Probiotic Compositions for the Treatment of COVID-19

Abstract

A probiotic composition is provided, comprising Lactobacillus plantarum CECT 30292, Lactobacillus plantarum CECT 7484, Lactobacillus plantarum CECT 7485, and Pediococcus acidilactici CECT 7483. The probiotic composition is useful in treating, preventing, or ameliorating COVID-19 and other infectious diseases. It is also useful in enhancing immune responses.

Description

[Technical field]

[0001] The present invention relates to the fields of medicine and microbiology, in particular to a probiotic composition that benefits human and livestock health, and is particularly useful in the treatment of COVID-19 disease and other infectious diseases, and increases antibodies against the SARSCoV-2 virus. [Background technology]

[0002] At the filing date of this application, the webpage clinicaltrials.gov (https: / / clinicaltrials.gov / ct2 / show / NCT04517422) discloses the design of a clinical trial with the title "Efficacy of L. plantarum and P. acidilactici in adults with SARS-CoV-2 and COVID-19" and the identification number NCT04517422. This disclosure was published on August 18, 2020. The sponsor and responsible party (provider of information) is AB-Biotics, SA. The applicant of this application is AB-Biotics, SA. The disclosure on the discussed webpage can therefore be considered a so-called "inventor-origin disclosure" (i.e., the subject matter in the public disclosure must be attributable to the inventor, one or more co-inventors, or another person who has acquired the subject matter directly or indirectly from the inventor or co-inventors).

[0003] The above web page disclosure states the following (emphasis added): "A randomized controlled trial (RCT) to evaluate the safety and efficacy of Lactobacillus plantarum CECT 7481, Lactobacillus plantarum CECT 7484, Lactobacillus plantarum CECT 7485 and P. acidilactici CECT 7483, administered once daily, to reduce the risk of developing moderate or severe disease in subjects with mild COVID-19." Description of intervention model: Randomized controlled trial. 300 subjects (aged 18-60 years) will be assigned to receive a combination of Lactobacillus plantarum CECT7481, Lactobacillus plantarum CECT 7484, Lactobacillus plantarum CECT 7485, and P. acidilactici CECT 7483 (branch 1) or placebo (branch 2). "Experiment: Probiotics The activity tested products include four lactic acid bacteria strains with Qualified Presumption of Safety (QPS) status by the European Food Safety Authority (EFSA): Lactobacillus plantarum CECT 7481, Lactobacillus plantarum CECT 7484, Lactobacillus plantarum CECT 7485, and Pediococcus acidilacti-ci CECT 7483..."

[0004] The present application relates to the combination of Lactobacillus plantarum CECT 30929 (also referred to herein as DSM 33765), Lactobacillus plantarum CECT 7484, Lactobacillus plantarum CECT 7485 and Pediococcus acidilactici CECT 7483. This combination is not disclosed in the above publications at the filing date of this patent application.

[0005] Regarding the use of probiotic compositions for the treatment of COVID-19, the above mentioned publications do not disclose any significant experimental data, i.e. the statements relating to the combination can be seen as mere statements not supported by any significant verifiable experimental data.

[0006] WO 2011 / 092261 A1 discloses a probiotic composition formed by L. plantarum CECT 7484, L. plantarum CECT 7485 y P. acidilactici CECT 7483 for the treatment of gastrointestinal diseases or symptoms, such as inflammatory bowel disease, irritable bowel syndrome or abdominal bloating and flatulence.

[0007] Coronavirus type 19 (COVID-19) is caused by the novel coronavirus type 2 (SARS-CoV-2, previously known as 2019-nCoV). SARS-CoV-2 infection can be asymptomatic or can cause a wide range of symptoms, ranging from mild (e.g., mild to moderate respiratory abnormalities, fever and / or headache, non-pneumonia or mild pneumonia) to severe (e.g., severe respiratory abnormalities, arterial blood oxygen partial pressure to inspired oxygen partial pressure ratio <300, and / or severe pneumonia), and sometimes severe symptoms (e.g., respiratory failure, septic shock, and / or multiple organ dysfunction or failure leading to death). Specifically, low arterial blood oxygen saturation has been repeatedly shown to be a risk factor for disease progression and subsequent death, regardless of other symptoms.

[0008] It is therefore of great interest to ensure rapid remission of mild individuals, who constitute the majority of cases, in order to avoid progression to more severe disease. However, effective treatment for people with mild to moderate disease has been more ambiguous. For example, the anti-inflammatory effect of corticosteroids has been found to be beneficial in severe COVID-19, but it is thought that their effect may actually worsen clinical outcomes in patients with mild or moderate disease.

[0009] L. plantarum strains have previously been shown to be beneficial in respiratory infections [Chong et al. 2019]. However, the strength of the effect of L. plantarum on immune cells has a wide variability among different strains [Meijerink et al. 2010].

[0010] A recent study reported that drug therapy (hydroxychloroquine, antibodies, and tocilizumab, alone or in combination) along with a probiotic mixture reduced the overall duration of symptoms in COVID-19 positive patients who were already moderate to severely ill and required hospitalization, although the study was not randomized and no effect on disease remission, reduction of viral load, or enhancement of antibody responses was reported in the study [D'Ettorre et al. 2020].

[0011] Also, the strain P. acidilactici K15 has recently been studied for cold and influenza prevention in preschool children during the cold and influenza season. However, limited efficacy was reported in the study, as the probiotic was unable to shorten the duration of fever or the incidence of influenza in the entire study population, but only shortened the duration of fever in subjects who consumed little fermented foods. Furthermore, the probiotic was unable to boost the levels of influenza-specific antibodies. [Hishiki et al. 2020]

[0012] Probiotic characteristics are strain-dependent, even among bacteria of the same species. It is therefore important to find strains that perform better in all probiotic requirements. In particular, immunological effects are rare in probiotics and are therefore considered to be highly strain-specific [Hill et al. 2014].

[0013] In view of the above, it is of great interest to find novel treatment methods for subjects suffering from mild to moderate symptoms of COVID-19 to avoid progression to more severe disease. Summary of the Invention

[0014] The Examples herein provide detailed experimental data demonstrating significant positive effects for the use of the probiotic compositions disclosed herein for the treatment of COVID-19 in humans.

[0015] Example 2 shows that the probiotic composition disclosed herein significantly increases the complete remission rate of the disease (day 30 of intervention). Furthermore, symptomatic remission is achieved faster in patients who received the probiotic composition. Specifically, the probiotic composition significantly reduces the viral load in nasopharyngeal swabs during the intervention, reducing it by an average of 9-fold (i.e., nearly 1 log) at day 30 of intervention. Furthermore, the probiotic composition enhances the production of antibodies, resulting in significantly higher concentrations of IgG and IgM. Finally, the duration of various specific symptoms of COVID-19 (e.g., fever, cough, headache, shortness of breath, bodily pain, hypoxemia, and diarrhea) is significantly reduced in patients who received the probiotic composition.

[0016] Example 3, on the other hand, shows that serum concentrations of interferon-β increased significantly in the probiotic group compared to placebo after both 15 and 30 days of intervention. Type I interferons (interferon α and interferon β) are proteins that have the ability to limit viral replication in vertebrate cells. In addition to this direct antiviral effect, type I interferons also have an indirect antiviral effect by stimulating B lymphocytes (B cells) and natural killer lymphocytes (NK cells). B cells produce virus-specific antibodies that bind to viral particles and thus prevent viral spread.

[0017] Type I interferons therefore have a broad-spectrum antiviral activity. Impaired type I interferon responses in humans have therefore been associated with more severe illness from pulmonary infections caused by various viruses, particularly coronaviruses such as SARS-CoV2, SARS-CoV1 (SARS) and MERS-CoV (MERS), but also influenza virus, human rhinovirus or respiratory syncytial virus, as well as non-pulmonary viral infections such as hepatitis C virus or brain infections (encephalitis) caused by herpes simplex virus [Channapannavar et al. 2019; Haagmans et al. 2004].

[0018] Thus, one skilled in the art will recognize the utility of the probiotic compositions provided herein that can increase interferon-β in patients to treat diseases in which high type I interferon may be beneficial, particularly viral infections, particularly respiratory viral infections, more particularly pulmonary viral infections.

[0019] Meanwhile, Example 3 also shows that serum concentrations of CXCL13 were significantly reduced over time in the probiotic group compared to placebo. CXCL13 is a protein that is induced by type I interferon during viral infection and helps initiate B cell recruitment to the site of infection. However, CXCL13 has potent pro-inflammatory effects and is associated with lung damage across a wide range of conditions, from COVID-19 to pulmonary fibrosis and chronic obstructive pulmonary disease. Notably, blocking CXCL13 has been shown to limit such lung damage.

[0020] Viral respiratory pathogens known to induce CXCL13 include SARS-CoV2, influenza, and respiratory syncytial virus. Thus, inducing type I interferon while limiting CXCL13 levels over time may be useful in such viral infections to stimulate antiviral responses while preventing excessive inflammation, particularly aging of lung tissue due to infection with SARS-CoV2, influenza virus, or respiratory syncytial virus. Since CXCL13 is known to be induced by type I interferon, this increase in type I interferon and concomitant decrease in CXCL13 are surprising effects of the probiotic compositions provided herein. Notably, this decrease in CXCL13 did not impair B cell activity, as shown by the increased levels of immunoglobulins IgG and IgM shown in Example 2. Moreover, these results correlate with a short duration of clinical symptoms and viral clearance, as shown in Example 3.

[0021] Example 4 shows that L. plantarum CECT 30292 significantly overexpresses the plantaricin G (plnG) gene compared to the WCFS1 type strain and compared to all other strains tested. Plantaricin G is a bacterial surface protein found in some strains of the species Lactobacillus plantarum. This protein is specifically involved in the mechanism of inhibiting the growth of competing pathogens. Importantly, among the proteins commonly produced by L. plantarum in general, plnG is specifically recognized by antigen-presenting cells (APCs), immune cells responsible for searching for potential pathogens. APCs release interferons and other immunoregulatory signals upon activation, thus helping to activate other immune cells such as antibody-producing B cells and natural killer (NK) cells.

[0022] As noted above with respect to the web page disclosure at clinicaltrials.gov, the statements in this web page disclosure may be regarded as mere statements not supported by any significant verifiable experimental data, and it is clear that based on this web page disclosure it was not plausible / reliable that the positive effects associated herein may be obtained in human patients.

[0023] Based on the knowledge of the prior art, a person skilled in the art could not foresee the significant positive effect experimentally described herein with reasonable expectation of success.Notably, clinical results exceed the predictions made by the initial research design hypothesis.Therefore, the significant positive effect experimentally described herein is unexpected for a person skilled in the art and can be considered as a major contribution to the art.

[0024] Thus, one aspect of the present invention is a method for producing a method for treating a cancer cell comprising the steps of: Lactobacillus plantarum strain deposited under the Budapest Treaty at the Spanish Type Culture Collection (CECT) under accession number CECT 30292, or a bacterial strain derived therefrom; a Lactobacillus plantarum strain deposited under the Budapest Treaty in the CECT under accession number CECT 7484, or a bacterial strain derived therefrom; A Lactobacillus plantarum strain deposited under the Budapest Treaty in the CECT under accession number CECT 7485, or a bacterial strain derived therefrom; and A probiotic composition comprising the strain Pediococcus acidilactici deposited under the Budapest Treaty in the CECT under accession number CECT 7483, or a bacterial strain derived therefrom, Each of the bacterial strains derived therefrom (a) has a genome that is at least 99.90% identical to the genome of the corresponding deposited strain; and (b) retain at least one capability of the corresponding deposited strain, which capability is selected from the group consisting of: -Increasing remission of COVID-19; -Reducing the duration of COVID-19 symptoms; - Reduce SARSCoV-2 viral load; - Stimulate the production of antibodies against the SARSCoV-2 virus (i.e., increase antibody levels); - Improve blood oxygen saturation; and -Reducing the duration of low oxygen saturation in the blood (i.e., hypoxemia).

[0025] Another aspect relates to the Lactobacillus plantarum strain deposited under the Budapest Treaty at the Spanish Culture Collection (CECT) under the accession number CECT 30292, or a bacterial strain derived therefrom, which comprises (a) has a genome that is at least 99.90% identical to the genome of the corresponding deposited strain; and (b) retain at least one capability of the corresponding deposited strain, which capability is selected from the group consisting of: -Increasing remission of COVID-19; -Reducing the duration of COVID-19 symptoms; - Reduce SARSCoV-2 viral load; - Stimulate the production of antibodies against the SARSCoV-2 virus (i.e., increase antibody levels); - Improve blood oxygen saturation; and -Reducing the duration of low oxygen saturation in the blood (i.e., hypoxemia).

