Formulations of segmented filamentous bacteria-induced metabolites and methods of use thereof

Abstract

Pharmaceutical formulations containing one or more metabolites, and a pharmaceutically acceptable carrier are disclosed. The one or more metabolites in the pharmaceutical formulation can be one or more amino acid derivatives, one or more sugars, one or more flavonoids, one or more organic acids, or a combination thereof. The pharmaceutical formulation can be in a form suitable for intranasal or oral administration. Also described are methods of using the pharmaceutical formulations for reducing and / or treating a respiratory disease or disorder, for example a respiratory infection, in a subject in need thereof. Following administration of the formulation, the total amount of the metabolites administered to the subject is effective to decrease viral or bacterial titer measured in a biological sample of the subject, compared to a control sample of the subject prior to the administration of the pharmaceutical formulation.

Description

[0001] ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT

[0002] FORMULATIONS OF SEGMENTED FILAMENTOUS BACTERIA-INDUCED

[0003] METABOLITES AND METHODS OF USE THEREOF

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims benefit of and priority to U.S. Provisional Application No. 63 / 770,524, filed March 12, 2025, and U.S. Provisional Application No. 63 / 699,619, filed September 26, 2024, both of which are specifically incorporated by reference herein in their entireties.

[0006] REFERENCE TO THE SEQUENCE LISTING

[0007] The Sequence Listing submitted as an XML file named “GSURF2025-005- 03PCT_ST26.xml” created on September 23, 2025, and having a size of 34,625 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.834(c)(1).

[0008] FIELD OF THE INVENTION

[0009] The disclosed invention is generally in the field of compositions and methods of use thereof for the treatment of respiratory diseases and / or disorders.

[0010] BACKGROUND OF THE INVENTION

[0011] Annual and sporadic influenza virus (IAV) outbreaks and the SARS / CoV-2 pandemic highlight that respiratory viral infection (RVI) remains a scourge of humanity, affecting all societies and strata therein. Yet, the impacts of RVI are highly heterogeneous. For instance, individuals with similar genetics and / or viral exposures often have different rates of infection, with some people exhibiting minimal disease and others becoming severely ill. The heterogeneity of RVI is poorly understood, primarily limited to observations that the elderly and those with various chronic health conditions (e.g. obesity and diabetes), are at greater risk of severe disease.

[0012] Accordingly, there remains a need for more effective measures for treating respiratory diseases and / or disorders, for example respiratory viral infections, such as infection by SARS-CoV- 2 and variants thereof.

[0013] Therefore, it is an object of the invention to provide pharmaceutical formulations containing segmented filamentous bacteria-induced metabolites which can reduce, minimize and / or treat respiratory diseases and / or disorders.

[0014] It is also an object of the invention to provide improved methods of treating a respiratory disease or disorder in a subject in need thereof.

[0015] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these

[0016] 45764921.1 1 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

[0017] BRIEF SUMMARY OF THE INVENTION

[0018] Disclosed are pharmaceutical formulations containing one or more metabolites and one or more pharmaceutically acceptable carrier. Optionally, the pharmaceutical formulations contain two or more metabolites, such as including taurine and sinapic acid. In some forms, the one or more metabolites are induced by segmented filamentous bacteria. The one or more metabolites in the pharmaceutical formulation can be one or more amino acid derivatives, one or more sugars, one or more flavonoids, or one or more organic acids, or a combination thereof. The disclosed formulations are suitable for treating one or more symptoms associated with a respiratory disease or disorder in a subject. Generally, methods for treating one or more symptoms associated with a respiratory disease or disorder in a subject in need thereof using the disclosed pharmaceutical formulation including: (i) administering to the subject the pharmaceutical formulation. Optionally, the administration step is repeated one or more times.

[0019] The one or more metabolites in the pharmaceutical formulation can be one or more amino acid and / or amino acid derivatives, one or more sugars, one or more flavonoids, or one or more organic acids, or a combination thereof. In some forms, the one or more amino acid derivatives are sulfur-containing amino acids. Exemplary sulfur-containing amino acids include, but are not limited to methionine, cysteine, homocysteine, taurine, and cysteamine. Optionally, the pharmaceutical formulation contains one or more amino acid derivatives, such as taurine and / or cysteamine. In some forms, the one or more sugars can be N-acetylglucosamine. Exemplary organic acids include but are not limited to nicotinic acid, aminolaevulinic acid, sinapic acid, and malonic acid, and combinations thereof. The one or more flavonoids can be isoflavonoids, neoflavonoids, and / or bioflavonoids. Exemplary flavonoids include but are not limited to isoflavones, genistein, daidzein, and formononetin, and combinations thereof.

[0020] In some forms, the pharmaceutical formulation includes one or more of the following: taurine, cysteamine, N-acetylglucosamine, nicotinic acid, aminolevulinic acid, sinapic acid, malonic acid, daidzein, formononetin, and genistein. In some forms, the pharmaceutical formulation includes taurine, cysteamine, N-acetylglucosamine, nicotinic acid, aminolevulinic acid, and malonic acid. In some forms, the pharmaceutical formulation includes taurine, cysteamine, N- acetylglucosamine, nicotinic acid, aminolevulinic acid, sinapic acid, malonic acid, daidzein, formononetin, and genistein.

[0021] In some forms, the pharmaceutical formulation includes taurine and cysteamine.

[0022] In some forms, the pharmaceutical formulation includes taurine and N-acetylglucosamine.

[0023] 45764921.1 2 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT

[0024] In some forms, the pharmaceutical formulation includes taurine and nicotinic acid.

[0025] In some forms, the pharmaceutical formulation includes taurine and aminolevulinic acid.

[0026] In some forms, the pharmaceutical formulation includes taurine and sinapic acid.

[0027] In some forms, the pharmaceutical formulation includes taurine and malonic acid.

[0028] In some forms, the pharmaceutical formulation includes taurine and daidzein.

[0029] In some forms, the pharmaceutical formulation includes taurine and formononetin.

[0030] In some forms, the pharmaceutical formulation includes taurine and genistein.

[0031] In some forms, the pharmaceutical formulation is in the form of a solid or a liquid. In some forms, the pharmaceutical formulation is in a form suitable for intranasal administration, oral administration, or parenteral administration. In some forms, the pharmaceutical formulation is in the form of a nasal spray.

[0032] In some forms, the pharmaceutical formulation further contains one or more active agents that are different from the one or more metabolites in the pharmaceutical formulation. Suitable active agents that can be used in the pharmaceutical formulation include, but are not limited to, antiinflammatory agents, antiviral agents, antibiotic agents, antipyretic agents, analgesic agents, blood glucose regulators, gastrointestinal agents, or respiratory tract agents, or a combination thereof.

[0033] In some forms, the respiratory disorder is a respiratory infection. In some forms, the respiratory infection is caused by a microorganism selected from a virus, a bacterium, a protozoan, and a fungus. Exemplary viruses causing the respiratory infection include, but are not limited to, orthomyxovirus, rhinovirus, paramyxovirus, coronavirus, adenovirus, human metapneumovirus (hMPV), enterovirus, and bocavirus. In some forms, the respiratory disease or disorder is cystic fibrosis, chronic obstructive pulmonary disease, pulmonary fibrosis, pneumonia, or asthma.

[0034] In some forms, when administered to a subject, the total amount of the one or more metabolites in the pharmaceutical formulation is effective, to reduce alveolar macrophage cell death, reduce viral levels in lung epithelial cells, and reduce lung viral titer, as indicated by the number of alveolar macrophages and / or TCIDso / g of virus present in a biological sample of the subject, compared to a control sample of the subject before administration. The biological sample can be a serum sample, for example a blood sample, or a bronchoalveolar lavage fluid sample.

[0035] In some forms, following the administration or optionally all of the administrations (when more than one administration is performed), the total amount of the metabolites administered to the subject is effective to reduce cell death of alveolar macrophages by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, as indicated by immunohistochemistry of a serum sample, optionally wherein the serum sample is a blood sample or a bronchoalveolar lavage fluid sample.

[0036] 45764921.1 3 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT

[0037] In some forms, following the administration or optionally all of the administrations (when more than one administration is performed), the total amount of the metabolites administered to the subject is effective to increase C1QA expression and / or reduce Notch4 expression by 5-fold, 10- fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, or 80-fold, as indicated by quantitative real-time PCR of a biological sample, such as a serum sample, a blood sample, or a bronchoalveolar lavage fluid sample.

[0038] In some forms, following the administration or optionally all of the administrations (when more than one administration is performed), the total amount of the metabolites administered to the subject is effective to reduce viral-induced expression of IFNP and / or IL-6 by 5-fold, 10-fold, 15- fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, or 80-fold, as indicated by quantitative real-time PCR of a biological sample, such as a serum sample, a blood sample, or a bronchoalveolar lavage fluid sample.

[0039] Also provided are methods of treating a respiratory disease or disorder (e.g., a respiratory infection, such as influenza, respiratory syncytial virus, coronavirus infection, or bacterial pneumonia) by administering alveolar macrophages (AMs) trained by the pharmaceutical formulations containing the metabolites to a subject in need thereof. For example, the method includes incubating the AMs with the pharmaceutical formulation containing the metabolites for a suitable period of time under suitable conditions to induce a trained or conditioned phenotype of the AMs. The trained or conditioned phenotype of the AMs are characterized by one or more of the following properties: an enhanced phagocytic activity, increased intracellular bacterial killing, increased production of reactive oxygen species, and altered chromatin accessibility, which promotes heightened responsiveness to respiratory pathogens. Following incubation of the AMs with the pharmaceutical formulation, the trained or conditioned phenotype of the AMs are administered (e.g., via transplantation) into the subject. For example, the metabolite-trained or conditioned phenotype of the AMs is administered to the subject via intranasal, intratracheal, aerosolized, or intravenous administration to the lung of the subject to repopulate or supplement the endogenous alveolar macrophages in the subject, and thereby reduce the viral or bacterial titer in the blood and / or bronchoalveolar fluid (BALF) of the subject.

[0040] BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figures 1A-1C show colonization of the intestine with SFB protects mice from Influenza respiratory infection. Figure 1A: “Excluded flora” (EF) and specific pathogen free (SPF) mice were inoculated with H1N1 influenza virus (CA09) (500 TCID50) as schematized. Figure IB: conventionally colonized SFB' mice received feces from a germfree (GF) or SFB -monoassociated (SFB-MA) mouse. 7 days post-transplant, mice were inoculated with CA09. Figure 1C: SFB' mice

[0042] 45764921.1 4 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT were untreated (naive) or administered SFB. Seven days later, mice were inoculated (or not) with CA09. Mice were euthanized 4-5 days later and whole lungs subjected to histopathologic or RNA- seq analysis. Data are a heatmap of genes that are significantly altered 2-fold in all treatment groups relative to the naive group. All experiments n = 4-5 per group. All data are presented as mean + SD. Statistical test: one-way ANOVA with Tukey’s multiple comparison test **P < 0.05; ***P < 0.001.

[0043] Figures 2A and 2B show SFB-mediated protection against IAV requires alveolar macrophages and alters them. Figure 2A: Mice were administered SFB and / or clodronate liposomes and then challenged with CA09. Figure 2B: As schematized, SFB’ CD45.1 mice were administered SFB or PBS. Seven days later, alveolar macrophages (AM) were isolated by FACS- sorting and transferred to SFB’ CD45.2 mice (250 x 103cells per mouse). One day post-transfer, mice were inoculated with CA09. 4.5 days later, AM was analyzed by flow cytometry. All experiments: n = 5 per group. All data are presented as mean ± SD. Statistical test: one-way ANOVA with Tukey’s multiple comparison test; *P < 0.5; **P < 0.05; ***P < 0.001, ****P < 0.0001.

[0044] Figures 3A and 3B show SFB+ AM exhibits inflammatory anergy and better disable IAV in vitro and in vivo. Figure 3A: SFB- and SFBA+ AM were studied ex vivo as schematized, i) basal gene expression via RNA seq, ii) lAV-induced gene expression by qRT-PCR. iii) IAV aliquots mixed with AM and infection titers in supernatants assayed 45 min later. Figure 3B: Mice administered PBS, SFB (d-7), SFB-AM, or SFB+ AM, and then challenged with CA09.

[0045] Figures 4A-4D show oral administration of fecal supernatant (FS), from SFB+ mice, partially recapitulates SFB’s protection against IAV. SFB+ mice were administered SFB, or FS from SFB+ or SFB- mice. 7 days later, mice were challenged with CA09. Weight loss / survival (Figure 4A), lung AM levels (Figure 4B), lung viral titer (Figure 4C), or lung type 1 epithelial cell viral levels (Figure 4D) measured on day 4. Open circle is no virus inoculum. Data mean + / - SD; ** p<0.01.

[0046] Figure 5 shows intranasal administration of SFB-MA fecal supernatant protects against IAV challenge. Mice were intranasally administered FS from GF or SFB-MA mice on indicated day prior to CA09 inoculation as schematized. Data are mean ± SD. * marks conditions of interest significantly difference from SFB- control.

[0047] Figures 6A-6I shows in vitro training of AM by SFB-derived fecal supernatant, serum, or lung homogenate recapitulated AM phenotype of SFB-colonized mice. Figure 6A is an experimental schematic. FACS-sorted alveolar macrophages (AM) from conventional SFB mice were incubated with fecal supernatant (FS), serum, or lung homogenate derived from SFB or SFB+mice. After 1 day, cells were washed and rested for 7 days in RPMI media containing GM-CSF,

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[0049] TGF-P, SP-A, and SP-D before downstream assays. Figure 6B is a graph showing viability of AM post-training measured by FACs. Figure 6C is a graph showing Clqa expression at basal level of trained AM over 15 days assayed by RT-qPCR). Figure 6D is a graph showing Ifnfi expression of AM exposed to UV-inactivated CA09 for 6 h, tracked over 15 days assayed by RT-qPCR. Figure 6E are bar graphs showing viral titers in supernatants Ih after AM exposure to live CA09 (MOI = 1) (left) and intracellular bacterial killing of AM treated with poly I:C (5d) and challenged with .S'. pneumoniae (MOI = 20). Figures 6F and 6G shows adoptive transfer of trained AMs into clodronate-treated SFB mice. Fecal SFB levels post-transfer quantified by RT-qPCR. Figure 6F are graphs showing one day post-transfer, mice were infected with lethal CA09; viral titers were measured at 4.5d. Figure 6G are graphs showing one day post- transfer, mice were sequentially infected with sublethal CA09 (50 TCIDso) and .S', pneumoniae (107CFU); bacterial titers were measured 3d after bacterial challenged. Figure 6H show AM were FACs-sorted from CD45.1 SFB and SFB+mice and transplanted to CD45.2 SFB-mice. Mice were euthanized at Id and 90d post transplanted, endogenous CD45.2 AM and transplanted CD45.1 AM composition quantified and sorted by FACs. Sorted AM were exposed to live CA09 (MOI = 1) and infectious titer were measured Ih post virus challenged. Figure 61 shows SFB' CD45.1 AM were FACs sorted and incubated with SFB+serum as in (A), and then transplanted to CD45.2 SFB'mice. Mice were euthanized at Id and 90d post transplanted, endogenous CD45.2 AM and transplanted CD45.1 AM composition quantified and sorted by FACs. Sorted AM were exposed to live CA09 (MOI = 1) and infectious titer were measured Ih post virus challenged. All experiments were performed with n = 5 per group. Results are shown as mean ± SD. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant.

[0050] Figures 7A-7F show identification of SFB-derived metabolites that reprogram lung alveolar macrophages. Figure 7A shows conventional SFB mice were administered SFB (oral) or SFB FS (intranasal). After 7 days, feces, serum, and lung tissues were analyzed by untargeted metabolomics screening. Shown are a venn diagram showing 9 overlapping metabolites significantly upregulated across feces, serum, and lung and volcano plots and heatmap highlighting these 9 common metabolites induced by SFB or SFB+FS. Figures 7B-7F show AMs from SFB mice were FACS- sorted and trained with individual metabolites identified by metabolomics (18-HEPE, daidzein, formononetin, genistein, linoleic acid, myristic acid, palmitoleic acid, sinapic acid, and taurine). After 1 day, AMs were washed and rested for 7 days in RPMI containing GM-CSF, TGF-P, SP-A, and SP-D before downstream assays. Figure 7C shows FACS analysis of AM viability posttraining (left), basal Clqa expression in trained AM measured by RT-qPCR (middle), and infectious titers Ih after challenge of trained AMs with live CA09 (right). Figure 7D shows 116 and

[0051] 45764921.1 6 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT

[0052] Ifn expression 6h after exposure of trained AM to UV-CA09. Figure 7E is a bar graph showing phagocytosis of IgG-latex beads by trained AMs (FACS). Figure 7F shows bacterial uptake (1 h) and intracellular killing (2 h) of 5. pneumoniae (MOI = 20) by trained AM. All experiments were performed with n = 5 per group. Results are shown as mean ± SD. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant.

[0053] Figures 8A and 8B show select SFB-induced metabolites partially recapitulate SFB+ AM phenotype. SFB+ AM were cultured for 7days in vitro to indicate metabolites (1-40 mM based on their level in FS). AM were then assayed for Clqa expression (Figure 8A) or exposed to UV-IAV, followed 6 hours later, by measure of IFNP by qRT-PCR (Figure 8B). SFB+ AM (i.e. trained in vivo) served as pos con. Metabolites boxed in red exhibited enhanced Clqa and reduced UV-IAV- induced IFN .

[0054] Figures 9A and 9B show AM trained, in vitro, by a metabolite cocktail, partially recapitulate SFB+ AM phenotype. AM from SFB+ mice were isolated by FACS and cultured with indicated metabolites as schematized. Such AM were then studied in vitro (Figure 9A) or via transplant into SFB+ mice (Figure 9B).

[0055] Figure 10 shows inflammatory anergy is not associated with reduction in proximal 1AV- induced signaling. AM, SFB- or SFB+, were exposed to UV-IAV. Level of phosphorylated STAT 1,3, and P38 were measured at indicated time by flow cytometry.

[0056] Figures 11A-11E show LPS-training potentiates lAV-induced inflammatory gene expression ex vivo and provides no benefit upon IAV challenge in vivo. SFB- AM were trained ex vivo (as in Figures 7 and 10) by PBS, EPS, or fecal sup from SFB- or SFB+ mice. AM were exposed in vitro to UV-IAV (Figure 11A), live IAV (Figure 11B), or transplanted into SFB- mice, which were then challenged with CA09 (Figures 11C-11E). Data means + / - SD, n=5.

[0057] Figure 12 shows SFB colonization protects against .S', pneumoniae. Mice, colonized or not with SFB, 7 days prior, were intranasally inoculated with 107CFU -S’, pneumoniae on day 0. Lung AM and CFU quantitated 2 days later. **P < 0.05; ***P < 0.001; ****P < 0.0001.

[0058] Figure 13 shows FACS -purified human AM exhibit quantifiable antiviral function and exhibit donor variability. Human AM was purified from BALF as indicated and then exposed to human Influenza virus strain CA09. Subject (S) 2 but not SI displayed the ability to neutralize the virus by about 70%. Both exhibited increased expression of IL-6 and IFNp.

[0059] Figure 14 shows IAV infection leads to depletion and replacement of AM but does not result in AM displaying “inflammatory anergy” phenotype. SFB+ mice were given a non-lethal IAV infection, which, as expected, acutely reduced tissue-resident (TR) AM and increased monocytic (Mo) AM. TR AM had recovered by 3 months, likely in part from Mo- AM acquiring TR

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[0061] AM phenotype but such new AM retained IAV responsiveness indicating IAV infection, itself does not alter AM responsiveness.

[0062] Figure 15 shows impeding SFB infection reduces generation of adaptive immunity.

[0063] Figure 16 shows data from transplant of feces from RV vaccine non-responders recapitulates phenotype.

[0064] Figure 17 shows data that C. Perfringens reduces RV infection and specific antibodies mRV in ASF mice.

[0065] Figures 18 -18G shows SFB in gut protects mice against influenza and that the protection lasts at least 3 months.

[0066] Figure 19 shows that SFB prevents entry of IAV into airway epithelial cells, thereby limiting viral replication.

[0067] Figure 20 is a bar graph showing gut SFB eliminated I AV-induced lung damage.

[0068] Figure 21 is a bar graph showing that gut SFB does not involve adaptive immunity, interferon, IL-17 / 22, ILC3.

[0069] Figure 22 shows that SFB has minimal direct impacts on lung leukocyte levels largely blocks impacts of IAV by blocking lAV-induced depletion of DC and AM.

[0070] Figure 23 shows that SFB has minimal direct impacts on lung leukocyte levels largely blocks impacts of IAV by blocking lAV-induced influx of several cell types.

[0071] Figures 24A-24D shows 4’ FIU blocks inflammatory cell recruitment but not loss of resident DC and AM.

[0072] Figures 25A-25J shows SFB’s protection against IAV does not require DC, Batf3- / - and requires alveolar macrophages (lost with clodronate or in CSF - / - mice.

[0073] Figure 26 shows AM transplanted from SFB+ resist lAV-induced depletion.

[0074] Figure 27 shows AM transplanted from SFB+ lack caspase 3 activation and fully resist depletion upon IAV infection.

[0075] Figure 28 shows impact of SFB colonization on AM gene expression.

[0076] Figure 29 shows SFB+ AM directly disable IAV reflecting enhanced phagocytosis and complement production.

[0077] Figure 30 shows transplant of AM from SFB+ mice confer protection against IAV.

[0078] Figures 31A-31D shows recapitulating SFB impacts on AM via transfer of fecal supernatant orally.

[0079] Figures 32A-32D shows LPS cannot train AM to impede IAV.

[0080] Figure 33 shows SFB increases taurine in feces, serum and BALF.

[0081] Figure 34 shows SFB increases gut bile salt hydrolase activity and serum taurine.

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[0083] Figures 35A and 35C shows the ability of serum from SFB+ mice to train AM to manage I AV is reduced by administration of a BSH inhibitor.

[0084] Figures 36A-36D shows intranasal taurine partially protects mice against 1AV challenge.

[0085] Figures 37A and 37B show SFB protects mice from secondary / co-bacterial infections after primary influenza virus infection. Figure 37A: (i) Experimental schema: Three- week-old C57BL / 6 SFB-mice received feces containing SFB but no other bacteria. Seven days post-transplant, mice were inoculated with 50 TCID50 of CA09. On day four post-CA09 inoculation, mice were inoculated intranasally with PBS or 1x107 CFU of S. pneumoniae, (ii) Body weight and survival rates were monitored daily for nine days following bacterial inoculation, (iii) Bacterial burden in lungs was evaluated four days post- inoculation, and (iv) lung sections were stained with H&E. Figure 37B: (i) Experimental schema: (i) Three-week-old C57BL / 6 SFB-mice were either colonized with SFB or not and inoculated with CA09 as described in Figure 37 A. Four days postvirus inoculation, the mice were inoculated with PBS, or 1x107 CFU of S. aureus, or H. influenzae, (ii) Body weight, survival, and lungs bacterial burden of mice inoculated with S. aureus, (iii) body weight, survival, and lung bacterial burden of mice inoculated with H. influenzae, (iv) Lung sections were stained with H&E. All experiments n = 5 per group. Data are representative of two independent experiments, yielding an identical pattern of results. Results are shown as mean ± SD. Statistical analysis: lung bacterial burden: one-way ANOVA or Student’s t test. Survival: log-rank Mental-Cox Test. *p < 0.05, **p < 0.01, ****p < 0.0001, ns not significant.

[0086] Figures 38A-38C show SFB protects mice from secondary / co-bacterial infections after primary influenza virus infection requires alveolar macrophages. Figure 38A: Three-week-old C57BL / 6 SFB-mice were either colonized with SFB or not and inoculated with CA09. Four days post-virus inoculation, the mice were inoculated with intranasally IxlO7CFU 5. pneumoniae. Mice were euthanized four days later, and lung assayed by flow cytometry. Representative flow plot, frequencies, and cell numbers of alveolar macrophages. Figure 38B: SFB-mice were either colonized with SFB or not for seven days, and clodronate liposomes were orally administered to mice two days prior to S. pneumoniae inoculation. Mice were euthanized on day four post bacterial inoculation. Body weight, survival rate and lung bacteria burden were measured. Figure 38C: SFB- mice were either colonized with SFB or not for seven days and clodronate liposome were administered to mice two days prior to CA09 inoculation. Four days post- virus inoculation, the mice were inoculated with intranasally IxlO7CFU S. pneumoniae. Mice were euthanized on day four post bacterial inoculation. Body weight, survival rate and lung bacteria burden were measured. All experiment n =5 per group. Data are representative of two experiments, yielding an identical pattern of results. Results are shown as mean ± SD. Statistical analysis: body weight: two-way

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[0088] ANOVA, lung bacterial burden: one-way ANOVA or Student’s t test. Survival: log-rank Mental- Cox Test. *p < 0.05, ** p< 0.01, ***p < 0.001, ****p < 0.001, ns not significant.

