| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,¶
* Institute of Bioorganic Chemistry of the Polish Academy of Sciences, Poznan, Poland;
Marcinkovski Academy of Medical Sciences, Department of Endocrinology, Poznan, Poland;
Marcinkovski Academy of Medical Sciences, Department of Pediatric, Poznan, Poland;
§ Institute of Biotechnology and Antibiotics, Warsaw, Poland; and
¶ Biotechnology Foundation Laboratories at Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA
1Correspondence: Biotechnology Foundation Laboratories at Thomas Jefferson University, Room M85 JAH, 1020 Locust St., Philadelphia, Pennsylvania 19107, USA.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSREFERENCES |
|---|
Key Words: oral vaccines • surface antigen (HBsAg) • transgenic lupin • lettuce and engineered plants
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSREFERENCES |
|---|
2
3
4
5
6
7)
and one report on
human ingestion of transgenic raw potatoes expressing Escherichia
coli enterotoxin (8)
. We obtained transgenic lupin
callus to express hepatitis B virus (HBV) surface antigen (HBsAg),
which was fed to mice. Mice fed the transgenic lupin tissue developed
significant levels of HBsAg-specific antibodies. Based on the
encouraging results of animal studies, we obtained transgenic lettuce
plants expressing HBsAg for human consumption. The high HBsAg-specific
immunoglobulin G (IgG) response of human volunteers in this study point
to the future of active immunization using dietary staples.
Transgenic lines of lupin callus and lettuce were obtained using
Agrobacterium tumefaciens C58 (pROK2S) and A.
tumefaciens L4404 (pROK2S), respectively. The lines of transgenic
lupin (callus) and lettuce (plants) in which the presence of HBsAg was
confirmed by polymerase chain reaction (data no shown) were further
analyzed for the accumulation of HbsAg, as determined in tissue
extracts from the transgenic lines (Fig. 1
). The estimated antigen content varied considerably in individual lines
of both transgenic lupin and lettuce leaves (from 11 ng/g in line LL-7
to 150 ng/g of fresh weight in line LL-10 of lupin callus and from 1
ng/g in line LT1 to 5. 5 ng/g in line LT-5 of fresh weight in
transgenic lettuce).
|
Callus tissue from the LL-10 line was used for oral immunization of
mice. In group A mice (see Materials and Methods), the second dose of
lupin callus elicited an HBsAg-specific IgG response that was up to
twofold higher than that observed in the sera of control mice (OD 0.36
vs. 0.15, P=0.0015) (Fig. 2
A). Sera from mice fed with transgenic callus over 5
consecutive days at 1 g per day showed no significant
HBsAg-specific antibody response after two feedings as compared with
sera from mice fed the control callus (Fig. 2B
). Based on
these results, human volunteers were fed HBsAg-expressing transgenic
lettuce leaves twice: first 200 g, and within 2 months, 150 g. Analysis of sera obtained from human volunteers 2 and 4 wk after the
first ingestion of HBsAg-expressing lettuce revealed no significant
levels of HBsAg-specific IgG (Fig. 3
). However, 2 wk after the second feeding, sera from all three
volunteers showed HBsAg-specific antibody titers above 3 IU/l 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 |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSREFERENCES |
|---|
. 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 |
|---|
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  X.-M. Lou, Q.-H. Yao, Z. Zhang, R.-H. Peng, A.-S. Xiong, and H.-K. Wang Expression of the Human Hepatitis B Virus Large Surface Antigen Gene in Transgenic Tomato Plants Clin. Vaccine Immunol., April 1, 2007; 14(4): 464 - 469. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Dubois, E. Botton, C. Meyer, A. Rieu, M. Bedu, B. Maisonneuve, and M. Mazier Systematic silencing of a tobacco nitrate reductase transgene in lettuce (Lactuca sativa L.) J. Exp. Bot., September 1, 2005; 56(419): 2379 - 2388. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Vitale and E. Pedrazzini Recombinant Pharmaceuticals from Plants: The Plant Endomembrane System as Bioreactor Mol. Interv., August 1, 2005; 5(4): 216 - 225. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Warzecha, H. S. Mason, C. Lane, A. Tryggvesson, E. Rybicki, A.-L. Williamson, J. D. Clements, and R. C. Rose Oral Immunogenicity of Human Papillomavirus-Like Particles Expressed in Potato J. Virol., August 15, 2003; 77(16): 8702 - 8711. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. S. Korban, S. F. Krasnyanski, and D. E. Buetow Foods as Production and Delivery Vehicles for Human Vaccines J. Am. Coll. Nutr., June 1, 2002; 21(90003): 212S - 217. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
