{"id":{"repo_id":"aachen","oai_identifier":"oai:publications.rwth-aachen.de:62074"},"canonical_url":"https://search.dev.ndltd.org/etd/aachen/oai:publications.rwth-aachen.de:62074","repository":{"repo_id":"aachen","name":"RWTH Aachen University","base_url":"https://publications.rwth-aachen.de/oai2d"},"display":{"title":"Characterization of the alfalfa mosaic virus as expression, presentation, delivery and screening system for potential epitopes derived from the respiratory syncytial virus fusion protein","abstract":"Plant viruses have gained sustainable interest as presentation and production systems for immunogenic peptides within the last decade and consequently their use as vaccines has become an increasingly viable proposition. In this study it was investigated if Alfalfa mosaic virus could be used to present (i) an RSV-F-derived peptide library, (ii) phage-selected mimics and (iii) anti-viral peptide fusion inhibitors. The library was then used to screen for B- and T-cell epitopes. The data obtained was used to establish AlMV as a screening tool and evaluate its performance in different applications. This is the first study in which a plant virus has been used to identify potential B and T-helper cell epitopes for vaccine development. Based on the performance of different AlMV-RSV-F constructs three factors were identified that influenced recombinant peptide expression as N-terminal AlMV CP fusion: (i) charge, (ii) hydrophobicity and (iii) size. Positively charged peptides and resulting chimeric CPs that had a charge and pI significant higher than wt, were found to be difficult or unexpressible. This was probably due to insufficient particle formation and a resulting induction of a plant HR. The performance of these constructs was improved by the addition of acidic amino acids that compensated the positive charge. High level of hydrophobicity decreased chimeric particle production as well. Peptides that contained more hydrophobic then polar aas were difficult to purify. This could be explained by the affinity of the hydrophobic peptides for the cell membrane and consequently their loss in the membrane fraction during sample preparation or virus purification. Compensating the hydrophobic amino acids with polar amino acids did improve the performance. Two phage-displayed random peptide libraries were successful screened for HNK20 binders. Several different phages showed good binding properties to HNK20 antibodies and two of them competed with RSV for binding. However, these properties were lost after expression of the mimics on AlMV particles. AlMV CP has already been used to successful express, present and deliver known B-cell epitopes from various pathogens including rabies virus, HIV and RSV. Furthermore, we have shown that all RSV-F derived constructs tested in mice generated a specific RSV IgG response out of which six covered known neutralization sites and are therefore likely to generate neutralizing abs. Chimeric AlMV particles were also successfully used as presentation, delivery and screening tool for T-helper cell epitopes using human dendritic cells for the assay. Out of a pool of 22 constructs at least 6 gave an RSV-F specific IFN-g response in one or more different donors. AlMV-RSV-F-24 (aa 390-413) gave a potential universal TH1 response when tested in 5 different donors, that covered the most common HLA-DR-types in the Caucasian population. Several other peptides also gave an IFN-gamma response. Our findings correlate well with those described in literature, thus indicating that the AlMV-CP presented RSV-F- derived peptides are processed correctly. No TH2 response could be detected, although it is possible that peptides presented on AlMV surface could have a TH1 bias. A comparison of the in vitro data with those generated in silico revealed that none of the programs used was able to predict T-helper cells correctly. However, consensus sequences of potential epitopes within the 24mer could be identified.","abstract_html":"Plant viruses have gained sustainable interest as presentation and production systems for immunogenic peptides within the last decade and consequently their use as vaccines has become an increasingly viable proposition. In this study it was investigated if Alfalfa mosaic virus could be used to present (i) an RSV-F-derived peptide library, (ii) phage-selected mimics and (iii) anti-viral peptide fusion inhibitors. The library was then used to screen for B- and T-cell epitopes. The data obtained was used to establish AlMV as a screening tool and evaluate its performance in different applications. This is the first study in which a plant virus has been used to identify potential B and T-helper cell epitopes for vaccine development. Based on the performance of different AlMV-RSV-F constructs three factors were identified that influenced recombinant peptide expression as N-terminal AlMV CP fusion: (i) charge, (ii) hydrophobicity and (iii) size. Positively charged peptides and resulting chimeric CPs that had a charge and pI significant higher than wt, were found to be difficult or unexpressible. This was probably due to insufficient particle formation and a resulting induction of a plant HR. The performance of these constructs was improved by the addition of acidic amino acids that compensated the positive charge. High level of hydrophobicity decreased chimeric particle production as well. Peptides that contained more hydrophobic then polar aas were difficult to purify. This could be explained by the affinity of the hydrophobic peptides for the cell membrane and consequently their loss in the membrane fraction during sample preparation or virus purification. Compensating the hydrophobic amino acids with polar amino acids did improve the performance. Two phage-displayed random peptide libraries were successful screened for HNK20 binders. Several different phages showed good binding properties to HNK20 antibodies and two of them competed with RSV for binding. However, these properties were lost after