Antiviral gene-activating polypeptide and lactobacillus johnsonii n6.2-derived extracellular vesicles enriched therewith

Abstract

A composition is provided herein, including purified proteins, genetically engineered proteins and extracellular vesicles (EVs) isolated from a naturally occurring probiotic Lactobacillus sp. wherein the EVs include specific proteins or polypeptides that induce production of antiviral genes. This includes genetically modified peptides to enhance the expression of antiviral genes in mammals and chicken macrophages. Also, the polypeptides or EVs can be packaged into an emulsion, or tablet such that the packaged polypeptides or EVs are protected in a gastrointestinal tract for transport to an intestine of a subject. In another embodiment, a method for preventing viral infections by administering compositions to a subject in need is presented.

Description

Attorney Ref. No.10457-591PC0 Antiviral gene-activating polypeptide and Lactobacillus johnsonii N6.2-derived extracellular vesicles enriched therewith GOVERNMENT SUPPORT STATEMENT

[0001] This invention was made with government support under Grant No. R01DK121130 awarded by National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0002] The importance of the microbiota as modulators of host immune responses has been established. However, a significant gap in knowledge is the identity of bacterial effector molecules that mediate specific immunological effects in the host.

[0003] Bacterial extracellular vesicles (EVs) have increasingly been in the forefront of research as mediators of host:microbe interactions. EVs are ubiquitously produced in all domains of life. Commensal microbiota derived EVs account for the largest and constant interactions with the host. Therefore, there is a significant need to elucidate the mechanisms involved in commensal- and probiotic bacteria derived EVs’ participation in interkingdom communication with the host.

[0004] It has been recently shown that the probiotic bacteria L. johnsonii N6.2 releases EVs with a differential composition of proteins, and lipids when compared to whole cells. L. johnsonii N6.2 is a probiotic bacterium that has been shown to mitigate type 1 diabetes in prone rodents (BBDP rat model) by maintaining euglycemic levels and reducing the inflammatory state (REF).

[0005] It is hypothesized that EVs play a central role in delivering bioactive molecules that may act as mechanistic effectors in immune modulation. We observed that the addition of EVs to the human pancreatic cell line βlox5 reduced cytokine-induced apoptosis (45). The role of EVs on beta cell function was further evaluated using primary human pancreatic islets. It was found that EVs significantly increased insulin secretion in the presence of high glucose concentrations. Through RNAseq analyses, increased expression of CYP1A1, CYP1B1, AHRR, and TIPARP genes in the aryl hydrocarbon receptor (AHR) pathways were found to be significantly induced in the presence of EVs. These increases positively correlated with increased GLUT6 and SREBF1 mRNA and positively correlated with reduced oxidative stress markers (45).Attorney Ref. No.10457-591PC0

[0006] Notably, it was found that incubation of βlox5 cells with L. johnsonii EVs also resulted in a significant induction of the 2’,5’-oligoadenylate synthetase (OAS). The OAS pathway is an IFN-stimulated antiviral response activated by viral or bacterial RNA (17). In mammals, the OAS family is composed of three enzymatically active enzymes, OAS1, OAS2 and OAS3, all of which were significantly induced in the presence of EVs, but not by purified membranes from L. johnsonii N6.2 (46). Similar results were observed for OASL, which is induced by RIG-1 and MAVS oligomerization and is crucial in stabilizing the OAS complex. Taken together, these findings suggested that nucleic acids present in NVs are sensed by the βlox5 cells. Other genes involved in the sensing and response to nucleic acids, such as IFI44L, MX1, MX2 and DDX60, were also induced in the presence of EVs.

[0007] The OAS pathway plays a crucial role in the cells’ antiviral response by creating 2′,5′- oligoadenylates that can activate the latent cellular RNase L causing the degradation of the RNA viral genome. To prevent the host response, some viruses have developed strategies to overcome the OAS pathway antiviral effects. For example, the NS1 protein in Influenza binds double stranded RNA inhibiting OAS activation.

[0008] Recent reports have shown that EVs of the commensals Enterobacter cloacae and Bacteroides thetaiotaomicron reduce the replication of murine norovirus through the induction of antiviral cytokines IL6, TNFα, IL1β and IFNɣ. A recent study has shown that the DNA from commensal Escherichia coli strain EVs can activate the cGAS-STING pathway to induce an antiviral response in mice. While a protective antiviral response was induced, these studies also showed that the commensal EV response culminated in the expression of pro-inflammatory cytokines, IFNβ. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1. L. johnsonii N6.2 secreted EVs inhibit MNV replication in RAW 264.7. A-B) Relative copies of MNV genome was determined by qRT-PCR at 18 hpi either in host cells (A) or RNA released into the supernatant (B). C) LHD quantification in supernatant. Activity is expressed as the percentage change (delta %) with respect to the untreated control. C-D) Effect of adding the EVs before (pre), with (co), or after (post) MNV inoculation was evaluated on MNV replication either after (C) 6 hpi and (D) 18 hpi in RAW 264.7 cells. (F-I) The analysis of the murine antiviral genes OAS1b, OASL1, MX1 and IFI44L was performed by qRT-PCR 6 hpi. Different letters onAttorney Ref. No.10457-591PC0 top of each bar indicates statistical significance of p≤0.001 from ANOVA analysis and post-hoc Tukey test performed on at least three biological replicates (with two qRT-PCR technical replicates each).

[0010] Figure 2. Administration of L. johnsonii N6.2 EVs alters the cytokine expression profile induced by MNV 18 hpi in RAW 264.7 cells. A-F) Analysis of mRNA levels of the murine cytokine genes IFNA1, IFNA4, IFNB, IFNL2, IFNL3 and IL10, respectively, by qRT-PCR. Different letters on top of each bar indicates statistical significance of p≤0.05 from ANOVA analysis and post-hoc Tukey test performed on three biological replicates (with two qRT-PCR technical replicates each).

[0011] Figure 3. The SH3B2 domain of Sdp stimulate the expression of MIP-2 and ISRE54 promoters in the RAW-DUAL KO-TLR4 cells. Proteins enriched in EVs were purified and tested at 1.5 ug / ml in RAW-DUAL KO-TLR4 cells. After 6 h of incubation, the stimulation of the MIP- 2 promoter fusion to supernatant alkaline phosphatase, SEAP) and interferon signaling (ISRE promoter fusion to Lucia luciferase) was evaluated. (A) SEAP activity is expressed as OD at 600 after 18 h. B) Lucia luciferase relative luminescence expressed as percentage relative to LPS stimulated cells used as negative control. Different letters on top of each bar indicates statistical significance of p≤0.05 from ANOVA analysis and post-hoc Tukey test performed on three to six biological replicates.

[0012] Figure 4. SH3B2 mitigates MNV-1 infection in a dose dependent manner. Increasing concentrations of purified SH3B2 (0.015 to 1.5 ug / ml) were added to RAW 264.7 cells and infected with MNV-1. 6 h dpi the cellular response as well as the viral levels were quantified by qRT-PCR. A) Cell lysis assessed by LHD quantification in supernatant. Activity is expressed as the percentage change (delta %) with respect to the untreated control. B) Relative copies of MNV genome; as well as C) Oas1b, and D) IL10 expression were quantified by qRT-PCR. Different letters on top of each bar indicates statistical significance of p≤0.05 from ANOVA analysis and post-hoc Tukey test performed on three to six biological replicates.

[0013] Figure 5. SH3B2 and EVs mitigates MNV-1 infection by stimulation of pathways mediated by the TLR2 / TLR4. Purified SH3B2 (1.5 ug / ml), EV10, and purification MOCK and buffer controls were added to RAW 264.7 cells. The stimulation of the expression of the mRNA levels of OAS1b and IL10 genes was evaluated after 6 h in wild type murine macrophages (A, G) as well as in macrophages derived from knockout mice in the TLR2 (B, H), TLR4 (C, I), TLR2 / TLR4 (D,Attorney Ref. No.10457-591PC0 J), TLR3 (E, K) and TLR9 (F, L), respectively. The same setup was infected with MNV-1 (M, N, O, P, Q, R, respectively), and the MNV-1 genome titers were quantified 18 hpi by qRT-PCR. Asterisks on top on top of each bar indicate statistical significance of p≤0.05 from ANOVA analysis and post-hoc Tukey test performed on three to six biological replicates.

[0014] Figure 6. SH3B2 and EVs mitigates MNV-1 infection by stimulating pathways mediated by the TRIF / TRAM and MYD88 adaptors. Purified SH3B2 (1.5 ug / ml), EV10, and purification MOCK and buffer controls were added to RAW 264.7 cells. The stimulation of the expression of the mRNA levels of OAS1b and IL10 genes was evaluated after 6 h in wild type murine macrophages (A, G) as well as in macrophages derived from knockout mice in the TRIF / TRAM (B, F), MAL (C, G) and MYD88 (D, J), IRF3 (E, K) and IRF7 (F, L) respectively. The same setup was infected with MNV-1 (M, N, O, P respectively), and the MNV-1 genome titer was quantified 18 hpi by qRT-PCR. Asterisks on top of each bar indicate statistical significance of p≤0.05 from ANOVA analysis and post-hoc Tukey test performed on three to six biological replicates.

[0015] Figure 7. In vivo evaluation of the efficacy of EV, SH3B2 and L. johnsonii N6.2 on MNV- 1 infection. Six-weeks old C57BL / 6 mice were orally administered L. johnsonii 108CFU, EV10, SH3B2, Liposomes or Liposomes loaded with SH3B2 daily for seven days (n=3 / group). Mice were then infected with MNV-1 or mock virus for 24 h. MNV was quantified by plaque assays (A) and by qRT-PCR (B). Asterisks on top of each bar indicate statistical significance of p≤0.05 from ANOVA analysis and post-hoc Tukey test performed on three to six biological replicates.

[0016] Figure 8. Dose-dependent activation of antiviral genes in RAW 264.7 cells by L. johnsonii N6.2 EVs after 6 h of incubation (A-F) or after MNV-1 infection (18 hpi). qRT-PCR analysis of the murine genes (A, G) OAS1b, (B, H) OAS2, (C, I) OASL1, (D, J) MX1, (E, K) MX2 and (F, L) IFI44L. Different letters on top of each bar indicate statistical significance of p≤0.05 from ANOVA analysis and post-hoc Tukey test performed on three biological replicates (with two qRT-PCR technical replicates each).

[0017] Figure 9. (A) Schematic representation of the location of the repetitive domains selected in Sdp and Muc. (B) SDS PAGE of the purified proteins.

[0018] Figure 10. Differential expression levels of OAS1 (A) and IL10 (B) in all the macrophage cell lines tested. The expression of the mRNA levels of OAS1b and IL10 genes was normalized to the Wild type (WT) macrophage cell line. C, effect of the deletions of cell receptors and pathway adaptor proteins on MNV-1 infection capabilities. The MNV-1 genome titer was quantified 18 hpiAttorney Ref. No.10457-591PC0 by qRT-PCR. Values were normalized t the WT macrophage cell line. Asterisks on top of each bar indicate statistical significance of p≤0.05 from ANOVA analysis and post-hoc Tukey test performed on three to six biological replicates.

