Tobacco plants were genetically transformed with the gene encoding hepatitis B surface antigen (HBsAg) linked to a nominally constitutive promoter. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed the presence of HBsAg in extracts of transformed leaves at levels that correlated with mRNA abundance. This suggests that there were no major inherent limitations of transcription or translation of this foreign gene in plants. Recombinant HBsAg was purified from transgenic plants by immunoafflnity chromatography and examined by electron microscopy. Spherical particles with an average diameter of 22 nm were observed in negatively stained preparations. Sedimentation of transgenic plant extracts in sucrose and cesium chloride density gradients showed that the recombinant HBsAg and human serum-derived HBsAg had similar physical properties. Because the HBsAg produced in transgenic plants is antigenically and physically similar to the HBsAg particles derived from human serum and recombinant yeast, which are used as vaccines, we conclude that transgenic plants hold promise as low-cost vaccine production systems.Hepatitis B virus infection is one ofthe most widespread viral infections of humans and causes acute and chronic hepatitis and hepatocellular carcinoma (1). The infectious viral particle (Dane particle) is a 43-nm double-shelled sphere that consists of a core containing the 3.2-kilobase (kb) DNA genome bound to the core protein, surrounded by the viral envelope containing phospholipids and the major surface antigen [hepatitis B surface antigen (HBsAg)] (2). In addition to Dane particles, the serum of infected individuals also contains 22-nm subviral particles in great excess over virions. These noninfectious particles contain the elements of the viral envelope, including the major 24-kDa peptide that occurs in glycosylated and unglycosylated forms (2).Because the host range of hepatitis B virus is limited to humans and chimpanzees, and since the virus cannot be propagated in cell culture, HBsAg for use in vaccines was purified from the serum of infected individuals until a recombinant form (rHBsAg) was produced in yeast (3). The immunogenic yeast-derived rHBsAg occurs in the form of spherical particles with an average diameter of 17 nm. Integration of the peptides into the phospholipid-containing particles greatly enhances their immunogenic properties (4). Subsequent work showed that the peptides present in the yeastderived particles were much less extensively disulfide-linked than in the human material but that such linking could be induced in vitro (5).Intramuscular injection of serum-derived HBsAg or yeastderived rHBsAg in healthy individuals results in effective immunization and protection from viral infection (6, 7). In many areas ofthe developing world, however, the expense of immunization programs for large segments of the population is prohibitive. This has led us to attempt the expression of rHBsAg in plants with the hope of developing a less ex...

Data are presented which suggest the existence of a light-harvesting pigment-protein complex which is functionally and structurally associated with photosystem I (PSI) reaction centers. These observations are based on techniques which aflow isolation of PSI using minimal concentrations of Triton X-100. Properties of density and self aggregation allowed purification of a "native" PSI complex. The isolated PSI particles appear as 106 A spherical subunits when viewed by freeze fracture microscopy. When incorporated into phosphatidyl choline vesicles, the particles lose self-aggregation properties and disperse uniformly within the lipid membrane. The isolated PSI preparation contains 110 ± 10 chlorophylls/P700 (Chi a/b ratio greater than 18); this represents a recovery of 27% of the original chloroplast membrane Chi. These particles were enriched in Chi a forms absorbing at 701 to 710 nm. Chi fluorescence at room temperature exhibited a maximum at 690 nm with a pronounced shoulder at 710 nm. At 77 K, peak fluorescence emission was at 736 am; in the presence of dithionite an additional fluorescence maximum at 695 nm was obtained at 77 K. This dual fluorescence emission peak for the PSI particles is evidence for at least two Chl populations within the PSI membrane subunit. The fluorescence emission observed at 695 nm was identified as arising from the core of PSI which contains 40 Chl/P700 (PSI40). This core complex, derived from native PSI particles, was enriched in Chi a absorbing at 680 and 690 nm and fluorescing with maximal emission at 694 am at 77 K. PSI particles consisting of the PSI core complex plus 20 to 25 Chi antennae (65 Chl/P700) could also be derived from native PSI complexes. These preparations were enriched in Chl a forms absorbing at 697 nm and exhibited a 77 K fluorescence emission maximum at 722 am. A comparison of native PSI particles which contain 110 Chl/P700 (PSI-110) and PSI particles containing 65 Chl/P700 (PSI-65) provides evidence for the existence of a peripheral Chi-protein complex tightly associated in the native PSI complex. The native PSI subunits contain polypeptides of 22,500 to 24,500 daltons which are not found in the PSI-65 or PSI-40 subfractions. It is suggested that these polypeptides function to bind 40 to 45 Chi per structural complex, including the Chi which emits fluorescence at 736 am. A model for the organization of Chli forms is presented in which the native PSI membrane subunit consists of a reaction center core complex plus two regions of associated light-harvesting antennae. The presence of energy "sinks" within the antennae is discussed.

