Note: Descriptions are shown in the official language in which they were submitted.
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Flavivirus Expression and Delivery System
The present invention generally relates to the field of gene expression and in
particular to Flavivirus gene expression and delivery systems and to virus
like
particles produced from such systems.
Improved methodologies far maximising recombinant gene expression are an on-
going effort in the art. Of particular interest is the development of
methodologies
that maximise recombinant expression of mammalian genes in safe vectors
suitable for producing commercially useful quantities of biologically active
proteins.
Currently, there are numerous expression systems available for the expression
of
genes. While procaryotic and yeast expression systems are extremely efficient
and easy to use, these systems suffer from a number of disadvantages,
including
an inability to glycosylate proteins, inefficient cleavage of "pre" or
"prepro"
sequences from proteins (eg., inefficient post translational modification),
and a
general inability to secrete proteins.
Another expression system widely available is the baculovirus expression
system.
This system is arguably one of the most efficient in protein production, but
is
limited only to use in insect cell lines. Unfortunately, insect cell lines
glycosylate
proteins differently from mammalian cell lines thus this system has not proven
useful for the production of many mammalian proteins. Another disadvantage of
this system is that it relies on the use of homologous recombination for the
construction of recombinant virus stocks. Thus, this system often proves very
laborious when large numbers of genetic variants have to be analysed.
In view of these problems the art has sought eucaryotic host systems,
typically
mammalian host cell systems, for mammalian protein production. One feature of
such systems is that the protein produced has a structure most like that of
the
natural protein species and purification often is easier since the protein can
be
secreted into the culture medium in a biologically active form.
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One of the most efficient mammalian cell expression systems is based on
Vaccinia virus. The main problem with this system, however, is that it uses
recombinant viruses that express the heterologous gene upon infection. Thus
there is no control over the virus once it has been release.
Recently researchers have started to explore the use of positive strand RNA
viruses such as Semliki Forest Virus (SFV), Sindbis (SIN) virus, and
poliovirus, as
vectors for expression of heterologous genes in vitro and in vivo. The success
of
these expression systems has been mainly based on each virus' ability to
produce high titer stocks of "pseudo" infectious particles containing
recombinant
replicon RNA packaged by structural proteins. In commercially available
Semliki
Forest virus (SFV) and Sindbis virus expression systems this is achieved by co-
transfection of replicon RNA with defective helper RNA(s) expressing
structural
genes, but lacking the packaging signal. Replicon RNA expression provides
enzymes for RNA replication and transcription of both RNA's, whereas helper
RNA supports the production of structural proteins for packaging of replicon
RNA
via expression of its subgenomic region. The main problem with these
expression
systems is that the viruses used in the expression system are cytopathic and
often compete out the host protein synthesis. Another major disadvantage of
these systems includes possible contamination with infectious particles
containing
packaged full-length genomic RNA (in other words, infectious virus) due to the
high probability of recombination between replicon and helper RNAs.
The present invention seeks to provide an improved expression and delivery
system that at least ameliorates some of the problems associated with prior
art
systems.
Throughout this specification, unless the context requires otherwise, the word
"comprise", or variations such as "comprises" or "comprising" will be
understood
to imply the inclusion of a stated integer or group of integers, but not the
exclusion of any other integer or group of integers including method steps.
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Summ~of_the Invention
The present invention provides a gene expression system comprising:
a) a replicon of flavivirus origin, which is adapted to receive at least a
nucleotide sequence without disrupting its replication capabilities and
which is unable to express at least part or all of a structural protein and or
another proteins) required for packaging of a flavivirus genome into a virus
particle; and
b) at least a second vector that is capable of expressing flavivirus
structural
proteins) and/or any other proteins required for packaging of the self-
replicating expression vector into flavivirus viral particles which vector is
engineered to prevent recombination with the self-replicating vector when
in its presence.
Any replicon (self-replicating expression vector) derived from any flavivirus
RNA
may be used in the present invention. The replicon should however encode a
sufficient amount of a flavivirus 5' UTR and at least a portion of the 5'
flavivirus
coding region for core protein, each of which is required for RNA replication.
Both
the 5' UTR and the 5' core protein coding region of a flavivirus genome
contains
regulatory elements that are required for flavivirus RNA replication. It will
be
appreciated that the flavivirus 5' UTR and the 5' core protein coding region
may
contain mutations or deletions in these regions and still be able to
replicate.
