COMPOSITION FOR USE IN THE TREATMENT OF RESPIRATORY INFECTION CAUSED BY CORONAVIRUS”

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a pharmaceutical composition comprising secretory anti-Coronavirus IgA class immunoglobulins and at least one pharmaceutically acceptable carrier, and a nasal or oronasal spray device comprising said composition, for use in the prevention and/or treatment of coronavirus viral infections. A preparation method of said pharmaceutical composition is also the subject of the invention.

BACKGROUND ART

Respiratory trait infections have a prevalence of viral origin and represent one of the major causes of illness and death in both children and adults worldwide.

Coronaviruses (CoVs) are a large family of respiratory viruses known for their ability to cause often minor airway infections, such as the common cold, but in some cases lifethreatening, such as pneumonia or bronchitis.

In particular, the infections caused by the new Coronavirus strain named SARS-CoV-2 (Severe Acute Respiratory Syndrome - Coronavirus 2), have caused an important pandemic currently underway characterized by fever, respiratory symptoms that in some subjects evolve into lethal pneumonia, known as COVID- 19 disease, which has caused the onset of very severe respiratory syndromes, with hundreds of thousands of deaths confirmed to date worldwide.

In the initial stages of infection, the virus spreads from the upper airways penetrating into the pulmonary alveoli: according to what is reported in the literature, there are indirect indications that an intense systemic antibody response correlates with a greater severity of the disease as, inside the lung tissue it can contribute to the onset of a cytokine storm (cytokine release syndrome or CRS). There is currently no etiological therapy, but it is reported in the literature that patients in the advanced stages of the disease benefit from assisted ventilation and/or the use of immunosuppressive therapies that limit CRS [Alzghari S.K. et al. J Clin Virol. 2020, 127: 104380],

To date, numerous studies have been conducted with the aim of identifying effective therapeutic interventions that can limit the impact of the infection. An example involves the possibility of collecting plasma from patients recovered from SARS-CoV-2 infection and administering it to patients with active infection. However, this approach has important limitations, as the intensity of the systemic antibody reaction could even be worsened by the administration of hyperimmune sera. As reported in the literature, (Perotti C et al Mortality reduction in 46 severe Covid-19 patients treated with hyperimmune plasma. A proof of concept single arm multicenter trial haematol.2020.261784; Doi: 10.3324/haematol.2020.261784 and Joyner et al Early safety indicators of COVID-19 convalescent plasma in 5,000 patients J Clin Invest. 2020 Jun 11: 140200. doi: 10.1172/JC1140200 Administration of hyperimmune plasma can induce severe lung injury typically referred to as transfusion-related acute lung injury Transfusion-related Acute Lung Injury " or TRALI), in which the clinical picture is identical or very similar to lung damage induced by covid-19.

In fact, as mentioned, a strong systemic antibody response is not always associated with a better prognosis. Conversely, there are indications that a reduced systemic antibody response correlates with a better prognosis: firstly, asymptomatic patients develop a reduced serum antibody response; secondly, patients with primary immunodeficiency and inability to produce antibodies do not develop the severe pulmonary consequences that require the use of assisted ventilation and targeted therapies to avoid CRS [Soresina A. et al. Pediatric Allergy Immunol. 2020; Quinti I. et al. J. Allergy Clin Immunol. 2020, 146 (1)].

The possibility of having different therapeutic options available becomes increasingly important when the possibility of obtaining a vaccine capable of conferring permanent immunity is questioned by the variability of the virus and by the possibility that not even natural infection can confer a permanent immunity [Sayak R. SN Compr. Clin. Med. 2020; SK Law et al. Hong Kong Med J. 2020, 26: 264-5; Kang H. J. Med Virol. 2020, 1- 3].

In light of the foregoing, there is therefore an extremely urgent need to have more effective and safe therapeutic solutions for the prevention and initial treatment of Coronavirus infections, and in particular SARS-CoV-2 infections.

SUMMARY OF THE INVENTION

The aim of the present invention is to provide an effective solution for the prevention and/or initial treatment of Coronavirus infections, and in particular for the prevention and/or treatment of SARS-CoV-2 infections.

Viruses, particularly RNA viruses such as Coronaviruses, are known to constantly evolve through mutations in their genome. While most of the mutations do not have a significant impact, some may give the virus some characteristics of particular relevance, such as a greater transmissibility, a greater pathogenicity with more severe forms of the disease or, above all, the possibility of circumventing immunity previously acquired by an individual either by natural infection or by vaccination.

In this context, therefore, the need to develop effective alternative solutions for the pharmacological treatment of these viral infections, which can be: combined with vaccination, used in the treatment of subjects with immunodeficiency conditions in which the response to an immunodeficiency active may be reduced or even absent, or even as a prevention in subjects who have been in close contact with a positive subject, becomes extremely important.

The solution object of the invention is represented by a pharmaceutical composition comprising anti-Coronavirus specific secretory immunoglobulins of the IgA class.

In addition to being present in serum, IgA class immunoglobulins are secreted within mucosal fluids, such as saliva, gastric juice, urine, breast milk. IgA dimers are secreted in association with a protein (secretory component) capable of making them more stable in the potentially hostile mucosal environment. The IgA useful for antiviral defense comprise two types: 1) natural immunity antibodies, present from birth, and 2) adaptive immunity antibodies, produced only following contact with non-self antigens. Both of these types of IgA class antibody are present in the saliva of patients recovered from Coronavirus infections, and in particular from SARS-CoV-2 infections, having contributed to overcome the infection. Advantageously, the anti-Coronavirus IgA class immunoglobulins present in the composition object of the present invention can therefore be isolated directly from a saliva sample previously obtained from one or more donor individuals, convalescing and/or healed from a Coronavirus infection.

