WO2024015522A1

WO2024015522A1 - Methods for analyzing extracellular vesicles and microbes

Info

Publication number: WO2024015522A1
Application number: PCT/US2023/027653
Authority: WO
Inventors: Daniela Beccati; Tanmoy GANGULY; Aylwin Ng
Original assignee: Evelo Biosciences, Inc.
Priority date: 2022-07-13
Filing date: 2023-07-13
Publication date: 2024-01-18

Abstract

Compositions and methods are provided for characterizing microbes and extracellular vesicles derived therefrom. The characterization of compositions of microbes and extracellular vesicles derived therefrom are also provided.

Description

METHODS FOR ANALYZING EXTRACELLULAR VESICLES AND MICROBES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/388,853, filed on July 13, 2022, the content of which is hereby incorporated by reference in its entirety.

BACKGROUND

[0002] Bacteria and microbial extracellular vesicles (mEVs) that are derived from bacteria can be used for a variety of purposes, including therapeutic applications. Accordingly, it is important to be able to: determine the presence of bacteria in a sample; identify bacteria in a sample; monitor the quality of bacteria in a sample; quantify the amount of bacteria in a sample; determine the presence of mEVs in a sample; identify mEVs in a sample; monitor the quality of mEVs in a sample; and quantify the amount of mEVs in a sample.

SUMMARY

[0003] The disclosure provides methods and compositions useful for evaluating microbes (e.g., bacteria) and/or microbial extracellular vesicles (mEVs, such as secreted mEVs (smEVs) or processed mEVs (pmEVs)), e.g., in a sample. The methods provided herein utilize one or more lectins, such as one or more plant lectins, to evaluate the bacteria and/or mEVs.

[0004] In certain aspects, provided herein are methods for analyzing bacteria or mEVs in a sample, the method comprising: contacting the sample comprising the bacterium or the mEV with one or more lectins; and detecting the binding of the bacterium or the mEV to one or more lectins, thereby analyzing the bacteria or the mEV in the sample.

[0005] In certain aspects, provided herein are methods useful in analyzing microbes (e.g., bacteria) and/or microbial extracellular vesicles (mEVs, such as secreted mEVs (smEVs) or processed mEVs (pmEVs)), such as by determining the presence of, identifying, and quantifying, microbes (e.g., bacteria) and/or microbial extracellular vesicles (mEVs, such as secreted mEVs (smEVs) or processed mEVs (pmEVs)). The methods provided herein utilize one or more lectins, such as one or more non-human lectins, such as plant lectins. For example, in some embodiments provided herein are various methods utilizing one or more lectins to determine the presence (e.g., present or absence); identify the type (e.g., genus, species or strain) and/or quantify the amount (e.g., an amount of a particular type (e.g., genus, species or strain) or a total amount) of bacteria or mEVs in a sample. For example, the methods can be used to determine the presence of, identify and/or quantify bacteria and/or mEVs in a sample, such as a sample containing excipients and bacteria and/or mEVs (such as a sample obtained at a particular stage or time of a process; from a fermentation culture, a biomass, or a drug substance (DS); and/or containing further excipients with a DS (such as a sample from a drug product (DP); for example, a sample, such as a sample of DS or DP can be reconstituted or resuspended in a liquid). In some embodiments provided herein, the various methods utilizing one or more lectins to determine the presence, identify and/or quantify bacteria or mEVs in a sample are used, for example, to identify the sample as a sample comprising the bacteria or as a sample comprising the mEVs derived from the bacteria. In some embodiments, the method identifies the sample as comprising the bacteria substantially free of mEVs derived therefrom. In some embodiments, the method identifies the sample as comprising the mEVs substantially free of the bacteria from which the mEVs were derived.

[0006] In some embodiments, methods of utilizing one or more lectins to detect the lectin-binding profile of bacteria or mEVs are provided herein. In some embodiments, the methods of utilizing one or more lectins to detect the lectin-binding profile of bacteria or mEVs may be used, for example, for quality control. In some embodiments provided herein, the method for detecting the lectin-binding profile of bacteria or mEVs may be used to monitor a change in the lectin-binding profile of the bacteria and/or the mEVs across samples, for example, samples obtained at different stages or times of a process as an in- process control. In some embodiments, monitoring a change in the lectin-binding profile of the bacteria and/or the mEVs comprises comparing the lectin-binding profiles of more than one sample, for example, samples obtained at different stages or times of a process as an in- process control, or after different storage conditions (e.g., time, temperature, and/or humidity). In some embodiments, monitoring a change in the lectin-binding profile of the bacteria and/or the mEVs comprises comparing the lectin-binding profiles of more than one sample, for example, samples obtained at a particular stage or time of a process; from a fermentation culture, a biomass, or a drug substance (DS); and/or containing further excipients with a DS (such as a sample from a drug product (DP)). In some embodiments, monitoring the change in the lectin-binding profile is used on samples after conditions for preparing or storing a material containing bacteria or mEVs change (e.g., temperature, humidity, time, and/or packaging). In some embodiments, monitoring the change in the lectin-binding profile is used on samples after the conditions for growing bacteria (e.g., media components, time, temperature, density, etc.), or for downstreaming processing of bacteria or mEVs, are altered.

[0007] In some embodiments, methods of utilizing one or more lectins to detect the glycosidic profile of bacteria or mEVs are provided herein. As used herein, the glycosidic profile refers to a profile of the glycans (e.g., glycoproteins, capsular polysaccharides, glycolipids, and peptidoglycans) decorating (e.g., present on) the surface of bacteria or mEVs. In some embodiments provided herein, the method for detecting the glycosidic profile of bacteria or mEVs may be used to monitor a change in the glycosidic profile of the bacteria and/or the mEVs across samples, for example, samples obtained at different stages or times of a process as an in-process control, or after different storage conditions (e.g., time, temperature, and/or humidity). In some embodiments, monitoring the change in the glycosidic profile of the bacteria and/or the mEVs comprises comparing the lectin-binding profiles of the more than one samples, for example, samples obtained at different stages or times of a process as an in-process control, or after different storage conditions (e.g., time, temperature, and/or humidity). In some embodiments, monitoring the change in the glycosidic profile of the bacteria and/or the mEVs comprises comparing the glycosidic profiles of the more than one samples, for example, samples obtained after different storage conditions (e.g., time, temperature, and/or humidity). In some embodiments provided herein, comparing the glycosidic profiles of the bacteria or the mEVs in each sample reveals the change in the glycosidic profile of the bacteria or the mEVs from sample to sample. In some embodiments, monitoring the change in the glycosidic profile is used on samples from a particular stage or time of a process; from a fermentation culture, a biomass, or a drug substance (DS); and/or containing further excipients with a DS (such as a sample from a drug product (DP)). In some embodiments, monitoring the change in the glycosidic profile is used on samples after conditions for preparing or storing a material containing bacteria or mEVs change (e.g., temperature, humidity, time, and/or packaging). In some embodiments, monitoring the change in the glycosidic profile is used on samples after the conditions for growing bacteria (e.g., media components, time, temperature, density, etc.), or for downstreaming processing of bacteria or mEVs, are altered. [0008] In some embodiments provided herein, the one or more lectins are nonhuman lectins. For example, in some embodiments, the non-human lectins are bacterial, plant, fungal, or non-human animal lectins. In some embodiments, the non-human lectins are plant lectins. In some embodiments, the non-human lectins are bacterial lectins. In some embodiments, about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the lectins are plant lectins. In some embodiments, more than 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the lectins are plant lectins. In some embodiments, the one or more lectins comprise the lectins BanLec and/or CSL3. In some embodiments, the one or more lectins comprise one or more of the lectins listed in Table 6. In some embodiments, the one or more lectins (e.g., the one or more plant lectins) comprise one or more of the following lectins: AAL, ACL, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA. In some embodiments, the one or more lectins (e.g., the one or more plant lectins) comprise the following lectins: AAL, ACL, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA. In some embodiments, the one or more lectins (e.g., the one or more plant lectins) comprise one or more of the following lectins: AAL, SJA, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA. In some embodiments, the one or more lectins (e.g., the one or more plant lectins) comprise the following lectins: AAL, SJA, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA. In some embodiments, the one or more lectins further comprise the lectins BanLec and/or CSL3. In some embodiments, the one or more lectins consist of the following lectins: AAL, ACL, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA, WGA, BanLec and CSL3. In some embodiments, the one or more lectins consist of the following lectins: AAL, SJA, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LT A, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA, WGA, BanLec and CSL3.

[0009] In some embodiments provided herein, the one or more lectins (e.g., the one or more plant lectins) comprise lectins that represent most known glycan-binding epitopes. [0010] In some embodiments provided herein, the one or more lectins (e.g., the one or more plant lectins) comprise one or more of: BPL and WGA. In some embodiments provided herein, the one or more lectins (e.g., the one or more plant lectins) comprise one or more of: BPL, RSL, VVL and WGA.

[0011] In some embodiments provided herein, the one or more lectins (e.g., the one or more plant lectins) comprise BPL and WGA. In some embodiments provided herein, the one or more lectins (e.g., the one or more plant lectins) comprise BPL, RSL, VVL and WGA.

[0012] In some embodiments provided herein, the one or more lectins (e.g., the one or more plant lectins) comprise one or more of: AIA, GSLB3, Morniga G and VVL. In some embodiments provided herein, the one or more lectins (e.g., the one or more plant lectins) comprise one or more of: AIA, BPL, ECL, GSLB4, HP A, Morniga G, SBA, VVL and WGA.

[0013] In some embodiments provided herein, the one or more lectins (e.g., the one or more plant lectins) comprise AIA, GSLB3, Morniga G and VVL. In some embodiments provided herein, the one or more lectins (e.g., the one or more plant lectins) comprise AIA, BPL, ECL, GSLB4, HP A, Morniga G, SBA, VVL and WGA.

[0014] In some embodiments provided herein, the one or more lectins (e.g., the one or more plant lectins) comprise one or more of: AIA, GNA, GSLB4, HHL, Morniga G, NPA and RCA-I. In some embodiments provided herein, the one or more lectins (e.g., the one or more plant lectins) comprise one or more of: AAL, AIA, BPL, ECL, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL, SBA, VVL and WFA.

[0015] In some embodiments provided herein, the one or more lectins (e.g., the one or more plant lectins) comprise AIA, GNA, GSLB4, HHL, Morniga G, NPA and RCA-I. In some embodiments provided herein, the one or more lectins (e.g., the one or more plant lectins) comprise AAL, AIA, BPL, ECL, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL, SBA, VVL and WFA. [0016] In some embodiments provided herein, the one or more lectins (e.g., the one or more plant lectins) comprise one or more of: GNA, HHL, HP A, LCA, NPA, PSA, and RSL. In some embodiments provided herein, the one or more lectins (e.g., the one or more plant lectins) comprise one or more of: AAL, BC2L-C Calsepa, ConA, GNA, GSII, HAA, HHL, HP A, LCA, LEL, NPA, PSA, RCA-I, RSL, STL and WGA.

[0017] In some embodiments provided herein, the one or more lectins (e.g., the one or more plant lectins) comprise GNA, HHL, HP A, LCA, NPA, PSA, and RSL. In some embodiments provided herein, the one or more lectins (e.g., the one or more plant lectins) comprise AAL, BC2L-C Calsepa, ConA, GNA, GSII, HAA, HHL, HP A, LCA, LEL, NPA, PSA, RCA-I, RSL, STL and WGA.

[0018] In some embodiments provided herein, the one or more lectins (e.g., the one or more plant lectins) comprise one or more of: AAL, AIA, Calsepa, GNA, HHL, LCA, NPA, PSA and RCA-I. In some embodiments provided herein, the one or more lectins (e.g., the one or more plant lectins) comprise one or more of: AAL, AIA, BC2L-C, Calsepa, ConA, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL and WGA. [0019] In some embodiments provided herein, the one or more lectins (e.g., the one or more plant lectins) comprise AAL, AIA, Calsepa, GNA, HHL, LCA, NPA, PSA and RCA-I. In some embodiments provided herein, the one or more lectins (e.g., the one or more plant lectins) comprise AAL, AIA, BC2L-C, Calsepa, ConA, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL and WGA.

[0020] In some embodiments provided herein, the one or more lectins comprise at least 1 lectin. In some embodiments provided herein, the one or more lectins comprise at least 5 lectins. In some embodiments provided herein, the one or more lectins comprise at least 10 lectins. In some embodiments provided herein, the one or more lectins comprise at least 15 lectins. In some embodiments provided herein, the one or more lectins comprise at least 20 lectins. In some embodiments provided herein, the one or more lectins comprise 1 to 45 lectins. In some embodiments provided herein, the one or more lectins comprise 1 to 20 lectins. In some embodiments provided herein, the one or more lectins comprise 1 to 15 lectins. In some embodiments provided herein, the one or more lectins comprise 1 to 10 lectins. In some embodiments provided herein, the one or more lectins comprise 1 to 5 lectins.

[0021] In some embodiments provided herein, the one or more lectins comprise at least 1 non-human lectin. In some embodiments provided herein, the one or more lectins comprise at least 5 non-human lectins. In some embodiments provided herein, one or more lectins comprise at least 10 non-human lectins. In some embodiments provided herein, the one or more lectins comprise at least 15 non-human lectins. In some embodiments provided herein, the one or more lectins comprise at least 20 non-human lectins. In some embodiments provided herein, the one or more lectins comprise 1 to 45 non-human lectins. In some embodiments provided herein, the one or more lectins comprise 1 to 20 non-human lectins. In some embodiments provided herein, the one or more lectins comprise 1 to 15 non-human lectins. In some embodiments provided herein, one or more lectins comprise 1 to 10 non-human lectins. In some embodiments provided herein, one or more lectins comprise 1 to 5 non-human lectins.

[0022] In some embodiments provided herein, the one or more lectins comprise at least 1 plant lectin. In some embodiments provided herein, the one or more lectins comprise at least 5 plant lectins. In some embodiments provided herein, the one or more lectins comprise at least 10 plant lectins. In some embodiments provided herein, the one or more lectins comprise at least 15 plant lectins. In some embodiments provided herein, the one or more lectins comprise at least 20 plant lectins. In some embodiments provided herein, the one or more lectins comprise 1 to 45 plant lectins. In some embodiments provided herein, the one or more lectins comprise 1 to 20 plant lectins. In some embodiments provided herein, the one or more lectins comprise 1 to 15 plant lectins. In some embodiments provided herein, the one or more lectins comprise 1 to 10 plant lectins. In some embodiments provided herein, the one or more lectins comprise 1 to 5 plant lectins.

[0023] In some embodiments provided herein, a panel of lectins (also referred to as a lectin panel) (i.e., comprising one or more lectins, such as one or more plant lectins) is provided. In some embodiments, the panel of lectins is used in the methods described herein. In some embodiments, about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the lectins in the lectin panel are non-human lectins. In some embodiments, more than 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the lectins in the lectin panel are non-human lectins. In some embodiments provided herein, the lectins in the lectin panel are non-human lectins. For example, in some embodiments, the non-human lectins are bacterial, plant, fungal, or non-human animal lectins. In some embodiments, the non-human lectins are plant lectins. In some embodiments, the non-human lectins are bacterial lectins. In some embodiments, about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the lectins in the lectin panel are plant lectins. In some embodiments, more than 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the lectins in the lectin panel are plant lectins. In some embodiments, the lectin panel comprises the lectins BanLec and/or CSL3. In some embodiments, the lectin panel comprises one or more of the lectins listed in Table 6. In some embodiments, the lectin panel comprises one or more of the following lectins: AAL, ACL, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LT A, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA. In some embodiments, the lectin panel comprises the following lectins: AAL, ACL, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA. In some embodiments, the lectin panel comprises one or more of the following lectins: AAL, SJA, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA. In some embodiments, the lectin panel comprises the following lectins: AAL, SJA, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA. In some embodiments, the lectin panel further comprises the lectins BanLec and/or CSL3. In some embodiments, the lectin panel consists of the following lectins: AAL, ACL, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA, WGA, BanLec and CSL3. In some embodiments, the lectin panel consists of the following lectins: AAL, SJA, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA, WGA, BanLec and CSL3. [0024] In some embodiments provided herein, the lectin panel comprises lectins that represent most known glycan-binding epitopes. [0025] In some embodiments provided herein, the lectin panel comprises one or more of: BPL and WGA. In some embodiments provided herein, the lectin panel comprises one or more of: BPL, RSL, VVL and WGA.

[0026] In some embodiments provided herein, the lectin panel comprises BPL and WGA. In some embodiments provided herein, the lectin panel comprises BPL, RSL, VVL and WGA.

[0027] In some embodiments provided herein, the lectin panel comprises one or more of: AIA, GSLB3, Morniga G and VVL. In some embodiments provided herein, the lectin panel comprises one or more of: AIA, BPL, ECL, GSLB4, HP A, Morniga G, SBA, VVL and WGA.

[0028] In some embodiments provided herein, the lectin panel comprises AIA, GSL B3, Morniga G and VVL. In some embodiments provided herein, the lectin panel comprises AIA, BPL, ECL, GSLB4, HP A, Morniga G, SBA, VVL and WGA.

[0029] In some embodiments provided herein, the lectin panel comprises one or more of: AIA, GNA, GSLB4, HHL, Morniga G, NPA and RCA-I. In some embodiments provided herein, the lectin panel comprises one or more of: AAL, AIA, BPL, ECL, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL, SBA, VVL and WFA. [0030] In some embodiments provided herein, the lectin panel comprises AIA, GNA, GSLB4, HHL, Morniga G, NPA and RCA-I. In some embodiments provided herein, the lectin panel comprises AAL, AIA, BPL, ECL, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL, SBA, VVL and WFA.

[0031] In some embodiments provided herein, the lectin panel comprises one or more of: GNA, HHL, HP A, LCA, NPA, PSA, and RSL. In some embodiments provided herein, the lectin panel comprises one or more of: AAL, BC2L-C Calsepa, ConA, GNA, GSII, HAA, HHL, HP A, LCA, LEL, NPA, PSA, RCA-I, RSL, STL and WGA.

[0032] In some embodiments provided herein, the lectin panel comprises GNA, HHL, HP A, LCA, NPA, PSA, and RSL. In some embodiments provided herein, the lectin panel comprises AAL, BC2L-C Calsepa, ConA, GNA, GSII, HAA, HHL, HP A, LCA, LEL, NPA, PSA, RCA-I, RSL, STL and WGA.

[0033] In some embodiments provided herein, the lectin panel comprises one or more of: AAL, AIA, Calsepa, GNA, HHL, LCA, NPA, PSA and RCA-I. In some embodiments provided herein, the lectin panel comprises one or more of: AAL, AIA, BC2L-C, Calsepa, ConA, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA- I, RSL and WGA.

[0034] In some embodiments provided herein, the lectin panel comprises AAL, AIA, Calsepa, GNA, HHL, LCA, NPA, PSA and RCA-I. In some embodiments provided herein, the lectin panel comprises AAL, AIA, BC2L-C, Calsepa, ConA, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL and WGA.

[0035] In some embodiments provided herein, the lectin panel comprises at least 1 lectin. In some embodiments provided herein, the lectin panel comprises at least 5 lectins. In some embodiments provided herein, the lectin panel comprises at least 10 lectins. In some embodiments provided herein, the lectin panel comprises at least 15 lectins. In some embodiments provided herein, the lectin panel comprises at least 20 lectins. In some embodiments provided herein, the lectin panel comprises 1 to 45 lectins. In some embodiments provided herein, the lectin panel comprises 1 to 20 lectins. In some embodiments provided herein, the lectin panel comprises 1 to 15 lectins. In some embodiments provided herein, the lectin panel comprises 1 to 10 lectins. In some embodiments provided herein, the lectin panel comprises 1 to 5 lectins. In some embodiments provided herein, the lectin panel comprises 25 to 45, 30 to 43, 35 to 42 or 39 to 42 lectins. In some embodiments, the lectin panel comprises 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 lectins.

[0036] In some embodiments provided herein, the lectin panel comprises at least 1 non-human lectin. In some embodiments provided herein, the lectin panel comprises at least 5 non-human lectins. In some embodiments provided herein, the lectin panel comprises at least 10 non-human lectins. In some embodiments provided herein, the lectin panel comprises at least 15 non-human lectins. In some embodiments provided herein, the lectin panel comprises at least 20 non-human lectins. In some embodiments provided herein, the lectin panel comprises 1 to 45 non-human lectins. In some embodiments provided herein, the lectin panel comprises 1 to 20 non-human lectins. In some embodiments provided herein, the lectin panel comprises 1 to 15 non-human lectins. In some embodiments provided herein, the lectin panel comprises 1 to 10 non-human lectins. In some embodiments provided herein, the lectin panel comprises 1 to 5 non-human lectins. In some embodiments provided herein, the lectin panel comprises 25 to 45, 30 to 43, 35 to 42 or 39 to 42 non-human lectins. In some embodiments, the lectin panel comprises 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 non-human lectins. [0037] In some embodiments provided herein, the lectin panel comprises at least 1 plant lectin. In some embodiments provided herein, the lectin panel comprises at least 5 plant lectins. In some embodiments provided herein, the lectin panel comprises at least 10 plant lectins. In some embodiments provided herein, the lectin panel comprises at least 15 plant lectins. In some embodiments provided herein, the lectin panel comprises at least 20 plant lectins. In some embodiments provided herein, the lectin panel comprises 1 to 45 plant lectins. In some embodiments provided herein, the lectin panel comprises 1 to 20 plant lectins. In some embodiments provided herein, the lectin panel comprises 1 to 15 plant lectins. In some embodiments provided herein, the lectin panel comprises 1 to 10 plant lectins. In some embodiments provided herein, the lectin panel comprises 1 to 5 plant lectins. In some embodiments provided herein, the lectin panel comprises 25 to 45, 30 to 43, 35 to 42 or 39 to 42 plant lectins. In some embodiments, the lectin panel comprises 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 plant lectins.

[0038] In some embodiments provided herein, the one or more lectins (such as in a lectin panel) are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the lectins are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each lectin in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of lectins. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16-subarray format. In some embodiments, one or more lectins are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0039] In some embodiments provided herein, bacteria or mEVs are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the bacteria or mEVs are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each of the bacteria or mEV sample in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of bacteria or mEVs. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16- subarray format. In some embodiments, bacteria or mEVs are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization. [0040] In some embodiments, the bacteria or the mEVs are detectably labeled. Examples of detectable labels include, but are not limited to, fluorescent moieties, radioactive moieties, paramagnetic moieties, luminescent moieties and/or colorimetric moieties. In some embodiments, the detectable label comprises a fluorescent moiety. Examples of fluorescent moieties include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa

Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa

Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa

Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine,

Venus, YPet, Emerald, Cerulean, Cy3 and CyPet. In some embodiments, the fluorescent moiety is Alexa Fluor555.

[0041] In some embodiments, the one or more lectins (such as in a lectin panel) are detectably labeled. Examples of detectable labels include, but are not limited to, fluorescent moieties, radioactive moieties, paramagnetic moieties, luminescent moieties and/or colorimetric moieties. In some embodiments, the detectable label comprises a fluorescent moiety. Examples of fluorescent moieties include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa

Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine,

[0042] In some embodiments provided herein, the one or more lectins (such as in a lectin panel) can be used to determine the presence, identify and/or quantify bacteria of the genus Prevotella, Fournierella, Harryflintia, Veillonella and/or Subdoligi mulum and/or mEVs derived from such bacteria. In some embodiments, the one or more lectins (such as in a lectin panel) can be used to determine the presence, identify and/or quantify bacteria of the species Prevotella histicola, Fournierella massiliensis, Harryflintia acelispora. Veillonella parvula and/or Subcloligranuliim var labile and/or mEVs derived from such bacteria. In some embodiments, the one or more lectins (such as in a lectin panel) can be used to determine the presence, identify and/or quantify bacteria of the strain Prevotella Strain B 50329 (NRRL accession number B 50329 and also referred to herein as “P. histicola 1”), Prevotella histicola ATCC designation number PTA-126140 (referred to herein as “P. histicola 2”), Fournierella massiliensis Strain A (ATCC Deposit Number PTA- 126696 and also referred to herein as “P massiliensis’"'), Harryflintia acetispora Strain A (ATCC Deposit Number PTA-126694 and referred to herein as “P. acetispora"), Veillonella parvula Strain A (ATCC Accession Number PTA-125691 and also referred to herein as “E parvula” , and/or Subdoligranulum variabile (referred to herein as “5. variabile”) and/or mEVs derived from such bacteria.

[0043] In some embodiments provided herein, the one or more lectins (such as in a lectin panel) can be used to detect or monitor the lectin-binding profile of bacteria and/or mEVs derived from such bacteria of the genus: Prevotella, Fournierella, Harryflintia, Veillonella and/or Subdoligranulum. In some embodiments, the one or more lectins (such as in a lectin panel) can be used to detect or monitor the lectin-binding profile of bacteria and/or mEVs derived from such bacteria of the species: Prevotella histicola, Fournierella massiliensis, Harryflintia acetispora, Veillonella parvula and/or Subdoligranulum variabile. In some embodiments, the one or more lectins (such as in a lectin panel) can be used to detect or monitor the lectin-binding profile of bacteria and/or mEVs of such bacteria of the strain: Prevotella Strain B 50329 (NRRL accession number B 50329 and also referred to herein as “P. histicola 1”), Prevotella histicola ATCC designation number PTA- 126140 (referred to herein as “P. histicola 2”), Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696 and also referred to herein as “P massiliensis” , Harryflintia acetispora Strain A (ATCC Deposit Number PTA-126694 and referred to herein as “P acetispora”), Veillonella parvula Strain A (ATCC Accession Number PTA-125691 and also referred to herein as “E parvula”), and/or Subdoligrctmiliim. variabile (referred to herein as “5. variabile”).

[0044] In some embodiments provided herein, the one or more lectins (such as in a lectin panel) can be used to detect or monitor the glycosidic profile of bacteria and/or mEVs derived from such bacteria of the genus: Prevotella, Fournierella, Harryflintia, Veillonella and/or Subdoligranulum. In some embodiments, the one or more lectins (such as in a lectin panel) can be used to detect or monitor the glycosidic profile of bacteria and/or mEVs derived from such bacteria of the species: Prevotella histicola, Fournierella massiliensis, Harryflintia acetispora, Veillonella parvula and/or Subdoligranulum variabile. In some embodiments, the one or more lectins (such as in a lectin panel) can be used to detect or monitor the glycosidic profile of bacteria and/or mEVs of such bacteria of the strain: Prevotella Strain B 50329 (NRRL accession number B 50329 and also referred to herein as “ . histicola 1”), Prevotella histicola ATCC designation number PTA-126140 (referred to herein as “ . histicola 2”), Fournierella massiliensis Strain A (ATCC Deposit Number PTA- 126696 and also referred to herein as “P. massiliensis’"'), Harryflintia acetispora Strain A (ATCC Deposit Number PTA-126694 and referred to herein as “77. acelispora"), Veillonella parvula Strain A (ATCC Accession Number PTA-125691 and also referred to herein as “F. parvula” , and/or Subdoligramdum variabile (referred to herein as “A variabile” ).

[0045] In certain aspects, provided herein is a method of determining the presence of a genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs) in a sample. In some embodiments, the method comprises: (a) contacting the sample comprising the bacteria or the mEVs with one or more lectins (such as in a lectin panel) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); and (b) detecting the binding of the bacteria or the mEVs to one or more lectins (e.g., detecting with a detectable label), thereby determining the presence of a genus, species, and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs).

[0046] In some embodiments, the bacteria or the mEVs are detectably labeled prior to step (a). In some embodiments, the bacteria or the mEVs are detectably labeled after step (a) and prior to step (b).

[0047] In some embodiments, the one or more lectins are detectably labeled prior to step (a). In some embodiments, the one or more lectins are detectably labeled after step (a) and prior to step (b).

[0048] In some embodiments provided herein, the one or more lectins (such as in a lectin panel) are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the lectins are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each lectin in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of lectins. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16-subarray format. In some embodiments, one or more lectins are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization. [0049] In some embodiments provided herein, bacteria or mEVs are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the bacteria or mEVs are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each of the bacteria or mEV sample in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of bacteria or mEVs. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16- subarray format. In some embodiments, bacteria or mEVs are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0050] In certain aspects, provided herein is a method of identifying the genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs) in a sample. In some embodiments, the method comprises: (a) contacting the sample comprising the bacteria or the mEVs with one or more lectins (such as in a lectin panel) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); and (b) detecting the binding of the bacteria or the mEVs to one or more lectins (e.g., detecting with a detectable label), thereby identifying the genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs).

[0051] In some embodiments, the bacteria or the mEVs are detectably labeled prior to step (a). In some embodiments, the bacteria or the mEVs are detectably labeled after step (a) and prior to step (b).

[0052] In some embodiments, the bacteria or the mEVs are detectably labeled, for example with a fluorescent moiety. Examples of detectable labels include, but are not limited to, fluorescent moieties, radioactive moieties, paramagnetic moieties, luminescent moieties and/or colorimetric moieties. Examples of fluorescent moieties include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean, Cy3 and CyPet. In some embodiments, the fluorescent moiety is Alexa Fluor555. [0053] In some embodiments, the bacteria or the mEVs are detectably labeled after step (a), contacting the sample comprising the bacteria or the mEVs with one or more lectins (such as in a lectin panel), and prior to step (b), detecting the binding of the bacteria or the mEVs to one or more lectins. In some embodiments, the bacteria or the mEVs are detectably labeled after step (a) and prior to step (b) by contacting the bacteria or the mEVs bound to one or more lectins , first with a biotinylated monoclonal antibody specific for the bacteria or the mEVs followed by a fluorescent moiety-conjugated streptavidin, such as streptavidin-Cy3. In some embodiments, the bacteria or the mEVs are labeled after step (a) and prior to step (b) by contacting the bacteria or the mEVs bound to the lectins, first with a rabbit polyclonal antibody specific for the bacteria or the mEVs followed by an anti-(rabbit) IgG labeled with a fluorescent moiety, such as Cy3. In some embodiments, the monoclonal antibody or the rabbit polyclonal antibody specifically binds to an antigen on the bacteria or bacterial mEVs. In some embodiments, the antibody or an antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Prevotella (e.g., bacteria of the species Prevotella histicola, such as bacteria of the strain Prevotella Strain B 50329 or strain Prevotella histicola ATCC designation number PTA-126140) or mEVs derived therefrom. In some embodiments, the antibody or the antigen binding fragment is a 17B01- 02G10 mAb or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments, the antibody or the antigen binding fragment is a 02F09-02B04 mAB or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Fournierella (e.g., bacteria of the species Fournierella massiHensis. such as bacteria of the strain Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696)) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Harryflintia (e.g., bacteria of the species Harryflintia acelispora. such as bacteria of the strain Harryflintia acetispora Strain A) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Veillonella (e.g., bacteria of the species Veillonella parvula, such as bacteria of the strain Veillonella parvula Strain A (ATCC Accession Number PTA-125691)) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Subdoligranulum (e.g., bacteria of the species Subdoligi mithmi ven -labile) or mEVs derived therefrom.