[0026] The present invention also relates to a probiotic composition comprising Lactobacillus plantarum strain CECT 30292, or a bacterial strain derived therefrom having the above mentioned characteristics.

[0027] Another aspect relates to a probiotic composition comprising the Lactobacillus plantarum strain deposited under the Budapest Treaty at the Spanish Culture Collection (CECT) under the accession number CECT 30292, or a bacterial strain derived therefrom, which bacterial strain comprises (a) has a genome that is at least 99.90% identical to the genome of the corresponding deposited strain; and (b) retain at least one capability of the corresponding deposited strain compared to a control, which capability is selected from the group consisting of: - Stimulate the production of IgG and IgM antibodies against the SARSCoV-2 virus (specifically, at least a doubling of IgG antibody levels); -increasing interferon-β levels (specifically, increasing interferon-β levels by at least 20%); - Decrease CXCL13 levels; - having at least the number of copies of the plnG gene of the deposited strain; and - overexpressing plantaricin G (specifically, plantaricin G is overexpressed at least 4-fold).

[0028] Another embodiment relates to the Lactobacillus plantarum strain deposited under the Budapest Treaty at the Spanish Culture Collection (CECT) under accession number CECT 30292.

[0029] Another aspect relates to a method for obtaining a mutant strain of Lactobacillus plantarum strain deposited under CECT 30292, said method comprising the steps of: (i): Producing a mutant of L. plantarum CECT 30292; (ii): analyzing the mutants obtained in step (i) and identifying mutant strains that retain or improve at least one of the properties of the deposited strain; and (iii): Isolating the mutant strain identified in step (ii), thereby obtaining a mutant strain of L. plantarum CECT 30292, wherein the mutant strain retains or improves at least one ability of the deposited strain.

[0030] "Probiotic composition(s)" is used herein to refer to various compositions according to various aspects and embodiments of the invention.

[0031] The probiotic compositions described herein can be used for various medical applications / uses as detailed herein. Alternatively, all uses described herein can be formulated as the use of any of the compositions described herein for the manufacture of a pharmaceutical, veterinary, medicine, food product, dietary supplement, or dietary product for the prevention and / or treatment of a disease disclosed herein. Alternatively, it can also be formulated as a method of treating or preventing a disease described herein or its symptoms and / or complications in a subject in need thereof, which method comprises administering to the subject a probiotic composition described herein.

[0032] One aspect of the present invention relates to a probiotic composition for use as a medicine (i.e., for use in the treatment or prevention of disease). Alternatively, this aspect can be formulated as a method for probiotic treatment, which method comprises administering to a subject in need of such treatment an effective amount of a probiotic composition of the present invention. As used herein, the term "probiotic" refers to live microorganisms that, when administered in appropriate amounts, confer a health benefit to the host.

[0033] One aspect of the invention relates to a probiotic composition according to the invention for use in a method of treating, preventing or ameliorating COVID-19 or its symptoms, complications and / or sequelae. Alternatively, this aspect may be formulated as a method of treating, preventing or ameliorating COVID-19 or its symptoms and / or complications in a subject in need thereof, the method comprising administering to the subject a probiotic composition as described herein.

[0034] Another aspect relates to a probiotic composition according to the invention for use in a method for treating, preventing or ameliorating an infectious disease or its symptoms, complications and / or sequelae. Alternatively, this aspect may be formulated as a method of treating, preventing or ameliorating an infectious disease or its symptoms and / or complications in a subject in need thereof, the method comprising administering to the subject a probiotic composition as described herein.

[0035] Another aspect relates to a probiotic composition according to the invention for use in enhancing an immune response (i.e. for use in promoting, stimulating or boosting an immune response or immunity). Alternatively, this aspect may be formulated as a method of enhancing an immune response in a subject in need thereof, the method comprising administering to the subject a probiotic composition as described herein.

[0036] Another aspect relates to a dietary supplement comprising the probiotic composition described herein and at least one acceptable excipient.

[0037] Another aspect relates to a pharmaceutical composition comprising an effective amount of a probiotic composition described herein and at least one pharma- ceutically acceptable excipient.

[0038] Another aspect relates to an edible product comprising the probiotic composition described herein and at least one edible ingredient.

[0039] The terms used in the claims and aspects of the present invention are understood in their broad and general sense herein. Nevertheless, some are defined below in the detailed description of the invention.

[0040] Throughout the specification and claims, the word "comprises" and variations thereof are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon examination of this specification or may be learned by practice of the present invention. Furthermore, the present invention encompasses all possible combinations of specific and preferred embodiments described herein. The following examples and figures are provided herein for illustrative purposes and are not intended to be limiting of the present invention. [Brief description of the drawings]

[0041] [Figure 1] A tree analysis based on ANI (average nucleotide identity) shows that L. plantarum DSM 33765 belongs to the same clade as CECT 7484 (ANI of 99.71%), but is more distantly related to the reference strain WCFS1, as well as ATCC 202195 and CECT 7485 (ANI values ​​of 98.7-98.8%). For further details, see the Examples herein. [Diagram 2] Pulse-field gel electrophoresis (PFGE) shows that the L. plantarum DSM 33765 strain (two replicates rep1 and rep2) has a reproducible PFGE profile that is distinct from that of the closely related strain L. CECT 7484. Specifically, compared to CECT 7484, the DSM 33765 strain lacks the 97 kDa band but has an additional band between 48.5 and 32.1 kDa. Thus, although closely related, both strains are shown to be distinct from each other. For further details, see the Examples herein. [Diagram 3]Figure 1 shows the concentration of SARS-CoV-2 viral load in nasopharyngeal swabs on days 0, 15 and 30 of the intervention for the probiotic and placebo groups. On day 30, the difference was an average of 0.9 log, thus the concentration was reduced by an average of 9-fold. For further details, see the Examples herein. [Figure 4] Figure 1 shows concentrations of IgG and IgM antibodies against SARS-CoV-2 on days 0, 15, and 30 of the intervention for the probiotic and placebo groups. On day 30, subjects receiving probiotics had IgG levels two times higher than subjects in the placebo group. For further details, see the Examples herein. [Diagram 5] Serum concentrations of interferon-β1a (INFb) are shown for the probiotic and placebo groups on days 0, 15, and 30 of the intervention. On day 30, subjects receiving probiotics had significantly increased INFb levels relative to the placebo group. For further details, see the Examples herein. [Figure 6] Serum concentrations of CXCL13 for the probiotic and placebo groups are shown on days 0, 15, and 30 of the intervention. Concentrations in the probiotic group were significantly lower than those in the placebo group on both days 15 and 30. For further details, see the Examples herein. [Figure 7] Figure 1 shows the doubling of plantaricin G (PlnG) protein in L. plantarum strains CECT 30292, CECT 7483, CECT 7484, CECT 7485, CECT 7481,299v and WCFS1. L. plantarum CECT 30292 significantly overexpressed the plnG gene compared to WCFS1 type strains and compared to all other strains tested. See the Examples herein for further details. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] definition Derived: As used herein, the terms "derived," "derivative," "variant," "mutant" (e.g., "mutant"), or any grammatical variation thereof, refer to a component that is isolated from or made using a particular molecule (e.g., a strain of the present disclosure). For example, a bacterial strain derived from a first bacterial strain (e.g., a deposited strain) can be a strain identical or substantially similar to the first strain. In the case of a bacterial strain, for example, the derived strain can be obtained by naturally occurring mutagenesis (artificially induced mutagenesis or artificially random mutagenesis).

[0043] Excipient: The terms "excipient" and "carrier" are used interchangeably and refer to an inert substance added to, for example, a pharmaceutical composition to further facilitate administration of a compound, for example, a bacterial strain of the present disclosure.

[0044] Identity: As used herein, the term "identity" refers to the overall conservation of monomer sequence between polymerized molecules (e.g., between DNA molecules and / or RNA molecules). The term "identical" without any additional qualifier means that the sequences are 100% identical (100% sequence identity). Describing two sequences as, for example, "70% identical" is equivalent to describing them as having, for example, "70% sequence identity."

[0045] The calculation of the percent identity of two polymer molecules, e.g., polynucleotide sequences, can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced into one or both of the first and second polynucleotide sequences). In certain embodiments, the length of the sequence aligned for comparison purposes is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the length of the reference sequence. Then, in the case of polynucleotides, the bases at corresponding base positions are compared.

[0046] The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, and it can be determined using a mathematical algorithm. Suitable software programs can be utilized for alignment of both protein and nucleotide sequences. One suitable program for determining percent sequence identity is bl2seq, which uses either the BLASTN (used to compare nucleic acid sequences) or BLASTP (used to compare amino acid sequences) algorithms to perform a comparison between two sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Match-er, which are part of the EMBOSS site of bioinformatics programs. Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (ClustalW, ClustalX, or Clustal Omega), MUSCLE, MAUVE, MUMMER, RAST, etc.

[0047] In certain embodiments, the percentage of identity (%ID) between a first sequence and a second sequence is calculated as %ID=100×(Y / Z), where Y is the number of amino acid residues or nucleobases scored as identical matches in an alignment of the first and second sequences (e.g., as aligned by visual inspection or a specific sequence alignment program), and Z is the total number of residues in the second sequence. When comparing complete or near-complete genomic nucleobase sequences, %ID is sometimes referred to as ANI (average nucleotide identity).

[0048] Prevent: As used herein, the terms "prevent", "preventing", "prevention" and variations thereof refer, for example, to the following: (i) partially or completely delaying the onset of the diseases, disorders and / or conditions disclosed herein; (ii) partially or completely delaying the onset of one or more symptoms, characteristics, or clinical manifestations, complications, or sequelae of the specific diseases, disorders, and / or conditions disclosed herein; (iii) partially or completely delaying the onset of one or more symptoms, characteristics, or symptoms, complications, or sequelae of the specific diseases, disorders, and / or conditions disclosed herein; (iv) partially or completely delaying progression from certain diseases, disorders and / or conditions disclosed herein; and / or (v) reducing the risk of developing a medical condition associated with a disease, disorder, and / or condition disclosed herein.

[0049] Subject: The terms "subject," "patient," "individual," and "host," and variations thereof, are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired, including, but not limited to, humans, domestic animals (e.g., dogs, cats, etc.), livestock (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.). The methods described herein are applicable to both human therapy and veterinary applications.

[0050] Subject in need thereof: As used herein, the phrase "subject in need thereof" includes a subject, such as a mammalian subject, that would benefit from the administration of a composition of the present disclosure.

[0051] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" is an amount of a composition of the present disclosure sufficient to produce a desired therapeutic, pharmaceutical and / or physiological effect in a subject in need thereof.

[0052] Treatment: As used herein, the terms "treat", "treatment", and "therapy" refer to, for example, reducing the severity of a disease or condition disclosed herein; ameliorating or eliminating one or more symptoms, complications, or sequelae associated with a disease disclosed herein (e.g., a coronavirus disease such as COVID-19); providing a beneficial effect to a subject having a condition / disease disclosed herein without necessarily curing the disease or condition. The term also includes prophylaxis or prevention of a disease or condition or symptom, complications, or sequelae thereof.

[0053] The term includes clinical or nutritional interventions to prevent a disease or condition, cure a disease or condition, delay the onset of a disease or condition, delay the onset of a symptom, complication or sequelae, reduce the severity of a disease or condition, reduce the severity of a symptom, complication or sequelae, ameliorate one or more symptoms, ameliorate one or more complications, ameliorate one or more sequelae, prevent one or more symptoms, prevent one or more complications, prevent one or more sequelae, delay one or more symptoms, delay one or more symptoms, delay one or more complications, delay one or more sequelae, ameliorate one or more symptoms, ameliorate one or more complications. "improvement" refers to improving or reducing the severity of a disease or condition, improving one or more sequelae, shortening the duration of one or more symptoms, shortening the duration of one or more complications, shortening the duration of one or more sequelae, reducing the frequency of one or more symptoms, reducing the frequency of one or more complications, reducing the frequency of one or more sequelae, reducing the severity of one or more symptoms, reducing the severity of one or more complications, reducing the severity of one or more sequelae, improving quality of life, increasing survival, preventing recurrence of a disease or condition, delaying the recurrence of a disease or condition, or any combination thereof, for example, as compared to that expected in the absence of treatment with a composition of the present disclosure.

