Background Art There are quite solid scientific evidences that a special class of proteins-the so-called Heat Shock Proteins (HSPs)-are accumulating in fresh herbaceous plant cells when exposed to heat. The molecular mechanism of this universal phenomenon was also circumscribed (Sorger 1991). Within minutes in heat shock condition, Heat Shock Factors (HSF) are coupling with Heat Shock Elements (HSE) inside of stress gene promoters. And thus, starts the intracellular scale up of transcription-translation of HSPs. This biochemical network swiftly amplifies the intracellular HSPs level at heat shock, i. e. between 36°C-39°C, and some of them like HSP72, HSP90 and small molecular mass HSP55-6 may reach up to 1% of total plant proteins. This 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).

The strong decline of heat stress genes function when cells are ageing must also be mentioned (Heidary at al 1994). And this synthesis decreasing pattern of ageing or wilting cells to provide HSPs was explained either by a disorder in the entirety of membranes/cytosol in cells or by the inability of HSF to assemble in trimmers, as the only transducers of heat shock signals to HSE which is the imperative element of promoters of heat shock inducible genes. So, the intracellular content of such ancient essential micronutrient as plant origin HSPs may oscillate according to age of plant cells and environmental conditions. In this respect, it is essential to point out the total loss of plant cells capability to provide HSPs synthesis after the processing of fresh plant biomas into conventional food/feed. And in the main, freshness (entirety/turgid) and

age of plant cells have appeared to be critical to initiate the functioning of heat shock genes (Nover at al 1991-99; Burke et al, 1985; Burke 1990; Kimpel, Key 1985; Singla et al 1997; Hernandez, Vierling 1993; Vierling 1990-91; Jaliashvili 1989-1999 ; Pollock at al 1993).

Originally, to our regret, plant derived HSPs pivotal role was limited exceptionally to heat tolerance. Now it is clear, that plant origin HSPs are those ancient essential micronutrients with an extraordinary chaperone synbiotic function for the consumers of fresh plant feed/food.

Except from speaking about, the role of plant origin HSPs as essential micronutrients for the well being of herbivores and humans has appeared practically not investigated up to this day.

Consequently, unfamiliarity with the role of plant origin HSPs as essential micronutrients has resulted in misunderstanding their role in building edible preventive vaccines against feed/food contaminated microbes. And such causative agents of feed/food derived diseases, are always a great many in fresh herbaceous plants. No doubt, during ingesting of fresh plant feed/food which are always contaminated with myriad of pathogenic microbes there occurs, in gut of mammalians, a build up of natural immunogenic mixtures as the base for oral binary vaccines with a preventive and curative function. And the imperative building blocks of such natural immunogenic mixtures in gut of herbivores and Homo may particularly be: -Non-Immunogenic essential micronutrients, such as plant origin HSPs, with high degree of conservation of 1D structures (Gupta 1995-98), Immunogenic cocktail of microbial origin antigens, with variable 1D 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.

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. No doubt, that the binary mixture: non-immunogenic plant origin HSPs and highly immunogenic microbial antigens, was the clue used for millenniums by heterotrophic consumers for their maintenance in the ocean of decomposers. And such pyramids-like cross relations between Life Levels may be connected with the fundamental role of autotrophic plants as the source of such essential micronutrients as

HSPs with extraordinary chaperone synbiotic functions for the well-being of at least such consumers as herbivores and Homo: -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.

And as a corroboration of this unnoticed process of building binary mixtures as imperative base of edible vaccines in gut of herbivores, here are some academic indications on the implication of plant origin HSP70: 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.

The sequences of plant origin HSP70, mostly inducible at 36°C-39°C, has appeared to stay highly conserved, whatever the species of fresh herbaceous plants which have been consumed as fresh food/feed (Gupta 1995-98; Vierling, Sun 1989; Feder, Hoffmann 1999; Macario at al 1999) ; The 1D structures of both plant cell origin HSP70 and human/animal cells derived HSP70, mostly inducible at 36°C-39°C, have appeared almost identical (Nover 1991; Numann 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; Panjwani at al 1999); APC with receptors to HSP70, particularly are abandoned in gut of herbivores and humans (Gaskin 1996); Daily norm of HSP70 as essential micronutrients may reach in gut up to 1% of total plant proteins at 36°C-39°C, only in the case of consuming fresh plants as food/feed ; Daily norm of HSP70 as essential micronutrients strongly depends on consumed fresh plant species integral bio-mass (Jaliashvili 1989-1996 unpublished results);

Therefore, the invention represented here is the World first attempt to mimic the immunogenic mixture which daily builds up in gut of almost all herbivores and Homo during the digestion of any fresh plant as feed/food, contaminated with innumerable micro-organisms. And render the precise synbiotic role of plant origin HSP70 in gut where APC cells are localised, and which are in reality the strongest preventive immune defence system of mammalians against feed/food derived diseases (Gaskin 1996; Hanson at al 1977-79; Brandtzaeg 1989; Miller- Schoop, Good 1975; Mowat 1987 ; Stokes 1984).

Plant origin HSPs-an ancient preventive synbiotics No doubt, an artificial composition of modern feed for industrialised livestock has arisen epidemic of TSE, multi-antibiotic resistant pathogens, etc., and has worsened the quality of meat and milk products. Slaughterhouses waste use as feed was identified as the main reason of the Mad Cow disease (or BSE) outbreak in EU (Prusiner 1991-1992; Chernoff a all 1995; European Commission, Directorate General for Research 2000). And this catastrophe, induced by non- physiological feed while consumed by industrialised live-stock, has once more re-confirmed that herbivores cannot remain healthy without grazing fresh herbaceous plants, in spite of the fact that in field condition fresh plant feed/food are contaminated with far more microbes than conventional feed. Although live-stock while grazing and chewing only fresh herbaceous plants are healthier and producing health-risk free products with perfect nutrient quality with longer shelf storage than the present feeding by conventional feed with probiotics, antibiotics, synthetic amino acids and vitamins (Hawken at al 2000).

So, a master-key question arises: -What are the enigmatic substance (s) which are supplied by fresh plant cells to live- stock in addition to known macro-and micro-nutrients, which may strengthen their stamina against myriad of pathogens? As a reminder, here are the all known macro-and micronutrients of fresh plant cells in the case of herbaceous forage grass-alfalfa: A) 80% of moisture (composed by cytoplasm, capillary, sub-structural and molecular water); B) 20% of solids, which is the integral indicator of the tremendously variable components of the following macro-and micro-nutrients: B1) Macro-nutrients such as: cellulose, lignin, pectins, fat/lipids, DNA/RNA, proteins;

B2) Micro-nutrients such as: vitamins, amino acids, nucleotides/nucleosides, flavanoids, alkaloid, antioxidants, bio-minerals, cathechins, oligosacharides, ribose/desoxiribose, lectines, saponins, resins, glycosides, essential oils.

And the only natural organic compounds that have urgently required to complete this classical list of plant cells components are: stress proteins, newly discovered essential micronutrients. Therefore, an attempt was done to classify plant cell containing stress sensible genes and their transcript-translate products, according to the stress factors, and some of which are additive in induction of stress proteins synthesis, e. g. drought and heat. Here is a short summary of discovered plant cell derived stress proteins: -Heat Stress Proteins (HSPs), -Cold Stress Proteins (CSPs), -Wound Stress Proteins (WSPs) or Protease Inhibitors (PI); -Drought Stress Proteins (DSPs), etc.

The main function of all kind of stress proteins is to defend plant cells from abiotic or biotic stress factors. And in comparison to genes, which permanently produce ordinary proteins in plant cells, these special set of stress genes start function only under appropriate stress condition e. g. heat, cold, wound, drought, etc. It is obvious now that fresh herbaceous plants are supplying to herbivore myriad of combinations of stress proteins in different climate conditions and seasonal periods. Every class of stress proteins possesses its own specific mode of action, and the most important and multi-panel appear chaperone function of HSPs and which doubtless may strengthen the stamina of herbivores. So, according to new scientific discoveries there is need to add to the above mentioned common organic compounds of plant cells, the special class of such essential micro-nutrients as: STRESS PROTEINS, and particularly the full spectrum of HSPs-the role of which for the subsistence of herbivores or Homo is practically not yet investigated.

As recently discovered, 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). Thus, 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.

There are great possibilities of additive synbiotic effects of different plant origin HSPs from combining themselves, as well as synergies together with any other class of stress proteins.

There are solid scientific research results, which are confirming the co-chaperoning ability of different molecular mass HSPs (van Eden, Young 1996; Morimoto at al 1990; Hsieh at al 1992; Jakob at al 1993 ; Wehmeyer at al 1996). The comprehension of plant origin HSPs as ancient non-toxic essential micronutrients, with chaperone function is at the origin of our invention represented hereafter: A) to reproduce plant origin HSPs as the crucial building block of essential micronutrients cocktail in gut of herbivores and Homo; B) to renovate plant origin HSPs natural content in conventional feed/food in order to partly restore their prophylactic edible vaccine function; C) to constitute the binary mixtures of plant origin HSP70 and immunogenic antigens combinations from selected pathogens as the base for the manufacture of a health-risk free new generation of Functional Processed Food (FPF) with preventive and/or curative vaccine function.

