WO2007129951A1

WO2007129951A1 - Virus susceptibility by plant nucleosomes

Info

Publication number: WO2007129951A1
Application number: PCT/SE2007/000421
Authority: WO
Inventors: Lars-Olof Hedén; Uif Rothman
Original assignee: Svenska Miljöbolaget SVV AB
Priority date: 2006-05-05
Filing date: 2007-05-03
Publication date: 2007-11-15
Also published as: EP2020858A1; EP2020858A4; CA2651239A1

Abstract

' The present invention relates to the use of a proteinaceous component isolated from plant chromatin, after dissociation of the same, as an antiviral agent. The proteinaceous plant component is produced by means of a method comprising the steps of homogenizing a plant material in order to expose its plant chromatin, dissociating the plant chromatin with a dissociating agent under hydrophobic conditions, and separating the dissociated plant chromatin into individual fractions, one comprising the proteinaceous plant component, by means of a hydrophobic interaction separation procedure. The invention further encompasses a method for treating viral infections in technical as well as pharmaceutical applications.

Description

TITLE

VIRUS SUSCEPTIBILITY BY PLANT NUCLEOSOMES

DESCRIPTION

Technical field

The present invention relates to the use of a proteinaceous component isolated from plant chromatin. More precisely, the invention relates to the use of a proteinaceous component isolated from plant chromatin, after dissociation of the same, as an antiviral agent, as well as a method of producing the same.

Background of the invention

Most eukaryotic organisms produce a wide variety of protective mechanisms directed towards infectious agents. Several mechanisms are based on those fundamental differ- ences, which exist in membrane composition and organization between microbes and cells of complex multicellular organisms, i.e. they are directed towards outer membranes of sensitive microbes. It is also known that basic peptides also inhibit the DNA/RNA/protein synthesis. These membranes are composed of lipids having negatively charged head groups facing outwards and the microbes apparently find it difficult to counteract the effects by altering their membrane composition and organization. Thus, the substances responsible for the antibiotic action are presumable candidates as substitutes for antibiotics.

One example is the phospholipid transfer proteins, which are able to transfer phospholipids between membranes. Antimicrobial phospholipid transfer proteins have been reported from a range of plant species including cereals, and these proteins vary in their activity against different pathogens. For example, in US 5 698 200 it is shown that a plant part can be protected from a plant pathogenic bacterium by means of an aqueous extract obtained from malted cereal grain.

However, the most studied class of protective agents is the antimicrobial peptides. They are found in all species of life, ranging from plants and insects to animals, including molluscs, crustaceans, amphibians, birds, fish, mammals, including humans.

These peptides interact directly with bacteria and kill them. They are termed antimicrobial because they have unusually broad spectra of activity including the ability to kill or neutralize Gram-negative and Gram-positive bacteria fungi (including yeast), parasites (including planaria and nematodes), cancer cells, and even enveloped viruses like HIV and herpes simplex virus. In general, these agents range in length from as few as 12 amino acids to molecules with over 70 residues. More than 500 such peptides have been discovered.

The mode of antimicrobial action of the almost always cationic antimicrobial peptides has been studied in detail among such peptides as melittin, magainin, gramicidin, cecropin, and defensins. The antimicrobial molecules also generally damage the membranes of the organisms that they attack. The cationic antimicrobial peptides have been found to possess bactericidal activity in vitro as well as in vivo. They kill very rapidly, do not easily select resistant mutants, are synergistic with conventional antibiotics, other peptides as well as lysozyme, and are able to kill bacteria in animal models.

As a consequence, antimicrobial peptides of animal origin are now developed as new antibiotic drug. Examples are the synthetic version of magainin (pexiganan) and the analogue of a protegrin, an antimicrobial peptide initially isolated from pig neutrophils.

However, natural sources have not proved to be economically profitable for the production of new alternative antibiotics. The only exception is the antimicrobial peptide nisin, which can be effectively produced in a Lactococcus lactis strain with high resistance to the substance.

An increasing number of larger proteins or fragments thereof have also been found to exhibit antimicrobial activities. For example, a murine macrophage protein, ubiquicidin, appears to be the same as the ribosomal protein S30. Also, two of the antimicrobial peptides in the stomach of bullfrog {Rana catesbeina) are derived from the N-terminus of pepsinogen. Likewise, a biocide peptide, named buforin I, has been isolated from stomach tissue of an Asian toad (BBRC 218:408, 1996). The amino acid sequence of the 39 amino acid long peptide was found to be identical with 37 of the 39 amino- terminal residues of the Xenopus histone H2A.

In addition, the whole protein molecule can exhibit an antimicrobial potential. Antimicrobial activity has been detected in acid extracts of liver, intestine, and stomach of atlantic salmons (BBRC 284:549, 2001). The corresponding antimicrobial protein can be isolated from salmon liver using acid extraction followed by ammonium sulfate precipitation, large-scale gel chromatography (gel filtration), reverse-phase HPLC, and size exclusion HPLC. The salmon antimicrobial (SAM) protein was found to have a molecular mass of 27.7 kD and was identified as the histone H1 protein. In WO 200110901 , the mammalian histone H1 protein from bovine thymus is used in antimicrobial compositions for treating microbial infections in different eukaryotic organisms. Thus, proteins having other well-established functions appear to exhibit a second property by being antimicrobial.

However, the use of bovine proteins, especially proteins from bovine thymus, should be avoided since such a material can be contaminated with deleterious virus, especially hepatitic viruses, or other pathogenic agents, for example priones. Bovine material - whether contaminated or not - must be subjected to extremely strict tests when intended to be used in connection with humans.

Furthermore, the isolation of new alternative antibiotics involves the collection of specified animal organs or tissue, followed by complex purification procedures in order to obtain a product that can be used in connection with human beings or domestic animals.

