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Identification of efficacious delivery systems for recombinant and nucleic acid construct vaccines (EFFICACIOUS DELIVERY)

Deliverables

A SYBR-green Assay was established for measuring IL-4, IL-2, IFN-g and IL-5, with the housekeeping genes Cyclophilin and GAPDH. It gave similar results to the multiplex RT-PCR, and was also appropriate to measure changes in mRNA-levels of cytokines upon stimulation of PBMCs with CSFV.
Prototype novel DNA vaccines against CSFV were generated. The eukaryotic expression vectors encoded the viral structural protein E2 and the non-structural protein NS3 (pcDNA4-E2/NS3). These expression vectors were supplemented with DNAs encoding the interleukins IL-12, IL-18 and the CD40 ligand for improvement in efficacy tests. In classical prime/boost vaccination, CSFV-E2 and/or -NS3 encoding plasmid DNA was only partially protective in stringently lethal challenge experiments with swine. This contrasted with the E2 expressing ORFV vector vaccine, which mediated solid protection and prevented CSFV shedding. Additional vaccination/challenge experiments tested the efficacy of DNA-mediated co-delivery of cytokines and CD154. These showed that co-delivery of IL-18 and CD154 induced an earlier appearance of serum antibodies, reduced B-cell deficiency after infection, and protected pigs against a lethal CSFV challenge. In contrast, co-delivery of IL-12 led to a reduced titre of neutralizing antibodies and protection, in comparison to pigs immunized with an E2 encoding plasmid DNA alone. For the required rapid efficacy of an intervention vaccination, a prime and multiple boost regime with the DNA vaccines achieved protection. Consequently, the consortium concludes that DNA vaccination against CSFV requires two booster applications for protection of swine and induction of CSFV-neutralizing antibodies. When piglets are immunized with the pcDNA4-E2-IL18 and pcDNA4-E2-CD40L plasmids, they are protected against lethal CSFV challenge and don�t show clinical signs.
Recombinant parapoxvirus Orf virus (ORFV) has been developed as a safe and immuno-stimulatory vector expressing the CSFV protein E2 (ORFV D1701VrVE2). Since the NS3 subunit of CSFV turned out to be non-protective neither when applied as coding plasmid DNA nor as E. coli expressed protein the construction of NS3 expressing recombinant ORFV was not included. Instead ORFV D1701VrVE2 vaccine application and delivery experiments were the main focus of the project with respect to this TIP. ORFV D1701VrVE2 was shown to be completely avirulent for pigs. The vector is a potent stimulator of IFN-a and VSV antiviral activity in porcine PBMC. In several animal immunisation experiments with swine ORFV D1701VrVE2 induced CSFV-neutralizing serum antibodies already after a single application. Immunized piglets were protected against lethal CSFV challenge and did not develop fever. Vaccinated and challenged animals recovered from the characteristic CSFV-induced B-cell reduction. The distribution of the vaccine dose over four intra-muscular injection sites (multi-site application) led to a rapid induction of neutralizing antibodies and to solid protection from CSF after a single vaccination. In contrast to single site-vaccinated animals, multi-site vaccinated piglets did not transmit the challenge virus to a naive sentinel. Two successive vector virus applications in a homologous prime-boost regimen did not provoke IFN-? producing cells among PBMC. However, a heterologous prime-boost regimen as a combination of prime with baculovirus expressed glycoprotein E2 followed by boost with the parapoxvirus vector induced high numbers of IFN-? producing cells. A similar beneficial effect became evident when the challenge infection mimicked the booster vaccination after a single vector prime. In contrast when the challenge CSFV was applied after a homologous prime with modified live CSFV vaccine the immunised piglets responded with lower numbers of IFN-? producing cells as well as significantly lower titres of CSFV-neutralizing serum antibodies. In conclusion, the project discovered that ORFV D1701VrVE2 was an efficient vaccine, inducing solid protection and preventing virus shedding. It was completely avirulent for pigs, and the vector is a potent stimulator of IFN-a. CSFV-neutralizing serum antibodies were induced after a single application, and immunized piglets were completely protected against lethal CSFV challenge. A multi-site application of the vaccine led to the most rapid induction of neutralizing antibodies plus solid protection, and prevented transmission of the challenge virus to naive sentinels.
