Plant-derived antigens as mucosal vaccines.

During the last two decades, researchers have developed robust systems for recombinant subunit vaccine production in plants. Stably and transiently transformed plants have particular advantages that enable immunization of humans and animals via mucosal delivery. The initial goal to immunize orally by ingestion of plant-derived antigens has proven difficult to attain, although many studies have demonstrated antibody production in both humans and animals, and in a few cases, protection against pathogen challenge. Substantial hurdles for this strategy are low-antigen content in crudely processed plant material and limited antigen stability in the gut. An alternative is intranasal delivery of purified plant-derived antigens expressed with robust viral vectors, especially virus-like particles. The use of pattern recognition receptor agonists as adjuvants for mucosal delivery of plant-derived antigens can substantially enhance serum and mucosal antibody responses. In this chapter, we briefly review the methods for recombinant protein expression in plants, and describe progress with human and animal vaccines that use mucosal delivery routes. We do not attempt to compile a comprehensive list, but focus on studies that progressed to clinical trials or those that showed strong indications of efficacy in animals. Finally, we discuss some regulatory concerns regarding plant-based vaccines.

Plant-derived recombinant F1, V, and F1-V fusion antigens of Yersinia pestis activate human cells of the innate and adaptive immune system.

Plague is still endemic in different regions of the world. Current vaccines raise concern for their side effects and limited protection, highlighting the need for an efficacious and rapidly producible vaccine. F1 and V antigens of Yersinia pestis, and F1-V fusion protein produced in Nicotiana benthamiana administered to guinea pigs resulted in immunity and protection against an aerosol challenge of virulent Y. pestis. We examined the effects of plant-derived F1, V, and F1-V on human cells of the innate immunity. F1, V, and F1-V proteins engaged TLR2 signalling and activated IL-6 and CXCL-8 production by monocytes, without affecting the expression of TNF-alpha, IL-12, IL-10, IL-1beta, and CXCL10. Native F1 antigen and recombinant plant-derived F1 (rF1) and rF1-V all induced similar specific T-cell responses, as shown by their recognition by T-cells from subjects who recovered from Y. pestis infection. Native F1 and rF1 were equally well recognized by serum antibodies of Y. pestis-primed donors, whereas serological reactivity to rF1-V hybrid was lower, and that to rV was virtually absent. In conclusion, plant-derived F1, V, and F1-V antigens are weakly reactogenic for human monocytes and elicit cell-mediated and humoral responses similar to those raised by Y. pestis infection.

Selective modulation of ligand-gated P2X purinoceptor channels by acute hypoxia is mediated by reactive oxygen species.

Purinergic excitatory synapses use ATP to mediate fast synaptic transmission via activation of P2X receptor cation channels, and this response can be altered by acute hypoxia. This study examined the effect of acute hypoxia on cloned homo- and heteromeric P2X2 and P2X3 receptors expressed in human embryonic kidney 293 cells. In cells expressing homomeric P2X2 receptors, perfusion of 5 microM ATP (EC25) induced an inward whole-cell current that showed little desensitization during repeated exposures under continuously normoxic conditions. Exposure to a hypoxic ATP solution (pO2, 25-40 mm Hg) significantly reduced the whole-cell current to 49% of normoxic control. This hypoxic inhibition of P2X2-mediated inward current was maintained across all potentials when a voltage-step protocol was applied. In contrast, currents mediated by homomeric P2X3 receptors or heteromeric P2X(2/3) receptors were insensitive to an acute hypoxic challenge. One mechanism whereby hypoxia may modulate P2X2 channels is via the production of reactive oxygen species (ROS). H2O2 (1.8 mM) reversibly reduced homomeric P2X2 whole-cell currents to 38% of control. Furthermore, H2O2 attenuated the effect of hypoxia on homomeric P2X2 whole-cell currents. Inhibitors of the mitochondrial electron transport chain that reduce (rotenone and myxothiazol) or increase (antimycin A) the production of ROS altered the magnitude of P2X2-mediated currents. In summary, this is the first report indicating that acute hypoxia is able to regulate the activity of any ligand-gated ion channel. Furthermore, our data show that acute hypoxia selectively modulates the P2X2 receptor and that the response of P2X2 receptor subunits to hypoxia is mediated through the mitochondrial production of ROS.

