New Generation Self-Replicating RNA Vaccines Derived from Pestivirus Genome.

While mRNA vaccines have shown their worth, they have the same failing as inactivated vaccines, namely they have limited half-life, are non-replicating, and therefore limited to the size of the vaccine payload for the amount of material translated. New advances averting these problems are combining replicon RNA (RepRNA) technology with nanotechnology. RepRNA are large self-replicating RNA molecules (typically 12-15 kb) derived from viral genomes defective in at least one essential structural protein gene. They provide sustained antigen production, effectively increasing vaccine antigen payloads over time, without the risk of producing infectious progeny. The major limitations with RepRNA are RNase-sensitivity and inefficient uptake by dendritic cells (DCs), which need to be overcome for efficacious RNA-based vaccine design. We employed biodegradable delivery vehicles to protect the RepRNA and promote DC delivery. Condensing RepRNA with polyethylenimine (PEI) and encapsulating RepRNA into novel Coatsome-replicon vehicles are two approaches that have proven effective for delivery to DCs and induction of immune responses in vivo.

Differential effects of the stress peptides PACAP and CRF on sleep architecture in mice.

Stress produces profound effects on behavior, including persistent alterations in sleep patterns. Here we examined the effects of two prototypical stress peptides, pituitary adenylate cyclase-activating polypeptide (PACAP) and corticotropin-releasing factor (CRF), on sleep architecture and other translationally-relevant endpoints. Male and female mice were implanted with subcutaneous transmitters enabling continuous measurement of electroencephalography (EEG) and electromyography (EMG), as well as body temperature and locomotor activity, without tethering that restricts free movement, body posture, or head orientation during sleep. At baseline, females spent more time awake (AW) and less time in slow wave sleep (SWS) than males. Mice then received intracerebral infusions of PACAP or CRF at doses producing equivalent increases in anxiety-like behavior. The effects of PACAP on sleep architecture were similar in both sexes and resembled those reported in male mice after chronic stress exposure. Compared to vehicle infusions, PACAP infusions decreased time in AW, increased time in SWS, and increased rapid eye movement sleep (REM) time and bouts on the day following treatment. In addition, PACAP effects on REM time remained detectable a week after treatment. PACAP infusions also reduced body temperature and locomotor activity. Under the same experimental conditions, CRF infusions had minimal effects on sleep architecture in either sex, causing only transient increases in SWS during the dark phase, with no effects on temperature or activity. These findings suggest that PACAP and CRF have fundamentally different effects on sleep-related metrics, and provide new insights into the mechanisms by which stress disrupts sleep.

Coatsome-replicon vehicles: Self-replicating RNA vaccines against infectious diseases.

Herein, we provide the first description of a synthetic delivery method for self-replicating replicon RNAs (RepRNA) derived from classical swine fever virus (CSFV) using a Coatsome-replicon vehicle based on Coatsome® SS technologies. This results in an unprecedented efficacy when compared to well-established polyplexes, with up to ∼65 fold-increase of the synthesis of RepRNA-encoded gene of interest (GOI). We demonstrated the efficacy of such Coatsome-replicon vehicles for RepRNA-mediated induction of CD8 T-cell responses in mice. Moreover, we provide new insights on physical properties of the RepRNA, showing that the removal of all CSFV structural protein genes has a positive effect on the translation of the GOI. Finally, we successfully engineered RepRNA constructs encoding a porcine reproductive and respiratory syndrome virus (PRRSV) antigen, providing an example of antigen expression with potential application to combat viral diseases. The versatility and simplicity of modifying and manufacturing these Coatsome-replicon vehicle formulations represents a major asset to tackle foreseeable emerging pandemics.

Self-Amplifying Pestivirus Replicon RNA Encoding Influenza Virus Nucleoprotein and Hemagglutinin Promote Humoral and Cellular Immune Responses in Pigs.