In one of its embodiments, the present invention advantageously provides isolating the specific secretory IgA present in salivary samples obtained from convalescent patients or patients recovered from Coronavirus infections, concentrating said immunoglobulins and standardizing them in such a way as to provide a particularly effective pharmaceutical composition for the treatment of the early stages of respiratory trait infections caused by these pathogens.

Alternatively, the immunoglobulins thus isolated can be characterized and reproduced according to standard molecular biology methods known to those skilled in the art.

The composition object of the invention offers the advantage of being suitable for administration by inhalation, aerosol, nebulization, or spray; therefore in a simple and easy way, even at home, directly into the respiratory trait of patients with infection at the initial stage. This mode of administration is effective in limiting or preventing the spread of the virus in lung tissue, where the onset of CRS has been observed in numerous patients with Covid-19. Thanks to the presence of the highly vascularized nasal mucosa, the administration by inhalation of the composition object of the invention not only allows the activity of therapeutic IgA at the mucosa level, but also involves greater effectiveness at lower doses of antibodies and with reduced side effects. .

Therefore, the object of the present invention are:

A pharmaceutical composition comprising anti-Coronavirus IgA class secretory immunoglobulins and at least one pharmaceutically acceptable carrier; - A pharmaceutical composition comprising anti-Coronavirus IgA class secretory immunoglobulins and at least one pharmaceutically acceptable carrier, for use in the prevention and/or treatment of Coronavirus infections;

- A device for the delivery by inhalation or by nasal or oronasal spray comprising a composition according to any of the embodiments herein described;

1. A process for the preparation of a pharmaceutical composition comprising IgA class secretory immunoglobulins and at least a pharmaceutically acceptable carrier, comprising the following steps: a. isolating said anti-Coronavirus secretory IgA immunoglobulins from a saliva sample previously obtained from one or more donor individuals; b. concentrating said IgA immunoglobulins; c. suspending the obtained product in suitable pharmaceutically acceptable excipients; and

2. A process for the preparation of a pharmaceutical composition comprising IgA class secretory immunoglobulins and at least a pharmaceutically acceptable carrier, comprising the following steps: a. expressing said anti-Coronavirus IgA secretory immunoglobulins in a host cell and/or organism; b. isolating said IgA class immunoglobulins expressed in step a. c. concentrating said IgA class immunoglobulins; d. suspending the obtained product in suitable pharmaceutically acceptable excipients.

Altri vantaggi e caratteristiche della presente invenzione risulteranno evidenti nella descrizione dettagliata. Other advantages and features of the present invention will become apparent in the detailed description.

GLOSSARY

The terms used in this description are as generally understood by the skilled person, unless otherwise indicated.

With the term "secretory immunoglobulins of IgA class ", also known by the acronym "S-lgA", in the context of the present description, refer to antibodies of IgA class or to an antigen-binding portion thereof, present in nasal secretions and mucosal, such as saliva, tears, gastric secretions or breast milk, and secreted by plasma cells adjacent to mucosal epithelial cells.

A single IgA molecule is composed, like all immunoglobulins, of a pair of heavy chains (H) and a pair of light chains (L). The latter comprise two Ig-type domains: a first variable domain (VL) and a constant domain (CL). The heavy chains are of type a, and each contain a variable Ig domain (VH or Va) and three constant domains (CH1/2/3 or Ca1/2/3). Two IgA subclasses with similar structure and functions have been identified, lgA1 and lgA2, which differ in the heavy chain, which can be encoded by two different genes a1 and a2.

The secretory immunoglobulins of the IgA class include IgA in mainly dimeric form, but also tri- or tetrameric. Having a greater resistance to proteolytic activity, lgA2 represents the main subclass in secretions, although both subclasses of IgA can form dimeric structures.

Dimers are formed by the covalent interaction of a small polypeptide structure known as the J junction chain (“joining") at the terminal constant region of the Fc portion.

The secretory dimeric forms of IgA immunoglobulins generally possess a glycoprotein, known as the “secretory component” (or SC fragment), i.e. the secreted component of the polymeric immunoglobulin receptor (poly-lg receptor, plgR) located on the basolateral surface of the mucosal epithelial cells. This secretory component is able to protect IgA from the action of bacteria and proteolytic enzymes in the mucosal environment.

The expression "antigen-binding portion" refers, in the context of the present invention, to a fragment or portion of the IgA class antibody that competes with the antibody for specific binding to the antigen. IgA class antibody fragments can be generated, for example, by a partial enzymatic digestion of immunoglobulins.

Common examples of antigen-binding antibody fragments and/or portions include Fab, Fab ', F (ab ¹ ) ² , Fc, Fd, Fv, scFv or single-chain fragments Fv, dAb, and all polypeptides which contain at least a portion of an antibody sufficient to give the polypeptide a specific binding to the antigen.

The expression "bind specifically" in the context of this specification refers to the ability of an antibody, in particular a secretory immunoglobulin of the IgA class, to bind more strongly to a target, such as a specific epitope, than to another target. An antibody is able to bind a first target more strongly than a second target, if it binds to the first target, for example, with a dissociation constant (Kd) that is less than the dissociation constant for the second target.

