TW202304485A - Methods and compositions for enhancing immunity - Google Patents

Abstract

The present invention provides for novel compositions and methods for enhancing immunity in an individual.

Description

Methods and compositions for enhancing immunity

The present invention relates to methods and compositions for enhancing immunity. RELATED APPLICATIONS This application claims priority to US Provisional Patent Application No. 63/173,947, filed April 12, 2021, the contents of which are hereby incorporated by reference in their entirety for all purposes.

In recent years, viral and bacterial infections have become increasingly common worldwide and present serious public health threats. For example, the coronavirus-2019 (COVID-19) global pandemic, the respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected nearly 1.2 billion people worldwide, including more than 2.6 million deaths , the threat to public health is exacerbated by the large number of asymptomatic carriers. Several potential therapeutic agents are currently being actively researched and developed for prophylaxis or treatment of COVID-19, thereby preventing or ameliorating its detrimental effects in affected patients, while experimental vaccines are being widely distributed to the general population .

Therefore, there is an urgent need for new, meaningful therapeutic approaches to control viral and bacterial infections and to alleviate or eliminate their associated effects. The aim of this study was to identify gut microbiome functional pathways and microbial metabolites for preventing severe infection outcomes and treating infection symptoms. Stimulation of these gut microbial functional pathways or direct supplementation with beneficial microbial metabolites is a potentially effective means of enhancing immunity, treating viral or bacterial infections (including COVID-19) and promoting recovery from these viral or bacterial diseases. The present invention fulfills this need and by illustrating the alterations in the gut microbiota during viral or bacterial infection and the potential role of therapeutic microorganisms and bacterial metabolites in the prevention, treatment and recovery of diseases and conditions caused by such infections. Other related needs.

In the context of infectious diseases caused by viral or bacterial pathogens and the prevention, treatment and recovery from these diseases, the present inventors conducted extensive studies on gut microbial functional pathways and microbial metabolites. The associated pathways and metabolites have been identified, and they now provide new ways and means to enhance or improve an individual's immunity and prevent or treat (e.g., reduce disease severity) and facilitate recovery from infectious diseases such as COVID-19. combination.

In a first aspect, the invention provides methods of enhancing immunity, preventing or treating a viral or bacterial infection, such as COVID-19, or reducing the symptoms or severity of a disease in an individual. The method includes the step of administering to the individual an effective amount of a composition comprising at least one short chain fatty acid, L-isoleucine, or one or more metabolites shown in Table 3, or producing a short chain fatty acid or L-isoleucine or such metabolites, or any combination thereof, provided that the subject does not otherwise require treatment with short-chain fatty acids or L-isoleucine or such metabolites. In some embodiments, the administering step comprises oral ingestion of the composition, e.g., in the form of a tablet, capsule, solution/suspension, emulsion, paste, powder, or in the form of a food or beverage item or an additive thereof . In some embodiments, the composition comprises at least one short chain fatty acid, optionally a combination of two or more different short chain fatty acids. In some embodiments, the composition comprises at least one short chain fatty acid and L-isoleucine. In some embodiments, the composition comprises at least one bacterium that produces short-chain fatty acids or L-isoleucine or such metabolites, optionally a combination of two or more different bacterial species, e.g. Mycobacterium and Coprococcus comes . In some embodiments, the short chain fatty acid is acetic acid, propionic acid, butyric acid, or isobutyric acid. In some embodiments, the short chain fatty acid may be butyric acid, butyrate, or butyrin. In some embodiments, the bacteria are butyrate-producing Gram-positive anaerobic bacteria. In some embodiments, the bacterium is Flostridium praussatus. In some embodiments, the bacterium is a species of the genus Bifidobacterium, such as Bifidobacterium adolescentis or Faecococcus chaperones, however in some cases other than Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve Bacillus and B. infantis, or at least not two, three or more of B. adolescentis, B. bifidum, B. longum, B. breve and B. infantis combination. In some embodiments, the subject has been diagnosed with COVID-19 or is at risk of becoming infected with SARS-CoV2. In some embodiments, particularly when the subject is seeking treatment (e.g., to reduce symptoms or disease severity) or to prevent COVID-19, the method further comprises administering to the subject an agent known to effectively inhibit SARS-CoV2 and One or more agents that promote recovery from COVID-19 (for example, ivermectin, vitamin C, vitamin D, quercetin, melatonin, zinc, azithromycin, hydroxychloroquine, fluvoxamine, or fluoxetine, or any combination thereof), which may be in the same composition or in a second separate composition. In some embodiments, the subject, while recovering from COVID-19, still suffers from long-lasting symptoms 4 weeks or more after the end of the active phase of the disease.

In a related aspect, the present invention provides novel uses of compositions for improving immunity in a subject. The composition comprises an effective amount of (1) at least one, possibly two or more active ingredients of: short-chain fatty acids, L-isoleucine, metabolites shown in Table 3, and A bacterium of said short-chain fatty acid or L-isoleucine or said metabolite; and (2) a physiologically acceptable excipient. In some embodiments, the compositions consist essentially of the active ingredient plus one or more excipients. In some embodiments, the subject is diagnosed with COVID-19 or the subject is at risk of infection with SARS-CoV-2, thereby seeking prevention or treatment of the disease (eg, reducing disease severity or symptoms). In some embodiments, the composition is formulated for oral ingestion, e.g., in the form of a tablet, capsule, solution/suspension, emulsion, paste, powder, or in a food or beverage item or additive form. In some embodiments, the composition comprises a short chain fatty acid and L-isoleucine, or a combination of at least two or more short chain fatty acids, with or without L-isoleucine. In some embodiments, the physiologically acceptable excipient in the composition is starch, inulin, oat bran, wheat bran, cellulose, guar gum, or pectin. In some embodiments, especially when the composition is used to prevent or treat COVID-19, the composition may further comprise one or more agents known to be effective in inhibiting SARS-COV2 and promoting recovery of patients from COVID-19 (e.g., ethanol vitamin C, vitamin D, quercetin, melatonin, zinc, azithromycin, hydroxychloroquine, fluvoxamine, or fluoxetine, or any combination thereof). In some embodiments, the bacterium is a butyrate-producing Gram-positive anaerobic bacterium. In some embodiments, the bacterium is Flostridium praussatus. In some embodiments, the bacterium is a species of the genus Bifidobacterium, such as Bifidobacterium juveniles or Faecococcus chaperones. In some cases, the bacteria does not include any of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium infantis, or the bacteria does not include Bifidobacterium adolescentis , Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve and Bifidobacterium infantis two or three or more.

In a second aspect, the present invention provides an agent for increasing immunity in a subject, for preventing or treating a viral or bacterial infection such as COVID-19 in a subject, or for reducing the symptoms or severity of a disease box. The kit comprises at least two, possibly more containers, each containing a composition comprising an effective amount of one or more active ingredients, which may be short chain fatty acids, L-isoleucine or epitope Compounds named in 3, or bacteria producing short chain fatty acids or L-isoleucine or said compounds, or any combination of the above. For example, the first and second containers contain two different compositions comprising two different short chain fatty acids or two different bacterial species producing the same or different short chain fatty acids, e.g. Gram-positive anaerobic bacteria that produce butyrate. The kit may further comprise one or more agents (e.g., ivermectin, vitamin C, vitamin D, quercetin, melatonin, Zinc, azithromycin, hydroxychloroquine, fluvoxamine, or fluoxetine, or any combination thereof). In addition, a user instruction manual can be included with the kit to describe the proper use of the kit.

definition

" Short chain fatty acids (SCFA) " are fatty acids having fewer than 6 carbon atoms. As used herein, SCFA includes formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid and 2-methylbutyric acid and their chemical derivatives such as salts, esters and the like.

The term " SARS-CoV-2 or severe acute respiratory syndrome coronavirus 2" as used herein refers to the virus that causes coronavirus disease 2019 (COVID-19). It is also known as the "COVID-19 virus".

The term " inhibiting " or " inhibition " as used herein refers to any detectable negative effect on a target biological process in a subject, such as RNA/protein expression of a target gene, biological activity of a target protein , cell signal transduction, cell proliferation, presence/level of organisms, especially microorganisms. Typically, inhibition is reflected in at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater reduction. "Inhibition" also includes a 100% reduction, ie complete abolition, prevention or clearance of a target biological process or signal. Other relative terms such as "suppressing", "suppression", "reducing" and "reduction" are used in a similar manner in this disclosure to refer to reduction to different levels (e.g. , at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction compared to the control level) up to complete elimination of the target biological process or signal. On the other hand, terms such as "activate", "activating", "activation", "increase", "increasing", "promoting" are used in this disclosure )", "promoting", "enhance", "enhancing" or "enhancement" encompasses different levels (e.g., at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or higher, such as 3, 5, 8, 10, 20-fold increase) Variety.

