Probiotic bacterial strains for use in preventive or adjunctive treatment of viral respiratory infections - Patents.com

Abstract

The present invention relates to specific and selected bacterial strains and compositions thereof for use in the treatment of viral respiratory tract infections, preferably coronavirus infections such as severe acute respiratory syndrome (COVID-19) caused by coronaviruses.

Description

[Technical field]

[0001] The present invention relates to specific and selected bacterial strains and compositions thereof for use in prophylactic treatments / methods of preventing or inhibiting or reducing viral respiratory tract infections, preferably coronavirus virus infections such as coronavirus severe acute respiratory syndrome (COVID-19), by receptor inhibition of viral transmission, e.g. ACE2 receptor inhibition. [Background technology]

[0002] Viral respiratory tract infections, as the name suggests, are infectious diseases caused by bacteria or viruses that affect the upper and / or lower respiratory tract (nose, trachea, bronchi, lungs).

[0003] Among viral respiratory diseases, infections associated with RNA viruses have received more attention than those caused by DNA viruses. Five families of RNA viruses that cause upper and lower respiratory tract infections have been identified: Orthomyxoviridae (which includes influenza viruses A, B, C, or D), Paramyxoviridae and Pneumoviridae (which include parainfluenza viruses, respiratory syncytial viruses, and human metapneumoviruses), Picornaviridae (which includes rhinoviruses A, B, and C, respiratory enteroviruses A, B, C, and D), and Coronaviridae (which includes human coronaviruses, severe acute respiratory syndrome coronaviruses, and Middle East respiratory syndrome coronaviruses).

[0004] The Coronaviridae family is a very important family of RNA viruses that cause respiratory infections such as the epidemic caused by SARS-CoV (severe acute respiratory syndrome coronavirus), MERS-CoV (Middle East respiratory syndrome coronavirus), and the SARS-CoV-2 pandemic (severe acute respiratory syndrome coronavirus 2) that began in 2020 and is still ongoing.

[0005] Coronaviruses are known to primarily target the human respiratory system, as previously observed with SARS-CoV and MERS-CoV. Phylogenetic and computational genomic analyses suggest that SARS-CoV-2 shares the same human cellular receptor (angiotensin-converting enzyme 2, ACE2) as SARS-CoV to enter host cells. Specifically, when an organism is infected with a virus belonging to the Coronaviridae family, such as SARS-CoV-2, the viral spike protein binds to the ACE2 receptor (a homolog of angiotensin-converting enzyme (ACE)) present on the organism's cell surface.

[0006] The spike protein (S protein, SPIKE) is a type I membrane glycoprotein that facilitates the attachment of viruses belonging to the coronavirus family to cellular receptors and subsequent entry into host cells. The protein consists of a short cellular tail, a transmembrane anchor, and a large ectodomain. The ectodomain contains two subunits, S1 (the N-terminal surface unit) and S2 (the C-terminal transmembrane unit). The distal S1 subunit contains the receptor-binding domain (RBD) responsible for binding to the host cell receptor and contributes to stabilizing the prefusion state of the membrane-anchored S2 subunit, which contains the functional elements required for membrane fusion.

[0007] ACE2 is a homolog of angiotensin-converting enzyme (ACE) and is mainly expressed on type II pneumocytes in the lungs and weakly on the surface of epithelial cells of the oral and nasal mucosa and nasopharynx, indicating that the lungs are the primary target of viruses belonging to the coronavirus family, such as SARS-CoV-2. Thus, the binding of the viral spike protein to the ACE2 receptor is fundamental for the initiation of viral infection of the airways.

[0008] The most effective way to control this type of infectious disease is probably vaccination, which is a type of prophylactic drug that serves to immunize the population against viral infections, such as the current SARS-CoV-2 infection. However, there are not always vaccines available for all types of viral respiratory tract infections, and they can be difficult to obtain or very expensive.

[0009] Once an infection has been contracted, there are certain treatments that can be administered, such as antiviral drugs, which are known in the art, however, these treatments are not always as effective as desired and may have significant side effects.

[0010] Therefore, a need exists for new products for the prevention, prevention, or amelioration of viral respiratory infections.

[0011] Following intensive research and development activity carried out by the Applicant, the present invention addresses and solves the technical problem described herein by providing specific and selected bacterial strains (e.g. probiotic bacterial strains or derivatives thereof, parabiotics or postbiotics), mixtures and compositions comprising at least one of said bacterial strains (probiotics or parabiotics or postbiotics thereof) for use in a prophylactic method of treating or inhibiting or otherwise alleviating said respiratory infections of a viral nature.

[0012] In particular, the applicant has surprisingly found that the bacterial strains described herein and compositions comprising them may be effectively used in the prophylactic treatment of viral respiratory tract infections.

[0013] Furthermore, the Applicant has surprisingly found that since the formation of a bond between the viral spike protein and the ACE2 receptor is fundamental for the initiation of viral respiratory tract infections, the bacterial strains described herein and compositions comprising them may be effectively used to treat viral respiratory tract infections at an early stage of infection.

[0014] Furthermore, the applicant has surprisingly found that the bacterial strains described herein and compositions comprising them may be effectively used in combination with anti-viral treatments.

[0015] Without wishing to be bound by any particular scientific theory, it is hypothesized that this positive effect may also result from the fact that probiotic bacteria have an effect on the regulation of the intestinal flora and on the immune response, particularly those occurring in the upper / lower respiratory tract, and thus may improve the host response, particularly against respiratory viral infections.

[0016] Particular bacterial strains (e.g., probiotic bacterial strains or derivatives thereof) and compositions thereof according to the present invention can advantageously significantly inhibit and / or reduce the binding between ACE2 and the RBD (receptor binding domain) of the spike protein.

[0017] The bacterial strains of the invention, as well as the mixtures and compositions of the invention comprising said bacterial strains, exhibit a high safety profile, can be used in all categories of subjects, are easy to prepare and are cost-effective.

[0018] These objectives, as well as others that will become apparent from the following detailed description, are achieved by the bacterial strains, mixtures and compositions of the present invention, thanks to the technical features present in the present specification and the appended claims. Summary of the Invention

