Therapeutic and prophylactic compositions

Abstract

Compositions and methods for preparing same are provided, comprising bee-derived components and marine plasma, particularly where the bee-derived components comprise or are derived from Zabrus. These compositions may be used in the treatment or prevention of disease, particularly bacterial, viral, fungal and / or parasitic disease.

Description

[Technical field]

[0001] The present invention is in the field of therapeutic compositions and methods for producing the same. [Background technology]

[0002] Despite lacking the antibody-based adaptive immune system found in mammals, bees and other insects have well-developed innate immune systems. In fact, more recent evidence suggests that the "innate" immune system of insects is stronger and more specific than the name commonly used to describe it. The concept of "immune priming" in insects describes how immune protection can be triggered in response to challenge with dead, harmless, or sublethal microorganisms and can be maintained and persisted for a long time, and even for many generations in bees (Cooper and Eleftherianos, "Memory and Specificity in the Insect Immune System: Current Perspectives and Future Challenges" Front. Immunol., 09 May 2017). This is clearly similar to the concept of specificity and memory of the supposedly intrinsic mammalian adaptive immune system and vaccination.

[0003] Honeybee activity is thought to result in the uptake of environmental contaminants, including pathogenic and genetic material from other sources. The transfer of these pathogenic molecules has been shown to be biologically active, causing gene knockdown and immunity that persists into adulthood. Transposable elements, non-coding RNA as well as bacteria, fungi and viruses give honeybees the unique ability to share immune-related things between individuals and generations. Findings suggest that this may play a role in social immunity and communication between hive members.

[0004] Antiviral defense mechanisms in bees are diverse in the pathways and cellular and extracellular effectors they use, including RNA interference (RNAi), endocytosis, melanization, encapsulation, autophagy, and conserved immune pathways including the Jak / STAT (Janus kinase / signal transducer and activator of transcription) pathway, the JNK (c-Jun N-terminal kinase) pathway, the MAPK (mitogen-activated protein kinase) pathway, and the NF-κB-mediated Toll and Imd (immunodeficiency) pathway (McMenamin et al, “Honey Bee and Bumble Bee Antiviral Defense”, Viruses 2018).

[0005] Solutions formed from seawater and / or its dilutions, sometimes called sea plasma or marine plasma, have been used therapeutically for over 125 years (Passebecq and Soulier, “Comparative Study of the Therapeutic Properties of Seawater Preparations” https: / / oceanplasma.org / documents / passbecsoulier-e.html). Evidence for the efficacy of saltwater in various therapeutic applications includes recent trials showing that saline irrigation and gargling are effective against the common cold (Ramalingam et al. “A pilot, open labelled, randomised controlled trial of hypertonic saline nasal irrigation and gargling for the common cold” Scientific Reports 2019).

[0006] Preventive approaches, such as vaccination, to prevent pathogen invasion or improve the body's response to such an attack are a preferred means of reducing the impact of infectious diseases and are particularly effective against many bacterial and viral pathogens. However, in some cases, vaccines are unable to provide the immunity required for protection, for example against human immunodeficiency virus (HIV), eukaryotes such as malaria parasites, and more generally in immunocompromised populations such as the elderly. Summary of the Invention [Problem to be solved by the invention]

[0007] There is a need to provide methods, compositions and methods of producing compositions for antimicrobial use. In particular, it is desirable to provide a means for preventing, alleviating or reducing the symptoms of viral, bacterial, fungal or parasitic infections in humans or animals. Preferably, these methods should be associated with minimal side effects, be tailored against specific pathogens and reduce the risk of resistance development. [Means for solving the problem]

[0008] In one aspect, the present invention provides a composition comprising a bee-derived component and marine plasma.

[0009] The bee-derived component may comprise a product of beekeeping, such as one or more of zabrus, honeycomb, bee venom, honey, royal jelly, propolis and pollen, preferably zabrus. The bee-derived component may be derived from a product of beekeeping, such as one or more of zabrus, honeycomb, honey, royal jelly, propolis and pollen, preferably zabrus. The bee-derived component may comprise lysozyme, typically lysozyme derived from zabrus. The bee-derived component may have a reduced content of hydrophobic components compared to the input zabrus, honeycomb, bee venom, honey, royal jelly, propolis and / or pollen. The bee-derived component may have a reduced content of sugars compared to the input zabrus, honeycomb, bee venom, honey, royal jelly, propolis and / or pollen.

[0010] The compositions may be formulated as a nasal spray, injectable serum, skin patch, eye drops and / or for oral administration.

[0011] The marine plasma may include seawater diluted with water, which may be in a ratio of 25% to 30% seawater and 70% to 75% water, preferably about 29% seawater and 71% water.

[0012] In one aspect, the invention provides a method of making a composition comprising providing a bee-derived component and combining the bee-derived component with a liquid comprising marine plasma.

[0013] The method may further include diluting the seawater with water to produce a liquid containing marine plasma. The method may further include obtaining seawater from below one or more plankton blooms in the ocean, typically from about 25 to about 35 meters, preferably about 30 meters below such plankton blooms. The seawater may be filtered, preferably microfiltered, typically dual-cooled microfiltered. Filtration may be through a porcelain filter with a pore size of 0.22 microns. The seawater may be diluted with purified water, distilled water or reverse osmosis water, or lightly mineralized spring water. The ratio of seawater to water in the marine plasma may be about 29% seawater and 71% water.

[0014] The bee-derived ingredients may include one or more of zabrus, honeycomb, bee venom, honey, royal jelly, propolis and pollen. The bee-derived materials may be derived from one or more of zabrus, honeycomb, bee venom, honey, royal jelly, propolis and pollen as raw materials. The method may include processing one or more raw materials to remove at least a portion of their hydrophobic components. Such removal may include disintegrating one or more raw materials in water and centrifuging the resulting suspension. The method may include processing one or more raw materials to remove at least a portion of their sugar content. Such removal may include precipitating sugars from the processed zabrus, honeycomb, bee venom, honey, royal jelly, propolis and / or pollen using a polar solvent, typically the polar solvent includes acetone.

[0015] The method may further include rearing bees as a source of bee-derived components. The bees may be provided with a nectar diet including farnesol. The bees may be provided with a diet including linden nectar. The bees may be reared in beehives, which may be treated with the enrichment method. The enrichment method may involve application of rosin or a coating including rosin to the exterior of the hive. In some embodiments, one or more applications of 30% turpentine and 70% rosin are used for hive enrichment. The coating may be applied as soon as the hive is placed in a suitable location.

[0016] The method may further comprise obtaining one or more of Zabrus, bee venom, honey, royal jelly, propolis and pollen, preferably Zabrus, from captive bees or their environment such as a beehive.

[0017] In a further aspect, the present invention provides compositions comprising beekeeping products, such as bee-derived components from Sabrus zabrus, and methods of producing such compositions. Such compositions may be further defined or produced as described for other aspects above. Such compositions may further comprise water, saline solution and / or marine plasma.

[0018] In a further aspect, the present invention provides a method for treating or preventing a disease comprising treating a subject in need thereof with one or more of the compositions described above or one or more compositions produced by the methods described above.

[0019] In a further aspect, the composition as described above or a composition produced by the method as described above may be for use in medicine, typically for use in a method of treating or preventing disease.

[0020] In a further aspect, the present invention provides the use of a composition as described above, or a composition produced by a method as described above, in the manufacture of a medicament.

[0021] In the above aspects, the disease to be treated or prevented comprises a bacterial, viral, fungal or parasitic disease, typically a viral or bacterial disease. The viral disease to be treated may be mediated by poliovirus, adenovirus, coronavirus or herpes virus.

[0022] The compositions may be formulated as a nasal spray, injectable serum, skin patch, eye drops and / or for oral administration. [Brief description of the drawings]

[0023] [Figure 1] 1 shows an exemplary method of preparing an extract for use in accordance with an embodiment of the present invention. [Diagram 2] The results of a virus inactivation test using an extract according to the invention, using as raw material inoculated zabrus from a hive inoculated with human coronavirus OC43, are shown in Figures 2A, B and C, respectively, showing a positive control of an A549 culture infected with HCoV-OC43, a culture not infected with virus (negative control), and a culture infected with HCoV-OC43 virus mixed with an inoculated zabrus preparation in isotonic marine plasma. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present disclosure provides therapeutic compositions comprising bee-derived components and marine plasma and / or bee-derived components derived from Sabrus. The compositions are intended to improve immunogenicity and promote induction of a desired immune response against infectious diseases from pathogens such as viruses, including influenza, poliovirus, and coronaviruses such as SARS-CoV-2, as well as other microbial parasites, bacteria, and fungi. As such, the compositions described herein may be considered as antibacterial compositions, antiviral compositions, antibacterial compositions, and / or compositions having antiviral and / or antibacterial properties. The compositions may be used to treat, alleviate symptoms of, or act prophylactically as a prophylactic against, diseases induced by pathogens. The compositions may be used, for example, to promote induction of a sustained ability of the recipient's immune system to thwart a particular pathogen, as an adjunct to a vaccine response.

[0025] Bees and bee-derived ingredients Bee keeping and products derived from beehives, also called beekeeping products, have been used throughout human history for their antibacterial and other medicinal properties using ingredients such as honey, royal jelly, propolis and pollen (Denisow, Denisow-Pietrzyk. “Biological and therapeutic properties of bee pollen: a review”. J Sci Food Agric. 2016; Ahmad et al “New Insights into the Biological and Pharmaceutical Properties of Royal Jelly” Int J Mol Sci. 2020; Russian Patent No. 2376978C2). The compositions of the present invention include one or more bee-derived ingredients, i.e., ingredients made by or obtained from bees, their hives or their activities or ingredients produced from such materials. Given the widespread agricultural use of bees, bee-derived ingredients can be easily transported to laboratories and other production facilities, can be cost-effective for large-scale production, and can be easily scaled up.

[0026] Biologically active substances occur naturally in bee products and include enzymes, vitamins, essential amino acids and essential fatty acids which, even at low concentrations, may have radioprotective, cardiotonic and immunostimulatory effects as well as beneficial effects against disturbances in metabolic cycles.

[0027] Honey matured in honeycomb cells is sealed by a wax cap or lid, which the beekeeper cuts off before extracting the honey. "Zaburus" is the term used to refer to these caps or tops that contain high quality wax, pollen, propolis, honey, enzymes and other ingredients. For example, enzymes include lysozyme, which bees add to the caps to protect the honey inside the cap from bacterial or other microbial contamination. Due to its low specific gravity compared to other components of honey, lysozyme is mainly concentrated in the upper part of the honey-containing cells, and without wishing to be bound by theory, this may explain the higher concentration of lysozyme in the zaburus compared to the extracted or sold honey or other parts of the honeycomb. Organic acids, free fatty acids, minerals, vitamins such as carotene, essential oils, proteins, fats, balsams and resins have also been identified in the zaburus. Beekeepers themselves do not necessarily consider it necessary to sell Zabrus, some even discarding this material as waste, and as a result the present invention also provides a use for this material.

[0028] Zabulus has natural histamine blocking properties, meaning that it can be used by individuals who are allergic to bees or bee products. Zabulus has previously been used to boost immunity in individuals with respiratory, nasopharyngeal, digestive and oral diseases. Furthermore, when the active bee-derived ingredient used in the composition is isolated, such as when lysozyme is extracted from bee products, potential allergens such as pollen or venom can be eliminated.

[0029] The decline of bee populations is of great importance to ecosystems worldwide, especially with regard to human activities related to the pollination of plants, such as food crops. One of the causes of the decline is believed to be the reduced ability of bees to protect their colonies from infectious diseases. With these considerations in mind, the compositions described herein may be obtained using certain beehive enrichment techniques and / or certain diets, as described below.

