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A plant-derived edible vaccine against hepatitis B
virus. J. Kapusta, A. Modelska, M. Figlerowicz, T. Pniewski, M.
Letellier, O. Lisowa, V. Yusibov, H. Koprowski, A. Plucienniczak, and
A.B. Legocki (1999) FASEB J., 1796–1799. The legends for
figures 1
,2
,3
,4
of this article were published under the wrong figures of
the original article. The two pages are reproduced correctly here. The
editors apologize for the error.
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and sera
from two of these volunteers had an HBsAg-specific antibody level higher
than 10 IU/l, accepted as a minimum protective level against HBV. Four
weeks after the second ingestion, HBsAg-specific antibody levels
declined (1.7 IU/l, Fig. 3
), although no further decline was observed
by 12 wk (Fig. 3)
. Serum IgA production was not detected. No noticeable
side effects were observed in the volunteers who ingested transgenic
lettuce expressing HBsAg during 20 wk after first ingestion.
The present study shows that antigens expressed in plants administered orally can induce a specific anti-HBsAg antibody response in mice as well as in humans. Mice that received 5 g of transgenic lupin callus over the course of 1 day developed a better immune response to HBV than those fed in multiple doses of 1 g of the tissue.
Two of the three human volunteers mounted a significant immune response after a second ingestion of transgenic lettuce. It remains unclear why one of the three volunteers did not develop a significant HBV response to the orally administered HBsAg. Vaccination of this subject with a standard HBV vaccine may clarify whether he is capable of developing significant immune response to HBV antigen at all. At 4 wk after the second ingestion of transgenic lettuce, serum HBsAg-specific antibody levels in all three volunteers had decreased considerably, although no further decline was observed at 12 wk Further monitoring of the HBV-specific antibody levels in these three subjects may provide additional information about the nature of the immune response and about the potential need for multiple feedings of transgenic plant-based vaccine.
One of the concerns regarding the plant-derived edible vaccines was related to the fact that humans ingesting transgenic plant expressing a foreign gene may not respond to immunization because the same species of plants are part of their regular diet. The results of this study suggest that an immune response against HBsAg can be elicited by ingestion of transgenic lettuce in humans who are consuming nontransgenic lettuce in their diet.
Two of three human volunteers who ingested transgenic lettuce leaves expressing HBsAg developed a serum antibody response at levels considered protective, thus suggesting that humans may be immunized orally against HBV with plants expressing the viral antigen.
MATERIALS AND METHODS
DNA cloning
All enzymatic digestions, ligations, cell
transformations, and other manipulations with DNA were done according
to Sambrook et al. (9). Plasmid pROK was used as a shuttle
vector to incorporate HBsAg coding sequences into the Ti-plasmid of
A. tumefaciens. HBsAg was cloned from pHB614 containing the
full genome of HBV adw subtype in order to obtain pROK2S
(Fig. 4
). Plasmid pROK2S carrying the HBsAg coding sequence was electroporated
(10) into A. tumefaciens strains C58 and
LBA4404 (10).
Plant transformation
Four-day-old seedlings of yellow lupin (Lupinus
luteus L.) were used as primary explants. After removing the
distal parts, cotyledons were transformed using A.
tumefaciens C58 (pROK2S). After 2 days of cultivation on
antibiotic-free, modified Murashige-Skoog medium (T. Pniewski, J.
Kapusta, and A. B. Legocki, unpublished results), the explants
were transferred to special medium (11) to stimulate
callus growth. Transgenic lines were selected in medium supplemented
with kanamycin and carbenicillin (T. Pniewski, J. Kapusta, and A. B. Legocki, unpublished results). Calli resistant to kanamycin were
isolated and cultured further.
Cotyledons isolated from 2-day-old seedlings of lettuce (Lactuca sativa L.) cv. Burpee Bibb were inoculated with A. tumefaciens LBA4404 (pROK2S). Transgenic lettuce plants were obtained as described (11).
