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.

Glycan-binding profile of DC-like cells.

Modification of vaccine carriers by decoration with glycans can enhance binding to and even targeting of dendritic cells (DCs), thus augmenting vaccine efficacy. To find a specific glycan-"vector" it is necessary to know glycan-binding profile of DCs. This task is not trivial; the small number of circulating blood DCs available for isolation hinders screening and therefore advancement of the profiling. It would be more convenient to employ long-term cell cultures or even primary DCs from murine blood. We therefore examined whether THP-1 (human monocyte cell line) and DC2.4 (immature murine DC-like cell line) could serve as a model for human DCs. These cells were probed with a set of glycans previously identified as binding to circulating human CD14low/-CD16+CD83+ DCs. In addition, we tested a subpopulation of murine CD14low/-CD80+СD11c+CD16+ cells reported as relating to the human CD14low/-CD16+CD83+ cells. Manα1-3(Manα1-6)Manß1-4GlcNAcß1-4GlcNAcß bound to both the cell lines and the murine CD14low/-CD80+СD11c+CD16+ cells. Primary cells, but not the cell cultures, were capable of binding GalNAcα1-3Galß (Adi), the most potent ligand for binding to human circulating DCs. In conclusion, not one of the studied cell lines proved an adequate model for DCs processes involving lectin binding. Although the glycan-binding profile of BYRB-Rb (8.17)1Iem mouse DCs could prove useful for assessing human DCs, important glycan interactions were missing, a situation which was aggravated when employing cells from the BALB/c strain. Accordingly, one must treat results from murine work with caution when seeking vaccine targeting of human DCs, and certainly should avoid cell lines such as THP-1 and DC2.4 cells.

Dendritic Cell Sensing of Hydrophobic Di- and Triacylated Lipopeptides Self-Assembled within Synthetic Virus-like Particles.

Dendritic cells (DCs) play critical roles in developing immune defenses. One important aspect is interaction with pathogen-associated molecular patterns (PAMPs)/danger-associated molecular patterns, including di- and triacylated lipopeptides. Isolated or synthetic lipopeptides are potent vaccine adjuvants, interacting with cell surface TLR2 heterodimers. In contrast, deep embedment within bacteria cell walls would impair lipopeptide interaction with cell surface TLR2, requiring degradation for PAMP recognition. Accordingly, DC processing in the absence of surface TLR2 ligation was defined using synthetic virus-like particles (SVLPs) carrying hydrophobic TLR2 PAMPs within di- and triacylated lipopeptide cores (P2Cys-SVLPs and P3Cys-SVLPs) compared with SVLPs lacking immunomodulatory lipopeptides. DCs rapidly and efficiently internalized SVLPs, which was dominated by slow endocytic processing via macropinocytosis, although some caveolar endocytosis was implicated. This delivered SVLPs primarily into macropinosomes often interacting with EEA-1+ early endosomes. Although endoplasmic reticulum association was occasionally noted, association with recycling/sorting structures was not observed. Involvement of LysoTracker+ structures slowly increased with time, with SVLPs present in such structures ultimately dominating. Only SVLPs carrying di- and triacylated lipopeptide cores induced DC activation and maturation independently of surface TLR2 ligation. Intracellular recognition of SVLP TLR2 ligands was confirmed by observing SVLPs' association with internal TLR2, which had similar kinetics to SVLP association with LysoTracker. This related to inflammatory cytokine induction by SVLP+ DCs, with adaptive immune response activation ex vivo/in vivo. Importantly, particular DCs, not monocytes, recognized intracellular exposure of the TLR2 PAMPs carried by di- and triacylated SVLP cores, which indicates subset-distinct recognition of functional internal TLR2 ligands. Thus, vaccines carrying hydrophobic TLR2 ligands would interact with particular DCs for efficient induction of specific immunity in the absence of additional adjuvant.

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.

Virus replicon particles expressing porcine reproductive and respiratory syndrome virus proteins elicit immune priming but do not confer protection from viremia in pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most devastating and economically significant viral disease of pigs worldwide. The vaccines currently available on the market elicit only limited protection. Recombinant vesicular stomatitis virus (VSV) replicon particles (VRP) have been used successfully to induce protection against influenza A virus (IAV) in chickens and bluetongue virus in sheep. In this study, VSV VRP expressing the PRRSV envelope proteins GP5, M, GP4, GP3, GP2 and the nucleocapsid protein N, individually or in combination, were generated and evaluated as a potential vector vaccine against PRRSV infection. High level expression of the recombinant PRRSV proteins was demonstrated in cell culture. However, none of the PRRSV antigens expressed from VRP, with the exception of the N protein, did induce any detectable antibody response in pigs before challenge infection with PRRSV. After challenge however, the antibody responses against GP5, GP4 and GP3 appeared in average 2 weeks earlier than in pigs vaccinated with the empty control VRP. No reduction of viremia was observed in the vaccinated group compared with the control group. When pigs were co-vaccinated with VRP expressing IAV antigens and VRP expressing PRRSV glycoproteins, only antibody responses to the IAV antigens were detectable. These data show that the VSV replicon vector can induce immune responses to heterologous proteins in pigs, but that the PRRSV envelope proteins expressed from VSV VRP are poorly immunogenic. Nevertheless, they prime the immune system for significantly earlier B-cell responses following PRRSV challenge infection.