[0019] Figure 11. The SH3B2 domain of Sdp stimulate the expression of MIP-2 and ISRE54 promoters in the RAW-DUAL KO-TLR4 cells. Wild type SH3b2 or mutant proteins were purified and tested at 0.6 ug / ml in RAW-DUAL KO-TLR4 cells. After 6 h of incubation, the stimulation of the MIP-2 promoter fusion to supernatant alkaline phosphatase, SEAP) and interferon signalling (ISRE promoter fusion to Lucia luciferase) was evaluated. (A) Activity is expressed as arbitrary units. B) Relative luminescence expressed as percentage relative to LPS stimulated cells used as negative control. Different letters on top of each bar indicate statistical significance of p≤0.05 from ANOVA analysis and post-hoc Tukey test performed on three to six biological replicates.

[0020] Figure 12 is a graph showing the expression of the innate response antiviral genes DHX58, OASL, MDA5, MAVS, MX1 and IRF7 in vitro using chicken-derived macrophages exposed to SH3b2. It was found that the addition of 0.6 ug / well of purified SH3b2 significantly induces the expression of OASL, MDA5, MX1 and IRF7. DETAILED DESCRIPTION

[0021] According to certain embodiments, disclosed is a method for treating an infection (e.g. viral infection) that involves administering a therapeutically effective amount of a pharmaceutical composition comprising at least one polypeptide comprising at least one sequence selected from SEQ ID NOs: 1-6, or fragments or variants thereof, to a subject in need. In a specific embodiment the polypeptide comprises SEQ ID NO: 2, or fragments thereof, or variants thereof, or optionally, wherein the polypeptide comprises SEQ ID NO. 2, a fragment thereof, or variant having at least 90% or 95% identity therewith. In a more specific embodiment, the pharmaceutical composition comprises extracellular vesicles comprising the at least one polypeptide. In certain examples, the pharmaceutical composition may be administered orally or parenterally.

[0022] In certain embodiments, the therapeutically effective amount is an amount sufficient to induce increased expression of an OAS1b, OAS2, OASL, MX1, MX2 and / or IFI44L gene in theAttorney Ref. No.10457-591PC0 subject. In specific embodiments, the therapeutically effective amount of the composition comprises EVS at 0.0001 μg / kg to 1.0 mg / kg the subject's body weight. Alternatively, or additionally, the pharmaceutical composition is formulated into a capsule containing the EVs. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. In a more specific embodiment, the pharmaceutical composition comprises EVs produced by a Lactobacillus sp, optionally L. johnsonii, and wherein the EVs are enriched for EVs that comprise an increased amount of sdp or a polypeptide comprising a sequence of SEQ ID NOs 1- 6.

[0023] Other embodiments disclosed herein are directed to a pharmaceutical composition comprising an isolated polypeptide comprising an amino acid sequence of SEQ ID NOs: 1-6, or fragment or variant thereof, and optionally a pharmaceutically acceptable carrier. The pharmaceutical composition may be formulated for parenteral administration or oral administration. In specific embodiments, the pharmaceutical composition comprises an isolated polypeptide having SEQ ID NO: 2, or a fragment or variant thereof.

[0024] Another embodiment relates to an isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 2, or a fragment or variant thereof.

[0025] Yet further embodiments pertain to a composition comprising EVs from a Lactobacillus sp., produced by: culturing Lactobacillus sp cells in media; centrifuging the media containing Lactobacillus sp. cells to produce a supernatant; retrieving EVs from the supernatant to obtain an EV sample; and enriching the EV sample for EVs having a higher sdp or sh3b domain content; and optionally, packaging the EVs in a pharmaceutically acceptable vehicle. In one example, the Lactobacillus sp. comprises Lactobacillus johnsonii. In a more specific embodiment, the Lactobacillus johnsonii is Lactobacillus johnsonii N6.2 strain. Overview

[0026] Based on our trafficking results where we observed that L. jonhnsonii are uptaken and released in the cytoplasm of the host cell within 30 min, we hypothesized that a preemptive activation of the OAS pathway by the EVs from L. johnsonii N6.2 could hinder a viral insult. To test this hypothesis, in this work the murine norovirus (MNV) was used as an RNA virus modelAttorney Ref. No.10457-591PC0 of infection. As L. johnsonii N6.2 is a probiotic that resides and shed its EVs in the gastrointestinal tract, the MNV provides a biological relevant model of infection as norovirus infects the gastrointestinal tract leading to severe diarrhea and other complications in individuals that are immunocompromised. Furthermore, based on our previous proteomic analyses, we propose that an enriched protein the EVs, act as the mediator of antiviral immunity observed. To this end we purified several proteins that are enriched in the EV of L. johnsonii N6.2 to identify the SH3B2 domain in Sdp as an activator of innate immune responses in vitro and in vivo. Definitions:

[0027] The term “apoptotic bodies” is used herein with reference to a type of extracellular vesicle with a diameter of from 1000 to 5000 nm that are specifically released by cells undergoing apoptosis.

[0028] As used herein, by the term “effective amount,” “amount effective," “therapeutically effective amount,” or the like, it is meant an amount effective at dose / dosages and for period(s) of time necessary to achieve the desired result. These terms refers to an amount of an enumerated agent, which, when administered or co-administered in a proper dosing regimen, is sufficient to reduce or ameliorate the severity, duration, or progression of the disorder being treated (e.g.,viral infection), prevent the advancement of the disorder being treated (e.g.,viral infection), cause the regression of the disorder being treated (e.g.,viral infection), or enhance or improve the prophylactic or therapeutic effects(s) of another therapy. The full therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations per day for successive days. In a specific example, the therapeutic amount is a dose / dosages and for period(s) of time to increase expression of endogenous antiviral genes in a subject. Examples of antiviral genes include OAS1b, OAS2, OASL1, MX1, MX2, IFI44L, INFalpha, INFbeta, INFLambda

[0029] The term “exosome” is used herein with reference to an extracellular vesicle of heterogeneous multivesicular origin that are from 20 nm to 100 nm in diameter and contain mRNA, miRNA, DNA and proteins.

[0030] The term “extracellular vesicle” or “EV”, is used herein with reference to a heterogeneous membrane vesicle of plasma membrane origin ranging from 30 to 5,000 nm inAttorney Ref. No.10457-591PC0 size. EVs includes exosomes, microvesicles, and apoptotic bodies. EVs are released under physiological conditions, upon cellular activation, senescence, and apoptosis.

[0031] The term “fragment” as used in the context of amino acid sequences refers to segment of contiguous amino acid residues of a given amino acid sequence. Fragments may be at least from 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 120, 130, or 140 amino acids in length, or greater. Such fragments may be used to produce chimeric agents as described in more detail below.

[0032] The term “microvesicle” or “MV” is used herein with reference to extracellular vesicles of plasma membrane origin that are 100 to 1000 nm in diameter and contain mRNA, miRNA, DNA, and proteins. For bacteria, microvesicles are often referred to as “bacterial extracellular vesicle” or bEV or simply EV

[0033] “Parenteral administration” means administration by a manner other than through the digestive tract. Parenteral administration includes topical administration, subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g. intrathecal or intracerebroventricular administration. Administration can be continuous, or chronic, or short or intermittent. The term “parenteral formulation” encompasses sterile liquid formulations of Sdp or sh3b domain (or fragments or variants thereof) intended for parenteral administration.

[0034] “Percent (%) sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0035] “Pharmaceutical composition” means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition can comprise one or more active agents and a sterile aqueous solution.Attorney Ref. No.10457-591PC0

[0036] “Pharmaceutically acceptable carrier” means a medium or diluent that does not interfere with the structure or function of the oligonucleotide. Certain, of such carries enable pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspension and lozenges for the oral ingestion by a subject. Certain of such carriers enable pharmaceutical compositions to be formulated for injection or infusion. For example, a pharmaceutically acceptable carrier can be a sterile aqueous solution.

[0037] The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The term also includes variants on the traditional peptide linkage joining the amino acids making up the polypeptide. Preferred “peptides”, “polypeptides”, and “proteins” are chains of amino acids whose alpha carbons are linked through peptide bonds. The terminal amino acid at one end of the chain (amino terminal) therefore has a free amino group, while the terminal amino acid at the other end of the chain (carboxy terminal) has a free carboxyl group. As used herein, the term “amino terminus” (abbreviated N-terminus) refers to the free α-amino group on an amino acid at the amino terminal of a peptide or to the α-amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the peptide. Similarly, the term “carboxy terminus” refers to the free carboxyl group on the carboxy terminus of a peptide or the carboxyl group of an amino acid at any other location within the peptide. Peptides also include essentially any polyamino acid including, but not limited to peptide mimetics such as amino acids joined by an ether as opposed to an amide bond.

[0038] The terms “treat”, “treating” or “treatment of” as used herein refers to providing any type of medical management to a subject. Treating includes, but is not limited to, administering a composition to a subject using any known method. for purposes such as curing, reversing, alleviating, reducing the severity of, inhibiting the progression of, or reducing the likelihood of a disease, disorder, or condition or one or more symptoms or manifestations of a disease, disorder or condition.

[0039] The term “variant” as used herein in the context of a nucleic acid sequence or amino acid sequence that has at least 80% identity to that sequence based on amino acid identity. More preferably, the polypeptide has at least 85% or 90% and more preferably at least 95%, 97% orAttorney Ref. No.10457-591PC0 99% identity based on amino acid identity to the amino acid sequence. There may be at least 80%, for example at least 85%, 90% or 95%, amino acid identity over a stretch of 40 or more, for example 60, 80, 100, 120, 140 or 160 or more, contiguous amino acids (“hard homology”). Sequences