[0054] In some embodiments, the one or more lectins are detectably labeled prior to step (a). In some embodiments, the one or more lectins are detectably labeled after step (a) and prior to step (b).

[0055] In some embodiments, the one or more lectins are detectably labeled, for example with a fluorescent moiety. Examples of detectable labels include, but are not limited to, fluorescent moieties, radioactive moieties, paramagnetic moieties, luminescent moieties and/or colorimetric moieties. Examples of fluorescent moieties include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean, Cy3 and CyPet. In some embodiments, the fluorescent moiety is Alexa Fluor555.

[0056] In some embodiments provided herein, the one or more lectins (such as in a lectin panel) are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the lectins are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each lectin in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of lectins. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16-subarray format. In some embodiments, one or more lectins are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0057] In some embodiments provided herein, bacteria or mEVs are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the bacteria or mEVs are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each of the bacteria or mEV sample in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of bacteria or mEVs. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16- subarray format. In some embodiments, bacteria or mEVs are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0058] In some aspects, provided herein is a method of determining the presence of a genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs) in the sample, the method comprising: (a) labeling the bacteria or the mEVs with a detectable label; (b) contacting the sample comprising the bacteria or the mEVs with one or more lectins (such as in a lectin panel) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); and (c) detecting the binding of the bacteria or the mEVs to one or more lectins (e.g., detecting with a detectable label), thereby determining the presence of a genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs).

[0059] In some embodiments, the bacteria or the mEVs are detectably labeled, for example with a fluorescent moiety. Examples of detectable labels include, but are not limited to, fluorescent moieties, radioactive moieties, paramagnetic moieties, luminescent moieties and/or colorimetric moieties. Examples of fluorescent moieties include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean, Cy3 and CyPet. In some embodiments, the fluorescent moiety is AlexaFluor555.

[0060] In some embodiments provided herein, the one or more lectins (such as in a lectin panel) are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the lectins are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each lectin in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of lectins. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16-subarray format. In some embodiments, one or more lectins are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization. [0061] In some embodiments provided herein, bacteria or mEVs are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the bacteria or mEVs are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each of the bacteria or mEV sample in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of bacteria or mEVs. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16- subarray format. In some embodiments, bacteria or mEVs are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0062] In some aspects, provided herein is a method of determining the presence of a genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs) in the sample, the method comprising: (a) labeling one or more lectins with a detectable label; (b) contacting the sample comprising the bacteria or the mEVs with the one or more lectins (such as in a lectin panel) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); and (c) detecting the binding of the bacteria or the mEVs to one or more lectins (e.g., detecting with a detectable label), thereby determining the presence of a genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs). In some embodiments, the one or more lectins are detectably labeled, for example with a fluorescent moiety. Examples of detectable labels include, but are not limited to, fluorescent moieties, radioactive moieties, paramagnetic moieties, luminescent moieties and/or colorimetric moieties. Examples of fluorescent moieties include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430,

Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555,

Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647,

Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790,

EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean, Cy3 and CyPet. In some embodiments, the fluorescent moiety is AlexaFluor555. [0063] In some embodiments provided herein, the one or more lectins (such as in a lectin panel) are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the lectins are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each lectin in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of lectins. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16-subarray format. In some embodiments, one or more lectins are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0064] In some embodiments provided herein, bacteria or mEVs are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the bacteria or mEVs are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each of the bacteria or mEV sample in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of bacteria or mEVs. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16- subarray format. In some embodiments, bacteria or mEVs are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0065] In certain aspects, provided herein is a method of determining the presence of a genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs) in a sample, the method comprising: (a) contacting the sample comprising the bacteria or the mEVs with one or more lectins (such as in a lectin panel) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); (b) contacting the bacteria or the mEVs bound to one or more lectins with an antibody; (c) detectably labeling the antibody; and (d) detecting the binding of the bacteria or the mEVs to one or more lectins (e.g., detecting the detectable label), thereby determining the presence of a genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs). In some embodiments provided herein, the bacteria or the mEVs bound to one or more lectins are first contacted with a biotinylated monoclonal antibody specific for the bacteria or the mEVs (step (b)), followed by a fluorescent moiety-conjugated streptavidin, such as streptavidin-Cy3 (step (c)). In some embodiments provided herein, the bacteria or the mEVs bound to one or more lectins are first contacted with a rabbit polyclonal antibody specific for the bacteria or the mEVs (step (b)), followed by an anti- (rabbit) IgG labeled with detectable label such as a fluorescent moiety, such as Cy3 (step (c)). In some embodiments, the monoclonal antibody or the rabbit polyclonal antibody specifically binds to an antigen on bacteria or bacterial mEVs. In some embodiments, the antibody or an antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Prevotella (e.g., bacteria of the species Prevotella histicola, such as bacteria of the strain Prevotella Strain B 50329 or strain Prevotella histicola ATCC designation number PTA-126140) or mEVs derived therefrom. In some embodiments, the antibody or the antigen binding fragment is a 17B01-02G10 mAb or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments, the antibody or the antigen binding fragment is a 02F09-02B04 mAB or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Fournierella (e.g., bacteria of the species Fournierella massiliensis, such as bacteria of the strain Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696)) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Harryflintia (e.g., bacteria of the species Harryflintia acelispora. such as bacteria of the strain Harryflintia acetispora Strain A) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Veillonella (e.g., bacteria of the species Veillonella parvula, such as bacteria of the strain Veillonella parvula Strain A (ATCC Accession Number PTA-125691)) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Subdoligranulum (e.g., bacteria of the species Subdoligranulum variabile) or mEVs derived therefrom. In some embodiments provided herein, the bacteria or the mEVs bound to one or more lectins are first contacted with a biotinylated monoclonal antibody specific for one or more lectins (step (b)), followed by a fluorescent moiety-conjugated streptavidin, such as streptavidin- Cy3 (step (c)). In some embodiments provided herein, the bacteria or the mEVs bound to one or more lectins are first contacted with a rabbit polyclonal antibody specific for the one or more lectins (step (b)), followed by an anti-(rabbit) IgG labeled with detectable label such as a fluorescent moiety, such as Cy3 (step (c)). In some embodiments, the monoclonal antibody or the rabbit polyclonal antibody specifically binds to an epitope on one or more lectins. [0066] In some embodiments provided herein, the one or more lectins (such as in a lectin panel) are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the lectins are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each lectin in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of lectins. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16-subarray format. In some embodiments, one or more lectins are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0067] In some embodiments provided herein, bacteria or mEVs are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the bacteria or mEVs are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each of the bacteria or mEV sample in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of bacteria or mEVs. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16- subarray format. In some embodiments, bacteria or mEVs are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0068] In some aspects, provided herein is a method of identifying the genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs) in the sample, the method comprising: (a) labeling the bacteria or the mEVs with a detectable label; (b) contacting the sample comprising the bacteria or the mEVs with one or more lectins (such as in a lectin panel) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); and (c) detecting the binding of the bacteria or the mEVs to one or more lectins (e.g., detecting with a detectable label), thereby identifying the genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs). In some embodiments, the bacteria or the mEVs are detectably labeled, for example with a fluorescent moiety. Examples of detectable labels include, but are not limited to, fluorescent moieties, radioactive moieties, paramagnetic moieties, luminescent moieties and/or colorimetric moieties. Examples of fluorescent moieties include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean, Cy3 and CyPet. In some embodiments, the fluorescent moiety is AlexaFluor555.

[0069] In some embodiments provided herein, the one or more lectins (such as in a lectin panel) are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the lectins are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each lectin in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of lectins. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16-subarray format. In some embodiments, one or more lectins are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0070] In some embodiments provided herein, bacteria or mEVs are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the bacteria or mEVs are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each of the bacteria or mEV sample in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of bacteria or mEVs. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16- subarray format. In some embodiments, bacteria or mEVs are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0071] In some aspects, provided herein is a method of identifying the genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs) in the sample, the method comprising: (a) labeling one or more lectins with a detectable label; (b) contacting the sample comprising the bacteria or the mEVs with the one or more lectins (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); and (c) detecting the binding of the bacteria or the mEVs to one or more lectins (e.g., detecting with a detectable label), thereby identifying the genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs). In some embodiments, the one or more lectins are detectably labeled, for example with a fluorescent moiety. Examples of detectable labels include, but are not limited to, fluorescent moieties, radioactive moieties, paramagnetic moieties, luminescent moieties and/or colorimetric moieties. Examples of fluorescent moieties include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514,

Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594,

Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680,

Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean, Cy3 and CyPet. In some embodiments, the fluorescent moiety is AlexaFluor555.

[0072] In some embodiments provided herein, the one or more lectins (such as in a lectin panel) are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the lectins are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each lectin in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of lectins. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16-subarray format. In some embodiments, one or more lectins are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0073] In some embodiments provided herein, bacteria or mEVs are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the bacteria or mEVs are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each of the bacteria or mEV sample in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of bacteria or mEVs. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16- subarray format. In some embodiments, bacteria or mEVs are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization. [0074] In some aspects, provided herein is a method of identifying the genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs) in a sample, the method comprising: (a) contacting the sample comprising the bacteria or the mEVs with one or more lectins (such as in a lectin panel); (b) contacting the bacteria or the mEVs bound to one or more lectins with an antibody; (c) detectably labeling the antibody; and (d) detecting the binding of the bacteria or the mEVs to one or more lectins (e.g., fluorescence microarray), thereby identifying the genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs). In some embodiments provided herein, the bacteria or the mEVs bound to one or more lectins are first contacted with a biotinylated monoclonal antibody specific for the bacteria or the mEVs (step (b)), followed by a fluorescent moiety-conjugated streptavidin, such as streptavidin-Cy3 (step (c)). In some embodiments provided herein, the bacteria or the mEVs bound to one or more lectins are first contacted with a rabbit polyclonal antibody specific for the bacteria or the mEVs (step

(b)), followed by an anti-(rabbit) IgG labeled with a fluorescent moiety, such as Cy3 (step

(c)). In some embodiments, the monoclonal antibody or the rabbit polyclonal antibody specifically binds to an antigen on a type of bacteria or bacterial mEVs. In some embodiments, the antibody or an antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Prevotella (e.g., bacteria of the species Prevotella histicola, such as bacteria of the strain Prevotella Strain B 50329 or strain Prevotella histicola ATCC designation number PTA-126140) or mEVs derived therefrom. In some embodiments, the antibody or the antigen binding fragment is a 17B01-02G10 mAb or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments, the antibody or the antigen binding fragment is a 02F09-02B04 mAB or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Fournierella (e.g., bacteria of the species Fournierella massiliensis, such as bacteria of the strain Fournierella massiliensis Strain A (ATCC Deposit Number PTA- 126696)) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Harryflintia (e.g., bacteria of the species Harryflintia acelispora. such as bacteria of the strain Harryflintia acetispora Strain A) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Veillonella (e.g., bacteria of the species Veillonella parvula, such as bacteria of the strain Veillonella parvula Strain A (ATCC Accession Number PTA-125691)) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Subdoligranulum (e.g., bacteria of the species Subdoligranuliim variabile) or mEVs derived therefrom. In some embodiments provided herein, the bacteria or the mEVs bound to one or more lectins are first contacted with a biotinylated monoclonal antibody specific for the one or more lectins (step (b)), followed by a fluorescent moiety-conjugated streptavidin, such as streptavidin-Cy3 (step (c)). In some embodiments provided herein, the bacteria or the mEVs bound to one or more lectins are first contacted with a rabbit polyclonal antibody specific for the one or more lectins (step (b)), followed by an anti- (rabbit) IgG labeled with a fluorescent moiety, such as Cy3 (step (c)). In some embodiments, the monoclonal antibody or the rabbit polyclonal antibody specifically binds to an epitope on one or more lectins.

[0075] In some embodiments provided herein, the one or more lectins (such as in a lectin panel) are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the lectins are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each lectin in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of lectins. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16-subarray format. In some embodiments, one or more lectins are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0076] In some embodiments provided herein, bacteria or mEVs are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the bacteria or mEVs are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each of the bacteria or mEV sample in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of bacteria or mEVs. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16- subarray format. In some embodiments, bacteria or mEVs are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0077] In some embodiments provided herein, the various methods may also be used, for example, to identify a sample as comprising the bacteria substantially free of (e.g., substantially isolated from) the mEVs derived therefrom. In some embodiments, a sample comprising bacteria substantially free of the mEVs derived therefrom comprises less than about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1 x 10³, 2 x 10³, 3 x 10³, 4 x 10³, 5 x

10³, 6 x 10³, 7 x 10³, 8 x 10³, 9 x 10³, 1 x 10⁴, 2 x 10⁴, 3 x 10⁴, 4 x 10⁴, 5 x 10⁴, 6 x 10⁴, 7 x

10⁴, 8 x 10⁴, 9 x 10⁴, 1 x 10⁵, 2 x 10⁵, 3 x 10⁵, 4 x 10⁵, 5 x 10⁵, 6 x 10⁵, 7 x 10⁵, 8 x 10⁵, 9 x

10⁵, 1 x 10⁶, 2 x 10⁶, 3 x 10⁶, 4 x 10⁶, 5 x 10⁶, 6 x 10⁶, 7 x 10⁶, 8 x 10⁶ or 9 x 10⁶ mEV particles per bacterium. In some embodiments, in a sample comprising bacteria substantially free of the mEVs derived therefrom, more than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of total particles in the sample are bacteria. In some embodiments, in a sample comprising bacteria substantially free of the mEVs derived therefrom, less than 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% of the total particles in the sample are mEVs.

[0078] In some embodiments provided herein, the various methods may also be used, for example, to identify a sample as comprising mEVs substantially free of the bacteria from which the mEVs were derived. In some embodiments, a sample comprising mEVs substantially free of the bacteria from which the mEVs were derived comprises less than 1 bacterium for about every 1 x 10³, 2 x 10³, 3 x 10³, 4 x 10³, 5 x 10³, 6 x 10³, 7 x 10³,

8 x 10³, 9 x 10³, 1 x 10⁴, 2 x 10⁴, 3 x 10⁴, 4 x 10⁴, 5 x 10⁴, 6 x 10⁴, 7 x 10⁴, 8 x 10⁴, 9 x 10⁴,

1 x 10⁵, 2 x 10⁵, 3 x 10⁵, 4 x 10⁵, 5 x 10⁵, 6 x 10⁵, 7 x 10⁵, 8 x 10⁵, 9 x 10⁵, 1 x 10⁶, 2 x 10⁶,

3 x 10⁶, 4 x 10⁶, 5 x 10⁶, 6 x 10⁶, 7 x 10⁶, 8 x 10⁶, 9 x 10⁶, 1 x 10⁷, 2 x 10⁷, 3 x 10⁷, 4 x 10⁷,

5 x 10⁷, 6 x 10⁷, 7 x 10⁷, 8 x 10⁷, 9 x 10⁷, 1 x 10⁸, 2 x 10⁸, 3 x 10⁸, 4 x 10⁸, 5 x 10⁸, 6 x 10⁸,

7 x 10⁸, 8 x 10⁸, 9 x 10⁸, 1 x 10⁹, 2 x 10⁹, 3 x 10⁹, 4 x 10⁹, 5 x 10⁹, 6 x 10⁹, 7 x 10⁹, 8 x 10⁹,

9 x 10⁹, 1 x IO¹⁰, 2 x IO¹⁰, 3 x IO¹⁰, 4 x IO¹⁰, 5 x IO¹⁰, 6 x IO¹⁰, 7 x IO¹⁰, 8 x IO¹⁰, 9 x IO¹⁰, 1 x 10¹¹, 2 x 10¹¹, 3 x 10¹¹, 4 x 10¹¹, 5 x 10¹¹, 6 x 10¹¹, 7 x 10¹¹, 8 x 10¹¹, 9 x 10¹¹ or 1 x 10¹² mEV particles. In some embodiments, in a sample comprising mEVs substantially free of the bacteria from which the mEVs were derived, more than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of total particles in the sample are mEVs. In some embodiments, in a sample comprising mEVs substantially free of the bacteria from which the mEVs were derived, less than 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% of the total particles in the sample are bacteria.

[0079] In certain aspects, provided herein is a method of quantifying bacteria or mEVs derived from bacteria (bacterial mEVs) in a sample. In some embodiments, the method comprises: (a) contacting the sample comprising the bacteria or mEVs with one or more lectins (such as in a lectin panel) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); (b) detecting the binding of the bacteria or mEVs to one or more lectins (e.g., detecting with a detectable label); and (c) comparing at least one lectin-binding signal of the sample to a lectin-binding signal of the same lectin for a reference sample comprising the bacteria or mEVs, wherein the particle count of bacteria or mEVs in the reference sample is known, thereby quantifying the bacteria or mEVs in the sample based on a change in lectin-binding signal intensity between the reference sample and the sample.

[0080] In some embodiments, the bacteria or mEVs are detectably labeled prior to step (a). In some embodiments, the bacteria or mEVs are detectably labeled after step (a) and prior to step (b). In some embodiments, the bacteria or mEVs are labeled after step (a) and prior to step (b) by contacting the bacteria or mEVs bound to one or more lectins , first with a biotinylated monoclonal antibody specific for the bacteria or mEVs followed by a fluorescent moi ety-conjugated streptavidin, such as streptavidin-Cy3. In some embodiments, the bacteria or mEVs are labeled after step (a) and prior to step (b) by contacting the bacteria or mEVs bound to one or more lectins , first with a rabbit polyclonal antibody specific for the bacteria or mEVs followed by an anti-(rabbit) IgG labeled with a fluorescent moiety, such as Cy3. In some embodiments, the monoclonal antibody or the rabbit polyclonal antibody specifically binds to an antigen on a type of bacteria or bacterial mEVs. In some embodiments, the antibody or an antigen binding fragment thereof specifically binds to an antigen on a type of bacteria or bacterial mEVs of the genus Prevotella (e.g., bacteria or bacterial mEVs of the species Prevotella histicola, such as bacteria or bacterial mEVs of the strain Prevotella Strain B 50329 or strain Prevotella histicola ATCC designation number PTA-126140). In some embodiments, the antibody or the antigen binding fragment is a 17B01-02G10 mAb or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments, the antibody or the antigen binding fragment is a 02F09-02B04 mAB or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria or bacterial mEVs of the genus Fournierella (e.g., bacteria or bacterial mEVs of the species Fournierella massiHensis. such as bacteria or bacterial mEVs of the strain Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696)). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria or bacterial mEVs of the genus Harryflintia (e.g., bacteria or bacterial mEVs of the species Harryflintia acelispora. such as bacteria or bacterial mEVs of the strain Harryflintia acetispora Strain A). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria or bacterial mEVs of the genus Veillonella (e.g., bacteria or bacterial mEVs of the species Veillonella parvula, such as bacteria or bacterial mEVs of the strain Veillonella parvula Strain A (ATCC Accession Number PTA-125691)). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria or bacterial mEVs of the genus Subdoligranulum (e.g., bacteria or bacterial mEVs of the species Subdoligranulum variabile).

[0081] In some embodiments, the one or more lectins are detectably labeled prior to step (a). In some embodiments, the one or more lectins are detectably labeled after step (a) and prior to step (b). In some embodiments, the one or more lectins are labeled after step (a) and prior to step (b) by contacting the bacteria or mEVs bound to one or more lectins, first with a biotinylated monoclonal antibody specific for the one or more lectins followed by a fluorescent moi ety-conjugated streptavidin, such as streptavidin-Cy3. In some embodiments, the one or more lectins are labeled after step (a) and prior to step (b) by contacting the bacteria or mEVs bound to one or more lectins, first with a rabbit polyclonal antibody specific for the one or more lectins followed by an anti-(rabbit) IgG labeled with a fluorescent moiety, such as Cy3. In some embodiments, the monoclonal antibody or the rabbit polyclonal antibody specifically binds to an epitope on one or more lectins.

[0082] In some embodiments provided herein, the one or more lectins (such as in a lectin panel) are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the lectins are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each lectin in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of lectins. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16-subarray format. In some embodiments, one or more lectins are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0083] In some embodiments provided herein, bacteria or mEVs are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the bacteria or mEVs are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each of the bacteria or mEV sample in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of bacteria or mEVs. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16- subarray format. In some embodiments, bacteria or mEVs are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0084] In some aspects, provided herein is a method of quantifying bacteria or mEVs derived from bacteria (bacterial mEVs) in a sample, the method comprises: (a) labeling the bacteria or mEVs with a detectable label; (b) contacting the sample comprising the bacteria or mEVs with one or more lectins (such as in a lectin panel) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); (c) detecting the binding of the bacteria or mEVs to one or more lectins (e.g., detecting the detectable label); and (d) comparing at least one lectin-binding signal of the sample to a lectin-binding signal of the same lectin for a reference sample comprising the bacteria or mEVs, wherein the bacteria or mEV particle count in the reference sample is known, thereby quantifying the bacteria or mEVs in the sample based on the change in the lectin- binding signal intensity between the reference sample and the sample.

[0085] In some embodiments, the bacteria or mEVs are detectably labeled, for example with a fluorescent moiety. Examples of detectable labels include, but are not limited to, fluorescent moieties, radioactive moieties, paramagnetic moieties, luminescent moieties and/or colorimetric moieties. Examples of fluorescent moieties include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean, Cy3 and CyPet. In some embodiments, the fluorescent moiety is AlexaFluor555. In some embodiments wherein the bacteria or mEVs are detectably labeled with a fluorescent moiety, step (d) comprises comparing a relative fluorescence intensity of binding of the sample to at least one lectin to a fluorescence intensity of binding of the reference sample to the same lectin(s) and quantifying the bacteria or mEVs in the sample based on the change in the relative fluorescence intensity of binding to the at least one lectin between the reference sample and the sample, wherein the bacteria or mEV particle count in the reference sample is known.

[0086] In some embodiments provided herein, the one or more lectins (such as in a lectin panel) are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the lectins are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each lectin in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of lectins. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16-subarray format. In some embodiments, one or more lectins are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0087] In some embodiments provided herein, bacteria or mEVs are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the bacteria or mEVs are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each of the bacteria or mEV sample in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of bacteria or mEVs. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16- subarray format. In some embodiments, bacteria or mEVs are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0088] In certain aspects, provided herein is a method of quantifying bacteria or mEVs derived from bacteria (bacterial mEVs) in a sample, the method comprises: (a) contacting the sample comprising the bacteria or mEVs with one or more lectins (such as in a lectin panel) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); (b) contacting the bacteria or mEVs bound to one or more lectins with an antibody; (c) detectably labeling the antibody; (d) detecting the binding of the bacteria or mEVs to one or more lectins (e.g., detecting the detectable label); and (e) comparing at least one lectin-binding signal of the sample to a lectin-binding signal of the same lectin for a reference sample comprising the bacteria or mEVs, wherein the bacteria or mEV particle count in the reference sample is known, thereby quantifying the bacteria or mEVs in the sample.

[0089] In some embodiments, the antibody is detectably labeled, for example with a fluorescent moiety. Examples of detectable labels include, but are not limited to, fluorescent moieties, radioactive moieties, paramagnetic moieties, luminescent moieties and/or colorimetric moieties. Examples of fluorescent moieties include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean, Cy3 and CyPet. In some embodiments wherein the antibody is detectably labeled with a fluorescent moiety, step (d) comprises comparing a relative fluorescence intensity of binding of the sample to at least one lectin to a fluorescence intensity of binding of the reference sample to the same lectin(s) and quantifying the bacteria or mEVs in the sample based on the change in the relative fluorescence intensity of binding to the at least one lectin between the reference sample and the sample, wherein the bacteria or mEV particle count in the reference sample is known.

[0090] In some embodiments provided herein, the bacteria or mEVs bound to one or more lectins are first contacted with a biotinylated monoclonal antibody specific for the bacteria or mEVs (step (b)), followed by a fluorescent moiety-conjugated streptavidin, such as streptavidin-Cy3 (step (c)). In some embodiments provided herein, the bacteria or mEVs bound to one or more lectins are first contacted with a rabbit polyclonal antibody specific for the bacteria or mEVs (step (b)), followed by an anti-(rabbit) IgG labeled with a fluorescent moiety, such as Cy3 (step (c)). In some embodiments, the monoclonal antibody or the rabbit polyclonal antibody specifically binds to an antigen on a type of bacteria or bacterial mEVs. In some embodiments, the antibody or an antigen binding fragment thereof specifically binds to an antigen on bacteria or bacterial mEVs of the genus Prevotella (e.g., bacteria or bacterial mEVs of the species Prevotella histicola, such as bacteria or bacterial mEVs of the strain Prevotella Strain B 50329 or strain Prevotella histicola ATCC designation number PTA-126140). In some embodiments, the antibody or the antigen binding fragment is a 17B01-02G10 mAb or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments, the antibody or the antigen binding fragment is a 02F09-02B04 mAB or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria or bacterial mEVs of the genus Fournierella (e.g., bacteria or bacterial mEVs of the species Fournierella massiHensis. such as bacteria or bacterial mEVs of the strain Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696)). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria or bacterial mEVs of the genus Harryflintia (e.g., bacteria or bacterial mEVs of the species Harryflintia acelispora. such as bacteria or bacterial mEVs of the strain Harryflintia acetispora Strain A). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria or bacterial mEVs of the genus Veillonella (e.g., bacteria or bacterial mEVs of the species Veillonella parvula, such as bacterial mEVs of the strain Veillonella parvula Strain A (ATCC Accession Number PTA-125691)). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria or bacterial mEVs of the genus Subdoligranulum (e.g., bacteria or bacterial mEVs of the species Subdoligranulum variabile).

[0091] In some embodiments provided herein, the one or more lectins (such as in a lectin panel) are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the lectins are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each lectin in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of lectins. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16-subarray format. In some embodiments, one or more lectins are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0092] In some embodiments provided herein, bacteria or mEVs are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the bacteria or mEVs are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each of the bacteria or mEV sample in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of bacteria or mEVs. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16- subarray format. In some embodiments, bacteria or mEVs are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0093] In certain aspects, provided herein is a method of detecting a lectin-binding profile of a sample comprising bacteria or microbial extracellular vesicles (mEVs). In some embodiments, the method comprises: (a) contacting the sample comprising the bacteria or the mEVs with one or more lectins (such as in a lectin panel) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); and (b) detecting the binding of the bacteria or the mEVs to one or more lectins (e.g., detecting with a detectable label), thereby detecting the lectin-binding profile of the sample. In some embodiments, the bacteria or the mEVs are detectably labeled prior to step (a). In some embodiments, the bacteria or the mEVs are detectably labeled after step (a) and prior to step (b). In some embodiments, the one or more lectins are detectably labeled prior to step (a). In some embodiments, the one or more lectins are detectably labeled after step (a) and prior to step (b).

[0094] In some embodiments provided herein, the one or more lectins (such as in a lectin panel) are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the lectins are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each lectin in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of lectins. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16-subarray format. In some embodiments, one or more lectins are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0095] In some embodiments provided herein, bacteria or mEVs are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the bacteria or mEVs are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each of the bacteria or mEV sample in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of bacteria or mEVs. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16- subarray format. In some embodiments, bacteria or mEVs are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0096] In certain aspects, provided herein is a method of monitoring a change in a lectin-binding profile in more than one sample comprising bacteria or microbial extracellular vesicles (mEVs). In some embodiments, the method comprises: (a) contacting each sample comprising the bacteria or the mEVs with one or more lectins (such as in a lectin panel) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); (b) detecting the binding of the bacteria or the mEVs to one or more lectins (e.g., detecting with a detectable label); and (c) comparing the lectin-binding profiles of the bacteria or the mEVs in each sample, thereby monitoring the change in the lectin-binding profile of the samples. In some embodiments provided herein, the method of monitoring the change in the lectin-binding profile is used as an in-process control. In some embodiments provided herein, the method of monitoring the change in the lectin-binding profile is used to monitor a change in the lectin-binding profile of multiple samples from one source, wherein the multiple samples were stored under different storage conditions (e.g., time, temperature, and/or humidity). In some embodiments, monitoring a change in the lectin- binding profile of the bacteria and/or the mEVs comprises comparing the lectin-binding profiles of more than one sample, for example, samples obtained at a particular stage or time of a process; or from a fermentation culture, a biomass, or a drug substance (DS); and/or containing further excipients with a DS (such as a sample from a drug product (DP)). In some embodiments, monitoring the change in the lectin-binding profile is used on samples after conditions for preparing or storing a material containing bacteria or mEVs change (e.g., temperature, humidity, time, and/or packaging). In some embodiments, monitoring the change in the lectin-binding profile is used on samples after the conditions for growing bacteria (e.g., media components, time, temperature, density, etc.), or for downstreaming processing of bacteria or mEVs, are altered. In some embodiments provided herein, comparing the lectin-binding profiles of the samples reveals change in the quality of the bacteria or the mEVs from sample to sample. In some embodiments, the bacteria or the mEVs are detectably labeled prior to step (a). In some embodiments, the bacteria or the mEVs are detectably labeled after step (a) and prior to step (b). In some embodiments, the one or more lectins are detectably labeled prior to step (a). In some embodiments, the one or more lectins are detectably labeled after step (a) and prior to step (b).