The term " treatment " or " treating " as used herein includes both curative and prophylactic measures taken to address the presence of a disease or condition or the risk of subsequent occurrence of such a disease or condition. It encompasses therapeutic or prophylactic measures for alleviating ongoing symptoms, inhibiting or slowing disease progression, delaying the onset of symptoms or eliminating or reducing side effects resulting from such diseases or conditions. Preventive measures and their variants in this context need not eliminate 100% of the occurrence of an event; rather, they refer to inhibiting or reducing the likelihood or severity of such occurrence or delaying such occurrence.

The term " severity " of a disease refers to the level and degree to which the development of the disease adversely affects the well-being and health of the patient suffering from the disease, such as short-term and long-term physical, mental and psychological impairment, up to and including the death of the patient . Severity of disease can be reflected in the nature and amount of curative and maintenance measures necessary, the duration required for patient recovery, the likely extent of recovery, the percentage of patients who fully recover, the percentage of patients requiring long-term care, and mortality .

A " patient " or " subject " receiving a composition or method of treatment of the present invention is a human, usually an adult, of any age, sex, and ethnic background, who may not be diagnosed with any particular disease or condition but is in need of enhanced immunity (e.g., at risk for viral or bacterial infection), or who has been diagnosed with a disease or condition involving a viral or bacterial infection such as COVID-19, exhibits one or more associated symptoms, or has been diagnosed with Diagnosed with such a disease or condition involving a viral or bacterial infection (such as COVID-19) and have substantially recovered from the disease, while continuing to experience residual symptoms (such as fatigue, headache, nausea, dizziness, muscle or joint pain) or generally debilitating) for a prolonged period of time (eg, at least 4 weeks after the end of the active phase of the disease, ie, the presence of viral or bacterial pathogens is no longer detectable as indicated by negative nucleic acid/PCR tests). Typically, a patient or subject receiving treatment according to the methods of the invention to enhance immunity need not otherwise be treated with the same therapeutic agent. For example, if a patient with COVID-19 is being treated in accordance with the claimed method, the patient does not have another disease unrelated to COVID-19 (e.g., cardiovascular disease, diabetes, gastrointestinal disorders and diseases such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS) or colitis treatable by butyrate). Although the patient may be of any age, in some instances the patient is at least 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85 years old; in some instances the patient may be 40 to 45 years old , or 50 to 65 years old, or 65 to 85 years old.

As used herein, the term " effective amount " refers to the amount of a substance administered to produce a therapeutic effect. Such effects include preventing, correcting or inhibiting to any detectable extent the progression of symptoms of the disease/condition and associated complications, such as one or more symptoms of viral or bacterial infections and associated disorders (eg, COVID-19). The exact amount will depend on the purpose of the treatment and will be determined by those skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding ( 1999); and Pickar, Dosage Calculations (1999)).

The term " about " when used in reference to a given value means encompassing a range of ±10% of that value.

A " pharmaceutically acceptable " or " pharmacologically acceptable " excipient is a substance that is not biohazardous or otherwise undesirable, i.e., the excipient can be administered with a biologically active agent in individual without causing any unwanted biological effects. The excipients also do not interact in a deleterious manner with any component of the composition in which they are contained.

The term " excipient " refers to any substance of an auxiliary nature that may be present in the final dosage form of the composition of the invention. For example, the term "excipient" includes vehicles, binders, disintegrants, fillers (diluents), lubricants, glidants (flow enhancers), compression aids, colorants, sweeteners, preservatives agent, suspending/dispersing agent, film-forming/coating agent, flavoring agent and printing ink.

The term " antibacterial agent " refers to any substance capable of inhibiting, suppressing or preventing the growth or proliferation of bacterial species. Known agents with antibacterial activity include various antibiotics that generally inhibit the proliferation of a broad spectrum of bacterial species, as well as agents that inhibit the proliferation of specific bacterial species, such as antisense oligonucleotides, small inhibitory RNAs, and the like. The term "antibacterial agent" is similarly defined to include agents with broad-spectrum activity in killing virtually all bacterial species as well as agents that specifically inhibit the proliferation of target bacteria.

When used in the context of describing a composition containing an active ingredient, the term " consisting essentially of ... refers to the fact that the composition does not contain any similar or related biological activity of the active ingredient or is capable of enhancing Or other ingredients that suppress activity, while one or more inactive ingredients such as physiologically or pharmaceutically acceptable excipients may be present in the composition. For example, a composition consisting essentially of an agent (e.g., SCFA or L-isoleucine or one or more compounds shown in Table 3) effective in treating a disease such as COVID-19 or reducing the severity of a disease does not contain any Combinations of other agents that may have any detectable prophylactic or therapeutic effect on the same disorder (eg, COVID-19) or may enhance or decrease the prophylactic or therapeutic effect of the active ingredient on the disorder. Detailed Description of the Invention I. Introduction

SARS-CoV-2 infection is associated with altered gut microbiota composition. Phylogenetic groups of gut bacteria involved in short-chain fatty acid metabolism were reduced in SARS-CoV-2 infection. This study aimed to characterize the functional spectrum of the gut microbiome in COVID-19 patients before and after disease resolution. Shotgun metagenomic sequencing was performed on stool samples from 55 antibiotic-naïve patients with COVID-19 and 70 non-COVID-19 controls. Serial stool samples (up to 6 time points) were collected during hospitalization and until 1 month after SARS-CoV-2 viral clearance. Fecal microbial pathways were assessed in conjunction with disease severity and blood immune and inflammatory markers. Changes in microbial function in fecal samples before and after disease resolution were also measured and metabolite target analysis was used to validate these functions.

Compared with non-COVID-19 controls, COVID-19 patients showed significant changes (p < 0.05) in gut microbiome function, characterized by genes for short-chain fatty acid (SCFA) or L-isoleucine biosynthesis Impaired capacity of the gut microbiome and enhanced capacity for urea production. Metabolite target analysis revealed significantly lower fecal concentrations of SCFA and L-isoleucine in fecal samples from COVID-19 patients. Impaired SCFA and L-isoleucine biosynthesis in stool samples persisted for up to 1 month after recovery in COVID-19 patients. Deficiency in SCFA and L-isoleucine biosynthesis and increased urea production were significantly associated with disease severity and plasma CXCL-10 concentration, platelet count, albumin and hemoglobin levels (all p < 0.05).

The gut microbiome of COVID-19 patients exhibits impaired SCFA and L-isoleucine biosynthetic capacity that persists even after disease resolution. These two microbial functions correlate with host immune responses, emphasizing the importance of gut microbial function in the pathogenesis and outcome of SARS-CoV-2 infection.

In addition to the beneficial effects on SCFA and L-isoleucine, this study also revealed that the degradation of L-1,2-propanediol, bifidobacterial shunt or 4-deoxy-L-threo-hexyl Beneficial effects of 4-deoxy-L-threo-hex-4-enopyranuronate, and negative effects on the urea cycle pathway. These findings translate to administration of SCFAs (such as acetate, propionate, butyrate/isobutyrate, valerate/isovalerate, and caproate) or L-isoleucine (including SCFA- or L-isoleucine-producing bacteria) , compounds that enhance L-1,2-propanediol degradation, bifidobacteria shunt or 4-deoxy-L-threo-hex-4-ene pyralate (ester), or inhibit, suppress or destroy the patient's Compounds of the urea cycle pathway for enhancing immunity, such as for preventing, treating or improving recovery from COVID-19. II. Pharmaceutical Composition and Administration

The present invention also provides a pharmaceutical composition comprising an effective amount of short-chain fatty acids (SCFA), L-isoleucine, any one of the metabolites shown in Table 3, or producing SCFA or L-isoleucine Bacteria of acids or metabolites of table 3 for use in prophylactic and/or prophylactic applications to enhance the immunity of humans or to reduce the risk of diseases involving viral or bacterial infections, for the treatment of such diseases or conditions (including reducing disease severity and promoting recovery), such as COVID-19. The pharmaceutical compositions of the present invention are suitable for use in various drug delivery systems. Suitable formulations for use in the present invention can be found in Remington's Pharmaceutical Sciences , Mack Publishing Company, Philadelphia, PA, 17th Edition (1985). For a brief review of drug delivery methods, see Langer, Science 249 : 1527-1533 (1990).