Preferably, the replicon should contain 5' UTR and at least about between 60
and
80 nucleotides from the 5' coding region for flavivirus core protein. The
relative
number of nucleotides from the 5' core protein coding region that will be
required
in the replicon for RNA replication wilt largely depend on the type of
flavivirus
used in the vector. For example when the replicon is derived from Kunjin virus
it
must contain at least 60 nucleotides of the 5' core protein coding region.
In one particular embodiment of the invention there is provided a gene
expression
system comprising:
a) a replicon of flavivirus origin which includes the nucleotide sequence for
a flavivirus 5' untranslated region (UTR), at least a portion of the 5' coding
region for flavivirus core protein, the nucleotide sequence coding for the
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flavivirus non-structural proteins, and part or all of the 3'-terminal
sequence
of a flavivirus 3'UTR, required for self-replication of flavivirus genomic
material, which vector is adapted to receive at least a nucleotide sequence
without disrupting its replication capabilities and which is unable to express
at least part or all of a structural proteins) region and or a proteins) or
part
thereof required for packaging of a flavivirus genome into a virus-like
particle; and
b) at least a second vector that is capable of expressing flavivirus
structural
proteins) and/or any other proteins required for packaging of the self-
replicating expression vector into flavivirus viral particles which vector is
engineered to prevent recombination with the self-replicating vector when
in its presence.
According to the present invention, the replicon of flavivirus origin is
adapted to
receive at least a nucleotide sequence. Insertion of such a nucleotide
sequence,
into the replicon rnay be achieved at any point in the replicon that does not
effect
processing of flavivirus proteins. For example, heteroiogous genes may ~e
inserted into the 3' UTR of the flavivirus replicon, within a structural gene
or within
the locality of deleted structural genes. Preferably, heterologous genes are
inserted into structural genes or in place of deleted structural genes since
such
insertions generally produce higher levels of expression and generally do not
affect replication efficiency of the replicon. If, however, the nucleotide
sequences) are inserted into the 3'UTR they may be preceded by an internal
ribosomal entry site (IRES) sequence. In an embodiment of the invention, the
3'
UTR is used only for insertion of IRES-Neo (neomycin transferase) or IRES-pac
(puromycin N-acetyl transferase) sequences. Such insertions allow the
generation
of stable cell lines persistently expressing foreign genes via antibiotic (eg
Geneticin or puromycin) selection.
In another preferred embodiment of the invention there is provided a gene
expression system comprising:
a) a replicon of flavivirus origin which includes a nucleotide sequence for a
flavivirus 5'UTR, at least a portion of a 5' coding region for flavivirus core
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Received 10 September 1999
protein, a nucleotide sequence coding for a flavivirus non-structural
proteins, the complete or most of the 3'-terminal region of a flavivirus
3'UTR required for self-replication of the genomic material and the
nucleotide coding sequence for flavivirus structural proteins, wherein (i)
the vector is adapted to receive at least a nucleotide sequence without
disrupting the replication capabilities of the vector, (ii) the nucleotide
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Received 24 November 1999
sequence is inserted into the vector in a manner which deactivates
expression of at least a gene that would otherwise code for a flavivirus
structural protein and (iii) the inserted nucleotide sequence does not
encode for the structural protein sequence that it deactivates; and
b) at least a second vector that is (i) capable of expressing the flavivirus
structural proteins) that is not expressed by the replicon and (ii)
engineered to prevent recombination with the self-replicating vector when
in its presence.
When the nucleotide sequence is inserted into the replicon it should be
introduced into the vector in a manner which avoids a frame shift in the open
reading frame of the vector coding sequence. This may be achieved by either
adapting the foreign nucleotide sequence or the vector to ensure the reading
frame of the vector coding sequence is maintained. In an alternative
arrangement foreign nucleotide sequence can be inserted without preserving
open reading frame of the vector if it is followed by a termination codon and
an
internal ribosomal entry site (IRES) sequence to ensure initiation of
translation of
the vector's nonstructural proteins.
