WO2021191904A1 - Methods for preventing and treating viral infection - Google Patents

Abstract

The present invention provides methods, compositions and tools for preventing and treating viral infections, and specifically viral infections caused related severe acute respiratory syndrome, and more specifically infection of the human SARS-CoV-2, its variants and related viruses.

Description

METHODS FOR PREVENTING AND TREATING VIRAL INFECTION

TECHNOLOGICAL FIELD

The present invention generally concerns methods for preventing and treating viral infections, and specifically viral infections related to acute respiratory syndrome, and more specifically infection with SARS-CoV-2 and variants thereof.

BACKGROUND ART

At the late 2019 and the beginning of 2020 a new public health crisis threatened the world with the emergence and spread of a novel coronavirus disease (COVID-19), which was traced to Wuhan, Hubei province, China. The disease is likely to have originated in bats and was transmitted to humans through yet unknown secondary intermediary host.

Early on it became apparent that the causative agent of COVID-19 in humans is SARS-CoV-2, and the disease is predominantly transmitted by inhalation or contact with SARS-CoV-2 contaminated surfaces or contact with droplets from lung and nasal discharge of patients infected with SARS-CoV-2, with an incubation period of 2-14 days. We know now that SARS-CoV-2 is also present in saliva of infected individuals, and that the viral levels in saliva are strongly indicative of a more severe disease.

The COVID-19 symptoms are complex. They usually include fever, dry cough, difficulty to breath, loss of smell, headache, fatigue, among others. While in most cases and especially in young patients the disease is mild or asymptomatic [1], in certain risk groups that are associated with age and comorbidities such as cardiovascular disease, diabetes, chronic respiratory disease and cancer the disease can progress to pneumonia, acute respiratory distress syndrome (ARDS), multi-organ dysfunction and even death. More recently with the emergence of new SARS-CoV-2 variants, severe presentations of COVID-19 and increased mortality have been observed outside those risk groups.

Several laboratory tests are available for the detection of SARS-CoV-2. Being a positive-sense single-stranded RNA, most tests are based on a type of nucleic acid amplification in nasopharyngeal swabs (e.g., reverse transcription polymerase chain reaction (RT-PCR), transcription-mediated amplification (TMA), or loop-mediated isothermal amplification). Detection of past infection is possible with serological tests. After a year of relentless research, the treatment of COVID-19 remains an acute problem, especially in hospitalized patients who are at risk of fast deterioration or non- hospitalized patients who are at risk for developing severe illness. A particular problem is with patients who develop a hyper-immune response to the viral infection (cytokine storm). Even today, the predictability of COVID-19 clinical outcomes still suffers from significant limitations.

In critically ill patients, apart from supportive care with oxygen and more advanced respiratory support such as ventilation, treatments with various corticosteroids (dexamethasone, prednisone, methylprednisolone) are being used to reduce the length of time on a ventilator and reduce mortality. Several antiviral treatments have been approved by the FDA for COVID-19 (remdesivir and baricitinib in combination with remdesivir). All the above, however, constitute non-specific treatments and are predominantly used in advanced stages of the disease.

In November 2020, the FDA granted emergency use authorization to two monoclonal antibody treatments (bamlanivimab, made by Eli Filly; and a combination of casirivimab and imdevimab, made by Regeneron). Both treatments have been approved for non-hospitalized adults and children over 12 with mild to moderate COVID-19 symptoms who are at risk for developing severe COVID-19 or being hospitalized for it. These therapies, however, must be given intravenously soon after developing symptoms.

As per WHO data from February 2021, at least seven different vaccines across three platforms have been introduced in various countries, and more than 200 additional vaccine candidates are in development. At this stage, however, there is a common agreement that some groups, such as children, immunocompromised patients and potentially other individuals, will remain unvaccinated, especially since the vaccination will not be available to the same extent to all populations worldwide. In other words, as much safe and effective vaccines are a game changer, for the foreseeable future we must still continue wearing masks, physically distancing ourselves, and avoiding crowds.

Therefore, rapid detection of SARS-CoV-2 through the entire range of its variants, and prevention of their propagation in populations that are naive to the vims are the biggest challenges the society faces today. A question therefore arises whether there is a safe, efficient and affordable way to interfere with the entry of this virus through the respiratory pathways. Many viruses require cell surface heparan sulfates proteoglycans (HSPGs) as an assisting cofactor for cell entry. HSPGs are highly sulfated and are composed of unbranched negatively charged heparan sulfate (HS) polysaccharides attached to a variety of cell surface or extracellular matrix proteins. Due to this property, HSPGs are broadly employed by a range of pathogens, and especially viruses, for cell entry. As a general concept, HSPGs serve as anchor sites that facilitate initial contact between a virus and host cells and assist in accumulation of viral particles on the cell surface [2, 3, 4].

Certain studies suggested that lactoferrin, a multifunctional protein that is present in external secretions, is an important host defense molecule during infant development. Several mechanisms were suggested to explain its protective function, i.e., either by binding of lactoferrin to HSPGs and interfering with viral attachment [5] and/or by inhibiting viral replication and enhancing host immunity [6].

Another interesting example is carrageenan, a family of linear sulfated polysaccharides that are extractable from red edible seaweeds. It was shown that administration of carrageenan nasal spray in children and in adults suffering from virus- related common cold or influenza reduced the duration of disease, increased viral clearance and reduced relapses of symptoms [7].

These two examples highlight the need to continue exploring HSPGs as an attractive target in the context of COVID-19 and other viral infections.

REFERENCES

[1] Singhal T et al., 2020, Indian J Pediatr 87(4): 281-286.

[2] Belting M, 2003, Trends Biochem Sci 28: 145-151.

[3] Cagno V et al, 2019, Viruses 11(7): 596.

[4] Lang K et al, 2011, PLos One 6(8) e23710.

[5] Jenssen H and Hancock EW, 2009, Biochimie 91: 19-29.

[6] Wakabayashi H et al, 2014, J Infect Chemother 20(11): 666-671.

[7] Koenighofer M’ et al, 2014, Multidiscip Respir Med 9(1):57.

GENERAL DESCRIPTION

The present invention essentially seeks to provide a quick and accessible solution to wide spread infections, such as COVID-19 and other airborne or waterborne viruses. At the core of the invention is the hypothesis that Goat Milk Proteins (GMP), alone or in combination with other natural substances, can interfere with the viral attachment to biological and non-biological surfaces, and thus may have a potential as multi-purpose antiviral agents and viral decontaminants.

The hypothesis stems from a number of findings. A specific example is shown in EXAMPLE 1, where GMP and lactoferrin interfered with the entry of SARS-CoV-2 pseudovirus into HEK293 cells (human embryonic kidney) in vitro , as reflected in the reduction or inhibition of the pseudovirus associated bioluminescence (see also Fig. 1). This can be explained by non-specific interaction of the viral spike (S) protein with HSPGs on the effector cells. On the other hand, other studies have shown that HSPGs facilitate the interaction between SARS-CoV-2 and its specific receptor, Angiotensin- Converting Enzyme 2 (ACE2), which is also expressed on the same cells. According to the current understanding, the SARS-CoV-2 spike protein can interact with both, the HSPGs and ACE2, and that HSPGs function either as adhesion molecules to facilitate viral attachment or as anchors to facilitate viral endocytosis via the specific receptor.

In addition, additional studies have suggested that this interference is not necessarily rooted in the interaction between GMP and/or lactoferrin and the cells, but rather in the interaction between GMP and/or lactoferrin and the vims. More specifically, a recent study has revealed that GMP inhibits the infection of Coxsackievirus a9. (Cox A9, an Enterovirus causing Hand-Foot-and-Mouth Disease) cells in vitro. Nother study using scanned electron microscope imaging suggested that GMP directly binds to yet another type of virus, the DNA virus Herpes Simplex Virus 1 (HSV-1), most likely via electrostatic interaction (see EXAMPLE 3 and Figs 3-4).

Notwithstanding, without being bound by any theory, present findings of interference between GMP and/or lactoferrin and SARS-CoV-2 and other viruses may offer new tools for combating COVID-19 and other widespread viral infections.