[0097] In some embodiments provided herein, the one or more lectins (such as in a lectin panel) are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the lectins are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each lectin in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of lectins. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16-subarray format. In some embodiments, one or more lectins are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0098] In some embodiments provided herein, bacteria or mEVs are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the bacteria or mEVs are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each of the bacteria or mEV sample in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of bacteria or mEVs. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16- subarray format. In some embodiments, bacteria or mEVs are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0099] In certain aspects, provided herein is a method of detecting a glycosidic profile of a sample comprising bacteria or microbial extracellular vesicles (mEVs). In some embodiments, the method comprises: (a) contacting the sample comprising the bacteria or the mEVs with one or more lectins (such as in a lectin panel) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); and (b) detecting the binding of the bacteria or the mEVs to one or more lectins (e.g., detecting with a detectable label), thereby detecting the glycosidic profile of the sample. In some embodiments, the bacteria or the mEVs are detectably labeled prior to step (a). In some embodiments, the bacteria or the mEVs are detectably labeled after step (a) and prior to step (b). In some embodiments, the one or more lectins are detectably labeled prior to step (a). In some embodiments, the one or more lectins are detectably labeled after step (a) and prior to step (b).

[0100] In some embodiments provided herein, the one or more lectins (such as in a lectin panel) are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the lectins are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each lectin in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of lectins. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16-subarray format. In some embodiments, one or more lectins are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0101] In some embodiments provided herein, bacteria or mEVs are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the bacteria or mEVs are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each of the bacteria or mEV sample in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of bacteria or mEVs. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16- subarray format. In some embodiments, bacteria or mEVs are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0102] In certain aspects, provided herein is a method of monitoring a change in a glycosidic profile in more than one sample comprising bacteria or microbial extracellular vesicles (mEVs). In some embodiments, the method comprises: (a) contacting each sample comprising the bacteria or the mEVs with one or more lectins (such as in a lectin panel) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); (b) detecting the binding of the bacteria or the mEVs to one or more lectins (e.g., detecting with a detectable label); and (c) comparing the lectin-binding profiles of the bacteria or the mEVs in each sample, thereby monitoring the change in the glycosidic profile of the samples. In some embodiments provided herein, the method of monitoring the change in the glycosidic profile is used as an in-process control. In some embodiments provided herein, the change in the lectin-binding profile across samples is used to monitor a change in the glycosidic profile of multiple samples from one source, wherein the multiple samples were stored under different storage conditions (e.g., time, temperature, and/or humidity). In some embodiments, monitoring the change in the glycosidic profile of the bacteria and/or the mEVs comprises comparing the glycosidic profiles of the more than one samples, for example, samples obtained after different storage conditions (e.g., time, temperature, and/or humidity). In some embodiments provided herein, comparing the glycosidic profiles of the bacteria or the mEVs in each sample reveals the change in the lectin-binding profile of the bacteria or the mEVs from sample to sample. In some embodiments, monitoring the change in the glycosidic profile is used on samples from a particular stage or time of a process; from a fermentation culture, a biomass, or a drug substance (DS); and/or containing further excipients with a DS (such as a sample from a drug product (DP)). In some embodiments, monitoring the change in the glycosidic profile is used on samples after conditions for preparing or storing a material containing bacteria or mEVs change (e.g., temperature, humidity, time, and/or packaging). In some embodiments, monitoring the change in the glycosidic profile is used on samples after the conditions for growing bacteria (e.g., media components, time, temperature, density, etc.), or for downstreaming processing of bacteria or mEVs, are altered. In some embodiments provided herein, comparing the glycosidic profiles of the bacteria or the mEVs in each sample reveals the change in the glycosidic profiles of the bacteria or the mEVs from sample to sample. In some embodiments, the bacteria or the mEVs are detectably labeled prior to step (a). In some embodiments, the bacteria or the mEVs are detectably labeled after step (a) and prior to step (b). In some embodiments, the one or more lectins are detectably labeled prior to step (a). In some embodiments, the one or more lectins are detectably labeled after step (a) and prior to step (b).

[0103] In some embodiments provided herein, the one or more lectins (such as in a lectin panel) are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the lectins are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each lectin in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of lectins. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16-subarray format. In some embodiments, one or more lectins are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0104] In some embodiments provided herein, bacteria or mEVs are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the bacteria or mEVs are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each of the bacteria or mEV sample in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of bacteria or mEVs. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16- subarray format. In some embodiments, bacteria or mEVs are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0105] In some aspects, provided herein is a composition comprising a. Prevotella histicola mEV, the composition exhibiting a lectin-binding profile (e.g., a fluorescence lectin-binding profile) to lectins BPL, RSL, VVL and WGA. In some embodiments, the lectin-binding profile of the Prevotella histicola mEV composition comprises: (a) stronger relative intensities (e.g., fluorescence intensities) of binding to BPL and WGA; and (b) weaker relative intensities (e.g., fluorescence intensities) of binding to RSL and VVL. In some embodiments, the relative intensities of binding to BPL and WGA are the dominant peaks of the lectin-binding profile (e.g., fluorescence lectin-binding profile). In some embodiments provided herein, the Prevotella histicola mEV is P. histicola 1 mEV. In some embodiments provided herein, the Prevotella histicola mEV is P. histicola 1 mEV, wherein the lectin-binding profile further comprises WFA and wherein the lectin-binding profile further comprises no significant relative intensity (e.g., fluorescence intensity) of binding to WFA compared to BPL, RSL, VVL and WGA. In some embodiments provided herein, the Prevotella histicola mEV is P. histicola 2 mEV, wherein the lectin-binding profile further comprises lectins: AAL, BC2L-C, GNA, HHL, HP A, NPA, PSA, RCA-I and WFA and wherein the lectin-binding profile further comprises weaker relative intensities (e.g., fluorescence intensities) of binding to AAL, BC2L-C, GNA, HHL, HP A, NPA, PSA, RCA- I and WFA compared to BPL and WGA.

[0106] In some aspects, provided herein is a composition comprising a. Fournierella massiliensis mEV, the composition exhibiting a lectin-binding profile (e.g., fluorescence lectin-binding profile) to lectins: AIA, BPL, ECL, GSLB4, HP A, Morniga G, SBA, VVL, and WGA lectins, wherein the lectin-binding profile comprises: (a) stronger relative intensities (e.g., fluorescence intensities) of binding to AIA, GSLB3, Morniga G and VVL; and (b) weaker relative intensities (e.g., fluorescence intensities) of binding to BPL, ECL, HP A, SBA and WGA, and wherein the intensities of binding to AIA, GSLB4, and Morniga G are the dominant peaks of the lectin-binding profile (e.g., fluorescence lectin-binding profile). In some embodiments, the Fournierella massiliensis mEV is Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696) mEV.

[0107] In some aspects, provided herein is a composition comprising a Harryflintia acetispora mEV, the composition exhibiting a lectin-binding profile (e.g., a fluorescence lectin-binding profile) to lectins: AAL, AIA, BPL, ECL, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL, SBA, VVL and WFA, wherein the lectin-binding profile comprises: (a) stronger relative intensities (e.g., fluorescence intensities) of binding to AIA, GNA, GSLB4, HHL, Morniga G, NPA and RCA-I; and (b) weaker relative intensities (e.g., fluorescence intensities) of binding to AAL, BPL, ECL, HP A, LCA, PSA, RSL, SBA, VVL and WFA, and wherein the intensities of binding to AIA and Morniga G are the dominant peaks of the lectin-binding profile (e.g., fluorescence lectin-binding profile). In some embodiments provided herein, the Harryflintia acetispora mEV is Harryflintia acetispora Strain A mEV.

[0108] In some aspects, provided herein is a composition comprising a Veillonella parvula mEV, the composition exhibiting a lectin-binding profile (e.g., a fluorescence lectin-binding profile) to lectins: AAL, BC2L-C Calsepa, ConA, GNA, GSII, HAA, HHL, HP A, LCA, LEL, NPA, PSA, RCA-I, RSL, STL and WGA, wherein the lectin-binding profile comprises: (a) stronger relative intensities (e.g., fluorescence intensities) of binding to GNA, HHL, HP A, LCA, NPA, PSA, and RSL; and (b) weaker relative intensities (e.g., fluorescence intensities) of binding to AAL, BC2L-C, Calsepa, ConA, GSII, HAA, LEL, RCA-I, STL and WGA, and wherein the intensity of binding to HPA is the dominant peak of the lectin-binding profile (e.g., fluorescence lectin-binding profile). In some embodiments provided herein, the Veillonella parvula mEV is Veillonella parvula Strain A (ATCC Accession Number PTA-125691) mEV.

[0109] In some aspects, provided herein is a composition comprising a Subdoligranulum variabile mEV, the composition exhibiting a lectin-binding profile (e.g., a fluorescence lectin-binding profile) to lectins: AAL, AIA, BC2L-C, Calsepa, ConA, GNA, GSI-B4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL and WGA, wherein the lectin-binding profile comprises: (a) stronger relative intensities (e.g., fluorescence intensities) of binding to AAL, AIA, Calsepa, GNA, HHL, LCA, NPA, PSA and RCA-I; and (b) weaker relative intensities (e.g., fluorescence intensities) of binding to BC2L-C, GSLB4, HP A, Morniga G and WGA, and wherein the lectin-binding profile also shows relative intensities (e.g., fluorescence intensities) of binding to Con A and RSL.

[0110] In certain aspects, the disclosure provides a lectin panel (i.e., comprising one or more lectins) for use in determining the presence, identifying, and/or quantifying microbes (e.g., bacteria) and microbial extracellular vesicles (mEVs), and/or for detecting and/or monitoring a lectin-binding profile and/or a glycosidic profile of bacteria or mEVs. In some embodiments, about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the lectins in the lectin panel are non-human lectins. In some embodiments, more than 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the lectins in the lectin panel are non-human lectins. In some embodiments, all of the lectins are non-human lectins. In some embodiments, the non-human lectins are bacterial, plant, fungal, or non-human animal lectins. In some embodiments, the non-human lectins are plant lectins. In some embodiments, the non-human lectins are bacterial lectins. In some embodiments, about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the lectins in the lectin panel are plant lectins. In some embodiments, more than 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the lectins in the lectin panel are plant lectins. In some embodiments, the lectin panel comprises the lectins BanLec and/or CSL3. In some embodiments, the lectin panel comprises one or more of the lectins listed in Table 6. In some embodiments, the lectin panel comprises one or more of the following lectins: AAL, ACL, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LT A, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA. In some embodiments, the lectin panel comprises the following lectins: AAL, ACL, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSI-B4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA. In some embodiments, the lectin panel comprises one or more of the following lectins: AAL, SJA, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA. In some embodiments, the lectin panel comprises the following lectins: AAL, SJA, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA. In some embodiments, the lectin panel further comprises the lectins BanLec and/or CSL3. In some embodiments, the lectin panel comprises the following lectins: AAL, ACL, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA, WGA, BanLec and CSL3. In some embodiments, the lectin panel comprises the following lectins: AAL, SJA, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA, WGA, BanLec and CSL3.

[OHl] In some embodiments provided herein, the lectin panel comprises one or more of: BPL and WGA. In some embodiments provided herein, the lectin panel comprises one or more of: BPL, RSL, VVL and WGA.

[0112] In some embodiments provided herein, the lectin panel comprises BPL and WGA. In some embodiments provided herein, the lectin panel comprises BPL, RSL, VVL and WGA.

[0113] In some embodiments provided herein, the lectin panel comprises one or more of: AIA, GSLB3, Morniga G and VVL. In some embodiments provided herein, the lectin panel comprises one or more of: AIA, BPL, ECL, GSLB4, HP A, Morniga G, SBA, VVL and WGA.

[0114] In some embodiments provided herein, the lectin panel comprises AIA, GSL B3, Morniga G and VVL. In some embodiments provided herein, the lectin panel comprises AIA, BPL, ECL, GSLB4, HP A, Morniga G, SBA, VVL and WGA. [0115] In some embodiments provided herein, the lectin panel comprises one or more of: AIA, GNA, GSI-B4, HHL, Morniga G, NPA and RCA-I. In some embodiments provided herein, the lectin panel comprises one or more of: AAL, AIA, BPL, ECL, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL, SBA, VVL and WFA. [0116] In some embodiments provided herein, the lectin panel comprises AIA, GNA, GSLB4, HHL, Morniga G, NPA and RCA-I. In some embodiments provided herein, the lectin panel comprises AAL, AIA, BPL, ECL, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL, SBA, VVL and WFA.

[0117] In some embodiments provided herein, the lectin panel comprises one or more of: GNA, HHL, HP A, LCA, NPA, PSA, and RSL. In some embodiments provided herein, the lectin panel comprises one or more of: AAL, BC2L-C Calsepa, ConA, GNA, GSII, HAA, HHL, HP A, LCA, LEL, NPA, PSA, RCA-I, RSL, STL and WGA.

[0118] In some embodiments provided herein, the lectin panel comprises GNA, HHL, HP A, LCA, NPA, PSA, and RSL. In some embodiments provided herein, the lectin panel comprises AAL, BC2L-C Calsepa, ConA, GNA, GSII, HAA, HHL, HP A, LCA, LEL, NPA, PSA, RCA-I, RSL, STL and WGA.

[0119] In some embodiments provided herein, the lectin panel comprises one or more of: AAL, AIA, Calsepa, GNA, HHL, LCA, NPA, PSA and RCA-I. In some embodiments provided herein, the lectin panel comprises one or more of: AAL, AIA, BC2L-C, Calsepa, ConA, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA- I, RSL and WGA.

[0120] In some embodiments provided herein, the lectin panel comprises AAL, AIA, Calsepa, GNA, HHL, LCA, NPA, PSA and RCA-I. In some embodiments provided herein, the lectin panel comprises AAL, AIA, BC2L-C, Calsepa, ConA, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL and WGA.

[0121] In some embodiments provided herein, the lectin panel comprises at least 1 lectin. In some embodiments provided herein, the lectin panel comprises at least 5 lectins. In some embodiments provided herein, the lectin panel comprises at least 10 lectins. In some embodiments provided herein, the lectin panel comprises at least 15 lectins. In some embodiments provided herein, the lectin panel comprises at least 20 lectins. In some embodiments provided herein, the lectin panel comprises 1 to 45 lectins. In some embodiments provided herein, the lectin panel comprises 1 to 20 lectins. In some embodiments provided herein, the lectin panel comprises 1 to 15 lectins. In some embodiments provided herein, the lectin panel comprises 1 to 10 lectins. In some embodiments provided herein, the lectin panel comprises 1 to 5 lectins. In some embodiments, the lectin panel comprises 25 to 45, 30 to 43, 35 to 42 or 39 to 42 lectins. In some embodiments, the lectin panel comprises 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 lectins.

[0122] In some embodiments provided herein, the lectin panel comprises at least 1 non-human lectin. In some embodiments provided herein, the lectin panel comprises at least 5 non-human lectins. In some embodiments provided herein, the lectin panel comprises at least 10 non-human lectins. In some embodiments provided herein, the lectin panel comprises at least 15 non-human lectins. In some embodiments provided herein, the lectin panel comprises at least 20 non-human lectins. In some embodiments provided herein, the lectin panel comprises 1 to 45 non-human lectins. In some embodiments provided herein, the lectin panel comprises 1 to 20 non-human lectins. In some embodiments provided herein, the lectin panel comprises 1 to 15 non-human lectins. In some embodiments provided herein, the lectin panel comprises 1 to 10 non-human lectins. In some embodiments provided herein, the lectin panel comprises 1 to 5 non-human lectins. In some embodiments provided herein, the lectin panel comprises 25 to 45, 30 to 43, 35 to 42 or 39 to 42 non-human lectins. In some embodiments, the lectin panel comprises 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 non-human lectins.

[0123] In some embodiments provided herein, the lectin panel comprises at least 1 plant lectin. In some embodiments provided herein, the lectin panel comprises at least 5 plant lectins. In some embodiments provided herein, the lectin panel comprises at least 10 plant lectins. In some embodiments provided herein, the lectin panel comprises at least 15 plant lectins. In some embodiments provided herein, the lectin panel comprises at least 20 plant lectins. In some embodiments provided herein, the lectin panel comprises 1 to 45 plant lectins. In some embodiments provided herein, the lectin panel comprises 1 to 20 plant lectins. In some embodiments provided herein, the lectin panel comprises 1 to 15 plant lectins. In some embodiments provided herein, the lectin panel comprises 1 to 10 plant lectins. In some embodiments provided herein, the lectin panel comprises 1 to 5 plant lectins. In some embodiments provided herein, the lectin panel comprises 25 to 45, 30 to 43, 35 to 42 or 39 to 42 plant lectins. In some embodiments, the lectin panel comprises 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 plant lectins. [0124] In some embodiments, the lectins of the lectin panel are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the lectins are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each lectin in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of lectins. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16-subarray format. In some embodiments, one or more lectins are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0125] In some aspects, provided herein is a method of analyzing bacteria or mEVs in a sample, the method comprising: contacting the sample comprising the bacterium or the mEV with a lectin panel provided herein; and detecting the binding of the bacterium or the mEV to one or more lectins in the lectin panel, thereby analyzing the bacteria or the mEV in the sample, e.g., as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0126] Figures 1A and IB show the layout of an exemplary panel of 39 lectins arranged on an array (Figure 1A) and the corresponding IDs (identities) of each of the lectins in the array (Figure IB).

[0127] Figure 2 shows an exemplary 16-subarray microarray chip and the components added to each of the 16 arrays in three steps of the assay.

[0128] Figures 3A and 3B show the binding profiles of bacterial biomass (Figure 3A) and mEVs (Figure 3B) from P. histicola 1 to a lectin microarray.

[0129] Figures 4A and 4B show the binding profiles of bacterial biomass (Figure 4A) and washed bacterial biomass (Figure 4B) of P. histicola 1 to a lectin microarray. [0130] Figures 5A-5F show the lectin-binding profiles of mEVs from different bacteria (Figures 5A-5F showing binding profiles of mEVs of: P. histicola 1, F. massi Hensis. H. acelispora. V. parvula, S. variabile, and P. histicola 2, respectively). [0131] Figures 6A-D show images of the lectin microarray assay results of mEVs from different bacteria (Figures 6A-6D showing results of mEVs of: P. histicola 1, F. massi Hensis. H. acelispora. and V. parvula, respectively). [0132] Figures 7A and 7B show multivariable analysis, non-Arsinh transformed data (Figure 7A) and Arsinh-transformed data (Figure 7B), of the lectin-binding profiles of mEVs from: (A) P. histicola 1, (B) F. massiHensis. (C) H. acelispora. (D) V. parvula, (E) S’. variabile, and (F) P. histicola 2.

[0133] Figures 8A and 8B show the lectin-binding profiles of two fermentation lots of P. histicola 1 bacteria, grown in media containing porcine hemoglobin (Figure 8A) or spirulina (Figure 8B).

[0134] Figures 9A-9C show the lectin-binding profiles of three lots of P. histicola 1 mEVs (Figures 9A-9C) manufactured and isolated under the same conditions and procedures.

[0135] Figures 10A-10C show the lectin-binding profiles of P. histicola 1 mEVs with and without excipients (Figure 10A /< histicola 1 mEVs sample without excipients, Figure 10B . histicola 1 mEVs sample with 95% w/w of excipients and Figure 10C P. histicola 1 mEVs sample with 99.5% w/w of excipients).

DETAILED DESCRIPTION

General

[0136] Bacteria and microbial extracellular vesicles (mEVs) that are derived from bacteria can have therapeutic uses. Accordingly, it is important to be able to determine the presence of, accurately identify the type (e.g., genus, species or strain) and quantify the bacteria and/or mEVs in a sample. Standard methods used to determine the identity of whole bacteria are nucleic acid based, and therefore may not be useful in assessing the identity of mEVs. Moreover, the use of Nanoparticle Tracking Analysis (NTA) to quantify mEVs in solution may require steps such as determining a sufficient number of data points for a particular particle to calculate the particle size and/or concentration. In certain aspects, provided herein are methods in which one or more lectins (such as non-human lectins, such as plant lectins) are used to determine the presence of, identify and/or quantify bacteria and mEVs in a sample (e.g., in solution or suspension), and in particular to identify the bacteria or the mEVs as from a particular bacterial genus, species or strain. The methods utilizing one or more lectins may also be used to identify a sample as comprising bacteria substantially free of the mEVs derived therefrom. The methods utilizing one or more lectins may also be used to identify a sample as comprising the mEVs substantially free of the bacteria from which the mEVs were derived. In certain aspects, provided herein are methods utilizing lectins to detect and/or monitor the lectin-binding profile of the bacteria or the mEVs derived from such bacteria in a sample, for use, such as in production, for example, as an in-process control or after different storage conditions (e.g., time, temperature, and/or humidity). In certain aspects, provided herein are methods utilizing lectins to detect and/or monitor the glycosidic profile of the bacteria or the mEVs derived from such bacteria in a sample, for use, such as in production, for example, as an in-process control or after different storage conditions (e.g., time, temperature, and/or humidity). [0137] The surface of microbes and mEVs contain glycans that, among other functions, mediate host-microbe interactions and stimulate recognition by the host immune system. The glycan profile of a microbe can change as microbes (e.g., bacteria) respond dynamically to their environment, which can also change the glycan profile of mEVs obtained from the microbes. Microbial glycans are a complex population of glycoproteins, capsular polysaccharides, glycolipids, and peptidoglycans. Isolation of single structures can be challenging, time consuming, and may not be able to identify surface-exposed structures readily accessible for binding. Use of lectins (such as a lectin microarray based on a panel of lectins immobilized on an array such as a microchip) can allow high-throughput profiling of glycans without the need for isolation of single structures. Given the high specificity of non-human lectins toward glycan structures, non-human lectin microarrays can identify glycan motifs exposed on the surface of bacteria and mEVs. Moreover, non-human lectins, particularly plant lectins, are easily purified, stable and commercially available.

[0138] As disclosed herein, in certain aspects, one or more lectins, such as non- human lectins, can surprisingly be used to determine the presence of, identify, quantify and otherwise characterize bacteria or bacterial mEVs. For example, as described herein, panels of lectins comprising 39-42 non-human lectins, a large majority of which are plant lectins and covering most carbohydrate-binding epitopes, showed specific and distinct lectin- binding profiles for mEVs of Prevotella histicola, Fournierella massiHensis. Harryflintia acelispora. Veillonella parvula and Subdoligranulum variabile strains. One or more lectins (such as in a lectin panel) may also be used to characterize and differentiate between compositions comprising bacteria and mEVs derived therefrom. One or more lectins (such as a lectin panel) may be used to identify a sample as comprising bacteria substantially free of the mEVs derived therefrom. One or more lectins (such as a lectin panel) may also be used to identify a composition as comprising mEVs substantially free of the bacteria from which the mEVs were derived. One or more lectins (such as a lectin panel) may be used to detect the lectin-binding profile or the glycosidic profile of the bacteria or the mEVs, such as in a sample. One or more lectins (such as a lectin panel) may also be used to monitor changes in the glycosidic profile of the bacteria or the mEVs in a sample, for example, as an in-process control or after different storage conditions (e.g., time, temperature, and/or humidity). In some embodiments, one or more of the lectins listed in Table 6 are one or more of the non-human lectins used in the methods and compositions provided herein.

Definitions

[0139] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a," "an," and "the" are understood to be singular or plural.

[0140] The term “about” when used before a numerical value indicates that the value may vary within a reasonable range, such as within ± 10%, ± 5% or ± 1% of the stated value.

[0141] As used herein, the term “antibody” may refer to both an intact antibody and an antigen binding fragment thereof. Intact antibodies are glycoproteins that include at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain includes a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. As used herein, CDRH1, CDRH2, and CDRH3 respectively refer to CDR1, CDR2 and CDR3 of the heavy chain, while CDRL1, CDRL2, and CDRL3 respectively refer to CDR1, CDR2 and CDR3 of the light chain. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The term “antibody” includes, for example, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies and antigen-binding antibody fragments.

[0142] An “antigen,” as used herein, refers to a molecule that is specifically recognized by an antibody. In some embodiments, an antigen is a surface molecule. [0143] The terms “antigen binding fragment” and “antigen-binding portion” of an antibody, as used herein, refer to one or more fragments of an antibody that retain the ability to bind to an antigen. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabodies, single-chain antibodies, NANOBODIES®, isolated CDRH3, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. These antibody fragments can be obtained using conventional recombinant and/or enzymatic techniques and can be screened for antigen binding in the same manner as intact antibodies.

[0144]

[0145] The term “decrease” or “deplete” means a change, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1/100, 1/1000, 1/10,000, 1/100,000, 1/1,000,000 or undetectable after treatment when compared to a pre-treatment state. Properties that may be decreased include the number of immune cells, bacterial cells, stromal cells, myeloid derived suppressor cells, fibroblasts, metabolites; the level of a cytokine; or another physical parameter (such as ear thickness (for example, in a DTH animal model) or tumor size (for example, in an animal tumor model)).

[0146] The term “drug substance” or “therapeutic agent” refers to an agent for therapeutic use and comprising bacteria and/or microbial extracellular vesicles (mEVs) (such as smEVs and/or pmEVs), e.g., that can be used to treat and/or prevent a disease and/or condition. In some embodiments, the therapeutic agent is a pharmaceutical agent. In some embodiments, a medicinal product, medical food, a food product, or a dietary supplement comprises a therapeutic agent. For example, the therapeutic agent disclosed herein may be a powder comprising bacteria and/or microbial extracellular vesicles (mEVs) (such as smEVs and/or pmEVs). In some embodiments, the therapeutic agent may further comprise an excipient.

[0147] The term “drug product” or “therapeutic composition” refers to a composition that comprises a therapeutically effective amount of a therapeutic agent. In some embodiments, the therapeutic composition is (or is present in) a medicinal product, medical food, a food product, or a dietary supplement. For example, the therapeutic composition may be a tablet or capsule comprising the therapeutic agent. In some embodiments, the therapeutic composition may be a powder comprising the therapeutic agent and additional excipients. In some embodiments, a therapeutic compositioncomprises a therapeutic agent and an additional excipient.

[0148] The term “epitope” means a protein determinant capable of specific binding to an antibody or T cell receptor. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains. Certain epitopes can be defined by a particular sequence of amino acids to which an antibody is capable of binding. [0149] The term “binds to the same epitope” with reference to two or more antibodies means that the antibodies bind to the same segment of amino acid residues, as determined by a given method. Techniques for determining whether antibodies bind to the "same epitope on ENPP1" with the antibodies described herein include, for example, epitope mapping methods, such as, x-ray analyses of crystals of antigemantibody complexes which provides atomic resolution of the epitope and hydrogen/deuterium exchange mass spectrometry (HDX-MS). Other methods monitor the binding of the antibody to antigen fragments or mutated variations of the antigen where loss of binding due to a modification of an amino acid residue within the antigen sequence is often considered an indication of an epitope component. In addition, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. Antibodies having the same VH and VL or the same CDR1, 2 and 3 sequences are expected to bind to the same epitope.

[0150] “Identity” between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0151] The term “increase” means a change, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 4- fold, 10-fold, 100-fold, 10^A3 fold, 10^A4 fold, 10^A5 fold, 10^A6 fold, and/or 10^A7 fold greater after treatment with an agent (e.g., mEVs) when compared to a pre-treatment state. Properties that may be increased include the number of immune cells, bacterial cells, stromal cells, myeloid derived suppressor cells, fibroblasts, metabolites; the level of a cytokine; or another physical parameter (such as ear thickness (e.g., in a DTH animal model) or tumor size). [0152] The term “isolated” or “enriched” encompasses a microbe (such as a bacterium), an mEV (such as an smEV and/or pmEV) or other entity or substance that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, purified, and/or manufactured by the hand of man. Isolated microbes or mEVs may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some embodiments, isolated microbes or mEVs are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. The terms “purify,” “purifying” and “purified” refer to a microbe or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated (e.g., whether in nature or in an experimental setting), or during any time after its initial production. A microbe or a microbial population or mEVs may be considered purified if it is isolated at or after production, such as from a material or environment containing the microbe or microbial population, and a purified microbe or microbial population may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered “isolated.” In some embodiments, purified microbes or microbial population or mEVs are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. In the instance of microbial compositions provided herein, the one or more microbial types present in the composition can be independently purified from one or more other microbes produced and/or present in the material or environment containing the microbial type. Microbial compositions and the microbial components thereof are generally purified from residual habitat products.

[0153] In some embodiments, the antibodies provided herein can be of any isotype. As used herein, "isotype" refers to the antibody class (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE) that is encoded by the heavy chain constant region genes. In some embodiments, the antibodies provided herein are IgG isotype antibodies (IgGl, IgG2, IgG3 and IgG4 in humans, and IgGl, IgG2a, IgG2b and IgG3 in mice). [0154] “Microbial extracellular vesicles” (mEVs) can be obtained from microbes such as bacteria, archaea, fungi, microscopic algae, protozoans, and parasites. In some embodiments, the mEVs are obtained from bacteria. In a preferred embodiment, a purified mEV composition may be substantially free of its originating or associated microbe (e.g., bacteria). mEVs include secreted microbial extracellular vesicles (smEVs) and processed microbial extracellular vesicles (pmEVs). “Secreted microbial extracellular vesicles” (smEVs) are vesicles naturally produced by microbes. smEVs are comprised of microbial lipids and/or microbial proteins and/or microbial nucleic acids and/or microbial carbohydrate moieties, and are isolated from culture supernatant. The natural production of these vesicles can be artificially enhanced (e.g., increased) or decreased through manipulation of the environment in which the bacterial cells are being cultured (e.g., by media or temperature alterations). Further, smEV compositions may be modified to reduce, increase, add, or remove microbial components or foreign substances to alter efficacy, immune stimulation, stability, immune stimulatory capacity, stability, organ targeting (e.g., lymph node), absorption (e.g., gastrointestinal), and/or yield (e.g., thereby altering the efficacy). As used herein, the term “purified smEV composition” or “smEV composition” refers to a preparation of smEVs that have been separated from at least one associated substance found in a source material (e.g., separated from at least one other microbial component) or any material associated with the smEVs in any process used to produce the preparation. It can also refer to a composition that has been significantly enriched for specific components. In a preferred embodiment, a purified smEV composition may be substantially free of its originating or associated microbe (e.g., bacteria). “Processed microbial extracellular vesicles” (pmEVs) are a non-naturally-occurring collection of microbial membrane components that have been purified from artificially lysed microbes (e.g., bacteria) (e.g., microbial membrane components that have been separated from other, intracellular microbial cell components), and which may comprise particles of a varied or a selected size range, depending on the method of purification. A pool of pmEVs is obtained by chemically disrupting (e.g., by lysozyme and/or lysostaphin) and/or physically disrupting (e.g., by mechanical force) microbial cells and separating the microbial membrane components from the intracellular components through centrifugation and/or ultracentrifugation, or other methods. The resulting pmEV mixture contains an enrichment of the microbial membranes and the components thereof (e.g., peripherally associated or integral membrane proteins, lipids, glycans, polysaccharides, carbohydrates, other polymers), such that there is an increased concentration of microbial membrane components, and a decreased concentration (e.g., dilution) of intracellular contents, relative to whole microbes. For gram -positive bacteria, pmEVs may include cell or cytoplasmic membranes. For gram -negative bacteria, a pmEV may include inner and outer membranes. pmEVs may be modified to increase purity, to adjust the size of particles in the composition, and/or modified to reduce, increase, add or remove, microbial components or foreign substances to alter efficacy, immune stimulation, stability, immune stimulatory capacity, stability, organ targeting (e.g., lymph node), absorption (e.g., gastrointestinal), and/or yield (e.g., thereby altering the efficacy). pmEVs can be modified by adding, removing, enriching for, or diluting specific components, including intracellular components from the same or other microbes. As used herein, the term “purified pmEV composition” or “pmEV composition” refers to a preparation of pmEVs that have been separated from at least one associated substance found in a source material (e.g., separated from at least one other microbial component) or any material associated with the pmEVs in any process used to produce the preparation. It can also refer to a composition that has been significantly enriched for specific components. In a preferred embodiment, a purified pmEV composition may be substantially free of its originating or associated whole microbe (e.g., bacteria).