WO 03/017769 relates to the use of a proteinaceous component isolated from plant chromatin, after dissociation of the same, as an antimicrobial agent, the proteinaceous component having an apparent molecular weight between 10 and 20 kD. The proteinaceous plant component is produced by means of a method comprising the steps of homogenizing a plant material in order to expose its plant chromatin, dissociating the plant chromatin with a dissociating agent under hydrophobic conditions, and separating the dissociated plant chromatin into individual fractions, one comprising the proteinaceous plant component, by means of a hydrophobic interaction separation procedure.

The said WO 03/017769 relates to antimicrobial treatment, where the microbes are bacteria, or fungus (yeasts).

Summary of the present invention

The present invention relates to an antiviral agent, and in particular to the use of a proteinaceous component isolated from any karyotic chromatins after dissociation of the same, as an antiviral agent, the very proteinaceous nucleosomal main component and its subfractions. Thus the purpose of the invention is to provide a new antiviral agent, whereby the above-mentioned problems are eliminated. Another purpose of the invention is to provide an antiviral agent, whereby the risk is avoided of passing on infectious agents in the food chain or being pathogenic to man and/or animals and/or plants.

Still another purpose is to provide an antiviral agent that is tasteless when used in connection with food.

A further purpose of the invention is to provide a method of producing an antiviral agent, in which cheap starting materials are utilized.

Yet a further purpose is to provide a method of producing an antiviral agent in a practically unlimited scale.

Still yet a further purpose is to provide a method of producing an antiviral agent, which does not require investments manufacturing in plants for microbiological fermentation.

These objects are accomplished by the use as well as the method according to the invention as claimed.

According to the invention a method is provided in which in a simple and rational way allows for the production of a proteinaceous component which can be used as an antiviral product, for example as a drug, a full preserving agent during manufacturing and transport, a functional food, such as a probiotic or prebiotic food, and/or neutraceutical additive as well as an animal feed additive.

A proteinaceous component can be prepared with surprisingly ease from an initially inert starting material comprising plant chromatin. In the inventive method, DNA is separated from basic nuclear plant chromatin. Preferably, the plant chromatin is obtained from plant grains, embryos or seeds. Preferably such plant material is obtained from the commercial commodities oat, wheat, barley, rye, com, rye wheat, rice, rape, soy bean, millet, sorghum, milo, or buck wheat. According to the invention any chromatin containing plant material (cauliflower, spinach, green grass), including marine plants, and weeds, edible vegetables, leftovers from the vegetable oil industry, gardening or forestry waste can be used for the preparation of an antiviral proteinaceous component on a large scale. Another important source is the commercial yeasts. However, fresh plant material is seldom optimal for the isolation and recovery of plant chromatin.

The plant chromatin used for the isolation of the proteinaceous component should be a heterochromatin (silent chromatin or "junk" DNA. The heterochromatin is hypoacet- ylated (deacetylated) chromatin, which assumes a more condensed structure than hyperacetylated chromatin due to a higher electropositive charge.

In addition, the choice of chromatin starting material for further specific protein extraction is also dependent on plant cell tissue location and state of differentiation. For example, a tissue comprised of small cells will have a higher cell density, and therefore is likely to contain more nucleic acids and accompanying antibacterial proteinaceous component than another same amount of tissue comprising larger cell size.

Likewise, many plants carry very large basal genome sizes due to high heterochromatin content, further enhanced by a polyploidy possibility. In this connection the amount of DNA per haploid cell as measured in the number of base pairs (the c-value) is referred to. The variation in DNA content of an organism is reflected by its DNA c-value or basal genome size. The c-value is defined as the content of DNA as measured by weight or number of nucleotides in a single copy of the entire sequence of DNA found within cells of that organism. It is the amount of nuclear DNA in its unreplicated haploid or gametic nucleus, irrespective of the ploidy level of the taxon. Thus, the c-value equals the genome size in diploid species, but always exceeds genome size in polyploid species.

It is also preferred that self-renewing undifferentiated stem cells of plants are utilized. These are found in meristems, regions that provide new growth at shoot and root tips. Thus, plant seedling root-tips are the superior starting material for chromatin extraction and subsequent downstream isolation of the proteinaceous component according to the invention. Such raw material is readily available in unlimited quantities, as being a waste product during the manufacturing of brewery malt and wheat germ oil.

Other plant raw materials of mitotically dividing cells under optimal growing conditions are also suitable for the preparation of a proteinaceous component according to the invention. Any germinating sprouts and rootlets or germs in germination phase can be used. Preferably, grains of one of the four kinds of cereals are used, which are allowed to germinate. A cost effective raw material to be used according to the present invention is what is called green malt, which is a starting material for beer production. The brewery industry produces green malt from barley, which after moisturizing is allowed to germinate for six days (malting). This industrially produced green malt, or by-products thereof (rootlings), can according to the invention be used for the production of an antiviral proteinaceous component.

Accordingly, rootlings of diploid corn and barley (DNA c-value of 5,000 Mbp) as well as onion (DNA c-value of 18,000 Mbp) are suitable starting extraction materials.

Preferably, the antibacterial proteinaceous component is extracted from a chromatin source, the DNA c-value of which exceeds 3,000 Mbp.

It is especially preferred that the starting material of the purification procedure comprises plant chromatin isolated from proliferating plant cells in S-phase. Germinating seeds (grains) with their rootlings as well as young leaves thus contain a large number of cells in S-phase.

In the inventive method of producing a proteinaceous plant component having antiviral activity, the method comprises the steps of

(i) homogenizing a plant material in order to expose its plant chromatin;

(ii) dissociating the plant chromatin with a dissociating agent under hydrophobic conditions; and

(iii) separating the dissociated plant chromatin into individual fractions, one comprising the proteinaceous plant component, by means of a hydrophobic interaction separation procedure.

