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

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

Exosomes from Plasmodium yoelii-Infected Reticulocytes Protect Mice from Lethal Infections

Exosomes are 30–100-nm membrane vesicles of endocytic origin that are released after the fusion of multivesicular bodies (MVBs) with the plasma membrane. While initial studies suggested that the role of exosomes was limited to the removal of proteins during the maturation of reticulocytes to erythrocytes, recent studies indicate that they are produced by different types of cells and are involved in promoting inter-cellular communication and antigen presentation. Here, we describe the isolation and characterization of exosomes from peripheral blood of BALB/c mice infected with the reticulocyte-prone non-lethal Plasmodium yoelii 17X strain. Importantly, proteomic analysis revealed the presence of parasite proteins in these vesicles. Moreover, immunization of mice with purified exosomes elicited IgG antibodies capable of recognizing P. yoelii-infected red blood cells. Furthermore, lethal challenge of immunized mice with the normocyte-prone lethal P. yoelii 17XL strain caused a significant attenuation in the course of parasitaemia, increased survival time, and altered the cell tropism to reticulocytes. These results were obtained also when the exosomes were isolated from a P. yoelii-infected reticulocyte culture indicating that reticulocyte-derived exosomes carry antigens and are involved in immune modulation. Moreover, inclusion of CpG ODN 1826 in exosome immunizations elicited IgG2a and IgG2b antibodies and promoted survival, clearance of parasites and subsequent sterile protection of 83% of the animals challenged with P. yoelli 17XL. To our knowledge, this is the first report of immune responses elicited by exosomes derived from reticulocytes opening new avenues for the modulation of anti-malaria responses

Increased expression levels of the pvcrt-o and pvmdr1 genes in a patient with severe Plasmodium vivax malaria

<p>Abstract</p> <p>Background</p> <p>There are increasing reports of severe clinical cases exclusively associated with <it>Plasmodium vivax </it>infections. Notably, this severity has been recently suggested to be associated with chloroquine resistance.</p> <p>Patients</p> <p>Two different patients presented at the Hospital Clinic in Barcelona with <it>P. vivax </it>malaria episodes. One patient had severe symptoms and the other mild symptoms. Both patients traveled through the Brazilian Amazon (Manaus) in 2007. For both patients the current diagnosis of malaria was the first. Two other patients with mild symptoms presented to the "Centro de Pesquisa em Medicina Tropical", also in the Brazilian Amazon (Rondônia) in 2000.</p> <p>Methods</p> <p>To exclude the possibility that the patient's severe symptoms were due to <it>Plasmodium falciparum</it>, a nested PCR was performed. A magnetic method was used to purify <it>P. vivax </it>free of human leukocytes. Quantitative real-time PCR was performed to compare the transcript levels of two main transporters likely to be involved in chloroquine resistance in <it>P. vivax</it>, namely the <it>P. vivax </it>chloroquine resistance transporter, <it>pvcrt-o</it>, and the <it>P. vivax </it>multidrug resistance transporter, <it>pvmdr 1</it>.</p> <p>Results</p> <p>Results demonstrated that the severe clinical symptoms were exclusively due to <it>P. vivax</it>. The patient presented acute respiratory conditions requiring admission to the intensive care unit. The magnetic method showed highly purified infected-reticulocytes with mature stages. In addition, it was found that parasites obtained from the severe patient had up to 2.9-fold increase in <it>pvmdr1 </it>levels and up to 21.9-fold increase in <it>pvcrt-o </it>levels compared to expression levels of parasites from the other patients with mild symptoms.</p> <p>Conclusion</p> <p>This is the first clinical case of severe disease exclusively associated with vivax malaria in Spain. Moreover, these findings suggest that clinical severity could be associated with increased expression levels of parasite genes likely involved in chloroquine resistance. It is necessary to further explore the potential of <it>pvmdr1 </it>and particularly <it>pvcrt-o </it>expression levels as molecular markers of severe disease in <it>P. vivax</it>.</p

Development of a genetic tool for functional screening of anti-malarial bioactive extracts in metagenomic libraries