The term "Coronavirus" refers, in the context of the present invention, to a virus subfamily of the Coronaviridae family. These include phylogenetically compact genotypes of wrapped, positive-sense, single-stranded RNA with a helically symmetrical nucleocapsid.

The name "coronavirus" takes its origin from the Latin "corona" and refers to the characteristic superficial glycoproteins S (or "Spike", from the English "tip", "spike") that coat the virion forming structures similar to "spinules" ’’Visible under the electron microscope. Among the most recently discovered strains of Coronavirus are the SARS- CoV strain, discovered in 2002, the MERS-CoV strain in 2012, and SARS-CoV-2 in 2019, responsible for serious outbreaks of respiratory trait infections.

The term "SARS-CoV-2" in this description refers to Coronavirus 2 from severe acute respiratory syndrome (from the English "severe acute respiratory syndrome coronavirus 2), a viral strain of the SARS-related Coronavirus species, belonging to the Coronaviridae family, responsible for the Coronavirus disease 2019 (COVID- 19). The outermost layer of the SARS-CoV-2 virus is characterized by a coating of S glycoproteins assembled to form trimeric structures that constitute the characteristic “crown” that surrounds the virion. S-glycoproteins determine the specificity of the virus for the epithelial cells of the respiratory trait. The ectodomain of Spike proteins is characterized by two distinct domains: an N-terminal domain, named S1, containing the human receptor binding domain-RBD (ACE2), and a C-terminal domain, named S2, responsible of the merger.

The outermost layer of the virus includes a second coating protein, known as the hemagglutinin esterase (HE) dimer, smaller than the S protein, which plays an important role in promoting the release of the virus within host cells. The virus is also constituted of membrane proteins that cross the outer coating, interacting within the virion with the RNA-protein complex. The genome of the virus consists of a positive polarity single strand RNA of large size (from 27 to 32 kb in the different viruses), associated with the N protein, which increases its stability.

With the acronym COVID-19 (from the English acronym Coronavirus Disease 19), or acute respiratory disease from SARS-CoV-2, an infectious respiratory disease caused by the SARS-CoV-2 virus is meant.

Acute lung injury associated with transfusion Transfusion-related Acute Lung Injury ”or TRALI.

CRS cytokine release syndrome or cytokine storm.

Secretory component: it is a protein that is secreted together with the IgA present in the mucous membranes which are generally dimeric and which serves to stabilize the Ig A in the extracellular environment.

For the purposes of this description, the term prevention means prevention of SARS- Cov-2 infection or also prevention of the onset of the cytokine storm (CRS) when the composition of the invention is administered in the early stages of the disease. Early treatment for the purposes of the present invention, means a treatment of the patient infected with SARS-Cov-2 at a stage of the infection preceding the onset of cytokine storm (CRS) triggered by the aforementioned infection.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned, the aim of the present invention is to provide an effective and safe pharmaceutical composition for the prevention and/or treatment of viral infections of the respiratory trait caused by Coronavirus.

A first aspect of the invention therefore refers to a pharmaceutical composition comprising anti-Coronavirus IgA class secretory immunoglobulins and at least one pharmaceutically acceptable carrier.

IgA-class secretory immunoglobulins play an important role in the immune response at the level of mucosal surfaces, limiting or disfavouring the adhesion of viral particles to the surface of epithelial cells and their subsequent penetration into the cells.

The anti-Coronavirus IgA secretory immunoglobulins according to the invention can be IgA class antibodies of any subclass, such as lgA1 or lgA2 antibodies. The anti- Coronavirus IgA class secretory immunoglobulins, according to any embodiment provided in the present description, can be in monomeric and/or polymeric form. According to one aspect of the invention, said anti-Coronavirus IgA secretory immunoglobulins are in dimeric form, i.e. comprising two IgA molecules linked together through a J chain. The secretory IgA may optionally comprise a secretory component (SC).

The term "IgA class antibodies" used in this description also comprises fragments and/or portions of said anti-Coronavirus IgA class secretory immunoglobulins binding the antigen of interest, as defined in the glossary. According to the present invention, the fragments and/or portions of IgA secretory immunoglobulins are fragments and/or portions which maintain the anti-coronavirus activities of the aforementioned immunoglobulins. According to one aspect of the invention, the anti-Coronavirus IgA secretory immunoglobulins or fragments and/or portions thereof present in the composition object of the invention have a neutralizing activity against viruses belonging to the Coronavirus family. "Neutralizing activity", in the context of the present invention, means the ability of the antibody or one of its antigen-binding fragments to neutralize or block the biological effect of the target antigen. For example, an antibody with neutralizing activity is an antibody capable of binding a specific viral antigen by inhibiting the binding of said antigen with the target cell.

According to an aspect of the invention, said anti-Coronavirus IgA secretory immunoglobulins or fragments and/or portions thereof are anti-Coronavirus Spike (S) protein IgA immunoglobulins or fragments and/or portions thereof, or immunoglobulins able to recognize and/or specifically bind a domain of the Coronavirus Spike protein or fragments and/or portions thereof, inhibiting or disfavouring the binding of said protein to a cellular receptor.

According to a preferred aspect of the invention, said secretory IgA class immunoglobulins or fragments and/or portions thereof are anti-SARS-CoV-2 IgA class immunoglobulins.

