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Novel Approaches to Oral Vaccines: Delivery of Antigens by Edible Plants.

Advances in genetic engineering in the past decade have accelerated the expression, in plants, of foreign proteins with industrial and pharmaceutical value. Antigens from infectious bacterial or viral diseases have been introduced into plants through plant virus-mediated infection or Agrobacterium tumefaciens-mediated stable transformation methods. Oral immunization with transgenic plant tissues that contain vaccine antigen proteins stimulates both systemic and mucosal immune responses in animals. Plant-based vaccines can provide significant levels of protection against challenge by viral or bacterial pathogens.

Journal Article↗

Immunity to potato mop-top virus in Nicotiana benthamiana plants expressing the coat protein gene is effective against fungal inoculation of the virus.

Nicotiana benthamiana stem tissue was transformed with Agrobacterium tumefaciens harboring a binary vector containing the potato mop-top virus (PMTV) coat protein (CP) gene. PMTV CP was expressed in large amounts in some of the primary transformants. The five transgenic lines which produced the most CP were selected for resistance testing. Flowers on transformed plants were allowed to self-fertilize. Transgenic seedlings selected from the T1 seed were mechanically inoculated with two strains of PMTV. Virus multiplication, assayed by infectivity, was detected in only one transgenic plant of 98 inoculated. T1 plants were also highly resistant to graft inoculation; PMTV multiplied in only one plant of 45 inoculated. Transgenic T1 seedlings were challenged in a bait test in which they were grown in soil containing viruliferous spores of the vector fungus Spongospora subterranea. In these tests only two plants out of 99 became infected. Of the five transgenic lines tested, plants of three lines were immune to infection following manual, graft, or fungal inoculation.

Base Sequence↗

Protection against tetanus toxin using a plant-based vaccine.

Plant-expressed vaccines may provide a unique opportunity for generating anti-pathogen immunity, especially in countries where cold storage is lacking. In the following study, we show that soluble protein from tobacco leaves expressing fragment C of tetanus toxin protected mice against a lethal tetanus toxin challenge. More importantly, we show that a single intranasal (i.n.) vaccination was as efficient as oral delivery, inducing high levels of activated CD4(+) T cells and anti-toxin antibody. Unlike the oral route, i.n. delivery did not require the presence of adjuvant (cholera toxin). Indeed, addition of cholera toxin induced bystander immune responses to plant proteins as well. This is the first study documenting protective immunity by a single i.n. dose of plant vaccine. Plant-based vaccines are promising because they are more heat stable, are easy to produce, cheap and do not require needles.

Animals↗

Protective immune response to foot-and-mouth disease virus with VP1 expressed in transgenic plants.

It has been reported recently that genes encoding antigens of bacterial and viral pathogens can be expressed in plants in a form in which they retain native immunogenic properties. The structural protein VP1 of foot-and-mouth disease virus (FMDV), which has frequently been shown to contain critical epitopes, has been expressed in different vectors and shown to induce virus-neutralizing antibodies and protection in experimental and natural hosts. Here we report the production of transformed plants (Arabidopsis thaliana) expressing VP1. Mice immunized with leaf plant extracts elicited specific antibody responses to synthetic peptides representing amino acid residues 135 to 160 of VP1, to VP1 itself, and to intact FMDV particles. Additionally, all of the immunized mice were protected against challenge with virulent FMDV. To our knowledge, this is the first study showing protection against a viral disease by immunization with an antigen expressed in a transgenic plant.

Animals↗

Natural variation of immune epitopes reveals intrabacterial antagonism.

Plants and animals detect biomolecules termed microbe-associated molecular patterns (MAMPs) and induce immunity. Agricultural production is severely impacted by pathogens which can be controlled by transferring immune receptors. However, most studies use a single MAMP epitope and the impact of diverse multicopy MAMPs on immune induction is unknown. Here, we characterized the epitope landscape from five proteinaceous MAMPs across 4,228 plant-associated bacterial genomes. Despite the diversity sampled, natural variation was constrained and experimentally testable. Immune perception in both Arabidopsis and tomato depended on both epitope sequence and copy number variation. For example, Elongation Factor Tu is predominantly single copy, and 92% of its epitopes are immunogenic. Conversely, 99.9% of bacterial genomes contain multiple cold shock proteins, and 46% carry a nonimmunogenic form. We uncovered a mechanism for immune evasion, intrabacterial antagonism, where a nonimmunogenic cold shock protein blocks perception of immunogenic forms encoded in the same genome. These data will lay the foundation for immune receptor deployment and engineering based on natural variation.