Self-amplifying replicon RNA (RepRNA) promotes expansion of mRNA templates encoding genes of interest through their replicative nature, thus providing increased antigen payloads. RepRNA derived from the non-cytopathogenic classical swine fever virus (CSFV) targets monocytes and dendritic cells (DCs), potentially promoting prolonged antigen expression in the DCs, contrasting with cytopathogenic RepRNA. We engineered pestivirus RepRNA constructs encoding influenza virus H5N1 (A/chicken/Yamaguchi/7/2004) nucleoprotein (Rep-NP) or hemagglutinin (Rep-HA). The inherent RNase-sensitivity of RepRNA had to be circumvented to ensure efficient delivery to DCs for intracellular release and RepRNA translation; we have reported how only particular synthetic delivery vehicle formulations are appropriate. The question remained concerning RepRNA packaged in virus replicon particles (VRPs); we have now compared an efficient polyethylenimine (PEI)-based formulation (polyplex) with VRP-delivery as well as naked RepRNA co-administered with the potent bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP) adjuvant. All formulations contained a Rep-HA/Rep-NP mix, to assess the breadth of both humoral and cell-mediated defences against the influenza virus antigens. Assessment employed pigs for their close immunological relationship to humans, and as natural hosts for influenza virus. Animals receiving the VRPs, as well as PEI-delivered RepRNA, displayed strong humoral and cellular responses against both HA and NP, but with VRPs proving to be more efficacious. In contrast, naked RepRNA plus c-di-AMP could induce only low-level immune responses, in one out of five pigs. In conclusion, RepRNA encoding different influenza virus antigens are efficacious for inducing both humoral and cellular immune defences in pigs. Comparisons showed that packaging within VRP remains the most efficacious for delivery leading to induction of immune defences; however, this technology necessitates employment of expensive complementing cell cultures, and VRPs do not target human cells. Therefore, choosing the appropriate synthetic delivery vehicle still offers potential for rapid vaccine design, particularly in the context of the current coronavirus pandemic.

Mucosal Administration of Cycle-Di-Nucleotide-Adjuvanted Virosomes Efficiently Induces Protection against Influenza H5N1 in Mice.

The need for more effective influenza vaccines is highlighted by the emergence of novel influenza strains, which can lead to new pandemics. There is a growing population of susceptible subjects at risk for severe complications of influenza, such as the elderly who are only in part protected by current licensed seasonal vaccines. One strategy for improving seasonal and pandemic vaccines takes advantage of adjuvants to boost and modulate evoked immune responses. In this study, we examined the capacity of the recently described adjuvant cyclic di-adenosine monophosphate (c-di-AMP) to serve as an adjuvant for improved mucosal influenza vaccines, and induce effective protection against influenza H5N1. In detail, c-di-AMP promoted (i) effective local and systemic humoral immune responses, including protective hemagglutination inhibition titers, (ii) effective cellular responses, including multifunctional T cell activity, (iii) induction of long-lasting immunity, and (iv) protection against viral challenge. Furthermore, we demonstrated the dose-sparing capacity of the adjuvant as well as the ability to evoke cross-clade protective immune responses. Overall, our results suggest that c-di-AMP contributes to the generation of a protective cell-mediated immune response required for efficacious vaccination against influenza, which supports the further development of c-di-AMP as an adjuvant for seasonal and pandemic influenza mucosal vaccines.

Self-replicating RNA vaccine functionality modulated by fine-tuning of polyplex delivery vehicle structure.

The major limitations with large and complex self-amplifying RNA vaccines (RepRNA) are RNase-sensitivity and inefficient translation in dendritic cells (DCs). Condensing RepRNA with polyethylenimine (PEI) gave positive in vitro readouts, but was largely inferior to virus-like replicon particles (VRP) or direct electroporation. In the present study, we improved such polyplex formulation and determined that fine-tuning of the polyplex structure is essential for ensuring efficacious translation. Thereby, three parameters dominate: (i) PEI molecular weight (MW); (ii) RepRNA:PEI (weight:weight) ratio; and (iii) inclusion of cell penetrating peptides (CPPs). Seven commercially available linear PEIs (MW 2,500-250,000) were classified as strong, intermediate or low for their aptitude at complexing and protecting RepRNA for delivery into porcine blood DCs. Inclusion of (Arg)9 or TAT(57-57) CPPs further modified the translation readouts, but varied for different gene expressions. Dependent on the formulation, translation of the gene of interest (GOI) inserted into the RepRNA (luciferase, or influenza virus hemagglutinin or nucleoprotein) could decrease, while the RepRNA structural gene (E2) translation increased. This was noted in the porcine SK6 cell line, as well as both porcine and, for the first time, human DCs. Two formulations - [Rep/PEI-4,000 (1:3)] and [Rep/PEI-40,000 (1:2)/(Arg)9] were efficacious in vivo in mice and pigs, where specific CD8+ T and CD4+ T-cell responses against the GOI-encoded antigen were observed for the first time. The results demonstrate that different polyplex formulations differ in their interaction with the RepRNA such that only certain genes can be translated. Thus, delivery of these large self-replicating RNA molecules require definition with respect to translation of different genes, rather than just the GOI as is the norm, for identifying optimal delivery for the desired immune activation in vivo.