[0054] Symptom: As used herein, the term "symptom" refers to a subjective or physical sign, indication, or evidence of a disease or physical disorder observed by a subject. In general, the term refers to any pathological phenomenon or deviation from normal in structure, function, or sensation experienced by a patient and indicative of a disease. A symptom is felt or noticed by the individual experiencing the symptom, but may not be easily noticed by others. In some aspects, a symptom may be a mild symptom, a moderate symptom, or a severe symptom. As used herein, the term "mild symptom" refers to a symptom that is not life-threatening and does not require intensive care (e.g., in a hospital ICU). As used herein, the term "moderate symptom" refers to a symptom that may be life-threatening and may require, for example, hospitalization, and therefore requires monitoring. As used herein, the term "severe symptom" refers to a symptom that is life-threatening and requires, for example, in a hospital ICU.

[0055] Complication: As used herein, the term "complication" refers to a pathological process or event that occurs during a disease or condition that is not an essential part of the disease or condition and may result from the disease / condition or from an independent cause. Thus, the term complication refers to a medical / clinical problem observed in a subject diagnosed with a disease or condition disclosed herein, such as COVID-19. In some embodiments, a complication may be temporary. In some embodiments, a complication may be chronic or permanent. As used herein, the term "sequelae" refers to a long-term, chronic, or permanent complication.

[0056] Probiotic Composition As mentioned above, one aspect of the present invention relates to a probiotic composition comprising the Lactobacillus plantarum strain deposited under accession number CECT 30292, or a bacterial strain derived therefrom.

[0057] In one embodiment, the Lactobacillus plantarum strain is the strain deposited under accession number CECT 30292.

[0058] In one embodiment, the probiotic composition further comprises Lactobacillus plantarum CECT 7484, Lactobacillus plantarum CECT 7485, and Pediococcus acidilactici CECT 7483.

[0059] Lactobacillus plantarum CECT 30292 (also referred to herein as DSM 33765) was isolated from humans (fecal samples) and from chicory (cichorium intybus). The strain was deposited at the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) with the address Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroogranismen und ZellKulturen GmbH, Inhoffenstr, 7B D-38124 Braunschweig, Germany. The deposited strain received the accession number DSM 33765 and the date of deposit 27 January 2021 (27.01.2021). This strain was also deposited on January 26, 2021 (26.01.2021) at the Spanish Culture Collection (CECT, Parc Cientific de la Universitat de Valencia, Carrer del Catedratic Agustin Escardino Benlloch, 9, 46980 Paterna, Valencia, Spain) under the accession number CECT 30292. Both deposits were made under the conditions of the Budapest Treaty, are viable and retain all of their characteristics associated with their deposits. They were deposited by the same applicant.

[0060] A particular embodiment relates to the Lactobacillus plantarum strain deposited under the Budapest Treaty at the Spanish Culture Collection (CECT) under accession number CECT 30292. The present invention also relates to a probiotic composition comprising the Lactobacillus plantarum strain CECT 30292.

[0061] Lactobacillus plantarum strains CECT 7485 and CECT 7484 and Pediococcus acidilactici strain CECT 7483 are described and protected in WO 2011 / 092261 (A1), the contents of which are incorporated herein by reference in their entirety. These three strains were deposited on February 4, 2009 (04.02.2009) at the Spanish Culture Collection (Parc Cientific de la Universitat de Valencia, Carrer del Catedratic Agustin Escardino Benlloch, 9, 46980 Paterna, Valencia, Spain) by the same applicant of the present application.

[0062] As will be understood by those skilled in the art in this context, the bacterial strain is isolated from its natural environment, i.e. it is not present in its natural environment and therefore does not contain other organisms and substances present in the natural environment.

[0063] Since Lactoplantibacillus plantarum (L. plantarum) was previously known as Lactobacillus plantarum, the terms Lactoplantibacillus plantarum and Lactobacillus plantarum are used interchangeably herein.

[0064] A wide variety of lactic acid bacteria species have a long history of apparently safe use. The European Food Safety Authority has developed a system to grant "Qualified Presumption of Safety" (QPS) status to taxonomic units that have a long and proven history of apparently safe use. The strains P. acidi-lactici CECT 7483, L. plantarum CECT 7484 and L. plantarum CECT 7485 and L. plantarum DSM 33765 belong to bacterial species with QPS status.

[0065] The emergence and spread of resistance to antibacterial agents in bacteria poses a threat to human and animal health and increases major financial and social costs. Whole genome sequence analysis revealed that the novel strain L. plantarum DSM 33765 does not carry antibiotic resistance genes. Strains CECT 7483, CECT 7484 and CECT 7485 also do not carry antibiotic resistance. Overall, these results rule out the risk of potential transfer of antibiotic resistance to pathogenic species.

[0066] By using the deposited strain as starting material, it is clear to the skilled artisan that further variants or mutants thereof can be routinely obtained by conventional mutagenesis or re-isolation techniques (i.e., re-cultivation) that retain or enhance the relevant characteristics and advantages described herein of the strains forming the compositions of the invention. Thus, the present invention also relates to variants of the strains disclosed herein. As used herein, the term "variant" or "mutant" of a strain refers to any naturally occurring or specifically developed strain derived from the deposited reference strain, mainly by mutation, that retains at least one of the above-mentioned capabilities of the deposited strain.

[0067] Example 5 shows the variability between the genomes of two different batches of the same strain (DSM 33765). It has been demonstrated that two isolates of the same strain can differ by 0.01% of average nucleotide identity (ANI). Thus, a strain that shows 99.99% ANI to the genome of the corresponding deposited strain is considered to be the same deposited strain CECT 30292.

[0068] In certain embodiments, a bacterial strain derived from a deposited strain has a genome that is at least 99.90% identical to the genome of the corresponding deposited strain, more particularly, the % identity is 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98% or 99.99%. Specifically, the % identity is at least 99.95%.

[0069] In a particular embodiment, the variant is obtained by using recombinant DNA techniques. In another embodiment, the variant is obtained by random mutagenesis. In another embodiment, the variant is a naturally occurring variant. In another embodiment, the variant is obtained by recultivating the deposited strain. Thus, another aspect of the invention relates to a method for obtaining a strain derived from the deposited strain, in particular L. plantarum CECT 30292, comprising using the deposited strain as starting material and applying mutagenesis or recultivation, the variant or mutant obtained further retaining or enhancing at least one capability of the deposited strain.

[0070] Alternatively, this can be expressed as a method for obtaining mutant strains of the deposited strain (in particular L. plantarum CECT 30292), which method comprises the following steps: (i): Producing a mutant of L. plantarum CECT 30292; (ii): analyzing the mutants obtained in step (i) and identifying mutant strains that retain or improve at least one of the properties of the deposited strain; and (iii): Isolating the mutant strain identified in step (ii), thereby obtaining a mutant strain of L. plantarum CECT 30292, wherein the mutant strain retains or improves at least one ability of the deposited strain.

[0071] In some embodiments, the resulting mutant retains (or improves) at least one capability of the corresponding deposited strain compared to a control, which capability is selected from the group consisting of: -Increasing remission of COVID-19; -Reducing the duration of COVID-19 symptoms; - Reduce SARSCoV-2 viral load; - stimulates the production of antibodies against the SARSCoV-2 virus (in particular stimulates the production of IgG and IgM antibodies against the SARSCoV-2 virus; more specifically, at least a doubling of IgG antibody levels); - Improve hypoxemia; -Reducing the duration of hypoxemia; -increasing interferon-β levels (specifically, increasing interferon-β levels by at least 20%); - Decrease CXCL13 levels; - having at least the number of copies of the plnG gene or more copies of the gene of the deposited strain; and Plantaricin G is overexpressed (specifically, plantaricin G is overexpressed at least four-fold).

[0072] A control is understood as a placebo or control strain that does not have such a specific ability.

[0073] In certain embodiments, the resulting mutant retains (or improves) at least one capability of the corresponding deposited strain, which capability is selected from the group consisting of: - enhance remission of COVID-19;- reduce the duration of COVID-19 symptoms;- reduce SARSCoV-2 viral load;- stimulate the production of antibodies against the SARSCoV-2 virus; (particularly stimulate the production of IgG and IgM antibodies against the SARSCoV-2 virus; more particularly, at least double the IgG antibody levels);- improve hypoxemia and reduce the duration of hypoxemia. In certain embodiments, the control for such ability is a placebo.

[0074] In certain embodiments, the resulting mutants retain (or improve) at least one ability of the corresponding deposited strain to stimulate the production of antibodies against the SARSCoV-2 virus, in particular to stimulate the production of IgG and IgM antibodies against the SARSCoV-2 virus, more particularly at least a doubling of IgG antibody levels. In certain embodiments, the control for such ability is a placebo.

[0075] In a particular embodiment, the resulting mutant retains (or improves) at least one ability of the corresponding deposited strain, which is to increase interferon-β levels, particularly, the interferon-β levels are increased by at least 20%, in a particular embodiment, the control for such ability is a placebo.

[0076] In certain embodiments, the resulting mutant retains (or improves) at least one ability of the corresponding deposited strain, which is to reduce CXCL13 levels, and in certain embodiments, the control for such ability is a placebo.

[0077] In certain embodiments, the resulting mutant retains (or improves) at least one capability of the corresponding deposited strain, which capability is at least the copy number of the plnG gene of the deposited strain or a higher number of copies of the gene. In certain embodiments, the control for such capability is a strain having only one copy of the plnG gene, such as, for example, strain WCSF1.

[0078] In a particular embodiment, the resulting mutant retains (or improves) at least one ability of the corresponding deposited strain, which is to overexpress plantaricin G, in particular, plantaricin G is overexpressed at least 4-fold. In a particular embodiment, the control for such ability is, for example, strain WCSF1.

[0079] The skilled artisan will determine the appropriate methods to be used to determine the performance of the resulting mutants relative to the corresponding deposited strain. Examples of possible methods for measuring these performances are given in the Examples section.

[0080] Example 6 shows that strain CECT 30292 has an additional copy of the plnG gene in a plasmid. Thus, one skilled in the art can attempt to increase the copy number of the plasmid containing the plnG gene or to insert additional copies of the plnG gene into the bacterial DNA of the deposited strain. Thus, in one embodiment, a bacterial strain derived from the deposited strain is obtained therefrom by inserting additional copies of the plnG gene into either the bacterial chromosome or a plasmid.

[0081] It is clear that obtaining mutant strains of CECT 30292 is a routine task for those skilled in the art, even without knowledge of the genomic sequence.

[0082] The strain that forms part of the composition of the present invention may be in the form of living cells. Alternatively, the strain may be in the form of non-viable cells. This may include microorganisms that are thermally killed, or that are killed by exposure to altered pH, sonication, radiation or high pressure. Product preparation with non-viable cells is simpler, since cells can be easily incorporated into dietary, pharmaceutical or edible products, and storage requirements are much less restrictive than those of viable cells.

[0083] The strains disclosed herein are produced by culturing (or fermenting) bacteria under suitable conditions in a suitable artificial medium. The expression "artificial medium" for microorganisms is understood to be a medium containing natural substances and, in some cases, synthetic chemicals such as the polymer polyvinyl alcohol that can reproduce some of their functions. A common suitable artificial medium is a nutrient broth that contains elements necessary for bacterial growth, including a carbon source (e.g., glucose), a nitrogen source (e.g., amino acids and proteins), water and salts. The growth medium can be in liquid form or is often mixed with agar or another gelling agent to obtain a solid medium. These strains can be cultured alone to form pure cultures, or can be cultured as mixed cultures together with other microorganisms, or can be cultured by culturing different types of bacteria separately and then combining them in the desired ratio. After cultivation, depending on the final preparation, the strains can be used as purified bacteria, or the bacterial culture or cell suspension can be used either as it is or after suitable post-treatment. In this specification, the term "biomass" is understood to be the bacterial strain culture obtained after cultivation (or fermentation, a term synonymous with cultivation).

[0084] In a particular embodiment, the strain is fermented in an artificial medium and subjected to a post-treatment after fermentation to obtain bacterial cells, the bacterial cells being in a liquid medium or in a solid form, specifically, the post-treatment is selected from the group consisting of drying, freezing, lyophilization, fluidized bed drying, spray drying and refrigeration in a liquid medium, more specifically, lyophilization.

[0085] The term "post-treatment" should be understood in this context as any treatment carried out on a biomass with the aim of obtaining storable bacterial cells. The purpose of the post-treatment is to reduce the metabolic activity of the cells in the biomass and thus slow down the rate of adverse cellular reactions. As a result of the post-treatment, the bacterial cells can be in solid or liquid form. In solid form, the stored bacterial cells can be in powder or granules. In any case, both solid and liquid forms containing bacterial cells do not exist in nature.