Expression of the B subunit of Escherichia coli heat-labile enterotoxin as a fusion protein in transgenic tomato.

Epitopes often require co-delivery with an adjuvant or targeting protein to enable recognition by the immune system. This paper reports the ability of transgenic tomato plants to express a fusion protein consisting of the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) and an immunocontraceptive epitope. The fusion protein was found to assemble into pentamers, as evidenced by its ability to bind to gangliosides, and had an average expression level of 37.8 microg g(-1) in freeze-dried transgenic tissues. Processing of selected transgenic fruit resulted in a 16-fold increase in concentration of the antigen with minimal loss in detectable antigen. The species-specific nature of this epitope was shown by the inability of antibodies raised against non-target species to detect the LTB fusion protein. The immunocontraceptive ability of this vaccine will be tested in future pilot mice studies.

Modulation of Kv1.5 currents by protein kinase A, tyrosine kinase, and protein tyrosine phosphatase requires an intact cytoskeleton.

The regulation of cardiac delayed rectifier potassium (Kv) currents by cAMP-dependent protein kinase (PKA) contributes to the control of blood pressure and heart rate. We investigated the modulation by PKA and protein phosphatases of cloned Kv1.5 channels expressed in Xenopus laevis oocytes. Exposure of oocytes to activators of PKA (100 nM forskolin, 1 mM 8-bromo-cAMP, or 1 mM 3-isobutyl-1-methylxanthine) had no effect on the amplitude of Kv1.5 currents. Inhibition of PKA by injection of protein kinase A inhibitor peptide or exposure to myristoylated protein kinase A inhibitor peptide (M-PKI; 100 nM) reduced currents mediated by Kv1.5. M-PKI also reduced the amplitude of currents mediated by mutated Kv1.5 channels in which the COOH terminal PKA phosphorylation sites and PSD-95, Disc-large, and ZO-1-binding domain were removed. The reduction of Kv1.5 currents by M-PKI was attenuated by inhibition of actin polymerization by 1 microM cytochalasins B and D, but was not affected by 10 microM phalloidin (stabilizes actin filaments) or 50 microM colchicine (disrupts microtubules). Treatment of oocytes with antisense oligonucleotides against alpha-actinin-2 abolished the reduction in Kv1.5 current by M-PKI. These observations suggest that Kv1.5 currents are activated by endogenous PKA in "resting" oocytes and that inhibition of PKA activity reveals the action of endogenous phosphatases. Indeed, injection of alkaline phosphatase reduced currents mediated by Kv1.5. Further preincubation of oocytes with 1 mM sodium orthovanadate (a protein tyrosine phosphatase inhibitor) abolished the reduction in Kv1.5 currents by M-PKI. We conclude that currents encoded by Kv1.5 are regulated by PKA and protein tyrosine phosphatase and that this regulation requires an intact actin cytoskeleton and alpha-actinin-2.

Expression of recombinant human acetylcholinesterase in transgenic tomato plants.

Enzyme therapy for the prevention and treatment of organophosphate poisoning depends on the availability of large amounts of cholinesterases. Transgenic plants are being evaluated for their efficiency and cost-effectiveness as a system for the bioproduction of therapeutically valuable proteins. Here we report production of a recombinant isoform of human acetylcholinesterase in transgenic tomato plants. Active and stable acetylcholinesterase, which retains the kinetic characteristics of the human enzyme, accumulated in tomato plants. High levels of specific activity were registered in leaves (up to 25 nmol min(-1) mg protein(-1)) and fruits (up to 250 nmol min(-1) mg protein(-1)).

Oral immunization with hepatitis B surface antigen expressed in transgenic plants.