The protein fractions of bovine and goat milk are qualitatively similar. The main difference between the two resides in quantitative differences and the amounts of caseins. For example, the hypoallergenicity of goat milk has been related to low levels of asl-casein (asl-cn). GMP are more digestible and have higher levels of 6 out 10 essential amino acids. Upon digestion or technological processing, GMP yield a unique composition of peptides that have been related to antimicrobial, immunomodulatory, antioxidant, antithrombotic, hypocholesterolemic and antiallergic activities. Caseins and lactoferrin are important components of GMP. The present examples demonstrate that GMP and lactoferrin, independently, are capable of interfering and inhibiting the entry of viruses into host cells ( see EXAMPLE 1 and Fig 1). As has been mentioned, lactoferrin can inhibit internalization of certain viruses via non-specific binding to HSPGs, by attaching to the receptors and preventing subsequent binding of the vims. In addition, lactoferrin can reduce the phenomenon known as cytokine storm via its inhibitory effect on the release of IL-6, a mediator of inflammation. Cytokine storm or hypercytokinemia is one of the main concerns in severe presentations of COVID-19.

Carrageenans offer yet another attractive candidate that can be obtained from a natural source. Carrageenans have been related to inhibition of certain viral infection, one example is their protective effect against common cold influenza in children and adults. Unlike GMP and lactoferrin, carrageenans are obtained from a vegetable source, and exclusively from edible red seaweed commonly known as Irish Moss. Carrageenans are also widely used in the food industry, mostly as thickeners and gelling agents.

All three actives, GMP, lactoferrin and carrageenan have been recognized as safe, by the FDA, i.e., actives in the GRAS category.

With the goal of finding safe and effective solution to COVID-19 and viral infections, in general, the inventors have chosen these natural-based actives, GMP, lactoferrin and/or carrageenans, as the basis for several prototype compositions and formulations ( see EXAMPLES 4, 5).

The inventors have further hypothesized that all three actives are likely to act through non-specific interactions with viruses and/or effector cells. Carrageenans are structurally similar to HS and are therefore likely to bind and interfere with viruses. More generally, the inventors hypothesized that that the protective effect of these actives against viral entry into the host could be generalized and applied to other RNA and DNA viruses, and potentially to viruses attached to non-biological surfaces.

The above concept has led to design and development of various applications: i. Compositions, methods and tools for administering GMP, lactoferrin and/or carrageenan(s) to mucosal tissues, i.e., the respiratory, digestive, or reproductive mucosa, being the first target of the attachment and infection of viral pathogens. ii. Compositions, methods and tools using these actives for interfering with viral interaction or contamination on biological and non-biological surfaces, such as medical and non-medical instruments, surfaces inside and outside health facilities, houses, vehicles and offices.

Within this framework, the inventors have developed and tested several formulations ( see EXAMPLES 4, 5), specifically:

- a line of oral compositions comprising combinations of GMP, lactoferrin and carrageenan. Additional compositions are currently under development to include combinations with specific GMP components (e.g., specific types and proportions of caseins) and specific types of carrageenans (e.g., kappa-carrageenan).

- a film forming nasal spray with mucoadhesive properties to produce a protective barrier against viral infections. The spray is formulated as a liquid and is converted to a gel upon contact with nasal mucosa. Special attention is given to the concentration of salts and pH as triggers to the conversion of the formulation from liquid to gel.

- a mucoadhesive tablet for sublingual administration adapted for sustained or controlled release of actives (e.g., within the time span of 5 h). This technology relies on previous experience of the inventor with formulations developed in the context of halitosis or aphthous stomatitis. The formulation uses a surface carbomer component, owing to which the table adheres to the mouth mucosa.

- a dietary supplement on the basis of Gelcarin carrageenan gel to include specific combinations of actives (e.g., types and proportions of caseins) and specific types of carrageenans (e.g., kappa-carrageenan).

- a spray for decontamination of biological and non-biological surfaces (also medical and non-medical surfaces). These compositions are formulated as a liquid or an aerosol, and are delivered via a dispenser, a nebulizer or an atomizer.

- a kit for in-house use of the formulations of the invention.

- a device for mucosal delivery of the compositions of the invention (e.g., nasal dispenser, or an inhaler or aspirator for upper respiratory airway).

The three main actives in the above composition are currently being tested for safety and cytotoxicity ( see EXAMPLE 5). So far, all three actives have proven to be relatively safe in a form of a mucoadhesive film forming nasal spray. Additional clinical trials in humans are currently ongoing ( see EXAMPLE 6). In summary, the invention provides a series of preventive and therapeutic tools for fighting against viral epidemics and pandemics, such as COVID-19 and epidemics of other viruses. The main actives included in the compositions of the invention originate from natural sources, and therefore can be considered as relatively safe for application in the clinical as well as in-house settings.

More importantly, and especially in view of the problems with vaccination against new SARS-CoV-2 variants, the compositions and methods of the invention can offer additional level of protection against the currently emerging and future variants, and the sole level of protection for the sub-groups and populations that cannot be vaccinated.

BRIEF DESCRIPTION OF DRAWINGS

To better understand the subject matter and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings.

Fig 1 illustrates the inhibitory effect of GMP and lactoferrin on SARS-CoV-2 pseudovirus entry into HEK293 cells as revealed by the luciferase activity assay. The actives are used in non-cytotoxic concentrations ( see Figs 2A-2B).

Figs 2A-2B illustrate studies of the cytotoxicity of GMP (2A) and lactoferrin (2B) in vitro using concentrations of actives as 0.1%, 1% and 5% (w/w).

Figs 3A-3C illustrate the inhibitory effect of GMP on the CoxA9 infection in BGM cells as revealed by the number of viral plaques in cells exposed to CoxA9 and GMP (3A), exposed to CoxA9 only (3B) negative controls (3C) plated on agar.

Figs 4A-4B illustrate direct interaction between GMP and HSV-1 viral particles as revealed by PTA staining and scanning in electron microscope of HSV-1 particles unexposed to GMP (4A) and exposed to GMP (4B).

DETAILED DESCRIPTION

Thus, in the most general sense, the present invention can be articulated in terms of compositions and methods to provide preventive and therapeutic solutions for widespread infections of airborne and waterborne viruses, notable examples of which are respiratory viruses and viruses of the digestive system such as SARS-CoV-2, influenza and viruses associated with gastroenteritis in children and adults. In one of the main aspects the invention provides compositions and methods using Goat Milk Protein (GMP), alone or in combination with carrageenan for interfering with viral interaction with or within a mucosal tissue in a mammalian respiratory, nasal, ocular, digestive, or reproductive tracts.

In numerous embodiments composition and methods of the invention apply to human subjects.

The terms 'interfering' or 'interference' as used herein imply a wide range of interactions between the actives and virus, which are not necessarily direct or specific and can involve interactions of the actives with the surface of mucosa cells via HSPGs or interaction of the actives with the virus via electrostatic forces.

The outcome of these interactions is a reduction in adhesion or binding of the vims to mucosa cells and a reduction in the density of the vims on the cell surface, and as a result, a reduction in the endocytosis and entry of the vims to the mucosa cells.

In certain embodiments such as a film forming nasal spray of the invention, the spray produces a physical barrier that reduces or prevents the viruses from reaching the mucosa, and thereby reducing the adhesion or binding of the vims to the mucosa cells and reducing the density of the vims on the cell surface.

The reduction can be expressed in the terms of up to about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 100% reduction in the density of vims on the cell surface compared to the cells not exposed to these actives.

It can be further expressed in term of up to about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 100% reduction in the attachment of vims to the cell surface compared to the cells not exposed to these actives.

It can be further expressed in term of up to about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 100% reduction in the rate of endocytosis of vims or vims entry into the cells compared to the cells not exposed to these actives.

The same aspect can be further articulated as compositions and methods using Goat Milk Protein (GMP), alone or in combination with carrageenan for preventing and/or treating subjects at risk of being infected or already infected with the vims. The virus can be an RNA or DNA virus infecting and/or replicating in a mucosal tissue of the human respiratory, nasal, ocular, and digestive or reproductive tracts.