The following scientific evidences are confirming this innovative idea of plant origin HSP70 use as the base building block of oral binary vaccine: * The spectacular similarity of heat stress these genes top activity at 36°C-39°C range in almost all fresh herbaceous plant cells; The induction of HSP70 inside of the widest spectrum of fresh plant cells at 36°C-39°C caused by environmental heat stress or herbivores'gut temperature while grazing and chewing fresh feed/food; The almost identity of 1D structure of plant cell derived HSP70 with 1D structure of human cells and animal cells derived HSP70 (according to the amino acid sequences); The universal capability of HSP70 to associate with a wide spectrum of antigenic peptides at 37°C ;

* An astonishing efficiency of vaccines based on the complex of HSP70: antigens manufactured in vitro while tested against cancer, bacterial or viral diseases in laboratory research and in clinical I-11-III trials; 'The discovery of 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; The discovery of the failure to produce HSP70 by conventional feeds (pellets, silos, silage, hay) at 36°C-39°C, and the imperative need to restore the daily norm of at least plant origin HSP70 in feed of livestock.

The increase of feed derived disease cases among industrialised livestock particularly in winter when fresh grass as the only source of HSP70 is not available.

And so the urgent need is confirmed to re-learn the enigmatic natural composition of the stress proteins cocktail and their role in keeping the stamina of herbivores. And the time is just right to consolidate scientists for defining the network of HSPs as essential micronutrients and particularly their extraordinary chaperone synbiotic function in gut for keeping the stamina of herbivores and Homo. And subsequently, the inevitable investigation on HSPs enigmatic synergies with other classes of stress proteins. No doubt, that HSPs will be high on the imperative World scientific agenda within the first part of XXI century.

Here is our innovative attempt to restore the daily norm of at least HSP70 in processed feed/food as the most desired natural synbiotics and particularly now when antibiotics wide use as feed additives has been restricted in EU to prevent the accelerated creation of multi-drug resistant pathogens in live-stock. No doubt, substitution of antibiotics by plant origin HSP70 in feed may constitute the basis to produce human health risk free meat and milk products. As, it is obvious, that any innovation devoted to plant cells derived HSP70 applications in feed industry and in Life Sciences will be in line with huge social demands to restore harmlessness in conventional feed. Thereafter, the application of plant origin HSP70 will induce the research of other stress proteins as new natural prophylactics and therapeutics. And here are World new technological innovations, which are able to manage the scale up manufacture of a full spectrum of HSPs as the cheapest and the finest prophylactic against feed/food derived diseases.

The herbaceous fresh plant cells origin HSPs of the present invention that can be used include, but are not limited to, Heat Shock Proteins (HSP), which are also referred to inter- changeably World wide as Heat Shock Proteins (HSP) according to the below listed

classification with the recently defined scope of their chaperone functions (According to Neumann et al., 1989 ; Singla et al 1997; Thain, Hickman 2000).

Plant origin HSPs : classification and chaperone function Plant cell origin Heat Shock Proteins (HSP) are a special class of stress proteins whose intracellular level increases when herbaceous fresh plant cells are exposed to long-term heat stress or short-term heat shock conditions in the particular temperature range from 25°C up to 55°C and capable of chaperone multi-panel functions. Plant cell derived all HSPs are subdivided according to their molecular mass into 6 major sub-classes, each of which is composed of sub- isomers with tangible variation in molecular mass (in kDa): 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.

Among all these plant cell derived sub-classes of HSPs, we have considered the HSP70 sub-class as the most probable typical block of the gut derived mixtures that are able to build an edible vaccine function. Here is the 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 HSP100 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.

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 ; -In transporting of proteins across intra-cellular membranes (Haucke, Schatz 1997); - In buffering of external and internal stress signals via coating enzymes, receptors, etc.; -In correcting stress damaged abnormal 3D structures of enzymes, receptors, structural proteins; -In chaperoning each other to establish an integral and still enigmatic network of HSPs orchestra; -In enhancing cells immune efficiency against virus, bacteria, parasites, etc.

The general condition of any origin HSPs use in vaccine Dr. P. Srivastava (1986-2000) has established the only basic criteria of any origin HSP70/90 sub-classes for their use in vaccines: -35% homology is the lowest margin in 1D 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.

As a reminder, the homology of 1D 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.

And precisely this range of 1D structures homology between HSP70/90 relevant sub- classes, has 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.

The inventions for construction of HSPs based vaccine Wide spectrum of small peptides which are fingerprints of immunogenic proteins, are incompetent to induce strong immune reactions without HSPs or MHC of neither B cell nor T cell lymphocytes. That was the principal base to postulate: HSP70/90 sub-classes are the imperative building blocks of the permanent self-healing network in all somatic cells. And after the identification of specific receptors of the 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. And the clue of HSP70/90 sub-classes serving as molecular vectors was authenticated by the fact that these sub- classes 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;).

Thus 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). There are two known technological inventions for the use in vitro of any origin HSP70/90 sub-classes for the manufacture of vaccines: to associate immunogenic antigens with HSP70/90 sub-classes either covalently or non-covalently. Here are our short analysis of each of these methods, both of which have widely used for production of immunogenic complexes on the base of any origin HSPs : -The method of covalent association in vitro of immune selective antigenic peptides with HSPs. These resulting immunogenic complexes being used by StressGen

Biotechnologies Corp., as injected vaccines up to recently, when Antigenics Inc., endeavoured to cancel their right to use this invention covered by their patents'claims.

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. Thereby, almost all those immunoglobulin superfamily of glycoproteins, secreted by mature B cell lymphocytes cloned by influence of such complex may as well cross indenture with endogenic HSPs what eventually will induce severe side effects. And injection of such complex, no doubt, it will ultimately distort the normal network of endogenic HSPs chaperone orchestral function and consequently may induce non-predictable pathological syndromes (van Eden at al 1988 ; Wicker 1995; zur Nieden at al 1995; Zimermann at al 1988 ; Sarge at al 1995; Pockley 1998).

-The method of non-covalent association in vitro of immune selective antigenic peptides with any origin HSP70/90 sub-classes. And this immunogenic complex, in parallel with a ready like complex in vivo which was obtained from cancer tissue, being used by Antigenics Inc., as injected or oral vaccines against cancer and infectious diseases in laboratory testing and clinical I-II-III trials up to this day.

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. Thereby, 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 sub- classes of HSPs able to release antigens under the influence of ATP or low pH. Doubtless, this complex will have no or far less side effects in comparison with the complex wherein HSPs are covalently coupled with antigens. This method of non-covalent association of immune selective antigens with HSPs in vitro has allowed altogether to use the molecular chaperone flexibility and

adjuvant capabilities of HSP70/90 sub-classes (see US Patents: N6,030,618 February 29,2000 and N5, 935,576 August 10,1999 and N5, 750,119 May 12,1998).

Although this invention has achieved to manufacture the complex both in vitro and in vivo, wherein immunogenic antigens were non-covalently associated with each of HSP70/90 sub-classes, it is faced with technological dilemmas that have attracted our attention. And here is scope of different dilemmas in manufacturing the complex either in vivo or in vitro wherein immunogenic antigens are non-covalently associated with HSP70/90 sub-classes.

A). The dilemmas to obtain an immunogenic complex constituted IN VIVO: In US Patents: N 6,030,618, February 29,2000 and N 5,837,251 November 17,1998 have been described the extraction and purification of immunogenic complexes wherein antigens are non-covalently associated in vivo with each of HSP70/90 sub-classes. In order to acquire such immunogenic complexes there is the need to purify wholly HSP70 or HSP90 sub-classes from entirely malformed or infected human/animal somatic cells. During homogenisation of malformed tissues, centrifugation and careful purification of the entire HSP70 sub-class on ADP-chromatography and of HSP90 sub-class on Con A Sepharose, none of these sub-classes lose the selected immunogenic antigens with which they are non-covalently associated inside of only malformed cells. And at the final stage of this invention, the yield of the immunogenic complex for each sub-classis of HSPs was extrapolated from Ig of human malformed tissue as follows: lmg of HSP70; 0. lmg HSP90 and O. Olmg gp96.

Unfortunately, in none of these US Patents was mentioned any dilemmas for such immunogenic complexes production wherein in vivo antigens are non-covalently associated with each of HSP70/90 sub-classes and particularly: a). Impossibility at early stages of cancer or infectious diseases to obtain critical mass of malformed tissues for the extraction of therapeutic doses of HSPs with cancer selective antigens; b). Demand of immune specific antigens standard load of HSPs in the case of different cancers or joint invasion of different intra-cellular pathogens e. g. virus (HIV) and bacteria (tuberculosis); c). Overload of HSPs with pseudo-antigens in artificially infected tissue cultures and low immune efficiency and therapeutic miss-dosage of such complexes; d). Impossibility to distinguish the dividing line between malformed and normal cells to obtain immune-selective antigens using HSPs as vectors, in order to use this complex for therapy of cancers or infectious diseases.

B). The dilemmas to obtain an immunogenic complex constituted IN VITRO.

US Patents: N 5,935,576 August 10,1999 and N6,048,530 April 11,2000 have described a procedure of construction the complexes wherein immune selective antigens are non- covalently associated with any origin HSP70 and HSP90 sub-classes in vitro. According to this invention the immunogenic complexes can be obtained in vitro when there is a problem at early stages of infection or malformed cells. Thus, 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: A). Pre-treatment with either ATP or low pH in order to remove any antigens or peptides that may associated with HSP70/90 sub-classes of interest, just after their purification from natural sources. And particularly when the ATP procedure is used, excess ATP is removed from the preparation by the addition of apyranase. While the low pH procedure is used, the buffer is readjusted to neutral pH by the addition of pH modifying reagents. However, there was not any indication the need of additional separation procedure of HSPs from either apyranase or pH- modified reagents and the consequent loss of such expensive HSP70/90 sub-classes that are an imperative building block of the immunogenic complexes.

