An interspecific Nicotiana hybrid as a useful and cost-effective platform for production of animal vaccines

The use of transgenic plants to produce novel products has great biotechnological potential as the relatively inexpensive inputs of light, water, and nutrients are utilised in return for potentially valuable bioactive metabolites, diagnostic proteins and vaccines. Extensive research is ongoing in this area internationally with the aim of producing plant-made vaccines of importance for both animals and humans. Vaccine purification is generally regarded as being integral to the preparation of safe and effective vaccines for use in humans. However, the use of crude plant extracts for animal immunisation may enable plant-made vaccines to become a cost-effective and efficacious approach to safely immunise large numbers of farm animals against diseases such as avian influenza. Since the technology associated with genetic transformation and large-scale propagation is very well established in Nicotiana, the genus has attributes well-suited for the production of plant-made vaccines. However the presence of potentially toxic alkaloids in Nicotiana extracts impedes their use as crude vaccine preparations. In the current study we describe a Nicotiana tabacum and N. glauca hybrid that expresses the HA glycoprotein of influenza A in its leaves but does not synthesize alkaloids. We demonstrate that injection with crude leaf extracts from these interspecific hybrid plants is a safe and effective approach for immunising mice. Moreover, this antigen-producing alkaloid-free, transgenic interspecific hybrid is vigorous, with a high capacity for vegetative shoot regeneration after harvesting. These plants are easily propagated by vegetative cuttings and have the added benefit of not producing viable pollen, thus reducing potential problems associated with bio-containment. Hence, these Nicotiana hybrids provide an advantageous production platform for partially purified, plant-made vaccines which may be particularly well suited for use in veterinary immunization programs.     

NMR-based metabonomics: a useful platform of oncology research

Cancer threatens human health, thus research focusing on oncology has great significance. Metabonomics is the global quantitative assessment of the dynamic metabolic response of a biological system to some exogenous or genetic pathophysiological perturbation. The metabolites are detected in tissues or fluids by various analytical methods, such as nuclear magnetic resonance (NMR) and mass spectroscopy. Metabonomics, as a tool, can provide a link between the laboratory and clinic. NMR-based metabonomics offers a useful tool to understand tumour biochemistry and may also has some potentials for tumour diagnosis and prognosis, even when some other disease processes are present. Here, we review NMR-based metabonomics principles and their applications in oncology research.     

Transgenic plants expressing a Clostridium difficile spore antigen as an approach to develop low-cost oral vaccines

Gastrointestinal infections caused by Clostridium difficile lead to significant impact in terms of morbidity and mortality, causing from mild symptoms, such as a low-grade fever, watery stools, and minor abdominal cramping as well as more severe symptoms such as bloody diarrhea, pseudomembrane colitis, and toxic megacolon. Vaccination is a viable approach to fight against C. difficile and several efforts in this direction are ongoing. Plants are promising vaccine biofactories offering low cost, enhanced safety, and allow for the formulation of oral vaccines. Herein, the CdeM protein, which is a spore antigen associated with immunoprotection against C. difficile, was selected to begin the development of plant-based vaccine candidates. The vaccine antigen is based in a fusion protein (LTB-CdeM), carrying the CdeM antigen, fused to the carboxi-terminus of the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) as a mucosal immunogenic carrier. LTB-CdeM was produced in plants using a synthetic optimized gene according codon usage and mRNA stability criteria. The obtained transformed tobacco lines produced the LTB-CdeM antigen in the range of 52–90 μg/g dry weight leaf tissues. The antigenicity of the plant-made LTB-CdeM antigen was evidenced by GM1-ELISA and immunogenicity assessment performed in test mice revealed that the LTB-CdeM antigen is orally immunogenic inducing humoral responses against CdeM epitopes. This report constitutes the first step in the development of plant-based vaccines against C. difficile infection.     