The terms 'mucosal tissue' or 'mucous membrane' generally imply a layer of cells that surrounds body organs and body orifices. It is a protective epithelial layer containing or secreting mucus and line part of the body that exposed to external environment, and are at risk of being infected by pathogens such as bacteria or viruses. There are many different mucous membranes (or mucosa), such as mucosa lining the respiratory, nasal, ocular, digestive, or reproductive systems. Specific examples are mucosa of the oral and nasal cavities, mucosa of the eye or surrounding the eye, mucosa of the gut, mucosa of the rectal or vaginal cavities, and mucosa of the urogenital system.

With respect to the main actives, GMP herein encompass the sum total of all proteins that can be extracted from goat milk, irrespective of breed, origin and climate (e.g., Alpine La Mancha, Nubian, Saanen breeds) and irrespective of the extract method. The total protein content in goat milk can vary (e.g., from 2.6 to 4.1 g/1).

In certain embodiments GMP can be enriched in specific components or modified to include specific components in certain concentrations and proportions.

There are 6 main goat milk proteins that are similar to the cow milk proteins in their general classification: aSl -casein (asl-CN), aS2-casein (as2-CN), b-casein (b-CN), K-casein (K-CN), b- Lactoglobulin (b-LG), and a-lactalbumin (a-LA), with CNs constituting approximately 80% of the total milk protein fraction.

Goat milk is characterized in that it has a natural whey protein to CN ratio of about 20:80. Goat milk is further characterized in the absence or low levels of asl-CN, owing to which it is less allergenic than the cow milk. Table 1 below shows characteristic CN profiles in the cow and goat milk.

Table 1. Protein composition of cow and goat skim milks

ITEM COW GOAT

The concentration of b-CN is 43% higher in goat milk than in cow milk, which has direct effect on b-CN recovery from GMP. Furthermore, b-CN can be extracted more efficiently from goat rennet casein. As a result, the extraction yield of b-CN can be increased up to 53% from goat milk relative to cow milk.

In numerous embodiments the at least one GMP in the compositions of the invention can be b-CN, alone or in combination with at least one another GMP.

In numerous embodiments the compositions can be further enriched in b-CN to a concentration up to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 100% of the total CN content, or in the range of at least about 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-100% of the total CN content. //-CN can be isolated directly from renneted skim milk, based on the preferential solubilization thereof at low temperature.

The same is true for the other CN components, namely the at least one GMP can be enriched in casein (asl-CN), aS2-casein (as2-CN), b-casein (b-CN), k-casein (K-CN), and sodium caseinate. Sodium caseinate refers to the sodium salt of casein.

For example, in numerous embodiments the compositions of the invention can be further enriched in K-CN to a concentration up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100% of the total CN content.

In other embodiments the compositions can be enriched in as2-CN to a concentration up to 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100% of the total CN content.

Yet in other specific embodiments the compositions can be enriched in asl-CN to a concentration up to 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100% of the total CN content. It should be noted that, in many cases, asl-CN is allergenic.

Further, the exact GMP profile can be further altered by respective extractions of GMP from various breeds. Genetic polymorphisms in different goat breeds have been related to various proportions and functional properties of CN fractions such as stability, solubility, and digestibility, e.g., asl-CN and K-CN. Additional modifications depend on post-translational modifications, such as glycosylation, phosphorylation, acetylation, and proteolytic cleavage. Additional important active in the compositions of the invention is lactoferrin. Lactoferrin herein refers to an iron-binding glycoprotein and is considered a major part of the non-specific disease resistance complex in the mammary gland. Goat colostrum lactoferrin concentration is higher than in cow's milk, i.e., from 455 to 2058 mg/dl in goat colostrum vs. 575 mg/dl in bovine. In addition, goat milk lactoferrin has been related to anticancer and antibacterial activities.

Therefore, in numerous embodiments the lactoferrin in the compositions of the invention constitutes one of the components of GMP.

Yet in other embodiments lactoferrin can be provided or supplemented from other sources, such as bovine and human sources.

The exact amount of lactoferrin in the compositions of the invention can vary depending on its origin, e.g., goat milk or colostrum, and can be enriched to a concentration up to 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 100% of the total GMP content, or in the range of at least about 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%- 70%, 70%-80%, 80%-90% and 90%-100% of the total GMP content.

In certain embodiments the compositions can comprise genetic variants of lactoferrin, depending on its origin, and lactoferrins with one or more post-translational modifications, such as glycosylation, phosphorylation, acetylation, and proteolytic cleavage.

Thus, in certain embodiments the methods and compositions of the invention can comprise at least one GMP which is a casein or lactoferrin.

Thus, in certain embodiments the methods and compositions of the invention can comprise at least one GMP which is a casein and lactoferrin.

In other embodiments the methods and compositions of the invention can comprise at least one GMP which is b-casein (b-CN) and lactoferrin either from goat and/or bovine source.

Yet another important active in the compositions of the invention is carrageenan. The term 'carrageenan' encompasses herein a family of linear sulfated polysaccharides having the general formula C₂₃H₂₃FN₄0₇Zn and molecular weight of 551.8 g/mol, and molecules referred to as Carrageenan MIV-150/ZA/CG MIV- 150/zinc acetate carrageenan 9000-07-1 zinc l-(5-cyano-2-pyridyl)-3-[(lS,2S)-2-(6-fluoro-2-hydroxy-3- propanoyl-phcnyljcyclopropylj urea diacetate . This term further encompasses herein the three basic types of carrageenans: iota (i-), kappa (K-) and lambda (l-) carrageenans. Molecular structures of the three carrageenans are shown below.

Thus, in numerous embodiments the methods and compositions of the invention can comprise one or more carrageenans selected from iota-carrageenan, kappa- carrageenan and lambda-carrageenan.

In some embodiments the carrageenan is a heteropolymer (e.g., a carrageenan comprising subunits of at least two carrageenans selected from kappa-carrageenan, iota- carrageenan and lambda carrageenan).

The choice of a specific carrageenan can further depend on the application or formulation of the composition. In the presence of calcium, for example, i-carrageenan forms a soft gel and k-carrageenan forms a stiff and brittle gel, but in the presence of potassium salts, k-carrageenan forms very firm and elastic gels.

Potassium and calcium ions are present in certain concentrations in nasal mucosa. Therefore, in certain embodiments and especially applications involving administering into the nasal cavity can use kappa-carrageenan. Yet in other embodiments the methods and compositions of the invention can use i-carrageenan.

In numerous embodiments the methods and compositions of the invention use carrageenan is in the form of a salt. Carrageenans are negatively charged molecules and have tendency to react with positively charged ions, e.g., potassium, to form salts.

Although carrageenans have been used hundreds of years in traditional food preparations, there is a current controversy as to the safety and clinical applicability of these agents, as some of them have been related certain inflammatory and toxic effects.

With respect to the main applications, in the broadest sense the compositions and methods of the invention provide prevention and/or treatment of viral infections. The term 'prevention and/or treatment of viral infection' encompasses prevention of inhibition of the attachment of virus to mucosal cells and thereby prevention or inhibition of virus entry into the cells. It further implies minimizing replication of the virus in the host cells and inhibiting viral titer in the body, and ultimately reducing the manifestation of symptoms of viral infection and infectivity of the treated individual.

Such prevention and/or treatment can be measured basing on direct measurements of the viral titer in the serum or blood, or in mucosal discharges of affected individuals, and by assessment of clinical symptoms of viral infection as per clinical criteria.

Viral titer can be further correlated to additional diagnostic indices obtained by molecular testing for the vims, specific viral antigens and specific antibodies, using PCR, RT-PCR, serological tests.

Thus, in numerous embodiments the prevention and/or treatment effects of the compositions and methods of the invention are apparent by the reduction of viral titer or respective correlates up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% of the initial level in the same individual (i.e., the level before treatment).

