Assessing protein immunogenicity with a dendritic cell line-derived endolysosomal degradome
The growing number of novel candidate molecules for the treatment of allergic diseases imposed a dramatic increase in the demand for animal experiments to select immunogenic vaccines, a pre-requisite for efficacy. Because no in vitro methods to predict the immunogenicity of a protein are currently a...
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| Veröffentlicht in: | PloS one 2011-02, Vol.6 (2), p.e17278-e17278 |
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| creator | Egger, Matthias Jürets, Alexander Wallner, Michael Briza, Peter Ruzek, Silke Hainzl, Stefan Pichler, Ulrike Kitzmüller, Claudia Bohle, Barbara Huber, Christian G Ferreira, Fátima |
| description | The growing number of novel candidate molecules for the treatment of allergic diseases imposed a dramatic increase in the demand for animal experiments to select immunogenic vaccines, a pre-requisite for efficacy. Because no in vitro methods to predict the immunogenicity of a protein are currently available, we developed an in vitro assay that exploits the link between a protein's immunogenicity and its susceptibility to endolysosomal proteolysis.
We compared protein composition and proteolytic activity of endolysosomal fractions isolated from murine bone marrow- and human blood- derived dendritic cells, and from the dendritic cell line JAWS II. Three groups of structurally related antigen variants differing in their ability to elicit immune responses in vivo (Bet v 1.0101 and Bet v 1.0401, RNases A and S, holo- and apo-HRP) were subjected to in vitro simulated endolysosomal degradation. Kinetics and patterns of generated proteolytic peptides were evaluated by gel electrophoresis and mass spectrometry.
Antigens displaying weak capacity of T cell priming in vivo were highly susceptible to endolysosomal proteases in vitro. As proteolytic composition, activity, and specificity of endolysosomal fractions derived from human and murine dendritic cells were comparable, the JAWS II cell line could be used as a substitute for freshly isolated human or murine cells in in vitro degradation assays.
Endolysosomal fractions prepared from the JAWS II cell line provide a reliable tool for in vitro estimation of protein immunogenicity. The rapid and simple assay described here is very useful to study the immunogenic properties of a protein, and can help to replace, reduce, and refine animal experiments in allergy research and vaccine development in general. |
| doi_str_mv | 10.1371/journal.pone.0017278 |
| format | Article |
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We compared protein composition and proteolytic activity of endolysosomal fractions isolated from murine bone marrow- and human blood- derived dendritic cells, and from the dendritic cell line JAWS II. Three groups of structurally related antigen variants differing in their ability to elicit immune responses in vivo (Bet v 1.0101 and Bet v 1.0401, RNases A and S, holo- and apo-HRP) were subjected to in vitro simulated endolysosomal degradation. Kinetics and patterns of generated proteolytic peptides were evaluated by gel electrophoresis and mass spectrometry.
Antigens displaying weak capacity of T cell priming in vivo were highly susceptible to endolysosomal proteases in vitro. As proteolytic composition, activity, and specificity of endolysosomal fractions derived from human and murine dendritic cells were comparable, the JAWS II cell line could be used as a substitute for freshly isolated human or murine cells in in vitro degradation assays.
Endolysosomal fractions prepared from the JAWS II cell line provide a reliable tool for in vitro estimation of protein immunogenicity. The rapid and simple assay described here is very useful to study the immunogenic properties of a protein, and can help to replace, reduce, and refine animal experiments in allergy research and vaccine development in general.