Immune dysfunctionality of replicative senescent mesenchymal stromal cells is corrected by IFNγ priming.

Industrial-scale expansion of mesenchymal stromal cells (MSCs) is often used in clinical trials, and the effect of replicative senescence on MSC functionality is of mechanistic interest. Senescent MSCs exhibit cell-cycle arrest, cellular hypertrophy, and express the senescent marker ß-galactosidase. Although both fit and senescent MSCs display intact lung-homing properties in vivo, senescent MSCs acquire a significant defect in inhibiting T-cell proliferation and cytokine secretion in vitro. IFNγ does not upregulate HLA-DR on senescent MSCs, whereas its silencing did not reverse fit MSCs' immunosuppressive properties. Secretome analysis of MSC and activated peripheral blood mononuclear cell coculture demonstrate that senescent MSCs are significantly defective in up (vascular endothelial growth factor [VEGF], granulocyte colony-stimulating factor [GCSF], CXCL10, CCL2) or down (IL-1ra, IFNγ, IL-2r, CCL4, tumor necrosis factor-α, IL-5) regulating cytokines/chemokines. Unlike indoleamine 2,3 dioxygenase (IDO), silencing of CXCL9, CXCL10, CXCL11, GCSF, CCL2, and exogenous addition of VEGF, fibroblast growth factor-basic do not modulate MSCs' immunosuppressive properties. Kynurenine levels were downregulated in senescent MSC cocultures compared with fit MSC counterparts, and exogenous addition of kynurenine inhibits T-cell proliferation in the presence of senescent MSCs. IFNγ prelicensing activated several immunomodulatory genes including IDO in fit and senescent MSCs at comparable levels and significantly enhanced senescent MSCs' immunosuppressive effect on T-cell proliferation. Our results define immune functional defects acquired by senescent MSCs, which are reversible by IFNγ prelicensing.

Self-Replicating RNA Vaccine Delivery to Dendritic Cells.

Most current vaccines are either inactivated pathogen-derived or protein/peptide-based, although attenuated and vector vaccines have also been developed. The former induce at best moderate protection, even as multimeric antigen, due to limitations in antigen loads and therefore capacity for inducing robust immune defense. While attenuated and vector vaccines offer advantages through their replicative nature, drawbacks and risks remain with potential reversion to virulence and interference from preexisting immunity. New advances averting these problems are combining self-amplifying replicon RNA (RepRNA) technology with nanotechnology. RepRNA are large self-replicating RNA molecules (12-15 kb) derived from viral genomes defective in at least one structural protein gene. They provide sustained antigen production, effectively increasing vaccine antigen payloads over time, without the risk of producing infectious progeny. The major limitation with RepRNA is RNase-sensitivity and inefficient uptake by dendritic cells (DCs)-absolute requirements for efficacious vaccine design. We employed biodegradable delivery vehicles to protect the RepRNA and promote DC delivery. Encapsulating RepRNA into chitosan nanoparticles, as well as condensing RepRNA with polyethylenimine (PEI), cationic lipids, or chitosans, has proven effective for delivery to DCs and induction of immune responses in vivo.

Polyethylenimine-based polyplex delivery of self-replicating RNA vaccines.