[0019] It is an object of the present invention to provide a bacterial strain for use in a method of treatment to prevent or inhibit or alleviate respiratory viral infections and associated diseases or symptoms in a subject in need thereof, wherein the bacterial strain is selected from the group comprising or consisting of: (i) a bacterial strain belonging to the genus Lactobacillus brevis selected from the group consisting of or including the bacterial strain identified as Lactobacillus brevis LBR01 (DSM 23034); (ii) a bacterial strain belonging to the genus Lactobacillus crispatus selected from the group comprising or consisting of Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus crispatus LCR04 (DSM 33487), and mixtures thereof; (iii) a bacterial strain belonging to the genus Limosilactobacillus fermentum (formerly Lactobacillus fermentum) selected from the group consisting of or comprising Limosilactobacillus fermentum (formerly Lactobacillus fermentum) LF10 (DSM 19187), Limosilactobacillus fermentum (formerly Lactobacillus fermentum) LF15 (DSM 26955), and mixtures thereof; (iv) a bacterial strain belonging to the genus Lactobacillus gasseri selected from the group consisting of or including the bacterial strain identified as Lactobacillus gasseri LGS01 (DSM 18299); (v) Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP01 (LMG P-21021), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP02 (LMG P-21020), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP09 (DSM 25710), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP14 (DSM 33401), and mixtures thereof, or a bacterial strain belonging to the species Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) selected from the group consisting of: (vi) Limosilactobacillus reuteri (formerly Lactobacillus reuteri) LRE02 (DSM 23878), Limosilactobacillus reuteri (formerly Lactobacillus reuteri) LRE03 (DSM 23879), Limosilactobacillus reuteri (formerly Lactobacillus reuteri) LRE11 (DSM 33827), and mixtures thereof, or selected from the group consisting of: reuteri) species; (vii) a bacterial strain belonging to the genus Lactobacillus salivarius subspecies salivarius selected from the group consisting of: a bacterial strain identified as Lactobacillus salivarius subspecies salivarius (DSM 22776); (viii) a bacterial strain belonging to the species Bifodobacterium adolescentis selected from the group consisting of Bifodobacterium adolescentis BA02 (EI-15) (DSM 18351), and mixtures thereof; (ix) a bacterial strain belonging to the genus Bifidobacterium bifidum selected from the group consisting of Bifidobacterium bifidum BB01 (DSM 22892), Bifidobacterium bifidum BB10 (DSM 33678), and mixtures thereof; (x) a bacterial strain belonging to the genus Bifidobacterium breve selected from the group consisting of Bifidobacterium breve BR03 (DSM 16604), Bifidobacterium breve B632 (DSM 24706), and mixtures thereof; (xi) a bacterial strain belonging to the genus Bifidobacterium infantis selected from the group consisting of Bifidobacterium infantis BI02 (DSM 24687); (xii) a bacterial strain belonging to the genus Bifidobacterium lactis selected from the group consisting of Bifidobacterium lactis BS01 (LMG P-21384); (xiii) Bifidobacterium longum selected from the group consisting of Bifidobacterium longum BL03 (DSM 16603), Bifidobacterium longum BL04 (DSM 23233), Bifidobacterium longum DLBL07 (DSM 25669), Bifidobacterium longum DLBL08 (DSM 25670), Bifidobacterium longum DLBL 10 (DSM 25672), Bifidobacterium longum DLBL 11 (DSM 25673) and mixtures thereof. Bacterial strains belonging to the genus Bifidobacterium longum; A bacterial strain selected from the group comprising or consisting of:

[0020] The object of the present invention is also a mixture for use in a subject in need thereof in a method of treatment for preventing, inhibiting or alleviating respiratory viral infections and associated diseases or symptoms, having the characteristics as defined in the appended claims.

[0021] An object of the present invention is to provide a composition for use in a method of treatment for preventing or inhibiting respiratory viral infections and associated diseases or symptoms in a subject, comprising: - a bacterial strain as claimed in the accompanying claims or a mixture of bacterial strains as claimed in the accompanying claims, - food grade or pharma- ceutically acceptable additives or excipients; It is also a composition comprising:

[0022] definition In the context of the present invention, the terms "respiratory tract" and "respiratory apparatus" refer to the set of organs and tissues dedicated to the breathing process. The main anatomical elements of the respiratory apparatus are the nose and its cavities, the mouth, the pharynx, the nasopharynx, the larynx, the trachea, the bronchi, the bronchioles, the lungs, the diaphragm and the intercostal respiratory muscles.

[0023] In the context of the present invention, the term "respiratory tract infections" refers to infectious diseases affecting the respiratory tract (nose, trachea, bronchi and lungs).

[0024] In the context of the present invention, the term "coronavirus" refers to the Coronaviridae family; the Coronavirinae subfamily; the Betacoronavirus genus; and a coronavirus species selected from the following strains: (I) Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) (isolated and first identified in 2002), (II) Severe Acute Respiratory Syndrome coronavirus-2 (SARS-CoV-2) (isolated and first identified in 2019), and (III) Severe Acute Respiratory Syndrome coronavirus-like (SARS-CoV-like) (Severe Acute Respiratory Syndrome coronavirus or Severe Acute Respiratory Syndrome-related coronavirus (in short SARSr-CoV or SARS-coronavirus or coronavirus)). Detailed Description of the Invention

[0025] An object of the present invention is to provide a bacterial strain for use in a method of treatment to prevent, inhibit or alleviate respiratory viral infections and associated diseases or symptoms in a subject in need thereof, the bacterial strain being selected from the group comprising or consisting of: (i) a bacterial strain belonging to the species Lactobacillus brevis, preferably a bacterial strain identified as Lactobacillus brevis LBR01 (DSM 23034). (ii) a bacterial strain belonging to the genus Lactobacillus crispatus, preferably selected from the group consisting of Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus crispatus LCR04 (DSM 33487), and mixtures thereof. (iii) A bacterial strain belonging to the genus Lactobacillus fermentum (formerly Lactobacillus fermentum), preferably selected from the group consisting of Lactobacillus fermentum LF10 (DSM19187), Lactobacillus fermentum LF15 (DSM 26955), and mixtures thereof. (iv) a bacterial strain belonging to the genus Lactobacillus gasseri, preferably a bacterial strain identified as Lactobacillus gasseri LGS01 (DSM 18299). (v) a bacterial strain belonging to the genus Lactiplantibacillus plantarum (formerly Lactobacillus plantarum), preferably selected from the group consisting of LP01 (LMG P-21021), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP02 (LMG P-21020), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP09 (DSM 25710), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP14 (DSM 33401), and mixtures thereof. (vi) A bacterial strain belonging to the genus Lactobacillus reuteri (formerly Lactobacillus reuteri), preferably selected from the group consisting of Lactobacillus reuteri (formerly Lactobacillus reuteri) LRE02 (DSM 23878), Lactobacillus reuteri (formerly Lactobacillus reuteri) LRE03 (DSM 23879), Lactobacillus reuteri (formerly Lactobacillus reuteri) LRE11 (DSM 33827), and mixtures thereof. (vii) a bacterial strain belonging to the species Lactobacillus salivarius subsp. salivarius, preferably a bacterial strain identified as Lactobacillus salivarius subsp. salivarius LS03 (DSM 22776). (viii) a bacterial strain belonging to the genus Bifidobacterium adolescentis, preferably selected from the group consisting of Bifidobacterium adolescentis BA02 (EI-15) (DSM 18351), Bifidobacterium adolescentis BA05 (EI-18) (DSM 18352), and mixtures thereof. (ix) A bacterial strain belonging to the genus Bifidobacterium bifidum, preferably selected from the group consisting of Bifidobacterium bifidum BB01 (DSM 22892), Bifidobacterium bifidum BB10 (DSM 33678), and mixtures thereof. (x) a bacterial strain belonging to the genus Bifidobacterium breve, preferably selected from the group consisting of Bifidobacterium breve BR03 (DSM 16604), Bifidobacterium breve B632 (DSM 24706), and mixtures thereof. (xi) A bacterial strain belonging to the species Bifidobacterium infantis, preferably the bacterial strain identified as Bifidobacterium infantis BI02 (DSM 24687). (xii) A bacterial strain belonging to the species Bifidobacterium lactis, preferably the bacterial strain identified as Bifidobacterium lactis BS01 (LMG P-21384). (xiii) a bacterial strain belonging to the genus Bifidobacterium longum, preferably selected from the group consisting of Bifidobacterium longum BL03 (DSM 16603), Bifidobacterium longum BL04 (DSM23233), Bifidobacterium longum DLBL07 (DSM 25669), Bifidobacterium longum DLBL08 (DSM 25670), Bifidobacterium longum DLBL 10 (DSM 25672), Bifidobacterium longum DLBL 11 (DSM 25673), and mixtures thereof.