The pharmaceutical compositions of the present invention may be administered by various routes, such as systemic administration by oral ingestion or local delivery using rectal suppositories. A preferred way of administering the pharmaceutical composition is with about 0.1-20, about 0.14-14 g, about 0.5-5 g, about 1-3 g, for example about 1-2 or about 1.4 g of SCFA or L-iso Daily doses of leucine or metabolites, or live bacteria capable of producing equivalent amounts of SCFA or L-isoleucine or metabolites were administered orally. A suitable dose may be administered in a single daily dose or in divided doses given at appropriate intervals, for example in sub-doses of two, three, four or more times per day.

For the preparation of pharmaceutical compositions containing SCFA, L-isoleucine, any one of the compounds shown in Table 3 or bacterial strains producing SCFA or L-isoleucine or the compounds shown in Table 3 or any combination thereof , using one or more inert and pharmaceutically acceptable carriers. Pharmaceutical carriers can be solid or liquid. Solid form preparations include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders or tablet disintegrating agents; it can also be an encapsulating material.

In powders, the carrier is usually a finely divided solid which is in admixture with the finely divided active ingredient, for example a SCFA such as butyric acid or a strain of Gram-positive anaerobic bacteria which produce SCFA. In tablets, the active ingredient is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

To prepare a pharmaceutical composition in the form of a suppository, a low melting wax such as a mixture of fatty acid glycerides and cocoa butter is first melted and the active ingredient is dispersed therein by, for example, stirring. The molten homogeneous mixture is then poured into suitably sized molds and allowed to cool and solidify.

Powders and tablets preferably contain from about 5% to about 70% by weight active ingredient (eg, one or more SCFAs, such as butyric acid or a Gram-positive anaerobic bacterial strain that produces at least one SCFA). Suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, Cocoa butter, etc.

The pharmaceutical composition may comprise a formulation of an active ingredient, e.g., one or more SCFAs, such as butyric acid or L-isoleucine, a metabolite in Table 3 or capable of producing at least one SCFA, L-isoleucine, Table 3 Gram-positive anaerobic bacterial strains of metabolites or any combination thereof, wherein the encapsulating material acts as a carrier to provide a capsule in which the active ingredient (with or without other carriers) is surrounded by the carrier so that the carrier is thus associated with the active ingredient. In a similar manner, sachets may also be included. Tablets, powders, sachets, and capsules can be used as solid dosage forms suitable for oral administration.

Liquid pharmaceutical compositions include, for example, solutions, suspensions and emulsions suitable for oral administration or topical delivery. Active components (e.g., one or more SCFAs, such as butyrate, or L-isoleucine, or metabolites named in Table 3, or capable of producing at least one SCFA, L-isoleucine, or said metabolites Gram-positive anaerobic bacterial strains) or sterile solutions of the active ingredient in solvents including water, buffered water, saline, PBS, ethanol or propylene glycol are liquid or semi-liquid compositions suitable for rectal administration instance. The composition may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents and the like.

Sterile solutions can be prepared by dissolving the active ingredient (e.g., one or more SCFAs or L-isoleucine or a compound shown in Table 3) in the desired solvent system and passing the resulting solution through a membrane filter. Sterile, or alternatively, prepared by dissolving the sterile active ingredient in a previously sterile solvent under aseptic conditions. The resulting aqueous solutions can be packaged for use as such, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the formulation is usually 3-11, more preferably 5-9, most preferably 7-8.

Pharmaceutical compositions containing active agents may be administered for prophylactic and/or therapeutic treatment. In therapeutic applications, the composition is administered to persons already suffering from a disease or condition caused by a viral or bacterial infection, such as COVID-19, in an amount sufficient to prevent, cure, reverse, or at least partially slow or arrest the symptoms of the condition and its complications of patients. An amount sufficient to accomplish this is defined as "therapeutically effective dose". Amounts effective for this use will depend on the severity of the disease or condition and the patient's weight and general state, but generally range from about 0.05 g to about 5 g of active agent (e.g., SCFA or L-isoleuron) per day for a 70 kg patient amino acid or any of the compounds shown in Table 3, or bacterial strains capable of producing them), more usually at a dose of about 0.05 g to about 1 g of active agent per day for a 70 kg patient.

In prophylactic applications, a pharmaceutical composition containing an active agent is administered to a patient susceptible to a disease or condition caused by a viral or bacterial infection such as COVID-19 or otherwise in an amount sufficient to delay or prevent the onset of symptoms Patients at risk of a disease or condition caused by a viral or bacterial infection, such as COVID-19. Such an amount is defined as a "prophylactically effective dose". In such use, the precise amount of active agent will again depend on the patient's state of health and weight, but will typically be from about 0.05 g to about 5 g of active agent for a 70 kg patient/day, more typically about 5 g of active agent for a 70 kg patient/day. 10mg to about 1g.

Single or multiple administrations of the compositions can be carried out at the dosage level and pattern selected by the treating physician. In any event, the pharmaceutical formulation should provide the active agent in an amount sufficient to ameliorate, alleviate or reverse the associated symptoms or delay the onset of symptoms in the patient, either therapeutically or prophylactically. III. Other therapeutic agents

Other known therapeutic agent(s) may be combined with an active agent (e.g., SCFA, L-isoleucine, one or more of the compounds shown in Table 3 or compounds capable of producing SCFA or L-isoleucine) in the practice of the invention. amino acids or live bacterial strains of said compounds) for use in preventing, reducing the risk of, treating, reducing the severity of, and promoting recovery from a disease or condition caused by a viral or bacterial infection, such as COVID-19. In prophylactic or therapeutic applications, one or more of these previously known therapeutic agents may be administered to a patient simultaneously in a single composition or separately in two or more different compositions together with an effective amount of the active agent.

For example, drugs and supplements known to be effective in preventing or treating COVID-19 include ivermectin, vitamin C, vitamin D, melatonin, quercetin, zinc, hydroxychloroquine, fluoxetine, or fluvoxa The drug, along with one or more antibiotics such as azithromycin or doxycycline, may be used in combination with one or more bacteria such as Bifidobacterium adolescentis or Faecalicoccus chaperones. However, in some cases, the composition does not include any of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium infantis, or does not include Bifidobacterium adolescentis , Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve and Bifidobacterium infantis any two, three or more. They can be used in combination with the active agents of the present invention to reduce the risk of SARS-COV2 infection in an individual and by reducing disease severity (including morbidity and mortality), alleviate one or more symptoms of the disease, and promote recovery from the disease to treat COVID. In particular, the combination of zinc, hydroxychloroquine, and azithromycin/doxycycline, and the combination of ivermectin, vitamin C, vitamin D, melatonin, quercetin, and zinc all showed high efficacy in COVID prevention and treatment. Therefore, these known drug/supplement combinations can be combined with SCFA, L-isoleucine, one or more of the metabolites shown in Table 3, or be able to produce SCFA or L-isoleucine or said metabolites Active agents for live bacterial cells of the present invention are used in the methods of the present invention.

Two exemplary compositions comprising (1) SCFA, L-isoleucine, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum (optionally with xylooligosaccharides, fructooligosaccharides and anti- and (2) zinc acetate, calcium acetate, Bifidobacterium breve, Bifidobacterium bifidum, fructooligosaccharides, maltodextrin. IV . Kit

The present invention also provides kits for improving the immunity of individuals or treating infectious diseases such as COVID-19 according to the methods disclosed herein. The kit typically includes a plurality of containers, each containing a composition comprising SCFA, L-isoleucine, or a live bacterial strain capable of producing SCFA or L-isoleucine, or a combination thereof. Optionally, an additional container may be included in the kit providing a composition comprising another compound that is therapeutically effective or known to be effective in the prevention and/or treatment of disease, including amelioration of symptoms and alleviation of disease and combinations of compounds that promote recovery from the disease, such as those described in the previous section or known in the relevant art. In some variations of the kit, a single container may contain a pharmaceutical composition comprising two or more ingredients effective in treating a disease (e.g., COVID-19), such as SCFA, L-isoleucine, or SCFA capable of producing or L-isoleucine plus live bacterial strains as described in the previous section or those known in the relevant research field. The kit can further include informational material providing instructions on how to dispense the pharmaceutical composition, including a description of the type of patient that can be treated (e.g., has received a diagnosis of an infectious disease (e.g., COVID-19) or has been considered to have human patients at high risk of contracting the disease, e.g. due to a risky occupation, e.g. medical care), and types of patients not included in the claimed method (e.g., suffering from gastrointestinal diseases such as inflammatory bowel disease, intestinal irritable syndrome or colitis), the dosage, frequency and manner of administration, and the like. Example

The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize various noncritical parameters that can be changed or modified to produce substantially the same or similar results. Example 1: Pathways and metabolites associated with COVID-19 Introduction

The coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) primarily infects the respiratory system but also affects other organs, including the gastrointestinal (GI) ^tract1-4 . Recent studies have reported alterations in the gut microbiome in SARS-CoV-2 infection5-7 characterized by beneficial (butyrate-producing) bacteria such ^as those from the families Ruminococcaceae and Lachnospiraceae ), and opportunistic pathogens, including Streptococcus , Rothia , Veillonella , and Actinomyces , showed ^enrichment8 ; these changes Even after recovering from COVID-19. Representation of several gut commensals with known immunomodulatory potential, including faecalibacterium prausnitzii , Eubacterium rectale , and bifidobacteria , in fecal samples from COVID-19 patients Insufficient host immune responses reflect disease severity and dysfunction ⁹ .