Epitopes↗

Crude saponins improve the immune response to an oral plant-made measles vaccine.

Millions of people live in areas where infectious diseases, such as measles, are endemic and resources are scarce. Heat-stable vaccines that are delivered orally will greatly enhance vaccination programs in these areas. A stumbling block in the development of oral vaccines is the availability of safe and effective mucosal adjuvants, especially for use with subunit vaccines. The experiments presented here examine the ability of CTB/CT, LT(R192G) and crude Quillaja saponin extracts to stimulate MV-specific immune responses in mice, following oral immunisation with plant-made measles virus hemagglutinin (MV-H) protein. LT(R192G) and crude saponin extracts both functioned as potent mucosal adjuvants when ad-mixed with plant-made MV-H protein, and were more effective than CTB/CT. MV-H protein supplemented with saponin extract induced the strongest MV-specific responses, in the greatest number of mice. Crude saponins are routinely used by the food and beverage industry at concentrations greater than those required for adjuvanticity, and as such, they have a better safety profile than bacterial enterotoxins. This study demonstrates their potential as adjuvants for use with oral plant-made vaccines.

Adjuvants, Immunologic↗

Immunoglobulin G specifically binding plant N-glycans with high affinity could be generated in rabbits but not in mice.

Xylosylated and core alpha1,3-fucosylated N-glycans from plants are immunogenic, and they play a still obscure role in allergy and in the field of plant-made protein pharmaceuticals. We immunized mice to generate monoclonal antibodies (mAbs) binding plant N-glycans specifically via the epitope containing either the xylose or the core alpha1,3-fucose residue. Splenocytes expressing N-glycan-specific antibodies derived from C57BL/6 mice previously immunized with plant glycoproteins were preselected by cell sorting to generate hybridoma lines producing specific antibodies. However, we obtained only mAbs unable to distinguish fucosylated from xylosylated N-glycans and reactive even with the pentasaccharide core Man3GlcNAc2. In contrast, immunization of rabbits yielded polyclonal sera selectively reactive with either fucosylated or xylosylated N-glycans. Purification of these sera using glyco-modified neoglycoproteins coupled to a chromatography matrix provided polyclonal sera suitable for affinity determination. Surface plasmon resonance measurements using sensor chips with immobilized glyco-modified transferrins revealed dissociation constants of around 10(-9) M. This unexpectedly high affinity of IgG antibodies toward carbohydrate epitopes has repercussions on our conception of the binding strength and significance of antiglycan IgE antibodies in allergy.

Animals↗

A comparison of the capacity of six cold-pressed plant oils to support development of acquired immune competence in the weanling mouse: superiority of low-linoleic-acid oils.

The objective of this investigation was to compare, at several levels of intake, the capacity of diverse cold-pressed plant oils to support development of acquired immune competence assessed in vivo in the weanling mouse. Safflower, maize, soyabean, rapeseed, flaxseed and olive oils were selected to represent widely differing 18 : 1n-9, 18 : 2n-6 and 18 : 3n-3 contents, and each oil was fed at three dietary levels (40, 80 and 160 g/kg) as the exclusive source of fat. C57BL/6J mice, ten males and ten females, had free access to each diet for 28 d beginning at 19 d of age. The primary serum haemagglutinin response to sheep red blood cells and the primary cutaneous delayed hypersensitivity response to dinitrochlorobenzene were used to assess humoral and cell-mediated competence respectively, on day 28. A zero-time control group, assessed immunologically at 19 d of age, was also included (n 32). Independently of dietary oil level, flaxseed, rapeseed, olive and soyabean oils supported development of a more vigorous antibody response than safflower (a useful point of reference, being rich in 18 : 2n-6 but low in 18 : 1n-9 and 18 : 3n-3), whereas only flaxseed oil supported development of cell-mediated responsiveness exceeding that of safflower-fed mice. Independently of oil type, development of both immunological indices correlated negatively with intake of 18 : 2n-6, and development of humoral competence varied inversely with dietary oil level. A low content of 18 : 2n-6, perhaps less than 20 g/100 g fatty acids, appears important to the capacity of a plant oil to support development of acquired immune competence in the young.

