Institute of Virology and Immunoprophylaxis


Institute of Virology and Immunoprophylaxis

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Summerfield A.,Institute of Virology and Immunoprophylaxis
Veterinary Immunology and Immunopathology | Year: 2012

Since type I interferon (IFN-I) is considered a potent antiviral defence mechanism, it is not surprising that during evolution viruses have development of various mechanisms to counteract IFN-I induction or release. Despite this, certain virus infections are associated with very high levels of systemic IFN-I. One explanation for this observation is the presence of plasmacytoid dendritic cells (pDC), which are able to produce high levels of IFN-I despite the presence of viral IFN-I antagonists. Examples of virus infection in pigs including classical swine fever virus, influenza virus, foot-and-mouth disease virus, and porcine circo virus type 2 highlight factors involved in controlling such responses and illustrate potential negative and positive effects for the host. Based on published data, we propose that in addition to the ability to activate pDC, the ability to spread systemically, and the tropism for lymphoid tissue also represent important factors contributing to strong systemic IFN-I responses during certain virus infections. © 2011 Elsevier B.V.

Thomann-Harwood L.J.,Institute of Virology and Immunoprophylaxis | Kaeuper P.,Medipol SA | Rossi N.,Medipol SA | Milona P.,Institute of Virology and Immunoprophylaxis | And 2 more authors.
Journal of Controlled Release | Year: 2013

Dendritic cells (DCs) play crucial roles in initiating and promoting immune defences, providing a pivotal target for vaccines. Although nanoparticle/nanogel-based delivery vehicles are showing potential for delivering vaccines to the immune system, there is little information on their characteristics of interaction with DCs. While particle uptake by DCs has been shown, the mechanism of cell targeting has not been studied. Moreover, it is still unclear how particle surface decoration influences the handling of such vaccines by DCs. Accordingly, chitosan nanogels carrying a model antigen, ovalbumin (ova), were analysed for interaction with and processing by DCs. Nanogel surfaces decorated with alginate (alg) or mannosylated alginate (alg-man), were used for targeting particular DC receptors. DC uptake of particles was observed, being dependent on endosomal-based processes. Inhibiting PI3-kinase or lipid raft activities impaired the uptake, which was only reduced, indicating the involvement of more than one endocytic pathway; notably, this was observed with both nanogel-delivered or free ova. Importantly, surface decoration of particles was less influential on particle uptake, contrasting with the ova cargo which played the major role. Such influence of the vaccine cargo has to date been largely ignored. When receptors interacting directly with ova were blocked, this altered the uptake of alg-nanogels and alg-man-nanogels carrying ova. The nanogels did have an influential role, in that modulation of DC functional activity owed more to the nanogel structure. Using an in vitro restimulation assay with ova-specific lymphocytes, nanogel-delivered and free ova were similarly effective at inducing specific antibody. Nanogel-delivered ova with mannose surface decoration was superior to free ova for inducing interferon-γ production by T-lymphocytes. Together, the data demonstrates that particle-based vaccine delivery should consider the influences of both the surface decoration and the vaccine cargo; each can influence different aspects of the interaction with DCs. Such combined influences are likely to impinge on the characteristics of the immune response induced. © 2012 Elsevier B.V.

McCullough K.C.,Institute of Virology and Immunoprophylaxis | Bassi I.,Institute of Virology and Immunoprophylaxis | Demoulins T.,Institute of Virology and Immunoprophylaxis | Thomann-Harwood L.J.,Institute of Virology and Immunoprophylaxis | Ruggli N.,Institute of Virology and Immunoprophylaxis
Therapeutic Delivery | Year: 2012