A replicon which encodes flavivirus structural and non-structural proteins may
be
either RNA or DNA based provided it is capable of self-replication and encodes
flavivirus structural and non-structural protein coding information. Where the
replicon is an RNA sequence the flavivirus genome is first reverse transcribed
into complementary DNA sequence and cloned into appropriate plasmid vector
containing procaryotic (bacteriophage) DNA-dependent RNA polymerise
promotor. The nucleotide sequence is then inserted into the resulting plasmid
containing replicon complementary DNA sequence and the genomic sequence is
then transcribed back into RNA prior to delivery to a host cell. Where the
vector
is DNA based the flavivirus genome is first reverse transcribed into
complementary DNA sequence and cloned into appropriate plasmid vector
containing eucaryotic expression promotor. A nucleotide sequence can then be
inserted into the resulting plasmid containing replicon complementary DNA
sequence, which is then introduced into a host cell as plasmid DNA.
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Received 24 November 1999
While the replicon will in most circumstances be prepared from a single strain
of
flavivirus it should be appreciated that in some circumstances nucleotide
sequences from more than one flavivirus strain may be brought together in a
single vector. Preferably the replicon is derived from the genomic sequence of
a
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single flavivirus species. Most preferably the replicon is derived from a
single
flavivirus species (such as Kunjin virus (KUN)) and includes the entire or a
substantial portion of the genome of that strain, the genome being modified in
at
least one of its structural proteins to accept a nucleotide sequence such that
the
insertion of the nucleotide sequence into the structural protein nucleotide
sequence disrupts coding for part or all of the structural protein.
Nucleotide sequences that may be inserted into the replicon include, for
example,
parts of flavivirus or non-flavivirus cDNA gene sequences. Nucleotide
sequences) that are inserted into the replicon must, however, disrupt the
expression of at least a structural protein thus preventing viral genome
packaging.
Desirably the inserted nucleotide sequence is a non-flavivirus nucleotide
sequence (hereinafter referred to as a "heterologous nucleotide sequence").
The
heterologous nucleotide sequence is not limited only to a sequence that
encodes
an amino acid sequence, but may also include sequences appropriate for
promoting replication and or expression of a sequence that encodes an amino
acid sequence.
Insertion of a heterologous nucleotide sequence into the replicon rnay occur
at
any point in a flavivirus structural proteins) or in any region of the
nucleotide
sequence where such a protein would normally be expressed in the native
flavivirus sequence had the protein not been deleted. In one embodiment of the
invention the heterologous nucleotide sequence is inserted into at least one
of the
structural genes deactivating that gene. In another embodiment at least a
structural gene is deleted from the vector and the deletion site is adapted to
serve
as the insertion site for heterologous genetic sequences. Most preferably, the
nucleotide sequence is inserted into the locality from where at least a
structural
gene was deleted.
By positioning heterologous nucleotide sequences within the locality of one or
more sites in the replicon that might otherwise code for structural genes in a
native flavivirus, the replicon is unable to produce structural proteins for
viral
packaging.
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Received 10 September 1999
To induce viral packaging the invention employs a second vector that is
engineered to prevent recombination with the replicon. Preferably, the second
vector is heterologous in origin to the origin of the replicon. Any non-
flavivirus
vector that is engineered to prevent recombination with the replicon may be
employed in the expression system to deliver the flavivirus structural protein
that
is deactivated in the replicon. For example, if a KUN replicon is used as the
self-
replicating expression vector, then the second vector may be derived from a
virus
other than a flavivirus. For example, the second vector could be derived from
an
alphavirus such as SFV or SIN, or from DNA virus such as adenovirus, fowlpox
virus, or vaccinia virus. Those of ordinary skill in the field will know other
vectors
that may be employed in this role. In a highly preferred form of the invention
the
replicon is derived from KUN while the second vector is derived from SFV to
take
account of the impossible recombination between KUN RNA and SFV RNA.
In an alternative embodiment of the invention the second vector may be a
plasmid DNA expression vector. For example, highly efficient packaging may be
achieved by inserting structural genes into CMV based DNA expression
cassettes which are inserted into baculovirus expression vectors which provide
very efficient delivery of the cassettes into mammalian cells (see for example
Shoji et al, (1997) J.Gen.Virol., 78: 2657-2664 and pBacMam-1 vector described
on the Novagen homepage). In another example the second vector may be an
inducible plasmid DNA expression vector (for example tetracycline inducible
vector (Clontech)) allowing selection of packaging cell lines expressing KUN
structural proteins in response to addition or removal of tetracycline in the
incubation medium.