In other embodiments the prevention and/or treatment effects of the compositions and methods of the invention are apparent by the reduction of clinical symptoms of infection up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% of the initial severity, number and/or recurrence of symptoms in the same individual.

In other embodiments the prevention and/or treatment effects of the compositions and methods of the invention are apparent by the reduction of viral titer or its correlates or clinical symptoms of the infection in a population-based sample up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 100% of their respective or average values in the population sample infected with the vims.

All these examples pertain to the prevention and/or treatment of already existing infection in an individual or a population.

Yet in numerous other embodiments the compositions and methods of the invention can be applied to a naive (or healthy) population, i.e., non-infected individuals who are at risk of being infected due to general prevalence of the infection in the same population.

In such case, the prevention and/or treatment effects of the compositions and methods of the invention are apparent by comparing the viral titer or its correlates or clinical symptoms of the infection in treated population group vs. untreated groups, yielding up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 100% reduction of their respective or average values.

In other words, this application can reduce the risk of being infected up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 100% in a naive uninfected individual or a population.

From another point of view, this application can reduce the risk of transmitting infection up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 100% to another naive uninfected individual or a population.

With respect to the relevant viruses, due to the non-specific interaction between viruses and the actives, it is presumed that the invention can be applicable to a wide range of RNA or DNA viruses that are infecting and/or replicating in a mucosal tissue of the human respiratory, nasal, ocular, and digestive or reproductive tracts.

In numerous embodiments the methods and compositions of the invention are applicable to respiratory viruses, or viruses or replicating in mucosal tissue of the human respiratory, nasal, ocular tracts. Respiratory viruses are usually transmitted through direct contact with an infected person, contact with contaminated mucosal discharges or droplets. These viruses are usually airborne, notable examples are influenza and coronaviruses. The respiratory viruses, apart from adenovirus Human bocavirus (HboV), have an RNA genome. The rhinovirus and adenovirus are non-enveloped viruses, while the other are enveloped viruses that belong to several different families, i.e., orthomyxoviruses, paramyxoviruses, and coronaviruses. Further, the orthomyxoviruses (influenza is the only member) differ from the paramyxoviruses in that they have a segmented RNA genome (8 segments), whereas the latter consists of a single segment.

In other words, although respiratory viruses are quite different in their structure and genome, the feature that unites all these viruses is their ability to infect mucosal cells of the respiratory tract and cause clinical symptoms in humans. Certain prominent respiratory viruses are shown in Table 2 below.

Table 2. The main families and members of respiratory viruses.

FAMILY (NUCLEIC ACID) REPRESENTATIVE VIRUS

Diagnosis and identification of specific viruses can be made with a high degree of confidence and specificity using various PCR-based assays on respiratory secretions.

Thus, in numerous embodiments the composition and methods of the invention are applicable to respiratory viruses belonging to members of families Orthomyxoviridae, Paramyxoviridae, Picornaviridae, Coronaviridae , Parvoviridae or Adenoviridae .

In certain embodiments the composition and methods of the invention can be applicable to viruses diagnosed as an Influenza vims, a Respiratory syncytial virus (RSV), an Enterovirus, a Coronavirus, an Adenovirus, a Human bocavirus (HBoV). In further embodiments the composition and methods of the invention can be applicable to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). This type of applications has been presently exemplified ( see EXAMPLE 1).

Due to the wide range of potential interactions between the actives and viruses, the term 'SARS-CoV-2' encompasses herein the entire range of viral mutants and variants that have been identified so far, as well as future variants with respect to the four structural proteins of SARS-CoV-2 (S, E, M, and N) and 16 non- structural proteins (Nspl-16, e.g., Nspl mediates RNA processing and replication, Nsp2 modulates the survival signaling pathway of host cell, Nsp3 is believed to separate the translated protein). This term further encompasses variants with mutations in the intergenic and regulatory regions.

By the same token, in other embodiments the composition and methods of the invention can be applicable to Coxsackieviruses. This type of applications has been presently exemplified ( see EXAMPLE 3).

Thus, and without being bound by any theory, it can be presumed that the compositions and methods of the invention can be effective against other viruses that have propensity to adhere to other mucosal tissues, such as mucosa of the gut and digestive system.

Thus, in certain embodiments the compositions and methods of the invention can be effective against viruses of digestive system or viruses causing acute gastroenteritis. This type of infections is particularly prevalent in children. They are transmitted either through direct contact with contaminated surfaces or through contaminated food.

In many cases the viruses are waterborne.

Thus, in numerous embodiments the compositions and methods of the invention can be applicable to viruses from families Reoviridae, Caliciviridae, Coronaviridae or Astroviridae .

In this context, SARS-CoV-2 is also relevant as gastrointestinal symptoms have been observed in certain patients with COVID-19. Further, specific staining of viral nucleocapsid protein was detected in gastric, duodenal, and rectal epithelia cells.

Thus, in certain embodiments the compositions and methods of the invention are applicable to SARS-CoV-2 infections in the gastrointestinal tract.

It should be noted that the compositions of the invention can further comprise carriers, excipients and additives to improve performance and effectiveness of actives. From yet another important aspect, the invention to provides compositions and methods for interfering with viral interaction with biological and non-biological surfaces (also medical and non-medical surfaces.

This specific aspect can be further articulated in the sense of interfering with the attachment of the virus to at least one biological or non-biological surface in a medical or non-medical setting.

In other words, the compositions of the invention with GMP, CN, lactoferrin and/or carrageenan, can act as natural decontaminants applied to biological as well as non-biological surfaces.

In certain embodiments the compositions and methods of the invention can be applied to the skin of a subject or a part thereof.

In yet other embodiments the compositions and methods of the invention can be applied to any surface in a healthcare facility or a part thereof.

In other embodiments the compositions and methods of the invention can be applied to any surface of a medical instrument or a part thereof.

In other embodiments the compositions and methods of the invention can be applied to non-medical instruments, or any surface in a house, a vehicle or an office.

More specifically, in some embodiments the compositions can be formulated to be applicable onto solid surfaces and sanitary items in order to decrease transmission and reduce viral spread via, e.g., hand-to-hand contact (e.g., embedded in wipes, gloves, hygiene tissues, nasal tissues and handkerchiefs).

In some embodiments the compositions can be formulated to be applicable onto any part of the human skin. In such case, the compositions can be formulated as an ointment, lotion, cream, or wipes to provide a topical barrier that inhibits attachment of the virus to the skin.

In some embodiment the compositions can be formulated to be applicable to a surface of medical instruments. Non-limiting examples of medical instruments include surgical instruments (e.g., forceps), medical imaging machines, infusion pumps, life support equipment (e.g., respirators, ventilators), medical lasers, medical laboratory equipment, medical monitors, stethoscopes, defibrillators, sterilization trays and hospital racks. An exemplary use of such compositions is for disinfecting life support equipment prior to use in a hospital or clinic, e.g., such that viruses can be wiped off with the herein described compositions.

In some embodiments the compositions can be formulated to be applicable to a surface of non-medical instruments. Non-limiting examples of non-medical instruments include house and garden furniture, kitchenware, cabinets and bookshelves, trash cans, recycling baskets, magazine and coat racks, lamps and lighting equipment, doorknobs, handrails, elevator switches and public transformation vehicles.

The term 'composition' herein encompasses composite compositions and formulations comprising therapeutically effective amounts of actives of the invention (e.g., GMP, CN, lactoferrin and/or carrageenan), and optionally other suitable additives such as diluents, preservatives, solubilizers, emulsifiers, adjuvant and/or carriers. The compositions may be liquids or semi solids lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCl, acetate, citrate, phosphate, Sorenson’s Phosphate Buffer, borate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, Pluronic 127, bile acid salts), solubilizing agents (e.g., tween 80, glycerol, propylene glycol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimerosal, m-cresol, benzyl alcohol, parabens, phenethyl alcohol, caprylyl glycol), flavoring (e.g., menthol, lemon) when the composition is in the form of a lozenge or troche or a mucoadhesive tablet, suspenders (e.g., silica), mucoadhesive agents and thickeners (e.g., Gellan gum, carbomer) and others.