Real time RT-PCR cytokine assays with samples gained from vaccinated and challenged swine has been developed. One assay established allows the simultaneous detection of 9 sequences. 3 different sets of 3 targets each are measured in a triplex-format using TaqMan-probes. Six targets can be selected from the following lymphokines: IL-2, IL-4, IL-5, IL-10, and IFN-gamma, and have the proinflammatory cytokines IL-1alpha and IL-6. Simultaneously the most suitable combination of 3 out of the four housekeeping genes (beta-actin, HPRT, GAPDH, and cyclophilin) can be selected, and their averaged expression values constitute a normalisation factor. The raw data of all targets of interest is than calculated relative to this normalisation factor, making also eventual changes of the relative expression level of the single housekeeping genes controllable, and quantifiable. Performance and suitability for the real time PCR was controlled.
A new adjuvant for subunit recombinant protein vaccines for application in pigs was tested. This was based on Pseudomonas aeruginosa outer membrane lipoprotein (OprI). It was studied in combination with recombinant viral E2 protein. The OprI was found to activate porcine DCs and effect a co-stimulatory action on T-cells. This was dependent on the OprI lipid tail. Thus, OprI is a potent immunostimulator of DC activity, and can enhance T-cell responses against CSFV. Due to these results, the adjuvant potential of OprI was tested with an E2 vaccine. OprI did not increase the efficacy of an optimal dose of E2, but was effective with sub-optimal doses. In the latter case, OprI promoted the formation of CSFV-neutralizing antibodies, but did not increase the number of IFN-gamma producing cells. In other trial, OprI in combination with E2 caused an increase in antibody titers and in interferon-gamma production. Altogether, these results confirm that OprI has interesting immunostimulatory activities.
Background: The development of dendritic cell (DC)-based vaccines using antigen-encoding mRNA requires identification of the critical parameters for efficient ex vivo loading of DCs. It has been reported that exogenously delivered mRNA can induce DC-activation, but the molecular mechanisms involved are unknown. The aim of the present study was to identify the means by which mRNA-dependent activation of DCs occurs. Methods: In vitro transcribed mRNA molecules were delivered into porcine monocyte-derived DCs (MoDCs) using the non-viral gene transfer procedures of electroporation, lipofection and passive pulsing. Using the green fluorescent protein (GFP) as reporter gene, as well as rhodamine-labelled RNA, intracellular delivery and transfection efficiency were assessed by laser-scanning confocal microscopy and flow cytometry. DC-activation was monitored in terms of MHC class II and CD80/86 upregulation, as well as the production of type I interferon (IFN-a/b). Results: Lipofection and electroporation of mRNA into MoDCs allowed 90% cell viability (48 hours after gene transfer), The transfection efficiency was 6 to 18% for lipofection, compared with 80% efficiency for electroporation. Passive pulsing resulted in <1% transfection efficiency. Importantly, mRNA-lipofected MoDCs produced type I IFN and upregulated MHC class II and CD80/86. Computational analysis of the mRNA molecules showed highly ordered secondary structures forming double-stranded RNA (dsRNA). This dsRNA was also detectable by immunofluorescence in mRNA-lipofected cells, using antibody specific for dsRNA. Digestion of the mRNA prior to lipofection with a double-strand specific RNase, but not a single-strand specific RNase, abrogated DC activation. Impairment of protein kinase R (PKR) with 2-aminopurine also interfered with the activation. Conclusions: Double-stranded secondary structures on mRNA delivered by lipofection can activate MoDCs. This could have important implications for mRNA-based immunomodulation of DCs, DC-based immunotherapy, and formulation of RNA-based vaccines. In addition, this report describes the first in vitro steps towards development of a novel large animal model system to evaluate DC-based vaccines against infectious diseases.