Self-amplifying replicon RNA (RepRNA) are large molecules (12-14 kb); their self-replication amplifies mRNA template numbers, affording several rounds of antigen production, effectively increasing vaccine antigen payloads. Their sensitivity to RNase-sensitivity and inefficient uptake by dendritic cells (DCs) - absolute requirements for vaccine design - were tackled by condensing RepRNA into synthetic, nanoparticulate, polyethylenimine (PEI)-polyplex delivery vehicles. Polyplex-delivery formulations for small RNA molecules cannot be transferred to RepRNA due to its greater size and complexity; the N:P charge ratio and impact of RepRNA folding would influence polyplex condensation, post-delivery decompaction and the cytosolic release essential for RepRNA translation. Polyplex-formulations proved successful for delivery of RepRNA encoding influenza virus hemagglutinin and nucleocapsid to DCs. Cytosolic translocation was facilitated, leading to RepRNA translation. This efficacy was confirmed in vivo, inducing both humoral and cellular immune responses. Accordingly, this paper describes the first PEI-polyplexes providing efficient delivery of the complex and large, self-amplifying RepRNA vaccines. FROM THE CLINICAL EDITOR: The use of self-amplifying replicon RNA (RepRNA) to increase vaccine antigen payloads can potentially be useful in effective vaccine design. Nonetheless, its use is limited by the degradation during the uptake process. Here, the authors attempted to solve this problem by packaging RepRNA using polyethylenimine (PEI)-polyplex delivery vehicles. The efficacy was confirmed in vivo by the appropriate humoral and cellular immune responses. This novel delivery method may prove to be very useful for future vaccine design.

Synthetic virus-like particles target dendritic cell lipid rafts for rapid endocytosis primarily but not exclusively by macropinocytosis.

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.

Chicken cells sense influenza A virus infection through MDA5 and CARDIF signaling involving LGP2.

Avian influenza viruses (AIV) raise worldwide veterinary and public health concerns due to their potential for zoonotic transmission. While infection with highly pathogenic AIV results in high mortality in chickens, this is not necessarily the case in wild birds and ducks. It is known that innate immune factors can contribute to the outcome of infection. In this context, retinoic acid-inducible gene I (RIG-I) is the main cytosolic pattern recognition receptor known for detecting influenza A virus infection in mammalian cells. Chickens, unlike ducks, lack RIG-I, yet chicken cells do produce type I interferon (IFN) in response to AIV infection. Consequently, we sought to identify the cytosolic recognition elements in chicken cells. Chicken mRNA encoding the putative chicken analogs of CARDIF and LGP2 (chCARDIF and chLGP2, respectively) were identified. HT7-tagged chCARDIF was observed to associate with mitochondria in chicken DF-1 fibroblasts. The exogenous expression of chCARDIF, as well as of the caspase activation and recruitment domains (CARDs) of the chicken melanoma differentiation-associated protein 5 (chMDA5), strongly activated the chicken IFN-ß (chIFN-ß) promoter. The silencing of chMDA5, chCARDIF, and chIRF3 reduced chIFN-ß levels induced by AIV, indicating their involvement in AIV sensing. As with mammalian cells, chLGP2 had opposing effects. While overexpression decreased the activation of the chIFN-ß promoter, the silencing of endogenous chLGP2 reduced chIFN-ß induced by AIV. We finally demonstrate that the chMDA5 signaling pathway is inhibited by the viral nonstructural protein 1. In conclusion, chicken cells, including DF-1 fibroblasts and HD-11 macrophage-like cells, employ chMDA5 for sensing AIV.

Propagation of classical swine fever virus in vitro circumventing heparan sulfate-adaptation.

Amplification of natural virus isolates in permanent cell lines can result in adaptation, in particular enhanced binding to heparan sulfate (HS)-containing glycosaminoglycans present on most vertebrate cells. This has been reported for several viruses, including the pestivirus classical swine fever virus (CSFV), the causative agent of a highly contagious hemorrhagic disease in pigs. Propagation of CSFV in cell culture is essential in virus diagnostics and research. Adaptation of CSFV to HS-binding has been related to amino acid changes in the viral E(rns) glycoprotein, resulting in viruses with altered replication characteristics in vitro and in vivo. Consequently, a compound blocking the HS-containing structures on cell surfaces was employed to monitor conversion from HS-independency to HS-dependency. It was shown that the porcine PEDSV.15 cell line permitted propagation of CSFV within a limited number of passages without adaptation to HS-binding. The selection of HS-dependent CSFV mutants was also prevented by propagation of the virus in the presence of DSTP 27. The importance of these findings can be seen from the altered ratio of cell-associated to secreted virus upon acquisition of enhanced HS-binding affinity, a phenotype proposed previously to be related to virulence in the natural host.

Porcine circovirus type 2 DNA influences cytoskeleton rearrangements in plasmacytoid and monocyte-derived dendritic cells.