[0086] Because they are the result of artificial post-treatment processes. The post-treatment process may require the use of one or more so-called post-treatment agents in certain embodiments. In the context of the present invention, the expression "post-treatment agent" refers to a compound used to carry out the post-treatment process described herein. Among the post-treatment agents, there are included, but are not limited to, dehydrating agents, bacteriostatic agents, cryoprotectants (cryoprotectants), inert fillers (also known as lyoprotectants), carrier materials (also known as core materials), etc., used alone or in combination.

[0087] There are two basic approaches to reduce the metabolic activity of bacterial cells and therefore two approaches to post-processing. The first method is to reduce the rate of all chemical reactions, which can be done by lowering the temperature by refrigeration or freezing using refrigerators, mechanical freezers, and liquid nitrogen freezers. Alternatively, reducing the rate of all chemical reactions can be achieved by adding substances that inhibit the growth of bacterial cells, i.e. bacteriostatic agents (abbreviated as Bstatic).

[0088] The second approach to carrying out post-treatment is to remove water from the biomass, a process that may involve sublimation of water using a freeze dryer. Suitable techniques for removing water from biomass are drying, freeze drying, spray drying or fluidized bed drying. Post-treatments that result in a solid form may be drying, freezing, freeze drying, fluidized bed drying, or spray drying.

[0089] The post-treatment is specifically freeze-drying, which involves the removal of water from the frozen bacterial suspension by sublimation under reduced pressure. The process consists of three steps: pre-freezing the product to form a frozen structure, primary drying to remove most of the water, and secondary drying to remove bound water. Due to the purpose and expected variability of the industrial process for the production and isolation of freeze-dried bacterial cultures, the latter generally contain a certain amount of inert filler, also known as a lyoprotectant. Its role is to standardize the content of live probiotic bacteria in the product. The following inert fillers in commercial freeze-dried cultures are used: sucrose, saccharose, lactose, trehalose, glucose, maltose, maltodextrin, corn starch, inulin, and other pharma- ceutically acceptable non-hygroscopic fillers. If necessary, other stabilizers or cryoprotectants, such as ascorbic acid, are also used to form a viscous paste, which is then subjected to freeze-drying. In either case, the material so obtained can be ground to a suitable size, including a powder.

[0090] As an alternative to storing the biomass in solid form, it may be stored in liquid form, either by adding a bacteriostatic agent to the culture medium as described above to stop bacterial growth, or by an intermediate step of harvesting the cells and resuspending the pellet in a saline solution containing a bacteriostatic agent, and optionally refrigerating it.

[0091] Sometimes, probiotic preparations are subjected to immobilization and / or coating or encapsulation processes to improve shelf life and / or functionality, e.g., as described above in the fluidized bed drying process. Several techniques for immobilization, coating or encapsulation of bacteria are known in the art.

[0092] The effective amount of colony forming units (cfu) for each strain in the composition is determined by one of skill in the art and depends on the final formulation. The term "colony forming unit" ("cfu") is defined as the number of bacterial cells revealed by microbial counting on an agar plate.

[0093] In some embodiments, for example in edible products (e.g., yogurt, candy), the strains are 5 cfu / g~10 12 cfu / g, more specifically 10 7 cfu / g~10 11 In some embodiments, for example in dietary supplements (e.g., tablets), the strains are present in amounts of 10 cfu / g per dose (e.g., tablet). 6 ~10 12 cfu, specifically 10 per dose 8 ~10 11 It is present in amounts ranging from cfu.

[0094] In a particular embodiment, the probiotic composition is composed of 50% strain L. plantarum CECT 30292 (e.g., 1 billion cfu) and 50% mixture of L. plantarum CECT 7485, CECT 7484, and P. acidilactici CECT 7483 (e.g., a mixture having equal proportions of the three strains and a total of 1 billion cfu).

[0095] As known to those skilled in the art, the effective amount of colony units can also be measured by the effective amount of active fluorescent units (afu). The term "active fluorescent units" (afu) is defined as the number of bacterial cells revealed by flow cytometry counting in a gate specific to the fluorescent properties of presumed live cells. Therefore, those skilled in the art would consider the above-specified amount of colony forming units (cfu) to be approximately the same as the amount of active fluorescent units (afu).

[0096] In one embodiment, the probiotic composition is a solid composition.

[0097] In another embodiment, the probiotic composition comprises about 10 5 cfu ~ approx. 10 12 The solid composition comprises a freeze-dried bacterial biomass containing each strain of cfu and a cryoprotectant.

[0098] Specifically, the probiotic composition includes at least one cryoprotectant that is an allergen-free cryoprotectant.

[0099] In some embodiments, the probiotic composition comprises at least one cryoprotectant selected from the group consisting of maltose, trehalose, mannitol (particularly d-mannitol), sucrose, lectose, dextrose, sodium ascorbate, or sodium citrate.

[0100] More specifically, the composition further comprises a pharma- ceutically acceptable carrier selected from an emulsion, a suspension, a gel, a paste, a granule, a powder, and a gum. Particularly, the carrier is an allergen-free carrier.

[0101] In some embodiments, the probiotic composition comprises one or more carriers selected from the group consisting of maltodextrin, cellulose, various types of starch, inulin, lactose, or reduced water activity carriers.

[0102] Medical Uses of Probiotic Compositions - Methods of Treating, Preventing, or Ameliorating COVID-19 or Its Symptoms, Complications, and / or Sequelae In some embodiments, administration of the probiotic composition results in the treatment, prevention, or amelioration of at least one symptom of COVID-19 selected from the group consisting of: - Fever; - Cough, especially a dry cough; -headache; -shortness of breath; -Body pain, especially muscle pain; - low oxygen saturation in the blood (hypoxemia), in particular an oxygen saturation SaO2 or SpO2 below 92%; and -diarrhea.

[0103] As known to those skilled in the art, SaO2 is an alternative measure of oxygen saturation in the blood and refers to the oxygen saturation measured in the arteries and thereafter by pulse oximetry. SaO2 measurements, which are equivalent to SpO2 measurements, can be used interchangeably in this description.

[0104] In some embodiments, administration of the probiotic composition results in at least one result (i.e., effect) selected from the group consisting of: - Remission of COVID-19; - Reduction in duration of COVID-19 symptoms; A reduction in SARS-CoV-2 viral load; - stimulation of antibody production (i.e., an increase in antibody levels) against the SARS-CoV-2 virus (in particular, stimulation of IgG and IgM antibody production against the SARSCoV-2 virus; more specifically, at least a doubling of IgG antibody levels); -Improvement of blood oxygen saturation (hypoxemia); and -Decreased duration of low oxygen saturation in the blood (i.e., hypoxemia).

[0105] In some embodiments, administration of the probiotic composition results in at least one result (i.e., effect) selected from the group consisting of: - an increase in interferon-β levels, specifically, an increase in interferon-β levels of 20%; and - Decreased CXCL13 levels.

[0106] In some embodiments, administration of the probiotic composition to a subject can enable the subject to avoid or reduce hospitalization, avoid ICU admission (i.e., eliminate the need for ICU treatment), delay the need for ICU treatment, reduce (shorten) ICU treatment time, reduce patient mortality, increase patient survival, reduce the likelihood of patient death, increase the likelihood of patient survival, reduce the risk of patient death, reduce the severity of at least one symptom, reduce the severity of at least one complication, reduce the severity of at least one sequelae, reduce the duration of at least one symptom, reduce the duration of at least one complication, reduce the duration of at least one sequelae, or any combination thereof. Thus, the present disclosure provides a method for avoiding or reducing hospitalization, reducing (shortening) ICU treatment time, delaying the need for ICU treatment, avoiding ICU admission (i.e., eliminating the need for ICU treatment), reducing patient mortality, increasing patient survival, reducing the likelihood of patient death, increasing the likelihood of patient survival, reducing the risk of patient death, reducing the severity of at least one symptom, reducing the severity of at least one complication, reducing the severity of at least one sequelae, reducing the duration of at least one symptom, reducing the duration of at least one complication, reducing the duration of at least one sequelae, or any combination thereof in a subject, which comprises administering a probiotic composition to a subject.

[0107] Medical uses of probiotic compositions - Methods for treating, preventing, or ameliorating an infectious disease or its symptoms, complications and / or sequelae.

[0108] As discussed herein, the probiotic composition shows high efficacy in inducing remission in COVID-19 patients, as well as shortening the duration of symptoms (e.g., shortness of breath, hypoxemia, diarrhea) and increasing virus-specific antibodies. Furthermore, the composition showed significant beneficial effects in reducing the duration of low oxygen saturation in the blood (hypoxemia).

[0109] Thus, the experimental data herein provide supporting evidence that the probiotic composition has a significant positive effect in other infectious diseases, particularly those characterized by one or more of the respiratory and / or gastrointestinal symptoms described above.

[0110] In particular, the experimental data herein provides evidence that the probiotic composition may have significant positive effects in the treatment of other viral respiratory infections accompanied by shortness of breath and hypoxemia (especially SpO2 less than 92%), such as SARS, MERS, Human Adenovirus 55, Influenza A, particularly H1N1, or novel emerging viruses.

[0111] In particular, hypoxemia occurs in respiratory infections associated with viral bronchiolitis infection of the lungs in infants and children. Viral bronchiolitis is most typically caused by respiratory syncytial virus, but can also be caused by rhinovirus, adenovirus, pneumovirus, human bocavirus, influenza virus, or parainfluenza virus. Concurrent bronchiolitis due to two or more viruses in the same individual is often reported. Hypoxemia has also been identified as a risk factor for death from viral influenza. The probiotic composition described herein exhibits high efficacy in reducing symptoms associated with respiratory tract infection. In particular, the experimental data herein provides supporting evidence that the probiotic composition has a significant positive effect in treating other respiratory infections (e.g., viral) that cause shortness of breath or hypoxemia (especially SaO2 or SpO2 less than 92%), such as severe influenza or viral bronchiolitis. Specifically, the experimental data herein provides supporting evidence that the probiotic composition has a significant positive effect in treating low oxygen saturation in the blood (especially SpO2 below 92%).

[0112] Thus, in some embodiments, the infectious disease is caused by a bacterial, viral, parasitic and / or fungal infection.

[0113] In a particular embodiment, the infectious disease is caused by a viral infection. More specifically, the viral infection is selected from the group consisting of adenovirus, viral hepatitis (particularly hepatitis C), viral encephalitis (particularly herpes simplex encephalitis), human bocavirus, human rhinovirus, influenza virus, particularly influenza A virus (particularly H1N1 and its variants), parainfluenza virus, pneumovirus, coronavirus, particularly β-coronavirus (more particularly SARS-CoV1, SARS-COV2 or MERS-CoV), respiratory syncytial virus, rhinovirus and novel or emerging viruses. More specifically, the viral infection is a coronavirus, influenza or respiratory syncytial virus infection.

[0114] In certain embodiments, the viral infection is a coronavirus infection. Coronaviruses are large, enveloped, single-stranded RNA viruses found in humans and other mammals. Coronaviruses (CoVs) induce acute and chronic diseases. Coronaviruses cause respiratory, gastrointestinal, and neurological diseases. There are seven known coronaviruses that cause disease in humans, while 229E, OC43, NL63, and HKU1 typically produce cold symptoms that are not easily separable from symptoms caused by other respiratory viruses, with no shortness of breath and complications in immunocompromised individuals. Thus, in some embodiments, the coronavirus is, for example, 229E, OC43, NL63, and HKU1 or a variant thereof.

[0115] On the other hand, SARS-CoV-1, MERS-CoV and SARS-CoV-2 (all three belonging to the β-coronavirus subgroup) can cause more severe illness ranging from dyspnea (shortness of breath) and low blood oxygen saturation (hypoxemia) to death. Notably, SARS-CoV-2 is the third coronavirus to cause severe disease in humans and spread globally in the past two decades. These three types of CoV are CoV-associated severe acute respiratory syndrome (SARS-CoV-1 and SARS-CoV-2) and CoV-associated Middle East respiratory syndrome (MERS-CoV).

[0116] In certain embodiments, the antibody is a monoclonal antibody.

[0117] In certain embodiments, the coronavirus is a SARS-CoV-2 virus, a SARS-CoV-1 virus, or a MERS-CoV virus.

[0118] In some embodiments, the viral infection is caused by the SARS (Severe Acute Respiratory Syndrome) virus or SARS-CoV-1, a virus identified in 2003. SARS was first reported in Asia in February 2003. The disease has spread to more than two dozen countries in North and South America, Europe, and Asia before the SARS pandemic of 2003. No known cases of SARS have been reported anywhere in the world since 2004. In some embodiments, the coronavirus infection is caused by the MERS (Middle East Respiratory Syndrome) virus or MERS-CoV. MERS is a viral respiratory disease that is new to humans. It was first reported in Saudi Arabia in 2012 and has since spread to several other countries, including the United States. Most people infected with MERS-CoV develop severe respiratory illness, including fever, cough, and shortness of breath.