B). Incubation of purified and pre-treated HSPs with immune selective antigens in special buffer with pH 7.2-7.5 and reagents from 25°C up to 65°C. And particularly after purification and above mentioned pre-treatment HSPs (9 ug) was incubated with immune selective antigens (1 ut) in a suitable binding buffer such as one containing 20mM sodium phosphate, 350 mM NaCI, 3mM MgCI2 and 1mM phenyl methyl sulfonyl fluoride (PMSF) during 3 hr at room temperature.

However, there was not indication the influence of each component of this incubation buffer on the loss of HSPs chaperone capability neither on the reactivity of amino acids groups of different immune selective antigens during incubation at mentioned above temperatures and corresponding times. There is no explanation why it has been removed PMSF from incubation

buffer while HSPs were incubated with immune selective antigens at 37°C for 1 hr or 60°C-65°C for 5-20 minutes.

C). Centrifugation to separate the complex from unbound HSPs and antigens via special UF membranes (CENTRICON 10). Under the influence of sedimentation coefficient, UF- membrane can separate complexes from not-associated immunogenic peptides having medium and small molecular masses, below lOkDa molecular mass. Consequently, we pay attention to such tech-artefacts that absorption/filtration stage of incubation medium via UF-membrane having lOkDa retention will inevitably collect both HSPs associated and non-associated with immune selective antigens. As it is clear that during incubation at any temperature there is no chance to manage 100% non-covalent association of immune selective antigens with HSPs. In contrast, this UF-membrane with lOkDa permeating capability will concentrate also HSP70/HSP90/gp96 that are not associated with antigens and possess 7-and 9-fold higher than 10kDa molecular masses.

And this is a very dangerous technical drawback, as it is a well-known fact: the absence of any preventive or curative effects of HSPs alone. And in reality there is no standard complex of HSPs with immunogenic antigens. Thus, at the end of this technological stage an index is absolutely required to indicate the correlation between the complex HSP: antigens and non- associated HSPs separately, for each temperature range 25°C-37°C and 60°C-65°C.

D). Removing the complexes from UF membranes was not mentioned in this invention. It is clear that the complexes cannot be used in injected vaccines before they will not be de- adsorbed from UF-membranes. There is no indication of an additional stage needed for washing out the complexes from UF-membranes. No doubt, de-adsorption also will induce a remarkable decrease in the immunogenic complex yield such as HSP: antigen.

E) 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.

It is doubtless, that all these tech-stages: filtration via UF-membrane of the complex and its de-adsorption/desiccation are the most wasteful ones. Thereby all these stages of HSPs based vaccine production need further refinement and improvement in order to decrease the significant loss of both most valuable and expensive organic compounds of vaccines.

F). Monitoring the yield of the immunogenic complex constructed in vitro was assayed by an expensive procedure via the separation of antigens and HSPs and their complex with the help of SDS-PAGE.

It should be underlined that this technological procedure is unable to indicate which HSPs are in complexes and which are not, as SDS reagent itself accelerates the dissociation of the complexes.

Moreover, SDS-PAGE technology could not monitor the small difference in molecular mass between non-associated and associated HSPs with antigens.

Neither will SDS-PAGE point out the real scenario of loading immune selective antigens having different amino acid sequence, for the reason of its inability to separate such low molecular mass immune selective peptides which are constituted by 5-15 amino acids.

It is obvious that SDS-PAGE monitoring technology will not allow to point exactly the real complex yield, neither the correlation index between associated and non associated HSPs with antigens.

G) Administration of this immunogenic complex: in addition to injection, oral arrangement was advocated too.

And in contrast to accepted postulates of reference, 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.

In these above cited US Patents, all of which are devoted to any origin HSPs application in pharmacy, nothing was mentioned that this invention for the immunogenic complex production in vitro has dilemmas such as: How to completely separate non-covalently associated HSP70/90 sub-classes with immune selective antigens from not at all associated HSP70/90 sub-classes after incubation in vitro?

How to monitor the yield of real immunogenic HSPs: antigens complexes from non-immunogenic HSPs alone? * How to achieve the optimal dosage of efficient vaccines if there are no entirely immunogenic complexes at the end of such a cost additive technological procedure? As mentioned above, after all related technological problems and dilemma, this immunogenic complex may originate very risky application drawbacks due to dose dependant opposite effects. And particularly these complexes, when used as injected vaccine in: Small doses primes lymphocytes to attack causative agents or malformed somatic cells, but, Over doses persuade lymphocytes to ignore the same antigens.

That was the reason why Dr. C. Slingluff, a cancer immunologist at University of Virginia has made such a brisk remark:"HSP are very reasonable approach, but Antigenics' technique is like giving a crude plant extract instead of a purified drug. To refine therapy, Antigenics would need to know what the native ingredients are-which it doesn't" (see New Scientists 24 February 2001, N2279, p. 41).

And if for the same goal, Nature has achieved in gut a daily build of mixtures with irreproachable edible preventive vaccine function and wherein the imperative building binary blocks are such essential micro-nutrients as plant origin HSPs with a high degree of conservation in 1D structures and daily fresh feed/food contaminated microbes'derived immune selective antigens with variable 1D structures, has raised the crucial question: -Is there a real need of all these cost additive and incredibly wasteful masterminding in vitro to achieve such non-denominational immunogenic complexes and at last to disintegrate them in stomach after oral administration? All these academic suggestions and the clue of the new invention: the mixture of plant origin HSPs and immune selected antigens were used as the base to manufacture in vitro edible binary vaccines, with monitoring easily attainable.

And there is doubtlessly an urgent need for such a simple alternative to the above described inventions which are advocating oral vaccines on the base of the same composites, but in complexes, the construction of which needs highly cost additive and over-complicated technological stages.

Additional dilemmas of HSPs use in vaccine

Given the universal property of HSP70/90 sub-classes, of any origin, to chaperone an efficient representation of immune selective antigens to lymphocytes, the dilemma remains as to how to use this bright clue to tune the colossal destructive capacity of an orchestra of almost 3 trillion immune competent cells. As none of the existing tech-inventions were able to overcome the scarcity of immune selective antigens in order to achieve the targeting of molecular vectors, such as HSP70/90 sub-classes, for priming lymphocytes.

All existing tech-inventions, which have already achieved, either in vivo or in vitro, the complex wherein molecular vector-carriers are HSP70/90 sub-classes of any origin, are absolutely useless as there is no immune selective antigens, since it is practically impossible to obtain them, when: . Detect the aetiology of starting diseases at their early phase, when virus, bacteria or any other causative agents or malformed cells are utterly present in minute numbers in the body;' Establish the correct diagnosis of the spectrum of causative agents of diseases when there are synergies between several viruses and bacteria; Determine the real causes of symptoms of diseases, when crossed influences occur in such a wide spectrum of abiotic causative factors as: agrochemicals, medical and narcotic drugs, heavy and toxic metals, fungal toxins, alcohol, high radiation, psycho-emotional trauma, free radicals, xenobiotics, environmental pollution, etc.

Ascertain which somatic ageing cells and particularly when they get out of control.

Furthermore, severe problems should be underlined during the extraction and purification of human origin HSPs and/or HSPs: antigen complexes, e. g.: a) Yields from human cell origin in HSPs solely and/or complexes of HSPs: peptides, from 1 g 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): -HSP70 is around 1000 lug, -HSP90 is about 150-200 ng ; -gp96 is only 10-20 ; j. g.

Such low yields in HSP70/90 sub-classes from human tissues will always face the additional limit due to real sizes of cancer or tumour tissues. Furthermore, as HSPs derived from normal tissues have neither prophylactic nor therapeutic effects, so, there is as acute need to

strongly determine the exact size of cancer/tumour tissues for the extraction of HSPs-antigens complexes. For that reason, when the size of cancer/tumour at the early phase of their development in human body is too small, there is no chance to extract HSPs from human cells with immune selective antigens. b) There are no common antigens between cancer patients. Such strong personifications of cancer antigens have limited the production generalisation of the HSPs-antigens complexes from human cell origin to treat other patients (Paladino at al 1987). That is the reason why the production cost of the HSPs-antigen complex as personal vaccines ranges from US$10 000 to 20 000 (see Antigenics Inc-white paper. doc 14/08/2001, by B. Goldman). c) The extraction of HSPs-antigens complexes from human cell origin, with volunteers, is faced with ethic and moral problems. Moreover, this complex is absolutely non-practical for curing other patients neither for the same disease nor diseases having another aetiology. d) The employment of artificial cultures of malformed human/animal origin tissues/cells with any causative agents in order to purify HSPs-peptides complexes for preventive vaccination of populations may raise serious risks of prions, amiloid beta and other proteins miss-folding in malformed cells. It is obvious that abnormally formed prions and amiloid beta proteins will associate with HSPs (Chernoff at al 1995; Perez at al 1991; Telling at al 1994). And in the inevitable case of vaccines contamination with the complexes of HSPs: prions or amiloid beta may cause the accelerating development of such mental disorders like Creutzfeld-Jakob disease, Alzheimer disease, etc. e) The application of artificially infected human/animal origin tissues/cells cultures with viruses, e. g. human immune-deficiency virus (HIV) for a targeted association of HSPs with HIV specific antigens, will never be universally cross effective against all HIV-1 Group M families e. g. A, B, C, D, F, H, J, or against HIV-1 Group N or Group O and etc. (Ward 1999; see International AIDS Vaccine Initiative-Scientific Blueprint 2000).