[0155] In some embodiments, the antibodies provided herein are monoclonal antibodies. The term “monoclonal antibody,” as used herein, refers to an antibody that displays a single binding specificity and affinity for a particular epitope or a composition of antibodies in which all antibodies display a single binding specificity and affinity for a particular epitope. Accordingly, the term “human monoclonal antibody” refers to an antibody or antibody composition that display(s) a single binding specificity and which has variable and optional constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0156] As used herein, “specific binding” refers to the ability of an antibody to bind to a predetermined antigen or the ability of a polypeptide to bind to its predetermined binding partner. Typically, an antibody or polypeptide specifically binds to its predetermined antigen or binding partner with an affinity corresponding to a KD of about 10'⁷ M or less, and binds to the predetermined antigen/binding partner with an affinity (as expressed by KD) that is at least 10 fold less, at least 100 fold less or at least 1000 fold less than its affinity for binding to a non-specific and unrelated antigen/binding partner (e.g., BSA, casein). Alternatively, specific binding applies more broadly to a two-component system where one component is a protein, lipid, or carbohydrate or combination thereof and engages with the second component which is a protein, lipid, carbohydrate or combination thereof in a specific way.

[0157] Strain” refers to a member of a bacterial species with a genetic signature such that it may be differentiated from closely-related members of the same bacterial species. The genetic signature may be the absence of all or part of at least one gene, the absence of all or part of at least one regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the absence (“curing”) of at least one native plasmid, the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one foreign gene (a gene derived from another species), the presence at least one mutated regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the presence of at least one non-native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof. Genetic signatures between different strains may be identified by PCR amplification optionally followed by DNA sequencing of the genomic region(s) of interest or of the whole genome. In the case in which one strain (compared with another of the same species) has gained or lost antibiotic resistance or gained or lost a biosynthetic capability (such as an auxotrophic strain), strains may be differentiated by selection or counter-selection using an antibiotic or nutrient/metabolite, respectively.

[0158] As used herein, a "type" of bacteria may be distinguished from other bacteria by: genus, species, sub-species, strain or by any other taxonomic categorization, whether based on morphology, physiology, genotype, protein expression or other characteristics known in the art.

Lectin Panel

[0159] In some aspects, one or more lectins (e.g., non-human lectins, e.g., plant lectins) are in a lectin panel.

[0160] In some embodiments, a lectin panel comprises one or more lectins. [0161] In some embodiments provided herein, the one or more lectins in a lectin panel are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the lectins are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each lectin in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of lectins. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16-subarray format.

[0162] In some embodiments, one or more lectins are immobilized on a sensorchip, such as for surface plasmon resonance (SPR) characterization.

[0163] In some embodiments, the lectins of the lectin panel represent most known glycan-binding epitopes.

[0164] The binding of a sample comprising bacteria or mEVs to one or more lectins in the lectin panel can provide the lectin-binding profile of the bacteria or mEVs.

[0165] The binding of a sample comprising bacteria or mEVs to one or more lectins in the lectin panel can provide the glycosidic profile (profile of glycan motifs) of the bacteria or mEVs.

[0166] In some embodiments, the lectin panel comprises non-human lectins. In some embodiments, about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the lectins in the lectin panel are non-human lectins. In some embodiments, more than 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the lectins in the lectin panel are non-human lectins. In some embodiments, the non-human lectins are bacterial, plant, fungal, or non-human animal lectins. In some embodiments, the non-human lectins are plant lectins. In some embodiments, the non-human lectins are bacterial lectins. In some embodiments, the non-human lectins in the lectin panel are plant lectins. In some embodiments, about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the lectins in the lectin panel are plant lectins. In some embodiments, more than 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the lectins in the lectin panel are plant lectins. In some embodiments, the lectin panel comprises one or more of the lectins listed in Table 6. In some embodiments, the lectin panel comprises BanLec and/or CSL3. In some embodiments, the lectin panel comprises: AAL, ACL, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SB A, SNA, STL, UEA-I, VVL, WFA and WGA. In some embodiments, the lectin panel comprises: AAL, SJA, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LT A, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA. In some embodiments, the lectin panel further comprises BanLec and/or CSL3. In some embodiments, other lectins may be used in various combinations (optionally with the lectins provided herein) to provide a glycosidic profile of bacteria and/or the mEVs, such as in a sample.

[0167] In some embodiments provided herein, the lectin panel comprises one or more of: BPL and WGA. In some embodiments provided herein, the lectin panel comprises one or more of: BPL, RSL, VVL and WGA.

[0168] In some embodiments provided herein, the lectin panel comprises BPL and WGA. In some embodiments provided herein, the lectin panel comprises BPL, RSL, VVL and WGA.

[0169] In some embodiments provided herein, the lectin panel comprises one or more of: AIA, GSLB3, Morniga G and VVL. In some embodiments provided herein, the lectin panel comprises one or more of: AIA, BPL, ECL, GSLB4, HP A, Morniga G, SBA, VVL and WGA.

[0170] In some embodiments provided herein, the lectin panel comprises AIA, GSL B3, Morniga G and VVL. In some embodiments provided herein, the lectin panel comprises AIA, BPL, ECL, GSLB4, HP A, Morniga G, SBA, VVL and WGA.

[0171] In some embodiments provided herein, the lectin panel comprises one or more of: AIA, GNA, GSLB4, HHL, Morniga G, NPA and RCA-I. In some embodiments provided herein, the lectin panel comprises one or more of: AAL, AIA, BPL, ECL, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL, SBA, VVL and WFA. [0172] In some embodiments provided herein, the lectin panel comprises AIA, GNA, GSLB4, HHL, Morniga G, NPA and RCA-I. In some embodiments provided herein, the lectin panel comprises AAL, AIA, BPL, ECL, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL, SBA, VVL and WFA.

[0173] In some embodiments provided herein, the lectin panel comprises one or more of: GNA, HHL, HP A, LCA, NPA, PSA, and RSL. In some embodiments provided herein, the lectin panel comprises one or more of: AAL, BC2L-C Calsepa, ConA, GNA, GSII, HAA, HHL, HP A, LCA, LEL, NPA, PSA, RCA-I, RSL, STL and WGA. [0174] In some embodiments provided herein, the lectin panel comprises GNA, HHL, HP A, LCA, NPA, PSA, and RSL. In some embodiments provided herein, the lectin panel comprises AAL, BC2L-C Calsepa, ConA, GNA, GSII, HAA, HHL, HP A, LCA, LEL, NPA, PSA, RCA-I, RSL, STL and WGA.

[0175] In some embodiments provided herein, the lectin panel comprises one or more of: AAL, AIA, Calsepa, GNA, HHL, LCA, NPA, PSA and RCA-I. In some embodiments provided herein, the lectin panel comprises one or more of: AAL, AIA, BC2L-C, Calsepa, ConA, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA- I, RSL and WGA.

[0176] In some embodiments provided herein, the lectin panel comprises AAL, AIA, Calsepa, GNA, HHL, LCA, NPA, PSA and RCA-I. In some embodiments provided herein, the lectin panel comprises AAL, AIA, BC2L-C, Calsepa, ConA, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL and WGA.

[0177] In some embodiments provided herein, the lectin panel comprises at least 1 lectin. In some embodiments provided herein, the lectin panel comprises at least 5 lectins. In some embodiments provided herein, the lectin panel comprises at least 10 lectins. In some embodiments provided herein, the lectin panel comprises at least 15 lectins. In some embodiments provided herein, the lectin panel comprises at least 20 lectins. In some embodiments provided herein, the lectin panel comprises 1 to 45 lectins. In some embodiments provided herein, the lectin panel comprises 1 to 20 lectins. In some embodiments provided herein, the lectin panel comprises 1 to 15 lectins. In some embodiments provided herein, the lectin panel comprises 1 to 10 lectins. In some embodiments provided herein, the lectin panel comprises 1 to 5 lectins. In some embodiments provided herein, the lectin panel comprises 25 to 45, 30 to 43, 35 to 42 or 39 to 42 lectins. In some embodiments, the lectin panel comprises 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 lectins.

[0178] In some embodiments provided herein, the lectin panel comprises at least 1 non-human lectin. In some embodiments provided herein, the lectin panel comprises at least 5 non-human lectins. In some embodiments provided herein, the lectin panel comprises at least 10 non-human lectins. In some embodiments provided herein, the lectin panel comprises at least 15 non-human lectins. In some embodiments provided herein, the lectin panel comprises at least 20 non-human lectins. In some embodiments provided herein, the lectin panel comprises 1 to 45 non-human lectins. In some embodiments provided herein, the lectin panel comprises 1 to 20 non-human lectins. In some embodiments provided herein, the lectin panel comprises 1 to 15 non-human lectins. In some embodiments provided herein, the lectin panel comprises 1 to 10 non-human lectins. In some embodiments provided herein, the lectin panel comprises 1 to 5 non-human lectins. In some embodiments provided herein, the lectin panel comprises 25 to 45, 30 to 43, 35 to 42 or 39 to 42 non-human lectins. In some embodiments, the lectin panel comprises 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 non-human lectins.

[0179] In some embodiments provided herein, the lectin panel comprises at least 1 plant lectin. In some embodiments provided herein, the lectin panel comprises at least 5 plant lectins. In some embodiments provided herein, the lectin panel comprises at least 10 plant lectins. In some embodiments provided herein, the lectin panel comprises at least 15 plant lectins. In some embodiments provided herein, the lectin panel comprises at least 20 plant lectins. In some embodiments provided herein, the lectin panel comprises 1 to 45 plant lectins. In some embodiments provided herein, the lectin panel comprises 1 to 20 plant lectins. In some embodiments provided herein, the lectin panel comprises 1 to 15 plant lectins. In some embodiments provided herein, the lectin panel comprises 1 to 10 plant lectins. In some embodiments provided herein, the lectin panel comprises 1 to 5 plant lectins. In some embodiments provided herein, the lectin panel comprises 25 to 45, 30 to 43, 35 to 42 or 39 to 42 plant lectins. In some embodiments, the lectin panel comprises 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 plant lectins.

[0180] some embodimentsln some embodiments provided herein, the lectins are immobilized on (e.g., tethered to) a surface. In some embodiments, the surface is a microchip. In some embodiments, the lectins are immobilized on the microchip in an array. In some embodiments, the array is a microarray. In some embodiments, each lectin in the array or microarray is immobilized in quadruplicate (repeated in 4 wells). In some embodiments, the microchip comprises a multi-subarray format, wherein each subarray unit comprises an array of lectins. In some embodiments, the microchip comprises an 8-subarray format. In some embodiments, the microchip comprises a 16-subarray format.

[0181] A lectin panel can be used in the methods provided herein.

[0182] In some aspects, provided herein is a method of analyzing bacteria or mEVs in a sample, the method comprising: contacting the sample comprising the bacterium or the mEV with a lectin panel provided herein; and detecting the binding of the bacterium or the mEV to one or more lectins in the lectin panel, thereby analyzing the bacteria or the mEV in the sample, e.g., as described herein.

Antibodies

[0183] Certain methods provided herein comprise the use of antibodies.

[0184] In some embodiments provided herein, the antibody or an antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Prevotella (e.g., bacteria of the species Prevotella histicola, such as bacteria of the strain Prevotella Strain B 50329 or strain Prevotella histicola ATCC designation number PTA-126140) or mEVs derived therefrom. In some embodiments, the antibody or the antigen binding fragment is a 17B01-02G10 mAb or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments, the antibody or the antigen binding fragment is a 02F09-02B04 mAB or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Fournierella (e.g., bacteria of the species Fournierella massiHensis. such as bacteria of the strain Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696)) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Harryflintia (e.g., bacteria of the species Harryflintia acelispora. such as bacteria of the strain Harryflintia acetispora Strain A) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Veillonella (e.g., bacteria of the species Veillonella parvula, such as bacteria of the strain Veillonella parvula Strain A (ATCC Accession Number PTA-125691)) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Subdoligramilum (e.g., bacteria of the species Subdoligranulum var labile) or mEVs derived therefrom.

[0185] As reported herein, antibodies that specifically bind an antigen or particular epitope (e.g., of an extracellular vesicle or a particular bacterial protein or other biomolecule (e.g., carbohydrate or lipid)) are useful in the methods provided herein, including methods for determining the presence, identifying and/or quantifying bacteria and/or mEVs from particular genera, species and/or strains. [0186] Antibodies typically bind specifically to their cognate antigen with high affinity, reflected by a dissociation constant (KD) of 10'⁵ to 10'¹¹ M or less. Any KD greater than about 10'⁴ M is generally considered to indicate nonspecific binding. As used herein, an antibody that "binds specifically" to an antigen refers to an antibody that binds to the antigen and substantially identical antigens with high affinity, which means having a KD of 10'⁷ M or less, preferably 10'⁸ M or less, even more preferably 5 x 10'⁹ M or less, and most preferably between 10'⁸ M and IO'¹⁰ M or less, but does not bind with high affinity to unrelated antigens. An antigen is "substantially identical" to a given antigen if it exhibits a high degree of sequence identity to the given antigen, for example, if it exhibits at least 80%, at least 90%, preferably at least 95%, more preferably at least 97%, or even more preferably at least 99% sequence identity to the sequence of the given antigen.

[0187] In some embodiments, provided herein are antigen-binding fragments of antibodies disclosed herein. The term “antigen-binding fragment” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding fragment” of an antibody, described herein, include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) roc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigenbinding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0188] In some embodiments, the antibodies provided herein are monoclonal antibodies. The term “monoclonal antibody,” as used herein, refers to an antibody that displays a single binding specificity and affinity for a particular epitope or a composition of antibodies in which all antibodies display a single binding specificity and affinity for a particular epitope.

[0189] In some embodiments, the antibodies provided herein are polyclonal antibodies. The term “polyclonal antibody,” as used herein, refers to a heterologous group of antibodies or a composition of heterologous antibodies that displays affinity for an antigen.

[0190] As used herein, the terms “specific binding,” “selective binding,” “selectively binds,” and “specifically binds,” refer to antibody binding to an epitope on a predetermined antigen. Typically, the antibody (i) binds with an equilibrium dissociation constant (K_D) of approximately less than 10'⁷ M, such as approximately less than 10 "⁸ M, 10'⁹ M or 10'¹⁰ M or even lower when determined by, e.g., surface plasmon resonance (SPR) technology in a BIACORE 2000 instrument using the predetermined antigen, as the analyte and the antibody as the ligand, or Scatchard analysis of binding of the antibody to antigen positive cells, and (ii) binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen.

[0191] In some embodiments, the antibodies provided herein are detectably labeled. Examples of detectable labels include, but are not limited to, fluorescent moieties, radioactive moieties, paramagnetic moieties, luminescent moieties and/or colorimetric moieties. In some embodiments, the targets described herein (e.g., bacteria or mEVs) are linked to, comprise and/or are bound by a fluorescent moiety. Examples of fluorescent moieties include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430,

EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean, Cy3 and CyPet. [0192] In some embodiments, the antibody is biotinylated.

Bacteria

[0193] In some embodiments, the methods and compositions provided herein can be used to identify and/or quantify a type of bacteria or bacterial mEVs, e.g., in a sample. As used herein, a "type" of bacteria may be distinguished from other bacteria by: genus, species, sub-species, strain or by any other taxonomic classification, whether based on morphology, physiology, genotype, protein expression or other characteristics known in the art.

[0194] Examples of taxonomic groups (e.g., class, order, family, genus, species or strain) of bacteria that can be used as a source of bacteria and/or mEVs (such as smEVs and/or pmEVs) are provided herein (e.g., listed in Table 1, Table 2, Table 3, and/or Table 4). Antibodies binding to a bacterial strain, species or genus, or mEVs from which they are derived (bacterial mEVs) listed herein may be produced and used in a method described herein to identify, and/or quantify the bacteria or bacterial mEVs.

[0195] In some embodiments, the bacterial strain from which bacteria or mEVs are obtained (e.g., derived) is a bacterial strain having a genome that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to a strain listed herein. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are oncotrophic bacteria. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are immunomodulatory bacteria. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are immunostimulatory bacteria. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are immunosuppressive bacteria. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are immunomodulatory bacteria. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are generated from a combination of bacterial strains provided herein. In some embodiments, the combination is a combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50 bacterial strains. In some embodiments, the combination includes the bacteria or the bacteria from which the mEVs are obtained (e.g., bacterial strains listed herein and/or bacterial strains having a genome that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to a strain listed herein (e.g., listed in Table 1, Table 2, Table 3, and/or Table 4). In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are generated from a bacterial strain provided herein. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are generated from one bacterial strain provided herein. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are from a bacterial strain listed herein ((e.g., listed in Table 1, Table 2, Table 3, and/or Table 4) and/or a bacterial strain having a genome that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to a strain listed herein (e.g., listed in Table 1, Table 2, Table 3, and/or Table 4).

[0196] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are gram-negative bacteria.

[0197] In some embodiments, the gram-negative bacteria belong to the class Negativicutes. The Negativicutes represent a unique class of microorganisms as they are the only diderm members of the Firmicutes phylum. These anaerobic organisms can be found in the environment and are normal commensals of the oral cavity and GI tract of humans. Because these organisms have an outer membrane, the yields of mEVs from this class were investigated. It was found that on a per cell basis these bacteria produce a high number of vesicles (10-150 mEVs/cell). The mEVs from these organisms are broadly stimulatory and highly potent in in vitro assays. Investigations into their therapeutic applications in several oncology and inflammation in vivo models have shown their therapeutic potential. The Negativicutes class includes the families Veillonellaceae, Setenomonadaceae.

Acidaminococcaceae. and Sporomusaceae . The Negativicutes class includes the genera Megasphaera, Setenomonas. Propionospora, and Acidaminococcus. Exemplary Negativicutes species include, but are not limited to, Megasphaera sp., Selenomonas felix, Acidaminococcus intestine, and Propionospora sp.

[0198] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are gram-positive bacteria.

[0199] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are aerobic bacteria.

[0200] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are anaerobic bacteria. In some embodiments, the anaerobic bacteria comprise obligate anaerobes. In some embodiments, the anaerobic bacteria comprise facultative anaerobes.

[0201] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are acidophile bacteria.

[0202] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are alkaliphile bacteria.

[0203] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are neutralophile bacteria.

[0204] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are fastidious bacteria.

[0205] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are nonfastidious bacteria.

[0206] In some embodiments, the bacteria or the mEVs themselves are lyophilized.

[0207] In some embodiments, the bacteria or the mEVs themselves are gamma irradiated (e.g., at 17.5 or 25 kGy).

[0208] In some embodiments, the bacteria or the mEVs themselves are UV irradiated.

[0209] In some embodiments, the bacteria or the mEVs themselves are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).

[0210] In some embodiments, the bacteria or the mEVs themselves are acid treated.

[0211] In some embodiments, the bacteria or the mEVs themselves are oxygen sparged (e.g., at 0.1 vvm for two hours).

[0212] The phase of growth can affect the amount or properties of bacteria and/or mEVs produced by bacteria. For example, mEVs can be isolated, e.g., from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.

[0213] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained from obligate anaerobic bacteria. Examples of obligate anaerobic bacteria include gram-negative rods (including the genera of Bacteroides, Prevotella, Porphyromonas, Fusobacterium, Bilophila, and Sutterella spp. gram -positive cocci (primarily Peptostreptococcus spp. gram -positive spore-forming (Clostridium spp. non-sporeforming bacilli (Actinomyces, Propionibacterium, Eubacterium, Lactobacillus and Bifidobacterium spp. and gram -negative cocci (mainly Veillonella spp. ). In some embodiments, the obligate anaerobic bacteria are of a genus selected from the group consisting of Agathobaculum, Alopobium. Blautia, Burkholderia, Dielma, Longicatena, Paraclostridium, Tiiricibacler. and Tyzzerella.

[0214] The Negativicutes class includes the families Veillonellaceae, Selenomonadaceae, Acidctminococcaceae. and Sporomusaceae . The Negativicutes class includes the genera Megasphaera. Selenomonas. Propionospora, and Acidaminococcus. Exemplary Negativicutes species include, but are not limited to, Megasphaera sp., Selenomonas felix, Acidaminococcus inleslini. and Propionospora sp.

[0215] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the Negativicutes class.

[0216] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the Veillonellaceae family.

[0217] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the Selenomonadaceae family.

[0218] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the Acidaminococcaceae family.

[0219] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the Sporomusaceae family.

[0220] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the Megasphaera genus.

[0221] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the Selenomonas genus.

[0222] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the Propionospora genus.

[0223] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the Acidaminococcus genus.

[0224] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Megasphaera sp. bacteria.

[0225] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Selenomonas felix bacteria.

[0226] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Acidaminococcus intestini bacteria. [0227] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Propionospora sp. bacteria.

[0228] The Oscillospiraceae family within the Clostridia class of microorganisms are common commensal organisms of vertebrates.

[0229] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the Clostridia class.

[0230] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the Oscillospiraceae family.

[0231] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the Faecalibacterium genus.

[0232] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the Fournierella genus.

[0233] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the Harryflintia genus.

[0234] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the Subdoligranulum genus.

[0235] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the Agathobaculum genus.

[0236] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Faecalibacterium prausnitzii (e.g., Faecalibacterium prausnitzii Strain A) bacteria.

[0237] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Fournierella massiliensis (e.g., Fournierella massiliensis Strain A) bacteria.

[0238] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Harryflintia acetispora (e.g., Harryflintia acetispora Strain A) bacteria.

[0239] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Subdoligranulum variabile bacteria.

[0240] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Agathobaculum sp. (e.g., Agathobaculum sp. Strain A) bacteria.

[0241] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are bacteria of a genus selected from the group consisting of Escherichia, Klebsiella, Lactobacillus, Shigella, and Staphylococcus. [0242] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are a species selected from the group consisting of Blautia massiliensis, Paraclostridium benzoelyticum, Dielma fastidiosa, Longicatena caecimuris. Lactococcus lactis cremoris. Tyzzerella nexilis, Hungatella effluvia, Klebsiella quasipneumoniae subsp. Similipneumoniae, Klebsiella oxyloca, and Veillonella tobetsuensis.

[0243] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are a Prevotella bacteria selected from the group consisting of Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea, Prevotella tannerae, Prevotella timonensis, Prevotella jejuni, Prevotella aurantiaca, Prevotella baroniae, Prevotella colorans, Prevotella corporis, Prevotella dentasini, Prevotella enoeca, Prevotella falsenii, Prevotella fusca, Prevotella heparinolytica, Prevotella loescheii, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella oryzae, Prevotella paludivivens, Prevotella pleuritidis, Prevotella ruminicola, Prevotella saccharolytica, Prevotella scopos, Prevotella shahii, Prevotella zoogleoformans, and Prevotella veroralis. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are a Prevotella histicola bacteria.

[0244] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are a strain of bacteria comprising a genomic sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the genomic sequence of the strain of bacteria deposited with the ATCC Deposit number as provided in Table 3. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are a strain of bacteria comprising a 16S sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the 16S sequence of the strain of bacteria deposited with the ATCC Deposit number as provided in Table 3.

[0245] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Agathobaculum sp. (e.g., Agathobaculum sp. Strain A) bacteria.

[0246] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are a strain of Agathobaculum sp. In some embodiments, the Agathobaculum sp. strain is a strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Agathobaculum sp. Strain A (ATCC Deposit Number PTA- 125892). In some embodiments, the Agathobaculum sp. strain is the Agathobaculum sp. Strain A (ATCC Deposit Number PTA- 125892).

[0247] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the class Bacteroidia [phylum Bacteroidota\. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are bacteria of order Bacteroidales. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the family Porphyromonadaceae. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the family Prevotellaceae . In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are bacteria of the class Bacteroidia wherein the cell envelope structure of the bacteria is diderm. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are bacteria of the class Bacteroidia that stain Gram negative. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are bacteria of the class Bacteroidia wherein the bacteria is diderm and the bacteria stain Gram negative.

[0248] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are bacteria of the class Clostridia [phylum Firmicutes\. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the order Eubacteriales. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the family Oscillispiraceae . In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the family Lachnospiraceae . In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the family Peptostreptococcaceae . In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the family Clostridiales family XIII/ Incertae sedis 41. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the class Clostridia wherein the cell envelope structure of the bacteria is monoderm. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the class Clostridia that stain gram-negative. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the class Clostridia that stain grampositive. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the class Clostridia wherein the cell envelope structure of the bacteria is monoderm and the bacteria stain gram-negative. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the class Clostridia wherein the cell envelope structure of the bacteria is monoderm and the bacteria stain gram-positive.

[0249] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the class Negativicutes [phylum Firmicutes\. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the order Veillonellales. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the family Veillonelloceae. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the order Selenomonadales. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are bacteria of the family Selenomonadaceae . In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the family Sporomusaceae . In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the class Negativicutes wherein the cell envelope structure of the bacteria is diderm. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the bacteria or the bacteria from which the mEVs are obtained are the mEVs are from bacteria of the class Negativicutes wherein the cell envelope structure of the bacteria is diderm and the bacteria stain Gram negative.

[0250] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the class Synergistia [phylum Synergistota\. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the order Synergistales. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the family Synergistaceae . In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the class Synergistia wherein the cell envelope structure of the bacteria is diderm. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the class Synergistia that stain gram -negative. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the class Synergistia wherein the cell envelope structure of the bacteria is diderm and the bacteria stain gram-negative.

[0251] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are from one strain of bacteria, e.g., a strain provided herein.

[0252] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are from one strain of bacteria (e.g., a strain provided herein) or from more than one strain provided herein.

[0253] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Lactococcus lactis cremoris bacteria, e.g., a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Lactococcus lactis cremoris Strain A (ATCC designation number PTA-125368). In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Lactococcus bacteria, e.g., Lactococcus lactis cremoris Strain A (ATCC designation number PTA-125368).

[0254] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Prevotella bacteria, e.g., a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329) o Prevotella histicola ATCC designation number PTA-126140. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Prevotella bacteria, e.g., Prevotella Strain B 50329 (NRRL accession number B 50329) ox Prevotella histicola ATCC designation number PTA-126140.

[0255] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Bifidobacterium bacteria, e.g., a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Bifidobacterium bacteria deposited as ATCC designation number PTA-125097. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Bifidobacterium bacteria, e.g., Bifidobacterium bacteria deposited as ATCC designation number PTA-125097.

[0256] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Veillonella bacteria, e.g., a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Veillonella bacteria deposited as ATCC designation number PTA-125691. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Veillonella bacteria, e.g., Veillonella bacteria deposited as ATCC designation number PTA-125691. [0257] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Ruminococcus gnavus bacteria. In some embodiments, the Ruminococcus gnavus bacteria are a strain comprising at least 90% (or at least 97%) genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Ruminococcus gnavus bacteria deposited as ATCC designation number PTA-126695. In some embodiments, the Ruminococcus gnavus bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Ruminococcus gnavus bacteria deposited as ATCC designation number PTA-126695. In some embodiments, the Ruminococcus gnavus bacteria are Ruminococcus gnavus bacteria deposited as ATCC designation number PTA-126695.

[0258] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Megasphaera sp. bacteria. In some embodiments, the Megasphaera sp. bacteria are a strain comprising at least 90% (or at least 97%) genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Megasphaera sp. bacteria deposited as ATCC designation number PTA-126770. In some embodiments, the Megasphaera sp. bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Megasphaera sp. bacteria deposited as ATCC designation number PTA-126770. In some embodiments, the Megasphaera sp. bacteria are Megasphaera sp. bacteria deposited as ATCC designation number PTA-126770.

[0259] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Fournierella massiliensis bacteria. In some embodiments, the Fournierella massiliensis bacteria are a strain comprising at least 90% (or at least 97%) genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Fournierella massiliensis bacteria deposited as ATCC designation number PTA-126696. In some embodiments, the Fournierella massiliensis bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Fournierella massiliensis bacteria deposited as ATCC designation number PTA-126696. In some embodiments, the Fournierella massiliensis bacteria are Fournierella massiliensis bacteria deposited as ATCC designation number PTA-126696. [0260] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Harryflintia acetispora bacteria. In some embodiments, the Harryflintia acetispora bacteria are a strain comprising at least 90% (or at least 97%) genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Harryflintia acetispora bacteria deposited as ATCC designation number PTA-126694 (also known as Harryflintia acetispora Strain A). In some embodiments, the Harryflintia acetispora bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Harryflintia acetispora bacteria deposited as ATCC designation number PTA-126694. In some embodiments, the Harryflintia acetispora bacteria are Harryflintia acetispora bacteria deposited as ATCC designation number PTA-126694.

[0261] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Subdoligranulum variabile bacteria. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are bacteria that produce metabolites, e.g., the bacteria produce butyrate, iosine, proprionate, or tryptophan metabolites.

[0262] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are bacteria that produce butyrate. In some embodiments, the bacteria are from the genus Blautia, Christensella, Copracoccus, Eubacterium, Lachnosperacea, Megasphaera, or Roseburia.