The gut microbiota is known to modulate host immune responses to respiratory viral infections ^10-12 . Metabolites secreted by the gut microbiome, such as tyrosine, can protect against influenza through type I interferons and inflammasome ^- dependent cytokines produced by lung cells13,14. Whether microbial-derived metabolites also modulate host immune responses to SARS-COV-2 infection remains unknown. Interestingly, in vitro experiments showed that butyrate downregulated genes essential for SARS-CoV-2 infection such as angiotensin-converting enzyme 2 (ACE2) and upregulated TLR (Toll-like receptor) resistance in intestinal epithelial organoids. Viral Pathways ¹⁵ .

Significant microbial activity is a reflection of the cumulative function of the entire community of gut microbiota, and the balance of this community and its output determine the net contribution to health or disease ¹⁶ . Deciphering the role of microbially derived butyrate or other metabolites in SARS-CoV-2 pathogenesis and severity may shed light on possible mechanistic links between gut microbiota and host defense against SARS-CoV-2 understand.

In this study, 55 antibiotic-naïve patients with COVID-19 were prospectively investigated and followed up from admission until 30 days after discharge. Using metagenomic and metabolomic analyses, changes in gut microbiome function and longitudinal dynamics in relation to disease severity and immune response were characterized. This finding was further validated using targeted metabolomics analysis to examine alterations in fecal microbial metabolites. Methods Subject recruitment and sample collection

This study was approved by the Clinical Research Ethics Committee (reference number 2020.076), and all patients provided written informed consent. Patients with COVID-19 were recruited from February to May 2020 at the Prince of ^Wales Hospital and United Christian Hospital in Hong Kong as described in previous studies. Patients were divided into ^four severity groups according to the symptoms reported by Wu et al. Briefly, patients were classified as mild if there were no radiographic findings of pneumonia, moderate if pneumonia was detected with fever and respiratory symptoms, and oxygen saturation if respiratory rate ≥30 breaths/min 93% (when breathing ambient air), or PaO2/FiO2 ≤ 300 mmHg, the patient was classified as severe, and if respiratory failure required mechanical ventilation or organ failure required intensive care, the patient was classified as critical. Blood and stool from hospitalized patients were collected by hospital staff, while discharged patients provided stool during follow-up. Samples were stored at -80°C until processing. stool DNA extraction

Detailed methods are described in Zuo et al ^{. 17} . Briefly, approximately 0.1 g of fecal samples were prewashed with 1 ml _ddH2O and pelleted by centrifugation at 13,000 xg for 1 min. Fecal DNA was subsequently extracted from the pellet using the Maxwell® RSC PureFood GMO and Authentication Kit (Promega, Madison, Wisconsin) according to the manufacturer's instructions. Shotgun metagenomic sequencing and profiling

Sequencing libraries were prepared from the extracted DNA using the Nextera DNA Flex Library Prep Kit (Illumina, California USA) and sequenced on the Illumina NovaSeq 6000 system at the Gut Microbiota Research Center at the Chinese University of Hong Kong. An average of 26 million ± 3.3 million readings (6G data), 32 million ± 4.6 million readings were obtained per sample.

Raw sequence ^reads19 were filtered and quality trimmed using Trimmomatic v0.36 as follows: 1. Trim low quality bases (quality score < 20), 2. Remove reads shorter than 50bp, 3. Track and cut sequencing adapters. Contaminated human reads were filtered using Kneaddata v0.7.3 (website: bitbucket.org/biobakery/kneaddata/wiki/Home, reference repository: GRCh38p12) with default parameters.

Maps of bacterial taxa and functional ^{composition20} were extracted from metagenomes of fungal-enriched DNA (Franzosa, 2018 #6) using humann2 v0.11.1 , which included mapping reads to clade-specific ^markers21 via Bowtie2 v2. 3.5 ²² referenced the ChocoPhlAn database to annotate species pan-genomes, performed a translational search of unmapped reads with DIAMOND v2.0.4 ²³ against the UniRef90 universal protein reference database ²⁴ and collected pathways from the resulting gene lists with reference to the Metacyc database ²⁵ , Taxonomic identification via MetaPhlAn2 . Plasma cytokine measurement

Whole blood samples collected in anticoagulant-treated tubes were centrifuged at 2,000 x g for 10 min and the supernatant collected. Cytokine Chemokine concentrations were measured using a MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel - Immunology Multiplex Assay (Merck Millipore, Massachusetts USA) on a Bio-Plex 200 system (Bio-rad laboratories, California, USA). The concentration of NT-proBNP was measured using the Human NT-proBNP ELISA kit (Abcam, Cambridge, UK). Quantification of fecal metabolites

Detection of short-chain fatty acids (SCFAs) by GC-MS/MS analysis, including acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, caproic acid. An Agilent 7890B gas chromatograph with a 7000D mass spectrometer (30 m long x 0.25 mm i.d. x 0.25 μm film thickness, J&W Scientific, USA) with a DB-5MS column was used. Helium was used as the carrier gas at a flow rate of 1.2 mL/min. Injections were performed in splitless mode with an injection volume of 2 μL. Keep the oven temperature at 90°C for 1 minute, increase to 100°C at a rate of 25°C/min, increase to 150°C at a rate of 20°C/min, and hold at 150°C for 0.6 minutes, increase at a rate of 25°C/min The rate was further increased to 200°C and held at 200°C for 0.5 minutes. After running for 3 minutes, all samples were analyzed in multiple reaction monitoring mode. The temperature of the injector inlet and transfer line was maintained at 200°C and 230°C, respectively.

L-isoleucine, urea and L-arginine were detected by LC-MS analysis. LC-ESI-MS/MS system (UPLC, ExionLC AD, website: sciex.com.cn/; MS, QTRAP® 6500+ system, website: sciex.com/) was used for analysis. The analysis conditions of L-isoleucine and L-arginine are as follows: HPLC: column, Waters ACQUITY UPLC HSS T3 C18 (100 mm × 2.1 mm id, 1.8 µm); solvent system, water containing 0.05% formic acid (A ), acetonitrile (B) containing 0.05 % formic acid. Gradient starts at 5% B (0-10 minutes), increases to 95% B (10-11 minutes), and gradually returns to 5% B (11-14 minutes); flow rate: 0.35 mL/min; temperature, 40°C ; Injection volume: 2 μL. The analysis conditions of urea are as follows: HPLC: column, Waters ACQUITY BEH Amide (100 mm × 2.1 mm id, 1.7μm); solvent system, water containing 0.1% formic acid (A), acetonitrile containing 0.1% formic acid (B); gradient Start at 95% B (0-4 minutes), decrease to 50% B (4-5 minutes), and gradually return to 95% B (5.1-10 minutes); flow rate: 0.35 mL/min; temperature, 40°C; Injection volume: 2 μL. ESI source operating parameters are as follows: ion source, turbo spray; source temperature 550 °C; ion spray voltage (IS) 5500 V (positive), -4500 V (negative); DP and CE for a single MRM transition with further DP and CE optimization is done. statistical analysis

Access data on abundance and function of bacterial taxa to Rv3.5.1. Data on bacterial taxa were normalized by sum scale (TSS), and data on functionality were normalized by Deseq2 (v1.26.0) based on relative log expression (RLE). Non-metric multidimensional scaling (NMDS) analysis based on Bray-Curtis differences was performed using the vegan package (v2.5-3). Differential microbial functional pathways between COVID-19 patients and non-COVID-19 controls were identified using Deseq2 (v 1.26.0). Associations of microbial pathways with disease severity were identified using the Multivariate Linear Analysis Model (MaAsLin) statistical framework implemented in the Huttenhower Lab Galaxy instance (website: huttenhower.sph.harvard.edu/galaxy/). Spearman correlations between microbial pathways and patient plasma parameters were evaluated using the cor and cor.test functions. Heatmaps were generated using the pheatmap package (v1.0.10). data availability