Accordingly, the plant material is first homogenized. In this connection the term homogenization means a disruption of the plant material cell walls in such a way that the chromatin of the plant is exposed and a homogenate is obtained as a slurry. The cell walls may be disrupted by any of a number of methods known to those skilled in the art including, but not limited to, high shear mixing, sonication, mechanical disruption, explosion by pressure etc. The cell walls are disrupted by means of a suitable device, whereby a homogenate is obtained. The plant chromatin in the homogenate is then dissociated by means of a dissociating agent in an aqueous solution thereof under hydrophobic conditions. Such conditions are those that promote hydrophobic interactions.

Suitable dissociating agents are urea, guanidinium chloride, and a chloride salt. Preferably, the chloride salt is sodium chloride of high ionic strength. In a preferred embodiment the histones are isolated as nucleosomes in a solution having a chloride concentration of above 0.3 M, preferably 0.5 M or higher. This nucleosome mixture may be inert with regard to microbiological effect, but will become activated by diluting the solution to a chloride concentration of 0.3 M or below. Thereby free histones will dissociate and go into solution.

It is an advantage if the homogenizing of the plant material is performed in the dissociating agent. In this way the purification procedure is simplified and the number purification steps are reduced.

The purification procedure of green malt is commenced by the homogenisation of the malt in an almost saturated salt solution comprising 4 M sodium chloride. The high ionic strength dissociates the chromatin as well as nucleosomes, a simultaneous degradation of proteinaceous material by proteases at the same time being prevented. Preferably, the homogenisation is performed in the presence of a hydrophobic matrix.

The homogenate can then be sieved on a sieve or a wire net or the like in order to remove cell debris or other particles from the plant chromatin, which are retained thereon. In this way a solution is obtained that facilitates a subsequent purification of the dissociated plant chromatin.

The dissociated plant chromatin is then separated into individual fractions, one comprising the proteinaceous plant component having antiviral activity. The separation is preferably performed by means of a hydrophobic interaction separation procedure. Preferably, the hydrophobic interaction separation procedure is hydrophobic chromatography.

Thus 2x100 g wheat germ is homogenized in an Ultra-Turrax homogenizer 2x1 minutes in 400 ml each of TBT (50 mM Tris-HCI, pH 8.07/0.5% Triton X-100/15 mM β- mercaptoethanol/50 mM Na-bisulfite)/50 mM NaHSO₃ with 100 μl 1-butanol (as an antifoam agent). The homogenates are combined and another 200 ml TBT/NaHSO₃ is added and left at +4⁰C for 10 minutes on a magnetic stirrer. The homogenate is centrifuged at 4,000xg for 5 minutes at 4⁰C. The supernatants are combined and passed through four consecutive nylon fabrics with defined mesh (2 mm, 1 mm, 0.5 mm, and finally 0.3 mm). The volume is adjusted to 1 ,000 ml and 3 M ammonium sulphate is added dropwise to a final concentration of 50 mM with stirring to precipitate the chromatin. Leave on a magnetic stirrer at +4⁰C for 10 minutes. Recover the chromatin by centrifugation at 10,000xg for 10 minutes at +4⁰C. The precipitate is homogenized in 400 ml TBT+50 mM ammonium sulfate (=TBM). Centrifuge at 10,000xg for 10 minutes at +4⁰C. The pellet is homogenized in 200 ml TBM and centrifuged as above. The homogenization and centrifugation is repeated four times. Finally, the precipitated material is homogenized in 200 ml 0.1xSSC+5 mM NaHSO₃. The histones in the chromatin preparation are extracted by dropwise addition of 0.5 M H₂SO₄ with stirring to a final concentration of 0.1 M. Leave at +4⁰C with stirring for 4 hours. Centrifuge at 10,000xg, +4⁰C for 10 minutes and recover the supernatant. The pellet is reextracted with 50 ml 0.1 M H₂SO₄ with stirring at+4°C for 4 hours and centrifuged as above. The supernatants are combined and concentrated with polyethylene glycol 6,000 and dialyzed against 0.1% acetic acid using dialysis tubing with a molecular cut off of 3,000 Dalton. The dialyzed material is stored at -2O⁰C

Other examples of suitable separation procedures are partition in polymeric systems, such as partition chromatography, counter current distribution, and gas aphron partition. The separation of the dissociated chromatin components can alternatively be performed on columns with metal chelate gels or immobilized heparin.

The functional ligand of the matrix used for the hydrophobic interaction and/or separation procedure should be an ether, an isopropyl, a butyl, or an octyl group. A phenyl group should be avoided. Preferably, the functional ligand is a butyl group on an agarose matrix which is cross linked to 4%. A ligand density of 40-50 μmol/ml is the achieved, which results in a binding capacity of 7 mg IgG per ml.

For example, after screening of the green malt homogenate as a slurry, a hydrophobic matrix is added batch wise to the solution obtained, the hydrophobic matrix being an hydrophobic interaction chromatography gel (HIC) containing active butyl groups. Suitable matrixes are Novarose® S-Butyl 1000/40 from Inovata AB, Bromma, Sweden, and Butyl Sepharose® 4 from Amersham Pharmacia Biotech, Sweden. The hydrophobic matrix is then washed with the high ionic strength salt solution, DNA being washed out. Then the matrix is poured into a column and subjected to a stepwise gradient elution with decreasing ionic strength of sodium chloride. A distinct antiviral proteinaceous component is eluted at a concentration of 1 M NaCI.

The proteinaceous component can be further purified by means of a conventional method suitable for purification of peptides/proteins. Such methods include centrifugation, precipitation at the isoelectric point, phase separations, ultrafiltration, gel chromatography (size exclusion chromatography), ion exchange chromatography or HPLC, as well as a combination of such methods. Preferably, the subsequent separation procedure is gel chromatography or ion exchange chromatography.

