Prototype of mucosal vaccine against hepatitis E virus resists gastrointestinal conditions in vitro

Autores
Müller, Melisa Florencia; Raya Tonetti, María Fernanda; Arce, Lorena Paola; Villena, Julio Cesar; Vizoso Pinto, María Guadalupe
Año de publicación
2022
Idioma
inglés
Tipo de recurso
documento de conferencia
Estado
versión publicada
Descripción
The Hepatitis E virus causes hepatitis; its principal port of entry is the gastrointestinal mucosa. Its genome consists in ORF1 that encodes for a polyprotein needed for replication, ORF2 the viral capsid and ORF3 a multifunctional phosphoprotein. The LysM domains are ubiquitous small domains, which mediate attachment of enzymes to bacterial peptidoglycan or to fungal chitin. Bacterium-like-particles (BLP) are non-live bacteria treated with heat and acid, which conserve their shape but lose proteins and DNA. The aim of our study was to generate fusion proteins and displayed them on the surface of BLP, to generate a mucosal vaccine which combines carrier and adjuvant properties for oral administration. In this work we cloned and expressed a chimeric protein with 5 LysM domains (LysM5) and ORF2 most immunogenic domain(O2P2). We obtained the protein under native and denaturing conditions purificated by NiNTA chromatography. BLP derived from Lactiplantibacillus plantarum IBL027, previously reported to have adjuvant activity on mucosa, were used for evaluating antigen display on its surface. Briefly, we put in contact bacterial lysates or purified proteins and BLP for an hour in rotation at room temperature and then washed them with PBS to get LysM5O2P2-BLP027. These complexes were tested on solutions simulating saliva, gastric and intestinal juices and incubated in 37°C for 5 minutes, 1 and 3 hours, respectively. Protein integrity was checked by SDS-PAGE. We tested several conditions for optimal expression of LysM5O2P2 in E. coli. Surprisingly, after purification, the protein did not bind to BLP but when we tested crude supernatants (under native and denaturing conditions), it bound at different proportions. Then, we tested the resistance of complexes to gastrointestinal conditions; both were resistant to artificial saliva (pH:7.2, lysozyme 100ppm) and simulated gastric juice (pH:2.5) but BLP exposing the native protein was resistant to simulated intestinal juice (pH:7.2; pancreatin: protease>1900USP) and BLP exposing the denatured protein was not. SDSPAGE revealed LysM5O2P2 was split in two by pancreatin, which is consistent with the presence of a trypsin recognition site in the protein sequence. Capsid proteins of enteric viruses must resist gastrointestinal conditions to reach their target cells. We postulate that the proper folding of LysM5O2P2 protects it from digestion. Some authors reported that native O2P2 dimerization protects it against trypsin digestion. The next step will be to study oral immunization protocols with the prototype vaccine LysM5O2P2-BLP.
Fil: Müller, Melisa Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina; Argentina
Fil: Raya Tonetti, María Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina; Argentina
Fil: Arce, Lorena Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina; Argentina
Fil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina
Fil: Vizoso Pinto, María Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina; Argentina
LVII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; XVI Annual Meeting of the Argentinean Society for General Microbiology
Ciudad Autónoma de Buenos Aries
Argentina
Sociedad Argentina de Investigación en Bioquímica y Biología Molecular
Sociedad Argentina de Microbiología General
Materia
HEPATITIS E
BACERIUM LIKE PARTICLES
LYSM DOMAIN
MUCOSAL VACCINE
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/271343
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/271343
