Deliverables Construction of single-chain mini-antibodies with neutralizing activity against rotavirus and coronavirus Starting from virus-neutralizing monoclonal antibodies anti-TGEV (Transmissible Gastroenteritis Virus, a member of the Coronavirus family) and anti-Rotavirus protein VP7, we applied the recombinant techniques already developed in our laboratory to generate anti-TGEV and anti-Rotavirus SIPs. SIPs (Small Immuno Proteins) are recombinant proteins composed of a scFv unit (VL and VH) from a monoclonal antibody of interest joined to a dimerising domain from natural immunoglobulins, which allows the dimerisation of the single-chain unit and the generation of a bivalent molecule. Anti-viral SIPs have been generated as porcine alpha-SIP, using the alpha-CH3 domain from swine IgA. In addition, full-length chimeric porcinized IgA were also obtained. The ability of the recombinant antibodies to recognize their antigens have been confirmed in immunofluorescence and immunoenzymatic assays. They were active as well in neutralization of virus infection. In the view of a possible utilization of these anti-viral reagents in humans, further SIP versions were generated, using the human Ig domains, alpha-CH3 and epsilonS2-CH4 forms. Both of these molecules were demonstrated to be secreted as covalently stabilized dimers, by the formation of inter-chain disulfide bonds between cysteine residues enclosed in the carboxy-terminal tails of both alpha-CH3 and epsilonS2-CH4 domains. Development of an eukaryotic vector based on a single genome recombinant coronavirus for the expression of immunotherapeutic antibodies The result described next has been performed in collaboration with Fort-Dodge Veterinaria (Partner 5). An eukaryotic vector based on a single genome coronavirus was developed. In order to increase the cloning capacity of the vector, the non-essential genes 3a/b were deleted from the cDNA encoding an infectious coronavirus RNA genome (rTGEV-delta3). The reporter gene of green fluorescent protein (GFP) was inserted into the viral genome replacing the deleted 3a/b genes, under the transcription regulatory sequences (TRSs) of gene 3a, leading to high expression levels of GFP protein, around 40 microg/10exp6 cells, during at least 20 passages in cell cultures. Expression levels driven by this TRS were higher than those of an expression cassette under the control of regulating sequences engineered with the N gene TRS, suggesting that in this viral context, transcription is more efficiently driven by the native TRS3a than by the artificial TRSN. No significant differences in growing kinetics in cell cultures or in in vivo replication and tropism were observed between rTGEV-delta3 and rTGEV-delta3-GFP and the wild type vector rTGEV. To facilitate the genetic manipulation of the viral genome, genes were separated by duplication of transcription-regulating sequences and introduction of unique restriction endonuclease sites at the 5¿ end of each gene using an infectious cDNA clone. The recombinant TGEV (rTGEV) replicated in cell culture with similar efficiency to the wild-type virus, and stably maintained the modifications introduced into the genome. In contrast, the rTGEV replication level in the lungs and gut of infected piglets and virulence were significantly reduced. rTGEV in which gene 7 expression was abrogated (rTGEV-delta7) were recovered from cDNA constructs, indicating that TGEV gene 7 was a non-essential gene for virus replication. Interestingly, in vivo infections with rTGEV-delta7 showed an additional reduction in virus replication in the lung and gut, and in virulence, indicating that TGEV gene 7 influences virus pathogenesis. This information will be very useful for the optimization of the expression using the designed vector. In addition it has let to the selection of a collection of virus vectors with different attenuation degrees. Furthermore, since it has been shown that gene 7 is not essential more room for heterologous genes is available. Interestingly, immunization of pregnant sows with the viral vector rTGEV-delta3-GFP induced a strong antibody response against TGEV and also against GFP both in the serum and in colostrum and milk. Significant levels of antibodies specific for TGEV and GFP were detected in the serum of the piglets during lactation, demonstrating that the vector rTGEV-delta3-GFP had induced lactogenic immunity in the progeny and showing its potential to provide protection to piglets against mucosal infections. The developed viral vector has been used to express relevant antigens for protection against diseases of farm animals, such us porcine respiratory and reproductive syndrome virus (PRSSV). The PRRSV ORF5 expressed by the TGEV-derived vector has induced a strong antibody response in immunized pigs. Partial or complete protection against the challenge with PRRSV was observed in immunized animals, showing the effectiveness of the TGEV-derived vector for the development of vaccines. Overall these data indicate that we have successfully designed a safe vector for the expression of heterologous genes, such us immunotherapeutical antibodies, to protect against enteric diseases. The effects that the administration of the TGEV-derived vector produced in piglets has been studied by analysing the pathogenicity induced in the gastrointestinal tract and associated immune system. rTGEV-delta7 vector has