Most preferably, a preparative gel chromatography step is accomplished in a column packed with a gel having an exclusion limit of 100 kD. The column is equilibrated with distilled water before being loaded with the fraction of 1 M NaCI exhibiting antibiotic activity. The column is then eluted with distilled water or ammonium acetate. In this way a desalting and purification is obtained at the same time in one and the same step. Thus, the proteinaceous component can be concentrated to dryness, for example by means of lyophilization, without any further purification steps.

A protein fraction having an apparent molecular weight between 10 and 20 kD was isolated, which exhibited antiviral properties.

It will be appreciated by the skilled man within the art that the purification of the proteinaceous component may be accomplished by many other methods known to those skilled in the art, all of which are contemplated by this invention.

A complexing agent, such as heparin, alginic acid, phytic acid, polyphosphate or a vanadinium compound, can also be used as a dissociating agent, provided that it dissociates the plant chromatin into its individual components. Alginic acid is especially preferred as a dissociating agent, alginate complexes with the viricidal active proteinaceous component being formed. Such complexes can be used with the aim of purification or be used as such for slow release of antiviral activity therefrom.

Before the separation of the dissociated plant chromatin into individual fractions according to the invention by means of for example a hydrophobic interaction separation procedure, no antiviral activity at all could be found in the starting material of dissociated chromatin. When proteinaceous plant components are purified according to the invention, the heterochromatin will automatically be utilized. Thus, the biological activity is successively formed by the physical separation of chromatin components. Theoretically, the separation procedure could result in an altered molecular structure of the components.

According to the invention, other antiviral active proteinaceous components may be obtained from other plant materials by means of other purification procedures after the elution from the hydrophobic matrix. This is due to the fact that proteins from different biological materials exhibit different post-synthetical modification patterns that reflect cellular activities of the plant material. Thus, a separation pattern should be influenced by the degree of for example acetylation, phosphorylation, methylation, ubiquitination, glycosylation, as well as ADP-ribosylation of a proteinaceous component obtained according to the invention.

Correspondingly, the isolated proteinaceous component from plant chromatin can subsequently be chemically modified. Such modifications include changes in molecular weight and/or acetylating level and would result in preparation forms having a more specific biological activity.

The simple inventive purification method allows for the production of an antiviral proteinaceous component in a practically unlimited scale. The process yield is more than 1 g protein from 1 kg of raw material (for example rootlings). By using germinating seeds with maximum protein synthesis the yield can be maximized, as is shown for example by malting for six days. When proliferating plant cells in S-phase are used, the natural chromatin protein synthesis is maximal and can represent up to 80% of the total protein synthesized.

The proteinaceous component isolated from plant chromatin according to the invention as an antiviral agent can be intensified together with one or more antivirally synergistic agents.

When the proteinaceous component is to be used as an antiviral agent, it can be formulated in buffered aqueous media containing a variety of salts and buffers. Preferably, the salts are alkali and alkaline earth halides, e.g. sodium chloride, potassium chloride, or sodium sulphate. Various, such as buffers may be used, such as citrate, phosphate, HEPES, Tris or the like to the extent that such buffers are physiologically acceptable for its purpose.

Various excipients or other additives may be used, when the proteinaceous component is formulated as a lyophilized powder, for subsequent use in solution. The excipients may include various polyols, inert powders or other extenders.

The inventive use also includes a composition that comprises the purified proteinaceous component in a biocidal concentration and an amount effective to kill bacteria or fungi and a suitable carrier. Such compositions may be used in numerous ways to combat viruses, for example in household or laboratory antiviral formulations using carriers well-known in the art.

Different compositions will have different degrees of activities towards different viruses. Effective amounts to be used for killing harmful viruses may be readily determined by those skilled in the art.

The proteinaceous component according to the present invention may also be combined with other proteins to act as preservatives in order to protect the proteinaceous component against proteolytic degradation. Alternatively, the inventive proteinaceous component or compositions may be used as preservatives or disinfectants in a wide variety of formulations, such as contact lens solutions, ointments, shampoos, medicaments, foods, and the like. The amount of proteinaceous component may vary depending upon the nature of other components, the degree of antiviral protection required, and the intended use of the composition.

The proteinaceous component can for example be used together with a suitable carrier in a composition for disinfection and cold sterilization of surfaces and as an adjuvant in food high-pressure pasteurization as well as in a composition as a water conservation agent, e.g. in pisciculture or to protect fishes in aquaculture. The proteinaceous component can also be used in an amount effective to kill viruses when enclosed in packaging materials to be slowly released therefrom.

EXAMPLES The invention will now be further described and illustrated by reference to the following examples. It should be noted, however, that these examples should not be construed as limiting the invention in any way. Inhibition of non-enveloped virus multiplication by wheat germ histones.

INTRODUCTION. In two papers by Lehrer et al. (1 , 2), basic peptides from rabbit leukocytes and human granulocytes were found to inactivate some enveloped viruses (herpes simplex type 1 and 2, vesicular stomatitis virus, influenza virus) but not others (cytomegalovirus). None of the tested naked (non-enveloped) viruses (i.e. reovirus and echovirus) were susceptible to the peptides.

Histones prepared by acid extraction of wheat germ have been shown in laboratory to have antimicrobial activity against a large number of bacterial species, gram-positive as well as gram-negative strains. The wheat germ histone preparations were found to inactivate the non-enveloped bacteriophages ΦX174, λ, and MS2. The results also suggest that the wheat germ histones are able to inactivate the fish pathogenic virus IPNV (infectious pancreatic necrosis virus).

Bacteriophages and propagation of phage.

Bacteriophage ΦX174 (single stranded circular DNA genome) and λ (double stranded linear DNA genome) was propagated on Escherichia coli MM294 in liquid culture using LB at 37⁰C until visible lysis. Bacteriophage MS2 (single stranded linear RNA genome) was propagated on E. coli C66 (F⁺) in liquid culture as above. Bacteriophage concentrations were determined as plaque forming units (pfu) on nutrient agar (for ΦX174) or tryptose agar (for λ and MS2) using a top agar with 0.6% agar in tryptose broth. The strain used for the propagation of the phage was also used as indicator bacteria in the top agar.