The raw sequence data generated for this study is available in the Sequence Read Archive under BioProject accession number PRJNA689961. Results Clinical characteristics of COVID-19 patients

COVID-19 患者的功能性 腸 道 微生物 組 改 變 Fifty-five hospitalized patients [28 males; mean ± standard error (se), 28.8 ± 0.3 years old] with laboratory-confirmed SARS-CoV-2 infection and 70 controls were recruited. Demographics, clinical characteristics, and time of stool collection are presented in Table 1 and Figure 1 . The median length of hospital stay for COVID-19 patients was 27 days (IQR, 4-45 days). Eighteen patients were followed up to 1 month after discharge [median (IQR), 21 (14.5-23.6) days]. All patients were antibiotic naive, and 14 (25.4%) received at least one antiviral drug. On admission, 17 patients (30.9%) developed fever, 5 patients (9.1%) developed diarrhea, and 24 patients (43.6%) developed respiratory symptoms. According to the COVID-19 severity grading ^criteria26 , patients were classified as critical (1.8%), severe (3.6%), moderate (21.8%), and mild (72.7%).

Functional gut microbiome alterations in COVID-19 patients

To understand how SARS-CoV-2 infection affects the function of the gut microbiome of COVID-19 patients, functional profiles, including gene abundance and their corresponding pathways, were extracted from the fecal metagenome. The composition of the microbial pathways at baseline (first stool sample collected after admission) was first investigated in COVID-19 patients and non-COVID-19 controls. Non-metric multidimensional scaling (NMDS) analysis revealed that the composition of microbial pathways in COVID-19 patients was significantly different from non-COVID-19 controls ( Fig. 2a , PERMANOVA test, p < 0.001). SARS-CoV-2 infection and COVID-19 disease severity among all host factors examined (age, sex, comorbidities, COVID-19 disease severity, fecal SARS-CoV-2 viral load and antiviral drugs) All significantly affected the composition of gut microbiota, with COVID-19 disease severity showing the largest effect size (R2 = 0.052, p < 0.05, PERMANOVA test, Figure 2c ). Furthermore, COVID-19 patients showed significantly lower microbial pathway richness in their stool compared with non-COVID-19 controls ( Fig. 2b , p < 0.01). These results suggest that SARS-COV-2 infection is associated with altered functional profiles of the gut microbiome. Reduction in Beneficial Microbial Function and Its Association with COVID-19 Severity

糞 便 SCFA 與 L- 異 亮氨酸生物合成 與 血 漿測 量 結 果的 相 關 性 We next asked which microbial function primarily drives the differences between COVID-19 patients and controls. Differential analysis by DESeq2 showed that 28 microbial pathways were decreased and 11 pathways were enriched in stool samples from COVID-19 patients at baseline compared with non-COVID-19 controls ( Table 2 , FDR < 0.05) . In patients with COVID-19, 10 of the 28 decreased pathways were associated with carbohydrate degradation, suggesting that the ability of the SARS-CoV-2-infected gut microbiome to degrade carbohydrates is impaired. The L-1,2-propanediol degradation and bifidobacterial shunt pathways involved in the biosynthesis of propionate and acetate were reduced by 5.6 and 2.9 fold, respectively, in COVID-19 patients compared to non-COVID-19 controls ( Table 2 ) . In contrast, the urea cycle pathway was 1.9-fold more abundant in patients with COVID-19 than in controls. These data suggest that COVID-19 patients exhibit impaired short-chain fatty acid production and exhibit enhanced urea biosynthesis. 39 different pathways were then associated with COVID-19 disease severity by MaAsLin2 analysis. Including 4-deoxy-L-threo-hex-4-enepyralate degradation, L-isoleucine biosynthesis III and adenosylcobalamin from the colinolamide I pathway Three pathways including salvage showed a significant negative correlation with COVID-19 severity ( Fig. 3a , FDR q < 0.05), while L-lysine biosynthesis III, braidin biosynthesis, urea cycle, preQ0 biosynthesis and phosphopantothenic acid biosynthesis I showed a significant positive correlation with the severity of COVID-19 ( Fig. 3b , FDR q < 0.05). Among them, the 4 ^- deoxy-L-threo-hex-4-enepyralate degradation pathway is associated with the production of pyruvate, a key metabolite in the fermentation of short-chain fatty acids27,28. The L-isoleucine biosynthesis III pathway is involved in the biosynthesis of the essential amino acid L-isoleucine29; adenosylcobalamin salvage from the cobalolamide I pathway is involved in the production ^of vitamin ^B1230 . The end products of these microbial pathways are key mediators in microbiota-host crosstalk and play important roles in regulating host innate and adaptive immunity ^31-33 . Altered microbial pathways in the gut of COVID-19 patients may have significant effects on host physiology and function.

Correlation of fecal SCFA and L - isoleucine biosynthesis with plasma measurements

SARS-CoV-2 infection can induce a dysfunctional immune response and consequent cytokine storm syndrome in a subset of COVID-19 patients, leading to a more severe disease outcome ³⁴ . Here, the levels of cytokines, chemokines, and inflammatory markers were compared between COVID-19 patients with mild disease (n = 40) and COVID-19 patients with moderate-severe disease (n = 15). Plasma concentration. COVID-19 patients with moderate/severe disease had elevated levels of plasma CXCL10, lactate dehydrogenase (LDH), C-reactive protein (CRP), decreased platelet count (PLT), albumin, and hemoglobin (MaAsLin2, FDR q <0.05) ( Fig. 4a-f ). These findings are consistent with other reports suggesting that a dysfunctional immune response is associated with worse COVID-19 disease35 ^.

The gut microbiota is known to regulate the development and function of the innate and adaptive immune systems in the host ³⁶ . To explore the role of gut microbiota function in regulating the host immune response to SARS-CoV-2 infection, the relationship between microbial pathways and the severity of blood immune markers was further investigated. The L-isoleucine biosynthetic pathway was negatively correlated with plasma CXCL-10 levels (Rho = -0.48, p = 0.01, Fig. 4g ), whereas with platelets (Rho = 0.38, p = 0.01) and albumin (Rho = 0.36, p = 0.01) were positively correlated with blood levels ( Fig. 4g ). Furthermore, the 4-deoxy-L-threo-hex-4-enepyralate degradation pathway showed a positive correlation with hemoglobin levels (Rho = 0.31, p = 0.03) ( Fig. 4g ). CXCL10 is a pro-inflammatory chemokine known to be associated with adverse outcomes in COVID- ¹⁹³⁷ . In COVID-19, low platelet counts have also been associated with an increased risk of severe illness and mortality from COVID- ¹⁹³⁸ . Hemoglobin (HGB) and albumin levels in plasma decrease in response to ^{inflammation39,40} . These two pathways may be involved in preventing excessive developmental inflammation in COVID-19. In contrast, the urea cycle pathway showed an inverse correlation with blood levels of platelet count (Rho = -0.34, p = 0.02) and albumin (Rho = -0.30, p = 0.04). PreQ0 biosynthetic pathway abundance showed a positive correlation with CXCL-10 and a negative correlation with platelet count (Rho = -0.35, p = 0.03) (Fig. 5h). Therefore, overexpression of these two pathways may be associated with a more dysfunctional immune response. Taken together, these data highlight that the gut microbiome may functionally calibrate host immunity against SARS-CoV-2 infection, thereby influencing COVID-19 severity. Impaired and prolonged microbial function of SCFA and L- isoleucine biosynthesis after recovery from COVID-19

