title: Serum-derived exosomes from non-viremic animals previously exposed to the porcine respiratory and reproductive virus contain antigenic viral proteins.
abstract: PRRSV is the etiological agent of one of the most important swine diseases with a significant economic burden worldwide and limitations in vaccinology. Exosomes are 30-100 nm vesicles of endocytic origin. Remarkably, immunizations with exosomes containing antigens from tumors or pathogens are capable of eliciting protective immune responses, albeit variably, in cancer and infectious diseases. Here we describe the isolation, molecular composition and immunogenicity of serum-derived exosomes from naïve animals, from PRRSV viremic animals and from animals previously PRRSV infected but already free of viruses (non viremic). Exosomes were isolated through size exclusion chromatography and characterized by different methodologies. Exosome-enriched fractions from naïve and natural infected animals contained classical tetraspanin exosomal markers (CD63 and CD81) and high concentrations of particles in the size-range of exosomes as detected by nanoparticle tracking analysis and cryo-TEM. NanoLC-MS/MS was used to identify viral antigens associated to exosomes. PRRSV-proteins were detected in serum samples from only viremic animals and from animals previously infected already free of viruses (non-viremic), but not in controls. Moreover, immune sera from pigs previously exposed to PRRSV specifically reacted against exosomes purified from non-viremic pig sera in a dose-dependent manner, a reactivity not detected when naïve sera was used in the assay. To facilitate future studies, a scaling-up process was implemented. To the best of our knowledge, this is the first molecular characterization of serum-derived exosomes from naïve pigs and pigs actively or previously infected with PRRSV. The presence of antigenic viral proteins in serum-derived exosomes free of virus, suggest their use as a novel vaccine approach against PRRSV.
authors.affiliation: Departamento de Producción Animal, ETSEA, Universidad de Lleida, Avenida Alcalde Rovira Roure 191, Lleida, Spain.|Innovex Therapeutics SL, Badalona, Spain.
authors.forename: Sergio
authors.initials: S
authors.lastname: Montaner-Tarbes
authors.affiliation: Innovex Therapeutics SL, Badalona, Spain.|IVECAT Group, Germans Trias i Pujol Health Science Research Institute (IGTP), Can Ruti Campus, 08916, Badalona, Spain.
authors.forename: Francesc E
authors.initials: FE
authors.lastname: Borrás
authors.affiliation: The Pirbright Institute, Ash Road, Pirbright, Surrey, GU24 0NF, UK.
authors.forename: Maria
authors.initials: M
authors.lastname: Montoya
authors.affiliation: Departamento de Producción Animal, ETSEA, Universidad de Lleida, Avenida Alcalde Rovira Roure 191, Lleida, Spain. lorenzo.fraile@prodan.udl.cat.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Fraile
authors.affiliation: Innovex Therapeutics SL, Badalona, Spain. hernandoa.delportillo@isglobal.org.|ICREA at ISGlobal, Barcelona Ctr. Int. Health Res. (CRESIB), Hospital Clínic-Universitat de Barcelona, 08036, Barcelona, Spain. hernandoa.delportillo@isglobal.org.|ICREA at Institut d'Investigació Germans Trias i Pujol, Can Ruti Campus, 08916, Badalona, Spain. hernandoa.delportillo@isglobal.org.
authors.forename: Hernando A
authors.initials: HA
authors.lastname: Del Portillo
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000276:immunology; D002850:Chromatography, Gel; D020285:Cryoelectron Microscopy; D004797:Enzyme-Linked Immunosorbent Assay; D055354:Exosomes / Q000276:immunology / Q000821:virology*; D005434:Flow Cytometry; D019318:Porcine Reproductive and Respiratory Syndrome / Q000276:immunology / Q000378:metabolism*; D019316:Porcine respiratory and reproductive syndrome virus / Q000276:immunology / Q000378:metabolism*; D013552:Swine
chemical_list: D000956:Antigens, Viral