CN120265303A

CN120265303A - Corynebacterium strains, combinations thereof and freeze-dried preparations thereof for preventing viral infections

Info

Publication number: CN120265303A
Application number: CN202380081587.0A
Authority: CN
Inventors: 多拉·萨博; 拉斯洛·塔马斯; 埃兹特·奥斯托哈齐; 伊斯特万·安塔尔; 巴拉兹·斯特尔兹; 埃梅斯·贝尔塔兰内·巴洛格; 贝拉·梅克里
Original assignee: Somerset University
Current assignee: Somerset University
Priority date: 2022-10-12
Filing date: 2023-10-12
Publication date: 2025-07-04
Also published as: WO2024079492A1; EP4601662A1

Abstract

The present invention relates to corynebacterium strains and combinations thereof capable of reducing cathepsin expression for use in the prevention of viral infections, in particular SARS-CoV-2 infections, or for the prevention of infections of enveloped respiratory viruses in a subject, wherein the viruses enter cells of the subject using the cathepsin entry pathway. In particular, the virus is the SARS-CoV-2 omnikom variant. The invention also relates to a lyophilized formulation comprising a corynebacterium strain or a combination thereof and a cryoprotectant for use in preventing infection of an enveloped respiratory virus, preferably SARS-CoV-2 virus, in a subject. Preferably, the formulation is administered to the upper respiratory tract of the subject.

Description

Corynebacterium strain for preventing virus infection, combination thereof and freeze-dried preparation thereof

Technical Field

The present invention relates to Corynebacterium strains and combinations thereof capable of reducing cathepsin expression for use in the prevention of viral infections, in particular SARS-CoV-2 infections. The invention also relates to lyophilized formulations comprising these corynebacteria strains or combinations for use in the prevention of coronavirus infections.

Background

Epithelial cells of the upper and lower respiratory tract are the primary targets for airborne infections. These cells are covered by a complex bacterial community located mainly in the upper respiratory tract, which may interact directly or indirectly with coronaviruses. The idea that symbiotic bacteria can also prevent infection by modulating both innate and adaptive host immune responses has been proposed in the art. In addition, nasopharyngeal or oronasopharyngeal preparations (preparations) comprising bacteria have been proposed to alter the composition of the microbiota in the upper respiratory tract.

Lappan and Peacock[Lappan&Peacock,2019.Corynebacterium and Dolosigranulum:future probiotic candidates for upper respiratory tract infections.Microbiology Australia,40(4),172–177.] reviewed studies observing the presence of corynebacteria and crafty cocci in the microbiota of the upper respiratory tract, and these studies indicate that they are related to health status. The authors suggested that symbiotic strains of Corynebacterium diphtheriae (Corynebacterium pseudodiphtheriticum) and of the genus lazy (Dolosigranulum pigrum), which are considered pathogens of the upper respiratory tract, might have a protective effect on the nasopharyngeal health of children.

The paper published by Man et al in 2017 also concludes that crafty and corynebacteria are potentially critical roles in the microbiota of the upper respiratory tract, as "in several epidemiological and mechanistic studies they are closely related to respiratory health and the elimination of potential pathogens (most notably streptococcus pneumoniae"). [ Man et al ,2017.The microbiota of the respiratory tract:Gatekeeper to respiratory health.Nature Reviews Microbiology,15(5),259–270].

Recently, some studies have also raised the problem of correlation between microbiota health and severe infection with RNA viruses and other coronavirus infections.

The highly transmissible and pathogenic coronaviruses that occur at the end of 2019 (i.e., severe acute respiratory syndrome coronavirus 2 (severe acute respiratory syndrome coronavirus, sars-CoV-2) that caused coronavirus disease 2019 (coronavirus disease 2019, covd-19)) caused pandemics. The importance of this study is supported by the fact that by day 21 of 9 of 2022, WTO has been confirmed to have accumulated 6.09 hundred million diagnosed cases and 650 ten thousand deaths due to COVID-19 (https://www.who.int/publications/m/item/weekly-epidemiological-update-on-covid-19---21-september-2022).

SARS-CoV-2 infection appears to have the dual property of being extremely fatal to some people, being surprisingly mild to others, and even not being sensitive to the virus at all.

Infection by the SARS-CoV-2 virus is a serious problem. The main host of the virus (setle) is on the nasal mucosa and the infection then progresses to severe systemic disease. Infection and its course depend on the susceptibility of the individual.

Epithelial cells of the upper and lower respiratory tracts are the primary targets for infection and replication of the SARS-CoV-2 virus. Given that the invasive door is protected by the symbiotic microbiota of the nasopharynx, several authors have tested bacterial composition in infected and uninfected individuals.

Nardelli et al [ Nardelli et al ,2021.Nasopharyngeal Microbiome Signature in COVID-19Positive Patients:Can We Definitively Get a Role to Fusobacterium periodonticumFrontiers in Cellular and Infection Microbiology,11(February),1–7.] and Maio et al [ Maio et al ,2020.Nasopharyngeal Microbiota Profiling of SARS-CoV-2Infected Patients.Biological Procedures Online 22,18.] ] found no difference in nasopharyngeal microbiome composition (microbiome composition) in samples from COVID-19 patients and virus negative controls. In contrast, from their data it can be concluded that SARS-CoV-2 infection does not significantly alter the composition of the microbiome compared to that of the general population. A large number of patients were tested and the 16S rRNA method was used, rosas-Salazar et al [Rosas-Salazar et al.,2021.SARS-CoV-2infection and viral load are associated with the upper respiratory tract microbiome.JAllergy Clin Immunol.2021Apr;147(4):1226-1233.e2] reported a mixture of corynebacteria, with some strains being more abundant in SARS-CoV-2 infected patients, but others being more abundant in non-virus infected people.

In contrast to targeting methods, next generation metagenomic sequencing also provides valuable information about microbiota composition at the species level. The next generation metagenomic study of Mostafa et al [ Mostafa et al ,2020.Metagenomic next-generation sequencing of nasopharyngeal specimens collected from confirmed and suspect COVID-19patients.MBio,11(6),1–13.] ] describes a statistically significant decrease in the incidence of the symbiotic organism Corynebacterium crowded (Corynebacterium accolens) in samples of COVID-19 positive patients. The authors mention that there is evidence in the prior art that crowded corynebacteria are inversely related to the colonisation by Streptococcus pneumoniae, however, further studies are required to derive the effect of these associations in patients with COVID-19, and that there is no suggestion that there may be a negative correlation between the presence of crowded corynebacteria and SARS-CoV-2 infection. Indeed, a decrease in the level of C.crowded bacteria is likely to be the result of infection.

Susceptibility to SARS-CoV-2 infection and its clinical course remain unpredictable. Tchoupou Saha et al [ Tchoupou Saha et al ,2022.Profile of the Nasopharyngeal Microbiota Affecting the Clinical Course in COVID-19Patients.Front.Microbiol.13:871627.] ] the nasopharyngeal microbiota of COVID-19 patients and patients negative for viral testing were studied by 16S ribosomal ribonucleic acid (ribosomal ribonucleic acid, rRNA) sequencing and specific polymerase chain reaction (polymerase chain reaction, PCR) targeting pathogens. They found that in patients tested positive for SARS-CoV-2, 9 taxa increased, e.g., corynebacterium acidophilus (Corynebacterium propinquum)/Corynebacterium pseudodiphtheriae and Aphenanthropi berkovic (Afipia birgiae). They also demonstrated a reduction in Corynebacterium acidophilus in asymptomatic individuals compared to other COVID-19 positive patients. However, these authors also indicated that further studies are required to determine the exact role of Corynebacterium crowded and in particular Corynebacterium acidophilus/Corynebacterium pseudodiphtheriae in the clinical course of the disease.

COVID-19 epidemics remain a serious problem. One reason for this is that the evolution of SARS-CoV-2 has led to the emergence of several new varieties. Newly released SARS-CoV-2 omnikon variety has become the dominant spreading variety in many countries. Because of the large number of mutations, armurostome exhibits cell tropism and entry patterns compared to other SARS-CoV-2 variants. Unlike the original or other SARS-CoV-2 variants, the HMG variant uses the cathepsin B/L entry pathway in addition to the TMPRSS2 (transmembrane protease/serine subfamily member 2 ) cell entry pathway.

The present inventors have aimed at isolating bacterial strains that down-regulate the cathepsin pathway, which strains can be used as probiotics for the prevention of respiratory viral infections (respiratory viral infection), in particular SARS-CoV-2 infections, determining the ideal combination of these strains, and formulating a nasal spray (lyophilized formulation) comprising these strains or a combination together with suitable excipients.

Disclosure of Invention

In a first aspect, there is provided a corynebacterium culture for use in preventing or treating infection by an enveloped respiratory virus (enveloped respiratory virus), preferably a coronavirus, in a subject, wherein the corynebacterium culture is capable of reducing expression of a protein that promotes entry of the enveloped respiratory virus into cells of the subject, wherein the entry promoting protein (ENTRY FACILITATING protein) is a cathepsin. Preferably, a corynebacterium culture is provided for use in preventing or treating infection by an enveloped respiratory virus, preferably a coronavirus, in a subject, wherein the corynebacterium culture is capable of reducing expression of a cathepsin in cells of the subject. Alternatively, the corynebacterium culture is also capable of reducing the expression of other proteins selected from the group consisting of angiotensin converting enzyme 2 (angiotenin-converting enzyme 2, ace 2) and transmembrane protease/serine subfamily member 2 (transmembrane protease/serine subfamily member 2, tmprss 2) that promote entry of enveloped respiratory viruses, preferably coronaviruses, into cells of a subject.

In an embodiment, a corynebacterium culture for preventing or treating infection by an enveloped respiratory virus, preferably a coronavirus, in a subject is provided, wherein the corynebacterium culture is capable of reducing expression of at least one protein that promotes entry of the enveloped respiratory virus into cells of the subject, wherein the at least one entry promoting protein is selected from the group consisting of cathepsin, ACE2 and TMPRSS2, or any combination thereof, wherein preferably the entry promoting protein is a cathepsin, or wherein preferably the entry promoting protein is a cathepsin and ACE2, or wherein the entry promoting protein is a cathepsin, ACE2 and TMPRSS2.

Preferably, the enveloped respiratory virus is a coronavirus, preferably SARS-CoV-2 and more preferably a SARS-CoV-2 omnikom variant.

In a second aspect, there is provided a formulation for use in preventing or treating an infection by a coronavirus in a subject, wherein the formulation comprises a corynebacterium culture capable of reducing expression of a protein that promotes entry of the coronavirus into cells of the subject, said entry promoting protein being a cathepsin, and wherein optionally said corynebacterium culture is further capable of reducing expression of other proteins that promote entry of the coronavirus into cells of the subject, selected from ACE2 and TMPRSS2. Preferably, the formulation is a probiotic composition (probiotic composition). Preferably, the formulation is (part of) a medical device. Preferably, the formulation is delivered by a medical device. Preferably, the formulation is a pharmaceutical formulation. Preferably, the formulation further comprises at least one excipient and/or pharmaceutically acceptable excipient suitable for use in probiotic formulations, when appropriate.

In a third aspect, a composition is provided comprising a corynebacterium culture capable of reducing expression of a protein that promotes entry of coronavirus into cells of a subject, said entry-promoting protein being a cathepsin, and wherein optionally said corynebacterium culture may also be capable of reducing expression of other proteins that promote entry of coronavirus into cells of a subject, selected from ACE2 and TMPRSS2. Preferably, the composition is a probiotic composition. Preferably, the composition is (part of) a medical device. Preferably, the composition is delivered by a medical device. Preferably, the composition is a pharmaceutical composition. Preferably, the composition is for use in the prevention or treatment of coronavirus infection. Preferably, the composition further comprises at least one excipient and/or pharmaceutically acceptable excipient suitable for use in probiotic compositions, when appropriate.

In a fourth aspect, there is provided a method for preventing or treating infection by coronavirus, the method comprising administering to a subject in need thereof a corynebacterium culture, or a formulation or composition comprising a corynebacterium culture, wherein the corynebacterium culture is capable of reducing expression of a protein that promotes entry of coronavirus into cells of the subject, said entry-promoting protein being a cathepsin, and wherein optionally the corynebacterium culture is also capable of reducing expression of other proteins that promote entry of coronavirus into cells of the subject, selected from ACE2 and TMPRSS2.

In any of the aspects of the invention, preferably, the corynebacterium culture is used for preventing infection by coronavirus, and thus also for preventing diseases caused by coronavirus.

In any of the aspects of the invention, preferably the corynebacterium culture is further capable of inhibiting the binding between Spike (Spike, S) protein of coronavirus, preferably the receptor binding domain (receptor binding domain, RBD) of S protein, and ACE 2.

In any one of the aspects of the present invention, preferably, the corynebacterium genus culture includes a viable corynebacterium selected from the group consisting of Corynebacterium crowded, corynebacterium propionicum (near Corynebacterium, corynebacterium propinquum) and Corynebacterium tuberculosis stearic acid (Corynebacterium nucleatum, corynebacterium tuberculostearicum). Preferably, the corynebacterium genus culture includes strains of Corynebacterium crowded, corynebacterium propionicum, and Corynebacterium tuberculosis stearate. Preferably, the corynebacterium culture comprises at least one strain of corynebacterium crowded. Preferably, the corynebacterium culture comprises at least one strain of Corynebacterium propionicum. Preferably, the corynebacterium culture comprises at least one strain of corynebacterium tuberculosis stearate.

