The present invention concerns compositions having aspecific immunostimulant properties useful for the prevention and treatment of viral infections, particularly of infections of the respiratory tract caused by corona virus and/or influenza virus.

Background of the invention

The administration of bacterial live or attenuated cells has been extensively studied as an immunotherapy tool for the prevention and treatment of viral or tumor pathologies.

In particular, Mycobacteria and Corynebacteria have been used in a number of preclinical and clinical studies.

The immunotherapeutic approach remains the preferred and sometimes the only approach available for infections caused by viruses against which no specific drug has been developed.

For instance, while effective antiviral drugs have been recently developed for the treatment of infections by human immunodeficiency virus (HIV), hepatitis C virus and Herpes virus, no drug has yet proven effective against the most common viruses responsible of respiratory infections (e-.g. rhinovirus, respiratory syncytial virus and influenza virus) and against viruses such as Coronaviruses responsible of the recent outspread of infections in China or against viruses responsible of some of the recent cases of zoonosis.

EP 681479 discloses an immunotherapeutic composition comprising a water insoluble fraction of the cell wall of bacteria, particularly of Mycobacterium phlei , optionally in combination with a glycosaminoglycan such as hyaluronic acid. No specific disclosure has however been made of hyaluronic acid and no information has been provided as to its possible role.

The antibacterial and antiviral properties of hyaluronic acid (HA) have been reported by Cermelli, Palmieri et al, in Virol J. 2011; 8: 141. The Authors concluded that “the spectrum of the antiviral activity exhibited by HA against both RNA and DNA viruses, known to have different structures (with or without envelope) and replication strategies, suggests a non-specific mechanism of action, probably involving cell membrane-virus interaction steps. The results of the kinetic experiments support this hypothesis. ” The prior art as a whole does not provide however a positive conclusion about the possibility of successfully treating viral infections by combining bacteria and glycosaminoglycan.

There is therefore the need for improved compositions which may prevent and protect from the viral infections, accelerating and attenuating the clinical course, reducing the death rate and the damages to parenchymal tissues of organs such as lungs, liver, spleen, kidneys, lymph nodes, etc.).

Description of the invention

It has now been found that the immunostimulant activity of Corynebacterium cells is remarkably improved by including them in a delivery system comprising hyaluronic acid or salts thereof which may be of natural or biosynthetic origin, linear or cross-linked or in form of complexes of fractions having different molecular weight (low molecular weight, typically from 10 to 300 kDa) and high molecular weight, typically from 1000 to 6000 kDa). Sodium hyaluronans are the most commonly used hyaluronic acid salts.

The invention accordingly provides antiviral compositions comprising inactivated cells of Corynebacterium parvum included in a matrix comprising hyaluronic acid or salts thereof.

In particular, it has been found that hyaluronic acid exerts a mechanism of positive chemiotaxis attracting theCD44 ⁺ cells (usually leucocytes, immunocompetent cells and fibroblasts) of a subject treated with the compositions of the invention. Additionally, hyaluronic acid, in view of its hydrophilic characteristics, attracts in its structure water molecules with a consequent swelling promoting the immune cells viability and the contact with the viral particles entrapped within the matrix. The virucidal effect is thereby enhanced by the close contact of the virus, the immune cells and the immunogenic Corynebacterium cells.

The selection of the kind of hyaluronic acid or of salts thereof (molecular weight, linear or cross-linked) allows the control of the residence time within the organism, for instance from a few days to several weeks, enhancing the clinical efficacy and promoting the continuous afflux of new immunocompetent cells destroying the viral units.

Hyaluronic acid, in the compositions of the invention, differs from the conventional adjuvants used in the preparation of vaccines since it also exerts by its own an antiviral activity, acting as a booster co-operating synergistically with Corynebacterium cells determining optimal protective/preventive results in short times, even allowing the cure of the full-blown infection.

Detailed description of the invention

The preferred strain of Corynebacterium parvum has the deposit number NCTC 10390, available from the obtained from the National Collection of Type Cultures, Colindale, London. Bacteria Collection: NTC Number:0387.

National Collection of Type Cultures, Porton Down, Salisbury, UK as well as from the ATCC. One or more strains may be advantageously used contemporaneously. As an alternative to whole cells or in combination thereto, fractions of the bacterial cell wall may also be used, for instance the fraction named p40 disclosed in EP 2900274, or bacterial extracts comprising DNA or RNA fractions.

The immunostimulant activity of C. parvum may further be enhanced by adjuvants such microcrystalline 1-tyrosine or polymers thereof, mucopolysaccharides, selenium and other oligoelements.

The cells are preferably inactivated by conventional methods as treatment with phenol or formaldehyde or by thermal or osmotic shock. The cell counts in each dose of the composition will typically contain from 2 x 10 ⁶ to 5 x 10 ⁹ cells, generally corresponding to 2-7 mg of bacterial mass. Lower counts will preferably be used for the inhalatory route.