Transgenic plants for the production of veterinary vaccines

The expression of antigens in transgenic plants has been increasingly used in the development of experimental vaccines, particularly oriented to the development of edible vaccines. Hence, this technology becomes highly suitable to express immunogenic proteins from pathogens. Foot and mouth disease virus, bovine rotavirus and bovine viral diarrhoea virus are considered to be the most important causative agents of economic loss of cattle production in Argentina, and they are thus optimal candidates for alternative means of immunization. Here, we present a review of our results corresponding to the expression of immunogenic proteins from these three viruses in alfalfa transgenic plants, and we discuss the possibility of using them for the development of plant-based vaccines.     

转基因番木瓜作为抗结核植物口服疫苗的初步研究

转基因植物疫苗的研制,可改变传统的疫苗接种方式和接种手段,大大降低疫苗的生产成本,具有广阔的应用前景。结核病的泛滥使抗结核的免疫预防急需开发新型的疫苗,研制抗结核的转基因植物疫苗具有重大的理论和实践意义。本文对具有良好免疫原性的结核杆菌分泌蛋白ESAT—6基因,进行了修饰改造,构建了带潮霉素选择抗性基因的植物表达载体和根癌农杆菌工程菌;采用叶盘法转化热带水果番木瓜,获得了13株抗性植株,通过PCR、Southern blot和RNA dot blot分子检测,确认了4株为转基因植株。番木瓜ESAT—6转基因植株的获得,为进一步的结核病植物口服疫苗的免疫研究和新型抗结核疫苗的研制与开发奠定了基础。     

Transgenic papaya plants from Agrobacterium-mediated transformation of somatic embryos

Summary Transgenic papaya (Carica papaya L.) plants were regenerated from embryogenic cultures that were cocultivated with a disarmed C58 strain of Agrobacterium tumefaciens containing one of the following binary cosmid vectors: pGA482GG or pGA482GG/cpPRV-4. The T-DNA region of both binary vectors includes the chimeric genes for neomycin phosphotransferase II (NPTII) and ß-glucuronidase (GUS). In addition, the plant expressible coat protein (cp) gene of papaya ringspot virus (PRV) is flanked by the NPTII and GUS genes in pGA482GG/cpPRV-4. Putative transformed embryogenic papaya tissues were obtained by selection on 150 g·ml^–1 kanamycin. Four putative transgenic plant lines were obtained from the cp gene^– vector and two from the cp gene⁺ vector. GUS and NPTII expression were detected in leaves of all putative transformed plants tested, while PRV coat protein expression was detected in leaves of the PRV cp gene⁺ plant. The transformed status of these papaya plants was analyzed using both polymerase chain reaction amplification and genomic blot hybridization of the NPTII and PRV cp genes. Integration of these genes into the papaya genome was demonstrated by genomic blot hybridizations. Thus, like numerous other dicotyledonous plant species, papayas can be transformed with A. tumefaciens and regenerated into phenotypically normal-appearing plants that express foreign genes.Journal Series no. 3757 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Azido-coronatine: a useful platform for "click chemistry"-mediated probe synthesis for bioorganic studies

We report on the development of azide-coronatine as a useful platform for azide alkyne cycloaddition ("click chemistry")-mediated synthesis of molecular probes. (+)-Azido-coronatine was synthesized in 10 steps with 11% yield using improved synthesis of coronafacic acid, in which the highly exo-selective Diels-Alder reaction (endo:exo > 1:25) is the key step. Azido coronatine was as effective as the original coronatine in a stomatal opening assay, and was easily modified to a fluorescein isothiocyanate (FITC)-labeled probe with high yield.     