[0040] Provided below are sequences that are related to composition and use embodiments described herein: sh3b domain amino acid sequences (bolded area indicates the SH3 domain). >sh3b1 (SEQ ID NO: 1) PAEKPNTTTNVNSDWTKQNGVFVTGGAINLRTGASTDSKVITQLPANSEVKYDAYRTIGQ YTWLRQPRANNEYGYLVGRDNGQAWGTFKEGSATTAKPAETKPANKPVQPTETKPVEK >sh3b2 (SEQ ID NO: 2) PSTNTNVNSDWTKQNGVFVTGGAINLRTGASTNSKVIAMLPTNTEVKYDAYRTEGQYTWL RQPRANNEYGYLVGRDNGQAWGTFKEGSAATKPAQTTNKPSQPAEK >sh3b3 (SEQ ID NO: 3) PNITTNVNSDWTKQNGVFVTGGAINLRTGASTNSKVIAMLPTNTEVKYDAYRTEGQYTWL RQPRANGQYGYLVGRDNGQAWGTFKEGSAATKPAQTTNKPSQPAEK >sh3b4 (SEQ ID NO: 4) PNITTNVNSDWTKQNGVFVTGGAINLRTGASTNSKVIAMLPTNTEVKYDAYRTEGQYTWL RQPRANGQYGYLVGRDNGQAWGTFKEGSAATKPAQTTNKPSQPTEK >sh3b5 (SEQ ID NO: 5) PNITTNVNSDWTKQNGVFVTGGAINLRTGASTDSKVIAMLPTNTEVKYDAYRTEDQYTWL RQPRANNEYGYLVGRDNGQAWGTFKEGSAKATNAVANKPVRQTT >sh3b6 (SEQ ID NO: 6) PKQVTTNNNASWTKQNGSFITGGAINLRTGASTTSPIIETLPINTVIKYDAYYRSGNYVW LRQPRANGQYGYLVGRLNNQAWGTYR- >T285_RS00875 (SEQ ID NO: 7) complete gene sequence of Sdp. The spaces are only entered to indicate the sequence components but each of sequences 64-72 are linked to form SEQ ID NO: 7. SEQ ID NO: 64 relates to the signal pep4de region, and SEQ ID NO: 65 encodes for the lysozyme / muramidase domain. SH3b Domains: SEQ ID NOs: 67-72 encode for SH3b domains 1, 2, 3, 4, 5, and 6, respec4vely.Attorney Ref. No.10457-591PC0 ATGCGTTCAACAAATAAGAAATTTATTAGTGCATTAGCTTGTGCTAGTGCTATGACAGCTTTAGCAATTGTAGA TCCTATGGGAGTTACTAAAACTCATCAAGTAGCACAAGCTGCA (SEQ ID NO: 64) ACTGATACTGTACCTGCAAAGTCTACAGGTGTAGATGTTTCAAGTTGGCAAGGTACCAACTTGGATCAACAA GCCAAATCTGGGGCACAATTTGCTGCCGTTAAAGTATCTGAAGGTACTAACTACCAAAACCCTAGAGCTCA AGGCCAAATCCAGAGTGCTGAACAAAATAATATGATGGCTATGGGTTACCACTACACTCATTTTGGCTCTAA CAGCAATCAAGCAGTTCAAGAAGGAAACTATGCAGTTAATTCAGCACAACAAGCTGGTTTACCACAAGGTT CATACTTAGCAACTGATTGGGAACAAGACGCTAACAATAATACTAATGGTAGTGTTGAAGCTAATACTAATGC AATCACTAACTTCATGGATACTGTCCATGATGGTGGATACAATCCAATGCTTTATTCTAGTGAATGGTTACTAA AGAATAAGGTTGATACTAATAAGATTTCTGAAAAGTATCCAAATGCATTATGGGTAGCTAAGTACAAAACTAAT GGTCGTGAAGATAATCCTGACTACAATTACTTCCCATCAATGGATAATGTAGCTATTTGGCAATATACGCAAA ATTGGCGTGGCCAAAATGTT (SEQ ID NO: 65) GACGGAAATGTTAATGTAGTTCCTCTTTCAAATAAGCCTACTACTGATACTAACAACACCTCAAATAACGCTG CTAATACCAATAATTCCAATGCTAATAACGGTCAAAGCAGTAGTCAAGCACCTGCAAATCCAATTACTAACA ATAGTAATGATACTGCTAAACCAAATAATAACAGTACACAACCAGCACAACCAACTGAAACTAAG (SEQ ID NO: 66) CCAGCAGAAAAGCCAAACACAACCACTAATGTAAACAGTGATTGGACTAAGCAAAACGGTGTCTTTGTAA CAGGTGGAGCAATTAACTTGAGAACTGGTGCAAGTACTGATAGTAAAGTCATTACTCAATTACCAGCAAACT CAGAAGTTAAATACGATGCTTACCGTACAATTGGCCAATACACATGGTTAAGACAACCACGTGCGAACAAC GAATACGGCTACTTAGTAGGACGTGATAATGGTCAAGCATGGGGAACATTCAAAGAAGGTTCAGCAACCA CTGCTAAACCAGCCGAAACTAAACCTGCTAATAAACCAGTACAACCAACTGAAACTAAGCCAGTAGAAAA G (SEQ ID NO: 67) CCAAGTACAAACACTAACGTAAATAGTGACTGGACTAAGCAAAACGGTGTCTTTGTAACTGGCGGCGCAAT CAACTTGAGAACTGGAGCAAGCACTAACAGTAAAGTAATTGCAATGCTTCCAACAAATACTGAAGTTAAGTA CGATGCATACCGTACGGAAGGCCAATACACATGGTTAAGACAACCACGTGCAAATAATGAATATGGTTACT TAGTAGGACGTGATAATGGCCAAGCATGGGGAACATTCAAGGAAGGTTCTGCAGCAACTAAGCCAGCTC AAACTACTAACAAACCAAGTCAACCAGCAGAAAAG (SEQ ID NO: 68) CCAAACATAACCACTAATGTAAACAGTGACTGGACTAAGCAAAATGGTGTCTTTGTAACAGGTGGCGCAAT CAACTTGAGAACTGGAGCAAGCACTAACAGTAAAGTAATTGCAATGCTTCCAACAAATACTGAAGTTAAGTA CGATGCATATCGTACGGAAGGCCAATACACATGGTTGAGACAACCACGTGCAAATGGACAATATGGTTACT TAGTAGGACGTGATAATGGCCAAGCATGGGGAACATTCAAGGAAGGTTCTGCAGCAACTAAGCCAGCTC AAACTACTAACAAACCAAGTCAACCAGCAGAAAAG (SEQ ID NO: 69) CCAAACATAACCACTAATGTAAACAGTGACTGGACTAAGCAAAATGGTGTCTTTGTAACAGGTGGCGCAAT CAACTTGAGAACTGGAGCAAGCACTAACAGTAAAGTAATTGCAATGCTTCCAACAAATACTGAAGTTAAGTA CGATGCATATCGTACGGAAGGCCAATACACATGGTTAAGACAACCACGTGCAAATGGACAATATGGTTACT TAGTAGGACGTGATAATGGCCAAGCATGGGGAACATTCAAGGAAGGTTCTGCAGCAACTAAGCCAGCTC AAACTACTAACAAACCAAGTCAACCAACAGAAAAG (SEQ ID NO: 70) CCAAACATAACCACTAACGTAAACAGTGATTGGACTAAGCAAAACGGTGTCTTTGTAACTGGCGGCGCAA TCAACTTGAGAACTGGAGCAAGCACTGATAGTAAAGTAATTGCAATGCTTCCAACAAATACTGAAGTTAAGT ACGATGCATACCGTACGGAAGACCAATACACATGGTTAAGACAACCACGTGCAAATAATGAATATGGTTAC TTAGTAGGACGTGATAATGGTCAAGCATGGGGAACATTCAAAGAAGGTTCTGCTAAAGCCACTAATGCAGT AGCTAATAAGCCTGTTCGACAAACTACT (SEQ ID NO: 71) CCTAAGCAAGTAACTACTAATAACAATGCTAGTTGGACTAAGCAAAATGGTTCTTTCATTACTGGTGGAGCA ATTAACTTAAGAACAGGTGCAAGTACTACTAGCCCAATTATTGAAACTTTACCAATTAATACTGTAATTAAATACAttorney Ref. No.10457-591PC0 GATGCATACTACCGTTCTGGTAACTATGTATGGTTAAGACAACCACGTGCAAATGGTCAATACGGTTATTTAG TTGGTCGTTTGAACAACCAAGCATGGGGAACTTACAGATAA (SEQ ID NO: 72) >T285_00875 Protein SEQ ID NO: 8 (amino acid sequence encoded by SEQ ID NO: 7) MRSTNKKFISALACASAMTALAIVDPMGVTKTHQVAQAATDTVPAK (SEQ ID NO: 73) STGVDVSSWQGTNLDQQAKSGAQFAAVKVSEGTNYQNPRAQGQIQSAEQNNMMAMGYHYTHFGSNSN QAVQEGNYAVNSAQQAGLPQGSYLATDWEQDANNNTNGSVEANTNAITNFMDTVHDGGYNPMLYSSEW LLKNKVDTNKISEKYPNALWVAKYKTNGREDNPDYNYFPSMDNVAIWQYTQNWRGQNVDGNVNVV (SEQ ID NO: 74) PLSNKPTTDTNNTSNNAANTNNSNANNGQSSSQAPANPITNNSNDTAKPNNNSTQPAQPTETK (SEQ ID NO: 75) PAEKPNTTTNVNSDWTKQNGVFVTGGAINLRTGASTDSKVITQLPANSEVKYDAYRTIGQYTWLRQPRANNE YGYLVGRDNGQAWGTFKEGSATTAKPAETKPANKPVQPTETKPVEK (SEQ ID NO: 1) PSTNTNVNSDWTKQNGVFVTGGAINLRTGASTNSKVIAMLPTNTEVKYDAYRTEGQYTWLRQPRANNEYGYL VGRDNGQAWGTFKEGSAATKPAQTTNKPSQPAEK (SEQ ID NO: 2) PNITTNVNSDWTKQNGVFVTGGAINLRTGASTNSKVIAMLPTNTEVKYDAYRTEGQYTWLRQPRANGQYGYL VGRDNGQAWGTFKEGSAATKPAQTTNKPSQPAEK (SEQ ID NO: 3) PNITTNVNSDWTKQNGVFVTGGAINLRTGASTNSKVIAMLPTNTEVKYDAYRTEGQYTWLRQPRANGQYGYL VGRDNGQAWGTFKEGSAATKPAQTTNKPSQPTEK (SEQ ID NO: 4) PNITTNVNSDWTKQNGVFVTGGAINLRTGASTDSKVIAMLPTNTEVKYDAYRTEDQYTWLRQPRANNEYGYL VGRDNGQAWGTFKEGSAKATNAVANKPVRQTT (SEQ ID NO: 5) PKQVTTNNNASWTKQNGSFITGGAINLRTGASTTSPIIETLPINTVIKYDAYYRSGNYVWLRQPRANGQYGYLV GRLNNQAWGTYR (SEQ ID NO: 6) CLUSTAL O(1.2.4) multiple sequence alignment SH3b6 ---PKQVTTNNNASWTKQNGSFITGGAINLRTGASTTSPIIETLPINTVIKYDAYYRSGN SH3b1 PAEKPNTTTNVNSDWTKQNGVFVTGGAINLRTGASTDSKVITQLPANSEVKYDAYRTIGQ SH3b5 ----PNITTNVNSDWTKQNGVFVTGGAINLRTGASTDSKVIAMLPTNTEVKYDAYRTEDQ SH3b2 ----PSTNTNVNSDWTKQNGVFVTGGAINLRTGASTNSKVIAMLPTNTEVKYDAYRTEGQAttorney Ref. No.10457-591PC0 SH3b3 ----PNITTNVNSDWTKQNGVFVTGGAINLRTGASTNSKVIAMLPTNTEVKYDAYRTEGQ SH3b4 ----PNITTNVNSDWTKQNGVFVTGGAINLRTGASTNSKVIAMLPTNTEVKYDAYRTEGQ . .** *:.****** *:************* * :* ** *: :***** .: SH3b6 YVWLRQPRANGQYGYLVGRLNNQAWGTYR----------------------------- SH3b1 YTWLRQPRANNEYGYLVGRDNGQAWGTFKEGSATTAKPAETKPANKPVQPTETKPVEK SH3b5 YTWLRQPRANNEYGYLVGRDNGQAWGTFKEGSAKATNAV---AN-KPVRQTT------ SH3b2 YTWLRQPRANNEYGYLVGRDNGQAWGTFKEGSAATKPAQ---TTNKPSQPAEK----- SH3b3 YTWLRQPRANGQYGYLVGRDNGQAWGTFKEGSAATKPAQ---TTNKPSQPAEK----- SH3b4 YTWLRQPRANGQYGYLVGRDNGQAWGTFKEGSAATKPAQ---TTNKPSQPTEK----- *.********.:******* *.*****::Mutagenesis Experiment: Sdp Protein Sequence (Complete): SEQ ID NO: 8 is provided below with point mutations in SH3b2 underlinedAttorney Ref. No.10457-591PC0 MRSTNKKFISALACASAMTALAIVDPMGVTKTHQVAQAATDTVPAKSTGVDVSSWQGTNLDQQAKSGAQFA AVKVSEGTNYQNPRAQGQIQSAEQNNMMAMGYHYTHFGSNSNQAVQEGNYAVNSAQQAGLPQGSYLAT DWEQDANNNTNGSVEANTNAITNFMDTVHDGGYNPMLYSSEWLLKNKVDTNKISEKYPNALWVAKYKTNG REDNPDYNYFPSMDNVAIWQYTQNWRGQNVDGNVNVVPLSNKPTTDTNNTSNNAANTNNSNANNGQS SSQAPANPITNNSNDTAKPNNNSTQPAQPTETKPAEKPNTTTNVNSDWTKQNGVFVTGGAINLRTGASTDSK VITQLPANSEVKYDAYRTIGQYTWLRQPRANNEYGYLVGRDNGQAWGTFKEGSATTAKPAETKPANKPVQPT ETKPVEKPSTNTNVNSDWTKQNGVFVTGGAINLRTGASTNSKVIAMLPTNTEVKYDAYRTEGQYTWLRQPRA NNEYGYLVGRDNGQAWGTFKEGSAATKPAQTTNKPSQPAEKPNITTNVNSDWTKQNGVFVTGGAINLRTGA STNSKVIAMLPTNTEVKYDAYRTEGQYTWLRQPRANGQYGYLVGRDNGQAWGTFKEGSAATKPAQTTNKPS QPAEKPNITTNVNSDWTKQNGVFVTGGAINLRTGASTNSKVIAMLPTNTEVKYDAYRTEGQYTWLRQPRANG QYGYLVGRDNGQAWGTFKEGSAATKPAQTTNKPSQPTEKPNITTNVNSDWTKQNGVFVTGGAINLRTGAST DSKVIAMLPTNTEVKYDAYRTEDQYTWLRQPRANNEYGYLVGRDNGQAWGTFKEGSAKATNAVANKPVRQT TPKQVTTNNNASWTKQNGSFITGGAINLRTGASTTSPIIETLPINTVIKYDAYYRSGNYVWLRQPRANGQYGYL VGRLNNQAWGTYRPoint Mutation positions in SH3b2: N (Asn) = 470 E (Glu) = 472 G (Gly) = 501Attorney Ref. No.10457-591PC0 D (Asp) = 503 Q (Gln) = 506 W (Trp) = 508 T (Thr) = 510