[0030] For optimal production of the bee-derived ingredients and beekeeping products used in the present invention, one or more techniques can be used in the husbandry of the bees themselves to protect and care for the bees and hives, and to encourage the formation of resistant bee colonies with strong resistance to most pathogens. In some embodiments, bees are fed a specific nectar diet that contains farnesol, an organic compound that is a 15-carbon acyclic sesquiterpene alcohol and exhibits antiseptic effects (Derengowski et al. "Antimicrobial effect of farnesol, a Candida albicans quorum sensing molecule, on Paracoccidioides brasiliensis growth and morphogenesis." Ann Clin Microbiol Antimicrob. 2009). Farnesol is produced by many plants and animals and can be added directly to bee diet as a supplement, or bees can be raised in an environment where a source of diet that naturally contains farnesol is available.

[0031] In some embodiments, the bee feed comprises or consists essentially of linden nectar, i.e. nectar from trees of the linden genus, such as Tilia cordata. As noted above, linden nectar contains farnesol and is used (mainly as linden honey) as a treatment for disorders such as influenza, tonsillitis, and sore throat. The feed can be provided to the bees in any suitable manner known in the art.

[0032] In some embodiments, beehives may be treated by hardening them with a coating. The hive hardening method preferably involves the application of a coating containing rosin or rosin. Rosin is obtained from wood. In some embodiments, one or more applications of about 30% turpentine and about 70% rosin are used for hive hardening. Such coatings are used to protect wooden beehives from electromagnetic fields and pathogens. The hive hardening technique used allows the bees to make their own wax, further protecting the bees from potentially harmful electromagnetic frequencies. In some embodiments, the coating is applied as soon as the hive is placed in a suitable location, such as in a field, as the bees need to adapt to the material.

[0033] As mentioned above, insect immune mechanisms, especially in bees, may be more specific and long-lived than previously thought (Cooper and Eleftherianos, 2017). It is possible to inoculate or expose bee colonies to specific pathogens, such as viruses, bacteria or parasites. This may reflect exposure to pathogens that bees encounter in nature, for example as a result of pollination activities. Thus, in some embodiments, bee colonies or hives may be exposed to one or more pathogens prior to collection of bee-derived components. This is believed to increase the specificity of the final bee-derived components against said pathogens and / or contribute to an increase in the production of antiviral components by the colony, thus increasing the efficacy of the final composition.

[0034] Inoculation of the colony may typically be performed by spraying the hive or comb with a suspension of the particular pathogen in a sucrose solution. The pathogen used for inoculation may be a bacterium, a virus, a fungus, a parasite such as a unicellular eukaryote or a multicellular parasite, or an immunogenic part or fragment thereof. For example, a suspension of the virus may be mixed with a sucrose solution and sprayed on both sides of all the combs. This procedure may be repeated 2, 3, 4, 5 or more times, at intervals of 1, 2, 3, 4, 5 or 6 days or repeated weekly. Collection of material from the hive for further use may be performed after inoculation (or the last inoculation), for example 1, 2, 3, 4, 5, 6, 7, 8, 10 or 14 days after inoculation. Typically, collection is performed 11 to 28 days after the first inoculation. Criteria for collection may include sealing of the hive lid and / or infiltration of the lid with immune factors. Preparations of the pathogen may be produced by any suitable means known in the art.

[0035] In some embodiments, the bee-derived component is or is derived from Zabrus. In some embodiments, the bee-derived component is or is derived substantially only from Zabrus, i.e., Zabrus is isolated from the main body of the honeycomb upon collection. For example, to isolate Zabrus, the honeycomb caps can be collected in any suitable manner, for example by cutting with a sharp knife. Zabrus can be treated and / or purified to isolate or concentrate certain components or remove other components. For example, Zabrus can be treated to remove waxes or other substances with low water solubility (i.e., hydrophobic components) to improve the water solubility of the resulting product. Agar diffusion methods can be used for confirmation, and cationic methods can be used for extraction of Zabrus subcomponents, such as lysozyme. Exemplary methods for the preparation of suitable extracts are discussed below.

[0036] The bee-derived ingredient may be or be derived from the honeycomb, which may be obtained with or without the attached cap (zabrus). Again, the honeycomb may be processed as described for the zabrus, typically with hydrophobic components removed, to prepare useful extracts.

[0037] In some embodiments, the bee-derived component comprises or is derived from one or more of honey, royal jelly, propolis, and pollen.

[0038] The bee-derived ingredients themselves, derived from the described raw materials, may have at least a portion of the hydrophobic components of the raw materials removed, for example, the bee-derived ingredients may have 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or substantially all of the hydrophobic components removed compared to the raw materials.

[0039] The bee-derived ingredients, which are themselves derived from the raw materials described, may have at least a portion of the sugar content of the raw materials removed, for example the bee-derived ingredients may have 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or substantially all of the sugar content removed compared to the raw materials.

[0040] Honey has many biologically active components. Polyphenols in honey are believed to have antioxidant, antibacterial and immunostimulatory properties. The main antibacterial activity is against bacteria, fungi, parasites and viruses. It has been reported that honey stimulates the proliferation of T lymphocytes in cell cultures and activates neutrophils (Al-Waili and Haq “Effect of honey on antibody production against thymus-dependent and thymus independent antigens in primary and secondary immune responses” 2004 Journal of Medicinal Food; Abuharfeil, Al Oran, Abo-Shehada “The effects of bee honey on the proliferative activity of human B and T lymphocytes and activity of phagocytes” 2008 Food and Agricultural Immunology; Bakr et al “Characteristics of Bioyoghurt Fortified With Fennel Honey” 2015 Int J Curr Micr App Sci).

[0041] Pollen contains polyphenols, vitamins B3, A and E, minerals and sterols (Rzepecka-Stojko, Anna et al. “Polyphenols from Bee Pollen: Structure, Absorption, Metabolism and Biological Activity.” Molecules (Basel, Switzerland) vol. 20, 12 21732-49.4 Dec. 2015).

[0042] Humans use bee pollen as a functional food with many biological properties, the main ones being sports performance and its antioxidant and antibacterial properties. In the context of this disclosure, pollen may refer to plant pollen extracts derived from the center of the pollen grain, rather than the whole pollen grain. It can be obtained from beehives.

[0043] Royal jelly, a glandular secretion used to feed larvae and queens, is thought to have antiviral effects against herpes and coxsackie viruses. Some studies have suggested immune stimulating activity in animals or cell cultures, including increasing white blood cell counts (Wang Bailong et al “Previously Unknown Gamma Globulin in Royal Jelly With Immune Stimulating Properties / Increasing Life Expectancy Of Lab Animals By 30%” 1987).

[0044] Propolis, a resinous mixture produced by bees from a mixture of exudates collected from plant sources, saliva and beeswax, is believed to have antiviral effects. For example, the effects of both poplar and baccharis propolis may be demonstrated against pathogenic viruses, including adenoviruses, coronaviruses, coxsackieviruses, herpes simplex (HSV-1, HSV-2, human T-lymphotropic virus-1 (HTLV-1). Quercetin and luteolin (constituents of poplar propolis) have antiviral activity against the SARS-CoV virus, the causative agent of SARS. The effect of propolis on the protein kinase PAK1 was also demonstrated. Abnormal activation of PAK1 is thought to be involved in the pathology of diverse diseases such as cancer, inflammation, viral infections, malaria, and immunosuppression. Therefore, propolis may be useful in blocking coronavirus-induced pulmonary fibrosis and stimulating the immune system. The immune-stimulating properties of propolis have been demonstrated in (Sforcin, “Propolis and the immune system: a review” Journal of Ethnopharmacology, 2007).

[0045] In some embodiments, the bee-derived component is bee venom, which has been shown to have antiviral activity (Uddin et al “Inhibitory effects of bee venom and its components against viruses in vitro and in vivo” Journal of Microbiology 2016; Mansour et al “Evaluation of Antiviral Activity of Bee Venom, Phospholipase A-2 (PLA-2) and Propolis against DNA and RNA Virus Models” IJSRP 2016). The main component, melittin, has many biological properties, including antiviral activity. Bee venom shows activity against adenovirus, enterovirus, herpesvirus (HPV16, 18), HIV, picornavirus, influenza A (PR8), leukemia virus, vesicular stomatitis (VSV), respiratory syncytial virus (RSV), enterovirus-71 (EV-71), and coxsackie (H3) viruses. An immunostimulatory effect has also been demonstrated, which may be due to phospholipase A264. It has also been proposed that by increasing the immune response, bee venom may help the body to cope with swine influenza A (H1N1)87 (PSI). Russian apicotherapists have shown that five to six prophylactic treatments of bee venom can significantly reduce the risk of PSI.

[0046] In some embodiments, the bee-derived component comprises or consists of lysozyme, also known as muramidase, which may be derived from Sabrus or other sources of bee-derived components such as honey. Lysozyme is an antibacterial enzyme present in many animals that plays a role in the innate immune system. Lysozyme is present in bodily secretions such as mucus, tears, breast milk and saliva, as well as animal-derived components such as egg white. Lysozyme is present in the lungs, digestive tract and liver, and is stable over a wide pH range. The antibacterial action of lysozyme is thought to result from hydrolyzing bonds in peptidoglycan cell walls and lysing bacteria. However, it has been reported that the antibacterial action occurs as a result of amino acid mutations within the enzyme active site, suggesting that other mechanisms of action are possible. Several studies have shown antiviral activity for lysozyme (Ferrari et al “Antiviral Activity of Lysozyme” Nature 1959; Malaczewska et al “Antiviral effects of nisin, lysozyme, lactoferrin and their mixtures against bovine viral diarrhoea virus” BMC Vet Res. 2019). Lysozyme is added to elderly gastrointestinal treatments and infant formula to promote digestibility. Lysozyme is used in skin care to treat and prevent acne. In Japan, lysozyme is used as a prescription to treat headaches, colds, and throat infections.

[0047] Lysozyme is also thought to have potential effects based on piezoelectric activity (Stapleton et al. “The direct piezoelectric effect in the globular protein lysozyme.” Applied Physics Letters, 2017). Piezoelectricity is an electrical charge that accumulates in biological materials such as bone, DNA, various proteins and viruses. This, combined with the low molecular weight of lysozyme, is thought to improve its compatibility with cell ion channels.

[0048] Any suitable method known in the art may be used to isolate lysozyme if this is desired. In general, methods for extracting and purifying lysozyme from egg white have been known for many years (Alderton Ward and Fevold, "Isolation of lysozyme from egg white", JBC 1944). These may include (a) adsorption of lysozyme on bentonite (montmorillonite clay), (b) elution of inactive contaminating proteins from the clay by successive washes with phosphate buffer (pH 7-8) and 5% aqueous pyridine, and (c) elution of the active substance with a pyridine-sulfuric acid solution at pH 5.0. The eluate may be dialyzed and dried in the frozen state. A white powder containing 85-90% of the lysozyme contained in the egg white may be obtained.

[0049] More recently developed methods are also known (Dekina et al, "Isolation and Purification of Lysozyme from the Hen Egg White" Biotechnologia 2015), which involve, for example, differential heat denaturation of the protein by changing the pH value of the medium, followed by neutralization, dialysis and further purification by gel chromatography. Agar diffusion or turbidimetry or any other method suitable for obtaining the presence of lysozyme in bee products can be used as determinants.

[0050] Other factors that may be present in the bee-derived ingredients include sulfhydryl enzymes and balsam. More generally, the bee-derived ingredients may include vitamins, enzymes, essential amino acids and fatty acids.

[0051] Other compounds within the bee-derived ingredients that have antibacterial and antiviral activity include essential oils, flavonoids, benzoic acid, abscisic acid, and 10-oxydecenoic acid. Without wishing to be bound by theory, it is believed that the remarkable antiviral activity of the compositions described and demonstrated herein is aided by the presence of several components: phenoxidase (controlled by the prophenoloxidase (proPO) activation system), lysozyme, defensins, flavonoid compounds, glucose oxidase, methylglyoxal, phenolic acids, hymenoptaecin, and apidaecin. Hymenoptaecin and apidaecin are antibacterial polypeptides produced by insects and other animals. The compositions according to the invention may include one or more of these components.

