Serum-derived exosomes from non-viremic animals previously exposed to the porcine respiratory and reproductive virus contain antigenic viral proteins

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

Targeted-pig trial on safety and immunogenicity of serum-derived extracellular vesicles enriched fractions obtained from Porcine Respiratory and Reproductive virus infections

The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is the etiological agent of one of the most important swine diseases with a significant economic burden worldwide. Unfortunately, available vaccines are partially effective highlighting the need of novel approaches. Previously, antigenic viral proteins were described in serum-derived extracellular vesicles (EVs) from pigs previously infected with PRRSV. Here, a targeted-pig trial was designed to determine the safety and immunogenicity of such extracellular vesicles enriched fractions. Our results showed that immunizations with EV-enriched fractions from convalescence animals in combination with montanide is safe and free of virus as immunizations with up-to two milligrams of EV-enriched fractions did not induce clinical symptoms, adverse effects and detectable viral replication. In addition, this vaccine formulation was able to elicit specific humoral IgG immune response in vaccinated animals, albeit variably. Noticeably, sera from vaccinated animals was diagnosed negative when tested for PRRSV using a commercial ELISA test; thus, indicating that this new approach differentiates vaccinated from infected animals. Lastly, after priming animals with EV-enriched fractions from sera of convalescence animals and boosting them with synthetic viral peptides identified by mass spectrometry, a distinctive high and specific IFN-γ response was elicited. Altogether, our data strongly suggest the use of serum EV-enriched fractions as a novel vaccine strategy against PRRSV.Anti-CD9, Anti-CD63 and anti-CD81 antibodies were kindly donated by Francisco Sánchez-Madrid and Maria Yañez-Mo, Hospital de la Princesa, Madrid, Spain. The authors wish to particularly thank Glòria Abella for her collaboration in conducting the field study and to Marta Alcobé, Miriam Moron Font and Paula Crego Mendez for technical assistance. This study received support from Innovex Therapeutics S.L., Pinsos del Segre SA, Granja Casanyé, Grup de Sanejament Porci (GSP, Lleida, Spain) and the FEDER project (COMRDI16-1-0035-03). Sergio Montanter-Tarbes is an industrial doctorate awarded by the Government of Catalonia, Spain (No. 2014 DI 044). ISGlobal and IGTP are members of the CERCA Programme, Generalitat de Catalunya

Exosome based vaccines: pros and cons in the world of animal health

Due to the emergence of antibiotic resistance and new and more complex diseases that affect livestock animal health and food security, the control of epidemics has become a top priority worldwide. Vaccination represents the most important and cost‐effective measure to control infectious diseases in animal health, but it represents only 23% of the total global animal health market, highlighting the need to develop new vaccines. A recent strategy in animal health vaccination is the use of extracellular vesicles (EVs), lipid bilayer nanovesicles produced by almost all living cells, including both prokaryotes and eukaryotes. EVs have been evaluated as a prominent source of viral antigens to elicit specific immune responses and to develop new vaccination platforms as viruses and EVs share biogenesis pathways. Preliminary trials with lymphocytic choriomeningitis virus infection (LCMV), porcine reproductive and respiratory syndrome virus (PRRSV), and Marek's disease virus (MDV) have demonstrated that EVs have a role in the activation of cellular and antibody immune responses. Moreover, in parasitic diseases such as Eimeria (chickens) and Plasmodium yoelii (mice) protection has been achieved. Research into EVs is therefore opening an opportunity for new strategies to overcome old problems affecting food security, animal health, and emerging diseases. Here, we review different conventional approaches for vaccine design and compare them with examples of EV‐based vaccines that have already been tested in relation to animal health

Key Gaps in the Knowledge of the Porcine Respiratory Reproductive Syndrome Virus (PRRSV)

The porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important swine diseases in the world. It is causing an enormous economic burden due to reproductive failure in sows and a complex respiratory syndrome in pigs of all ages, with mortality varying from 2 to 100% in the most extreme cases of emergent highly pathogenic strains. PRRSV displays complex interactions with the immune system and a high mutation rate, making the development, and implementation of control strategies a major challenge. In this review, the biology of the virus will be addressed focusing on newly discovered functions of non-structural proteins and novel dissemination mechanisms. Secondly, the role of different cell types and viral proteins will be reviewed in natural and vaccine-induced immune response together with the role of different immune evasion mechanisms focusing on those gaps of knowledge that are critical to generate more efficacious vaccines. Finally, novel strategies for antigen discovery and vaccine development will be discussed, in particular the use of exosomes (extracellular vesicles of endocytic origin). As nanocarriers of lipids, proteins and nucleic acids, exosomes have potential effects on cell activation, modulation of immune responses and antigen presentation. Thus, representing a novel vaccination approach against this devastating disease