[0026] All bacterial strains referred to in this invention have been deposited under the provisions of the Budapest Treaty. The depositors and owners of the bacterial strains described and / or claimed in this patent application hereby agree to make available all such strains for the entire life of this patent.

[0027] The species nomenclature of bacterial strains belonging to the genus Lactobacillus has been revised after the reclassification of the genus as reported in the paper by Zheng et al., Int. J. Syst. Evol. Microbiol., 70(4):2782-2858 (2020). In the context of the present invention, the species nomenclature adopted after the reclassification is adopted, and the name according to the old classification is indicated in parentheses.

[0028] The following are deposit details of the bacterial strains used in this context:

[0029] Lactobacillus brevis LBR01 It was deposited under the provisions of the Budapest Treaty by the Probiotic Società per Azioni on October 13, 2009 with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 23034.

[0030] Lactobacillus crispatus LCR01 It was deposited under the provisions of the Budapest Treaty by the Probiotic Società per Atzioni on March 2, 2011 with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 24619.

[0031] Lactobacillus crispatus LCR04 It was deposited in accordance with the provisions of the Budapest Treaty with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 33487 on April 2, 2020 by the Probiotic Società per Azioni.

[0032] Rimosil Lactobacillus Fermentum (formerly Lactobacillus Fermentum) LF10 It was deposited in accordance with the provisions of the Budapest Treaty with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 19187 on 20 March 2007 by AniDral Esse Ehre Ehre (now Probiotica Società per Atzioni).

[0033] Rimosil Lactobacillus fermentum (formerly Lactobacillus fermentum) LF15 It was deposited under the provisions of the Budapest Treaty by the Probiotic Società per Azioni on March 1, 2013 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 26955.

[0034] Lactobacillus gasseri LGS01 It was deposited in accordance with the provisions of the Budapest Treaty with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 18299 on May 24, 2006 by AniDral Esse Ehre Ehre (now Probiotica Società per Azioni).

[0035] Lactiprantibacillus plantarum (formerly Lactobacillus plantarum) LP01 It was deposited in accordance with the provisions of the Budapest Treaty at the Belgian Coordinated Collections of Microorganisms (BCCM) Laboratorium voor Microbiologie - Bacterienverzameling (LMG) on 16 October 2001 by Mofin Esse-Ehle-Ehle under the deposit number LMG P-21021.

[0036] Lactiprantibacillus plantarum (formerly Lactobacillus plantarum) LP02 It was deposited in accordance with the provisions of the Budapest Treaty at the Belgian Coordinated Collections of Microorganisms (BCCM) Laboratorium voor Microbiologie - Bacterienverzameling (LMG) on 16 October 2001 by Mofin Esse-Ehle-Ehle under the deposit number LMG P-21020.

[0037] Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP09 It was deposited under the provisions of the Budapest Treaty by the Probiotic Società per Azioni on 24 February 2012 with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 25710.

[0038] Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP14 It was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under the deposit number DSM 33401 on January 16, 2020 by the Probiotic Società per Azioni in accordance with the provisions of the Budapest Treaty.

[0039] Rimosil Lactobacillus reuteri (previously Lactobacillus reuteri) LRE02 It was deposited under the provisions of the Budapest Treaty by the Probiotic Società per Azioni on August 5, 2010 with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 23878.

[0040] Rimosil Lactobacillus reuteri (previously Lactobacillus reuteri) LRE03 It was deposited under the provisions of the Budapest Treaty by the Probiotic Società per Atzioni on August 5, 2010 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 23879.

[0041] Rimosil Lactobacillus reuteri (formerly Lactobacillus reuteri) LRE11 It was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 33827 on 15 February 2021 by the Probiotic Società per Azioni in accordance with the provisions of the Budapest Treaty.

[0042] Lactobacillus salivarius subsp. salivarius LS03 It was deposited under the provisions of the Budapest Treaty by the Probiotic Società per Azioni on July 23, 2009 with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 22776.

[0043] Bifidobacterium adolescentis BA02 (EI-15) It was deposited in accordance with the provisions of the Budapest Treaty with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 18351 on 15 June 2006 by AniDral Esse Ehre Ehre (now Probiotica Società per Atzioni).

[0044] Bifidobacterium adolescentis BA05 (EI-18) It was deposited in accordance with the provisions of the Budapest Treaty with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 18352 on 15 June 2006 by AniDral Esse Ehre Ehre (now Probiotica Società per Atzioni).

[0045] Bifidobacterium bifidum BB01 (DSM 22892) It was deposited in accordance with the provisions of the Budapest Treaty with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under the deposit number DSM 19818 on 30 October 2007 by AniDral Esse Ehre Ehre (now Probiotica Società per Atzioni).

[0046] Bifidobacterium bifidum BB10 It was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under the deposit number DSM 33678 on 22 October 2020 by the Probiotic Società per Azioni in accordance with the provisions of the Budapest Treaty.

[0047] Bifidobacterium breve BR03 It was deposited in accordance with the provisions of the Budapest Treaty with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 16604 on 20 July 2004 by AniDral Esse Ehre Ehre (now Probiotica Società per Atzioni).

[0048] Bifidobacterium breve B632 It was deposited under the provisions of the Budapest Treaty by the Probiotic Società per Azioni on April 7, 2011 with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 24706.

[0049] Bifidobacterium infantis BI02 It was deposited under the provisions of the Budapest Treaty by the Probiotic Società per Azioni on March 29, 2011 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 24687.

[0050] Bifidobacterium lactis BS01 It was deposited in accordance with the provisions of the Budapest Treaty by the Anidral Esse Ehre Ehre (now Probiotica Società per Azioni) at the Belgian Coordinated Collections of Microorganisms (BCCM) Laboratorium voor Microbiologie - Bacterienverzameling (LMG) on 31 January 2002 under the deposit number LMG P-21384.

[0051] Bifidobacterium longum BL03 It was deposited in accordance with the provisions of the Budapest Treaty with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 16603 on 20 July 2004 by AniDral Esse Ehre Ehre (now Probiotica Società per Atzioni).

[0052] Bifidobacterium longum BL04 It was deposited under the provisions of the Budapest Treaty by the Probiotic Società per Azioni on January 12, 2010 with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 23233.

[0053] Bifidobacterium longum DLBL07 It was deposited under the provisions of the Budapest Treaty by the Probiotic Società per Azioni on 16 February 2012 with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 25669.

[0054] Bifidobacterium longum DLBL08 It was deposited under the provisions of the Budapest Treaty by the Probiotic Società per Azioni on 16 February 2012 with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 25670.