[0119] In some embodiments, the viral infection is caused by SARS-CoV-2. As used herein, the terms "SARS-CoV-2," "SARS-CoV-2 virus," and "COVID-19 virus" are used interchangeably to refer to the coronavirus that causes COVID-19 (coronavirus disease 2019). As used herein, the term "COVID-19" refers to a viral infection caused by SARS-CoV-2. COVID-19 is a new infectious disease caused by the severe acute respiratory syndrome 2 coronavirus (SARS-CoV-2). COVID-19 disease was first described on December 1, 2019 and became a global pandemic within a few weeks. Studies demonstrate that SARS-CoV-2 induces a cytokine storm that is associated with the severity of COVID-19 disease and its prognosis. COVID-19 patients may develop mild (localized symptoms in the upper respiratory tract with fever, myalgia, etc.), moderate (mild pneumonia) or severe disease, including severe pneumonia, acute respiratory distress syndrome (ARDS), sepsis and septic shock. Nevertheless, most individuals infected with SARS-CoV-2 may not have any illness or may develop mild illness, but SARS-CoV-2 infection is not just a cold.

[0120] In some embodiments, the viral infection may be caused by other viruses, such as, for example, human adenovirus 55, influenza virus (eg, influenza A, particularly H1N1), or respiratory syncytial virus.

[0121] In one embodiment, the infection results in (or induces) a respiratory disease.

[0122] In certain embodiments, the respiratory disorder is characterized by shortness of breath.

[0123] In certain embodiments, the respiratory disorder is characterized by hypoxemia (particularly SaO2 or SpO2 less than 92%).

[0124] In certain embodiments, the respiratory disease affects the upper respiratory tract.

[0125] In certain embodiments, the respiratory disease affects the lower respiratory tract (ie, lungs, alveoli).

[0126] In a particular embodiment, the respiratory disease is pneumonia, particularly mild or severe pneumonia.

[0127] In certain embodiments, the respiratory disease is ARDS.

[0128] As used herein, the terms "acute respiratory distress syndrome" and "ARDS" refer to a pulmonary disease or condition in which infection (e.g., viral infection) or injury to the lungs leads to lung inflammation, fluid retention in the alveolar air sacs, low blood oxygen levels, and difficulty breathing. Thus, in some embodiments, the present disclosure provides a method of treating ARDS comprising administering a probiotic composition of the present disclosure.

[0129] ARDS is a life-threatening condition in which severe damage occurs to most or all of both lungs. ARDS is the rapid onset of progressive insufficiency of the lungs, especially with respect to their ability to take in oxygen, and is usually associated with the insufficiency of other organs. ARDS is characterized by diffuse pulmonary microvascular injury that leads to increased permeability and thus noncardiogenic pulmonary edema. The fundamental abnormality in ARDS is the destruction of the normal alveolar-capillary barrier. Alternative terms frequently used to refer to ARDS include adult hyaline membrane disease, adult respiratory distress syndrome, congestive atelectasis, hemorrhagic pulmonary syndrome, Da Nang lung disease, stiff lung disease, shock lung, white lung disease, and wet lung disease, among others.

[0130] In some embodiments, a patient is considered to be suffering from "ARDS" if the following symptoms are present: 1) Clinical evidence of respiratory distress. 2) Chest x-ray revealing diffuse bilateral airspace disease ("pulmonary edema"). 3) Hypoxemia difficult to correct with supplemental oxygen. 4) Hemodynamic evidence of pulmonary artery obstruction (wedge) pressure <18 mmHg. 5) Static chest compliance of less than 40 mL / cm of water.

[0131] In some embodiments, the cause of ARDS is, for example, a severe widespread infection (sepsis), severe low blood pressure (shock), pneumonia, inhaling food into the lungs (inhalation), injury to the lungs from breathing high concentrations of oxygen, pulmonary embolism, chest injury, burns, drug overdose, or disseminated intravascular coagulation.

[0132] In some embodiments, sequelae arising from the lungs in ARDS patients include reduced lung volume, compliance, and large intrapulmonary shunts (blood perfusing the unventilated parts of the lungs). Reduced lung volume (residual volume) contributes to ventilation / perfusion imbalance. Reduced lung compliance is caused by increased pulmonary recoil pressure in edematous lungs, which clinically increases the work of breathing and leads to respiratory muscle fatigue. ARDS affects the pulmonary vasculature. Pulmonary hypertension not associated with hypoxemia is very common in patients with ARDS. Pulmonary hypertension is caused by a 3-5-fold increase in pulmonary vascular resistance (PVR) and is associated with increased work of the right ventricle.

[0133] Common symptoms of ARDS include dyspnea, tachipnea, dry cough, retrosternal discomfort, and flail. In some embodiments, patients exhibit moderate to severe dyspnea and may have purpura. In some embodiments, lung examination reveals harsh crackles and bronchial breath sounds. In some embodiments, patients require assisted mechanical ventilation.

[0134] SARS-CoV-2 may also have gastrointestinal disorders due to microinflammation, vascular dams, and dysbiosis of the gastrointestinal tract. As a result, 9% to 34% of COVID-19 patients suffer from diarrhea. As discussed herein, the probiotic composition of the present invention significantly shortens the duration of diarrhea in COVID-19 patients. The mean duration of diarrhea in patients who received the probiotic composition was less than half of the mean duration of the placebo group. Thus, the experimental data herein provide supporting evidence that the probiotic composition has a significant positive effect on the duration of gastrointestinal symptoms, such as diarrhea, caused by diseases and infections other than COVID-19.

[0135] Thus, in one embodiment, the infection results in (ie, induces) gastrointestinal disease.

[0136] In one embodiment, the gastrointestinal disorder is characterized by microinflammation, vascular damage and / or dysbiosis of the gastrointestinal tract.

[0137] In some embodiments, in connection with infectious diseases and the resulting respiratory and / or gastrointestinal diseases, administration of the probiotic composition treats, prevents, or ameliorates at least one symptom selected from the group consisting of: -Fever, - Cough, especially a dry cough -headache, -shortness of breath, -Body pain, especially muscle pain, - low oxygen saturation in the blood (hypoxemia), especially oxygen saturation SaO2 or SpO2 < 92%, and -diarrhea.

[0138] In some embodiments, administration of the probiotic composition results in at least one result (i.e., effect) selected from the group consisting of: - remission of infection, - Reduction in the duration of symptoms, - Reduction in viral load, - stimulation of antibody production (i.e., an increase in antibody levels), in particular stimulation of IgG and IgM antibody production, more particularly at least a doubling of IgG antibody levels; -Improvement of blood oxygen saturation (hypoxemia), and -Decreased periods of low oxygen saturation in the blood (i.e., hypoxemia).

[0139] In some embodiments, administration of the probiotic composition results in at least one result (i.e., effect) selected from the group consisting of: - an increase in interferon-β levels, specifically, an increase in interferon-β levels of 20%; and - Decreased CXCL13 levels.

[0140] In some embodiments, the present disclosure provides a method of selecting a subject having a disease or condition disclosed herein (e.g., COVID-19) for treatment with a probiotic composition according to the present invention, comprising determining whether a biomarker is present selected from the group consisting of lymphopenia, neutrophilia, activation of coagulation (e.g., as indicated by increased D-dimer levels), acute kidney injury (e.g., as indicated by increased blood urea and creatinine), increased levels of IL-6, increased plasma cardiac troponin I, increased plasma lactate dehydrogenase, increased plasma ferritin, increased levels of IL-10, increased levels of TNFα, decreased levels of CD4+ T cells, decreased levels of CD8+ T cells, leukocytosis, increased levels of procalcitonin, decreased expression of IFNγ in CD4+ T cells, increased levels of cytokines produced by inflammatory macrophages, or a combination thereof, wherein the presence of the biomarker indicates that the subject may be treated with the probiotic composition disclosed herein.

[0141] Medical Use of Probiotic Compositions for Enhancing Immune Responses Protection against COVID-19 is largely mediated by immune responses against the SARS-CoV-2 spike (S) protein. The S protein is involved in virus-cell binding and is the target of virus-neutralizing antibodies. Functional neutralizing antibodies specific for SARS-CoV-2 (anti-spike glycoprotein and anti-RBD), produced after infection, vaccination, or both, are believed to be important for virus neutralization and clearance, and are quantified by the use of in vitro neutralization assays. Therefore, enhancing the production of SARS-CoV-2-specific antibodies is of great interest to achieve long-term immunity derived from both early infection and / or vaccination. In particular, IgA-type antibodies dominate the immune response against SARS-CoV-2 early in infection but rapidly fade after the third week of infection, while IgG-type antibodies have been shown to significantly increase their positivity rate.

[0142] As discussed herein, the probiotic composition of the present invention enhances the immune response of patients, resulting in a significant increase in the concentration of IgG and IgM antibodies (p<0.001).Notably, on the 30th day of intervention, subjects receiving the probiotic composition had IgG levels two times higher than subjects in the placebo group.Therefore, the experimental data herein provide evidence supporting that the probiotic composition has a significant positive effect on stimulating the immune response of patients, particularly in the production of antibodies derived from both early infection and / or vaccine.

[0143] Furthermore, the above evidence supports that the probiotic composition also has a significant positive effect in stimulating the immune response (e.g., increasing the production of antibodies) from other infections (e.g., bacterial or viral infections) or vaccines, particularly viral infections and / or viral vaccines.

[0144] Thus, in some embodiments, the probiotic compositions are used to enhance, accelerate and prolong immune system responses.

[0145] In certain embodiments, administration of the probiotic composition stimulates the production of antibodies, particularly IgG and / or IgM.

[0146] In one embodiment, the immune system response includes the production of antibodies (particularly IgG and / or IgM), particularly IgG antibody levels are at least doubled.

[0147] In other embodiments, the probiotic composition is used as an adjuvant, immunostimulant, immunoenhancing agent, or immunomodulator.

[0148] In some embodiments, the probiotic compositions disclosed herein can be administered in combination with other therapeutic and / or prophylactic treatments (e.g., anticoagulants, anti-inflammatory agents, or blood pressure regulating agents). The probiotic composition and the other treatment can be formulated for separate, sequential, simultaneous administration, or mixed.

[0149] In some embodiments, the probiotic composition is used in conjunction with other treatments used for respiratory infections, especially respiratory infections of viral origin causing hypoxemia, especially COVID-19, such as, but not limited to, corticosteroids (e.g., dexamethasone, prednisone, methylprednisolone, or hydrocortisone), antipyretics (e.g., acetaminophen or nonsteroidal anti-inflammatory drugs, e.g., ibuprofen and similar compounds), virus-specific neutralizing antibodies (e.g., REGN-COV2), vaccines (e.g., vaccines using RNA vaccines, recombinant chimeric viruses or peptide vaccines), molecules with antiviral activity (e.g., interferons, plitidepsin, remdesivir, oseltamivir and similar compounds) or cytokine signaling blockade (e.g., tocilizumab).

[0150] In some embodiments, the probiotic composition is administered in combination with a vaccine. In one embodiment, the probiotic composition is for use in combination with a vaccine.

[0151] In some embodiments, the probiotic composition is administered in combination with an anti-inflammatory drug, such as an anti-inflammatory corticosteroid. Examples of corticosteroids are methylprednisolone, fludrocortisone, hydrocortisone, prednisone, prednisolone, flunisolide, fluticasone, beclomethasone, budesonide, ciclesonide, mometasone, and any combination thereof. In one embodiment, the probiotic composition is for use in combination with an anti-inflammatory drug.

[0152] In one embodiment, the probiotic composition is administered in combination with an antipyretic medication.

[0153] Products Containing Probiotic Compositions In some embodiments, the bacterial strains described herein can be formulated for separate, sequential, simultaneous administration, or mixed. The administration regimen and form of administration are determined by those skilled in the art. The administration of the bacterial strains can be administered separately (after a time interval between the administration of the first and second bacterial strains), sequentially (one after the other), concomitantly (simultaneously), or mixed (together). Thus, they can be administered as separate forms or as part of a single composition. When the products are administered in separate dosage forms, the dosage forms can be in the same or different containers, or, for example, capsules or pills. In certain embodiments, the strains described herein are administered in a single composition, for example, tablets.