[0052] Method for preparing the extract The following describes an exemplary method of preparing an extract for use in accordance with an embodiment of the present invention, as further illustrated in Figure 1. Suitably, the extract comprises an aqueous extract comprising a mixture of hydrophilic components obtainable from beekeeping products such as honeycomb and / or zabrus.

[0053] As raw material for the extraction process, honeycomb can be obtained from beehives, which can be treated, inoculated and / or enriched as described elsewhere herein by any suitable method. Depending on the desired part to be used, the entire honeycomb with the sealing cap can be used or the sealing cap can be cut off from the honeycomb by any suitable method, typically using a sharp knife, i.e. to isolate the zabulus. The sealing cap or a part thereof can also be used as material for the subsequent steps. The material may or may not be contaminated with residual honey. The material can be frozen at this stage for ease of transportation.

[0054] The material can be ground or pulverized by any suitable method. A suspension of the hydrophobic components of the raw material (mainly waxes and water-insoluble proteins, vitamins or enzymes, essential oils, carotenoids, etc.) in water is then prepared (101). This can be done by adding the ground material to a blender, typically a high-speed (>30000 rpm) blender, together with demineralized water or preferably double-distilled water. The material is blended, for example, for at least 5 minutes, at least 10 minutes, at least 15 minutes, or at least 1 hour.

[0055] The prepared suspension / emulsion is then separated (102), for example by filtration, centrifugation and / or evacuation under vacuum, to produce a precipitate. Evacuation can be carried out through a sintered glass flat filter.

[0056] The resulting precipitate may be broken up by blending with additional water as above to form a further suspension / emulsion, followed by separation also as above (103). These steps of suspension and separation may be repeated for a total of 2, 3, 4, 5 or more cycles. The objective is to separate the hydrophilic phase components (the "sugar" phase) from the hydrophobic (or "wax") phase components as completely as possible. Repetition of the procedure facilitates separation of the water-soluble components from the hydrophobic wax fraction.

[0057] Once this is done, the resulting hydrophilic and hydrophobic phases may each be further processed separately to extract desired components, although it is contemplated that in some embodiments only one of the phases may be processed.

[0058] The water-soluble fractions (e.g., filtrate or supernatant) prepared in the previous step may be combined at this point. At this stage, the resulting fractions may be concentrated, for example, by vacuum evaporation, to produce a thick, viscous, hydrophilic product / solution (104). This is understood to include many water-soluble natural products, including honey sugar, which has an average content of about 40% glucose, 40% fructose, 3-4% maltose, and 5% polysaccharides, as well as lysozyme, as described above.

[0059] In some embodiments, a step of precipitating sugars from the product may be included to increase the concentration of lysozyme and / or other bactericidal substances (105). This may be accomplished by precipitation using a polar solvent, typically a bipolar liquid in which monosaccharides are slightly or completely insoluble, such as acetone, absolute ethyl alcohol or 2-propanol or DMSO, although other alcohols are believed to be suitable. As it is desirable to preserve the structure of any protein components, particularly lysozyme, solvents that do not denature such components are preferred, particularly acetone.

[0060] Therefore, this precipitation step can be carried out using a mechanical stirrer containing the described polar solvent and gradually introducing the hydrophilic fraction together with water to produce solution I (106). A milky suspension is obtained that layers an upper solvent phase and a lower aqueous phase.

[0061] Water is used to at least partially extract lysozyme and other components from a suspension of the sugar fraction in acetone. Both phases are centrifuged together, after which the acetone solution (Solution II) and the aqueous solution (Solution III) become completely separate and are typically isolated separately in a separatory funnel (108).

[0062] The acetone fraction can be typically evaporated in a vacuum evaporator (109) to produce an aqueous solution of monosaccharides and other hydrophilic substances (Solution IV) and, among other things, the expected lysozyme and / or other components. Previously established data on the solubility of chicken lysozyme indicate that this cationic polypeptide is completely soluble in a mixture of 10 ml of acetone and 5.2 ml of water.

[0063] The hydrophobic wax fraction produced in the initial suspension and separation step can also be further processed (110). This can be done by heating with a non-polar solvent such as cyclohexane and filtration to separate the high melting point waxes and produce a solution of the low melting point waxes in cyclohexane (or an equivalent solvent) and an aqueous fraction containing the remainder of the water soluble materials. For example, some or all of the hydrophobic fraction is suitably heated to its melting point (above 65° C.) with distilled water and cyclohexane in a glass vessel. After stirring, the hot mixture is typically filtered by vacuum filtration through a sintered glass plate, which can itself be heated prior to use or can be heated using an electric heating mantle (112).

[0064] The immiscible liquids in the resulting mixture can be separated, for example, by a glass separatory funnel, again typically at elevated temperatures (e.g., above 70° C.) to avoid solidification (113). These fractions can be further concentrated, for example, by vacuum concentration (114). The separated aqueous fraction (Solution V), with or without concentration in a vacuum evaporator, can be added to one or more of the fractions isolated in other steps (Solutions III and / or IV), or can be processed as a separate fraction containing the bioactive molecules.

[0065] Similarly, the cyclohexane fraction can be purified by dropwise addition to an excess of acetone to produce a waxy precipitate. The resulting mixture can be separated by vacuum suction or centrifugation, and the resulting filtrate is a solution of the impurities in a mixture of cyclohexane and acetone. This can be further separated by distillation to isolate the impurities as a mixture of liquid and solid components.

[0066] Further extraction methods may be preferred in various circumstances, for example to comply with GMP or other requirements and / or to ensure collection of various components, for example, it may be preferred to avoid the use of acetone in the extraction process to avoid loss or damage to various potentially biocidal components.

[0067] Thus, further methods of preparing extracts for use in accordance with embodiments of the present invention may include some or all of the following steps: All containers and equipment used in these processes must be sterilized and cleaned before the extraction process begins.

[0068] The raw material is extracted from hives / comb caps with honey or crushed combs with honey as described above. The extracted raw material is stored at 3-8°C to avoid the growth of mold and / or Gram(+) bacteria.

[0069] A specified amount of sterile, distilled / demineralized water is poured into the mixer / blender, preferably at a rotation speed of about 30000-35000 rpm. Pre-measured ingredients are then dispensed in portions into the running mixer. This disintegration process of disintegrating ingredients in water is preferably carried out for no more than 4-8 minutes, depending on the weight of ingredients being added.

[0070] The resulting suspension in the mixer (typically a cloudy white-beige liquid) is centrifuged. For example, depending on the scale, this can be in a bucket centrifuge or a decanter-type high-speed flow-through centrifuge at a rotation speed of >16000-20000 rpm. Centrifugation to remove the hydrophobic suspension, mainly wax, preferably takes more than 10 minutes. The liquid centrifuged from the precipitate is transferred to a container such as a glass buffer tank.

[0071] The disintegration process is then repeated with the centrifuged hydrophobic precipitate. The mixer / blender is preferably operated at a speed of >16000-20000 rpm with sterile water, distilled / demineralized water. Small amounts of precipitate extracted from the centrifuge precipitate are added to the blender at regular intervals (e.g., every 5-8 seconds) and the precipitate blending process is preferably continued for no more than 4-5 minutes after the entire amount of precipitate has been introduced.

[0072] The second suspension of hydrophobic material obtained therein is centrifuged again as described above, for example at a rotation speed of >16000-20000 rpm. The resulting liquid is added to the above-mentioned container. If necessary for thorough removal of the water-soluble bioactive material from the precipitate, the disintegration and centrifugation steps can be repeated one or more more times.

[0073] Optionally, if the fractions of the centrifuged liquid collected in the container indicate the presence of hydrophobic substances in the liquid (e.g., as evidenced by a slight turbidity of the liquid forming these fractions), the liquid can be subjected to further centrifugation at high speed and for a longer period than the previous centrifugation, and / or filtered, for example through a fritted filter / Shotta / Type G-4 / or a filtration membrane, which would be conventional to remove such substances from the solution.

[0074] The resulting material is a clear liquid, slightly yellow in color, obtained after centrifugation and possible purification. It can be transferred to a vacuum evaporator for concentration, for example, in a stripping flask at 38-40° C., a pressure of about 0.15-0.20 B (15 kPa-20 kPa) and 120 rpm. This can be run until no further water droplets are discharged from the condenser or the like. The resulting product is typically a very viscous, dark red-brown material, to which water or other solvents can be added in order to remove it from the distillation flask.

[0075] The product is soluble in water as well as in aqueous solutions such as marine plasma, saline solutions, etc. However, the solubility of the end product in other solvents such as ethanol, some ethers, etc. is significantly reduced by phenomena such as precipitation of sugars from the end product by these solvents.

[0076] If storage or transportation is required, the concentrated aqueous solution obtained in the small volume distillation flask is poured into the final container, which is placed in a water bath at 36-38°C or under an infrared radiator, and the residual water is then removed by a stream of nitrogen. This can be done with occasional shaking to eliminate the solid skin that forms during the application of this method. Advantageously, the container with the concentrated aqueous solution can be placed in a thermostatically controlled shaker, which is fully realized during the production stage to obtain it.

[0077] After complete evaporation, the container can be closed while maintaining a nitrogen atmosphere to facilitate storage and prevent the development of microbial colonies. Prior to dissolution in the product for use, the concentrated extract can be sterilized in any suitable manner, for example by microfiltration and / or brief gamma irradiation.

[0078] As an example of quantities, for 100 g of typical bee-derived raw material, 800 ml of HO can be used during the first process to obtain a suspension. For the second and any further steps of decomposition, 160 ml of HO can be used.

[0079] Marine Plasma Most pharmaceutical compositions, such as vaccines, are administered in aqueous solutions, suspensions or contain a lot of water, with the active ingredient making up only a very small percentage of the administered preparation. In some embodiments of the present invention, such as those involving compositions containing bee-derived components from Zabrus, standard pharmaceutical excipients such as water or saline, gels, suspensions, emulsions, etc., can be used.

[0080] In aspects of the invention, marine plasma is used in place of water, saline, or other solvents or excipients. For purposes of this specification, marine plasma refers to a liquid that includes seawater. In some embodiments, marine plasma consists of or consists essentially of seawater. In some embodiments, seawater is diluted with water until the seawater is substantially isotonic with bodily fluids such as plasma. Thus, marine plasma that includes seawater diluted to such levels can be referred to as "isotonic" marine plasma, while marine plasma that consists of or consists essentially of seawater or less diluted seawater can be referred to as "hypertonic" marine plasma. By using marine plasma instead of water, it is believed that the use of additional components that are typically used in pharmaceutical compositions, such as preservatives, e.g., formaldehyde, is not required. This may allow for lower costs in production as well as providing a more natural product that lacks the potentially harmful effects of such chemicals, such as allergic and / or toxic reactions. This may allow for the composition to be used more frequently and / or prophylactically than compositions that have such potentially harmful chemicals. Thus, in some embodiments, the composition is substantially free of pharmaceutical preservatives, such as formaldehyde.

[0081] Other typical components of pharmaceutical compositions include antibiotics, emulsifiers, stabilizers, adjuvants, and acidity regulators. While the compositions of the present invention may in fact include such components, it is contemplated that the use of marine plasma may allow one or more of these to be omitted entirely, again with the advantage of reducing costs and potentially harmful effects. Thus, in some embodiments, the compositions are substantially free of one or more of antibiotics, preservatives, emulsifiers, stabilizers, adjuvants, and / or acidity regulators.