[0055] Bifidobacterium longum DLBL 10 It was deposited under the provisions of the Budapest Treaty by the Probiotic Società per Azioni on 16 February 2012 with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 25672.

[0056] Bifidobacterium longum DLBL 11 It was deposited under the provisions of the Budapest Treaty by the Probiotic Società per Azioni on 16 February 2012 with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 25673.

[0057] The strains used in this context, or mixtures thereof, may be viable probiotic bacterial strains, or derivatives thereof such as parabiotic or postbiotic. Preferably, the bacterial strains used in this context are probiotic / viable / live bacterial strains, or cell extracts or supernatants of said bacterial strains.

[0058] In the context of the present invention, the term "probiotic" refers to live, viable bacterial strains that, when administered in appropriate amounts, confer a health benefit to the host (or the official FAO and WHO 2002 definition).

[0059] In the context of the present invention, the term "derivative" of a viable probiotic bacterial strain means a paraprobiotic, or postbiotic, or any other derivative of the bacterial strain that has the ability to provide a benefit (similar to the viable bacteria from which they are derived) to the organism to which they are administered.

[0060] In the context of the present invention, the term "paraprobiotics" refers to non-viable (i.e., replicatively incompetent) bacterial cells (intact or not), or cell extracts, e.g., crude cell extracts, which, when administered in appropriate amounts, provide a health benefit to the host (similar to the viable bacterial strains from which they are derived). Examples of paraprobiotics include bacterial strains inactivated by heat (e.g., intermittently sterilized bacterial strains), sonication (ultrasound), gamma irradiation (gamma rays), or lysates of bacterial strains or cell extracts of bacterial strains.

[0061] In the context of the present invention, the term "postbiotics" refers to any substance released or produced through the metabolic and catabolic activity of a viable probiotic bacterial strain, which, when administered in appropriate amounts, provides a health benefit to the host (similar to the viable bacterial strain from which they are derived). Examples of postbiotics are exopolysaccharides, wall fractions, metabolic products or metabolic bioproducts, or supernatants obtained from the bacterial strain.

[0062] The term "subject" in the context of the present invention means mammals (animals and humans), preferably human subjects.

[0063] The term "therapeutically effective amount" refers to that amount of a mixture or compound or formulation that elicits a biological or medicinal response in a tissue, system or subject as sought and defined by experts in the field.

[0064] The bacterial strains described in this context act through preventive antiviral effects and restoration of the intestinal microbiome.

[0065] Furthermore, the bacterial strains described herein, as well as mixtures and compositions comprising them, are for use in the therapeutic treatment of viral infections of the respiratory tract at an early stage of infection, preferably between days 1 and 14 after viral infection, preferably between days 1 and 10 after viral infection.

[0066] Furthermore, the applicant has surprisingly found that the bacterial strains described herein and compositions comprising them may be effectively used in combination with anti-viral treatments.

[0067] Furthermore, the bacterial strains described herein are readily available, easy to administer (orally), safe, and inexpensive compared to antiviral and immunomodulatory drugs.

[0068] The strains, mixtures and compositions described in the present invention are for use in prophylactic treatment methods, i.e. making it possible to limit / reduce the risk of contracting a viral infection by preventing it or by reducing the likelihood of contracting it.

[0069] The strains, mixtures and compositions described in the present invention are for use in therapeutic treatment methods and are intended as a treatment method that can limit / reduce the spread of viral infection by reducing / alleviating early symptoms at an early stage after viral infection, preferably within 14 days after infection.

[0070] Preferably, the mixtures described herein are for use in a method of prophylactic treatment, or inhibition or alleviation of viral infection of the respiratory tract by a coronavirus, preferably the SARS-CoV-2 virus, and associated diseases or symptoms in a subject in need thereof.

[0071] Preferably, the compositions described herein are for use in a method of prophylactic treatment, or in inhibiting or alleviating viral infection of the respiratory tract by a coronavirus, preferably the SARS-CoV-2 virus, and associated diseases or symptoms in a subject in need thereof.

[0072] The strains, mixtures and compositions described in the present invention are for use in adjunctive treatment methods that may be administered in combination with antiviral therapy to enhance its effectiveness and help strengthen the health of the immune system and microbiota of subjects suffering from viral respiratory tract infections.

[0073] An object of the present invention is to provide a bacterial strain for use in a method of treatment to prevent, inhibit or alleviate respiratory viral infections and associated diseases or symptoms in a subject in need thereof, the bacterial strain being selected from the group comprising or consisting of: Lactobacillus brevis LBR01 (DSM 23034) Lactobacillus crispatus LCR01 (DSM 24619) Lactobacillus crispatus LCR04 (DSM 33487) Limosil Lactobacillus fermentum (formerly Lactobacillus fermentum) LF10 (DSM19187) Limosil Lactobacillus fermentum (formerly Lactobacillus fermentum) LF15 (DSM 26955) Lactobacillus gasseri LGS01 (DSM 18299) Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP01 (LMG P-21021) Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP02 (LMG P-21020) Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP09 (DSM 25710) Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP14 (DSM 33401) Rimosil Lactobacillus reuteri (formerly Lactobacillus reuteri) LRE02 (DSM 23878) Rimosil Lactobacillus reuteri (formerly Lactobacillus reuteri) LRE03 (DSM 23879) Rimosil Lactobacillus reuteri (formerly Lactobacillus reuteri) LRE11 (DSM 33827) Lactobacillus salivarius subsp. salivarius LS03 (DSM 22776) Bifidobacterium adolescentis BA02 (EI-15) (DSM 18351) Bifidobacterium bifidum BB01 (DSM 22892) Bifidobacterium bifidum BB10 (DSM 33678) Bifidobacterium breve BR03 (DSM 16604) Bifidobacterium breve B632 (DSM 24706) Bifidobacterium infantis BI02 (DSM 24687) Bifidobacterium lactis BS01 (LMG P-21384) Bifidobacterium longum BL03 (DSM 16603) Bifidobacterium longum BL04 (DSM23233) Bifidobacterium longum DLBL07 (DSM 25669) Bifidobacterium longum DLBL08 (DSM 25670) Bifidobacterium longum DLBL 10 (DSM 25672) Bifidobacterium longum DLBL 11 (DSM 25673) and mixtures thereof.

[0074] Preferably, the bacterial strain is for use in a prophylactic treatment method for preventing or inhibiting or alleviating viral respiratory tract infections and associated diseases or symptoms in a subject in need thereof, the bacterial strain being selected from the group comprising or consisting of: Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus crispatus LCR04 (DSM 33487), rimosyllactobacillus fermentum (formerly Lactobacillus fermentum) LF15 (DSM 26955), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP01 (LMG P-21021), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP02 (LMG P-21020), Lactobacillus salivarius subsp. salivarius LS03 (DSM 22776), Bifidobacterium bifidum BB10 (DSM 33678), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP09 (DSM 25710), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP14 (DSM 33401), Bifidobacterium longum BL03 (DSM 16603), Lactobacillus gasseri LGS01 (DSM 18299) and mixtures thereof.