[0154] The probiotic compositions described herein can be formulated as food, pharmaceutical or veterinary products, in which the strains are the only active agent or are mixed with one or more other active agents and / or with pharma- ceutically or veterinarily acceptable excipients (in the case of pharmaceutical or veterinary products) or suitable additives (in the case of food products). Preferably, the additional active agent(s) are other probiotic bacteria that are not antagonistic to the strains forming the composition of the invention. Depending on the formulation, the strains can be added as purified bacteria, as bacterial cultures, as part of bacterial cultures, as post-treated bacterial cultures, alone or together with suitable carriers or ingredients. Prebiotics such as fructooligosaccharides (especially inulin), galactooligosaccharides, xylooligosaccharides, arabinoxylan oligosaccharides, pectin, β-glucan or partially hydrolyzed guar gum can also be added.

[0155] In some embodiments, the probiotic composition is in the form of a pharmaceutical composition comprising an effective amount of the probiotic composition and at least one pharma- ceutically acceptable excipient.

[0156] In certain embodiments, the composition of the present invention is formulated as a pharmaceutical product, in particular as a dietary supplement.The term "pharmaceutical product" is understood herein in its broadest sense and includes any composition that contains active ingredient, in this case, in particular the strain described herein in the form of composition together with pharmaceutically acceptable excipients.This term is not limited to medicine.

[0157] The term "pharmacologically acceptable" is recognized in the art and includes those compounds, materials, compositions, carriers, vehicles and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g., a human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a satisfactory benefit / risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in the standard pharmaceutical technical literature. Some non-limiting examples of substances which can serve as pharma- ceutically acceptable carriers include: sugars (e.g., lactose, glucose, and sucrose), starches (e.g., corn starch, potato starch, and the like), cellulose and its derivatives (e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate), powdered tragacanth, malt, gelatin, talc, cocoa butter and suppository waxes, oils (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), glycols (e.g., propylene glycol), polyols (e.g., glycerin, sorbitol, mannitol, polyethylene glycol), esters (e.g., ethyl oleate, ethyl laurate), agar, buffers (magnesium hydroxide, aluminum hydroxide), alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, phosphate buffer solutions, and other non-toxic compatible substances used in pharmaceutical formulations.

[0158] The excipients are selected from the group including, but not limited to, fillers / diluents / bulking agents, binders, anti-adherents, disintegrants, coating agents, anti-caking agents, antioxidants, lubricants, sweeteners, flavoring agents, coloring agents, surfactants and other classes of pharma- ceutical and veterinarily acceptable excipients.

[0159] The filler is selected from the group including, but not limited to, inulin, oligofructose, pectin, modified pectin, microcrystalline cellulose, lactose, starch, maltodextrin, saccharose, glucose, fructose, mannitol, xylitol, non-crystalline sorbitol, calcium carbonate, dicalcium phosphate, other inert inorganic and organic pharma- ceutically acceptable fillers, and mixtures of these substances. In oral suspension dosage forms, the filler or diluent is selected from the group including vegetable oils, oleic acid, oleyl alcohol, liquid polyethylene glycols, other pharma-ceutically acceptable inert liquids, or mixtures of these substances.

[0160] Binders are used in solid dosage forms, for example, to hold the ingredients in a tablet together, to ensure that tablets and granules can be formed with the required mechanical strength, and to provide bulk to low-active-dose tablets. Binders in solid dosage forms such as tablets are lactose, sucrose, maize starch, modified starch, microcrystalline cellulose, modified cellulose (e.g., hydroxypropyl methylcellulose (HPMC) and hydroxyethyl cellulose), other water-soluble cellulose ethers, polyvinylpyrrolidone (PVP), also known as povidone, polyethylene glycol, sorbitol, maltitol, xylitol, and dibasic calcium phosphate, other suitable pharma- ceutically acceptable binders, or mixtures of these substances.

[0161] Anti-adherents are used to reduce adhesion between the powder (granules) and the punch surfaces, thus preventing sticking to the tablet punch. They are also used to help protect the tablet from sticking. The most commonly used is magnesium stearate.

[0162] The following materials may be used as disintegrants and superdisintegrants in solid dosage forms such as tablets and capsules, but are not limited to: cross-linked polyvinylpyrrolidone, sodium starch glycolate, sodium carboxymethylcellulose, calcium carboxymethylcellulose, and formaldehyde-casein, other suitable pharma- ceutically acceptable disintegrants and superdisintegrants, or mixtures thereof.

[0163] Coatings for solid dosage forms such as tablets and capsule-filling granules protect the ingredients from degradation by moisture in the air, make large, unpleasant-tasting tablets easier to swallow, and / or, in the case of enteric coatings, allow complete passage through the strongly acidic medium of gastric juices (pH about 1) and release in the duodenum or ileum (small intestine). Most coated tablets use a cellulose ether hydroxypropyl methylcellulose (HPMC) film coating. In some cases, other coating materials are used, such as synthetic polymers and copolymers such as polyvinylacetate phthalate (PVAP), methyl acrylate-methacrylic acid copolymers, methyl methacrylate-methacrylic acid copolymers, shellac, corn protein zein or other polysaccharides, waxes or wax-like substances (e.g., beeswax), higher fatty alcohols such as stearic acid, cetyl or stearyl alcohol, solid paraffin, glycerol monostearate, glycerol distearate, or combinations thereof. Capsules are coated with gelatin or hydroxypropyl methylcellulose.

[0164] Enteric coatings control the rate of drug release and determine where in the digestive tract the drug is released. Materials used in enteric coatings include fatty acids, waxes, shellac, plastics, and vegetable fibers and mixtures thereof, and may be combined with other coatings mentioned above.

[0165] Anti-caking agents are additives added to powdered or granular materials to prevent the formation of lumps (caking) and facilitate packaging, shipping, and consumption. The following are used as anti-caking agents in solid dosage forms such as tablets, capsules, or powders: magnesium stearate, colloidal silica, talc, other pharma- ceutically acceptable anti-caking agents, or mixtures thereof.

[0166] Lubricants are used in solid dosage forms, especially tablets and capsules, to prevent ingredients from clumping together and from sticking to tablet punches or capsule filling machines, as well as in hard capsules. As lubricants, talc or silica, and fats such as vegetable stearin, magnesium stearate or stearic acid, and mixtures thereof, are the most frequently used lubricants in tablets or hard gelatin capsules.

[0167] Sweeteners are added to make ingredients more palatable, especially in solid dosage forms, such as chewable tablets, as well as in liquid dosage forms, such as cough syrups.Sweeteners can be selected from artificial, natural or synthetic or semi-synthetic sweeteners.Non-limiting examples of sweeteners are aspartame, acesulfame potassium, cyclamate, sucralose, saccharin, sugar or any mixture thereof.

[0168] Flavorings can be used to mask unpleasant tasting active ingredients in any dosage form. Flavorings can be natural (e.g., fruit extracts) or artificial. For example, the following improvements can be made: (1) in bitter products, mint, cherry or anise can be used; (2) in salty products, peach or apricot or liquorice can be used; (3) in sour products, raspberry can be used; (4) in overly sweet products, vanilla can be used.

[0169] Except for the auxiliary agents from the class of excipients, the formulations from the present invention may contain other pharma- ceutically active substances or nutritional substances, such as, but not limited to, vitamins, such as vitamin D (calciferol) in pharma- ceutically acceptable chemical form, salts or derivatives, minerals in pharma- ceutically and nutritionally acceptable chemical form, and L-amino acids. As for the preparation of the formulations of the present invention, it is within the scope of the skilled artisan and depends on the formulation to be finally administered. For example, but not limited to, when the final dosage form is an oral solid dosage form, such as a tablet, capsule, powder, granule, oral suspension, the process for the preparation of the solid dosage form of the formulation may be as follows: The method comprises (1) homogenizing an active ingredient, comprising at least one or more post-treated probiotic bacteria of the present invention in an effective amount, with (2) one or more excipients to form a homogenous mixture, which is lubricated, for example with magnesium stearate or other lubricants as required, to obtain a powdered final dosage form. Such homogenous powder is filled into a normal gelatin capsule or into a gastro-resistant capsule. In the case of tablets, they are produced by direct compression or granulation. In the first case, a homogenous mixture of the active ingredient and a suitable excipient (e.g. anhydrous lactose, amorphous sorbitol, etc.) is prepared. In the second case, tablets are processed from the mixture in granular form. Granules are prepared by a process of granulating the active ingredient of the formulation with suitable fillers, binders, disintegrants, and a small amount of purified water. The granules thus prepared are sieved and dried until the moisture content is <1% w / w.

[0170] Regarding the method of preparing liquid dosage forms (e.g., oral suspensions), the method comprises homogenizing the active ingredients of the formulation, comprising an effective amount of at least one or more post-processed probiotic bacteria of the present invention, in an inert liquid diluent (filler), such as various vegetable oils, such as sunflower oil, soybean oil, or olive oil, oleic acid, oleyl alcohol, liquid polyethylene glycols, such as PEG 200, PEG 400, or PEG 600, or other inert pharma- ceutically acceptable liquids. The method further comprises treating the homogenized mixture by one or more processes selected from the group including: (1) stabilizing the formulation by adding suspension stabilizers such as beeswax, colloidal silica, and homogenizing, (2) sweetening the formulation by adding sweeteners and homogenizing, and (3) flavoring the formulation by adding flavorings and homogenizing. Such forms of formulations can also contain other excipients or ingredients commonly used in the art.

[0171] Pharmaceutical products can adopt different forms or names depending on the product approval route and depending on the country. For example, a medicine is a specific pharmaceutical product. A medical food is another specific pharmaceutical product. The term "medical food" or "food for special medical purposes" is used in some countries to refer to foods that are specially formulated and intended for the dietary management of diseases with unique nutritional requirements that cannot be met by normal diet alone. These are defined in regulations such as the Food and Drug Administration's 1988 Orphan Drug Act Amendments in the United States and the Commission Directive 1999 / 21 / EC in Europe. Medical foods are different from the broader categories of dietary supplements and traditional foods with health claims. Thus, in certain embodiments, the strains of the present invention are formulated as medical foods.

[0172] In some embodiments, the probiotic composition is in the form of a dietary supplement comprising the probiotic composition and at least one acceptable excipient.

[0173] Specifically, the probiotic composition is in the form of a capsule, tablet, pill, sachet, or suspension (e.g., in oil). Specifically, the capsule, tablet, or pill has a weight of about 350 mg to about 450 mg, more specifically 400 mg. In particular, the sachet has a weight of about 2 g to about 6 g.

[0174] In certain embodiments, the probiotic composition is in the form of a vegetable capsule and comprises HMPC.

[0175] Dietary supplements, also known as dietary supplements or nutritional supplements, are considered as another specific pharmaceutical product. They are preparations or products intended to supplement the diet, made from compounds normally used in food, that provide nutrients or beneficial components that may not normally be taken in or consumed in sufficient amounts in the normal diet. In most cases, dietary supplements are considered as foods, but sometimes they are also defined as drugs, natural health products, or dietary supplements. In the sense of the present invention, dietary supplements also include nutraceuticals. Dietary supplements are usually sold "over the counter (OTC)", i.e. without a prescription. When dietary supplements take the form of pills, capsules, tablets or powders, they contain the same excipients as those used in medicines. However, dietary supplements can also take the form of foods (e.g. bars or yogurt) that are enriched with some nutrients.

[0176] Thus, in certain embodiments, the probiotic composition is formulated as a dietary or nutritional supplement. The dietary supplement can be administered on its own, mixed with a suitable drinking liquid such as water, yogurt, milk or fruit juice, or mixed with solid or liquid foods. In this context, the dietary supplement can be in the form of tablets or lozenges, pills, capsules, granules, powders, suspensions, sachets, sweeteners, bars, syrups, and corresponding dosage forms, usually in the form of unit doses.

[0177] In some embodiments, the probiotic composition is in the form of an oil suspension administered alone or mixed with a liquid. The oil suspension comprises at least one edible oil, such as olive oil, corn oil, soybean oil, linseed oil, sunflower oil or rice oil. The oil is present in an amount of at least 70% (w / w). In certain embodiments, the oil suspension also comprises at least one excipient that is an emulsifier, stabilizer or anti-caking agent in an amount of 0.1-15% w / w. Suitable agents are silica, silica gel, colloidal silica, precipitated silica, talc, magnesium silicate, lecithin, pectin, starch, modified starch, konjac gum, xanthan gum, gellan gum, carrageenan, sodium alginate, fatty acid mono- or diglycerides such as glycerol monostearate or glycerol monooleate, and citric acid esters of mono- or diglycerides.