Dendritic cells (DCs) are essential to many aspects of immune defense development and regulation. They provide important targets for prophylactic and therapeutic delivery. While protein delivery has had considerable success, RNA delivery is still expanding. Delivering RNA molecules for RNAi has shown particular success and there are reports on successful delivery of mRNA. Central, therein, is the application of cationic entities. Following endocytosis of the delivery vehicle for the RNA, cationic entities should promote vesicular membrane perturbation, facilitating cytosolic release. The present review explains the diversity of DC function in immune response development and control. Promotion of delivered RNA cytosolic release is discussed, relating to immunoprophylactic and therapeutic potential, and DC endocytic machinery is reviewed, showing how DC endocytic pathways influence the handling of internalized material. The potential advantages for application of replicating RNA are presented and discussed, in consideration of their value and development in the near future. © 2012 Future Science Ltd.

Guzylack-Piriou L.,French National Institute for Agricultural Research | Alves M.P.,Institute of Virology and Immunoprophylaxis | McCullough K.C.,Institute of Virology and Immunoprophylaxis | Summerfield A.,Institute of Virology and Immunoprophylaxis
Developmental and Comparative Immunology | Year: 2010

Based on the known importance of Flt3 ligand (Flt3L) for the development of mouse dendritic cells (DCs), the present study compared the phenotype and function of DC derived from porcine bone marrow haematopoietic cells using either granulocyte-macrophage colony-stimulating factor or Flt3L (GMCSF-DC and Flt3L-DC, respectively). To this end, porcine Flt3L was cloned resulting in the identification of three isoforms of Flt3L. Compared to GMCSF-DC which were uniformly CD14+, Flt3L-DC had a more diverse phenotype comprised of CD172a-CD11a- progenitor cells, CD172a+CD14-CD163- DC and CD172a+CD14+CD163+ DC. In addition, only the Flt3L-DC contained interferon-producing plasmacytoid DC, although their frequency was low. Only the CD14- Flt3L-DC responded to TLR2, -3, -4, -7 and -9 agonists by upregulating CD80/86. This population of DC was also more potent in T-cell stimulation assays when compared to the CD14+ counterpart. Interestingly, Flt3 was not only highly expressed on DC precursors, but also found on Flt3L-DC but not on GMCSF-DC or monocyte-derived DC. Furthermore, also DC circulating in the blood but not monocytes or other leukocytes expressed this receptor. Taken together, our study demonstrates that Flt3L-DCs are more suitable to study the interaction of pathogens with DC. Moreover, we show that also in the pig Flt3 remains expressed in a restricted manner on DC originating from a bone marrow DC precursors, typically representing steady-state DC in lymphoid tissue and blood. © 2009 Elsevier Ltd. All rights reserved.

Saiz J.-C.,Instituto Nacional Of Investigacion Y Tecnologia Agraria Y Alimentaria Inia | McCullough K.C.,Institute of Virology and Immunoprophylaxis
Virus Research | Year: 2012

Virus infection of host cells requires that entry into the cell results in efficient genome release leading to translation and replication. These initial steps revolving around the entry and genomic release processes are crucial for viral progeny generation. Despite the variety of receptors used by viruses to initiate entry, evidence from both enveloped and non-enveloped viral infections is highlighting the important role played by intracellular acidic compartments in the entry of many viruses. These compartments provide connecting nodes within the endocytic network, presenting multiple viral internalization pathways. Endosomal compartments employing an internal acidic pH can trigger molecular mechanisms leading to disassembly of viral particles, thus providing appropriate genome delivery. Accordingly, viruses have evolved to select optimal intracellular conditions for promoting efficient genome release, leading to propagation of the infectious agent. This review will address the implications of cellular compartment involvement in virus infectious processes, and the roles played by the viruses' own machinery, including pH sensing mechanisms and the methodologies applied for studying acid-dependent viral entry into host cells. © 2012 Elsevier B.V.