There is also a need to underline the severe problems which gene-technology faces when recombinant HSPs are used for the scale up manufacture in vitro of the HSPs: antigens complexes, and particularly: f) All recombinant HSPs of human/animal cells origin expressed in E. coli will have a far more severe immune response in the case of their use in vaccines. And the high probability of this assumption occurring can be supported by the fact that E. coli has no Golgi apparatus. The Golgi apparatus is a highly specialised sub-cellular system that provides post-translation

modifications to almost all newly synthesised proteins, which avoids them to be recognised by the own immune system. Golgi apparatus exists only in human/animal and plant cells. That is the reason why the use of microorganisms as"Single Cell Production Factories", which lack of Golgi apparatus, will produce recombinant HSPs which lack natural post-translation modification. So the recombinant HSPs in vaccines, or such"undressed HSPs", will be far easier recognised by the human immune system. Consequently, the efficiency of vaccines on the base of recombinant HSPs may significantly decrease, as antibodies will eliminate almost all of them.

Moreover, antibody against recombinant HSPs will recognise also appropriate endogenic HSPs and initiate severe autoimmune diseases.

The evident failure to represent microorganisms as"Single Cell Factory"as equal to human or animal or plant cells has shifted World biotech industries from microorganisms towards the far better"equipped"plant cells. According to 1998 report of Mr. W. S. White, vice- president of Monsanto, the use of plant cells as advanced and most desired"natural factories", just became a commercially viable reality for the production of any kind of therapeutically valuable proteins, and among them HSP70 too.

And the crucial question arises: is there a real need to produce recombinant HSP70/90 sub-classes of human cell origin at all, just after the invention which makes the idea of recombinant any origin HSP70/90 sub-classes still-born, as it allows a non-waste and scale up manufacture of HSP70/90 sub-classes of herbaceous plant origin which possess an almost identical 1D structure with HSP70/90 sub-classes of human/animal cells origin.

The significance of post-translation modification of proteins is in attributing their natural functions. In addition, it mysteriously defends them from their own immune system attack, as post-translation modification of proteins is provided within sub-cellular structures: Golgi apparatus and Endoplasmic Reticulum (ER).

That is the reason why almost all recombinant therapeutically valuable proteins expressed in E. coli, have already faced the major obstacle of additional cost additive post-translation modification for obtaining the solubility and the attribution of natural functions. Moreover, as a rule, all recombinant therapeutically valuable proteins without natural post-translation modification may result in autoimmune disorders. Recently, this dangerous trend of crops re- engineering as sources of vaccines was pointed out and condemned (see Resurgence N127-128, 2001, pp. 19-37). And here is the scope of old tendencies and all the new trends in the construction of injected and oral vaccines.

New trends in the construction of vaccines There are two imperative methods in vaccination: a) inoculation and b) immunization, in order to tune the colossal destructive power of immune competent cells when primed with immune selective antigens: a). The method of inoculation: An injection of minute living infective pathogens into humans or domestic animals is usually followed by a mild but non-fatal infection in the patient, with a resulting immunity to the virulent pathogens. Live pathogens inoculations always induce strong responses from both B cell and T cell lymphocytes. But, nowadays, the method of inoculation of live pathogens is rarely used, considered as very treacherous, and the method of immunisation by killed or attenuated pathogens is being very much preferred, as far less hazardous. b). The method of immunisation : Active or Passive immunities and Vaccines types.

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. Fortunately HSP70/90 sub- classes 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 (antiserum) to detect infected pathogens only in lymph and blood stream. But there is the problem of low efficiency of ready antibodies as they are recognised by B cell lymphocytes as common antigens, which should be neutralised.

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. Here are major vaccines subtypes and some predicaments while they prime mammalian immune cells.

. Classical Vaccines comprise : a) Killed pathogen, or b) Live-attenuated pathogen.

There are several problems for the scale up production and use of classical vaccines: * Killed or attenuated pathogens could not mimic live ones in infecting somatic cells and they are much safer to use in vaccines. But, there is no 100% guarantee for either killing or attenuating all pathogens during their scale up their manufacture as the only base of vaccines.

Killed or attenuated pathogens based vaccines mainly induce cloning of B cell lymphocytes. And the antibody-mediated immune response neutralises pathogens only in lymph/blood, but are practically useless against the portion of pathogens that have already infected somatic cells.

The need of large amounts of human/animal tissues for pathogenic micro- organisms scale up cultivation before their killing or attenuation, has raised ethic and ecological problems to obtain such large volumes of human/animal tissue sources and the waste disposal after their production ; Animal tissues use, as a base of cultivation medium, always contain myriad of undesirable retroviruses, which may be co-purified with cultivated selected pathogens, resist killing or attenuation procedures and infect humans or animals during vaccination; The cultivation of selected strains of pathogens never gives those spectrums of natural antigens, for the reason that live pathogens are permanently mutating and changing.

Thus, antigens derived from single strains of long-term cultivated pathogens are far less effective than those derived from live pathogens.

* Genetically engineered vaccines: 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.

These 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.

In order to achieve the process of infection-like scenario, synthetic genes were inserted inside of so-called health-risk free vectors, which may be either non-pathogenic live microorganisms or crops. There are several vectors for 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. There are several solid technical problems in the scale up manufacture of such vaccines and particularly: 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. Thereby, they could not target T cell lymphocytes on live pathogen infected somatic cells. There is also the increased ecological danger if genetically modified live vectors will change common hosts species, as has happened with vaccinia viruses used as vector for the human small pox eradication campaign and which established themselves in a new reservoir, namely the buffalo. The unpredictability of living recombinant micro-organisms should be also pointed out, for the reason of widening the spectrum of recombination between viruses. A severe virulence jump was already reported after the insertion of foreign genes into non-virulent virus.

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.

Nonetheless, in spite of such visible advantages of gene engineered edible vaccines on the base of crop vectors as: no syringes, no refrigeration, reduced costs of immunisation and vaccines production (as there is no need of antigens purification), nobody can predict their acceptance by the European society, which has practically rejected the first generation of genetically modified crops.

Furthermore, there are technical and ecological problems which almost overlap all the above mentioned advantages and particularly: * The missing standard dosage of gene engineered edible vaccines, as none of the crop vectors used so far come in standard size, The declining efficiency of edible vaccines during the storage of mature crops; The gut inactivation immunogenic proteins of edible vaccines; The necessary addition of adjuvants such as cholera toxin to enhance the uptake of antigens and increase the effectiveness of edible vaccines; The genes antagonism, plants don't grow well when they produce foreign proteins ; The ecological problems e. g. cross pollination between same food and vaccine crops; The health risk for Homo e. g. food induced allergy, etc.

And once again the incisive question arises: Is there a real need for non-predictable and cost-additive genotype engineering of food and feed crops, if the opportunity is developed to produce Functional Processed Food (FPF) with edible vaccine function ? * How myriad of herbivores consuming only fresh plants have escaped millenniums in the ocean of micro-organisms and what are the building blocks of such natural edible vaccines? Hereafter, the attempt is made to clarify what are such highly probable natural edible vaccines from fresh herbaceous plant, which were solely used by herbivores for millenniums.

* Edible Natural Vaccines:

The kingdom of herbivores and partly Homo sapiens permanently use fresh herbaceous plants as natural daily feed/food and as a rule drink water thereafter, both of which are contaminated by myriad of bacteria, virus, protozoa, fungi, parasites. Therefore, the survival rate and life longevity of herbivores or Homo directly depend on a network of first line immune defence which adapts their stamina to permanent destructive pressure from feed/food and water and air innumerable contaminated micro-organisms (Mestecky, McGhee 1989 ; Service 1994).

This first line of the immune bastion is represented by secondary lymphoid tissues, whose location in body spreads out between 3 structures, in their major part within: Unencapsulated lymphoid regions of the gut submucosa, and the minor part among: Unencapsulated lymphoid regions of the respiratory tract, and Unencapsulated lymphoid regions of the urogenital tract.

And 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. 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).

There are astonishing synergies in the orchestra of immune cells action between both primary lymphoid and myeloid tissues instituted within secondary lymphoid tissues, total the number of which is estimated around 3 trillions. Here are scientific evidences that have indicated the vital role of HSPs in the astonishingly selective and synchronic network of lymphoid and myeloid tissues derived immune competent cells in their capacity to distinguish body's own normal cells from infected or abnormal or foreign cells. Now it is clear that both lineage of immune defence cells are providing highly selective rejection and annihilation of what appears foreign or malformed via the HSPs conducted integral network of the following super-families of special proteins: * Immune globulin dg') 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.

With the help of lymphokines, 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. But none of these lymphokines e. g. Interferon (INFa- (3-y) or Interleukins (IL1-8) are able to prime selectively any lymphocytes like HSP70. And it has appeared that 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. In addition, HSP70 may tune the expression of several lymphokines, particularly interleukin-lp, interleukin-6, tumour necrosis factor-a, 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).

