-expressing plant lines by measuring their GUS enzymic activity, as with previous reports that showed a positive correlation between
GUS activity and accumulation of the fused vaccine peptides (Dus Santos et al., 2002
; Gil et al., 2001
).
Our results showed that plant-derived eBRV4GUS was able to induce a rotavirus-specific antibody response in mice when administered either intraperitoneally or orally. In addition, oral immunization was achieved not only by feeding the animals with fresh transgenic alfalfa leaves, but also by intragastric immunization with crude extracts from transgenic plants (data not shown).
Most notable was the observation that immunization of female mice induced not only an anti-BRV humoral response, but also elicited a specific secretory antibody response in those mice that they were able to transfer to their offspring through colostrum. Consequently, suckling mice presented specific rotavirus antibodies in their serum at levels comparable to those in their respective dams. Depending on the route of immunization of the dams, 71 or 76 % of the experimentally challenged pups were protected from virus infection. In contrast, for those animals born to mothers immunized with control plant antigens, protection was 15 or 21 % when compared with unvaccinated controls.
The VP4-derived eBRV4 epitope has been shown to provide protection from virulent BRV either by binding to the target cell in vitro (Ruggeri & Greenberg, 1991) or in vivo (Ijaz et al., 1998
), or by inducing antibodies that are able to prevent virus binding to the gut epithelial cells (Ijaz et al., 1991
). Passive protection to BRV challenge using this peptide has also been previously reported in mice (Ijaz et al., 1991
), and in cattle by immunization with a BRV VP8* protein fragment subunit antigen (Lee et al., 1995
). The same antigen has also been expressed in plants via a plant virus-based vector and it was also able to induce passive protection in a murine model by intraperitoneal immunization of the dams (D. M. Perez-Filgueira and others, unpublished).
We believe that our results demonstrate the feasibility of this strategy, since we could provide reasonable protection to susceptible pups through a mucosal immune response induced by an orally administered vaccine formulation obtained from transgenic plants. The results presented here also support the concept of using transgenic plants as a novel and safe system for vaccine production, which could become a very attractive alternative in animal health. The fact that feeding animals with transgenic plants expressing a recombinant antigen caused a systemic immune response demonstrates the prospect of using recombinant plants as both an expression and delivery system for oral vaccines, even against antigens for which a systemic immune response is required. The establishment of food plant-based oral immunization methods for microbial antigens may represent an inexpensive alternative to conventional fermentation systems for vaccine production, as food plants can be grown inexpensively in large quantities and provide a practical and feasible delivery system.
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ACKNOWLEDGEMENTS |
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Received 17 September 2003;
accepted 13 February 2004.
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