Formulation of a microparticle carrier for oral polyplex-based DNA vaccines

Oral induction of a disseminated mucosal immune response with polyplex-based DNA vaccines requires the delivery of intact polyplexes (polyelectrolyte complexes formed by self-assembly of plasmid DNA with a cationic polymer) to subepithelial lymphoid tissue (e.g. Peyer's patches) within the gastrointestinal tract. This work describes the formulation of a microparticle polyplex carrier allowing the potential of this approach to be realised. PEGylated PEI/DNA polyplexes (DNA concentration 20 microg/ml) formed at N/P 5:0 (defined as the ratio of polycation amino groups to DNA phosphates) were stable to salt-induced aggregation and could be concentrated to a final DNA concentration of 1 mg/ml without polyplex size increase. Polyplexes containing 1:1 polyethylene glycol (PEG)/polyethylenimine (PEI) ratio (mass/mass) gave similar levels of luciferase gene expression in B16F10 cells compared to non-PEG complexes. Poly-(D,L-lactide-co-glycolide) (PLGA) microparticles containing PEGylated polyplexes (approximately 17% DNA encapsulation efficiency) were formulated using a modified double emulsion solvent evaporation method. The microencapsulation and release of intact polyplexes from the microparticle carrier was demonstrated using polyanion (heparin sulfate and poly(aspartic acid) (PAA)) displacement techniques and electron microscopy. Microparticles containing PEGylated polyplexes (24 microg beta-galactosidase DNA) were given orally to Wistar rats. Significant transgene expression (compared to background) was found in peripheral tissue (spleen) 72 h after administration. This work demonstrates the potential application of microparticle carriers for mucosal polyplex-based vaccination.     

Preparation of multifunctional glyconanoparticles as a platform for potential carbohydrate-based anticancer vaccines

A novel platform for anticancer vaccines has been prepared using glyconanotechnology recently developed in our laboratory. Ten different multifunctional gold glyconanoparticles incorporating sialylTn and Lewis(y) antigens, T-cell helper peptides (TT) and glucose in well defined average proportions and with differing density have been synthesised in one step and characterised using NMR and TEM. Size and nature of the linker were crucial to control kinetics of S-Au bond formation and to achieve the desired ligand ratio on the gold clusters. The technology presented here opens the way for tailoring polyvalent anticancer vaccines candidates and drug delivery carriers with defined average chemical composition.     

Exploiting cholera vaccines as a versatile antigen delivery platform

The development of safe, immunogenic and protective cholera vaccine candidates makes possible their use as a versatile antigen delivery platform. Foreign antigens can be delivered to the immune system with cholera vaccines by expressing heterologous antigens in live attenuated vectors, as fusion proteins with cholera toxin subunits combined with inactivated Vibrio cholerae whole cells or by exposing them on the surface of V. cholerae ghosts. Progress in our understanding of the genes expressed by V. cholerae during infection creates unprecedented opportunities to develop an improved generation of vaccine vectors to induce immune protection against a broad range of pathogenic organisms.     

Plant-based vaccines: Expression and oral immunogenicity

Summary The use of plants for production and delivery of vaccine proteins has shown promise in research conduced during the past decade. Genes encoding bacterial and viral antigens are faithfully expressed, processed, and assembled in plant cells to form immunogenic proteins. Studies in animals and humans showed that ingestion of transgenic potato containing vaccine proteins caused production of antigen-specific antibodies in serum and mucosal secretions. Future studies must aim to improve antigen expression and to develop processes to formulate plant material for antigen stability and convenient oral delivery. Furthermore, production methods should ensure the containment of pharmaceutical crops and prevent contamination of the food supply.     

An oral cancer biobank initiative: a platform for multidisciplinary research in a developing country

Identification of diagnostic markers for early detection and development of novel and therapeutic agents for effective patient management are the main motivation for cancer research. Biological specimens from large cohort and case-control studies which are crucial in providing successful research outcomes are often the limiting factor that hinders research efforts, especially in developing countries. Therefore, the Malaysian Oral Cancer Database and Tissue Bank System (MOCDTBS) were established to systematically collect large number of samples with comprehensive sociodemographic, clinicopathological, management strategies, quality of life and associated patient follow-up data to facilitate oral cancer research in Malaysia. The MOCDTBS also promotes sharing among researchers and the development of a multidisciplinary research team. The following article aims to describe the process of setting-up and managing the MOCDTBS.     