In any of the aspects of the present invention, preferably, the corynebacterium genus culture does not include corynebacterium pseudodiphtheriae.

In any one of the aspects of the invention, preferably, the corynebacterium culture comprises at least one corynebacterium strain selected from the group consisting of:

A c.crowded corynebacterium SU001 strain deposited at NCAIM (National Collection of Agricultural and Industrial Microorganisms, national institute of agriculture and industrial microbiological deposit, institute of food science and technology, university of agriculture and life sciences, H-1118, budapest, som loway 14-16, hungary) under accession number NCAIM P (B) 001495 or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and optionally the derivative, variant or mutant thereof is also capable of reducing ACE2 expression and/or TMPRSS2 expression and/or inhibiting the binding of ACE2 and S proteins and/or the derivative, variant or mutant thereof expresses LipS1 lipase and/or comprises a sequence encoding LipS lipase;

A corynebacterium propionicum SU002 strain deposited under accession number NCAIM P (B) 001496 at month 22 of 2021 or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing expression of cathepsin and optionally the derivative, variant or mutant thereof is also capable of reducing expression of ACE2 and/or expression of TMPRSS2 and/or inhibiting binding of ACE2 and S proteins;

A corynebacterium propionicum SU003 strain deposited under accession number NCAIM P (B) 001497 at month 22 of 2021 or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and optionally the derivative, variant or mutant thereof is also capable of reducing ACE2 expression and/or TMPRSS2 expression and/or inhibiting ACE2 and S protein binding;

A crowded corynebacterium SU004 strain deposited under accession number NCAIM P (B) 001500 at 1 month 17 of 2022 or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and optionally the derivative, variant or mutant thereof is also capable of reducing ACE2 expression and/or reducing TMPRSS2 expression and/or inhibiting the binding of ACE2 and S proteins and/or expressing a lipase and/or comprising a sequence encoding a lipase;

A corynebacterium tuberculosis stearic acid SU005 strain deposited under accession number NCAIM P (B) 001501 at month 4 and 27 of 2022 or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of decreasing expression of cathepsin and optionally the derivative, variant or mutant thereof is also capable of decreasing expression of ACE2 and/or decreasing expression of TMPRSS2 and/or inhibiting binding of ACE2 and S proteins;

A crowded corynebacterium SU006 strain deposited under accession number NCAIM P (B) 001502 at month 4 of 2022 or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and optionally the derivative, variant or mutant thereof is also capable of reducing ACE2 expression and/or reducing TMPRSS2 expression and/or inhibiting the binding of ACE2 and S proteins and/or the derivative, variant or mutant thereof expresses a lipase and/or comprises a sequence encoding a lipase;

A corynebacterium propionicum SU007 strain deposited under accession number NCAIM P (B) 001504 at month 27 of 2022 or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and optionally the derivative, variant or mutant thereof is also capable of reducing ACE2 expression and/or reducing TMPRSS2 expression and/or inhibiting the binding of ACE2 and S proteins;

A crowded corynebacterium SU008 strain deposited under accession number NCAIM P (B) 001505 at 4/27 of 2022 or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and optionally the derivative, variant or mutant thereof is also capable of reducing ACE2 expression and/or reducing TMPRSS2 expression and/or inhibiting the binding of ACE2 and S proteins and/or the derivative, variant or mutant thereof expresses a lipase and/or comprises a sequence encoding a lipase;

A crowded corynebacterium SU009 strain deposited under accession number NCAIM P (B) 001506 at 5.25 of 2022 or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing expression of a cathepsin and optionally the derivative, variant or mutant thereof is also capable of reducing expression of ACE2 and/or reducing expression of TMPRSS2 and/or inhibiting binding of ACE2 and S proteins and/or the derivative, variant or mutant thereof expresses a lipase and/or comprises a sequence encoding a lipase;

A corynebacterium propionicum SU010 strain deposited under accession number NCAIM P (B) 001507 at 5.25 of 2022 or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing expression of cathepsin and optionally the derivative, variant or mutant thereof is also capable of reducing expression of ACE2 and/or reducing expression of TMPRSS2 and/or inhibiting binding of ACE2 and S proteins.

A strain of c.crowded corynebacterium SU004 deposited with NCAIM (national institute of agriculture and industrial microbiology, food science and technology, university of agriculture and life sciences, H-1118, budapest, somalox road 14-16, hungarian) under accession number NCAIM P (B) 001500 or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and, optionally, the derivative, variant or mutant thereof is further capable of reducing ACE2 expression and/or TMPRSS2 expression and/or inhibiting the binding of ACE2 and S proteins and/or the derivative, variant or mutant thereof expresses a lipase and/or comprises a sequence encoding a lipase;

a strain of corynebacterium crowded SU004 deposited with NCAIM (national institute of agriculture and industrial microbiology, food science and technology, university of agriculture and life sciences, H-1118, budapest, som loy 14-16, hungarian) under accession number NCAIM P (B) 001500, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and, optionally, the derivative, variant or mutant thereof is also capable of reducing ACE2 expression and/or TMPRSS2 expression and/or inhibiting ACE2 and S protein binding;

A crowded corynebacterium SU006 strain deposited under accession number NCAIM P (B) 001502 at 4/27 of 2022 or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and optionally the derivative, variant or mutant thereof is also capable of reducing ACE2 expression and/or reducing TMPRSS2 expression and/or inhibiting the binding of ACE2 and S proteins;

a crowded corynebacterium SU008 strain deposited under accession number NCAIM P (B) 001505 at 4/27 of 2022 or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and optionally the derivative, variant or mutant thereof is also capable of reducing ACE2 expression and/or reducing TMPRSS2 expression and/or inhibiting the binding of ACE2 and S proteins;

A crowded corynebacterium SU009 strain deposited under accession number NCAIM P (B) 001506 at 5.25 of 2022 or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and optionally the derivative, variant or mutant thereof is also capable of reducing ACE2 expression and/or reducing TMPRSS2 expression and/or inhibiting the binding of ACE2 and S proteins;

A strain SU004 of corynebacterium crowded with accession number NCAIM P (B) 001500, deposited at NCAIM (national institute of agriculture and industrial microbiology, institute of food science and technology, university of agriculture and life sciences, H-1118, budapest, somnolway 14-16, hungarian) or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression;

A corynebacterium tuberculosis SU005 strain deposited under accession number NCAIM P (B) 001501 at month 4 and 27 of 2022, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of decreasing cathepsin expression;

a crowded corynebacterium SU006 strain deposited under NCAIM accession number NCAIM P (B) 001502, 4, 27, 2022, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression;

A corynebacterium propionicum SU007 strain deposited under accession number NCAIM P (B) 001504 at time 27, 4, 2022, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression;

A strain SU008 of corynebacterium crowded with accession number NCAIM P (B) 001505 deposited at NCAIM, 4, 27, 2022, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression;

a strain SU009 of corynebacterium crowded with accession number NCAIM P (B) 001506 deposited at NCAIM, 5.25, 2022, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression;

A corynebacterium propionicum SU010 strain deposited under accession number NCAIM P (B) 001507 at 5.25 of 2022 or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant is capable of reducing cathepsin expression.

In another aspect, the invention relates to a crowded coryneform SU001 strain deposited on day 22 of 2022 under accession number NCAIM P (B) 001495, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and optionally the derivative, variant or mutant thereof is also capable of reducing ACE2 expression and/or TMPRSS2 expression and/or inhibiting the binding of ACE2 and S proteins and/or the derivative, variant or mutant thereof expresses LipS1 lipase and/or comprises a sequence encoding LipS1 lipase, and to use as a medicament for the treatment of cancer, in a human. The invention also relates to a corynebacterium culture comprising or consisting essentially of corynebacterium crowded SU 001. Preferably, the strain of Corynebacterium crowded SU001, or a culture comprising or consisting essentially of the strain of Corynebacterium crowded SU001, is used to prevent infection of an enveloped respiratory virus, preferably coronavirus, more preferably SARS-CoV-2 virus, in a subject. In embodiments, the SARS-CoV-2 virus is a variant of SARS-CoV-2 obrykon.

In another aspect, the invention relates to a strain SU002 of corynebacterium propionicum deposited under accession number NCAIM P (B) 001496 at month 22 of 2021 or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and optionally the derivative, variant or mutant thereof is also capable of reducing ACE2 expression and/or TMPRSS2 expression and/or inhibiting ACE2 and S protein binding. The invention also relates to a corynebacterium culture comprising or consisting essentially of Corynebacterium propionicum SU 002. Preferably, the strain of Corynebacterium propionicum SU002, or a culture comprising or consisting essentially of the strain of Corynebacterium propionicum SU002, is used to prevent infection of an enveloped respiratory virus, preferably a coronavirus, more preferably a SARS-CoV-2 virus, in a subject. In embodiments, the SARS-CoV-2 virus is a variant of SARS-CoV-2 obrykon.

In another aspect, the invention relates to a strain SU003 of c.propionicum deposited under accession number NCAIM P (B) 001497 at month 22 of 2021 or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and optionally the derivative, variant or mutant thereof is also capable of reducing ACE2 expression and/or TMPRSS2 expression and/or inhibiting the binding of ACE2 and S proteins. The invention also relates to a corynebacterium culture comprising or consisting essentially of Corynebacterium propionicum SU 003. Preferably, the strain of Corynebacterium propionicum SU003, or a culture comprising or consisting essentially of the strain of Corynebacterium propionicum SU003, is used to prevent infection of an enveloped respiratory virus, preferably a coronavirus, more preferably a SARS-CoV-2 virus, in a subject. In embodiments, the SARS-CoV-2 virus is a variant of SARS-CoV-2 obrykon.

In another aspect, the invention relates to a strain SU004 of corynebacterium crowded with accession number NCAIM P (B) 001500 deposited on month 17 of 2022 or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and optionally the derivative, variant or mutant thereof is also capable of reducing ACE2 expression and/or reducing TMPRSS2 expression and/or inhibiting the binding of ACE2 and S proteins and/or the derivative, variant or mutant thereof expresses a lipase and/or comprises a sequence encoding a lipase. The invention also relates to a corynebacterium culture comprising or consisting essentially of corynebacterium crowded SU 004. Preferably, the strain of Corynebacterium crowded SU004, or a culture comprising or consisting essentially of the strain of Corynebacterium crowded SU004, is used to prevent infection of the subject with enveloped respiratory viruses, preferably coronaviruses, more preferably SARS-CoV-2 virus. In embodiments, the SARS-CoV-2 virus is a variant of SARS-CoV-2 obrykon.

In another aspect, the invention relates to a corynebacterium tuberculosis stearic acid SU005 strain deposited under accession number NCAIM P (B) 001501 at month 27 of 2022, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and optionally the derivative, variant or mutant thereof is also capable of reducing ACE2 expression and/or reducing TMPRSS2 expression and/or inhibiting the binding of ACE2 and S proteins. The invention also relates to a corynebacterium culture comprising or consisting essentially of Corynebacterium tuberculosis stearic acid SU 005. Preferably, the corynebacterium tuberculosis SU005 strain, or a culture comprising or consisting essentially of the corynebacterium tuberculosis SU005 strain, is used to prevent infection of an enveloped respiratory virus, preferably coronavirus, more preferably SARS-CoV-2 virus, in a subject. In embodiments, the SARS-CoV-2 virus is a variant of SARS-CoV-2 obrykon.

In another aspect, the invention relates to the crowded corynebacterium SU006 strain deposited on month 27 of 2022 under accession number NCAIM P (B) 001502, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and optionally the derivative, variant or mutant thereof is also capable of reducing ACE2 expression and/or reducing TMPRSS2 expression and/or inhibiting the binding of ACE2 and S proteins and/or the derivative, variant or mutant thereof expresses a lipase and/or comprises a sequence encoding a lipase. The invention also relates to a corynebacterium culture comprising or consisting essentially of corynebacterium crowded SU 006. Preferably, the strain of Corynebacterium crowded SU006, or a culture comprising or consisting essentially of the strain of Corynebacterium crowded SU006, is used to prevent infection of an enveloped respiratory virus, preferably coronavirus, more preferably SARS-CoV-2 virus, in a subject. In embodiments, the SARS-CoV-2 virus is a variant of SARS-CoV-2 obrykon.

In another aspect, the invention relates to the strain SU007 of corynebacterium propionicum deposited on month 27 of 2022 under accession number NCAIM P (B) 001504, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and, optionally, the derivative, variant or mutant thereof is also capable of reducing ACE2 expression and/or reducing TMPRSS2 expression and/or inhibiting ACE2 and S protein binding. The invention also relates to a corynebacterium culture comprising or consisting essentially of Corynebacterium propionicum SU 007. Preferably, the strain of Corynebacterium propionicum SU007, or a culture comprising or consisting essentially of the strain of Corynebacterium propionicum SU007, is used to prevent infection of an enveloped respiratory virus, preferably a coronavirus, more preferably a SARS-CoV-2 virus, in a subject. In embodiments, the SARS-CoV-2 virus is a variant of SARS-CoV-2 obrykon.