Inhibition of bacteriophage multiplication by wheat germ histones.

The different phages were diluted to the appropriate concentration in phosphate buffered saline (PBS for ΦX174 and MS2, λ-buffer for phage λ) or distilled water. The acid extracted histones were used either in 0.1 M acetic acid or dialyzed against several changes of PBS and diluted in PBS or in distilled water. Bacteriophage and wheat germ histones were mixed on ice in plastic tubes and incubated in a water bath set to the appropriate temperature and for the time indicated below. After the incubation, the number of pfu was determined as described above. The agar plates were incubated over night at 3O⁰C for ΦX174 and at 37⁰C for λ and MS2. Inhibition of IPNV infection of salmon cells in vitro. The salmon fish cell line Xxx was grown in micro titre plates with YYY minimal medium supplemented with 5% heat inactivated fetal calf serum. The cells were grown at 2O⁰C in a CO₂ incubator (10% CO₂). The virus IPNV and the wheat germ histones were diluted in PBS. Virus and histones were mixed in micro titre plate wells and incubated at 37° C for two hours after which 20 μl was added to micro titre plate wells with fish cells and 200 μl growth medium and incubated as for propagation of the fish cells. Viral infection of the cells was determined as cytophatogenic effect using a microscope. The cells were observed daily for three days and finally fixed as described by Carlsson et al. (3).

RESULTS.

Inhibition of bacteriophage λ multiplication.

Bacteriophage λ was diluted to 2x10⁴ pfu/ml. Equal volumes of the diluted phage and acid extracted histone (4,530 μg/ml) in 0.1 IVl acetic acid was mixed and incubated at room temperature. At different time points, pfu/ml was determined. As a control, the same volume of diluted phage was mixed with an equal volume of 0.1 M acetic acid.

The results presented in Fig. 1 show that the wheat germ histones lowered the pfu

1 ,000-fold in 128 minutes at room temperature. Only a marginal decrease in pfu was observed for the control.

To further show that the inactivation of the viral reproduction is due to the histones a dose response experiment was done. The histone preparation was diluted in 0.1 M acetic acid and equal amounts of diluted histones and phage λ (2x10⁴ pfu/ml) were mixed on ice and incubated at room temperature for 60 minutes. Pfu was determined as above and the results are presented in Fig. 2.

Inhibition of phage ΦX174 multiplication by wheat germ histones.

To expand the possible antiviral spectrum of the wheat germ histones, the sensitivity of the single-stranded small icosahedral bacteriophage ΦX174 was analysed. Preliminary experiments showed that ΦX174, unlike phage λ, is sensitive to 0.05 M acetic acid (results not shown). The acid extracted histones were therefore dialyzed against several changes of PBS before use. A time course experiment was done by mixing, on ice, 2.7 ml ΦX174 diluted to about 10⁶ pfu/ml in PBS and 0.3 ml of the dialyzed wheat germ histones diluted in PBS to 453 μg/ml. The mixture was incubated at 37⁰C. At time points, samples were taken and the number of pfu was determined as described above. The results in Fig. 3 show a rapid decrease in pfu with time although not as pronounced as for phage λ.

Previous experiments have shown that the antibacterial activity of wheat germ histones is enhanced under low ionic conditions. The ability of histones to prevent multiplication of phage ΦX174 was therefore analysed by diluting the virus as well as the acid extracted and PBS dialyzed wheat germ histone in distilled water before incubation. Diluted wheat germ histones and phage ΦX174 (~ 4x10⁶ pfu/ml), both diluted in distilled water were mixed on ice in the proportion 1/10 and incubated at 37⁰C for 60 minutes. Plaque forming units was determined as described above. The results presented in Table 1 show a 1 , 000-fold reduction in the number of plaque forming units at a final concentration of 1.13 μg/ml of wheat germ histones.

In a time course experiment, 0.5 ml wheat germ histones diluted in water to a concentration of 45.3 μg/ml was mixed with 4.5 ml of phage ΦX174 (~ 4x10⁶ pfu/ml) giving a final concentration of histones of 4.53 μg/ml. The mixture was incubated at 37⁰C and samples were taken at intervals for determination of pfu. The results presented in Fig. 4 show a rapid decrease in the number of plaque forming units within 4 minutes. Size heterogeneity of the ΦX174 plaques was observed in the samples exposed to histones as shown in Fig. 5 with the majority of the plaques being smaller than normal.

The temperature dependence of the phage inhibiting properties of the histones was analyzed by mixing phage ΦX174 diluted to ~ 4x10⁶ pfu/ml in distilled water and different concentrations of wheat germ histone diluted in water. Incubations were done for 60 minutes at different temperatures before determination of pfu. The results presented in Table 2 show that at 10⁰C sixteen times more histones is needed to achieve the same degree of phage reduction compared to at 3O⁰C in 60 minutes. However, a 5, 000-fold reduction of pfu was obtained at a concentration of 22.7 μg/ml of histones when incubated at 1O⁰C for 60 minutes.

Inactivation of the RNA phage MS2 by wheat germ histone.

To eliminate the possibility that the antiviral activity of wheat germ histones is specific for DNA viruses, the antiviral activity was tested on the small icosahedral RNA phage MS2. Since MS2 is a male specific phage, i.e. uses sex pili as the receptor, the F⁺ strain E. coli C66 was used for the propagation of the virus as well as for the determination of pfu. Phage MS2 was diluted to 4x10⁶ pfu/ml in PBS. Diluted phage (0.4 ml) was mixed on ice with 0.1 ml wheat germ histpne at different concentrations. The mixtures were incubated at 37⁰C for 60 minutes after which the number of pfu was determined as described above. The results presented in Fig. 6 show a 100-fold reduction in plaque forming units at 45.3 μg/ml of wheat germ histones. A 50-fold reduction was obtained at 4.53 μg/ml of the histones.