* Cluster of Differentiation (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. Several of these glycoproteins e. g. CD14 on the surface of monocytes ; CD91 on the surface of monocytic lineage as well as on the surface of hepatocytes, fibroblasts, karatinocytes ; and CDllb+ 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. It was well demonstrated that CD-receptor- mediated endocytosis by HSP70/90 sub-classes of any origin, are able to prime with as little as 0.001-0.01 pg of immune selective small peptides the whole orchestra of monocytes, macrophages, APC and CD8 T cell (Binder at al 2000 ; Asea at al 2000 ; Arnold-Schild at al 1999).

Major Histocampatibilitv Complex (MHC) defines both intra-and extra-cellular antigens that are processed in cytosol and particularly within Endoplasmic Reticulum (ER) and Golgi Apparatus (Golgi) of professional antigen presenting cells or any somatic cells in body.

There are two major classes of MHC: Class I and Class II, which help T cells to distinguish normal cells from malformed or infected somatic cells. MHC class I binds intra-cellular origin antigenic peptides in ER outside of the Golgi and display them abundantly on the surface of cells. While 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. There is need to underline that T cells are accepting MHC class I represented peptides on the surface of somatic cells as an indication of intracellular disorders, while 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. There is still a lack of knowledge on the precise molecular mechanism in the processing and protection of immune selective antigens from an attack by exo-and endo-peptidases in ER and Golgi up to their display to MHC Class I and MHC Class II. And recent solid scientific research results have pointed out that 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 (Arnold-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).

This phenomenal network of the first line of immune bastion in gut relies for a great part on the nature of feed/food derived composites. It is doubtless that there is no such single universal antigen against myriad of pathogenic micro-organisms, which are contaminating fresh herbaceous plant feed/food and water. So, it is clear that ready edible natural vaccines do not exist in Nature and they should result from the mixtures that are building daily in gut. And fresh herbaceous plant species and water, which are always contaminated with innumerable strains of micro-organisms, are only what most herbivores were using as daily feed/food for millenniums.

These academic considerations lead to assume that the base of natural edible vaccine is the mixture which is daily constructing in gut while herbivores are ingesting natural fresh plant feed/food and water which are contaminated by diverse types of micro-organisms. And we

presume that the imperative building blocks of this regular immunogenic mixture in gut should be: A) Molecular vector-carrier: one or more with unvaried 1D structure, of plant origin. and B) Immune selective antigen : several with varied 1D structures, of microbial origin.

And face such daily emergencies, the gut localised the first line of immune bastion predominantly use this immunogenic mixture to build daily requirements of edible binary vaccines in order to prevent fresh plant feed/food and water derived causative agents of diseases.

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). So, 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. And 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). b) Coincidentally gastric acid is attenuating a great part of the microbes, which are contaminating fresh plant feed/food daily ingested by herbivores. And 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. And in these immunogenic mixtures, wherein HSPs are predominantly plant origin, capable to protect microbial immune selective antigens from further digestion in gut. These immunogenic mixtures are forwarded from stomach to small intestine where pH is favourable for plant origin HSPs to chaperone immune selective microbial antigens towards APC, which are abundant in this part of gut. It is clear that such ubiquitous essential micronutrient, as the plant origin HSPs are should be an imperative building block of natural daily edible vaccines in gut of herbivores. And as a affirmation of this assumption, there are such scientific evidences as: 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. No doubt, 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.

Therefore, plant cell derived HSP72 may remarkably enhance the efficiency of this first line of immune defence instituted in gut. There are also scientific evidences which confirm the relevant molecular vector and super-cytokines functions for exogenous 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 (Fujihara, Nadler 1999; Asea at al 2000). d) 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. Since 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.

And within minutes, 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 1. 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.

So, 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.

Now it is obvious that the herbivores'gut constructed immunogenic mixtures, with edible binary vaccine function, should be among the ideal ones as provided after millenniums of protection of all herbivores against multiple diseases. And the nature of the imperative building blocks of this regular immunogenic mixtures is also clear, which is daily generated in gut while herbivores ingest fresh plant feed/food and water contaminated with countless microbes. Here is the blueprint of natural edible vaccines: Molecular vector-carrier: one or more with unvaried 1D 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 micro- organisms. For this reason, 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 1D 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.

And highly inducible plant origin 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. As a confirmation of this our postulate, is the astounding similarity between 1D structures of HSP72/73 sub-classes derived from plant and human/animal cells. 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.

Similar beliefs by Fujihara and Nadler (1999) have validated for 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".

And the following irrevocable advantages that: a) 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.

Here are some indications of therapeutic doses and the spectrum of 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 sub- classes as molecular vectors, range from 0.01-1.0 mg/kg of body weight, for the injected immunisation of mammalians. On the base of laboratory and clinical research results, it has been established that extremely low quantities of such vaccine are sufficient to stimulate both B cell and the cytotoxic T cell responses. And according to US Patent 5,837, 251, issued November 17, 1998; the recommendation of a single therapeutic dose 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".

That was an astonishing and sensational discovery in the field of art methods for determining drug dosage levels, that therapeutic doses of HSPs based vaccines is the same for 25g mouse with a body surface of 0.0066Sqm as for a 60 kg human adult with body surface area of 1.6 Sqm. Two years later another US Patent N 6,048,530, issued April 11,2000; is recommending the same single therapeutically needed dose of a given HSP-antigen complex: in the range of 0.1-0.5 mg/kg, while for almost 80% positive curative rate, e. g. in the case of cancer, the total quantity used is up to 6 mg of HSP70 per 75kg-patient, which should be divided into three or five injections, with one or two weeks intervals.

These extremely low quantities of injected vaccine which used 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 sub- classes (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-1 and HIV-11, 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.

The use of 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.

Disclosure of the Invention 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.

There is proposed 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. These vaccines are used as healing or prophylactic means for human being.

There is proposed 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.: HSP110, HSP90, HSP70, HSP60, HSPs 55-6 kDa and/or by their combination.

There is proposed 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.

There is proposed 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.: HSP110, HSP90, HSP70, HSP60, HSPs 55-6 kDa and/or by their combination.

There is proposed the 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.

There is proposed the 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.

There is also proposed the method for designing an immunogenic mixture constituted either in situ or in vitro by mixing herbaceous plant cells derived HSPs with integral individual antigen derived from human urine containing proteins, polypeptides, peptides, lipopolysaccharides.

There is also proposed the 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.

The obtained composition is used as edible binary vaccine for a strong individual healing of causative agents of opportunistic diseases.

There is also provided the 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.

There is also provided the method for constituting of 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.

There is also provided the use of 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.

There is also provided the 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 There is proposed the method of producing the integral internal antigen from a single patient promotes to collect its urine in a sterile container, concentrating and identifying proteins, polypeptides, peptides, lipopolysaccharides by known methods of gel-chromatography or affinity chromatography or ion-selective chromatography, etc., with subsequent computerising them as finger-prints of causative agents of patient's diseases.

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 There is proposed the new 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 micro- organisms or their synthetic analogues, the origin of exterior antigen is the culture of malformed somatic cells of animals which get out of control under the influence of senescence or of abiotic or biotic stresses or their synthetic analogues.

These 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..

There is proposed 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.

There is also proposed 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.

There is proposed 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 There is proposed 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 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..

There is proposed 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.

There is proposed the 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.

There is proposed also the combination of 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. And 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. And on the discovery that 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.

Immune selective microbial peptides from any causative agents of diseases derived from feed/food and water, presumably are the antigenic complementary blocks of this immunogenic mixture. While 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; * Grazed fresh grass mass is only mixing with mucus in the rumen and quite a large part of the herbaceous grass pulp can be returned to the mouth as cud for re-chewing and optimisation of the endo-biochemical exothermic reaction in this plant bio-mass; The full digestion of the major part of proteins derived from grazed fresh grass cells occurs in the abomassum, the true stomach of ruminants.

We are giving account of these feeding specificity of ruminants and keeping in mind the lowest intracellular level of HSPs in non heat shocked grass plant, and have attempted to establish if the rate of HSPs synthesis will increase when grazed grass undergoes a heat shock between 36°C-38°C in the rumen. We consider this our approach as the only way to determine, on the base of experimental ruminants, the real daily natural norm of such peculiar essential micro-nutrients as HSPs, derived from fresh herbaceous plant cells with their phenomenal molecular vector-carrier function. As a control, in comparison to fresh herbaceous grass volume, we have used 4 times more of the same plant species biomass in the form of pellets or hay or silage or silos.

On the base of these feeding peculiarities of ruminants, we have considered that investigating the intracellular level of HSPs in fresh grass, before and after grazing by experimental cows, should be provided particularly on the level of feed digestion in rumen, in order to point out if fresh grass undergoes a heat shock in the rumen far before full digestion of proteins starts in the abomassum.

Therefore, we have estimated the influence on the intracellular level of HSPs in fresh grass cells under"incubation at 36°C-38°C probable range"in the rumen of experimental cow in

field condition. We express our gratitude to Prof. Dr. A. Bakuraze for advises in providing surgery on rumen for extracting permanently chewed bio-mass in experimental cows. Before grass pulp was returned to the mouth as cud for chewing, samples of around 100 grams of grass biomass were taken out from rumen for HSPs analyses. The experiments were done in monoculture field condition where only alfalfa (Medicago sativa) was cultivated and also in grassland where more than 5 grass species were growing. The samples of fresh herbaceous plant biomass were taken out from experimental rumen every 30 minutes after starting grazing immediately weighed and frozen before the HSPs spectrum was examined. For comparison, we have collected manually the same quantity of fresh grass biomass from the same grassland, and which was also immediately frozen after harvesting before HSPs extraction and research.