[0041] In one example, structure-based site directed mutagenesis was used to change conserved residues to either alanine or a differently charged amino acid. Each mutation was verified by sequencing. To evaluate the impact of each of the proteins or domains on stimulation of innate immune responses, RAW-Dual™ KO-TLR4 Cells were utilized. The impact of the mutations was evaluated by following the activity of two reporter genes. The use of this cell line allowed the quantification of the signalling through NF-κB (MIP-2 promoter fusion to supernatant alkaline phosphatase, SEAP) and interferon signalling (ISRE promoter fusion to Lucia luciferase). It was found that the Sdp domain SH3B2, used as positive control, significantly induced the expression of the MIP-2 by increasing SEAP activity. In contrast, mutations on SH3b2 glutamic acid 472 to valine (E472V), glycine 501 to aspartic acid (G501D) and tryptophan 510 to alanine (W510A) resulted in proteins that with a significant reduction in the stimulation of both SEAP and Lucia luciferase activity.

[0042] Interestingly, as shown in Figure 11, a significant increase in the activity was observed for the mutant in glutamic 472 to alanine (E472A) for SEAP and Lucia activity. Increased activity was also observed for glycine 501 to alanine (G501A), aspartic acid 503 to alanine (D503A) and glutamine 506 to alanine (Q506A) for the interferon pathway reporter gene. These results indicate that these mutant proteins have the potential to be more effective therapeutics when compared to the wild type protein.

[0043] For the purposes of comparing two closely-related polypeptide or polynucleotide sequences, the “% sequence identity” (or “% identity”, used interchangeably herein) between a first sequence and a second sequence may be calculated. Polypeptide or polynucleotide sequences are said to be the same as or identical to other polypeptide or polynucleotide sequences, if they share 100% sequence identity over their entire length. Residues in sequences are numbered from left to right, i.e. from N- to C-terminus for polypeptides; from 5′ to 3′ terminus for polynucleotides. The terms “identical” or percentage “identity”, in the context of two or more polypeptide sequences, refer to two or more sequences or sub-sequences that are theAttorney Ref. No.10457-591PC0 same or have a specified percentage of amino acid residues that are the same (i.e., 70% identity, optionally 75%, 80%, 85%, 90%, 95%, 98% or 99% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition also refers to the compliment of a test sequence. Optionally, the identity exists over a region that is at least 250 amino acids in length, such as 300 amino acids or 350 amino acids. Suitably, the comparison is performed over a window corresponding to the entire length of the reference sequence (as opposed to the derivative sequence).

[0044] For sequence comparison, one sequence acts as the reference sequence, to which the test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percentage sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0045] A “comparison window”, as used herein, refers to a segment in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well- known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman 1981, by the homology alignment algorithm of Needleman & Wunsch 1970, by the search for similarity method of Pearson & Lipman 1988, by computerised implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al. 1995).

[0046] One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendrogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle 1987. The method used is similar to theAttorney Ref. No.10457-591PC0 method described by Higgins & Sharp 1989. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid coordinates for regions of sequence comparison and by designating the program parameters. Using PILEUP, a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al. 1984). Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. 1977 and Altschul et al. 1990, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (website at www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al., supra). These initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, anAttorney Ref. No.10457-591PC0 expectation (E) or 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff 1989) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

[0047] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001. A “difference” between sequences refers to an insertion, deletion or substitution of a single residue in a position of the second sequence, compared to the first sequence. Two sequences can contain one, two or more such differences. Insertions, deletions or substitutions in a second sequence which is otherwise identical (100% sequence identity) to a first sequence result in reduced % sequence identity. For example, if the identical sequences are 9 residues long, one substitution in the second sequence results in a sequence identity of 88.9%. If the identical sequences are 17 amino acid residues long, two substitutions in the second sequence results in a sequence identity of 88.2%.

[0048] Alternatively, for the purposes of comparing a first, reference sequence to a second, comparison sequence, the number of additions, substitutions and / or deletions made to the first sequence to produce the second sequence may be ascertained. An addition is the addition of one residue into the first sequence (including addition at either terminus of the first sequence). A substitution is the substitution of one residue in the first sequence with one different residue. A deletion is the deletion of one residue from the first sequence (including deletion at either terminus of the first sequence).

[0049] Suitably, a substitution may be conservative. A ‘conservative’ substitution is an amino acid substitution in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which is expected to have little influence on the function, activity or other biological properties of the polypeptide. Such conservative substitutions suitably areAttorney Ref. No.10457-591PC0 substitutions in which one amino acid within the following groups is substituted by another amino acid residue from within the same group: Group Amino acid residue Non-polar aliphatic Glycine Alanine Valine Leucine Isoleucine Aromatic Phenylalanine Tyrosine Tryptophan Polar uncharged Serine Methionine Cysteine Threonine Asparagine Glutamine Negatively charged Aspartate Glutamate Positively charged Lysine Arginine Histidine

[0050] A polypeptide as described herein may include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or more contiguous amino acids, or any range derivable therein, of SEQ ID NO:1-6. The polypeptides described herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more variant amino acids within at least, or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,Attorney Ref. No.10457-591PC0 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or more contiguous amino acids, or any range derivable therein, of SEQ ID NO:1-6. Extracellular Vesicles

[0051] As part of the formation and release of EVs, unwanted molecules are eliminated from cells. However, cytosolic and plasma membrane proteins are also incorporated during these processes into the EVs, resulting in EVs having particle size properties, lipid bilayer functional properties, and other unique functional properties that allow the EVs to potentially function to carry their payload.

[0052] According to one embodiment, EVs are isolated from a naturally occurring probiotic Lactobacillus sp. In a specific embodiment, the Lactobacillus species is Lactobacillus johnsonii. In an even more specific embodiment, EVs are isolated from a strain of Lactobacillus johnsonii, Lactobacillus johnsonii N6.2. As used herein, the term “isolating,” or “to isolate,” as it relates to EVs refers to any artificial (i.e., not naturally occurring) process for obtaining EVs from a common source material (e.g. a sample of Lactobacillus sp), where the process results in a more useful form of a EVs. The “more useful form” of the EVs can be characterized in a variety of ways, no one of which is limiting. For example a Lactobcillus sp population of cells can be manipulated to produce EVs having a higher amount of a molecule of interest compared to cells not manipulated. Alternatively, or additionally, the invention provides methods for isolating EVs from a sample containing a Lactobacillus sp. that results in isolating EVs containing a higher amount of a molecule of interest (e.g. polypeptide, protein or other biomolecule), for example at least 5-50 percent higher amount of a molecule of interest, compared to other EVs in a population of EVs from a common source material. Isolating can involve obtaining EVs with a higher amount of a molecule of interest such that EVs containing the molecule of interest below the threshold percentage are completely removed from the EVs with the molecule of interest above the threshold percentage (purified). Moreover, isolating can involve obtaining EVs with a higher amount of a molecule of interest such that EVs containing the molecule of interest below the threshold percentage are reduced compared to EVs with the molecule of interest above the threshold percentage, i.e. higher ratio of EVs with higher amount of molecule of interest vs. those with a lower amount of molecule of interest (enriched). Accordingly, a more useful form pertains to a purified or enriched population of EVs.Attorney Ref. No.10457-591PC0 Sdp or sh3b domain polypeptides and compositions