Said IgA-class secretory immunoglobulins or anti-SARS-CoV-2 fragments and/or portions thereof comprise, according to one embodiment, anti-SARS-CoV-2 protein S IgA immunoglobulins or fragments and/or portions thereof, i.e. IgA or fragments and/or portions thereof, that are capable of specifically binding a region of the SARS-CoV-2 Spike protein and/or of inhibiting, at least partially, the binding of said protein to a receptor.

According to an embodiment of the present invention, said anti- SARS-CoV-2 protein S secretory IgA immunoglobulins or fragments and/or portions thereof are able to inhibit the binding between said protein S and the human ACE2 receptor, preventing the subsequent entry of viral particles into cells. Non-limiting examples of anti- SARS-CoV-2 protein S secretory IgA immunoglobulins or fragments and/or portions thereof comprise, for example, anti-SARS-CoV-2 S1 and/or S2 subunit of S protein immunoglobulins or fragments and/or portions thereof, or IgA or fragments and/or portions thereof able to bind the trimeric form of the SARS- CoV-2 protein S in its pre- or post-fusion state.

According to one aspect of the invention, said anti-Coronavirus IgA class secretory immunoglobulins or fragments and/or portions thereof are human IgA, or recombinant S-lgA or fragments and/or portions thereof.

According to an aspect of the invention, the composition object of the invention comprises an amount of secretory immunoglobulins of the IgA class or fragments and/or portions thereof sufficient to exert a protective and/or therapeutic effect against Coronavirus infections such as SARS-Cov-2 or other coronoaviruses. According to an embodiment of the invention, the composition contains anti-Coronavirus IgA class secretory immunoglobulins in therapeutically (or preventively) effective concentrations, such as for example a concentration of between 100 and 250 mg/ml, such as for example about 200 mg/ml, or about 180 mg/ml, or about 160 mg/ml, or about 140 mg/ml, or about 120 mg/ml, or about 100 mg/ml, or about 80 mg/ml.

In one embodiment, the pharmaceutical composition of the invention comprising anti- Coronavirus IgA secretory immunoglobulins or fragments and/or portions thereof comprises at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of IgA.

The composition may further comprise other proteins normally present in saliva such as ptyalin, mucin and possibly lysozyme.

In one embodiment, the unit dose administered may range from 1 to 5 ml, such as for example 1, 2, 3, 4 or 5 ml of composition, depending on the type of administration selected from those described.

Pharmaceutically acceptable carriers suitable for the realization of the composition of the invention are those commonly used for preparations for administration by air, such as buffered solutions or other suitable carriers known to those skilled in the art. An example of a suitable carrier can be represented by the PBS.

According to the present invention, any embodiment of the composition as herein described can be administered for the prevention of coronavirus infections, in particular for the prevention of SARS-Cov-2 infections. The preventive activity carried out by the formulation herein described is inherent to the very concept of secretory IgA, and the administration by inhalation of the aforementioned formulation in any of the modes herein described, distributes said secretory IgA precisely on the respiratory mucous membranes protecting them, in fact, from infection by part of the virus.

The term prevention in the present invention also comprises the prevention of severe forms of SARS-Cov-2 infection, represented by the onset of CRS. The secretory IgA present in the formulation of the invention have in fact the function of preventing the spread of the virus in the lung tissue and the consequent triggering of CRS.

The therapeutic treatment, according to the present invention, is preferably the treatment of the early stages of the infection, that is, the stage of the disease prior to the onset of CRS.

Object of the invention is therefore also a method of prevention or therapy of coronavirus infections, in particular from SARS-Cov-2, wherein the composition as described in any embodiment provided in the present description, is administered in therapeutically effective doses to a subject in need thereof.

Therapeutically effective doses are to be understood as doses and frequency of administration sufficient to exert a preventive effect of infection or a therapeutic effect in which CRS is avoided or reduced.

Individuals particularly suitable for administration for preventive purposes can for example be relatives of infected subjects (in particular of subjects in home isolation) or even health workers, i.e. individuals who are more at risk of infection.

A stronger preventive effect can be obtained by taking the composition of the invention before coming into contact with the patient suffering from Covid- 19 or with environments in which there have been patients affected by Covid-19. Another preventive use of the composition of the invention may be particularly desirable for individuals who have to use public means of transportation or in any case means shared with other subjects for travel, such as buses, trains, airplanes, ferries or other.

Also in this case, taking the composition before (and possibly during) such displacements allows to exert an infection prevention effect in case of presence of individuals, symptomatic or not, vectors of SARS-Cov-2, in said means of transport.

From the therapeutic point of view, as mentioned above, taking the composition according to the present description is particularly recommended for patients infected with SARS-Cov-2, especially in order to prevent the acute phases of the disease, in particular in order to prevent the CRS.

Thanks to the ease of collection of the sample, the accessibility, safety, and non- invasiveness of the collection, according to a preferred embodiment of the invention, said anti-Coronavirus IgA secretory immunoglobulins or fragments and/or portions thereof can be isolated directly from a salivary sample previously obtained from one or more donor subjects. The isolation of secretory IgA from said sample can be carried out using any of the techniques known in the field for the purification of IgA class secretory immunoglobulins starting from a salivary sample, and/or using a process as described below in this application.

"Donor subjects" means, for example, convalescent and/or recovered from Coronavirus infections, in particular from SARS-CoV-2 infections subjects, who have therefore developed specific secretory IgA antibodies in response to viral infection.