Analysis of Variance↗

Immunization of chickens with VP2 protein of infectious bursal disease virus expressed in Arabidopsis thaliana.

Transgenic plants represent a safe, effective, and inexpensive way to produce vaccines. The immunogenicity of VP2 protein of an infectious bursal disease (IBD) virus variant E isolate expressed in transgenic Arabidopsis thaliana was compared with a commercial vaccine in specific-pathogen-free broiler chickens. The VP2 coding sequence was isolated and integrated into A. thaliana genome by Agrobacterium tumefaciens-mediated transformation. Soluble VP2 expressed in transgenic plants was used to immunize chickens. Chickens receiving oral immunization with plant-derived VP2 at 1 and 3 wk of age had an antibody response using enzyme-linked immunosorbent assay and 80% protection against challenge infection at 4 wk. Chickens primed with a commercial vaccine at 1 wk followed by an oral booster with VP2 expressed in plants at 3 wk of age showed 90% protection. Chickens immunized with a commercial vaccine at 1 and 3 wk showed 78% protection. Results supported the efficacy of plant-produced VP2 as a vaccine against IBD.

Age Factors↗

Immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants.

Transgenic plants expressing recombinant proteins from pathogenic microorganisms provide an inexpensive edible vaccine for induction of local immunity. Three transgenic plant lines were generated expressing the spike (S) protein of transmissible gastroenteritis virus (TGEV), a protein crucial for establishing mucosal immunity. All three of them were driven by a strong plant promoter. One construct contained the 3.7 kb 5' end of the native S gene sequence. In the second construct part of the S gene, from nucleotide 49 to 1785, was modified for optimal plant recognition and was fused to a plant signal peptide coding sequence. The third construct contained the D epitope-coding region of the S gene, from nucleotide 1201 to 1591, which was fused to the alfalfa beta-amylase gene. The S gene products were detected by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Antigens from all three transgenic plant lines induced TGEV-specific immune responses in pigs as determined by virus neutralization and ELISA, and the resultant antibody titers for all three constructs were similar.

Animals↗

[The characteristics of the immune response of the body in poultry plant workers with extrinsic allergic alveolitis].

There have been studied parameters characterizing cellular and humoral immunity such as T-lymphocytes, subpopulations of theophylline-sensitive (Ths-E-RFL), theophylline-resistant (Thr-E-RFL) lymphocytes, blood serum content of immunoglobulins A, M in the integrated poultry farm workers with exogenous allergic alveolitis (EAA) with regard to their work record. The results obtained pointed to the reduction of the blood serum content of T-lymphocytes, Ths-E-RFL and Thr-E-RFL in the integrated poultry farm workers with service duration ranging from 1 to 20 years, this evidencing inhibition of cellular immunity in EAA. The level of serum immunoglobulins A, M was found to be elevated in workers with a work record of 1-20 years in EAA. In summery, study into the immunologic parameters such as T-lymphocytes, Ths-E-RFL, immunoglobulins A, M levels in the blood serum of the workers with service duration 1-20 years showed reduction in T-lymphocytes, Ths-E-RFL, Thr-E-RFL and rise in immunoglobulins A, M, suggesting inhibition of the cellular link of immunity and stimulation of humoral immunity.

Adult↗

Induction of resistance of wheat plants to pathogens by pretreatment with N-methylated substances.

It has been established that the application of N-methylated compounds as N, N-dimethyl-L-tyrosine and glycine-betaine can induce resistance to biotrophic fungi (Erysiphe graminis, Puccinia recondita) in wheat (Yubileynaya) at two different pretreatment concentrations. This dose-dependent "double immune response" of plants is the basic phenomenon of the biochemical immunization. These N-methylated compounds are potential formaldehyde generators and the formaldehyde formed can take part in the induction of resistance of wheat plants. It seems that the dose-dependent "double immune response" of plants is in correlation with the biotransformation steps of formaldehyde cycle as a fundamental biochemical pathway.