expression of the mimics on AlMV particles. AlMV CP has already been used to successful express, present and deliver known B-cell epitopes from various pathogens including rabies virus, HIV and RSV. Furthermore, we have shown that all RSV-F derived constructs tested in mice generated a specific RSV IgG response out of which six covered known neutralization sites and are therefore likely to generate neutralizing abs. Chimeric AlMV particles were also successfully used as presentation, delivery and screening tool for T-helper cell epitopes using human dendritic cells for the assay. Out of a pool of 22 constructs at least 6 gave an RSV-F specific IFN-g response in one or more different donors. AlMV-RSV-F-24 (aa 390-413) gave a potential universal TH1 response when tested in 5 different donors, that covered the most common HLA-DR-types in the Caucasian population. Several other peptides also gave an IFN-gamma response. Our findings correlate well with those described in literature, thus indicating that the AlMV-CP presented RSV-F- derived peptides are processed correctly. No TH2 response could be detected, although it is possible that peptides presented on AlMV surface could have a TH1 bias. A comparison of the in vitro data with those generated in silico revealed that none of the programs used was able to predict T-helper cells correctly. However, consensus sequences of potential epitopes within the 24mer could be identified.","abstract_has_math":false,"creators":["Munz, Georg"],"institution":"Publikationsserver der RWTH Aachen University","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Fischer, Rainer"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2005,"date_issued":"2005","date_published":"2005","updated_at":"2026-07-30T19:43:19Z","subjects":["info:eu-repo/classification/ddc/570","RS-Virus","Epitop","Peptidbibliothek","Virusimpfstoff","Alfalfa-Mosaik-Virus","Biowissenschaften, Biologie","AlMV","RSV","Fusion Protein","epitope vaccine"],"languages":["eng"],"rights":["info:eu-repo/semantics/openAccess"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123670%22"],"render_values":[{"text":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123670%22","href":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123670%22","code":true}]}]},"links":{"outbound_url":"https://publications.rwth-aachen.de/record/62074","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Fischer, Rainer"]},{"key":"dc:creator","label":"Author","values":["Munz, Georg"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:coverage","label":"Dc Coverage","values":["DE"]},{"key":"dc:date","label":"Dc Date","values":["2005"]},{"key":"dc:publisher","label":"Institution","values":["Publikationsserver der RWTH Aachen University"]},{"key":"dc:relation","label":"Dc Relation","values":["info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-20050235"]},{"key":"dc:type","label":"Dc Type","values":["info:eu-repo/semantics/doctoralThesis","info:eu-repo/semantics/publishedVersion"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["info:eu-repo/classification/ddc/570","RS-Virus","Epitop","Peptidbibliothek","Virusimpfstoff","Alfalfa-Mosaik-Virus","Biowissenschaften, Biologie","AlMV","RSV","Fusion Protein","epitope vaccine"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["info:eu-repo/semantics/openAccess"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/record/62074","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123670%22"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Plant viruses have gained sustainable interest as presentation and production systems for immunogenic peptides within the last decade and consequently their use as vaccines has become an increasingly viable proposition. In this study it was investigated if Alfalfa mosaic virus could be used to present (i) an RSV-F-derived peptide library, (ii) phage-selected mimics and (iii) anti-viral peptide fusion inhibitors. The library was then used to screen for B- and T-cell epitopes. The data obtained was used to establish AlMV as a screening tool and evaluate its performance in different applications. This is the first study in which a plant virus has been used to identify potential B and T-helper cell epitopes for vaccine development. Based on the performance of different AlMV-RSV-F constructs three factors were identified that influenced recombinant peptide expression as N-terminal AlMV CP fusion: (i) charge, (ii) hydrophobicity and (iii) size. Positively charged peptides and resulting chimeric CPs that had a charge and pI significant higher than wt, were found to be difficult or unexpressible. This was probably due to insufficient particle formation and a resulting induction of a plant HR. The performance of these constructs was improved by the addition of acidic amino acids that compensated the positive charge. High level of hydrophobicity decreased chimeric particle production as well. Peptides that contained more hydrophobic then polar aas were difficult to purify. This could be explained by the affinity of the hydrophobic peptides for the cell membrane and consequently their loss in the membrane fraction during sample preparation or virus purification. Compensating the hydrophobic amino acids with polar amino acids did improve the performance. Two phage-displayed random peptide libraries were successful screened for HNK20 binders. Several different phages showed good binding properties to HNK20 antibodies and two of them competed with RSV for binding. However, these properties were lost after expression of the mimics on AlMV particles. AlMV CP has already been used to successful express, present and deliver known B-cell epitopes from various pathogens including rabies virus, HIV and RSV. Furthermore, we have shown that all RSV-F derived constructs tested in mice generated a specific RSV IgG response out of which six covered known neutralization sites and are therefore likely to generate neutralizing abs. Chimeric AlMV particles were also successfully used as presentation, delivery