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WO2002024220A2 - Procede de production de vaccin et de melange immunogene base sur des proteines de choc thermique (hsp) provenant de cellules de plantes herbacees - Google Patents

Procede de production de vaccin et de melange immunogene base sur des proteines de choc thermique (hsp) provenant de cellules de plantes herbacees Download PDF

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WO2002024220A2
WO2002024220A2 PCT/GE2001/000004 GE0100004W WO0224220A2 WO 2002024220 A2 WO2002024220 A2 WO 2002024220A2 GE 0100004 W GE0100004 W GE 0100004W WO 0224220 A2 WO0224220 A2 WO 0224220A2
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hsps
antigen
origin
cells
derived
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PCT/GE2001/000004
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WO2002024220A3 (fr
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Tengiz Jaliashvili
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Tengiz Jaliashvili
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Priority to AU2001295814A priority Critical patent/AU2001295814A1/en
Priority to EP01976546A priority patent/EP1326637A2/fr
Publication of WO2002024220A2 publication Critical patent/WO2002024220A2/fr
Publication of WO2002024220A3 publication Critical patent/WO2002024220A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides

Definitions

  • the invention relates generally to the biochemistry, more particularly to food/feed industries, fitness and clinical nutrition.
  • HSPs Heat Shock Proteins
  • HSF Heat Shock Factors
  • HSE Heat Shock Elements
  • HSPs scale up synthesis may last up to 9 days, but then declines up to the basal low level and some, e.g. HSP72, may even disappear in plant cells. It was well established that such an increase of HSPs synthesis in cells helps plant tissues to withstand a broad range of environmental stresses, from high heat up to chilling temperatures. It needs to be underlined that, in non-stress condition, heat inducible HSPs are at low levels in plant cells or some even are not detectable (Neumann at al., 1989; Nover 1991; Pollock at al, 1993; Petersen 1990; Singla at al 1997; Vierling 1990-1991; DeRocher at al 1991; Peterson 1990).
  • Non-Immuno genie essential micronutrients such as plant origin HSPs, with high degree of conservation of ID structures (Gupta 1995-98),
  • Immunogenic cocktail of microbial origin antigens with variable ID structures. This way to build up daily in gut the mixture of plant origin HSPs and a cocktail of immunogenic microbial antigens becomes partly observable in nature as the base of natural oral binary vaccines.
  • This discovery has a tremendous practical value for building selective immune natural mixtures as the evident base for the manufacture of health-risk free innovative Functional Processed Food (FPF) to be used as edible efficient vaccines with targeted preventive function.
  • FPF Functional Processed Food
  • This new generation of FPF on the base of the binary mixtures of plant origin HSPs and selected immunogenic antigens may well prevent the invasion of antigen specific pathogens via the remarkably enhanced efficiency of Antigen Presenting Cells (APC) localised in gut - the most powerful preventive system within the first line of immune defence.
  • APC Antigen Presenting Cells
  • Decomposers Level which are heterotrophic bacteria, fungi, etc., based on Consumers Level, which are heterotrophic herbivores, Homo, etc., based on - Producers Level, which are autotrophic herbaceous plants - the universal vital sources of natural macronutrients and essential micronutrients within HSPs for the well being of herbivores and Homo.
  • An immunogenic mixture which is frequently building in gut while ingesting daily norm of fresh plant feed/food contaminated with innumerable microbes, is always represented with a composition of plant origin HSP70 and highly variable microbial origin antigens.
  • HSP70 mostly inducible at 36°C-39°C, have appeared almost identical (Nover 1991; Nuniann at al 1989; Nicchitta 1998);
  • HSP70 possess receptors on the surface of professional Antigen Presenting Cells (APC), which amplify around 10,000-fold the immune cells targeted attack only against microbes or malformed cells specific to antigens which HSP70 display to these type of lymphocytes (Arnold-Schild at al 1999; Pa ⁇ jwani at al 1999);
  • APC Antigen Presenting Cells
  • Bl Macro-nutrients such as: cellulose, lignin, pectins, fat/lipids, DNA/RNA, proteins; B2) Micro-nutrients such as: vitamins, amino acids, nucleotides/nucleosides, flavanoids, alkaloids, antioxidants, bio-minerals, cathechins, oligosacharides, ribose/desoxiribose, lectines, saponins, resins, glycosides, essential oils.
  • Micro-nutrients such as: vitamins, amino acids, nucleotides/nucleosides, flavanoids, alkaloids, antioxidants, bio-minerals, cathechins, oligosacharides, ribose/desoxiribose, lectines, saponins, resins, glycosides, essential oils.
  • CSPs Cold Stress Proteins
  • WSPs Wound Stress Proteins
  • PI Protease Inhibitors
  • DSPs Drought Stress Proteins
  • fresh herbaceous plant cells contain heat stress sensitive genes which dramatically increase HSPs synthesis above 15°C, with an optimum for growth and particularly swiftly in the range of 36°C-39°C (Pollock at al 1993; Singla at al 1997).
  • plant intracellular level of HSPs may increase up to 1% of total proteins at this heat shock range of temperature in gut of herbivores - far before the full digestion of fresh plant feed/food. That was the reason why we have considered that fresh herbaceous plant cells derived HSPs, the intracellular level of which may increase particularly swiftly at gut temperature, are essential micronutrients with chaperone synbiotic function for consumers. It is obvious, that fresh plant cells derived stress proteins and among them HSPs are non-toxic essential micronutrients and harmless for wild herbivores, which have daily consumed their cocktail for millenniums.
  • HSP70 receptors on the surface of a wide spectrum of immune competent cells, which may increase by 10,000-fold their response to antigens displayed to them by HSP70;
  • HSP Heat Shock Proteins
  • HSP Heat Shock Proteins
  • Plant origin HSPs classification and chaperone function
  • HSP Heat Shock Proteins
  • HSP 110 families cover the range HSP 115-HSP99;
  • HSP90 families cover the range HSP96-HSP82; HSP70 families cover the range HSP78- HSP68; HSP60 families cover the range HSP65-HSP55; HSP45-6 families: cover the range of more than 20 different small molecular mass HSP sub-class and
  • Ubiquitins 6-10 families which are subunits of an ATP dependent multi-enzyme complex located within both cytoplasm and nucleus involved in selective proteins degradation /apoptosis.
  • HSP70 sub-class As the most probable typical block of the gut derived mixtures that are able to build an edible vaccine function.
  • HSP70 sub-class that is composed by:
  • HSP73 or HSC70 or BiP sub-isomers that are moderately heat stress inducible.
  • HSP73 synthesises constitutively and is present in both the cytosol and nucleus.
  • HSP73 transiently chaperone many proteins in an ATP dependent manner to facilitate their proper folding.
  • Spinach Endoplasmic Reticulum luminal HSC73 is expressed in all tissues, with the exception of dry seed.
  • HSC73 is involved together with HSP 100 in the proper assembly and folding of newly synthesised proteins.
  • HSC73 intracellular level is not elevated dramatically like HSP72 either by cold or water stresses or heat shock, neither by their combinations.
  • HSP72 or HSP70B' sub-isomer which is absent in unstressed cells as its coding genes have no basal expression.
  • HSP72 coding genes promoters are strictly inducible at heat shock condition and HSP72 may reach within minutes up to 1% of intracellular total proteins level.
  • HSP72 genes are encoding more basic amino acids than HSP73 genes. And both these genes sequences are very similar in human/animal cells and plant cells, e.g. HSP72 gene in human is 77% similar to HSP73 genes, while both are 74% similar to soybean HSP72 and 63% similar to maize HSP72.
