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.

Production of a fusion protein consisting of the enterotoxigenic Escherichia coli heat-labile toxin B subunit and a tuberculosis antigen in Arabidopsis thaliana.

Transgenic plants are potentially safe and inexpensive vehicles to produce and mucosally deliver protective antigens. However, the application of this technology is limited by the poor response of the immune system to non-particulate, subunit vaccines. Co-delivery of therapeutic proteins with carrier proteins could increase the effectiveness of the antigen. This paper reports the ability of transgenic Arabidopsis thaliana plants to produce a fusion protein consisting of the B subunit of the Escherichia coli heat-labile enterotoxin and a 6 kDa tuberculosis antigen, the early secretory antigenic target ESAT-6. Both components of the fusion protein were detected using GM1-ganglioside-dependent enzyme-linked immunosorbant assay. This suggested the fusion protein retained both its native antigenicity and the ability to form pentamers.

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.

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)).

Avicins, a family of triterpenoid saponins from Acacia victoriae (Bentham), suppress H-ras mutations and aneuploidy in a murine skin carcinogenesis model.

We tested the ability of avicins, a family of triterpenoid saponins obtained from Acacia victoriae (Bentham) (Leguminosae: Mimosoideae), to inhibit chemically induced mouse skin carcinogenesis. Varying doses of avicins were applied to shaved dorsal skin of SENCAR mice 15 min before application of 100 nmol of 7,12-dimethylbenz[a]anthracene (DMBA) twice a week for 4 weeks (complete carcinogenesis model). The dorsal skin of a second group of mice was treated with one dose of 10 nmol of DMBA. Avicins were then applied 15 min before repetitive doses of 2 microg of phorbol 12-tetradecanoate 13-acetate (TPA) twice a week for 8 weeks (initiation/promotion model). At 12 weeks, avicins produced a 70% decrease in the number of mice with papillomas and a greater than 90% reduction in the number of papillomas per mouse in both protocols. We also observed a 62% and 74% reduction by avicins in H-ras mutations at codon 61 in the DMBA and DMBA/TPA models, respectively, as well as a significant inhibition of the modified DNA base formation (8-OH-dG) in both protocols. Marked suppression of aneuploidy occurred with treatment at 16 weeks in the initiation/promotion experiment. These findings, when combined with the proapoptotic property of these compounds and their ability to inhibit hydrogen peroxide (H(2)O(2)) generation, nuclear factor-kappaB (NF-kappaB) activation, and inducible nitric oxide synthase (iNOS) induction reported elsewhere, suggest that avicins could prove exciting in reducing oxidative and nitrosative stress and thereby suppressing the development of human skin cancer and other epithelial malignancies.

Avicins, a family of triterpenoid saponins from Acacia victoriae (Bentham), inhibit activation of nuclear factor-kappaB by inhibiting both its nuclear localization and ability to bind DNA.

Triterpenoid saponins, which are present in leguminous plants and some marine animals, possess a broad range of biological actions. We have earlier reported the extraction of avicins, a family of triterpenoid saponins obtained from the Australian desert tree Acacia victoriae (Leguminosae: Mimosoideae) that inhibit tumor cell growth and induce apoptosis, in part, by perturbing mitochondrial function. These saponins have also been found to prevent chemical-induced carcinogenesis in mice. This study examines the effect of a triterpene mixture (F094) and a single molecular species (avicin G) isolated from the mixture on tumor necrosis factor (TNF)-induced activation of nuclear transcription factor-kappaB (NF-kappaB) in Jurkat cells (human T cell leukemia). Both F094 and avicin G were found to be potent inhibitors of TNF-induced NF-kappaB. Treatment of Jurkat cells with avicin G resulted in a much slower accumulation of the p65 subunit of NF-kappaB into the nucleus whereas the degradation of IkappaBalpha was unaffected. Avicin G also impaired the binding of NF-kappaB to DNA in in vitro binding assays. Treatment of cells with DTT totally reversed the avicin G-induced inhibition of NF-kappaB activity, suggesting that sulfhydryl groups critical for NF-kappaB activation were being affected. Avicin G treatment resulted in decreased expression of NF-kappaB-regulated proteins such as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2). Thus, the avicins may prove important for reducing both oxidative and nitrosative cellular stress and thereby suppressing the development of malignancies and related diseases.

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.

Avicins: triterpenoid saponins from Acacia victoriae (Bentham) induce apoptosis by mitochondrial perturbation.

Anticancer agents target various subcellular components and trigger apoptosis in chemosensitive cells. We have recently reported the tumor cell growth inhibitory properties of a mixture of triterpenoid saponins obtained from an Australian desert tree (Leguminosae) Acacia victoriae (Bentham). Here we report the purification of this mixture into two biologically pure components called avicins that contain an acacic acid core with two acyclic monoterpene units connected by a quinovose sugar. We demonstrate that the mixture of triterpenoid saponins and avicins induce apoptosis in the Jurkat human T cell line by affecting the mitochondrial function. Avicin G induced cytochrome c release within 30-120 min in whole cells and within a minute in the cell-free system. Caspase inhibitors DEVD or zVAD-fmk had no effect on cytochrome c release, suggesting the direct action of avicin G on the mitochondria. Activation of caspase-3 and total cleavage of poly(ADP-ribose) polymerase (PARP) occurred between 2 and 6 h posttreatment with avicins by zVAD-fmk. Interestingly, in the treated cells no significant changes in the membrane potential preceded or accompanied cytochrome c release. A small decrease in the generation of reactive oxygen species (ROS) was measured. The study of these evolutionarily ancient compounds may represent an interesting paradigm for the application of chemical ecology and chemical biology to human health.