Tappeiner C.,Inselspital | Flueckiger F.,Institute of Virology and Immunoprophylaxis | Boehnke M.,Institute of Ophthalmology | Goldblum D.,University of Basel | Garweg J.G.,University of Bern
Journal of Cataract and Refractive Surgery | Year: 2012

Purpose: To assess the impact of topical anesthetic agents and ethanol on ocular surface wound healing using an ex vivo whole-globe porcine model. Setting: Department of Ophthalmology, Inselspital, University of Bern, Bern, Switzerland. Design: Experimental study. Methods: Standardized corneoepithelial lesions (5.0 mm diameter, 40 μm depth) were created with excimer laser light in freshly enucleated porcine eyes. The globes (6 per group) were exposed to different concentrations of ethanol (2.0% to 99.0%), cocaine (2.0% to 10.0%), procaine hydrochloride (0.4%), tetracaine (0.5% to 1.0%), or lidocaine (2.0%), 3 drops/hour for 3 hours. Control solutions were physiologic saline, balanced salt solution, and tissue-culture medium. After 20 to 26 hours, wound-healing response was compared by measuring the diameter of each corneoepithelial lesion. Results: The mean diameter of corneoepithelial lesions exposed to physiologic saline decreased from 4.78 mm ± 0.19 (SD) to 4.44 ± 0.17 mm between 20 and 26 hours. After 24 hours, the mean lesion size, compared with physiological saline, was larger after cocaine 5.0% (5.20 ± 0.26 mm) and 10.0% (5.39 ± 0.12 mm), tetracaine 0.5% (5.59 ± 0.35 mm) and 1.0% (5.55 ± 0.27 mm), and procaine hydrochloride 0.4% (5.76 ± 0.12 mm), but not after lidocaine 2.0% (5.01 ± 0.17 mm). Balanced salt solution, tissue-culture medium, ethanol 2.0% to 99.0%, and cocaine 2.0% did not inhibit the wound-healing response. Conclusions: In an ex vivo whole-globe porcine model, lidocaine 2.0% and cocaine 2.0% were the least toxic anesthetic agents. At all concentrations, ethanol had no impact on wound healing. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned. © 2012 ASCRS and ESCRS.

Haines F.J.,Animal Health and Veterinary Laboratories Agency | Hofmann M.A.,Institute of Virology and Immunoprophylaxis | King D.P.,The Pirbright Institute | Drew T.W.,Animal Health and Veterinary Laboratories Agency | Crooke H.R.,Animal Health and Veterinary Laboratories Agency
PLoS ONE | Year: 2013

A single-step, multiplex, real-time polymerase chain reaction (RT-PCR) was developed for the simultaneous and differential laboratory diagnosis of Classical swine fever virus (CSFV) and African swine fever virus (ASFV) alongside an exogenous internal control RNA (IC-RNA). Combining a single extraction methodology and primer and probe sets for detection of the three target nucleic acids CSFV, ASFV and IC-RNA, had no effect on the analytical sensitivity of the assay and the new triplex RT-PCR was comparable to standard PCR techniques for CSFV and ASFV diagnosis. After optimisation the assay had a detection limit of 5 CSFV genome copies and 22 ASFV genome copies. Analytical specificity of the triplex assay was validated using a panel of viruses representing 9 of the 11 CSFV subgenotypes, at least 8 of the 22 ASFV genotypes as well as non-CSFV pestiviruses. Positive and negative clinical samples from animals infected experimentally, due to field exposure or collected from the UK which is free from both swine diseases, were used to evaluate the diagnostic sensitivity and specificity for detection of both viruses. The diagnostic sensitivity was 100% for both viruses whilst diagnostic specificity estimates were 100% for CSFV detection and 97.3% for ASFV detection. The inclusion of a heterologous internal control allowed identification of false negative results, which occurred at a higher level than expected. The triplex assay described here offers a valuable new tool for the differential detection of the causative viruses of two clinically indistinguishable porcine diseases, whose geographical occurrence is increasingly overlapping. © 2013 Haines et al.