Ajuts: Departamento Administrativo de Ciencias, Tecnología e Innovación (Colciencias), República de Colombia; Convocatoria 489 - 2009, Código 657048925406, Contrato de financiación RC. 427 - 2009 Colciencias - CorpoGen; Programa de Asistencias Graduadas de Universidad de los Andes, Bogotá, Colombia; i Programa Jóvenes Investigadores de ColcienciasBACKGROUND: The chemical treatment of Plasmodium falciparum for human infections is losing efficacy each year due to the rise of resistance. One possible strategy to find novel anti-malarial drugs is to access the largest reservoir of genomic biodiversity source on earth present in metagenomes of environmental microbial communities. METHODS: A bioluminescent P. falciparum parasite was used to quickly detect shifts in viability of microcultures grown in 96-well plates. A synthetic gene encoding the Dermaseptin 4 peptide was designed and cloned under tight transcriptional control in a large metagenomic insert context (30 kb) to serve as proof-of-principle for the screening platform. RESULTS: Decrease in parasite viability consistently correlated with bioluminescence emitted from parasite microcultures, after their exposure to bacterial extracts containing a plasmid or fosmid engineered to encode the Dermaseptin 4 anti-malarial peptide. Here, a new technical platform to access the anti-malarial potential in microbial environmental metagenomes has been develope

Pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of Plasmodium vivax in human patients

BACKGROUND: Plasmodium vivax is the most widely distributed human malaria, responsible for 70–80 million clinical cases each year and large socio-economical burdens for countries such as Brazil where it is the most prevalent species. Unfortunately, due to the impossibility of growing this parasite in continuous in vitro culture, research on P. vivax remains largely neglected. METHODS: A pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of P. vivax was performed. To do so, 1,184 clones from a cDNA library constructed with parasites obtained from 10 different human patients in the Brazilian Amazon were sequenced. Sequences were automatedly processed to remove contaminants and low quality reads. A total of 806 sequences with an average length of 586 bp met such criteria and their clustering revealed 666 distinct events. The consensus sequence of each cluster and the unique sequences of the singlets were used in similarity searches against different databases that included P. vivax, Plasmodium falciparum, Plasmodium yoelii, Plasmodium knowlesi, Apicomplexa and the GenBank non-redundant database. An E-value of <10(-30 )was used to define a significant database match. ESTs were manually assigned a gene ontology (GO) terminology RESULTS: A total of 769 ESTs could be assigned a putative identity based upon sequence similarity to known proteins in GenBank. Moreover, 292 ESTs were annotated and a GO terminology was assigned to 164 of them. CONCLUSION: These are the first ESTs reported for P. vivax and, as such, they represent a valuable resource to assist in the annotation of the P. vivax genome currently being sequenced. Moreover, since the GC-content of the P. vivax genome is strikingly different from that of P. falciparum, these ESTs will help in the validation of gene predictions for P. vivax and to create a gene index of this malaria parasite

Naturally-acquired humoral immune responses against the N- and C-termini of the Plasmodium vivax MSP1 protein in endemic regions of Brazil and Papua New Guinea using a multiplex assay

<p>Abstract</p> <p>Background</p> <p>Progress towards the development of a malaria vaccine against <it>Plasmodium vivax</it>, the most widely distributed human malaria parasite, will require a better understanding of the immune responses that confer clinical protection to patients in regions where malaria is endemic.</p> <p>Methods</p> <p>Glutathione <it>S</it>-transferase (GST) and GST-fusion proteins representing the N- terminus of the merozoite surface protein 1 of <it>P. vivax</it>, PvMSP1-N, and the C-terminus, PvMSP1-C, were covalently coupled to BioPlex carboxylated beads. Recombinant proteins and coupled beads were used, respectively, in ELISA and Bioplex assays using immune sera of <it>P. vivax </it>patients from Brazil and PNG to determine IgG and subclass responses. Concordances between the two methods in the seropositivity responses were evaluated using the Kappa statistic and the Spearman's rank correlation.</p> <p>Results</p> <p>The results using this methodology were compared with the classical microtitre enzyme-linked immnosorbent assay (ELISA), showing that the assay was sensitive, reproducible and had good concordance with ELISA; yet, further research into different statistical analyses seems desirable before claiming conclusive results exclusively based on multiplex assays. As expected, results demonstrated that PvMSP1 was immunogenic in natural infections of patients from different endemic regions of Brazil and Papua New Guinea (PNG), and that age correlated only with antibodies against the C-terminus part of the molecule. Furthermore, the IgG subclass profiles were different in these endemic regions having IgG3 predominantly recognizing PvMSP1 in Brazil and IgG1 predominantly recognizing PvMSP1 in PNG.</p> <p>Conclusions</p> <p>This study validates the use of the multiplex assay to measure naturally-acquired IgG antibodies against the merozoite surface protein 1 of <it>P. vivax</it>.</p