Identification of a Newly Conserved SLA-II Epitope in a Structural Protein of Swine Influenza Virus

Despite the role of pigs as a source of new Influenza A Virus viruses (IAV) potentially capable of initiating human pandemics, immune responses to swine influenza virus (SwIV) in pigs are not fully understood. Several SwIV epitopes presented by swine MHC (SLA) class I have been identified using different approaches either in outbred pigs or in Babraham large white inbred pigs, which are 85% identical by genome wide SNP analysis. On the other hand, some class II SLA epitopes were recently described in outbred pigs. In this work, Babraham large white inbred pigs were selected to identify SLA II epitopes from SwIV H1N1. PBMCs were screened for recognition of overlapping peptides covering the NP and M1 proteins from heterologous IAV H1N1 in IFNγ ELISPOT. A novel SLA class II restricted epitope was identified in NP from swine H1N1. This conserved novel epitope could be the base for further vaccine approaches against H1N1 in pigs.info:eu-repo/semantics/publishedVersio

Assessment of extracellular vesicles and their applicability in diseases of veterinary importance

Extret el capítol 3.3 de la tesi per convenis de confidencialitat amb empreses i per possibilitat de generar patents.Les vacunes continuen sent l’estratègia més important pel control i l’erradicació de malalties infeccioses en salut pública i animal. El virus del Síndrome Respiratori i Reproductiu Porcí (SRRP) és una de les malalties porcines més importants en el món. Causa una enorme afectació econòmica degut als problemes reproductius en les femelles i al síndrome respiratori en porcs de totes les edats, en les cuals les vacunes existents indueixen una reposta immune i protectora limitada. Les vesícules extracel·lulars estan al punt de mira com a font important d’antígens i interactors entre patògens i hostes. En aquest estudi, hem caracteritzat vesícules extracel·lulars per a cercar antigens per la vacunació d’aquesta malatia porcina. En primer lloc, les vesícules extracel·lulars derivades de sèrum porcí han estat avaluades utilitzant diferents metodologies d’aïllament, caracterització i concentració, trobant proteïnes virals específiques i immunogèniques. A més a més, quan les vesícules derivades del sèrum de porcs infectats amb SRRP han estat testades en un assaig clínic de vacunació usant una estratègia prime-boost, les vesícules han demostrat ser segures, sense causar infecció ni efectes adversos i induïnt una resposta específica d’anticossos i secreció de IFN-γ. D’altra banda, els animals vacunats amb vesícules extracel·lulars van mostrar una càrrega viral inferior i uns millors resultats clínics comparats amb aquells vacunats amb el mètode clàssic de vacunes peptídiques, en els quals es van observar majors efectes adversos (major càrrega viral). Per probar la utilitat d’aquesta metodologia en altres virus una altra malaltia porcina important va ser sel·leccionada, el virus de la Febre Africana Porcina, i es va evaluar el contigunt de vesícules extracel·lulars, trobant proteïnes virals específiques similars a aquelles obtingudes en SRRP. En conclusió, les vesícules extracel·lulars representen una font important per a la identificació d’antígens immunogènics que podrien ser usats com a una nova estratègia de vacunació per induïr protecció contra malalties virals porcines tals com SRRP i podent ser extrapolades a altres malalties porcines virals.La vacunación continúa siendo una de las estrategias más importantes para el control y la erradicación de enfermedades infeccionas en salud pública y salud animal. El Síndrome Reproductivo y Respiratorio Porcino (PRRSV) es una de las enfermedades porcinas más importantes en el mundo, causante de grandes pérdidas económicas debido a las fallas en cerdas reproductoras, un síndrome respiratorio complejo en cerdos de todas las edades y en el cual las vacunas disponibles inducen una respuesta inmune y protectora limitada. Las vesículas extracelulares (EVs) son actualmente consideradas como importantes fuentes de antígenos en múltiples relaciones patógeno-hospedador. En el presente estudio, se evaluaron las vesículas extracelulares como una fuente de antígenos adecuados para vacunación en esta enfermedad de importancia veterinaria. En primer lugar, se valoraron las EVs utilizando distintas metodologías para su aislamiento, caracterización y concentración, mediante los cuales fue posible detectar proteínas virales especificas e inmunogénicas en su contenido. Además, cuando las EVs derivadas de suero de animales infectados con PRRSV se probaron en un ensayo de vacunación en cerdos utilizando una estrategia de vacunación Prime-Boost, estas demostraron ser seguras al no causar infección ni efectos secundarios, y fueron capaces de inducir una respuesta de anticuerpos y secreción de IFN-γ especifica en los animales vacunados. Conjuntamente, los animales vacunados con EVs mostraron una menor carga viral y un mejor desarrollo clínico cuando se comparó su estado con animales vacunados con un enfoque clásico de vacunas de péptidos en el cual se observó un aumento de la carga viral y otros síntomas clínicos. Para probar la aplicabilidad de esta metodología en otros virus, se selecciono la Peste Porcina Africana (PPA), otra enfermedad porcina de gran importancia como modelo para evaluar el contenido de las EVs en esta enfermedad, encontrando resultados similares a los obtenidos con PRRSV en donde se detectaron proteínas virales especificas en las fracciones enriquecidas con EVs. En conclusión, las vesículas extracelulares representan una importante fuente para la identificación de antígenos inmunogénicos que pueden ser utilizados como una nueva estrategia de vacunación para inducir protección en enfermedades porcinas virales como PRRSV y además pudiendo extrapolarse a otras enfermedades virales en el cerdo.Vaccination continues to be the most important strategy for control and eradication of infectious diseases in public and animal health. Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is one of the most important swine diseases in the world, causing an enormous economic burden due to reproductive failure in sows and a complex respiratory syndrome in pigs of all ages, in which available vaccines induce limited immune responses and protection. Extracellular vesicles (EVs) are now in the scope as important source of antigens and interactors in several pathogen-host relations. In the present research work, extracellular vesicles were evaluated as suitable antigen source for vaccination in this important swine disease. First, EVs derived from swine sera were assessed using different methodologies for isolation, characterization and concentration finding that contained specific and immunogenic viral proteins. Moreover, when PRRSV-sera derived EVs were tested in a targeted-pig vaccination trial using a prime-boost strategy, EVs demonstrated to be safe causing no infection neither secondary effects and were able to induce specific antibody and cell mediated IFN-γ secretion. In addition, EVs vaccinated animals showed less viral load and best clinical outcome when compared to those vaccinated with a classical approach of viral peptides in which adverse effects (increased viral load) were observed. To test the applicability of this approach in other viruses, African Swine fever, another important swine disease was selected and EVs content evaluated, finding similar results to those obtain in PRRSV, where specific viral proteins were associated to EVs enriched fractions. In conclusion, extracellular vesicles represent an important source to identify immunogenic antigens that could be used as a novel vaccination strategy to induce protection for porcine viral diseases such as PRRSV and can be extrapolated to other swine viral diseases