Pharming vaccines for hepatitis and cytomegalovirus: Towards the development of multivalent and subunit vaccines for oral delivery of antigens

A plant based high fidelity vaccine production system is being developed with emphasis on producing antigens capable of being orally delivered in multivalent or subunit plant packets. Plant-based edible vaccines may provide an attractive, safe and inexpensive alternative to conventional vaccine production. Edible plant tissues are not normally antigenic in nature. However, foreign antigens from common infectious organisms like hepatitis-B virus (HBV) can be produced along with naturally occurring storage proteins in DNA-transformed plants. Upon administration via the oral route, these transgenic plant tissues may mobilize the protective humoral and mucosal immune responses to challenge the natural infectious agent. When tobacco, carrot and rice plants were transformed with the truncated version of the HBV nucleocapsid gene expression construct, non-infective hepatitis B viral core particles were observed via electron microscopy. A second plant codon-optimised HBV expression construct was designed that included the extensin signal sequence for augmented HBV particle accumulation. Upon transformation of tobacco plants with the codon-optimised construct, over 4 times more transgenic plants with high levels of expression of the HBV nucleocapsid protein were generated in comparison with a similar vector containing the unmodified wild-type HBV gene codon sequence. Further analysis via Western blotting confirmed the presence of the viral antigen in the total protein extracts from transgenic tobacco leaves and seeds. Electron microscopy showed that the expressed protein self-assembled into viral-like particles of 25–30 nm in diameter. To develop an edible subunit vaccine in plant seeds, a third plant transformation construct was used for the synthesis of the human cytomegalovirus glycoprotein B (HCMV gB) subunit. The gB protein derived from tobacco seeds retained critical structural features including epitopes for neutralizing antibodies and was targeted to the protein storage vesicles of tobacco seed endosperm. Two different strains of mice were orally immunized with tobacco seeds containing low concentrations of HCMV gB, with varying dosages, but without adjuvant. No anti-gB response was detected in intestinal or serum samples. However, a systemic immune response to normal tobacco seed proteins was observed in both strains of mice. While higher expression levels of antigens in seeds must be achieved, seeds may provide an effective and immunostimulatory vehicle for delivering edible vaccines to the intestinal mucosa. One of the outstanding challenges includes defining optimum conditions of antigen presentation, dosage and immunization schedules that will induce strong mucosal and/or systemic immune responses in heterogeneous populations. Here we review the different strategies being employed to produce specific oral antigens in plant tissues.     

Transgenic mouse models of Alzheimer's disease: how useful have they been for therapeutic development?

Transgenic mice have been created in an attempt to generate models of human Alzheimer's disease, but success has been partial and unpredictable. The overall aim of this paper is to illustrate how genomics can be used in translational research, turning genetic information in the form of pathogenic mutations into clinically useful drugs against a major human disease. This paper will illustrate how genetic information allows researchers to dissect the aetiology of a disease and then replicate the disease in vivo through the process of transgenesis. The limitations of recreating a condition like Alzheimer's disease in a transgenic mouse, how far the mice have advanced understanding of the disease and how useful they have been for the development of therapeutics will then be discussed.     

Characterization of diacetylenic liposomes as carriers for oral vaccines

In order to evaluate liposomes as vehicle for oral vaccines the characterization and stability of polymerized and non-polymerized liposomes were examined. Mixtures of 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3 phosphocholine) (DC8,9PC) with saturated 1,2-dimiristoyl-sn-glycero-3-phosphocholine in molar ratio 1:1 were used. Saturated and non-saturated lipids were combined to give a chemically modified membrane by UV polymerization derived from DC8,9PC. Characterization was carried out by electronic microscopy, differential scanning calorimetry (DSC) and by hydrophobicity factor (HF). The stability towards the digestive tract (including saliva): acidic solutions, bile and pancreatin are compared to buffer pH 7.4, measuring the release of Glucose-6-phosphate or bovine plasma albumin entrapment. The polymerized liposomes showed further augmentation of the HF and the size. DSC showed phase separation and lower Tt if compared to data obtained for DC8,9PC. The HF, as main factor is discussed in relation to in vitro stability, suggesting that polymerized and non-polymerized liposomes would serve effectively as an oral delivery vehicle.     