In another aspect, the invention relates to a strain SU008 of corynebacterium crowded with accession number NCAIM P (B) 001505 deposited on month 27 of 2022, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and optionally the derivative, variant or mutant thereof is also capable of reducing ACE2 expression and/or reducing TMPRSS2 expression and/or inhibiting the binding of ACE2 and S proteins and/or expressing a lipase and/or comprising a sequence encoding a lipase. The invention also relates to a corynebacterium culture comprising or consisting essentially of corynebacterium crowded SU 008. Preferably, the strain of Corynebacterium crowded SU008, or a culture comprising or consisting essentially of the strain of Corynebacterium crowded SU008, is used to prevent infection of a subject with enveloped respiratory viruses, preferably coronaviruses, more preferably SARS-CoV-2 virus. In embodiments, the SARS-CoV-2 virus is a variant of SARS-CoV-2 obrykon.

In another aspect, the invention relates to a strain SU009 of corynebacterium crowded with accession number NCAIM P (B) 001506 deposited on 5.2022 or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and optionally the derivative, variant or mutant thereof is also capable of reducing ACE2 expression and/or reducing TMPRSS2 expression and/or inhibiting ACE2 and S protein binding and/or the derivative, variant or mutant thereof expresses a lipase and/or comprises a sequence encoding a lipase. The invention also relates to a corynebacterium culture comprising or consisting essentially of corynebacterium crowded SU 009. Preferably, the strain of Corynebacterium crowded SU009, or a culture comprising or consisting essentially of the strain of Corynebacterium crowded SU009, is used to prevent infection of a subject with enveloped respiratory viruses, preferably coronaviruses, more preferably SARS-CoV-2 virus. In embodiments, the SARS-CoV-2 virus is a variant of SARS-CoV-2 obrykon.

In another aspect, the invention relates to a strain SU010 of corynebacterium propionicum deposited under accession number NCAIM P (B) 001507 at 5.25 of 2022 or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and optionally the derivative, variant or mutant thereof is also capable of reducing ACE2 expression and/or reducing TMPRSS2 expression and/or inhibiting the binding of ACE2 and S proteins. The invention also relates to a corynebacterium culture comprising or consisting essentially of Corynebacterium propionicum SU 010. Preferably, the corynebacterium propionicum SU010 strain, or a culture comprising or consisting essentially of the corynebacterium propionicum SU010 strain, is used to prevent infection of an enveloped respiratory virus, preferably a coronavirus, more preferably a SARS-CoV-2 virus, in a subject. In embodiments, the SARS-CoV-2 virus is a variant of SARS-CoV-2 obrykon.

In another aspect, the invention relates to a corynebacterium strain selected from the group consisting of:

A strain SU009 of Corynebacterium crowded with accession number NNCAIM P (B) 001506 deposited at NCAIM, 5.25, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression and optionally the derivative, variant or mutant thereof is also capable of reducing ACE2 expression and/or reducing TMPRSS2 expression and/or inhibiting the binding of ACE2 and S proteins and/or the derivative, variant or mutant thereof expresses a lipase and/or comprises a sequence encoding a lipase, and

Preferably, the corynebacterium culture consists essentially of corynebacterium, preferably essentially of corynebacterium crowded and/or corynebacterium propionicum and/or corynebacterium tuberculosis stearate.

In particular, the corynebacterium culture includes a corynebacterium strain that is viable in a medium.

In a preferred embodiment of the invention, the corynebacterium culture comprises a viable corynebacterium strain selected from the group consisting of Corynebacterium crowded, corynebacterium propionicum and Corynebacterium tuberculosis stearic acid.

In any one of the aspects of the present invention, preferably, the corynebacterium culture includes at least two corynebacterium strains selected from the group consisting of corynebacterium strains SU001 to SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of the above-mentioned cathepsin. Corynebacterium cultures comprising at least two different corynebacterium strains are also referred to as combinations.

Preferably, the combination comprises at least two viable corynebacterium strains selected from the group of strains consisting of Corynebacterium crowded, corynebacterium propionicum and Corynebacterium tuberculosis stearate. Preferably, the combination comprises at least two strains, wherein the strains belong to the same species selected from the group consisting of Corynebacterium crowded, corynebacterium propionicum and Corynebacterium tuberculosis stearate. Preferably, the combination comprises at least two strains, wherein the at least two strains belong to different species selected from the group consisting of Corynebacterium crowded, corynebacterium propionic acid and Corynebacterium tuberculosis stearic acid. Preferably, the combination comprises at least two strains selected from the group consisting of Corynebacterium crowded SU001, corynebacterium propionicum SU002, corynebacterium propionicum SU003, corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium crowded SU006, corynebacterium propionicum SU007, corynebacterium crowded SU008, corynebacterium crowded SU009, corynebacterium propionicum SU010, and any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. Preferably, the combination comprises at least two strains selected from the group consisting of Corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium crowded SU006, corynebacterium propionicum SU007, corynebacterium crowded SU008, corynebacterium crowded SU009, corynebacterium propionicum SU010, and any derivatives, variants or mutants thereof capable of reducing cathepsin expression.

Preferably, the combination comprises at least three viable corynebacterium strains selected from the group of Corynebacterium crowded, corynebacterium propionicum and Corynebacterium tuberculosis stearate. Preferably, the combination comprises at least three strains, wherein at least two strains belong to different species selected from the group consisting of Corynebacterium crowded, corynebacterium propionic acid and Corynebacterium tuberculosis stearic acid. Preferably, the combination comprises at least three strains selected from the group consisting of Corynebacterium crowded SU001, corynebacterium propionicum SU002, corynebacterium propionicum SU003, corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium crowded SU006, corynebacterium propionicum SU007, corynebacterium crowded SU008, corynebacterium crowded SU009, corynebacterium propionicum SU010, and any derivatives, variants or mutants thereof capable of reducing cathepsin expression. Preferably, the combination comprises at least three strains selected from the group consisting of Corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium crowded SU006, corynebacterium propionicum SU007, corynebacterium crowded SU008, corynebacterium crowded SU009, corynebacterium propionicum SU010, and any derivatives, variants or mutants thereof capable of reducing cathepsin expression.

Preferably, the combination comprises four viable Corynebacterium strains selected from the group of Corynebacterium crowded, corynebacterium propionicum and Corynebacterium tuberculosis stearic acid. Preferably, the combination comprises four strains, wherein at least two of the strains belong to different species selected from the group consisting of Corynebacterium crowded, corynebacterium propionic acid and Corynebacterium tuberculosis stearic acid. Preferably, the combination comprises four strains, wherein at least one strain is Corynebacterium crowded, at least one strain is Corynebacterium propionicum and at least one strain is Corynebacterium tuberculosis stearate. Preferably, the combination comprises four strains selected from the group consisting of Corynebacterium crowded SU001, corynebacterium propionicum SU002, corynebacterium propionicum SU003, corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium crowded SU006, corynebacterium propionicum SU007, corynebacterium crowded SU008, corynebacterium crowded SU009, corynebacterium propionicum SU010, and any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. Preferably, the combination comprises four strains selected from the group consisting of Corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium crowded SU006, corynebacterium propionicum SU007, corynebacterium crowded SU008, corynebacterium crowded SU009, corynebacterium propionicum SU010, and any derivatives, variants or mutants thereof capable of reducing cathepsin expression.

In a preferred embodiment, the combination comprises at least two strains, wherein the at least two strains are Corynebacterium crowded SU004 and Corynebacterium tuberculosis SU005, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least two strains, wherein the at least two strains are Corynebacterium crowded SU004 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least two strains, wherein the at least two strains are Corynebacterium crowded SU004 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least two strains, wherein the at least two strains are Corynebacterium tuberculosis SU005 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least two strains, wherein the at least two strains are Corynebacterium tuberculosis SU005 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least two strains, wherein the at least two strains are Corynebacterium crowded SU009 and Corynebacterium propionicum SU010, or any derivative, variant or mutant thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least two strains, wherein the at least two strains are Corynebacterium crowded SU004 and Corynebacterium crowded SU006, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least two strains, wherein the at least two strains are Corynebacterium tuberculosis SU005 and Corynebacterium crowded SU006, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least two strains, wherein the at least two strains are Corynebacterium crowded SU006 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least two strains, wherein the at least two strains are Corynebacterium crowded SU004 and Corynebacterium propionicum SU007, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least two strains, wherein the at least two strains are Corynebacterium crowded SU004 and Corynebacterium crowded SU008, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least two strains, wherein the at least two strains are Corynebacterium tuberculosis SU005 and Corynebacterium propionicum SU007, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least two strains, wherein the at least two strains are Corynebacterium tuberculosis SU005 and Corynebacterium crowded SU008, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least two strains, wherein the at least two strains are Corynebacterium propionicum SU007 and Corynebacterium crowded SU008, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least two strains, wherein the at least two strains are Corynebacterium crowded SU006 and Corynebacterium propionicum SU007, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least two strains, wherein the at least two strains are Corynebacterium crowded SU006 and Corynebacterium crowded SU008, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least two strains, wherein the at least two strains are Corynebacterium crowded SU006 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least two strains, wherein the at least two strains are Corynebacterium propionicum SU007 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least two strains, wherein the at least two strains are Corynebacterium propionicum SU007 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least two strains, wherein the at least two strains are Corynebacterium crowded SU008 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least two strains, wherein the at least two strains are Corynebacterium crowded SU008 and Corynebacterium propionicum SU010, or any derivative, variant or mutant thereof capable of reducing the expression of cathepsin.

In a preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU004, corynebacterium tuberculosis SU005 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU004, corynebacterium tuberculosis SU005 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU004, corynebacterium crowded SU009 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium tuberculosis SU005, corynebacterium crowded SU009 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU004, corynebacterium tuberculosis SU005 and Corynebacterium crowded SU006, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU004, corynebacterium crowded SU006 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium tuberculosis SU005, corynebacterium crowded SU006 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU004, corynebacterium tuberculosis SU005 and Corynebacterium propionicum SU007, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU004, corynebacterium tuberculosis SU005 and Corynebacterium crowded SU008, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU004, corynebacterium propionicum SU007, and Corynebacterium crowded SU008, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium tuberculosis SU005, corynebacterium propionicum SU007 and Corynebacterium crowded SU008, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU004, corynebacterium crowded SU006 and Corynebacterium propionicum SU007, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. in another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU004, corynebacterium crowded SU006 and Corynebacterium crowded SU008, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU004, corynebacterium crowded SU006 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU004, corynebacterium propionicum SU007 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU004, corynebacterium propionicum SU007, and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU004, corynebacterium crowded SU008 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU004, corynebacterium crowded SU008 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium tuberculosis SU005, corynebacterium crowded SU006 and Corynebacterium propionicum SU007, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium tuberculosis SU005, corynebacterium crowded SU006 and Corynebacterium crowded SU008, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium tuberculosis SU005, corynebacterium crowded SU006 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium tuberculosis SU005, corynebacterium propionicum SU007 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium tuberculosis SU005, corynebacterium propionicum SU007 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium tuberculosis SU005, corynebacterium crowded SU008 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium tuberculosis SU005, corynebacterium crowded SU008 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU006, corynebacterium propionicum SU007 and Corynebacterium crowded SU008, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU006, corynebacterium propionicum SU007 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU006, corynebacterium propionicum SU007 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU006, corynebacterium crowded SU008 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU006, corynebacterium crowded SU008 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU006, corynebacterium crowded SU009 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium propionicum SU007, corynebacterium crowded SU008 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium propionicum SU007, corynebacterium crowded SU008 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium propionicum SU007, corynebacterium crowded SU009 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises at least three strains, wherein the at least three strains are Corynebacterium crowded SU008, corynebacterium crowded SU009 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression.

In a preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium crowded SU006 and Corynebacterium propionicum SU007, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium crowded SU006 and Corynebacterium crowded SU008, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium crowded SU006 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium crowded U006 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium propionicum SU007 and Corynebacterium crowded SU008, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium propionicum SU007 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium propionicum SU007 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium crowded SU008 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium crowded SU008 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium crowded SU009 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium crowded SU006, corynebacterium propionicum SU007, and Corynebacterium crowded SU008, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium crowded SU006, corynebacterium propionicum SU007 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium crowded SU006, corynebacterium propionicum SU007 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium crowded SU006, corynebacterium crowded SU008 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium crowded SU006, corynebacterium crowded SU008 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium crowded SU006, corynebacterium crowded SU009 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. in another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium propionicum SU007, corynebacterium crowded SU008 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium propionicum SU007, corynebacterium crowded SU008 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium propionicum SU007, corynebacterium crowded SU009 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium crowded SU008, corynebacterium crowded SU009 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium tuberculosis SU005, corynebacterium crowded SU006, corynebacterium propionicum SU007 and Corynebacterium crowded SU008, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium tuberculosis SU005, corynebacterium crowded SU006, corynebacterium propionicum SU007 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium tuberculosis SU005, corynebacterium crowded SU006, corynebacterium propionicum SU007 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium tuberculosis SU005, corynebacterium crowded SU006, corynebacterium crowded SU008 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium tuberculosis SU005, corynebacterium crowded SU006, corynebacterium crowded SU008 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium tuberculosis SU005, corynebacterium crowded SU006, corynebacterium crowded SU009 and Corynebacterium propionicum SU010, or any derivative, variant or mutant thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium tuberculosis SU005, corynebacterium propionicum SU007, corynebacterium crowded SU008 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium tuberculosis SU005, corynebacterium propionicum SU007, corynebacterium crowded SU008 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. in another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium tuberculosis SU005, corynebacterium propionicum SU007, corynebacterium crowded SU009 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium tuberculosis SU005, corynebacterium crowded SU008, corynebacterium crowded SU009 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU006, corynebacterium propionicum SU007, corynebacterium crowded SU008 and Corynebacterium crowded SU009, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU006, corynebacterium propionicum SU007, corynebacterium crowded SU008 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU006, corynebacterium propionicum SU007, corynebacterium crowded SU009 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. In another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU006, corynebacterium crowded SU008, corynebacterium crowded SU009 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing cathepsin expression. in another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium propionicum SU007, corynebacterium crowded SU008, corynebacterium crowded SU009 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin.