With respect to the Gellan gum in particular, Gellan gum herein refers to a water- soluble anionic polysaccharide produced by the bacterium Sphingomonas elodea. It is typically used as a food additive for binding, stabilizing and texturing purposes. Mucoadhesive properties of Gellan gum have been previously suggested on the basis of studies of nasal spray formulations containing Gellan gum. It has been shown that such formulations have a viscosity sufficient to spray out from the device and elasticity great enough to adhere to the mucosal membrane. Various modifications of Gellan gum may further increase the adherence time on mucosal surfaces, and the overall retention of the drugs at the site uptake. Thus, in certain embodiments the compositions of the invention can be formulated with Gellan gum. This type of formulation is particularly applicable to nasal spray of the invention.

In further embodiments the formulations with Gellan gum can provide controlled release of the actives (see below).

In certain embodiments the compositions are pharmaceutical composition in a form suitable for administration to a human subject.

In certain embodiments and particularly those involving dispersion, the compositions of the invention are formulated as a liquid or an aerosol, and are delivered via an aerosol dispenser, a nebulizer, an atomizer.

The term 'aerosol' herein encompasses a suspension of fine solid particles or liquid droplets in air or another gas. Aerosols can be natural or anthropogenic. Examples of natural aerosols are fog, mist, dust, forest exudates and geyser steam.

Yet again, the choice of pharmaceutically acceptable carriers comprised in the nasal and oral dosage form of the composition depends upon the exact formulation, e.g., an emulsion, a solution, a suspension, an ointments/lubricant or a gel.

The compositions of the invention can be adapted for various applications, i.e.: administered orally (e.g., via a lozenge, troche or a gargle solution), nasally (e.g., via drops, lubricant, viscous gel, irrigation, nebulizer aerosol, spray), into the eye (e.g., via drops or ointment), buccally, vaginally or rectally (e.g., using a suppository). For example, for intranasal applications, the composition may be formed as a nasal spray, an aerosol, nasal drops, a nasal irrigation or a nasal lubricant (e.g., gel).

More specifically, in some embodiments, the compositions are administered into the oral cavity or oral mucosa in the form of a lozenge, troche, a tablet or a mucoadhesive tablet. Such forms can be further presented as chewable lozenge, troche, a tablet and can further comprise one or more sweeteners, flavorings, or physical binders.

Further, lozenge and troches can comprise the active ingredient, a flavor, a sweetener as sucralose or xylitol or mannitol or sucrose. Pastilles or a mucoadhesive tablets can comprise active ingredient in an inert base, such as gelatin and glycerin, or sucralose or xylitol or mannitol sucrose and, emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art. Lozenges and troches provide an efficient mucosal delivery mode for the composition's constituents (GMO, CN, lactoferrin and/or carrageenan) which enable attachment of the constituents to the oral mucosal surface.

In some embodiments, the compositions are administered into the nasal cavity or nasal mucosa in the form of a nasal spray, nasal drops, a nasal viscous gel, a nasal ointment, nasal powder a nebulizer or an aerosol.

In some embodiments the compositions are administered into the eye. Ocular delivery of the composition of the present invention may be carried out using suitable mode, such as topical administration (e.g., using drops, viscous solution, gel or an ointment), periocular administration, tissue specific microinjection, intraorbital administration or intravitreal injection.

In some embodiments the compositions are formulated as a liquid, a semiliquid, a solid, a powder, an ointment, a gel.

According to the present invention, the appropriate dosages of the composition of the present invention are administered to human subjects that are at risk of being infected with a vims (e.g., by either nasal spray or oral lozenge).

Of particular relevance is the concept of 'therapeutically effective dose'. Generally, the term 'therapeutically effective dose' to an amount or a concentration of active that induces the desired clinical effect or change in the relevant clinical condition as measured by respective definition criteria. In this case, the term applies to viral titer or its correlates or clinical symptoms of the respective viral infection.

A person of skill in the art would readily realize that the specific dose and frequency of dose administrations that are suitable for specific viral infection or a subject, and whether it is meant to be preventive or therapeutic.

In numerous embodiments a therapeutically effective dose can be determined using 'trial-and-error' considering, apart from the amount actives, also age, weight, health condition, sex, diet of individual patient. This constitutes a personalized approach.

In other embodiments a therapeutically effective dose can be determined on populational basis in human clinical trials.

Of relevance in this context are consideration of cytotoxicity of each active (e.g., GMO, CN, lactoferrin and/or carrageenan). Such considerations have been presently exemplified (see EXAMPLE 5). In certain embodiments the oral composition and methods of the invention are meant to provide sustained or controlled release of actives. The terms 'controlled release', 'sustained release' or 'modified release' generally relate to tailor-made formulations designed to release a drug at a predetermined rate. This kind of properties can be provided by various types of coating and additives.

To that end, controlled release can be achieved by dissolution-mediated controlled release formulations, including reservoir and matrix types. Matrix dissolution systems are the most used technique with the active being homogeneously distributed throughout a polymer matrix. As the polymer matrix dissolves (typically via an erosion-mediated process), drug molecules are released into the external environment. In this system, the size of the formulation (and hence matrix) decreases over time, thereby resulting in a non-linear drug release.

Reservoir systems involve a drug core that is coated with a rate-limiting polymer. Using this system, drug release is determined by the thickness and the dissolution rate of the polymer membrane surrounding the drug core. Once the coated polymer membrane dissolves, the drug will be released similarly to an immediate release preparation.

In certain embodiments the compositions of the invention can comprise coating with hydroxypropyl methylcellulose (HPMC) polymer.

In yet another aspect the invention provides a kit comprising the compositions of invention and instructions of use. Specifically, in terms of actives the kit of the invention comprises at least one GMP, alone or in combination with carrageenan.

In certain embodiments in terms of actives the kit can comprise at least one GMP that is CN and/or lactoferrin and/or at least one carrageenan.

As discloses herein, casein (CN) generally refers to a phosphoprotein that is commonly found in mammalian (e.g., cow, goat, sheep, or buffalo) milk. CN may be a genetic variant of b-CN and may also be in the form of sodium caseinate.

In some embodiments, the casein comprises at least one CN selected from aSi- CN, aS₂-CN, b-CN and k- CN.

In some embodiments, the CN is a CN derived from goat milk.

As used herein, lactoferrin generally refers to a multifunctional protein of the transferrin family that is a globular glycoprotein with a molecular mass of about 80 kDa and which is widely represented in various secretory fluids, such as milk, saliva, tears, and nasal secretions. Lactoferrin can be isolated from mammalian (e.g., goat, sheep, buffalo) milk or produced by recombinant methods.

Specific features and various combinations of said actives have been previously discussed.

More generally, the components of the present composition (i.e., GMP, CN, lactoferrin and/or carrageenan) may act synergistically to prevent, mitigate, or decrease symptoms of viral infection.

The synergistic effect in the combination of CN, lactoferrin and/or carrageenan included in the present compositions or formulations may or may not be dose dependent.

In the context of kit, the kit can include containers such as vials, bottles, cans, pressurized cans, dispenser, packages, compartments or others, for preserving and storing the compositions of the invention in predetermined amounts and forms.

In certain embodiments the containers (e.g., nasal spray, aerosol) contain predetermined amounts of the compositions of the invention. The composition can be dispensed in a spray liquid or solid, an aerosol, or in a liquid form or semi-solid form. The containers can have spray, pump, or squeeze mechanisms.

The instructions for use may include variations that can be implemented and explanations on how to apply the compositions and the components of kit (e.g., apply the drops or nasal spray into the nostrils or eyes).

It is yet another aspect of the invention to provide a device for mucosal delivery of the present compositions. Specific features and requirements of such devices have been previously discussed. Some non-limiting examples include, although non-limited to, aerosol dispensers, nebulizers and atomizers for formulations to be applied to nasal and oral mucosa. Additional examples can include specific applicators to the eye.