Transgenic papaya plants from Agrobacterium-mediated transformation of petioles of in vitro propagated multishoots

Summary In order to establish a model system for introduction of foreign genes into papaya (Carica papaya L.) plants by Agrobacterium-mediated transformation, petioles from multishoots were used as explant source and bacterial neomycin phosphotransferase II (NPT II) gene and -glucuronidase (GUS) gene were used as a selection marker and a reporter, respectively. Cross sections of papaya petioles obtained from multishoots micropropagated in vitro were infected with A. tumefaciens LBA4404 containing NPTII and GUS genes and co-cultured for 2 d. The putative transformed calluses were identified by growth on the selective medium containing kanamycin and carbenicillin, and consequently regenerated to plants via somatic embryogenesis. Thirteen putative transgenic lines were obtained from a total of 415 petiole fragments treated. Strong GUS activity was detected in the selected putative transgenic calli or plants by fluorogenic assay. Western blot analysis using GUS antiserum confirmed that the GUS protein was expressed in putative transformed papaya cells and transgenic plants. The presence of the GUS gene in the papaya tissues was detected by PCR amplification coupled with Southern blot.     

Newcastle disease virus-like particles as a platform for the development of vaccines for human and agricultural pathogens

Vaccination is the single most effective way to control viral diseases. However, many currently used vaccines have safety concerns, efficacy issues or production problems. For other viral pathogens, classic approaches to vaccine development have, thus far, been unsuccessful. Virus-like particles (VLPs) are increasingly being considered as vaccine candidates because they offer significant advantages over many currently used vaccines or developing vaccine technologies. VLPs formed with structural proteins of Newcastle disease virus, an avian paramyxovirus, are a potential vaccine candidate for Newcastle disease in poultry. More importantly, these VLPs are a novel, uniquely versatile VLP platform for the rapid construction of effective vaccine candidates for many human pathogens, including genetically complex viruses and viruses for which no vaccines currently exist.     

Controlling endemic cholera with oral vaccines

Background

Although advances in rehydration therapy have made cholera a treatable disease with low case-fatality in settings with appropriate medical care, cholera continues to impose considerable mortality in the world''s most impoverished populations. Internationally licensed, killed whole-cell based oral cholera vaccines (OCVs) have been available for over a decade, but have not been used for the control of cholera. Recently, these vaccines were shown to confer significant levels of herd protection, suggesting that the protective potential of these vaccines has been underestimated and that these vaccines may be highly effective in cholera control when deployed in mass immunization programs. We used a large-scale stochastic simulation model to investigate the possibility of controlling endemic cholera with OCVs.

Methods and Findings

We construct a large-scale, stochastic cholera transmission model of Matlab, Bangladesh. We find that cholera transmission could be controlled in endemic areas with 50% coverage with OCVs. At this level of coverage, the model predicts that there would be an 89% (95% confidence interval [CI] 72%–98%) reduction in cholera cases among the unvaccinated, and a 93% (95% CI 82%–99%) reduction overall in the entire population. Even a more modest coverage of 30% would result in a 76% (95% CI 44%–95%) reduction in cholera incidence for the population area covered. For populations that have less natural immunity than the population of Matlab, 70% coverage would probably be necessary for cholera control, i.e., an annual incidence rate of ≤ 1 case per 1,000 people in the population.

Conclusions

Endemic cholera could be reduced to an annual incidence rate of ≤ 1 case per 1,000 people in endemic areas with biennial vaccination with OCVs if coverage could reach 50%–70% depending on the level of prior immunity in the population. These vaccination efforts could be targeted with careful use of ecological data.