[0263] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are bacteria that produce iosine. In some embodiments, the bacteria are from the genus Bifidobacterium, Lactobacillus, or Olsenella.

[0264] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are bacteria that produce proprionate. In some embodiments, the bacteria are from the genus Akkermansia, Bacteriodes, Dialister, Eubacterium, Megasphaera, Parabacteriodes, Prevotella, Ruminococcus, or Veillonella.

[0265] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are bacteria that produce tryptophan metabolites. In some embodiments, the bacteria are from the genus Lactobacillus or Peptostreptococcus.

[0266] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are bacteria that produce inhibitors of histone deacetylase 3 (HDAC3). In some embodiments, the bacteria are from the species Bariatricus massiliensis, Faecalibacterium prausnitzii, Megasphaera massiliensis, or Roseburia intestinalis. [0267] In some embodiments, the bacteria are from the genus Alloiococcus, Bacillus, Catenibacterium, Corynebacterium, Cupriavidus, Enhydrobacter, Exiguobacterium, Faecalibacterium, Geobacillus, Methylobacterium, Micrococcus, Morganella, Proteus, Pseudomonas, Rhizobium, or Sphingomonas. In some embodiments, the bacteria are from the genus Cutibacterium. In some embodiments, the bacteria are from the species Cuixbaclerium avidum. In some embodiments, the bacteria are from the genus Lactobacillus. In some embodiments, the bacteria are from the species Lactobacillus gasseri. In some embodiments, the bacteria are from the genus Dysosmobacter . In some embodiments, the bacteria are from the species Dysosmobacter welbionis.

[0268] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the genus Alloiococcus, Bacillus, Catenibacterium, Corynebacterium, Cupriavidus, Enhydrobacter, Exiguobacterium, Faecalibacterium, Geobacillus, Methylobacterium, Micrococcus, Morganella, Proteus, Pseudomonas, Rhizobium, or Sphingomonas.

[0269] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the Cutibacterium genus. In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Cutibacterium avidum bacteria.

[0270] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the genus Leuconostoc.

[0271] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the genus Lactobacillus.

[0272] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are of the genus Akkermansia, Bacillus, Blautia, Cupriavidus, Enhydrobacter, Faecalibacterium, Lactobacillus, Lactococcus, Micrococcus, Morganella, Propionibacterium, Proteus, Rhizobium, or Streptococcus.

[0273] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Leuconostoc holzapfelii bacteria.

[0274] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Akkermansia muciniphila, Cupriavidus metallidurans, Faecalibacterium prausnitzii, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus sakei, or Streptococcus pyogenes bacteria. [0275] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Lactobacillus casei. Lactobacillus plantarum, Lactobacillus paracasei. Lactobacillus plantarum, Lactobacillus rhamnosus, or Lactobacillus sakei bacteria.

[0276] In some embodiments, the mEVs described herein are obtained from a genus selected from the group consisting of Acinetobacter, Deinococcus,' Helicobacter, Rhodococcus,' Weissella cibar ia, Alloiococcus,' Atopobiunr, Catenibacteriunr, Corynebacterium,' Exiguobacteriunr, Geobacillus,' Methylobacteriunr, Micrococcus,' Morganella, Proteus,' Rhizobiunr, Rothia, Sphingomonas,' Sphingomonas,' and Leuconostoc. [0277] In some embodiments, the mEVs described herein are obtained from a species selected from the group consisting of Acinetobacter baumanii,' Deinococcus radiodurans,' Helicobacter pylori,' Rhodococcus equi,' Weissella cibaria, Alloiococcus otitis,' Atopobium vaginae,' Catenibacterium mituokai,' Corynebacterium glutamicunr, Exiguobacterium aurantiacunr, Geobacillus stearothermophilus,' Methylobacterium jeotgali,' Micrococcus luteus,' Morganella morganii,' Proteus mirabilis,' Rhizobium leguminosarum,' Rothia amarae,' Sphingomonas paucimobilis,' and Sphingomonas koreens.

[0278] In some embodiments, the mEVs are from Leuconostoc holzapfelii bacteria. In some embodiments, the mEVs are from Leuconostoc holzapfelii Ceb-kc-003 (KCCM11830P) bacteria.

[0279] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Megasphaera sp. bacteria (e.g., from the strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387).

[0280] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Megasphaera massiliensis bacteria (e.g., from the strain with accession number NCIMB 42787, NCIMB 43388 or NCIMB 43389).

[0281] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Megasphaera massiliensis bacteria (e.g., from the strain with accession number DSM 26228).

[0282] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Parabacteroides distasonis bacteria (e.g., from the strain with accession number NCIMB 42382).

[0283] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Megasphaera massiliensis bacteria (e.g., from the strain with accession number NCIMB 43388 or NCIMB 43389), or a derivative thereof. See, e.g., WO 2020/120714. In some embodiments, the Megasphaera massiliensis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, and/or CRISPR sequence) of Megasphaera massiliensis bacteria from the strain with accession number NCIMB 43388 or NCIMB 43389. In some embodiments, the Megasphaera massiliensis bacteria is the strain with accession number NCIMB 43388 or NCIMB 43389.

[0284] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Megasphaera massiliensis bacteria strain deposited under accession number NCIMB 42787, or a derivative thereof. See, e.g., WO 2018/229216. In some embodiments, the Megasphaera massiliensis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, and/or CRISPR sequence) of the Megasphaera massiliensis bacteria strain deposited under accession number NCIMB 42787. In some embodiments, the Megasphaera massiliensis bacteria is the strain deposited under accession number NCIMB 42787.

[0285] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Megasphaera spp. bacteria from the strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387, or a derivative thereof. See, e.g., WO 2020/120714. In some embodiments, the Megasphaera sp. bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, and/or CRISPR sequence) of the Megasphaera sp. from a strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387. In some embodiments, the Megasphaera sp. bacteria is the strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387. [0286] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Parabacteroides distasonis bacteria deposited under accession number NCIMB 42382, or a derivative thereof. See, e.g., WO 2018/229216. In some embodiments, the Parabacteroides distasonis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, and/or CRISPR sequence) of the Parabacteroides distasonis bacteria deposited under accession number NCIMB 42382. In some embodiments, the Parabacteroides distasonis bacteria is the strain deposited under accession number NCIMB 42382.

[0287] In some embodiments, the bacteria or the bacteria from which the mEVs are obtained are Megasphaera massiliensis bacteria deposited under accession number DSM 26228, or a derivative thereof. See, e.g., WO 2018/229216. In some embodiments, the Megasphaera massiliensis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, and/or CRISPR sequence) of Megasphaera massiliensis bacteria deposited under accession number DSM 26228. In some embodiments, the Megasphaera massiliensis bacteria is the strain deposited under accession number DSM 26228.

Table 1: Bacteria by Class

Table 2: Exemplary Bacterial Strains

Table 3: Exemplary Bacterial Strains

Table 4. Exemplary Bacterial Strains

[0288] In some embodiments, the bacteria or the mEVs provided herein are detectably labeled. Examples of detectable labels include, but are not limited to, fluorescent moieties, radioactive moieties, paramagnetic moieties, luminescent moieties and/or colorimetric moieties. In some embodiments, the targets described herein are linked to, comprise and/or are bound by a fluorescent moiety. Examples of fluorescent moieties include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridininchlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa

Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa

Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean and

CyPet.

Production of Processed Microbial Extracellular Vesicles (pmEVs)

[0289] In certain aspects, the pmEVs described herein can be prepared using any method known in the art.

[0290] In some embodiments, the pmEVs are prepared without a pmEV purification step. For example, in some embodiments, bacteria from which the pmEVs described herein are released are killed using a method that leaves the bacterial pmEVs intact, and the resulting bacterial components, including the pmEVs, are used in the methods and compositions described herein. In some embodiments, the bacteria are killed using an antibiotic (e.g., using an antibiotic described herein). In some embodiments, the bacteria are killed using UV irradiation.

[0291] In some embodiments, the pmEVs described herein are purified from one or more other bacterial components. Methods for purifying pmEVs from bacteria (and optionally, other bacterial components) are known in the art. In some embodiments, pmEVs are prepared from bacterial cultures using methods described in Thein, et al. (J. Proteome Res. 9(12):6135-6147 (2010)) or Sandrini et al. (Bio-protocol 4(21): el287 (2014)), each of which is hereby incorporated by reference in its entirety. In some embodiments, the bacteria are cultured to high optical density and then centrifuged to pellet bacteria (e.g., at 10,000- 15,000 x g for 10- 15 min at room temperature or 4°C). In some embodiments, the supernatants are discarded and cell pellets are frozen at -80°C. In some embodiments, cell pellets are thawed on ice and resuspended in 100 mM Tris-HCl, pH 7.5 supplemented with 1 mg/mL DNase I. In some embodiments, cells are lysed using an Emulsiflex C-3 (Avestin, Inc.) under conditions recommended by the manufacturer. In some embodiments, debris and unlysed cells are pelleted by centrifugation at 10,000 x g for 15 min at 4°C. In some embodiments, supernatants are then centrifuged at 120,000 x g for 1 hour at 4°C. In some embodiments, pellets are resuspended in ice-cold 100 mM sodium carbonate, pH 11, incubated with agitation for 1 hour at 4°C, and then centrifuged at 120,000 x g for 1 hour at 4°C. In some embodiments, pellets are resuspended in 100 mM Tris-HCl, pH 7.5, recentrifuged at 120,000 x g for 20 min at 4°C, and then resuspended in 0.1 M Tris-HCl, pH 7.5 or in PBS. In some embodiments, samples are stored at -20°C.

[0292] In certain aspects, pmEVs are obtained by methods adapted from Sandrini et al. 2014. In some embodiments, bacterial cultures are centrifuged at 10,000-15,500 x g for 10-15 min at room temp or at 4°C. In some embodiments, cell pellets are frozen at -80°C and supernatants are discarded. In some embodiments, cell pellets are thawed on ice and resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA supplemented with 0.1 mg/mL lysozyme. In some embodiments, samples are incubated with mixing at room temp or at 37°C for 30 min. In some embodiments, samples are re-frozen at -80°C and thawed again on ice. In some embodiments, DNase I is added to a final concentration of 1.6 mg/mL and MgCb to a final concentration of 100 mM. In some embodiments, samples are sonicated using a QSonica Q500 sonicator with 7 cycles of 30 sec on and 30 sec off. In some embodiments, debris and unlysed cells are pelleted by centrifugation at 10,000 x g for 15 min. at 4°C. In some embodiments, supernatants are then centrifuged at 110,000 x g for 15 min at 4°C. In some embodiments, pellets are resuspended in 10 mM Tris-HCl, pH 8.0, 2% Triton X-100 and incubated 30-60 min with mixing at room temperature. In some embodiments, samples are centrifuged at 110,000 x g for 15 min at 4°C. In some embodiments, pellets are resuspended in PBS and stored at -20°C. [0293] In certain aspects, a method of forming (e.g., preparing) isolated bacterial pmEVs, described herein, comprises the steps of: (a) centrifuging a bacterial culture, thereby forming a first pellet and a first supernatant, wherein the first pellet comprises cells; (b) discarding the first supernatant; (c) resuspending the first pellet in a solution; (d) lysing the cells; (e) centrifuging the lysed cells, thereby forming a second pellet and a second supernatant; (f) discarding the second pellet and centrifuging the second supernatant, thereby forming a third pellet and a third supernatant; (g) discarding the third supernatant and resuspending the third pellet in a second solution, thereby forming the isolated bacterial pmEVs.

[0294] In some embodiments, the method further comprises the steps of: (h) centrifuging the solution of step (g), thereby forming a fourth pellet and a fourth supernatant; (i) discarding the fourth supernatant and resuspending the fourth pellet in a third solution. In some embodiments, the method further comprises the steps of: (j) centrifuging the solution of step (i), thereby forming a fifth pellet and a fifth supernatant; and (k) discarding the fifth supernatant and resuspending the fifth pellet in a fourth solution. [0295] In some embodiments, the centrifugation of step (a) is at 10,000 x g. In some embodiments the centrifugation of step (a) is for 10-15 minutes. In some embodiments, the centrifugation of step (a) is at 4°C or room temperature. In some embodiments, step (b) further comprises freezing the first pellet at -80°C. In some embodiments, the solution in step (c) is 100 mM Tris-HCl, pH 7.5 supplemented with 1 mg/ml DNasel. In some embodiments, the solution in step (c) is 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, supplemented with 0.1 mg/ml lysozyme. In some embodiments, step (c) further comprises incubating for 30 minutes at 37°C or room temperature. In some embodiments, step (c) further comprises freezing the first pellet at -80°C. In some embodiments, step (c) further comprises adding DNase I to a final concentration of 1.6 mg/ml. In some embodiments, step (c) further comprises adding MgCb to a final concentration of 100 mM. In some embodiments, the cells are lysed in step (d) via homogenization. In some embodiments, the cells are lysed in step (d) via emulsiflex C3. In some embodiments, the cells are lysed in step (d) via sonication. In some embodiments, the cells are sonicated in 7 cycles, wherein each cycle comprises 30 seconds of sonication and 30 seconds without sonication. In some embodiments, the centrifugation of step (e) is at 10,000 x g. In some embodiments, the centrifugation of step (e) is for 15 minutes. In some embodiments, the centrifugation of step (e) is at 4°C or room temperature. [0296] In some embodiments, the centrifugation of step (f) is at 120,000 x g. In some embodiments, the centrifugation of step (f) is at 110,000 x g. In some embodiments, the centrifugation of step (f) is for 1 hour. In some embodiments, the centrifugation of step (f) is for 15 minutes. In some embodiments, the centrifugation of step (f) is at 4°C or room temperature. In some embodiments, the second solution in step (g) is 100 mM sodium carbonate, pH 11. In some embodiments, the second solution in step (g) is 10 mM Tris-HCl pH 8.0, 2% triton X-100. In some embodiments, step (g) further comprises incubating the solution for 1 hour at 4°C. In some embodiments, step (g) further comprises incubating the solution for 30-60 minutes at room temperature. In some embodiments, the centrifugation of step (h) is at 120,000 x g. In some embodiments, the centrifugation of step (h) is at 110,000 x g. In some embodiments, the centrifugation of step (h) is for 1 hour. In some embodiments, the centrifugation of step (h) is for 15 minutes. In some embodiments, the centrifugation of step (h) is at 4°C or room temperature. In some embodiments, the third solution in step (i) is 100 mM Tris-HCl, pH 7.5. In some embodiments, the third solution in step (i) is PBS. In some embodiments, the centrifugation of step (j) is at 120,000 x g. In some embodiments, the centrifugation of step (j) is for 20 minutes. In some embodiments, the centrifugation of step (j) is at 4°C or room temperature. In some embodiments, the fourth solution in step (k) is 100 mM Tris-HCl, pH 7.5 or PBS.

[0297] pmEVs obtained by methods provided herein may be further purified by size based column chromatography, by affinity chromatography, and by gradient ultracentrifugation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. If filtration was used to concentrate the filtered supernatant, the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column. Samples are applied to a 35- 60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C. Briefly, using an Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 35% Optiprep in PBS. In some embodiments, if filtration was used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep. Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C. [0298] In some embodiments, to confirm sterility and isolation of the pmEV preparations, pmEVs are serially diluted onto agar medium used for routine culture of the bacteria being tested, and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 pm filter to exclude intact cells. To further increase purity, isolated pmEVs may be DNase or proteinase K treated.

[0299] In some embodiments, the sterility of the pmEV preparations can be confirmed by plating a portion of the pmEVs onto agar medium used for standard culture of the bacteria used in the generation of the pmEVs and incubating using standard conditions.

[0300] In some embodiments select pmEVs are isolated and enriched by chromatography and binding surface moi eties on pmEVs. In other embodiments, select pmEVs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins or other methods known to one skilled in the art.

[0301] The pmEVs can be analyzed, e.g., as described in Jeppesen et al. (2019) Cell 177:428.

[0302] In some embodiments, pmEVs are lyophilized.

[0303] In some embodiments, pmEVs are gamma irradiated (e.g., at 17.5 or 25 kGy).

[0304] In some embodiments, pmEVs are UV irradiated.

[0305] In some embodiments, pmEVs are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).

[0306] In some embodiments, pmEVs are acid treated.

[0307] In some embodiments, pmEVs are oxygen sparged (e.g., at 0.1 vvm for two hours).

[0308] The phase of growth can affect the amount or properties of bacteria. In the methods of pmEV preparation provided herein, pmEVs can be isolated, e.g., from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.

Production of Secreted Microbial Extracellular Vesicles (smEVs)

[0309] In certain aspects, the smEVs described herein can be prepared using any method known in the art. [0310] In some embodiments, the smEVs are prepared without an smEV purification step. For example, in some embodiments, bacteria described herein are killed using a method that leaves the smEVs intact and the resulting bacterial components, including the smEVs, are used in the methods and compositions described herein. In some embodiments, the bacteria are killed using an antibiotic (e.g., using an antibiotic described herein). In some embodiments, the bacteria are killed using UV irradiation. In some embodiments, the bacteria are heat-killed.

[0311] In some embodiments, the smEVs described herein are purified from one or more other bacterial components. Methods for purifying smEVs from bacteria are known in the art. In some embodiments, smEVs are prepared from bacterial cultures using methods described in S. Bin Park, et al. PLoS ONE. 6(3):el7629 (2011) or G. Norheim, et al. PLoS ONE. 10(9): e0134353 (2015) or Jeppesen, et al. Cell 177:428 (2019), each of which is hereby incorporated by reference in its entirety. In some embodiments, the bacteria are cultured to high optical density and then centrifuged to pellet bacteria (e.g., at 10,000 x g for 30 min at 4°C, at 15,500 x g for 15 min at 4°C). In some embodiments, the culture supernatants are then passed through filters to exclude intact bacterial cells (e.g., a 0.22 pm filter). In some embodiments, the supernatants are then subjected to tangential flow filtration, during which the supernatant is concentrated, species smaller than 100 kDa are removed, and the media is partially exchanged with PBS. In some embodiments, filtered supernatants are centrifuged to pellet bacterial smEVs (e.g., at 100,000-150,000 x g for 1-3 hours at 4°C, at 200,000 x g for 1-3 hours at 4°C). In some embodiments, the smEVs are further purified by resuspending the resulting smEV pellets (e.g., in PBS), and applying the resuspended smEVs to an Optiprep (iodixanol) gradient or gradient (e.g., a 30-60% discontinuous gradient, a 0-45% discontinuous gradient), followed by centrifugation (e.g., at 200,000 x g for 4-20 hours at 4°C). smEV bands can be collected, diluted with PBS, and centrifuged to pellet the smEVs (e.g., at 150,000 x g for 3 hours at 4°C, at 200,000 x g for 1 hour at 4°C). The purified smEVs can be stored, for example, at -80°C or -20°C until use. In some embodiments, the smEVs are further purified by treatment with DNase and/or proteinase K.

[0312] For example, in some embodiments, cultures of bacteria can be centrifuged at 11,000 x g for 20-40 min at 4°C to pellet bacteria. Culture supernatants may be passed through a 0.22 pm filter to exclude intact bacterial cells. Filtered supernatants may then be concentrated using methods that may include, but are not limited to, ammonium sulfate precipitation, ultracentrifugation, or filtration. For example, for ammonium sulfate precipitation, 1.5-3 M ammonium sulfate can be added to filtered supernatant slowly, while stirring at 4°C. Precipitations can be incubated at 4°C for 8-48 hours and then centrifuged at 11,000 x g for 20-40 min at 4°C. The resulting pellets contain bacteria smEVs and other debris. Using ultracentrifugation, filtered supernatants can be centrifuged at 100,000- 200,000 x g for 1-16 hours at 4°C. The pellet of this centrifugation contains bacteria smEVs and other debris such as large protein complexes. In some embodiments, using a filtration technique, such as through the use of an Amicon Ultra spin filter or by tangential flow filtration, supernatants can be filtered so as to retain species of molecular weight > 50 or 100 kDa.

[0313] Alternatively, smEVs can be obtained from bacteria cultures continuously during growth, or at selected time points during growth, for example, by connecting a bioreactor to an alternating tangential flow (ATF) system (e.g., XCell ATF from Repligen). The ATF system retains intact cells (> 0.22 pm) in the bioreactor, and allows smaller components (e.g., smEVs, free proteins) to pass through a filter for collection. For example, the system may be configured so that the < 0.22 pm filtrate is then passed through a second filter of 100 kDa, allowing species such as smEVs between 0.22 pm and 100 kDa to be collected, and species smaller than 100 kDa to be pumped back into the bioreactor. Alternatively, the system may be configured to allow for medium in the bioreactor to be replenished and/or modified during growth of the culture. smEVs collected by this method may be further purified and/or concentrated by ultracentrifugation or filtration as described above for filtered supernatants.

[0314] smEVs obtained by methods provided herein may be further purified by size-based column chromatography, by affinity chromatography, by ion-exchange chromatography, and by gradient ultracentrifugation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. If filtration was used to concentrate the filtered supernatant, the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column. Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C. Briefly, using an Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in PBS and 3 volumes of 60% Optiprep are added to the sample. In some embodiments, if filtration was used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep.

Samples are applied to a 0-45% discontinuous Optiprep gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C, e.g., 4-24 hours at 4°C.

[0315] In some embodiments, to confirm sterility and isolation of the smEV preparations, smEVs are serially diluted onto agar medium used for routine culture of the bacteria being tested, and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 pm filter to exclude intact cells. To further increase purity, isolated smEVs may be DNase or proteinase K treated.

[0316] In some embodiments, for preparation of smEVs used for in vivo injections, purified smEVs are processed as described previously (G. Norheim, et al. PLoS ONE.

10(9): e0134353 (2015)). Briefly, after sucrose gradient centrifugation, bands containing smEVs are resuspended to a final concentration of 50 pg/mL in a solution containing 3% sucrose or other solution suitable for in vivo injection known to one skilled in the art. This solution may also contain adjuvant, for example aluminum hydroxide at a concentration of 0-0.5% (w/v). In some embodiments, for preparation of smEVs used for in vivo injections, smEVs in PBS are sterile-filtered to < 0.22 pm.

[0317] In some embodiments, to make samples compatible with further testing (e.g., to remove sucrose prior to TEM imaging or in vitro assays), samples are buffer exchanged into PBS or 30 mM Tris, pH 8.0 using filtration (e.g., Amicon Ultra columns), dialysis, or ultracentrifugation (200,000 x g, > 3 hours, 4°C) and resuspension.

[0318] In some embodiments, the sterility of the smEV preparations can be confirmed by plating a portion of the smEVs onto agar medium used for standard culture of the bacteria used in the generation of the smEVs and incubating using standard conditions.

[0319] In some embodiments, select smEVs are isolated and enriched by chromatography and binding surface moi eties on smEVs. In other embodiments, select smEVs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins or other methods known to one skilled in the art.

[0320] The smEVs can be analyzed, e.g., as described in Jeppesen, et al. Cell 177:428 (2019).

[0321] In some embodiments, smEVs are lyophilized. [0322] In some embodiments, smEVs are gamma irradiated (e.g., at 17.5 or 25 kGy).

[0323] In some embodiments, smEVs are UV irradiated.

[0324] In some embodiments, smEVs are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).

[0325] In some embodiments, smEVs s are acid treated.

[0326] In some embodiments, smEVs are oxygen sparged (e.g., at 0.1 vvm for two hours).

[0327] The phase of growth can affect the amount or properties of bacteria and/or smEVs produced by bacteria. For example, in the methods of smEV preparation provided herein, smEVs can be isolated, e.g., from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.

[0328] The growth environment (e.g., culture conditions) can affect the amount of smEVs produced by bacteria. For example, the yield of smEVs can be increased by an smEV inducer, as provided in Table 5.

Table 5: Culture Techniques to Increase smEV Production

[0329] In the methods of smEVs preparation provided herein, the method can optionally include exposing a culture of bacteria to an smEV inducer prior to isolating smEVs from the bacterial culture. The culture of bacteria can be exposed to an smEV inducer at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.

Methods of Determining the Presence of Bacteria and/or mEVs

[0330] In certain aspects, provided herein is a method of determining the presence of bacteria or microbial extracellular vesicles (mEVs) derived from the bacteria in a sample. For example, a sample is obtained from a particular stage or time of a process; from a fermentation culture, a biomass, or a drug substance (DS); and/or containing further excipients with a DS (such as a sample from a drug product (DP)). As another example, conditions for preparing or storing a material containing bacteria or mEVs may change (e.g., temperature, humidity, time, and/or packaging). As another example, the conditions for growing bacteria (e.g., media components, time, temperature, density, etc.), or for downstreaming processing of bacteria or mEVs, may be altered. For example, a sample, such as a sample of a powder, DS or DP, can be reconstituted or resuspended in a liquid prior to use in the method. The effects of such treatment on the bacteria and/or mEVs can be evaluated using one or more lectins (e.g., evaluating binding to the one or more lectins). [0331] The method comprises: (a) contacting the sample comprising the bacteria or the mEVs with one or more lectins (such as a panel of lectins) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); and (b) detecting the binding of the bacteria or the mEVs to the one or more lectins (e.g., detecting with a detectable label), thereby determining the presence of bacteria or mEVs derived from such bacteria in the sample.

[0332] In certain aspects, provided herein is a method of determining the presence in a sample of a genus, species and/or strain of bacteria or microbial extracellular vesicles (mEVs) derived from the bacteria. The method comprises: (a) contacting the sample comprising the bacteria or the mEVs with one or more lectins (such as a panel of lectins) (e.g., under conditions that allow interaction between the bacteria or the mEVs with one or more lectins); and (b) detecting the binding of the bacteria or the mEVs to the one or more lectins (e.g., detecting with a detectable label), thereby determining the presence of a genus, species and/or strain of bacteria or mEVs derived from such bacteria in the sample.

[0333] In some embodiments, the bacteria or the mEVs are detectably labeled prior to step (a).

[0334] Examples of detectable labels include, but are not limited to, fluorescent moieties, radioactive moieties, paramagnetic moieties, luminescent moieties and/or colorimetric moieties. In some embodiments, the bacteria or the mEVs are labeled with a fluorescent moiety. Examples of fluorescent moieties include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514,

Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean and CyPet. In some embodiments, the fluorescent moiety is AlexaFluor555.

[0335] In some embodiments, the bacteria or the mEVs are detectably labeled after step (a) and prior to step (b).

[0336] In some embodiments, the bacteria or the mEVs are labeled prior to step (b) by contacting the bacteria or the mEVs bound to the one or more lectins, first with a biotinylated monoclonal antibody specific for the bacteria or the mEVs followed by a fluorescent moiety conjugated streptavidin, such as streptavidin-Cy3. In some embodiments, the bacteria or the mEVs are labeled prior to step (b) by contacting the bacteria or the mEVs bound to the one or more lectins, first with a rabbit polyclonal antibody specific for the bacteria or the mEVs followed by an anti-(rabbit) IgG labeled with a fluorescent moiety, such as Cy3. In some embodiments, the monoclonal antibody or the rabbit polyclonal antibody specifically binds to an antigen on a type of bacteria or bacterial mEVs.

[0337] In some embodiments, the antibody or antigen binding fragment thereof is an antibody or antigen binding fragment thereof that specifically binds to an antigen on a type of bacteria or bacterial mEVs. In some embodiments, the antibody or an antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Prevotella (e.g., bacteria of the species Prevotella histicola, such as bacteria of the strain Prevotella Strain B 50329 or strain Prevotella histicola ATCC designation number PTA- 126140) or mEVs derived therefrom. In some embodiments, the antibody or the antigen binding fragment is a 17B01-02G10 mAb or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments, the antibody or the antigen binding fragment is a 02F09-02B04 mAB or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Fournierella (e.g., bacteria of the species Fournierella massihensis. such as bacteria of the strain Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696)) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Harryflintia (e.g., bacteria of the species Harryflintia acelispora. such as bacteria of the strain Harryflintia acetispora Strain A) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Veillonella (e.g., bacteria of the species Veillonella parvula, such as bacteria of the strain Veillonella parvula Strain A (ATCC Accession Number PTA-125691)) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Subdoligranulum (e.g., bacteria of the species Subdoligranulum variabile, such as bacteria of the strain Subdoligranulum variabile) or mEVs derived therefrom.

[0338] In some embodiments, the lectins are detectably labeled prior to step (a). [0339] Examples of detectable labels include, but are not limited to, fluorescent moieties, radioactive moieties, paramagnetic moieties, luminescent moieties and/or colorimetric moieties. In some embodiments, the lectins are labeled with a fluorescent moiety. Examples of fluorescent moieties include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean and CyPet. In some embodiments, the fluorescent moiety is AlexaFluor555.

[0340] In some embodiments, the lectins are detectably labeled after step (a) and prior to step (b).

[0341] In some aspects, provided herein is a method of determining the presence of a genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs) in the sample, the method comprises: (a) labeling the bacteria or the mEVs with a detectable label; (b) contacting the sample comprising the bacteria or the mEVs with one or more lectins (such as a panel of lectins); and (c) detecting the binding of the bacteria or the mEVs to the one or more lectins (e.g., fluorescence microarray), thereby determining the presence of a genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs).

[0342] In some embodiments, the bacteria or the mEVs are detectably labeled, for example with a fluorescent moiety. Examples of a fluorescent moiety include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, Ypet, Emerald, Cerulean, Cy3 and CyPet. In some embodiments, the fluorescent moiety is AlexaFluor555. [0343] In some embodiments, the lectins are detectably labeled, for example with a fluorescent moiety. Examples of a fluorescent moiety include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, Ypet, Emerald, Cerulean, Cy3 and CyPet. In some embodiments, the fluorescent moiety is AlexaFluor555.

[0344] In certain aspects, provided herein is a method of determining the presence of a genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs) comprising: (a) contacting the sample comprising the bacteria or the mEVs with one or more lectins (such as a panel of lectins); (b) contacting the bacteria or the mEVs bound to the one or more lectins with an antibody; (c) detectably labeling the antibody; and (d) detecting the binding of the bacteria or the mEVs to the one or more lectins (e.g., fluorescence microarray), thereby determining the presence of a genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs). In some embodiments provided herein, the bacteria or the mEVs bound to the one or more lectins are contacted with a biotinylated monoclonal antibody or a rabbit polyclonal antibody specific for the bacteria or the mEVs.