  • Lymphocytes possess on their surface accessory proteins so called Cluster of Differentiation (CD) and some of them may associate with HSP70 and HSP90 separately and facilitate internalising each of them inside of endosomal structures, where the low pH could provoke the dissociation of peptides from them. And these receptors mediated presentation of immunogenic peptides by HSP70 or HSP90 to professional Antigen Presenting Cells (APC) has been shown to be up to 10,000-fold more efficient than in the case when APC have taken the same peptides by phagocytosis. HSP72 possess the capability to carry far more and a wider spectrum of antigenic peptides than HSP90.
  • CD Cluster of Differentiation
  • HSPs Plant origin HSPs of different molecular masses, redistributed inside of sub-cellular structures of plant cells (Vierling 1990-91; Neuman at al 1989; Singla at al 1997; Boston at al 1996). Any origin HSPs are fulfilling miscellaneous chaperone functions, which may be expressed in the following particular ways: - In folding of all kinds of just synthesised proteins into their best functional 3D structures;
  • 35% homology is the lowest margin in ID structures of any origin HSP70/90 sub- classes to be used in vaccines, while homology from 55% to 75% or more, on the base of amino acid sequences, is preferable.
  • the homology of ID structures between herbaceous plant cells derived HSP70/90 sub-classes and human animal cells origin HSP70/90 pertinent sub-classes are in the range of 55%-85%, on the base of as well amino acids as nucleotides sequences.
  • HSP70/90 relevant sub- classes have offered the clue that any fresh herbaceous plants are the preferable sources for their induction, non- waste extraction and purification in order to construct in vitro the cheapest binary mixtures World wide, as the most obvious base for the scale up manufacture of natural FPF with the function of efficient preventive edible vaccines.
  • HSP70/90 sub-classes On the surface of a wide variety of lymphocytes, it was presumed that natural or recombinant HSP70/90 sub-classes might be used as universal carriers for immune selective antigenic peptides.
  • HSP70/90 sub-classes serving as molecular vectors was authenticated by the fact that these subclasses per se lack targeted immune selectivity after their extraction from either human/animal normal cells or plant cells or any micro-organisms and their purification from contaminated peptides (Srivastava at al 1986-2000; Falk at al 1990; Rotzschke at al 1990; Arnold-Schild at al 1999; Panjwani at al 1999; Binder at al 2000; Assea at al 2000; Fujihara, Nadler 1999; Udono, Srivastava 1994; Palladino at al 1987; Mehlert, Young 1989; ).
  • HSP70/90 sub-classes are accepted World wide as almost ideal molecular vectors for displaying health-risk free antigenic peptides to lymphocytes (Fujihara Nadler 1999; Heike at al 1996; Kaufmann, Schoel 1994; Csermely at al 1998).
  • This tech-invention to associate covalently immunogenic antigens with any origin HSPs has several disadvantages. Since immune selective antigens are covalently cross-linked with any origin HSPs, the chaperone capabilities of HSPs are totally abolished and partly an affinity to receptors, too. And the biggest disadvantage of such complexes wherein antigens are covalently associated with HSPs is the lost natural molecular flexibility of HSPs to release antigens inside of immune cells under the influence of either ATP or at low pH condition. Therefore, in the case of using such complexes as injected vaccines will inevitably induce the production of antibodies against HSPs, for such unmistakable reason that HSPs will be accepted by B cell lymphocytes as part of antigens.
  • HSP70/90 sub-classes This tech-invention of immune selective antigens non-covalent association in vitro with any origin HSP70/90 sub-classes is far more advanced than the other method as it mimics exactly the natural chaperone capability of HSPs to associate non-covalently inside of cells with proteins, polypeptides and peptides.
  • Antigenics Inc. have used both possibilities to obtain the complexes wherein immune selective antigens are non-covalently associated to any of HSP70/90 sub-classes, either in vivo or in vitro.
  • Within such immunogenic complexes both subclasses of HSPs able to release antigens under the influence of ATP or low pH.
  • the immunogenic complexes can be obtained in vitro when there is a problem at early stages of infection or malformed cells.
  • this method was represented as far easy way to obtain therapeutically acceptable doses of HSP7/90 sub-classes associated with immune selective antigens.
  • Invention uses the universal chaperone capability of almost any origin of HSP70/90 sub-classes to non-covalently associate in vitro with immune selective proteins, polypeptides, peptides. And this in vitro procedure for the construction of immunogenic complexes is easily avoiding most of the problems indicated by us above. But, this in vitro technology of manufacturing the immunegenic complexes requires a complicated technological process, needing a series of cost additive and wasteful stages and particularly:
  • Desiccation needs the immunogenic complexes after washing out from UF- membranes in order to standardize the therapeutic doses. There is no indication of this technological stage either.
  • SDS-PAGE technology could not monitor the small difference in molecular mass between non-associated and associated HSPs with antigens.
  • HSPs inevitably will lose non- covalently associated antigens at low pH of gastric acid in stomach, while this complex was recommended to use in oral delivery. It is doubtful, that any of those pharmaceutically acceptable additives, which were advocated by the principal inventor, can completely prevent the complexes from disintegration in stomach by gastric acids. Unfortunately, there is no indication on the complex deterioration index in stomach after oral administration. There is neither comparative investigation on efficiency fluctuation of the immunogenic complexes during oral administration in laboratory or clinical trial, nor any manifestation of differences between injected and orally administrated immunogenic complexes.
  • Antigenics would need to know what the native ingredients are - which it doesn't” (see New Engineers 24 February 2001, N2279, p. 41).
  • HSPs and/or HSPs:antigen complexes e.g.: a) Yields from human cell origin in HSPs solely and/or complexes of HSPs:peptides, from lg of human cancer/tumour or normal tissue, are around 0.01-0.1% and the separate yields of HSP70/90 sub-classes are the following (see US Patents: N 5, 935, 576 August 10, 1999 and N6,048,530 April 11, 2000):
  • - HSP90 is about 150-200 ⁇ g
  • HIV human immune-deficiency virus
  • Active immunity may be conferred with the use of a classical vaccine, which contains natural antigens from either killed or attenuated pathogens and in addition some types of adjuvants.
  • Classical vaccines mostly induce B cells, but stimulate T cell lymphocytes either not at all or at very small rates (see below).
  • the dilemma is that killed or attenuated pathogens could not infect cells. Therefore, without entering cells, vaccines based on them could not use those intracellular vectors that used by live pathogens.
  • the lack of knowledge on such intracellular vectors was the stumbling block to display immune selective antigens to T cells in order to prime their destructive role against infected or malformed somatic cells.
  • HSP70/90 subclasses and MHC have just appeared to be this imperative part of intracellular molecular vectors which prime both B and T cell lymphocytes after an invasion by live pathogens. It is clear those HSP70/90 sub-classes, as ideal universal molecular vectors have tremendously widened the horizons of health-risk free immunisation. Passive immunity may be achieved through the injection of appropriate ready antibodies
  • Vaccines are the finest therapeutic instruments for focusing immune competent cells on causative agents of disease.
  • the vaccine action mimics the learning process of human or animal body's B cell mediated immune defence system exactly in the same manner when live pathogenic microorganisms start their invasion.
  • Vaccines leave behind memory B cells on alert, ready to swiftly generalise their huge immune potential of antibody-mediated destruction of live pathogens as soon as they find their way into the lymph or blood.
  • Classical Vaccines comprise: a) Killed pathogen, or b) Live-attenuated pathogen.
  • the scientific clue to achieve the safety of vaccines is to use genes from selective pathogens to produce a single viral or bacterial protein for the manufacture of the so-called subunit vaccines. It can be achieved by employing genes in the form of DNA or RNA for the manufacture of natural immunogenic antigens, rather than using pathogens themselves.
