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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230629	2/9	Haemophilus influenzae	Rd KW20	(d)(U)C DG Filtration	Roier S	2015	56%
Study summary Full title A basis for vaccine development: Comparative characterization of Haemophilus influenzae outer membrane vesicles. All authors Roier S, Blume T, Klug L, Wagner GE, Elhenawy W, Zangger K, Prassl R, Reidl J, Daum G, Feldman MF, Schild S Journal Int J Med Microbiol Abstract Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released fro (show more...)Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Pure outer membrane vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Protein markers EV: None non-EV: RpoA Proteomics yes Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches)/ vaccine development Sample Species Haemophilus influenzae Sample Type Cell culture supernatant EV-producing cells Rd KW20 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 240 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 144000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Protein Yield (µg) per mL/OD490 Western Blot Not detected contaminants RpoA Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field

	EV230629	4/9	Haemophilus influenzae	Hib strain Eagan	(d)(U)C DG Filtration	Roier S	2015	56%
Study summary Full title A basis for vaccine development: Comparative characterization of Haemophilus influenzae outer membrane vesicles. All authors Roier S, Blume T, Klug L, Wagner GE, Elhenawy W, Zangger K, Prassl R, Reidl J, Daum G, Feldman MF, Schild S Journal Int J Med Microbiol Abstract Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released fro (show more...)Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Pure outer membrane vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Protein markers EV: None non-EV: RpoA Proteomics yes Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches)/ vaccine development Sample Species Haemophilus influenzae Sample Type Cell culture supernatant EV-producing cells Hib strain Eagan EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 240 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 144000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Protein Yield (µg) per mL/OD490 Western Blot Not detected contaminants RpoA Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field

	EV230629	6/9	Haemophilus influenzae	NTHi 2019-R	(d)(U)C DG Filtration	Roier S	2015	56%
Study summary Full title A basis for vaccine development: Comparative characterization of Haemophilus influenzae outer membrane vesicles. All authors Roier S, Blume T, Klug L, Wagner GE, Elhenawy W, Zangger K, Prassl R, Reidl J, Daum G, Feldman MF, Schild S Journal Int J Med Microbiol Abstract Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released fro (show more...)Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Pure outer membrane vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Protein markers EV: None non-EV: RpoA Proteomics yes Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches)/ vaccine development Sample Species Haemophilus influenzae Sample Type Cell culture supernatant EV-producing cells NTHi 2019-R EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 240 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 144000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Protein Yield (µg) per mL/OD490 Western Blot Not detected contaminants RpoA Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field

	EV230629	1/9	Haemophilus influenzae	Rd KW20	(d)(U)C Filtration	Roier S	2015	33%
Study summary Full title A basis for vaccine development: Comparative characterization of Haemophilus influenzae outer membrane vesicles. All authors Roier S, Blume T, Klug L, Wagner GE, Elhenawy W, Zangger K, Prassl R, Reidl J, Daum G, Feldman MF, Schild S Journal Int J Med Microbiol Abstract Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released fro (show more...)Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections. (hide) EV-METRIC 33% (74th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Outer membrane vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: None non-EV: RpoA Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches)/ vaccine development Sample Species Haemophilus influenzae Sample Type Cell culture supernatant EV-producing cells Rd KW20 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 240 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 144000 Density gradient Type Discontinuous Number of initial discontinuous layers 8 Lowest density fraction 20% Highest density fraction 50% Total gradient volume, incl. sample (mL) 9.8 + sample Sample volume (mL) not spec Orientation top-down Speed (g) 150000 Duration (min) 1020 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction not spec Pelleting: duration (min) 240 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 144000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per mL/OD490 Western Blot Not detected contaminants RpoA Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 89

	EV230629	3/9	Haemophilus influenzae	Hib strain Eagan	(d)(U)C Filtration	Roier S	2015	33%
Study summary Full title A basis for vaccine development: Comparative characterization of Haemophilus influenzae outer membrane vesicles. All authors Roier S, Blume T, Klug L, Wagner GE, Elhenawy W, Zangger K, Prassl R, Reidl J, Daum G, Feldman MF, Schild S Journal Int J Med Microbiol Abstract Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released fro (show more...)Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections. (hide) EV-METRIC 33% (74th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Outer membrane vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: None non-EV: RpoA Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches)/ vaccine development Sample Species Haemophilus influenzae Sample Type Cell culture supernatant EV-producing cells Hib strain Eagan EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 240 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 144000 Density gradient Type Discontinuous Number of initial discontinuous layers 8 Lowest density fraction 20% Highest density fraction 50% Total gradient volume, incl. sample (mL) 9.8 + sample Sample volume (mL) not spec Orientation top-down Speed (g) 150000 Duration (min) 1020 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction not spec Pelleting: duration (min) 240 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 144000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per mL/OD490 Western Blot Not detected contaminants RpoA Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 152