As a double control to fresh alfalfa with a moisture of around 78%-82%, we have used the same alfalfa species in the form of dehydrated pellets with a moisture of 10%-12% and hay with a moisture ranging between 22%-25% or silage or silos with moisture of around 55%-65 %.

All these conventional feeds were given to experimental cows as control instead of fresh grass in the same field condition manually inserted in common paper bags.

All experiments were provided in field condition in the early morning, 5-7 a. m., at air temperature around 8°C-14°C during the harvesting season of fresh alfalfa (May-October).

Rumen temperature was monitored before every time 100 grams of grass biomass taken from rumen. And a common range of temperature fluctuation in experimental rumen was around 36°C-38°C. Grass pulp from rumen was immediately weighed and frozen before homogenisation and extraction of HSPs under low pH of 5.0-5.5.

And after mechanical extraction and dehydration of the plant cells HSPs enriched fraction, we have investigated the comparative spectrum of HSPs in heat shocked fresh alfalfa cell and heat shocked conventional feed. Both spectrum 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. We have examined the full spectrum of herbaceous plant cells derived HSPs sub-classes, with standard procedures to determine proteins molecular weights with gel- chromatography on TSK-50 and further identification of one or two dimensional gel- electrophoresis and HPLC (Jaliashvili 1989-2000, see PCT/GE 00/00003, Issued 19 May, 2000).

As alternative method for purification of HSP70/90 sub-classes we have used two step purification indicated by Denis (1988).

All research results of the comparative analyses of HSPs contents in fresh alfalfa and processed bio-masses have pointed out that only fresh alfalfa cells can sense a heat stress at 36°C-38°C and increase HSPs synthesis within the rumen of experimental cows. And hardly detectable traces of HSP70/90 sub-classes content were found in 200-400 g bio-mass of non heat shocked fresh alfalfa which was harvested directly from grassland at 8°C-14°C air temperature.

Comparative analyses of HSPs content inside of heat shocked and non heat shocked alfalfa cells -has doubtlesly demonstrated a remarkable increase in synthesis of almost all HSPs sub-classes in samples taken from rumen between 30-60 minutes. Maximum rates of HSPs synthesis were reached already within 30-120 minutes of starting grazing and chewing fresh alfalfa biomass.

While none of the conventional feeds cells were able to provide a synthesis of any HSPs sub- classes during the same period of ingestion in rumen. We were unable to detect any of the HSPs sub-classes content from bio-mass samples even four time larger, either from pellets or hay or silos or silage, taken from rumen 30-240 minutes after starting the digestion of any of these conventional feeds.

These results obtained on experimental cows in field condition fully coincides with the capability of fresh alfalfa to promptly increase HSPs synthesis in green-house and field conditions at 36°C-38°C air temperature (Jaliashvili 1989-1996, unpublished results).

Now it is clear that grazed fresh alfalfa cells sense heat shock at 36°C-38°C in rumen and within 10-60 minutes and is significantly increasing HSPs synthesis up to 1% of total biomass proteins. Part of speech, we have monitored with the help of different plant origin pigment (yellow and red) that experimental cow after grazing fresh alfalfa pulp was kept in rumen for quite a long period. Some part of the pulp was moving as cud for re-chewing up and down mouth-rumen-mouth-rumen, before sending fresh grass feed to abomassum. Therefore, it is obvious that the time scale to establish a heat shock condition in rumen for grazed fresh plant cells at 36°C-38°C should be considered over 60 minutes. Such a long incubation time of fresh grass plants in rumen at 36°C-38°C has, no doubt, managed the maximal increase in intracellular HSPs level in fresh grass intact cells, up to 1 % of total plant bio-mass proteins.

And in order to establish a correlation between intact and disrupted plant cells, we have taken out from rumen samples for cytological investigation of fresh alfalfa biomass after grazing and chewing by experimental cows in field condition. Cytological analyses of alfalfa pulp from rumen have well verified that up to 80-90% plant cells remain intact in rumen, during at least the first 30-60 minutes, after starting grazing and chewing fresh alfalfa in mono-culture field

condition. It has appeared that the process of grazing only cuts fresh plant in short fragments but does not promptly disrupt all cells. So in alfalfa undamaged cells at 36°C-38°C, the intracellular level of almost all HSPs sub-classes is dramatically increasing within minutes, and particularly remarkably the HSP72 sub-class and the small molecular mass HSPs.

In addition we are giving account to another very probable reason why the HSPs synthesis increase in plant cells occurs at heat shock condition in rumen. It was defined that just after the experimental cows start grazing fresh grass in field condition, plant cells undergo both a heat shock at 36°C-38°C and pH stress in animal mouth and rumen. Moreover, herbaceous grass pulp can generate an endo-biochemical exothermic reaction that increases temperature up to 41°C. And in order to optimise the temperature of the grazed biomass, grass pulp is returned to the mouth as cud for re-chewing. And re-chewed plant cells biomass already contains polymorphous proteins partially denatured 3D structure at such heat shock temperature. We consider that when high temperature sensitive proteins are losing their 3D structure, this may very strongly propagate the rate of synthesis of HSPs in grazed and chewed fresh alfalfa intact cells. All these research results made it obvious that fresh herbaceous plant intact cells at 36°C- 38°C temperature of rumen will increase the HSPs synthesis far before all cells are disrupted by chewing and gastric acid in abomassum.

We have attempted to approximate maximal quantity of plant cells derived HSPs that 100-g of fresh alfalfa may supply to experimental cows. In the early morning, 100g of fresh alfalfa contain around 80% (or 80 milliliter) of moisture and 20% (or 20 grams) of solids. We are considering 20 g solids as 100% of dry matter of 100g fresh alfalfa, within which the ratio of total proteins were approximately 18%-22% during the harvesting season of alfalfa. These our research results of total proteins 3.6g-4.4g contents in fresh alfalfa coincides with an accepted World wide opinion that 100 g fresh alfalfa may supply to herbivores around 4 g of total proteins (Kohler, Knuckles 1977 ; McKenie at 1988). Nobody, so far, mentioned this daily norm of such essential micronutrients as HSPs are, in 100 g of fresh alfalfa (Levit 1980; Russelle 2001). So, our research results have pointed out that 100g fresh alfalfa may supply to herbivores up 40 mg HSPs at 36°C-38°C heat shock condition of rumen. It must be strongly underlined that we have not detected this quantity of HSPs before grazing-chewing of fresh alfalfa biomass. And this amount of HSPs, approximately 1% of total proteins, has reappeared in fresh alfalfa biomass only within the gut at 36°C-38°C. Since cows are ingesting daily around 100 kg of feed, we have considered that a daily natural norm of plant cells derived HSPs for cows may reach up to

1% of total proteins which was estimated above for this specific fresh plant bio-mass. However, we were faced with some problems described below in approximating precise daily natural norms of fresh herbaceous plant cells derived HSPs for herbivores.

Problems in approximating daily natural norms of herbaceous plant cell derived HSPs We have managed to demonstrate that in rumen heat shock condition at 36°C-38°C, 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 100g of fresh alfalfa intact cells, in the followingmanner: 2.8-12.6 mg of HSPs were found in association with around 0.3 g of soluble alfalfa proteins, the content of which inside of plant cell cytoplasm we estimated to be around 7% of total proteins, and 27. 4-37.2 mg of HSPs were extracted from around 3.7 g of non-soluble structural proteins inside of alfalfa cells.

And we have observed that the main part of plant origin HSPs can easily release from their associated forms at low pH during extraction and purification, as in experimental scenario model in animal stomach. So, during grazing, chewing and ingestion of 100 g of fresh alfalfa bio-mass, 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.

We have considered this major part of HSPs associated with non-soluble structural plant cells membrane proteins, as the hardly extractable form, and so to be the most suitable for a direct application in feed industry. And after the epidemic of BSE and CJD due to the use of conventional feed and the restriction of antibiotics use in live-stock-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.

Regarding the 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. Thereby, there is the need to strongly underline that soluble protein-associated HSPs derived from herbaceous plant are a strategic resource which may well satisfy an increasing dernand of HSPs in Food industry and Life Sciences. From this point of view the, yield of 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 0n5mg HSP90 sub- classes for use in Food industry and Life Sciences. And as a reminder, the full scope of World opportunity for HSPs yield: during almost 8 000 years alfalfa remained a major forage crop, with a harvest 10 t per hectare (and 3-5 harvests per season) and about 40 million hectares are grown each year world-wide (Russele 2001 ; McKenie at al 1988).

As a comparative example, 100 g of human cancer tissue or 10 ml of human recombinant cells (pellet) may supply around 1. 0 me HSP72/73 and 0, 2 mg HSP90 for use in Pharmacy.

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).

With regard to this strategic task and giving account to the only known alternative to antibiotics which plant origin HSP are, it is just time to restore, at least a minimum daily norm of plant origin HSP in feed for industrial live-stock and in processed foods which are used in clinical nutrition World-wide.