Protein extraction and analysis
Protein was extracted as described (2)
from transgenic tissue homogenized in phosphate buffer. The homogenate
was centrifuged at 30,000 x g for 15 min to remove
nonhomogenized cell debris. HBsAg in the supernatant was quantitated
using an Auszyme monoclonal diagnostic kit (Abbott Lab., North Chicago,
Ill.) according to the manufacturer’s instructions. The amount of
antigen in plant extracts was calculated using a standard curve based
on different concentrations of purified HBsAg.
Immunization of mice
Six- to 8-wk-old male BALB/c mice (five in a group)
were immunized by feeding with transgenic lupin callus containing
HBsAg. Group A mice received 5 g of callus tissue for 1 day, and
group B mice were fed 1 g of callus on each of 5 consecutive days;
the respective feeding protocols were repeated once after a 1 month
interval. Callus tissue fed to each mouse was equivalent to ~750 ng
of HBsAg. Transgenic callus was administrated without any additional
feeding. Control mice were fed with transgenic callus containing vector
without HBsAg, using the same schedule of administration. Serum samples
were collected at the following times: 2 days before the first feeding
(preimmune) from all the mice; 2 wk after the first and second
administrations for mice receiving 5 g of callus tissue on 1 day;
and 9 days after each of the two immunizations for mice fed with the
callus over 5 consecutive days.
Immunization of human volunteers
Five adult volunteers (male and female), 25–59 years
of age and in good health, were enrolled in the study after signing
consent forms. The volunteers had no previous HBV vaccination, no
history of HBV infection, and no detectable anti-HBs or anti-HBc serum
antibodies. Three individuals received transgenic lettuce leaves twice:
200 g at first, and within 2 month, 150 g. The amount of
HBsAg in the lettuce plants varied from 0.1 to 0.5 µg/100 g of fresh
tissue. Two control individuals were given the same amount of
nontransgenic lettuce according to the same schedule. Volunteers
consumed no food or liquids for 1 h before and after ingesting
lettuce. Leaves were washed and were eaten without additives. Blood
samples from all volunteers were collected before the first ingestion
(preimmune), 2 and 4 wk after the first ingestion, and 2, 4, and 12 wk
after the second lettuce ingestion.
ELISA
Mouse and human serum samples were analyzed for the
presence of anti-HBsAg-specific antibodies by enzyme-linked
immunoabsorbent assay (ELISA), as described (12), using
96-well plates (Nunc-Immuno PolySorp, Roskilde, Denmark) coated with
100 µl per well of HBsAg (2 µg/ml, Biodesign, Kennebunkport, Maine)
overnight at 4°C. Peroxidase-conjugated goat anti-mouse IgG
(
-chain-specific, Sigma, St. Louis, Mo.) and IgA (
chain-specific, Sigma) were used to detect anti-HBsAg-specific
antibodies in sera from immunized mice. Anti-HBsAg-specific antibodies
in human sera were detected using peroxidase-conjugated goat anti-human
IgG (-chain specific, Sigma) and IgA ( chain specific,
Sigma). The Hepanostika anti-HBs diagnostic kit (Organon Teknika)
was used to estimate the level of antibodies in human sera. The assay
was carried out according to the diagnostic kit protocol and the
instructions for Organon Teknika Microelisa Comp system. The
concentration of anti-HBsAg antibody in the test sera was calculated
using WHO standardized sera provided by the manufacturer. One of
the standard serum samples contained no anti-HBsAg antibodies. Two
others had anti-HBsAg antibody titers equal to 10 IU/l (low positive
control) and 100 IU/l (high positivecontrol).
ACKNOWLEDGMENTS
The authors thank Dr. Maurice Hilleman for his critical reading of this manuscript. The research at the Institute of Bioorganic Chemistry of the Polish Academy of Sciences was partially funded by a grant from the State Committee for Scientific Research of Poland (Research project No. 6 PO4B 010 09). The research at the Biotechnology Foundation Laboratories is supported by a grant from the Commonwealth of Pennsylvania. A collaborative grant from NATO supported the research of both institutions.
REFERENCES
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