In a more preferred embodiment, the corynebacterium culture comprises four corynebacterium strains selected from the group consisting of:

(i) Corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium crowded SU009 and corynebacterium propionicum SU010, or any derivative, variant or mutant thereof capable of decreasing the expression of cathepsin;

(ii) Corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium crowded SU006 and corynebacterium propionicum SU010, or any derivative, variant or mutant thereof capable of reducing expression of cathepsin, and

(Iii) Corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium propionicum SU007 and corynebacterium crowded SU008, or any derivative, variant or mutant thereof capable of decreasing the expression of cathepsin.

In a preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium crowded SU009 and Corynebacterium propionicum SU010, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. (examples such combinations are referred to as combination K1.) in another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium crowded SU006 and Corynebacterium propionicum SU010, or any derivative, variant or mutant thereof capable of reducing the expression of cathepsin. (such combination is referred to as combination K2. In the examples.) in another preferred embodiment, the combination comprises four strains, wherein the four strains are Corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium propionicum SU007 and Corynebacterium crowded SU008, or any derivatives, variants or mutants thereof capable of reducing the expression of cathepsin. (such a combination is referred to as combination K3 in the examples.)

Preferably, the corynebacterium culture comprising at least two corynebacterium strains consists essentially of corynebacterium, preferably essentially of corynebacterium crowded and/or corynebacterium propionicum and/or corynebacterium tuberculosis stearate.

In particular, the corynebacterium culture comprising at least two corynebacterium strains comprises at least two viable corynebacterium strains in a medium. Preferably, the corynebacterium culture comprising at least two corynebacterium strains comprises at least three viable corynebacterium strains in a medium. More preferably, the corynebacterium culture comprising at least two corynebacterium strains comprises four viable corynebacterium strains of the medium.

In a preferred embodiment of the present invention, the corynebacterium culture comprising at least two corynebacterium strains comprises at least two viable corynebacterium strains selected from the group of strains consisting of Corynebacterium crowded, corynebacterium propionicum and Corynebacterium tuberculosis. In a preferred embodiment of the present invention, the corynebacterium culture comprising at least two corynebacterium strains comprises at least three viable corynebacterium strains selected from the group of strains consisting of Corynebacterium crowded, corynebacterium propionicum and Corynebacterium tuberculosis. In a preferred embodiment of the present invention, the corynebacterium culture comprising at least two corynebacterium strains comprises four viable corynebacterium strains selected from the group of strains consisting of Corynebacterium crowded, corynebacterium propionicum and Corynebacterium tuberculosis.

In any of the aspects of the present invention, the corynebacterium genus culture does not include corynebacterium crowded SU001 and/or corynebacterium propionicum SU002 and/or corynebacterium propionicum SU003. In embodiments, the corynebacterium culture does not include corynebacterium crowded SU001. In another embodiment, the corynebacterium culture does not include Corynebacterium propionicum SU002. In another embodiment, the corynebacterium culture does not include Corynebacterium propionicum SU003.

Preferably, in any aspect of the invention, the corynebacterium strain or corynebacterium culture capable of decreasing expression of a cathepsin is a corynebacterium strain or corynebacterium culture, respectively, that decreases relative expression of cathepsin mRNA after 24 hours of incubation with Caco-2 cells. In embodiments, the bacterial co-culture assays described in the examples are used to determine whether a corynebacterium strain or a corynebacterium culture reduces the relative expression of cathepsin mRNA.

The invention also relates to a pharmaceutical composition comprising a corynebacterium culture capable of reducing cathepsin expression in a subject, and a pharmaceutically acceptable excipient or pharmaceutically acceptable carrier.

Preferably, the formulation or composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient. Preferably, the formulation or composition is a probiotic formulation or composition. Preferably, the formulation or composition is (part of) a medical device. Preferably, the formulation or composition is delivered by a medical device. Preferably, the formulation or composition is for inhaled administration (INHALATIVE ADMINISTRATION), more preferably in the form of an inhalable powder, aerosol mixture, oral inhalation solution or suspension, nasal drops, nasal spray, nasopharyngeal spray or nasal mist (NASAL MIST). Preferably, the formulation or composition is a nasal spray.

The invention also relates to preparations comprising a corynebacterium culture for use according to the invention. The formulations of the invention include a pharmaceutically acceptable excipient, such as a carrier. Preferably, the formulation comprises one or more components that support the survival or, if desired, the growth of corynebacteria, and such components are optionally considered excipients. Any excipient is preferably physiologically compatible with the upper airway epithelial cells.

Preferably, the formulation is for the upper respiratory tract of a subject.

In embodiments, the formulation is for nasal administration, more preferably nasal drops or nasal sprays.

In embodiments, the formulation is for inhaled administration, more preferably in the form of an inhalable powder, aerosol mixture, oral inhalation solution or suspension, nasal spray, nasal aerosol.

In embodiments, the formulation is in the form of an intranasal formulation for topical administration, preferably in the form of a viscous liquid, liquid suspension, paste, gel or ointment (paste, ointment).

Preferably, the formulation is a formulation for delivery to the upper respiratory tract. Preferably, the formulation is delivered by a device capable of delivering the formulation to the upper respiratory tract.

Preferably, the formulation is in the form of a nasal formulation, for example a nasal cream, nasal gel, nasal ointment, nasal drops (including solutions, suspensions, emulsions, lyophilized powders for suspensions, powders for solutions), nasal powders, nasal sprays (including solutions, suspensions, emulsions, lyophilized powders for suspensions, powders for solutions), nasal washes, nasal patches or nasopharyngeal sprays. Alternatively, the formulation is in the form of a skin and nose ointment, an intra-sinus solution, an intra-sinus lotion (including a suspension), a powder for an intra-sinus solution, a powder for a nasal spray solution, or an oral mucosal solution, a nasal/oral mucosal solution, or a nasal/oral mucosal spray.

Preferably, the formulation is in the form of an oropharyngeal formulation, such as an oropharyngeal spray, an oropharyngeal spray solution, an oropharyngeal suspension, an oropharyngeal emulsion, an oropharyngeal powder, an oropharyngeal gel or an oropharyngeal lotion.

Alternatively, the formulation is in the form of an ocular-nasal article, such as an effervescent tablet for ocular-nasal suspension, a lyophilisate for ocular-nasal suspension (including use in drinking water), or ocular-nasal suspension.

Preferably, the formulation is in the form of an oral mucosal preparation, for example, buccal films, buccal tablets, compressed lozenges, gargle concentrates, oral mucosal solution concentrates, skin solutions, skin/oral mucosal sprays, skin/oral mucosal/oral solutions, effervescent buccal tablets, gargles (including powder for solution, tablet for solution), gargles/mouthwashes, gargles/collutory, gum gels, gum pastes (GINGIVAL PASTE), gum solutions, laryngopharynx sprays, lozenges, medicated chewing gums, mouthwashes (including powder for solution, tablet for solution), mucoadhesive buccal tablets, nasal sprays and oral mucosal solutions, nasal/oral mucosal solutions, nasal/oral mucosal sprays, oral mucosal capsules, oral mucosal creams, oral mucosal drops, oral mucosal patches, oral mucosal gels, oral mucosal ointments, oral mucosal pastes, oral mucosal patches, oral mucosal sachets, oral mucosal solutions, oral mucosal sprays (including emulsions, solutions, suspensions), oral mucosal suspensions, oral mucosal/laryngopharynx solutions, oral mucosal/laryngopharynx sprays, lozenges, pills, gingival gel powders, mouthwash powders, sublingual films, sublingual lyophilized powders, sublingual sprays (including emulsions, solutions, suspensions) or sublingual tablets.

Preferably, the formulation is in the form of a pulmonary article, such as an aerosol, a concentrate for nebulizer solution, a bronchopulmonary instillation (including solutions, suspensions, powders for solutions, and powders for suspensions), an inhalation gas, an inhalation infusion pad, an inhalation powder (including hard capsules, pre-dispensing, and tablets), an inhalation solution, an inhalation vapor (including capsules, effervescent tablets, emulsions, infusion pads, infusion plugs, liquids, ointments, powders, solutions, and tablets), a liquefied dental gas, a medical gas (including compression, low temperature and liquefaction), a nebulizer emulsion, a nebulizer solution, a nebulizer suspension, an oral solution/concentrate for a nebulizer solution, a powder/injection/infusion solution for a nebulizer solution, a powder for a nebulizer suspension, a pressurized inhalant (including emulsions, solutions, and suspensions), a metered inhalation.

The invention also relates to a lyophilized formulation comprising a corynebacterium culture capable of reducing cathepsin expression in a subject and a cryoprotectant.

The invention also relates to a formulation comprising any of the corynebacteria cultures (including combinations) defined above, wherein the formulation is a lyophilized formulation. The lyophilized formulation comprises any of the above corynebacterium cultures (including combinations) and a cryoprotectant.

In embodiments, the cryoprotectant comprises a hydrocolloid polymer (hydrocolloid polymer), and/or a carbohydrate or carbohydrate derivative in the lyophilized formulation. Preferably, in the lyophilized formulation, the cryoprotectant comprises a hydrocolloid polymer, and a carbohydrate or carbohydrate derivative.

Preferably, in the lyophilized formulation, the carbohydrate or carbohydrate derivative is selected from trehalose, sucrose, glucose, lactose, mannitol, sorbitol, inulin, maltodextrin and isomaltulose, preferably from maltodextrin and isomaltulose. Preferably, in the lyophilized formulation, the carbohydrate is isomaltulose.

Preferably, in the lyophilized formulation, the carbohydrate or carbohydrate derivative is maltodextrin at a concentration of 0.1g/100mL to 3g/100mL, preferably at a concentration of 0.5g/100mL to 2.5g/100mL, or isomaltulose at a concentration of 0.1g/100mL to 3g/100mL, preferably at a concentration of 0.5g/100mL to 2.5g/100 mL. Preferably, in the lyophilized formulation, the carbohydrate is isomaltulose at a concentration of 0.5g/100mL to 2.5g/100mL, more preferably at a concentration of 0.5g/100 mL.

Preferably, in the lyophilized formulation, the hydrocolloid polymer is selected from hydroxypropyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, guar gum, carrageenan and xanthan gum, preferably from kappa-carrageenan (KAPPA CARRAGEENAN) and xanthan gum. Alternatively, the hydrocolloid polymer is a pH-adjusting hydrocolloid polymer (e.g., alginic acid/alginate, polygalactomannuronic acid (poly-galactomannuronic acid)), in which case a buffer is added to the formulation.

Preferably, in the lyophilized formulation, the hydrocolloid polymer is kappa-carrageenan in a concentration of 0.05g/100mL to 1g/100mL, preferably in a concentration of 0.1g/100mL to 0.5g/100mL, or xanthan gum in a concentration of 0.01g/100mL to 0.5g/100mL, preferably in a concentration of 0.05g/100mL to 0.25g/100 mL. Preferably, in the lyophilized formulation, the hydrocolloid polymer is xanthan gum at a concentration of 0.05g/100mL to 0.25g/100mL, more preferably at a concentration of 0.05g/100 mL. Preferably, in the lyophilized formulation, the hydrocolloid polymer is kappa-carrageenan at a concentration of 0.1g/100mL to 0.5g/100mL, more preferably at a concentration of 0.1g/100 mL.

In a preferred embodiment, the lyophilized formulation comprises a corynebacterium culture comprising four corynebacterium strains selected from the group consisting of:

(Iii) Corynebacterium crowded SU004, corynebacterium tuberculosis SU005, corynebacterium propionicum SU007 and corynebacterium crowded SU008, or any derivative, variant or mutant thereof capable of reducing expression of cathepsin, and

Comprising a carbohydrate or carbohydrate derivative, and a hydrocolloid polymer;

preferably, wherein the carbohydrate or carbohydrate derivative is maltodextrin or isomaltulose, and wherein the hydrocolloid polymer is kappa-carrageenan or xanthan gum.

Preferably, the lyophilized formulation comprises any of the corynebacterium cultures (including combinations) defined above, wherein the corynebacterium cultures comprise 10 ³ CFU/mL to 10 ¹¹ CFU/mL of corynebacterium strain. Preferably, the corynebacterium culture comprises 10 ⁴ CFU/mL to 10 ¹¹ CFU/mL, preferably 10 ⁵ CFU/mL to 10 ¹¹ CFU/mL, Preferably 10 ⁶ CFU/mL to 10 ¹¹ CFU/mL, preferably 10 ⁷ CFU/mL to 10 ¹¹ CFU/mL, Preferably 10 ⁸ CFU/mL to 10 ¹¹ CFU/mL, preferably 10 ⁸ CFU/mL to 10 ¹⁰ CFU/mL, Preferably 10 ⁹ CFU/mL to 10 ¹¹ CFU/mL, Preferably 10 ⁹ CFU/mL to 10 ¹⁰ CFU/mL, more preferably 10 ⁸ CFU/mL or 10 ⁹ CFU/mL. Preferably, the corynebacterium culture comprises at least two, at least three or four corynebacterium strains, wherein the culture comprises 10 ⁴ CFU/mL to 10 ¹¹ CFU/mL of each strain. More preferably, the corynebacterium culture comprises 10 ⁷ CFU/mL to 10 ¹¹ CFU/mL, preferably 10 ⁸ CFU/mL to 10 ¹¹ CFU/mL, Preferably 10 ⁸ CFU/mL to 10 ¹⁰ CFU/mL, preferably 10 ⁹ CFU/mL to 10 ¹¹ CFU/mL, Preferably 10 ⁹ CFU/mL to 10 ¹⁰ CFU/mL, preferably 10 ⁸ CFU/mL or 10 ⁹ CFU/mL.