[0178] Specifically, the probiotic composition is in the form of an infant nutritional supplement in the form of an oil suspension. In a particular embodiment, the oil suspension comprises sunflower oil and colloidal silica (specifically 1% by weight), and bacterial cells.

[0179] In another embodiment, the oil suspension comprises sunflower oil, an agent selected from lecithin, mono- or diglycerides of fatty acids, carrageenan, and sodium alginate, and bacterial cells.

[0180] In some embodiments, the probiotic composition is in the form of an edible product comprising the probiotic composition and at least one edible ingredient. In particular, the edible product may be a dairy product, milk, yogurt, or curd.

[0181] The compositions of the invention can also be included in various food or edible compositions such as dairy products (yogurt, cheese, fermented milk, milk powder, milk-based fermented products, ice cream, fermented cereal-based products, milk-based powders), breads, bars, spreads, biscuits and cereals, beverages, different types of oils, or dressings. The term "food" is used herein in its broadest sense and includes any type of product in any presentation form that can be ingested by an animal, but excludes pharmaceutical and veterinary products. Examples of other foods are meat products, chocolate spreads, fillings and frostings, chocolates, confectionery, baked goods, sauces and soups, fruit juices and coffee whiteners. Foods of particular interest are dietary supplements and infant formulas. Foods include in particular carrier materials such as oatmeal porridge, lacto-fermented foods, resistant starch, dietary fiber, carbohydrates, proteins and glycosylated proteins. In a particular embodiment, the strains of the invention are encapsulated or coated. In particular, the milk can be of animal or vegetable origin.

[0182] In one embodiment, the probiotic composition is in the form of a vegetable capsule filled with a powder containing L. plantarum DSM 33765 (e.g., 1 billion freeze-dried colony forming units (CFU)) along with L. plantarum CECT 7484, L. plantarum CECT 7485 and P. acidilactici CECT 7483 (e.g., 1 billion freeze-dried colony forming units (CFU)) together with a carrier (e.g., maltodextrin, potato starch, dextrose).

[0183] In another embodiment, the probiotic composition is in the form of an aluminum sachet containing the bacterial strains (e.g., in the amounts described above), a carrier (e.g., maltodextrin, dextrose, rice fiber, potato starch), a sweetener (e.g., sucralose, sodium saccharin) and a flavoring agent.

[0184] In another embodiment, the probiotic composition is in the form of a bottle containing the bacterial strains (eg, in the same amounts as above) suspended in sunflower oil to be administered as drops.

[0185] In some embodiments, the probiotic composition of the present disclosure is administered shortly after the onset of a disease disclosed herein or after detection of an infection, hi some embodiments, additional doses of the composition of the present disclosure are then administered after the initial dose.

[0186] In some embodiments, the probiotic composition is administered in a single dose or in repeated doses at specific time intervals, for example, daily for a specific number of days, or according to a specific dosing schedule. Specifically, the probiotic composition is administered for 10 to 90 days. More specifically, it is administered for 10 to 60 days or 15 to 45 days, more specifically for 30 days.

[0187] In some embodiments, the probiotic composition is administered from once every three days to three times a day, particularly once a day.

[0188] In some embodiments, the probiotic composition of the present disclosure can be administered, for example, orally, pulmonary, intravenously, intramuscularly, or subcutaneously. Specifically, the probiotic composition is administered orally. In other embodiments, the probiotic composition can be administered by nasal inhalation, oral spray, or nasogastric route. EXAMPLES

[0189] Example 1: Strain L. plantarum CECT 30292 (DSM 33765) The deposit numbers CECT 30292 and DSM 33765 are used interchangeably.

[0190] 1.1. Strain isolation The novel strain of L. plantarum, deposited as DSM 33765, was isolated from both humans (fecal samples) and chicory (cichorium intybus) by dilution of plant extracts on phosphate-buffered saline (PBS) followed by plating on Man-Rogosa-Sharpe agar plates (Man-Rogosa-Sharpe, MRS) and incubation at 37°C under microaerobic conditions (5% CO2).

[0191] 1.2. Whole genome sequencing Strain L. plantarum DSM 33765 was grown in MRS liquid broth for 8 h at 37°C and cells were harvested by centrifugation in early logarithmic phase. Total genome and plasmid were extracted using QIAamp DNA Mini Kit (Quiagen) according to the manufacturer's instructions with an additional lysis step of incubating cells for 10 min at 37°C in lysis buffer supplemented with 20 mg / mL lysozyme (Sigma Aldrich, Steinheim am Albuch, Germany) and 40 U / mL mutanolysin (Sigma Aldrich). Template DNA was prepared using Sequel II Binding Kit 2.0 (Pacific Bioscience) and sequenced using PacBioRSII at TAKARA BIO INC (Kaneka, Japan) and assembled using the software SMRT Link v8.0.0 (Pacific Bioscience).

[0192] Chromosomal alignments between DSM 33765 and the genome of the reference L. plantarum strain WCSF1 [Kleerebezem et al. 2003], as well as the genomes of L. plantarum CECT 7484, CECT 7485 and ATCC 202195, were performed using the progressive MAUVE algorithm [Darling et al. 2004].

[0193] The results of the whole genome comparison (Table 1) unambiguously show that strain DSM 33765 is the same L. plantarum species, as it is clearly higher than the 95% species boundary based on an average nucleotide identity (ANI) across the whole genome of >98.5% [Jain et al. 2018].

[0194] [Table 1]

[0195] Furthermore, tree analysis based on ANI (Figure 1 ) indicates that this strain belongs to the same clade as CECT 7484 (ANI of 99.71%) but is more distantly related to the reference strain WCFS1, as well as ATCC 202195 and CECT 7485 (ANI values ​​of 98.7–98.8%).

[0196] 1.3. Molecular Fingerprinting DSM 33765 strain was tested according to a previously described protocol [Rodas et al. 2005] with minor modifications. Strains were grown on MRS agar plates and incubated for 18 h at 37 °C and 5% CO2. Cells were harvested and washed three times in 8 mL PET (10 mM Tris pH 7.6, 1 M NaCl) and then centrifuged at 6000 rpm for 10 min. The pellet was resuspended in 700 mL lysis buffer (6 mM Tris, 1 M NaCl, 0.1 M EDTA, 0.5% SLS, 0.2% deoxycholate, 1 mg / mL lysozyme, 40 U / mL mutanolysin, 20 mg / mL RNase). An equal volume of 1.6% low melting point agarose (FMC BioProducts, Rockland, ME, USA) was added to the resuspended cells and allowed to solidify for 1 h at 4 °C. The inserts were transferred to 2 mL of lysis buffer II (0.5 M EDTA pH 9.2, 1% N-lauryl sarcosine and 1 mg / mL pronase) and incubated for 48 h at 50° C. The inserts were then washed with TE buffer (10 mM Tris, 1 mM EDTA pH 8.0) at room temperature. Total DNA digestion was performed with Sma-I restriction enzyme (Roche Diagnostics).

[0197] Pulsed-field gel electrophoresis (PFGE) was performed in duplicate using a CHEF DRIII apparatus (BioRad Laboratories). Inserts were loaded onto 1% agarose gels (SeaKem ME agarose, FMC Bio-Products, ME, USA). DNA MW (molecular weight) markers were Lambda ladder PFG marker and Low Range PFG marker (New England Biolabs). After electrophoresis, gels were stained with ethidium bromide and UV using the GelDoc System (BioRad).

[0198] The results show that L. plantarum strain DSM 33765 has a reproducible PFGE profile that differs from that of the closely related strain L. plantarum CECT 7484. Specifically, compared to CECT 7484, strain DSM 33765 lacks the 97 kDa band but has additional bands between 48.5 and 32.1 kDa, thus indicating that although closely related, both strains are distinct from each other.

[0199] 1.4. Genome annotation General genome annotation was performed using the software DFAST [Tanizawa et al. 2018], which includes tRNAscan and Prodigal features, and the Conserved Domain Database [Shennan et al. 2020]. Plasmids were identified using PlasmidDB software [Galata et al. 2019].

[0200] To identify the presence of possible antibiotic resistance (AR) genes, L.DSM 33765 was compared against the final version of the Comprehensive Antibiotic Resistance Database (CARD), a bioinformatics database of resistance genes, their products, and associated phenotypes [Alcock et al. 2020], using the Resistance Gene Identifier (RGI) which supports the analysis of the whole genome using default parameters. As a double check, the genome sequence was also compared against the final version of the ResFinder database [Bortolaia et al. 2020].

[0201] The results are shown in Table 2. L. plantarum DSM 33765 yielded four contigs containing 3,322,082 bp. The larger contig (3,237,983 bp) was identified as a chromosomal sequence, whereas three further contigs (41,908 bp, 35,007 bp and 7,184 bp) were found to correspond to different L. plantarum plasmids. No antibiotic resistance genes were found in the bacterial chromosome or in any of the three plasmids. Two pilin genes and ten mucin-binding genes were identified, highlighting the adaptation of the strain to bind to mucus and epithelium and thus supporting its ability to colonize the intestine.

[0202] [Table 2]

[0203] 1.5. Resistance of CECT 30292 strain to gastric conditions An artificial stomach medium was prepared containing 7.3 g / L sodium chloride, 0.5 g / L potassium chloride, 3.8 g / L sodium bicarbonate and 3 g / L pepsin in distilled water. Samples of the simulated medium were adjusted to pH 2.3 and pH 3 with 1 N hydrogen chloride. 0.1 mL of an overnight culture of CECT 30292 strain was inoculated into 10 mL of artificial stomach medium at pH 2.3 or pH 3, mixed thoroughly and incubated at 37°C for 30 or 90 minutes. 10-fold serial dilutions were then prepared in buffered saline medium. Both 0.1 mL of the overnight culture and 0.1 mL of the various serial dilutions were plated on different MRS agar plates. After 48 hours of incubation at 37°C under anaerobic conditions, colonies were counted. Experiments were performed in quadruplicate.

[0204] The results in Table 3 below show that this strain is highly resistant to exposure to gastric medium.

[0205] [Table 3]

[0206] Example 2: Clinical Trials 2.1. Test Products One billion freeze-dried cfu of L. plantarum CECT 7484, L. plantarum CECT 7485 and P. acidilactici CECT 7483 were used in vegetable capsules (hydroxymethylpropyl-cellulose, HMPC) along with one billion freeze-dried cfu of the novel strain L. plantarum DSM 33765, mixed with maltodextrin carrier. The CFU load of the capsules was verified by serial dilution in PBS buffer followed by plating in MRS agar and counting the colonies after 48 h incubation at 37° C. under microaerobic conditions.

[0207] Study design A quadruple-blind (patients, caregivers, outcome assessors and principal investigator) randomized clinical trial against placebo was conducted to evaluate the safety and efficacy of the strain combination (L. plantarum DSM 33765 and strains L. plantarum CECT 7484, L. plantarum CECT 7485 and P. acidilactici CECT 7483 at a total dose of 2 billion CFU administered once daily in subjects with mild COVID-19. The trial was performed in accordance with the ethical provisions of the Declaration of Helsinki.

[0208] The first objective was to improve COVID-19 severity according to the World Health Organization scale [WHO Working Group 2020]. The second objective was to evaluate the effect of the combination in reducing the frequency and severity of individual COVID-19 symptoms, reducing viral load, and increasing the levels of immunoglobulin antibodies IgG and IgM.

[0209] Three hundred adults aged 18–60 years with positive nasopharyngeal detection of SARSCoV-2 by real-time polymerase chain reaction (PCR), COVID-19 symptom resolution (according to the WHO COVID-19 severity scale), and providing written informed consent were randomized. Individuals with blood oxygen saturation (SpO2) below 90%, taking immunosuppressants, acute pancreatitis, or regular use of other probiotics within 2 weeks of study initiation were excluded from participation. Randomization was performed according to a computer-generated list, and all study participants (patients, caregivers, outcome assessors, and principal investigator) were blinded as to whether they were assigned to placebo or probiotic capsules. Probiotic and placebo capsules were indistinguishable from each other. The computer-printed list was kept in a locked, sealed envelope by a pharmacist not participating in the study.

[0210] Individuals randomized to either probiotic or placebo received one capsule of their signature intervention once daily for 30 consecutive days. COVID-19 symptoms were assessed every 5 days. Quantification of SARSCoV-2 viral load in nasopharyngeal swabs was performed on days 0, 15, and 30 of the intervention. Quantification of specific IgG and IgM antibodies to SARSCoV-2 was performed in blood samples on days 0, 15, and 30 of the intervention.

[0211] 2.3. Characteristics of subjects recruited at baseline The probiotic and placebo groups in the study were balanced with regard to baseline clinical and demographic characteristics (Table 4).