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(1) and expression of the capsid protein in recombinant baculovirus-infected insect cells (4) have facilitated the study of the virus and the development of a candidate vaccine (5). Expression of the Norwalk virus capsid protein (NVCP) in insect cells yields a protein with an apparent Mr of 58,000 that self-assembles into insect cell-derived Norwalk virus-like particles (i-rNVs) lacking viral RNA, which are reactive with sera from Norwalk virus-infected humans (4). Electron cryomicroscopy of i-rNV shows that the 38-nm empty capsid is composed of 90 dimers of NVCP that form arch-like capsomeres (6). The particles are morphologically and antigenically similar to authentic virus particles, stable on storage at 4°C, stable after lyophilization, and resistant to pH 3.0 treatment (4, 7). These qualities make i-rNV attractive for use as a potential vaccine against Norwalk virus. Recent studies showed that oral immunization of mice with as little as 50 ,ug of i-rNV per dose resulted in the production of serum and mucosal antibodies against NVCP (5). This result is striking in view of the fact that i-rNV is a nonreplicating vaccine and no cholera toxin (CT) adjuvant is needed to achieve immunization.We have experimented with the use of plants as an economical alternative for expression and delivery of recombinant vaccines (8-10). Hepatitis B surface antigen (HBsAg) expressed in tobacco leaves forms subviral particles (8) that are similar to the recombinant yeast-derived antigen, which is licensed for parenteral immunization (11). The plant-derived HBsAg retains both B-and T-cell epitopes when studied in a mouse model (10). Furthermore, the Escherichia coli heatlabile enterotoxin B-subunit expressed in potato tubers and fed to mice without preparation (other than slicing) stimulates serum and gut mucosal antibodies against E. coli labile enterotoxin B-subunit (9). These studies provide proof that recombinant antigens can be produced in transgenic plants, and, at least in some cases, these antigens are orally immunogenic.We report here the expression of recombinant NVCP in transgenic tobacco leaves and potato tubers. The NVCP from tobacco leaves self-assembles into tobacco-derived virus-like particles (t-rNVs) that are morphologically and physically similar to i-rNVs. Further, we show that either partially purified t-rNV given orally or potato tubers expressing NVCP fed directly to mice stimulate the production of antibodies against NVCP. We conclude that a plant-derived edible vaccine for Norwalk virus is feasible. Together with our previous studies, these findings bolster the concept of using transgenic plants for a novel, safe vaccine production and delivery system for developing countries. MATERIALS AND METHODSConstruction of Plant Expression Vectors. A 2.4-kbp DNA fragment containing the gene encoding NVCP was obtained by partial EcoRI digestion of pUCNV4145 (1) and subcloned into pBluescript-KS (Stratagene) at the EcoRI site. One clone (pKSNV2.4) was digested with SmaI and SstI; the resulting 1.9-kbp fragmen...

The binding subunit of Escherichia coli heat-labile enterotoxin (LT-B) is a highly active oral immunogen. Transgenic tobacco and potato plants were made with the use of genes encoding LT-B or an LT-B fusion protein with a microsomal retention sequence. The plants expressed the foreign peptides, both of which formed oligomers that bound the natural ligand. Mice immunized by gavage produced serum and gut mucosal anti-LT-B immunoglobulins that neutralized the enterotoxin in cell protection assays. Feeding mice fresh transgenic potato tubers also caused oral immunization.