[0053] In certain embodiments, provided are compositions that include isolated Sdp or a sh3b domain thereof, or a polypeptide comprising an amino acid sequence of one or more of SEQ ID NOs: 1-6 and a pharmaceutically acceptable carrier. In the context of protein or polypeptide molecules, the terms (1) “isolated” refers to the state of the molecule after the starting material has been subjected to a method for isolating the molecule of interest (that is to say, isolating a molecule of interest from a starting material will produce an isolated molecule); (2) the term “purifying” or “to purify” refers to a process for removing at least one impurity or contaminant from a starting material (for example, purifying a molecule of interest from a starting material refers to a process for removing at least one impurity from the starting material to produce a relatively more pure form of the molecule of interest); (3) the term “substantially purified” refers to protein or polypeptide molecules that are removed from their natural environment or from a starting material (i.e., they are isolated) and where they are largely free from other components with which they are naturally associated or substantially free of other components that may render future use or study sub-optimal, difficult or impossible; (4) the terms “purified” or “partially purified” refers to protein or polypeptide molecules of interest that are removed from either (i) their natural environment, or from (ii) a starting material (i.e., they are isolated), and where (a) at least one impurity from the starting material has been removed, or (b) at least one component with which the molecule is naturally associated has been removed (a “purified” or “partially purified” molecule may still contain additional components that may render future use or study of the molecule sub-optimal, difficult or impossible); (5) the term “enriching” (and “enriched” and the like) refers to a process whereby a molecule of interest that is in a mixture has an increased ratio of the amount of that molecule to the amount of other undesired components in that mixture after the enriching process as compared to before the enriching process; (6) the term “concentrating” refers to a process whereby a protein or polypeptide molecule that is in a mixture that has been subjected to that process has a greater concentrationAttorney Ref. No.10457-591PC0 after the process as compared to the concentration of the molecule in the mixture before the process; and (7) the term “depleted” refers to a mixture containing an undesirable component, where that undesirable component has been (i) completely removed from the mixture, (ii) sufficiently removed from the mixture to be undetectable, or (iii) partially removed from the mixture such that its concentration in the mixture is significantly reduced. EV Compositions

[0054] Embodiments of the present invention include pharmaceutical compositions comprising EVs isolated from Lactobacillus sp, such as Lactobacillus johnsonii. In certain embodiments, EVs can include EVs derived from a Lactobacillus sp cells that synthesize and express sdp or a sh3b domain thereof, and packages same into an exosome and / or microvesicle. Such pharmaceutical compositions comprise a therapeutically effective amount of EVs, and optionally a pharmaceutically acceptable carrier. Pharmaceutical Compositions and Administration

[0055] With respect to protein or polypeptide compositions and EV containing compositions, the term “pharmaceutically acceptable” means approved by a regulatory agency of government or listed in the US Pharmacopeia, the European or UK Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the protein, polypeptide, and / or EVs are administered. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the EVs, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0056] One or more other excipients may be included in the composition of this invention to (1) impart satisfactory processing and compression characteristics to the composition (e.g., adjust the flowability, cohesion and other characteristics of the composition) and (2) give additional desirable physical characteristics to the tables (e.g. color, stability, hardness, disintegration).Attorney Ref. No.10457-591PC0 Mostly the excipients aid in the delayed release of the drug from the composition to achieve regional delivery to the lower GI. As used herein, the term "excipient" may include all excipients present in the dosage form, including all components other than the drug entity and the hydrocolloid gum from higher plants. A plurality of excipient substances may be present in any dosage form, and may include multiple substances having similar pharmaceutical function (e.g., lubricants, binders, diluents) or similar structure (e.g., a mixture of monosaccharides). Preferably the fewer excipients present the better. Such excipients are present in an amount sufficient to provide the composition with the desired delayed release / regional delivery characteristics, hardness rating and handling characteristics and will generally be present at a level of about 2% by weight to about 50% by weight, preferably about 2% by weight to about 40% by weight and more preferably about 2% to about 10% by weight. Excipients may be selected from many categories known in the pharmaceutical arts. The excipients used will be chosen to achieve the desired object of the invention keeping in mind the activity of the drug being used, as well as its physical and chemical characteristics such as water solubility and possible interactions with the excipients to be used.

[0057] For example with drugs that are more water soluble, generally a lower percentage by weight of excipients will be used, i.e., less than about 20% or from about 2% to about 15% by weight, preferably no more than about 10% by wt, while for drugs that are less water soluble a higher percentage by weight may be used, e.g., about 20% up to about 40% by wt. These levels may be adjusted to achieve the desired hardness and porosity of the final tablet composition to obtain the delayed release profile.

[0058] From the foregoing discussion, it is seen that one aspect of this invention is a particle mass of a solid dosage form that can be administered orally as a tablet. Thus, such composition is neither a liquid nor a gas, but a solid tablet having an amount of drug as a unit dosage. Generally, this unit dosage will be an amount that can be swallowed by a human subject and may vary from a total of about 100 milligrams to about 1500 mg, preferably no more than about 1200 mg and particularly no more than about 800 mg. For children, the size of the tablet may be significantly less than for adults, and for elderly patients who have difficulty swallowing, the total amount may be less than what would be viewed as a normal amount for adults. It is to be understood that the tablets of this invention may be designed as a single tablet having a unit dosage amount orAttorney Ref. No.10457-591PC0 several smaller tablets, e.g. 2-5, may be combined in a capsule for oral administration. The composition used to prepare the tablet may be granulated.

[0059] In a preferred embodiment, the pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for oral administration to humans.

[0060] The amount of the Lactobacillus sp. EVs containing Sdp or an sh3b domain thereof, or pharmaceutical compositions comprising a polypeptide comprising amino acid sequence of one or more of SEQ ID NOs: 1-6, or fragments or variants thereof, which will be effective in the treatment of a particular disease or disorder (e.g. viral infection) will depend on the nature of the disease or disorder, and can be determined by standard clinical techniques. In addition, in vitro and in vivo assays may optionally be employed to help identify optimal dosage ranges. The dosage will depend on the body weight of the subject. However, in one example suitable dosage ranges for oral administration or parenteral administration may be about 10 pg to 100 mg, 20 pg to 50 mg, 0.1 mg to 20 mg, or 0.5 mg to 10 mg (calculated either per kg body weight or as total dose per individual). Suitable dosage ranges for intranasal administration are generally about 0.01 pg / kg body weight to 1 mg / kg body weight. Effective doses may be extrapolated from dose- response curves derived from in vitro or animal model test systems. Suppositories generally contain an active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.

[0061] One aspect of this invention is an orally-deliverable tablet having an inner composition optionally surrounded by a pharmaceutically-acceptable coating. The tablet preferentially delivers a therapeutically effective amount of a composition comprising enriched EVs or isolated polypeptides from a naturally occurring probiotic Lactobacillus sp. to the GI tract downstream from the stomach, without significant release of the composition in the stomach upon oral administration of the composition to a subject in need thereof. The composition or pharmaceutical composition of the invention can be coated with an enteric coating. The EVs and / or polypeptide(s) can be provided in a tablet or capsule and can be released in a burst or in a controlled fashion. The EVs and / or polypeptide(s) can also be encapsulated in a microsphere, a liposome, a nanosphere or a microemulsion, or be provided in the form of pellets or minitablets for delivery to and release in the intestine.Attorney Ref. No.10457-591PC0

[0062] The general approaches to delivering drugs to the lower GI tract (e.g. small intestine and colon) for interaction with immune cells in the mucosa of the lower GI tract include: 1) enteric coating designed to release drug in the more alkaline environment of the gastrointestinal tract, 2) bioerodible coatings and matrices, 3) prodrugs, 4) timed-release systems and, 5) enteric polymeric material-based release systems that release drug after they transit through the stomach and reach the intestines. A general discussion of these approaches and others may be found in PCT Patent application No. PCT / US91 / 03014 by Sintov and Rubinstein.

[0063] Capsules containing EVs and / or polypeptides can be prepared according to known techniques. See for example US Pat. Pub. 20170368049 incorporated by reference. For preparing solid compositions such as tablets, the principal active ingredient may be mixed with a pharmaceutical carrier, e.g. conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g. water. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the intestine or lower GI tract, or to otherwise be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, acetyl alcohol and cellulose acetate.

[0064] In preparing the tablet compositions of this invention one may use pharmaceutical compression or molding techniques, preferably the former due to its adaptability to large scale production methods. Using techniques known in the art, the tablets of the invention may take any appropriate shape such as discoid, round, oval, oblong, cylindrical, triangular, hexagonal, and the like. The tablets may be coated or uncoated. If coated they may be sugar-coated (to cover objectionable tastes or odors and to protect against oxidation), film coated (a thin film of water soluble matter for similar purposes), or enteric coated (to resist dissolution in gastric fluid but allow disintegration of the coating in the small intestine-- as discussed herein before). Depending on whether the tablet is a uniform matrix tablet, an active core tablet or a concentration gradient tablet, the process for preparation will vary slightly.Attorney Ref. No.10457-591PC0

[0065] In order to ensure tablet hardness and uniformity of weight, content and other items, it is preferable to prepare the tablets having the composition of this invention by using a pre- granulation technique. In general, the granulation techniques can include the wet granulation method, the fluid bed granulation method, the dry granulation method or direct compression.

[0066] Once the tablets are appropriately formed, they can then be coated by any of the necessary coating techniques as discussed in Chapter 90 of Remington's. For example, the tablets may be sugar-coated in accordance with the procedure discussed therein or film coated or preferably enterically coated. Enteric coating is preferred in the tablets of this invention to minimize the release of any of the drug in the upper GI and assure the release to the lower GI particularly the colon. As much as pertinent of the Remington's sections of Chapters 88 and 90 is incorporated herein by reference.

[0067] In certain embodiments, compositions comprising Sdp or a domain thereof (e.g. SEQ ID NOs: 1, 2, 3, 4, 5, and / or 6, or fragment or variant thereof), or EVs comprising same, are ideally ready-to-use or ready to administer for parenteral administration, optionally, without the need for reconstitution. In certain embodiments, a ready-to-use formulation is a liquid stored in a pharmaceutically suitable container, for example, a glass vial or plastic intravenous bag. A ready-to-use or ready-to-administer composition is a sterile, liquid injectable composition not requiring reconstitution before use, for example, a lyophilized powder, such that the formulation can directly administered or further diluted if present as a concentrated solution. For example, a ready-to-administer composition can be include the required concentration and volume in the final container such as a syringe or injector whereas a ready-to-use preparation can be at the required concentration and a volume in a container that may be transferred to a final administration device such as a syringe or infusion bag for administration to a patient. Parenteral administration may be performed by subcutaneous injection, intramuscular injection, or intravenous injection by means of a syringe, optionally a pen-like syringe. Diluents can include, for instance, fluids suitable for parenteral administration such as water for injection, sodium chloride or dextrose solutions.

[0068] In some embodiments, the composition comprising Sdp or one or more sh3b domains thereof (e.g. SEQ ID Nos 1-6, or fragments or variants thereof), or enriched EVs containing same, as described herein will be administered orally to a mammalian subject in need thereof using a therapeutically effective amount of the composition that is sufficient to provide theAttorney Ref. No.10457-591PC0 desired physiological effect. The mammalian subject may be a domestic animal or pet but preferably is a human subject. The level of composition needed to give the desired physiological result is readily determined by one of ordinary skill in the art. Other parameters that may be taken into account in determining dosage for the pharmaceutical composition embodiments described herein may include disease state of the subject or age of the subject.