	EV230629	5/9	Haemophilus influenzae	NTHi 2019-R	(d)(U)C Filtration	Roier S	2015	33%
Study summary Full title A basis for vaccine development: Comparative characterization of Haemophilus influenzae outer membrane vesicles. All authors Roier S, Blume T, Klug L, Wagner GE, Elhenawy W, Zangger K, Prassl R, Reidl J, Daum G, Feldman MF, Schild S Journal Int J Med Microbiol Abstract Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released fro (show more...)Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections. (hide) EV-METRIC 33% (74th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Outer membrane vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: None non-EV: RpoA Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches)/ vaccine development Sample Species Haemophilus influenzae Sample Type Cell culture supernatant EV-producing cells NTHi 2019-R EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 240 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 144000 Density gradient Type Discontinuous Number of initial discontinuous layers 8 Lowest density fraction 20% Highest density fraction 50% Total gradient volume, incl. sample (mL) 9.8 + sample Sample volume (mL) not spec Orientation top-down Speed (g) 150000 Duration (min) 1020 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction not spec Pelleting: duration (min) 240 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 144000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per mL/OD490 Western Blot Not detected contaminants RpoA Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 82

	EV230629	7/9	Haemophilus influenzae	NTHi 1479-R	(d)(U)C Filtration	Roier S	2015	14%
Study summary Full title A basis for vaccine development: Comparative characterization of Haemophilus influenzae outer membrane vesicles. All authors Roier S, Blume T, Klug L, Wagner GE, Elhenawy W, Zangger K, Prassl R, Reidl J, Daum G, Feldman MF, Schild S Journal Int J Med Microbiol Abstract Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released fro (show more...)Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections. (hide) EV-METRIC 14% (44th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Outer membrane vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches)/ vaccine development Sample Species Haemophilus influenzae Sample Type Cell culture supernatant EV-producing cells NTHi 1479-R EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 240 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 144000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Characterization: Protein analysis None Protein Concentration Method Bradford Protein Yield (µg) per mL/OD490 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 99

	EV230629	8/9	Haemophilus influenzae	NTHi 3198-R	(d)(U)C Filtration	Roier S	2015	14%
Study summary Full title A basis for vaccine development: Comparative characterization of Haemophilus influenzae outer membrane vesicles. All authors Roier S, Blume T, Klug L, Wagner GE, Elhenawy W, Zangger K, Prassl R, Reidl J, Daum G, Feldman MF, Schild S Journal Int J Med Microbiol Abstract Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released fro (show more...)Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections. (hide) EV-METRIC 14% (44th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Outer membrane vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches)/ vaccine development Sample Species Haemophilus influenzae Sample Type Cell culture supernatant EV-producing cells NTHi 3198-R EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 240 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 144000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Characterization: Protein analysis None Protein Concentration Method Bradford Protein Yield (µg) per mL/OD490 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 101

	EV230629	9/9	Haemophilus influenzae	NTHi 9274-R	(d)(U)C Filtration	Roier S	2015	14%
Study summary Full title A basis for vaccine development: Comparative characterization of Haemophilus influenzae outer membrane vesicles. All authors Roier S, Blume T, Klug L, Wagner GE, Elhenawy W, Zangger K, Prassl R, Reidl J, Daum G, Feldman MF, Schild S Journal Int J Med Microbiol Abstract Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released fro (show more...)Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections. (hide) EV-METRIC 14% (44th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Outer membrane vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches)/ vaccine development Sample Species Haemophilus influenzae Sample Type Cell culture supernatant EV-producing cells NTHi 9274-R EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 240 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 144000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Characterization: Protein analysis None Protein Concentration Method Bradford Protein Yield (µg) per mL/OD490 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 93

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EV-TRACK ID	EV230629
species	Haemophilus influenzae
sample type	Cell culture
cell type	Rd KW20	Hib strain Eagan	NTHi 2019-R	Rd KW20	Hib strain Eagan	NTHi 2019-R	NTHi 1479-R	NTHi 3198-R	NTHi 9274-R
condition	Control condition	Control condition	Control condition	Control condition	Control condition	Control condition	Control condition	Control condition	Control condition
separation protocol	dUC/ Density gradient/ Filtration	dUC/ Density gradient/ Filtration	dUC/ Density gradient/ Filtration	dUC/ Filtration	dUC/ Filtration	dUC/ Filtration	dUC/ Filtration	dUC/ Filtration	dUC/ Filtration
vesicle related term	Pure outer membrane vesicle	Pure outer membrane vesicle	Pure outer membrane vesicle	Outer membrane vesicle	Outer membrane vesicle	Outer membrane vesicle	Outer membrane vesicle	Outer membrane vesicle	Outer membrane vesicle
Exp. nr.	2	4	6	1	3	5	7	8	9
EV-METRIC %	56	56	56	33	33	33	14	14	14

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