[0345] In some embodiments provided herein, the antibody is detectably labeled with a fluorescent moiety conjugated streptavidin, such as streptavidin-Cy3, or anti-(rabbit) IgG labeled with a fluorescent moiety, such as Cy3. Examples of a fluorescent moiety include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridininchlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, Ypet, Emerald, Cerulean, Cy3 and CyPet.

[0346] In some embodiments, the antibody specifically binds to an antigen on a type of bacteria or bacterial mEVs. In some embodiments, the antibody or an antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Prevotella (e.g., bacteria of the species Prevotella histicola, such as bacteria of the strain Prevotella Strain B 50329 or strain Prevotella histicola ATCC designation number PTA- 126140) or mEVs derived therefrom. In some embodiments, the antibody or the antigen binding fragment is a 17B01-02G10 mAb or part thereof (U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments, the antibody or the antigen binding fragment is a 02F09-02B04 mAB or part thereof (U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Fournierella (e.g., bacteria of the species Fournierella massiHensis. such as bacteria of the strain Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696)) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Harryflintia (e.g., bacteria of the species Harryflintia acelispora. such as bacteria of the strain Harryflintia acetispora Strain A) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Veillonella (e.g., bacteria of the species Veillonella parvula, such as bacteria of the strain Veillonella parvula Strain A (ATCC Accession Number PTA-125691)) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Subdoligranulum (e.g., bacteria of the species Subdoligranulum variab e. such as bacteria of the strain Subdoligranulum variabile) or mEVs derived therefrom.

[0347] In some embodiments, the lectins are detectably labeled, for example with a fluorescent moiety. Examples of a fluorescent moiety include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514,

Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, Ypet, Emerald, Cerulean, Cy3 and CyPet. In some embodiments, the fluorescent moiety is AlexaFluor555. Methods of Identifying Bacteria and/or mEVs

[0348] In certain aspects, provided herein is a method of identifying in a sample the genus, species and/or strain of bacteria or the genus, species and/or strain of bacteria from which a microbial extracellular vesicle (mEV) is derived. In some embodiments, the method may further identify a sample as comprising bacteria substantially free of the mEVs derived therefrom. In some embodiments, the method may further identify a sample comprising mEVs as substantially free of the bacteria from which the mEVs were derived. The method comprises: (a) contacting the sample comprising the bacteria or the mEVs with one or more lectins (such as a panel of lectins) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); and (b) detecting the binding of the bacteria or the mEVs to the one or more lectins (e.g., detecting with a detectable label), thereby identifying the genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs).

[0349] In some embodiments, the bacteria or the mEVs are detectably labeled prior to step (a).

[0350] Examples of detectable labels include, but are not limited to, fluorescent moieties, radioactive moieties, paramagnetic moieties, luminescent moieties and/or colorimetric moieties. In some embodiments, the bacteria or the mEVs are labeled with a fluorescent moiety. Examples of fluorescent moieties include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514,

[0351] In some embodiments, the bacteria or the mEVs are detectably labeled after step (a) and prior to step (b).

[0352] In some embodiments, the bacteria or the mEVs are labeled prior to step (b) by contacting the bacteria or the mEVs bound to one or more lectins, first with a biotinylated monoclonal antibody specific for the bacteria or the mEVs followed by a fluorescent moiety conjugated streptavidin, such as streptavidin-Cy3. In some embodiments, the bacteria or the mEVs are labeled prior to step (b) by contacting the bacteria or the mEVs bound to one or more lectins, first with a rabbit polyclonal antibody specific for the bacteria or the mEVs followed by an anti-(rabbit) IgG labeled with a fluorescent moiety, such as Cy3. In some embodiments, the monoclonal antibody or the rabbit polyclonal antibody specifically binds to an antigen on a type of bacteria or bacterial mEVs.

[0353] In some embodiments, the antibody or antigen binding fragment thereof is an antibody or antigen binding fragment thereof that specifically binds to an antigen on a type of bacteria or bacterial mEVs. In some embodiments, the antibody or an antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Prevotella (e.g., bacteria of the species Prevotella histicola, such as bacteria of the strain Prevotella Strain B 50329 or strain Prevotella histicola ATCC designation number PTA- 126140) or mEVs derived therefrom. In some embodiments, the antibody or the antigen binding fragment is a 17B01-02G10 mAb or part thereof (U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments, the antibody or the antigen binding fragment is a 02F09-02B04 mAB or part thereof (U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Fournierella (e.g., bacteria of the species Fournierella massiliensis, such as bacteria of the strain Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696)) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Harryflintia (e.g., bacteria of the species Harryflintia acelispora. such as bacteria of the strain Harryflintia acetispora Strain A) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Veillonella (e.g., bacteria of the species Veillonella parvula, such as bacteria of the strain Veillonella parvula Strain A (ATCC Accession Number PTA-125691)) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Subdoligranulum (e.g., bacteria of the species Subdoligranulum variabile) or mEVs derived therefrom.

[0354] In some embodiments, the lectins are detectably labeled prior to step (a). [0355] Examples of detectable labels include, but are not limited to, fluorescent moieties, radioactive moieties, paramagnetic moieties, luminescent moieties and/or

I l l colorimetric moi eties. In some embodiments, the bacteria or the mEVs are labeled with a fluorescent moiety. Examples of fluorescent moieties include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean and CyPet. In some embodiments, the fluorescent moiety is AlexaFluor555.

[0356] In some embodiments, the lectins are detectably labeled after step (a) and prior to step (b).

[0357] In some aspects provided herein is a method of identifying the genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs) in a sample, the method comprises: (a) labeling the bacteria or the mEVs with a detectable label; (b) contacting the sample comprising the bacteria or the mEVs with one or more lectins (such as a panel of lectins) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); and (c) detecting the binding of the bacteria or the mEVs to one or more lectins (e.g., detecting the detectable label), thereby identifying the genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs).

[0358] In some embodiments, the bacteria or the mEVs are detectably labeled, for example with a fluorescent moiety. Examples of a fluorescent moiety include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, Ypet, Emerald, Cerulean, Cy3 and CyPet. In some embodiments, the fluorescent moiety is AlexaFluor555.

[0359] In some embodiments, the lectins are detectably labeled, for example with a fluorescent moiety. Examples of a fluorescent moiety include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, Ypet, Emerald, Cerulean, Cy3 and CyPet. In some embodiments, the fluorescent moiety is AlexaFluor555.

[0360] In certain aspects, provided herein is a method of identifying the genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs) comprising: (a) contacting the sample comprising the bacteria or the mEVs with one or more lectins (such as a panel of lectins) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); (b) contacting the bacteria or the mEVs bound to one or more lectins with an antibody; (c) detectably labeling the antibody; and (d) detecting the binding of the bacteria or the mEVs to one or more lectins (e.g., detecting the detectable label), thereby identifying the genus, species and/or strain of bacteria or mEVs derived from such bacteria (bacterial mEVs). In some embodiments provided herein, the bacteria or the mEVs bound to one or more lectins are contacted with a biotinylated monoclonal antibody or a rabbit polyclonal antibody specific for the bacteria or the mEVs. In some embodiments provided herein, the antibody is detectably labeled with a fluorescent moiety conjugated streptavidin, such as streptavidin-Cy3, or anti-(rabbit) IgG labeled with a fluorescent moiety, such as Cy3. Examples of a fluorescent moiety include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, Ypet, Emerald, Cerulean, Cy3 and CyPet.

[0361] In some embodiments, the antibody specifically binds to an antigen on a type of bacteria or bacterial mEVs. In some embodiments, the antibody or an antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Prevotella (e.g., bacteria of the species Prevotella histicola, such as bacteria of the strain Prevotella Strain B 50329 or strain Prevotella histicola ATCC designation number PTA- 126140) or mEVs derived therefrom. In some embodiments, the antibody or the antigen binding fragment is a 17B01-02G10 mAb or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments, the antibody or the antigen binding fragment is a 02F09-02B04 mAB or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Fournierella (e.g., bacteria of the species Fournierella massiHensis. such as bacteria of the strain Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696)) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Harryflintia (e.g., bacteria of the species Harryflintia acelispora. such as bacteria of the strain Harryflintia acetispora Strain A) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Veillonella (e.g., bacteria of the species Veillonella parvula, such as bacteria of the strain Veillonella parvula Strain A (ATCC Accession Number PTA-125691)) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Subdoligranulum (e.g., bacteria of the species Subdoligranulum variabile) or mEVs derived therefrom.

Methods of Quantifying Bacteria and/or mEVs

[0362] In certain aspects, provided herein is a method of quantifying bacteria or mEVs derived from bacteria (bacterial mEVs) in a sample. In some embodiments, the method comprises: (a) contacting the sample comprising the bacteria or the mEVs with one or more lectins (such as a panel of lectins) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); (b) detecting the binding of the bacteria or the mEVs to one or more lectins (e.g., detecting with a detectable label); and (c) comparing at least one lectin-binding signal of the sample to a lectin-binding signal of the same lectin for a reference sample comprising the bacteria or mEVs, wherein the particle count of bacteria or mEVs in the reference sample is known, thereby quantifying the bacteria or mEVs in the sample based on a change in lectin-binding signal intensity between the reference sample and the sample. In some embodiments, the bacteria or mEVs are detectably labeled prior to step (a).

[0363] In some embodiments, the bacteria or the mEVs are detectably labeled after step (a) and prior to step (b). [0364] In some embodiments, the bacteria or the mEVs are labeled after step (a) and prior to step (b) by contacting the bacteria or the mEVs bound to the lectin, first with a biotinylated monoclonal antibody specific for the bacteria or the mEVs followed by a fluorescent moi ety-conjugated streptavidin, such as streptavidin-Cy3. In some embodiments, the bacteria or the mEVs are labeled after step (a) and prior to step (b) by contacting the bacteria or the mEVs bound to the lectins, first with a rabbit polyclonal antibody specific for the mEVs followed by an anti-(rabbit) IgG labeled with a fluorescent moiety, such as Cy3. In some embodiments, the monoclonal antibody or the rabbit polyclonal antibody specifically binds to an antigen on a type of the bacteria or the mEVs. In some embodiments, the antibody or an antigen binding fragment thereof specifically binds to an antigen on bacteria or mEVs of the genus Prevotella (e.g., bacteria or mEVs of the species Prevotella histicola, such as bacteria or mEVs of the strain Prevotella Strain B 50329 or strain Prevotella histicola ATCC designation number PTA-126140). In some embodiments, the antibody or the antigen binding fragment is a 17B01-02G10 mAb or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments, the antibody or the antigen binding fragment is a 02F09-02B04 mAB or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria or mEVs of the genus Fournierella (e.g., bacteria or mEVs of the species Fournierella massiHensis. such as bacteria or mEVs of the strain Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696)). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria or mEVs of the genus Harryflintia (e.g., bacteria or mEVs of the species Harryflintia acelispora. such as bacteria or mEVs of the strain Harryflintia acetispora Strain A). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria or mEVs of the genus Veillonella (e.g., bacteria or mEVs of the species Veillonella parvula, such as bacteria or mEVs of the strain Veillonella parvula Strain A (ATCC Accession Number PTA-125691)). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria or mEVs of the genus Subdoligramdum (e.g., bacteria or mEVs of the species S bdoligf'anulum variabile). [0365] In some aspects, provided herein is a method of quantifying bacteria or mEVs derived from bacteria (bacterial mEVs) in the sample, the method comprising: (a) labeling the bacteria or the mEVs with a detectable label; (b) contacting the sample comprising the bacteria or the mEVs with one or more lectins (such as a panel of lectins) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); (c) detecting the binding of the bacteria or the mEVs to one or more lectins (e.g., detecting the detectable label); and (d) comparing at least one lectin-binding signal of the sample to a lectin-binding signal of the same lectin for a reference sample comprising the bacteria or mEVs, wherein the particle count of bacteria or mEVs in the reference sample is known, thereby quantifying the bacteria or mEVs in the sample based on a change in lectin- binding signal intensity between the reference sample and the sample. In some embodiments, the bacteria or the mEVs are detectably labeled, for example with a fluorescent moiety. Examples of detectable labels include, but are not limited to, fluorescent moieties, radioactive moieties, paramagnetic moieties, luminescent moieties and/or colorimetric moieties. Examples of fluorescent moieties include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514,

[0366] In certain aspects, provided herein is a method of quantifying bacteria or mEVs derived from bacteria (bacterial mEVs) in a sample. In some embodiments, the method comprises: (a) contacting the sample comprising the bacteria or the mEVs with one or more lectins (such as a panel of lectins) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); (b) contacting the bacteria or the mEVs bound to one or more lectins with an antibody; (c) detectably labeling the antibody; (d) detecting the binding of the bacteria or the mEVs to one or more lectins (e.g., detecting the detectable label); and (e) comparing at least one lectin-binding signal of the sample to a lectin-binding signal of the same lectin for a reference sample comprising the bacteria or mEVs, wherein the particle count of bacteria or mEVs in the reference sample is known, thereby quantifying the bacteria or mEVs in the sample based on a change in lectin-binding signal intensity between the reference sample and the sample. In some embodiments provided herein, the bacteria or the mEVs bound to one or more lectins are first contacted with a biotinylated monoclonal antibody specific for the bacteria or the mEVs (step (b)), followed by a fluorescent moiety-conjugated streptavidin, such as streptavidin-Cy3 (step (c)). In some embodiments provided herein, the bacteria or the mEVs bound to one or more lectins are first contacted with a rabbit polyclonal antibody specific for the bacteria or the mEVs (step (b)), followed by an anti-(rabbit) IgG labeled with a fluorescent moiety, such as Cy3 (step (c)). In some embodiments, the monoclonal antibody or the rabbit polyclonal antibody specifically binds to an antigen on a type of bacteria or mEVs. In some embodiments, the antibody or an antigen binding fragment thereof specifically binds to an antigen on bacteria or mEVs of the genus Prevotella (e.g., bacteria or mEVs of the species Prevotella histicola, such as bacteria or mEVs of the strain Prevotella Strain B 50329 or strain Prevotella histicola ATCC designation number PTA-126140). In some embodiments, the antibody or the antigen binding fragment is a 17B01-02G10 mAb or part thereof (U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments, the antibody or the antigen binding fragment is a 02F09-02B04 mAB or part thereof (U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria or mEVs of the genus Fournierella (e.g., bacteria or mEVs of the species Fournierella massiHensis, such as bacteria or mEVs of the strain Fournierella massiHensis Strain A (ATCC Deposit Number PTA-126696)). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria or mEVs of the genus Harryflintia (e.g., bacteria or mEVs of the species Harryflintia acelispora. such as bacteria or mEVs of the strain Harryflintia acetispora Strain A). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria or mEVs of the genus Veillonella (e.g., bacteria or mEVs of the species Veillonella parvula, such as bacteria or mEVs of the strain Veillonella parvula Strain A (ATCC Accession Number PTA-125691)). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria or mEVs of the genus Subdoligranulum (e.g., bacteria or mEVs of the species Subdoligranulum variabile). Other Methods of Quantifying Bacteria and/or mEVs Particles

[0367] In some aspects, compositions and samples described herein comprise a certain ratio of bacteria particles to mEV particles. The number of bacteria particles can be based on actual particle number or (if the bacteria is live) the number of CFUs. The particle number can be established by combining a set number of purified mEVs with a set number of purified bacteria, by modifying the growth conditions under which the bacteria are cultured, or by modifying the bacteria itself to produce more or fewer mEVs.

[0368] To quantify the numbers of mEVs and/or bacteria present in a sample, for example in a reference sample in the quantifying method utilizing lectins as described above, electron microscopy (e.g., EM of ultrathin frozen sections) can be used to visualize the mEVs and bacteria and count their relative numbers. Alternatively, combinations of nanoparticle tracking analysis (NTA), Coulter counting, and dynamic light scattering (DLS) or a combination of these techniques can be used. NTA and the Coulter counter count particles and show their sizes. DLS gives the size distribution of particles, but not the concentration. Bacteria frequently have diameters of 1-2 pm. The full range is 0.2-20 pm. Combined results from Coulter counting and NTA can reveal the number of bacteria in a given sample. Coulter counting reveals the number of particles with diameters of 0.7-10 pm. NTA reveals the number of particles with diameters of 50-1400 nm. For most bacterial samples, the Coulter counter alone can reveal the number of bacteria in a sample. mEVs are generally 20-250 nm in diameter. NTA will allow us to count the number of particles that are 50-250 nm in diameter. DLS reveals the distribution of particles of different diameters within an approximate range of 1 nm- 3 pm.

Methods of Detecting and Monitoring Lectin-Binding Profiles of Bacteria and/or mEVs [0369] Methods of detecting a lectin-binding profile of a sample comprising bacteria or microbial extracellular vesicles (mEVs) are provided herein. A lectin-binding profile refers to the binding affinity of a sample (e.g., and/or of bacteria and/or microbial extracellular vesicles (mEVs) therein) to one or more lectins, such as in a panel of lectins. As described herein, bacteria and mEVs can show strain-specific fingerprint-like lectin- binding profiles to one or more lectins, such as in a panel of lectins.

[0370] In some embodiments, the method of detecting a lectin-binding profile of a sample comprising bacteria or mEVs comprises: (a) contacting the sample comprising the bacteria or the mEVs with one or more lectins (such as a panel of lectins) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); and (b) detecting the binding of the bacteria or the mEVs to one or more lectins (e.g., detecting with a detectable label), thereby detecting the lectin-binding profile of the sample.

[0371] In some embodiments, the bacteria or the mEVs are detectably labeled. In some embodiments, the bacteria or the mEVs are detectably labeled prior to step (a). In some embodiments, the bacteria or the mEVs are detectably labeled after step (a) and prior to step (b).

[0372] In some embodiments, the lectins are detectably labeled. In some embodiments, the lectins are detectably labeled prior to step (a). In some embodiments, the lectins are detectably labeled after step (a) and prior to step (b).

[0373] Methods of monitoring a change in a lectin-binding profile of bacteria and/or mEVs across more than one sample are also provided herein. A change in a lectin-binding profile may be an increase or decrease in the binding signal to one or more lectins (e.g., of a panel of lectins) or a change in the overall pattern of the binding signals to one or more lectins, e.g., in a panel. Monitoring a change includes results where there is no change in the lectin-binding profile of bacteria or mEVs. In some embodiments, monitoring the change in the lectin-binding profile is used as an in-process control. The lectin-binding profile of samples comprising bacteria or mEVs may be used to monitor, for example, fermentation conditions and/or lot to lot variability. In some embodiments, monitoring the change in the lectin-binding profile is used on samples after different storage conditions (e.g., time, temperature, and/or humidity). In some embodiments provided herein, comparing the lectin-binding profiles of the bacteria or the mEVs in each sample reveals the change in the lectin-binding profile of the bacteria or the mEVs from sample to sample. In some embodiments, monitoring the change in the lectin-binding profile is used on samples from a particular stage or time of a process; from a fermentation culture, a biomass, or a drug substance (DS); and/or containing further excipients with a DS (such as a sample from a drug product (DP)). In some embodiments, monitoring the change in the lectin-binding profile is used on samples after conditions for preparing or storing a material containing bacteria or mEVs change (e.g., temperature, humidity, time, and/or packaging). In some embodiments, monitoring the change in the lectin-binding profile is used on samples after the conditions for growing bacteria (e.g., media components, time, temperature, density, etc.), or for downstreaming processing of bacteria or mEVs, are altered. The effects of such changes and alterations on the bacteria and/or mEVs can be monitored using one or more lectins (e.g., evaluating binding to one or more lectins).

[0374] In certain aspects, provided herein is a method of monitoring a change in the lectin-binding profile of more than one sample comprising bacteria or microbial extracellular vesicles (mEVs). In some embodiments, the method comprises: (a) contacting each sample comprising the bacteria or the mEVs with one or more lectins (such as a panel of lectins) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); (b) detecting the binding of the bacteria or the mEVs to one or more lectins (e.g., detecting with a detectable label); and (c) comparing the lectin-binding profiles of the bacteria or the mEVs in each sample, thereby monitoring the change in the lectin-binding profile of the samples.

[0375] In some embodiments, the bacteria or the mEVs are detectably labeled prior to step (a). In some embodiments, the bacteria or the mEVs are detectably labeled after step (a) and prior to step (b).

[0376] In some embodiments, the bacteria or the mEVs are detectably labeled prior to step (a), for example with a fluorescent moiety.

[0377] Examples of a fluorescent moiety include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean, Cy3 and CyPet. In some embodiments, the fluorescent moiety is AlexaFluor555. In some embodiments wherein the detectable label is a fluorescent moiety, a change in the lectin-binding profile may be a change in the relative fluorescence intensity of binding to one or more lectin of the lectin panel, such as, for example, a change in the relative fluorescence unit (RUF) of a particular lectin or a change in the overall pattern of fluorescence intensities of binding to the lectins in the lectin panel. For example, one sample may exhibit a lectin-binding profile with a stronger relative fluorescence intensity of binding to one lectin as compared to the other lectins in the panel while another sample may show a weaker relative fluorescence intensity of binding to the same lectin compared to the other lectins in the same lectin panel. [0378] In some embodiments, detecting the binding of the bacteria or the mEVs to the lectins involves the use of an antibody prior to step (b). In some embodiments provided herein, the bacteria or the mEVs bound to one or more lectins are contacted with a biotinylated monoclonal antibody or a rabbit polyclonal antibody specific for the bacteria or the mEVs. In some embodiments provided herein, the antibody is detectably labeled with a fluorescent conjugated streptavidin, such as streptavidin-Cy3, or anti-(rabbit) IgG labeled with a fluorescent moiety, such as Cy3. In some embodiments, the antibody specifically binds to an antigen on bacteria or bacterial mEVs. In some embodiments, the antibody or an antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Prevotella (e.g., bacteria of the species Prevotella histicola, such as bacteria of the strain Prevotella Strain B 50329 or strain Prevotella histicola ATCC designation number PTA- 126140) or mEVs derived therefrom. In some embodiments, the antibody or the antigen binding fragment is a 17B01-02G10 mAb or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments, the antibody or the antigen binding fragment is a 02F09-02B04 mAB or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Fournierella (e.g., bacteria of the species Fournierella massihensis. such as bacteria of the strain Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696)) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Harryflintia (e.g., bacteria of the species Harryflintia acelispora. such as bacteria of the strain Harryflintia acetispora Strain A) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Veillonella (e.g., bacteria of the species Veillonella parvula, such as bacteria of the strain Veillonella parvula Strain A (ATCC Accession Number PTA-125691)) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Subdoligranulum (e.g., bacteria of the species Subdoligranulum variabile) or mEVs derived therefrom.

[0379] In some embodiments, the lectins are detectably labeled prior to step (a). In some embodiments, the lectins are detectably labeled after step (a) and prior to step (b). [0380] In some embodiments, the lectins are detectably labeled prior to step (a), for example with a fluorescent moiety.

[0381] Examples of a fluorescent moiety include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean, Cy3 and CyPet. In some embodiments, the fluorescent moiety is AlexaFluor555. In some embodiments wherein the detectable label is a fluorescent moiety, a change in the lectin-binding profile may be a change in the relative fluorescence intensity of binding to one or more lectin of the lectin panel, such as, for example, a change in the relative fluorescence unit (RUF) of a particular lectin or a change in the overall pattern of fluorescence intensities of binding to the lectins in the lectin panel. For example, one sample may exhibit a lectin-binding profile with a stronger relative fluorescence intensity of binding to one lectin as compared to the other lectins in the panel while another sample may show a weaker relative fluorescence intensity of binding to the same lectin compared to the other lectins in the same lectin panel.

[0382] In some embodiments, detecting the binding of the bacteria or the mEVs to the lectins involves the use of an antibody prior to step (b).

Methods of Detecting and Monitoring Bacteria and/or mEV Surface Glycans [0383] Methods of detecting a glycosidic profile of a sample comprising bacteria or microbial extracellular vesicles (mEVs) are provided herein. A glycosidic profile refers to the surface glycans of bacteria and/or mEVs revealed by the binding affinity of the bacteria and/or mEVs to one or more lectins, such as a panel of lectins.

[0384] In some embodiments, the method of detecting a glycosidic profile of a sample comprising bacteria or mEVs comprises: (a) contacting the sample comprising the bacteria or the mEVs with one or more lectins (such as a panel of lectins) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); and (b) detecting the binding of the bacteria or the mEVs to one or more lectins (e.g., detecting with a detectable label), thereby detecting the glycosidic profile of the sample. [0385] In some embodiments, the bacteria or the mEVs are detectably labeled prior to step (a). In some embodiments, the bacteria or the mEVs are detectably labeled after step (a) and prior to step (b).

[0386] In some embodiments, the lectins are detectably labeled prior to step (a). In some embodiments, the lectins are detectably labeled after step (a) and prior to step (b). [0387] Methods of monitoring a change in a glycosidic profile of bacteria and mEVs across more than one sample are also provided herein. A change in a glycosidic profile is revealed by a change in binding to one or more lectins, such as an increase or decrease in the binding signal to one or more lectins or a change in the overall pattern of the binding signals toone or more lectins (e.g., a change in the binding profile). Monitoring a change includes results where there is no change in the binding profile of bacteria or mEVs. [0388] In some embodiments, monitoring the change in the glycosidic profile is used as an in-process control. In some embodiments, monitoring the change in the glycosidic profile is used after different storage conditions (e.g., time, temperature, and/or humidity). The glycosidic profile derived from the lectin-binding signal of a panel of lectins may be used to monitor, for example, fermentation conditions and/or lot to lot variability. In some embodiments, monitoring the change in the glycosidic profile is used on samples after different storage conditions (e.g., time, temperature, and/or humidity). In some embodiments provided herein, comparing the glycosidic profiles of the bacteria or the mEVs in each sample reveals the change in the glycosidic profile of the bacteria or the mEVs from sample to sample. In some embodiments, monitoring the change in the glycosidic profile is used on samples from a particular stage or time of a process; from a fermentation culture, a biomass, or a drug substance (DS); and/or containing further excipients with a DS (such as a sample from a drug product (DP)). In some embodiments, monitoring the change in the glycosidic profile is used on samples after conditions for preparing or storing a material containing bacteria or mEVs change (e.g., temperature, humidity, time, and/or packaging). In some embodiments, monitoring the change in the glycosidic profile is used on samples after the conditions for growing bacteria (e.g., media components, time, temperature, density, etc.), or for downstreaming processing of bacteria or mEVs, are altered. The effects of such changes and alterations on the bacteria and/or mEVs can be monitored using one or more lectins (e.g., evaluating binding to one or more lectins). In some embodiments provided herein, comparing a change in the binding signals of the bacteria or the mEVs in each sample reveals the change in the glycosidic profile of the bacteria or the mEVs from sample to sample.

[0389] In certain aspects, provided herein is a method of monitoring a change in the glycosidic profile of more than one sample comprising bacteria or microbial extracellular vesicles (mEVs). In some embodiments, the method comprises: (a) contacting each sample comprising the bacteria or the mEVs with one or more lectins (such as a panel of lectins) (e.g., under conditions that allow interaction between the bacteria or the mEVs with a lectin); (b) detecting the binding of the bacteria or the mEVs to one or more lectins (e.g., detecting with a detectable label); and (c) comparing the lectin binding signals of the bacteria or the mEVs in each sample, thereby monitoring the change in the glycosidic profile of the samples.

[0390] In some embodiments, the bacteria or the mEVs are detectably labeled prior to step (a). In some embodiments, the bacteria or the mEVs are detectably labeled after step (a) and prior to step (b).

[0391] In some embodiments, the bacteria or the mEVs are detectably labeled prior to step (a), for example with a fluorescent moiety. Examples of a fluorescent moiety include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridininchlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean, Cy3 and CyPet. In some embodiments, the fluorescent moiety is AlexaFluor555. In some embodiments wherein the detectable label is a fluorescent moiety, a change in the glycosidic profile may be derived from a change in the relative fluorescence intensity of binding to one or more lectin of the lectin panel, such as, for example, a change in the relative fluorescence unit (RUF) of a particular lectin or a change in the overall pattern of fluorescence intensities of binding to the lectins in the lectin panel. For example, one sample may exhibit a stronger relative fluorescence intensity of binding to one lectin as compared to the other lectins in the panel while another sample may show a weaker relative fluorescence intensity of binding to the same lectin compared to the other lectins in the same lectin panel. [0392] In some embodiments, detecting the binding of the bacteria or the mEVs to one or more lectins involves the use of an antibody, for example, prior to step (b). In some embodiments provided herein, the bacteria or the mEVs bound to one or more lectins are contacted with a biotinylated monoclonal antibody or a rabbit polyclonal antibody specific for the bacteria or the mEVs. In some embodiments provided herein, the antibody is detectably labeled with a fluorescent conjugated streptavidin, such as streptavidin-Cy3, or anti-(rabbit) IgG labeled with a fluorescent moiety, such as Cy3. In some embodiments, the antibody specifically binds to an antigen on bacteria or bacterial mEVs. In some embodiments, the antibody or an antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Prevotella (e.g., bacteria of the species Prevotella histicola, such as bacteria of the strain Prevotella Strain B 50329 or strain Prevotella histicola ATCC designation number PTA-126140) or mEVs derived therefrom. In some embodiments, the antibody or the antigen binding fragment is a 17B01-02G10 mAb or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments, the antibody or the antigen binding fragment is a 02F09-02B04 mAB or part thereof (see U.S. Provisional Application No. 63/286,934 and PCT Publication No. WO 2023/107537). In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Fournierella (e.g., bacteria of the species Fournierella massiliensis, such as bacteria of the strain Fournierella massiliensis Strain A (ATCC Deposit Number PTA- 126696)) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Harryflintia (e.g., bacteria of the species Harryflintia acelispora. such as bacteria of the strain Harryflintia acetispora Strain A) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Veillonella (e.g., bacteria of the species Veillonella parvula, such as bacteria of the strain Veillonella parvula Strain A (ATCC Accession Number PTA-125691)) or mEVs derived therefrom. In some embodiments provided herein, the antibody or antigen binding fragment thereof specifically binds to an antigen on bacteria of the genus Subdoligranulum (e.g., bacteria of the species Subdoligranulum variabile,) or mEVs derived therefrom.