[0082] The composition of seawater is similar in many ways to the fluids of the human or mammalian body, especially blood plasma, as understood since the late 1800s and the work of Rene Quinton. This reduces any negative reaction of the internal environment to the marine plasma used in the compositions of the present invention. Marine plasma is believed to be the result of a combination of factors generated by whirlpool plankton blooms, cyclones due to oceanographic and biological phenomena, resulting from specific conditions such as light, temperature, ocean currents and weather patterns. These can provide an abundance of zooplankton that secrete a rich and bioactive fluid or serum in the process of feeding on the nutrients and marine flora (e.g., phytoplankton). When combined with the surrounding seawater, this fluid contains inorganic salts, amino acids, DNA, RNA, antioxidants, polysaccharides, essential fatty acids, vitamins and phytochemicals in bioactive form, in addition to numerous major and trace minerals.

[0083] The marine plasma present in the compositions described herein or obtained in the methods of making such compositions may be produced by specific extraction processes and protocols used to ensure that specific concentrations of components are present and maintain their integrity. Such protocols include taking seawater to be used as or to make the marine plasma from below a plankton bloom, for example from about 10 to about 35 meters below such plankton bloom, typically about 25 to 35 meters below, and preferably about 30 meters below. Plankton blooms can be detected by satellite monitoring to identify color changes in affected areas. Thus, in some embodiments, the marine plasma is seawater taken below a plankton bloom, which may be further processed as described elsewhere herein and / or used in hypertonic or isotonic form as described above.

[0084] Further processing, if applicable, is carried out after harvesting the seawater and before or after dilution to the required osmolality. This preferably involves microfiltration of the seawater at about 0.22 micron filtration, typically double chilled microfiltration. Strict controls are in place to ensure its purity. The pharmaceutical manufacturing equipment is compliant with ISO9001, ISO14001, FDA Dietary Supplement GMP, GMP and GMP / NCF Cosmetics (ISO22716) standards.

[0085] Ocean water has approximately three times the osmolality (solute concentration) of bodily fluids. To achieve an osmolality suitable for administration to a subject, seawater is typically diluted with water suitable for therapeutic use, typically purified water, distilled water or reverse osmosis water, to generate the marine plasma. Typically, when the marine plasma comprises seawater diluted with water, the final concentration comprises 25%-30% seawater and 70%-75% water, preferably about 29% seawater and 71% water.

[0086] Other elements that may be present in the composition include fucoidan and "marine peptides," also derived from the marine environment. Fucoidan is believed to have antiviral, neuroprotective and immunomodulatory properties. Marine-derived proteins and bioactive peptides have potential for use as functional ingredients in functional foods and pharmaceuticals due to their effectiveness in both preventing and treating disease. Marine peptides are believed to be responsible for the activity of the compositions described herein, such as promoting antioxidant, antihypertensive, anticoagulant, antiproliferative, anti-human immunodeficiency virus, calcium binding, anti-obesity and anti-diabetic effects, including against parasites (Ngo et al "Biological Activities of Marine Bioactive Peptides" 2013; Cheung et al. "Marine Peptides: Bioactivities and Applications." Mar Drugs. 2015).

[0087] Typically, the marine plasma used in the compositions according to the invention may have a pH of about 6-7. The conductivity may be about 14-18 mS / cm. The total dissolved solids may be about 7-9 g / l. The salinity may be about 8.5-10 g / l.

[0088] It has been demonstrated that cell membranes contain proteins that play a role in filtration. Minerals and trace elements must be in ionic form to cross cell membranes and reach the cell nucleus and activate genes. It is believed that the minerals present in marine plasma as defined herein and their presence in ionic form allow for improved uptake into cells, if necessary. Minerals present in marine plasma play many roles in the functioning of the body and may have beneficial epigenetic effects.

[0089] Tables 1 and 2 show the possible mineral content of the marine plasma used.

[0090] [Table 1]

[0091]

Table 2

[0092] Further advantages of the use of marine plasma in the present invention include antibacterial and especially antiviral effects. For example, butylated hydroxytoluene (BHT) is an organic antioxidant that naturally occurs in marine plasma in trace amounts, especially when isolated under plankton blooms as described above, and butylated hydroxytoluene is produced by phytoplankton (Babu and Wu, "Production of Natural Butylated Hydroxytoluene as an Antioxidant by Freshwater Phytoplankton", J Phycol. 2008). Research has shown that small amounts of BHT can knock out outbreaks of lipid enveloped viruses such as herpes and cytomegalovirus (CMV) (Freeman et al "Treatment of recurrent herpes simplex labialis with topical butylated hydroxytoluene" Clin Pharmacol Ther. 1985; Kim et al "Inactivation of cytomegalovirus and Semliki Forest virus by butylated hydroxytoluene". The Journal of Infectious Diseases 1978). Lipid enveloped viruses include the pathogenic organism SARS-CoV-2 responsible for the Covid-19 pandemic. Thus, marine plasma and compositions containing marine plasma may contain BHT as discussed herein, which may explain some of the demonstrated antiviral activity. BHT has also demonstrated other properties against a range of conditions, such as cardiovascular disease, cancer and brain injury, and has also been suggested to slow aging.(Bjorkhem et al.“The antioxidant butylated hydroxytoluene protects against atherosclerosis.”Arterioscler Thromb.1991;Hocman“Chemoprevention of cancer:phenolic antioxidants(BHT,BHA).”Int J Biochem.1988;Crews et al“BHT blocks NF-kappaB activation and ethanol-induced brain damage.”Alcohol Clin Exp Res.2006;Harman“Free radical theory of aging:effect of free radical reaction inhibitors on the mortality rate of male LAF1 mice”J Gerontology.1968)。

[0093] Further components that may be present in marine plasma include naturally abundant silica. The naturally high silica content in marine plasma confers antiviral properties, and studies have shown that silica reduces the efficiency with which viruses infect other organisms and can even inactivate viruses when silica surrounds them (Laidler et al. “Reversible Inactivation and Desiccation Tolerance of Silicified Viruses”, J Virol 2013; https: / / www.nature.com / scitable / blog / viruses101 / viruses_coated_in_silica_exhibit / ; Maruyama et al. “Possibility for controlling global warming by launching nanoparticles into the stratosphere” Journal of Thermal Science and Technology 2015; Martin KR “The chemistry of silica and its potential health benefits” J Nutr Health Aging. 2007; Pati et al “Nanoparticle Vaccines Against Infectious Diseases” Front. Immunol 2018).

[0094] Silica nanoparticles have been shown to have adjuvant activity (Skrastina et al, "Silica Nanoparticles as the Adjuvant for the Immunisation of Mice Using Hepatitis B Core Virus-Like Particles" PLOS One 2014), which may be the basis for the immune enhancing effect of the compositions described herein. Again, this adjuvant effect arising from naturally occurring components means that in some embodiments, additional adjuvants such as aluminum salts can be omitted from the composition. This has advantages such as reducing any potentially toxic effects of added adjuvants. However, in some embodiments, further adjuvants may be added.

[0095] Formulation and Administration The composition according to the present invention can be formulated and administered in any suitable manner. In particular, it is envisaged that the composition can be formulated as a nasal spray, an injectable serum, a skin patch, eye drops and / or oral administration. Thus, administration can be intranasal, intramuscular, intravenous, intraarterial, subcutaneous, intraperitoneal, topical (including to the surface of the eye), transmucosal and / or oral, as appropriate.

[0096] The marine plasma bee-derived ingredient composition may be an aqueous solution, any suitable emulsion (e.g., oil-in-water, water-in-oil, microemulsion, multiple emulsion, nanoemulsion) or aqueous gel. The bee-derived ingredients may be dissolved, suspended, emulsified or carried by the marine plasma. Similar formulations may be used in embodiments where marine plasma is not used.

[0097] Information regarding specific formulations and routes of administration and considerations therefor can be found below.

[0098] Nasal sprays. The absorption of nasal sprays is influenced by the residence time on the epithelial tissue in the mucosa. In this method of administration, mucociliary clearance can inhibit the delivery of the formulation to the absorption site. Waxes and oils from bee products can be added to increase viscosity and reduce clearance. Increased mucociliary clearance reduces the absorption of the product. Apart from that, nasal spray formulations can be developed by suitable means known in the art. Such formulations are particularly useful against microorganisms that invade via the respiratory tract and associated tissues. Deposition of the active ingredient in the anterior and posterior regions of the nose affects the absorption of the active ingredient in nasal formulations. For example, the SARS-CoV-2 virus is believed to enter cells primarily through the ACE2 receptor. The nasal cavity (and the conjunctival mucosa that is anatomically associated with the nasal cavity) has one of the highest levels of ACE2 receptors in the human body, making the nasal mucosa the most important route for coronavirus translocation. The tear film (the liquid layer of the eye) contains many antibacterial and antiviral compounds, one of which is lysozyme, which functions continuously to promote the elimination of viral pathogens. Nasal sprays can enhance lysozyme substances (in embodiments in which lysozyme is included) to reach the nose and eyes and replenish the tear film. As a result, nasal sprays (and other formulations) can be used prophylactically as virus mitigation / protection formulations.

[0099] Skin patch. A patch may be used to allow the composition to be absorbed through the skin. Such a patch may include a backing layer that serves as the outer surface of the patch during use, a reservoir layer containing the composition described herein, an adhesive for fixing the patch to the skin of a subject, and a release liner that is peeled off to expose the reservoir layer and the adhesive. The adhesive may suitably be an organic adhesive and may be included in the reservoir layer and / or the composition itself. The reservoir layer may further include a penetration enhancer that promotes percutaneous absorption. The penetration enhancer may include bee venom (either as a bee-derived component or with another) or one or more of its components, such as melittin. Melittin is a peptide and is the main component of bee venom. Melittin induces membrane permeabilization and lyses cells. Bee venom also has biologically active amines, such as histamine, epinephrine, dopamine, norepinephrine, and enzymes such as phospholipase A2, hyaluronidase, acid phosphomonoesterase, lysophospholipase, etc. Other components of bee venom include lipids, carbohydrates and free amino acids. In a patch formulation, it may be particularly advantageous to formulate the composition as an aqueous gel, colloidal gel or hydrogel by any suitable means so that the composition is more easily contained within the patch and held against the skin. Thus, the composition may further comprise hydrophilic polymer chains.

[0100] Injectable compositions. As mentioned above, the compositions may be formulated for injection, which may be intramuscular, intravenous, intraarterial, subcutaneous or intraperitoneal, preferably intravenous. Typically, such compositions may be formulated at a ratio of one thousandth of a gram of bee-derived component, e.g., lysozyme, to 0.5 ml of marine plasma or other vehicle. Other bee-derived components may be present in microgram quantities, if desired.

[0101] Oral Compositions. Compositions for oral administration are similar to the injectable forms described above. However, more formulations may be used since the oral mucosal epithelium is the main site of most pathogens and viruses. This may be achieved by increasing the concentration of bee-derived ingredients and / or including additional bee-derived ingredients or marine products described elsewhere herein. Oral compositions may be formulated in liquid, gel or capsule form or in any suitable manner. In particular, the compositions described herein may be delivered through food, for example by adding them to food, beverages or other nutritional products.

[0102] Topical ophthalmic compositions. The compositions described herein can be formulated for delivery by application (such as eye drops) to the ocular surface in both humans and animals. Intraocular vaccines in both humans and animals have disadvantages, such as contributing only to local immunity and having the risk of cross-reactivity with other reactions and potential side effects. Lysozyme, which is naturally present in the lacrimal gland and nasal mucus of both humans and animals, is also the main active moiety of certain embodiments of the compositions described herein, making the compound non-foreign to the subject to be vaccinated.

[0103] Other possibilities for formulation or administration are also contemplated.

[0104] Uses and Effects As described above, both the bee-derived components and the marine plasma are believed to have a variety of antimicrobial activities, including antibacterial, antiviral, antifungal and antiparasitic activities. The combination of the bee-derived components and the marine plasma is particularly advantageous for a number of reasons. The combination of antimicrobial activities from the components leads to coverage of multiple potential microbial targets. In addition, if the bee-derived components are selected or produced for efficacy against specific microbial targets, as described elsewhere herein, the combination of the specific activity from the bee-derived components and the general activity of the marine plasma may allow for action against specific targets without sacrificing the general immune supportive effect. Furthermore, as described, the use of marine plasma in place of typical inert solvents or carriers (such as water or saline) in such formulations allows for antimicrobial activity to be provided in parts of the composition that are usually bare of nothing.