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0017278</identifier><identifier>PMID: 21359181</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Allergic diseases ; Allergies ; Allergy ; Animal experimentation ; Animal research ; Animals ; Antibody Formation - physiology ; Antigen presentation ; Antigens ; Assaying ; Bet v 1 antigen ; Biology ; Bone composition ; Bone marrow ; Bone Marrow Cells - immunology ; Bone Marrow Cells - metabolism ; Cell Line ; Degradation ; Dendritic cells ; Dendritic Cells - immunology ; Dendritic Cells - metabolism ; Dendritic structure ; Experiments ; Gel electrophoresis ; Genes, p53 ; Humans ; Hypersensitivity ; Immune response ; Immunogenicity ; Immunology ; Immunotherapy ; In vitro methods and tests ; Kinetics ; Laboratories ; Ligands ; Lymphocytes ; Lymphocytes T ; Lysosomes - immunology ; Lysosomes - metabolism ; Mass spectrometry ; Mass spectroscopy ; Medical research ; Medical treatment ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Knockout ; Molecular biology ; Peptides ; Priming ; Proteases ; Protein composition ; Protein Processing, Post-Translational - immunology ; Protein Processing, Post-Translational - physiology ; Proteins ; Proteins - immunology ; Proteins - metabolism ; Proteolysis ; Ribonuclease ; T cell receptors ; T cells ; Vaccine development ; Vaccines ; Vaccines - biosynthesis</subject><ispartof>PloS one, 2011-02, Vol.6 (2), p.e17278-e17278</ispartof><rights>COPYRIGHT 2011 Public Library of Science</rights><rights>2011 Egger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>Egger et al. 2011</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c691t-86cd81d06b393f41ad2a2f44ee3eaba8ba9bf2a368123652515dfad85d0bdadf3</citedby><cites>FETCH-LOGICAL-c691t-86cd81d06b393f41ad2a2f44ee3eaba8ba9bf2a368123652515dfad85d0bdadf3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3040223/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3040223/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2102,2928,23866,27924,27925,53791,53793,79600,79601</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21359181$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Câmara, Niels</contributor><creatorcontrib>Egger, Matthias</creatorcontrib><creatorcontrib>Jürets, Alexander</creatorcontrib><creatorcontrib>Wallner, Michael</creatorcontrib><creatorcontrib>Briza, Peter</creatorcontrib><creatorcontrib>Ruzek, Silke</creatorcontrib><creatorcontrib>Hainzl, Stefan</creatorcontrib><creatorcontrib>Pichler, Ulrike</creatorcontrib><creatorcontrib>Kitzmüller, Claudia</creatorcontrib><creatorcontrib>Bohle, Barbara</creatorcontrib><creatorcontrib>Huber, Christian G</creatorcontrib><creatorcontrib>Ferreira, Fátima</creatorcontrib><title>Assessing protein immunogenicity with a dendritic cell line-derived endolysosomal degradome</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>The growing number of novel candidate molecules for the treatment of allergic diseases imposed a dramatic increase in the demand for animal experiments to select immunogenic vaccines, a pre-requisite for efficacy. Because no in vitro methods to predict the immunogenicity of a protein are currently available, we developed an in vitro assay that exploits the link between a protein's immunogenicity and its susceptibility to endolysosomal proteolysis.
We compared protein composition and proteolytic activity of endolysosomal fractions isolated from murine bone marrow- and human blood- derived dendritic cells, and from the dendritic cell line JAWS II. Three groups of structurally related antigen variants differing in their ability to elicit immune responses in vivo (Bet v 1.0101 and Bet v 1.0401, RNases A and S, holo- and apo-HRP) were subjected to in vitro simulated endolysosomal degradation. Kinetics and patterns of generated proteolytic peptides were evaluated by gel electrophoresis and mass spectrometry.
Antigens displaying weak capacity of T cell priming in vivo were highly susceptible to endolysosomal proteases in vitro. As proteolytic composition, activity, and specificity of endolysosomal fractions derived from human and murine dendritic cells were comparable, the JAWS II cell line could be used as a substitute for freshly isolated human or murine cells in in vitro degradation assays.
Endolysosomal fractions prepared from the JAWS II cell line provide a reliable tool for in vitro estimation of protein immunogenicity. The rapid and simple assay described here is very useful to study the immunogenic properties of a protein, and can help to replace, reduce, and refine animal experiments in allergy research and vaccine development in general.