Ultimately, it is another intention of the invention to provide use of at least one GMP, alone or in combination with carrageenan in the manufacture or preparation of a medicament preventing or treating viral infection, wherein the virus is an RNA or DNA vims that infects and/or replicates in a mucosal tissue of the respiratory, digestive or reproductive tract.

Specific features and advantages of the compositions and methods pertaining to this aspect have been previously discussed. EXAMPLES EXAMPLE 1

Antiviral effect of GMP and lactoferrin on SARS-CoV2 pseudovirus in vitro

The study used the pseudovirus luciferase platform designed for SARS-CoV-2 with pseudovirus particles expressing the viral spike (S) protein and the luciferase bioluminescence assay in HEK293 (human embryonic kidney) effector cells.

Samples containing 1% Goat Milk Proteins (GMP) or 0.1% lactoferrin in PBS (w/w) or control (PBS only) were incubated with SARS-CoV-2 pseudovirus and added to HEK293 cells. These concentrations were determined as non-toxic for HEK293 cells in a prior experiment. Luciferase activity, expressed in relative light units (RLUs), was determined according to the manufacturer’s instructions. The inhibition rate of the tested actives was calculated from luciferase luminescence values.

Experimental procedure

HEK 293T cells were seeded at 50% confluency in a flat 96 well plate.

Day 0: SARS-CoV-2 pseudoviruses (5μl per well, Cat# 79992-1 BPS Bioscience) were incubated with 1% GMP or 0.1% lactoferrin in PBS (w/w) or control (PBS only) for 30 min. Following incubation, the mixture was added to the HEK 293T cells.

Day 1: RPMI medium was added to all wells to increase cell viability.

Day 2: The One-Step Luciferase Assay System (BPS Bioscience #60690-1) was used to measure luminescence in the infected cells using luminometer as per manufacturer’s instructions.

Results

The luciferase luminescence values are shown in Fig. 1 and Table 3 below.

Table 3. Inhibition rate of the SARS-CoV-2 pseudovirus in HEK 293T SAMPLE LUMINESCENCE UNIT % VIRUS

INHIBITION

The results show significantly lower luminescence values with GMP and lactoferrin, i.e., up to 50% lower with 1% GMP and up to 70% lower with 0.1% lactoferrin, suggesting that the two actives interfere with the pseudovirus entry into the cells.

Conclusions

The pseudovirus SARS-CoV-2 particles express the viral spike (S) protein. On the other hand, HEK293 cells express HSPGs and Angiotensin-Converting Enzyme 2 (ACE2) cell surface receptors. ACE2 was proposed as a receptor for HCoV-NL63, another type of human coronavirus known since 2004, and more recently, a receptor for the new SARS-CoV-2. According to current understanding, the SARS-CoV-2 spike protein interacts with both, HSPGs and ACE2, and HSPGs function either as adhesion molecules to facilitate binding of the virus or as anchors to facilitate viral endocytosis via its specific receptor.

GMP and lactoferrin have demonstrated significant inhibition effect on the SARS- CoV-2 pseudovirus entry into HEK293 cells. Irrespective of the mechanism of action, these actives proved to be successful candidates for inhibiting SARS-CoV-2 infection.

EXAMPLE 2

Cytotoxic studies on GMP and lactoferrin in vitro

Cytotoxic effect of GMP and lactoferrin was studied in HEK293 cells. The actives were tested in serial concentrations in using PBS.

Experimental procedure

Day 0: HEK 293T cells were seeded in a flat 96 well plate to the density of 30,000 cells per plate. The experiment was performed in triplicates.

Day 1: Actives and controls were introduced into the wells (5μl per well): GMP at a concentration of 0.01%, 0.1%, 1% and 5% in PBS (w/w); lactoferrin at a concentration of 0.01%, 0.1%, 1% and 5% in PBS (w/w); and PBS and water controls.

Day 2: Cells were extracted from the wells, stained with Trypan blue and counted under the light microscope.

Results

Cell viability values (%) are shown in Table 4 below and Figs 2A-2B. Table 4. Cytotoxicity of GMP, lactoferrin and carrageenan in HEK 293T

CONCENTRATION

The results show that GMP and lactoferrin were well tolerated up to the concentrations of 1% and 0.1% (w/w), respectively, to achieve the threshold of 70% viability.

Conclusions

In terms of cytotoxicity, GMP and lactoferrin can be considered relatively safe up to the concentrations of 1% and 0.1% (w/w). Antiviral effects of the two actives at the same concentrations were demonstrated in EXAMPLE 1 above.

EXAMPLE 3

Antiviral effect on other viruses in vitro

The antiviral effect of GMP was further tested in BGM (African green monkey) cells plated on agar and infected with Coxsackievirus a9 (Cox A9).

Experimental procedure

BGM cells were plated on agar plates and cultured until creating a uniform layer. Cells were incubated with Cox A9 (100 pfu) with and without 50% GMP for 1 h at 37°C. Cells were washed and incubated at 37°C, 5% CO₂ for 72 h until plaques were visible.

HSV viral particles were concentrated 10 min at 1000 rpm in 4°C, twice, separated by ultracentrifugation for 2 h at 6000 rpm at 4°C. Concentrated virus was incubated with and without 50% GMP for 45 min at 37 °C. Samples were stained with PTA negative staining and studies under Transmission Electron Microscope (TEM) under magnification X 40,000. Results

Antiviral activity of GMP on CoxA9 was demonstrated by the number of plaques in plated BMG cells, whereby cells exposed to the GMP treatment had a significantly lesser number of plaques (31 plaques) compared to the non-treated cells (63 plaques)

(see Figs 3A-3C).

Direct interaction between GMP and HSV viral particles was demonstrated by TEM images showing HSV-1 particles coated or 'decorated' with GMP as opposed to negative controls (see Figs 4A-4B).

Conclusions

These findings indicate that GMP has inhibitory effect on additional types of viruses, beyond SARS-CoV-2. In addition, they support the notion of direct interaction between the vims and GMP, or casein as its predominant component, most likely via electrostatic forces and not via a specific receptor.

EXAMPLE 4

Prototype formulations of GMP, lactoferrin and carrageenan

Two prototype formulations have been developed to include the actives of the invention, i.e., GMP, lactoferrin and carrageenan: i. Nasal spray containing mucoadhesive liquid formulations with the respective actives. Upon contact with nasal mucosa, the liquid formulation is converted to a gel, thereby producing a protective layer inside the nasal cavity and inhibiting viral entry. The currently developed formulations contain GMP, lactoferrin and carrageenan as a compound active in various concentrations. Additional formulations are under development.

Several feasibility studies are currently ongoing, including studies to determine the effective concentration of actives in the final product and studies for optimizing stability and the content of various additives (preservatives, osmolarity agents and others). Special attention is given to the concentration of salts and pH as triggers of conversion of the formulation from liquid to gel (e.g., Gellan gum). ii. Mucoadhesive tablet containing formulation(s) for sustained and/or controlled release of the respective actives. This technology relies on previous experience of the inventor with formulations developed in the context of halitosis or aphthous stomatitis. The formulation uses a surface carbomer component that facilitates adherence to the mouth mucosa. Upon contact with mouth mucosa and hydration with saliva, the actives are released from the tablet. Current studies focus on optimization of controlled or sustained release of actives in the oral and sublingual milieu.

EXAMPLE 5

Extended cytotoxicity test in vitro using MTT assay in L929 cells

Objective

To evaluate the cytotoxicity of the Film Forming Nasal Spray of the invention using MTT assay in L929 mouse fibroblast cells. The threshold of 70% viability was considered as non-cytotoxic.

Methodological procedure

The specifications of the test item are provided below:

Film forming nasal spray

Actives 1 % GMP, 1 % lactoferrin, 0.2 % Carageenan

Storage conditions Ambient (+15 to +25°C) Category Surface device: Mucosal membrane Contact Duration Prolonged (>24 hour to 30 days) and Permanent (> 30days)

Solutions and serial dilutions of the item and controls were prepared as follows:

Film forming nasal spray stock: 20% solution was prepared by dissolving 2 mL of Film Forming Nasal Spray in 8 mL PBS. The stock was serially diluted to 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156% in culture media. The stock and the dilutions were tested.