[0089] Figure 39 shows SFB+AM have increases bactericidal via phagocytosis and complements activation, (i) Schematic design for ex vivo studies of AM. (ii) SFB moderately enhanced AM phagocytosis of bacteria ex vivo. AM from SFB+and SFB" mice were FACs-sorted and incubated with either S. pneumoniae, or .S'. aureus, or H. influenzae for sixty minutes or one hundred twenty minutes. After incubation period, supernatants were harvested and CFU of bacteria remaining in supernatant were assayed, (iii) SFB increases AM bactericidal ex vivo. SFB mice were either colonized with SFB or not and inoculated with CA09. On day four post CA09 inoculation, mice were euthanized, and AM were FACs-sorted. FACs-sorted AM were incubated with indicated bacterial. Antibiotic protection assay results displayed as Percent CFU remaining = p* j g q r v i s 1

[0090] — ^-%100. (iv) SFB+AM enhances phagocytosis and bactericidal require Clq complements. AM from SFB+and SFB" mice were FACs-sorted and incubated with UV-CA09 overnight and then further incubated with or without Cl qa / b / c neutralization or isotype antibody for one hour. After incubation time, supernatants were removed and replaced with new media. AM were then subject to antibiotic protection assay as in (iii) and results shown as Percent CFU remaining. All experiments n = 5 per group. Data are representative of two independent experiments, yielding an identical pattern of results. Results are shown as mean ± SD. Statistical analysis: student’s t test (ii), Oneway ANOVA (iii, iv). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant.

[0091] Figures 40A and 40B show adoptive transfer SFB+AM mitigates secondary bacterial infection severity. Figure 40A: As schematized, SFB mice were administered SFB or PBS. Seven days later, AM were FACs-sorted and adoptive transfer to a clodronate-mediated-AM-depletion- SFB mice. Mice were administered 5-6xl05cells per mouse intranasally. One day post adoptive transfer, mice were inoculated with 5OTCIDso CA09 and four days later with IxlO7-S'. pneumoniae. Figure 40B: As schematized, SFB mice were inoculated with 50TCIDso CA09. On day four post CA09 inoculation, mice were received 6xl05AM that FACs-sorted from SFB+and SFB" mice. Two days post adoptive AM transfer, mice were inoculated with IxlO7S. pneumoniae. Mice were euthanized for 4 days post bacteria inoculation for lung bacteria titer, gross lung and histology assessment or monitored for survival and body weight. Mice were euthanized for 4 days post bacteria inoculation for lung bacteria titer, gross lung and histology assessment or monitored for survival and body weight. All experiments n = 5 per group. Data are representative of two independent experiments, yielding an identical pattern of results. Results are shown as mean ± SD. Statistical analysis: lung bacterial burden: Student’s t test. Survival: log-rank Mental-Cox Test. *p < 0.05, **p < 0.01

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[0093] Figure 41 shows optimizing A / CA / 07 / 2009 dose for secondary / co-bacterial infections. Three- week-old C57BL / 6 mice were inoculated with the A / CA / 07 / 2009 (H1N1) influenza virus (CA09) at different doses: 500 TCID50, 50 TCID50, and 5 TCID50. (i) Survival and body weight were monitored for fourteen days, (ii) A representative flow plot, the frequency and cell numbers of alveolar macrophages (AM), were analyzed on day 4.5 post-CA09 inoculation. All experiments n = 5 per group. Results are shown as mean ± SD. Statistical analysis: One-way ANOVA. ****p < 0.0001.

[0094] Figure 42A and 42B shows SFB protects mice from secondary / co-bacterial infection by enhance mucociliary clearance. Figure 42A: Bacterial titer in bronchoalveolar fluid (BALF), blood and spleen of mice in figure 2A. Figure 42B: (i) Enrichment plot from gene set of mucociliary clearance calculated by GSEA analysis from total lung RNA-seq of SFB+CA09 / CA09. (ii) Three- week-old C57BL / 6 SFB-mice were either colonized with SFB or not and inoculated with CA09. Four days post- virus inoculation, the mice were inoculated with intranasally 500 CFU .S’. pneumoniae. Two hours after bacterial inoculation, BALF harvest bacterial remaining CFU in CFU in BALF

[0095] BALF was calculated. % remaining CFU in BALF = — x 100. All experiments n = 5 per group. Data are representative of two experiments, yielding an identical pattern of results. Results are shown as mean ± SD. Statistical analysis: one-way ANOVA. *p < 0.05, ****p < 0.0001, ns not significant.

[0096] Figure 43A and 43B show SFB protects mice from secondary / co-bacterial infection that is long lasting. Figure 43A: (i) Experimental schema: Three-week-old C57BL / 6 SFB’mice were either colonized with SFB or not and inoculated with CA09. At Two days, four days or ten days post-virus inoculation, the mice were inoculated with intranasally IxlO7CFU .S'. pneumoniae. Mice were either monitored for body weight loss and survival rate or euthanized on day four post bacterial inoculation, (ii) Survival rate, body weight, and lung bacterial titer, (iii) Representative flow plot, frequency, and cell numbers of alveolar macrophage. Figure 43B: SFB mice were either colonized with SFB or not and inoculated with CA09. At four days, twenty days, thirty days or ninety days post-virus inoculation, the mice were inoculated with intranasally IxlO7CFU .S'. pneumoniae euthanized four days later. Lung bacterial burden and lung histology were assessed. All experiment n = 3-5 per group. Results are shown as mean ± SD. Statistical analysis: lung bacterial burden: one-way ANOVA or Student’s t test. Survival: log-rank Mental-Cox Test. *p < 0.05, ***p < 0.001, ****p < 0.0001.

[0097] Figures 44A and 44B show SFB prevents influenza virus induces temporary immune paralysis / reduced anti-microbial peptides during secondary / co-bacterial infection. SFB’mice were either colonized with SFB or not and inoculated with CA09. Four days post-virus inoculation, the

[0098] 45764921.1 11 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT mice were inoculated with intranasally IxlO7CFU .S'. pneumoniae. Mice were euthanized four days later. Figure 44A: Gene expression of immune cells chemo- attractants, and anti-microbial peptides were assayed by RT-qPCR shown as a heatmap. Figure 44B: Frequencies and cell numbers of innate immune cells. All experiment n = 3-5 per group. Results are shown as mean ± SD. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0099] Figures 45A-45C show SFB+AM exhibits increase phagocytosis, and complement activation. Figure 45A: Enrichment plot from gene set of positive regulation of phagocytosis, phagocytosis engulfment, complement activation, and antimicrobial humoral responses calculated by GSEA analysis from RNA-seq of SFB+AM / SFB"AM. Figure 45B: The volcano of RNA-seq and RT-qPCR shows Clqa, Clqb and Clqc are up-regulated in SFB+AM Figure 45C: C57BL / 6 SFB mice were either colonized with SFB or inoculated with CA09 or received PBS for five days and AM were FACs-sorted and exposed to pHrodo Green S. aureus BioParticle for 30mins. Degree of phagocytosis were assayed by FACs. Antibiotic protection assay logioCFU counts of Figure 3iii. Bacterial in supernatants post antibiotics, Uptake, and Survival, and %live macrophages of .S'. pneumonia, S. aureus, and H. influenzae. All experiments n > 4 per condition. Results are shown as mean ± SD. Statistical analysis: One-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant.

[0100] Figures 46A-46J show SFB-derived fecal supernatant protects against respiratory infection. Figure 46A is a schematic showing conventional SFB mice were administered SFB or PBS orally, or intranasally administered SFB+-derived fecal supernatant (SFB+FS) or SFB -derived fecal supernatant (SFB FS). SFB" AM, SFB+AM, SFB FS, and SFB+FS AMs were FACS-sorted seven days post-administration. Figure 46B are bar graphs showing AMs were exposed to live CA09 (MOI = 1); viral titers in the supernatant were assayed 1 h post-infection (left), AMs were exposed to S. pneumoniae (MOI = 20); intracellular bacterial killing was assayed 2 h post- infection (middle), and AMs were treated with poly I:C for 5 days and then exposed to S. pneumoniae (MOI = 20); intracellular bacterial killing was assayed 2 h post-infection (right). AMs were exposed to UV-inactivated CA09; RNA was harvested 6 h later and gene expression was assayed by RT-qPCR, shown as a heatmap. Seahorse metabolic assays of AMs energy phenotypes under the indicated conditions. Figure 46C is a collection of plots showing AMs were either labeled with CellTrace Violet or left unlabeled, co-cultured together, and exposed to live CA09. Live AMs were measured by FACS at the indicated timepoints. Figure 46D is a collection of bar graphs showing ROS, glutathione, DNA damage (yH2AX), and cleaved caspase-3 were measured 6 h post-CA09 infection by FACS and shown as MFI. Figures 46E is a schematic showing SFB mice were intranasally administered SFB FS or SFB+FS and, 7 days later, inoculated with CA09. Figure 46F

[0101] 45764921.1 12 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT shows mice were euthanized 4.5 days post-inoculation for gross lung assessment and viral titers, monitored for survival, body weight, and temperature for 14 days. Figure 46G is a representative FACS plots and quantification AM frequencies and absolute numbers. Figure 46H is a schematic showing SFB mice were intranasally administered SFB FS or SFB+FS and, 7 days later, inoculated with 50 TCIDso of CA09. On day 4, mice were intranasally administered vehicle (PBS) or 1 x 107CFU of S’, pneumoniae. Figure 461 shows body weight (left) and survival (right) were monitored daily. Figure 46J shows quantification of bacterial CFUs in lung, blood, and spleen was performed on day 3 post-. S', pneumoniae inoculation. All experiments were performed with n = 5 per group. Results are shown as mean ± SD. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant.

[0102] Figures 47A-47L show synthetic mixtures of SFB elicited metabolites recapulates SFB+ AM in phenotype in mouse and human AM. Figure 47A is a schematic showing AMs from SFB mice were FACS-sorted and either SFB+FS or with cocktail of metabolites containing daidzein, formononetin, genistein, sinapic acid, and taurine or with control vehicle (DMSO). After 1 day, AMs were washed and rested for 7 days in RPMI containing GM-CSF, TGF-0, SP-A, and SP-D before downstream assays. Figure 47B shows FACS analysis of AM viability post-training (left), basal Clqa expression in trained AM measured by RT-qPCR (middle), and infectious titers Ih after challenge of trained AMs with live CA09 (right). Figure 47C is a collection of bar graphs showing 116 and Ifn > expression 6h after exposure of trained AM to UV-CA09. Phagocytosis of IgG-latex beads by trained AMs measured FACs. Figure 47D is a collection of bar graphs showing bacterial uptake (1 h) and intracellular killing (2 h) of S. pneumoniae (MOI = 20) by trained AM. Figure 47E is a schematic showing FACs verification of human AM (hAM) and experiment schematic: human AM were trained with SFB-FS, SFB+FS, DMSO, or 5-mix metabolites as in (A). Figure 47F shows infectious titers Ih after challenge of trained AMs with live CA09 (left) and phagocytosis of IgG-latex beads by trained AMs measured FACs (right). Figure 47G is a collection of bar graphs showing 116 and Ijn[J> expression 6h after exposure of trained AM to UV-CA09, and bacterial uptake (1 h) and intracellular killing (2 h) of .S'. pneumoniae (MOI = 20) by trained AM. Figure 47H shows trained AM were treated with poly I:C and 5d later challenged with .S'. pneumonaie (MOI = 20), bacterial uptake and intracellular bacterial killing were assayed as in (vii). Figure 471 shows trained AM were either labeled with CellTrace Violet or CellTrace Far Red and co-culture together as follow: SFB-FS trained hAM with SFB+FS trained hAM and DMSO trained hAM with 5-mix trained hAM. Live AM were measured by FACs at Oh, 6h, and 12h post CA09 inoculation. Figures 47J-47L are graphs of results from seahorse metabolic assay of hAM as indicated conditions. All experiments were performed with n = 4-5 per group. Results are shown as

[0103] 45764921.1 13 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT mean ± SD. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant.

[0104] Figures 48A-48J show rational simplification of SFB-derived metabolites into a 3-mix cocktail. Figures 48A-48C are graphs showing combinatorial testing of SFB-derived metabolites identifies minimal mixes required for anti-inflammatory and antiviral activity. AM FACs sorted SFB mice were trained with different combinations of taurine, sinapic acid, daidzein, formononetin, and genistein, rested for 7 d, and then challenged with either UV-CA09 to assessed of 116 gene expression or live CA09 for virus neutralization capability. Figure 48D is a schematic of an experimental timeline. SFB mice mice were administered intranasally with either DMSO, or SFB+FS, 5-mix containing taurine / sinapic acid / daidzein / formononetin / genistein, or 3-mix D containing taurine / sinapic acid / daidzein, or 3-mix G taurine / sinapic acid / genistein. 7 days later, inoculated with CA09. Mice were euthanized 4.5 days post-inoculation. Gross lung assessment was done. Figure 48E is a pair of heatmaps showing lung immune cells assessed by FACs and displaying as heatmap with each cell types normalized independently to its own minimum and maximum value (range 0-100) (left) and inflammatory cytokines the lungs measured by RT-qPCR (right). Figures 48F and 48G show survival, body weight changes, temperature, and viral titers. Figure 48H is a pair of bar graphs showing lung AM frequency (left) and cells numbers (right) measured by FACs. Figure 481 show ROS, glutathione, and cleaved caspase of AM shown as MFI. Figure 48J shows an experimental schematic: SFB-mice were treated as in treated as in Figures 48D-48I prior to pathogen inoculation. Mice were then inoculated with 50 TCIDso of CA09. On day 4, mice were intranasally administered vehicle (PBS) or 1 x 107CFU of .S’, pneumoniae. Shown are body weight and survival as well as quantification of bacterial CFUs in lung, blood, and spleen was performed on day 3 post-5, pneumoniae inoculation. All experiments were performed with n = 4-5 per group. Results are shown as mean ± SD. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant.

[0105] Figures 49A-49S show SFB mediated level taurine by influencing microbial bile salt hydrolyze and genistein by upregulates host lactase phlorizin hydrolase activities. As schematized in Figure 49A, SFB mice were colonized with SFB or not for seven days prior to virus inoculation. On day 7, mice were challenged with CA09 at sublethal (5OTCIDso), and lethal dose (5OOTCIDso). Blood and bronchoalveolar fluid (BALF) were collected at the indicated timepoints. Figure 49B are graphs showing taurine level was quantified in blood (left) and BALF (right). Figure 49C shows body weight change over the course of the experiment. Figure 49D is an experimental timeline showing feces were collected from CD45.1 SFB mice prior to oral administered SFB and bile acid inhibitor GR7. AMs were FACS-sorted from CD45.1 mice and trained in vitro with FS for 1 day,

[0106] 45764921.1 14 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT then washed and cultured for 7 days in GM-CSF / SP-A / SP-D supplemented media. Distinct trained AM populations (SFB GR7', SFB+GR7 , SFB+GR7+) were studied in vitro with live or UV- inactivated CA09, or transplanted into clodronate-treated mice or SFB CD45.2 recipient mice. Mice treated clodronate were monitored for clinical signs and euthanized at 4.5-day post inoculation to measured lung viral tier. In CD45.2 recipients, donor and host compositions of AM were measured by FACs (CD45.1 vs. CD45.2) and assessed for oxidative stress and caspase-3 activation. Figure 49E are graphs showing survival rate, body weight change, and temperature. Figure 49F are graphs showing lung viral titer, and percent of lung epithelial cells containing CA09 measured by FACs, and inflammatory cytokines and chemokines in lung tissues assayed by RT- qPCR. Figure 49G are graphs showing frequency and cell number of AM (left), and cell numbers of inflammatory monocytes and neutrophils in the lung (right). Figure 49H is an experimental timeline showing ASF mice were fed either with grain-base chow (GBC), defined diet for 7 days, followed by SFB colonization for another 7 days. FACs sorted SFB AM were trained in vitro with fecal supernatant, serum, and lung homogenate collected from GBC / SFB+, CDD / SFB', and CDD / SFB+mice. Trained AM were inoculated with UV-CA09 or live CA09. Figures 49I-49J are a collection of bar graphs showing AM viability post training measured by FACs (left), expression of 116. I n / 3 6h post UV-CA09 inoculation and infectious tier Ih post live CA09 inoculation. Figure 49K shows LoglO TCID50 / mL in FS, serum, and lung supplemented samples. Figure 49L is a schematic showing how genistein and daidzein generated from the precursors genistin and daidizin. The isoflavone glycosides genistein and daidzein are hydrolyzed by host- or microbiota-derived 0- glucosidase / phlorizin hydrolase into their aglycone forms, genistein and daidzein. These metabolites either transfer to lung via blood stream or undergo glucuronidation and sulfation in the liver, generating conjugated forms that circulate systemically. In the lung, conjugated genistein and daidzein are taken up and deconjugated by local 0-glucosidases and sulfatases, regenerating active genistein and daidzein. Figure 49M shows quantification of small intestine of SFB+ / SFB‘ mice highlighted differential expression of glycosidase-related genes (Gba2, Gba, Let) significantly altered by SFB colonization. Shown are RT-qPCR Gba2, Gba, Let gene expression the small intestine of SFB+and SFB' mice, quantification of lactate phlorizin hydrolase activity in the small intestine of mice that SFB+and SFB' mice in the presence of 2F-DNP-Glc, a lactate phlorizin hydrolase inhibitor (iv). quantification of serum genistein (v). Also shown are quantification of 0- glucosidase activities in the small intestine and feces of conventional SFB+and SFB' mice (left), and quantification of 0-glucosidase in feces of germ-free SFB+and SFB' mice (right). Figure 49N is an experimental timeline showing feces that were collected from SFB'mice prior to oral administered SFB and 2F-DNP-Glc, an LPH inhibitor. AM were FACS-sorted from mice and

[0107] 45764921.1 15 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT trained in vitro with fecal supernatant for 1 day, then washed and cultured for 7 days in GM- CSF / SP-A / SP-D supplemented media. Distinct trained AM populations were studied in vitro with live or UV-inactivated CA09, or transplanted into clodronate-treated mice or conventional SFB recipient mice and inoculated with CA09. Figure 490 are graphs showing survival rate, body weight change, and temperature. Figures 49P-49S are graphs showing lung viral titer (left), inflammatory cytokines and chemokines in lung tissues assayed by RT-qPCR (middle), and number of AM, neutrophils, and inflammatory monocytes (right). All experiments were performed with n = 4-5 per group. Results are shown as mean ± SD. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant.

[0108] Figures 50A-50N show intranasal administration of taurine, genistein and sinapic acid protect against respiratory infection. As schematized in Figure 50A, mice received oral or intranasal administration of vehicle, SFB, SFB+FS, or metabolite combinations (genistein + taurine, genistein + sinapic acid, taurine + sinapic acid) or 3-mix genistein / taurine / sinapic acid (TGS) from day 0. On day 7, mice were infected with influenza virus CA09 (SOOTCIDso) and monitored for clinical signs. Mice were euthanized at day 4.5 post inoculation. AM were FACs sorted and challenged with 5. pneumoniae ex vivo. Figure 50B shows survival, body weight change, and temperature. Figure 50C shows lung virus titer (left) and lung inflammatory cytokines and chemokines assayed by RT-qPCR (right). Figure SOD shows frequency, and cell numbers of lung AM and bacteria uptake. Figure 50E shows intercellular bacteria killing of AM. Figure 50F is an experimental timeline and survival curve showing SFB mice were inducing diabetes using STZ and were pre-treated with TGS for seven days and then mice were inoculated with CA09. Figure 50G shows body weight change, temperature, and lung viral titer as day 4.5 post CA09 inoculation. Figure 50H is an experimental timeline and survival curve showing SFB mice fed with high fat diet and were pre-treated TGS and inoculated with CA09 as in Figure 50E and 50F. Figure 501 shows body weight change, temperature, and lung viral titer as day 4.5 post CA09 inoculation. Figure 50 J is an experimental timeline showing SFB mice were pre-treated intranasally with TGS at 7d, 30d, 60d, 90d, and 120d prior to CA09 inoculation. Mice were inoculated with CA09 and monitored for clinical signs. Mice were euthanized at day 4.5 post inoculation for lung viral titer assessment and AM were FACs sorted and challenged with 5. pneumoniae ex vivo. Figure 50K is a collection of graphs showing survival rate and lung virus titer, lung AM cell numbers quantified by FACs, and intercellular bacterial of killing of AM. Figure SOL is an experimental timeline showing SFB mice were pre-treated intranasally with TGS. After 120d, mice were either received DMSO or a second dose of TGS followed 7 days later by CA09 inoculation. Mice were euthanized at day 4.5 post inoculation for lung viral titer assessment and AM were FACs sorted and challenged with .S’.

[0109] 45764921.1 16 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT pneumoniae ex vivo. Figure 50M and 50N is a collection of graphs showing survival rate and lung virus titer, lung AM cell numbers quantified by FACs, and intercellular bacterial of killing of AM. All experiments were performed with n = 5 per group. Results are shown as mean ± SD. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant.

[0110] Figures 51A-51C are graphs showing that SFB impacts a wide range of respiratory pathogens, including viral pathogens (Figure 51A), bacterial pathogens (Figure 51B), and combination of viral and bacterial pathogens (Figure 51C).

[0111] Figures 52A-52G are graphs showing SFB+serum trained AMs retain their activity after freeze-thaw and provide time-dependent therapeutic benefits during influenza infection and influenza / bacterial co-infections. Figure 52A is an experimental timeline showing AM were FACS- sorted from SFB mice and trained for 1 day with serum from SFB+or SFB mice, then washed and rested for 7 days. Trained AMs were frozen in -80°C, stored for 10 days, and thawed for downstream experiment. For ex vivo experiments: Thawed trained AM were measured Clqa expression at basal level or challenged with UV-inactivated CA09 or live CA09. For in vivo experiments: Thawed trained AM were adoptively transferred into SFB'clodronate-treated B6 mice. One day later, recipients were challenged with CA09. Lung viral titers, AM levels, and innate immune responses were measured 4 days post-infection. Figure 52B is a collection of bar graphs showing Clqa expression at basal level of trained freeze / thawed AM measured by RT-qPCR (left), Ifn / 3 expression at after AM were exposed to UV-CA09 for 4h (middle), and virus infectious titer after AM were inoculated with live CA09 for Ih (right). Figure 52C is a collection of graphs showing survival, body weight change, temperature, lung viral titers, and FACs plot representative and frequency of lung epithelial cells containing CA09. As schematized in Figure 52D, SFB mice were intranasally inoculated with 5OOTCIDso CA09. Mice were received adoptive transfer SFB-FS trained AM or SFB+FS trained AM at 12h, 24h, 36h, 48h post CA09 inoculation. Clinical signs were monitored daily through day 14. Lung viral titers and FACs analysis of immune populations were performed at day 4 post-infection. Figure 52E is a collection of graphs showing survival rate, body weight change, temperature, and lung viral titers. As schematized in Figure 52F, SFB mice were intranasally inoculated with 5OTCIDso CA09. At day 4 post CA09 inoculation, mice were inoculated with S. pneumonaie and received adoptive transfer SFB-FS trained AM or SFB+FS trained AM at 12h, 24h, 36h, 48h post bacterial inoculation. Clinical signs were monitored daily, and lung bacterial titer were measured at day 3 post inoculation. Figure 52G is a collection of graphs showing survival rate, body weight change, and bacterial viral titers, inflammatory cytokines and chemokines were assayed by RT-qPCR.

[0112] 45764921.1 17 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT

[0113] Figures 53A-53D shows in vitro dose optimization of taurine, genistein, and sinapic acid. AM isolated from SFB mice were trained for 24 h to varying concentrations of taurine, genistein, or sinapic acid, then washed and rested for 7 days. Trained AM were checked for cell viability by FACs and subsequently challenged with UV-inactivated CA09 for Ifnfi induction assessed by RT- qPCR) or live CA09. Shown are taurine (Figure 53B), sinapic acid (Figure 53C), genistein (Figure 53D).