[0069] The compositions may take the form of suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. In some embodiments, the composition embodiments described herein may be administered orally or intravenously via parenteral nutritional therapy to a subject via an emulsion. The emulsion may include, in some embodiments, an aqueous continuous phase and a dispersed phase. The boundary between the phases called the "interface". The present emulsions are adapted for application to a mucosal surface of a vertebrate animal, preferably a mammal, including humans. These compositions improve the permeability and bioavailability of active compounds after application to a mucous surface. Mucosal surfaces of interest include the intestinal mucosa. Use of bioadhesive polymers in pharmaceutical emulsions affords enhanced delivery of drugs in bioadhesive polymer-coated suspensions, in some examples. Bioadhesive pharmaceutical emulsions may be used to deliver the EVs isolated from Lactobacillus sp. described herein to: a) prolong the residence time in situ, thereby decreasing the number of drug administrations required per day; and b) may be localized in the specified region to improve and enhance targeting and bioavailability of delivered drugs.

[0070] The ability to retain and localize a drug delivery emulsion in a selected region leads to improved bioavailability, especially for drugs exhibiting a narrow window of adsorption due to rapid metabolic turnover or quick excretion. Intimate contact with the target absorption membrane improves both the extent and rate of drug absorption.

[0071] Bioadhesion is the characteristic of certain natural and synthetic polymers of binding to various biological tissues. Of particular interest are polymers which bind to the mucous lining that covers the surface of many tissues which communicate directly or indirectly with the external environment, such as the gut, for example. Mucus binding polymers may be referred to as mucoadhesive. Several bioadhesive, and specifically mucoadhesive, polymers are known. The chemical properties of the main mucoadhesive polymers are summarized as follows: a. strong H-bonding groups (--OH, --COOH) in relatively high concentration;Attorney Ref. No.10457-591PC0 b. strong anionic charges; c. sufficient flexibility of polymer backbone to penetrate the mucus network or tissue crevices; d. surface tension characteristics suitable for wetting mucus and mucosal tissue surfaces; and e. high molecular weight.

[0072] Bioadhesive polymers may be used in the pharmaceutical composition embodiments described herein, examples of bioadhesive polymers currently used in pharmaceutical preparations include: carboxymethylcellulose (CMC), hydroxypropylmethylcellulose (HPMC), polyacrylic and polymethacrylic acid and their derivatives, pectin, alginic acid, chitosan, polyvinylpyrrolidone, hyaluronic acid, and polyvinyl alcohol. The most frequently used polymer is Carbopol (Carbomer), which is a high molecular weight polyacrylic acid polymer. It is used in many formulations for bioadhesive drug delivery systems, as a suspending agent, as a tablet coating, and in ocular suspensions.

[0073] Pharmaceutical composition embodiments described herein may include the composition comprising Sdp or one or more sh3b domains thereof (e.g. SEQ ID Nos 1-6, or fragments or variants thereof), or enriched EVs containing same isolated from Lactobacillus sp., are incorporated into inert lipid carriers such as oils, surfactant dispersions, emulsions, liposomes etc. In a specific embodiment, liposomes may be made from lipids purified from Lactobacillus johnsonii N6.2 Self-emulsifying formulations are ideally isotropic mixtures of oils, surfactants and co-solvents that emulsify to form fine oil in water emulsions when introduced in aqueous media. Fine oil droplets would pass rapidly from stomach and promote wide distribution of drug throughout the GI tract, thereby overcome the slow dissolution step typically observed with solid dosage forms. These embodiments may provide control release self-emulsifying pellets, microspheres, tablets, capsules etc. that increase the use of "self-emulsification.”.

[0074] In further embodiments, composition embodiments described herein may be microencapsulated for delivery to a subject. Microencapsulation (ME) offers the potential to reduce the adverse effects on probiotic viability in the gastrointestinal (GI) tract environment. ME separates microorganism cells from their environment until they are released. Controlled release of the MV’s is a particular benefit of ME. It is beneficial for encapsulated probioticAttorney Ref. No.10457-591PC0 microorganisms to be released in the small intestine where the Peyer's patches exist to activate the immune system, in some embodiments.

[0075] Oral delivery will be the most straightforward mode of administration to deliver the composition comprising Sdp or one or more sh3b domains thereof (e.g. SEQ ID Nos 1-6, or fragments or variants thereof), or enriched EVs containing same, to the gut mucosa. However, in alternative embodiments, other methods of administration are contemplated. Accordingly, suitable methods for administering the composition comprising Sdp or one or more sh3b domains thereof (e.g. SEQ ID Nos 1-6, or fragments or variants thereof), or enriched EVs containing same, in accordance with the methods of the presently-disclosed subject matter include, but are not limited to, systemic administration, parenteral administration (including intravascular, intramuscular, and / or intraarterial administration), oral delivery, buccal delivery, rectal delivery, subcutaneous administration, intraperitoneal administration, inhalation, dermally (e.g., topical application), intratracheal installation, surgical implantation, transdermal delivery, local injection, intranasal delivery, and hyper-velocity injection / bombardment. Where applicable, continuous infusion can enhance drug accumulation at a target site (see, e.g., U.S. Pat. No. 6,180,082). In some embodiments of the therapeutic methods described herein, the therapeutic compositions are administered orally, intravenously, intranasally, or intraperitoneally to thereby treat a disease or disorder. In further aspects, a composition may be administered more than one time to the subject, and may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more times. EXAMPLES Material and Methods Bacterial Growth and EV Isolation

[0076] L. johnsonii N6.2 was grown in exosome depleted de Man, Rogosa, Sharpe media (ED-MRS) media at 37°C under static condition as previously described (Harrison et al). The vesicles suspended in PBS were subsequently quantified using a NanoSight 300 (Malvern instruments Ltd, Malvern, UK) at the University of Florida ICBR Flow Core Facility, RRID:SCR_019119. Cell Line Propagation

[0077] All cell lines were grown in complete growth media containing Dulbecco’s Modified Eagle Medium (DMEM) (4.5 g / L glucose, 4mM L-glutamine, and 1 mM sodium pyruvate) supplemented withAttorney Ref. No.10457-591PC0 10% heat inactivated FBS (Sigma-Aldrich, Saint Louis, MO, USA), 1% penicillin and streptomycin solution containing 10,000 units of penicillin and 10 mg of streptomycin / ml (Sigma-Aldrich, Saint Louis, MO, USA). Initial experiments and viral challenge were measured in the murine Mφ cell line RAW 264.7. To further elucidate the mechanisms by which EVs initiate the host sensory response, knockout murine Mφs cell lines were used. The following reagents have been obtained from BEI Resources, NIAD, NIH: Mφ cell line derived from wild type mice NR-9456 (WT), and the Mφ cell lines derived from following knockout Mice: ΔTLR2 (NR-9457), ΔTLR3 (NR-19974), ΔTLR4 (NR-9458), ΔTLR2 / TLR 4 (NR-19975), ΔTLR7 (NR-15634), and ΔTLR 9 (NR-9569). The following adaptor proteins will also be evaluated: Mφ cell line derived from: ΔTRIF / TRAM (NR-9568), ΔMyD88 (NR-15633), ΔMAL (NR- 9459); as well as the ΔIRF3 (NR-15635), and ΔIRF7 (NR-15636) (BEI). RAW-DUAL KO-TLR4 reporter cell line was obtained from (InvivoGen, San Diego, CA) . Murine Norovirus Production

[0078] Murine norovirus was produced as previously described (Mosby et al., 2023). The pSPMNV-1. CW3 plasmid (5 μg) was transfected into HEK 293T cells to express the recombinant murine norovirus-1 genome. The supernatant from transfection (MOI=0.05) was used to infect RAW264.7 cells. At 36-48 hours post-infection (hpi), the cells were checked for ~90% cytopathological effect. The supernatant containing viruses was harvested ultracentrifuged through a 25% sucrose cushion to obtain purified viral pellets. Viral pellets were resuspended in PBS and titrated using TCID50 assay. All virus stocks were aliquoted and stored at -80°C upon receipt and were thawed on ice for 1 hr prior to use. Mock inoculum was generated by transfecting HEK 293T cells with the pSP.CW3 plasmid not containing the MNV-1 genome and following the same viral isolation procedure stated above. Inoculation of Cells with MNV and L. johnsonii N6.2 EVs

[0079] RAW 264.7 cells were plated 5x105cells / mL in a 12 well plate and grown for 48 hrs to reach confluence (~1x106cells) before infections. The cells were treated with L. johnsonii N6.2 EVs at a ratio of 1:10,000 cell:EVs (1 x 108, EV8; 1 x 109, EV9; 1 x 1010, EV10) in RAW 264.7 media. The MNV-1 inoculum was prepared as 500 μL solution of MNV-1 (MOI=5) in media and incubated at 37°C for 1 h with gentle rotation prior to infection. Plated cells were then inoculated with 500 μL of the MNV-1 solution into each well then rocked gently to mix and incubated at 37°C with 5% CO2for 1 hr. The inoculum was removed from each well, and wells were washed with PBS twice. Complete media was added to the wells, and the cells were grown for 6 or 18 hpi at 37°C with 5% CO2. The supernatant was then collected for LDH analysis, MNV-1 quantification, and cells were lysed for RNA isolation and qRT- PCR analysis.Attorney Ref. No.10457-591PC0 mRNA Extraction and qRT-PCR

[0080] RNA was extracted from eukaryotic cell lines using the Qiagen RNeasy Miniprep following manufacture’s specifications (QIAGEN, Germantown, MD). The RNA extracted were treated with DNase using the DNase Turbo kit (Thermo Scientific, Waltham, MA) and assessed for concentration and purity using a NanoDrop One Microvolume UV-vis spectrophotometer (Thermo Scientific, Waltham, MA) and visually analyzed for degradation using a 1% agarose gel. qRT-PCR was performed using a QuantStudio 6 Flex (Thermo Scientific, Waltham, MA) as previously described. Briefly, the cDNA is synthesized from the mRNA extracted then the genes expression is quantified by the fluorescent signal of the PowerUp SYBR green (Thermo Scientific, Waltham, MA) due to the amplification from primer designed for the genes of interest and the endogenous control gene expression (GAPDH). Cycle Threshold (CT) values were used to calculate the relative change in expression compared to control conditions. Primer sequences used are listed in Table 1. MNV-1 genome was quantified in a similar manner by amplifying the MNV-1 cDNA genome using specific primers for the MNV-1 genome. MNV-1 levels were expressed as either relative expression compared to a control condition or absolute MNV-1 genome count by creating a standard curve. Lactate Dehydrogenase Cytotoxicity Assay

[0081] Viral induced cytotoxicity was evaluated in RAW 264.7 macrophages by following the leakage of the cytoplasmic enzyme Lactate Dehydrogenase (LDH) to the supernatant. Supernatant LDH activity was measured using the CyQUANT LDH Cytotoxicity assay kit (Thermo Scientific, Waltham, MA) following manufacturer’s specification. The percent difference (Δ%) was calculated by normalizing to the control group not treated with either the MNV or L. johnsonii N6.2. Mice infection

[0082] C57BL / 6 mice were housed in the University of Florida animal facilities. The animal protocols were approved by the Institutional Animal Care and Use Committees at the University of Florida. Mice were orally fed daily for seven days. The treatments (N=3) were: PBS for the mock and MNV only groups, 1x108colony forming units of L. johnsonii N6.2, 1x1010EVs, 1x1010liposomes, 3 ug of SH3B2 and 1x1010liposomes loaded with 3 ug of SH3B2. On the seventh day, all groups of mice (except the mock group) were orally inoculated with 1x107MNV-1 or mock inoculum.24 hours post infection (hpi) mice were euthanized and viral titers of the intestines were analyzed in ileum tissues by qRT-PCR as well as plaque assay as described earlier (Mosby et al., 2023). The experiment was repeated twice.Attorney Ref. No.10457-591PC0 Gene overexpression and protein purification. Cloning of proteins Sdp (locus tag T285_RS00825, domains SH3B2, SH3B6, SH3B1-SH3B2, Lys), Muc (locus tag T285_RS08930, domains Muc1, Muc3, Muc4, Muc5), LexA, PepD, PepC, P8875 and Eno3.