  • Synthetic DNA RNA vaccines consist of bacterial plasmids with a strong viral promoter, the gene of interest and a polyadenylation/transcriptional termination sequence.
  • plasmids are multiplied in growing bacteria e.g. E. coli, yeast, insect or mammalian cells, purified, dissolved in a saline solution and injected into the host. These vaccines tend to give good B cell responses, but weak T cell activation.
  • Health-risk free vectors which may be either non-pathogenic live microorganisms or crops.
  • pathogens' specific genes display inside of human/animal bodies: Live vectors are genetically engineered non-virulent virus e.g. vaccinia, inside of which a synthetic gene of selected pathogen was inserted in order to produce immunogenic proteins after vaccination.
  • live vectors are genetically engineered non-virulent virus e.g. vaccinia, inside of which a synthetic gene of selected pathogen was inserted in order to produce immunogenic proteins after vaccination.
  • the genome of live vector strongly represses foreign gene function.
  • Health-risk free live vectors never will infect somatic cells like the pathogens whose genes they are delivering.
  • Shuttle vectors were genetically engineered from naked short pieces of DNA without the virus "shell” and constructed to break species barriers. This small DNA fragments can be taken up by cells and multiply and mutate indefinitely. They may transfer selected genes horizontally and may be harmful due to random insertions of gene-assemblies into cellular genomes in non targeted species. They may persist longer in the environment after a release or escape to target places and induces unpredictable biohazards.
  • Crop vectors were genetically engineered in edible food/ feed plants, where synthetic genes of selected pathogens were inserted in order to produce immunogenic proteins.
  • the crops that are mostly used as delivery systems for subunit vaccines include: bananas, potatoes, tomatoes, lettuce, rice, wheat, soybeans, corn. Boyce Thompson Institute at Cornell University provided clinical trial of potatoes containing the Norwalk virus DNA, indicating they caused vomiting and diarrhoea. And such potatoes have elicited an immune response in 19 out of 20 volunteers fed by this edible vaccine.
  • Feed plant also have been used as edible vaccines e.g. alfalfa, maize.
  • An expression of the viral structural protein VP1 in alfalfa was already managed in order to induce a protective antibody response to the foot and mouth disease virus.
  • these secondary lymphoid tissues armament is constituted by primary lymphoid and myeloid tissues derived from different lineages of immune competent cells.
  • Primary lymphoid tissue (adult bone marrow, thymus) perpetually supply non-maturated lymphocytes and which are enigmatically converting - only after migration to secondary lymphoid tissues- into maturated Antigen Presenting Cells (APC), B cell and T cell lymphocytes.
  • APC Antigen Presenting Cells
  • B cell and T cell lymphocytes While myeloid tissues supply distinct lineage of immune cells e.g. monocytes, basophils, neutrophils, eosinophils, descendant macrophages, mast cells, megakaryocytes and their platelets (Thain, Hickman 2000).
  • Immuno lobulin Ig and Lymphokines.
  • Immunoglobulins IgA, IgD, IgE, IgG, IgM are all exercising an antibody activity detecting pathogens in blood and lymph.
  • B cells may easily become antibody-producing plasma cells, if any of the professional antigen presenting immune cells such as macrophages or Dendritic cells or APC will prime B cells with immune selective antigens. Every B cell may be cloned with a single antigen and plasma cell will produce highly selective antibodies towards only those fragments that have primed B cells cloning. As a rule surface proteins of live pathogens and their fragments are highly preferable for vaccination.
  • lymphocytes are communicating and building an orchestral network of different subdivisions of immune competent cells.
  • Certain lymphokines may well tune the re-production rate of highly selective antibodies by plasma cells and prime macrophages and T cell killer lymphocytes for the destruction of infected or malformed cells.
  • none of these lymphokines e.g. Interferon (INF ⁇ - ⁇ - ⁇ ) or Interleukins (IL1-8) are able to prime selectively any lymphocytes like HSP70.
  • HS70 may play a dual role: to prime indirectly both T cell and B cell lymphocytes via displaying immune selective antigens to APC, on the surface of which are receptors for HSP70/90 sub-classes.
  • HSP70 may tune the expression of several lymphokines, particularly interleukin-l ⁇ , interleukin-6, tumour necrosis factor- ⁇ , which are multifunction cytokines with effects on host resistance to parasites and malignancy. So, HSP70 in addition to intra- and extra-cellular molecular vector functions may also act as a powerful super-cytokine (Fujihara, Nadler 1999; Asea at al 2000).
  • CD antigens are membrane glycoproteins of the Ig super-family, which are regulating the adhesion between T cell and macrophages, dendritic cells, Antigen-Presenting Cells (APC), etc. They include accessory receptors e.g. CD2, CD4, CD8, CD28, CD40, B7, TCR, etc., each of which helps T cell lymphocyte to discriminate normal cells from infected or malformed or transplanted ones.
  • accessory receptors e.g. CD2, CD4, CD8, CD28, CD40, B7, TCR, etc.
  • CD14 on the surface of monocytes CD91 on the surface of monocytic lineage as well as on the surface of hepatocytes, fibroblasts, karatinocytes; and CDl lb ⁇ on the surface of macrophages have appeared specific receptors and/or co-receptors for HSP70/90 sub-classes. And with the help of these accessory receptors such as CD surface glycoproteins may increase by 10,000-fold the efficiency of the first line of immune defence in gut.
  • MHC Major Histocampatibility Complex
  • MHC class I binds infra-cellular origin antigenic peptides in ER outside of the Golgi and display them abundantly on the surface of cells.
  • MHC class II binds extra-cellular origin antigenic peptides inside of Golgi and display them on the surface of lymphoid B cells or macrophages.
  • MHC class I occur in low levels on most body cells and display bound peptides on the surface of cells, where they are mostly accepted by T cells lymphocytes.
  • T cells are accepting MHC class I represented peptides on the surface of somatic cells as an indication of intracellular disorders
  • MHC class II represented immune selective antigens on the surface of APC act as a signal to start searching among somatic cells which are infected.
  • HSP70 sub-class may chaperone both an intra- and extra-cellular proteins processing inside of any somatic cell or lymphocytes.
  • HSP70 also protect the optimal immune selective size of peptides from protease attack and chaperone them towards either MHC Class I or MHC Class II (Amold-Schild at al 1999; Binder at al 2000; Panjwani at al 1999; Asea at al 2000; Chiang at al 1989; Harris at al 1989; Wearsch, Nicchitta 1997).
  • A) Molecular vector-carrier one or more with unvaried ID structure, of plant origin, and B) Immune selective antigen: several with varied ID structures, of microbial origin.
  • This frequent immunogenic mixture with the help of a universal molecular vector may remarkably enhance the efficiency of a wide spectrum of immune competent cells existing in gut via their priming with variable immune selective antigens. Therefore, daily creating this immunogenic mixture in gut generates the Active Induced Natural Immunity (AINI).
  • AINI Active Induced Natural Immunity
  • this still enigmatic network provided in gut for building the immunogenic mixture with edible binary vaccine function may be divided into the following highly integral central occurrences: a) During ingesting of a the daily norm, fresh plant feed/food undergo heat shock at gut temperature, what increases the synthesis of HSPs within minutes, particularly HSP72 the intracellular level of which dramatically increases up to 1% of total plant proteins, just after herbivores start grazing and chewing fresh plant bio-mass. And according to the rate of fresh plant feed/food disintegration in gut, heat shocked fresh plant cells are gradually releasing in stomach essential micronutrients, among which HSPs. Plant origin HSPs astonishingly resist to protease attack and low pH in stomach.