[0105] As such, the compositions described herein or produced by the methods described may be used as antibacterial, antiviral, antibacterial, antifungal and / or antiparasitic compositions.

[0106] As a result, methods of treatment or prevention of various conditions are possible using these compositions. A method of treating bacterial, viral, fungal or parasitic diseases, typically viral or bacterial diseases, can include administering a therapeutically effective amount of the composition described herein or the composition produced by the method described herein to a subject in need thereof. The composition can be formulated as described above (e.g., as a nasal spray, an injectable serum, a skin patch, eye drops and / or an oral formulation) and / or administration can be by one or more of the methods described above.

[0107] The method of treating disease may be carried out through one or more of a variety of methods. The antibacterial effect of the components of the composition detailed above may have a direct effect on disease-causing pathogens. Similarly, the immunogenic or immunostimulatory activity of the components of the composition described above may enhance or support the immune system in preventing disease. The compositions described herein may also act to relieve symptoms of various diseases, such as cough, sore throat, rhinitis, rashes, and other symptoms, and may act in such a way as, for example, viral alleviation.

[0108] It is also contemplated that the compositions described herein and compositions produced by the methods described herein can be used in methods of preventing disease, such as bacterial, viral, fungal, or parasitic diseases, typically viral or bacterial diseases. Again, such methods can include administering a therapeutically effective amount of the compositions described herein or compositions produced by the methods described herein to a subject in need thereof. The compositions can be formulated as described above (e.g., as a nasal spray, injectable serum, skin patch, eye drops, and / or oral formulation), and / or administration can be by one or more of the methods described above.

[0109] Methods of preventing disease may be similarly performed in one or more of a variety of ways. The antimicrobial action of the components of the compositions detailed above may have a direct effect on disease-causing pathogens, killing or inactivating such pathogens before they enter the body, or may reduce the infectious dose so that the pathogen cannot establish itself. Such preventative activity may act by preventing the entry of pathogens into the body by providing a physical barrier, such as a protective layer over the mucous membranes and tear film of the eye. For example, when administered as a nasal spray or eye drops, the composition may prevent the entry of pathogens that access the body through the relevant mucous membranes or respiratory tract by physically blocking the pathogen or killing or inactivating the pathogen. Thus, the compositions described herein may be particularly effective in preventing and / or treating respiratory diseases such as influenza, SARS-CoV and SARS-CoV-2. Similarly, the immunogenic or immunostimulatory activity of the components of the compositions described above may enhance or support the immune system of the subject to whom the composition is administered, such that the composition may be used as an immune support or booster.

[0110] The provision of lysozyme and other bioactive factors can act to replenish the limited amount of bioactive agents produced by the human or animal body, for example in the lacrimal duct, oral cavity and tracheobronchial airways of the lungs. This can have the effect of lowering the amount of virus and / or the number of particles ingested, which can help prevent infection. Even in infected individuals, this can reduce the amount of virus that invades while stimulating the immune response, thereby alleviating symptoms, mitigating the course of the disease and increasing the effectiveness of the body's defenses.

[0111] More particularly, it is envisioned that the compositions described herein or compositions produced by the methods described herein may be used to improve an ongoing immune response in a subject against a particular pathogen, thus providing the subject with long-lasting immunity against that pathogen, similar to a vaccine response. As noted above, it is possible to inoculate a bee colony with a particular pathogen in order to induce an immune response against such a target. If bee-derived components obtained from such treated bees have specific activity against that pathogen, it is contemplated that compositions comprising such bee-derived components may be used to promote induction of an immune response in a subject against that pathogen, either directly against that pathogen and / or via administration to the subject.

[0112] It is also believed that the compositions of the present invention may improve or correct certain common problems with oral vaccines. There is consensus that the failure of oral vaccines is mainly due to oral resistance. Oral vaccination is particularly difficult due to many factors, including the harsh gastrointestinal environment, which means that the delivered antigens are difficult to survive, and the mechanism of oral resistance, which reduces the immune response to ingestion. The compositions of the present invention aim to avoid the difficult problems of administration by the commonly performed oral route. Oral vaccination by this method offers the possibility of stimulating two immune responses, humoral and cellular, at systemic and mucosal sites. Naturally occurring peptides in marine plasma may induce and improve oral tolerance, thus increasing the chances of oral vaccine success.

[0113] Potential pathogens and associated diseases that the described compositions may be used to treat, mitigate and / or prevent include bacterial, viral, fungal or parasitic diseases. For example, these include viral diseases caused by pathogenic organisms such as influenza, coronaviruses such as SARS-CoV-2 and its variants, and rhinoviruses. Bacterial targets may include Salmonella enteritidis, Escherichia coli, Staphylococcus aureus, and the like.

[0114] Furthermore, due to differences in the geographic distribution of pathogens and the ability of bees to combat pathogens in their surroundings, the present invention can easily provide customized products for any region of the world by locating source hives in specific locations and / or providing the hives with customized pathogen compositions.

[0115] Some of the mechanisms of antibacterial action of certain components in the compositions described herein are known, such as the enzymatic action of lysozyme on bacterial cell walls, while others are less well understood. It is believed that epigenetic effects may underlie some of the effects. A discussion of possible effectors of insect immunity can be found in the art (Chan et al. "The innate immune and systemic response in honeybees to a bacterial pathogen, Paenibacillus larvae." BMC Genomics. 2009).

[0116] Lysozyme's well-documented antibacterial effects help protect against secondary infections following viral exposure. Lysozyme is highly stable and can withstand the acidic environment of the stomach, making it safe for oral administration.

[0117] The compositions described herein may be used in human subjects, but also in non-human animals, such as non-human mammals, and livestock animals raised for meat or other purposes. In particular, it is believed that poultry, especially domestic poultry, may be subjects of the compositions described herein.

[0118] Economic losses caused by major infectious diseases in poultry in the past decade have had a significant impact on both commercial and public sectors. Poultry diseases can also be transmitted to humans, i.e. they are zoonotic and pose a threat to public health. Infectious diseases in chickens that are transmissible to humans range from viruses (e.g. avian influenza, Newcastle disease), bacteria (e.g. Salmonella enteritidis, Escherichia coli) that can be food-borne, to parasites, fungi, etc.

[0119] Livestock health crises affect large parts of the world. For example, in the MENA region, such problems included H5N8, H9N2, Gumboro, Newcastle virus, infectious laryngotracheitis, IB 4-91, and avian infectious bronchitis virus strain IB Ma5. Some of these infectious diseases had no vaccines developed at the time, leading to large-scale livestock burning programs and the closure of farms.

[0120] The advantage of the compositions of the present invention as prophylactic antimicrobial compositions in livestock animals is that they can replace the use of antibiotics, which run the risk of encouraging the evolution of resistant microbial strains that can be transmitted to humans.In addition, certain farming practices may mean that the use of antibiotics is prohibited.

[0121] Similarly, the advantage of all compositions described herein is that they contain natural ingredients, exclude harmful chemicals, and can minimize side effects. In particular, this means that such compositions can be used over long periods of time without concern for the accumulation of potentially harmful components. This is particularly useful when used as a preventative and / or immune-boosting composition, as it allows the composition to be taken during daily life and / or during periods of high disease risk, such as winter and epidemics / pandemics, and maintain its benefits. The natural source of the composition's components can also reduce the risk of drug resistance developing in the microorganisms against which the drug is directed, and allow the prevention of resistance to standard treatments that may have already developed. As a result, the compositions described herein can be used with other treatments, again with the advantage of a low risk of side effects. EXAMPLES

[0122] Preparation of viral suspension: For preparation of viral suspension, HeLa cells were cultured at 175 cm with Dulbeco's minimum essential medium with Earle's BSS and 10% fetal bovine serum (FBS). 3 The cells were cultured in flasks (NEST SCIENTIFIC Biotechnology, New Jersey, USA). Poliovirus type 1, LSc 2ab (picornavirus), and non-enveloped RNA viruses (stock virus suspensions) were added to the monolayer for 1 h at 37 °C, with gentle shaking every 15 min. After the cells showed a cytopathic effect, the cells were frozen (-80 °C) and thawed three times, followed by low-speed centrifugation (10 min, 1500 × g) to sediment the cell debris. After aliquoting, the virus titer was measured and a Median Tissue Culture Infectious Dose (TCID ) of 10–12 was determined. 50 ) was determined. The virus suspension was then divided into tubes in 1 ml volumes and given to the nest boxes for inoculation.

[0123] Six additional viruses were generated in a similar manner (Adenovirus 5, Adenovirus 36, Coronavirus OC43, Herpes simplex and Equine herpesvirus type 1).

[0124] Bee colony management and inoculation: Bee colonies of the species Carniolan (Apis mellifera carnica) from the experimental academic apiary of the University of Life Sciences in Wroclaw (UPWr) in Słojec were used for certain experiments, i.e., for inoculation with polioviruses, discussed below. The colonies were healthy, in good condition, and inhabited two sections of the hive. For other experiments, colonies of the wild bee, the Northern European dark bee (Apis mellifera mellifera) (Augustow strain), inhabiting wooden hives, were used.

[0125] After assessing the swarms for brood population, colonies were selected for specific inoculations. During this stage, no treatment with chemicals (e.g., acaricides for treatment of mites and / or ticks) was performed on the colonies, which could result in increased predation among the swarms. Bees had constant access to food during the experiment.

[0126] Inoculation of colonies with virus preparations was performed by spraying all hives from both sides. 5 ml of virus suspension was mixed with 95 ml of 50% sucrose solution to produce the composition for spraying. For each virus, this was performed by spraying every 4 days for a total of 4 times. 9 days after the last application, material was taken from the colonies for further use.

[0127] Processing of material: Material isolated from bee colonies for further processing was processed into separate extracts, including: Inoculated nests - crushed honeycombs without sealing caps (i.e. without the zabulus discussed herein) from colonies inoculated with the viruses described; Inoculated Zabulus - A fragment of a sealed cap from a colony inoculated with the described virus, contaminated with some honey and wax; Uninoculated nests - crushed hives without sealing caps (without Zabrus) from colonies that were not inoculated with the virus; Uninoculated Zabulus - A fragment of a sealed cap from a colony that was not inoculated with virus.

[0128] These materials were added in portions to a blender containing sterile water and distilled / demineralized water, respectively, for 4-8 minutes at >30,000 rpm to break down the ingredients and produce a milky suspension of the hydrophobic components of the ingredients in water.

[0129] The resulting suspension in the mixer (a cloudy beige liquid) was transferred to a bucket centrifuge at a rotation speed of >16000-20000 rpm for 10 min. The liquid separated from the sediment was transferred to a glass buffer tank.

[0130] The disintegration and centrifugation steps were performed on the centrifuged hydrophobic precipitate at least one more time with a blender speed of >16000-20000 rpm for 4-5 minutes, and the centrifuged liquid was again transferred to the glass buffer tank. If the presence of hydrophobic material was subsequently evident in the liquid collected in the buffer tank (e.g., a slight milky color of the liquid forming these fractions), the liquid was subsequently filtered and / or centrifuged as described above.

[0131] The resulting clear liquid preparation was evaporated in a stripping flask at 38-40°C under vacuum, 0.15-0.20 B pressure and 120 rpm until no water droplets were discharged from the condenser into the receiver, yielding a very thick viscous hydrophilic product, from which water was removed and subsequently processed for storage at the desired location by removing the remaining water under a nitrogen stream as described elsewhere herein.