Agrobacterium -mediated transformation of embryogenic cell suspensions of the banana cultivar Rasthali (AAB).

A protocol was developed for establishing embryogenic suspension cultures from in vitro-grown, thin shoot-tip sections of the banana cultivar Rasthali. The best medium for callus induction was an MS-based medium supplemented with 2 mg/l 2,4-D and 0.2 mg/l zeatin. The callus was transferred to liquid medium to establish embryogenic cell suspensions. These cultures were subsequently used for Agrobacterium-mediated transformation. The Agrobacterium tumefaciens strain EHA105 containing the binary vector pVGSUN with the als gene as a selectable marker and an intron-containing the gusA gene as a reporter gene was used for transformations. The herbicide Glean was used as a selection agent. Two hundred putative transformants were recovered, of which a set of 16 was tested by histochemical analysis for GUS expression and by Southern blot analysis with a probe for the gusA gene. The plants were positive for GUS expression and integration of the gusA gene. Two of the transformants were grown to maturity under greenhouse conditions. Bananas were harvested to test GUS expression by histochemical analysis. The fruit from both transgenics tested positive for GUS expression.

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.

Plants for delivery of edible vaccines.

Over the past decade, scientific advances in molecular biology and immunology have improved understanding of many diseases and led to the development of novel strategies for vaccination. The development of plants expressing vaccine antigens is a particularly promising approach. Plant-derived antigenic proteins have delayed or prevented the onset of disease in animals and have proven to be safe and functional in human clinical trials. Future areas of research should further characterize the induction of the mucosal immune system and appropriate crop species for delivery of animal and human vaccines.

A tool for functional plant genomics: chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations.

Self-complementary chimeric oligonucleotides (COs) composed of DNA and modified RNA residues were evaluated as a means to (i) create stable, site-specific base substitutions in a nuclear gene and (ii) introduce a frameshift in a nuclear transgene in plant cells. To demonstrate the creation of allele-specific mutations in a member of a gene family, COs were designed to target the codon for Pro-196 of SuRA, a tobacco acetolactate synthase (ALS) gene. An amino acid substitution at Pro-196 of ALS confers a herbicide-resistance phenotype that can be used as a selectable marker in plant cells. COs were designed to contain a 25-nt homology domain comprised of a five-deoxyribonucleotide region (harboring a single base mismatch to the native ALS sequence) flanked by regions each composed of 10 ribonucleotides. After recovery of herbicide-resistant tobacco cells on selective medium, DNA sequence analyses identified base conversions in the ALS gene at the codon for Pro-196. To demonstrate a site-specific insertion of a single base into a targeted gene, COs were used to restore expression of an inactive green fluorescent protein transgene that had been designed to contain a single base deletion. Recovery of fluorescent cells confirmed the deletion correction. Our results demonstrate the application of a technology to modify individual genetic loci by catalyzing either a base substitution or a base addition to specific nuclear genes; this approach should have great utility in the area of plant functional genomics.

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.

The abundant 31-kilodalton banana pulp protein is homologous to class-III acidic chitinases.

We have identified and characterized the abundant protein from the pulp of banana fruit (Musa acuminata cv. Grand Nain), and have isolated a cDNA clone encoding this protein. Comparison of the amino terminal sequence of the purified 31 kDa protein (P31) suggests that it is related to plant chitinases. Western analyses utilizing rabbit anti-P31 antiserum demonstrate that this protein is pulp-specific in banana. A full-length cDNA clone homologous to class III acidic chitinase genes has been isolated from a pulp cDNA library by differential screening. The identity of this clone as encoding P31 was verified by comparisons between the amino-terminal peptide sequence and the cDNA sequence and cross-hybridization of the translation product of the cDNA clone with P31 antiserum. Northern and western blot analyses of RNA and protein isolated from banana pulp at different stages of ripening indicate that the cDNA and protein are expressed at high levels in the pulp of unripe fruit, and that their abundance decreases as the fruit ripens. Based on its expression pattern and deduced amino acid sequence and composition, we hypothesize that the physiological role of P31 is not for plant protection, but as a storage protein in banana pulp.

Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice.

Alternatives to cell culture systems for production of recombinant proteins could make very safe vaccines at a lower cost. We have used genetically engineered plants for expression of candidate vaccine antigens with the goal of using the edible plant organs for economical delivery of oral vaccines. Transgenic tobacco and potato plants were created that express the capsid protein of Norwalk virus, a calicivirus that causes epidemic acute gastroenteritis in humans. The capsid protein could be extracted from tobacco leaves in the form of 38-nm Norwalk virus-like particles. Recombinant Norwalk virus-like particle (rNV) was previously recovered when the same gene was expressed in recombinant baculovirus-infected insect cells. The capsid protein expressed in tobacco leaves and potato tubers cosedimented in sucrose gradients with insect cell-derived rNV and appeared identical to insect cell-derived rNV on immunoblots of SDS/polyacrylamide gels. The plant-expressed rNV was orally immunogenic in mice. Extracts of tobacco leaf expressing rNV were given to CD1 mice by gavage, and the treated mice developed both serum IgG and secretory IgA specific for rNV. Furthermore, when potato tubers expressing rNV were fed directly to mice, they developed serum IgG specific for rNV. These results indicate the potential usefulness of plants for production and delivery of edible vaccines. This is an appropriate technology for developing countries where vaccines are urgently needed.