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.