[0083] Standard methods were used for L. johnsonii N6.2 genomic DNA isolation (QIAGEN DNeasy Blood and Tissue Kit, Germantown, Maryland, USA), restriction enzyme digestion, agarose gel electrophoresis, ligation and transformation (Gardner et al., 2016; Pagliai et al., 2010). The primers are listed in Table 2. PCR fragments were cloned into p15TVL (GenBank accession EF456736) vector. Upon transformation into Escherichia coli DH5α, recombinant plasmids were confirmed by sequencing with T7 universal primers. His-tagged fusion genes were transformed into E. coli BL21 (DE3). For protein purification, cells were grown in Luria Broth at 37°C to an optical density of 0.8. Genes were overexpressed with 0.5 mM isopropyl-tiol-β-D-galactopyranoside (IPTG) and incubated at 17°C for 16 h. Cells were harvested by centrifugation at 7,800 x g for 20 min. Next, the cell pellet was resuspended in binding buffer (500 mM NaCl, 5% glycerol, 50 nM Tris, 5 mM imidazole, pH 8.0) and lysed using a French press. The lysates were then centrifuged at 35,000 x g for 45 min and the supernatant was applied to a Ni2+affinity column. The column was washed with 200 mL of wash buffer (binding buffer with 25 mM imidazole) and the proteins were eluted with elution buffer (binding buffer with 250 mM imidazole). The purified proteins were dialyzed against 10 mM Tris (pH=8), 2.5% glycerol, 500 mM NaCl and 0.5 mM TCEP, and stored at -80°C (Gardner et al., 2016; Pagliai et al., 2010). Protein concentrations were measured by Bradford assay (Bio-Rad). Bovine serum albumin (BSA; Gold BioTechnology, St. Louis, MO, USA) was used as the standard.

[0084] Mock was prepared following the same protein purification protocol except that the p15TVL plasmid was not transformed into BL21(DE3). Secreted Embryonic Alkaline Phosphatase (SEAP) and Lucia Luciferase (Lucia) Assays

[0085] RAW-DUAL KO-TLR4 reporter cell line (InvivoGen, San Diego, Ca) was treated in triplicates with EVs at 1:10,000 ratio, Proteins at concentrations of 0.015, 0.15, 1.5 ug / mL.100 ng / mL Lipopolysaccharide was included as the negative control. After 24h of incubation at 37°C, cell culture supernatants were collected and analyzed. Quantification of SEAP activity as NF-κB reporter activity was evaluated using QUANTI-Blue solution (InvivoGen, USA) following the manufacture’s protocol. IRF induction was evaluated by luminescence using QUANTI-Luc 4 solution (InvivoGen, USA) following the manufacturer’s protocol. Bacterial lipid extraction and liposome formationAttorney Ref. No.10457-591PC0

[0086] For total lipid extraction L. johnsonii N6.2 cultures were grown and harvested as described above. Cells were washed twice with 1% (w / v) NaCl then frozen at -80°C overnight and freeze-dried (Labconco, Kansas City, MO, USA) for 24h. Total lipids were extracted using a modified Bligh and Dyer method (Lewis et al., 2000, Da Silva et al., 2023; Cuaycal et al., 2023). The dried total lipid extract was then resuspended in 1mL of 1x PBS. The PBS lipid suspension is then extruded through a 0.1µm polycarbonate membrane filter (Whatman-Cytiva, Marlborough, MA, USA) an Avanti Mini-extruder following manufacturer’s protocol (Avanti Polar Lipids, AL, USA). For liposomes containing proteins, total lipids were mixed with 3 ug of the purified proteins, extruded again and quantified using Nanosight (Harrison et al., 2021). Statistical Analysis

[0087] GraphPad Prism 9.491 software (GraphPad Software, La Jolla, CA, United States) and Origin 9.7.0.188 (OriginLab Corporation, Northampton, MA, USA) were used for data analysis and visualization. Statistical tests were performed using one-way analysis of variance (ANOVA) to evaluate the effects of treatments, followed by a Tukey post-hoc test. Results were summarized as means ± standard deviation, and significance of model terms and treatment comparisons were considered significant at a level of α=0.05, or otherwise specified. Results L. johnsonii N6.2 EVs reduced the replication of MNV-1 genome and reduce cytotoxicity in RAW 264.7 Mφs.

[0088] First, we evaluated whether L. johnsonii N6.2 EVs can elicit a similar antiviral response in the murine RAW 264.7 Mφ cell line. To this end RAW 264.7 cells were treated with increasing concentrations of EVs (1 x 108, EV8; 1 x 109, EV9; 1 x 1010, EV10) and incubated for 6h. It was found that the expression of OAS1b (the murine OAS1 homolog), OAS2, OASL, MX1 were upregulated in presence of EVs in dose dependent manner. Moreover, the RAW 264.7 cells also upregulated the expression of IFI44L, in agreement with our previous results in in human cell lines in response to EVs (Fig.8). Next, we evaluated whether the observed antiviral response induced by L. johnsonii N6.2- derived EVs has an inhibitory effect against an RNA viral insult, using the MNV as the viral infection model. MNV is a positive-sense RNA virus that can infect the murine Mφ cell line RAW 264.7. To evaluate the effect of EVs on MNV infection, RAW 264.7 cells were challenged with MNV in presence or absence of increasing concentrations of EVs. The MNV relative replication was assessed by qRT-PCR 18 hpi, and the viral titer shed was quantified from the supernatant (Fig.1 A, B). It was found that RAWAttorney Ref. No.10457-591PC0 264.7 Mφs pretreated with EVs significantly decreased the amount of MNV-1 genome in a dose dependent manner. The highest inhibitory effect was observed using 1 x 1010, EV10. It was tested whether the timing of addition of EVs had a significant effect of MNV infection. EVs were either added 5 h prior to viral inoculation, co-inoculated with the MNV, or the EVs were added 1 hr after the viral inoculation. MNV-1 replication and RNA sensing pathway in the host were assessed after 6- or 18-hpi. It was found that the L. johnsonii N6.2 EVs treatments were equally effective at decreasing MNV replication (Fig.1 D,E). These results indicate that the time of EVs exposure does not play a crucial role in the hinderance to the viral replication and that L. johnsonii N6.2 EVs may not inhibit MNV-1 entry. These results may be explained by the quick uptake of L. johnsonii N6.2 EVs into eukaryotic cells. We recently reported that L. johnsonii N6.2 EVs are actively internalized into the endosome within 15 min of exposure (da Silva et al., 2023).

[0089] The impact of MNV-1 replication and EVs on the expression of RNA sensing pathway in Mφ were assessed after 6- or 18-hpi. The reduction in MNV replication were positively correlated with an increase in expression of OAS1b, OAS2, OASL, MX1, MX2 and IFI44L at 18 hpi (Fig.1 F-I). Interestingly, the induction levels of these genes were by EVs or MNV added individually reached similar levels, while the pretreatment of RAW 264.7 with EVs followed by the MNV-1 challenge reached significantly higher levels of OAS1b, OAS2, OASL, MX1, MX2 and IFI44L in presence of EV10. These results indicated that similar pathways of entry and host signaling are shared between the MNV-1 and L. johnsonii N6.2-derived EVs.

[0090] To elucidate if a shorter incubation time would differentially affect the induction of RNA sensing genes that may limit MNV replication, RAW 264.7 cells were challenged with MNV in presence or absence of increasing concentrations of EVs and they were evaluated after 6 hpi. It was found that the expressions of OAS1b, OASLI, IF44L, and MX1 were not induced by MNV-1 alone at 6 hpi (Fig.1 F-I), in contrast to the results obtained after 18 hpi (Fig.8). EVs alone or EVs pre-incubation for 5 hrs followed by MNV-1 infection showed similar levels of induction in all the host genes tested, while the addition of EVS during MNV infection or 1 hr after infection resulted in significantly higher induction levels of the RNA sensing genes tested. These results indicate that the quick uptake of EVS is responsible for eliciting the activation of the host antiviral response resulting in reduced MNV-1 replication.

[0091] It was expected that a decrease in viral genome replication mediated by EVs would be correlated with decrease viral induced cytotoxicity and lyses. To this end, lactate dehydrogenase (LDH) activity was measured. LDH is an intracellular enzyme and the activity of this enzyme in the supernatant indicates cell membrane disruption. RAW 264.7 cells challenged with MNV-1 in presence or absence of increasing concentrations of EVs and LDH activity evaluated. Compared to the negative infection control, there wasAttorney Ref. No.10457-591PC0 approximately a 60.0% (± 24.5%, p=0.0042) increase in LDH activity in the supernatant of MNV infection control (Fig.1 C). Interestingly, all concentrations of EVs were able to prevent cell lysis. These findings indicate that the antiviral response induced by the L. johnsonii N6.2 EVs prevents viral induced cytotoxicity by decreasing the overall viral genome available for viral formation and cell lysis. L. johnsonii N6.2 EVs elicit an antiviral response triggering a tolerogenic profile of cytokine expression.