[0393] In some embodiments, the lectins are detectably labeled prior to step (a). In some embodiments, the lectins are detectably labeled after step (a) and prior to step (b). [0394] In some embodiments, the lectins are detectably labeled prior to step (a), for example with a fluorescent moiety. Examples of a fluorescent moiety include, but are not limited to, Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean, Cy3 and CyPet. In some embodiments, the fluorescent moiety is AlexaFluor555. In some embodiments wherein the detectable label is a fluorescent moiety, a change in the glycosidic profile may be derived from a change in the relative fluorescence intensity of binding to one or more lectin of the lectin panel, such as, for example, a change in the relative fluorescence unit (RUF) of a particular lectin or a change in the overall pattern of fluorescence intensities of binding to the lectins in the lectin panel. For example, one sample may exhibit a stronger relative fluorescence intensity of binding to one lectin as compared to the other lectins in the panel while another sample may show a weaker relative fluorescence intensity of binding to the same lectin compared to the other lectins in the same lectin panel.

Exemplary Embodiments

1. A method of identifying a bacterium or a microbial extracellular vesicle (mEV) in a sample, the method comprising:

(a) contacting the sample comprising the bacterium or the mEV with one or more lectins; and

(b) detecting the binding of the bacterium or the mEV to one or more lectins, thereby identifying the bacteria or the mEV in the sample.

2. The method of embodiment 1, wherein an mEV is identified.

3. The method of embodiment 1, wherein a bacterium is identified.

4. The method of any one of embodiments 1 to 3, wherein the method further comprises identifying the sample as comprising the mEV substantially free of the bacterium from which the mEV was derived.

5. A method of determining the presence of (e.g., detecting) a bacterium or a microbial extracellular vesicle (mEV) in a sample, the method comprising: (a) contacting the sample comprising the bacterium or the mEV with one or more lectins; and

(b) detecting the binding of the bacterium or the mEV to one or more lectins, thereby determining the presence of (e.g., detecting) the bacterium or the mEV in the sample.

6. The method of embodiment 5, wherein an mEV is present.

7. The method of embodiment 5, wherein a bacterium is present.

8. The method of embodiments 5, wherein the mEV is derived from the bacterium.

9. The method of any one of embodiments 1 to 8, wherein the sample comprises an excipient.

10. The method of any one of embodiments 1-9, wherein the one or more lectins comprise non-human lectins.

11. The method of any one of embodiments 1-9, wherein the one or more lectins consist of non-human lectins.

12. The method of any one of embodiments 1 to 11, wherein the non-human lectins comprise bacterial, plant, fungal, or non-human animal lectins.

13. The method of any one of embodiments 1 to 12, wherein more than 50% of the non- human lectins are plant lectins.

14. The method of any one of embodiments 1 to 12, wherein more than 75% of the non- human lectins are plant lectins.

15. The method of any one of embodiments 1 to 12, wherein more than 85% of the non- human lectins are plant lectins.

16. The method of any one of embodiments 1 to 12, wherein more than 90% of the non- human lectins are plant lectins.

17. The method of any one of embodiments 1 to 12, wherein more than 95% of the non- human lectins are plant lectins.

18. The method of any one of embodiments 1 to 12, wherein all of the non-human lectins are plant lectins.

19. The method of any one of embodiments 1 to 18, wherein the panel comprises 1 to 45 lectins.

20. The method of any one of embodiments 1 to 19, wherein the one or more lectins comprise one or more of the lectins listed in Table 6. 21. The method of any one of embodiments 1 to 20, wherein the one or more lectins comprise BanLec.

22. The method of any one of embodiments 1 to 20, wherein the one or more lectins comprise CSL3.

23. The method of any one of embodiments 1 to 20, wherein the one or more lectins comprise: AAL, ACL, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA.

24. The method of any of one of embodiments 1 to 20, wherein the one or more lectins comprise: AAL, SJA, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA.

25. The method of any of one of embodiments 1 to 20, wherein the one or more lectins comprise one or more of: BPL and WGA.

26. The method of any of one of embodiments 1 to 20, wherein the one or more lectins comprise one or more of: BPL, RSL, VVL and WGA.

27. The method of any of one of embodiments 1 to 20, wherein the one or more lectins comprise BPL and WGA.

28. The method of any of one of embodiments 1 to 20, wherein the one or more lectins comprise BPL, RSL, VVL and WGA.

29. The method of any of one of embodiments 1 to 20, wherein the one or more lectins comprise one or more of: AIA, GSLB3, Morniga G and VVL.

30. The method of any of one of embodiments 1 to 20, wherein the one or more lectins comprise one or more of: AIA, BPL, ECL, GSLB4, HP A, Morniga G, SBA, VVL and WGA.

31. The method of any of one of embodiments 1 to 20, wherein the one or more lectins comprise AIA, GSLB3, Morniga G and VVL.

32. The method of any of one of embodiments 1 to 20, wherein the one or more lectins comprise AIA, BPL, ECL, GSLB4, HP A, Morniga G, SBA, VVL and WGA.

33. The method of any of one of embodiments 1 to 20, wherein the one or more lectins comprise one or more of: AIA, GNA, GSLB4, HHL, Morniga G, NPA and RCA-I. 34. The method of any of one of embodiments 1 to 20, wherein the one or more lectins comprise one or more of: AAL, Al A, BPL, ECL, GN A, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL, SBA, VVL and WFA.

35. The method of any of one of embodiments 1 to 20, wherein the one or more lectins comprise AIA, GNA, GSLB4, HHL, Morniga G, NPA and RCA-I.

36. The method of any of one of embodiments 1 to 20, wherein the one or more lectins comprise AAL, AIA, BPL, ECL, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL, SBA, VVL and WFA.

37. The method of any of one of embodiments 1 to 20, wherein the one or more lectins comprise one or more of: GNA, HHL, HP A, LCA, NPA, PSA, and RSL.

38. The method of any of one of embodiments 1 to 20, wherein the one or more lectins comprise one or more of: AAL, BC2L-C Calsepa, ConA, GNA, GSII, HAA, HHL, HP A, LCA, LEL, NPA, PSA, RCA-I, RSL, STL and WGA.

39. The method of any of one of embodiments 1 to 20, wherein the one or more lectins comprise GNA, HHL, HP A, LCA, NPA, PSA, and RSL.

40. The method of any of one of embodiments 1 to 20, wherein the one or more lectins comprise AAL, BC2L-C Calsepa, ConA, GNA, GSII, HAA, HHL, HP A, LCA, LEL, NPA, PSA, RCA-I, RSL, STL and WGA.

41. The method of any of one of embodiments 1 to 20, wherein the one or more lectins comprise one or more of: AAL, AIA, Calsepa, GNA, HHL, LCA, NPA, PSA and RCA-I.

42. The method of any of one of embodiments 1 to 20, wherein the one or more lectins comprise one or more of: AAL, AIA, BC2L-C, Calsepa, ConA, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL and WGA.

43. The method of any of one of embodiments 1 to 20, wherein the one or more lectins comprise AAL, AIA, Calsepa, GNA, HHL, LCA, NPA, PSA and RCA-I.

44. The method of any of one of embodiments 1 to 20, wherein the one or more lectins comprise AAL, AIA, BC2L-C, Calsepa, ConA, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL and WGA.

45. The method of any one of embodiments 1 to 44, wherein the lectins are immobilized on a surface.

46. The method of embodiment 45, wherein the surface is a microchip.

47. The method of embodiment 46, wherein the microchip comprises a microarray.

48. The method of embodiment 45, wherein the surface is a sensorchip. 49. The method of any one of embodiments 1 to 44, wherein the bacterium or the mEV is immobilized on a surface.

50. The method of embodiment 49, wherein the surface is a microchip.

51. The method of embodiment 50, wherein the microchip comprises a microarray.

52. The method of embodiment 49, wherein the surface is a sensorchip.

53. The method of any one of embodiments 1 to 52, wherein the bacterium or the mEV is detectably labeled.

54. The method of any one of embodiments 1 to 52, wherein the lectin is detectably labeled.

55. The method of any one of embodiments 1 to 53, wherein the bacterium or the mEV is detectably labeled prior to step (a).

56. The method of embodiment 55, wherein the bacterium or the mEV is detectably labeled with a fluorescent moiety.

57. The method of embodiment 56, wherein the fluorescent moiety is AlexaFluor555.

58. The method of any one of embodiments 1 to 53, wherein the bacterium or the mEV is detectably labeled after step (a) and prior to step (b).

59. The method of embodiment 58, wherein the bacterium or the mEV is labeled by contacting the bacterium or the mEV bound to one or more lectins first with a biotinylated monoclonal antibody specific for the bacterium or the mEV followed by a streptavidin-Cy3.

60. The method of embodiment 58, wherein the bacterium or the mEV is labeled by contacting the bacterium or the mEV bound to one or more lectins first with a rabbit polyclonal antibody specific for the bacterium or the mEV followed by an anti-(rabbit) IgG labeled with Cy3.

6E The method of any one of embodiments 1 to 52 or 54, wherein the lectin is detectably labeled prior to step (a).

62. The method of embodiment 61, wherein the lectin is detectably labeled with a fluorescent moiety.

63. The method of embodiment 62, wherein the fluorescent moiety is AlexaFluor555.

64. The method of any one of embodiments 1 to 52 or 54, wherein the lectin is detectably labeled after step (a) and prior to step (b).

65. The method of any one of embodiments any one of embodiments 1 to 64, wherein the binding in step (b) is detected with a fluorescence scanner. 66. The method of any one of embodiments 1 to 65, wherein identifying the bacterium or the mEV comprises visual interpretation of the binding of the bacterium or the mEV to one or more lectins.

67. The method of any one of embodiments 1 to 65, wherein determining the presence of the bacterium or the mEV comprises visual interpretation of the binding of the bacterium or the mEV to one or more lectins.

68. A method of detecting a glycosidic profile of a bacterium or a microbial extracellular vesicle (mEV) in a sample, the method comprising:

(a) contacting the bacterium or the mEV with one or more lectins; and

(b) detecting the binding of the bacteria or the mEV to one or more lectins, thereby detecting the glycosidic profile of the bacteria or the mEV.

69. A method of monitoring a change in a glycosidic profile of a first sample and a second sample each comprising a bacterium or a microbial extracellular vesicle (mEV), the method comprising:

(a) separately contacting the first sample and the second sample comprising the bacterium or the mEV with one or more lectins;

(b) detecting the binding of the bacterium or the mEV in the first sample and the second sample to one or more lectins; and

(c) comparing the lectin-binding profiles of the bacterium or the mEV in the first sample and the second sample, thereby monitoring the change in the glycosidic profile of the first sample and the second sample.

70. A method of detecting a lectin-binding profile of a sample comprising a bacterium or a microbial extracellular vesicle (mEV), the method comprising:

(a) contacting the bacterium or the mEV with one or more lectins; and

(b) detecting the binding of the bacterium or the mEV to one or more lectins, thereby detecting the lectin-binding profile of the sample.

71. A method of monitoring a change in a lectin-binding profile in a first sample and a second sample each comprising a bacterium or a microbial extracellular vesicle (mEV), the method comprising:

(b) detecting the binding of the bacteria or the mEV in the first sample and the second sample to one or more lectins; and (c) comparing the lectin-binding profiles of the bacterium or the mEV in the first sample and the second sample, thereby monitoring the change in the lectin-binding profile of the first sample and the second sample.

72. The method of any one of embodiments 68 to 71, wherein the method is used as an in- process control.

73. The method of any one of embodiments 1 to 72, wherein the sample or first and second samples comprise bacterial biomass comprising the bacterium and mEV derived from the bacterium.

74. The method of any one of embodiments 1 to 73, wherein an mEV is present.

75. The method of any one of embodiments 1 to 74, wherein a bacterium is present.

76. The method of any one of embodiments 1 to 75, wherein the sample or first and second samples comprise the mEV substantially free of the bacterium from which the mEV was derived.

77. The method of any one of embodiments 1 to 76, wherein the sample or first and second samples comprise an excipient.

78. The method of any one of embodiments 1 to 77, wherein the first and second samples both comprise bacterium and/or mEV derived from the bacterium of the same genus.

79. The method of any one of embodiments 1 to 77, wherein the first and second samples both comprise bacterium and/or mEV derived from the bacterium of the same species.

80. The method of any one of embodiments 1 to 77, wherein the first and second samples both comprise bacterium and/or mEV derived from the bacterium of the same strain.

81. The method of any one of embodiments 68 to 80, wherein the one or more lectins comprise non-human lectins.

82. The method of any one of embodiments 68 to 80, wherein the one or more lectins consist of non-human lectins.

83. The method of any one of embodiments 68 to 82, wherein the non-human lectins comprise one or more of the following: bacterial, plant, fungus, or non-human animal lectins.

84. The method of any one of embodiments 68 to 83, wherein more than 50% of the non- human lectins are plant lectins.

85. The method of any one of embodiments 68 to 83, wherein more than 75% of the non- human lectins are plant lectins. 86. The method of any one of embodiments 68 to 83, wherein more than 85% of the nonhuman lectins are plant lectins.

87. The method of any one of embodiments 68 to 83, wherein more than 90% of the nonhuman lectins are plant lectins.

88. The method of any one of embodiments 68 to 83, wherein more than 95% of the nonhuman lectins are plant lectins.

89. The method of any one of embodiments 68 to 83, wherein all of the non-human lectins are plant lectins.

90. The method of any one of embodiments 68 to 89, wherein the panel comprises 1 to 45 lectins.

91. The method of any one of embodiments 68 to 90, wherein the one or more lectins comprise one or more of the lectins listed in Table 6.

92. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise BanLec.

93. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise CSL3.

94. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise: AAL, ACL, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA.

95. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise: AAL, SJA, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA.

96. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise one or more of: BPL and WGA.

97. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise one or more of: BPL, RSL, VVL and WGA.

98. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise BPL and WGA. 99. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise BPL, RSL, VVL and WGA.

100. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise one or more of: AIA, GSI-B3, Morniga G and VVL.

101. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise one or more of: AIA, BPL, ECL, GSLB4, HP A, Morniga G, SB A, VVL and WGA.

102. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise AIA, GSLB3, Morniga G and VVL.

103. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise AIA, BPL, ECL, GSLB4, HP A, Morniga G, SBA, VVL and WGA.

104. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise one or more of: AIA, GNA, GSLB4, HHL, Morniga G, NPA and RCA-I.

105. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise one or more of: AAL, AIA, BPL, ECL, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL, SBA, VVL and WFA.

106. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise AIA, GNA, GSLB4, HHL, Morniga G, NPA and RCA-I.

107. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise AAL, AIA, BPL, ECL, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL, SBA, VVL and WFA.

108. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise one or more of: GNA, HHL, HP A, LCA, NPA, PSA, and RSL.

109. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise one or more of: AAL, BC2L-C Calsepa, ConA, GNA, GSII, HAA, HHL, HP A, LCA, LEL, NPA, PSA, RCA-I, RSL, STL and WGA.

110. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise GNA, HHL, HP A, LCA, NPA, PSA, and RSL.

111. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise AAL, BC2L-C Calsepa, ConA, GNA, GSII, HAA, HHL, HP A, LCA, LEL, NPA, PSA, RCA-I, RSL, STL and WGA.

112. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise one or more of: AAL, AIA, Calsepa, GNA, HHL, LCA, NPA, PSA and RCA-I. 113. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise one or more of: AAL, AIA, BC2L-C, Calsepa, ConA, GNA, GSI-B4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL and WGA.

114. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise AAL, AIA, Calsepa, GNA, HHL, LCA, NPA, PSA and RCA-I.

115. The method of any one of embodiments 68 to 91, wherein the one or more lectins comprise AAL, AIA, BC2L-C, Calsepa, ConA, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL and WGA.

116. The method of any one of embodiments 68 to 115, wherein the one or more lectins are immobilized on a surface.

117. The method of embodiment 116, wherein the surface is a microchip.

118. The method of embodiment 117, wherein the microchip comprises a microarray.

119. The method of embodiment 116, wherein the surface is a sensorchip.

120. The method of any one of embodiments 68 to 115, wherein the bacterium or the mEV is immobilized on a surface.

121. The method of embodiment 120, wherein the surface is a microchip.

122. The method of embodiment 121, wherein the microchip comprises a microarray.

123. The method of embodiment 120, wherein the surface is a sensorchip.

124. The method of any one of embodiments 68 to 123, wherein the bacterium or the mEV is detectably labeled.

125. The method of any one of embodiments 68 to 123, wherein the lectin is detectably labeled.

126. The method of any one of embodiments 68 to 124, wherein the bacterium or the mEV is detectably labeled prior to step (a).

127. The method of embodiment 126, wherein the bacterium or the mEV is detectably labeled with a fluorescent moiety.

128. The method of embodiment 127, wherein the fluorescent moiety is AlexaFluor555.

129. The method of any one of embodiments 68 to 124, wherein the bacterium or the mEV is detectably labeled after step (a) and prior to step (b).

130. The method of embodiment 129, wherein the bacterium or the mEV is labeled by contacting the bacteria or the mEV bound to one or more lectins first with a biotinylated monoclonal antibody specific for the bacterium or the mEV followed by a streptavidin-Cy3. 131. The method of embodiment 129, wherein the bacteria or the mEV is labeled by contacting the bacterium or the mEV bound to one or more lectins first with a rabbit polyclonal antibody specific for the bacterium or the mEV followed by an anti-(rabbit) IgG labeled with Cy3.

132. The method of any one of embodiments 68 to 123 or 125, wherein the lectin is detectably labeled prior to step (a).

133. The method of embodiment 132, wherein the lectin is detectably labeled with a fluorescent moiety.

134. The method of embodiment 133, wherein the fluorescent moiety is AlexaFluor555.

135. The method of any one of embodiments 68 to 123 or 125, wherein the lectin is detectably labeled after step (a) and prior to step (b).

136. The method of any one of embodiments 68 to 135, wherein the binding in step (b) is detected with a fluorescence scanner.

137. The method of any one of embodiments 68 to 135, wherein detecting the binding of the bacterium or the mEV to one or more lectins comprises a visual detection.

138. The method of embodiment 69 or 71, wherein comparing the lectin-binding profiles of the bacterium or the mEV in each sample comprises a visual comparison.

139. A method of quantifying the amount of a bacterium or a microbial extracellular vesicle (mEV) in a sample, the method comprising:

(a) contacting the sample comprising the bacterium or mEV with one or more lectins;

(b) detecting the binding of the bacterium or mEV to one or more lectins; and

(c) comparing at least one lectin-binding signal of the sample to a lectin-binding signal of the same lectin for a reference sample comprising the bacterium or mEV, wherein the particle count of bacterium or mEV in the reference sample is known, thereby quantifying the bacterium or mEV in the sample based on a change in lectin-binding signal intensity between the reference sample and the sample.

140. The method of embodiment 139, wherein an mEV is present.

141. The method of embodiment 139 or 140, wherein a bacterium is present.

142. The method of embodiment 139, wherein the mEV is derived from a bacterium.

143. The method of any one of embodiments 139 to 142, wherein the one or more lectins comprise non-human lectins. 144. The method of any one of embodiments 139 to 142, wherein the one or more lectins consist of non-human lectins.

145. The method of any one of embodiments 139 to 144, wherein the non-human lectins comprise one or more of the following: bacterial, plant, fungus, or non-human animal lectins.

146. The method of any one of embodiments 139 to 145, wherein more than 50% of the non-human lectins are plant lectins.

147. The method of any one of embodiments 139 to 145, wherein more than 75% of the non-human lectins are plant lectins.

148. The method of any one of embodiments 139 to 145, wherein more than 85% of the non-human lectins are plant lectins.

149. The method of any one of embodiments 139 to 145, wherein more than 90% of the non-human lectins are plant lectins.

150. The method of any one of embodiments 139 to 145, wherein more than 95% of the non-human lectins are plant lectins.

151. The method of any one of embodiments 139 to 145, wherein all of the non-human lectins are plant lectins.

152. The method of any one of embodiments 139 to 151, wherein the panel comprises 1 to 45 lectins.

153. The method of any one of embodiments 139 to 152, wherein the one or more lectins comprise one or more of the lectins listed in Table 6.

154. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise BanLec.

155. The method of any one of embodiments 139 to 153, wherein the one or more lectins compriseCSL3.

156. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise: AAL, ACL, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA.

157. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise: AAL, SJA, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA.

158. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise one or more of: BPL and WGA.

159. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise one or more of: BPL, RSL, VVL and WGA.

160. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise BPL and WGA.

161. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise BPL, RSL, VVL and WGA.

162. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise one or more of: AIA, GSLB3, Morniga G and VVL.

163. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise one or more of: AIA, BPL, ECL, GSLB4, HP A, Morniga G, SBA, VVL and WGA.

164. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise AIA, GSLB3, Morniga G and VVL.

165. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise AIA, BPL, ECL, GSLB4, HP A, Morniga G, SBA, VVL and WGA.

166. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise one or more of: AIA, GNA, GSLB4, HHL, Morniga G, NPA and RCA-I.

167. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise one or more of: AAL, AIA, BPL, ECL, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL, SBA, VVL and WFA.

168. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise AIA, GNA, GSLB4, HHL, Morniga G, NPA and RCA-I.

169. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise AAL, AIA, BPL, ECL, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL, SBA, VVL and WFA.

170. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise one or more of: GNA, HHL, HP A, LCA, NPA, PSA, and RSL. 171. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise one or more of: AAL, BC2L-C Calsepa, ConA, GNA, GSII, HAA, HHL, HP A, LCA, LEL, NPA, PSA, RCA-I, RSL, STL and WGA.

172. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise GNA, HHL, HP A, LCA, NPA, PSA, and RSL.

173. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise AAL, BC2L-C Calsepa, ConA, GNA, GSII, HAA, HHL, HP A, LCA, LEL, NPA, PSA, RCA-I, RSL, STL and WGA.

174. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise one or more of: AAL, AIA, Calsepa, GNA, HHL, LCA, NPA, PSA and RCA-I.

175. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise one or more of: AAL, AIA, BC2L-C, Calsepa, ConA, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL and WGA.

176. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise AAL, AIA, Calsepa, GNA, HHL, LCA, NPA, PSA and RCA-I.

177. The method of any one of embodiments 139 to 153, wherein the one or more lectins comprise AAL, AIA, BC2L-C, Calsepa, ConA, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL and WGA.

178. The method of any one of embodiments 139 to 177, wherein the lectins are immobilized on a surface.

179. The method of embodiment 178, wherein the surface is a microchip.

180. The method of embodiment 179, wherein the microchip comprises a microarray.

181. The method of embodiment 178, wherein the surface is a sensorchip.

182. The method of any one of embodiments 139 to 177, wherein the bacterium or the mEV is immobilized on a surface.

183. The method of embodiment 182, wherein the surface is a microchip.

184. The method of embodiment 183, wherein the microchip comprises a microarray.

185. The method of embodiment 182, wherein the surface is a sensorchip.

186. The method of any one of embodiments 139 to 185, wherein the bacterium or the mEV is detectably labeled.

187. The method of any one of embodiments 139 to 185, wherein the lectin is detectably labeled. 188. The method of any one of embodiments 139 to 186, wherein the bacterium or the mEV is detectably labeled prior to step (a).

189. The method of embodiment 188, wherein the bacterium or the mEV is detectably labeled with a fluorescent moiety.

190. The method of embodiment 189, wherein the fluorescent moiety is AlexaFluor555.

191. The method of any one of embodiments 139 to 186, wherein the bacterium or the mEV is detectably labeled after step (a) and prior to step (b).

192. The method of embodiment 191, wherein the bacterium or the mEV is labeled by contacting the bacterium or the mEV bound to one or more lectins first with a biotinylated monoclonal antibody specific for the bacterium or the mEV followed by a streptavidin-Cy3.

193. The method of embodiment 191, wherein the bacterium or the mEV is labeled by contacting the bacteria or the mEV bound to one or more lectins first with a rabbit polyclonal antibody specific for the bacterium or the mEV followed by an anti-(rabbit) IgG labeled with Cy3.

194. The method of any one of embodiments 139 to 185 or 187, wherein the lectin is detectably labeled prior to step (a).

195. The method of embodiment 194, wherein the lectin is detectably labeled with a fluorescent moiety.

196. The method of embodiment 195, wherein the fluorescent moiety is AlexaFluor555.

197. The method of any one of embodiments 139 to 185 or 187, wherein the lectin is detectably labeled after step (a) and prior to step (b).

198. The method of any one of embodiments any one of embodiments 139 to 197, wherein the binding in step (b) is detected with a fluorescence scanner.

199. The method of any one of embodiments 1 to 198, wherein the mEV is a secreted mEV (smEV).

200. The method of any one of embodiments 1 to 198, wherein the mEV is a processed mEV (prnEV).

201. The method of any one of embodiments 1 to 200, wherein the bacterium or mEV is of the genus Prevotella.

202. The method of any one of embodiments 1 to 200, wherein the bacterium or mEV is of the species Prevotella histicola. 203. The method of any one of embodiments 1 to 200, wherein the bacterium or mEV is of the species Prevotella histicola and the monoclonal antibody comprises 17B01-02G10 or 02F09-02B04.

204. The method of embodiment 203, wherein the monoclonal antibody is 17B01-02G10.

205. The method of embodiment 203, wherein the monoclonal antibody is 02F09-02B04.

206. The method of any one of embodiments 1 to 200, wherein the bacterium or mEV is of the strain Prevotella Strain B 50329 (NRRL accession number B 50329).

207. The method of any one of embodiments 1 to 200, wherein the bacterium or mEV is of the strain Prevotella histicola ATCC designation number PTA-126140.

208. The method of any one of embodiments 1 to 200, wherein the bacterium or mEV is of the genus Fournierella.

209. The method of any one of embodiments 1 to 200, wherein the bacterium or mEV is of the species Fournierella massiliensis.

210. The method of any one of embodiments 1 to 200, wherein the bacterium or mEV is of the strain is Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696).

211. The method of any one of embodiments 1 to 200, wherein the bacterium or mEV is of the genus Harryflintia.

212. The method of any one of embodiments 1 to 200, wherein the bacterium or mEV is of the species Harryflintia acetispora.

213. The method of any one of embodiments 1 to 200, wherein the bacterium or mEV is of the strain is Harryflintia acetispora Strain A (ATCC Deposit Number PTA-126694).

214. The method of any one of embodiments 1 to 200, wherein the bacterium or mEV is of the genus Veillonella.

215. The method of any one of embodiments 1 to 200, wherein the bacterium or mEV is of the species Veillonella parvula.

216. The method of any one of embodiments 1 to 200, wherein the bacterium or mEV is of the strain is Veillonella parvula Strain A (ATCC Accession Number PTA-125691).

217. The method of any one of embodiments 1 to 200, wherein the bacterium or mEV is of the genus Subdoligranulum.

218. The method of any one of embodiments 1 to 200, wherein the bacterium or mEV is of the species Subdoligranulum variabile. 219. A composition comprising a Prevotella histicola mEV, the composition exhibiting a fluorescence lectin-binding profile to: BPL, RSL, VVL and WGA, wherein the lectin- binding profile comprises: stronger relative fluorescence intensities of binding to BPL and WGA; and weaker relative fluorescence intensities of binding to RSL and VVL, and wherein the intensities of binding to BPL and WGA are the dominant peaks of the fluorescence lectin-binding profile.

220. A composition comprising a Fournierella massiliensis mEV, the composition exhibiting a fluorescence lectin-binding profile to: AIA, BPL, ECL, GSLB4, HP A, Morniga G, SBA, VVL, and WGA, wherein the lectin-binding profile comprises: stronger relative fluorescence intensities of binding to AIA, GSLB3, Morniga G and VVL; and weaker relative fluorescence intensities of binding to BPL, ECL, HP A, SBA and WGA, and wherein the intensities of binding to AIA, GSLB4, and Morniga G are the dominant peaks of the fluorescence lectin-binding profile.

221. A composition comprising a Harryflintia acetispora mEV, the composition exhibiting a fluorescence lectin-binding profile to: AAL, AIA, BPL, ECL, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL, SBA, VVL and WFA, wherein the lectin- binding profile comprises: stronger relative fluorescence intensities of binding to AIA, GNA, GSLB4, HHL, Morniga G, NPA and RCA-I; and weaker relative fluorescence intensities of binding to AAL, BPL, ECL, HP A, LCA, PSA, RSL, SBA, VVL and WFA, and wherein the intensities of binding to AIA and Morniga G are the dominant peaks of the fluorescence lectin-binding profile.

222. A composition comprising a Veillonella parvula mEV, the composition exhibiting a fluorescence lectin-binding profile to: AAL, BC2L-C Calsepa, ConA, GNA, GSII, HAA, HHL, HP A, LCA, LEL, NPA, PSA, RCA-I, RSL, STL and WGA, wherein the lectin- binding profile comprises: stronger relative fluorescence intensities of binding to GNA, HHL, HP A, LCA, NPA, PSA, and RSL; and weaker relative fluorescence intensities of binding to AAL, BC2L-C, Calsepa, ConA, GSII, HAA, LEL, RCA-I, STL and WGA, and wherein the intensity of binding to HPA is the dominant peak of the fluorescence lectin- binding profile.

223. A composition comprising a Subdohgranuliim variabile mEV, the composition exhibiting a fluorescence lectin-binding profile to: AAL, AIA, BC2L-C, Calsepa, ConA, GNA, GSLB4, HHL, HPA, LCA, Morniga G, NPA, PSA, RCA-I, RSL and WGA, wherein the lectin-binding profile comprises: stronger relative fluorescence intensities of binding to AAL, AIA, Calsepa, GNA, HHL, LCA, NPA, PSA and RCA-I; and weaker relative fluorescence intensities of binding to BC2L-C, GSLB4, HPA, Morniga G and WGA, and wherein the profile also shows relative fluorescence intensities of binding to Con A and RSL.

224. A lectin panel, wherein the panel comprises one or more lectins in Table 6.

225. A lectin panel, wherein the panel comprises AAL, ACL, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HPA, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA.

226. A lectin panel, wherein the panel comprises AAL, SJA, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HPA, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA.

