Targeting of the Nasal Mucosa by Japanese Encephalitis Virus for Non-Vector-Borne Transmission.

The mosquito-borne Japanese encephalitis virus (JEV) causes severe central nervous system diseases and cycles between Culex mosquitoes and different vertebrates. For JEV and some other flaviviruses, oronasal transmission is described, but the mode of infection is unknown. Using nasal mucosal tissue explants and primary porcine nasal epithelial cells (NEC) at the air-liquid interface (ALI) and macrophages as ex vivo and in vitro models, we determined that the nasal epithelium could represent the route of entry and exit for JEV in pigs. Porcine NEC at the ALI exposed to with JEV resulted in apical and basolateral virus shedding and release of monocyte recruiting chemokines, indicating infection and replication in macrophages. Moreover, macrophages stimulated by alarmins, including interleukin-25, interleukin-33, and thymic stromal lymphopoietin, were more permissive to the JEV infection. Altogether, our data are important to understand the mechanism of non-vector-borne direct transmission of Japanese encephalitis virus in pigs.IMPORTANCE JEV, a main cause of severe viral encephalitis in humans, has a complex ecology composed of a mosquito-waterbird cycle and a cycle involving pigs, which amplifies virus transmission to mosquitoes, leading to increased human cases. JEV can be transmitted between pigs by contact in the absence of arthropod vectors. Moreover, virus or viral RNA is found in oronasal secretions and the nasal epithelium. Using nasal mucosa tissue explants and three-dimensional porcine nasal epithelial cells cultures and macrophages as ex vivo and in vitro models, we determined that the nasal epithelium could be a route of entry as well as exit for the virus. Infection of nasal epithelial cells resulted in apical and basolateral virus shedding and release of monocyte recruiting chemokines and therefore infection and replication in macrophages, which is favored by epithelial-cell-derived cytokines. The results are relevant to understand the mechanism of non-vector-borne direct transmission of JEV.

Self-Amplifying Replicon RNA Delivery to Dendritic Cells by Cationic Lipids.

Advances in RNA technology during the past two decades have led to the construction of replication-competent RNA, termed replicons, RepRNA, or self-amplifying mRNA, with high potential for vaccine applications. Cytosolic delivery is essential for their translation and self-replication, without infectious progeny generation, providing high levels of antigen expression for inducing humoral and cellular immunity. Synthetic nanoparticle-based delivery vehicles can both protect the RNA molecules and facilitate targeting of dendritic cells-critical for immune defense development. Several cationic lipids were assessed, with RepRNA generated from classical swine fever virus encoding nucleoprotein genes of influenza A virus. The non-cytopathogenic nature of the RNA allowed targeting to dendritic cells without destroying the cells-important for prolonged antigen production and presentation. Certain lipids were more effective at delivery and at promoting translation of RepRNA than others. Selection of particular lipids provided delivery to dendritic cells that resulted in translation, demonstrating that delivery efficiency could not guarantee translation. The observed translation in vitro was reproduced in vivo by inducing immune responses against the encoded influenza virus antigens. Cationic lipid-mediated delivery shows potential for promoting RepRNA vaccine delivery to dendritic cells, particularly when combined with additional delivery elements.

Glycan recognition by human blood mononuclear cells with an emphasis on dendritic cells.

Dendritic cells (DCs) play crucial roles in innate and adaptive immune response, for which reason targeting antigen to these cells is an important strategy for improvement of vaccine development. To this end, we explored recognition of DCs lectins by glycans. For selection of the glycan "vector", a library of 229 fluorescent glycoprobes was employed to assess interaction with the CD14low/-CD16+CD83+ blood mononuclear cell population containing the DCs known for their importance in antigen presentation to T-lymphocytes. It was found that: 1) the glycan-binding profiles of this CD14low/-CD16+CD83+ subpopulation were similar but not identical to DCs of monocyte origin (moDCs); 2) the highest percentage of probe-positive cells in this CD14 low/-CD16+CD83+ subpopulation was observed for GalNAcα1-2Galß (Adi), (Neu5Acα)3 and three mannose-reach glycans; 3) subpopulation of CD14low/-CD16+ cells preferentially bound 4'-O-Su-LacdiNAc. Considering the published data on specificity of DCs binding, the glycans showing particular selectivity for the CD14 low/-CD16+CD83+ cells are likely interacting with macrophage galactose binding lectin (MGL), siglec-7 and dectin-2. In contrast, DC-SIGN is not apparently involved, even in case of mannose-rich glycans. Taking into consideration potential in vivo competition between glycan "vectors" and glycans within glycocalyx, attempting to target vaccine to DCs glycan-binding receptors should focus on Adi and (Neu5Acα)3 as the most promising vectors.