[0114] Figures 54A-54Q show in vivo dose optimization of taurine, genistein, and sinapic acid. As shown in Figure 54A, mice were treated intranasally with taurine at escalating doses (0.2, 2, 20, or 200 mg / kg) or vehicle control 7d prior to influenza virus CA09. Outcomes were assessed at day 4 post-infection, including lung viral titers, histology, AM profiling by FACS, and host gene expression by RT-qPCR. Survival and clinical monitoring continued until day 14. Figure 54B shows survival and weight loss in mice pretreated with taurine prior to CA09 challenge. Figures 54C and 54D is a collection of graphs showing survival rate, body weight change, temperature, and lung viral titer at day 4 post CA09 inoculation. Figure 54E are bar graphs showing frequency, cell numbers of AM. Figure 54F are bar graphs showing frequency of lung epithelial cells and ciliated airway cells containing CA09. As shown in Figure 54G, mice were treated intranasally with genistein at escalating doses (0.02, 0.2, 2, 20, or 200 mg / kg) or vehicle control 7d prior to influenza virus CA09. Outcomes were assessed at day 4 post-infection, including lung viral titers, histology, AM profiling by FACS, and host gene expression by RT-qPCR. Survival and clinical monitoring continued until day 14. Figure 54H show survival and weight loss in mice pretreated with genistein prior to CA09 challenge. Figures 541 and 54J show survival rate, body weight change, temperature, and lung viral titer at day 4 post CA09 inoculation. Figure 54K shows frequency, cell numbers of AM. Figure 54L shows frequency of lung epithelial cells and ciliated airway cells containing CA09. As shown in Figure 54M, mice were treated intranasally with sinapic acid at escalating doses (0.01, 0.1, 1, 10, or 100 mg / kg) or vehicle control 7d prior to influenza virus CA09. Outcomes were assessed at day 4 post-infection, including lung viral titers, histology, AM profiling by FACS, and host gene expression by RT-qPCR. Survival and clinical monitoring continued until day 14. Figure 54N shows survival and weight loss in mice pretreated with sinapic acid prior to CA09 challenge. Figures 540 and 54P shows survival rate, body weight change, temperature, and lung viral titer at day 4 post CA09 inoculation. Figure 54Q shows frequency, cell numbers of AM.

[0115] Figures 55A-55H shows SFB-derived 3-mix (TGS) metabolites confer protection against IAV under pre-existing conditions by preserving and reprogramming AM. Figures 55A-55D shows SFB mice were induced with diabetes using STZ and were pre-treated with TGS for seven days and

[0116] 45764921.1 18 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT then mice were inoculated with CA09. Figure 55A shows body weight change, resting glucose, and glucose tolerance test prior to CA09 inoculation. Figure 55B shows frequency of lung epithelial cells and ciliated cells containing CA09. Figure 55C shows frequency, and cell numbers of lung AM. Figure 55D shows ROS, Glutathione, DNA damage, and cleaved caspase-3 of AM measured by FACs. Figures 55E-55H shows SFB mice were induced with metabolic syndrome by fed them with high-fat diet for 60days, and were pre-treated with TGS for seven days and then mice were inoculated with CA09. Figure 55E shows body weight change, food intake, prior to CA09 inoculation. Figure 55F shows frequency of lung epithelial cells and ciliated cells containing CA09. Figure 55G shows frequency, and cell numbers of lung AM. Figure 55H shows ROS, Glutathione, DNA damage, and cleaved caspase-3 of AM measured by FACs.

[0117] DETAILED DESCRIPTION OF THE INVENTION

[0118] The disclosed method and compositions can be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.

[0119] I. DEFINITIONS

[0120] The use of the terms "a," "an," "the," and similar referents in the context of describing the presently claimed invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0121] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0122] Use of the term "about" is intended to describe values either above or below the stated value in a range of approx. + / - 10%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 5%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 2%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in

[0123] 45764921.1 19 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0124] The term "titer" refers to the concentration of a virus or a biological product in a given sample. Specifically, it indicates the amount of virus, measured in milligrams per liter (mg / L) or TCIDso / g, that is present in the culture or sample from a subject. TCIDso (50% tissue culture infectious dose) is an endpoint dilution assay used to measure infectious viral titer.

[0125] The term “variant” or “mutant,” as used herein refer to an artificial outcome that has a pattern that deviates from what occurs in nature.

[0126] The terms “inhibit” or “reduce” in the context of inhibition, mean to reduce, or decrease in activity and quantity. This can be a complete inhibition or reduction in activity or quantity, or a partial inhibition or reduction. Inhibition or reduction can be compared to a control or to a standard level. Inhibition can be measured as a % value, e.g., from 1% up to 100%, such as 5%, 10, 25, 50, 75, 80, 85, 90, 95, 99, or 100%. For example, pharmaceutical formulations containing one or more metabolites may inhibit or reduce the activity and / or quantity of one or more viruses or variants thereof by about 10%, 20%, 30%, 40%, 50%, 75%, 85%, 90%, 95%, or 99% from subjects that did not receive or were not treated with the formulations. In some forms, the inhibition and reduction are compared according to the level of mRNAs, proteins, cells, tissues, and organs.

[0127] A weight percent of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

[0128] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0129] II. PHARMACEUTICAL FORMULATIONS

[0130] Disclosed are pharmaceutical formulations containing one or more metabolites, preferably two or more metabolites, and optionally one or more pharmaceutically acceptable carrier and / or excipients. In some forms, the composition containing the metabolite(s) can be formulated as nutraceutical compositions. A skilled person would understand that the descriptions of pharmaceutical formulations containing the metabolite(s) herein can be applied to the nutraceutical compositions, as well.

[0131] In some forms, the metabolites in the pharmaceutical formulation are generated by segmented filamentous bacteria, such as taurine, genistein, and sinapic acid. The one or more metabolites in the pharmaceutical formulation can include one or more amino acids and / or amino acid derivatives, one or more sugars, one or more flavonoids, one or more organic acids, or a

[0132] 45764921.1 20 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT combination thereof. For example, the metabolites in the pharmaceutical formulation include at least taurine and sinapic acid.

[0133] The disclosed pharmaceutical formulations are based on the discovery that gut microbiota composition, specifically segmented filamentous bacteria (SFB) can strongly influence respiratory viral infection (RVI) outcomes. Data in the non-limiting Examples demonstrated that SFB increased a variety of metabolites in biological samples, for example amino acid derivatives such as taurine and sinapic acid, in biological samples (Figure 33 and Figure 48A-48B) and that administration of such SFB-induced metabolites can protect against respiratory disorders, for example, respiratory infections such as influenza viral infection (IAV) (Figure 34).

[0134] The disclosed pharmaceutical formulations are particularly useful for treating one or more symptoms associated with a respiratory disease or disorder in a subject, such as by increasing the number of alveolar macrophages in lung tissue, reducing AM cell death in lung tissue, and / or inhibiting or reducing viral replication in lung tissue. In some forms, the disclosed pharmaceutical formulations are useful for increasing survival in a subject having a severe respiratory disease / disorder.

[0135] In some forms, the pharmaceutical formulation includes one or more of the following: taurine, cysteamine, N-acetylglucosamine, nicotinic acid, aminolevulinic acid, sinapic acid, malonic acid, daidzein, formononetin, and genistein. In some forms, the pharmaceutical formulation includes taurine, cysteamine, N-acetylglucosamine, nicotinic acid, aminolevulinic acid, and malonic acid. In some forms, the pharmaceutical formulation includes taurine, cysteamine, N- acetylglucosamine, nicotinic acid, aminolevulinic acid, sinapic acid, malonic acid, daidzein, formononetin, and genistein. In some forms, the pharmaceutical formulation includes taurine, daidzein, formononetin, genistein, sinapic acid, and taurine. In some forms, the pharmaceutical formulation includes (1) taurine, (2) sinapic acid, and (3) geinistein and / or daidzein. For example, the pharmaceutical formulation includes taurine, genistein, and sinapic acid. In some forms, the pharmaceutical formulation includes at least taurine and sinapic acid. Optionally, the pharmaceutical formulation can further include one or more additional metabolites, e.g., daidzein, genistein, and formononetin.

[0136] In some forms, the pharmaceutical formulation includes taurine and cysteamine.

[0137] In some forms, the pharmaceutical formulation includes taurine and N-acetylglucosamine.

[0138] In some forms, the pharmaceutical formulation includes taurine and nicotinic acid.

[0139] In some forms, the pharmaceutical formulation includes taurine and aminolevulinic acid.

[0140] In some forms, the pharmaceutical formulation includes taurine and sinapic acid.

[0141] In some forms, the pharmaceutical formulation includes taurine and malonic acid.

[0142] 45764921.1 21 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT

[0143] In some forms, the pharmaceutical formulation includes taurine and daidzein.

[0144] In some forms, the pharmaceutical formulation includes taurine and formononetin.

[0145] In some forms, the pharmaceutical formulation includes taurine and genistein.

[0146] A. Amino Acids and Amino Acid Derivatives

[0147] The pharmaceutical formulation disclosed herein may contain one or more amino acid and / or amino acid derivatives. In some forms, the pharmaceutical formulation disclosed herein can contain at least one amino acid derivative. In other forms, the pharmaceutical composition disclosed herein can contain a combination of two or more amino acid derivatives. For example, the pharmaceutical formulation can contain a combination of two, three, four, or more amino acid derivatives. In some forms, the amino acids and amino acid derivatives in the pharmaceutical formulation are sulfur-containing amino acids and amino acid derivatives. Exemplary amino acid and amino acid derivatives that contain sulfur include, but are not limited to methionine, cysteine, homocysteine, taurine, and cysteamine, and derivatives thereof. In some forms, the pharmaceutical formulation contains taurine and / or cysteamine. In some forms, the pharmaceutical formulation contains taurine.

[0148] The precise amount of the one or more amino acid derivatives in the pharmaceutical formulation can vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the respiratory disease or disorder, e.g., virus infection or symptoms being treated, as well as the pharmacokinetics of the agents being administered. In some forms, the total amount of the one or more amino acid derivatives in the pharmaceutical formulation is effective, when administered to a subject, to reduce alveolar macrophage cell death, reduce viral levels in lung epithelial cells, and reduce lung viral titer, as indicated by the number of alveolar macrophages and level of viral RNA measured in a biological sample of the subject, compared to a control sample of the subject before administration. For example, the reduced alveolar macrophage cell death can be measured by the number of %GFP+ cells in a serum sample, for example, a blood sample or a bronchoalveolar lavage fluid sample. In another example, the level of viral RNA in a biological sample such a serum sample, for example, a blood sample or a bronchoalveolar lavage fluid sample can be measured via quantitative real-time PCR.

[0149] The one or more amino acids can be present in the pharmaceutical formulation at a dosage ranging from about 0.01 mg / kg to about 250 mg / kg. The dosage of the one or more amino acids is calculated using the sum of the weights of all amino acids in the pharmaceutical formulation. In some forms, the one or more amino acids can be present in the pharmaceutical formulation at a dosage ranging from about 0. 1 mg / kg to about 250 mg / kg body weight, from about 0.01 mg / kg to about 200 mg / kg body weight, from about 0.1 mg / kg to about 200 mg / kg body weight, from about

[0150] 45764921.1 22 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT

[0151] 0.01 mg / kg to about 150 mg / kg body weight, from about 0.1 mg / kg to about 150 mg / kg body weight, from about 0.01 mg / kg to about 100 mg / kg body weight, from about 0.1 mg / kg to about 100 mg / kg body weight, from about 0.01 mg / kg to about 50 mg / kg body weight, from about 0.1 mg / kg to about 50 mg / kg body weight, from about 0.01 mg / kg to about 10 mg / kg body weight, from about 0.1 mg / kg to about 10 mg / kg body weight, or from about 0.5 mg / kg to about 50 mg / kg body weight. For example, the one or more amino acids is present in the pharmaceutical formulation at a dosage ranging from about 0.2 mg / kg to about 200 mg / kg body weight.

[0152] B. Sugars

[0153] The pharmaceutical formulation disclosed herein may contain one or more sugars. In other forms, the pharmaceutical composition disclosed herein can contain a combination of two or more sugars. For example, the pharmaceutical formulation can contain a combination of two, three, four, or more sugars. An exemplary sugar that can be used in the pharmaceutical formulation is N- acetylglucosamine.

[0154] The precise amount of the sugar in the pharmaceutical formulation can vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the respiratory disease or disorder, e.g., virus infection or symptoms being treated, as well as the pharmacokinetics of the agents being administered. In some forms, the total amount of the one or more sugars in the pharmaceutical formulation is effective, when administered to a subject, to reduce alveolar macrophage cell death, reduce viral levels in lung epithelial cells, and reduce lung viral titer, as indicated by the number of alveolar macrophages and level of viral RNA measured in a biological sample of the subject, compared to a control sample of the subject before administration. For example, the reduced alveolar macrophage cell death can be measured by the number of %GFP+ cells in a serum sample, for example, a blood sample or a bronchoalveolar lavage fluid sample. In another example, the level of viral RNA in a biological sample such a serum sample, for example, a blood sample or a bronchoalveolar lavage fluid sample can be measured via quantitative real-time PCR.

[0155] The one or more sugars can be present in the pharmaceutical formulation at a dosage ranging from about 0.01 mg / kg to about 250 mg / kg. The dosage of the one or more sugars is calculated using the sum of the weights of all sugars in the pharmaceutical formulation. In some forms, the one or more sugars is present in the pharmaceutical formulation at a dosage ranging from about 0.1 mg / kg to about 250 mg / kg body weight, from about 0.01 mg / kg to about 200 mg / kg body weight, from about 0. 1 mg / kg to about 200 mg / kg body weight, from about 0.01 mg / kg to about 150 mg / kg body weight, from about 0.1 mg / kg to about 150 mg / kg body weight, from about 0.01 mg / kg to about 100 mg / kg body weight, from about 0.1 mg / kg to about 100 mg / kg body weight,

[0156] 45764921.1 23 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT from about 0.01 mg / kg to about 50 mg / kg body weight, from about 0.1 mg / kg to about 50 mg / kg body weight, from about 0.01 mg / kg to about 10 mg / kg body weight, from about 0. 1 mg / kg to about 10 mg / kg body weight, or from about 0.5 mg / kg to about 50 mg / kg body weight. For example, the one or more sugars is present in the pharmaceutical formulation at a dosage ranging from about 0.2 mg / kg to about 200 mg / kg body weight.

[0157] C. Organic Acids

[0158] The pharmaceutical formulation disclosed herein may contain one or more organic acids. In some forms, the pharmaceutical formulation disclosed herein can contain at least one organic acid. In other forms, the pharmaceutical composition disclosed herein can contain a combination of two or more organic acids. In some forms, the pharmaceutical composition disclosed herein can contain a combination of two or more phenolic acids. For example, the pharmaceutical formulation can contain a combination of two, three, four, or more organic acids. Exemplary organic acids that can be used in the pharmaceutical formulation include, but are not limited to, nicotinic acid, aminolaevulinic acid, sinapic acid, and malonic acid, and combinations thereof.

[0159] The precise amount of the organic acid (s) in the pharmaceutical formulation can vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the respiratory disease or disorder, e.g., virus infection or symptoms being treated, as well as the pharmacokinetics of the agents being administered. In some forms, the total amount of the one or more organic acids in the pharmaceutical formulation is effective, when administered to a subject, to reduce alveolar macrophage cell death, reduce viral levels in lung epithelial cells, and reduce lung viral titer, as indicated by the number of alveolar macrophages and level of viral RNA measured in a biological sample of the subject, compared to a control sample of the subject before administration. For example, the reduced alveolar macrophage cell death can be measured by the number of %GFP+ cells in a serum sample, for example, a blood sample or a bronchoalveolar lavage fluid sample. In another example, the level of viral RNA in a biological sample such a serum sample, for example, a blood sample or a bronchoalveolar lavage fluid sample can be measured via quantitative real-time PCR.

[0160] The one or more organic acids can be present in the pharmaceutical formulation at a dosage ranging from about 0.01 mg / kg to about 250 mg / kg. The dosage of the one or more organic acids is calculated using the sum of the weights of all organic acids in the pharmaceutical formulation. In some forms, the one or more organic acids is present in the pharmaceutical formulation at a dosage ranging from about 0.1 mg / kg to about 250 mg / kg body weight, from about 0.01 mg / kg to about 200 mg / kg body weight, from about 0.1 mg / kg to about 200 mg / kg body weight, from about 0.01 mg / kg to about 150 mg / kg body weight, from about 0.1 mg / kg to about 150 mg / kg body weight,

[0161] 45764921.1 24 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT from about 0.01 mg / kg to about 100 mg / kg body weight, from about 0.1 mg / kg to about 100 mg / kg body weight, from about 0.01 mg / kg to about 50 mg / kg body weight, from about 0. 1 mg / kg to about 50 mg / kg body weight, from about 0.01 mg / kg to about 10 mg / kg body weight, from about 0.1 mg / kg to about 10 mg / kg body weight, or from about 0.5 mg / kg to about 50 mg / kg body weight. For example, the one or more organic acids is present in the pharmaceutical formulation at a dosage ranging from about 0.2 mg / kg to about 100 mg / kg body weight.

[0162] D. Flavonoids

[0163] The pharmaceutical formulation disclosed herein may contain one or more flavonoids. In some forms, the pharmaceutical formulation disclosed herein can contain at least one flavonoid. In other forms, the pharmaceutical composition disclosed herein can contain a combination of two or more flavonoids. For example, the pharmaceutical formulation can contain a combination of two, three, four, or more flavonoids. The one or more flavonoids in the pharmaceutical formulation can be isoflavonoids, neoflavonoids, and / or bioflavonoids. Exemplary flavonoids that can be used in the pharmaceutical formulation include but are not limited to isoflavones, genistein, daidzein, and formononetin, and combinations thereof. For example, the pharmaceutical formulation can contain genistein, daidzein, and / or formononetin.

[0164] E. Additional Active Agents

[0165] In some forms, the pharmaceutical formulation further contains one or more active agent, in addition to the one or more metabolites, such as an anti-inflammatory agent, an antioxidant, antiviral agent, an antimalaria agent, or a biologic agent, or a combination thereof. The additional active agents are different from the metabolites in the pharmaceutical formulation. The one or more additional active agents can be formulated into the same pharmaceutical formulation containing one or more of the metabolites as a single dosage unit or as multiple dosage units for coordinated, combination, or concomitant administration, or into separate pharmaceutical compositions for combinational therapy.

[0166] Alternatively, the one or more additional active agents may be formulated as separate pharmaceutical compositions, for example, as a single dosage unit or as multiple dosage units, for co-administration with the pharmaceutical formulation containing one or more of the metabolites described herein.

[0167] Examples of anti-inflammatory agents that can be formulated in the pharmaceutical composition containing the disclosed metabolites or in a separate pharmaceutical composition include, but are not limited to, steroids, such as clobetasol, halobetasol, halcinonide, amcinonide, betamethasone, desoximetasone, diflucortolone, fluocinolone, fluocinonide, mometasone, clobetasone, desonide, hydrocortisone, prednicarbate, and triamcinolone, salts thereof, and

[0168] 45764921.1 25 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT combinations thereof; non-steroidal anti-inflammatory drugs, such as aceclofenac, aspirin, celecoxib, clonixin, dexibupafen, dexketoprofen, diclofenac, diflunisal, droxicam, etodolac, etoricoxib, fenoprofen, flufenamic acid, flurbiprofen, ibuprofen, indomethacin, isoxicam, ketoprofen, ketorolac, licofelone, lomoxicam, loxoprofen, lumiracoxib, meclofenamic acid, mefenamic acid, meloxicam, nabumetone, naproxen, nimesulide, oxaprozin, parecoxib, phenylbutazone, piroxicam, rofecoxib, salsalate, sulindac, tenoxicam, tolfenamic acid, tolmetin, and valdecoxib, and combinations thereof.

[0169] Examples of antioxidants that can be formulated in the pharmaceutical composition containing the disclosed metabolites or in a separate pharmaceutical composition include, but are not limited to, vitamin C, vitamin E, beta-carotene, carotenoids, and minerals, such as selenium and manganese, and combinations thereof.

[0170] Examples of antiviral agents that can be formulated in the pharmaceutical composition containing the disclosed metabolites or in a separate pharmaceutical composition include, but are not limited to, remdesivir (e.g., Veklury®), favipiravir (e.g., Avigan®), lopinavir / ritonavir (e.g., Kaletra®, Aluvia®), nitazoxanide (e.g., Alinia®), danoprevir (e.g., Ganovo®), ASC-09, umifenovir (e.g., Arbidol®), nafamostat, brequinar, AT-527, ABX464, merimepodib, molnupiravir, opaganib (e.g., Yeliva®), ivermectin (e.g., Soolantra®, Stromectol®, Skiice®), and hydroxychloroquine, and combinations thereof.

[0171] Examples of antimalaria agents that can be formulated in the pharmaceutical composition containing the disclosed metabolites or in a separate pharmaceutical composition include, but are not limited to, hydroxychloroquine and chloroquine.

[0172] Exemplary antibiotic agents that can be formulated in the pharmaceutical formulation containing the disclosed metabolites or in a separate pharmaceutical composition include, but are not limited to, Cephalosporins, Penicillin, Macrolides, quinolones, sulfonamides, and tetracyclines.

[0173] Exemplary respiratory tract agents that can be formulated in the pharmaceutical formulation containing the disclosed metabolites or in a separate pharmaceutical composition include, but are not limited to, antihistamines, antileukotrienes, bronchodilators, mast cell stabilizers, and pulmonary antihypertensives.

[0174] Exemplary gastrointestinal agents that can be formulated in the pharmaceutical formulation containing the disclosed metabolites or in a separate pharmaceutical composition include, but are not limited to, antispasmodics, irritable bowel syndrome agents, and proton pump inhibitors.

[0175] Exemplary blood glucose regulators that can be formulated in the pharmaceutical formulation containing the disclosed metabolites or in a separate pharmaceutical composition include, but are not limited to, antidiabetic agents such as alpha glucosidase inhibitors, biguanides,

[0176] 45764921.1 26 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT incretin mimetics, and meglitinides; insulins such as rapid- acting insulin, short acting insulin, insulin mixtures, and analogs thereof; and glycemic agents.

[0177] When formulated into a pharmaceutical composition that is separated from the pharmaceutical composition containing the disclosed metabolites, the one or more additional active agents may be formulated in various formulations, for example, injectable formulations, lyophilized formulations, liquid formulations, or oral formulations. The formulation can be selected based upon the suitable administration route.

[0178] F. Pharmaceutically Acceptable Carriers and Excipients

[0179] In some forms, the pharmaceutical formulation disclosed herein contains a pharmaceutically acceptable carrier, optionally more than one pharmaceutically acceptable carrier, depending on the form of the formulation.

[0180] Suitable pharmaceutically acceptable carriers and excipients are generally recognized as safe (GRAS), and may be administered to an individual without causing undesirable biological side effects or unwanted interactions.

[0181] Representative carriers and excipients include solvents (including buffers), diluents, pH modifying agents, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tonicity agents, and stabilizing agents, and a combination thereof.

[0182] Excipients can be added to a liquid or solid pharmaceutical composition (for in vivo or in vitro applications) to assist in sterility, stability (e.g. shelf-life), integration, and to adjust and / or maintain pH or isotonicity of the metabolites in the pharmaceutical composition, such as diluents, pH modifying agents, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tonicity agents, and stabilizing agents, and a combination thereof.

[0183] Metabolites for administering to a subject in need thereof, such as a mammal, can be dissolved or suspended in a suitable carrier to form a liquid pharmaceutical formulation, such as sterile saline, phosphate buffered saline (PBS), balanced salt solution (BSS), viscous gel, or other pharmaceutically acceptable carriers for administration. The pharmaceutical formulation may also be a sterile solution, suspension, or emulsion in a nontoxic, parenterally acceptable diluent or solvent.

[0184] G. Forms of Pharmaceutical Formulations

[0185] The disclosed metabolites can be formulated into a pharmaceutical formulation, in a liquid form or a solid form, for oral administration, mucosal administration (e.g. intranasal administration), or parenteral administration (e.g. intramuscular administration, intravenous administration, intraperitoneal administration, and subcutaneous administration) to a subject.

[0186] 45764921.1 27 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT

[0187] 1. Oral Formulations

[0188] The pharmaceutical formulation containing one or more metabolites may be provided in a form suitable for oral administration to a subject, such as a mammal (i.e., an oral composition). Oral administration may involve swallowing, so that the metabolites, optionally including additional active agent(s) in the pharmaceutical composition, enter the gastrointestinal tract, or buccal or sublingual administration may be employed by which the one or more metabolites enter the blood stream directly from the mouth.

[0189] Compositions suitable for oral administration include solid compositions such as tablets, capsules containing particulates, liquids, powders, lozenges (including liquid-filled lozenges), chews, multi- and nano-particulates, gels, solid solutions, liposomes, films, ovules, sprays, and liquid compositions.