The difference in rate of phage λ inhibition in comparison to phage ΦX174 can be ascribed to the different temperatures used. The assay of ΦX174 as described in Fig. 3 was performed at 37⁰C while the λ inhibition experiment was performed at room temperature. The temperature dependency for inactivation is further illustrated by the data in Table 2 below.

Previous experiments on the antibacterial activity of wheat germ histones have shown that including EDTA in the assay buffer enhances the activity. This suggests that cations are competing with the histones for binding to the target on the bacterial cell.

An even more pronounced antimicrobial effect was obtained if the histones and the bacteria were diluted in water, again suggesting a competition for binding to the bacteria by some component in the buffer used (PBS). The results in Fig. 3 and Fig. 4 show that the rate of phage inhibition is much more rapid in distilled water than in buffer suggesting that cations may compete with the histones for binding to the phage particles.

The difference in plaque morphology as shown in Fig. 5 was unexpected. Phage ΦX174 normally forms large plaque due to the small size of the phage particle. The plaque morphology suggests that the virus infection in the small sized plaques is due to a delay of the infection. This can be explained by a mechanism where the histones bound to the phage particles are "delivered" to the cells where the histones slow down the metabolic activity of the bacterial cells. This inhibition of metabolism is overcome with time provided that a sufficient low amount of histones have been introduced into the cells.

Thus it is shown that the whole mixture of plant histones works as an antiviral agent, and not only the part components, i.e., the core histones and the H1 histone. At the preparation of the histones these are soluble during acid extraction but natural nucleosomes are reconstituted while the ion strength declines during the final dialysing phase. This means that the product will obtain a stable form which can be stored and transported. The antiviral effect might be due to a structural change on virus capsidal level changing the docketing ability of the virus.

The molecular weight of the H1 is between 40 and 45 kDa when analysing the molecular weight using SDS-PAGE. As the histones are negatively charged an acidic urea gel electrophoresis may provide a lower molecular weight.

TABLES.

Table 1. Dose dependent inhibition of ΦX174 plaque forming units, bbbbb ΦX174 diluted in distilled water to about 3x10⁶ pfu/ml was mixed with wheat germ histones (dialyzed against PBS) diluted in distilled water and incubated at 37⁰C for 60 minutes. Plaque forming units in the different mixtures were done as described in Material and Methods section.

Table 2. Temperature dependence of phage ΦX174 inactivation by acid extracted wheat germ histones. Phage ΦX174 diluted in distilled water to about 3x10^e pfu/ml was mixed with wheat germ histones (dialyzed against PBS) diluted in distilled water and incubated at different temperatures for 60 minutes. Plaque forming units in the different mixtures were done as described in Material and Methods section above.

Inhibition of non-enveloped virus multiplication by wheat germ histones. Extended study including the mammalian Adeno-virus.

The above reported antiviral activity of acid extracted wheat germ histones has further been extended to include the single-stranded DNA bacteriophage M 13 and the mammalian Adenovirus Ad2. The toxicity of the histones on mammalian HeLa cells has been analyzed by measuring the metabolic activity of cells exposed to histones. The antiviral and antibacterial activities of heat-treated histones were analyzed.

MATERIAL AND METHODS.

Bacteriophage and propagation of phage.

Bacteriophage M13mp18 (Yanisch-Perron et al., 1985) is a filamentous single-stranded circular DNA virus using F-pili as the receptor in the infection of bacteria. The phage was propagated using Escherichia coli JM103 (Yanisch-Perron et al., 1985) as the host strain.

Inhibition of phage M13 multiplication by wheat germ histones. Phage M13mp18 was diluted in10 mM sodium phosphate buffer, pH 7.0 (NAPB) to about 3x10⁵ plaque forming units/ml (pfu/ml). From this dilution, 450 μl was mixed with 50 μl wheat germ histones diluted in NAPB. After 60 minutes incubation at 37⁰C the mixture was chilled on ice and plaque forming units was determined as described before (Yanisch-Perron et al., 1985) with exponentially growing E. coli JM103 as the indicator strain. As a control, 50 μl NAPB replaced wheat germ histones.

Inhibition of Adeno virus multiplication by wheat germ histones.

Costar (Corning Inc.) 48 wells microtiter plates were seeded with about 2x10⁴ HeLa S3 cells in Eagle's minimal essential medium with 5% fetal bovine serum and incubated at 37⁰C in 5% CO₂ to about 80% confluency. Adenovirus type 2 (Ad2) (1.7x10¹² virions/ml, about 3% infectious) is diluted in NAPB to a multiplicity of infection of 4 (see below). Equal volumes (40 μl) of diluted virus and wheat germ histones diluted in NAPB are mixed in polypropylene tubes (final MOI=2, see below) and incubated at 37⁰C for 2 hours. From the incubated mixtures, 20 μl is added to the HeLa cells giving a final MOI of 2, i.e. two infectious virions/HeLa cell. After 1 hour at 37⁰C for infection the medium with virions and histones is replaced with fresh medium. After 48 hours of incubation at 37⁰C the cells are lysed by repeated freezing and thawing of cells and medium and finally by adding 4 μl 10% SDS/well. Adenovirus antigen was quantified using an ELISA with rabbit anti-Ad2 antibodies (primary antibody) and alkaline phosphatase- 00421

conjugated goat anti-rabbit antibodies (secondary antibody). p-Nitrophenyl phosphate /1 mg/ml) was used as the substrate for the alkaline phosphatase. Absorbance at 405 nm was recorded after two hours at 37⁰C.

Effect of wheat germ histones on HeLa cells.