been established as the best candidate for in vivo infections since a significant reduction in its virulence has been shown. To evaluate the potential of the viral vector in immunotherapy against rotavirus infections, a challenge model for rotavirus has been developed in CD/CD piglets. In addition, recombinant antibodies with the variable modules from a TGEV-neutralizing monoclonal antibody and constant modules from porcine IgA were successfully expressed in tissue cultures, showing a TGEV-specific binding and neutralizing activity. Interestingly, protection against TGEV infection of cell cultures was demonstrated by stably transforming susceptible cells with cDNA constructs encoding the rIgA. Production of edible plant material containing functional epsilon-SIP molecules Two constructs, pBinP-YP-1 and YP2, containing the sequences of alpha-SIP and epsilon-SIPs, respectively, in CPMV RNA-2 were used to agroinoculate cowpea (Vigna unguiculata) plants in the presence of the CPMV RNA-1 construct, pBinP-S1NT. Samples of fresh leaf tissue were provided to Partners 1a and 1b for the preparation of extracts and the testing of the ability of the SIPs contained in them to bind and neutralise TGEV. Though no binding or neutralisation was found in the extracts from plants expressing the alpha-SIP, very encouraging results for both binding and neutralisation were obtained with the extracts containing the epsilon-SIPs. Western blots of protein extracts, which had been reduced prior to electrophoresis showed the presence of a predominant band of 41KDa, the size expected for the monomeric SIP. Under non-reducing conditions, approximately 70% of the SIP molecules ran as dimers, consistent with efficient dimerisation being promoted by the C-terminal cysteine residue present in the epsilon-SIPs sequence. It was estimated that the expression levels may be up to 1% of soluble protein. Preliminary in vivo tests have shown the ability of the epsilon-SIPs -expressing extracts to protect piglets against TGEV infection. Production of high levels of foreign proteins in plants A combined transgene/virus vector approach for the production of high levels of foreign proteins in plants has been developed by the PLANTIBODYTHERAPY project. The project has demonstrated that integration of full-length cDNA copies of CPMV RNA-1 and -2 into the genome of Nicotiana benthamiana gives rise to a phenotype which mimics virus infection. Integration of the individual components gives rise to plants which are phenotypically normal. However, the infected phenotype can be regenerated by crossing lines transgenic for RNA-1 and RNA-2 or by inoculating lines transgenic for RNA-2 with RNA-1. When a cDNA copy of RNA-2 harbouring the marker gene, GFP, was used to transform N. benthamiana only very low levels of basal GFP expression were found. However large quantities of GFP could be obtained either by inoculating the RNA-2-GFP plants with RNA-1 or by crossing with RNA-1 transgenic lines. These results showed that CPMV could be developed into a combined transgene/virus approach for the expression of foreign proteins in plants. To exploit this system, plasmids pBinP-YP-1 and YP-2 were used to transform N. benthamiana. A number of plants were regenerated and shown to contain the transgene. When the transgenic lines were crossed with N. benthamiana transgenic for the CPMV RNA 1 construct, symptoms of virus infection were seen in the progeny of both YP-1 and YP-2 plants. Production of plant material containing functional recombinant antibodies with anti-coronavirus neutralizing activity The genes encoding the intact chimerical L and H chains (LC and HC) derived from the mAb 6A.C3 specific for the S protein of the TGEV (Transmissible Gastroenteritis Virus) coronavirus, as well as genes encoding small immunopeptides derived from the same antibody, in the form alpha-SIP and epsilon-SIP, have been cloned in the monopartite PPV- and PVX-derived vectors. A sequence encoding the endoplasmic reticulum (ER) retention signal KDEL was linked to the 3-end of the chimeric HC gene. Either direct application of plasmid DNA (PPV-derived constructs) or infiltration with Agrobacterium tumifaciens harbouring binary system plasmids (PPV- and PVX- derived plasmids) were used to inoculate Nicotiana clevelandii plants. All the constructs were shown to be infectious, although infection rates were low for some PPV-derived clones. In order to produce assembled rIgAs, independent bacteria expressing the LC and HC genes were agroinfiltrated simultaneously. Western blot analyses have shown the accumulation of alpha-SIP and epsilon-SIP when they are expressed with the PVX vector. Oligomerization of the SIPs expressed in plants was also observed in western blot under non-reducing conditions. TGEV-specific ELISA data showed high TGEV-binding activity of extracts of plants infected with the mixture of PVX chimera expressing LC and HC. Binding to TGEV was also detected in plants infected with SIP-expressing PPV- or PVX- derived chimera, although binding activity was higher when the SIPs were expressed from the PVX vector. Samples that were positive in ELISA showed rather high TGEV neutralization titers. First in vivo tests indicate that extracts of plants expressing the recombinant antibodies are able to protect piglets against TGEV. Searching for OpenAIRE data... 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