According to one aspect of the invention, said donor subjects are mammals, in particular human beings immunized against a Coronavirus infection.

A salivary sample suitable for the isolation of specific anti-Coronavirus IgA or fragments and/or portions thereof present in the composition object of the invention is a salivary sample obtained from a donor subject using any of the methods known in the field for collecting salivary samples. Non-limiting examples of suitable methods for collecting salivary fluids include:

- drainage method (“draining”): saliva is made to drip from the lower lip of a subject into a pre-weighed or graduated test tube equipped with a funnel;

- spitting method: saliva is accumulated at the base of the oral floor of the subject and spit inside a pre-weighed or graduated test tube and/or container, at defined time intervals;

- aspiration method: saliva is continuously aspirated from the oral floor of a subject into a test tube using a saliva expeller or an aspirator;

- absorbent method: saliva is collected (absorbed) by a pre-weighed swab, a cotton roll or a gauze sponge placed in the oral cavity at the orifices of the main salivary glands.

As known in the art, saliva samples obtained from donor subjects include two types of IgA class immunoglobulins: 1) natural immunity antibodies, present since birth, and 2) adaptive immunity antibodies, produced only following contact with non-self antigens.

Therefore, according to an aspect of the invention, a pharmaceutical composition according to any of the variants described above can also comprise immunoglobulins of the IgA class that are not specific for the viral antigen, for example secretory immunoglobulins of the IgA class produced by cells of the innate immune system but which can recognize structures characteristic of groups of microorganisms (pathogen associated molecular patterns: PAMPs).

Alternatively, anti-Coronavirus IgA secretory immunoglobulins or fragments and/or portions thereof isolated from a sample obtained from one or more donor subjects can be characterized, for example by means of protein sequencing techniques, and reproduced according to standard methods, so to obtain high quantities of the same to be used in a composition object of the invention.

Examples of standard methods known in the art include, for example, methods for the production of recombinant IgA class monoclonal antibodies comprising at least the following steps: (i) amplifying and cloning the genes encoding the specific IgA class antibodies or fragments and/or portions thereof within a suitable vector, (ii) introducing the vector into host cells and/or a host organism, and (iii) inducing the expression of antibodies within the host cells and/or organism. The immunoglobulins or fragments and/or portions thereof thus expressed can be recovered from the cells and/or the host organism using standard methods of protein purification.

Therefore, according to an aspect of the invention, the anti-coronavirus IgA secretory immunoglobulins or fragments and/or portions thereof suitable for use in a composition object of the invention, are recombinant antibodies or fragments and/or portions thereof, in particular monoclonal antibodies of recombinant IgA class, i.e. produced recombinantly inside host cells using standard methods known in the field.

By way of example only, said recombinant anti-Coronavirus IgA secretory immunoglobulins or fragments and/or portions thereof can be produced by: (i) transfecting or transformating of host cells with an expression vector comprising at least one nucleotide sequence coding for said anti-secretory IgA Coronavirus or fragments and/or portions thereof; (ii) cultivating of host cells for a period of time sufficient to allow the expression of the antibody in the host cells, or, alternatively, the secretion of the antibody in the culture medium used; (iii) recovering IgA or fragments and/or portions thereof from host cells and/or from the culture medium using standard protein purification methods.

Fragments and/or portions of anti-Coronavirus secretory IgA can alternatively also be produced by enzymatic or chemical cleavage of intact secretory IgA immunoglobulins.

The nucleotide sequences and/or genes encoding anti-Coronavirus secretory IgA or fragments and/or portions thereof suitable to be used for the expression and/or production of anti-Coronavirus recombinant monoclonal secretory IgA or fragments and/or portions thereof according to the present invention, can be obtained by standard cloning techniques known in the field, starting from/from: - sequencing of anti-Coronavirus IgA secretory immunoglobulins isolated from a saliva sample obtained from one or more donor subjects as mentioned above; and/or

- - amplification and sequencing of anti-Coronavirus IgA encoding genes isolated from cells of said donor subjects, such as human memory B cells.

Said gene and/or protein sequencing procedures and said amplification procedures can be carried out using any of the techniques known in the field of cell biology, genetic engineering, synthetic biology, protein engineering or the like.

Alternatively, said nucleotide sequences and/or genes can be selected from a specific gene-library coming from donors previously immunized against a Coronavirus infection.

According to an aspect of the invention, the host cells that can be used for the expression and/or production of said recombinant secretory immunoglobulins of anti- Coronavirus IgA class or fragments and/or portions thereof are preferably human cells, or are hybridoma cells .

A skilled person, depending on the host cells and the organism of interest, will have sufficient information in the art to select an optimal expression vector for the production of recombinant antibodies as previously indicated. Non-limiting examples of organisms that can be used for the expression and/or for the production of recombinant IgA anti- Coronavirus secretory immunoglobulins or fragments and/or portions thereof according to the invention, comprise mammals and/or plants.

Pharmaceutically acceptable carriers suitable for use in a pharmaceutical composition according to any of the embodiments described therein, comprise, but are not limited to, solvents, dispersion media, antibacterial and antifungal agents, diluents, thickening agents, salts, adjuvants, or surfactants, provided they are physiologically compatible.

Some examples of pharmaceutically acceptable carriers comprise, for example, water, saline, phosphate buffered saline, glycerol, mixture of water and ethanol and the like, as well as combinations thereof. In some cases it is preferable to include in the composition object of the invention also isotonic agents, for example sugars or polyalcohols, such as mannitol or sorbitol.