[0132] To produce the final extract, the above products were combined with marine plasma, specifically isotonic or hypertonic marine plasma obtained from Quinton Medical, product numbers PT200 and PT201, respectively (Laboratoires Quinton International, SL, Alicante, Spain). The initial solution has the viscosity of honey. They were suspended in marine plasma (hypertonic or isotonic). The final concentration produced depends on the initial weight of the extract obtained, the concentrations being discussed below. For example, a concentration of about 1-3% was prepared by suspending 0.6g-0.9g of extract in 10ml of marine plasma.

[0133] Example 1 - Determination of the effectiveness of the preparation against poliovirus type 1 (PV-1) The aim of this study was to evaluate the virus inactivation properties against poliovirus type 1 of the described extract preparations obtained from hives inoculated or not inoculated with poliovirus type 1 using the quantitative suspension assay according to PN-EN 14476+A2:2019-08. This standard describes a quantitative suspension test for the determination of virus inactivation activity in the pharmaceutical field by mixing 1 volume part of test virus suspension, 1 volume part of interfering substance and 8 volume parts of disinfectant. Aliquots are taken at the specified contact times and the remaining infectivity is determined.

[0134] Preparation of test virus suspension: For preparation of the test virus suspension, HeLa cells were cultured at 175 cm with Dulbeco's minimum essential medium with Earle's BSS and 10% fetal bovine serum (FBS). 3 The cells were cultured in flasks (NEST SCIENTIFIC Biotechnology, New Jersey, USA). Poliovirus type 1 (stock virus suspension) was added to the monolayer for 1 h at 37°C with gentle shaking every 15 min. After the cells showed a cytopathic effect, the cells were frozen (-80°C) and thawed three times, followed by low-speed centrifugation (10 min, 1500 x g) to sediment the cell debris. After sorting, the test virus suspension was stored in aliquots at -80°C.

[0135] Infectivity assay: Infectivity was determined by titration at the endpoint, by transferring 0.1 ml of each dilution into 8 wells of a microplate, starting with the highest dilution. This was followed by transfer of 0.1 ml of freshly trypsinized HeLa cells (10–15 × 10 per well). 3 The microplates were incubated at 37°C in a 5% CO2 atmosphere. The plates were observed daily (for 7 days) and the cytopathic effect was read by using an inverted microscope (Axio Observer, Carl Zeiss MicroImaging GmbH). Infectious dose TCID 50 / ml was calculated according to the Spearman i Karber method using the following formula.

number

[0136] Inactivation assay: Measurement of virus inactivation activity was performed. The prepared extract was examined as a 0.1% solution. To prepare the extract for use, 0.690 g of the extract prepared from the polio-inoculated hive described above was resuspended in water. Half of the resulting suspension was diluted in hypertonic marine plasma and half in isotonic marine plasma to produce the extract for use. 800 microliters of extract were then mixed with 100 microliters of virus and 100 microliters of PBS. 50 microliters of this mixture was inoculated into 50 microliters of cell culture. The final amount of extract was 0.1%. The contact time was 60 minutes. For easier operation, the volumes in this assay were 0.1 ml test virus suspension, 0.1 ml interfering substance (PBS) and 0.8 ml test product (extract (0.17%) + isotonic marine plasma; or extract (0.17%) + hypertonic marine plasma). Immediately after the end of the contact time, the bactericidal activity was evaluated by 10 -12 The titration was stopped by dilution to 0. Virus control titrations were performed at 0 and 60 min contact times.

[0137] Cytotoxicity measurements were performed with 1 ml test product to determine the concentration of product at which no cytotoxicity was detected. -1 ~10 -8 This is done by making a series of dilutions at or above dilution factors (depending on the virus) which are then inoculated into cell cultures to determine how toxic the test product is and whether the changes in the cultured cells caused by toxicity are similar to those caused by the virus.

[0138] Cell susceptibility For the cell susceptibility control, 2 volumes of double distilled water were mixed with 8 volumes of the highest apparently non-cytotoxic dilution of the product in PBS. This mixture was added to a large volume of 2-fold concentrated cell suspension. After 1 hour at 37°C, the cells were centrifuged and resuspended in medium. Finally, a comparative titration of the virus test suspension was performed on treated and untreated (PBS) cells.

[0139] Efficacy control for inhibition of bactericidal activity. A mixture of cold DMEM + 2% FCS and the preparation was incubated in an ice bath for 30 min and incubated for 10 min. -12 A dilution series was made up to 100%. The virus titer was determined and compared to the test titer.

[0140] Reference virus inactivation test A 1.4% formaldehyde solution was included as a reference for the measurement of virus inactivation activity. The cytotoxicity of the formaldehyde test solution was 10 -5 was determined by dilution to

[0141] Validation The following criteria were assessed: a. The titer of the test virus suspension is ≥ 4 log 10 This allowed the measurement of the decrease. b. The cytotoxicity of the test product (0.1%) is 4 log of the viral titer 10 This makes it possible to detect the decline. c. The starting concentration of the product (0.1%) increases by 4 logs within 60 minutes. 10 The results showed a reduction in virus titer of more than 100%. d. Comparative titration on treated (1:10000 dilution of preparation) and untreated cells showed acceptable differences for the virus ( <log 10 ). e. Product inhibition efficiency control was ≦0.5 log. It was determined that these criteria were met and, as a result, testing to PN-EN 14476+A2:2019-08 is warranted.

[0142] Results: Results of virus inactivation tests using extracts from inoculated, uninoculated and uninoculated zabrus as raw materials, prepared as described above, and formulated into either isotonic or hypertonic marine plasma. In these examples, the hypertonic marine plasma was undiluted and the isotonic plasma was diluted three-fold with spring water. Each condition was performed in replicates of 8 cell culture units.

[0143] The results are shown in Table 3. In the table, a score of 0 indicates no detected viral activity and a score of 1-4 indicates the detection of the presence of virus (degree of cytopathic effect / CPE). For ease of visualization, conditions where CPE was not detected in any replicate test are shaded in grey.

[0144] Determination of the toxicity of the products under the same conditions (not shown) did not reveal any notable toxicity at any of the dilutions tested. Similarly, formaldehyde control experiments, not shown, showed cytotoxicity at dilution levels 1 and 2, with little or no viral activity at higher dilutions.

[0145] Summary The mean reduction in poliovirus activity was as follows: Inoculated Zabrus - Isotonic 5.3125log Inoculated Zabulus - Hypertonic 5.0log Unvaccinated Zabrus - Isotonic 4.6875log Zabulus not vaccinated - Hypertonic 5.0log Uninoculated nest - isotonic 3.75log Uninoculated nests - Hypertonic 3.75 logs

[0146] According to the standard, the preparation must have a viral titer of at least 4 log after the recommended exposure time. 10 If the reduction in the viral inactivation property is greater than 99.99% (inactivation > 99.99%), it is considered to be a viral inactivation property. Inoculated Zabrus extract (from hives inoculated with poliovirus type 1) in isotonic or hypertonic marine plasma was tested at 0.1% for an exposure time of 60 minutes. After this time, a reduction in the viral inactivation property of > 4 log equates to a potency of > 99.99%. 10 A reduction in the titer of poliovirus type 1 was determined. Therefore, the formulation can be considered as a preparation with virus inactivation properties against non-enveloped poliovirus type 1.

[0147] Viral inactivation potency tests against poliovirus type 1 were performed on sarcoids that were not inoculated with poliovirus type 1 and on nests that were not inoculated with poliovirus type 1. In both cases, the efficacy of viral inactivation properties for extracts suspended in hypertonic marine plasma was -4 log (>99.99% viral reduction) and 3.5 log (99.95% viral reduction) for extracts suspended in isotonic marine plasma. Equivalent tests using only marine plasma were performed but showed only a slight reduction in viral activity (not shown).

[0148] Example 2 - Determination of the effectiveness of the preparation against Equine Herpesvirus Type 1 (EHV-1) The objective of this study was to determine the viral inactivation properties against Equine Herpesvirus type 1 of inoculated Zabrus preparations obtained from hives inoculated with Equine Herpesvirus type 1 using a quantitative suspension assay according to PN-EN 14476+A2:2019-08.

[0149] Preparation of the test virus suspension was carried out essentially as in Example 1, except that RK-13 ​​cells and a stock virus suspension of Equine Herpesvirus Type 1 were used.

[0150] Infectivity assays were carried out essentially as in Example 1, except that RK-13 ​​cells were used.

[0151] Inactivation assay Tests for the determination of virus inactivation properties were subsequently carried out. The inoculated Zabulus preparations were examined as 0.1% solutions. The contact time was 30 minutes at a temperature of 10°C. The volumes in the assay were 0.1 test virus suspension, 0.1 ml interfering substance (PBS) and 0.8 ml test product (inoculated Zabulus extract (0.17%) and isotonic marine plasma). Immediately after the end of the contact time, the activity of the germicides was assessed by 10 -8 The reaction was stopped by diluting to 0.

[0152] The steps of measurement of cytotoxicity, cytosensitivity and reference virus inactivation tests were carried out substantially as in Example 1. The validation criteria were met, namely: a. The titer of the test virus suspension is ≥ 4 log 10 This allowed the measurement of the decrease. b. The difference in titer between the control virus and the virus in the 30 min inactivation assay was log-0.5 to log-2.5. C. The cytotoxicity of the test product (0.1%) is 4 log of the viral titer. 10 This makes it possible to detect the decline. d. The starting concentration of the product (0.1%) increased by 4 logs within 30 minutes. 10 The results showed a reduction in virus titer of more than 100%. e. Comparative titration on treated (1:10,000 dilution of the preparation) and untreated cells showed acceptable differences for the virus ( <log 10 ). It was determined that these criteria were met and, as a result, testing to PN-EN 14675 was warranted.

[0153] Results The results of the viral inactivation tests using inoculated Zabrus as raw material and extracts prepared as described above and formulated with isotonic marine plasma are shown in Table 4. In the table, a score of 0 indicates no detected viral activity, while a score of 1-4 indicates detection of the presence of virus (degree of cytopathic effect / CPE). t indicates detection of cytotoxicity. For ease of visualization, conditions where CPE was not detected in any replicate test have been shaded in grey.

[0154] Determination of the toxicity of the products under the same conditions (not shown) did not show any significant toxicity at any of the dilutions tested. Similarly, formaldehyde control experiments, not shown, showed cytotoxicity at dilution levels 1 and 2, and no viral activity at higher dilutions.

[0155] Summary According to the standard, the preparation should produce a virus titer of at least 4 log after the recommended exposure time.10 If the reduction in the concentration of the virus is greater than 99.99%, it is considered to be a virus inactivating property. Inoculated Zabulus extract (from a hive inoculated with Equine Herpesvirus Type 1) in isotonic marine plasma was tested at a concentration of 0.1%. The exposure time was 30 minutes at a temperature of 10°C. After this time, a reduction in the concentration of the virus inactivating property of ≥ 4 logs equates to a potency of ≥ 99.99%. 10 A reduction in the titer of 1000 mg / kg / day was determined. Therefore, the formulation can be considered to have virus-inactivating properties against enveloped equine herpesvirus type 1.

[0156] Example 3 - Determination of the effectiveness of the preparation against adenovirus type 36 (Adenoviridae) The aim of this study was to determine the viral inactivation properties against adenovirus type 36 of inoculated Zabrus preparations obtained from hives inoculated with adenovirus type 36 using a quantitative suspension assay according to PN-EN 14476+A2:2019-08.

[0157] Preparation of the test virus suspension was carried out substantially as in Example 1, except that A549 cells and a stock virus suspension of Adenovirus type 36 were used. Similarly, the infectivity assay was carried out substantially as in Example 1, except that A549 cells were used.

[0158] The inactivation assays were carried out substantially as in Example 1. Similarly, the steps of measuring efficacy controls and reference virus inactivation tests for inhibition of cytotoxicity, cell susceptibility, bactericidal activity were carried out substantially as in Example 1.