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.

Self-replicating Replicon-RNA Delivery to Dendritic Cells by Chitosan-nanoparticles for Translation In Vitro and In Vivo.

Self-amplifying replicon RNA (RepRNA) possesses high potential for increasing antigen load within dendritic cells (DCs). The major aim of the present work was to define how RepRNA delivered by biodegradable, chitosan-based nanoparticulate delivery vehicles (nanogel-alginate (NGA)) interacts with DCs, and whether this could lead to translation of the RepRNA in the DCs. Although studies employed virus replicon particles (VRPs), there are no reports on biodegradable, nanoparticulate vehicle delivery of RepRNA. VRP studies employed cytopathogenic agents, contrary to DC requirements-slow processing and antigen retention. We employed noncytopathogenic RepRNA with NGA, demonstrating for the first time the efficiency of RepRNA association with nanoparticles, NGA delivery to DCs, and RepRNA internalization by DCs. RepRNA accumulated in vesicular structures, with patterns typifying cytosolic release. This promoted RepRNA translation, in vitro and in vivo. Delivery and translation were RepRNA concentration-dependent, occurring in a kinetic manner. Including cationic lipids with chitosan during nanoparticle formation enhanced delivery and translation kinetics, but was not required for translation of immunogenic levels in vivo. This work describes for the first time the characteristics associated with chitosan-nanoparticle delivery of self-amplifying RepRNA to DCs, leading to translation of encoded foreign genes, namely influenza virus hemagglutinin and nucleoprotein.

Alginate-coated chitosan nanogels differentially modulate class-A and class-B CpG-ODN targeting of dendritic cells and intracellular delivery.

CpG-oligodeoxynucleotides (CpG-ODNs) interact with dendritic cells (DCs), but evidence is less clear for CpG-ODN admixed with or incorporated into vaccine delivery vehicles. We loaded alginate-coated chitosan-nanogels (Ng) with class-A or class-B CpG-ODN, and compared with the same CpG-ODNs free or admixed with empty Ng. Experiments were performed on both porcine and human blood DC subpopulations. Encapsulation of class-A CpG-ODN (loading into Ng) strongly reduced the CpG-ODN uptake and intracellular trafficking in the cytosol; this was associated with a marked deficiency in IFN-α induction. In contrast, encapsulation of class-B CpG-ODN increased its uptake and did not influence consistently intracellular trafficking into the nucleus. The choice of CpG-ODN class as adjuvant is thus critical in terms of how it will behave with nanoparticulate vaccine delivery vehicles. The latter can have distinctive modulatory influences on the CpG-ODN, which would require definition for different CpG-ODN and delivery vehicles prior to vaccine formulation. FROM THE CLINICAL EDITOR: This basic science study investigates the role of class-A and class-B CpG-oligodeoxynucleotides loaded into alginate-coated chitosan nanogels, demonstrating differential effects between the two classes as related to the use of these nanoformulations as vaccine delivery vehicles.

Self-Amplifying Replicon RNA Vaccine Delivery to Dendritic Cells by Synthetic Nanoparticles.