</description><subject>Allergic diseases</subject><subject>Allergies</subject><subject>Allergy</subject><subject>Animal experimentation</subject><subject>Animal research</subject><subject>Animals</subject><subject>Antibody Formation - physiology</subject><subject>Antigen presentation</subject><subject>Antigens</subject><subject>Assaying</subject><subject>Bet v 1 antigen</subject><subject>Biology</subject><subject>Bone composition</subject><subject>Bone marrow</subject><subject>Bone Marrow Cells - immunology</subject><subject>Bone Marrow Cells - metabolism</subject><subject>Cell Line</subject><subject>Degradation</subject><subject>Dendritic cells</subject><subject>Dendritic Cells - immunology</subject><subject>Dendritic Cells - metabolism</subject><subject>Dendritic structure</subject><subject>Experiments</subject><subject>Gel electrophoresis</subject><subject>Genes, p53</subject><subject>Humans</subject><subject>Hypersensitivity</subject><subject>Immune response</subject><subject>Immunogenicity</subject><subject>Immunology</subject><subject>Immunotherapy</subject><subject>In vitro methods and tests</subject><subject>Kinetics</subject><subject>Laboratories</subject><subject>Ligands</subject><subject>Lymphocytes</subject><subject>Lymphocytes T</subject><subject>Lysosomes - immunology</subject><subject>Lysosomes - metabolism</subject><subject>Mass spectrometry</subject><subject>Mass spectroscopy</subject><subject>Medical research</subject><subject>Medical treatment</subject><subject>Mice</subject><subject>Mice, Inbred BALB C</subject><subject>Mice, Inbred C57BL</subject><subject>Mice, Knockout</subject><subject>Molecular biology</subject><subject>Peptides</subject><subject>Priming</subject><subject>Proteases</subject><subject>Protein composition</subject><subject>Protein Processing, Post-Translational - immunology</subject><subject>Protein Processing, Post-Translational - physiology</subject><subject>Proteins</subject><subject>Proteins - immunology</subject><subject>Proteins - metabolism</subject><subject>Proteolysis</subject><subject>Ribonuclease</subject><subject>T cell receptors</subject><subject>T cells</subject><subject>Vaccine development</subject><subject>Vaccines</subject><subject>Vaccines - biosynthesis</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>DOA</sourceid><recordid>eNqNk1uL1DAUx4so7rr6DUQLguLDjLk1074Iw-JlYGHB24sP4TRJO1nSZDZpV-fbmzrdZSr7IHlIOPmd_7kkJ8ueY7TEdIXfXfkhOLDLnXd6iRBekVX5IDvFFSULThB9eHQ-yZ7EeIVQQUvOH2cnBNOiwiU-zX6uY9QxGtfmu-B7bVxuum5wvtXOSNPv81-m3-aQK-1UML2RudTW5tY4vVA6mBut8nTl7T766DuwiWwDKN_pp9mjBmzUz6b9LPv-8cO388-Li8tPm_P1xULyCveLkktVYoV4TSvaMAyKAGkY05pqqKGsoaobApSXmFBekAIXqgFVFgrVClRDz7KXB92d9VFMfYkCk4ozVuAKJWJzIJSHK7ELpoOwFx6M-GvwoRUQUm1WC0BS1ZJVqCYrBgqAyKKWkrMxyUaTpPV-ijbUnVZSuz6AnYnOb5zZitbfCIoYIoQmgTeTQPDXg4696EwcmwpO-yGKsmAFL8tiDPXqH_L-4iaqhZS_cY1PYeWoKdZsxSvE6KpM1PIeKi2lOyPTH2pMss8c3s4cEtPr330LQ4xi8_XL_7OXP-bs6yN2q8H22-jt0Bvv4hxkB1AGH2PQzV2PMRLjCNx2Q4wjIKYRSG4vjt_nzun2z9M_hOkEJQ</recordid><startdate>20110216</startdate><enddate>20110216</enddate><creator>Egger, Matthias</creator><creator>Jürets, Alexander</creator><creator>Wallner, Michael</creator><creator>Briza, Peter</creator><creator>Ruzek, Silke</creator><creator>Hainzl, Stefan</creator><creator>Pichler, Ulrike</creator><creator>Kitzmüller, Claudia</creator><creator>Bohle, Barbara</creator><creator>Huber, Christian G</creator><creator>Ferreira, Fátima</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20110216</creationdate><title>Assessing protein immunogenicity with a dendritic cell line-derived endolysosomal degradome</title><author>Egger, Matthias ; Jürets, Alexander ; Wallner, Michael ; Briza, Peter ; Ruzek, Silke ; Hainzl, Stefan ; Pichler, Ulrike ; Kitzmüller, Claudia ; Bohle, Barbara ; Huber, Christian G ; Ferreira, Fátima</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c691t-86cd81d06b393f41ad2a2f44ee3eaba8ba9bf2a368123652515dfad85d0bdadf3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Allergic diseases</topic><topic>Allergies</topic><topic>Allergy</topic><topic>Animal experimentation</topic><topic>Animal research</topic><topic>Animals</topic><topic>Antibody Formation - physiology</topic><topic>Antigen presentation</topic><topic>Antigens</topic><topic>Assaying</topic><topic>Bet v 1 antigen</topic><topic>Biology</topic><topic>Bone composition</topic><topic>Bone marrow</topic><topic>Bone Marrow Cells - immunology</topic><topic>Bone Marrow Cells - metabolism</topic><topic>Cell Line</topic><topic>Degradation</topic><topic>Dendritic cells</topic><topic>Dendritic Cells - immunology</topic><topic>Dendritic Cells - metabolism</topic><topic>Dendritic structure</topic><topic>Experiments</topic><topic>Gel electrophoresis</topic><topic>Genes, p53</topic><topic>Humans</topic><topic>Hypersensitivity</topic><topic>Immune response</topic><topic>Immunogenicity</topic><topic>Immunology</topic><topic>Immunotherapy</topic><topic>In vitro methods and tests</topic><topic>Kinetics</topic><topic>Laboratories</topic><topic>Ligands</topic><topic>Lymphocytes</topic><topic>Lymphocytes T</topic><topic>Lysosomes - immunology</topic><topic>Lysosomes - metabolism</topic><topic>Mass