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Prototype of mucosal vaccine against hepatitis E virus resists gastrointestinal conditions in vitroMüller, Melisa FlorenciaRaya Tonetti, María FernandaArce, Lorena PaolaVillena, Julio CesarVizoso Pinto, María GuadalupeHEPATITIS EBACERIUM LIKE PARTICLESLYSM DOMAINMUCOSAL VACCINEhttps://purl.org/becyt/ford/3.3https://purl.org/becyt/ford/3The Hepatitis E virus causes hepatitis; its principal port of entry is the gastrointestinal mucosa. Its genome consists in ORF1 that encodes for a polyprotein needed for replication, ORF2 the viral capsid and ORF3 a multifunctional phosphoprotein. The LysM domains are ubiquitous small domains, which mediate attachment of enzymes to bacterial peptidoglycan or to fungal chitin. Bacterium-like-particles (BLP) are non-live bacteria treated with heat and acid, which conserve their shape but lose proteins and DNA. The aim of our study was to generate fusion proteins and displayed them on the surface of BLP, to generate a mucosal vaccine which combines carrier and adjuvant properties for oral administration. In this work we cloned and expressed a chimeric protein with 5 LysM domains (LysM5) and ORF2 most immunogenic domain(O2P2). We obtained the protein under native and denaturing conditions purificated by NiNTA chromatography. BLP derived from Lactiplantibacillus plantarum IBL027, previously reported to have adjuvant activity on mucosa, were used for evaluating antigen display on its surface. Briefly, we put in contact bacterial lysates or purified proteins and BLP for an hour in rotation at room temperature and then washed them with PBS to get LysM5O2P2-BLP027. These complexes were tested on solutions simulating saliva, gastric and intestinal juices and incubated in 37°C for 5 minutes, 1 and 3 hours, respectively. Protein integrity was checked by SDS-PAGE. We tested several conditions for optimal expression of LysM5O2P2 in E. coli. Surprisingly, after purification, the protein did not bind to BLP but when we tested crude supernatants (under native and denaturing conditions), it bound at different proportions. Then, we tested the resistance of complexes to gastrointestinal conditions; both were resistant to artificial saliva (pH:7.2, lysozyme 100ppm) and simulated gastric juice (pH:2.5) but BLP exposing the native protein was resistant to simulated intestinal juice (pH:7.2; pancreatin: protease>1900USP) and BLP exposing the denatured protein was not. SDSPAGE revealed LysM5O2P2 was split in two by pancreatin, which is consistent with the presence of a trypsin recognition site in the protein sequence. Capsid proteins of enteric viruses must resist gastrointestinal conditions to reach their target cells. We postulate that the proper folding of LysM5O2P2 protects it from digestion. Some authors reported that native O2P2 dimerization protects it against trypsin digestion. The next step will be to study oral immunization protocols with the prototype vaccine LysM5O2P2-BLP.Fil: Müller, Melisa Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina; ArgentinaFil: Raya Tonetti, María Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina; ArgentinaFil: Arce, Lorena Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina; ArgentinaFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Vizoso Pinto, María Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina; ArgentinaLVII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; XVI Annual Meeting of the Argentinean Society for General MicrobiologyCiudad Autónoma de Buenos AriesArgentinaSociedad Argentina de Investigación en Bioquímica y Biología MolecularSociedad Argentina de Microbiología GeneralTech Science Press2022info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/conferenceObjectReuniónJournalhttp://purl.org/coar/resource_type/c_5794info:ar-repo/semantics/documentoDeConferenciaapplication/pdfapplication/vnd.openxmlformats-officedocument.wordprocessingml.documentapplication/pdfhttp://hdl.handle.net/11336/271343Prototype of mucosal vaccine against hepatitis E virus resists gastrointestinal conditions in vitro; LVII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; XVI Annual Meeting of the Argentinean Society for General Microbiology; Ciudad Autónoma de Buenos Aries; Argentina; 2021; 180-1810327-95451667-5746CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.techscience.com/biocell/v46nSuppl.1/46213/pdfInternacionalinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T11:47:12Zoai:ri.conicet.gov.ar:11336/271343instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-22 11:47:12.495CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Prototype of mucosal vaccine against hepatitis E virus resists gastrointestinal conditions in vitro