  • Plant origin HSPs pre-incubation with gastric acids eventually releases any peptides adsorbed by them.
  • plant origin HSPs may chaperone attenuated microbes derived immune selective antigens as well across to membranes of endothelial cells which line he submucosa, as directly priming with antigens APC, macrophages via those CD surface receptors which possess a high affinity towards HSP70/90 sub-classes (Jaliashvili 1989-1998 unpublished results).
  • gastric acid is attenuating a great part of the microbes, which are contaminating fresh plant feed/food daily ingested by herbivores.
  • gut proteases in dissimilar ways breakdown attenuated microbes' surface proteins into peptides. It is certain that almost all of these microbial origin peptides are ideal natural antigens with an immune selectivity to those microbes, that are existing in daily norms of herbaceous fresh plant feed/food and water which are ingested by herbivores. It is obvious that plant origin HSPs may chaperone to APC predominantly the peptides derived from surface proteins of only gastric acid attenuated microbes after their attack by gut proteases. c) And plant origin HSPs and microbial peptides are daily building immunogenic mixtures in gut of herbivores in field condition.
  • HSP72 sub-class dramatically increase up to 1% of total plant proteins during the ingestion of fresh plant bio-mass at gut temperature; they possess a high structural homology with human/animal cell origin HSP72/73, and some CD receptors on APC surface possess high affinity to HSP72/73 sub-class.
  • plant origins HSPs may chaperone also accelerate transfer of microbial immune selective antigens across the membranes of epithelial cells within the submucosa and lymphoid nodes. Each lymphoid node has its own autonomy of antigens making process: mostly APC dependent and partly via macrophages.
  • plant cell derived HSP72 may remarkably enhance the efficiency of this first line of immune defence instituted in gut.
  • HSP70 which targets immature dendritic cells to increase phagocytosis in vitro, or elicits integral ( attack of macrophages, T cell and dentritic cells towards malformed somatic cells.
  • Macrophages engulf feed/food contaminated live microbes and after their digestion display microbial HSP70/90/65 sub-classes to B cells and T cell lymphocytes.
  • microbial derived HSPs are ready complexes with immune selective microbial antigens they are immunodominant antigens. And such phagocytosis-mediated presentation of immune selective microbial proteins and peptides has appeared 10,000-fold less effective than priming of APC, mediated by HSPs receptor. And comparing to the APC network, the major pathway of gut first line immune defence, the macrophages immune protection can be considered as rendering just a service (Asea at al 2000; Kauffman, Schoel 1994, Arnold-Schild at al 1999). e) Gut epithelial cells within submucosa undergo an invasion of live microbes, which have escaped the gastric acid and protease attack during ingestion of fresh plant feed/food.
  • epithelial cells infected by virulent microbes enhance the synthesis of their own HSPs in order to chaperone extra-cellular microbial origin proteins processing and display to MHC Class I. So, epithelial cells origin HSPs may generalise the alarm to eradicate any infected cells in gut submucosa.
  • Epithelial cells derived HSP70/90 sub-classes may build complexes with immune selective microbial antigens of invaders and prime with this complex directly APC or dendritic cells. And these professional antigen-presenting cells will prime T cell lymphocytes and macrophages to eradicate any somatic cells showing a sign of such infection.
  • epithelial cells as part of the first line defence bastion of gut submucosa, via a self-sacrificial process, are building mostly a T cell mediated curative immune defence against virulent micro- organisms which are contaminating fresh plant Feed/ food.
  • Molecular vector-carrier one or more with unvaried ID structure, of plant origin presumably inducible at 36°C-39°C in fresh herbaceous plant cells, the derived HSP72 sub-class, which may be co-chaperoned by other sub-classes.
  • Plant origins HSP70 are ubiquitous in gut during digestion by herbivores of fresh herbaceous plant. Plant origins HSP70 possess the distinct post-translation modification absent in microbial HSP70. That feature provides APC to easily distinguish, with the help of molecular vector-carriers either from plant or microbial origin, how many immune selective peptides are derived from attenuated or live microorganisms.
  • plant origin HSP70 as molecular vector-carriers, will inevitably display only immune selective peptides derived from attenuated pathogens. Therefore, plant origin HSP70 will help the first line of immune defence to define the microbes' contamination indexes in daily norms of herbaceous feed/food. That is the clue which mobilises the first line of gut defence to exactly required lymphocytes numbers to prevent and eradicate causative agents of disease, far before they invade somatic cells.
  • Immune selective antigen several with varied ID structures, of microbial origin doubtless fresh plant feed/food or water contaminated microbes derived peptides, and particularly surface proteins from those which were attenuated by gastric acid and broken down by gut proteases.
  • HSP72 sub-class which may reach up to 1% of total plant proteins at 36°C-39°C while herbivores ingest fresh herbaceous plants in field condition, may be, although unnoticed up to now, one of the domineering natural blocks of edible binary vaccines.
  • HSP72/73 sub-classes derived from plant and human/animal cells are the vast majority of this and other sub-classes of plant origin HSPs released in gut. Therefore, such a remarkable quantity of this and other sub-classes of plant origin HSPs released in gut may significantly enhance the preventive efficiency of herbivores' immune defence against feed/food derived diseases, via the display of immune selective microbial antigens to APC localised in gut.
  • HSP70 human cells origin HSP70 e.g.: "In addition, recent reports describing the ability of peptide-bound HSP70 molecules to induce antitumor or antiviral immunity as well as the development of memory CTLs support the notion that these proteins might function to convey a protective immune response by providing an antigen-presentation function".
  • plant origin HSP72 are ubiquitous in gut during digestion of fresh herbaceous plant by herbivores; b) plant origin HSP72 possess a distinct post-translation modification from microbial one, constitute the clue of plant origin HSP72 sub-class use as harmless universal molecular vector-carriers for the building the immunogenic mixtures with function of preventive binary vaccines.
  • HSP70/90 sub-classes based vaccines mostly used by experts of almost all Scientific-Medical Centres and private firms for their injected administration in laboratory or clinical I-II-III phase trials, World wide.
  • the therapeutic doses and spectrum of HSPs based vaccines The therapeutic doses of a new generation of vaccines, which are using HSP70/90 subclasses as molecular vectors, range from 0.01-1.0 mg/kg of body weight, for the injected immunisation of mammalians.
  • HSP70/90 subclasses as molecular vectors
  • HSP70/90 sub-classes based vaccine is "..an amount of HSP70- and/or gp96-antigenic molecule complexes is administered in the range of about 10 micrograms to about 600 micrograms for human patient, the preferred human dosage being the same as used in 25 g mouse, i.e., in the rage of 10-100 micrograms".
  • HSP70/90 sub-classes as molecular vectors for priming lymphocytes with immune selective antigens either obtained in vivo or in vitro from malformed or infected somatic cells generally have no those side effects that have conventional vaccines. And almost all scientists Worldwide have accepted any origin HSP70/90 sub-classes as health-risk free and ideal molecular vectors for diverse immune selective antigens. Likewise, there is no limitation to the therapeutic spectrum in the case of HSP70/90 sub-classes use as imperative building blocks of injected or oral vaccines, whatever the origin of HSP70/90 subclasses (see US Patent 6,048,530, issued April 11, 2000). Therefore, a valid claim was made on any origin HSP70/90 sub-classes as universal molecular vectors, which are able to prime lymphocytes with immune selective antigens against appropriate causative agents of diseases and which may be from:
  • Viruses e.g. HIV-I and HIV-II, hepatitis type-A/B/C, papilloma, polio, varicella, adenovirus, measles, etc. - bacteria e.g. tuberculoses, shigella dysentery, gastroentritis, typhoid, meningitis, etc.; protozoa e.g. Kokzidioa, Leishmania, Trypanosoma, etc. parasites e.g. Rickettsia, Chlamidia, etc.