Oral immunogenicity of recombinant hepatitis B surface antigen (HBsAg) derived from yeast (purified product) or in transgenic potatoes (uncooked unprocessed sample) was compared. An oral adjuvant, cholera toxin, was used to increase immune responses. Transgenic plant material containing HBsAg was the superior means of both inducing a primary immune response and priming the mice to respond to a subsequent parenteral injection of HBsAg. Electron microscopy of transgenic plant samples revealed evidence that the HBsAg accumulated intracellularly; we conclude that natural bioencapsulation of the antigen may provide protection from degradation in the digestive tract until plant cell degradation occurs near an immune effector site in the gut. The correlate of protection from hepatitis B virus infection is serum antibody titers induced by vaccination; the protective level in humans is 10 milliunits/ml or greater. Mice fed HBsAg-transgenic potatoes produced HBsAg-specific serum antibodies that exceeded the protective level and, on parenteral boosting, generated a strong long-lasting secondary antibody response. We have also shown the effectiveness of oral delivery by using a parenteral prime-oral boost immunization schedule. The demonstrated success of oral immunization for hepatitis B virus with an "edible vaccine" provides a strategy for contributing a means to achieve global immunization for hepatitis B prevention and eradication.

Functional and molecular expression of a voltage-dependent K(+) channel (Kv1.1) in interstitial cells of Cajal.

1. Located within the gastrointestinal (GI) musculature are networks of cells known as interstitial cells of Cajal (ICC). ICC are associated with several functions including pacemaker activity that generates electrical slow waves and neurotransmission regulating GI motility. In this study we identified a voltage-dependent K(+) channel (Kv1.1) expressed in ICC and neurons but not in smooth muscle cells. 2. Transcriptional analyses demonstrated that Kv1.1 was expressed in whole tissue but not in isolated smooth muscle cells. Immunohistochemical co-localization of Kv1.1 with c-kit (a specific marker for ICC) and vimentin (a specific marker of neurons and ICC) indicated that Kv1.1-like immunoreactivity (Kv1.1-LI) was present in ICC and neurons of GI tissues of the dog, guinea-pig and mouse. Kv1.1-LI was not observed in smooth muscle cells of the circular and longitudinal muscle layers. 3. Kv1.1 was cloned from a canine colonic cDNA library and expressed in Xenopus oocytes. Pharmacological investigation of the electrophysiological properties of Kv1.1 demonstrated that the mamba snake toxin dendrotoxin-K (DTX-K) blocked the Kv1.1 outward current when expressed as a homotetrameric complex (EC(50) = 0.34 nM). Other Kv channels were insensitive to DTX-K. When Kv1.1 was expressed as a heterotetrameric complex with Kv1.5, block by DTX-K dominated, indicating that one or more subunits of Kv1.1 rendered the heterotetrameric channel sensitive to DTX-K. 4. In patch-clamp experiments on cultured murine fundus ICC, DTX-K blocked a component of the delayed rectifier outward current. The remaining, DTX-insensitive current (i.e. current in the presence of 10(-8) M DTX-K) was outwardly rectifying, rapidly activating, non-inactivating during 500 ms step depolarizations, and could be blocked by both tetraethylammonium (TEA) and 4-aminopyridine (4-AP). 5. In conclusion, Kv1.1 is expressed by ICC of several species. DTX-K is a specific blocker of Kv1.1 and heterotetrameric channels containing Kv1.1. This information is useful as a means of identifying ICC and in studies of the role of delayed rectifier K(+) currents in ICC functions.

Novel voltage-dependent non-selective cation conductance in murine colonic myocytes.

1. Two components of voltage-gated, inward currents were observed from murine colonic myocytes. One component had properties of L-type Ca(2+) currents and was inhibited by nicardipine (5 x 10(-7) M). A second component did not 'run down' during dialysis and was resistant to nicardipine (up to 10(-6) M). The nicardipine-insensitive current was activated by small depolarizations above the holding potential and reversed near 0 mV. 2. This low-voltage-activated current (I(LVA)) was resolved with step depolarizations positive to -60 mV, and the current rapidly inactivated upon sustained depolarization. The voltage of half-inactivation was -65 mV. Inactivation and activation time constants at -45 mV were 86 and 15 ms, respectively. The half-recovery time from inactivation was 98 ms at -45 mV. I(LVA) peaked at -40 mV and the current reversed at 0 mV. 3. I(LVA) was inhibited by Ni(2+) (IC(50) = 1.4 x 10(-5) M), mibefradil (10(-6) to 10(-5) M), and extracellular Ba(2+). Replacement of extracellular Na(+) with N-methyl-D-glucamine inhibited I(LVA) and shifted the reversal potential to -7 mV. Increasing extracellular Ca(2+) (5 x 10(-3) M) increased the amplitude of I(LVA) and shifted the reversal potential to +22 mV. I(LVA) was also blocked by extracellular Cs(+) (10(-4) M) and Gd(3+) (10(-6) M). 4. Warming increased the rates of activation and deactivation without affecting the amplitude of the peak current. 5. We conclude that the second component of voltage-dependent inward current in murine colonic myocytes is not a 'T-type' Ca(2+) current but rather a novel, voltage-gated non-selective cation current. Activation of this current could be important in the recovery of membrane potential following inhibitory junction potentials in gastrointestinal smooth muscle or in mediating responses to agonists.