[0075] Preferably, the bacterial strain is selected from the group comprising or consisting of: Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus crispatus LCR04 (DSM 33487), Rimosilactobacillus fermentum (formerly Lactobacillus fermentum) LF15 (DSM 26955), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP01 (LMG P-21021), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP02 (LMG P-21020), Lactobacillus salivarius subsp. salivarius LS03 (DSM 22776), Bifidobacterium bifidum BB10 (DSM 33678), and mixtures thereof.

[0076] Alternatively, the bacterial strain is selected from the group comprising or consisting of: Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus crispatus LCR04 (DSM 33487), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP01 (LMG P-21021), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP02 (LMG P-21020), Lactobacillus salivarius subsp. salivarius LS03 (DSM 22776), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP09 (DSM 25710), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP14 (DSM 33401), Bifidobacterium longum BL03 (DSM 16603), Lactobacillus gasseri LGS01 (DSM 18299), and mixtures thereof.

[0077] Advantageously, the bacterial strains described herein may be in the form of viable / probiotic strains or cell extracts or supernatants obtainable from said bacterial strains.

[0078] The present invention further relates to a supernatant obtainable from a bacterial strain according to any one of the embodiments described herein, preferably the supernatant is obtainable from a bacterial strain selected from the group comprising or consisting of: Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus crispatus LCR04 (DSM 33487), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP01 (LMG P-21021), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP02 (LMG P-21020), Lactobacillus salivarius subsp. salivarius LS03 (DSM 22776), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP09 (DSM 25710), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP14 (DSM 33401), Bifidobacterium longum BL03 (DSM 16603), Lactobacillus gasseri LGS01 (DSM 18299), and mixtures thereof.

[0079] In one embodiment the bacterial strain is in the form of a live / probiotic strain and is preferably selected from the group comprising or consisting of: Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus crispatus LCR04 (DSM 33487), Rimosilactobacillus fermentum (formerly Lactobacillus fermentum) LF15 (DSM 26955), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP01 (LMG P-21021), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP02 (LMG P-21020), Lactobacillus salivarius subsp. salivarius LS03 (DSM 22776), Bifidobacterium bifidum BB10 (DSM 33678), and mixtures thereof.

[0080] In one embodiment the bacterial strain for use according to the present invention is selected from the group comprising or consisting of: (ii) a bacterial strain belonging to the Lactobacillus crispatus species selected from the group consisting of: Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus crispatus LCR04 (DSM 33487), and mixtures thereof. (iii) A bacterial strain belonging to the genus Rimosilactobacillus fermentum (formerly Lactobacillus fermentum) selected from the group consisting of Rimosilactobacillus fermentum (formerly Lactobacillus fermentum) LF15 (DSM 26955), and mixtures thereof. (v) A bacterial strain belonging to the genus Lactobacillus reuteri (formerly Lactobacillus reuteri) selected from the group consisting of Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP01 (LMG P-21021), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP02 (LMG P-21020), and mixtures thereof. (vii) a bacterial strain belonging to the species Lactobacillus salivarius subsp. salivarius selected from the group consisting of a bacterial strain identified as Lactobacillus salivarius subsp. salivarius LS03 (DSM 22776). (ix) A bacterial strain belonging to the species Bifidobacterium bifidum selected from the group consisting of: Bifidobacterium bifidum BB01 (DSM 22892), Bifidobacterium bifidum BB10 (DSM 33678), and mixtures thereof.

[0081] An object of the present invention is also a mixture comprising or consisting of at least one bacterial strain described herein for use in a method for the treatment of respiratory viral infections and associated diseases or symptoms in a subject in need thereof.

[0082] The mixture of the present invention preferably comprises at least two bacterial strains, or at least three bacterial strains, or at least four bacterial strains, or at least five bacterial strains selected from the group comprising or consisting of: Lactobacillus brevis LBR01 (DSM 23034) Lactobacillus crispatus LCR01 (DSM 24619) Lactobacillus crispatus LCR04 (DSM 33487) Limosil Lactobacillus fermentum (formerly Lactobacillus fermentum) LF10 (DSM19187) Limosil Lactobacillus fermentum (formerly Lactobacillus fermentum) LF15 (DSM 26955) Lactobacillus gasseri LGS01 (DSM 18299) Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP01 (LMG P-21021) Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP02 (LMG P-21020) Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP09 (DSM 25710) Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP14 (DSM 33401) Rimosil Lactobacillus reuteri (formerly Lactobacillus reuteri) LRE02 (DSM 23878) Rimosil Lactobacillus reuteri (formerly Lactobacillus reuteri) LRE03 (DSM 23879) Rimosil Lactobacillus reuteri (formerly Lactobacillus reuteri) LRE11 (DSM 33827) Lactobacillus salivarius subsp. salivarius LS03 (DSM 22776) Bifidobacterium adolescentis BA02 (EI-15) (DSM 18351) Bifidobacterium bifidum BB01 (DSM 22892) Bifidobacterium bifidum BB10 (DSM 33678) Bifidobacterium breve BR03 (DSM 16604) Bifidobacterium breve B632 (DSM 24706) Bifidobacterium infantis BI02 (DSM 24687) Bifidobacterium lactis BS01 (LMG P-21384) Bifidobacterium longum BL03 (DSM 16603) Bifidobacterium longum BL04 (DSM23233) Bifidobacterium longum DLBL07 (DSM 25669) Bifidobacterium longum DLBL08 (DSM 25670) Bifidobacterium longum DLBL 10 (DSM 25672) Bifidobacterium longum DLBL 11 (DSM 25673)

[0083] Preferably, the mixture comprises at least two strains selected from the group comprising or consisting of: Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus crispatus LCR04 (DSM 33487), Limosilactobacillus fermentum (formerly Lactobacillus fermentum) LF15 (DSM 26955), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP01 (LMG P-21021), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP02 (LMG P-21020), Lactobacillus salivarius subsp. salivarius LS03 (DSM 22776), Bifidobacterium bifidum BB10 (DSM 33678), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP09 (DSM 25710), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP14 (DSM 33401), Bifidobacterium longum BL03 (DSM 16603), Lactobacillus gasseri LGS01 (DSM 18299).

[0084] In one embodiment the mixture comprises: Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus crispatus LCR04 (DSM 33487), Limosilactobacillus fermentum (formerly Lactobacillus fermentum) LF15 (DSM 26955), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP01 (LMG P-21021), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP02 (LMG P-21020), Lactobacillus salivarius subsp. salivarius LS03 (DSM 22776), Bifidobacterium bifidum BB10 (DSM 33678), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP09 (DSM 25710), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP14 (DSM 33401), Bifidobacterium longum BL03 (DSM 16603), Lactobacillus gasseri LGS01 (DSM 18299).

[0085] The mixtures described in this context may also comprise at least one prebiotic ingredient or prebiotic fibre, preferably selected from the group comprising or consisting of inulin, fructooligosaccharides (FOS), galactooligosaccharides (GOS), guar gum and mixtures thereof, and preferably inulin.

[0086] The object of the present invention is also a composition comprising at least one bacterial strain as described herein, or a mixture of bacterial strains as described herein, and additives and / or excipients of pharma- ceutical or food acceptable grade, which composition may be advantageously used in a method of prophylactic treatment, or inhibition, or reduction of respiratory viral infections and associated pathologies or symptoms in a subject in need thereof.

