Background and objectives

The use of recombinant viruses in human medicine is taking on increasing importance. Coronaviruses have several advantages for use as vectors for delivery of foreign DNA to cells. Firstly, integration of the virus genome into the host cell chromosome is unlikely. Secondly, coronaviruses have the largest RNA genome known for an RNA virus, offering, in principle, possible space for the insertion of large foreign genes. Thirdly, coronaviruses can infect the enteric and respiratory mucosa, and thus may be used to induce a strong secretory immune response. In addition, the tropism (specificity of infection) of coronaviruses for lungs or intestine may be modified by manipulation of the spike (S) protein. Finally, non-pathogenic coronavirus strains infecting most species of interest, are available for the development of expression systems.

The objective of this project is to test the stability and biosafety of recombinant porcine coronavirus-derived expression systems, for biotechnological applications. Major objectives are to construct a new human-specific version of the vector, and to generate a transgenic swine model to assess biosafety for future applications in human medicine.


Approach and methodology

Two types of expression systems have been developed based on porcine transmissible gastroenteritis coronavirus (TGEV) genomes: a helper-dependent expression system, and a single genome that is modified either by targeted recombination or by engineering a cDNA encoding an infectious RNA. The genetic stability of the helper-dependent expression system was studied in cell culture. Tissue tropism, recombination by modifying the S gene, and expression of the foreign gene were investigated in vivo in 2 day old piglets. A fully functional, infectious virulent TGEV, able to infect both the enteric and respiratory tract, was engineered. Three steps were then followed to obtain the single genome: construction of the full-length cDNA was initiated from a defective minigenome (DI); a two-step amplification system that combines transcription in the nucleus from the cytomegalovirus (CMV) immediate early promoter, with a second amplification in the cytoplasm driven by the viral polymerase; cloning of the cDNA as a bacterial artificial chromosome (BAC).
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Main findings and outcome

A helper-dependent expression vector, which has a tropism for and delivers antigens to the pig’s respiratory or enteric mucosa, was developed. TGEV and human coronavirus HCoV-229E, were used as expression systems. In addition beta-glucuronidase (GUS), the ORF5 of the porcine respiratory and reproductive syndrome virus (PRRSV) were expressed, and high expression levels were obtained. Helper virus and minigenome were detected in about 7% of the cells in vitro, using three independent methods. The limitation of helper-dependent expression systems involving two components, is their instability and the restricted number of co-infected cells. This system maintained expression of the introduced viral vector for about ten passages in cell culture, however starting at passage five, deleted forms of the minigenome were prevalent and their proportion increased with passage number and time. Using TGEV-derived minigenomes, expression was highly dependent on the nature of the heterologous gene. Expression of GUS, ORF5 or PRSSV using TGEV minigenomes was increased until passage three, but new, lower sized mRNA bands occurred after passage five. In general, the insertion of a heterologous gene such as GUS into TGEV derived minigenomes, led to a 50-fold reduction in the levels of the minigenome RNA. RNA from helper virus and minigenomes could be detected by PCR, both in lungs and in the intestine of piglets infected on post natal day 2. By means of in situ hybridisation, it was possible to reveal helper virus replication in a large number of pneumocytes and enterocytes, but minigenome seemed to only be present in pneumocytes. Improving the vector as a single genome system should substantially increase its efficiency in vivo.

Targeted recombination mediated by cross-over, allowed the replacement of the S gene of a respiratory strain of TGEV, by the S gene of enteric TGEV strain PUR-C11. This led to the isolation of viruses with modified tropism and virulence. Recombinants were selected in vivo using their new tropism in piglets.

A full-length infectious TGEV cDNA clone was constructed. Using TGEV cDNA, the 0.72 kb green fluorescent protein (GFP) was cloned into the RNA genome by replacing the non-essential 3a and 3b genes, giving an engineered genome with high stability. TGEV cDNA could be the basis for a tissue-specific expression system that can be used in four species: human, porcine, canine and feline by replacing the S gene included in the cDNA with that of the coronavirus infecting the target species.
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Conclusions

Two expression systems for the introduction of genetically modified elements into animals were developed - one helper-dependent and the other based on single genomes constructed by targeted recombination or by using an infectious cDNA. These model systems provide a suitable system in which the biosafety of introduced genetically modified organisms (GMOs) can be examined. High-level expression of heterologous antigens was obtained, and these in vitro expression levels should be sufficient to elicit protective immune responses in vivo. Efficient single genome, coronavirus vectors have been constructed expressing foreign genes. Thus, a new avenue with high potential has been opened for coronaviruses, making them of great interest as expression vectors for vaccine development and gene therapy. Further expression experiments of the single genome in animals are in progress and will show the in vivo applicability of this new vector system. These model systems provide a suitable system in which the biosafety of introduced GMOs can be examined.