Vehicle control stock was prepared in culture media (MEM, 10% HS) and PBS in 1:1 ratio was used as the vehicle control.

Positive control stock was prepared in DPBS. 2mg/mL solution was prepared by diluting 0.5 mL stock in 5.0 mL culture media (2.0 mg/mL final). Concentrations of 0.0062, 0.0125, 0.025, 0.050, 0.075, 0.1, 0.15 and 0.2 mg/mL were prepared in diluent media maintaining a final DMSO concentration at 0.5%. Seeding cells (Day 1)

Cells were removed by trypsinization, centrifuged at 1000 rpm for 5 min and re- suspended in culture medium. Cell concentration was adjusted to lxlO⁵ cells/mL. 100 μL culture medium was added into the peripheral wells of a 96-well microtiter plate. 100 μL cell suspension was added to the wells subjected to the treatment and controls. Cells were incubated for 24 h (5% CO2, 37°C) to form a half-confluent monolayer. Samples were tested in triplicates.

Treatment (Day 2)

After 24 h incubation, plates were examined under phase contrast microscope. Culture medium was replaced with 100 μL vehicle control in designed wells (no treatment). In the remaining wells, 100 μL dilutions of the test item or positive controls were added. Cells were incubated for 24 h (5% CO2, 37°C).

MTT assay (Day 3)

Plates were examined under phase contrast microscope. Changes in cell morphology due to cytotoxic effect were recorded.

Cells were washed with 100 μL DPBS. 100 μL MTT solution (1 mg/mL) was added and plates were incubated at 37 °C, 5% CO2 for 2 h. MTT solution was washed with isopropanol while shaking. The absorption was measured at 570 nm vs. blank.

Cytotoxicity calculation

Relative cell viability was calculated as a percentage of vehicle control.

Reduction of cell viability by more than 30% was considered as cytotoxic.

Results and Conclusions

Morphological observations

Cells treated with the test item were comparable to the vehicle control.

Interpretation based on cell viability (above 70% threshold):

Cells treated with the test item at the concentrations of 5%, 2.5%, 1.25%, 0.625%, 0.312% and 0.156% were above the threshold viability (non-cytotoxic). Concentrations of 20% and 10% were below the threshold. EXAMPLE 6

Evaluation of safety and efficacy of the new formulations of the invention

This trial is still ongoing. Patients (N=50) are being recruited after respective diagnosis of COVID-19 based on clinical symptoms and laboratory diagnosis of SARS- CoV-2 using serological test for IgM/IgG antibodies.

Patients are being treated with daily administration of the Film Forming Nasal Spray (4 times) per day and GMP sustained release tablets (2 times, every 12 h) during the period of 1 month. Patients are subjected to medical evaluation every 10 days. The severity of symptoms is scored from 0 to 5 (from the lowest to the most severe symptoms). There is no control group.

Findings of reduction of symptoms can be indicative as to the therapeutic effect of the Film Forming Nasal Spray and the GMP sustained release tablet on the progression of disease in patients with COVID-19.

SPECIFIC EMBODIEMENTS

In one of its main aspects the invention provides a method for prevention and treatment of viral infection caused by a respiratory virus selected from coronaviridae/corona-virus, orthomyxoviridae, paramyxoviridae Coxsackie family of viruses and adenoviridae family in a subject in need thereof or who is at risk of being infected with the virus, the method comprising administering to a mucosal tissue or a membrane of the subject a composition comprising at least one casein, alone or in combination with at least one lactoferrin and/or at least one carrageenan, and a pharmaceutically acceptable carrier.

In certain embodiments, by the method of the invention the composition is administered into the oral cavity of a human subject in the form of a lozenge, troche or mucoadhesive tablet.

In other embodiments, the composition is administered into the nasal cavity of a human subject in a form of a nasal spray, nasal drops, a viscous gel, a nasal ointment, a nasal powder, a nebulizer or an aerosol.

Still in other embodiments, the composition is administered into the eye in the form of drops, a viscous solution, a gel or an ointment. In another aspect, the invention provides a composition comprising at least one casein, alone or in combination with at least one lactoferrin and at least one carrageenan for use in prevention and treatment of viral infection in a subject in need thereof or at risk of being infected with the virus, wherein the vims is selected from coronaviridae/corona- virus, orthomyxoviridae, paramyxoviridae Coxsackie family of viruses and adenoviridae family.

In certain embodiments, the composition of the invention can be used in preventing infection of coronavirus.

In specific embodiments, the coronavirus is a COVID-19 causing pathogen.

In further embodiments, the COVID-19 causing pathogen is SARS-CoV-2.

In certain embodiments, the at least one casein comprised in the composition of the invention is selected from al casein, ab -casein, aS2-casein, b casein, k-casein and sodium caseinate.

In certain embodiments, the composition of the invention comprises a mammalian milk comprising the lactoferrin and casein portion of the composition.

In further embodiments the mammalian milk in the composition is goat milk.

In certain embodiments, the carrageenan comprised in the composition of the invention is kappa-carrageenan.

In numerous embodiments, the carrageenan is provided in the form of a salt.

In yet another aspect the invention provides a composition comprising at least one casein, alone or in combination with at least one lactoferrin and at least one carrageenan for use in preventing the attachment of a vims selected from coronaviridae/coronavirus, orthomyxoviridae, paramyxoviridae and adenoviridae family onto a variety of medical or non-medical surfaces.

In certain embodiments, the medical surface to which the composition is applied is any part of a skin of a subject.

In other embodiments, the medical surface is a surface of a medical instrument.

Still in other embodiments, the medical surface is a surface of a non-medical instrument.

It is another aspect of the invention to provide a kit comprising the above compositions and instructions of use.

It is still another aspect to provide a device for mucosal delivery comprising the compositions of the invention that are used for clinical purposes in humans.

Claims

1. A composition comprising at least one Goat Milk Protein (GMP), alone or in combination with carrageenan for use in interfering with viral interaction with a mucosal tissue in a mammalian respiratory, nasal, ocular, digestive or reproductive tracts.

2. The composition of claim 1, wherein the interfering with viral interaction being for preventing and/or treating viral infection in a human subject at risk of being infected or already infected with the virus, the virus being an RNA or DNA virus infecting and/or replicating in the mucosal tissue.

3. The composition of claim 1 or 2, wherein the at least one GMP is a casein and/or lactoferrin.

4. The composition of any one of claims 1 to 3, further comprising lactoferrin from a non-goat source.

5. The composition of claim 2, wherein the virus is a respiratory virus belonging to Orthomyxoviridae , Paramyxoviridae, Picornaviridae, Coronaviridae, Parvoviridae or Adenoviridae families.

6. The composition of claim 5, wherein the virus is selected from an Influenza virus, a Respiratory syncytial virus (RSV), an Enterovirus, a Coronavirus, an Adenovirus, a Human bocavirus (HBoV).

7. The composition of claim 6, wherein the virus is Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

8. The composition of claim 6, wherein the virus is a Coxsackievirus.

9. The composition of claim 2, wherein the virus is a virus of digestive system belonging to Reoviridae, Caliciviridae, Coronaviridae or Astroviridae families.

10. The composition of claim 5 or 9, wherein the virus is SARS-CoV-2.

11. The composition of claim 3, wherein the casein is selected from casein (asl-CN), aS2-casein (as2-CN), b-casein (b-CN), k-casein (K-CN) and sodium caseinate.

12. The composition of claim 4, wherein the lactoferrin is a bovine lactoferrin.

13. The composition of claim 1 or 2, wherein the carrageenan is selected from iota- carrageenan, kappa-carrageenan and lambda-carrageenan.

14. The composition of claim 13, wherein the carrageenan is kappa-carrageenan.

15. The composition of claim 13 or 14, wherein the carrageenan is in the form of a salt.

16. The composition of claim 1, wherein the mammal is human.

17. A composition comprising at least one Goat Milk Protein (GMP), alone or in combination with carrageenan for use in interfering with viral interaction with biological and non-biologic al surfaces.