[0087] In the context of the present invention, a "composition" means a composition which may be a pharmaceutical composition (or Live Biotherapeutic Product), a medical device composition (e.g. Medical Device Regulation (EU) 2017 / 745 (MDR)), a food supplement and / or a food for special medical purposes (FSMP), or a cosmetic composition.

[0088] The at least one bacterial strain is present in the mixture M or composition of the invention at a concentration of 10x10 6 CFU to 10x10 12 CFU (e.g., 10x10 7 CFU or 10x10 8 CFU or 10x10 9 CFU or 10x10 10 CFU), preferably about 1x10 9 CFU to 10x10 9 They can be present in concentrations ranging from CFU (CFU: colony forming units).

[0089] When the mixture M or composition of the invention contains two or more bacterial strains of the bacterial strain of the invention, said two, three, four, five or six bacterial strains may be in a ratio expressed in CFU of about 1:1, or about 1:1:1, or about 1:1:1:1, or about 1:1:1:1:1, or about 1:1:1:1:1:1:1 (CFU: colony forming units).

[0090] To assess the number of viable bacteria in a composition or mixture M of the present invention, said composition or mixture M may be analyzed, for example, by flow cytometry to determine the AFU value and / or by plate counting to determine the CFU value.

[0091] The dosage units described above may be administered to a subject in need thereof once, twice, three or four times daily, preferably twice.

[0092] The compositions of the invention comprising at least one bacterial strain of the invention or comprising a mixture of bacterial strains according to any one of the described embodiments may be formulated for oral use (gastrointestinal or sublingual), for nasal inhalation (e.g., spray or drops), for oral inhalation (e.g., spray, dry powder for inhalation), or for topical use (e.g., skin, rectum, vagina or eye). Preferably, the compositions or mixtures of the invention are for oral use.

[0093] The compositions of the present invention may be formulated for oral use in a solid form, e.g. selected from tablets, chewable tablets, orosoluble tablets, capsules, granules, flakes, powders, soluble powders or granules (e.g. packaged in a sachet), orosoluble powders or granules (e.g. packaged in an orosoluble stick); or in a liquid form, e.g. selected from solutions, suspensions, emulsions, syrups, e.g. packaged in a drinkable vial, dispensable liquid in spray form; or in a semi-liquid form, e.g. selected from soft gels, gels, or creams; preferably, the compositions or mixtures of the present invention are for oral use in solid form, more preferably in soluble or water-soluble powder form (e.g. in water or an aqueous liquid).

[0094] Said at least one additive and / or excipient of pharmaceutical or food grade contained in the composition of the invention together with the mixture M comprising at least one probiotic bacterial strain or derivative thereof of the invention is a therapeutically inactive substance suitable for pharmaceutical or food grade use selected from auxiliary substances known to the skilled person, such as, for example, diluents, solvents, solubilizers, thickeners, sweeteners, flavorings, colorants, lubricants, surfactants, antimicrobial agents, antioxidants, preservatives, pH stabilizing buffers and mixtures thereof. EXAMPLES

[0095] method The bacterial strains of the invention used in the following exemplary experimental part of the invention are listed in Table 1.

[0096] All bacterial strains referred to in this invention have been deposited in accordance with the provisions of the Budapest Treaty. The depositor (Probiotica Sochieta per Atzioni) of the bacterial strains described and / or claimed in this patent application and its owner (Probiotica Sochieta per Atzioni) hereby agree to make available all such bacterial strains for the entire term of this patent.

[0097] [Table 1]

[0098] Probiotic strains were stored in 20% glycerol at −80°C before use.

[0099] The strains were thawed and cultured overnight at 37°C in de Man, Rogosa and Sharpe medium (MRS).

[0100] Because Bifidobacteria are more sensitive to oxygen concentrations than Lactobacilli, MRS was enriched with 1% by volume of cysteine ​​solution and thawed the following morning, and this fresh culture was used for the experiments in this context.

[0101] The efficacy in treating respiratory tract infections was tested not only on live bacteria, but also on thawed and frozen bacterial strains (cell extracts) and supernatants, and the results showed that not only live bacterial strains, but also supernatants and cell extracts were effective against viral respiratory tract infections.

[0102] Preparation of supernatants and extracts from bacterial strains

[0103] Bacterial strains that had been passaged 3–4 times after thawing were used in the experiments. All bacterial strains were 1 × 10 9 The amount of active fluorescent units (AFU) was verified to be greater than or equal to ml and counted by flow cytometry.

[0104] To prepare the supernatant, 5 ml of the bacterial culture was taken and centrifuged at 4000 rpm for 15 minutes. After centrifugation, the supernatant was collected and stored in a refrigerator at 4°C until use.

[0105] A thaw-freeze procedure was performed to obtain cell extracts from 5 ml of bacterial culture. The heat treatment consisted of a heating step in a microwave oven for 20 s at a power of 700 W, followed by a freezing step at -80 °C. This procedure was repeated a total of four times. At the end of the last cycle, the cell extracts thus obtained were counted by flow cytometry to ensure that there were no viable cells in the sample. At this point, each cell extract was read in a spectrophotometer to obtain an optical density (OD) close to 1 and resuspended in Dulbecco's buffered saline (DPBS) for use.

[0106] Inhibitor screening assay To evaluate the efficacy of the bacterial strains according to the invention in the prophylactic treatment of viral respiratory tract infections, the bacterial strains listed in Table 1 were evaluated for their ability to inhibit the binding of viruses belonging to the Coronaviridae family. Results are presented below for the SARS-CoV-2 virus, but similar results were obtained for the other viruses tested.

[0107] To test the ability to inhibit the binding of the SARS-CoV-2 virus to its human receptor, we used the SARS-CoV-2 Inhibitor Screening Assay (AG-48B-0001-KI01) from AdipoGen Life Sciences. This inhibitor screening assay is based on a colorimetric sandwich ELISA. Specifically, the kit measures the binding of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein to the human angiotensin-converting enzyme 2 (ACE2) receptor.

[0108] Measurements were performed five times and three different conditions (in triplicate) were tested: live strain, cell extract, and supernatant.

[0109] Each experimental set included the following experimental points: a negative control (to verify the binding between spike protein and ACE2), a positive inhibitory control (to verify the ability of anti-ACE2 antibodies to block the interaction between ACE2 and the RBD of the spike protein used), an experimental point to verify that the medium used for bacterial growth had no inhibitory ability between RBD and ACE2, and finally an experimental point to verify the inhibitory effect of the viable strain, its supernatant, and its cell extract on RBD-ACE2 binding.

[0110] 1. Preparation of Plates A 96-well plate was coated with 100 μl / well of antigen; in this case, the RBD of the SARS-CoV-2 spike protein (SPIKE) (1 μg / ml). Briefly, lyophilized SPIKE was reconstituted in 100 μl of deionized water to 0.1 mg / ml, and the reconstituted SPIKE was diluted in DPBS to a working concentration of 1 μg / ml. The plate was then covered with plastic film and left in a 4°C refrigerator overnight.