Dendritic cells (DC) play essential roles determining efficacy of vaccine delivery with respect to immune defence development and regulation. This renders DCs important targets for vaccine delivery, particularly RNA vaccines. While delivery of interfering RNA oligonucleotides to the appropriate intracellular sites for RNA-interference has proven successful, the methodologies are identical for RNA vaccines, which require delivery to RNA translation sites. Delivery of mRNA has benefitted from application of cationic entities; these offer value following endocytosis of RNA, when cationic or amphipathic properties can promote endocytic vesicle membrane perturbation to facilitate cytosolic translocation. The present review presents how such advances are being applied to the delivery of a new form of RNA vaccine, replicons (RepRNA) carrying inserted foreign genes of interest encoding vaccine antigens. Approaches have been developed for delivery to DCs, leading to the translation of the RepRNA and encoded vaccine antigens both in vitro and in vivo. Potential mechanisms favouring efficient delivery leading to translation are discussed with respect to the DC endocytic machinery, showing the importance of cytosolic translocation from acidifying endocytic structures. The review relates the DC endocytic pathways to immune response induction, and the potential advantages for these self-replicating RNA vaccines in the near future.

Expression and functional analyses of liver expressed antimicrobial peptide-2 (LEAP-2) variant forms in human tissues.

The antimicrobial peptide Liver Expressed Antimicrobial Peptide-2 (LEAP-2) is proposed to function as part of the vertebrate innate immune system. However, the highly conserved nature of the LEAP-2 peptide primary structure among vertebrates suggests more fundamental physiological roles. RT-PCR analyses confirmed expression of LEAP-2 mRNA variants in human gastro-intestinal (GI) epithelial tissues and THP-1 monocytes. Three cDNA products indicative of at least three different spliced transcripts were observed. Translation of the cDNA sequences supported synthesis of transcripts encoding the secreted LEAP-2 peptide and two variants lacking signal sequences suggesting intracellular localisation. The synthesis and cytoplasmic localisation of LEAP-2 peptides in epithelia was supported by immunohistochemical analyses. Functional data suggested that LEAP-2 is not involved in the physiological response of GI epithelia to iron, nor is it mitogenic for epithelial cells or chemotactic for THP-1 monocytes. However, changes in the LEAP-2 transcript patterns associated with the challenge of THP-1 monocytes with lipopolysaccharide (100ng/ml) were supportive of the peptides having multiple roles in the innate immune response.

The chicken host peptides, gallinacins 4, 7, and 9 have antimicrobial activity against Salmonella serovars.

The gallinacin genes clustered on chromosome 3 of the chicken (Gallus gallus domesticus) genome encode a group of cationic antimicrobial peptides characteristic of the beta-defensins. In this study, gallinacins 4, 7, and 9, all predicted to contain the conserved pattern of cysteines typical of beta-defensins but differing in their charge and hydrophobicity, were characterised for their in vivo gene expression patterns and in vitro antimicrobial activities against Salmonella serovars. Reverse-transcription PCR analyses of chicken epithelial tissues indicated gallinacin (Gal) 7 expression to be ubiquitous while Gal 4 and Gal 9 expression appeared localized to specific epithelial tissues including the ovary, trachea, and lung, respectively. In addition Gal 7, but neither Gal 4 nor Gal 9, expression was identified in tissues taken from the non-domesticated bird species, Parus caeruleus, Larus argentatus, and Columba palambus. Analysis of Gal 7 expression in chickens in response to an oral challenge with either Salmonella enterica serovar Typhimurium SL1344 or Salmonella enteriditis indicated no significant increase in small intestinal Gal 7 mRNA expression although a significant increase (p <0.05) was detected in the liver, suggesting that, in response to Salmonella infection Gal 7 expression is inducible in the liver. Neither Gal 4 nor Gal 9 expression was induced in the chicken small intestine in response to the oral Salmonella infection. The antimicrobial capabilities of Gals 4, 7, and 9 against Salmonella serovars including S. typhimurium SL1344 and S. enteriditis were investigated in vitro using recombinant His-tagged peptides and a time-kill assay. The antimicrobial activity data indicated the potency of the recombinant gallinacins against the Salmonella serovars to be in the order Gal 9> or=4>7, and provided evidence for the synergistic interaction of Gals 7 and 9 against S. enteriditis. These results support in silico data that Gals 4, 7, and 9 are part of the innate defences of the chicken and function in microbial killing activities.