Agents capable of increasing the shelf life of the composition and preserving the effectiveness of the antibodies can also be used in the composition object of the invention and comprise wetting agents, emulsifiers, preservatives or buffers.

The composition object of the invention in any of the embodiments described therein may also comprise stabilizing agents of the microbial load, such as benzalkonium chloride or the like.

The pharmaceutical compositions according to any of the embodiments described therein are sterile and stable under storage conditions.

Advantageously, according to an aspect of the invention, the pharmaceutical composition according to any of the previously described embodiments, can be administered by inhalation and/or via intra-nasal and/or oronasal route. The dispersion of a pharmaceutical composition according to the invention in very fine drops facilitates its transport through the respiratory trait, increasing the bioavailability of the active ingredients at the level of the mucosal membranes.

According to an aspect of the invention, the composition object of the invention can be made in any form as long as it is suitable inhalation and/or intra-nasal and/or oronasal administration, and in particular in a form selected from aerosol and/or nasal or oronasal spray, nebulized formulation, solution, emulsion, suspension, nasal drops, powder. The composition object of the invention can also be packaged both in multidose containers and in single-dose containers.

Non-limiting examples of generating devices that can be used for aerosol administration of a pharmaceutical composition according to the invention include a nebulizer (or small volume nebulizer, SVN), a metered-dose inhaler (or pressurized metered-dose inhaler, pMDI) , or a dry powder inhaler (DPI).

According to one embodiment, the IgA class secretory antibodies can be administered to a subject in the form of an aerosol composition from a nebulizer operated by a suitable propellant, such as air or oxygen, from a compressor, or from an electric power device. The selection of the most appropriate dispensing device for administering a composition object of the invention can be made in relation to the type of patient, the higher ease of use, or the frequency of administration.

Object of the invention is also a pharmaceutical composition according to any of the embodiments previously described, for use in the prevention and/or treatment of Coronavirus infections.

According to one aspect of the invention, said pharmaceutical composition can be used for the prevention and/or treatment of SARS-CoV, MERS-CoV infections, and in particular for the prevention and/or treatment of SARS-CoV-2 infections.

The composition object of the invention in any of the embodiments described herein is particularly indicated for the treatment of SARS-CoV-2 infections in an initial stage, or for the treatment of subjects presenting mild symptoms and/or asymptomatic subjects.

Also, object of the invention is an inhalation delivery device or a delivery device by means of a nasal or oronasal spray comprising a pharmaceutical composition according to any of the previously described embodiments.

An inhalation delivery device or a nasal or oronasal spray delivery device according to the present invention may be used by a subject suffering from, or at risk of contracting, a coronavirus infection, and in particular a SARS-CoV-2 infection, to prevent and/or treat said infection.

According to an embodiment of the invention, said nasal or oronasal spray delivery device can comprise a bottle and/or vial containing the pharmaceutical composition to be administered and a spray atomizer system connected to a terminal syringe, for example in the form of a spout, to allow the delivery of an optimal volume of the composition per dose directly into the nasal or oronasal cavity.

Through the generation of a thin local cloud of particles, said delivery device by means of a nasal or oronasal spray allows the delivery of an appropriate dose of the composition object of the invention rapdily and effectively within the nasal or oronasal cavity. The device can alternatively be equipped with an oronasal mask in order to allow delivery throughout the oropharyngeal cavity.

A further example of an inhaling delivery device according to the present invention is a pressurized metered dose inhaler device (MDI) comprising the pharmaceutical composition to be administered in a suitable dose.

Object of the invention is also a process for the production of a pharmaceutical composition according to any of the previously described embodiments, comprising the following steps: a. isolating IgA-class secretory immunoglobulins from a saliva sample previously obtained from one or more donor subjects; b. concentrating said IgA class immunoglobulins; c. suspending the product obtained and in suitable pharmaceutically acceptable excipients.

As previously mentioned, said one or more salivary samples can be obtained from each donor subject using any of the methods known in the art for the collection of salivary fluids.

The saliva samples obtained can be stored at room temperature up to a maximum of 30-90 minutes before proceeding with the isolation of the secretory IgA immunoglobulins. Alternatively, these samples can be stored at a temperature equal to or below -20 ° C until the time of analysis. Samples can possibly be stored at -80 ° C even for years to ensure minimal degradation.

The saliva sample or samples obtained from a donor can be preliminarily subjected to a diagnostic investigation aimed at determining the presence of specific secretory antiCoronavirus IgA antibodies in the sample, as well as verifying the absence of other harmful particles, such as pathogenic microorganisms, (ie EBV, CMV, HCV). This diagnostic investigation can be carried out using a saliva test selected from those commonly known in the state of the art and/or also using serological tests. Non-limiting examples of techniques that can be used for diagnostic purposes on a salivary sample to identify secretory anti-Coronavirus IgA and/or harmful particles include immunoassays, colorimetric assays and/or high resolution mass spectrometry.

An example of a colorimetric assay involves, for example, the collection of the saliva sample with an absorbent swab, the application of a chemical solution on the swab and the evaluation of a color change of the sample to indicate a positive or negative result regarding the presence of harmful particles.

The saliva samples may also be preliminarily subjected to a centrifugation and/or filtration step aimed at eliminating particulates, cell residues, microorganisms and/or other specific impurities possibly present in the sample, or aimed at reducing the viscosity of the starting sample.