[0190] Liquid compositions for oral administration include suspensions, solutions, syrups, and elixirs. Such oral compositions may be employed as fillers in soft or hard capsules and can contain one or more suitable carriers and / or excipients, for example, water, ethanol, polyethylene glycol, propylene glycol, chitosan polymers and chitosan derivatives (e.g. N-trimethylene chloride chitosan, chitosan esters, chitosan modified with hydrophilic groups, such as amino groups, carboxyl groups, sulfate groups, etc.), methylcellulose, a suitable oil, one or more emulsifying agents, and / or suspending agents. Liquid compositions for oral administration may also be prepared by the reconstitution of a solid, for example, from a sachet.

[0191] Optionally, the one or more metabolites are included in a fast-dissolving and / or fast-disintegrating dosage form.

[0192] For tablet or capsule dosage forms, in addition to the one or more metabolites described herein, tablets generally contain disintegrants, binders, diluents, surface active agents, lubricants, glidants, antioxidants, colourants, flavoring agents, preservatives, or taste masking agents, or a combination thereof.

[0193] Examples of suitable disintegrants for forming a table or capsule dosage form containing the one or more metabolites include, but are not limited to, sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinised starch and sodium alginate. Generally, the disintegrant can have a concentration in a range from about 1 wt% to about 25 wt%, from about 5 wt% to about 20 wt% of the tablet or capsule dosage form containing one or more metabolites.

[0194] Binders are generally used to impart cohesive qualities to a tablet composition containing one or more metabolites. Suitable binders for forming a tablet or capsule formulation containing the

[0195] 45764921.1 28 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT one or more metabolites include, but are not limited to, microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinised starch, chitosan polymers and chitosan derivatives (e.g. N- trimethylene chloride chitosan, chitosan esters, chitosan modified with hydrophilic groups, such as amino groups, carboxyl groups, sulfate groups, etc.), hydroxypropyl cellulose, and hydroxypropyl methylcellulose.

[0196] Suitable diluents for forming a table or capsule composition containing the one or more metabolites include, but are not limited to, lactose (as, for example, the monohydrate, spray-dried monohydrate or anhydrous form), chitosan polymers and chitosan derivatives (e.g. N-trimethylene chloride chitosan, chitosan esters, chitosan modified with hydrophilic groups, such as amino groups, carboxyl groups, sulfate groups, etc.), N-sulfonated derivatives of chitosan, quaternarized derivatives of chitosan, carbosyalkylated chitosan, microcrystalline chitosan, mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate.

[0197] Tablet or capsule composition containing the one or more metabolites may also contain surface active agents, such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc, in the coating. When present, surface active agents can have a concentration in a range from about 0.2 wt% to 5 wt% of the tablet or capsule formulation.

[0198] Tablet or capsule compositions containing the one or more metabolites also generally contain lubricants, such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulphate. Lubricants can have a concentration in a range from about 0.25 wt% to 10 wt%, from about 0.5 wt% to about 3 wt% of the tablet or capsule composition.

[0199] Other possible excipients included in a tablet or capsule formulation containing the one or more metabolites include glidants (e.g. Talc or colloidal anhydrous silica at about 0.1 wt% to about 3 wt% of the table or capsule formulation), antioxidants, colourants, flavouring agents, preservatives and taste-masking agents. When present, glidants can have a concentration in a range from about 0.2 wt% to 1 wt% of the tablet or capsule composition.

[0200] An exemplary tablet composition contains up to about 80 wt% of the one or more metabolites described herein, from about 10 wt% to about 90 wt% binder, from about 0 wt% to about 85 wt% diluent, from about 2 wt% to about 10 wt% disintegrant, and from about 0.25 wt% to about 10 wt % lubricant.

[0201] Tablet or capsule blends, including the one or more metabolites and one or more suitable excipients, may be compressed directly or by roller to form tablets. Tablet or capsule blends or portions of the blends may alternatively be wet-, dry-, or melt-granulated, melt congealed, or

[0202] 45764921.1 29 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT extruded before tableting. The final table or capsule composition may contain one or more layers and may be coated or uncoated; it may even be encapsulated in a particle, such as a polymeric particle or a liposomal particle.

[0203] Solid formulations containing the one or more metabolites for oral administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release formulations.

[0204] 2. Mucosal Formulations

[0205] The disclosed pharmaceutical formulation containing one or more metabolites can be formulated for pulmonary or mucosal administration. The administration can include delivery of the composition to the lungs, nasal, and oral (sublingual, buccal) mucosa. In some forms, the disclosed pharmaceutical formulation contains at least one liquid pharmaceutically acceptable carrier and is in a liquid form, such as an emulsion or a suspension. In some forms, the disclosed pharmaceutical formulation is a nasal formulation, for example a nasal spray.

[0206] For example, the pharmaceutical formulation containing one or more metabolites can also be administered intranasally or by oral inhalation, typically in the form of a dry powder (either alone, as a mixture, for example, in a dry blend with lactose, or as a mixed component particle, for example, mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurized container, pump, spray, atomizer (preferably an atomizer using electrohydrodynamics to produce a fine mist), or nebulizer, with or without the use of a suitable propellant, such as water, ethanol -water mixture, 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. For intranasal or oral inhalation use, the powder may contain a bioadhesive agent, for example, chitosan or cyclodextrin. The term “aerosol” as used herein refers to any preparation of a fine mist of particles, which can be in solution or a suspension, whether or not it is produced using a propellant. Aerosols can be produced using standard techniques, such as ultrasonication or high-pressure treatment.

[0207] The pressurized container, pump, spray, atomizer, or nebulizer contains a solution or suspension of a pharmaceutical formulation containing one or more metabolites including, for example, ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilizing, or extending release of the active, a propellant(s) as solvent and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.

[0208] Capsules (made, for example, from gelatin or hydroxypropylmethylcellulose), blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the pharmaceutical formulation containing one or more metabolites described herein, a suitable powder base such as lactose or starch and a performance modifier such as 1 -leucine, mannitol, or

[0209] 45764921.1 30 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT magnesium stearate. The lactose may be anhydrous or in the form of a monohydrate, preferably the latter. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose, and trehalose.

[0210] An exemplary solution formulation for use in an atomizer using electrohydrodynamics to produce a fine mist may contain from 1 pg to 10 mg total amount of the one or more metabolites per actuation and the actuation volume may vary from 1 pl to 100 pl. An exemplary formulation may contain a plurality of the one or more metabolites disclosed herein, propylene glycol, sterile water, ethanol and sodium chloride. Alternative solvents that may be used instead of propylene glycol include glycerol and polyethylene glycol.

[0211] Suitable flavors, such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, may be added to those formulations intended for inhaled / intranasal administration.

[0212] Formulations for inhaled / intranasal administration may be formulated to be immediate and / or modified release using for example, PGLA. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release formulations.

[0213] In the case of dry powder inhalers and aerosols, the dosage unit is determined by means of a valve which delivers a metered amount. Units in accordance with the pharmaceutical formulation are typically arranged to administer a metered dose or "puff". The overall daily dose will be administered in a single dose or, more usually, as divided doses throughout the day.

[0214] In some forms, the one or more metabolites can be formulated for pulmonary delivery, such as intranasal administration or oral inhalation. Carriers for pulmonary formulations can be divided into those for dry powder formulations and for administration as solutions. Aerosols for the delivery of therapeutic agents to the respiratory tract are known in the art. For administration via the upper respiratory tract, the metabolites can be formulated into an aqueous solution, e.g., water or isotonic saline, buffered or un-buffered, or as an aqueous suspension, for intranasal administration as drops or as a spray. Such aqueous solutions or suspensions may be isotonic relative to nasal secretions and of about the same pH, ranging e.g., from about pH 4.0 to about pH 7.4 or, from pH 6.0 to pH 7.0. Buffers should be physiologically compatible and include, simply by way of example, phosphate buffers. One skilled in the art can readily determine a suitable saline content and pH for an innocuous aqueous solution for nasal and / or upper respiratory administration.

[0215] In some forms, the aqueous solution is water, physiologically acceptable aqueous solutions containing salts and / or buffers, such as phosphate buffered saline (PBS), or any other aqueous solution acceptable for administration to an animal or human. Such solutions are well known to a person skilled in the art and include, but are not limited to, distilled water, de-ionized water, pure or

[0216] 45764921.1 31 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT ultrapure water, saline, phosphate-buffered saline (PBS). Other suitable aqueous vehicles include, but are not limited to, Ringer’s solution and isotonic sodium chloride. Aqueous suspensions may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

[0217] In some forms, solvents that are low toxicity organic (i.e. nonaqueous) class 3 residual solvents, such as ethanol, acetone, ethyl acetate, tetrahydrofuran, ethyl ether, and propanol may be used for the formulations. The solvent is selected based on its ability to readily aerosolize the formulation. The solvent should not detrimentally react with the metabolites. An appropriate solvent should be used that forms a suspension of the metabolites. The solvent should be sufficiently volatile to allow formation of an aerosol of the solution or suspension. Additional solvents or aerosolizing agents, such as freons, can be added as desired to increase the volatility of the solution or suspension.

[0218] In some forms, the pharmaceutical formulations may contain minor amounts of surfactants or other excipients well known to those of the art. In this context, “minor amounts” means no excipients are present that might affect or mediate penetration of the metabolites in tissues and that the excipients that are present in amount that do not adversely affect penetration of the metabolites in tissues.

[0219] In some forms, the pharmaceutical formulations containing one or more of the metabolites can be administered directly to the mucous membranes (including the surface membranes of the nose, lungs and mouth), such that metabolites cross the mucosal layer and enters the underlying tissues.

[0220] Such formulations for direct application on the mucous membrane generally contain a dermatologically acceptable carrier that is suitable for application to the mucous membrane, has good aesthetic properties, is compatible with the active agents and any other components, and will not cause any untoward safety or toxicity concerns.

[0221] The carrier can be in a wide variety of forms. For example, emulsion carriers, including, but not limited to, oil-in-water, water-in-oil, water-in-oil-in-water, and oil-in-water-in-silicone emulsions, are useful herein. These emulsions can cover a broad range of viscosities, e.g., from about 100 cps to about 200,000 cps. These emulsions can also be delivered in the form of sprays using either mechanical pump containers or pressurized aerosol containers using conventional propellants. These carriers can also be delivered in the form of a mousse or a transdermal patch.

[0222] Formulations for direct application on the mucous membrane may be formulated to be immediate and / or modified release. Modified release formulations include delayed, sustained,

[0223] 45764921.1 32 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT pulsed, controlled, targeted and programmed release formulations. Thus, the metabolites may be formulated as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot providing modified release of the active agents. Examples of such formulations include drug-coated stents.

[0224] Buffered solutions also affect the tonicity of the nasal formulations. For example, the total concentration of salts and / or buffering agents in the buffered solution is adjusted so that the nasal formulation is isotonic or hypertonic. The “total concentration of salts and / or buffering agents” refers to the total number of mole of the salts and / or buffering agents in the aqueous solution of the pharmaceutical formulation divided by the total volume of the aqueous solution of the pharmaceutical formulation. “Isotonic” refers to an osmotic pressure that is the same as the fluid present in the nasal membranes. “Hypertonic” refers to an osmotic pressure that is higher than the fluid present in the nasal membranes.

[0225] 3. Parenteral Formulations

[0226] Optionally, the pharmaceutical composition containing one or more of the metabolites is in a form suitable for administration directly into the blood stream, into muscle, or into an internal organ. Suitable routes for such parenteral administration include intravenous, intraperitoneal, intrathecal, intrasternal, intramuscular, and subcutaneous delivery. Suitable means for parenteral administration include needle (including microneedle) injectors, needle-free injectors, and infusion techniques.

[0227] For example, the pharmaceutical formulation containing one or more of the metabolites is in a form suitable for intramuscular administration, intravenous administration, intraperitoneal administration, or subcutaneous administration, or a combination thereof.

[0228] Parenteral formulations containing the metabolites described herein are typically aqueous solutions which can contain excipients such as salts, carbohydrates and buffering agents (e.g., from about pH 6.5 to about pH 8.0, from about pH 6.5 to about pH 7.4, from about pH 6.5 to about pH 7.0, from about pH 7.0 to pH 8.0, or from about pH 7.0 to about pH 7.4), but, for some applications, they may be more suitably formulated as a sterile aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.

[0229] The liquid compositions containing the metabolites for parenteral administration may be a solution, a suspension, or an emulsion.

[0230] The liquid pharmaceutically acceptable carrier forming the parenteral composition containing the metabolites can include one or more physiologically compatible buffers, such as a phosphate buffers. One skilled in the art can readily determine a suitable saline content and pH for an aqueous carrier for administration (e.g., from about pH 6.5 to about pH 8.0, from about pH 6.5 to

[0231] 45764921.1 33 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT about pH 7.4, from about pH 6.5 to about pH 7.0, from about pH 7.0 to pH 8.0, or from about pH 7.0 to about pH 7.4).

[0232] Liquid compositions containing the metabolites for parenteral administration may include one or more suspending agents, such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone, gum tragacanth, or lecithin. The liquid compositions may also include one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate.

[0233] Optionally, the liquid composition containing the metabolites contains one or more solvents that are low toxicity organic (i.e., nonaqueous) class 3 residual solvents, such as ethanol, acetone, ethyl acetate, tetrahydofuran, ethyl ether, and propanol, and a combination thereof. Any such solvents included in the liquid formulation should not detrimentally react with the metabolites or any additional active agents in the liquid composition. Solvents such as freon, alcohol, glycol, polyglycol, or fatty acid, can also be included in the liquid composition containing the metabolites as desired to increase the volatility of the solution or suspension.

[0234] Liquid compositions containing the metabolites for parenteral administration may also contain minor amounts of polymers, surfactants, or other pharmaceutically acceptable excipients known to those in the art. In this context, "minor amounts" means an amount that is sufficiently small to avoid adversely affecting uptake of the metabolites by the targeted cells, such as pituitary gonadotrophs.

[0235] The preparation of parenteral compositions containing the metabolites is typically under sterile conditions, for example, by lyophilization, which can be accomplished using standard pharmaceutical techniques known to those skilled in the art.

[0236] Compositions for parenteral administration containing the metabolites may be formulated to provide immediate and / or modified release of the active agent. Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release formulations.

[0237] H. Forms of Nutraceutical Formulations

[0238] The disclosed metabolite(s), e.g., taurine and sinapic acid, can be formulated as dietary supplements (also referred to as “nutraceuticals”). In some forms, the supplements can include a combination of two or more metabolites, e.g., taurine, sinapic acid, and genistein, each provided in an amount such as those described above.

[0239] Other nutraceuticals agents may also be included in the dietary supplement. Nutraceutical agents are natural, bioactive chemical compounds that have health promoting, disease preventing or medicinal properties. Examples of nutraceutical agents include, but are not limited to, Allium cepa, Allium sativum, Aloe vera, Angelica Species, Naturally Occurring Antioxidants, Aspergillus oryzae, barley grass, Bromelain, Carnitine, carotenoids and flavonoids, Catechin, Centella asiatica (Gotu

[0240] 45764921.1 34 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT kola), Coenzyme Q10, Chinese Prepared Medicines, Coleus forskohlii, Commiphora mukul, Conjugated Linoleic Acids (CLAs), Crataegus oxyacantha (Hawthorne), Curcuma longa (Turmeric), Echinacea Species (Purple Coneflower), Eleutherococcus senticosus (Siberian Ginseng), Ephedra Species, Dietary Fish Oil, Genistein, Ginkgo biloba, Glycyrrhiza (Licorice), Hypericum perforatum (St. John's Wort), Hydrastis (Goldenseal) and other Berberine-con taining plants, Lactobacillus, Lobelia (Indian Tobacco), Melaleuca allernifolia, Menaquinone, Mentha piperita, n-glycolylneuraminic acid (NGNA), Panax Ginseng, Pancreatic Enzymes, Piper mythisticum, Procyanidolic Oligomers, Pygeum africanum, Quercetin, Sarsaparilla species, Serenoa repens (Saw palmetto, Sabal serrulata), Silybum marianum (Milk Thistle), Rosemary / Lemon balm, Selenite, Tabebuia avellanedae (LaPacho), Taraxacum officinale, Tanacetum parthenium (Feverfew), Taxol, Uva ursi (Bearberry), Vaccinium myrtillus (Blueberry), Valerian officinalis, Viscum album (Mistletoe), Vitamin A, Beta-Carotene and other carotenoids, and Zingiber officinale (Ginger).

[0241] Several nutraceutical agents are used in treating viral disorders (e.g., Genistein (in soy / red clover), rosemary / lemon balm, selenite, barley grass, lauric acid, Phyllanthus amarus / niruri (see, e.g., Nicolson, G. (1998) J. Medicine 1:123-128; herein incorporated by reference in its entirety). Additional nutraceutical agents with anti-viral effects include, but are not limited to, catechins, flavonoids, especially luteolin, Echinacea, cascara, and n-glycolylneuraminic acid (NGNA). Optionally, NGNA is provided from sea cucumbers, e.g., an extract of sea cucumbers, or is prepared from chitin. In some forms, NGNA is prepared as described in WO 00 / 38967, incorporated by reference herein its entirety. For example, N-glycolylneuraminic acid can be purchased commercially from, for example, Sigma Chemical Company, St. Louis, Mo. N- glycolylneuraminic acid also can be synthesized. For example, CMP-N-acetylneuraminic acid hydroxylase can be used to synthesize N-glycolylneurarninic acid as its CMP-glycoside. See, Schlenzka et al., Glycobiolog, 1994, 4(5) :675-683. Non-enzymatic methods of synthesis include, for example, synthesis from N-acetylneuraminic acid using methanol or hydrochloric acid and benzylalcohol. Other synthesis methods are described in Choi et al., J. Org. Chem., 1996, 61:8 / 39 (from mannosamine), Faillard et al., J. Physiol. Chem.' 1965, 344: 167 (from glucosamine), U.S. Pat. Nos. 4,774,326 and 4,774,327, both of which are incorporated by reference herein in their entirety.

[0242] Dietary supplements containing the metabolites can contain, for example, a daily dosage of between 0.2 g and 20.0 g of the metabolites, e.g., taurine and sinapic acid. Furthermore, the dietary supplement can be provided in an amount sufficient to induce the physiological response desired, e.g., to reduce or alleviate cold symptoms, prevent the onset of colds, increase the energy of

[0243] 45764921.1 35 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT subjects, and / or increase the feeling of well-being of subjects. In some forms, the compositions are provided for use in inducing one of the foregoing responses, while in other forms, the compositions are provided for use in inducing two or more of the foregoing responses.

[0244] Dietary supplements containing the disclosed metabolites, e.g., taurine and sinapic acid, can be administered in any suitable form, e.g., pill, and food product (e.g., a beverage, bar, powder, or shake).

[0245] In some forms, the dietary supplements are in the form of a pill, tablet or capsule. The ingredients of the dietary supplement are contained in acceptable excipients and / or carriers for oral consumption. The carrier can be a liquid, gel, gelcap, capsule, powder, solid tablet (coated or noncoated), tea, or the like. For example, the dietary supplement can be in the form of a capsule such as a hard gelatin capsule. Suitable excipient and / or carriers include maltodextrin, calcium carbonate, dicalcium phosphate, tricalcium phosphate, microcrystalline cellulose, dextrose, rice flour, magnesium stearate, stearic acid, croscarmellose sodium, sodium starch glycolate, crospovidone, sucrose, vegetable gums, lactose, methylcellulose, povidone, carboxymethylcellulose, corn starch, and the like (including mixtures thereof). For example, the carrier includes calcium carbonate, magnesium stearate, maltodextrin, and mixtures thereof. The various ingredients and the excipient and / or carrier are mixed and formed into the desired form using conventional techniques. The tablet or capsule may be coated with an enteric coating that dissolves at a pH of about 6.0 to 7.0. A suitable enteric coating that dissolves in the small intestine but not in the stomach is cellulose acetate phthalate. Further details on techniques for formulation for and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

[0246] In other forms, the dietary supplement is provided as a powder or liquid suitable for adding by the consumer to a food or beverage. For example, in some forms, the dietary supplement can be administered to an individual in the form of a powder, for instance to be used by mixing into a beverage, or by stirring into a semi-solid food such as a pudding, topping, sauce, puree, cooked cereal, or salad dressing, for instance, or by otherwise adding to a food.

[0247] In some forms, the disclosed metabolites e.g., taurine and sinapic acid can be formulated into food products such as beverages or energy bars e.g., meal replacement bars. In these forms, the dietary supplements can optionally serve as meal or snack replacement and generally provide nutrient calories. These forms can include, but are not limited to, beverages e.g., soft drinks, milk and other dairy drinks, and diet drinks, baked goods, puddings, dairy products, confections, snack foods, frozen confections or novelties e.g., ice cream, milk shakes, prepared frozen meals, candy, snack products e.g., chips, soups, spreads, sauces, salad dressings, prepared meat products, cheese, yogurt and any other fat or oil containing foods, and food ingredients (e.g., wheat flour). In one

[0248] 45764921.1 36 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT form, the disclosed metabolites are formulated in yoghurt. Servings of the food product can contain between about 20 mg and about 20.0 g of the metabolites.

[0249] Dietary supplements in the form of food products e.g., energy bars can be formulated to provide carbohydrates, proteins, and fats in balanced amounts. Sources of protein to be incorporated into the dietary supplement can be any suitable protein utilized in food products and can include whey protein, whey protein concentrate, whey powder, egg, soy flour, soymilk soy protein, soy protein isolate, caseinate (e.g., sodium caseinate, sodium calcium caseinate, calcium caseinate, potassium caseinate), animal and vegetable protein and mixtures thereof. For example, the protein is a combination of whey protein concentrate and calcium caseinate.

[0250] The dietary supplement can also contain other ingredients, such as one or a combination of other vitamins, minerals, antioxidants, fiber and other dietary supplements (e.g., protein, amino acids, choline, lecithin, omega-3 fatty acids). Selection of one or several of these ingredients is a matter of formulation, design, and consumer preference. The amounts of these ingredients added to the dietary supplements are readily known to the skilled artisan. Guidance to such amounts can be provided by the U.S. RDA doses for children and adults. Further vitamins and minerals that can be added include, but are not limited to, calcium phosphate or acetate, tribasic; potassium phosphate, dibasic; magnesium sulfate or oxide; salt (sodium chloride); potassium chloride or acetate; ascorbic acid; ferric orthophosphate; niacinamide; zinc sulfate or oxide; calcium pantothenate; copper gluconate; riboflavin; beta-carotene; pyridoxine hydrochloride; thiamin mononitrate; folic acid; biotin; chromium chloride or picolonate; potassium iodide; sodium selenate; sodium molybdate; phylloquinone; vitamin D3; cyanocobalamin; sodium selenite; copper sulfate; vitamin A; vitamin C; inositol; potassium iodide.

[0251] Flavors, coloring agents, spices, nuts and the like can be incorporated into the product. Flavorings can be in the form of flavored extracts, volatile oils, chocolate flavorings, peanut butter flavoring, cookie crumbs, crisp rice, vanilla or any commercially available flavoring. Examples of useful flavoring include, but are not limited to, pure anise extract, imitation banana extract, imitation cherry extract, chocolate extract, pure lemon extract, pure orange extract, pure peppermint extract, imitation pineapple extract, imitation rum extract, imitation strawberry extract, or pure vanilla extract; or volatile oils, such as balm oil, bay oil, bergamot oil, cedarwood oil, walnut oil, cherry oil, cinnamon oil, clove oil, or peppermint oil; peanut butter, chocolate flavoring, vanilla cookie crumb, butterscotch or toffee. In one form, the dietary supplement contains cocoa or chocolate.

[0252] Emulsifiers can be added for stability of the final product. Examples of suitable emulsifiers include, but are not limited to, lecithin (e.g., from egg or soy), and / or mono- and di-glycerides.

[0253] 45764921.1 37 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT

[0254] Other emulsifiers are readily apparent to the skilled artisan and selection of suitable emulsifier(s) will depend, in part, upon the formulation and final product.

[0255] Preservatives can also be added to the nutritional supplement to extend product shelf life. Preferably, preservatives such as potassium sorbate, sodium sorbate, potassium benzoate, sodium benzoate or calcium disodium EDTA are used.

[0256] III. METHODS OF USE

[0257] The disclosed pharmaceutical formulations are particularly useful for treating respiratory diseases and disorders. In some forms, the respiratory disorder can be a respiratory infection caused by a respiratory virus, for example a coronavirus such as SARS-CoV-2, and RSV.