[0111] The next day, the liquid remaining in the coated wells was removed by aspiration and inversion of the plate and blotted onto clean absorbent paper. 200 μl of blocking buffer was then added to block the SPIKE protein on the plate and incubated for 2 hours at room temperature. The coated wells were then aspirated and washed three times with 300 μl of 1X wash buffer. After the final wash, the plate was inverted and blotted onto clean absorbent paper to remove any remaining wash solution.

[0112] At this point, three wells corresponding to the negative control were treated with 200 μl of biotinylated ACE2 (0.5 μg / ml). Briefly, lyophilized ACE2 was reconstituted in 100 μl of deionized water to a concentration of 0.1 mg / ml. It was then diluted and mixed with ELISA buffer 1X to a final concentration of 0.5 μg / ml. In the second negative control well, 100 μl of MRS medium was also added, and in the third negative control well, 100 μl of DPBS was also added. Meanwhile, three wells corresponding to the positive control were treated with 200 μl of anti-ACE2 antibody (human), mAb (blocking) (AC384) (INHIB, 1 μg / ml). INHIB is an antibody that inhibits the ACE2 receptor and prevents its interaction with SPIKE and the enzyme peroxidase. Therefore, no enzymatic reaction should occur in the positive control. INHIB at 1 mg / ml was diluted in ACE2 (0.5 μg / ml) to obtain a final concentration of 1 μg / ml. As was done for the negative control, 100 μl of MRS medium was also added to the second well of the positive control, and 100 μl of DPBS was also added to the third well. In each of the remaining wells, 100 μl / well of bacterial strain (100x10 6 AFU), supernatant, and cell extract according to the present invention (previously prepared), and 100 μl / well of biotinylated ACE2 (0.5 μg / ml) was added.

[0113] Finally, the plates were covered with plastic film and incubated overnight at 37°C in a humidified atmosphere of 5% CO2.

[0114] At the beginning of the third and final day, plates were washed by adding 300 μl of 1X Wash Buffer, and this procedure was repeated three times. After the final wash, residual 1X Wash Buffer was removed by inverting the plates and blotting them onto clean absorbent paper.

[0115] After adding 100 μl of streptavidin-conjugated horseradish peroxidase (HRP), the plate was covered with plastic film and incubated at room temperature for 2 h. HRP can bind to the biotin of ACE2 because the streptavidin bound to HRP has four binding sites with high affinity for biotin.

[0116] After the incubation was completed, three washes and removal of the 1X wash buffer were repeated as above.

[0117] Next, 100 μl / well of K-Blue aqueous tetramethylbenzidine (TMB) was added and incubated for 5 minutes at room temperature. TMB is a 100% solvent-free substrate that contains 3,3',5,5' tetramethylbenzidine and hydrogen peroxide, which develops a deep blue color in the presence of conjugated peroxidase.

[0118] Finally, 50 μl / well of stop solution (H2SO4, 2M) was added to inhibit the enzymatic activity of peroxidase. The addition of sulfuric acid not only inhibited the color development but also converted the blue oxidation product of TMB into a yellow derivative with a significantly higher molar absorbance at 450 nm. The absorbance of this colored solution was quantified spectrophotometrically (λ=450 nm).

[0119] statistical analysis Results are expressed as the mean ± standard deviation of the mean of at least three different independent experiments. Statistical analysis was performed using Student's t test. p < 0.05 was considered significant.

[0120] result The bacterial strains tested modulate the binding of the RBD to the human ACE2 receptor.

[0121] As previously described, we used the "SARS-CoV-2 inhibitor screening assay" to identify and characterize the effect of bacterial strains on inhibiting the RBD binding of the SARS-CoV-2 spike protein to the human ACE2 receptor. Three different conditions were tested: probiotic bacterial strains (live bacteria), their supernatants normalized by cell number, and cell extracts.

[0122] Plates were coated with the RBD domain of the spike protein. Negative control wells were treated with ACE2, which can bind to the RBD. In contrast, positive control wells were treated with ACE2 and an anti-ACE2 human monoclonal antibody to measure the ability of the anti-ACE2 antibody to block the interaction between ACE2 and the RBD. The remaining wells were treated with ACE2 and different bacterial strains.

[0123] The final results were expressed as the percentage inhibition of binding between ACE2 and RBD compared to the negative control (-).

[0124] The positive control gave an inhibition rate of 51.73±11.28%, while the negative control gave 0%.

[0125] Interestingly, all the tested bacterial strains showed different abilities to inhibit the binding of ACE2 to the RBD, as shown in Table 2.

[0126] [Table 2-1] [Table 2-2] [Table 2-3]

[0127] The human monoclonal antibody used as a positive control was commercially available as anti-ACE2 (human) (SARS receptor, angiotensin-converting enzyme 2, ACEH; metalloprotease MPROT15; SARS-Cov-2 receptor) mAb (AC384) and purchased from Adipogen International (product code AG-20A-0037).

[0128] The results show that all the tested bacterial strains were effective. In particular, excellent results were obtained with the LCR01, LCR04, LF15, LP01, LP02, LS03, and BB10 bacterial strains. These values ​​were very similar to those obtained with the positive control, indicating that these bacterial strains can inhibit the binding between ACE2 and the RBD of the spike protein.

[0129] As shown in Table 2, the bacterial strains LCR01, LCR04, LGS01, LP01, LP02, LP09, LP14, LS03 and BL03 are similarly effective.

[0130] Analysis of the results shows that many of the supernatants also exhibited the ability to inhibit the binding of ACE2 receptor to RBD by more than 70% (Table 2).

[0131] The effect of the bacterial strain according to the invention, its supernatants and cell extracts on inhibiting the binding of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein with the human ACE2 receptor, in particular the binding associated with viral entry into the host cell, has been demonstrated.

[0132] Analyzing this result, without wishing to be bound to any particular scientific theory, one can speculate that this inhibitory activity may be due to the release of certain metabolites of the probiotics that may inhibit the binding of RBD to the ACE2 receptor.

[0133] From what has been shown above, it can be concluded that the bacterial strain described in this context is an effective solution for the treatment of viral airway infections, in particular those caused by COVID-19. This type of treatment can be used alone or in combination with newly developed treatments or even to support vaccination programs.