According to an aspect of the invention, said step a) of isolating of the secretory antiCoronavirus IgAs of IgA class or fragments and/or portions thereof from a sample of saliva, can be carried out by any of the techniques known in the field or combinations thereof. Non-limiting examples of techniques that can be used for the isolation of said secretory IgAs from a salivary sample include immunoprecipitation techniques (also known as immuno-affinity or pull-down techniques), fractional precipitation, affinity chromatography techniques, and/or ion exchange chromatography and/or size exclusion chromatography, or combinations thereof.

According to an embodiment of the invention, said step a) can be carried out by affinity chromatography, for example by using a resin of specific affinity for IgA class immunoglobulins. Non-limiting examples of commercially available affinity resins for purification of IgA class immunoglobulins include, for example, the resin "CaptureSelect ™ IgA Affinity Matrix" (ThermoFisher) based on a fragment of llama antibody capable of recognizing human IgA, or the resin "LigaTrap® Human IgA Resin" (LigaTrap).

In one embodiment, said anti-Coronavirus IgA class secretory immunoglobulins can be isolated from a salivary sample using an affinity chromatography technique and in particular using a recombinant bacterial IgA-binding protein, IgA-bp, as an affinity reagent. Said protein is able to specifically bind IgA class immunoglobulins, constituting an ideal reagent for a quick and simple purification of these immunoglobulins.

In case it is desired to purify in a specific way class lgA1 secretory immunoglobulins, it is possible alternatively to use Jacalin, an alpha-D-galactose binding lectin extracted from jack fruit seeds (Artocarpus integrifoglia), as an affinity reagent which is able to specifically bind lgA1 and not lgA2. lgA1 molecules bound to the Jacalin reagent can be eluted by competitive displacement using a buffer containing galactose.

An example of a commercially available affinity resin for purification of lgA1 class immunoglobulins is the “PierceTM Jacalin Agarose” resin (ThermoFisher).

As an alternative to the use of affinity chromatography techniques, it is possible to use conventional chromatography techniques for the purification of IgA from salivary samples. Generally, the protocols in this case provide for the combination of two or more purification techniques, for example ion exchange chromatography and size exclusion chromatography. Said protocols can also provide for a preliminary fractionation step, for example by means of ammonium sulphate.

Non-limiting examples of chromatographic resins that can be used to isolate IgA immunoglobulins from a saliva sample include DEAE cellulose, Sephadex G-200, DEAE-Sephadex A-50, Jacalin-Sepharose, Sepharose-protein G, Fastflow-S Sepharose, superose 6, Sepharose 4B activated with cyanogen bromide conjugated to an anti-lgA antibody.

According to an aspect of the invention, said process can comprise an additional washing step of the immunoglobulin fractions obtained following step a).

Isolated anti-Coronavirus IgA secretory immunoglobulins can be characterized using standard methods known in the art, such as immunoprecipitation, immunohistochemistry, Western Blot (WB), ELISA, immunofluorescence, immunocytochemistry, protein sequencing techniques or a combination thereof. According to an aspect of the invention, said step b) of concentration of IgA class immunoglobulins can be carried out using any of the techniques known in the field for the concentration of a protein extract.

In an embodiment of the invention, said antibody concentration step can be carried out by precipitation of the IgA with ammonium sulphate, followed by re-solubilization of the proteins in a suitable carrier, and/or by a dialysis and/or filtration step, to remove excess salt.

The concentration of IgA immunoglobulins in the final composition can be determined using any of the techniques known in the art, for example by means of an enzyme immunoassay, i.e. a quantitative ELISA assay, colorimetric assay, chromatographic analysis.

According to a further aspect of the invention, the process object of the invention can include a fourth step d) comprising the adjustment of the pH of the composition and/or the addition of at least one pharmaceutically acceptable carrier selected from the compounds indicated above.

The present invention also relates to a process for the production of a pharmaceutical composition according to any of the previously described embodiments, comprising the following steps: a. expressing anti-Coronavirus IgA secretory immunoglobulins in a host cell and/or organism; b. isolating said IgA class immunoglobulins expressed in step b. c. concentrating said IgA class immunoglobulins; d. suspending the product obtained and in suitable pharmaceutically acceptable excipients.

Step (a) of the process just described allows the production of recombinant anti¬

Coronavirus IgA secretory immunoglobulins in high quantities that can be used for the production of a composition object of the invention sufficient to treat a large number of patients.

Both the processes described above will be followed by or will comprise appropriate steps for sterilizing the product.

According to an aspect of the invention, the nucleotide sequences and/or the genes and/or the vectors suitable for the expression of said immunoglobulins according to step (a) of said process can be obtained using standard methods for cloning and/or amplifying nucleic acids, starting from/from:

- sequencing of anti-Coronavirus IgA secretory immunoglobulins or fragments and/or portions thereof isolated from a sample obtained from one or more donor subject according to the methods described in this document; and/or

- amplifying and sequencing of anti-Coronavirus secretory IgA coding genes or fragments and/or portions thereof isolated from cells of said donor individuals, such as human memory B cells.

According to one aspect of the invention, said nucleotide sequences and/or genes can be selected from a specific "gene library" coming from subjects previously immunized against Coronavirus infections.