[0258] It will be appreciated the disclosed methods can be methods of treatment of the symptoms and conditions described herein. “Treatment” refers to the medical management of a patient with the intent to cure, ameliorate, or stabilize, a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. An effective amount or therapeutically effective amount means a dosage and / or other element (e.g., amount of time) sufficient to treat, inhibit, or alleviate one or more symptoms of a disease state being treated or to otherwise provide a desired pharmacologic and / or physiologic effect. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being administered.

[0259] A pharmaceutical or nutraceutical composition containing the metabolites is suitable for preventing the increase in viral or bacterial replication associated with a respiratory disease / disorder in a subject. In some forms, the metabolites are present in the pharmaceutical or nutraceutical composition in an amount effective to reduce or limit the increase in viral titer in blood and / or bronchoalveolar lavage fluid (BALF) by about 5% to about 70% within 48 hours of administration, relative to a control. For example, the metabolites are present in the pharmaceutical or nutraceutical composition in an amount effective to reduce the viral or bacterial titer in blood and / or BALF by about 35%, thereby limiting the spread and severity of the viral or bacterial infection and preventing

[0260] 45764921.1 38 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT further exacerbation of symptoms. In some forms, the metabolites in the pharmaceutical or nutraceutical composition are present in an amount effective to prevent the increase in viral or bacterial titer when administered to the subject prior to infection (e.g., within about 24 hours to about 7 days), thereby limiting early viral or bacterial replication in the subject.

[0261] In some forms, the control is a reference point for the average viral or bacterial replication levels observed in the population without treatment, serving as a baseline for comparing the effect of the composition on reducing viral or bacterial replication. In other forms, the control is a subject or group that receives no treatment or receives a placebo, thereby allowing for a comparison of the effects of the composition on viral or bacterial replication under similar conditions.

[0262] The method generally includes administering the pharmaceutical or nutraceutical composition to a subject in need thereof. The pharmaceutical or nutraceutical composition can be administered by any suitable routes, such as by mucosal administration (e.g., intranasal administration), oral administration, intramuscular administration, intravenous administration, intraperitoneal administration, or subcutaneous administration, or a combination thereof. In some forms, the pharmaceutical formulation is administered intranasally or orally.

[0263] The administration step can be performed once or repeated one or more times, at a regular time interval or irregularly. For example, the administration step is repeated once, twice, or three times, per day, for a period of one day, three days, one week, two weeks, one month, three months, six months, or one year.

[0264] In some forms, the method further includes administering one or more additional active agents prior to, during, and / or subsequent to step (i), the administration step. The active agent can be any one of those described above, such as an antiviral agent or anti-inflammatory agent, or a combination thereof.

[0265] Also provided are methods of treating a respiratory disease or disorder (e.g., a respiratory infection, such as influenza, respiratory syncytial virus, coronavirus infection, or bacterial pneumonia) by administering alveolar macrophages (AMs) trained by the pharmaceutical formulations containing the metabolites to a subject in need thereof. For example, the method includes incubating the AMs with the pharmaceutical formulation containing the metabolites for a suitable period of time under suitable conditions to induce a trained or conditioned phenotype of the AMs. The trained or conditioned phenotype of the AMs are characterized by one or more of the following properties: an enhanced phagocytic activity, increased intracellular bacterial killing, increased production of reactive oxygen species, and altered chromatin accessibility, which promotes heightened responsiveness to respiratory pathogens. The incubation can be performed ex vivo in culture medium supplemented with one or more metabolites (e.g., taurine, genistein, and / or

[0266] 45764921.1 39 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT sinapic acid), serum fractions, cytokines, or other immunomodulatory agents that modulate chromatin accessibility, enhance intracellular bacterial killing, or augment reactive oxygen species production.

[0267] Following incubation of the AMs with the pharmaceutical formulation, the trained or conditioned phenotype of the AMs are administered (e.g., via transplantation) into the subject. For example, the trained or conditioned phenotype of AMs can be washed, concentrated, and formulated with a pharmaceutically acceptable carrier suitable for transplantation into a recipient subject. In some forms, the trained or conditioned phenotype of AMs is administered directly to the lung, for example via intranasal, intratracheal, or aerosolized delivery, to repopulate or supplement the endogenous alveolar macrophage compartment, and thereby reduce the viral or bacterial titer in the blood and / or bronchoalveolar fluid (BALF) of the subject. In some forms, the treated AMs are introduced systemically, such as by intravenous infusion, to traffic to the pulmonary environment.

[0268] In some forms, the methods include both autologous transplantation, in which a subject’s own AMs are collected, trained or conditioned with the pharmaceutical formulation containing the metabolites, and re- administered to the subject. In some forms, the methods include allogeneic transplantation, in which AMs derived from a donor source are similarly trained or conditioned, and then administered to a subject in need thereof. The methods are applicable to treatment or prophylaxis of infectious and inflammatory respiratory conditions, including but not limited to viral infections, bacterial pneumonias, and acute respiratory distress syndrome (ARDS).

[0269] A. Diseases / Disorders to be Treated

[0270] The pharmaceutical formulations described herein are particularly suitable for treating a respiratory disease or disorder in a subject in need thereof. In some forms, the respiratory disorder is a respiratory infection, such as a viral infection (e.g., influenza, coronavirus, respiratory syncytial virus (RSV), and methicillin-resistant Staphylococcus aureus (MRSA)). In some forms, the respiratory disorder is a genetic disorder, for example, cystic fibrosis. In some forms, the pharmaceutical formulations are suitable for administering to treat a disease or condition associated with a respiratory infection e.g., pneumonia, bronchitis, acute respiratory distress syndrome (ARDS), and / or post-infectious inflammatory complications.

[0271] In some forms, the disclosed pharmaceutical formulations containing one or more metabolites can be used to treat a respiratory infection in a subject. The respiratory infection can be caused by a microorganism, for example, a virus, a bacterium, a protozoan, and a fungus.

[0272] In some forms, the respiratory infection is caused by a virus. Exemplary respiratory viruses include, but are not limited to, orthomyxovirus, rhinovirus, paramyxovirus, coronavirus, adenovirus, human metapneumovirus (hMPV), enterovirus, and bocavirus. Exemplary

[0273] 45764921.1 40 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT orthomyxoviruses include but are not limited to influenza A, influenza B, and influenza C, and variants thereof. Exemplary paramyxoviruses include but are not limited to Respiratory syncytial virus (RSV) and parainfluenza virus, and variants thereof. Exemplary coronaviruses include but are not limited to SARS-CoV, MERS-CoV, and SARS-CoV-2 (COVID-19), and variants thereof.

[0274] In some forms, the disclosed pharmaceutical formulations containing one or more metabolites can be used to treat a respiratory disease or disorder in a subject. Exemplary respiratory diseases include but are not limited to cystic fibrosis, chronic obstructive pulmonary disease, pulmonary fibrosis, pneumonia, emphysema, acute respiratory distress syndrome, and asthma.

[0275] In these forms, the method generally includes administering the pharmaceutical formulation to a subject in need thereof. The pharmaceutical formulation can be administered by any suitable routes, such as by mucosal administration (e.g., intranasal administration), oral administration, intramuscular administration, intravenous administration, intraperitoneal administration, or subcutaneous administration, or a combination thereof.

[0276] Also disclosed are methods of preventing a secondary infection following a primary disease or disorder (such as an infection caused by a virus or bacteria, diabetes, etc.) in a subject. In some forms, the method includes (i) administering to the subject, a pharmaceutical composition containing one or more metabolites, optionally wherein the administration step is repeated one or more times. In other forms, the method includes administering a pharmaceutical formulation containing one or more segmented filamentous bacteria (SFB) to the subject, optionally wherein the administration step is repeated one or more times. The pharmaceutical formulation can be administered by any suitable routes, such as by mucosal administration (e.g., intranasal administration), oral administration, intramuscular administration, intravenous administration, intraperitoneal administration, or subcutaneous administration, or a combination thereof.

[0277] In some forms, the pharmaceutical formulation is administered after the subject shows one or more symptoms of the primary disease or disorder, optionally within a week, 3 days, 1 day, 12 hours, 5 hours, 2 hours, or 1 hour after the onset of the one or more symptoms of the primary disease or disorder.

[0278] In some forms, the primary disease or disorder is caused by a virus, bacteria, or ailment (e.g., diabetes or cystic fibrosis). In some forms, the secondary infection is independently caused by a virus or bacteria, optionally wherein the virus or bacteria is Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae.

[0279] The administration step can be performed once or repeated one or more times. For example, the administration step is repeated once, twice, or three times, per day, for a period of one day, three days, one week, two weeks, one month, three months, six months, or one year. For example, the

[0280] 45764921.1 41 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT pharmaceutical formulation can be administered once every week, once every two weeks, once a month, once every two months, once every three months, once every four months, once every five months, or once every six months, for a time period from 1 day to 1 year, from 1 day to 9 months, from 1 day to 6 months, from 1 day to four weeks, from 1 day to three weeks, from 1 day to two weeks, from 1 day to 1 week, or from 1 day to 3 days. In some forms, the pharmaceutical formulation is administered irregularly for at least two time, at least three times, at least four times, at least five times, at least ten times, at least 20 times, or at least 30 times, within a time period of one week, one month, six months, 1 year, 2 years, 3 years, or 5 years.

[0281] Typically, following the administration or all of the administrations of the disclosed pharmaceutical formulation, an effective amount of the metabolites is administered to the subject to prevent or reduce symptoms associated with a respiratory disease or disorder caused by the virus, such as RSV or SARS-CoV-2. For example, following the administration or all of the administrations of the disclosed pharmaceutical formulation, the subject shows no symptom or less symptoms associated with a disease or disorder caused by the respiratory disease or disorder, such as headache, persistent loss of smell, persistent loss of taste, memory loss, brain fog (difficulty concentrating, sense of confusion or disorientation), dizziness, anxiety, depression, earache, hearing loss, ringing in ears (tinnitus), fatigue, or gastrointestinal issues, or a combination thereof, compared to a control. The control is a subject administered with the pharmaceutical formulation without the metabolites, such as a normal saline nasal formulation. In some forms, the control is the subject being administered the pharmaceutical formulation containing the metabolites, prior to the administration of the pharmaceutical formulation containing the metabolites.

[0282] B. Dosages and Effective Amounts

[0283] Pharmaceutical formulations typically contain an effective amount of metabolites and one or more pharmaceutically acceptable carriers and / or excipients. As used herein, the term “effective amount” means any amount of the metabolites that is sufficient to achieve the desired therapeutic, prophylactic, and / or diagnostic effect on a biological sample or in a subject to which it is administered. Depending on the condition to be treated and / or the route of administration, such an effective amount of the metabolites can be from about 0.01 mg / kg to about 250 mg / kg body weight, from about 0.1 mg / kg to about 250 mg / kg body weight, from about 0.01 mg / kg to about 200 mg / kg body weight, from about 0.1 mg / kg to about 200 mg / kg body weight, from about 0.01 mg / kg to about 150 mg / kg body weight, from about 0.1 mg / kg to about 150 mg / kg body weight, from about 0.01 mg / kg to about 100 mg / kg body weight, from about 0.1 mg / kg to about 100 mg / kg body weight, from about 0.01 mg / kg to about 50 mg / kg body weight, from about 0.1 mg / kg to about 50 mg / kg body weight, from about 0.01 mg / kg to about 10 mg / kg body weight, from about 0.1 mg / kg

[0284] 45764921.1 42 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT to about 10 mg / kg body weight, or from about 0.5 mg / kg to about 50 mg / kg body weight, of the subject for each administration.

[0285] For example, the effective amount of the metabolites is from about 0. 1 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 110 mg / kg, about 120 mg / kg, about 130 mg / kg, about 140 mg / kg, about 150 mg / kg, about 160 mg / kg, about 170 mg / kg, about 180 mg / kg, about 190 mg / kg, about 200 mg / kg, about 210 mg / kg, about 220 mg / kg, about 230 mg / kg, about 240 mg / kg, or about 250 mg / kg, of the subject per day, for each administration. The amount of the metabolites administered, the route of administration, and the further treatment regimen can be determined by the treating clinician or testing technologies, depending on factors such as the age, gender and general condition of the subject, the nature and severity of the disease / symptoms being prevented, treated, or diagnosed, and / or the samples being tested.

[0286] In some forms, the pharmaceutical composition is in a unit dosage form, and can be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or in any other suitable singledose or multi-dose holder or container (which can be properly labeled); optionally with one or more leaflets containing product information and / or instructions for use. Generally, such unit dosages can contain between 0.1 and 1000 mg, and usually between 0.1 and 500 mg, between 5 and 500 mg, between 0.1 and 100 mg, or between 1 and 100 mg, of the disclosed metabolites, e.g., about 0.1, 1, 5, 10, 25, 50, 100, 200, 300 or 400 mg per unit dosage.

[0287] The effect of the pharmaceutical formulations can be compared to a control. Suitable controls are known in the art and include, for example, an untreated subject, or a placebo-treated subject. Typical control is a comparison of a condition or symptom of a subject prior to and after administration of the pharmaceutical formulation. The condition or symptom can be a biochemical, molecular, physiological, or pathological readout. For example, the effect of the formulation on a particular symptom, pharmacologic, or physiologic indicator can be compared to an untreated subject, or the condition of the subject prior to treatment. In some forms, the symptom, pharmacologic, or physiologic indicator is measured in a subject prior to treatment, and again one or more times after treatment is initiated. In some forms, the control is a reference level, or average determined based on measuring the symptom, pharmacologic, or physiologic indicator in one or more subjects that do not have the disease or condition to be treated (e.g., healthy subjects). In some forms, the effect of the treatment is compared to a conventional treatment that is known the art. Suitable control subjects are unvaccinated subjects, or subjects receiving the same amount of a therapeutic, prophylactic and / or diagnostic agent in the absence of pharmaceutical formulations including one or more metabolites.

[0288] 45764921.1 43 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT

[0289] In some forms, following the administration or optionally all of the administrations (when more than one administration is performed), the total amount of the metabolites administered to the subject is effective to reduce cell death of alveolar macrophages by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, as indicated by immunohistochemistry of a biological sample of the subject, such as a serum sample, a blood sample, or a bronchoalveolar lavage fluid sample, compared to a control sample of the subject. A “control sample” is referred to a sample of the same type obtained from the subject before administration of the pharmaceutical formulation. For example, the treatment effect is evaluated using a bronchoalveolar lavage fluid sample obtained from the subject before the administration or the first administration of the pharmaceutical formulation and after the administration or all of the administration of the pharmaceutical formulation, where the bronchoalveolar lavage fluid sample obtained before the administration or the first administration is the control sample.

[0290] For example, human alveolar macrophage-like (AML) cells can be extracted from peripheral blood mononuclear cells or purified monocytes. This process involves isolating cells, incubating them in a cocktail of pulmonary surfactant and lung-associated cytokines, and then analyzing their phenotype using immunohistochemical staining. For example, alveolar macrophages can be measured in bronchoalveolar lavage (BAL) fluid, which can be obtained by inserting a bronchoscope into the airways and washing the lungs with a sterile saline solution. The fluid sample can then be suctioned back and analyzed for cells and other components.

[0291] In some forms, following the administration or optionally all of the administrations (when more than one administration is performed), the total amount of the metabolites administered to the subject is effective to increase C1QA expression and / or reduce Notch4 expression by 5-fold, 10- fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, or 80-fold, as indicated by quantitative real-time PCR of a biological sample of the subject, such as a serum sample, a blood sample, or a bronchoalveolar lavage fluid sample, compared to a control sample of the subject.

[0292] In some forms, following the administration or optionally all of the administrations (when more than one administration is performed), the total amount of the metabolites administered to the subject is effective to reduce viral-induced expression of IFNP and / or IL-6 by 5-fold, 10-fold, 15- fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, or 80-fold, as indicated by quantitative real-time PCR of a biological sample of the subject, such as a serum sample, a blood sample, or a bronchoalveolar lavage fluid sample, compared to a control sample of the subject.

[0293] In some forms, following the administration or optionally all of the administrations (when more than one administration is performed), the total amount of the metabolites administered to the subject is effective to reduce viral infectivity of a biological sample of the subject, such as a serum

[0294] 45764921.1 44 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT sample, a blood sample, or a bronchoalveolar lavage fluid sample, by >30%, >40%, >50%, >60%, 70%, >80%, >85%, >90%, >95%, >98%, >99%, >99.9%, or >99.9999%, compared to a control sample of the subject, e.g., a biological sample of the same type obtained from the subject before administration of the pharmaceutical formulation. In some forms, following administration or optionally all of the administrations (when more than one administration is performed), the total amount of the metabolites administered to the subject is effective to reduce viral infectivity by at least 2 logic, at least 3 logic, at least 4 logic, at least 5 logic, or at least 6 logic, as indicated in biological sample of the subject, such as a serum sample, a blood sample, or a bronchoalveolar lavage fluid sample, compared to a control sample of the subject, e.g., a biological sample of the same type obtained from the subject before administration of the pharmaceutical formulation.

[0295] In some forms, following the administration or optionally all of the administrations (when more than one administration is performed), the total amount of the metabolites administered to the subject is effective to increase gut microbiota of the subject, as indicated by an increase in the number of segmented filamentous bacteria measured in a biological sample of the subject, compared to a control sample of the subject prior to the administration of the pharmaceutical formulation. In some forms, the biological sample is a fecal sample. In some forms, the biological sample is a serum sample, a blood sample, or a bronchoalveolar lavage fluid sample.

[0296] In some forms, following the administration, or optionally all of the administrations, the total amount of the SFB administered to the subject is effective to prevent a secondary infection by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, as indicated by quantitative realtime PCR of a serum sample, optionally wherein the serum sample is a blood sample or a bronchoalveolar lavage fluid sample of the subject, compared to a control (e.g., a subject having the same primary disease / disorder but not administered with the pharmaceutical formulation).

[0297] In some forms, following the administration or optionally all of the administrations (when more than one administration is performed), the total amount of the SFB administered to the subject is effective to prevent a secondary infection in the subject, as indicated by the absence of symptoms associated with the secondary infections, compared to a control (e.g., a subject that is infected with the same primary disease / disorder, but not administered with the pharmaceutical formulation).

[0298] C. Subjects

[0299] A subject in need of treatment is typically a subject having a respiratory disease or disorder, for example a subject having a respiratory viral infection such as a coronavirus infection or an RSV infection. Exemplary diseases associated with respiratory infections include but are not limited to pneumonia, bronchitis, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), multisystem inflammatory syndrome in children (MIS-C), and / or multisystem inflammatory

[0300] 45764921.1 45 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT syndrome in adults (MIS-A). In some forms, the subject is a mammal, including, but not limited to, murines, simians, humans, mammalian farm animals and livestock, mammalian sport animals, and mammalian pets. Preferably, the subject is human.

[0301] A subject having a respiratory infection includes a subject that has been exposed to a respiratory virus and has acute or chronic detectable levels of the respiratory virus in his / her body or has signs and symptoms associated with infection of the virus. Methods of assessing and detecting respiratory infections in a subject are known by those of ordinary skill in the art. A subject at risk of having a respiratory infection is a subject that may be expected to come in contact with a virus as described above. Examples of such subjects are medical workers or those traveling to parts of the world where the incidence of infection is high. In some forms, the subject is at an elevated risk of an infection because the subject has one or more risk factors to have an infection. Examples of risk factors to be infected and / or develop mild, moderate, and / or severe symptoms include immunosuppression, immunocompromised, age (advanced or very young), and surgery. The degree of risk of infection depends on the multitude and the severity or the magnitude of the risk factors that the subject has. Risk charts and prediction algorithms are available for assessing the risk of an infection in a subject based on the presence and severity of risk factors. Other methods of assessing the risk of infection in a subject are known by those of ordinary skill in the art. In some forms, the subject who is at an elevated risk of an infection may be an apparently healthy subject. An apparently healthy subject is a subject who has no signs or symptoms of disease.

[0302] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0303] The disclosed compositions and methods can be further understood through the following numbered paragraphs.

[0304] Paragraph 1. A composition for treating one or more symptoms associated with a respiratory disease or disorder in a subject, the composition comprising one or more metabolites, optionally two or more metabolites, and one or more pharmaceutically acceptable carriers and / or excipients, wherein the one or more metabolites are in an effective amount to reduce viral or bacterial replication, in the subject by 5% to 70% within 48 hours of administration.

[0305] Paragraph 2. The composition of paragraph 1, wherein the one or more metabolites are one or more amino acids, one or more sugars, one or more flavonoids, or one or more organic acids, or a combination thereof, optionally wherein the one or more metabolites are selected from the group consisting of taurine, cysteamine, N-acetylglucosamine, nicotinic acid, aminolevulinic acid,

[0306] 45764921.1 46 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT malonic acid, sinapic acid, daidzein, formononetin, and genistein, optionally wherein one or more metabolites are taurine, cysteamine, N-acetylglucosamine, nicotinic acid, aminolevulinic acid, malonic acid, sinapic acid, daidzein, formononetin, and genistein.

[0307] Paragraph 3. The composition of paragraph 1 or 2, wherein the one or more metabolites comprise an amino acid, an organic acid, and an isoflavonoid,

[0308] Paragraph 4. The composition of paragraph 2 or 3, wherein the one or more amino acids comprise sulfur-containing amino acids (e.g., methionine, cysteine, homocysteine, taurine, cysteamine, etc.), optionally wherein the one or more amino acid comprise taurine and / or cysteamine.

[0309] Paragraph 5. The composition of any one of paragraphs 2-4, wherein the one or more sugar comprise N-acetylglucosamine.

[0310] Paragraph 6. The composition of any one of paragraphs 2-5, wherein the one or more organic acids comprise nicotinic acid, aminolaevulinic acid, sinapic acid, and / or malonic acid, optionally wherein the organic acid is sinapic acid.

[0311] Paragraph 7. The composition of any one of paragraphs 2-6, wherein the one or more flavonoids are isoflavonoids, neoflavonoids, and / or bioflavonoids.

[0312] Paragraph 8. The composition of paragraph 7, wherein the isoflavonoids are daidzein, formononetin, and / or genistein, optionally wherein the isoflavonoid is genistein.

[0313] Paragraph 9. The composition of any one of paragraphs 1-8, wherein the one or more metabolites comprise taurine and sinapic acid.

[0314] Paragraph 10. The composition of any one of paragraphs 1-8, wherein the one or more metabolites comprise taurine, sinapic acid, genistein.

[0315] Paragraph 11. The composition of any one of paragraphs 1-10, wherein the composition is in the form of a solid or a liquid.

[0316] Paragraph 12. The composition of any one of paragraphs 1-11, wherein the composition is in a form suitable for intranasal administration or oral administration.

[0317] Paragraph 13. The composition of any one of paragraphs 1-10, wherein the composition is in the form of a nasal spray.

[0318] Paragraph 14. The composition of any one of paragraphs 1-13, wherein when administered to a subject, the total amount of the one or more metabolites in the composition is effective to reduce alveolar macrophage cell death, reduce viral levels in lung epithelial cells, and reduce lung viral titer, as indicated by number of alveolar macrophages and TCIDso / mL of virus, respectively, measured in a biological sample of the subject, compared to a control sample of the subject before administration, optionally wherein the biological sample is a serum sample, optionally wherein the serum sample is a blood sample or a bronchoalveolar lavage fluid sample.

[0319] 45764921.1 47 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT

[0320] Paragraph 15. The composition of any one of paragraphs 1-14, further comprising one or more active agents that are different from the metabolites.

[0321] Paragraph 16. The composition of paragraph 15, wherein the one or more active agents are antiinflammatory agents, antiviral agents, antibiotic agents, antipyretic agents, analgesic agents, blood glucose regulators, gastrointestinal agents, or respiratory tract agents, or a combination thereof. Paragraph 17. A method for treating one or more symptoms associated with a respiratory disease or disorder in a subject in need thereof, comprising:

[0322] (i) administering to the subject the composition of any one of paragraphs 1-16, optionally wherein the administration step is repeated one or more times.

[0323] Paragraph 18. The method of paragraph 17, wherein following the administration or optionally all of the administrations, the total amount of the metabolites administered to the subject is effective to increase gut microbiota of the subject, as indicated by an increase in the number of segmented filamentous bacteria measured in a biological sample of the subject, compared to a control sample of the subject prior to the administration of the pharmaceutical formulation, optionally wherein the biological sample is a fecal sample or a serum sample.

[0324] Paragraph 19. The method of paragraph 17 or 18, wherein the respiratory disorder is a respiratory infection.