[0092] A canonical antiviral response culminates in the expression of type I interferons (IFN-I), IFNα and IFNβ, followed by the induction of the expression of innate immune response genes in nearby cells. To further elucidate the mechanisms by which the L. johnsonii N6.2 EVs elicits the antiviral response, the expression of murine type one interferons, IFNα (IFNA1 and IFNA4) and IFNβ (IFNB) genes, and type three interferons, IFNλ (IFNL2 and IFNL3) genes, were analyzed in the presence and absence of the EVs and / or MNV-118 hpi. It was found that MNV-1 induced the expression of IFNα genes, IFNA1 and IFNA4, as well as IFNB (Fig.2) while the other genes were not strongly induced by the MNV-1 infection (IFNL2 and IFNL3). The addition of EVs alone did not result in expression of IFNA1 and IFNA4 while the addition of EVs during MNV-1 infection resulted in significant decrease in the expression of both genes. Noteworthy, the expression of IFNA1 was reduced at all concentrations of EVs tested while IFNA4 was dose dependent and linked to the viral load. On the other hand, the RAW 264.7 cells had basal level of expression IFNB. IFNB expression increased in the presence of the EVs, but significantly lower than the positive MNV control. Nonetheless, the EV10 treatment of the RAW 264.7 cells inoculated with MNV showed a decrease in IFNB expression, as observed in the IFNα genes, when compared to the MNV infection control (Fig.2).

[0093] The expression of IFN-I as an outcome of viral infection, can limit viral infections, but when unchecked can lead to deleterious inflammation. IL10 has been reported to produce an immunosuppressive function that is induced by L. johnsonii N6.2 derived EVs. It was found that while the MNV positive control did not induce the expression of IL10, RAW 264.7 cells have a strong dose- dependent induction of the IL10 genes in the presence of the L. johnsonii N6.2 EVs (Fig.2 F). We propose that the strong induction of IL10 expression may limit the detrimental inflammatory effects produced by the induction of IFNβ expression. The SH3B2 domain of Sdp stimulate innate immune responses similar to EVs

[0094] Based on our initial proteomic analyses of L. johnsonii EVs, we selected and purified differentially enriched proteins in EVs (Harrison et al., 2021). Proteins Eno3, EV1875, EV8875, EV1390 were purified using Ni+affinity columns (Fig.9). In addition, selected domains of two large proteins enriched in the EV,Attorney Ref. No.10457-591PC0 Sdp (Lysin, SH3B1-SH3B2, SH3B2 and SH3B6) and Muc (Muc1, Muc3, Muc4 and Muc5) we purified and tested. To evaluate the impact of each of the proteins or domains on stimulation of innate immune responses, RAW-Dual™ KO-TLR4 Cells were utilized. The presence of a TLR4 knockout, IRF and MIP- 2 (NF-κB) allowed for the exclusion of effects by potential lipopolysaccharide contamination from protein purification, and the quantification of the signaling through NF-κB (MIP-2 promoter fusion to supernatant alkaline phosphatase, SEAP) and interferon signaling (ISRE promoter fusion to Lucia luciferase). It was found that 1.5 ug / ml of the Sdp domain SH3B2 significantly induced the expression of the MIP-2 and ISRE to a similar level as EVs while domain SH3B6 did not induce significant changes in the reporter genes (Fig.3 A). Likewise, other proteins enriched in the EVs did not induce the expression of the SEAP or Lucia reporters (Fig.3 B). L. johnsonii N6.2 EVs and SH3B2 utilize the TRIF / TRAM and MyD88 adaptor proteins pathways to initiate cellular response

[0095] To determine the mechanisms of EV and SH3B2 domain utilized in signal transduction, WT RAW 264.7 Mφ as well as the knockout murine derived Mφ (ΔTLR2, ΔTLR3, ΔTLR4, ΔTLR2 / TLR4, ΔTLR9, ΔTRIF / TRAM, ΔMyD88, ΔMAL, ΔIRF3, ΔIRF7, Table 3) of surface and / or endosomal receptors as well as signaling adaptor proteins were used. Cells were incubated with 0.6 ug of SH3B2 or EV10. Mock and buffer were used as control. Interestingly, EV and SH3B2 showed a similar pattern of response. It was found that MφΔTLR2, Mφ ΔTLR4, Mφ ΔTRIF / TRAM, and Mφ ΔMyD88 resulted in impaired responses to EVs as well as SH3B2. The other KO mutations tested did not resulted in a significant effect on their responses to EVs or SH3B2 (Fig.5, Fig.6). Using the expression of OAS1b as reporter of the innate antiviral responses, it was found that in the double knockout of the endosomal adaptor proteins ΔTRIF / TRAM had significant reduction in the OAS1b expression when incubated with SH3B2 or EVs (Fig.6). These results are in agreement with our previous report that preventing EV cellular uptake using endocytosis inhibitors decreased the cells expression of the antiviral genes in human pancreatic cells (Teixeira et al., 2022). In contrast, the expression of IL10 gene was reduced significantly in the ΔMyD88 and the ΔTLR2 cell lines for EV but only on ΔMyD88 in presence of SH3B2.

[0096] The impact of the impaired ability of the Mφ ΔTLR2, Mφ ΔTRIF / TRAM, and ΔMyD88 to sense SH3B2 or EVs was evaluated on MNV-1 infection assays. It was found that while the mutations tested resulted in cells that were less sensitive to MNV infection (Fig.10), Mφ ΔTRIF / TRAM, and Mφ ΔMyD88 behaved as the infection control in presence of EV or SH3B2 (Fig.6). These results indicateAttorney Ref. No.10457-591PC0 that SH2B2 and EVs signaling through TRIF / TRAM and ΔMyD88 is required to achieve full protection to MNV infection. L. johnsonii N6.2 EVs and SH3B2 lower MNV titers in vivo

[0097] C57BL / 6 mice were orally fed with L. johnsonii N6.2 at 10e8, EVs at 10e10 and 3 ug SH3B2. Additonally, 10e10 liposomes made of L. jhonsonii lipids as well as 10e10 liposomes loaded with 3 ug SH3B2 were tested. PBS Buffer carrier was administered to the control group. After seven days, all groups were challenged with MNV at 1x107viral particles per animal for 24 h. The MNV titers were quantified in distal ileum tissue. It was found that L. johnsonii N6.2, EVs and liposomes+SH3B2 significantly decreased MNV titers in distal ileum while liposomes alone showed no effect on MONV infection titer (Fig.7). In agreement with this observation, the mRNA MNV genome quantification from ileum tissue also showed a significant decrease in relative intracellular viral load (this is not available for the lip and lip+SH3B2). These results indicate that L. johnsonii N6.2, EVs as well as SH3B2 can help in control viral infection and potentially decrease transmissibility by lowering the viral titers shed by the host.Attorney Ref. No.10457-591PC0 Table 1 Primers utilized for qRT-PCR. Primer name Primer sequence (5'-3') SourceSEQ ID NOmurine GAPDH Forward AGTATGACTCCACTCACGGCAAAT *10original workAttorney Ref. No.10457-591PC0 * Table 2. Primers used amplification of genes from L. johnsonii N6.2 Locus tag Primer name ` Source SEQ ID NO: SH3B2 Harrison et al., 38Attorney Ref. No.10457-591PC0 Table 3. Bacterial and macrophage cell lines utilized. Bacterial Strains Genotype Reference or Source kAttorney Ref. No.10457-591PC0 β) / TRAM (TRIF-related adaptor molecule) double KO mice ΔM D88 M NR 15633 D i d f M D88 l id diff ti ti BEI R

[0098] In another example, the expression of the innate response antiviral genes DHX58, OASL, MDA5, MAVS, MX1 and IRF7 was evaluated in vitro using chicken-derived macrophages. It was found that the addition of 0.6 ug / well of purified SH3b2 significantly induce the expression of OASL, MDA5, MX1 and IRF7.

[0099] Based on our previous results that indicate that the stimulation of this pathway in vitro was translated to an in vivo prevention of norovirus infections, we are confident that this preliminary data will result in a therapeutic application to H5N1 (avian flu).

[0100] Although the present invention has been described in considerable detail with reference to certain preferred versions thereof, other versions are possible. Therefore, the spirit and scope of the appended claims should not be limited to the description of the preferred versions contained herein.

[0101] The reader’s attention is directed to all papers and documents which are filed concurrently with this specification and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0102] All the features disclosed in this specification (including any accompanying claims, abstract, and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0103] Any element in a claim that does not explicitly state “means for” performing a specified function, or “step for” performing a specific function, is not to be interpreted as a “means” or “step” clause as specified in 35 U.S.C §112, sixth paragraph. In particular, the use of “step of” in the claims herein is not intended to invoke the provisions of 35 U.S.C §112, sixth paragraph.

Claims

Attorney Ref. No.10457-591PC0 CLAIMS What is claimed is:

1. A method for treating an infection, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising at least one polypeptide comprising at least one sequence selected from SEQ ID NOs: 1-6, or fragments or variants thereof, to a subject in need.

2. The method of claim 1, wherein the polypeptide comprises SEQ ID NO: 2, or fragments thereof, or variants thereof.

3. The method of claim 1, wherein the pharmaceutical composition comprises extracellular vesicles comprising the at least one polypeptide.

4. The method of any of claims 1-3, wherein the polypeptide comprises SEQ ID NO. 2, a fragment thereof, or variant having at least 90% or 95% identity therewith.

5. The method of claim 4, wherein the polypeptide is SEQ ID NO:

2.

6. The method of any of claims 1-5, wherein the pharmaceutical composition is administered orally or parenterally.

7. The method of any of claims 1-6, wherein the infection is a viral infection.

8. The method of any of claims 1-7, wherein the therapeutically effective amount is an amount sufficient to induce increase expression of an OAS1b, OAS2, OASL, MX1, MX2 and / or IFI44L gene in the subject.

9. The method of any of claims 3-8, wherein the therapeutically effective amount of the composition comprises EVS at 0.0001 μg / kg to 1.0 mg / kg the subject's body weight.

10. The method of any of claims 3-8, wherein the pharmaceutical composition is formulated into a capsule containing the EVs.

11. The method of any of claims 1-10, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

12. The method of any of claims 3-8, wherein the pharmaceutical composition comprises EVs produced by a Lactobacillus sp, optionally L. johnsonii, and wherein the EVs are enriched for EVs that comprise an increased amount of sdp or a polypeptide comprising a sequence of SEQ ID NOs 1-6.Attorney Ref. No.10457-591PC0 13. A pharmaceutical composition comprising an isolated polypeptide comprising an amino acid sequence of SEQ ID NOs: 1-6, or fragment or variant thereof, and optionally a pharmaceutically acceptable carrier.

14. The pharmaceutical composition of claim 13 formulated for parenteral administration.

15. The pharmaceutical composition of claim 13 formulated for oral administration.

16. The pharmaceutical composition of any of claims 13-15, wherein the isolated polypeptide is SEQ ID NO: 2, or a fragment or variant thereof.

17. An isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 2, or a fragment or variant thereof.

18. A composition comprising EVs from a Lactobacillus sp., produced by: culturing Lactobacillus sp cells in media; centrifuging the media containing Lactobacillus sp. cells to produce a supernatant; retrieving EVs from the supernatant to obtain an EV sample; and enriching the EV sample for EVs having a higher sdp or sh3b domain content; and optionally, packaging the EVs in a pharmaceutically acceptable vehicle.

19. The composition of claim 18, wherein the Lactobacillus sp. comprises Lactobacillus johnsonii.

20. The composition of claim 18, wherein the Lactobacillus johnsonii is Lactobacillus johnsonii N6.2 strain.

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