of mucosa-specific RNA-vectors expressing foreign antigens and recombinant
antibodies for prevention of disease
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
Main findings and outcome
A helper-dependent expression vector, which has a tropism for and delivers
antigens to the pigs 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
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.
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.
Almazan F., Gonzalez J.M., Penzes Z., Izeta A., Calvo E., Plana-Duran
J. and Enjuanes L., "Engineering the largest RNA virus genome
as an infectious bacterial artificial chromosome".
Proc. Natl. Acad. Sci. USA, 97,
2000, pp. 5516-5521.
Alonso S., Izeta A., Sola I. and Enjuanes L., "Transcription
regulatory sequences in transmissible gastroenteritis coronavirus".
J. Virol., submitted.
Izeta A., Smerdou C., Alonso S., Penzes Z., Méndez A., Plana-Durán
J. and Enjuanes L., "Replication and packaging of transmissible
gastroenteritis coronavirus-derived synthetic minigenomes".
J. Virol., 73,
1999, pp. 1535-1545.
Sánchez C.M., Izeta A., Sánchez-Morgado J.M., Alonso
S., Sola I., Balasch M., Plana-Durán J. and Enjuanes L.,
"Targeted recombination demonstrates that the spike gene of
transmissible gastroenteritis coronavirus is a determinant of its
enteric tropism and virulence".
J. Virol., 73, 1999, pp. 7607-7618.
Sola I., Izeta A., González J.M. and Enjuanes L., Tissue
specific expression into the mucosal surface using a single genome
vector based on recombinant coronaviruses, submitted.
October 1998 September 2000
Federal Research Centre for Virus Diseases of Animals (FRCVDA)
Insel Riems (DE)
G. Brem, M. Müller
Veterinärmedizinische Fakultät der Universität Wien
Centro Nacional de Biotecnología
University of Bristol (UK)
J. Plana Durán
Fort Dodge Veterinaria
Vall de Bianya (ES)