The low molecular weight hyaluronic acid or hyaluronan has an average molecular weight Mw ranging from 10 to 300 kDa, preferably from 20 to 150 kDa, more preferably from 50 to 100 kDa.

The high molecular weight hyaluronic acid or hyaluronan has an average molecular weight Mw ranging from 1000 to 6000 kDa, preferably from 1000 to 45000 kDa, more preferably from 1000 to 3500 kDa.

In alternative to linear hyaluronic acids, cross-linked forms may also be used. Cross- linked hyaluronic acid derivatives are commercially available or may prepared by known methods, such as those disclosed in Carbohydrate polymers, 85, (2011), 469-489.

The complexes of high and low molecular weight hyaluronic acid disclosed in WO 2012032151 may also be conveniently used.

The doses of hyaluronic acid or of salts thereof may vary within wide limits and will be ultimately determined by the desired physical form of the composition (liquid, gel, powder, spray). The amount will generally range from 20 to 40 mg for the inhalatory route and from 40 to 100 mg/ml for liquid/gel form. Higher doses are possible in view of the high tolerability of hyaluronic acid.

The compositions may be in form of gel, suitable for intradermal administration, or in form of micronized powder, suitable for inhalation, prepared by dispersing the inactivated bacterial cells (freeze-dried or lyophilized) in a powder of micronized (20-40 micron) hyaluronic acid or hyaluronan.

The compositions in powder form may be inhaled by means of known devices such as those known as capsule based powder inhalers (Pill-haler®, Breezhaler®) and similar devices. The particulate mixture is inhaled in the medium and low respiratory airways sensitizing the cellular structures along the respiratory tree, from the tonsillar Waldeier ring, a structure comprising a chain of lymphocyte aggregates and lymphoid structures located in the oral cavity, to the trachea and lung alveoli.

The powder hydrated by the interstitial liquid forms on the mucosae an hydrophilic biofilm attracting the immunocompetent cells, immobilizing the free viral particles or the virus-infected cells, exposing them to the inactivation induced by Corynebacterium parvum through immuno-potentiation of dendritic cells as well as to their removal by phagocytosis and to the suppression of the viral particles by means of interferon secreted by the cell themselves. The powder inhalation allows, in prevention protocols, the formation of a coating over the respiratory mucosa, alerting the defense mechanisms and preventing the entry of the viruses into the cells of the respiratory epithelium, preventing thereby also the expansion of viral granulomatous pneumonia or the onset of inflammatory damages of other parenchymal tissues.

For preventive purposes an inhalation twice a day is recommended whereas three to six administrations are indicated for the therapy, lasting up to six days or more.

The compositions of the invention may also be absorbed on a sponge material suitable for insertion into the nasal cavity. An example of suitable material is lyophilized heterologous collagen.

The sponges may be inserted in the nostrils in each choana, where are shaped by means of an external massage of the outer nose skin. The sponge remains in situ until it is lysed and dissolved after having scavenged the living inflammatory cells transudated from the nasal sub-mucosa, neutralizing the viruses and preventing their diffusion towards other tissues/organs.

The compositions in form of gel, liquid or spray are administered by the intradermal or subcutaneous route so as to induce a wheal acting as a systemic stimulant of immune defenses.

The compositions of the invention may also be administered topically, in form of lotions, ointments, creams, gels, suppositories or ovules for the treatment of dermatitis or skin, vaginal, anal or ear diseases caused by viruses. Said topical compositions may also comprise keratolytic agents such as salicylic acid, vitamins, surfactants, glycols or other useful agents. In case of topical, skin or mucosae delivery to counteract localized viral infections, other suitable forms included cream, gel, fluid (milk) or spray or powder for exudating lesions or wet sweating skin areas (such as groin, axillae etc.).

If required, the compositions may be administered both by the inhalatory and by the parenteral route, either simultaneously or separately, the intradermal route being preferred when the maximal and persistent protection is desired. The inhalatory route is especially indicated in case of localized inflammatory virus infections such as tracheitis, bronchitis, interstitial pneumonia, alveolitis, laryngitis, tonsillitis, pharyngitis, in order to quick recovery the physiological functions of the respiratory functions. The compositions of the invention are characterized by outstanding efficacy in contrasting infections by different types of pathogenic viruses for human and animal species, as a consequence of a very marked incretion of interferon from the leukocytes after 24-72 hours from the administration. Likewise, the number and the activity of the killer T-cells and of macrophages is also increased. The compositions of the invention are also useful for the treatment of cutaneous or skin infections by bacteria, protozoa and mycetes.

The invention is described in more detail in the following example 1.

Example 1 - Preliminary clinical trials

17 patients affected by viral infections of different origin have been treated with a composition consisting of 5.000.000 cells of freeze-dried inactivated Cory neb acterium parvum NCTC 10390 cells in 1 ml of hyaluronic acid. The administration schedule and the results are reported in the following Table 1.

Table 1