and screening tool for T-helper cell epitopes using human dendritic cells for the assay. Out of a pool of 22 constructs at least 6 gave an RSV-F specific IFN-g response in one or more different donors. AlMV-RSV-F-24 (aa 390-413) gave a potential universal TH1 response when tested in 5 different donors, that covered the most common HLA-DR-types in the Caucasian population. Several other peptides also gave an IFN-gamma response. Our findings correlate well with those described in literature, thus indicating that the AlMV-CP presented RSV-F- derived peptides are processed correctly. No TH2 response could be detected, although it is possible that peptides presented on AlMV surface could have a TH1 bias. A comparison of the in vitro data with those generated in silico revealed that none of the programs used was able to predict T-helper cells correctly. However, consensus sequences of potential epitopes within the 24mer could be identified."]},{"key":"dc:source","label":"Dc Source","values":["Aachen : Publikationsserver der RWTH Aachen University 112 S. : Ill., graph. Darst. (2005). = Aachen, Techn. Univ., Diss., 2005"]},{"key":"dc:title","label":"Title","values":["Characterization of the alfalfa mosaic virus as expression, presentation, delivery and screening system for potential epitopes derived from the respiratory syncytial virus fusion protein"]}]}],"canonical_facts":{"dc:contributor":["Fischer, Rainer"],"dc:coverage":["DE"],"dc:creator":["Munz, Georg"],"dc:date":["2005"],"dc:description":["Plant viruses have gained sustainable interest as presentation and production systems for immunogenic peptides within the last decade and consequently their use as vaccines has become an increasingly viable proposition. In this study it was investigated if Alfalfa mosaic virus could be used to present (i) an RSV-F-derived peptide library, (ii) phage-selected mimics and (iii) anti-viral peptide fusion inhibitors. The library was then used to screen for B- and T-cell epitopes. The data obtained was used to establish AlMV as a screening tool and evaluate its performance in different applications. This is the first study in which a plant virus has been used to identify potential B and T-helper cell epitopes for vaccine development. Based on the performance of different AlMV-RSV-F constructs three factors were identified that influenced recombinant peptide expression as N-terminal AlMV CP fusion: (i) charge, (ii) hydrophobicity and (iii) size. Positively charged peptides and resulting chimeric CPs that had a charge and pI significant higher than wt, were found to be difficult or unexpressible. This was probably due to insufficient particle formation and a resulting induction of a plant HR. The performance of these constructs was improved by the addition of acidic amino acids that compensated the positive charge. High level of hydrophobicity decreased chimeric particle production as well. Peptides that contained more hydrophobic then polar aas were difficult to purify. This could be explained by the affinity of the hydrophobic peptides for the cell membrane and consequently their loss in the membrane fraction during sample preparation or virus purification. Compensating the hydrophobic amino acids with polar amino acids did improve the performance. Two phage-displayed random peptide libraries were successful screened for HNK20 binders. Several different phages showed good binding properties to HNK20 antibodies and two of them competed with RSV for binding. However, these properties were lost after expression of the mimics on AlMV particles. AlMV CP has already been used to successful express, present and deliver known B-cell epitopes from various pathogens including rabies virus, HIV and RSV. Furthermore, we have shown that all RSV-F derived constructs tested in mice generated a specific RSV IgG response out of which six covered known neutralization sites and are therefore likely to generate neutralizing abs. Chimeric AlMV particles were also successfully used as presentation, delivery and screening tool for T-helper cell epitopes using human dendritic cells for the assay. Out of a pool of 22 constructs at least 6 gave an RSV-F specific IFN-g response in one or more different donors. AlMV-RSV-F-24 (aa 390-413) gave a potential universal TH1 response when tested in 5 different donors, that covered the most common HLA-DR-types in the Caucasian population. Several other peptides also gave an IFN-gamma response. Our findings correlate well with those described in literature, thus indicating that the AlMV-CP presented RSV-F- derived peptides are processed correctly. No TH2 response could be detected, although it is possible that peptides presented on AlMV surface could have a TH1 bias. A comparison of the in vitro data with those generated in silico revealed that none of the programs used was able to predict T-helper cells correctly. However, consensus sequences of potential epitopes within the 24mer could be identified."],"dc:identifier":["https://publications.rwth-aachen.de/record/62074","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123670%22"],"dc:language":["eng"],"dc:publisher":["Publikationsserver der RWTH Aachen University"],"dc:relation":["info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-20050235"],"dc:rights":["info:eu-repo/semantics/openAccess"],"dc:source":["Aachen : Publikationsserver der RWTH Aachen University 112 S. : Ill., graph. Darst. (2005). = Aachen, Techn. Univ., Diss., 2005"],"dc:subject":["info:eu-repo/classification/ddc/570","RS-Virus","Epitop","Peptidbibliothek","Virusimpfstoff","Alfalfa-Mosaik-Virus","Biowissenschaften, Biologie","AlMV","RSV","Fusion Protein","epitope vaccine"],"dc:title":["Characterization of the alfalfa mosaic virus as expression, presentation, delivery and screening system for potential epitopes derived from the respiratory syncytial virus fusion protein"],"dc:type":["info:eu-repo/semantics/doctoralThesis","info:eu-repo/semantics/publishedVersion"]},"updated_at":"2026-07-30T19:43:19Z"}