Functional disruption of dendritic cells (DC) is an important strategy for viral pathogens to evade host defences. In this context, porcine circovirus type 2 (PCV2), a single-stranded DNA virus, impairs plasmacytoid DC (pDC) and conventional DC activation by certain viruses or Toll-like receptor (TLR) ligands. This inhibitory capacity is associated with the viral DNA, but the impairment does not affect all signalling cascades; TLR7 ligation by small chemical molecules will still induce interleukin-6 (IL-6) and tumour necrosis factor-α secretion, but not interferon-α or IL-12. In this study, the molecular mechanisms by which silencing occurs were investigated. PP2, a potent inhibitor of the Lyn and Hck kinases, produced a similar profile to the PCV2 DNA interference with cytokine secretion by pDC, efficiently inhibiting cell activation induced through TLR9, but not TLR7, ligation. Confocal microscopy and cytometry analysis strongly suggested that PCV2 DNA impairs actin polymerization and endocytosis in pDC and monocyte-derived DC, respectively. Altogether, this study delineates for the first time particular molecular mechanisms involved in PCV2 interference with DC danger recognition, which may be responsible for the virus-induced immunosuppression observed in infected pigs.

Immunogenic and replicative properties of classical swine fever virus replicon particles modified to induce IFN-α/ß and carry foreign genes.

Virus replicon particles (VRP) are genetically engineered infectious virions incapable of generating progeny virus due to partial or complete deletion of at least one structural gene. VRP fulfil the criteria of a safe vaccine and gene delivery system. With VRP derived from classical swine fever virus (CSF-VRP), a single intradermal vaccination protects from disease. Spreading of the challenge virus in the host is however not completely abolished. Parameters that are critical for immunogenicity of CSF-VRP are not well characterized. Considering the importance of type I interferon (IFN-α/ß) to immune defence development, we generated IFN-α/ß-inducing VRP to determine how this would influence vaccine efficacy. We also evaluated the effect of co-expressing granulocyte macrophage colony-stimulating factor (GM-CSF) in the vaccine context. The VRP were capable of long-term replication in cell culture despite the presence of IFN-α/ß. In vivo, RNA replication was essential for the induction of an immune response. IFN-α/ß-inducing and GM-CSF-expressing CSF-VRP were similar to unmodified VRP in terms of antibody and peripheral T-cell responses, and in reducing the blood levels of challenge virus RNA. Importantly, the IFN-α/ß-inducing VRP did show increased efficacy over the unmodified VRP in terms of B-cell and T-cell responses, when tested with secondary immune responses by in vitro restimulation assay.

Cellular processes essential for African swine fever virus to infect and replicate in primary macrophages.

The macrophage (Mø) is an essential immune cell for innate immunity. Such cells are targeted by African swine fever virus (ASFV). The early phases of infection with ASFV have been previously characterized in non-leukocyte cells such as Vero cells. Here, we report on several additional key parameters that ASFV utilizes during the infection of primary Mø. Related to virus infection, we established that receptor-mediated endocytosis of the virus by Mø is not the exclusive means of entry to infect the host cells. Analysis of the ensuing processes identified divalent cation-dependent activities to be particularly important, relating to the virus requirement for microtubule assembly needed for endocytic and endosomal processing. Actin-dependent endocytosis and endocytic flux involving microtubule activity are also implicated, pointing to entry via phagocytosis. Subsequently, the virus avoids terminal degradation by circumventing mature lysosome activities, including autophagosome-lysosome delivery. Nevertheless, the replicative cycle is apparently dependent on certain lysosomal functions, i.e. activities sensitive to propylamine are essential for the virus, whereas vinblastine- and leupeptin-sensitive functions only partially influence viral replication. The present work has identified cellular processes essential for ASFV to infect and replicate in the macrophage. These findings will improve our understanding of the cellular pathways employed by viruses infecting immune scavenger cells.

Porcine Flt3 ligand and its receptor: generation of dendritic cells and identification of a new marker for porcine dendritic cells.

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.

Hemagglutinin-dependent tropism of H5N1 avian influenza virus for human endothelial cells.