[0325] Paragraph 20. The method of paragraph 19, wherein the respiratory infection is caused by a microorganism selected from the group consisting of a virus, a bacterium, a protozoan, and a fungus.

[0326] Paragraph 21. The method of paragraphs 19 or 20, wherein the respiratory infection is caused by a virus selected from the group consisting of orthomyxovirus, rhino virus, paramyxovirus, coronavirus, adenovirus, human metapneumovirus (hMPV), enterovirus, and bocavirus.

[0327] Paragraph 22. The method of paragraph 21, wherein the orthomyxovirus is selected from the group consisting of influenza A, influenza B, and influenza C.

[0328] Paragraph 23. The method of paragraph 21, wherein the paramyxovirus is selected from the group consisting of Respiratory syncytial virus (RS V) and parainfluenza virus.

[0329] Paragraph 24. The method of paragraph 21, wherein the coronavirus is selected from the group consisting of SARS-CoV, MERS-CoV, and SARS-CoV-2 (COVID-19).

[0330] Paragraph 25. The method of paragraph 17 or 18, wherein the respiratory disease or disorder is selected from the group consisting of cystic fibrosis, chronic obstructive pulmonary disease, pulmonary fibrosis, pneumonia, and asthma.

[0331] Paragraph 26. The method of any one of paragraphs 17-25, further comprising a step of administering a second active agent prior to, during, or after step (i).

[0332] 45764921.1 48 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT

[0333] Paragraph 27. The method of any one of paragraphs 17-26, wherein the composition is administered once every week, once every two weeks, once a month, once every two months, once every three months, once every four months, once every five months, or once every six months, for a time period from 1 day to 1 year, from 1 day to 9 months, from 1 day to 6 months, from 1 day to four weeks, from 1 day to three weeks, from 1 day to two weeks, from 1 day to 1 week, or from 1 day to 3 days or wherein the composition is administered irregularly for at least two time, at least three times, at least four times, at least five times, at least ten times, at least 20 times, or at least 30 times, within a time period of one week, one month, six months, 1 year, 2 years, 3 years, or 5 years. Paragraph 28. The method of any one of paragraphs 17-27, wherein the composition is administered by parenteral injection, intranasal administration, or by oral administration. Paragraph 29. The method of any one of paragraphs 17-28, wherein the subject is a mammal. Paragraph 30. The method of any one of paragraphs 17-29, wherein the subject is immunocompromised.

[0334] Paragraph 31. The method of any one of paragraphs 17-30, wherein the composition administered to the subject is at a dose from 0.1 mg to 250 mg for each administration.

[0335] Paragraph 32. The method of any one of paragraphs 17-31, wherein following the administration or optionally all of the administrations, the total amount of the metabolites administered to the subject is effective to reduce cell death of alveolar macrophages by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, as indicated by immunohistochemistry of a serum sample, optionally wherein the serum sample is a blood sample or a bronchoalveolar lavage fluid sample. Paragraph 33. The method of any one of paragraphs 17-32, wherein following the administration or optionally all of the administrations, the total amount of the metabolites administered to the subject is effective to increase C1QA expression and / or reduce Notch4 expression by 5-fold, 10-fold, 15- fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, or 80-fold, as indicated by quantitative real-time PCR of a serum sample, optionally wherein the serum sample is a blood sample or a bronchoalveolar lavage fluid sample.

[0336] Paragraph 34. The method of any one of paragraphs 17-33, wherein following the administration or optionally all of the administrations, the total amount of the metabolites administered to the subject is effective to reduce viral-induced expression of IFNP and / or IL-6 by 5-fold, 10-fold, 15-fold, 20- fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, or 80-fold, as indicated by quantitative real-time PCR of a serum sample, optionally wherein the serum sample is a blood sample or a bronchoalveolar lavage fluid sample.

[0337] 45764921.1 49 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT

[0338] Paragraph 35. A method for preventing a secondary infection following a primary disease or disorder in a subject in need thereof, comprising:

[0339] (i) administering to the subject the composition of any one of paragraphs 1-16, optionally wherein the administration step is repeated one or more times.

[0340] Paragraph 36. A method for preventing a secondary infection following a primary disease or disorder in a subject in need thereof, comprising:

[0341] (i) administering to the subject a pharmaceutical formulation comprising one or more segmented filamentous bacteria (SFB), optionally wherein the administration step is repeated one or more times.

[0342] Paragraph 37. The method of paragraph 35 or 36, wherein the pharmaceutical formulation is administered after the subject shows one or more symptoms of the primary disease or disorder, optionally within a week, 3 days, 1 day, 12 hours, 5 hours, 2 hours, or 1 hour after the onset of the one or more symptoms of the primary disease or disorder.

[0343] Paragraph 38. The method of any one of paragraphs 35-37, wherein the primary disease or disorder is caused by a virus, bacteria, or ailment (e.g., diabetes or cystic fibrosis) and / or wherein the secondary infection is caused by a virus or bacteria, optionally wherein the virus or bacteria is Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae.

[0344] Paragraph 39. The method of any one of paragraphs 35-38, wherein following the administration, or optionally all of the administrations, the total amount of the SFB administered to the subject is effective to prevent the secondary infection by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, as indicated by quantitative real-time PCR of a serum sample, optionally wherein the serum sample is a blood sample or a bronchoalveolar lavage fluid sample of the subject, compared to a control (e.g., a subject having the same primary disease / disorder but not administered with the pharmaceutical formulation).

[0345] Paragraph 40. The method of any one of paragraphs 35-39, wherein following the administration or optionally all of the administrations (when more than one administration is performed), the total amount of the SFB administered to the subject is effective to prevent the secondary infection in the subject, as indicated by the absence of symptoms associated with the secondary infections, compared to a control (e.g., a subject that is infected with the same primary disease / disorder, but not administered with the pharmaceutical formulation).

[0346] The disclosed compositions and methods can be further understood through the following non-limiting examples.

[0347] 45764921.1 50 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT

[0348] Examples

[0349] Example 1: Colonization of the Intestine with SFB Protects Mice from Influenza Respiratory Infection

[0350] It is believed that gut microbiota composition might influence RVI severity. Proneness of “excluded flora (EF)” mice were compared, which lack a discrete panel of disease-modulating commensal microbes, which might or might not otherwise be present in colonies of “specific pathogen-free (SPF)” mice commonly used in biomedical research2. Mice were intranasally inoculated with 2009 pandemic A / CA / 07 / 2009 (H1N1) IAV strain, herein referred to as CA09, which recapitulates major clinical features of human disease in mice3. Compared to EF mice, SPF mice exhibited significantly reduced lung viral titers 4 days post CA09 inoculation (Figure 1A). Furthermore, SPF mice lacked hypothermia and weight loss observed in EF mice, and all animals of the SPF group survived. Of the organisms known to be absent in EF mice, segmented filamentous bacteria (SFB) stood out as a potential modulator of IAV proneness in that SFB, despite being a strict anaerobe and, thus, restricted to the intestinal luminal surface, is known to systemically impact T-cells4, 5. Furthermore, SFB mediates the spontaneous innate immune-mediated resistance to rotavirus (RV), an intestinal pathogen, that spontaneously arose in some mouse colonies6.

[0351] SFB is challenging to culture but can be isolated, maintained, and studied by administering it via fecal microbial transplantation (FMT). Hence, mice lacking SFB but otherwise carrying a complex SPF microbiota, herein referred to as SFB’ (SFB negative) mice, were administered FMT from germ-free (GF) mice or mice mono-associated with SFB (SFB-MA). Administering SFB in this manner fully recapitulated the IAV resistance of mice naturally colonized with SFB (Figure IB). Such protection against IAV infection was observed within a few days of SFB administration and persisted beyond 3 months even though SFB levels begin to decline rapidly 2 weeks postadministration1. SFB-mediated protection against CA09-induced disease associated with complete suppression of IAV lung histopathology, whereas SFB by itself had no effect on tissue appearance (Figure 1C). Consistent with previous observations that SFB only resides in the gut lumen or attached to the apical epithelial gut surface, SFB was readily detected by PCR in the ileum but was not found in the lung or spleen1. SFB-mediated protection against IAV was fully maintained in Ragl’ / _mice1, which lack most T and B lymphocytes, indicating that TH 17 cells or other components of adaptive immunity do not play a role. Use of other KO mice ruled out an array of candidate mechanisms including those involving interferon (type I or 2), IL17, and IL-22.

[0352] Transcriptomic profiling of whole lungs was used to evaluate how SFB colonization protected against IAV. RNA-sequencing analysis revealed that SFB, by itself, had negligible impact on lung gene expression, whereas IAV infection, by itself, markedly remodeled the lung

[0353] 45764921.1 51 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT transcriptome. Strikingly, such lAV-induced changes in gene expression were nearly wholly absent in SFB-colonized mice, indicating that presence of SFB broadly ameliorated lAV’s impact on lung tissue.

[0354] Example 2: SFB’s protection against IAV infection associates with, and requires, preservation of alveolar macrophages.

[0355] Using flow cytometric analysis, it was found that SFB colonization prevented lAV-induced depletion of resident phagocytes, including AM and dendritic cells (DC). This was not mimicked by a direct-acting antiviral drug, indicating that SFB’s preservation of these cells potentially contributes to, rather than reflect, reduced viral loads. Potential roles for DC and / or AM in SFB- mediated protection against IAV were tested in mice harboring deficiencies in these cells.

[0356] It was observed that SFB -mediated protection against IAV was maintained in Batf3 ~ mice, which wholly lack cDCl7, but was greatly reduced in Csf2''' mice, which have a 70% reduction in basal AM levels8. The role of AM was further examined by depleting these cells with clodronate liposomes9, which specifically depleted AM and did not affect other lung innate immune cells1. Depletion of AM, by itself, did not significantly impact IAV loads or clinical signs of disease but eliminated the ability of SFB to lower IAV lung titers, ameliorate hypothermia and weight loss, and prevent death (Figure 2A). These results suggest that SFB-mediated impacts on AM are linked to its protection against RVI.

[0357] SFB-mediated maintenance of AM levels either reflect increased generation of these cells, which can occur from monocyte progenitors migrating to the lung or local self-renewal10, or that SFB colonization had somehow “emboldened” AM to resist death upon IAV infection. It was observed that SFB markedly prevented the lAV-induced increase in levels of cleaved caspase-3, which marks AM commitment to cell death11, thereby supporting the hypothesis that SFB colonization had somehow “emboldened” AM to resist death upon IAV infection. In SFB- mice, lAV-induced caspase-3 activation in AM associated with the presence of IAV components, which were undetectable in AM of SFB+ CA09-inoculated mice, indicating that AM had engulfed IAV- infected epithelial cells (EC)12, which were in markedly greater abundance in SFB- mice13. AM protect lung EC from IAV at early time points following lAV-infection, i.e. prior to their depletion14, leading to the prediction that depleting AM with clodronate, thus precluding their preservation by SFB, might ablate SFB-mediated protection of EC from IAV infection. Indeed, clodronate treatment increased IAV levels in EC and conducting airway cells in SFB+ mice. Collectively, these results indicate that SFB colonization resulted in AM resisting lAV-induced depletion, thereby enabling sustained AM-mediated protection of EC, likely contributing to lower IAV burden and ameliorated pathology.

[0358] 45764921.1 52 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT

[0359] Persistence of AM in CA09-infected SFB+ mice was potentially attributable to SFB altering the lung environment and / or reflect that AM themselves were intrinsically transformed to withstand 1AV challenge. To differentiate between these possibilities, we transferred FACS-sorted AM from SFB+ and SFB- mice to SFB- mice using donor mice with a CD45.1 subtype, thereby enabling distinction of endogenous and transplanted cells (Figure 2B). The degree to which AM harvested from SFB+ and SFB- mice, hereafter referred to as SFB+ and SFB- AM, engrafted following transplant was similar, comprising about 20% of the total AM in their new host. CA09 infection did not significantly alter relative abundance of SFB- AM but resulted in SFB+ AM becoming their host’s majority AM population. This shift in AM proportions reflected that endogenous AM in both recipient groups as well as SFB- AM were all depleted by CA09 infection, whereas SFB+ AM fully resisted CA09-induced depletion and, concomitantly, lacked Caspse-3 activation, despite having been transplanted into an SFB-free host. Thus, colonization of the gut with SFB altered AM to withstand lAV-induced depletion.

[0360] Example 3: SFB Colonization of Intestine Alters AM to Withstand and Better Manage IAV Infection.

[0361] Differences between SFB+ and SFB- AM were characterized ex vivo (Figure 3A). Profiling of basal gene expression (i.e. without IAV exposure) by RNA-seq found that only 24 of the 15,510 genes expressed by AM were differentially expressed between SFB- and SFB+ AM, 18 of which were both confirmed by PCR and biologically replicated. Six of the genes upregulated in SFB+ AM are associated with an M2 macrophage phenotype. The genes most prominently up- and down- regulated, respectively, in SFB+ AM were Tsc22d3 and Notch 4. which, respectively, function in suppressing and activating pro-inflammatory gene expression suggesting such signaling might be dampened in SFB+ AM. It was observed that SFB+ AM lacked the robust UV-irradiated LAV- induced activation of pro-inflammatory gene expression exhibited by SFB- AM. The small panel of genes upregulated in SFB+ AM also included those that function in antiviral resistance, including complement Clqa and Ifitml. These results suggest that SFB+ AM were not anergic to IAV per se but, rather, might have better virus neutralizing capacity. This possibility was evaluated by incubating CA09 stocks with AM for 45 minutes at which time AM were removed by centrifugation and supernatant infectious titers and IAV genomes quantified. It was found that, relative to SFB-AM, SFB+ AM lowered IAV titers by 150-fold more than SFB-AM, at 37°C but not 4°C. The increased disabling of CA09 by SFB+ AM was mediated by both Clqa and increased phagocytosis.

[0362] An additional series of AM transplants were conducted to determine the extent to which SFB-induced alterations in AM phenotype protected against IAV. It was observed that

[0363] 45764921.1 53 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT administration of SFB- AM to a host replete with endogenous AM modestly reduced IAV titers, gross lung damage, hypothermia, weight loss, and death (Figure 3B). Yet the administration of SFB+ AM provided markedly greater protection against IAV challenge, approximating that conferred by administration of SFB itself, as assessed by all assayed parameters. Follow up studies using various KO mice and antibody-mediated neutralizations revealed that both inflammatory anergy and increased antiviral function, mediated, in part by reduced Notch4 and increased Clqa, worked in synergy to mediated increased AM survival and reduced disease severity.

[0364] Example 4: Identification of Molecule (s) That Reprogram AM to Better Manage RVI.

[0365] It was hypothesized that gut SFB generate a soluble factor(s) (i.e. a metabolite, also referred to herein as a SFB-induced AM Remodeler or “SAR”) which translocate to the lung and reprogram AM. Potentially, SAR is secreted by SFB and / or the host but, in contrast to the change in AM phenotype induced by the BCG vaccine, would not likely be a consequence of generalized increased in gut permeability because, unlike BCG, SFB-induced changes do not require endogenous microbiota17. Fecal supernatants (FS) were generated from germfree mice and SFB- mono associated (MA) mice, and were orally administered to SFB- mice, which were then, 1 week later, challenged with IAV. As shown in Figure 4, 3 oral administrations of SFB-MA FS resulted in mice displaying a greater than 1-log reduction in IAV titer, relative to mice administered FS from germfree mice. This reduction in IAV titer is associated with reduced clinical features of disease and increased AM survival. A single dose of SFB-MA FS partially recapitulated these beneficial effects. Importantly, it was verified that FS administration did not transfer SFB itself. Administering a single dose of SFB-MA FS into the lung (i.e. intranasally), protected mice from lAV-induced AM depletion and disease (Figure 5). Such protection against IAV challenge began to decline by 2 months and was completely gone by 4 months, contrasting with protection provided by SFB colonization, which like SFB itself, seems to persist for the life of the mouse.

[0366] Phenotypic transformation of AM could also be recapitulated ex vivo (Figure 6). Specifically, exposure of AM to FS, serum, or BALF from SFB+, but not SFB-, mice resulted in AM increasing basal expression of Clqa, exhibiting inflammatory anergy to UV-IAV, and having greater capacity to disable IAV when incubated with this virus. Furthermore, transplant of such ex vzvo-trained AM into mice conferred protection against IAV challenge. Collectively, these results support the existence of a SAR, which translocate from the gut to the lung lumen, wherein it reprograms AM to better manage RVI.

[0367] The molecular components of SAR via non-targeted metabolomic analysis were evaluated. Specifically, FS from conventional and germfree mice before, and after, colonization with SFB was analyzed on the basis that molecules whose abundance was increased by SFB colonization in both

[0368] 45764921.1 54 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT states would be the strongest SAR candidates, his approach yielded 32 (of about 6000) candidate metabolites of which 18 could be readily commercially purchased (Figure 7). These molecules were tested for their ability to train AM in vitro, using concentrations based on literature and metabolomics data. None of the 18 molecules impacted AM viability. Two related molecules, namely taurine and cysteamine, both increased Clqa expression and attenuated UV-IAV-induced expression of IFNP and / or IL-6. N-acetylglucosamine increased Clqa expression while 3 molecules, namely nicotinic acid, aminolaevulinic acid, and malonic acid attenuated UV-IAV- induced expression of IFN and / or IL-6, suggesting a potential contribution to SAR (Figure 8).

[0369] The extent to which a combination of these molecules might recapitulate SAR was tested. AM were exposed ex vivo to a cocktail of these 6 molecules. One week later, such AM were examined ex vivo, or transplanted into mice, which were then inoculated with CA09. Both approaches demonstrated that AM trained by the cocktail ex vivo exhibited a phenotype that was reminiscent of, albeit less robust than, that of AM trained by colonization of SFB in vivo (Figure 9). These results demonstrate that components of SAR may be identified using the described approaches and that doing so may yield practical strategies to mitigate RVI risk.

[0370] Example 5: Mechanism and Consequences of AM Phenotype Changes which Result from SFB Colonization

[0371] AM from SFB colonized mice, herein referred to as SFB+AM exhibit inflammatory anergy and enhanced antiviral function, which confers increased AM survival, enabling their sustained protection of lung epithelial cells, thereby minimizing RVI severity. The SFB+ AM phenotype differs dramatically from other reported examples of AM training, raising the question of how it was working, which answered, may lead to improved RVI management strategies. Thus, this study investigated how the unique gene expression of pattern of SFB+ AM is mediated and evaluated how it impacts challenges beyond RVI.

[0372] The inflammatory anergy of SFB+ AM was potentially attributed to diminished signaling by the pattern recognition receptors that recognize IAV and / or that some pro-inflammatory genes were less available to be transcribed due to changes in chromatin structure. To investigate the first possibility, activation of proximal lAV-induced signaling cascades in SFB- and SFB+ AM were examined. It was observed, via flow cytometry, that exposure to IAV resulted in phosphorylation of STAT1, STAT3, and P38MAPK to similar extents in SFB- and SFB+ AM (Figure 10), indicating lack of differential proximal signaling. Assay of the NF-KB pathway by western blot observed similar trends (not shown), suggesting a role for chromatin modification in mediating SFB’s impacts on AM.

[0373] 45764921.1 55 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT

[0374] Epigenetic changes mediate impacts of other AM trainers, notably LPS and BCG17'23.

[0375] However, the published consequences of AM trained by LPS and BCG seem opposite to what was observed for SFB. The impacts of LPS and SFB on AM responsiveness to 1AV were directly compared. Briefly, SFB- AM were isolated and exposed, in vitro, to PBS, LPS or fecal supernatants (FS) from SFB- or SFB+ mice. Such AM were assayed in vitro for basal and lAV-induced gene expression by qPCR. The ability of such AM to reduce the infectious titers of IAV aliquots were also measured, with which they were incubated. Additionally, the trained AM were transplanted into mice, which were then intranasally challenged with IAV. LPS and SFB had opposing impacts on AM function in vitro (Figures 11A and 11B). Specifically, LPS did not share the ability of SFB- FS to elevate Clqa expression. Accordingly, in contrast to AM exposed to SFB-FS, LPS -treated AM did not robustly neutralize IAV. Furthermore, while SFB-FS induced inflammatory anergy to UV-IAV, LPS potentiated pro-inflammatory gene expression. Differential impacts of AM trained by LPS and SFB-FS were even more striking following AM transplant (Figures 11C-11E). Specifically, SFB-FS trained AM resisted lAV-induced depletion while LPS exacerbated it. Such persistence of SFB FS-trained AM associated with minimal IAV loads in epithelial cells and modest clinical-type disease indices. In contrast, administration of LPS-trained AM elevated IAV and exacerbated disease. These dramatically opposing impacts by LPS and SFB-derived products may ultimately prove relevant to understanding how distinct microbiotas can potentiate or reduce risk of severe RVI.

[0376] Example 6: Ability of SFB+ AM to Manage Non- Viral Challenges

[0377] An exemplary approach includes protecting individuals at risk for severe RVI for a few months via intranasally administered molecules that transform their AM into an SFB+-like AM phenotype and / or directly administered autologous SFB+-like AM. The impacts of transplanted SFB+ AM in well-characterized non-RVI lung disease models were examined. 6-8 week old male and female C57 BL / 6 mice, purchased from Jackson Labs and verified to be SFB- upon arrival, were intranasally administered PBS or 200x103AM, which were freshly isolated from the lungs of CD45.1 mice, colonized or not by SFB, enabling distinction of endogenous and transplanted AM. Irrespective of whether they are SFB- or SFB+ AM, the transplanted AM make up about 20% of the total AM population and persist at this level for at least 90 days, during which time SFB+ AM maintain inflammatory anergy and enhanced capacity to disable IAV. Exogenous SFB+ AM provides strong protection against IAV, approaching that provided by SFB itself (Figure 3).

[0378] The mice were intranasally administered 108CFU of Streptococcus pneumoniae or Hemophilus influenzae. Mice were euthanized 4 days later to assess lung CFU, histopathology and gene expression (pro-inflammatory and antibacterial). Lungs were subjected to flow cytometry,

[0379] 45764921.1 56 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT measuring levels of AM (endogenous and exogenous) other resident and inflammatory cells. Parallel sets of mice will be monitored for clinical indies of disease including weight loss, morbidity, and mortality. The results suggest that colonization by SFB itself results in better AM survival and reduced S. pneumo colonization (Figure 12).

[0380] Example 7 : Gut Microbiome Associates with, and is Capable of Influencing AM Phenotype and / or RVI Severity in Humans

[0381] While long-lasting changes in host defense posture have classically been considered the purview of the adaptive immune system, it has more recently become clear that some populations of innate immune cells, especially long-lived tissue resident cells such as AM have “memory” in that they can be “trained” in a manner that markedly alters their phenotype28. Potentially, one’s acquired AM phenotype influences severity of RVI. It was demonstrated that assessment of human AM function can be done using flow cytometric purified bronchiolar lavage fluid (BALF) AM. Specifically, BALF collected from healthy control subjects was used to obtain highly purified AM and it was found that the same assays used to measure functional responses in murine AM can be applied to human AM (Figure 13). Data from mice suggests that lAV-induced AM gene expression is not lastingly impacted by previous 1AV infection (Figure 14).

[0382] Example 8: Impeding SFB Infection Reduces Generation of Adaptive Immunity

[0383] It was found that impeding SFB infection reduced the generation of adaptive immunity (Figure 15). Transplanting feces from RV vaccine non-responders recapitulated phenotype (Figure 16). 16S analysis on feces samples at day 0 prior to rotavirus infection was performed, and Clostridium perfringens, an opportunistic bacterium, was detected in mice transplant with vaccine non-responders’ group i.e., RV vaccine-resistance FMT mice (data not shown). C. Perfringens reduces RV infection and specific antibodies mRV in ASF mice (Figure 17).

[0384] Whether the impacts of the gut microbiome on viral infection extended beyond the intestine was tested. It was found that SFB in the gut protected mice against influenza (Figures 18A-18C) and this protection lasted at least three months (Figures 18D-18G).

[0385] Since airway lung epithelial cells are main site for influenza virus infection and replication, it was observed that in the presence of SFB, the entry to all three types of lung epithelial cells were greatly reduced (Figures 19A-19C). lAV-induced lung damage was assayed 4 days post IAV challenge and it was observed that gut SFB eliminated lAV-induced lung damage (Figure 20).

[0386] Using a targeted approach to test whether SFB protection via adaptive immunity pathways. SFB-mediated protection against IAV was fully maintained in Ragl- / - mice, which lack most T and B lymphocytes. Although innate antiviral immunity typically involves interferon, SFB-mediated

[0387] 45764921.1 57 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT protection against IAV was maintained in Ifnar- / - and statl- / - mice, which lack type I interferon signaling and broadly compromised in IFN signaling. Ragl- / -H2rg- / -, which lacks type 3 ILC, and upon antibody-mediated neutralization of IL- 17 and IL-22, SFB-mediated protection against IAV (Figure 21).