  • Cancer cells e.g. fibrosarcoma, colon carcinoma, breast or prostate cancer cells, melanoma, meningioma, etc.
  • HSP72/73 sub-class herbaceous plant origin HSP72/73 sub-class, as harmless essential micronutrients and the cheapest natural molecular vector-carriers in feed/food industries and clinical nutrition are the clue of the invention we are representing herein.
  • the objective of the invention is - the method to manufacture of non-expensive, health risk-free and efficient edible vaccines on the base of the immunogenic mixture of HSPs and antigens.
  • the vaccine on the base of immunogenic mixture constituted either in situ or in vitro, composed of Heat Stress Proteins (HSPs) derived from fresh herbaceous plant cells, which are only mixed but not bound neither non-covalentlty nor covalently with exterior antigens derived from pathogenic micro-organisms or malformed somatic tissues/cells of Homo or their synthetic analogues, in which antigen is the culture of pathogenic micro-organisms and/or the culture of malformed somatic tissue/cells of Homo, virus, bacteria, protozoa, fungi, the culture of intra-cellular or extra-cellular pathogenic micro-organisms or their synthetic analogues, the culture of human malfunctioning somatic tissues/cells which get out of control under the influence of senescence or of abiotic or biotic stresses or their synthetic analogues.
  • HSPs Heat Stress Proteins
  • the vaccine on the base of immunogenic mixture constituted either in situ or in vitro, composed of plant origin HSPs, exterior antigens and the molecular vector- carrier represented by one molecular mass HSPs sub-class derived from herbaceous plant cells e.g.: HSP 110, HSP90, HSP70, HSP60, HSPs 55-6 kDa and/or by their combination.
  • the vaccine on the base of immunogenic mixture constituted either in situ or in vitro, composed of HSPs derived from fresh herbaceous plant cells, which are only mixed but not bound neither non-covalentlty nor covalently with interior antigen derived from malformed somatic tissues and/or urine, which were obtained directly from a patient or their synthetic analogues, in which interior antigen is malformed somatic tissue and/or urine obtained from a single human patient, virus, bacteria, protozoa .fungi, in which the source of interior antigen is malfunction somatic tissues/cells, obtained directly from a human being or their synthetic analogues, malfunction somatic tissues/cells, obtained directly from a human being or their synthetic analogues, a human patient's urine derived proteins, polypeptides, peptides, lipopolysaccharides, etc., or their synthetic analogues.
  • These vaccines are used as healing or prophylactic means for the same human patient.
  • the vaccine on the base of immunogenic mixture constituted either in situ or in vitro, composed of plant origin HSPs, interior antigen and the molecular vector-carrier represented by one molecular mass HSPs sub-class derived from herbaceous plant cells e.g.: HSP 110, HSP90, HSP70, HSP60, HSPs 55-6 kDa and/or by their combination.
  • composition of immunogenic mixture constituted either in situ or in vitro, designed from herbaceous plant cells derived HSPs and integral individual antigen derived from human urine containing proteins, polypeptides, peptides, lipopolysaccharides.
  • composition of immunogenic mixture constituted either in situ or in vitro, designed from herbaceous plant cells derived HSPs, integral individual antigen derived from human urine containing proteins, polypeptides, peptides, lipopolysaccharides and individual diet in clinical nutrition, which is used as healing or prophylactic individual means only for the patient whose urine was used as source of integral internal antigen or its synthetic analogues and also as an edible binary vaccine and/or base of Functional Processed Food (FPF) with a strong individual healing or prophylactic function.
  • FPF Functional Processed Food
  • composition of immunogenic mixture constituted either in situ or in vitro designed from plant origin HSPs and AIDS internal antigen derived from a patient's urine in combination with natural or synthetic external antigen of different strains of HIV and the method for designing this composition of immunogenic mixture constituted either in situ or in vitro by mixing plant origin HSPs and AIDS internal antigen derived from a patient's urine in combination with natural or synthetic external antigen of different strains of HIV, wherein the most preferable is equal molar ratio, but not restricting any other molar ratio, wherein each building block of an immunogenic mixture is in the form of powder, liquid or jelly and the storage of building blocks of immunogenic mixture as powders is provided at room temperature before their mixing.
  • composition of the immunogenic mixture comprising plant origin HSPs and internal antigen, which is used as a healing or prophylactic means only for the individual whose urine was used as source of integral internal antigen and as a healing or prophylactic means with any vehicles, which are fitting in pharmacy or in fitness or in food/feed industries.
  • immunogenic mixture either in situ or in vitro, promotes to mix of plant origin HSPs in the form of liquid or powder with internal or external antigens in the form of liquid or powder before using this mixture composition as a strong individual edible binary vaccine with or without vehicles, wherein the immunogenic mixture is preferably as powder and is stored at room temperature before its use as a strong individual edible binary vaccine with or without vehicles.
  • the lost HSPs during processing or storage of the plant food as fitness or food additives in order to restore the daily norm of immunogenic mixture in situ.
  • composition of immunogenic mixture on the base of herbaceous plant cells derived HSPs, antigen and probiotics, prebiotics, synbiotics which is used as clinical nutrition or in fitness or in food industry and also as building block of FPF and/or of any natural diets as the healing and prophylactic efficiency of clinical nutrition
  • This method promotes a concentration of individual immune selective internal antigen by the means of vacuum-evaporation in the range of 55°C-95°C or cross-flow membrane filtration (UF/RO) or lyophilisation or Zeodration, the identification of individual immune selective internal antigen by gel-chromatography in the range of 35°C-45°C during 35 minutes, the identification of individual immune selective internal antigen by gel-chromatography in the range of 45°C-75°C during 20 minutes
  • the vaccine on the base of immunogenic mixture constituted either in situ or in vitro, composed of HSPs derived from fresh herbaceous plant cells, which are only mixed but not bound neither non-covalentlty nor covalently with exterior antigens derived from pathogenic micro-organisms or malformed somatic cells or their synthetic analogues, wherein the origin of exterior antigen is virus, bacteria, protozoa, fungi, the source of immune selective exterior antigen is the culture of intra-cellular or extra-cellular pathogenic microorganisms or their synthetic ana
  • vaccines are used as a healing or prophylactic means for the farm animals including cattle, horses, sheep, goats, pigs, etc, household pets including: cats, dogs, etc. or zoo animals.
  • the vaccine on the base of immunogenic mixture constituted either in situ or in vitro, composed of HSPs derived from fresh herbaceous plant cells, exterior antigens derived from pathogenic micro-organisms or malformed somatic cells or their synthetic analogues and the molecular vector-carrier represented by one molecular mass HSPs sub-class derived from herbaceous plant cells e.g.: HSP110, HSP90, HSP70, HSP60, HSPs 55-6 kDa and/or by their combination.
  • the vaccine on the base of immunogenic mixture constituted either in situ or in vitro, composed of plant origin HSPs, antigen and the molecular vector-carrier represented by one or more sub-classes of HSPs derived from different herbaceous plant species and antigens derived from more than one pathogenic micro-organisms or malformed somatic cells of animals.