title Prototype of mucosal vaccine against hepatitis E virus resists gastrointestinal conditions in vitro
spellingShingle Prototype of mucosal vaccine against hepatitis E virus resists gastrointestinal conditions in vitro
Müller, Melisa Florencia
HEPATITIS E
BACERIUM LIKE PARTICLES
LYSM DOMAIN
MUCOSAL VACCINE
title_short Prototype of mucosal vaccine against hepatitis E virus resists gastrointestinal conditions in vitro
title_full Prototype of mucosal vaccine against hepatitis E virus resists gastrointestinal conditions in vitro
title_fullStr Prototype of mucosal vaccine against hepatitis E virus resists gastrointestinal conditions in vitro
title_full_unstemmed Prototype of mucosal vaccine against hepatitis E virus resists gastrointestinal conditions in vitro
title_sort Prototype of mucosal vaccine against hepatitis E virus resists gastrointestinal conditions in vitro
dc.creator.none.fl_str_mv Müller, Melisa Florencia
Raya Tonetti, María Fernanda
Arce, Lorena Paola
Villena, Julio Cesar
Vizoso Pinto, María Guadalupe
author Müller, Melisa Florencia
author_facet Müller, Melisa Florencia
Raya Tonetti, María Fernanda
Arce, Lorena Paola
Villena, Julio Cesar
Vizoso Pinto, María Guadalupe
author_role author
author2 Raya Tonetti, María Fernanda
Arce, Lorena Paola
Villena, Julio Cesar
Vizoso Pinto, María Guadalupe
author2_role author
author
author
author
dc.subject.none.fl_str_mv HEPATITIS E
BACERIUM LIKE PARTICLES
LYSM DOMAIN
MUCOSAL VACCINE
topic HEPATITIS E
BACERIUM LIKE PARTICLES
LYSM DOMAIN
MUCOSAL VACCINE
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.3
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv The Hepatitis E virus causes hepatitis; its principal port of entry is the gastrointestinal mucosa. Its genome consists in ORF1 that encodes for a polyprotein needed for replication, ORF2 the viral capsid and ORF3 a multifunctional phosphoprotein. The LysM domains are ubiquitous small domains, which mediate attachment of enzymes to bacterial peptidoglycan or to fungal chitin. Bacterium-like-particles (BLP) are non-live bacteria treated with heat and acid, which conserve their shape but lose proteins and DNA. The aim of our study was to generate fusion proteins and displayed them on the surface of BLP, to generate a mucosal vaccine which combines carrier and adjuvant properties for oral administration. In this work we cloned and expressed a chimeric protein with 5 LysM domains (LysM5) and ORF2 most immunogenic domain(O2P2). We obtained the protein under native and denaturing conditions purificated by NiNTA chromatography. BLP derived from Lactiplantibacillus plantarum IBL027, previously reported to have adjuvant activity on mucosa, were used for evaluating antigen display on its surface. Briefly, we put in contact bacterial lysates or purified proteins and BLP for an hour in rotation at room temperature and then washed them with PBS to get LysM5O2P2-BLP027. These complexes were tested on solutions simulating saliva, gastric and intestinal juices and incubated in 37°C for 5 minutes, 1 and 3 hours, respectively. Protein integrity was checked by SDS-PAGE. We tested several conditions for optimal expression of LysM5O2P2 in E. coli. Surprisingly, after purification, the protein did not bind to BLP but when we tested crude supernatants (under native and denaturing conditions), it bound at different proportions. Then, we tested the resistance of complexes to gastrointestinal conditions; both were resistant to artificial saliva (pH:7.2, lysozyme 100ppm) and simulated gastric juice (pH:2.5) but BLP exposing the native protein was resistant to simulated intestinal juice (pH:7.2; pancreatin: protease>1900USP) and BLP exposing the denatured protein was not. SDSPAGE revealed LysM5O2P2 was split in two by pancreatin, which is consistent with the presence of a trypsin recognition site in the protein sequence. Capsid proteins of enteric viruses must resist gastrointestinal conditions to reach their target cells. We postulate that the proper folding of LysM5O2P2 protects it from digestion. Some authors reported that native O2P2 dimerization protects it against trypsin digestion. The next step will be to study oral immunization protocols with the prototype vaccine LysM5O2P2-BLP.