To explore whether the function of the gut microbiome was restored in recovered patients, post-discharge stool samples were collected from 19 patients (days since discharge, median (IQR), 21 (16-23.5) days) ( Fig. 1 ). Nonmetric multidimensional scaling (NMDS) analysis indicated that the composition of microbial pathways in COVID-19 patients after recovery appeared to cluster away from patients' baseline samples (p < 0.05, baseline vs. Non-COVID-19 controls were comparable ( Fig. 5a ). Similarly, the richness of microbial pathways was higher in post-discharge COVID-19 patients compared with baseline and showed levels comparable to non-COVID-19 controls ( Fig. 5b ). These data suggest that the overall function of the gut microbiome can be restored after disease resolution. It was investigated whether different microbial functional pathways (shown in Table 2 ) were restored in COVID-19 patients after disease resolution. Sixteen microbial pathways that were underrepresented in the COVID-19 baseline microbiome showed sustained reductions after disease resolution ( Fig. 5c ). Interestingly, 62.5% (10 out of 16) of these pathways were involved in carbohydrate degradation, suggesting that COVID-19 patients display persistent impairment of microbial functions for carbohydrate degradation. Furthermore, bifidobacteria involved in SCFA and L-isoleucine shunt, L-1,2-propanediol degradation, 4-deoxy-L-threo-hex-4-enepyranal even after disease resolution Salt (ester) degradation and L-isoleucine biosynthesis III still showed a decrease, indicating that the capacity of SCFA biosynthesis and L-isoleucine biosynthesis continued to be impaired in COVID-19 patients. These data suggest that gut microbiome function in patients only partially recovers after COVID-19 subsides, while several specific microbial functions remain persistently impaired. Decreased fecal concentrations of SCFA and L- isoleucine in COVID-19

The fecal metabolome provides a functional readout of microbial activity and can serve as a mediator of host-microbiome interactions41 ^. Based on targeted metabolome analysis of fecal samples from COVID-19 patients and controls, gut microbial function was found to be associated with altered gut microbial metabolites. L-1,2-propanediol degradation, bifidobacterial shunt, and 4-deoxy-L-threo-hex-4-enepyranylate degradation pathways are associated with SCFA production, and in COVID-19 decreased in patients (Table 2). Fecal concentrations of SCFAs (including acetate, propionate, isobutyrate, butyrate) were significantly lower in COIVD-19 patients than in controls ( Fig . 6a ), further supporting that SARS-CoV-2 infection may affect the gut microbiome of SCFAs generating capacity. When COVID-19 patients were classified into mild disease and moderate/severe disease, more patients with moderate/severe disease showed lower concentrations of all types of SCFAs compared with mild disease and controls ( Fig. 6a ), suggesting that deficient SCFA production may be associated with more severe disease. The concentration of L-isoleucine was also lower in COVID-19 patients than in controls (p < 0.05, Fig. 6b). This result is consistent with the finding that the L-isoleucine biosynthesis III pathway is underrepresented at the microbial DNA level in patients with COVID-19 ( Table 2 ). SARS-CoV-2 infection may be associated with impaired capacity of the gut microbiome for L-isoleucine biosynthesis. Similarly, urea and L-arginine, involved in the urea cycle pathway, were overrepresented at the microbial DNA level and at the metabolite level in more severe patients compared with mild patients and controls. is also overrepresented ( Fig. 6b ).

It was examined which bacterial species contributed most to changes in these microbial pathways. None of the pathways were dominated by any single species, suggesting that changes in these pathways may result from community-level changes in their function ( Fig. 11 ). Notably, the bacterium Clostridium prausitidis is responsible for 4-deoxy-L-threo-hex-4-enepyranylate degradation pathway and L-isoleucine biosynthesis III ( Figure 11c and d ) The main contributors of both, suggesting a possible beneficial role of F. prausniella in combating SARS-CoV-2 infection. discuss

In this study, it was shown for the first time that COVID-19 patients have markedly altered gut microbial function with impaired short-chain fatty acid (SCFA) production capacity. Since there are significant inter-individual differences in microbial community composition, characterizing microbial functions or metabolites is important in understanding their contribution to disease pathogenesis. SCFAs, including butyrate, as well as propionate and acetate, can exert anti-inflammatory effects by priming anti-inflammatory immune cells and inhibiting inflammatory signaling pathways ⁴² . In addition, butyrate maintains the integrity of the intestinal barrier to prevent the translocation and recycling of intestinal endotoxins and bacteria, thereby reducing systemic inflammatory responses ⁴³ . Recently, butyrate was found to act in an intestinal epithelial organoid model by downregulating genes essential for SARS-CoV-2 infection, such as angiotensin-converting enzyme 2 (ACE2) and upregulating the TLR (Toll-like receptor) antiviral pathway, To protect the host from viral infection ¹⁵ . Two key butyrate-producing phyla, Fustridium pprasus and Eubacterium rectalum, were the most abundant bacteria in healthy colonic communities and have been shown to be ^reduced in COVID-19 stool samples. Importantly, this study provides evidence that COVID-19 patients, especially those with more severe symptoms, showed reduced SCFA (including butyrate, acetate, and propionate) in stool compared with controls concentration. These data highlight that SCFAs produced by gut microbiota may play an important role in COVID-19 pathogenesis and disease severity. Supplementation with SCFAs or probiotics that produce SCFAs may have the potential to improve disease outcomes, although this hypothesis requires further confirmation. Notably, impaired SCFA biosynthetic capacity in COVD-19 patients persisted until one month after disease resolution, which may cause long-term health complications due to the importance of SCFAs in host immunity and metabolism.

The gut microbiota also plays a key role in manipulating amino acid pools and profiles during amino acid digestion ^and absorption, thereby mediating aspects of host physiology44. The gut microbiota of COVID-19 patients were found to have attenuated L-isoleucine biosynthetic capacity, and they had lower fecal concentrations of L-isoleucine compared with controls. Recent studies have shown that L-isoleucine can induce the expression of host defense peptides (i.e., β-defensins), which can modulate host innate and adaptive immunity and alleviate the deleterious effects of pathogens on humans and animals32 ^{, 33} . Consistent with this, the inventors found that the L-isoleucine biosynthesis III pathway was inversely correlated with disease severity and pro-inflammatory chemokine (CXCL-10). These results collectively support that L-isoleucine produced by the gut microbiota may attenuate the severity of COVID-19 by modulating the host immune response to SARS-COV-2 infection. In addition, L-isoleucine is one of the branched-chain amino acids (BCAA), which is considered to increase fatigue substances (lactate, ammonia, and 5-HT), energy metabolites (glucose and free fatty acids) and muscle pain substances (LDH and CK) serum concentrations to improve central and muscular weakness with nutritional supplements ^45,46 . Interestingly, it was observed that patients after hospital discharge still had persistently impaired L-isoleucine biosynthesis. This may partly explain why COVID-19 survivors report persistent symptoms of fatigue and muscle weakness47 ^.

Furthermore, gut microbiota-dependent urea metabolism is known to be associated with host urea homeostasis and involved in disease progression ⁴⁸ . The urea cycle associated with the gut microbiome was found to be enriched in COVID-19 samples and positively correlated with disease severity. Patients with more severe symptoms showed higher fecal urea concentrations. Shen et al. also observed that COVID-19 patients had higher serum urea concentrations than non-COVID-19 cases. Altered gut microbiome may be associated with disruption of urea cycle function during COVID-19 infection. However, how disruption of the urea cycle affects disease outcome requires further study.

This study has several strengths. None of the subjects were on antibiotics (which are known to affect the microbiome) and were followed up with serial stool sample collection until after hospital discharge. A particularly striking finding is that a hallmark of human gut microbial function (i.e., impaired SCFAs) persists after viral clearance, and that these changes may contribute to symptoms of so-called "long COVID," including fatigue and muscular powerless. Although this is still speculative, it requires further research. Since the production of SCFA and L-isoleucine was affected by dietary fiber and protein, dietary records of patients were collected during hospitalization to eliminate the effect of diet. All patients received a standard hospital diet provided by the hospital, which was in line with the customary diet commonly used in Hong Kong, China. Therefore, it is unlikely that significant changes or alterations in diet would have affected the findings.

