Comparison of cellular assays for TLR activation and development of a species-specific reporter cell line for cattle

PRRs are sentinels of the innate immune system, with TLRs being the most important. Assays for TLR ligand interactions have been used to gain insights into their function and signaling pathways. As significant differences exist between species with regard to ligand recognition, it is necessary to adapt these tools for TLRs of other species. In the present work, we describe a species-specific cell-based assay adapted for the analysis of single PRRs. Human embryonic kidney 293T cells were stably transfected with the NF-κB-inducible reporter gene secreted embryonic alkaline phosphatase (SEAP) together with bovine TLR2. We compared the SEAP response with an existing luciferase NF-κB reporter assay for correlation with IL-8 production. A dose-dependent response was detected upon stimulation using both methods with good correlation to IL-8 secretion. Lower stimulant concentrations were detected by SEAP assay than IL-8 secretion. The luciferase assay produced high non-specific background for all ligand concentrations. Of all assays tested, we found the bovine-specific SEAP reporter assay to be the most convenient and delivered results in the shortest time. The developed reporter cell line would lend well to rapid, high-throughput TLR ligand screening for cattle

ORAL IMMUNOGENICITY OF A PLANT VIRUS VECTOR BASED PORCINE CIRCOVIRUS ANTIGEN

A recombinant cucumber mosaic virus based expression system has been developed for the production of an immunogenic porcine circovirus epitope. The resulting nanoparticle was shown to elicit specific immune response in mice and pigs, when administered parenterally. To evaluate the oral applicability of this vaccine candidate, two experiments were performed. In the first one, the resistance of the vector itself to mucosal environment was tested in mice. Cucumber mosaic virus particles fed to mice were able to elicit specific mucosal and serum antibody production. In the second experiment, recombinant cucumber mosaic virus fed to piglets resulted in the appearance of porcine circovirus specific serum antibodies. The vector proved to be able to survive in the gastrointestinal tract, so that an epitope expressed on its surface could induce specific immune response. These results indicate that the developed plant virus based expression system offers an effective method for mucosal vaccine production

Az állat-egészségügyi célú vakcina-elõállítás lehetõségei növények felhasználásával

Összefoglalás. A fertõzõ betegségek megelõzésének alapvetõ eszköze az állatorvosi gyakorlatban a specifi kus vakcinák kiterjedt és célzott használata. A vakcinafejlesztés az elmúlt évtizedekben látványosan megújult, és ennek a folyamatnak egy lehetséges fejlõdési irányát jelenti a növényekben elõállított antigének vakcinázási célú felhasználása. A szerzõk áttekintést nyújtanak a szûkebb terület eddigi eredményeirõl, a már megvalósított és várható kutatási és fejlesztési irányokról. A növényekkel történõ antigén-elõállítás egyik ígéretes lehetõsége az alegységvakcinák gyártásának. Mára számos, állat-egészségügyi szempontból jelentõs immunogén fehérje expressziója történt meg transzgenikus növényekben vagy növényeket fertõzõ vírusvektorok felhasználásával. A növényi expressziós rendszerek elõnye a gazdaságosság, valamint az, hogy alkalmasak lehetnek szájon át alkalmazható vakcinák elõállítására is. Summary. Widespread and targeted use of specifi c vaccines is an essential tool of the prevention and control of infectious diseases in the veterinary practice. Vaccine development went through a spectacular reform during the last decades and one potential direction of this progress is the use of antigens produced in plants for vaccination purposes. The authors provide a summary of the results of this fi eld, about the already accomplished and the expected research and development courses. Antigen expression in plants is a promising possibility for subunit vaccine production. To date, many immunogenic proteins of veterinary importance have been expressed in transgenic plants or using plant virus vectors. Among the advantages of plant expression systems are the possibility of inexpensive production and the potential of oral vaccine application, as well

A Cucumber Mosaic Virus Based Expression System for the Production of Porcine Circovirus Specific Vaccines

Potential porcine circovirus type 2 (PCV2) capsid protein epitopes, suitable for expression on the surface of cucumber mosaic virus (CMV) particles were determined by a thorough analysis of the predicted PCV capsid protein structure. The ab initio protein structure prediction was carried out with fold recognition and threading methods. The putative PCV epitopes were selected on the basis of PCV virion models and integrated into the plant virus coat protein, after amino acid position 131. The recombinants were tested for infectivity and stability on different Nicotiana species and stable recombinant virus particles were purified. The particles were tested for their ability to bind to PCV induced porcine antibodies and used for specific antibody induction in mice and pigs. The results showed that PCV epitopes expressed on the CMV surface were recognized by the porcine antibodies and they were also able to induce PCV specific antibody response. Challenge experiment with PCV2 carried out in immunized pigs showed partial protection against the infection. Based on these results it was concluded that specific antiviral vaccine production for the given pathogen was feasible, offering an inexpensive way for the mass production of such vaccines

Oral immunogenicity of a plant virus vector based porcine circovirus antigen — short communication

A recombinant cucumber mosaic virus based expression system has been developed for the production of an immunogenic porcine circovirus epitope. The resulting nanoparticle was shown to elicit specific immune response in mice and pigs, when administered parenterally. To evaluate the oral applicability of this vaccine candidate, two experiments were performed. In the first one, the resistance of the vector itself to mucosal environment was tested in mice. Cucumber mosaic virus particles fed to mice were able to elicit specific mucosal and serum antibody production. In the second experiment, recombinant cucumber mosaic virus fed to piglets resulted in the appearance of porcine circovirus specific serum antibodies. The vector proved to be able to survive in the gastrointestinal tract, so that an epitope expressed on its surface could induce specific immune response. These results indicate that the developed plant virus based expression system offers an effective method for mucosal vaccine production

CMV and PCV epitopes on the virion surface.

<p>Spatial localization of the epitopes on the modified CMV CP trimer (A, B) and on the predicted PCV2 pentamer surface (C, D). The epitope (PCV2_224-233) was inserted after the 131 aa position. The CMV subunits A, B and C are cyan, pink and gold, respectively. The epitope is illustrated in licorice representation. The basic amino acids are blue, acidic amino acids are red, the non-polar amino acids are gray and the polar amino acids are colored in green. Plan view of the external surface of the predicted PCV2 CP pentamer (C) and the side view of the pentamer showing the outer surface and the inner surface part (D). External PCV2 capsid protein epitope colors: PCV2_37-43 is ice cube, PCV2_90-96 is mauve, PCV2_126-145 is cyan, PCV2_169-186 is pink and PCV2_224-233 is colored with the above used color schemes.</p

X-ray structure of the PCV2 virion (PDB ID code: 3R0R).

<p>The last seven residues are not present in the X-ray structure but it is well visible that the C-terminal tail (red) of the PCV2 capsid protein is located at the edge of the CP pentamer.</p

Visualization of epitope insertion points in CMV.

<p>Localization of stable epitope insertion sites G83/S84 (blue), G131/S132 (red) and D176/I177 (green) in CMV. The first two sites are on the external surface of the virion while the third is located towards the inside of the virion (A). The CMV subunits A, B and C are cyan, pink and orange, respectively. The symmetrical distribution of the inserted epitope (red) is visible on the Van der Waals representation of the modified CMV virion surface (B).</p