  • the vaccine on the base of immunogenic mixture constituted either in situ or in vitro, composed of plant origin HSPs, which only mixed but not bound neither non- covalentlty nor covalently with interior integral antigen derived from somatic tissues/cells and/or urine of farm or zoo animals or their synthetic analogues, wherein the source of interior antigen is somatic tissues/cells and/or urine of farm or zoo animals and the origin of interior antigen is virus, bacteria, protozoa, fungi, intra-cellular and/or extra-cellular pathogenic micro-organisms or their synthetic analogues, animal malfunctioning somatic cells which get out of control under the influence of senescence or of abiotic or biotic stresses or their synthetic analogues
  • the vaccine on the base of immunogenic mixture constituted either in situ or in vitro, composed of plant origin HSPs, exterior antigens derived from pathogenic microorganisms or malformed somatic cells or their synthetic analogues and the molecular vector- carrier represented by one molecular mass HSPs sub-class derived from herbaceous plant cells e.g.: HSP110, HSP90, HSP70, HSP60, HSPs 55-6 kDa and/or by their combination.
  • the vaccine on the base of immunogenic mixture composed of plant origin HSPs and interior integral antigen derived from farm or zoo animals' urine containing proteins, polypeptides, peptides, lipopolysaccharides, etc., or their synthetic analogues.
  • composition of the immunogenic mixture copmprising herbaceous plant cells derived HSPs, interior and exterior integral antigens integration and vehicles used in feed industry e.g. cellulose, geo-minerals (zeolites, etc.), which is used as a healing and/or prophylactic means in veterinary and feed industry and also the method for constituting the immunogenic mixture by mixing of herbaceous plant cells derived HSPs and exterior or interior antigens in the form of powder or liquid, before its use as edible binary vaccine solely or in combination with any vehicles acceptable in veterinary or in feed industry.
  • feed industry e.g. cellulose, geo-minerals (zeolites, etc.
  • immunogenic mixture composed of herbaceous plant cell origin HSPs, integral interior or exterior antigens and probiotics, prebiotics, synbiotics used in veterinary and feed industry.
  • the present invention provides a method for a simple construction of an immunogenic mixture having the function of preventive binary vaccines.
  • the foundation used for this immunogenic mixture are the health risk free and the cheapest imperative building blocks: HSPs derived from herbaceous plant cells and an antigenic peptide with an immune selectivity to the causative agents of diseases.
  • This invention is grounded on the breakthrough finding: the phenomenal synthesis increase of HSPs at 36°C-39°C heat shock condition inside of fresh herbaceous plant cells, either in pasture or in gut.
  • herbivores are developing in their stomach an immunogenic mixture: plant origin HSPs and microbial antigens after ingesting the daily norm of fresh herbaceous plant as feed/food, contaminated by innumerable microbes.
  • These imperative building blocks of the immunogenic mixture in the stomach of herbivores are neither covalently nor non-covalently associated in low pH environment of gastric acids.
  • Wlrier plant cells derived HSPs as essential micronutrients, play only the function of molecular vector-carriers within an immunogenic mixture.
  • Plant cell origin HSPs approximation of daily natural norm
  • the vehicle of the immunogenic mixture, operating as an edible binary vaccine, is the daily natural diet used by herbivores for millenniums, and particularly such extraordinary essential micronutrients as plant origin HSPs derived from fresh herbaceous plant cells, possessing a molecular vector-carrier function.
  • Plant cell derived HSPs are a unique class within fresh herbaceous plants derived essential micro-nutrients, and the only ones which may increase dramatically their intracellular level at herbivores' 36°C-38°C gut temperature. Consequently, our paramount goal was to determine the daily natural norm of plant origin HSPs on the base of experimental herbivores.
  • the following feeding specificity of some herbivores (deer, antelopes, giraffes, cattle, sheep, goats) were used to achieve this goal: • Grazed fresh grass, after being swallowed into the rumen, undergoes only a partial anaerobic digestion by cellulase from a symbiotic bacterial flora;
  • HSPs enriched fraction we have investigated the comparative spectrum of HSPs in heat shocked fresh alfalfa cell and heat shocked conventional feed. Both spectrums of HSPs we were compared with non- heat shocked fresh alfalfa cells extracts that were harvested manually, directly from grassland at 8°C-14°C-air temperature.
  • HSPs content within fresh grass plant intact cells increases up to 1% of total plant proteins of grazed bio-mass, but it has appeared that almost all newly synthesised HSPs swiftly associate with as well soluble as non-soluble plant proteins. Therefore, there is need to underline that almost all newly synthesised HSPs sub-classes chaperone polymorphous enzymes and particularly RUBISCO, in order to defend them from stress factors within plant cells. So, approximately 40 mg of HSPs are distributed inside of lOOg of fresh alfalfa intact cells, in the following manner:
  • HSPs were extracted from around 3.7 g of non-soluble structural proteins inside of alfalfa cells.
  • HSPs can easily release from their associated forms at low pH during extraction and purification, as in experimental scenario model in animal stomach.
  • ruminants may receive, in addition to 4 g of total edible proteins, also heat stress genes generated products: minimum 2.8 mg of HSPs released from soluble proteins and maximum 37.2 mg HSPs which non-soluble proteins may release at low pH of hydrochloric acids in stomach.
  • HSPs associated with non-soluble structural plant cells membrane proteins
  • the most important task for Feed Industry is to restore herbaceous plant derived natural composition in feeds, and particularly such essential micro-nutrients as plant cell derived HSPs, with their molecular carrier- vectors function, in that seasonal period when fresh grass is not available for domestic animals.
  • HSPs sub-classes associated with soluble proteins and which are easily extractable by mechanical presses we have considered them as highly preferable source for applications HSPs in Food industry and Life Sciences.
  • HSP70/90 sub-classes from 100 g fresh alfalfa during the whole season may not be less than e.g. 2,8 mg HSP72/73 and 0,5mg HSP90 subclasses for use in Food industry and Life Sciences.
  • 100 g of human cancer tissue or 10 ml of human recombinant cells (pellet) may supply around 1.0 mg HSP72/73 and 0,2 mg HSP90 for use in Pharmacy.
  • human recombinant cells Pellet
  • There are ethical and moral and health risk problems in the cultivation of human tissues for the manufacture of HSPs see US Patent 5 948 646, Sep.7, 1999).
  • HSPs The percentage of release of associated HSPs at low pH in stomach of herbivores. Thereafter, we have directed our attention to major common factors, which may induce the fluctuation of the daily norm of fresh herbaceous plant cells origin HSPs and found out the following, and particularly:
  • the rate of HSPs synthesis differs separately in cells of leaves and in cells of stems;
  • Herbivores at various ages are grazing different amount of fresh grass per day, etc.
  • HSP70 sub-class found out their astonishing resistance to low pH and attack of proteases at 37°C, for at least 1-2 hrs incubation in vitro. This our results was re-confirmed the HSPs high resistance to heat shock, to extreme pH and a wide spectrum of proteases influence as these were already well known facts (Neumann at al., 1989; Vierling 1990/91; Singa at a., 1997; Pollock at al 1993).
  • This clue was used by the present invention as a simple way to counterbalance an inevitable loss in the capability to provide the synthesis of HSPs after processing fresh herbaceous plants as feed/food by the addition in the diet daily norm of ready made combination of immunogenic mixture, wherein plant origin HSPs are used as molecular vector-carriers and immune selective peptides as antigenic agents.
  • this immunogenic mixture was proposed as the innovative combination of a new generation of Functional Processed Food (FPF) in order to restore in diets the preventive edible binary vaccines function of fresh herbaceous plants. And FPF acquires the same preventive edible binary vaccine function like fresh plant.
  • FPF Functional Processed Food
  • This new generation of FPF will predominantly use as molecular vector-carriers the herbaceous plant origin HSP70 sub-class, but are not limits to their combination with other HSPs sub-classes or any stress proteins from plant origin.
  • This building block of innovative immunogenic mixture manages the efficient activation of abundant APC in gut, by displaying immune selective antigens to them. And the targeted APC may in turn, with the same immune selective antigens, prime both B cell and T cell lymphocytes and mobilise their destructive power to eradicate causative agents of diseases pointed by antigens, whatever their localisation either intra-cellular or extra-cellular.