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.

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.

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.

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.

Alginate-coated chitosan nanogel capacity to modulate the effect of TLR ligands on blood dendritic cells.

Biodegradable nanoparticles have been employed for vaccine delivery, frequently admixed with adjuvants. Surprisingly, there is little information on their modulation of immune responses, speculated to be negligible. We analyzed the immunomodulatory capacity of alginate-coated chitosan nanogels (Ng), on porcine and human blood dendritic cells (DCs), when applied with defined adjuvants targeting different DC subpopulations. DC maturation, cytokine production and cell migration were assessed. Ng differentially influenced the immunomodulatory characteristics of individual Toll-like receptor (TLR) ligands: Pam3Cys-SK4-induced IL-1ß was enhanced; CpG-oligodeoxynucleotides (CpG-ODN)-induced IFN-α, IL-6 and TNFα were impaired; CpG-ODN-induced CD86 and CCR7, and cell migration, were diminished-plasmacytoid DCs (pDCs) were particularly sensitive. Therein, the Ng influence on DC endocytosis of the TLR ligands was apparently a major contributory element. This demonstrates the importance of predefining the interplay between delivery vehicles and admixed immunostimulatory moieties, for ensuring appropriate immune activation and efficacious combinations. FROM THE CLINICAL EDITOR: Biodegradable nanoparticles have been utilized in vaccine delivery; however, there is little information available on their immunomodulatory properties, which are thought to be negligible. This study clearly demonstrates that nanogels do influence the developing immune response, which needs to be taken into consideration when utilizing these otherwise very efficacious vaccine delivery approaches.

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.

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.

Dendritic cell internalization of foot-and-mouth disease virus: influence of heparan sulfate binding on virus uptake and induction of the immune response.

Dendritic cells (DC), which are essential for inducing and regulating immune defenses and responses, represent the critical target for vaccines against pathogens such as foot-and-mouth disease virus (FMDV). Although it is clear that FMDV enters epithelial cells via integrins, little is known about FMDV interaction with DC. Accordingly, DC internalization of FMDV antigen was analyzed by comparing vaccine virus dominated by heparan sulfate (HS)-binding variants with FMDV lacking HS-binding capacity. The internalization was most efficient with the HS-binding virus, employing diverse endocytic pathways. Moreover, internalization relied primarily on HS binding. Uptake of non-HS-binding virus by DC was considerably less efficient, so much so that it was often difficult to detect virus interacting with the DC. The HS-binding FMDV replicated in DC, albeit transiently, which was demonstrable by its sensitivity to cycloheximide treatment and the short duration of infectious virus production. There was no evidence that the non-HS-binding virus replicated in the DC. These observations on virus replication may be explained by the activities of viral RNA in the DC. When DC were transfected with infectious RNA, only 1% of the translated viral proteins were detected. Nevertheless, the transfected cells, and DC which had internalized live virus, did present antigen to lymphocytes, inducing an FMDV-specific immunoglobulin G response. These results demonstrate that DC internalization of FMDV is most efficient for vaccine virus with HS-binding capacity, but HS binding is not an exclusive requirement. Both non-HS-binding virus and infectious RNA interacting with DC induce specific immune responses, albeit less efficiently than HS-binding virus.

Dendritic cells--at the front-line of pathogen attack.