In another aspect, the invention relates to a kit comprising any of the above lyophilized formulations and a suitable reconstitution agent (e.g., ringer's solution). A reconstitution agent (reconstitution agent) is used to reconstitute the lyophilized formulation into a formulation for the upper respiratory tract of a subject. Preferably, a reconstitution agent is used to reconstitute the lyophilized formulation into a nasal spray. The reconstitution agent is preferably a ringer's solution.

The present invention relates to a corynebacterium culture capable of reducing expression of a cathepsin in a subject, the corynebacterium culture being for use in medicine.

The present invention relates to corynebacterium cultures or lyophilized formulations for use in preventing or treating an infection by a respiratory virus in a subject, said corynebacterium cultures being capable of reducing expression of a cathepsin in the subject. In another embodiment, the invention relates to a corynebacterium culture or lyophilized formulation for use in preventing infection of an enveloped respiratory virus in a subject, wherein the virus enters cells of the subject using a cathepsin entry pathway, and wherein the corynebacterium culture is capable of reducing expression of a cathepsin in the subject. Preferably, the present invention relates to a corynebacterium culture or a lyophilized formulation for use in preventing or treating infection of an enveloped respiratory virus in a subject, said corynebacterium culture being capable of reducing expression of a cathepsin in the subject. The enveloped respiratory virus is a DNA or RNA virus, preferably an RNA virus. Preferably, the present invention relates to a corynebacterium culture or lyophilized formulation for use in preventing or treating an infection by a coronavirus in a subject, said corynebacterium culture being capable of reducing expression of a cathepsin in the subject, wherein the coronavirus is preferably SARS-CoV-2, more preferably mainly SARS-CoV-2 variant, most preferably SARS-CoV-2 omnique variant, by the cathepsin B/L entry pathway.

The invention also relates to a corynebacterium culture or lyophilized formulation for use in preventing or treating an infection of a respiratory virus, preferably an enveloped respiratory virus, more preferably a coronavirus, most preferably a SARS-CoV-2 virus in a subject, said corynebacterium culture being capable of reducing expression of a cathepsin in the subject, and optionally also capable of

-Reducing ACE2 expression in cells of a subject, and/or

-Reducing expression of TMPRSS2 in cells of a subject.

-Reducing ACE2 expression in cells of a subject, and/or

-Reducing expression of TMPRSS2 in cells of a subject, and/or

-Inhibiting the binding of ACE2 receptor to spike (S) proteins of viruses.

In an alternative embodiment, the invention relates to a corynebacterium culture (including combinations) or lyophilized formulation for use in thermostating the upper respiratory tract of a subject against pathogenic respiratory RNA viruses of said subject, said corynebacterium culture being capable of reducing expression of a cathepsin in cells of the subject. In another embodiment, the corynebacteria culture (including combinations) or lyophilized formulation is used to thermostate the upper respiratory tract of a subject against an enveloped respiratory virus, preferably a coronavirus, more preferably a SARS-CoV-2 virus.

In a preferred embodiment, the corynebacterium culture or lyophilized formulation is applied to the upper respiratory tract of a subject.

In embodiments, the corynebacterium culture further includes a nucleotide sequence encoding a lipase. Preferably, the corynebacterium culture is also capable of producing lipase.

The subject is a vertebrate subject selected from the group consisting of fish, amphibians, reptiles, birds and mammals, preferably selected from the group consisting of reptiles, birds and mammals. In embodiments, the subject is a warm subject selected from birds and mammals. Preferably, the subject is a mammalian subject, preferably a human subject.

In particular, the subject is a mammal or an avian (avian), preferably a mammal. In particular, the subject is a mammalian subject, preferably a farm animal, livestock or human, in particular a human subject.

Preferably, the pathogenic respiratory RNA virus or enveloped respiratory virus is a coronavirus. Preferably, the virus is SARS coronavirus. Preferably, the virus is selected from SARS-CoV-1, SARS-CoV-2 and MERS. Preferably, the coronavirus is SARS-CoV-2. More preferably, the coronavirus is a variant of SARS-CoV-2 that uses the cathepsin pathway of entry. More preferably, the coronavirus is a variant of SARS-CoV-2 that uses primarily the cathepsin B/L entry pathway. Most preferably, the coronavirus is the SARS-CoV-2 omnikon variant.

Preferably, the pathogenic respiratory RNA virus or enveloped respiratory virus is a virus that utilizes a cathepsin entry pathway to enter cells of a subject.

In particular, the coryneform bacterium culture is any of the coryneform bacterium cultures (including combinations) defined above.

In a preferred embodiment, the corynebacterium culture or the lyophilized preparation is used for preventing respiratory tract infection, respiratory tract colonization or respiratory tract disease. In a preferred embodiment, the corynebacterium cultures or lyophilized preparations are used for the prophylaxis of diseases caused by infectious agents which are host in the respiratory tract, preferably the upper respiratory tract.

In a preferred embodiment, the corynebacterium culture or lyophilized preparation is used for preventing diseases caused by respiratory RNA virus infection. Respiratory viruses cause respiratory infections.

In a preferred embodiment, the corynebacterium culture or lyophilized formulation used according to the present invention is used against enveloped coronaviruses with spike proteins, and wherein the corynebacterium culture is capable of reducing the expression of cathepsin. In a preferred embodiment, the corynebacterium cultures or lyophilized preparations used according to the invention are used against enveloped coronaviruses with spike proteins, wherein the corynebacterium cultures are capable of reducing the expression of cathepsins and are also capable of

-Reducing the expression of a receptor protein, preferably ACE2, in a host cell, preferably an epithelial cell, and/or

Reducing the expression of serine proteases in host cells, preferably epithelial cells, which activate spike proteins, preferably TMPRSS2, and optionally,

Reducing/inhibiting interactions between spike proteins and receptor proteins (preferably ACE 2) in host cells.

In a preferred embodiment, the RNA virus is an enveloped coronavirus with spike proteins (preferably a coronavirus selected from the group consisting of SARS-CoV-1, SARS-CoV-2 and MERS), and reducing expression of a cathepsin enzyme comprises reducing mRNA expression of the cathepsin enzyme and/or reducing expression of a cathepsin enzyme at a protein level. In a preferred embodiment, the RNA virus is an enveloped coronavirus with spike proteins (preferably a coronavirus selected from the group consisting of SARS-CoV-1, SARS-CoV-2 and MERS), and reducing expression of a cathepsin comprises reducing mRNA expression of the cathepsin and/or reducing expression of a cathepsin at a protein level, and optionally,

Reducing expression of a receptor protein, preferably ACE2, in a host cell comprises reducing mRNA expression of the receptor protein and/or reducing expression of the receptor protein at the protein level, and/or

Reducing the expression of serine protease that activates spike proteins (preferably TMPRSS 2) in a host cell comprises reducing mRNA expression of the receptor protein and/or reducing expression of the receptor protein at the protein level.

In a preferred embodiment of the invention, the corynebacterium cultures (including combinations) or lyophilized formulations are used for the treatment or prophylaxis of respiratory diseases of the upper respiratory tract. In a preferred embodiment, the corynebacterium cultures (including combinations) or lyophilized formulations are used for the treatment or prevention of respiratory infections, respiratory colonisation or respiratory diseases. Preferably, the respiratory tract infection, respiratory tract colonization or respiratory tract disease is of the upper respiratory tract. In a preferred embodiment, cultures (including combinations) or lyophilized formulations of corynebacteria are used to prevent diseases caused by infectious agents that are host in the respiratory tract, preferably the upper respiratory tract.

Preferably, the corynebacterium culture (including combination) or the lyophilized formulation is administered to the upper respiratory tract of the subject,

Preferably to the nasal cavity and the pharynx, preferably to the nasopharynx, the oropharynx, the oro/mesopharynx (oro/mesopharynx) and/or the laryngopharynx, in particular to the nasopharynx,

Preferably in the form of drops, sprays, inhalable formulations (inhalable formulation) or ointments.

Preferably, the corynebacterium culture (including combination) or lyophilized formulation is used for the treatment (including prevention) or preferably the prevention of respiratory diseases caused by coronavirus (preferably SARS virus) in a subject. Preferably, the coronavirus is selected from the group consisting of SARS-CoV-1, SARS-CoV-2 and MERS. More preferably, the coronavirus is a variant of SARS-CoV-2, preferably a variant of SARS-CoV-2 armstrong, using mainly the cathepsin B/L entry pathway. Preferably, the respiratory disease is COVID-19 disease. Preferably, the subject is a mammalian subject, preferably a human subject.

Preferably, the corynebacterium cultures or lyophilized preparations are used for the prevention of symptoms of diseases caused by infectious agents which are host in the respiratory tract, preferably the upper respiratory tract. Preferably, the corynebacterium culture or the lyophilized preparation is used for preventing symptoms of a disease caused by coronavirus. These symptoms include fever or chills, coughing, shortness of breath or dyspnea, fatigue, muscle or body pain, headache, loss of taste or smell, sore throat, nasal obstruction or runny nose, nausea, vomiting and diarrhea.

In a preferred embodiment, the corynebacterium culture or combination thereof, or lyophilized formulation thereof, is administered to the subject for about 5 to 7 days, once or twice a day. In a preferred embodiment, the corynebacterium culture or combination thereof, or lyophilized formulation thereof, is administered to the subject twice daily, preferably in the morning and evening. In the case of administration into the throat (e.g., by a mouthwash or mouthwash formulation), the corynebacterium culture or combination thereof, or lyophilized formulation thereof, is administered to the subject after a meal.

The invention also relates to a method for preventing infection of an enveloped respiratory virus, preferably a coronavirus, more preferably SARS-CoV-2 omnikow variety, the method comprising:

Administering to a subject in need thereof a corynebacterium culture as defined above, or a formulation as defined above, or a composition as defined above.

Preferably, the method comprises administering a corynebacterium culture comprising at least one or at least two, preferably at least three, more preferably four corynebacterium strains selected from the group consisting of

A strain SU004 of corynebacterium crowded with accession number NCAIM P (B) 001500, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression, deposited at NCAIM (national institute of agriculture and industrial microbiology, institute of food science and technology, university of agriculture and life sciences, H-1118, budapest, somnolway 14-16, hungarian) at 1 month 17 of 2022;

a corynebacterium propionicum SU005 strain deposited under accession number NCAIM P (B) 001501 at time 27, 4, 2022, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression;

Preferably, the method comprises administering a lyophilized formulation as described above.

Abbreviations (abbreviations)

ACE2 angiotensin converting enzyme 2

CFU colony forming units

COVID-19 coronavirus diseases 2019

EMEM eagle minimum basal medium

GAPDH 3-phosphoglyceraldehyde dehydrogenase

MALDI-TOF matrix assisted laser Desorption ionization time of flight

MERS middle east respiratory syndrome

NCAIM national agricultural and Industrial microbiological Collection center

PBS phosphate buffered saline

PCR polymerase chain reaction

RRNA ribosomal RNA

SARS-CoV-2 severe acute respiratory syndrome coronavirus 2

TMPRSS2 transmembrane protease/serine subfamily Member 2

Definition of the definition

As used herein, a bacterium is "viable" if it is capable of proliferating (growth "if the number of viable bacteria increases) under conditions that include appropriate media and nutrients, as well as appropriate temperatures.

As used herein, "culture" refers to the culture of bacteria in an artificial environment (i.e., in vitro). Culturing and thus the culture may include maintaining a viable form of the bacteria and/or proliferation of the bacteria. Thus, "culture" includes bacteria in a culture medium in which the bacteria are maintained in a viable form, including a dry (lyophilized) form, a culture on a solid medium, and a liquid form. The term culture also includes the meaning of the term "consortium (consortium)". As used herein, a "culture" may include only one strain, or may include more than one strain. Thus, as used herein, the term "corynebacterium culture" is a culture that includes a viable corynebacterium. The culture may comprise only one corynebacterium strain, or it may contain at least two (e.g., at least three, preferably four) different corynebacterium strains, unless otherwise indicated.

Preferably, the corynebacterium strain or corynebacterium culture "capable of decreasing expression of a protein" is a corynebacterium strain or corynebacterium culture, respectively, which decreases expression of the protein in mammalian cells in vitro co-culture of the mammalian cells and corynebacterium. Preferably, expression is determined by measuring the mRNA level of the protein, and protein expression is "reduced" when the mRNA level of the protein in the co-cultured mammalian cells is low compared to the mRNA level of the protein measured in a control mammalian cell (without bacteria). Preferably, the mammalian cell is a human cell, optionally a human colon epithelial cell line Caco-2. In an embodiment, the corynebacterium strain or corynebacterium culture capable of decreasing expression of a cathepsin is a corynebacterium strain or corynebacterium culture, respectively, that decreases relative expression of cathepsin mRNA after 24 hours of incubation with Caco-2 cells.