Wheat germ histones diluted in NAPB is added to HeLa S3 cells, in growth medium, in microtiter plate wells. One set of cells were left with the histones for the entire 48 hours incubation and in one set of cells, the histone containg medium was replaced with histone free medium after one hour at 37⁰C and the incubated at 37⁰C for 48 hours. To each well, 40 μl substrate (EZ4U, non-radioactive cell proliferation & cytotoxicity assay, Biomedica, Germany) was added. The absorbance at 450 nm was recorded after 60 minutes at 37⁰C. The method is based on that living cells can reduce the uncoloured tetrazodium salt into the intensely coloured formazan. This reduction process requires functional mitochondria, which are inactivated within minutes after cell death.

Heat treatment of wheat germ histones.

Histones at a concentration of about 8758 μg/ml in 0.1% acetic acid was incubated at 100⁰C for 60 minutes and chilled on ice. The antiviral activity after the heat treatment was analyzed by mixing 450 μl phage ΦX174 and 50 μl heat-treated or untreated histones diluted in NAPB. After 60 minutes at 37⁰C, the number of plaque forming units was determined with E. coli MM294 as indicator bacterium. As a control, 50 μl 0.1% acetic acid was diluted in NAPB as for the histones and used as with the histones. The antibacterial activity of wheat germ histones incubated at 100⁰C for different times was analyzed by radial diffusion assay as described before (Lehrer et al., 1991) with E. coli MM294 as the test organism..

RESULTS.

Inhibition of phage M13 multiplication by wheat germ histones.

The results in Figure 7 show a 10-fold reduction in the number of plaque forming units at a final concentration of 1.8 μg/ml of wheat germ histones. A 50-fold increase in the final histone concentration did not cause a further decrease in pfu/ml. Compared to other bacterial virus, this reduction in pfu is very low. The explanation for this limited activity may be found in the structure of the phage. Phage M13 is a filamentous "male- specific" phage (using F pili as the receptor) composed of about 3.000 molecules of g8p, the protein subunit making up the main "body" of the virus and about 5 molecules of the receptor recognition protein g3p. A plausible explanation for the obtained results is that there are two different "populations" of phage where in one population the receptor-recognition protein g3p is not accessible to interaction with the phage.

Inhibition of Adeno virus multiplication by wheat germ histones. The results in Figure 8 show that wheat germ histones efficiently inhibit synthesis of Adeno virus antigen. For this virus one has to take into account that only about 3% of the virions are infectious and that the non-infectious virions are most likely able to bind the histones. This explains the relatively high amount of histones required when compared to the experiments with bacterial viruses.

Effect of wheat germ histones on HeLa cells.

In Figure 9 the relative amount of formazan formed by HeLa cells in the presence of wheat germ histones is shown. The results show a marginal effect on the metabolism in the presence for one hour of up to 350 μg histones/ml. If the cells are exposed to the histones for 48 hours, a 50% reduction in formazan formation is shown. If this reflects a reduction of the metabolism in all cells or that a proportion of the cells are non-viable is not known presently. A staining for viable cells should be done to determine which possibility is true.

Heat treatment of wheat germ histones.

The antibacterial activity against E. coli MM294 of histones treated for different times at 100⁰C is shown in Figure 10. The antibacterial activity remained the same even after 60 minutes at 100⁰C. The results in Figure 11 show that also the antiviral activity was unaffected by incubation at 100⁰C for 60 minutes. A reduction of the number of plaque forming units by four orders of magnitude was obtained for both the untreated and heat-treated histones. The low concentration of acetic acid in the samples had no effect on the ability of the phage to infect the bacterial cells as shown in the control.

007/000421

LEGENDS TO FIGURES.

Figure 1. Time course inhibition of bacteriophage λ multiplication. Equal volumes of phage λ diluted in buffer to about 2x10⁴ pfu/ml and acid extracted wheat germ histones were mixed and incubated at room temperature. At the time intervals indicated, 0.1 ml was taken for determination of pfu as described in Material and Methods section.

Figure 2. Dose response of phage λ inactivation. Phage λ diluted to about 2x10⁴ pfu/ml and different dilutions of acid extracted wheat germ histones (dilutions made in 0.1 M acetic acid) were mixed and incubated at room temperature for 60 minutes. Plaque forming units were determined as described in Material and Methods section.

Figure 3. Kinetics of phage ΦX174 inactivation by wheat germ histones. Phage ΦX174 at a concentration of about 10⁶ pfu/ml in PBS was mixed with acid extracted wheat germ histone, dialyzed against PBS at a final concentration of 45.3 μg/ml and incubated at 37⁰C. At the indicated time points, samples were withdrawn for determination of pfu as described in Material and Methods section.

Figure 4. Kinetics of phage ΦX174 inactivation by wheat germ histones. Bacteriophage ΦX174 diluted to about 3x10⁶ pfu/ml in distilled water was mixed with wheat germ histone (dialyzed against PBS and diluted in distilled water) at a final concentration of 2.3 μg/ml and incubated at 37⁰C. At the indicated time points, samples were taken for determination of pfu. In the control experiment, histones were replaced by distilled water.

Figure 5. Size heterogeneity of phage ΦX174 plaques. The picture shows the plaque size heterogeneity in samples exposed to wheat germ histones.

Figure 6. Inactivation of phage MS2 by wheat germ histones. Phage MS2, diluted to about 10⁷ pfu/ml in PBS was mixed with acid extracted wheat germ histones dialyzed against PBS and diluted to different concentrations in PBS The mixtures were incubated at 37⁰C for 60 minutes after which the number of plaque forming units was determined. 007/000421

Figure 7. Inhibition of M13 infection by wheat germ histones. Phage M13mp18 diluted as described in Material and Methods was incubated with different concentrations of wheat germ histones. After 60 minutes at 37⁰C the number of plaque forming units was determined with exponentially growing E. coli JM103 as the indicator organism. As a control, the same number of phage was incubated with NAPB.

