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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.
Anti‑ CD63 and anti‑ CD81 antibodies were kindly donated by Francisco Sánchez‑Madrid and Maria Yañez‑Mo, Hospital de la Princesa, Madrid, Spain. We thank Miriam Morón Font for technical assistance and Inés Lozano and Marta Monguió ‑Tortajada for valuable scientific discussions. SMT is supported by an Industrial PhD Fellowship from the government of Catalonia (AGAUR) as part of a collaborative agreement between INNOVEX THERAPEUTICS SL and the University of Lleida (Id No 2014 DI 044) |