The present invention also provides a method for producing a stable cell line
capable of persistently producing replicon RNA's, comprising the steps of:
(i) introducing into a cell a replicon of flavivirus origin which is adapted
to receive at least a nucleotide sequence without disrupting its
replication capabilities and which is unable to express at least part
or all of a structural proteins) region and or a proteins) or part
thereof required for packaging of a flavivirus genome into a virus-
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like particle; and
(ii) culturing that cell line under conditions which permit cell growth and
replication.
Conditions that permit cell growth and replication will be known to those of
ordinary skill in the field. In particular the conditions will vary depending
on the
type of cell that is used in the method. To prepare such cell lines, the
described
vectors are preferably constructed in selectable form by inserting an IRES-Neo
or
IRES-pac cassette into the 3'UTR.
In another embodiment, the invention provides a method for producing a
flavivirus
like particles containing a replicon as herein described comprising the steps
of:
(i) introducing into a cell a replicon of flavivirus origin which is adapted
to receive at least a nucleotide sequence without disrupting its
replication capabilities and which is unable to express at least part
or all of a structural proteins) region and or a proteins) or part
thereof required for packaging of a flavivirus genome into a virus-
like particle;
(ii) introducing into a replicon containing cell a second vector that is
capable of expressing flavivirus structural proteins) and/or any
other proteins required for packaging of the self-replicating
expression vector into flavivirus viral particles which vector is
engineered to prevent recombination with the self-replicating vector
when in its presence; and
(iii) harvesting virus like particles containing the replicon.
Preferably the replicon containing virus like particles prepared by this
method are
purified from cellular and viral proteins and nucleic acids that may cause an
adverse immunological or physiological reaction when introduced into an
animal.
Methods for purifying such viral particles are known in the art. Most
preferably
the replicon containing virus like particles are 50%, 60%, 70%, 80%, 90%, 95%
or
99% free of all contaminating material including cellular and viral proteins,
lipids
and nucleic acids.
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In further embodiment, the invention provides a flavivirus like particles
containing
a flavivirus replicon that is adapted to receive at least a nucleotide
sequence
without disrupting its replication capabilities. Desirably the virus like
particles are
purified from cellular and viral nucleic acids and amino acid sequences that
may
cause an adverse immunological or physiological reaction when introduced into
an animal. Such particles may be used as a therapeutic agent. A person of
ordinary skill in the field will appreciate that the described virus particles
can be
used to deliver to a subject any nucleotide sequence that is inserted into the
replicon. For example the replicon within the virus like particles may be
employed
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to deliver to a cell a nucleotide sequence encoding one or more amino acid
sequences which are capable of inducing, for example, a protective immune
response to a subject.
In further embodiment, the invention provides a DNA based replicon of
flavivirus
origin that is adapted to receive at least a nucleotide sequence without
disrupting
its replication capabilities. The DNA based replicon may be introduced into a
cell
as a naked vector (i.e. flavivirus structural proteins do not surround it) or
alternatively used for preparation of virus like particles containing
encapsidated
replicon RNA in accordance with the described method. Whether the DNA based
replicon is prepared as a naked vector or in virus like particles it should be
purified from cellular and viral nucleic acids and amino acid sequences that
may
cause an adverse immunological or physiological reaction in an animal prior to
introduction into that animal. Such particles may be used as a therapeutic
agent.
A person of ordinary skill in the field will appreciate that the described
virus
particles can be used to deliver to a subject any nucleotide sequence that is
inserted into the replicon. In a particularly preferred form of the invention
the
replicon is prepared in DNA form and is used for preparation of virus like
particles
containing encapsidated replicon RNA for delivery into a cell via infection.
Detailed description of the invention
Although the present invention describes a means for producing proteins, the
term "protein" should be understood to include within its scope parts of
proteins
such as peptide and polypeptide sequences.
In use, the replicon is introduced into a host cell where gene expression and
hence protein production take place. Because the vector is capable of self-
replication, multiple copies of the replicon will also be generated. This
leads to an
exponential increase in the number of replicons in the host cell as well as an
exponential increase in the amount of protein that is produced.