Fil: Müller, Melisa Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina; Argentina
Fil: Raya Tonetti, María Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina; Argentina
Fil: Arce, Lorena Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina; Argentina
Fil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina
Fil: Vizoso Pinto, María Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina; Argentina
LVII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; XVI Annual Meeting of the Argentinean Society for General Microbiology
Ciudad Autónoma de Buenos Aries
Argentina
Sociedad Argentina de Investigación en Bioquímica y Biología Molecular
Sociedad Argentina de Microbiología General
description The Hepatitis E virus causes hepatitis; its principal port of entry is the gastrointestinal mucosa. Its genome consists in ORF1 that encodes for a polyprotein needed for replication, ORF2 the viral capsid and ORF3 a multifunctional phosphoprotein. The LysM domains are ubiquitous small domains, which mediate attachment of enzymes to bacterial peptidoglycan or to fungal chitin. Bacterium-like-particles (BLP) are non-live bacteria treated with heat and acid, which conserve their shape but lose proteins and DNA. The aim of our study was to generate fusion proteins and displayed them on the surface of BLP, to generate a mucosal vaccine which combines carrier and adjuvant properties for oral administration. In this work we cloned and expressed a chimeric protein with 5 LysM domains (LysM5) and ORF2 most immunogenic domain(O2P2). We obtained the protein under native and denaturing conditions purificated by NiNTA chromatography. BLP derived from Lactiplantibacillus plantarum IBL027, previously reported to have adjuvant activity on mucosa, were used for evaluating antigen display on its surface. Briefly, we put in contact bacterial lysates or purified proteins and BLP for an hour in rotation at room temperature and then washed them with PBS to get LysM5O2P2-BLP027. These complexes were tested on solutions simulating saliva, gastric and intestinal juices and incubated in 37°C for 5 minutes, 1 and 3 hours, respectively. Protein integrity was checked by SDS-PAGE. We tested several conditions for optimal expression of LysM5O2P2 in E. coli. Surprisingly, after purification, the protein did not bind to BLP but when we tested crude supernatants (under native and denaturing conditions), it bound at different proportions. Then, we tested the resistance of complexes to gastrointestinal conditions; both were resistant to artificial saliva (pH:7.2, lysozyme 100ppm) and simulated gastric juice (pH:2.5) but BLP exposing the native protein was resistant to simulated intestinal juice (pH:7.2; pancreatin: protease>1900USP) and BLP exposing the denatured protein was not. SDSPAGE revealed LysM5O2P2 was split in two by pancreatin, which is consistent with the presence of a trypsin recognition site in the protein sequence. Capsid proteins of enteric viruses must resist gastrointestinal conditions to reach their target cells. We postulate that the proper folding of LysM5O2P2 protects it from digestion. Some authors reported that native O2P2 dimerization protects it against trypsin digestion. The next step will be to study oral immunization protocols with the prototype vaccine LysM5O2P2-BLP.
publishDate 2022
dc.date.none.fl_str_mv 2022
dc.type.none.fl_str_mv info:eu-repo/semantics/publishedVersion
info:eu-repo/semantics/conferenceObject
Reunión
Journal
http://purl.org/coar/resource_type/c_5794
info:ar-repo/semantics/documentoDeConferencia
status_str publishedVersion
format conferenceObject
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/271343
Prototype of mucosal vaccine against hepatitis E virus resists gastrointestinal conditions in vitro; LVII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; XVI Annual Meeting of the Argentinean Society for General Microbiology; Ciudad Autónoma de Buenos Aries; Argentina; 2021; 180-181
0327-9545
1667-5746
CONICET Digital
CONICET
url http://hdl.handle.net/11336/271343
identifier_str_mv Prototype of mucosal vaccine against hepatitis E virus resists gastrointestinal conditions in vitro; LVII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; XVI Annual Meeting of the Argentinean Society for General Microbiology; Ciudad Autónoma de Buenos Aries; Argentina; 2021; 180-181
0327-9545
1667-5746
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://www.techscience.com/biocell/v46nSuppl.1/46213/pdf
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dc.coverage.none.fl_str_mv Internacional
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publisher.none.fl_str_mv Tech Science Press
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