The term "formulation" relates to the composition of matter, which includes at least one biologically active ingredient (preferably a medically active ingredient), an "active agent" and at least one other substance, e.g., a medium and/or excipient suitable for administration to a subject (e.g., mammal or bird), or both. Preferred formulations are those for the upper respiratory tract of the subject, preferably a thermostated subject. The terms "formulation" and the term "composition" are used interchangeably in the context of the present invention.

The term "administering" as used herein shall include the route by which the formulation of the invention is introduced or applied to a subject in need thereof to exert its intended function. In particular, administration as used herein relates to administration to the upper respiratory tract of a subject (e.g., mammal or bird).

The term "subject" as used herein shall mean a warm-blooded animal (mammalian or avian, preferably mammalian) subject, particularly a human. In particular, the medical use of the invention or the corresponding method of treatment is applicable to subjects in need of control or treatment of a condition or disease caused by an infectious agent that hosts the respiratory tract, preferably the upper respiratory tract.

The term "patient" includes subjects receiving healthcare, such as subjects receiving prophylactic (prophylactic) or therapeutic treatment.

As used herein, "prophylactic treatment" or "prevention" or "prophylaxis" includes measures on or with respect to a patient to prevent infection in a patient, including reducing the rate of infection or the severity of infection (number of infected cells). The patient may be an uninfected patient, or a patient who has been infected but is prevented from further infection or reinfection by the means. Preferably, the controlling or preventing comprises at least one or more or periodic applications to the target site, i.e. the upper respiratory tract, in particular the epithelium thereof.

As used herein, "treating (treating, treatment)", "treating" refers to ameliorating, alleviating, reducing, alleviating symptoms of a disease or disorder, such as symptoms of an infection. "treating", "treating" or "treating" also includes reducing the number of viruses that can enter cells of a patient.

"Upper respiratory tract" refers to the portion of the respiratory system that is located over the sternal angle (outside the chest), above the vocal cords, or above the cricoid cartilage. Preferably, the throat is sometimes included in the upper respiratory tract. The upper respiratory tract includes the nasal and paranasal sinuses, the pharynx (including nasopharynx/upper pharynx, oropharynx/middle pharynx and laryngopharynx/hypopharynx), and preferably includes the larynx. In a preferred embodiment, "upper respiratory tract" refers to the nasal cavity and nasopharynx.

The expression "infection" means unwanted proliferation of a virus within a cell of a subject.

The terms "comprises" or "comprising" are to be interpreted as having a non-exhaustive meaning and are to be interpreted as having any means to add or relate to a further feature or method step or component, including the recited feature or method step or component. Such terms may be limited to "consisting essentially of or" consisting essentially of, which is to be understood as consisting of the mandatory features or method steps or components listed in the list (e.g., in the claims) while allowing for additional inclusion of other features or method steps or components that do not materially affect the basic characteristics of the use, method, composition, or other subject matter.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents and should be construed to cover the meaning of "one or more" unless the context clearly dictates otherwise. In general, it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

"Formulation for the upper respiratory tract" includes any formulation delivered to the upper respiratory tract of a subject, including any of the nasal, nasopharyngeal, ocular nasal, oral mucosa, and pulmonary formulations listed above.

The term "Corynebacterium crowded SU001" means the Corynebacterium crowded SU001 strain deposited on NCAIM (national institute of agriculture and Industrial microorganisms, institute of food science and technology, university of Hungary agriculture and life sciences, H-1118, budapest, som Louis 14-16, hungary) accession No. NCAIM P (B) 001495 at 22, 2021.

The term "Corynebacterium propionicum SU002" refers to the strain Corynebacterium propionicum SU002 deposited on month 22 of 2021 under accession number NCAIM P (B) 001496.

The term "Corynebacterium propionicum SU003" refers to the strain Corynebacterium propionicum SU003 deposited on the year 22 of 2021 under the accession number NCAIM P (B) 001497.

The term "Corynebacterium crowded SU004" means a Corynebacterium crowded SU004 strain deposited with NCAIM (national institute of agriculture and Industrial microorganisms, institute of food science and technology, university of Hungary agriculture and life sciences, H-1118, budapest, som Louis 14-16, hungary) under accession No. NCAIM P (B) 001500 at day 1 of 2022.

The term "Corynebacterium tuberculosis SU005" refers to the strain Corynebacterium tuberculosis SU005 deposited on the accession number NCAIM P (B) 001501 at 2022, 4, 27.

The term "Corynebacterium crowded SU006" refers to the Corynebacterium crowded SU006 strain deposited on NCAIM under accession number NCAIM P (B) 001502, month 4, and 27.

The term "Corynebacterium propionicum SU007" refers to the strain Corynebacterium propionicum SU007 deposited on month 4 and 27 of 2022 under the accession number NCAIM P (B) 001504.

The term "Corynebacterium crowded SU008" means the Corynebacterium crowded SU008 strain deposited on the NCAIM under accession number NCAIM P (B) 001505, 4.27.

The term "Corynebacterium crowded SU009" refers to the strain Corynebacterium crowded SU009 deposited with NCAIM under accession number NCAIM P (B) 001506 at 5.25.

The term "Corynebacterium propionicum SU010" refers to the strain Corynebacterium crowded SU010 deposited on the accession number NCAIM P (B) 001507, 5, 25 of 2022.

Drawings

FIG. 1. Temperature during lyophilization.

FIG. 2A effect of different Corynebacterium strains (SU 001-SU 010) on the relative mRNA expression of cathepsins.

FIG. 2B effect of different Corynebacterium strains (SU 004-SU 010) on the relative mRNA expression of cathepsins.

FIG. 3 effects of different Corynebacterium strains (SU 001-SU 010) on the relative mRNA expression of cathepsin, ACE2 and TMPRSS 2.

FIG. 4 effects of different Corynebacterium strains (SU 004-SU 010) on the relative mRNA expression of cathepsin, ACE2 and TMPRSS 2.

FIG. 5 effect of different Corynebacterium strains (SU 001-SU 010) on ACE2-S1 protein binding activity (%).

FIG. 6 effect of different Corynebacterium strains (SU 004-SU 010) on ACE2-S1 protein binding activity (%).

FIG. 7 effect of K1 combination on relative expression of cathepsin, ACE2, TMPRSS 2.

FIG. 8 effect of K2 combination on relative expression of cathepsin, ACE2, TMPRSS 2.

FIG. 9. Effect of K3 combination on relative expression of cathepsin, ACE2, TMPRSS 2.

FIG. 10 effect of xanthan on ACE2, TMPRSS2 and cathepsin expression in combination with K1, K2 and K3.

FIG. 11 effect of carrageenan on ACE2, TMPRSS2 and cathepsin expression in combinations of K1, K2 and K3.

Detailed Description

The inventors determined that the normal flora of the nasopharynx affects the susceptibility to SARS-CoV-2 infection.

The presence of corynebacteria in the nasopharyngeal flora can reduce an individual's susceptibility to SARS-CoV-2 infection by a variety of mechanisms.

Corynebacterium strains can reduce an individual' S susceptibility to SARS-CoV-2 infection by a variety of mechanisms, by downregulating SARS-CoV-2 receptor, ACE2 and TMPRSS2, by inhibiting the binding of S1 protein to ACE2 receptor, and by lipase production. All of these mechanisms work together by inhibiting the binding of SARS-CoV-2 to host cells and by acting on the lipid envelope of the SARS-CoV-2 virus. These mechanisms can reduce the susceptibility of an individual to enveloped respiratory viral infection, particularly SARS-CoV-2.

The purpose of the present inventors is as follows. Their objective was to determine the effect of corynebacterium strains on the cathepsin pathway initiated by the omnirange variant in human cells. After testing the action of corynebacteria, they deposited several strains. Their objective was to determine the ideal combination of corynebacterium strains and also their prophylactic effect on SARS-CoV-2 infection for the purpose of probiotic use. Furthermore, their aim was to identify vectors for corynebacterium strains suitable for probiotic use, in addition to which the corynebacterium strains can retain their effectiveness against SARS-CoV-2 and their viability.

Whereas the entry of the SARS-CoV-2 virus into cells not only through ACE2 and TMPRSS2, but also the protease pathway has become increasingly important, particularly in the case of the Omikovia variant, the inventors isolated Corynebacterium strains and examined the effect of bacterial co-culture on protease expression. After 24 hours incubation of the isolated corynebacterium strain with Caco-2 cells, the relative expression of the cathepsin mRNA is significantly reduced to a different extent, thereby contributing to reduced ACE2 expression, reduced TMPRSS2 expression, ACE2-S protein inhibition and the presence of lipase, and reduced entry of SARS-CoV-2 virus, preferably the SARS-CoV-2 omnikow mutant, into the cells.

The inventors have demonstrated that corynebacteria are able to down-regulate cathepsin protein expression in human cells. The protease pathway is an important entry for the SARS-CoV-2 virus, and the currently widely spread variety of Omikovia is mainly entered by the protease pathway.

Of the corynebacteria studied by them, ten strains of corynebacteria have been deposited under the name SU001-SU 010. In preserving strains of Corynebacterium, they consider their effect on cathepsin expression in addition to other effects against SARS-CoV-2 infection.

Combinations K1, K2, K3 of deposited Corynebacterium strains containing four Corynebacterium strains were identified which, in addition to having the advantageous properties unique to Corynebacterium strains, further enhanced the down-regulation of ACE2, TMPRSS2 and cathepsin expression.

The inventors describe that the carrier xanthan does not affect the colony count of the lyophilized corynebacterium combinations K1, K2, K3.

They also describe that the combination of corynebacteria K1, K2, K3 with xanthan gum carrier maintained their viability (availability) and colony count for 14 days at room temperature after lyophilization. Furthermore, they describe that the combination of corynebacteria K1, K2, K3 with the carrier xanthan retains the effect of down-regulating ACE2, TMPRSS2 and cathepsins after lyophilization and may even produce a synergistic improvement.

Thus, the present inventors have unexpectedly found that xanthan gum not only acts as an excipient (carrier), but also as an adjuvant-it improves the effect of the corynebacteria combination, in particular the combination K2 (see fig. 10).

They describe that the carrier kappa-carrageenan does not affect colony counts of the corynebacterium combinations K1, K2, K3 after lyophilization. In addition, they describe that the combination of corynebacteria K1, K2, K3 with kappa-carrageenan vector maintains their viability and colony count for 14 days at room temperature after lyophilization. They also describe that the combination of corynebacteria K1, K2, K3 with the carrier kappa-carrageenan retains the effect of down-regulating ACE2, TMPRSS2 and cathepsins after lyophilization.

The combination of bacterial strains may be reconstituted in the form of a nasal spray or a pharyngeal spray after lyophilization. They retain their known and tested biological activity.

It is an important step to find not only the appropriate excipient for the lyophilized formulation but also the appropriate concentration of excipient.

The carbohydrate or carbohydrate derivative excipient in the lyophilized formulation is a cryoprotectant that protects the corynebacterium culture.

The concentration of hydrocolloid polymer in the lyophilized formulation controls the viscosity of the formulation. The lyophilized formulation should not be a gel but should be capable of forming sufficient blocks. In other words, the formulation should not be too viscous or too dense. Too high a concentration may result in gelation and too low a concentration may result in the formulation failing to form an appropriate mass during lyophilization.

The lyophilized formulation should also have a desirable osmolality range, preferably in the range of 290 to 500mOsmol/kg, more preferably in the range of 300 to 400 mOsm/kg. More preferably, the lyophilized formulation has an osmotic pressure of 320 to 370mOsmol/kg.

Preferably, the pH of the lyophilized formulation is from pH 5 to 7, more preferably from pH 5.5 to 6.5.

Another important aspect is that the lyophilized formulation must be physiologically tolerable and thus the excipients used and their concentrations should be carefully selected.

In the case of nasal sprays, it is also important that the droplet size distribution [Kulkarni&Shaw(2012).Formulation and characterization of nasal sprays.Inhalation Magazine,June 2012]. be larger, the droplets will emerge from the nose, while droplets smaller than 10 microns may enter the nasal cavity further and reach the lungs (which is not the intended delivery site). Therefore, it is necessary to minimize droplets smaller than 10 μm. For example, in a nasal spray, the droplet size may be 30-70 μm, up to 200 μm.

Examples

EXAMPLE 1 Corynebacterium strain

Materials and methods

Bacterial sample collection

Corynebacterium isolates with putative protective effects were collected from uninfected patients despite intimate contact with their COVID positive family members. Nasopharyngeal swab samples of COVID negative patients were inoculated on Columbia blood agar (Biolab, hungary) and incubated at 37℃in a humid atmosphere containing 5% CO ₂. The corynebacterium strain was selected from other bacterial participants using an antibacterial susceptibility test tray containing 50 μg of fosfomycin (Fosfomycin) (Oxoid, sweden). All bacteria cultured were identified by MALDI-TOF method (Bruker Daltonik, germany). Three previously isolated Corynebacterium strains (SU 001-003) were examined for their excellent properties and 7 additional Corynebacterium strains (SU 004-010) were isolated and biologically tested.