Figure 8. Determination of the relative amount of Adeno virus type 2 antigen in HeLa cells exposed to virions for 1 hour in the presence of different amounts of wheat germ histones. In the control wells, histones were replaced by NAPB. The number of infectious virions was about 2 per HeLa cell (MOI=2). The error bars represent the standard deviation from two experiments

Figure 9. HeLa S3 cells were exposed to increasing amounts of wheat germ histones for variable times. For one set of cells, the histone containing medium was replaced by histone free medium after one hour of incubation at 37⁰C. One set of cells was incubated at 37⁰C with histones for the entire 48 hour period. Reduction of tetrasodium salt was determined spectrophotometrically at 450 nm and expressed as the amount of formazan formed relative to control cells.

Figure 10. Heat-resistance of wheat germ histones antibacterial activity. Aliquots of wheat germ histones were incubated at 100⁰C for different times as indicated and chilled on ice. The antibacterial activity was analyzed by radial diffusion assay with E. coli MM294 and the activity relative to non-heated sample is shown.

Figure 11. Heat-resistance of wheat germ histones antiviral activity. Phage ΦX174 was incubated at 37⁰C for 60 minutes with non-heated or heated wheat germ histones before determination of infectivity as plaque forming units/ml.

REFERENCES.

1. Lehrer, R. I., K. Daher, T. Ganz, and M. E. Selsted (1985). Direct inactivation of viruses by MCP-1 and MCP-2, natural peptide antibiotics from rabbit leukocytes. J. Virol. 54:467-472.

2. Daher, K. A., M. E. Selsted, and R. I. Lehrer (1986). Direct inactivation of viruses by human granulocyte defensins. J. Virol. 60:1068-1074.

3. Carlsson, A., J. Kuznar, M. Varga, and E. Everitt (1994). Purification of infectious pancreatic necrosis virus by anion exchange chromatography increases the specific activity. J. Virol. Meth. 47:27-36.

4. Lehrer, R. I., M. Rosenman, S. S. S. L. Harwig, R. Jackson, and P. Eisenhauer. 1991. Ultrasensitive assays for endogenous antimicrobial polypeptides. J. Immunol. Meth. 137: 167-173.

5. Yanisch-Perron, C, J. Vieira, and J. Messing (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-119.

Claims

1. Non-therapeutic use of a proteinaceous component isolated from plant chromatin, after dissociation of the same, as an antiviral agent, said proteinaceous component.

2. Use as in claim 1 , wherein said plant chromatin is heterochromatin.

3. Use as in claim 1 or 2, wherein said plant chromatin has DNA c-value that exceeds 3,000 Mbp.

4. Use as in any of claims 1 -3, wherein said plant chromatin is obtained from any plant material.

5. Use as in claim 4, wherein said from plant seeds are selected from the group consisting of oat, wheat, barley, rye, corn, rice, rape, soy, millet, or buck wheat grains or embryos.

6. Use as in claim 4 or 5, wherein said plant chromatin is obtained from said grains during germination.

7. Use as in claim 1, wherein said antiviral agent is directed against DNA virus, single or double stranded ones.

8. Use as in claim 1 , wherein said antiviral agent is directed against RNA virus, single or double stranded ones.

9 .Use of a proteinaceous component as in any of claims 1-8 together with at least one antivirally synergistic agent.

10. Use as in claim 9, wherein said at least one antiviral synergistic agent is a nucleoside analogue.

11. Use of a proteinaceous component as in any of claims 1 -10 in complex with a complexing agent to be slowly released therefrom.

12. Use as in claim 11, wherein said complexing agent is alginic acid.

13. Use of a proteinaceous component as in any of claims 1-12 in an amount effective to prevent virus propagation, and a suitable carrier, in a composition as a food additive.

14. Use of a proteinaceous component as in any of claims 1-12, and a suitable carrier, in a composition as a growth promoting animal feed additive.

15. Use of a proteinaceous component as in any of claims 1-12, in an amount effective to prevent virus propagation, and a suitable carrier, in a composition for disinfection and cold sterilization of surfaces and as an adjuvant in food high-pressure pasteurization.

16. Use of a proteinaceous component as in any of claims 1-12, in an amount effective to prevent virus propagation enclosed in packaging materials to be slowly released therefrom.

17. Use of a proteinaceous component as in any of claims 1-12, in an amount effective to prevent virus propagation, and a suitable carrier, in a composition as a water conservation agent.

18. Use as in claim 18 in pisciculture

19. Use as in claim 18 in aquaculture.

20. Use of a proteinaceous component isolated from plant chromatin, after dissociation of the same, as an antiviral agent for treating virus infections, said proteinaceous component being derived from the nucleosomal compartment.

21. Use of a nuleosome fraction in solution having a chloride concentration of above 0.3 M, which nucleosome containing solution is made subject to dilution at the site where to effect its antiviral activity whereby the dilution has a chloride concentration of 0.3 M or less.

22. Use according to claim 20, wherein the virus infection is caused by a human adeno-virus infection.

23. Method of producing a proteinaceous plant component having antiviral activity, the method comprises the steps of (i) homogenizing a plant material in order to expose its plant chromatin; (ii) dissociating the plant chromatin with a dissociating agent under hydrophobic conditions; and

(iii) separating the dissociated plant chromatin into individual fractions, one comprising the proteinaceous plant component, by means of a hydrophobic interaction separation procedure, whereby the histones are isolated as nucleosomes in a solution having a chloride concentration of above 0.3 M, preferably 0.5 M or higher.

24. Method for treating a virus infection, whereby a therapeutic amount of an antiviral agent in the form of a proteinaceous component isolated from plant chromatin, after dissociation of the same, is administered to a mammal, including human suffering from a virus infection.

25. Method for treating a virus infection, whereby a growth inhibiting amount of an antiviral agent in the form of a proteinaceous component isolated from plant chromatin, after dissociation of the same, is administered to a product infected by a virus infection.