Efficient immune defence function is dependent on the role played by dendritic cells (DCs), particularly the interaction between conventional DC (cDC) and plasmacytoid DC (pDC), together with other monocytic cells. This functionality of immune defences is open to manipulation by viral pathogens infecting DC, a situation further complicated by the diversity of mechanisms employed by different viruses and the subset of DC involved. The present review uses two virus examples--classical swine fever virus (CSFV) and porcine circovirus type 2 (PCV2)--to demonstrate the complexity of this host-pathogen scenario. CSFV is a monocytotropic RNA virus infecting and replicating in both cDC and pDC. This virus employs its non-structural Npro protein for antagonizing the Type I interferon (IFN) induction pathway. The Npro protein promotes proteasomal degradation of interferon regulatory factor (IRF)3, particularly notable in cDC. In contrast, CSFV infection induces IFNalpha production by pDC, probably due to a lack of interference by the Npro protein with the IRF7 more prominent in pDC. Such ability of the virus to inhibit cDC while augmenting IFNalpha production by pDC might lead to an exaggerated pDC response, relating to the immunopathological characteristics of the disease. PCV2 is an ssDNA containing virus, which in contrast to CSFV is inefficient in its capacity to replicate in DC. Recent evidence suggests that virus replication occurs in endothelial cells, with the DC being more involved through their particularly elevated endocytosis of the virus. PCV2 can accumulate to high levels both in vitro and in vivo, a phenomenon dependent on the virus capsid protein, inferring that the viral capsid or genome impedes DC endocytic degradation of the virus. Nevertheless, the presence of PCV2 in cDC does not interfere with processing of other antigens. The immunoregulatory characteristics of PCV2 are manifest as impairment of "danger" recognition by cells of the innate defences. This varies dependent on the "danger" signal and the cells responding, especially when one compares cDC and pDC. Overall, the PCV2-induced immunomodulation contrasts with that of CSFV in being a property dependent on the viral genome, particularly the dsDNA replicative form, and with immunoregulatory capacity for both cDC and pDC. Moreover, PCV2 compromises immune defence development against other pathogens rather than itself. In conclusion, the DC family represents a critical immune defence element open to modulation by virus infection, with serious consequences for host resistance to disease. The characteristics of the immune modulation depend on the virus and the DC subsets involved. Overall, the roles played by the pDC can be decisive in shaping the outcome of the infection and the characteristics of the virus-induced immunocompromisation.

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.

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.

Mycoplasma contamination and viral immunomodulatory activity: dendritic cells open Pandora's box.

During in vitro investigations on the interaction of classical swine fever virus (CSFV)--an immunosuppressive viral pathogen--with monocyte-derived dendritic cells (MoDC) a soluble factor with a strong anti-proliferative activity for T lymphocytes was found. This activity, with an inhibitory dilution 50% (ID(50)) of 10(3)-10(7), was induced after virus infection of monocytes differentiating into DC. UV--inactivation of the supernatants and blocking experiments with a monoclonal antibody against the E2 envelope protein of CSFV initially indicated a virus-dependency. However, further investigations including filtration and centrifugation experiments as well as antibiotic treatment demonstrated the involvement of mycoplasma. This was confirmed by a Hoechst 33258 staining, PCR and mycoplasma cultures--Mycoplasma hyorhinis was identified as the contaminant. Elucidation of a mycoplasma presence occurred under conditions in which the original virus stocks prepared in SK6 cells were negative for mycoplasma using the above tests. Moreover, conventional passage of the virus on the SK6 cells used for this purpose did not reveal any mycoplasma. It was the passage of virus in MoDC rather than SK6 cells that was required to expose the contamination. Three passages of the anti-proliferative supernatants on MoDC cultures increased the ID(50) 10(3)-fold; only when these MoDC-derived supernatants were employed was the mycoplasma contaminant also detectable on SK6 cells. In conclusion, these data demonstrate that regular testing of cell lines and virus stocks for mycoplasma does not necessarily identify their presence, and that application of passage in MoDC cultures could prove an aid for identifying initially undetectable levels of mycoplasma contamination.

Porcine B-cell activating factor promotes anti-FMDV antibodies in vitro but not in vivo after DNA vaccination of pigs.

'B-cell activating factor belonging to the TNF family' (BAFF) represents a cytokine produced by antigen presenting cells promoting B-cell maturation, activation and immunoglobulin class switching. In the present study, we demonstrate expression of BAFF on cultured monocyte-derived dendritic cells, which is further enhanced by interferon-alpha or interferon-gamma treatment. From these cells, porcine BAFF was cloned and the recombinant protein was expressed in mammalian cells with and without a FLAG tag at the carboxyl terminus. Only the protein without the FLAG tag was bioactive in vitro, and promoted B-cell survival and the differentiation of foot-and-mouth disease virus (FMDV)-specific memory B cells into antibody producing cells. Based on this result it was tested whether BAFF can enhance FMDV antibody responses in the context of a DNA vaccination. To this end, pigs were immunised with the anti-FMDV DNA vaccine plasmid pcDNA3.1/P1-2A3C3D and a pCI plasmid expressing porcine BAFF. Using a needle-free transdermal application method, also referred to as 'jet injection', pigs were vaccinated three times and their humoral response quantified by ELISA and a virus neutralisation test. After the third vaccination, three out of six animals vaccinated with the pcDNA3.1/P1-2A3C3D alone but none of the animals that also received the BAFF expressing plasmid had seroconverted. These data suggest that BAFF is not appropriate as a genetic adjuvant when applied as a simple co-injection with the antigen-encoding plasmid.

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.

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.