[0212] [Table 4]

[0213] 2.4.Results During the 30-day intervention, only three subjects in the probiotic group and four subjects in the placebo group dropped out. No patients died or required intensive care during the 30-day intervention.

[0214] Complete remission of COVID-19: Complete remission of COVID-19 is defined as complete disappearance of symptoms accompanied by a negative PCR result (i.e., a score of 0 on the WHO COVID-19 severity scale). No patient showed complete remission on day 15 of the intervention. However, far more patients in the probiotic group than in the placebo group showed complete remission on day 30 of the intervention, and the difference was highly statistically significant (p-value < 0.001 by chi-square test). See Table 5.

[0215] Symptom remission: Symptom remission was defined as complete disappearance of symptoms regardless of PCR result (i.e., a score of 0 or 1 on the WHO COVID-19 severity scale). Patients receiving probiotics showed more remission in the early phases of the intervention (i.e., days 10, 15, and 20) than patients receiving placebo. See Table 5.

[0216] [Table 5]

[0217] Viral load: During the 30-day intervention, the concentration of viral load in nasopharyngeal swabs was reduced more in the probiotic group than in the placebo group, and the difference was highly statistically significant (p<0.001 by repeated measures ANOVA), as can be seen in Figure 3. At the end of the 30-day intervention, the difference was an average of 0.9 log, thus the concentration was reduced by 25 mean 9-fold.

[0218] Concentrations of IqG and IqM antibodies: During the 30-day intervention, concentrations of IgG and IgM antibodies against SARS-CoV-2 increased more in the probiotic group than in the placebo group, and the difference was highly statistically significant (p<0.001 by repeated measures ANOVA), as can be seen in Figure 4. At the end of the 30-day intervention, subjects receiving probiotics had IgG levels two times higher than subjects in the placebo group.

[0219] Statistical adjustments were performed to assess whether age, blood type, body mass index, concurrent diabetes, or concurrent hypertension modulated the effect of probiotics on remission rates, viral load, and antibody levels. Notably, none of these variables were found to modulate the effect of probiotic intervention. Thus, probiotics achieved their beneficial effects regardless of age, blood type, body mass index, concurrent diabetes, or concurrent hypertension.

[0220] Reduction of COVID-19 specific symptoms: During the 30-day intervention, the probiotic intervention was also found to significantly reduce the duration of various specific symptoms of COVID-19, as well as the number of days with blood oxygen saturation below 92% (hypoxemia), compared to placebo, the latter despite a higher proportion of subjects with a saturation below 92% at baseline in the probiotic group (all p<0.05 by Mann-Whitney test). See Table 6.

[0221] [Table 6]

[0222] Example 3: Measurement of serum concentrations of interferon-β (IFNb) and the chemokine CXCL13 3.1. Method 70 individuals were randomly selected from the 300 individuals described in Example 2. Randomization was stratified by intervention group to ensure that 35 corresponded to the placebo group and 35 to the probiotic group, by lung infiltration status at the start of the intervention, and by responder status at the end of the intervention to ensure that the selected subpopulation of 70 individuals had a similar distribution to the total population of 300 individuals.

[0223] Archived frozen serum samples (see Example 2) taken on days 0, 15 and 30 of the intervention were used to measure interferon-β (IFNb) and the chemokine CXCL13 using standard Enzyme-Linked ImmunoSorbent Assay (ELISA) laboratory kits. Digital lung x-ray scans taken on days 0, 15 and 30 were also retrieved from patient records and pulmonary inflammatory infiltrates, whenever present, were scored using a confirmed Brixia score (the higher the score, the more severe the inflammatory infiltrate) [Borghesi et al., 2020].

[0224] 3.2. Results Clinical characteristics of subjects: The probiotic and placebo groups in the subpopulation of 70 subjects were balanced with respect to clinical and demographic characteristics at baseline (Table 7).

[0225] [Table 7]

[0226] Serum concentrations of interferon-β: At baseline, the levels of IFNb were indistinguishable between the probiotic and placebo groups. During the 30-day intervention, the levels of IFNb increased significantly in the probiotic group compared to the placebo (p<0.001, repeated measures ANOVA), as can be seen in Figure 5. More precisely, the concentrations in the probiotic group were significantly higher both at days 15 and 30 compared to the placebo (p<0.05).

[0227] Serum concentrations of CXCL13: At baseline, CXCL13 levels were indistinguishable between the probiotic and placebo groups. During the 30-day intervention, CXCL13 levels significantly decreased over time in the probiotic group compared to placebo (p<0.001, repeated measures ANOVA), as seen in Figure 6. More precisely, concentrations in the probiotic group were significantly higher both at days 15 and 30 compared to placebo (p<0.05).

[0228] Resolution of Symptoms: The 70 subjects reported their symptoms daily in an electronic patient diary. The duration of some symptoms was significantly shorter in the probiotic group compared to the placebo group, as shown in Table 8.

[0229] [Table 8]

[0230] As further confirmation of the positive role of type I interferon against viral infections, higher levels of interferon-β correlated with fewer days with fever (Spearman correlation=-0.24, p-value=0.042) and fewer days with cough (Spearman correlation=-0.26, p-value=0.033) in the entire population of 70 subjects. A similar trend (i.e., p-value greater than 0.05 but less than 0.10) was observed for bodily pain (Spearman correlation=-0.21, p-value=0.075). Furthermore, lower levels of CXCL13 also correlated with fewer days with fever (Spearman correlation=0.27, p-value=0.024).

[0231] In the subgroup of patients with pulmonary inflammatory infiltrates at baseline (n=15 in probiotic and n=11 in placebo), the reduction in infiltrate correlated with higher interferon-β (Spearman correlation=0.46, p-value=0.018) and especially lower CXCL13 (Spearman correlation=0.69, p<0.001), consistent with a protective effect of lowering CXCL13 to reduce pulmonary inflammation.

[0232] Viral clearance: All 70 subjects were positive for SARS-CoV2 at the beginning of the intervention by PCR analysis (see Example 2). At the end of the 30-day intervention, 17 of 35 subjects (48.6%) in the probiotic group and 8 of 35 subjects (22.9%) in the placebo group achieved complete viral remission by PCR analysis. Thus, the effect of the probiotic was more than twice as high as that of the placebo, and this difference was statistically significant (chi-square test p-value = 0.026).

[0233] Example 4: Measurement of serum concentrations of plantaricin G 4.1. Method L. plantarum strains (CECT 30292, CECT 7483, CECT 7484, CECT 7485, CECT 7481, 299v, and WCFS1) were grown in MRS broth at 37° C. Bacterial cells were cultured at log phase (OD ) by centrifugation. 620 Approximately 0.6) and stationary phase (OD620 >1.5).

[0234] Total RNA was isolated from the pellet using the RNeasy Mini Kit according to the manufacturer's protocol. The RNA was then converted to cDNA using the NxGen Reverse Transcriptase Kit. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using SYBRGreen on an ABI7900 instrument, and cycle threshold (Ct) values ​​were recorded. Data were normalized with the housekeeping gene RecA, and gene expression was expressed using the delta-delta Ct method, using the expression of the WCSF1 strain as a reference. The RT-qPCR primers used in the experiment are listed in Table 9.

[0235] [Table 9]

[0236] 4.2. Results RT-qPCR experiments revealed that L. plantarum CECT 30292 significantly overexpresses the plnG gene compared to strain WCFS1 (4-fold increase, p<0.001) and to all other strains tested (p<0.05). This effect is consistent when the strain is in both exponential and stationary phase (Figure 7). Thus, strain L. plantarum CECT 30292 is more likely to be sensed by immune cells than other L. plantarum strains.

[0237] Example 5: Assessment of intra-strain variability 5.1. Method To assess intrastrain variability, whole genome sequencing was performed on two different batches of strain DSM 33765 using the same method already described in Example 1.

[0238] 5.2. Results The pairwise average nucleotide identity (ANI) between the genomes of two different batches of the same strain (DSM 33765) was 99.99%. Considering that the size of the genome of this strain is 3,322,082 base pairs, this difference of 0.01% amounts to approximately 330 nucleotide polymorphisms.

[0239] Example 6: Copy of the plnG gene 6.1. Method The sequence of the plantaricin G gene (plnG) was downloaded from the UniProt database (https: / / www.uniprot.org / uniprot / Q1WFD0, UniProtKB-Q1WFD0(Q1WFD0_LACPN), entry version 73, 29 Sep 2021) and searched against the genomes of L. plantarum strains CECT 7484, CECT 7485, DSM 33765, as well as the reference strain WCSF1 (see Example 1). The search was performed using the "tblastn" application from the BLAST program (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastDocs&DOC_TYPE=Download).

[0240] 6.2. Results The bacterial chromosome of the four analyzed L. plantarum strains carries one copy of the plantaricin G (plnG) gene. Notably, strain DSM 33765 was the only strain that also contained a plasmid carrying the plnG gene, as shown in Table 10 below. This could explain the significantly higher plantaricin G expression achieved by this strain (Example 4).

[0241] [Table 10]

[0242] References https: / / clinicaltrials.gov / ct2 / show / NCT04517422 WO2011092261A1 Chong et al.Lactobacillus plantarum DR7 improved upper respiratory tract infections via enhancing immune and inflammatory parameters:A randomized,double-blind,placebo-controlled study.J.Dairy Sci.2019.102:4783-4797 Meijerink et al.Identification of Genetic Loci in Lactobacillus plantarum That Modulate the Immune Response of Dendritic Cells Using Comparative Genome Hybridization.PLoS One 2010.5:e10632 D’Ettorre et al.Challenges in the Management of SARS-CoV2 Infection:The Role of Oral Bacterio-therapy as Complementary Therapeutic Strategy to Avoid the Progression of COVID-19.Front Med(Lausanne)2020.:389 Hishiki et al.A Double-Blind,Randomized,Placebo-Controlled Trial of Heat-Killed Pediococcus acidilactici K15 for Prevention of Respiratory Tract Infections among Preschool Children.Nutrients 2020.12(7):1989 Hill et al.The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic.Nat Rev Gastroenterol Hepatol 2014.11:506-14 Channapannavar et al.IFN-I response timing relative to virus replication determines MERS coronavirus infection outcomes.J Clin Invest 2019;129:3625-3639 Haagmans et al.Pegylated interferon-alpha protects type 1 pneumocytes against SARS corona-virus infection in ma-caques.Nat Med.2004;10290-3 Kleerebezem et al.Complete genome sequence of Lactobacillus plantarum WCFS1.Proc Natl Acad Sci USA.2003.100(4):1990-5 Darling et al.Mauve:multiple alignment of conserved genomic sequence with rearrangements.Genome Res 2004.14(7):1394-403 Jain et al.High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries Nat Commun.2018 Nov 30;9(1):5114 Rodas et al.Polyphasic study of wine Lactobacillus strains:taxonomic implications.Int J Syst Evol Microbiol 2005.55(Pt 1):197-207 Tanizawa et al.DFAST:a flexible prokaryotic genome annotation pipeline for faster genome publication.Bioinformatics 2018.34(6):1037-1039 Shennan et al.CDD / SPARCLE:the conserved domain database in 2020.Nucleic Acids Res 2020.48:D265-D8 Galata et al.PLSDB:a resource of complete bacterial plasmids.Nucleic Acids Res.2019.74:D195-D202 Alcock et al.CARD 2020:Antibiotic Resistome Surveillance with the Comprehensive Antibiotic Resistance Database.Nucleic Acids Research 2020.48:D517-D525 Bortolaia et al.ResFinder 4.0 for predictions of phenotypes from genotypes.Journal of Antimicrobial Chemotherapy 2020.75:3491-3500 WHO Working Group.A minimal common outcome measure set for COVID-19 clinical research.Lancet Infect Dis 2020.20:e192-e197 Borghesi et al.Chest X-ray severity index as a predictor of in-hospital mortality in coronavirus dis-ease 2019:A study of 302 patients from Italy.Int.J.Infect Dis 2020;96:291-293

Claims

1. Lactobacillus plantarum strain deposited under the Budapest Treaty at the Spanish Culture Collection (CECT) under accession number CECT 30292.

2. A method for obtaining a mutant strain of Lactobacillus plantarum deposited under CECT 30292, comprising the steps of: (i): Creating a mutant of L. plantarum CECT 30292; (ii): analyzing the mutants obtained in step (i) and identifying mutant strains that retain or improve at least one of the properties of the deposited strain; and (iii): isolating the mutant strain identified in step (ii), thereby obtaining a mutant strain of L. plantarum CECT 30292, wherein the mutant strain retains or improves at least one ability of the deposited strain.

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