In conclusion, this study demonstrates that gut microbial function is perturbed in COVID-19, including decreased SCAF and L-isoleucine biosynthesis and increased urea production. It highlights the correlation between impaired gut microbiome function and the severity of COVID-19. Further research is needed to better understand how impaired gut microbial function is involved in inflammation and disease outcome, as well as to explore the therapeutic potential of microbial functional products and metabolites against COVID-19. Example 2: Short-chain fatty acids associated with COVID-19 Methods Subject recruitment and sample collection

The pilot study included 25 subjects in the synbiotic group who received the synbiotic formulation in addition to standard care and 10 subjects in the control group (control) who received standard care only. From August 13, 2020 to October 9, 2020, hospitalized patients aged 18 years or older who had confirmed SARS- Diagnosis of CoV-2 infection. Subjects admitted to the intensive care unit or on a ventilator, with a known history of active endocarditis, on hemodialysis, or pregnant at the time of recruitment were excluded. Exclude subjects with known increased risk of infection due to immunosuppression, such as prior organ or hematopoietic stem cell transplantation, neutropenia (ANC < 500 cells/μl), or HIV and CD4 < 200 cells/μl . The synbiotic formulation used in this study contained a mixture of Bifidobacterium strains (25 billion CFU/capsule) and prebiotics (galacto-oligosaccharides, xylo-oligosaccharides, resistant dextrins) and was administered in two equal doses (4 capsules twice a day) administered. Capsules are prepared in-house at Prince of Wales Hospital and dispensed by our central Clinical Research Pharmacy. The probiotic species selected for this formula were obtained based on positive correlations with bacterial species reduced by COVID-19 in two independent healthy Chinese metagenomic data sets (N = 1400), and in 100 COVID-19 patients It was further validated in the macrogenome data ^{of 50} . The formulation is designed to replenish missing bacteria that have been reported to be associated with enhanced immunity in COVID-19 patients. The subjects were instructed to take synbiotic capsules (a total of 8 capsules per day) and food for 28 days. All patients have standard three meals a day provided by the hospital. COVID-19 positive subjects (controls) who were not treated with oral synbiotic formulations and admitted to the hospital during the same time period were also recruited. Blood and stool from in-hospital patients were collected by hospital staff, while discharged patients provided stool or self-sampled at home on the day of follow-up. Fecal DNA extraction and shotgun metagenomic sequencing and profiling

Same procedure as described in Example 1. Targeted detection of short- chain fatty acids (SCFA) in plasma

Add 50 μl of the inactivated plasma sample to a 1.5mL EP tube containing 100 μL of phosphoric acid (36% v/v) solution. Vortex the mixture for 3 min. Add 150 μL of MTBE (with internal control) solution, then vortex for 3 minutes and sonicate for 5 minutes on ice. Then, the mixture was centrifuged at 12000 r/min for 10 min at 4 °C. The supernatant was collected and used for gas chromatography-mass spectrometry (GC-MS/MS) analysis of six SCFAs (acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, caproic acid). An Agilent 7890B gas chromatograph with a 7000 D mass spectrometer (30 m length x 0.25 mm id use x 0.25 μm film thickness, J&W Scientific, USA) with a DB-5MS column was used to perform GC-MS/MS analysis of sugars. Helium was used as the carrier gas at a flow rate of 1.2 mL/min. Injections were performed in splitless mode with an injection volume of 2 μL. Keep the oven temperature at 90°C for 1min, increase to 100°C at a rate of 25°C/min, increase to 150°C at a rate of 20°C/min, maintain for 0.6min, and increase to 100°C at a rate of 25°C/min 200°C, hold for 0.5min after running for 3min. All samples were analyzed in multiple reaction monitoring mode. The injector inlet and transfer line temperatures were 200°C and 230°C, respectively. Detection of SARS - CoV-2 Viral Load in Nasopharyngeal Swabs

Measurement of SARS-CoV-2 viral load in nasopharyngeal swabs using a real-time RT-PCR assay. Viral RNA was extracted from nasopharyngeal swab samples using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). Quantification of SARS-CoV-2 RNA by real-time RT-PCR. Primer-probe set N1 (2019-nCoV_N1-F: 5'-GAC CCC AAA ATC AGC) designed by the US Centers for Disease Control and Prevention was purchased from Integrated DNA Technologies (Coralville, IA) GAA AT-3', 2019-nCoV_N1-R: 5'-TCT GGT TAC TGC CAG TTG AAT CTG-3' and 2019-nCoV_N1-P: 5'-FAM-ACC CCG CAT TAC GTT TGG ACC-BHQ1-3' ). The 1-step real-time RT-PCR reaction contained 10 μL of the extracted preparation, 4 μL of TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems, Foster City, CA), and the final reaction volume was 20 μL. Primer and probe concentrations were 0.5 μM and 0.125 μM, respectively. Cycle conditions were performed with StepOnePlus real-time PCR system (Applied Biosystems), 25°C for 2 minutes, 50°C for 15 minutes, 95°C for 2 minutes, followed by 45 cycles of 95°C for 15 seconds and 55°C for 30 seconds. Cytokine measurement _

Plasma samples were collected after centrifugation of whole blood at 1,500 xg for 10 minutes at room temperature (RT) without braking. Concentrations of cytokines and chemokines in plasma were measured using Custom Premix Human Cyto Panel A 47 Plex (Millipore, # HCYTA-60K-47C). Measure all samples on first thaw. Non-targeted measurement of plasma metabolites

For non-targeted metabolite measurements, add 50 µL of plasma and 300 µL of pure methanol. The mixture was vortexed for 3 minutes and centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was then collected and centrifuged at 12,000 rpm for 5 min at 4°C. After centrifugation, the mixture was frozen at -20°C for 30 minutes, and then centrifuged at 12000 r/min for 3 minutes at 4°C. Use 150 µL of supernatant for on-plate analysis. Sample extracts were analyzed using LC-ESI-MS/MS system. MS/MS spectra were obtained on an information-dependent basis using a triple TOF mass spectrometer. statistical analysis

Concentrations of SCFA, IL-6 and metabolites from different time points were compared using the paired Wilcoxon signed-rank test. The relative abundance of SARS-COV-2 bacteria or the CT value and the longitudinal correlation of SCFA and the correlation of IL-6 and metabolites were evaluated by repeated measurement correlations using the Hmim and rmcorr packages in R, respectively. All plots were visualized using ggplot2 or the corr suite. result

Plasma concentrations of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, caproic acid were evaluated. Among them, after treatment, the concentration of acetic acid was significantly increased in the synbiotic group (p < 0.001), but not in the control group (p = 0.65) ( Fig. 7 ). Repeated measures correlation analysis showed that the cycle threshold (CT) of SARS-CoV-2 in nasopharyngeal swabs was positively correlated with plasma acetate concentration (R = 0.48, p = 0.046), suggesting that plasma acetate concentration is associated with viral load negatively correlated ( Figure 8 ). After synbiotic treatment, the relative abundance of 11 species was significantly changed in patients in the synbiotic group. Among them, the relative abundance of Bifidobacterium adolescentis and Faecococcus chaperonii were positively correlated with the concentration of plasma acetic acid ( Fig . The relative abundance of biliary bacteria was negatively correlated with the concentration of plasma acetate ( Figure 10 ).

Untargeted metabolome analysis was performed on plasma samples from all 25 subjects in the SIM01 group and 10 subjects in the control group. A total of 743 compounds with identification information were identified, 20 of which were upregulated in the SIM01 group but not in the control group ( Fig. 12 , 13 ). Among these SIM01-related metabolites, 15 of them were significantly negatively correlated with IL-6 in the SIM01 group ( Table 3 ). IL-6 is a proinflammatory cytokine that can lead to a hyperinflammatory response that positively correlates with the severity of COVID-19. Therefore, supplementation with one or more compounds listed in Table 3 may be beneficial in reducing symptoms of COVID-19 and preventing severe illness in COVID-19 patients.

All patents, patent applications and other publications cited in this application, including GenBank accession numbers and equivalents, are hereby incorporated by reference in their entirety for all purposes.

[ Fig. 1] Schematic representation of stool sample collection and length of hospital stay in COVID-19 patients (n = 55). "CoV" means a patient with COVID-19. "D0" indicates the date of onset of the patient.

[ Fig. 2] Altered functional gut microbiome profiles in COVID-19 patients. a. Composition of microbial functional pathways in COVID-19 and non-COVID-19 individuals, as visualized by Bray-Curtis difference-based NMDS (non-metric multidimensional scaling) plots. b. Richness of microbial pathways in COVID-19 and non-COVID-19 controls, evaluated based on the Chao1 index. Significant p-values were determined by the Mann-Whitney test and expressed as ***p<0.001. c. Effect size of host factors on microbial pathway composition. Effect sizes and statistical significance were determined by PERMANOVA analysis. ***p<0.001, **p<0.01, *p<0.05.

[ Fig. 3] Correlation of microbial pathways with severity of COVID-19. a. Microbial pathways were significantly inversely associated with disease severity. b. Microbial pathways are positively correlated with disease severity. Patients with COVID-19 were classified into mild patients (n=40, including patients with mild COVID-19 symptoms) and non-mild patients (n=15, including patients with moderate, severe and critical COVID-19 symptomatic patients). Correlations between the abundance of microbial pathways and disease severity were assessed by microbiome multivariate association with linear model 2 (MaAsLin2).