Here we present data showing oral immunogenicity of recombinant hepatitis B surface antigen (HBsAg) in preclinical animal trials. Mice fed transgenic HBsAg potato tubers showed a primary immune response (increases in HBsAg-specific serum antibody) that could be greatly boosted by intraperitoneal delivery of a single subimmunogenic dose of commercial HBsAg vaccine, indicating that plants expressing HBsAg in edible tissues may be a new means for oral hepatitis B immunization. However, attainment of such a goal will require higher HBsAg expression than was observed for the potatoes used in this study. We conducted a systematic analysis of factors influencing the accumulation of HBsAg in transgenic potato, including 5' and 3' flanking elements and protein targeting within plant cells. The most striking improvements resulted from (1) alternative polyadenylation signals, and (2) fusion proteins containing targeting signals designed to enhance integration or retention of HBsAg in the endoplasmic reticulum (ER) of plant cells.

Incubation of Chlamydomonas reinhardii cells at light levels that are several times more intense than those at which the cells were grown results in a loss of photosystem II function (termed photoinhibition). The loss of activity corresponded to the disappearance from the chloroplast membranes of a lysine-deficient, herbicide-binding protein of 32,000 daltons which is thought to be the apoprotein of the secondary quinone electron acceptor of photosystem II (the QE; protein). In vivo recovery from the damage only occurred following de novo synthesis (replacement) of the chloroplast-encoded QB protein . We believe that the turnover of this protein is a normal consequence of its enzymatic function in vivo and is a physiological process that is necessary to maintain the photosynthetic integrity of the thylakoid membrane. Photoinhibition occurs when the rate of inactivation and subsequent removal exceeds the rate of resynthesis of the QB protein .A 32,000-dalton integral membrane polypeptide ofphotosystem II (PS 11)' is known to be the binding site for several families of herbicides, including the triazines (1). Competition studies using herbicides and quinone analogues (2) have supported the hypotheses that the herbicides act by displacement of a bound quinone (QB) which functions as the secondary quinone electron acceptor for PS II (3, 4). It has been accepted that the 32-kilodalton (kd) polypeptide shall be designated as the QB protein since it functions as the apoprotein of the bound quinone (formalized at the International Conference on Herbicides That Inhibit Photosynthesis, Wageningen, 1983; see reference 5). Pulse-labeling studies using Spirodella (6) and Chlamydomonas (7) indicate that the QB protein exhibits a very rapid turnover in the light.After transfer of dark-grown maize seedlings to light, the level of the mRNA coding for the QB protein becomes the most abundant message in the chloroplast (8) . For this reason the 32-kd protein has also been referred to as the product of a "photogene" (9) . In mature leaf tissue these high mRNA 'Abbreviations used in this paper: kd, kilodalton ; LDS, lithium dodecyl sulfate ; LHC, light-harvesting chlorophyll a/b-protein complex ; PS II, photosystem 11 .THE JOURNAL OF CELL BIOLOGY -VOLUME 99 AUGUST 1984 481-485 0 The Rockefeller University Press -0021-9525/84/08/0481/05 $1 .00 levels are maintained ; this corresponds to the continued high rate of synthesis (and corresponding turnover) of the QB protein .The reason for the high mRNA levels and rapid turnover rate of the QB protein in chloroplasts has not been known. Matoo et al. (10) have suggested that the rapid turnover may in some way be related to a control mechanism for PS II function. Alternatively, Arntzen et al. (1) hypothesized that the unusually high rate of turnover of the QB protein in the light could be a natural consequence of its in vivo function as the secondary acceptor of PS II. (This enzymatic function involves the stabilization of reactive quinone anions in the formation of the reduced ...

Compared with vaccine delivery by injection, oral vaccines offer the hope of more convenient immunization strategies and a more practical means of implementing universal vaccination programs throughout the world. Oral vaccines act by stimulating the immune system at effector sites (lymphoid tissue) located in the gut. Genetic engineering has been used with variable success to design living and non-living systems as a means to deliver antigens to these sites and to stimulate a desired immune response. More recently, plant biotechnology techniques have been used to create plants which contain a gene derived from a human pathogen; the resultant plant tissues will accumulate an antigenic protein encoded by the foreign DNA. In pre-clinical trials, we found that antigenic proteins produced in transgenic plants retained immunogenic properties when purified; if injected into mice the antigen caused production of protein-specific antibodies. Moreover, in some experiments, if the plant tissues were simply fed to mice, a mucosal immune response occurred. The present study was conducted as a proof of principle to determine if humans would also develop a serum and/or mucosal immune response to an antigen delivered in an uncooked foodstuff.