227. A lectin panel, wherein the panel comprises BanLec.

228. A lectin panel, wherein the panel comprises CSL3.

229. A lectin panel, wherein the panel comprises one or more of: BPL and WGA.

230. A lectin panel, wherein the panel comprises one or more of: BPL, RSL, VVL and WGA.

231. A lectin panel, wherein the panel comprises BPL and WGA.

232. A lectin panel, wherein the panel comprises BPL, RSL, VVL and WGA.

233. A lectin panel, wherein the panel comprises one or more of: AIA, GSLB3, Morniga G and VVL. 234. A lectin panel, wherein the panel comprises one or more of: AIA, BPL, ECL, GSLB4, HP A, Morniga G, SB A, VVL and WGA.

235. A lectin panel, wherein the panel comprises AIA, GSLB3, Morniga G and VVL.

236. A lectin panel, wherein the panel comprises AIA, BPL, ECL, GSLB4, HP A, Morniga G, SB A, VVL and WGA.

237. A lectin panel, wherein the panel comprises one or more of: AIA, GNA, GSLB4, HHL, Morniga G, NPA and RCA-I.

238. A lectin panel, wherein the panel comprises comprises one or more of: AAL, AIA, BPL, ECL, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL, SBA, VVL and WFA.

239. A lectin panel, wherein the panel comprises AIA, GNA, GSLB4, HHL, Morniga G, NPA and RCA-I.

240. A lectin panel, wherein the panel comprises AAL, AIA, BPL, ECL, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL, SBA, VVL and WFA.

241. A lectin panel, wherein the panel comprises one or more of: GNA, HHL, HP A, LCA, NPA, PSA, and RSL.

242. A lectin panel, wherein the panel comprises one or more of: AAL, BC2L-C Calsepa, ConA, GNA, GSII, HAA, HHL, HP A, LCA, LEL, NPA, PSA, RCA-I, RSL, STL and WGA.

243. A lectin panel, wherein the panel comprises GNA, HHL, HP A, LCA, NPA, PSA, and RSL.

244. A lectin panel, wherein the panel comprises AAL, BC2L-C Calsepa, ConA, GNA, GSII, HAA, HHL, HP A, LCA, LEL, NPA, PSA, RCA-I, RSL, STL and WGA.

245. A lectin panel, wherein the panel comprises one or more of: AAL, AIA, Calsepa, GNA, HHL, LCA, NPA, PSA and RCA-I.

246. A lectin panel, wherein the panel comprises one or more of: AAL, AIA, BC2L-C, Calsepa, ConA, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL and WGA.

247. A lectin panel, wherein the panel comprises AAL, AIA, Calsepa, GNA, HHL, LCA, NPA, PSA and RCA-I.

248. A lectin panel, wherein the panel comprises AAL, AIA, BC2L-C, Calsepa, ConA, GNA, GSLB4, HHL, HP A, LCA, Morniga G, NPA, PSA, RCA-I, RSL and WGA. 249. The lectin panel of any one of embodiments 224 to 248, wherein more than 50% of the non-human lectins are plant lectins.

250. The lectin panel of any one of embodiments 224 to 248, wherein more than 75% of the non-human lectins are plant lectins.

251. The lectin panel of any one of embodiments 224 to 248, wherein more than 85% of the non-human lectins are plant lectins.

252. The lectin panel of any one of embodiments 224 to 248, wherein more than 90% of the non-human lectins are plant lectins.

253. The lectin panel of any one of embodiments 224 to 248, wherein more than 95% of the non-human lectins are plant lectins.

254. The lectin panel of any one of embodiments 224 to 248, wherein all of the non-human lectins are plant lectins.

255. The lectin panel of any one of embodiments 224 to 254, wherein the panel comprises up to 45 lectins.

256. A method of analyzing bacteria or mEVs in a sample, the method comprising:

(a) contacting the sample comprising the bacteria or the mEVs with the lectin panel of any one of embodiments 224 to 255; and

(b) detecting the binding of the bacteria or the mEVs to one or more lectins in the lectin panel, thereby analyzing the bacteria or the mEVs in the sample.

257. A method of analyzing bacteria or mEVs in a sample, the method comprising:

(a) contacting the sample comprising the bacteria or the mEVs with one or more lectins; and

(b) detecting the binding of the bacteria or the mEVs to one or more lectins, thereby analyzing the bacteria or the mEV in the sample.

EXAMPLES

Example 1: Materials

Generation of polyclonal antibodies

[0395] Four rabbits (New Zealand white) were immunized periodically with a batch of F. massiliensis mEVs (gradient purified mEVs) in Complete Freund’s Adjuvant (CFA) using standard procedures. Serum was collected at each immunization (Day 0, Day 35, Day 56/58). Serum was also collected on Day 90. To enrich the specificity of the serum, the serum from Day 90 (Terminal serum) was used to generate purified IgG, using Protein A purification method. Polyclonal antibodies to H. acetispora mEVs were also generated in New Zealand white rabbits.

Generation of monoclonal antibodies

[0396] Monoclonal antibodies were generated against mEVs of P. histicola 1. Balbc mice were immunized with gradient purified P. histicola 1 mEVs using an optimized Rapid Immunization Protocol (RIMMS). Splenocytes were harvested from mice that showed high polyclonal antibody serum titers. Splenic B cells were fused with myeloma cells and grown in a selection medium (ultralow bovine immunoglobin FBS containing media). The supernatant collected from the fused cells was tested for specificity to /< histicola 1 mEVs and other closely related strain and genus mEVs to choose mixed clones (multi pure) specific to P. histicola 1 mEVs for further subcloning. Dilution cloning of the mixed clones generated two clones (17B01-02G10 clone and 02F09-02B04 clone) which were expanded for mAb isolation and purification.

[0397] The monoclonal antibodies (e.g., 17B01-02G10 clone and 02F09-02B04 clone) to /< histicola 1 are further described in U.S. Provisional Application serial number 63/286,934 and PCT Publication No. WO 2023/107537, hereby incorporated by reference in their entireties.

Example 2: Lectin Microarray

[0398] Commercially available lectin microarrays displaying 39 non-human lectins, the majority being plant lectins, were assayed. The lectins used represent most carbohydrate-binding epitopes and were characterized on glycan arrays before immobilization on the chip to confirm lectin specificity. In some experiments, we selected two additional lectins, Recombinant Oncorhynchus keta L-rhamnose-binding lectin (CSL3) and Musa Paradisiaca (Banana) Lectin (BanLec) for their specificity toward monosaccharide structures potentially present in bacterial preparations (L-rhamnose for CSL3; internal a(l,3) glucosyl- and mannosyl- residues or [3(1,3) and [3(1,6) glucosyl- structures for BanLec). These two lectins were added to the microarray and tested alongside the traditional panel of 39 lectins. The lectins immobilized on the microarray in the examples below, in various combinations, are reported in Table 6.

[0399] Table 6: Lectins immobilized on the microarray

Example 3: Lectin Microarray Analysis

[0400] Lectin microarrays were produced and commercialized by Z Biotech, LLC (12635 E. Montview Blvd., suite 214, Aurora, CO 80045, www.zbiotech.com). Each lectin microarray chip contained 16 subarrays and each array (a subarray on the chip) was functionalized with 39-41 lectins so that 16 samples could be assayed simultaneously.

[0401] Figures 1A and IB show an example of how 39 lectins are laid out in an array and the IDs (identities) of each of the lectins in the array. Figure 1A shows the layout of the array: each lectin (NL1 to NL39) is in quadruplicate (repeated in 4 wells); NC is a negative control printed buffer; PCI is a positive control (e.g., a biotinylated probe); and M is a marker. Figure IB shows the IDs of the lectins corresponding to each of NL1 to NL39. [0402] Single strains of bacteria or mEVs preparations were suspended in PBS buffer and analyzed by Z Biotech according to the User Manual posted on the company’s website

(nebula, wsimg.com/65e5934c8a5 ca0003281ecf9e9c5dd9f?AccessKeyId=B5CD53DB3740 9833427C&disposition=0&alloworigin=l), hereby incorporated by reference in its entirety. [0403] smEVs were used in these studies.

[0404] Before analysis, bacteria and mEVs were diluted to a concentration of 50-5 pg protein/ml and 10-0.5 pg protein/ml, respectively. Binding of bacteria and mEVs to the lectin microarray was detected (a) by incubation with biotinylated mouse monoclonal antibodies specifically raised against the bacteria or mEVs followed by incubation with streptavidin-Cy3; (b) by incubation with rabbit polyclonal antibodies specifically raised against the bacteria or mEVs followed by incubation with anti-(rabbit) IgG labeled with Cy3; or (c) by labeling bacteria or mEVs with the fluorescent moiety AlexaFluor555 (AF555).

[0405] An example 16-subarray assay layout on a lectin microarray chip is shown in Figure 2. Each box in Figure 2 corresponds to an array as shown in Figure 1A. In Figure 2, Step 1 shows sample lots of test material (e.g., EVI-29, EV2-18 and EV3-24 representing bacteria or mEVs; EV4-16-AF555 representing bacteria or mEVs labeled with AF555) or control added to each array. Step 2 shows antibodies added to certain arrays. Step 3 shows streptavidin-Cy3 or anti-(rabbit) IgG labeled with Cy3 added to certain arrays. Rows F-H serve as assay controls. [0406] The arrays were scanned at 532 nm wavelength with a microarray scanner. To control for non-specific binding, background values from assay control wells were subtracted from the sample values before data analysis and graphing.

Example 4: Selective Binding of Bacteria and mEVs to a Panel of Non-Human Lectins Immobilized on a Microarray

[0407] Following the procedure set out in Example 3, above, bacterial biomass and mEVs from P. histicola 1 were profiled with a lectin microarray. Bacteria and mEVs bound to the lectin microarray were detected with monoclonal antibodies raised against P. histicola 1 mEVs (monoclonal antibody 17B01-02G10), followed by incubation with streptavidin-Cy3. The x-axes of Figures 3A and 3B show the 41 lectins used in these arrays (see also Example 2, Table 6, above). The arrays were scanned at 532 nm wavelength.

[0408] smEVs were used in these studies.

[0409] As shown in Figure 3A, the array showed binding of P. histicola 1 bacterial biomass to lectins: AAL, BC2LC, BPL, RSL, and WGA. As shown in Figure 3B, P. histicola 1 mEVs showed binding to lectins: BPL, RSL, VVL and WGA. Comparison of Figure 3A and Figure 3B shows different relative signal intensities of binding to lectins: BPL, RSL and WGA. The data show that P. histicola 1 bacteria mainly present fucosylated structures, while P. histicola 1 mEVs are richer in glycans containing V-acetylated glucosamine or terminal galactose/V-acetylated galactosamine.

Example 5: Identification of smEVs in Bacterial Preparations with Lectin Microarray [0410] Following the procedure set out in Example 3, above, bacterial biomass from P. histicola 1 was analyzed on a lectin microarray. The x-axes of Figures 4A and4B show the 41 lectins used in the array (see also Example 2, Table 6, above). The array was scanned at 532 nm wavelength. The results of the bacterial biomass from P. histicola 1 are shown in Figure 4A.

[0411] As bacterial biomass contains smEVs generated during the fermentation process, bacteria were washed in PBS buffer and recovered by centrifugation to eliminate most of the smEVs. This procedure was repeated three times and the washed bacteria were reanalyzed by lectin microarray. Washed bacteria were detected with monoclonal antibodies raised against P. histicola 1 mEVs (monoclonal antibody 17B01-02G10) followed by incubation with streptavidin-Cy3. The array was scanned at 532 nm wavelength. The results of the washed bacteria are shown in Figure 4B.

[0412] As seen in Figure 4B, binding of bacteria to LEL was uncovered after washing the bacterial biomass extensively with PBS buffer. Washed microbes show a decrease in signals (e.g., BPL and WGA) previously identified in mEVs preparation from P. histicola 1 (see Figure 3B), indicating a different glycosylation pattern for smEVs versus microbes. This phenomenon can be due to preferential enrichment of selected glycan structures during smEVs formation.

Example 6: Quantification of smEVs in Bacterial Preparations by Lectin Microarrays [0413] Prior to and after each successive wash of the bacterial biomass in Example 5, above, measure the particle count of smEVs in the biomass by NTA (nanoparticle tracking analysis). Analyze the bacterial biomass from P. histicola 1 and each wash on a lectin microarray following the procedure set out in Example 3, above. Correlate the particle counts with change in lectin signal relative intensities of binding to quantify the amount of smEVs present in a sample.

Example 7: Differentiate mEVs from Different Bacterial Strains by Lectin Microarrays

[0414] mEVs from massiliensis, H. acetispora, V. parvula, S. variabile and P. histicola 1 and 2, two different strains of Prevotella histicola, were analyzed by lectin microarray as described in Example 3, above. F. massiliensis and H. acetispora mEVs were detected with polyclonal antibodies raised against each of the bacterial mEVs followed by incubation with anti-(rabbit) IgG labeled with Cy3. V. parvula and . variabile mEVs were labeled with fluorescent moiety AlexaFluor555 (AF555). P. histicola 1 and 2 mEVs were detected with monoclonal antibodies raised against P. histicola 1 mEVs (monoclonal antibody 17B01-02G10) followed by incubation with streptavidin-Cy3. The arrays were scanned at 532 nm wavelength.

[0415] smEVs were used in these studies.

[0416] The results are shown in Figures 5A-5F. The x-axes of Figures 5A-5E show the 39 lectins used in these arrays (see also Example 2, Table 6, above). Figure 5F shows the 41 lectins used in this array (see also Example 2, Table 6, above). Figure 5A shows the binding profile of P. histicola 1 mEVs. Figure 5B shows the binding profile of F. massiliensis mEVs. Figure 5C shows the binding profile of H. acetispora mEVs. Figure 5D shows the binding profile of V. parvula mEVs. Figure 5E shows the binding profile of S’, vanabile mEVs. Figure 5F shows the binding profile of P. histicola 2 mEVs. The lectins of Figure 5E are in the same order as NL1-NL39 of Figure 5F: AAL, ACL/ACA, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SB A, SNA, STL, UEA-I, VVL, WFA and WGA.

[0417] Figures 6A-6D show the visual results of the lectin microarray assays.

Figure 6A shows the assay result of P. histicola 1 mEVs. Figure 6B shows the assay result of F. massiliensis mEVs. Figure 6C shows the assay result of H. acetispora mEVs. Figure 6D shows the assay result of V. parvula mEVs. Each type of bacterial mEVs shows a unique lectin binding pattern (e.g., a lectin-binding profile).

[0418] The lectin binding affinities for the mEVs analyzed in this study showed significant differences, providing a strain-specific fingerprint of the glycosidic profiles that can be employed as an ID test to identify mEVs of different origin. Moreover, the unique pattern results of the assay may provide a quick visual confirmation of the type of mEVs in a sample.

Example 8: Generating Lectin-Binding Signatures by Applying Multivariable Analysis

[0419] Using the data in Example 7, above, in which lectin-binding across multiple samples (A to F, corresponding to the mEVs identified: P. histicola 1, F. massiliensis, H. acetispora, V. parvula, S. variabile, and P. histicola 2, respectively) were examined, we applied inverse hyperbolic (Arsinh) transformation and Z-score transformation to the lectin- binding values for each type of mEV sample (A-F). Arsinh approximates the natural logarithm (log) but allows the retention of zero-valued observations which are not possible with log-transformations, making it easier to approximate a normal distribution and reduce heteroskedasti city .

[0420] More importantly, Arsinh transformation provides a more comprehensive representation of lectin-binding signals by not only capturing dominant signals but also “up-weighting” the contribution of less-dominant ones, facilitating better resolution or definition between samples, especially closely clustered mEVs: (A and F), (B and C) and (D and E).

[0421] Thus, by analyzing both non-Arsinh and Arsinh-transformed data, we were able to not only identify closely related mEVs and bacteria, but also obtain a finer-grain distinguishing feature set (comprising lectins that bind) to stratify samples of closely related mEVs and bacteria.

[0422] Figures 7A and7B show multivariable analysis data of samples A to F in different concentrations; A to F corresponding to the mEVs identified in Example 7, above: (A) P. histicola 1, (B) F. massiliensis. (C) H. acelispora. (D) V. parvula, (E) S. variabile, and (F) P. histicola 2. Figure 7A shows dimensionality reduction plots and heat maps from multivariable analysis with non-Arsinh transformed data and reveals that samples binding to lectins WGA (NL39) and BPL (NL5) can identify mEV samples A and F from the others (B, C, D and E). Figure 7B shows dimensionality reduction plots and heat maps from multivariable analysis with Arsinh-transformed data and shows that additional differential lectin-binding by RSL (NL32), although weaker, constitute an important extended lectin- binding profile that can further distinguish between closely related mEVs A and F. This is made possible with Arsinh transformation.

Example 9: Effects of Fermentation Conditions on Bacteria Glycosylation Profiles [0423] Two lots of P. histicola 1 bacteria were grown under different conditions, i.e., the media used during bacterial growth were either enriched or depleted of components such as Tween, porcine hemoglobin, and/or spirulina. The lots were then analyzed by the lectin microarrays as described in Example 3, above. All the samples were diluted to approximately 5 pg protein/ml. Bacteria were detected with antibodies raised against P. histicola 1 mEVs (monoclonal antibody 17B01-02G10) followed by incubation with streptavidin-Cy3. The arrays were scanned at 532 nm wavelength.

[0424] Figure 8A shows the results of a fermentation lot grown with porcine hemoglobin. Figure 8B shows the results of a fermentation lot grown with spirulina. The x- axes of Figures 8A and 8B show the 41 lectins used in these arrays (see also Example 2, Table 6, above). Comparison of the results in Figures 8A and 8B show that changes in the fermentation components had minimal effect on the binding profiles. Changes in signal relative intensities of binding could be further analyzed to determine the relative ratio of the identified glycan motifs.

Example 10: Monitoring Lot-to-Lot Consistency Using Lectin Microarray

[0425] The lectin microarray described in Example 2 was assayed with three lots of mEVs from P. histicola 1 generated under the same manufacturing conditions and with the same isolation procedure (i.e., fermentation retentate, before addition of excipients). All the samples were diluted to approximately 50 pg protein/ml. mEVs were detected with antibodies raised against P. histicola 1 mEVs (monoclonal antibody 17B01-02G10) followed by incubation with streptavidin-Cy3. The arrays were scanned at 532 nm wavelength. smEVs were used in these studies.

[0426] Results are shown in Figures 9A-9C. The x-axes of Figures 9A-9C show the 39 lectins used in these arrays (see also Example 2, Table 6, above): AAL, ACA, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LTA, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SB A, SNA, STL, UEA-I, VVL, WFA and WGA. The binding events for three lots of mEVs from P. histicola 1 (Figures 9A-9C, respectively) are similar across the analyzed lots, showing stronger relative intensities of binding with BPL and WGA and weaker relative intensities of binding with ACA, RSL, and VVL. This confirms that the manufacturing process generates samples with consistent glycosidic profiles and the lectin microarray may be used to monitor lot-to-lot consistency.

Example 11: Binding Profiles of Formulated mEVs

[0427] The lectin-binding profile of mEVs from P. histicola 1 with different components: (A) without excipients, (B) containing 95% w/w of excipients and (C) containing 99.5% w/w of excipients were compared using the lectin microarray. Compositions (A) and (B), without excipients and containing 95% w/w of excipients respectively, were from the same lot of mEVs. Composition (C), containing 99.5% w/w of excipients, was from a different lot mEVs. Excipient stock solution comprising 20% trehalose and 80% mannitol were added to compositions (B) and (C) to form a low dose 95% w/w of excipients composition (B) and a high dose 99.5% w/w of excipients composition (C) drug substance (DS).

[0428] All the samples were diluted to approximately 50 pg protein/ml. mEVs were detected with antibodies raised against P. histicola 1 mEVs (monoclonal antibody 17B01- 02G10) followed by incubation with streptavidin-Cy3. The arrays were scanned at 532 nm wavelength. smEVs were used in these studies.

[0429] Results are shown in Figures 10A-10C. The x-axes of Figures 10A and 10B show the 39 lectins used in these arrays (see also Example 2, Table 6, above). The x- axes of Figure 10C shows the 40 lectins used in this array (see also Example 2, Table 6, above). Lectins NL1-NL39 are the same in Figures 10A-10C and are: AAL, ACL/ACA, AIA, BC2L-C, BPL, Calsepa, ConA, DBA, DSA, ECL, EEA, GNA, GSLB4, GSII, HAA, HHL, HP A, LCA, LEL, LT A, MAL-I, MAL-II, Morniga G, NPA, PHA-E, PHA-L, PNA, PSA, PTA, RCA-I, RPA, RSL, SBA, SNA, STL, UEA-I, VVL, WFA and WGA. The results show that the presence of excipient did not prevent mEVs from binding to the immobilized lectins, allowing direct analysis of formulated drug substance and drug products.

Example 12: Binding of Non-Human Lectins to EVs Immobilized on a Microarray [0430] The method of the present Example utilizes printing of EV preparations on a microarray to achieve immobilization, followed by incubation with non-human lectins. After washing, non-human lectins bound to the EVs are detected using fluorescently labeled primary or secondary antibodies. Arrays are then scanned at 532nm wavelength with a microarray scanner to measure fluorescence and detect lectins bound to the EVs. [0431] EVs microarrays are generated. Solutions containing 1E12 p/ml (particles per milliliter) of EVs are printed on a chip containing 16 subarrays. EVs from Prevotella histicola, Prevotella melaninogenica, and Prevotella jejuni are used. The EVs immobilized on the subarrays are incubated with commercially available non-human lectins (such as one or more lectins provided in Table 6), diluted at two different concentrations (10-0.1 pg/ml). Lectins are tagged with a tag such as an Fc or His tag that can be detected with an antibody specific for the tag. After washing, Fc-tagged lectins are detected by incubation with anti- Fc Cy3 antibody. His-tagged lectins are incubated with anti-His antibodies, followed by incubation with anti-sheep, anti-goat, or anti-rabbit IgG Cy3. smEVs are used in these studies.

[0432] The arrays are then scanned at 532nm wavelength with a microarray scanner. To control for non-specific binding, background values from antibody binding to control wells are subtracted from the sample values before data analysis and graphing. Binding profiles for EVs from Prevotella histicola, Prevotella melaninogenica, and Prevotella jejuni are reported.

Example 13: SPR Method to Measure Binding of EVs to Non-Human Lectins [0433] An SPR method is designed to measure affinity of non-human lectins for EVs preparations from Prevotella histicola. The method uses the phenomenon of surface plasmon resonance (SPR) to monitor interactions between a ligand immobilized on the surface of a sensor chip and an analyte in solution passing over the sensor chip. The analysis allows real-time, label -free monitoring of binding phenomena and affinity between the ligand and the analyte.

[0434] Experiment 1 : The method of the present Example is used to measure interactions of selected non-human lectins (such as one or more lectins provided in Table 6) immobilized on a sensorchip with EV preparations from Prevotella histicola injected across the lectin surface. EVs from Harryflintia acelispora. Veillonella parvula, and Subdoligranulum variabile are also tested at the same concentrations. smEVs are used in these studies.

[0435] Three different lots of EVs from Prevotella histicola are analyzed at the same concentration. The sensogram profiles can be compared across lots to establish if the lots have different activities toward the analyzed lectins.

[0436] Experiments are carried out on a Biacore 8k instrument. The surface of a Series S CM5 or XanTec CMD200L sensor chip is derivatized using an antibody capture kit. The anti-IgG (Fc) antibody is diluted to 12.5 pg/ml in sodium acetate pH 5.0 and immobilized using the amine coupling kit reagents according to the manufacturer suggestions. The anti-IgG (Fc) is injected for 360 sec, at 10 pl/min flow rate in the running buffer lOmM Hepes pH 7.4, 150mN NaCl.

[0437] Fc-tagged lectins are diluted in lOmM Hepes pH 7.4, 150mN NaCl to a 50 pM concentration and injected for 240s at lOul/min to reach an immobilization level of approximately 1,000-2,500 RU.

[0438] Analytes (EVs) are diluted in 25mM Tris pH 7.5, 150mM NaCl, 4mM CaCh at 5 different concentrations starting from 5E10 p/ml followed by 2-fold or 5-fold serial dilution and injected over the immobilized lectins at a flow rate of 30uL/min for a contact time of 30s, followed by 300s or 450s of dissociation time.

[0439] The running buffer is in 25mM Tris pH 7.5, 150mM NaCl, 4mM CaCh, 0.05% surfactant P20.

[0440] Surface regeneration is performed with 25mM Tris pH 8.0, 150mM NaCl, 50mM EDTA injected at a flow rate of 30uL/min for 60s of contact time, followed by 30 sec of stabilization time. A line wash and a carry over control step are included between each cycle. [0441] In all the presented data, sensograms from the reference cell are subtracted from sensograms from the flow cell to correct for non-specific binding.

[0442] Experiment 2: Experiments are carried out on a Biacore 8k instrument. One or more non-human lectins are immobilized on the surface of a Series S CM5 or XanTec CMD200L sensor chip via amine coupling according to manufacturer instructions to reach an immobilization level of approximately 4,000 RU.

[0443] For the immobilization procedure, lectins are diluted in lOmM Hepes pH

7.4, 150mN NaCl to a 50 pM concentration and injected on the surface for 360s at lOul/min to reach an immobilization level of approximately 1,000-2,500 RU.

[0444] Analytes (EVs) are diluted in 25mM Tris pH 7.5, 150mM NaCl, 4mM CaCh at 5 different concentrations starting from 5E10 p/ml followed by 2-fold or 5-fold serial dilution and injected over the immobilized lectins at a flow rate of 30uL/min for a contact time of 30s, followed by 300s or 450s of dissociation time.

[0445] The running buffer is in 25mM Tris pH 7.5, 150mM NaCl, 4mM CaCh, 0.05% surfactant P20.

[0446] Surface regeneration is performed with 25mM Tris pH 8.0, 150mM NaCl, 50mM EDTA injected at a flow rate of 30uL/min for 60s of contact time, followed by 30 sec of stabilization time. A line wash and a carry over control step are included between each cycle.

[0447] In all the presented data, sensograms from the reference cell are subtracted from sensograms from the flow cell to correct for non-specific binding.

Incorporation by reference

[0448] All publications, patents, patent applications and sequence accession numbers mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Equivalents

[0449] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:

1. A method of analyzing bacteria or mEVs in a sample, the method comprising:

(b) detecting the binding of the bacteria or the mEVs to one or more of the lectins, thereby analyzing the bacteria or the mEV in the sample.

2. A method of identifying a bacterium or a microbial extracellular vesicle (mEV) in a sample, the method comprising:

(b) detecting the binding of the bacterium or the mEV to one or more of the lectins, thereby identifying the bacteria or the mEV in the sample.

3. A method of determining the presence of (e.g., detecting) a bacterium or a microbial extracellular vesicle (mEV) in a sample, the method comprising:

(b) detecting the binding of the bacterium or the mEV to one or more of the lectins , thereby determining the presence of (e.g., detecting) the bacterium or the mEV in the sample.

4. A method of detecting a glycosidic profile of a bacterium or a microbial extracellular vesicle (mEV) in a sample, the method comprising:

(a) contacting the bacterium or the mEV with one or more lectins; and

(b) detecting the binding of the bacteria or the mEV to one or more of the lectins, thereby detecting the glycosidic profile of the bacteria or the mEV.

5. A method of monitoring a change in a glycosidic profile of a first sample and a second sample each comprising a bacterium or a microbial extracellular vesicle (mEV), the method comprising:

(b) detecting the binding of the bacterium or the mEV in the first sample and the second sample to one or more of the lectins; and (c) comparing the lectin-binding profiles of the bacterium or the mEV in the first sample and the second sample, thereby monitoring the change in the glycosidic profile of the first sample and the second sample.

6. A method of detecting a lectin-binding profile of a sample comprising a bacterium or a microbial extracellular vesicle (mEV), the method comprising:

(a) contacting the bacterium or the mEV with one or more lectins; and

(b) detecting the binding of the bacterium or the mEV to one or more of the lectins, thereby detecting the lectin-binding profile of the sample.

7. A method of monitoring a change in a lectin-binding profile in a first sample and a second sample each comprising a bacterium or a microbial extracellular vesicle (mEV), the method comprising:

(b) detecting the binding of the bacteria or the mEV in the first sample and the second sample to one or more of the lectins; and

(c) comparing the lectin-binding profiles of the bacterium or the mEV in the first sample and the second sample, thereby monitoring the change in the lectin-binding profile of the first sample and the second sample.

8. A method of quantifying the amount of a bacterium or a microbial extracellular vesicle (mEV) in a sample, the method comprising:

(b) detecting the binding of the bacterium or mEV to one or more of the lectins; and

9. The method of any one of claims 1 to 8, wherein the sample or first and second samples comprise an excipient.

10. The method of any one of claims 1 to 9, wherein the one or more lectins comprise nonhuman lectins.

11. The method of any one of claims 1 to 10, wherein the non-human lectins comprise one or more of the following: bacterial, plant, fungus, or non-human animal lectins.

12. The method of any one of claims 1 to 11, wherein the one or more lectins comprise 1 to 45 lectins.

13. The method of any one of claims 1 to 12, wherein the one or more lectins comprise one or more of the lectins listed in Table 6.

14. The method of any one of claims 1 to 13, wherein the one or more lectins are immobilized on a surface.

15. The method of any one of claims 1 to 13, wherein the bacterium or the mEV is immobilized on a surface.

16. The method of any one of claims 1 to 15, wherein the bacterium or the mEV is detectably labeled.

17. The method of any one of claims 1 to 15, wherein the lectin is detectably labeled.

18. The method of any one of claims 1 to 17, wherein the bacterium or mEV is of the genus Prevotella.

19. The method of any one of claims 1 to 17, wherein the bacterium or mEV is of the genus Fournierella.

20. The method of any one of claims 1 to 17, wherein the bacterium or mEV is of the genus Harryflintia.

21. The method of any one of claims 1 to 17, wherein the bacterium or mEV is of the genus Veillonella.

22. The method of any one of claims 1 to 17, wherein the bacterium or mEV is of the genus Subdoligranulum.

23. A lectin panel, wherein the panel comprises one or more lectins in Table 6.

24. The lectin panel of claim 23, wherein the one or more lectins comprise up to 45 lectins.

25. A method of analyzing bacteria or mEVs in a sample, the method comprising:

(a) contacting the sample comprising the bacteria or the mEVs with the lectin panel of claim 23 or 24; and detecting the binding of the bacteria or the mEVs to one or more of the lectins in the lectin panel, thereby analyzing the bacteria or the mEVs in the sample.