spectrometry</topic><topic>Mass spectroscopy</topic><topic>Medical research</topic><topic>Medical treatment</topic><topic>Mice</topic><topic>Mice, Inbred BALB C</topic><topic>Mice, Inbred C57BL</topic><topic>Mice, Knockout</topic><topic>Molecular biology</topic><topic>Peptides</topic><topic>Priming</topic><topic>Proteases</topic><topic>Protein composition</topic><topic>Protein Processing, Post-Translational - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Egger, Matthias</au><au>Jürets, Alexander</au><au>Wallner, Michael</au><au>Briza, Peter</au><au>Ruzek, Silke</au><au>Hainzl, Stefan</au><au>Pichler, Ulrike</au><au>Kitzmüller, Claudia</au><au>Bohle, Barbara</au><au>Huber, Christian G</au><au>Ferreira, Fátima</au><au>Câmara, Niels</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Assessing protein immunogenicity with a dendritic cell line-derived endolysosomal degradome</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2011-02-16</date><risdate>2011</risdate><volume>6</volume><issue>2</issue><spage>e17278</spage><epage>e17278</epage><pages>e17278-e17278</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>The growing number of novel candidate molecules for the treatment of allergic diseases imposed a dramatic increase in the demand for animal experiments to select immunogenic vaccines, a pre-requisite for efficacy. Because no in vitro methods to predict the immunogenicity of a protein are currently available, we developed an in vitro assay that exploits the link between a protein's immunogenicity and its susceptibility to endolysosomal proteolysis.
We compared protein composition and proteolytic activity of endolysosomal fractions isolated from murine bone marrow- and human blood- derived dendritic cells, and from the dendritic cell line JAWS II. Three groups of structurally related antigen variants differing in their ability to elicit immune responses in vivo (Bet v 1.0101 and Bet v 1.0401, RNases A and S, holo- and apo-HRP) were subjected to in vitro simulated endolysosomal degradation. Kinetics and patterns of generated proteolytic peptides were evaluated by gel electrophoresis and mass spectrometry.
Antigens displaying weak capacity of T cell priming in vivo were highly susceptible to endolysosomal proteases in vitro. As proteolytic composition, activity, and specificity of endolysosomal fractions derived from human and murine dendritic cells were comparable, the JAWS II cell line could be used as a substitute for freshly isolated human or murine cells in in vitro degradation assays.
Endolysosomal fractions prepared from the JAWS II cell line provide a reliable tool for in vitro estimation of protein immunogenicity. The rapid and simple assay described here is very useful to study the immunogenic properties of a protein, and can help to replace, reduce, and refine animal experiments in allergy research and vaccine development in general.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>21359181</pmid><doi>10.1371/journal.pone.0017278</doi><tpages>e17278</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | PloS one, 2011-02, Vol.6 (2), p.e17278-e17278 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1296445190 |
| source | MEDLINE; Public Library of Science; EZB Free E-Journals; DOAJ Directory of Open Access Journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Allergic diseases Allergies Allergy Animal experimentation Animal research Animals Antibody Formation - physiology Antigen presentation Antigens Assaying Bet v 1 antigen Biology Bone composition Bone marrow Bone Marrow Cells - immunology Bone Marrow Cells - metabolism Cell Line Degradation Dendritic cells Dendritic Cells - immunology Dendritic Cells - metabolism Dendritic structure Experiments Gel electrophoresis Genes, p53 Humans Hypersensitivity Immune response Immunogenicity Immunology Immunotherapy In vitro methods and tests Kinetics Laboratories Ligands Lymphocytes Lymphocytes T Lysosomes - immunology Lysosomes - metabolism Mass spectrometry Mass spectroscopy Medical research Medical treatment Mice Mice, Inbred BALB C Mice, Inbred C57BL Mice, Knockout Molecular biology Peptides Priming Proteases Protein composition Protein Processing, Post-Translational - immunology Protein Processing, Post-Translational - physiology Proteins Proteins - immunology Proteins - metabolism Proteolysis Ribonuclease T cell receptors T cells Vaccine development Vaccines Vaccines - biosynthesis |
| title | Assessing protein immunogenicity with a dendritic cell line-derived endolysosomal degradome |
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