From all these scientific investigations observation, we have considered that, although the level of HSPs increased in plant cells after heat stress at 36°C-38°C in gut of herbivores, the daily natural norm of plant origin HSPs directly depends on: -The percentage of disruption of fresh plant cells during chewing by herbivorous, and -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 degrees of lignin content in stems and leaves cells vary during the harvesting period; -The indexes of stems/leaves correlation in fresh herbaceous plants vary during season; -The rate of HSPs synthesis differs separately in cells of leaves and in cells of stems; -The intensity of fresh grass total mass grazed per day by herbivores is different,

-The temperature fluctuation in grassland during the day may influence on HSPs content and on hot afternoon, when content HSPs is maximal in plants cows stop grazing grass.

In spite of the tremendous complication in approximating the alfalfa cell derived HSPs daily natural norm for experimental cows in field condition, we have managed to extrapolate the results of our 10 years observations and have considered that every 100 g of fresh Alfalfa may supply the following daily dose induced by grazing and chewing by ruminants : -Minimum dose in late autumn (when leaves are almost fallen) around: 0.05-0.5 mg; -Medium dose in early spring around: 2-20 mg; -Maximum dose in summer around: 30-40 mg.

Afterwards, according to our additional observation in grassland real condition where herbivores graze more than 5 herbaceous grass species as fresh feed per day it was defined that: -total proteins and HSPs content changes in grazed fresh herbaceous grass plants; -the HSPs production ability is different in stems and in leaves, particularly in old herbaceousplants; -herbivores select enigmatically their grazing species, but prefer mostly fresh grass plants in pasture, -Herbivores at various ages are grazing different amount of fresh grass per day, etc.

And in spite of the above mentioned stumbling blocks in defining precise daily natural norms of plant origin HSPs for of herbivores in grassland variable condition, our discovery that only fresh herbaceous plant cells hold the capability to sense heat stress in the digestive tract of mammalians and increase HSPs synthesis within gut has, no doubt, pointed out that plant origin HSPs are extraordinary essential micro-nutrients. And plant origin HSPs should fall in those micronutrient categories that are lost during processing and storage of fresh herbaceous plants.

Therefore, an urgent task is to restore in conventional feed daily norms of these essential plant cells derived micronutrients.

And in order to prove that HSPs are imperative building blocks of the immunogenic mixture in stomach, we have investigated the influence of abomassum juice on plant origin HSP70 sub-class and 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).

The discovery of the increased synthesis HSPs as such ancient essential plant origin micronutrients in gut of mammalians has also an obvious direct impact on Food Industry and on Life Sciences, too. Part of speech, the role of plant origin HSPs in keeping the stamina of herbivores and Homo has appeared as a practically not investigated new scientific iceberg, only the upper part of which we were so lucky to uncover. That was the reason why we have developed a new theory"Plant origin HSPs-Natural Therapeutics". Where we have proposed a new vision of plant cell derived HSPs against feed/food derived diseases, on the base of the evidences given in the background of this invention. Hereunder, we propose a new innovative vision of those advantages offered by plant cells derived HSPs, with their molecular vector- carriers function within an immunogenic mixture, which may open a major progress predominantly in oral vaccination, but not restricting any other form of administration.

Functional Processed Food-vehicle of immunogenic mixture At present, old postulates in feed/food industries and clinical nutrition need a revision, since none of them takes into account: * to estimate losses of HSPs sub-classes as essential micro-nutrients, during processing and storage of fresh herbaceous plants bio-masses; The nature's postulate using health risk-free immunogenic mixture, wherein plant origin HSPs, represented as any their sub-class alone or in combination and which are not associated either covalently or non-covalently with immune selective antigens from causative agents of diseases.

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. And 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. 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. As a confirmation of this assumption, there is need to point out that internalised HSP70 within monocytes remained stable for 24 hr at 37°C (Fujihara, Nadler 1999).

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.

The best source for plant origin HSPs manufacture All species of the herbaceous plants are very suitable sources for non-waste manufacture of plant origin HSPs sub-classes and among them the best is 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 sub- classes. 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. to produce 1 ton of pellets with around 10% moisture needs to evaporate up to 70% of water in the total mass of 4 million tons of fresh alfalfa with 80% moisture. The energy cost for only evaporating this quantity of water from fresh alfalfa was according to production manager of France-Luzerne-around 140 FF/ton of pellets, with the use of 350-kg coal (2500kWh) or 85 FF/ton of pellets, if using 190 m3 of gas (1250 kWh). We have also given attention to such technological dilemma as the swift drying fresh alfalfa needing to use drying air temperatures as high as 850°-120°C, what is dramatically"burning"proteins'efficiency in pellets. Here is a scope of disadvantages of alfalfa pellets processing technology: -Pellets dehydration requires an enormous energy per unit: production cost is very high; -Pellets processing needs high temperature (850-120°C) : proteins'nutrient quality drops; -Pellets lose the ability to synthesise HSPs : daily norm of HSPs drops for livestock; -Pellets lose around 70% of moisture: prophylactic value of cytosol drops for livestock ; -Pellets adsorb water during high humidity in air: needs costly water protecting packing;

-Pellet production creates strong greenhouse gases: affects planet's life-support systems; -Pellets factories are highly specialized and work only several weeks per year : equipment depreciation is too irrational.

We consider, that the invented bio-rational technology claimed by PCT-Patent GE00/00003, Issued 19 May, 2000 may decrease up to 80% the energy consumption during feed processing from alfalfa and in addition produce HSPs enriched fraction with 65% purity.

The method to use fresh alfalfa as source of HSPs The dramatic increase in health-risk, since using modern feed in industrialised live-stock and the irrationality of pellets and hay processing, described in the background of the invention, has pointed out to the need of lean thinking by mimicking the bio-rational way existing in Nature (Hawken at al 2000). The new innovation is introducing a radical change in the way the use of fresh alfalfa for feed manufacture must be considered. Indeed, the innovative new method should be compared with the existing old technology of feed manufacture as follows: Old Technology of processing fresh Alfalfa with 80% moisture: 1 resource => 1 product :-Feed (Hay or Pellets or Silos or Silage) New technology of processing fresh Alfalfa with 80% moisture: 1 resource => 5 products:-Feed with HSPs (Hay or Pellet or Silos or Silage) -Feed Additives with HSPs -Cytoplasm water -Bio-Minerals -HSPs sub-classes enrich fraction with 65% purity With such a bio-rational technology we have proposed the modernisation of old pellets technology for an integral and cost effective production of better quality feed and in addition to obtain significant quantity of almost all HSPs sub-classes with natural post-translation modification and other natural products, too. The nutrient quality of such new types of feed and feed additives is higher than any conventional feed and feed additives as digestibility of edible proteins are improved since the new method avoids their burning at 850°C-120°C during pellet production. Moreover, in the new feed content of such ancient essential micronutrients as HSPs sub-classes was partly restored. Therefore, the new innovative method mimics the bio-rational technology to use the full plant natural values as done by herbivores and managed to: -Decrease by more than 50% the production cost of pellets in the existing technology;

-Improve pellets quality by the keeping nutrient value of alfalfa proteins and their enrichment by HSPs.

The induction, extraction and purification of all HSPs sub-classes of plant cells origin were done according to the recently published PCT-Patent:"Method for induction synthesis of heat stress proteins (HSP) and production from herbaceous plant" (see GE00/00003, Issued 19 May, 2000). Here is a short summary of it.

Induction of HSPs synthesis in fresh alfalfa As a reminder, only fresh grass plants possess the ability to sense heat stress and increase the synthesis of heat stress proteins. While none of the existing commercial feeds (pellets, hay, silos, silage, forage) possess such ability to induce and produce HSPs. On the base of this capability of fresh alfalfa to sense heat stress we have worked out a heat shock condition, which is able to stimulate a swift increase of HSPs synthesis in fresh harvested alfalfa biomass. This was managed by a short-term temperature shock in the range of 38°C-55°C, which is able to partly miss-fold the heat labile RUBISCO, the major protein in herbaceous plant leaves. And after the heat shock the intracellular content undergoes an increase in almost all HSPs sub- classes, up to 1% of total proteins of fresh harvested alfalfa.

We have integrated the short-term heat shock, just before harvesting, with heat stress special conditions, after harvesting alfalfa, which develops an endo-biochemical exothermic reaction in order to manage a partial miss-order of 3D structures of labile proteins within the intact plant cells. Both short-term heat shock and partial miss-folding of 3D structures of labile proteins in cytoplasm of plant cells are very strong inductors of HSPs genes transcription and translation of almost all HSPs sub-classes, only in fresh alfalfa. According to our experiences, 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). As a rule, 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 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. According to 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. Thereby, 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. Since 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. In this imunogenic mixture 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: A). 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; B ! 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. As 1D structure of herbaceous plant cells derived HSP72/73 sub- class is practically identical to 1D 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. Doubtless, this immunogenic mixture with edible binary vaccine function has opened tremendous opportunities for a wide use of the cheapest herbaceous plants cells

origin HSP70 sub-class as molecular vector-carriers of immune selective antigenic peptides.

Thus, 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.