[0388] SFB had minimal direct impacts on lung gene expression but largely blocks impacts of IAV (data not shown). SFB had minimal direct impacts on lung leukocyte levels and largely blocked impacts of IAV (Figures 22 and 23). For instance, SFB blocked lAV-induced depletion of DC and AM (Figure 22). SFB also blocked lAV-induced influx of neutrophils and NK cells (Figure 23). It was observed that 4’FIU administration blocked inflammatory cell recruitment but not loss of resident DC and AM (Figures 24A-24I). Although SFB’s protection against IAV did not require DC; Batf3- / - (Figures 25A-25D), SFB’s protection against IAV required AM (Figures 25E-25K).

[0389] AM transplanted from SFB+ mice resisted lAV-induced depletion (Figure 26). AM transplanted from SFB+ lacked caspase 3 activation and fully resisted depletion upon IAV infection (Figure 27). SFB+ AM directly disabled IAV (Figure 29). Transplanting AM from SFB+ mice conferred protection against IAV (Figure 30). SFB impacts on AM were recapitulated following oral administration of fecal supernatant (Figure 31).

[0390] SFB increased taurine in feces, serum and BALF (Figure 33). SFB increased gut bile salt hydrolase activity and serum taurine (Figure 34). Intranasal administration of taurine partially protected mice following IAV infection (Figure 36). The ability of serum from SFB+ mice to train AM to manage IAV was reduced by BSH inhibitor (Figure 35).

[0391] The results demonstrated that: (i) carriage of the commensal microbe segmented filamentous bacteria (SFB) protected against viral infection, for example, severe influenza viral (IAV), RSV and SARS-CoV-2 infection, as assessed by viral titers, histopathology, and clinical disease features (Figures 18-20), (ii) such protection was independent of interferon and adaptive immunity but requires basally resident alveolar macrophages (AM) (Figures 21 and 25); (iii) AM from SFB- colonized animals were altered to resist lAV-induced depletion and inflammatory signaling by directly disabling IAV via increased complement production and phagocytosis (Figure 29), and (iv) transplant of AM isolated from SFB-colonized hosts into SFB-free hosts recapitulated SFB- mediated protection against IAV thereby linking intestinal microbiota, AM phenotype, and RVI severity (Figure 30).

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[0393] Example 9: Reprogramming of alveolar macrophages by intestinal segmented filamentous bacteria protects mice from secondary infection by lethal bacterial pneumoniae following influenza infection

[0394] Materials and Methods

[0395] Mice

[0396] Wild-type C57BL / 6 (B6 WT) mice were purchased from The Jackson Laboratory and used at 3 weeks of age. Experiments were conducted using age- and gender-matched groups. The Jackson Laboratory does not routinely test for segmented filamentous bacteria (SFB) in mice; however, all mice obtained from this vendor were tested and confirmed to be SFB-negative upon arrival at GSU. Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Georgia State University.

[0397] Mono-associated SFB transplantation

[0398] Fecal samples were collected from germ-free donor mice, suspended in 20% glycerol PBS solution at 80 mg / mL concentration, passed through a 40 pm filter, and stored at -80°C. Frozen fecal suspensions were orally administered to recipient mice at 200 ml per mouse.

[0399] Virus and Bacteria strains

[0400] Streptococcus pneumoniae 6303 (strain designation C1P 104225), Methicillin-resistant Staphylococcus aureus (strain designation F-182), were obtained from the American Type Culture Collection (ATCC). Frozen bacterial stocks of 5. pneumoniae were streaked out on Trypticase Soy Agar plates containing 5% sheep blood. A single colony was inoculated into 5 ml of Todd-Hewitt broth with 0.5% added yeast extract and grown at 37°C with 5% CO2 without shaking. Haemophilus influenzae (strain designation AMC 36-A-5 624, NCTC 8469) was obtained from ATCC and cultured in ATCC medium 2167 Haemophilus Test Medium media 37 °C with 5% CO2 for 48 hours. Bacteria were centrifuged and resuspended in PBS and diluted to the appropriate intended inoculum. (IxlO7CFU or 500 CFU per mouse).

[0401] Virus and bacterial co-infection

[0402] For virus infections, animals were anesthetized with isoflurane and intranasally inoculated with 50pl of 50 TCID50 units / animal of recombinant A / CA / 07 / 2009 (H1N1) (CA09) GFP- expressing reporter virus. For secondary or co-bacterial infections, anesthetized mice were intranasally inoculated with 50 pL of PBS containing IxlO7CFU of .S'. pneumoniae, or S. aureus, or H. influenzae. Bacterial burdens in the lungs, BALF, and spleen were measured by euthanizing the infected mice on either day 4 or day 5 post-infection and plating serial 10-fold dilutions of each sample onto blood agar plates (S. pneumoniae and S. aureus) or chocolate agar plates (H. influenzae).

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[0404] Lung digestion and flow cytometry analysis of immune cells

[0405] Lungs were dissected from mice and minced with scissors before digestion in RPMI solution containing 5% FBS, DNase 1, and 1 mg / mL Collagenase type IV (Sigma) for 30 minutes at 37°C. During digestion, lungs were further homogenized by swishing with a 5 mL syringe every 10 minutes. For flow cytometry analysis, cells were blocked with 1 mg / million cells using antiCD 16 / anti-CD32 in 100 pL PBS for 15 minutes at 4°C, followed by washing with PBS to remove any residual blocking antibodies. Cells were then incubated with the following conjugated monoclonal antibodies: CD45 BV605, MHCII BV650, CDllb APC-Cy7, CDllc BV786, Ly6C PerCp-Cy5.5, Ly6G AF700, CD64 BV421, CD24 FITC, CD117 GV510, SiglecF PE-CF594, and Fcerl APC. Multi-parameter analysis was performed on a CytoFlex (Beckman Coulter) and data were analyzed using FlowJo software (Tree Star).

[0406] Antibiotic protection / Intracellular bactericidal assay

[0407] Alveolar macrophages were sorted, cultured, and incubated with S. pneumoniae, or S. aureus, or H. influenzae at a multiplicity of infection (MOI) of 20. The cultures were centrifuged at 500 x g for 10 minutes to increase macrophage-bacteria interaction, followed by a one-hour incubation for bacterial uptake. The media were then replaced with an antibiotic cocktail (RPMI with 5% FBS, 500 pg / mL gentamicin, 500 U / mL penicillin, and 500 pg / mL streptomycin) and incubated for 1 hour to eliminate extracellular bacteria. After washing with PBS, some wells were lysed with 0.1% Triton X-100 to collect bacterial CFUs, which were serially diluted and plated on blood agar plates or chocolate agar plates to measure bacterial uptake. The remaining wells were incubated for an additional hour in fresh media before lysis and CFU collection to assess bacterial survival. The percentage of CFU remaining was calculated as % CFU remaninig = CFU Survial . > _ - CFU Uptake X100

[0408] Histopathologic analysis

[0409] Lungs were perfused using 10% neutral-buffered formalin, dissected, and fixed. Formalin- fixed lungs were transferred to 70% EtOH, embedded in paraffin, sectioned, stained.

[0410] Adoptive transfer of alveolar macrophages

[0411] Lungs were harvested from C57BL / 6 mice and digested with Collagenase type IV as described previously. Alveolar macrophages (defined as CD45+MHCII+CDl lc+SiglecF+) were stained and FACS-sorted. 5-6xl05alveolar macrophages in 30 pL PBS were administered intranasally to recipient mice.

[0412] RNA-seq analysis. RNA-seq analysis were performed with data sets from (ref CHM paper). Total lung RNA-seq accession number: PRJEB71449 and alveolar macrophages RNA-seq

[0413] 45764921.1 60 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT accession number: PRJEB71689 from ENA European Nucleotide Archive. Pathway enrichment analysis was analyzed using GSEA software.

[0414] Quantitative real-time PCR

[0415] Total RNA from lungs or from FACs-sorted alveolar macrophages were isolated using the Qiagen RNeasy Mini Kit with on-column DNase digestion according to manufacturer’s protocol. cDNA was generated using the Superscript First Strand Synthesis kit for RT-PCR and random hexamer primers (Invitrogen). RT-qPCR was performed with SYBR Green using StepOnePlus PCR system (Applied Biosystem), and all genes expression was normalized to Gapdh. All primers are listed below.

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[0417] Table 1: RT-qPCR Primers

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[0419] Quantification and statistical analysis

[0420] Results were expressed as mean ± SEM. All data was plotted in GraphPad Prism version 10. Statistical significance was assessed by One-way ANOVA, Student’s t test, and Two-way ANOVA. Differences between experimental groups were considered significant if *p<0.005, **p<0.005, ***p<0.001, ****p<0.0001.

[0421] Experimental schematics and graphical abstract.

[0422] All experimental schematics and graphical abstract created using BioRender.

[0423] Results and Discussion

[0424] Infection of the respiratory tract by an array of viruses, including influenza virus (IAV), results in depletion of alveolar macrophages, thereby increasing proneness to bacterial pneumonia. Colonization of the intestine by segmented filamentous bacteria (SFB) prevents such depletion. It was hypothesized that SFB might protect against secondary bacterial infections occurring shortly after IAV infection. To test this hypothesis, experimental conditions were established in mice lacking SFB (i.e., SFB mice), where IAV infection depleted AM but was not lethal on its own. C57BL / 6 mice were intranasally administered 2009 pandemic A / CA / 07 / 2009 (H1N1) IAV, herein referred to as CA09, which recapitulates major features of human influenza in mice. A relatively moderate inoculum, namely TCIDsoo (roughly equivalent to 500 PFU), resulted in a more than 80% reduction in AM levels but was uniformly lethal thus precluding administration of a secondary bacterial challenge (Figure 41). Reducing the inoculum by 10- and 100-fold resulted in more moderate levels of AM depletion, 54% and 40%, and caused symptoms of disease, including weight loss, but not death. Thus, the former, TCIDso, was chosen for further study.

[0425] SFB- mice were orally administered vehicle or SFB, and thereafter referred to as SFB+ mice. 7 days later, mice were inoculated, or not, with CA09. 4 days following the inoculation, mice were nasally administered S. pneumoniae (107CFU). In the absence of CA09, S'. pneumoniae was uniformly non-lethal irrespective of SFB although, compared to SFB- mice, SFB+ mice displayed less weight loss and exhibited a more than 1-log reduction in lung CFU (Figure 37A), suggesting that SFB gut colonization conferred some protection against this lung pathogen. An analogous but much more striking pattern was observed when S', pneumoniae was administered following IAV infection. Specifically, CA09 / S. pneumoniae infection resulted in 100% and 0% lethality in SFB- and SFB+ mice, respectively. Such lethality, which occurred 5-6 days post S. pneumoniae administration, was, on day 4, associated with stark gross and histopathologically evident lung injury, which was not observed in CA09 / S. pneumoniae-in cXed SFB+ mice. The potentiation of S. pneumoniae-induced disease by prior CA09 infection associated with a 1-log increase in lung S. pneumoniae CFU on day 4. In contrast, CA09 infection did not increase this parameter in SFB+

[0426] 45764921.1 63 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT mice resulting in them displaying starkly lower levels of 6. pneumoniae CFU levels in lung, BALF, blood, and spleen (Figure 42A). A similar pattern of results was observed in secondary bacterial infection models in which inoculation of CA09 was followed by Hemophilus influenzae and Staphylococcus aureus (Figure 37B). These results indicate that SFB colonization protects against secondary bacterial infection.

[0427] It is likely that the reduced bacterial loads in SFB+ mice reflected increased clearance of the bacterial pathogen. To evaluate this, previously generated RNA-seq data from whole lungs of mice administered SFB and / or CA09, assayed at a time approximately corresponding to the secondary bacterial infection administered in this study, were analyzed. It was observed that SFB colonization resulted in a significant post-CA09 increase in the expression of genes involved in enhanced mucosal clearance of bacterial pathogens. (Figure 42B). To investigate this, post-CA09 -S'. pneumoniae infection was performed using only 500 CFU and bacteria that remained 2hours later was measured. Non-CA09 infected mice and SFB+ CA09-infected mice fully cleared this small inoculum. In contrast, 80% of the live inoculum was recovered in CA09-infected SFB- mice. These results indicate that SFB colonization enhanced post-CA09 infection bacterial clearance.

[0428] The hypothesis that AM mediated the resistance of SFB+ mice to secondary bacterial infection was tested. S' pneumoniae infection, by itself, modestly reduced AM levels and potentiated the much greater loss of these cells that occurred following CA09 infection (Figure 38A). In stark contrast, AM levels of SFB+ mice were similar to that of naive mice irrespective of CA09 and S’. pneumoniae. The temporal window of protection against secondary infection conferred by SFB also aligned it being AM-mediated. Specifically, SFB strongly protected against AM depletion and disease when S. pneumoniae was administered 2 d or 10 d following CA09 inoculation (Figure 43A) but provided only modest protection by d30 post-CA09, at which time the depleted AM have been replaced by recruited monocytic precursors and no protection by d90 (Figure 43B), at which point newly generated AM are known to have acquired full functionality. A role for AM was also supported by use of clodronate liposomes; this approach specifically depletes AM without altering levels of other lung immune cells. This means of AM depletion recapitulated the increased proneness to S. pneumoniae infection exhibited by CA09-infected mice as assessed by CFU, weight loss, and survival (Figure 38B). Moreover, clodronate resulted in SFB- and SFB+ mice becoming equally prone to .S’. pneumoniae infection. Furthermore, the relative resistance of SFB+ mice to CA09 / .S’. pneumoniae infection was eliminated by clodronate treatment, assessed by CFU, weight loss, and mortality (Figure 38C). These results suggest that alterations of AM in SFB+ CA09- infected were important and sufficient for their resistance to S. pneumoniae infection.

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[0430] While AM being present in greater numbers in SFB+ mice post-CA09 infection likely contributed to protection against 5. pneumoniae infection, additional mechanisms may also be involved. More specifically, that SFB+ AM are more adept at neutralizing IAV led to the hypothesis that SFB+ AM, not only better survive CA09 challenge, but may also have greater capacity per AM to eliminate bacteria they encounter. Re-analysis of RNA sequencing data supports this hypothesis. An untargeted comparison of gene expression of SFB- and SFB+ AM found the latter exhibited enhanced expression of genes involved in phagocytosis, complement activation, and secretion of antibacterial mediators. To investigate the functional consequences of this observation, AM were isolated from SFB and SFB+mice 3 days post-CA09 inoculation via FACS, and equal numbers of SFB and SFB+AM were exposed to modest bacterial challenges, namely S. pneumoniae, S. aureus, or H. influenzae (all at MOI = 20). In all cases, supernatants collected at 90 minutes lacked CFU indicating both AM populations had contained these challenges but, at the 60-minute time point, supernatants from SFB- AM had bacterial loads that were higher indicating greater per cell clearance of bacteria by SFB+ AM. In contrast, quantitation of intracellular CFU in AM, via use of cell-impermeant antibiotics, Ih post-exposure to bacteria showed greater numbers of viable bacteria in SFB+ AM. It appeared that such rapid removal of bacteria from the suspension by SFB+ AM coupled with increased intracellular CFU AM reflected greater phagocytosis. In accord with this notion SFB+ AM exhibited greater ability to internalize fluorescent particles that had been coated with .S'. aureus antigens.

[0431] SFB+ AM also demonstrated increased ability to kill the bacteria they had internalized. Specifically, despite SFB+AM ending the initial 1-hour assay period with 2-fold higher levels of intracellular bacteria, they reduced the number of viable intracellular bacteria by over 90% during the following 2 hours, whereas SFB AM only modestly reduced their intracellular bacterial load during this period. It was hypothesized that complement played a role in mediating the differences between SFB- and SFB+ AM. Measurement of complement gene expression by qPCR confirmed the RNA-seq finding that SFB gut colonization had indeed led to increased complement expression. The role of complement expression was investigated by administering AM a cocktail of neutralizing anti-complement antibodies during the initial Ih period during which they were incubated with bacteria. Antibody-mediated neutralization of complement reduced both the uptake and killing of bacteria by SFB+AM, while having no impact on these parameters in SFB AM. Thus, compared to SFB AM, SFB+AM exhibit elevated complement expression, resulting in an enhanced ability to uptake and kill bacteria they encounter.

[0432] Next, AM transplantation studies were conducted to assess the extent to which the changes in AM, induced by SFB gut colonization, were sufficient to protect against CA09 / .S'. pneumoniae

[0433] 45764921.1 65 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT infection. An approach in which transplanted AM would be present throughout CA09 and .S'. pneumoniae infection was utilized (Figure 40A). Specifically, SFB- mice were subjected to clodronate-mediated AM depletion and then administered SFB- or SFB+ AM. Such mice were then inoculated with CA09 and, 4 days later, with S. pneumoniae. Recipients of SFB and SFB+AM demonstrated significantly different outcomes following this regimen, with 0% and 100% survival, respectively, which correlated with a 3-log difference in S. pneumoniae CFU and marked differences in gross and histopathologic lung appearance 4 days post-inoculation. A significant portion of this difference likely reflected differential responsiveness of these groups of mice to CA09. SFB+AM resisted depletion following transplantation, and their recipients are relatively resistant to CA09 infection, as assessed by viral titers and disease symptoms. It was observed that recipients of SFB AM lost more weight, indicating they were already sicker, and concomitantly had far fewer AM at the time they were exposed to S. pneumoniae. Thus, the experimental scheme was adjusted to assess potential impacts of SFB+AM on secondary bacterial infection that were not directly related to their impact on CA09 infection. (Figure 40B). Specifically, SFB- mice were subjected to CA09 infection, under conditions that result in symptomatic disease and AM depletion, which is maximal 4d post-CA09 administration. Such mice were then, 6d post-CA09 inoculation, administered SFB- or SFB+ AM and then, 2d later, inoculated with P. pneumoniae. Compared to mice receiving SFB- AM, recipients of SFB+ AM displayed 3-fold lower levels of lung CFU 4d post P. pneumoniae inoculation. Moreover, recipients of SFB- AM uniformly succumbed within 6d of the secondary P. pneumoniae challenge while most (80%) of recipients of SFB+ AM survived and regained much of the weight lost in response to the primary CA09 challenge. The results suggested that reprogramming of AM by gut SFB colonization not only maintains the presence of AM following respiratory viral infection but confers those cells with enhanced ability to manage respiratory bacterial pathogens, which are often the ultimate cause of deaths occurring in viral pandemics.

[0434] Example 10: Gut Microbiota-Generated Metabolites Reprogram Lung Macrophages and Confers Broad Resistance to Respiratory Infections

[0435] Materials and Methods

[0436] The materials and methods for obtaining the results in this Example are as described above in Example 9.

[0437] Results and Discussion

[0438] Segmented filamentous bacteria (SFB) is a common gut microbiota constituent in many animals, potentially reflecting that its impacts on the immune system, which broadly increase host resistance to infection, confer a survival advantage. SFB is less common in humans, at least in the

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[0440] West, spurring our interest in leveraging it to decrease one’s proneness to infection. That SFB induces Thl7 cells and, consequently, promotes chronic inflammatory diseases including colitis, arthritis, and osteoporosis highlights the risks of administering SFB itself. The present study investigated the infection-resistance of SFB -colonized mice, herein referred to as SFB+mice, the results of which might uncover disease-mitigating strategies. One exemplary immunologic consequence of SFB colonization, namely its epigenetic reprogramming of lung-resident alveolar macrophages (AM) was investigated, which increases resistance of mice to respiratory infection in an array of model including influenza and post-influenza bacterial pneumonia. AM from SFB- colonized mice (i.e. SFB+AM) display increased pathogen neutralization and reduced pro- inflammatory gene expression, even in response to inactivated (non-replicating) pathogens. Furthermore, SFB+AM are resistant to pathogen-induced cell death and preferentially use oxidative phosphorylation (OXPHOS). The SFB+AM phenotype is associated with epigenetic changes and, concomitantly, cognate phenotypes of SFB+AM being maintained following transplant into SFB’ mice. Yet how SFB, which in accord with it being anaerobic is restricted to the luminal surface of the intestine, reprograms AM remained unknown.

[0441] The potential role for soluble metabolites in mediating SFB -induced AM reprogramming was evaluated. SFB" mice, were orally administered fecal supernatants (FS) from SFB+mice and, one week later, inoculated with 2009 pandemic influenza virus strain A / CA / 07 / 2009 (H1N1), herein referred to as CA09. While a single administration of SFB+FS did not have a clear impact, 3 doses of it, given every other day, partially recapitulated SFB’s protection against CA09 as assessed by lung viral titers and disease symptoms (data not shown). Such moderate protection against CA09 was not accompanied by appearance of fecal SFB genomes verifying that SFB+FS administration had not transferred SFB itself. The moderate protection conferred by oral SFB+FS led to experiments testing whether gut to lung translocation of SFB metabolites, at levels sufficient to reprogram AM, may require SFB’s prolonged presence and / or that SFB plays an active role in the translocation process, consistent with observations that SFB embeds into epithelial cells and alters vesicular trafficking therein. The extent to which administrating SFB+FS intranasally, increasing its access to the lung lumen, might recapitulate the SFB+AM phenotype. As schematized (Figure 46A) SFB" mice were left untreated (negative control), colonized with SFB (positive control), or intranasally administered SFB’ FS or SFB+FS. AM were isolated one week later and phenotypic ally examined, in vitro, in response to IAV, UV-irradiated IAV, and / or S. pneumoniae. AM isolated from SFB’FS-treated mice were phenotypically similar to those from PBS-treated mice for all parameters assayed. In contrast, AM from SFB+FS-treated mice were phenotypically similar to SFB+AM in that they exhibited increased capacity to neutralize CA09, minimal UV-IAV-induced

[0442] 45764921.1 67 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT expression of pro-inflammatory cytokines (i.e. they displayed inflammatory anergy), enhanced expression of complement Clqa, OXPHOS energy phenotypes, and better survival when exposed to CA09, potentially due to having less DNA damage (Figures 46B-46D). AM reprogrammed by SFB colonization or intranasal FS administration maintained their enhanced antiviral capacity following their transplant into the lung (data not shown). Furthermore, like bone fide SFB+AM, AM from SFB+FS-treated mice were highly resistant to type I interferon-induced loss of antibacterial function. Thus, a single intranasal dose of SFB+FS, which corresponded to about l / 400,hof the intestinal luminal content, largely recapitulated the SFB+AM phenotype (Figures 46A-46D).

[0443] Intranasal administration of SFB+FS also recapitulated the infection-resistance of SFB+mice. Specifically, relative to mice administered SFB" FS, mice receiving SFB+FS were resistant to primary CA09 challenge and post-CA09 S’, pneumoniae infection as indicated by pathogen loads, immune parameters, histopathology and disease symptoms including death (Figures 46E-46J, and data not shown). As shown in Figures 46A-46J, ATAC-seq analysis demonstrated that exposure to SFB-derived metabolites altered chromatin accessibility in alveolar macrophages. The accessibility patterns closely mirrored those observed in cells directly colonized with SFB, indicating that the metabolites recapitulate the epigenetic remodeling effects of the bacteria itself. An identical pattern of results was obtained when comparing nasal administration of FS from germfree and SFB- monoassociated mice. The prophylactic protection, conferred by a single intranasal dose of SFB+FS, was largely maintained for at least 60 days and completely diminished by 120d (data not shown) indicative of it being mediated by epigenetically altering AM. Furthermore, analysis of AM via ATAC-seq found that, like SFB+AM, AM from SFB+FS-treated mice exhibited increased accessibility of genes that mediate phagocytosis (e.g., Itgam, Cd68, Itgax, Fcgrl, Fcgr2b, Fcgr3, Marco, Mrcl, Mertk, Cd36, Axl, Msrl, Cdl63, Cd93), intracellular bacterial killing (e.g., Lampl, Lamp2, Hexa, Hexb, Ctsd, Ctse, Ctsb, Atg7, Atg3, Atg5, Nbrl, Sqstml, Scarb2), and oxidative phosphorylation (OXPHOS) (e.g., Cox6a2, Cox4il, Sdhb, Sdhd, Uqcrcl, Atp5d, Atp5al, Ndufc2, Ndufv2, NdufaQ, Ndujb4). (data not shown). Concomitantly, Seahorse analysis indicated that AM from SFB+FS-treated mice displayed a metabolic phenotype similar to that of SFB+AM, namely increased reliance on OXPHOS to manage primary viral infection and thus maintaining a glycolytic reserve to manage secondary bacterial infection (data not shown).