  • HSP70 sub-class The strongest assumptions for plant origin HSP70 sub-class use, as imperative building blocks of an innovative immunogenic mixture are the following: - Herbaceous plant cell derived HSP72/73 sub-class possesses the highest structural homology to human/animal cells derived HSP72/73 similar sub-class. With a 55%-85% range of homology, HSP70 sub-class can be invoked as the most preferable for their use in building of preventive and curative vaccines.
  • Herbaceous plant cell derived HSP70 sub-class possesses a distinct post- translation modification from human/animal cells derived or microbial HSP70 sub-class. This is a remarkable advantage of the plant origin HSP70 sub-class in their use as edible binary vaccines. As such distinct post-translation modification of plant ell derived HSPs may significantly decrease the risk of auto-immune diseases which may be induced by recombinant human HSP70 expressed in E. coli; - Herbaceous plant cells derived origin HSP70 sub-class possess a strong resistance to heat and acid treatment. Thus, there is no need to refrigerate an immunogenic mixture based preventive edible binary vaccine;
  • Herbaceous plant cells derived HSP72/73 sub-class are able to chaperone the widest spectrum of immune selective antigens and protect them from attack of gut proteases; - Herbaceous plant cells origin HSP70 sub-class may be manufactured industrially cost effectively and non-wastefully;
  • Herbaceous plant cells derived HSP70 sub-class may be co-chaperoned by the widest spectrum of plant cells derived HSPs and particularly by the small molecular mass HSPs;
  • the production cost of herbaceous plant cells origin HSP70 sub-class is far lower than human cells or microbial origin or even recombinant origin HSP70;
  • the manufacture herbaceous plant cells origin HSP70 sub-class may cluster- integrate agriculture with Life Sciences and stimulate the search and selection of environmental stress resistant plant species on the base of their screening for stress proteins production ability.
  • Alfalfa (Lucerne, Medicago sativa and falcata), the short-term renewable forage grass, which contains the full spectrum of all HSPs sub-classes. Alfalfa is the cheapest agricultural source for non-waste production of all HSPs subclasses. Alfalfa is well accepted worldwide as food e.g. alfalfa-sprouts account for about 7% of total USA alfalfa-seeds supply. Alfalfa has been used as fitness in preventing osteoporosis, cancer, heart diseases and menopausal symptoms.
  • Alfalfa is premier feed for livestock. Alfalfa is improving soil and dramatically reduces the need of nitrogen fertiliser and pest control technologies as well as crop rotations.
  • France-Luzerne produces more than 30 products from alfalfa foliage (Russelle 2001).
  • France-Luzerne a Cooperative of around 4500 French farmers who are cultivating alfalfa on 85 000 hectares, is the biggest centralised producer of alfalfa pellets in EU.
  • France-Luzerne is annually harvesting around 4,000,000 tons of fresh alfalfa and produces from it almost 1,000,000 tons of pellets, which they keep in centralised storehouses for commercialisation as premium feed.
  • Pellets production technology is extremely energy consuming and expensive, e.g.
  • the yield of the HSP72/73 sub-class with 85% purification rate is from 2.8 g up to 16g from different species of fresh alfalfa during the harvesting season (based on 100-kg of fresh alfalfa with 80% of moisture).
  • HSP72/73 sub-class has the highest yield among any other HSPs sub-classes, in spite of the great content variation in different species of herbaceous plants (PCT-Patent GE00/00003, Issued 19 May, 2000).
  • An edible binary vaccine is from 2.8 g up to 16g from different species of fresh alfalfa during the harvesting season (based on 100-kg of fresh alfalfa with 80% of moisture).
  • the present invention provides a method, which can be carried out easily and at low costs for the constitution of an immunogenic mixture with edible binary vaccine function.
  • the new invention there is no need of preliminary association neither covalently nor non- covalently of plant origin HSPs with any immune selective antigens in vitro.
  • this inventive method has remarkably increased the yield of edible binary vaccines since the process of constituting an immunogenic mixture within the proposed invention has no loss of such imperative building blocks as: plant origin HSP70 sub-class with molecular vector-carriers function and immune selective antigens from causative agents of diseases.
  • the immunogenic mixture has opened the opportunity to achieve an integral preventive effect of natural edible binary vaccines.
  • HSP70 sub-class as molecular vector-carriers may chaperone different types of immune selective antigens and display them to APC in gut of Homo and animals.
  • each component may be used in dehydrated powder forms what can remarkably prolong the shelf life and eliminate the need for refrigerating edible binary vaccines. Therefore, such preferable form of an immunogenic mixture is well suitable to compose many diets respecting as well the personal taste preferences (Benson at al 1978; Zhao at al 1997; Wittenberg 1972). None of the components of such edible vaccines will confront regional or national habits and customs. And here is the blueprint an the innovative compositions of a new generation of Functional Processed Food (FPF) with preventive function of edible binary vaccines:
  • FPF Functional Processed Food
  • Macro component 99% of FPF biomass — any natural plant origin diet and probiotics that is not increasing the secretion of proteases in gut of mammalians;
  • Micro-component 1% of FPF biomass - an immunogenic mixture with the following building blocks in any molar ratio: • invariable component: plant cells origin HSP70 sub-class as molecular vector- carriers alone or in synergistic combinations with other HSPs sub-classes and other types of plant origin stress proteins;
  • Variable component immune selective antigens chosen from causative agents of diseases.
  • the present invention main advantage is that the mixture composition of edible binary vaccines may open a far broader range of combinations of all kinds of stress proteins to reach those natural synergies, which exist in gut of herbivores and Homo during the digestion of fresh herbaceous plant feed/food.
  • ID structure of herbaceous plant cells derived HSP72/73 subclass is practically identical to ID structure of human/animal cells origin HSP72/73 sub-class; they are interchangeable in any form of oral vaccines, which uses HSP70 as molecular vector- carriers of antigens.
  • the new invention may build in vitro unlimited mixture combinations of plant origin HSP72/73 with favoured single or integral immune selective antigens, for syringe/needle free targeted preventive immunisation of any human being Worldwide.
  • the use of alfalfa cells derived HSP72/73 sub-class as molecular vector-carriers has a number of advantages and particularly:
  • Alfalfa cells derived HSP70 sub-class is an ancient essential micro-nutrient with distinct post-translation modification from human/animal cells origin HSP70 and have not those technological problems and side effects that recombinant human/animal HSP70 expressed in E.coli imply;
  • Alfalfa cells origin HSP70 while they chaperones immune selective antigens within an immunogenic mixture at 36°C-38°C at suitable intestine pH, may protect chosen immune selective antigens from aggressive proteases and deliver them efficiently to APC, which particularly are abundant in small intestine;
  • Alfalfa cells origin HSP72/73 sub-class is preferential a long-lived but completely biodegradable molecular vector-carrier of immune selective antigens.
  • Extra-cellular sources lymph, blood serum, urine, synovial fluid and cultures of causative agents of diseases;
  • Intra-cellular sources MHC complexes, HSPs complexes, malformed and infected tissue/cells.
  • synthetic immune selective antigens in any molecular ranges since small molecular mass peptides may easily synthesise with chemical methods, which was considered as the safest to use in vaccines.
  • chemical methods which was considered as the safest to use in vaccines.
  • high molecular mass proteins While with the method of genetic engineering there is no problem already to manufacture high molecular mass proteins. There is need to point out the low immunogenicity of small molecular mass peptides in comparison with high molecular mass proteins or lipopolysaccharides.