Although current H5N1 highly pathogenic avian influenza viruses (HPAIV) are inefficiently transmitted to humans, infected individuals can suffer from severe disease, often progressing rapidly to acute respiratory distress syndrome and multiorgan failure. This is in contrast with the situation with human influenza viruses, which in immunocompetent individuals usually cause only a respiratory disease which is less aggressive than that observed with avian H5N1 viruses. While the biological basis of inefficient transmission is well documented, the mechanisms by which the H5N1 viruses cause fatal disease remain unclear. In the present study, we demonstrate that human pulmonary microvascular endothelial cells (hPMEC) had a clearly higher susceptibility to infection by H5N1 HPAIV than to infection by human influenza viruses. This was measurable by de novo intracellular nucleoprotein production and virus replication. It was also related to a relatively higher binding capacity to cellular receptors. After infection of hPMEC, cell activation markers E-selectin and P-selectin were upregulated, and the proinflammatory cytokines interleukin-6 and beta interferon were secreted. H5N1 virus infection was also associated with an elevated rate of cell death. Reverse genetics analyses demonstrated a major role for the viral hemagglutinin in this cell tropism. Overall, avian H5N1 viruses have a particular receptor specificity targeting endothelial cells that is different from human influenza viruses, and this H5N1 receptor specificity could contribute to disease pathogenesis.

Innate immune responses against foot-and-mouth disease virus: current understanding and future directions.

Foot-and-mouth disease (FMD) represents one of the most economically important diseases of farm animals. The basis for the threat caused by this virus is the high speed of replication, short incubation time, high contagiousness, and high mutation rate resulting in constant antigenic changes. Thus, although protective immune responses against FMD virus (FMDV) can be efficacious, the rapidity of virus replication and spread can outpace immune defence development and overrun the immune system. FMDV can also evade innate immune responses through its ability to shut down cellular protein synthesis, including IFN type I, in susceptible epithelial cells. This is important for virus evolution, as FMDV is quite sensitive to the action of IFN. Despite this, innate immune responses are probably induced in vivo, although detailed studies on this subject are lacking. Accordingly, this interaction of FMDV with cells of the innate immune system is of particular interest. Dendritic cells (DC) can be infected by FMDV and support viral RNA replication, and viral protein synthesis but the latter is inefficient or abortive, leading most often to incomplete replication and progeny virus release. As a result DC can be activated, and particularly in the case of plasmacytoid DC (pDC), this is manifest in terms of IFN-alpha release. Our current state of knowledge on innate immune responses induced by FMDV is still only at a relatively early stage of understanding. As we progress, the investigations in this area will help to improve the design of current vaccines and the development of novel control strategies against FMD.

Targeting the porcine immune system--particulate vaccines in the 21st century.

During the last decade, the propagation of immunological knowledge describing the critical role of dendritic cells (DC) in the induction of efficacious immune responses has promoted research and development of vaccines systematically targeting DC. Based on the promise for the rational design of vaccine platforms, the current review will provide an update on particle-based vaccines of both viral and synthetic origin, giving examples of recombinant virus carriers such as adenoviruses and biodegradable particulate carriers. The viral carriers carry pathogen-associated molecular patterns (PAMP), used by the original virus for targeting DC, and are particularly efficient and versatile gene delivery vectors. Efforts in the field of synthetic vaccine carriers are focussing on decorating the particle surface with ligands for DC receptors such as heparan sulphate glycosaminoglycan structures, integrins, Siglecs, galectins, C-type lectins and toll-like receptors. The emphasis of this review will be placed on targeting the porcine immune system, but reference will be made to advances with murine and human vaccine delivery systems where information on DC targeting is available.

Porcine circovirus type 2 displays pluripotency in cell targeting.

Porcine circovirus type 2 (PCV2) is the causative agent of a multifactorial disease associated with immunocompromisation and co-infections. In vivo, viral DNA and antigens are found in monocytic, epithelial and endothelial cells. Of these, PCV2 replication has only been studied in monocytic cells, in which little or no replication was identified. Accordingly, PCV2 infection was studied in the endothelial cell line PEDSV.15, aortic endothelial cells, gut epithelial cells, fibrocytes and dendritic cells (DC). In all cells except DC PCV2 replication was detectable, with an increase in the levels of capsid and replicase protein. Variations in endocytic activity, virus binding and uptake did not relate to the replication efficiency in a particular cell. Furthermore, replication did not correlate to cell proliferation, although a close association of viral proteins with chromatin in dividing cells was observed. No alteration in the division rate of PCV2-infected cultures was measurable, relating to replicase expression in only a small minority of the cells. In conclusion, the broad cell targeting of PCV2 offers an explanation for its widespread tissue distribution.