Sharma R.,Institute of Virology and Immunoprophylaxis | Ghasparian A.,University of Zürich | Robinson J.A.,University of Zürich | McCullough K.C.,Institute of Virology and Immunoprophylaxis
PLoS ONE | Year: 2012

DC employ several endocytic routes for processing antigens, driving forward adaptive immunity. Recent advances in synthetic biology have created small (20-30 nm) virus-like particles based on lipopeptides containing a virus-derived coiled coil sequence coupled to synthetic B- and T-cell epitope mimetics. These self-assembling SVLP efficiently induce adaptive immunity without requirement for adjuvant. We hypothesized that the characteristics of DC interaction with SVLP would elaborate on the roles of cell membrane and intracellular compartments in the handling of a virus-like entity known for its efficacy as a vaccine. DC rapidly bind SVLP within min, co-localised with CTB and CD9, but not caveolin-1. In contrast, internalisation is a relatively slow process, delivering SVLP into the cell periphery where they are maintained for a number of hrs in association with microtubules. Although there is early association with clathrin, this is no longer seen after 10 min. Association with EEA-1+ early endosomes is also early, but proteolytic processing appears slow, the SVLP-vesicles remaining peripheral. Association with transferrin occurs rarely, and only in the periphery, possibly signifying translocation of some SVLP for delivery to B-lymphocytes. Most SVLP co-localise with high molecular weight dextran. Uptake of both is impaired with mature DC, but there remains a residual uptake of SVLP. These results imply that DC use multiple endocytic routes for SVLP uptake, dominated by caveolin-independent, lipid raft-mediated macropinocytosis. With most SVLP-containing vesicles being retained in the periphery, not always interacting with early endosomes, this relates to slow proteolytic degradation and antigen retention by DC. The present characterization allows for a definition of how DC handle virus-like particles showing efficacious immunogenicity, elements valuable for novel vaccine design in the future. © 2012 Sharma et al.

Lannes N.,Institute of Virology and Immunoprophylaxis | Summerfield A.,Institute of Virology and Immunoprophylaxis
PLoS ONE | Year: 2013

Plasmacytoid dendritic cells (pDC) are the most potent producers of type-I interferon (IFN) and represent the main interferon (IFN)-α source in response to many viruses. Considering the important roles played by type I IFN's, not only as antiviral effectors but also as potent alarming cytokine of the immune system, we investigated how such responses are regulated by various cytokines. To this end, we stimulated enriched pDC in the presence or absence of particular cytokines with a strong activator, CpG DNA, or a weak activator of pDC, foot-and-mouth disease virus (FMDV). Alternatively, we pre-incubated pDC for 16 h before stimulation. The pro-inflammatory cytokines tested Interleukin (IL)-6, IL17A, tumour necrosis factor (TNF)-α did not influence IFN-α responses except TNF-α, which promoted responses induced by FMDV. The haematopoietic cytokines Fms-related tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) had enhancing effects on pDC activation at least in one of the protocols tested. IFN-β and IFN-γ were the most potent at enhancing FMDV-induced IFN-α, up to 10-fold. Interestingly, also the Th2 cytokine IL-4 was an efficient promoter of pDC activity, while IL-10 was the only negative regulator of IFN-α in pDC identified. The cytokines enhancing IFN-α responses also promoted pDC survival in cell culture with the exception of GM-CSF. Taken together this work illustrates how the cytokine network can influence pDC activation, a knowledge of relevance for improving vaccines and therapeutic interventions during virus infections, cancers and autoimmune diseases in which pDC play a role. © 2013 Lannes, Summerfield.

Bruckner L.,Institute of Virology and Immunoprophylaxis
Journal of Comparative Pathology | Year: 2010

In Europe, two main regulatory texts regulate the immunogenicity requirements of vaccines, the European Pharmacopoeia and the guidelines of the European Medicines Evaluation Agency (EMEA). This article outlines these requirements and the regulatory challenges. © 2009 Elsevier Ltd. All rights reserved.

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