Regulation of BK(Ca) channels expressed in human embryonic kidney 293 cells by epoxyeicosatrienoic acid.

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites of cytochrome P450 monooxygenase, which are released from endothelial cells and dilate arteries. Dilation seems to be caused by activation of large-conductance Ca2+ activated K+ channels (BK(Ca)) leading to membrane hyperpolarization. Previous studies suggest that EETs activate BK(Ca) channels via ADP-ribosylation of the G protein Galphas with a subsequent membrane-delimited action on the channel [Circ Res 78:415-423, 1996; 80:877-884, 1997; 85:349-356, 1999]. The present study examined whether this pathway is present in human embryonic kidney (HEK) 293 cells when the BK(Ca) alpha-subunit (cslo-alpha) is expressed without the beta-subunit. 11,12-EET increased outward K+ current in whole-cell recordings of HEK293 cells. In cell-attached patches, 11,12-EET also increased the activity of cslo-alpha channels without affecting unitary conductance. This action was mimicked by cholera toxin. The ADP-ribosyltransferase inhibitors 3-aminobenzamide and m-iodobenxylguanidine blocked the stimulatory effect of 11,12-EET. In inside-out patches 11,12-EET was without effect on channel activity unless GTP was included in the bathing solution. GTP and GTPgammaS alone also activated cslo-alpha channels. Dialysis of cells with anti-Galphas antibody completely blocked the activation of cslo-alpha channels by 11,12-EET, whereas anti-Galphai/o and anti-Gbetagamma antibodies were without effect. The protein kinase A inhibitor KT5720 and the adenylate cyclase inhibitor SQ22536 did not reduce the stimulatory effect of 11,12-EET on cslo-alpha channels in cell-attached patches. These data suggest that EET leads to Galphas-dependent activation of the cslo-alpha subunits expressed in HEK293 cells and that the cslo-beta subunit is not required.

Production of hepatitis B surface antigen in transgenic plants for 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.

Human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes.

A new approach for delivering vaccine antigens is the use of inexpensive, plentiful, plant-based oral vaccines. Norwalk virus capsid protein (NVCP), assembled into virus-like particles, was used as a test antigen, to determine whether immune responses could be generated in volunteers who ingested transgenic potatoes. Twenty-four healthy adult volunteers received 2 or 3 doses of transgenic potato (n=20) or 3 doses of wild-type potato (n=4). Each dose consisted of 150 g of raw, peeled, diced potato that contained 215-751 microgram of NVCP. Nineteen (95%) of 20 volunteers who ingested transgenic potatoes developed significant increases in the numbers of specific IgA antibody-secreting cells. Four (20%) of 20 volunteers developed specific serum IgG, and 6 (30%) of 20 volunteers developed specific stool IgA. Overall, 19 of 20 volunteers developed an immune response of some kind, although the level of serum antibody increases was modest.

Process options in hepatitis B surface antigen extraction from transgenic potato.