Heterogeneous populations from in vitro cultures of antigen presenting cells in pigs

25 p. (Faltan las figuras)Dendritic cells (DCs) are the most potent antigen presenting cells (APCs). Because of the difficulty in obtaining these cells directly from tissues, different sources of DCs are frequently used for in vitro experimentation and many of their biological and functional characteristics were studied using these systems. Until recently, it was assumed that specific culture conditions polarized the differentiation of either DCs or macrophages (Macs); however, it was shown that some DC culture systems in other species generate heterogeneous cell populations that can be identified according to their CD11c and MHC class II (MHC-II) expression. Following this approach, porcine DCs were directly isolated from peripheral blood or differentiated in vitro by culturing bone marrow (BM) progenitor cells or blood monocytes treated with growth factors. Mostly homogeneous monocyte-derived DCs (MoDCs) were obtained with similar phenotype and phagocytic characteristics to that of blood DCs. On the contrary, BM-derived DC (BMDC) cultures generated two distinct heterogeneous populations identified as MHC-II+ and MHC-II++ cells. BMDCs MHC-II+ had similar phenotypic and phagocytic characteristics to those of MoDCs and blood DCs. However, BMDCs MHC-II++ population expressed a higher amount of surface markers and transcribed genes associated with Macs-lineage exhibiting a higher phagocytic capacity than all the other cells. Noteworthy, every cell system expressed different genetic signatures. These results will help interpreting and re-interpreting data obtained using in vitro systems.This work was financially supported by the UK’s BBSRC grants BBS/ E/I/00002067, BBS/E/I/00002014, core capability grant BBS/E/I/ 00007039 and ASF-STOP Cost action (CA15116). MP was recipient of the fellowship CONICYT 21130626 from the Chilean Goverment.Peer reviewe

Serum-derived exosomes from non-viremic animals previously exposed to the porcine respiratory and reproductive virus contain antigenic viral proteins

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