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.

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-016-0345-x) contains supplementary material, which is available to authorized users.

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.

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.

African swine fever is a devastating hemorrhagic infectious disease, which affects domestic and wild swines (Sus scrofa) of all breeds and ages, with a high lethality of up to 90–100% in naïve animals. The causative agent, African swine fever virus (ASFV), is a large and complex double-stranded DNA arbovirus which is currently spreading worldwide, with serious socioeconomic consequences. There is no treatment or effective vaccine commercially available, and most of the current research is focused on attenuated viral models, with limited success so far. Thus, new strategies are under investigation. Extracellular vesicles (EVs) have proven to be a promising new vaccination platform for veterinary diseases in situations in which conventional approaches have not been completely successful. Here, serum extracellular vesicles from infected pigs using two different ASFV viruses (OURT 88/3 and Benin ΔMGF), corresponding to a naturally attenuated virus and a deletion mutant, respectively, were characterized in order to determine possible differences in the content of swine and viral proteins in EV-enriched fractions. Firstly, EVs were characterized by their CD5, CD63, CD81 and CD163 surface expression. Secondly, ASFV proteins were detected on the surface of EVs from ASFV-infected pig serum. Finally, proteomic analysis revealed few specific proteins from ASFV in the EVs, but 942 swine proteins were detected in all EV preparations (negative controls, and OURT 88/3 and Benin ΔMGF-infected preparations). However, in samples from OURT 88/3-infected animals, only a small number of proteins were differentially identified compared to control uninfected animals. Fifty-six swine proteins (Group Benin) and seven proteins (Group OURT 88/3) were differentially detected on EVs when compared to the EV control group. Most of these were related to coagulation cascades. The results presented here could contribute to a better understanding of ASFV pathogenesis and immune/protective responses in the host.

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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.