The process conditions for recombinant hepatitis B surface antigen (HBsAg) extraction from transgenic potato were examined. The effects of temperature, the reducing agent beta-mercaptoethanol (BME), and proteinase inhibitors on the level of antigenic activity of recovered HBsAg were determined. Sedimentation profiles were performed to characterize HBsAg assembly into virus-like particles. Increasing the temperature of the sample for about 1 min increased the measured HBsAg antigenic activity. The optimum temperature was around 50 degrees C. A 3-fold enhancement of the antigenic activity was obtained in extract from transgenic potato expressing HBsAg, when monoclonal antibodies were used to assay for HBsAg. When antigenic activity was determined by polyclonal antibodies, no enhancement in the antigenic activity was obtained. Temperature may affect the conformation of the a epitope to which the monoclonal antibodies bind or alter the fluidity of surface lipid regions. BME increased the antigenic activity of HBsAg up to 4-fold when monoclonal antibodies directed against the a determinant were used, but there was no increase with polyclonal antibodies. This observation suggests that BME affects the structure or presentation of the a epitope. In the presence of BME and leupeptin, a proteinase inhibitor, higher antigenic activity was obtained. Leupeptin might protect the antigen, which might become more susceptible to proteolytic degradation after reduction, as a result of stimulation of sulfhydryl proteases. Although both temperature and BME increased the antigenic activity of HBsAg individually, when combined their interaction was antagonistic, resulting in reduced antigenic activity. Different proteinase inhibitors, including leupeptin, aprotinin, E-64, pefabloc, and pepstatin, had no significant effect on HBsAg from potato extract in a 2 h period in the absence of BME. The sedimentation profile of potato-produced HBsAg was determined in 5-30% sucrose gradients. Yeast-derived recombinant HBsAg was used as a positive control. The HBsAg from transgenic potato showed sedimentation and density properties that are very similar to the yeast-produced antigen, indicating assembly into virus-like particles. BME treatment did not change the sedimentation profile.

Kir3.1/3.2 encodes an I(KACh)-like current in gastrointestinal myocytes.

Expression of the Kir3 channel subfamily in gastrointestinal (GI) myocytes was investigated. Members of this K(+) channel subfamily encode G protein-gated inwardly rectifying K(+) channels (I(KACh)) in other tissues, including the heart and brain. In the GI tract, I(KACh) could act as a negative feedback mechanism to temper the muscarinic response mediated primarily through activation of nonselective cation currents and inhibition of delayed-rectifier conductance. Kir3 channel subfamily isoforms expressed in GI myocytes were determined by performing RT-PCR on RNA isolated from canine colon, ileum, duodenum, and jejunum circular myocytes. Qualitative PCR demonstrated the presence of Kir3.1 and Kir3.2 transcripts in all smooth muscle cell preparations examined. Transcripts for Kir3.3 and Kir3.4 were not detected in the same preparations. Semiquantitative PCR showed similar transcriptional levels of Kir3.1 and Kir3.2 relative to beta-actin expression in the various GI preparations. Full-length cDNAs for Kir3.1 and Kir3.2 were cloned from murine colonic smooth muscle RNA and coexpressed in Xenopus oocytes with human muscarinic type 2 receptor. Superfusion of oocytes with ACh (10 microM) reversibly activated a Ba(2+)-sensitive and inwardly rectifying K(+) current. Immunohistochemistry using Kir3.1- and Kir3.2-specific antibodies demonstrated channel expression in circular and longitudinal smooth muscle cells. We conclude that an I(KACh) current is expressed in GI myocytes encoded by Kir3.1/3.2 heterotetramers.

Laparoscopic drainage of giant lymphocele after renal transplantation.

Lymphocele is a relatively frequent complication of kidney transplantation. A 46-year-old man presented 2 years after kidney transplantation with a giant septated lymphocele. The patient underwent successful laparoscopic drainage of the collection and was discharged home on the day of the procedure. Laparoscopic drainage is a safe and effective treatment for complex lymphocele after kidney transplantation.

Cyclic GMP-dependent protein kinase activates cloned BKCa channels expressed in mammalian cells by direct phosphorylation at serine 1072.

NO-induced activation of cGMP-dependent protein kinase (PKG) increases the open probability of large conductance Ca2+-activated K+ channels and results in smooth muscle relaxation. However, the molecular mechanism of channel regulation by the NO-PKG pathway has not been determined on cloned channels. The present study was designed to clarify PKG-mediated modulation of channels at the molecular level. The cDNA encoding the alpha-subunit of the large conductance Ca2+-activated K+ channel, cslo-alpha, was expressed in HEK293 cells. Whole cell and single channel characteristics of cslo-alpha exhibited functional features of native large conductance Ca2+-activated K+ channels in smooth muscle cells. The NO-donor sodium nitroprusside increased outward current 2.3-fold in whole cell recordings. In cell-attached patches, sodium nitroprusside increased the channel open probability (NPo) of cslo-alpha channels 3.3-fold without affecting unitary conductance. The stimulatory effect of sodium nitroprusside was inhibited by the PKG-inhibitor KT5823. Direct application of PKG-Ialpha to the cytosolic surface of inside-out patches increased NPo 3.2-fold only in the presence of ATP and cGMP without affecting unitary conductance. A point mutation of cslo-alpha in which Ser-1072 (the only optimal consensus sequence for PKG phosphorylation) was replaced by Ala abolished the PKG effect on NPo in inside-out patches and the effect of SNP in cell attached patches. These results indicate that PKG activates cslo-alpha by direct phosphorylation at serine 1072.