[0159] The validation criteria were evaluated substantially as in Example 1, and it was determined that the criteria were met, so that the test according to PN-EN 14476+A2:2019-08 was valid.

[0160] Results Results of viral inactivation tests using extracts starting from inoculated Zabulus from hives inoculated with adenovirus type 36, prepared as described above, and formulated into isotonic marine plasma. Each condition was performed in replicates of eight cell culture units.

[0161] The results are shown in Table 5. In the table, a score of 0 indicates no detected viral activity, and a score of 1-4 indicates detection of the presence of virus (degree of cytopathic effect / CPE). t indicates detection of cytotoxicity. For ease of visualization, conditions in which CPE was not detected in any replicate test are shaded in grey.

[0162] Determination of the toxicity of the products under the same conditions (not shown) did not reveal any notable toxicity at any of the dilutions tested. Similarly, formaldehyde control experiments, not shown, showed cytotoxicity at dilution levels 1 and 2, with little or no viral activity at higher dilutions.

[0163] Summary According to the standard, the preparation should produce a virus titer of at least 4 log after the recommended exposure time. 10 A reduction in the viral inactivation properties is considered to be a viral inactivation property (inactivation ≥ 99.99%). The inoculated Zabrus preparation was tested against the obesity-associated adenovirus type 36 at a concentration of 0.1% and an exposure time of 60 minutes. After this time, a reduction of ≥ 4 log equates to an efficacy of viral inactivation properties of ≥ 99.99%. 10 A reduction in the titer of 100 μg / ml was determined. Therefore, the preparation can be considered to have virus-inactivating properties against non-enveloped adenovirus type 36.

[0164] Example 4 - Determination of the effectiveness of the preparation against herpes simplex type 1 (family Herpesviridae) The aim of this study was to determine the virus inactivation properties against herpes simplex type 1 (HSV1) of inoculated Zabrus preparations obtained from hives inoculated with HSV1 virus using a quantitative suspension assay according to PN-EN 14476+A2:2019-08.

[0165] Preparation of the test virus suspension was carried out substantially as in Example 1, except that a stock virus suspension of Herpes Simplex Virus Type 1 (HSV1) was used. Similarly, the infectivity assay was carried out substantially as in Example 1.

[0166] The inactivation assays were carried out substantially as in Example 1. Similarly, the steps of measuring efficacy controls and reference virus inactivation tests for inhibition of cytotoxicity, cytosensitivity, bactericidal activity were carried out substantially as in Example 1.

[0167] The validation criteria were evaluated substantially as in Example 1, and it was determined that the criteria were met, so that the test according to PN-EN 14476+A2:2019-08 was valid.

[0168] Results: Results of virus inactivation tests using extracts prepared as above and formulated with isotonic marine plasma, using as raw material inoculated zabulus from hives inoculated with HSV1. Each condition was performed in duplicate with 8 cell culture units. As a control, a virus inactivation effect test against Herpes simplex virus type 1 was similarly performed with isotonic marine plasma without the addition of hive extract.

[0169] The results are shown in Table 6. In the table, a score of 0 indicates no detected viral activity, and a score of 1-4 indicates detection of the presence of virus (degree of cytopathic effect / CPE). t indicates detection of cytotoxicity. For ease of visualization, conditions in which CPE was not detected in any replicate test are shaded in grey.

[0170] Determination of the toxicity of the products under the same conditions (not shown) did not reveal any notable toxicity at any of the dilutions tested. Similarly, formaldehyde control experiments, not shown, showed cytotoxicity at dilution levels 1 and 2, with little or no viral activity at higher dilutions.

[0171] Summary According to the standard, the preparation should produce a virus titer of at least 4 log after the recommended exposure time. 10 If there is a reduction (inactivation ≥ 99.99%), it is considered to have virus inactivation properties. The inoculated Zabrus preparation was tested against HSV1 at a concentration of 0.1% and an exposure time of 60 minutes. After this time, a viral inactivation potency of ≥ 4 log equates to a viral inactivation potency of ≥ 99.99%. 10 A reduction in the titer of 1.0001 was determined. Thus, the formulation can be considered as having viral inactivating properties against HSV1. As a control study, a viral inactivating properties efficacy test against Herpes Simplex Virus Type 1 was performed with isotonic marine plasma. The efficacy of viral inactivating properties was 2.75 log (99.75% viral reduction).

[0172] Example 5 – Determination of the effectiveness of the preparation against influenza virus A / H1N1 The aim of this study was to determine the viral inactivation properties against influenza virus A / H1N1 of inoculated Zabrus preparations obtained from hives inoculated with influenza virus A / H1N1 using the quantitative suspension assay according to PN-EN 14476+A2:2019-08 and embryonated chicken eggs according to the revised ISO 18184 standard.

[0173] The PN EN 14476+A2:2019-08 standard describes a quantitative suspension test for the determination of virus inactivation properties in the pharmaceutical field, in which 1 volume part of test virus suspension, 1 volume part of interfering substance and 8 volume parts of disinfectant are mixed (phase 2 / step 1). Aliquots are taken at specified contact times to determine the remaining infectivity.

[0174] The use of chicken embryos to test with influenza virus is described in the ISO 18184 standard. A suspension of the product to be tested and the virus is inoculated into the allantoic cavity of 10-day-old embryonated chicken eggs after the exposure time. The ECE are then incubated at 35°C for 2 days and the allantoic fluid is tested using the hemagglutination test. Hemagglutination indicates virus replication and therefore the lack of virus-inactivating properties of the test substance.

[0175] Preparation of the test virus suspension was carried out essentially as in Example 1, except that a stock virus suspension of influenza virus A / H1N1 was used.

[0176] Preparation of chicken embryos: 10-day-old chicken embryos from Rossa hens were used. Embryos were infected with 0.2 ml of virus suspension inoculated into the allantoic cavity of embryonated chicken eggs (ECE). Eggs were incubated in an incubator at 35°C for 3 days. After incubation, eggs were placed in a refrigerator to cool overnight and then allantoic fluid was collected. All fluids collected from eggs were examined by hemagglutination to confirm influenza virus replication. Immediately prior to testing, virus-containing allantoic fluids were diluted with 0.01% ethanol to obtain a virus concentration of 10 7 EID 50 The solution was diluted with phosphate buffered saline (PBS) (pH 7.2) to 0.2 ml.

[0177] Preparation of 0.5% suspension of chicken erythrocytes: 4.0 ml of chicken venous blood was collected using a syringe containing 1.0 ml of sterile 2% sodium citrate placed in a tube. The blood was centrifuged at 1,000 g for 10 min. The supernatant was then collected and the cells were soaked with PBS (5 ml). This was repeated twice, and then 0.5 ml of the cell pellet was suspended in 99.5 ml of PBS and the suspension was shaken.

[0178] Test procedure (ISO18184 / F7) and PN-EN14476+A2:2019-08: The test for the determination of virus inactivation activity was carried out according to PN-EN14476+A2:2019-08 (EN5.5.2). As raw material, a 0.1% solution of extract using inoculated Zabulus from a hive inoculated with influenza virus A / H1N1 in isotonic marine plasma was prepared as described above. To make the operation more convenient, the volumes used in the assay were 0.1 ml test virus suspension, 0.1 ml interfering substance (PBS) and 0.8 ml test product (extract from inoculated Zabulus and isotonic marine plasma). Immediately after the end of the contact time, the activity of the germicide was stopped by cooling.

[0179] Inoculation of 10-day-old embryonated chicken eggs with virus suspension according to ISO 18184: After a contact time of 60 minutes, 0.2 ml of the prepared suspension of virus containing the inoculated Zabrus preparation was inoculated into the allantoic cavity of three ECEs. The ECEs were incubated at 35°C for 2 days. After 2 days of incubation, the allantoic fluid was collected from the eggs and placed in tubes as described. A 0.5% suspension of chicken red blood cells was then added to the tubes. The tubes were observed for hemagglutination.

[0180] Calculation of viral titer: EID 50 Viral titers were calculated using the method of Reed and Muench. The 50% EID50 is calculated based on the cumulative number of infected embryos.

[0181] Efficacy values ​​were calculated based on the following formula: Mv=-lg(Vb / Va)=-[lg(Vb)-lg(Va)] where Mv is the value for antiviral efficacy; lg(Vb) is the EID20 of infected embryo allantoic fluid not contacted with the test sample after a given contact time. 50 lg(Va) is the EID 20 of infected allantoic fluid from embryos contacted with the sample after the indicated contact time. 50 Shows.

[0182] The results of the investigation are shown in Table 7. Viral infection (-): Red blood cell precipitation is observed, but no agglutination. Viral infection (+): Red blood cell agglutination is observed.

[0183] According to the standard, a preparation is considered to have virus-inactivating properties if the virus titer is reduced compared to the control virus after the recommended exposure time. The observed reduction was 6 logs. Therefore, the formulation can be considered to have virus-inactivating properties against enveloped influenza virus A / H1N1.

[0184] Example 6 – Determination of the effectiveness of the preparation against human coronavirus OC43 (HcoV-OC43) The aim of this study was to determine the viral inactivation properties against human coronavirus OC43 (HcoV-OC43) of inoculated Zabrus preparations obtained from hives inoculated with HcoV-OC43 using a quantitative suspension assay according to PN-EN 14476+A2:2019-08.

[0185] Preparation of the test virus suspension was carried out essentially as in Example 1, except that A549 cells and a stock virus suspension of betacoronavirus 1 strain OC43 (ATCC® VR-1558™) were used.

[0186] Infectivity assay: Infectivity was determined as an endpoint titration by transferring 0.1 ml of each dilution into 8 wells of a microplate, starting with the highest dilution. This was followed by transfer of 0.1 ml of freshly trypsinized A549 cells (10–15 × 10 per well). 3cells) were added. Due to insufficient cytopathic effect, the presence of virus was determined by indirect immunofluorescence. For this, the liquid above the cultures was removed, the cells were washed with PBS and then fixed with 4% PFA solution for 10 min. The cultures were washed twice with PBS. To permeabilize the cell membrane, a 0.3% Triton X solution was applied for 3 min, followed by two PBS washes. In the next step, nonspecific binding was applied for 40 min with 3% BSA, followed by the application of the primary antibody (anti-HCoV OC43 monoclonal antibody (CABT-B341), CD Creative Diagnostic) for 60 min. After two washes with PBS, the secondary antibody (goat anti-mouse IgG H&L (Alexa Fluor® 488) (ab150113), Abcam) was applied for 40 min. The plates were observed under a fluorescent inverted microscope (Axio Observer, Carl Zeiss MicroImaging GmbH). Infectious dose TCID 50 The amount per ml was calculated by the Spearman i Karber method as in Example 1.

[0187] Inactivation assays were carried out essentially as in Example 1, except that 3% solutions of inoculated Zabrus extracts were examined. Results were read by indirect immunofluorescence as described above.

[0188] The steps of measuring the efficacy controls for cytotoxicity, cytosensitivity, inhibition of biocidal activity and the reference virus inactivation test were carried out substantially as in Example 1. The validation criteria were assessed substantially as in Example 1 and the criteria were determined to be met, so that the test according to PN-EN 14476+A2:2019-08 is valid.

[0189] As raw material, inoculated zabrus from a hive inoculated with human coronavirus OC43 was used, with the results of a viral inactivation test using an extract prepared as described above and formulated in isotonic marine plasma. Each condition was performed in replicates of 8 cell cultures. The results are shown in Figures 2A, B and C, respectively, for A549 cultures infected with HcoV-OC43 as a positive control, cultures not infected with virus (negative control) and cultures infected with HcoV-OC43 virus mixed with an inoculated zabrus preparation in isotonic marine plasma as described. It can be seen that the viral signal is reduced or absent in the treated cultures. The results are also shown in Table 8. In the table, a score of 0 indicates no detected viral activity and a score of 1 to 4 indicates the detection of the presence of the virus (degree of cytopathic effect / CPE). t indicates the detection of cytotoxicity. To facilitate visualization, conditions in which CPE was not detected in any replicate test are shaded in grey.