Detection of Caco-2 cell ACE2, TMPRSS2 and cathepsin L expression after bacterial Co-culture

The human colon epithelial cell line Caco-2 was previously obtained and used in this study. Caco-2 cells were cultured in EMEM medium (Lonza Bioscience, USA) at 37℃under a humid atmosphere containing 5% CO ₂, with medium changes every two days. No antibiotics were added to allow co-cultivation of the bacteria. Caco-2 cells were cultured alone for four days, and then an overnight culture of Corynebacterium SU001-010 was added. Human Caco-2 cells and bacteria were co-cultured for an additional 24 hours.

Cells were washed with PBS and 0.25% trypsin and centrifuged. Total RNA was isolated using innuPREP RNA Mini kit 2.0 (Analytik Jena GmbH, germany) according to the manufacturer's instructions. RNA concentrations were determined using a NanoDrop 1000 spectrophotometer (sammer feichi technology, usa). RT-PCR tests were performed using 10-100ng RNA using PRIMESCRIPT RT kit (Takara Bio, USA) and the resulting cDNA was amplified on a qTOWER G (Analytik Jena GmbH, germany) instrument in the presence of selected primers.

The primers for ACE2 were 5'-GGG ATC AGA GAT CGG AAG AAG AAA-3' forward (SEQ ID NO: 1) and 5'-AGG AGG TCT GAA CAT CAT CAG TG-3' reverse (SEQ ID NO: 2). The primers for TMPRSS2 were 5'-AAT CGG TGT GTT CGC CTC TAC-3' forward (SEQ ID NO: 3) and 5'-CGT AGT TCT CGT TCC AGT CGT-3' reverse (SEQ ID NO: 4). The primers for cathepsin L are 5'-CTG GTG GTT GGC TAC GGA TT-3' CTSL forward (SEQ ID NO: 5) and 5'-CTC CGG TCT TTG GCC ATC TT-3' reverse (SEQ ID NO: 6). The primers for GAPDH are 5'-CTA CTG GCG CTG GCA AGG CTG T-3' forward (SEQ ID NO: 7) and 5'-GCC ATG AGG TCC ACC ACC CTG CTG-3' reverse (SEQ ID NO: 8).

Relative mRNA expression was calculated by the cyclic threshold change (ΔΔct) method and normalized to the geometric mean of housekeeping gene GAPDH. By calculating the difference between their cts, the basal mRNA levels of ACE2, TMPRSS2 and cathepsins were compared to the levels of housekeeping gene GAPDH.

Statistical analysis:

In ACE2, TMPRSS2 and cathepsin expression studies, the differences between mRNA levels measured in different bacterial co-culture groups were calculated by two-tailed student t-test.

Effect of Corynebacterium on ACE2, TMPRSS2 and cathepsin expression

In the case of SU001-010 strains, the inventors studied how they affected the relative ACE2, TMPRSS2 and cathepsin expression of Caco-2 cells cultured with each strain separately.

Results

Effect of Corynebacterium on cathepsin expression

Whereas the SARS-CoV-2 virus enters the cell not only through ACE2 and TMPRSS2, but the cathepsin pathway becomes increasingly important, especially in the case of the Omikovia variant, we examined the effect of bacterial co-culture on protease expression in the case of the previously isolated Corynebacterium strain. The corynebacterium strain significantly reduced the relative mRNA expression of cathepsins to a different extent after 24 hours incubation with Caco-2 cells (see FIGS. 2A and 2B), thereby contributing to reduced ACE2 expression, reduced TMPRSS2 expression, ACE2-S protein inhibition and lipase presence in addition to the previously described effect of SARS-CoV-2 virus on reduction of the entry of the Omikovia mutant into the cells.

The corynebacterium strain also reduced the relative mRNA expression of ACE2 and TMPRSS2 (see fig. 3 and 4). In addition, corynebacterium strains inhibited the binding of S1 protein to ACE2 receptor (see fig. 5 and 6).

EXAMPLE 2 combination of Corynebacterium strains

Materials and methods

Culture study of Corynebacterium combinations

The present inventors studied how individual corynebacterium strains mutually affect each other in proliferation. Pure bacterial cultures of each bacterial strain were suspended in saline, normalized to turbidity 0.5McFarland, and made up of the same proportion of mixtures containing 4-4 different groups of strains SU 001-010. 100. Mu.L of the mixture was inoculated onto Columbia blood agar (Biolab, hungary) and incubated at 37℃for 48 hours in a humid atmosphere containing 5% CO ₂. Depending on the culture, it may be determined which corynebacterium strains allow other strains to grow in combination with it, depending on the colony morphology. k1-K2-K3, three different combinations can be edited based on successful joint growth. Detection of ACE2, TMPRSS2 and cathepsin L expression in Caco-2 cells after bacterial Co-cultivation

The human colon epithelial cell line Caco-2 was previously obtained and used in this study. Caco-2 cells were cultured in EMEM medium (Lonza Bioscience, USA) at 37℃under a humid atmosphere containing 5% CO2, with medium changes every two days. No antibiotics were added to allow co-cultivation of the bacteria. Caco-2 cells were cultured alone for four days, and then overnight cultures of combinations of K1, K2 or K3 Corynebacterium strains were added. Human Caco-2 cells and bacteria were co-cultured for an additional 24 hours.

Statistical analysis:

Effects of different Corynebacterium combinations (K1, K2, K3) on their respective biological effects ACE2 expression, TMPRSS2 expression and cathepsin expression

In the case of the K1, K2 and K3 combinations, the inventors studied how they affected the relative ACE2, TMPRSS2 and cathepsin expression of Caco-2 cells cultured with each combination separately.

Results

During the course of the present inventors' studies, in order to examine the effect of Corynebacterium on the beneficial properties of previously individually examined strains, they were cultured 4-4 strains together and examined their combined effect.

A combination of (i) a combination K1 comprising Corynebacterium strains SU004, SU005, SU009 and SU010, (ii) a combination K2 comprising Corynebacterium strains SU004, SU005, SU006 and SU010, and (iii) a combination K3 comprising Corynebacterium strains SU004, SU005, SU007 and SU008 was used.

In all three combinations, their results show that the different corynebacterium strains used in the combination do not affect each other's individual effects during co-cultivation, and surprisingly, in some cases, an effect of enhancing the beneficial results is observed.

Effects of K1 combinations on mRNA expression of cathepsin, ACE2, TMPRSS2

The K1 combination contains two crowded corynebacterium strains SU004, SU009, a tuberculosis corynebacterium stearate SU005 and a propionicum SU010. When the K1 combination is used, the effect of the corynebacterium combination on cathepsin, ACE2 and TMPRSS2 expression is approximately the average of the individual effects of each bacterium (see fig. 7).

Effect of K2 combination on mRNA expression of cathepsin, ACE2 and TMPRSS2

The K2 combination contains two crowded corynebacterium strains SU004, SU006, a tuberculosis corynebacterium stearate SU005 and a propionicum SU010. When the K2 combination is used, the effect of the corynebacterium combination on cathepsin and TMPRSS2 expression is approximately the average of the effects of each bacterium alone, and in the case of ACE2 the relative expression rate is similar to SU004 strain (see fig. 8). Effect of K3 combination on mRNA expression of cathepsin, ACE2, TMPRSS2

K3 combination contains two crowded corynebacterium strains SU004, SU008, one Corynebacterium tuberculosis SU005 and one Corynebacterium propionicum SU007. When a K3 combination is used, the effect of the coryneform combination on cathepsin and ACE2 expression is very similar to the values of the most potent strains, however, in the case of TMPRSS2 expression, the K3 combination has a greater effect than each bacterium alone, and in the case of TMPRSS2 expression down-regulation, there is a synergy (see fig. 9).

EXAMPLE 3 lyophilized formulations

Materials and methods

Research on the frame formation properties of different hydrocolloid polymer and carbohydrate containing solutions for lyophilization

The following excipients and excipient combinations were tested (1) 0.05g xanthan gum/100 mL and vitamin A, (2) 0.1g/100mL kappa-carrageenan and 0.5g/100mL maltodextrin and vitamin A, (3) 0.1g/100mL kappa-carrageenan and 0.1g/100mL maltodextrin and vitamin A, (4) 0.05g xanthan gum/100 mL and 0.1g/100mL maltodextrin and vitamin A, (5) 0.05g xanthan gum/100 mL and 0.5g/100mL maltodextrin and vitamin A, (6) (also known as A solution) 0.1g/100mL kappa-carrageenan and 0.5g/100mL isomaltulose, (7) (also known as B solution) 0.05g xanthan gum/100 mL and 0.5g/100mL isomaltulose.

Early experiments showed that vitamin a reduced the effect of the excipient combination. Thus, the inventors excluded vitamin a from the excipient combination and selected combinations (6) and (7) (also referred to as a solution and B solution, respectively) for subsequent experiments.

Freeze-drying

One of the objectives of the present inventors was to formulate nasal spray probiotics and to examine the viability of the bacterial strains after two weeks of lyophilization and storage.

Stabilization of the bacterial strain in the dry solid state was performed using a freeze dryer (Scanvac Coolsafe 110-04 freeze dryer LaboGene ^TM, lingerie, denmark).

The lyophilization process generally consists of three steps, namely, freezing, primary drying and secondary drying. During freezing, water will crystallize and then the frozen ice will be removed by sublimation. In the final secondary drying step, the remaining moisture is removed to reach the final moisture content.

As cryoprotectants, hydrocolloid polymers and carbohydrates are used for lyophilization of probiotics, which after lyophilization has a satisfactory survival rate of the microorganisms. Typically, carbohydrates (e.g., trehalose, sucrose, glucose, lactose, maltodextrin) and polymers are used as cryoprotectants .[Meng XC,Stanton C,Fitzgerald GF,Daly C,Ross RP.(2008).Anhydrobiotics:The challenges of drying probiotic cultures.Food Chem.,106:1406-1416.]

It is technically important to combine these cryoprotectant compounds with a composition suitable for lyophilization to ensure bacterial viability, characterized by being reconfigurable into a stable physical form of sprayable liquid having the appropriate viscosity, pH (5-7) and osmolarity (300-400 mosm). Preferably, the pH of a suitable composition is from pH 5.5 to 6.5 and the osmotic pressure of a suitable composition is from 320 to 370mOsmol/kg.

Hydrocolloid polymers and carbohydrate derivatives are used as protective agents during the formation of the lyophilisate. 10 ⁸ CFU/mL of ringer solution from each bacterial strain was frozen at-70℃and stored for 24 hours. Lyophilization begins with a first drying at a temperature between-30 ℃ and-45 ℃ for a duration of no more than 16 hours. The secondary drying is performed until the proper moisture content is not more than 5%, and the sample temperature is kept not more than 10 ℃. The entire lyophilization process takes 24-36 hours .[Zayed,G.and Y.H.Roos.2004.Influence of trehalose and moisture content on survival of Lactobacillus salivarius subjected to freeze-drying and storage.Process Biochem.9:1081–1086.] and is further described in FIG. 1.

Shelf-life studies of different solutions comprising hydrocolloid polymers and carbohydrates

Two different solutions were prepared in ringer's solution containing (A) 0.1g/100mL kappa-carrageenan and 0.5g/100mL isomaltulose (A solution), or (B) 0.05g xanthan/100 mL and 0.5g/100mL isomaltulose (B solution).

Fractions of 2mL of A or B solution were inoculated with 10 ⁸ CFU (colony forming units)/mL of SU001-010 strain or with a combination of K1, K2, K3 strains. After 7 and 14 days of storage at room temperature, 100 μl of the solutions from the different species were inoculated onto columbia blood agar (Biolab, hungary) to determine the number of colony forming units as the number of viable corynebacterium strains.

The human colon epithelial cell line Caco-2 was previously obtained and used in this study. Caco-2 cells were cultured in EMEM medium (Lonza Bioscience, USA) at 37℃under a humid atmosphere containing 5% CO ₂, with medium changes every two days. No antibiotics were added to allow co-cultivation of the bacteria. Caco-2 cells were cultured alone for four days, and then overnight cultures of K1, K2 or K3 combinations in solution of Corynebacterium SU001-010 or A or B were added. Human Caco-2 cells and bacteria were co-cultured for an additional 24 hours.

Statistical analysis:

Effect of different hydrocolloid polymer and carbohydrate containing solutions on individual biological effects of SU001-010 strains or K1-K3 compositions on ACE2 expression, TMPRSS2 expression and cathepsin expression

In the case of SU001-SU010 strain, or K1, K2 and K3 in combination in a or B solution, the inventors studied how they affect the relative ACE2, TMPRSS2 and cathepsin expression with Caco-2 cells cultured with each strain or combination, respectively.

Results

Effect of different excipients (xanthan and kappa-carrageenan) on the combination of Corynebacterium strains K1, K2, K3

Various combinations K1, K2 and K3 found to be effective were studied with different excipients to determine the effect of the excipients on the bacterial combination. The inventors further investigated the effect of two excipients, xanthan gum, anionic polysaccharide and kappa-carrageenan, on the bioactivity of corynebacterium strains.

Investigation of the Effect of lyophilization on Corynebacterium growth

The inventors further examined the effect of lyophilization on different corynebacterium strains and on the combination of K1, K2 and K3, respectively. After lyophilization, the inventors determined colony counts for each bacteria and colony counts for combinations K1, K2, K3, respectively. Their findings indicate that all bacteria and combinations thereof retain their original bacterial numbers.

TABLE 1 effects of lyophilization

They further analyzed the survival rate of corynebacterium strains, and the corynebacterium strains survived for 14 days in all combinations K1, K2 and K3.