The steps of (a) expression, (b) isolation and (c) concentration of said anti-Coronavirus IgA secretory immunoglobulins or fragments and/or portions thereof according to the procedure described herein can be carried out using any of the methods known to the skilled person, including the methodologies previously described in this document.

According to a further aspect of the invention, the process object of the invention can include an additional step e) comprising the adjustment of the pH of the composition and/or the addition of at least one pharmaceutically acceptable carrier selected from the compounds indicated above.

All the sterilization steps described in this document can be carried out using sterilization techniques known in the art, such as, for example, sterilization by filtration. Also described herein is a method for the prevention and/or treatment of a viral infection of the respiratory trait caused by Coronavirus, and in particular by SARS-CoV- 2, comprising a step of administering, to a patient in need thereof, a pharmaceutical composition according to any of the embodiments described above.

EXAMPLES

Some non-limiting examples of the methodologies according to the present invention are reported below.

Collection of biological material (IgA) by means of Salviette

Salivary specimens from donor patients recovered from SARS-CoV-2 infection are collected using sterile Salivette® tubes according to the instructions provided by the manufacturer (Sarstedt). Salivette® cotton swabs saturated with saliva are placed in the appropriate Salivette® centrifugation containers. The samples are then subjected to centrifugation at 1000xg, at 10 ° C for 2 minutes. At the end of the centrifugation, the supernatants (recovered saliva) are collected from the Salivette® vial and transferred to an appropriate container. A cocktail of protease inhibitors is added to the sample (cOmpleteTM Mini, Roche). Samples are sterilized by filtration using a 0.22 pm filter. The samples are frozen in dry ice and stored at a temperature of -80 ° C until the time of analysis.

Purification of secretory IgA

To isolate secretory IgA, samples are subjected to an affinity chromatography purification step using an IgA-specific affinity resin (CaptureSelectTM IgA AffinityMatrix, ThermoFisher) with a capacity of> 8 mg IgA per mL of resin. A column with an internal diameter of 25 mm is packed up to a height of the resin bed egual to 7 mm, resulting in a column volume (CV) egual to 3.4 mL.

For affinity column loading, 375 mL (110 CV) of the human saliva sample is loaded into the column at 0.5 CV/min (1.7 mL/min). The column is washed with 5 CV of PBS at 0.5 CV/min. An elution buffer containing 0.1 M glycine in deionized water at pH 3 is used for the elution of the sample. The elution is carried out using 4 CV of elution buffer at 0.5 CV/min and fractions equal to 0.5 CV are collected. The pH of each eluted fraction is corrected to a value equal to 4.5-5.5 by adding 1 mL of 0.5 M Tris pH 8.0 per 20 mL of eluate.

Concentration of antibodies

The collected antibodies are concentrated by fractional precipitation with ammonium sulphate and re-solubilization of the IgA pellet in a suitable buffer. The mixture obtained is subjected to dialysis in order to remove the excess salt.

Characterization of the composition

The detection of anti-SARS-CoV-2 IgA secretory immunoglobulins is performed by ELISA assay using a commercial "NovaLisa® SARS-CoV-2 IgA ELISA" kit (Eurofins technologies), using a recombinant SARS-CoV-2 nucleocapsid antigen.

The total level of salivary IgA antibodies within the sample is determined using a quantitative indirect immunoassay provided by Salimetrics, LLC (State College, PA, USA). The method is based on the use of a goat anti-human IgA antibody conjugated to horseradish peroxidase. This enzyme-antibody complex is added in known quantities to samples containing standard amounts of secretory IgA or anti-Coronavirus secretory IgA samples isolated from saliva previously obtained.

The enzyme-antibody conjugate is able to bind slgA in standard samples or in the samples under examination: the quantity of free enzyme-antibody complex in solution is inversely proportional to the concentration of secretory IgA present in each starting sample. Following the incubation and mixing of the reagents, an equal volume of solution is taken from each sample analyzed and added in duplicate to a microtitre plate coated with human slgA. The peroxidase-antibody complex binds to the human slgA present in the plate. After incubation, the unbound components are washed away. The amount of the enzyme-antibody complex is then determined by reaction of the horseradish peroxidase enzyme in the presence of the substrate tetramethylbenzidine (TMB). This reaction produces a blue colour, while a yellow colour is formed following the interruption of the reaction with an acid solution. The amount of enzyme-antibody complex is detected by reading the optical density at 450 nm and is inversely proportional to the amount of anti-Coronavirus IgA secretory immunoglobulin originally present in the sample.

The total protein content of the salivary sample is determined by the Bradford method.

All samples and standards are analysed in triplicate. Determination of salivary IgA in Covid-19 patients and convalescent patients

A group of patients with Covid- 19 and convalescent patients, once signed the informed consent, was subjected to analysis according to the procedures described above, aimed at ascertaining the presence of neutralizing IgA in saliva samples.

The results reported in Table 1 confirm the actual presence of neutralizing igA (NSIgA) in the saliva of some (10/43) patients and convalescents.

In particular, NSIgA were detected in 7/10 patients over 80 days after the onset of symptoms (overall mean: 66 days).

The inventors have observed that the presence of NSIgA in saliva confers some protection against the progression of the disease to a more severe course. In fact, it was possible to observe that only 2/17 patients with NSIgA needed hospitalization in intensive care against 3/13 patients with absent NSIgA (P = 0.05 with Fisher's exact test). Furthermore, the Spearman coefficient between salivary IgA titre and neutralizing salivary IgA titre is equal to 0.89.

Table 1