[0190] Summary According to the standard, the preparation should produce a virus titer of at least 4 log after the recommended exposure time. 10 If there is a reduction in the viral inactivation properties (inactivation ≥ 99.99%), it is considered to be a viral inactivation property. The inoculated Zabrus preparation was tested against human coronavirus OC43 at a concentration of 3% and an exposure time of 60 minutes. After this time, a viral inactivation potency of ≥ 4 log is equivalent to a viral inactivation potency of ≥ 99.99%. 10 A decrease in the titer of OC43 was determined. Therefore, the preparation is considered to have a virus-inactivating effect against human coronavirus OC43.

[0191] Example 7 - Further determination of the effectiveness of the preparation against adenovirus type 36 (Adenoviridae) The aim of this study was to determine the viral inactivation properties against adenovirus type 36 of inoculated Zabrus preparations obtained from hives inoculated with adenovirus type 36 using a quantitative suspension assay according to PN-EN 14476+A2:2019-08.

[0192] Preparation of the test virus suspension was carried out substantially as in Example 1, except that A549 cells and a stock virus suspension of adenovirus type 36 were used. Similarly, the infectivity assay was carried out substantially as in Example 1, except that A549 cells cultured in Eagle's minimum essential medium with Earle's BSS and 10% fetal bovine serum were used.

[0193] The inactivation assays were carried out substantially as in Example 1, except that the prepared extracts were tested as 10% solutions. Similarly, the steps of measuring efficacy controls for cytotoxicity, cell susceptibility, inhibition of bactericidal activity and reference virus inactivation tests were carried out substantially as in Example 1.

[0194] The verification criteria were determined substantially as in Example 1, and as a result, it was determined that the test according to PN-EN 14476+A2:2019-08 met the criteria to be valid.

[0195] Results: As raw material, inoculated Zabrus from a hive inoculated with adenovirus type 36 was used, and results of virus inactivation tests using extracts prepared as described above and formulated with isotonic marine plasma were obtained. Each condition was performed in replicates of 8 cell culture units.

[0196] The results are shown in Table 9. In the table, a score of 0 indicates no detected viral activity, and a score of 1-4 indicates detection of the presence of virus (degree of cytopathic effect / CPE). t indicates detection of cytotoxicity. For ease of visualization, conditions where CPE was not detected in any replicate test are shaded in grey.

[0197] Product toxicity assessment under the same conditions (not shown) indicated toxicity at dilution levels of 1, 2, and 3. Similarly, formaldehyde control experiments, not shown, indicated cytotoxicity at dilution levels of 1 and 2, with little or no viral activity observed at higher dilutions.

[0198] Summary According to the standard, the preparation should produce a virus titer of at least 4 log after the recommended exposure time. 10 If the reduction is greater than 99.99% (inactivation > 99.99%), it is considered to be a viral inactivation property. The inoculated Zabrus preparation was tested against the obesity-associated adenovirus type 36 at a concentration of 10% and an exposure time of 60 minutes. After this time, a viral inactivation efficacy of > 4 log equals a viral inactivation efficacy of > 99.99%. 10 A reduction in the titer of 100 μg / ml was determined. Therefore, the preparation can be considered to have virus-inactivating properties against non-enveloped adenovirus type 36.

[0199] Example 8 - Further determination of the effectiveness of the preparation against herpes simplex type 1 (family Herpesviridae) The objective of this study was to determine the viral inactivation properties against HSV1 of an uninoculated Zabrus preparation obtained from hives not specifically inoculated with any virus, using a quantitative suspension assay according to PN-EN14476+A2:2019-08, i.e. to examine the properties of a composition prepared without a step of inoculating the hives, against the pathogen against which the composition is tested.

[0200] Preparation of the test virus suspension was carried out substantially as in Example 4. Similarly, the infectivity assay was carried out substantially as in Example 4.

[0201] Inactivation assay: The test for the measurement of virus inactivation activity was according to PN-EN 14476+A2:2019-08 (EN 5.5.2). Extracts from caps (uninoculated Zabrus) were prepared from uninoculated hives. Extract samples were suspended in marine plasma to obtain a 10% suspension. The 10% suspension was the starting preparation for the test and was determined as concentration 0 (C0). A series of 10-fold dilutions was prepared from C0. C0- 10% suspension of extract C1- 10% C0 C2- 1% C0 C3- 0.1% C0 C4 - 0.01% C0 The contact time was 60 minutes. For easier operation, the volumes in this assay were 0.1 ml test virus suspension, 0.1 ml interfering substance (PBS) and 0.8 ml test product. Immediately after the end of the contact time, the activity of the bactericide was evaluated by 10 -12 The titration of the virus control was performed with a contact time of 60 min.

[0202] The validation criteria were determined substantially as in Example 1, and as a result, it was determined that the criteria were met such that the test according to PN-EN 14476+A2:2019-08 was valid.

[0203] Results: As raw material, uninoculated Zabulus was used, and the results of the virus inactivation tests using extracts were prepared as described above and formulated with isotonic marine plasma. Each condition was performed in duplicate with 4 cell culture units. As a control, the virus inactivation effect test against Herpes simplex virus type 1 was similarly performed with isotonic marine plasma without the addition of hive extract.

[0204] The results are shown in Table 10. In the table, a score of 0 indicates no detected viral activity, and a score of 1-4 indicates detection of the presence of virus (degree of cytopathic effect / CPE). t indicates detection of cytotoxicity. For ease of visualization, conditions where CPE was not detected in any replicate test are shaded in grey.

[0205] Summary According to the standard, the preparation should produce a virus titer of at least 4 log after the recommended exposure time. 10 If there is a reduction (inactivation ≥ 99.99%), it is considered to be a viral inactivation property. Non-inoculated Zabrus preparations were tested against HSV1 at the concentrations indicated. The exposure time was 60 min. After this time, the titer reduction was determined. C0-3log 10 - Inactivation=99.9% C1-2log 10 - Inactivation=99% C2-2log 10- Inactivation=99% C3-0log 10 - Inactivation=0% C4-0log 10 - Inactivation=0%

[0206] The product at C0 concentration can be considered to have 99.9% virus inactivating properties against enveloped herpes simplex virus type 1. With increasing dilution factors, the virus inactivating activity of Beemar decreases. At C3 and C4 concentrations, the preparation does not show virus inactivating properties.

[0207] Example 9 - Further determination of the efficacy of the filtered preparation against herpes simplex type 1 (family Herpesviridae) The aim of this study was to determine the virus inactivation properties against HSV1 of uninoculated filtered Zabulus preparations obtained from hives not specifically inoculated with any virus, using a quantitative suspension assay according to PN-EN 14476+A2:2019-08, i.e. to examine the properties of the filtered composition.

[0208] Preparation of the test virus suspension was carried out substantially as in Example 8. Similarly, the infectivity assay was carried out substantially as in Example 8.

[0209] The inactivation assay was carried out essentially as in Example 8, except that the prepared extract was tested as a 10% solution (C0) and two concentrations were prepared. C0- 10% suspension of extract C1- 10% C0

[0210] The validation criteria were determined substantially as in Example 8, and as a result, it was determined that the criteria were met such that the test according to PN-EN 14476+A2:2019-08 was valid.

[0211] Results: Results of virus inactivation tests using extracts prepared as above, formulated with isotonic marine plasma and filtered, using uninoculated Zabrus as raw material. Each condition was performed in duplicate with 4 cell culture units. As a control, the virus inactivation properties efficacy test against Herpes Simplex Virus Type 1 was performed similarly with isotonic marine plasma without the addition of hive extract.

[0212] The results are shown in Table 11. In the table, a score of 0 indicates no detected viral activity, and a score of 1-4 indicates detection of the presence of virus (degree of cytopathic effect / CPE). t indicates detection of cytotoxicity. For ease of visualization, conditions where CPE was not detected in any replicate test are shaded in grey.

[0213] Summary According to the standard, the preparation should produce a virus titer of at least 4 log after the recommended exposure time. 10 If there is a reduction (inactivation ≥ 99.99%), it is considered to be a viral inactivation property. Non-inoculated filtered Sabrus preparations were tested against HSV1 at the concentrations indicated. The exposure time was 60 minutes. After this time, the titer reduction was determined. C0-4log 10 - Inactivation=99.99% C1- 4log 10 - Inactivation=99.99%

[0214] Both the C0 and C1 concentration products can be considered to have 99.99% virus inactivating properties against enveloped herpes simplex virus type 1, suggesting that the filtered preparation is more effective than the preparation used without filtration (see Example 8).

[0215] [Table 3] JPEG2024539667000006.jpg246156JPEG2024539667000007.jpg24547

[0216]

Table 4

[0217]

Table 5

[0218]

Table 6

[0219]

Table 7

[0220]

Table 8

[0221]

Table 9

[0222]

Table 10

[0223]

Table 11

Claims

1. A composition comprising a bee-derived component and marine plasma, The bee-derived component comprises or is derived from Zabrus; The marine plasma comprises seawater.

2. The composition of claim 1, wherein the bee-derived component has a reduced content of hydrophobic components and / or a reduced content of sugar components compared to the input Zabulus.

3. The composition of claim 1 , wherein the bee-derived component comprises lysozyme.

4. The marine plasma includes seawater diluted with water, and further includes:

2. The composition of claim 1, wherein the water-diluted seawater is present in relative amounts of about 25% to 30% seawater and 70% to 75% water, preferably 29% seawater and 71% water.

5. 10. The composition of claim 1, formulated as a nasal spray, injectable serum, skin patch, eye drops and / or for oral administration.

6. 1. A method of making a composition, the method comprising providing a bee-derived component and combining the bee-derived component with marine plasma, The bee-derived component comprises or is derived from Zabrus; The method, wherein the marine plasma comprises seawater.

7. 7. The method of claim 6, comprising diluting seawater with water to generate marine plasma, optionally the water being purified water, distilled water, or reverse osmosis water.

8. 7. The method of claim 6, comprising obtaining seawater from below one or more plankton blooms in the ocean, typically from about 25 to about 35 metres, preferably about 30 metres below said plankton blooms.

9. 7. The method of claim 6, comprising treating the Zabrus to remove at least a portion of its hydrophobic components and / or to remove at least a portion of its sugar content.

10. The composition described in claim 1 or the method described in claim 6, wherein the marine plasma has a mineral content according to Table 1 or Table 2.

11. The method of claim 6, wherein the method comprises rearing bees as a source of bee-derived components and obtaining Zabrus from the bees.

12. 12. The method of claim 11, comprising providing said bees with a diet comprising farnesol and / or linden nectar.

13. 12. The method of claim 11, comprising rearing the bees in beehives and treating the beehives with an enrichment method.

14. 14. The method of claim 13, wherein the fortification method comprises applying a rosin or rosin-containing coating to the exterior of the beehive, typically comprising one or more applications of 30% turpentine and 70% rosin.

15. 12. The method of claim 11, comprising inoculating the bees against one or more specific pathogens prior to obtaining the bee-derived components.

16. 10. A composition according to claim 1 or a composition produced by the method according to claim 6 for use in the treatment or prevention of a disease.

17. 17. A composition for use according to claim 16, for use in a method of treatment or prophylaxis against a bacterial, viral, fungal or parasitic disease, typically a viral or bacterial disease.

18. The composition for use according to claim 17, wherein the disease is a viral disease mediated by a poliovirus, an adenovirus, a coronavirus or a herpesvirus.

19. 17. The composition for use according to claim 16, wherein the method comprises intranasal, intramuscular, intravenous, intraarterial, subcutaneous, intraperitoneal, topical, transmucosal and / or oral administration.

20. 17. The composition for use according to claim 16, wherein the composition is a vaccine.

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