Influence of xanthan on mRNA expression of cathepsins, ACE2 and TMPRSS2

The inventors further analyzed the effect of xanthan alone and mixtures in combination with K1, K2 and K3 on the relative expression of ACE2, TMPRSS2 and cathepsins. However, xanthan itself reduced the expression of ACE2, TMPRSS2 and cathepsins to a lesser extent than the combination of K1, K2 and K3.

Interestingly, the use of K1 and K3 in combination with xanthan did not affect their original effects, however, surprisingly, in the case of K2 combination, a synergistic effect with xanthan was observed, as the presence of the combination of K2 and xanthan down-regulates ACE2, TMPRSS2 and cathepsin expression to a greater extent than alone (see fig. 10).

Effect of kappa-carrageenan on mRNA expression of cathepsins, ACE2 and TMPRSS2

They further analyzed the effect of kappa-carrageenan alone and in combination with K1, K2 and K3 mixtures on the relative expression of ACE2, TMPRSS2 and cathepsins. Kappa-carrageenan alone also significantly reduced ACE2, TMPRSS2 and cathepsin expression itself.

However, interestingly, no additional effect was produced in the case of K1 binding to kappa-carrageenan. Surprisingly, a synergistic effect was observed in the case of K2 and kappa-carrageenan, which when used together down-regulates the expression of all ACE2, TMPRSS2 and cathepsins to a greater extent. In the case of K3, kappa-carrageenan did not affect the down-regulating effect of ACE2, TMPRSS2 and cathepsin expression caused by K3 combinations (see fig. 11).

Industrial applicability

The corynebacterium culture or a combination thereof or a lyophilized preparation thereof according to the present invention is used for treating or preventing respiratory diseases of the upper respiratory tract. The corynebacterium cultures according to the present invention or combinations thereof or lyophilized preparations thereof are also used for the treatment or prevention of respiratory infections, respiratory colonisation or respiratory diseases. Preferably, the respiratory tract infection, respiratory tract colonization or respiratory tract disease is of the upper respiratory tract. The corynebacterium cultures according to the present invention or combinations thereof or lyophilized preparations thereof are used for preventing diseases caused by infectious agents of hosts in the respiratory tract, preferably the upper respiratory tract.

Reference to the literature

Kulkarni&Shaw(2012).Formulation and characterization of nasal sprays.Inhalation Magazine,June 2012Lappan&Peacock,2019.Corynebacterium and Dolosigranulum:future probiotic candidates for upper respiratory tract infections.Microbiology Australia,40(4),172–177.

De Maio F,Posteraro B,Ponziani FR,Cattani P et al.(2020).Nasopharyngeal Microbiota Profiling of SARS-CoV-2 Infected Patients.Biological Procedures Online 22,18.doi:10.1186/s12575-020-00131-7.

Man WH,de Steenhuijsen Piters WA,Bogaert D(2017).The microbiota of the respiratory tract:Gatekeeper to respiratory health.Nature Reviews Microbiology,15(5),259–270.doi:10.1038/nrmicro.2017.14.

Meng XC,Stanton C,Fitzgerald GF,Daly C,Ross RP.(2020).Anhydrobiotics:The challenges of drying probiotic cultures.Food Chem.,106:1406-1416.

Mostafa HH,Fissel JA,Fanelli B,Bergman Y et al.(2020).Metagenomic next-generation sequencing of nasopharyngeal specimens collected from confirmed and suspect COVID-19 patients.MBio,11(6),1–13.doi:10.1128/mBio.01969-20.

Nardelli C,Gentile I,Setaro M,Di Domenico C,et al.(2020).Nasopharyngeal Microbiome Signature in COVID-19 Positive Patients:Can We Definitively Get a Role to Fusobacterium periodonticumFrontiers in Cellular and Infection Microbiology,11(February),1–7.doi:10.1038/nrmicro.2017.14.

Rosas-Salazar C,Kimura KS,Shilts MH,Strickland BA et al.(2020).SARS-CoV-2 infection and viral load are associated with the upper respiratory tract microbiome.J Allergy Clin Immunol.2021;147(4):1226-1233.e2.doi:10.1038/nrmicro.2017.14.

Tchoupou Saha et al.,2022.Profile of the Nasopharyngeal Microbiota Affecting the Clinical Course in COVID-19 Patients.Front.Microbiol.13:871627.doi:10.3389/fmicb.2022.871627

Zayed,G.and Y.H.Roos.2004.Influence of trehalose and moisture content on survival of Lactobacillus salivarius subjected to freeze-drying and storage.Process Biochem.9:1081–1086.

Claims

1. A Corynebacterium culture for preventing coronavirus infection in a subject, wherein the Corynebacterium culture is capable of reducing the expression of cathepsin.

2. The Corynebacterium culture used according to claim 1, wherein the Corynebacterium culture capable of reducing the expression of cathepsin is a Corynebacterium culture that reduces the relative expression of cathepsin mRNA after incubation with Caco-2 cells for 24 hours.

3. A Corynebacterium culture for use in preventing coronavirus infection in a subject, wherein the coronavirus uses the cathepsin pathway, preferably mainly uses the cathepsin pathway, to enter the cells of the subject, and wherein the Corynebacterium culture is capable of reducing the expression of cathepsins,

Preferably, the Corynebacterium culture capable of reducing the expression of cathepsin is a Corynebacterium culture that reduces the relative expression of cathepsin mRNA after incubation with Caco-2 cells for 24 hours.

4. The Corynebacterium culture for use according to any of the preceding claims, wherein the coronavirus is SARS-CoV-2.

5. The Corynebacterium culture for use according to any of the preceding claims, wherein the coronavirus is SARS-CoV-2 variant Omicron.

6. The Corynebacterium culture for use according to any of the preceding claims, wherein the Corynebacterium culture comprises at least one or at least two, preferably at least three, more preferably four Corynebacterium strains selected from the group consisting of Corynebacterium congestum, Corynebacterium propionici and Corynebacterium tuberculostearic acid.

7. A Corynebacterium culture for preventing infection by SARS-CoV-2 virus in a subject, wherein the Corynebacterium culture comprises at least one or at least two, preferably at least three, more preferably four Corynebacterium strains selected from the following:

The Corynebacterium crowdii SU004 strain deposited on January 17, 2022 at NCAIM (National Collection of Agricultural and Industrial Microorganisms, Institute of Food Science and Technology, Hungarian University of Agricultural and Life Sciences, H-1118, Budapest, Somlói Str. 14-16, Hungary) with accession number NCAIM P(B)001500, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression;

The Corynebacterium tuberculosis stearic acid SU005 strain deposited with NCAIM on April 27, 2022 with accession number NCAIM P(B)001501, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression;

The strain SU006 of Corynebacterium crowdedii deposited with NCAIM on April 27, 2022 with accession number NCAIM P(B)001502, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression;

Corynebacterium propionici SU007 strain deposited with NCAIM on April 27, 2022 with accession number NCAIM P(B)001504, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression;

The strain SU008 of Corynebacterium crowdedii deposited with NCAIM on April 27, 2022 with accession number NCAIM P(B)001505, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression;

The strain SU009 of Corynebacterium crowdedii deposited with NCAIM on May 25, 2022 with accession number NCAIM P(B)001506, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression;

Corynebacterium propionici SU010 strain deposited with NCAIM on May 25, 2022 with accession number NCAIM P(B)001507, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression;

Preferably, any derivative, variant or mutant capable of reducing the expression of cathepsin is a derivative, variant or mutant respectively that reduces the relative expression of cathepsin mRNA after 24 hours of incubation with Caco-2 cells.

8. The Corynebacterium culture for use according to claim 3, wherein, preferably, the coronavirus is the SARS-CoV-2 virus, more preferably the SARS-CoV-2 variant Omicron, and the Corynebacterium culture comprises at least one of the following

The Corynebacterium crowdii SU001 strain deposited on June 22, 2021 at NCAIM (National Collection of Agricultural and Industrial Microorganisms, Institute of Food Science and Technology, Hungarian University of Agricultural and Life Sciences, H-1118, Budapest, Somlói Str. 14-16, Hungary) with accession number NCAIM P(B)001495, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression;

Corynebacterium propionici SU002 strain deposited with NCAIM on June 22, 2021 with accession number NCAIM P(B)001496, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression;

Corynebacterium propionici SU003 strain deposited with NCAIM on June 22, 2021 with accession number NCAIM P(B)001497, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression;

The strain SU004 of Corynebacterium crowdedii deposited with NCAIM on January 17, 2022 with accession number NCAIM P(B)001500, or a derivative, variant or mutant thereof, wherein the derivative, variant or mutant thereof is capable of reducing cathepsin expression;

9. A congested Corynebacterium SU004 strain, wherein the congested Corynebacterium SU004 strain was deposited in NCAIM on January 17, 2022, with the accession number of NCAIM P(B)001500.

10. A SU005 strain of Corynebacterium tuberculosis, wherein the SU005 strain of Corynebacterium tuberculosis was deposited in NCAIM on April 27, 2022, with the accession number of NCAIM P(B)001501.

11. A congested Corynebacterium SU006 strain, wherein the congested Corynebacterium SU006 strain was deposited in NCAIM on April 27, 2022, with the accession number of NCAIM P(B)001502.

12. A propionic acid Corynebacterium SU007 strain, wherein the propionic acid Corynebacterium SU007 strain was deposited in NCAIM on April 27, 2022, with the accession number of NCAIM P(B)001504.

13. A congested Corynebacterium SU008 strain, wherein the congested Corynebacterium SU008 strain was deposited in NCAIM on April 27, 2022, with the accession number of NCAIM P(B)001505.

14. A congested Corynebacterium SU009 strain, wherein the congested Corynebacterium SU009 strain was deposited in NCAIM on May 25, 2022, with the accession number of NCAIM P(B)001506.

15. A propionic acid Corynebacterium SU010 strain, wherein the propionic acid Corynebacterium SU010 strain was deposited in NCAIM on May 25, 2022, with the accession number of NCAIM P(B)001507.

16. The Corynebacterium strain according to any one of claims 9 to 15, or any derivative, variant or mutant thereof capable of reducing the expression of cathepsin, for use in preventing infection by SARS-CoV-2 virus in a subject.

17. The Corynebacterium culture for use according to any one of claims 1 to 7, wherein the Corynebacterium culture comprises four Corynebacterium strains selected from the group consisting of:

(i) Corynebacterium congestum SU004, Corynebacterium tuberculostearicum SU005, Corynebacterium congestum SU009 and Corynebacterium propionici SU010, or any derivative, variant or mutant thereof capable of reducing the expression of cathepsin;

(ii) Corynebacterium congestum SU004, Corynebacterium tuberculostearicum SU005, Corynebacterium congestum SU006 and Corynebacterium propionici SU010, or any derivative, variant or mutant thereof capable of reducing the expression of cathepsin; and

(iii) Corynebacterium congestum SU004, Corynebacterium tuberculostearicum SU005, Corynebacterium propionici SU007 and Corynebacterium congestum SU008, or any derivative, variant or mutant thereof capable of reducing the expression of cathepsin.

18. A Corynebacterium culture for preventing infection by SARS-CoV-2 virus in a subject, wherein the Corynebacterium culture comprises four Corynebacterium strains, and the four Corynebacterium strains are Corynebacterium congestum SU004, Corynebacterium tuberculostearic acid SU005, Corynebacterium propionici SU007, and Corynebacterium congestum SU008.

19. A lyophilized preparation for preventing coronavirus infection in a subject, wherein the lyophilized preparation comprises a culture or strain of Corynebacterium as defined in any one of the preceding claims, and a cryoprotectant.

20. The lyophilized preparation for use according to claim 19,

(i) wherein the coronavirus is SARS-CoV-2, and

(ii) wherein the lyophilized preparation comprises a culture or strain of Corynebacterium as defined in any one of claims 7 to 16, and

(iii) wherein the cryoprotectant comprises a hydrocolloid polymer and a carbohydrate or a carbohydrate derivative,

Preferably, the carbohydrate or carbohydrate derivative is selected from trehalose, sucrose, glucose, lactose, mannitol, sorbitol, inulin, maltodextrin and isomaltulose, preferably selected from maltodextrin and isomaltulose, and

Preferably, the hydrocolloid polymer is selected from hydroxypropyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, guar gum, carrageenan and xanthan gum, preferably selected from kappa-carrageenan and xanthan gum.

21. The lyophilized formulation for use according to claim 20, wherein the carbohydrate or carbohydrate derivative is isomaltulose at a concentration of 0.5 g/100 mL to 2.5 g/100 mL, and the hydrocolloid polymer is kappa-carrageenan at a concentration of 0.1 g/100 mL to 0.5 g/100 mL or xanthan gum at a concentration of 0.05 g/100 mL to 0.25 g/100 mL, and/or wherein the Corynebacterium culture comprises 10 ³ CFU/mL to 10 ¹¹ CFU/mL of Corynebacterium strains, preferably 10 ³ CFU/mL to 10 ¹¹ CFU/mL of each Corynebacterium strain.

22. The lyophilized formulation according to any one of claims 19 to 21, wherein:

The lyophilized preparation includes a Corynebacterium culture, the Corynebacterium culture includes four Corynebacterium strains, the four Corynebacterium strains are Corynebacterium congestum SU004, Corynebacterium tuberculostearic acid SU005, Corynebacterium congestum SU006, and Corynebacterium propionici SU010; and

The cryoprotectant includes kappa-carrageenan or xanthan gum.