Betaine↗

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.

Animals↗

Gene silencing-based disease resistance.

The definition of a disease is fundamentally difficult, even if one considers only genetically based diseases. In its broadest sense, disease can be defined as any deviation from the norm that results in a physiological disadvantage. Natural selection ensures that the norm for any given species is constantly changing. In addition, some disadvantages are latent and might only manifest under certain environmental conditions. Conversely, an apparent disadvantage can carry a benefit, for example, the disease sickle-cell anemia that is an advantage in malarial areas. Because of the difficulties in giving disease a precise definition, in this review, gene silencing-based disease resistance will be restricted to the description of gene inactivation processes that contribute to maintain the physical fitness of an organism. In this sense, we are concerned with the elimination of invasive nucleic acid expressing. In numerous organisms, a variety of severe diseases are caused by the attack of invasive nucleic acids such as viruses and retroviral or transposable elements. Organisms have developed diverse mechanisms to defend themselves against such attack that include immune responses and apoptosis. Fungi, plants, invertebrates and vertebrates also enlist gene silencing systems to counteract the harmful effects of invasive nucleic acids. In particular, plants that lack interferon and immune responses have established efficient transcriptional and post-transcriptional gene silencing systems. In this review, we describe how plants defend against invasive nucleic acids and focus on the continual evolutionary battle between plants and viruses. In addition, the importance of controlling transposon activity is outlined. Finally, gene silencing-related mechanisms of genomic imprinting and X-chromosome inactivation are discussed in the context of disease resistance.

Animals↗

Theoretical model of the three-dimensional structure of a disease resistance gene homolog encoding resistance protein in Vigna mungo.

Plant disease resistance (R) genes, the key players of innate immunity system in plants encode 'R' proteins. 'R' protein recognizes product of avirulance gene from the pathogen and activate downstream signaling responses leading to disease resistance. No three dimensional (3D) structural information of any 'R' proteins is available as yet. We have reported a 'R' gene homolog, the 'VMYR1', encoding 'R' protein in Vigna mungo. Here, we describe the homology modeling of the 'VMYR1' protein. The model was created by using the 3D structure of an ATP-binding cassette transporter protein from Vibrio cholerae as a template. The strategy for homology modeling was based on the high structural conservation in the superfamily of P-loop containing nucleoside triphosphate hydrolase in which target and template proteins belong. This is the first report of theoretical model structure of any 'R' proteins.

ATP-Binding Cassette Transporters↗

Medicinal and ethnoveterinary remedies of hunters in Trinidad.

BACKGROUND: Ethnomedicines are used by hunters for themselves and their hunting dogs in Trinidad. Plants are used for snakebites, scorpion stings, for injuries and mange of dogs and to facilitate hunting success. RESULTS: Plants used include Piper hispidum, Pithecelobium unguis-cati, Bauhinia excisa, Bauhinia cumanensis, Cecropia peltata, Aframomum melegueta, Aristolochia rugosa, Aristolochia trilobata, Jatropha curcas, Jatropha gossypifolia, Nicotiana tabacum, Vernonia scorpioides, Petiveria alliacea, Renealmia alpinia, Justicia secunda, Phyllanthus urinaria,Phyllanthus niruri,Momordica charantia, Xiphidium caeruleum, Ottonia ovata, Lepianthes peltata, Capsicum frutescens, Costus scaber, Dendropanax arboreus, Siparuma guianensis, Syngonium podophyllum, Monstera dubia, Solanum species, Eclipta prostrata, Spiranthes acaulis, Croton gossypifolius, Barleria lupulina, Cola nitida, Acrocomia ierensis (tentative ID). CONCLUSION: Plant use is based on odour, and plant morphological characteristics and is embedded in a complex cultural context based on indigenous Amerindian beliefs. It is suggested that the medicinal plants exerted a physiological action on the hunter or his dog. Some of the plants mentioned contain chemicals that may explain the ethnomedicinal and ethnoveterinary use. For instance some of the plants influence the immune system or are effective against internal and external parasites. Plant baths may contribute to the health and well being of the hunting dogs.

Animals↗