[ Fig. 4 ] Correlation between fecal SCFA and L-isoleucine biosynthesis and plasma measurement results. a, b, c. Plasma parameters are positively correlated with disease severity. d, e, f. Plasma parameters are inversely correlated with disease severity. Correlation analysis was determined by MaASLin2. Mild patients include those with mild COVID-19 symptoms, while non-mild patients include those with moderate, severe, and critical COVID-19 symptoms. g Spearman correlations of plasma parameters with microbial pathways. Blue circles and positive values indicate a positive correlation, and red circles and negative values indicate an inverse correlation. Size and shading indicate the magnitude of the correlation, with darker shades showing higher correlations than lighter shades.

[ Fig. 5] Impaired and prolonged microbial function of SCFA and L-isoleucine biosynthesis after recovery from COVID-19. a. Composition of microbial pathways in non-COVID-19 controls and COVID-19 patients at baseline and post-hospital discharge, as visualized by NMDS (Non-Metric Multidimensional Scaling) plots based on Bray-Curtis differences. Statistical significance between baseline samples and post-discharge samples was determined by PERMANOVA analysis. b. Abundance of microbial pathways in non-COVID-19 controls and COVID-19 patients at baseline and after hospital discharge, as assessed based on the Chao1 index. Significant p-values were determined by the Mann-Whitney test and expressed as *p<0.05. c. Heatmap summarizing the functional changes in the gut microbiome of COVID-19 patients after hospital discharge. Labels on the left of the plot indicate microbial pathways, and labels on the right indicate the parental classes of these microbial pathways. Pathways with higher relative abundance are colored red, while those with low relative abundance are colored blue. Module 1 represents microbial pathways that were enriched at baseline in COVID-19 patients and returned to non-COVID-19 controls after hospital discharge. Module 2 represents microbial pathways that exhibit a decrease in COVID-19 patients and return to non-COVID-19 controls after hospital discharge. Module 3 represents microbial pathways that exhibit a reduction in COVID-19 patients compared to non-COVID-19 controls while maintaining a sustained reduction after hospital discharge.

[ Fig. 6] Fecal concentrations of SCFA and L-isoleucine decreased in COVID-19. a Fecal concentrations of short-chain fatty acids including valeric acid, propionic acid, isovaleric acid, isobutyric acid, caproic acid, butyric acid, and acetate in COVID-19 patients and non-COVID-19 controls. b. Fecal concentrations of L-isoleucine, urea, and L-arginine in COVID-19 patients and non-COVID-19 controls. Mild patients include those with mild COVID-19 symptoms, while non-mild patients include those with moderate, severe, and critical COVID-19 symptoms. Statistical significance was determined by Welch's t-test. Significant differences between COVID-19 patients and non-COVID-19 controls are represented as *p<0.05, significant differences between non-mild patients and non-COVID-19 controls are represented as #p<0.05, ###p<0.01 and # ##p < 0.001.

[ Fig. 7] Comparison of plasma acetate concentrations at baseline and week 5 in COVID-19 patients in the control (n = 9) and synbiotics groups (n = 22). Between-group comparisons of changes in plasma acetate concentrations were analyzed by paired Wilcoxon signed-rank tests. Plasma acetate concentrations from the same patients at baseline and week 5 are illustrated. Points from the same patient are connected with gray lines.

[ Fig. 8] The correlation of plasma acetic acid concentration with the cycle threshold (CT) of real-time RT-PCR of SARS-CoV-2 virus from nasopharyngeal swabs of COVID-19 patients was analyzed by repeated measurement correlation. Each point represents the plasma acetate concentration and CT value from each sample. Samples from the same patient are indicated by the same color. Colored lines show the measured correlation fits for each patient's replicates.

[ Fig. 9] The correlation between plasma acetate concentrations from COVID-19 patients and the relative abundance of Bifidobacterium adolescentis and Faecalis chaperonii was analyzed by repeated measurement correlation. Each point represents plasma acetate concentration and relative abundance of B. adolescentis and Co. chaperones from each sample. Samples from the same patient are indicated by the same color. Colored lines show repeated measures correlation fits for each patient.

[ Fig. 10] Plasma acetate concentrations from COVID-19 patients were analyzed by repeated measurement correlation with unclassified Parabacteroides , Odoribacter splanchnicus , unclassified Bilophila ) and the relative abundance of Bilophila wadsworthia . Each point represents the plasma acetate concentration versus the relative abundance of unclassified Bacteroides, O. viscera, unclassified Biliophilus, and B. worschii from each sample. Samples from the same patient are indicated by the same color. Colored lines show repeated measures correlation fits for each patient.

[ Fig. 11] Contribution of organisms to microbial pathways in COVID-19 baseline and non-COVID-19 stool samples. Abundance of microorganisms was normalized by Deseq2 based on relative log expression (RLE).

[ Fig. 12] Volcano plot analysis showing fold change in plasma metabolite abundance between week 5 and baseline in COVID-19 patients receiving SIM01. Horizontal lines delineate thresholds for statistical significance, where p<0.05, and vertical lines indicate thresholds for [log2 fold change], which are >1. Red dots with markers refer to those metabolites that were significantly upregulated in the SIM01 group but not in the control group.

[ Fig. 13] The abundance of 20 metabolites that were significantly upregulated after SIM01 supplementation was identified by untargeted metabolomics analysis.

Claims (23)

A method for enhancing immunity, preventing or treating COVID-19, or preventing severe COVID-19 or alleviating its symptoms in a subject, comprising administering an effective amount of a composition to the subject, the composition A bacterium comprising short-chain fatty acid, L-isoleucine, a compound shown in Table 3, or producing said short-chain fatty acid or L-isoleucine or said compound, wherein said subject is otherwise No additional treatment with the short chain fatty acids or L-isoleucine or the compounds is required. The method according to claim 1, wherein the administering step comprises oral intake. The method according to claim 1, wherein the composition comprises short-chain fatty acids and L-isoleucine. The method according to claim 1, wherein the short-chain fatty acid is acetic acid, propionic acid, butyric acid or isobutyric acid. The method as claimed in claim 1, wherein the bacterium is Faecalibacterium prausnitzii . The method according to claim 1, wherein said bacteria is a species of the genus Bifidobacterium. The method as claimed in item 6, wherein the bacterium is Bifidobacterium adolescentis . The method according to claim 1, wherein the bacteria is Coprococcus comes . The method according to claim 6, wherein the bacteria are not Bifidobacterium adolescent, Bifidobacterium bifidum , Bifidobacterium longum , Bifidobacterium breve or Bifidobacterium infantis Bacillus ( bifidobacterium infantis ). The method of claim 1, wherein the subject has been diagnosed with COVID-19. The method according to claim 1, wherein the short-chain fatty acid is butyric acid, butyrate or glyceryl butyrate. A composition for improving immunity, preventing or treating COVID-19, or preventing severe COVID-19 or alleviating its symptoms in a subject, comprising an effective amount of (1) short-chain fatty acid, L-isoleucine Acids, compounds shown in Table 3, or bacteria producing short-chain fatty acids or L-isoleucine and (2) physiologically acceptable excipients. The composition of claim 12, wherein the subject is diagnosed with COVID-19. The composition according to claim 12, which is formulated for oral intake. The composition according to claim 12, wherein said composition comprises short-chain fatty acids and L-isoleucine. The composition according to claim 12, wherein the physiologically acceptable excipient is starch, inulin, oat bran, wheat bran, cellulose, guar gum or pectin. The composition as claimed in claim 12, wherein the bacterium is Clostridium prausniformum. The composition of claim 12, wherein the bacterium is a species of the genus Bifidobacterium. The composition as claimed in claim 18, wherein the bacterium is Bifidobacterium adolescentis. The composition as claimed in claim 12, wherein the bacterium is Faecococcus chaperonii. The composition according to claim 18, wherein the bacteria are not Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve or Bifidobacterium infantis. A kit for improving immunity, preventing or treating COVID-19, or preventing severe COVID-19 or alleviating its symptoms in a subject, comprising (1) a first composition comprising an effective amount of the first A short-chain fatty acid, L-isoleucine, a compound shown in Table 3, or a bacterium producing the short-chain fatty acid or L-isoleucine or the compound; and (2) a second composition, It comprises an effective amount of a second different short chain fatty acid, a second different compound shown in Table 3, or a second different short chain fatty acid producing bacterium, or a second different producing short chain fatty acid Bacteria with a second different compound are shown. The kit of claim 22, wherein the first composition and the second composition comprise two different butyrate-producing Gram-positive anaerobic bacteria.

Images