18. The composition of claim 17, wherein said interfering with viral interaction with biological and non-biologic al surfaces comprises interfering with the attachment of the virus to at least one biological or non-biological surface in a medical or non-medical setting.

19. The composition of claim 17, wherein the at least one GMP is a casein and/or lactoferrin.

20. The composition of claim 19, further comprising lactoferrin from a non-goat source.

21. The composition of claim 17 or 18, wherein the biological surface is at least one of the skin, mucosa and epithelium of a subject or a part thereof.

22. The composition of claim 17 or 18, wherein the non-biological surface in a medical setting is any surface in a healthcare facility or a part thereof.

23. The composition of claim 17 or 18, wherein the non-biological surface in a medical setting is any surface of a medical instrument or a part thereof.

24. The composition of claim 17 or 18, wherein the non-biological surface in a nonmedical setting is a surface of a non-medical instrument, or any surface in a house, a vehicle or an office.

25. The composition of claim 17 or 18, wherein the virus is a respiratory virus belonging to Orthomyxoviridae, Paramyxoviridae, Picornaviridae, Coronaviridae , Parvoviridae or Adenoviridae families.

26. The composition of claim 25, wherein the virus is selected from an Influenza virus, a Respiratory syncytial virus (RSV), an Enterovirus, a Coronavirus, an Adenovirus, a Human bocavirus (HBoV).

27. The composition of claim 26, wherein the virus is SARS-CoV-2.

28. The composition of claim 26, wherein the virus is a Coxsackievirus.

29. The composition of claim 19, wherein the casein is selected from casein (asl- CN), aS2-casein (as2-CN), b-casein (b-CN), k-casein (K-CN) and sodium caseinate.

30. The composition of claim 19, wherein the lactoferrin is a bovine (Bovidae) lactoferrin.

31. The composition of claim 17, wherein the carrageenan is selected from iota- carrageenan, kappa-carrageenan and lambda-carrageenan.

32. The composition of claim 31, wherein the carrageenan is kappa-carrageenan.

33. The composition of any one of claims 17 to 32, the composition is being formulated as a liquid or an aerosol, and is delivered via an aerosol dispenser, a nebulizer, an atomizer.

34. The composition of any one of claims 1 to 16, the composition is being formulated as a liquid, a semiliquid, a solid, a powder, an ointment, a gel.

35. The composition of claim 34, the composition being adapted for administering to the nasal mucosa.

36. The composition of claim 34, the composition being adapted for administering to the eye.

37. The composition of claim 34, the composition being adapted for administering to the oral mucosa.

38. The composition of claim 34, the composition being adapted for oral or sublingual administering.

39. The composition of claim 37 or 38 in the form of a lozenge, a troche, a tablet or a mucoadhesive tablet.

40. The composition of claim 37 or 38 in the form of aerosol.

41. The composition of claim 37 or 38, the composition is being formulated to provide sustained or controlled release of the at least one GMP, alone or in combination with lactoferrin and/or carrageenan that are comprised in the composition.

42. A method of interfering with viral interaction with a mucosal tissue in a mammalian respiratory, nasal, ocular, digestive or reproductive tracts, the method comprising administering to the mammal a therapeutically effective amount of at least one Goat Milk Protein (GMP), alone or in combination with carrageenan.

43. The method of claim 42, wherein the interfering with viral interaction further comprises preventing and/or treating viral infection in a human subject at risk of being infected or already infected with the virus, the virus being an RNA or DNA virus infecting and/or replicating in the mucosal tissue.

44. A method of interfering with viral interaction with biological and non-biological surfaces, the method comprising delivering a composition comprising at least one Goat Milk Protein (GMP), alone or in combination with carrageenan to a medical and nonmedical surface.

45. The method of claim 44, wherein said interfering with viral interaction with biological and non-biologic al surfaces comprises interfering with the attachment of the vims to at least one biological and non-biological surface in a medical or non-medical setting.

46. The method of any one of claims 42 to 44, wherein the at least one GMP is a casein and/or lactoferrin.

47. The method of 46, further comprising lactoferrin from a non-goat source.

48. The method of any one of claims 42 or 45, wherein the vims is a respiratory vims belonging to Orthomyxoviridae, Paramyxoviridae, Picornaviridae, Coronaviridae , Parvoviridae or Adenoviridae families.

49. The method of claim 48, wherein the vims is selected from an Influenza vims, a Respiratory syncytial vims (RSV), an Enterovirus, a Coronavims, an Adenovirus, a Human bocavims (HBoV).

50. The method of claim 49, wherein the vims is SARS-CoV-2.

51. The method of claim 49, wherein the vims is a Coxsackievims.

52. The method of claim 46, wherein the casein is selected from casein (asl-CN), aS2-casein (as2-CN), b-casein (b-CN), k-casein (K-CN) and sodium caseinate.

53. The method of claim 47, wherein the lactoferrin is a bovine lactoferrin.

54. The method of claim 42 or 44, wherein the carrageenan is selected from iota- carrageenan, kappa-carrageenan and lambda-carrageenan.

55. The method of claim 54, wherein the carrageenan is kappa-carrageenan.

56. The method of claim 42, wherein the mammal is human.

57. The method of claim 42 or 43, wherein said administering the composition is an oral or a nasal administering of the composition as a liquid or an aerosol composition via an aerosol dispenser, a nebulizer, an atomizer.

58. The method of claim 42 or 43, wherein said administering the composition is an oral, a nasal or an ocular administering of the composition as a liquid, a semiliquid, a solid, a powder, an ointment, a gel composition.

59. The method of claim 42 or 43, wherein said administering the composition is an oral or a sublingual administering of the composition as a lozenge, a troche, a tablet or a mucoadhesive tablet.

60. The method of claim 59, wherein said oral administering the composition further comprises sustained or controlled administering of the at least one GMP, alone or in combination with lactoferrin and/or carrageenan.

61. The method of claim 44 or 45, wherein the biological surface is at least one of the skin, mucosa and epithelium of a subject or a part thereof.

62. The method of claim 44 or 45, wherein the non-biological surface in a medical setting is any surface in a healthcare facility or a part thereof.

63. The method of claim 44 or 45, wherein the non-biological surface in a medical setting is any surface of a medical instrument or a part thereof.

64. The method of claim 44 or 45, wherein the non-biological surface in a nonmedical setting is a surface of a non-medical instrument, or any surface in a house, a public facility, a vehicle, an office or a part thereof.

65. The method of claim 44, wherein said delivering the composition to the medical or non-medical surfaces is delivering the composition as a liquid or an aerosol via an aerosol dispenser, a nebulizer, an atomizer.

66. The method of claim 42 or 43, wherein the virus is a virus of digestive system belonging to Reoviridae, Caliciviridae, Coronaviridae or Aslroviridae families.

67. The method of claim 66, wherein the virus is SARS-CoV-2.

68. A kit comprising the composition of any one of claims 1 to 41 and instructions of use.

69. A device for mucosal delivery comprising the composition of any one of claims 1 to 16.

70. Use of at least one Goat Milk Protein (GMP), alone or in combination with carrageenan in the manufacture or preparation of a medicament preventing or treating viral infection, wherein the virus is an RNA or DNA virus that infects and/or replicates in a mucosal tissue of the respiratory, nasal, ocular, digestive or reproductive tracts.

71. A composition comprising at least one casein, at least one lactoferrin and/or at least one carrageenan for use in preventing and/or treating viral infection in a human subject at risk of being infected or already infected with the virus, the virus being an RNA or DNA virus infecting and/or replicating in a mucosal tissue of the respiratory, nasal, ocular, digestive or reproductive tracts.

72. A composition comprising b-casein, alone, or in combination with lactoferrin and/or carrageenan for use in preventing and/or treating viral infection in a human subject at risk of being infected or already infected with the virus, the vims being an RNA or DNA virus infecting and/or replicating in a mucosal tissue of the respiratory, nasal, ocular, digestive or reproductive tracts.

73. The composition of claim 71 or 72, the composition further comprising Gellan gum and being formulated as a mucoadhesive nasal spray.