Angiotensin II type 1 receptor-mediated inhibition of K+ channel subunit kv2.2 in brain stem and hypothalamic neurons.

Angiotensin II (Ang II) has powerful modulatory actions on cardiovascular function that are mediated by specific receptors located on neurons within the hypothalamus and brain stem. Incubation of neuronal cocultures of rat hypothalamus and brain stem with Ang II elicits an Ang II type 1 (AT1) receptor-mediated inhibition of total outward K+ current that contributes to an increase in neuronal firing rate. However, the exact K+ conductance(s) that is inhibited by Ang II are not established. Pharmacological manipulation of total neuronal outward K+ current revealed a component of K+ current sensitive to quinine, tetraethylammonium, and 4-aminopyridine, with IC50 values of 21.7 micromol/L, 1.49 mmol/L, and 890 micromol/L, respectively, and insensitive to alpha-dendrotoxin (100 to 500 nmol/L), charybdotoxin (100 to 500 nmol/L), and mast cell degranulating peptide (1 micromol/L). Collectively, these data suggest the presence of Kv2.2 and Kv3.1b. Biophysical examination of the quinine-sensitive neuronal K+ current demonstrated a macroscopic conductance with similar biophysical properties to those of Kv2.2 and Kv3.1b. Ang II (100 nmol/L), in the presence of the AT2 receptor blocker PD123,319, elicited an inhibition of neuronal K+ current that was abolished by quinine (50 micromol/L). Reverse transcriptase-polymerase chain reaction analysis confirmed the presence of Kv2.2 and Kv3.1b mRNA in these neurons. However, Western blot analyses demonstrated that only Kv2.2 protein was present. Coexpression of Kv2.2 and the AT1 receptor in Xenopus oocytes demonstrated an Ang II-induced inhibition of Kv2.2 current. Therefore, these data suggest that inhibition of Kv2.2 contributes to the AT1 receptor-mediated reduction of neuronal K+ current and subsequently to the modulation of cardiovascular function.

A review of oral vaccination with transgenic vegetables.

Mucosal immunization of the gastrointestinal tract is an effective way to stimulate local and systemic immune responses. Oral vaccines must be formulated in such a way that antigens are protected as they pass through the adverse environment of the stomach and are delivered to the mucosal inductive sites. Vaccine antigens cloned into edible transgenic plants are a promising new delivery system for oral vaccines. Such vaccines could be safe, inexpensive, and multicomponent.

Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene.

The authors have designed and constructed a plant-optimize synthetic gene encoding the Escherichia coli heat-labile enterotoxin B subunit (LT-B), for use in transgenic plants as an edible vaccine against enterotoxigenic E. coli. Expression of the synthetic LT-B gene in potato plants under the control of a constitutive promoter yielded increased accumulation of LT-B in leaves and tubers, as compared to the bacterial LT-B gene. The plant-derived LT-B assembled into native pentameric structures as evidenced by its ability to bind ganglioside. The authors demonstrated immunogenicity by feeding mice the raw tubers and comparing the anti-LT-B serum IgG and faecal IgA to that produced in mice gavaged with bacterial LT-B. Mice were fed three weekly doses of 5 g tuber tissue containing either 20 or 50 micrograms LT-B, or gavaged weekly with 5 micrograms of LT-B from recombinant E. coli. One week after the third dose, mice immunized with potato LT-B had higher levels of serum and mucosal anti-LT-B than those gavaged with bacterial LT-B. Mice were challenged by oral administration of 25 micrograms LT, and protection assessed by comparing the gut/carcass mass ratios. Although none of the mice were completely protected, the higher dose potato vaccine compared favourably with the bacterial vaccine. These findings show that an edible vaccine against E. coli LT-B is feasible.

Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato.

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.