No doubt, cost effective FPF with harmless mixture of plant origin HSP70 and immune selective antigenic peptides may far better fulfil Medicine's ideal goal: health-risk free immunisation, than cost additive Genetically Modified Functional Food (GMFF) with treacherous mixture of bacterial and viral genes. And the most remarkable advantage of FPF is that the immunisation cost will be far less than with GMFF, as there is no need to change existing genome of crops or national tradition of diets. Part of speech, to produce very efficient FPF with edible binary vaccine function there is only need to add to a daily personal norm of nourishment the mixture with combination of two tasteless and health-risk free building blocks in such most preferable minute quantities of combination as: 0.01-10.0 mg of plant origin HSP72/73 sub-class and 0.03-30.0 mg immune selective antigens.

The sources of immune selective antigens There are two major classes of sources to obtain natural 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.

There is an additional possibility to obtain 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. 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. Even adjuvants, e. g.

Complete Freund's adjuvant (comprises killed M. tuberculosis suspended in oil and emulsified with the aqueous antigen preparation) was not very effective to elicit antigenicity of peptides.

Only the recent discovery of two imperative molecular vector-carriers of peptides, such as MHC and HSPs, has established a new horizon for increasing their use as the safest and efficient delivery system of peptides. But, it is far easier and preferable to use HSPs than MHC in vaccines (see US Patent 5,935,576 August 10,1999). And this universal vector-carrier capability of HSP70/90 sub-classes was the base of very effective attempts made by Antigenics Inc., to integrate the extraction of HSP70/90 with obtaining both classes of immune selective antigenic peptides: Intra-cellular genome origins from malformed cells and . Extra-cellular genome origin from infected cells.

And many World leading immunologists are striving to fight such calamities as: cancer and AIDS with the help of this phenomenal capability of HSPs to chaperone a wide spectrum of immune selective antigenic peptides to both B cell and T cell lymphocytes. Lately Dr. Bruce D.

Walker has announced:"I am very optimistic about the use of HSPs as an immune-specific delivery mechanism for HIV antigens. We hope that our prior data combined with HSPs'strong immune activation effects will accelerate development of an HIV vaccine, not only to prevent infection but also to boost effective immune responses in persons already infected" (see Antigenics Inc., News Release July 25,2001).

And in order to avoid any false expectation, there is need to underline that nowadays there is no ubiquitous antigens for all different strains of HIV. Mention must be made of the dilemmas of HSP70/90 complex extraction with immune selective antigens of all HIV strains or all causative agents of opportunistic diseases at any phase of AIDS. Since each of HIV strain's genome acquired antigens from cells infected by them camouflage specific antigens. And in addition, HIV invasion abolishes the network of

T cell lymphocytes against causative agents of opportunistic diseases, which in reality are the real reasons of AIDS lethality. Therefore, in order to avoid all these dilemmas and follow the prediction of the ingenious Dr. Jonas Salk-"There Must Be a Better Way" (see Cohen 2001, Shots in the Dark, p. 316), there must an effective passage-way: To build in vitro the mixture of plant origin HSP72/73 and selective antigen derived any strains of HIV or integral antigens from urine of individual, which are infected with HIV. The common vehicle of this immunogenic mixture may be FPF, in which 99% of the bio- mass may be designed from synergetic compounds and in addition satisfy the taste of patients from any social scales. Personalising of FPF with edible binary vaccine function is the only way to achieve an efficient exposure of HIV specific antigens to gut localised APC with the help of plant origin HSP72/73 sub-class. And the repeated daily priming of abundant APC in gut with HIV immune selective antigens by plant origin HSP72/73 may succeed to mobilise T cell lymphocytes to eradicate the somatic cells infected by HIV as well as causative agents of opportunistic diseases in the case of use an integral antigens.

Urine derived antigens Many leading hospitals World wide have verified an uncontested advantage of urine derived antigens, as a very effective and harmless curative medical means to treat cancer, allergy, tuberculosis, etc., (Eldor, http ://www. csen. com/theory/cancer. htm; Bjornejo 1956, Soda 1968; Thompson 1943; Rote at al 1980; Wilson, Lewis 1983; Owens 1982).

Here is a confirmation of an astonishing curative results of more than 50 century practice of oral auto-urine therapy and which has received a strong scientific-medical base after the finding of immune selective antigens in urine (Enomoto at al 1980; Rote at 1980; Starkey at al 1975; Stanley at 1970 ; Thomson 1943; Tietze 1996 ; Tsuji 1965; Weiss, Poirier 1995; Weiss at al 1996; Wilson, Levis 1983; Barnet 1988; Amstrong 1971; Christny 1994;).

It is well known that blood and lymph are continuously supplying food/oxygen to more than 230 different types of tissues and washing out metabolite waste from extra-cellular spaces within human/animal bodies. And this daily colossal service network of blood and lymph towards almost several trillion of somatic cells is finalised in kidney, where blood is releasing from metabolites of different tissues and among them myriad of proteins as well as peptides derived from broken down cells. All these proteins and peptides with molecular masses below 60 kDa are easily penetrating, under hydrostatic pressure, over a million nephrons'ultra-filtrating membranes of kidney. Afterwards, kidneys reabsorb as much as 80% of glomerular filtrate

across the cells of the proximal convoluted tubules into capillaries of the Vasa Recta. Finally, 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. Thus, 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. And 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"haR paid a particular attention to the composition of diet during urine therapy. In order to prevent E percentage increase of pseudo-antigens in urine he recommended to avoid starchy, pungent, sour, salty foods, which may easy disturb almost all nephrons network in kidney of patients. While tc prevent an increase of food-derived peptides in blood he suggested to restrict ingestion oi legumes, which are very rich with proteins. Therefore, the wisdom is obvious in such following warning by the author of this ancient manuscript".. during the process of the intake oi Shivambu, the following things should be strictly avoided : vegetables in the form of leaves flowers or legumes; grains that cause flatulency ; and starchy, pungent, sour and salty foods Sexual intercourse should also be avoided. This will help to accomplish the fruits of this method Behaving against these rules will put man in unexpected difficulties." (see Coen van der Kroon The complete guide to urine therapy, 1998, p. l 14-123).

There is no doubt, that antigens derived from urine may well substitute any immune selective antigenic peptides when the causative agents of diseases is unknown and define then diagnostically is impossible or very expensive. Urine derived antigens use in mixture with edible binary vaccine function will be also useful when causative agents of diseases are antibioti ( resistant strains of bacteria. The amino acid sequences of immune selective antigenic peptides o : interest may be determined by manual sequencing techniques in order to build information bant of personal efficient antigens.

Thereafter determination their antigenicity by antibody or ELISA (enzyme linkec immunosorbent assay) or other methods.

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 manage with integration of two conventional immunoaffinity and HPLC procedures. The amino aci (

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.

Human urine collected every morning in clean dry glass container, from 300 ml up to around 800 ml, immediately undergoes vacuum-evaporation at 55-75°C down to 50-100 ml.

Concentrated urine will instantly undergo gel filtration chromatography (TSK 40) at 35°C or 45°-55°C. 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 pSiemens/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.

Example II.

Immunogenic mixture: with edible binary vaccine function 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.

Preliminary weighed 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.

Once immunogenic mixture of plant cells origin HSPs with antigens are reconstructed, they can be characterised further on determination of immune respond using the administration protocols, as described in clinical nutrition.

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Planta vol. 196, pp. 530-538 INTERNET INFORMATION 103. Traavik T., University of Tromsô & Norvegian Institute of Gene Ecology, Tromso, Norway at"Environmental effects of genetically engineered vaccines" http ://www. twnside. org. sg./title/heal-cn. htm 104. Hansen M. K. Research Associate Consumer Policy Institute/Consumers Union "Genetic engineering is not an extension of conventional plant breeding", http ://www. consumersunion. org/food/widexpi200. htm 105. GRAIN : Seedling: December 2000:"Eat up your vaccines" http://www. grain. org/publications/dec001-en. c6n

106. Sowing diseases, new and old. Genetic engineering & the world health crisis. http://www. twnside. org. sg./title/vaccine. htm 107. The State of the World's Vaccines & Immunization Information Newsline. http://www. unicef. org/newsline/vpressr. htm PATENT DOCUMENTS: USA Patents: 108. Patent N 6,048,530"Stress protein-peptide complexes as prophylactic and therapeutic vaccines against intracellular pathogens", Issued on April 11, 2000; 109. Patent N 6,030,618"Therapeutic and prophylactic methods using heat shock proteins", Issued on February 29,2000; 110. Patent N 6,017,544"Composition compraiing immunogenic stress protein- peptide complexes against cancer and a cytoline", Issued on January 25,2000; 111. 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; 112. Patent N 6,007,821"Method and compositions for the treatment of autoimmune disease using heat shock proteins", Issued on December 28,2000; 113. Patent N 5,997,873"Method of preparation of heat shock protein 70-peptide complexes", Issued on December 7,1999; 114. Patent N 5,985,270"Adoptive immunotherapy using macrophages sensitized with heat shock protein-epitope complexes", Issued on November 16,1999; 115. Patent N 5,961,979"Stress protein-peptide cmplexes as prophylactic and therapeutic vaccines against intracellular pathogens", Issued on October 5,1999; 116. Patent N 5,948,646"Methods for preparation of vaccines agains cancer cmprising heat shock protein-peptide complexes", Issued on September 7,1999; 117. Patent N 5,935,576"Compositions and methods for the treatment and prevention of neoplastic diseases using heat shock proteins complexed-with exogenous antigens", Issued on August 10,1999; 118. 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;

120. 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 121. Patent N 5,750,119"Immunotherapeutic stress protein-peptide complexes against cancer", Issued on May 12,1998