Claims

1. A pharmaceutical composition for use in a method of treatment for preventing or inhibiting, or ameliorating, a respiratory viral infection and associated disease or symptoms, comprising a bacterial strain, the bacterial strain being: (i) a bacterial strain belonging to the genus Lactobacillus brevis selected from the group consisting of or comprising the bacterial strain identified as Lactobacillus brevis LBR01 (DSM 23034); (ii) a bacterial strain belonging to the genus Lactobacillus crispatus selected from the group comprising or consisting of Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus crispatus LCR04 (DSM 33487), and mixtures thereof; (iii) a bacterial strain belonging to the genus Limosilactobacillus fermentum (formerly Lactobacillus fermentum) selected from the group consisting of Limosilactobacillus fermentum (formerly Lactobacillus fermentum) LF10 (DSM 19187), Limosilactobacillus fermentum (formerly Lactobacillus fermentum) LF15 (DSM 26955), and mixtures thereof; (iv) a bacterial strain belonging to the genus Lactobacillus gasseri selected from the group consisting of or including the bacterial strain identified as Lactobacillus gasseri LGS01 (DSM 18299); (v) Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP01 (LMG P-21021), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP02 (LMG P-21020), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP09 (DSM 25710), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP14 (DSM 33401), and mixtures thereof; (vi) Limosilactobacillus reuteri (formerly Lactobacillus reuteri) LRE02 (DSM 23878), Limosilactobacillus reuteri (formerly Lactobacillus reuteri) LRE03 (DSM 23879), Limosilactobacillus reuteri (formerly Lactobacillus reuteri) LRE11 (DSM 33827), and mixtures thereof, reuteri) species; (vii) a bacterial strain belonging to the genus Lactobacillus salivarius sub. salivarius selected from the group consisting of a bacterial strain identified as Lactobacillus salivarius subspecies salivarius (Lactobacillus salivarius sub. salivarius) LS03 (DSM 22776); (viii) a bacterial strain belonging to the species Bifodobacterium adolescentis selected from the group consisting of Bifodobacterium adolescentis BA02 (EI-15) (DSM 18351), and mixtures thereof; (ix) a bacterial strain belonging to the genus Bifidobacterium bifidum selected from the group consisting of Bifidobacterium bifidum BB01 (DSM 22892), Bifidobacterium bifidum BB10 (DSM 33678), and mixtures thereof; (x) a bacterial strain belonging to the genus Bifidobacterium breve selected from the group consisting of Bifidobacterium breve BR03 (DSM 16604), Bifidobacterium breve B632 (DSM 24706), and mixtures thereof; (xi) a bacterial strain belonging to the genus Bifidobacterium infantis selected from the group consisting of Bifidobacterium infantis BI02 (DSM 24687); (xii) a bacterial strain belonging to the genus Bifidobacterium lactis selected from the group consisting of Bifidobacterium lactis BS01 (LMG P-21384); (xiii) Bifidobacterium longum selected from the group consisting of Bifidobacterium longum BL03 (DSM 16603), Bifidobacterium longum BL04 (DSM 23233), Bifidobacterium longum DLBL07 (DSM 25669), Bifidobacterium longum DLBL08 (DSM 25670), Bifidobacterium longum DLBL 10 (DSM 25672), Bifidobacterium longum DLBL 11 (DSM 25673) and mixtures thereof. Bacterial strains belonging to the genus Bifidobacterium longum; A pharmaceutical composition selected from the group comprising or consisting of:

2. 2. The pharmaceutical composition according to claim 1, wherein the bacterial strain is a live or killed or intermittently killed bacteria, preferably a viable probiotic bacterial strain, or a cell extract or supernatant of said viable bacterial strain.

3. Bacterial strains (i) a bacterial strain belonging to the Lactobacillus brevis species selected from the group consisting of or comprising the bacterial strain identified as Lactobacillus brevis LBR01 (DSM 23034); (ii) a bacterial strain belonging to the Lactobacillus crispatus species selected from the group consisting of Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus crispatus LCR04 (DSM 33487), and mixtures thereof; (iii) a bacterial strain belonging to the genus Rimosilactobacillus fermentum (formerly Rimosilactobacillus fermentum) selected from the group consisting of Rimosilactobacillus fermentum (formerly Rimosilactobacillus fermentum) LF10 (DSM 19187), Rimosilactobacillus fermentum (formerly Rimosilactobacillus fermentum) LF15 (DSM 26955), and mixtures thereof; (iv) a bacterial strain belonging to the Lactobacillus gasseri species selected from the group consisting of or comprising the bacterial strain identified as Lactobacillus gasseri LGS01 (DSM 18299); (v) a bacterial strain belonging to the genus Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) selected from the group consisting of Lactobacillus plantarum LP01 (LMG P-21021), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP02 (LMG P-21020), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP09 (DSM 25710), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP14 (DSM 33401), and mixtures thereof; (vii) a bacterial strain belonging to the species Lactobacillus salivarius subsp. salivarius selected from the group consisting of the bacterial strain identified as Lactobacillus salivarius subsp. salivarius LS03 (DSM 22776); (ix) a bacterial strain belonging to the species Bifidobacterium bifidum selected from the group consisting of Bifidobacterium bifidum BB01 (DSM 22892), Bifidobacterium bifidum BB10 (DSM 33678), and mixtures thereof; (xii) a bacterial strain belonging to the species Bifidobacterium lactis selected from the group consisting of Bifidobacterium lactis BS01 (LMG P-21384), and mixtures thereof; 2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is selected from the group comprising or consisting of:

4. The bacterial strain is in the form of a viable / probiotic strain, preferably Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus crispatus LCR04 (DSM 33487), Rimosilactobacillus fermentum (formerly Lactobacillus fermentum) LF15 (DSM 26955), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP01 (LMG P-21021), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP02 (LMG P-21020), Lactobacillus salivarius subsp. salivarius LS03 (DSM 22776), Bifidobacterium bifidum BB10 (DSM 33678) and mixtures thereof.

5. A supernatant obtainable from a bacterial strain according to claim 1, preferably Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus crispatus LCR04 (DSM 33487), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP01 (LMG P-21021), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP02 (LMG P-21020), Lactobacillus salivarius subsp. salivarius LS03 (DSM 22776), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP09 (DSM 25710), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP14 (DSM 33401), Bifidobacterium longum BL03 (DSM 16603), Lactobacillus gasseri LGS01 (DSM 18299), or mixtures thereof.

6. 2. A pharmaceutical composition comprising a mixture comprising at least two strains, preferably at least three strains, according to claim 1.

7. The mixture is selected from the group consisting of Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus crispatus LCR04 (DSM 33487), rimosilactobacillus fermentum (formerly Lactobacillus fermentum) LF15 (DSM 26955), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP01 (LMG P-21021), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP02 (LMG P-21020), Lactobacillus salivarius subsp. salivarius LS03 (DSM 22776), Bifidobacterium bifidum BB10 (DSM 33678), Lactobacillus plantarum LP09 (DSM 7. The pharmaceutical composition of claim 6, comprising Lactobacillus sp. Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) LP14 (DSM 33401), Bifidobacterium longum BL03 (DSM 16603), Lactobacillus gasseri LGS01 (DSM 18299), and mixtures thereof.

8. A pharmaceutical composition as described in claim 6, wherein the mixture further comprises at least one prebiotic ingredient or prebiotic fiber, preferably the at least one prebiotic ingredient or prebiotic fiber is selected from the group including or consisting of inulin, fructooligosaccharides (FOS), galactooligosaccharides (GOS), guar gum, and mixtures thereof, preferably inulin.

9. 9. A pharmaceutical composition according to any of claims 1 to 8 for use in a method of treatment to prevent or inhibit or reduce respiratory viral infections by a coronavirus, preferably the SARS-CoV-2 virus, and associated pathologies or symptoms.

10. A composition for use in a method of treatment for preventing or inhibiting viral respiratory infections and associated diseases or symptoms, comprising: A bacterial strain according to any one of claims 1 to 5 or a mixture of strains according to any one of claims 6 to 8, and food grade or pharma- ceutically acceptable grade additives or excipients; Preferably, the composition for use in a method of treatment for the prevention, or inhibition, or treatment of respiratory viral infections by a coronavirus, preferably the SARS-CoV-2 virus, and associated diseases or symptoms.