Erratum

Abstract

The FASEB JournalVolume 13, Issue 15 p. 2339-2340 ErratumFree Access Erratum This article corrects the following: 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, A. B. Legocki, Volume 13Issue 13The FASEB Journal pages: 1796-1799 First Published online: October 1, 1999 First published: 01 December 1999 https://doi.org/10.1096/fasebj.13.15.2339AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat 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 1–4 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. Figure 1Open in figure viewer Schematic representation of pROK2S binary vector carrying the S gene of HBV. RB and LB are the right and left borders, respectively, of the A. tumefaciens Ti-plasmid known to be required for integration of foreign genes into the plant genome. NOSp and NOSt are nopalin synthase promoter and transcription terminator from the A. tumefaciens Ti-plasmid, respectively. CaMV35Sp is a promoter originating from cauliflower mosaic virus. HBsAg is a surface antigen of HBV. 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 received5goftransgenic 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. Figure 2Open in figure viewer Evaluation of HBsAg accumulation in transgenic lupin callus and lettuce lines. Individual transgenics are indicated on the x axis. Lupin and lettuce plants transformed with A. tumefaciens vector without HBsAg were used as a control. Figure 3Open in figure viewer Serum antibody response in mice immunized orally with transgenic lupin callus containing HBsAg. Mice were fed with5gofcallus in one dose (A)orwith1goneach of 5 consecutive days (B). Control mice were fed the transgenic callus without HBsAg according to the same respective schedules. Serum samples were collected before the first immunization (preimmune) and 2 wk after the second immunization. Data represent the mean (+ sd) of five (HBsAg) or four (control) individual measurements. Figure 4Open in figure viewer Titer of antibodies in three individuals (1–3) immunized orally with transgenic lettuce harboring HBsAg and in two individuals (4, 5) fed control lettuce without HBsAg 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 × 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 received5gofcallus 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 receiving5gofcallus 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 (α 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 (γ-chain specific, Sigma) and IgA (α 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 1Hiatt, A. C., Cafferkey, R., and Bowdish K. (1989) Production of antibodies in transgenic plants. Nature (London) 342, 76– 78CrossrefCASPubMedWeb of Science®Google Scholar 2Mason, H. S., Lam, D. M.-K., and Arntzen, C.J. (1992) Expression of hepatitis B surface antigen in transgenic plants. Proc. Natl. Acad. Sci. USA 89, 11745– 11749CrossrefCASPubMedWeb of Science®Google Scholar Volume13, Issue15December 1999Pages 2339-2340 FiguresReferencesRelatedInformation