  • adjuvants e.g. Complete Freund's adjuvant (comprises killed M.
  • kidneys reabsorb as much as 80% of glomerular filtrate across the cells of the proximal convoluted tubules into capillaries of the Vasa Recta.
  • urine contains a cocktail of almost all peptides which are highly immune selective antigens towards malformed or infected or senescence cells or causative agents of diseases.
  • urine derived antigenic peptides well reflect the life status of every single cell in the body, even when the diagnosis is impossible at the early phases of pathogens invasion or miss-function of ageing cells.
  • this scientific clue was used for more than 50 centuries as the base of urine therapy to prime APC abundant in gut with most preferable antigens for individual immunisation. That was the reason why the ingenious author of 50-century old manuscript "Shivambu Kalpa Vidhi" has paid a particular attention to the composition of diet during urine therapy.
  • MHC derived antigens MHC Class I and Cass II may source of the immune selective antigenic peptides (Falk a al 1991).
  • the purification of immune selective antigenic peptides from MHC may easy managf with integration of two conventional immunoaffinity and HPLC procedures.
  • the amino acic sequences of immune selective antigenic peptides of interest may be determined by manual sequencing techniques described elsewhere.
  • Example I Purification of integral antigenic peptides from human urine.
  • Concentrated urine will instantly undergo gel filtration chromatography (TSK 40) at 35°C or 45°-55°C.
  • TSK 40 gel filtration chromatography
  • the time for separating urinary peptides from any kind of low molecular size substances contained in urine needs around 35 minutes at 35°C. It is worth underlining that chromatography at higher 45°-55°C, remarkably decreases the time needed for the separation and fractionation of urinary peptides on gel filtration (TSK 40) to approximately 20 min.
  • the process of peptides separation from low molecular substances is achieved by gel chromatography column (TSK40 15cmx2cm) with chemically pure water with conductivity around 0.3 ⁇ Siemens/cm, having 35°C or 45°C-55°C temperature and pH 5.5-5.8. All peptide fractions were monitored at 210 nm and, after separately collected, either kept under room temperature and dehydrated before use.
  • Urine derived antigenic peptides fractions are used as building blocks of immunogenic mixtures wherein the plant derived HSP72/73 sub-class is neither non-covalently nor covalently associated with urine derived antigens. It is preferable to keep and mix both building blocks of immunogenic mixtures in dehydrated powder forms in any molar ratio.
  • Immunogenic mixtures with edible binary vaccine function are easily prepared from combination of either the dehydrated powder or the solution of HSPs any sub-classes purified from fresh alfalfa cell and antigenic peptides isolated from any suitable natural sources or synthetic ones.
  • each component of combination may mix in vitro at room temperature before use as edible binary vaccine.
  • the most preferable combination of plant origin HSP70 sub-class and urine derived antigens are in the molar ratio - 1 :3.
  • HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone & cytokine Nature Medicine vol. 6, N4, p. 435-441
  • the 90kDa molecular chaperone family structure, function, & clinical application. A comprehensive review Pharmacol. Therap. Vol. 79, N2, p. 129-168 20. Denis M. 1988
  • Protein phylogenies & signature sequences A reappraisal of evolutionary relationships among Archaebacteria, Eubacteria & Eukaryotes Microbiol Mol Biol Rev vol. 62, p. 1435-1491
  • Interaction of endoplasmic reticulum chaperone GRP94 with peptide substrates is adenine nucleotide-indipendent.
  • a new urea Broth-Based test tp detect Helicobacter ylori presence in upper gastrointestinal Biopsies.
  • Patent N 6,048,530 Stress protein-peptide complexes as prophylactic and therapeutic vaccines against intracellular pathogens
  • Patent N 6,030,618 Therapeutic and prophylactic methods using heat shock proteins
  • Patent N 6,017, 544 Composition compraiing immunogenic stress protein- peptide complexes against cancer and a cytoline", Issued on January 25, 2000;
  • Patent N 6,017,540 Prevention and treatment of primary and metastatic neoplastic diseases and infectious diseases with heat shock/stress protein-peptide complexes", Issued on January 25, 2000;
  • Patent N 6,007, 821 Metal and compositions for the treatment of autoimmune disease using heat shock proteins", Issued on December 28, 2000;
  • Patent N 5,997,873 Metal of preparation of heat shock protein 70-peptide complexes, Issued on December 7, 1999;
  • Patent N 5,985,270 Alignment N 5,985,270 "Adoptive immunotherapy using macrophages sensitized with heat shock protein-epitope complexes", Issued on November 16, 1999;
  • Patent N 5,961,979 Stress protein-peptide cmplexes as prophylactic and therapeutic vaccines against intracellular pathogens
  • Patent N 5,948, 646 Metal-organic compound-derived protein-peptide complexes
  • Patent N 5,837,251 Compositions and methods using complexes of heat shock proteins and antigenic molecules for the treatment of neoplastic diseases", Issued on November 17, 1998
  • Patent N 5,830,464 Compositions and methods for the treatment and growth inhibition of cancer using heat shock/stress protein-peptide complexes in combination with adoptive immunotherapy", Issued on November 3, 1998
  • Patent N 5,750,119 "Immunotherapeutic stress protein-peptide complexes against cancer", Issued on May 12, 1998

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Abstract

Cette invention est basée sur la découverte que les cellules de plantes herbacées fraîches peuvent subir un choc thermique lors de leur digestion et apporter ainsi à leurs consommateurs une dose journalière d'un mélange de micronutriments essentiels tels que des protéines de choc thermique et des antigènes microbiens immunogènes. L'invention concerne le procédé de production de vaccin, de composition de mélange sur la base de protéines de choc thermiques dérivées de cellules de plantes herbacées et de combinaison d'antigène immunospécifique permettant d'améliorer la résistance immune des consommateurs contre des micro-organismes pathogènes.
PCT/GE2001/000004 2000-10-18 2001-10-16 Procede de production de vaccin et de melange immunogene base sur des proteines de choc thermique (hsp) provenant de cellules de plantes herbacees WO2002024220A2 (fr)

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AU2001295814A AU2001295814A1 (en) 2000-10-18 2001-10-16 Vaccine comprising heat stress proteins from herbaceous plants
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WO2014086994A1 (fr) * 2012-12-07 2014-06-12 Alfa Biogene International B.V. Protéines de choc thermique (hsp) pour le traitement d'effets secondaires liés à l'imiquimod
CN112163567A (zh) * 2020-10-28 2021-01-01 杭州西非电子信息技术有限公司 一种自动消毒的指纹信息采集装置

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WO1994029459A1 (fr) * 1993-06-04 1994-12-22 Whitehead Institute For Biomedical Research Proteines du stress et leurs utilisations
US5837251A (en) * 1995-09-13 1998-11-17 Fordham University Compositions and methods using complexes of heat shock proteins and antigenic molecules for the treatment and prevention of neoplastic diseases
GEP20002333B (en) * 1999-05-19 2000-09-10 Method for Inducing Synthesis of Heat Stress Proteins in Herbal Plants

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014086994A1 (fr) * 2012-12-07 2014-06-12 Alfa Biogene International B.V. Protéines de choc thermique (hsp) pour le traitement d'effets secondaires liés à l'imiquimod
US10159709B2 (en) 2012-12-07 2018-12-25 Alfa Biogene International B.V. HSP for use in treatment for imiquimod related side effects
US11052127B2 (en) 2012-12-07 2021-07-06 Alfa Biogene International B.V. HSP for use in treatment for imiquimod related side effects
CN112163567A (zh) * 2020-10-28 2021-01-01 杭州西非电子信息技术有限公司 一种自动消毒的指纹信息采集装置

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