[0444] Administration of SFB+FS up to 60h post-CA09 exposure protected against post-CA09 .S’. pneumonia infection (Figures 52F and 52G). Thus, metabolite(s) generated, or elicited by, SFB are capable of inducing the SFB+AM phenotype, resulting in better management of respiratory pathogens.

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[0446] SFB-induced AM reprogramming was recapitulated in vitro, to identify the metabolites involved and whether they act directly upon AM. SFB" and SFB+FS, serum, and bronchiolar lavage fluid (BALF) were used to “train” SFB" AM in vitro. Based on previously described AM training protocols, the AM were washed 24 h later and cultured for an additional 7d (Figures 6A-6I). The AM were then assayed in vitro or via transplant into AM-depleted SFB" mice. AM trained in this manner largely recapitulated phenotype of AM trained in vivo. Specifically, relative to AM exposed to SFB" fluids, AM trained in vitro by SFB+FS, serum or BALF displayed elevated expression of basal and UV-IAV-induced complement clqa and inflammation-suppressing tss22d3 and markedly reduced expression of an array of IFN / inflammatory genes (data not shown). Analogous to AM trained in vivo via intranasal SFB+FS, in vitro AM training could be achieved by relatively dilute specimens of SFB+FS (1:800) (data not shown). Furthermore, like bona fide SFB+AM, AM trained in vitro by SFB+FS, serum, and BALF exhibited increased capacity to neutralize CA09 in vitro. Most importantly, transplant of these AM into SFB" mice reduced their proneness to CA09 infection as assessed by disease symptoms and lung viral titers (Figures 6A-6G). These SFB+-like AM phenotypes differed markedly from that of AM trained in vitro by LPS in that the latter were hyper- inflammatory and potentiated CA09-induced disease (data not shown). Similar to intranasally administered SFB+FS, transplant of AM trained in vitro by SFB+FS also mitigated severity of post-CA09 -S', pneumoniae infection (Figures 6A-6G). When maintained in culture, in vitro-trained SFB+-like AM reverted back to an untrained phenotype within 2 weeks. In contrast, such AM maintained their phenotype for at least 90d when these cells were stored at -80°C (Figures 52A- 52C) or transplanted into SFB" mice (Figures 6H and 61). Thus, SFB generated metabolites can act directly on AM to increase their infection management capacity while the lung environment is important to maintain this phenotype.

[0447] The metabolites that drove SFB-induced AM reprogramming were investigated and identified. Live or pasteurized SFB was administered to mice. When mice received killed SFB, they lost the ability to train AM This indicates that SFB must be alive to generate the important mediators required for AM training (data not shown). A role for retinoic acid was first explored, because this molecule is produced by SFB and has been shown to have immune modulating properties. However, a pharmacologic inhibitor of retinoic acid signaling had no impact on SFB- induced AM training (data not shown) demonstrating that retinoic acid does not play a role in this case. Untargeted LC / MS-based metabolomic analysis was then performed (Figures 7A-7F and data not shown). SFB colonization of the intestine increased the abundance of hundreds of metabolites in feces, some of which required the presence of microbiota other than SFB in that they were not elicited by SFB colonization of germfree mice. SFB colonization induced fewer metabolites in

[0448] 45764921.1 69 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT serum, and fewer still in BALF. To identify which SFB-induced metabolites contributed to AM reprogramming, experiments were conducted based on the observation that SFB-induced changes in AM do not require a commensal microbiota and thus mediators of AM reprogramming should be induced by SFB colonization in both conventional and germfree mice. Additionally, alignment with results of the in vitro experiments, mediators of SFB-induced AM reprogramming were enriched in SFB+FS, serum, and BALF. Combining these criteria yielded 9 candidates, including those of presumed diet and host origin. Authentic versions of each of these metabolites (i.e., metabolites obtained from commercial sources), the extent to which they could, in vitro, by themselves, induce 6 specific features of the SFB+AM phenotype: 1) Increased CA09 neutralization. 2) Increased phagocytosis of beads and S. pneumoniae. 3) Increased S. pneumoniae killing. 4) Elevated clqa expression. 5-6) Reduced UV-IAV-induced IL-6 (5) and type I IFN expression (6). Metabolite concentrations were based on relative LC / MS signal intensity and previous studies of their bioactivity and / or serum concentration. Four of the tested metabolites showed no capacity to modulate AM by any assay tested while one, the isoflavone formononetin, moderately enhanced pathogen killing and UV-IAV-induced IL-6 expression (Figures 7B-7F). Two other candidate isoflavones, namely genistein and daidzein, which are closely related to each other, markedly increased phagocytosis and pathogen neutralization capacity while modestly suppressing IL-6. Conversely, sinapic acid starkly suppressed UV-IAV-induced type I IFN and modestly enhanced pathogen neutralization capacity. In contrast, taurine displayed significant, albeit not maximal, capacity to induce all features of SFB+AM. These results, combined with taurine being amongst most prominent SFB-induced metabolites in serum and BALF, and observations from others that reduced serum taurine levels mark severe viral infections in mice and humans, suggested taurine might be an important mediator of SFB-induced AM remodeling. Yet, that neither taurine, nor any of the other tested metabolites, fully recapitulated the ability of fluids from SFB+mice to train AM (Figures 6A-6G vs Figures 7A-7F) suggested involvement of a combination of SFB-elicited metabolites.

[0449] Training SFB" AM in vitro with a combination of taurine, genistein, daidzein, sinapic acid, and formononetin (5-mix) resulted in AM that were indistinguishable from AM trained by SFB FS based on both in vitro assays and AM transplant studies (Figures 47A-47D and data not shown). SFB-induced metabolites similarly trained mouse and human AM mice (Figures 47E-47L). Specifically, exposure of cultured human AM to 5-mix or SFB+FS, but not SFB" FS, resulted in increased pathogen neutralization capacity (CA09, S pneumoniae, and their combination), inflammatory anergy, increased phagocytosis, increased resistance to pathogen-induced death, and preferential use of OXPHOS. One factor precluding administration of SFB to humans is its

[0450] 45764921.1 70 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT promotion of Thl7 cells, which is driven, in part, by SFB’s surface antigens, evidenced by the observation that SFB promotes Thl7 cells even in vitro, wherein, being a strict anaerobe, it does not survive. In contrast to SFB itself, neither SFB FS nor the 5-metabolite fixture induced Thl7 cells in vitro Furthermore, neither SFB+FS nor 5-mix increased Thl7 cells in vivo (data not shown). Thus, SFB-generated metabolites, and authentic versions thereof, can serve to beneficially remodel AM without potentiating risk of Thl7 cell-mediated diseases.

[0451] Probing the consequence of removing each 5-mix component revealed that both taurine and sinapic acid were both needed for maximal inflammatory anergy while no individual component was important for increased pathogen neutralization (Figures 48A-48C). It was concluded that taurine and sinapic were important and then examined if addition of any of the other metabolites restored full activity. Addition of either genistein or daidzein, but not formononetin fulfilled this criterion indicating that 3 -metabolite mixtures consisting of taurine and sinapic acid and either genistein, or its close molecular relative, daidzein, could recapitulate benefits of SFB FS.

[0452] How SFB colonization resulted in increased abundance of AM-remodeling metabolites was evaluated. Measure of serum taurine by ELISA confirmed that SFB colonization increased levels of this metabolite within 3 days administration, consistent with the observation that SFB protects against IAV infection when administered as little as 2d prior to IAV inoculation. A sublethal, but not a lethal, dose of CA09 led to a marked increase in serum taurine in SFB" mice that was evident by 8d post-inoculation. Serum taurine returned to baseline levels day 12 post-IAV inoculation. In contrast, the elevated taurine levels exhibited by SFB+mice were not impacted by CA09 infection. A similar pattern of results was seen for BALF taurine levels (Figure 49 A). Collectively, these results accord with the notion that lAV-induced taurine helps resolve respiratory viral infection and, furthermore, contributes to the SFB-induced AM reprogramming that aids in its management.

[0453] That taurine-conjugated bile acids are abundant in the murine ileum in general and were slightly reduced by SFB colonization suggested a possible role for bile salt hydrolase (BSH), an enzyme possessed by SFB (and many other commensal bacteria), in mediating the SFB-induced increase in taurine levels. Consistent with this notion, colonizing mice with SFB led to an increase in fecal BSH activity that correlated with increased serum taurine, measured by ELISA (data not shown). Oral administration of a gut-restricted BSH inhibitor markedly lowered serum taurine levels in SFB+mice. Furthermore, BSH inhibition markedly reduced the extent to which SFB+FS was able to reprogram AM toward a SFB+AM phenotype in vitro, as assessed via in vitro readouts and AM-transplant studies. Thus, BSH-generated taurine is a key soluble mediator of SFB-induced AM reprogramming (Figure 49B and data not shown). Other than taurine, SFB-induced AM- reprogramming metabolites seemed likely to have derived from their standard grain-based chow

[0454] 45764921.1 71 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT

[0455] (GBC) diet. Indeed, major ingredients of GBC include soy and oats, which contain genistein, daidzein, and formononetin, respectively. It was tested whether feeding mice compositionally defined diets (CDD) made from highly refined ingredients expected to lack these phytochemicals might prevent SFB-induced AM training. Testing was not straight forward in that CDD feeding alters microbiota in a manner that strongly impedes SFB colonization, which would make lack of AM training in such mice uninformative. To surmount this hurdle, Altered Schaedler Flora (ASF) mice were used, which harbor a defined minimal microbiota that does not contain phyla that impact SFB. It was observed that SFB colonized ASF mice fed GBC or CDD at similar levels (Figure 49C). Such SFB colonization associated with similarly increased fecal taurine levels in ASF mice fed CDD and GBC, indicating that SFB-induced taurine was of host, rather than dietary, origin. In accord with taurine being an important mediator of inflammatory anergy, training of AM by FS, serum and BALF isolated from SFB+ASF reduced UV-IAV induced gene expression irrespective of diet. In contrast, SFB+specimens from CDD-fed mice did not increase capacity of AM to neutralize CA09, indicating an importantrole for diet-derived phytochemicals in SFB-induced AM reprogramming.

[0456] The capacity of the 3 SFB-induced metabolites (arbitrarily selecting genistein over daidzein) to protect mice against CA09 infection using dose ranges based on the metabolomics data and previous studies. SFB" mice were administered vehicle (PBS), taurine (0.2-200 mg / kg), genistein (0.02-200 mg / kg), or sinapic acid (0.01-100 mg / kg) (Figures 54A-54Q). 7d later, mice were inoculated with CA09. All mice in vehicle group and no mice given the higher doses of any of the 3 compounds, died by d4, at which time all mice were euthanized to permit in-depth assessment. A parallel experiment found that taurine also fully protected against CA09-induced death through day 14 (data not shown). The protection against mortality conferred by taurine but not genistein or sinapic acid associated with reduced hypothermia. Post-mortem analysis indicated that all 3 compounds ameliorated gross and microscopic lung pathology. Moreover, the benefits of the compounds correlated with reduced CA09-induced AM deficits. Minimal dose for optimal protection for taurine, genistein and sinapic acid was, respectively, 20, 2, and 1 mg / kg respectively. The compounds also varied in the extent to which they recapitulated specific aspects of SFB+mouse phenotype. Similar to the in vitro results, sinapic acid strongly suppressed CA09-induced lung pro-inflammatory gene expression but only slightly lowered lung viral titers. Genistein did the converse (stark lowering of titers and modestly reducing pro-inflammatory gene expression while taurine moderately impacted all of these parameters. Thus, similar to the in vitro studies, these results suggested that beneficial impacts of SFB colonization would best be recapitulated by a combination of metabolites.

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[0458] Intranasal administration of live SFB, or SFB+FS or or 3-metabolite mixtures containing taurine, sinapic acid, and genistein or daidzein fully recapitulated the ability of SFB+FS to protect mice against CA09 infection and approached the level of protection conferred by SFB colonization as assessed by pathogen loads, inflammation, symptoms, and mortality (Figures 50A-50C). Like SFB and SFB+FS, these metabolite mixtures provided stark protection against post-CA09 S. pneumoniae infection. Such protection by these mixtures of authentic metabolites mice was not specific to healthy mice but, rather, extended to mice with diabetes and metabolic syndrome, both of which increased risk for severe respiratory infection (Figures 5OD-5OF and Figures 55A-55H). 2- metabolite mixtures, namely taurine / sinapic, taurine / genistein, and sinapic / genistein were modestly to moderately less effective (Figures 50A-50C). Of note, orally given TGS at high dose can also mimicking the protective effect of SFB+FS (data not shown). The protection provided by a single administration of taurine, genistein and sinapic acid, henceforth referred to as TGS was fully maintained for 60d, partially evident for 90d, and gone by 120d (Figures 50E and 50F). This duration accords with the notion that the benefit was mediated by epigenetic reprogramming of AM and suggests the possible use of TGS to provide seasonal non-specific protection against respiratory infection. Administration of a second dose of TGS 120d following the initial dose provided protection similar to that conferred by the original dose. Such ability of a second TGS administration to restore protection was not impeded by CA09 infection occurring in the interim between the doses (Figure 50G, data not shown). As shown in Figures 51A-51C, mice pretreated with live SFB exhibited markedly reduced pathogen titers following challenge with infectious agents compared to PBS-treated controls. Notably, SFB-treated mice remained asymptomatic despite infection, consistent with enhanced pathogen resistance. Further, as demonstrated by the data in Figures 50A-50C, administration of TGS yielded a comparable protective effect, similarly lowering pathogen titers.

[0459] The lungs of young humans, and naive mice of any age maintained in SPF vivaria, are predominately derived from fetal liver cells. In contrast, many adults, whom, unlike naive SPF mice, have typically been exposed to a variety of respiratory pathogens over their lifetimes resulting in a major portion of their AM being of bone marrow origin. The extent to which TGS could protect a host that had been subjected to irradiation was investigated, followed by bone marrow administration, resulting in chimeric mice in which nearly all of the AM were of bone marrow origin (data not shown). Such mice were protected by TGS similar to naive mice. A variant of this experiment in which lungs were shielded from radiation resulted in mice retaining their endogenous AM irrespective of TGS administration indicating that, like SFB itself, TGS trains lung-resident AM and does not involve recruitment of bone marrow derived cells. Lastly, it was noted that in

[0460] 45764921.1 73 ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT such chimeras, an added macrophage surface label remained undiluted on AM over the course of TGS applied over a 120d period suggesting that the second TGS dose had retrained the same AM rather than trained their daughter cells. Figures 52A-52E show that AM trained by metabolites in vitro can hold their trained phenotype in freezers (Figures 52A-52C) or following transplant (Figures 52D and 52E). This demonstrated a role for therapeutic modalities that would involve AM transplant. Figures 53A-53D show in vitro concentration-dependence of AM training for the three compounds: taurine, genistein, and sinapic acid. This data reinforced that these metabolites acted on AM.

[0461] Figures 54A-54Q show dose-dependent protection by each compound in vivo. Figures 55A- 55H show oral efficacy of a combination of taurine, genistein, and sinapic acid (TGS) can recapitulate impacts of SFB.

[0462] In conclusion, colonization of the intestine by SFB, results in BSH-mediated generation of taurine and liberation of food-derived phytochemicals, including genistein, daidzein, and sinapic acid, which are translocated via circulation to the lung lumen wherein they epigenetically alter AM. Such alterations increase AM OXPHOS usage, phagocytosis, and complement expression while suppressing type 1 IFN / pro-inflammatory signaling. Such reprogramming markedly improves the capacity of AM to manage respiratory pathogens and avoid inflammatory pathology. A single intranasal administration of these SFB -generated metabolites can largely mimic the benefits of SFB colonization and thus might be developable as practical safe means of providing broad seasonal protection against an array of respiratory pathogens.

[0463] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0464] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

[0465] 45764921.1 74

Claims

ATTORNEY DOCKET NO. GSURF 2025-005-03 PCTCLAIMSWe claim:

1. A composition for treating one or more symptoms associated with a respiratory disease or disorder in a subject, the composition comprising one or more metabolites, optionally two or more metabolites, and one or more pharmaceutically acceptable carriers and / or excipients, wherein the one or more metabolites are in an effective amount to reduce viral or bacterial replication, in the subject by 5% to 70% within 48 hours of administration.

2. The composition of claim 1 , wherein the one or more metabolites are one or more amino acids, one or more sugars, one or more flavonoids, or one or more organic acids, or a combination thereof, optionally wherein the one or more metabolites are selected from the group consisting of taurine, cysteamine, N-acetylglucosamine, nicotinic acid, aminolevulinic acid, malonic acid, sinapic acid, daidzein, formononetin, and genistein, optionally wherein one or more metabolites are taurine, cysteamine, N-acetylglucosamine, nicotinic acid, aminolevulinic acid, malonic acid, sinapic acid, daidzein, formononetin, and genistein.

3. The composition of claim 1 or 2, wherein the one or more metabolites comprise an amino acid, an organic acid, and an isoflavonoid,4. The composition of claim 2, wherein the one or more amino acids comprise sulfur- containing amino acids (e.g., methionine, cysteine, homocysteine, taurine, cysteamine, etc.), optionally wherein the one or more amino acid comprise taurine and / or cysteamine.

5. The composition of claim 2, wherein the one or more sugar comprise N- acetylglucosamine.

6. The composition of claim 2, wherein the one or more organic acids comprise nicotinic acid, aminolaevulinic acid, sinapic acid, and / or malonic acid, optionally wherein the organic acid is sinapic acid.

7. The composition of claim 2, wherein the one or more flavonoids are isoflavonoids, neoflavonoids, and / or bioflavonoids.

8. The composition of claim 7, wherein the isoflavonoids are daidzein, formononetin, and / or genistein, optionally wherein the isoflavonoid is genistein.

9. The composition of claim 1, wherein the one or more metabolites comprise taurine and sinapic acid.

10. The composition of claim 1, wherein the one or more metabolites comprise taurine, sinapic acid, genistein.

11. The composition of claim 1, wherein the composition is in the form of a solid or a liquid.45764921.1 75ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT12. The composition of claim 1, wherein the composition is in a form suitable for intranasal administration or oral administration.

13. The composition of claim 1, wherein the composition is in the form of a nasal spray.

14. The composition of claim 1, wherein when administered to a subject, the total amount of the one or more metabolites in the composition is effective to reduce alveolar macrophage cell death, reduce viral levels in lung epithelial cells, and reduce lung viral titer, as indicated by number of alveolar macrophages and TCIDso / mL of virus, respectively, measured in a biological sample of the subject, compared to a control sample of the subject before administration, optionally wherein the biological sample is a serum sample, optionally wherein the serum sample is a blood sample or a bronchoalveolar lavage fluid sample.

15. The composition of claim 1, further comprising one or more active agents that are different from the metabolites.

16. The composition of claim 15, wherein the one or more active agents are anti-inflammatory agents, antiviral agents, antibiotic agents, antipyretic agents, analgesic agents, blood glucose regulators, gastrointestinal agents, or respiratory tract agents, or a combination thereof.

17. A method for treating one or more symptoms associated with a respiratory disease or disorder in a subject in need thereof, comprising:(i) administering to the subject the composition of any one of claims 1-16, optionally wherein the administration step is repeated one or more times.

18. The method of claim 17, wherein following the administration or optionally all of the administrations, the total amount of the metabolites administered to the subject is effective to increase gut microbiota of the subject, as indicated by an increase in the number of segmented filamentous bacteria measured in a biological sample of the subject, compared to a control sample of the subject prior to the administration of the pharmaceutical formulation, optionally wherein the biological sample is a fecal sample or a serum sample.

19. The method of claim 17, wherein the respiratory disorder is a respiratory infection.

20. The method of claim 19, wherein the respiratory infection is caused by a microorganism selected from the group consisting of a virus, a bacterium, a protozoan, and a fungus.

21. The method of claim 19, wherein the respiratory infection is caused by a virus selected from the group consisting of orthomyxovirus, rhinovirus, paramyxovirus, coronavirus, adenovirus, human metapneumovirus (hMPV), enterovirus, and bocavirus.

22. The method of claim 21, wherein the orthomyxovirus is selected from the group consisting of influenza A, influenza B, and influenza C.45764921.1 76ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT23. The method of claim 21, wherein the paramyxovirus is selected from the group consisting of Respiratory syncytial virus (RSV) and parainfluenza virus.

24. The method of claim 21, wherein the coronavirus is selected from the group consisting of SARS-CoV, MERS-CoV, and SARS-CoV-2 (COVID-19).

25. The method of claim 17, wherein the respiratory disease or disorder is selected from the group consisting of cystic fibrosis, chronic obstructive pulmonary disease, pulmonary fibrosis, pneumonia, and asthma.

26. The method of claim 17, further comprising a step of administering a second active agent prior to, during, or after step (i).

27. The method of claim 17, wherein the composition is administered once every week, once every two weeks, once a month, once every two months, once every three months, once every four months, once every five months, or once every six months, for a time period from 1 day to 1 year, from 1 day to 9 months, from 1 day to 6 months, from 1 day to four weeks, from 1 day to three weeks, from 1 day to two weeks, from 1 day to 1 week, or from 1 day to 3 days or wherein the composition is administered irregularly for at least two time, at least three times, at least four times, at least five times, at least ten times, at least 20 times, or at least 30 times, within a time period of one week, one month, six months, 1 year, 2 years, 3 years, or 5 years.

28. The method of claim 17, wherein the composition is administered by parenteral injection, intranasal administration, or by oral administration.

29. The method of claim 17, wherein the subject is a mammal.

30. The method of claim 17, wherein the subject is immunocompromised.

31. The method of claim 17, wherein the composition administered to the subject is at a dose from 0.1 mg to 250 mg for each administration.

32. The method of claim 17, wherein following the administration or optionally all of the administrations, the total amount of the metabolites administered to the subject is effective to reduce cell death of alveolar macrophages by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, as indicated by immunohistochemistry of a serum sample, optionally wherein the serum sample is a blood sample or a bronchoalveolar lavage fluid sample.

33. The method of claim 17, wherein following the administration or optionally all of the administrations, the total amount of the metabolites administered to the subject is effective to increase C1QA expression and / or reduce Notch4 expression by 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, or 80-fold, as indicated by quantitative real-45764921.1 77ATTORNEY DOCKET NO. GSURF 2025-005-03 PCT time PCR of a serum sample, optionally wherein the serum sample is a blood sample or a bronchoalveolar lavage fluid sample.

34. The method of claim 17, wherein following the administration or optionally all of the administrations, the total amount of the metabolites administered to the subject is effective to reduce viral-induced expression of IFNP and / or IL-6 by 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, or 80-fold, as indicated by quantitative real-time PCR of a serum sample, optionally wherein the serum sample is a blood sample or a bronchoalveolar lavage fluid sample.

35. A method for preventing a secondary infection following a primary disease or disorder in a subject in need thereof, comprising:(i) administering to the subject the composition of claim 1, optionally wherein the administration step is repeated one or more times.

36. A method for preventing a secondary infection following a primary disease or disorder in a subject in need thereof, comprising:(i) administering to the subject a pharmaceutical formulation comprising one or more segmented filamentous bacteria (SFB), optionally wherein the administration step is repeated one or more times.

37. The method of claim 35 or 36, wherein the pharmaceutical formulation is administered after the subject shows one or more symptoms of the primary disease or disorder, optionally within a week, 3 days, 1 day, 12 hours, 5 hours, 2 hours, or 1 hour after the onset of the one or more symptoms of the primary disease or disorder.

38. The method of claim 35 or 36, wherein the primary disease or disorder is caused by a virus, bacteria, or ailment (e.g., diabetes or cystic fibrosis) and / or wherein the secondary infection is caused by a virus or bacteria, optionally wherein the virus or bacteria is Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae.

39. The method of claim 35 or 36, wherein following the administration, or optionally all of the administrations, the total amount of the SFB administered to the subject is effective to prevent the secondary infection by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, as indicated by quantitative real-time PCR of a serum sample, optionally wherein the serum sample is a blood sample or a bronchoalveolar lavage fluid sample of the subject, compared to a control (e.g., a subject having the same primary disease / disorder but not administered with the pharmaceutical formulation).

40. The method of claim 35 or 36, wherein following the administration or optionally all of the administrations (when more than one administration is performed), the total amount of the45764921.1 78ATTORNEY DOCKET NO. GSURF 2025-005-03 PCTSFB administered to the subject is effective to prevent the secondary infection in the subject, as indicated by the absence of symptoms associated with the secondary infections, compared to a control (e.g., a subject that is infected with the same primary disease / disorder, but not administered with the pharmaceutical formulation).45764921.1 79

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