Upon introduction of the second vector, containing the structural genes
necessary to produce virus particles, structural proteins are produced. These
proteins encapsulate the replicon therein forming a "pseudo" recombinant virus
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that is only capable of producing heterologous protein inside another cell.
The
pseudo-virus
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can not however replicate to produce new viral particles because the genes
necessary for the production of the structural proteins are not provided in
the
replicon. Pseudo-virus stock will only be produced when co-transfection of the
replicon and the vector bearing the structural genes occurs.
Some advantages associated with the use of the present invention include:
(1) The flavivirus expression system has relatively high level of protein
expression in eukaryotic cell lines.
(2) The flavivirus expression system is capable of expressing proteins in a
wide variety of mammalian cell lines and cell types.
(3) The replicons used in the flavivirus expression system produce a long-
term non-cytopathic replication in host cells. There are no observable
effects on the host's translation process. This feature of flavivirus
replicons also allows selection of stable cell lines continuously
expressing other genes using a replicon vector expressing a gene
confirming resistance to an antibiotic (e.g. neomycin transferase (Neo),
puromycin N-acetyltransferase (pac), etc.)
{4) The flavivirus expression system is an RNA system that does not
permit integration of viral genomic material into a host's genomic
sequence.
The replication of flaviviruses is quite different from other viruses. For
example,
flaviviruses differ from alphaviruses (such as SFV and SIN) by their genome
structure (structural genes situated at the 5' end of the genome) and by the
absence of synthesis of subgenomic RNA. Furthermore, there are no data to
date on packaging of flavivirus RNA.
Substantial progress in the development of mammalian cell expression systems
has been made in the last decade, and many aspects of these systems' features
are well characterised. A detailed review of the state of the art of the
production
of foreign proteins in mammalian cells, including useful cell lines, protein
expression-promoting sequences, marker genes, and gene amplification
methods, is disclosed in Bendig, M., (1988) Genetic Engineering 7: 91-127.
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It will be appreciated that any replicon derived from any flavivirus RNA,
which is
lacking at least a structural gene and which is adapted to receive at least a
nucleotide sequence may be employed in the present invention. Preferably the
replicon used in the invention should be adapted to include part or all of the
following: at least, about the first 150 nucleotides of a flavivirus genome;
at least
about the last 60 nucleotides of E protein; substantially all of the
nonstructural
region; and part or all of the 3'UTR. Replication of a flavivirus genome is
dependent on the genes in the nonstructural region of the genome being present
during transcription and translation. Preferably any modification made to the
nonstructural region should not interfere with the functional activity of the
genes
within the nonstructural region of the genome. In a highly preferred form of
the
invention, the replicon is derived from KUN and includes the first 157
nucleotides
of the KUN genome, the last 66 nucleotides of E protein, the entire
nonstructural
region, and all of the 3'UTR.
Optimal flavivirus replicon design for transfection into eukaryotic cells
might also
include sequences inserted into the replicon such as: sequences to promote
expression of the heterologous gene of interest, including appropriate
transcription initiation, termination, and enhancer sequences; as well as
sequences that enhance translation efficiency, such as the Kozak consensus
sequence; internal ribosomal entry site (IRES) of picornaviruses; an
alphavirus
subgenomic 26S promoter to enhance expression of inserted genes if
cotransfection with alphavirus replicon RNA is used.
Flavivirus replicon RNA can be produced in in vitro transcription reaction
with
DNA-dependent RNA polymerase from corresponding plasmid cDNA constructs
incorporating a procaryotic (bacteriophage) promoter upstream of KUN genome
sequence. Such replicon constructs are referred to as RNA-based replicon
vectors. Resulting in vitro transcribed RNA can be delivered into the cell
cytoplasm by RNA transfection followed by its self-amplification and
translation
resulting in expression of heterologous genes.
Alternatively, flavivirus replicon RNA can be produced in cells (in vivo) by
the
cellular transcription machinery after transfection of corresponding plasmid
cDNA
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constructs incorporating a eucaryotic expression promoter upstream and
transcription termination signal downstream of the KUN replicon sequence.
These replicon constructs are referred to as DNA-based replicon vectors.
Production of replicon RNA from these DNA-based vectors occurs in the nucleus
of transfected cells by RNA polymerise II, followed by the transport of RNA
into
thecytoplasm where its amplification and translation takes place.
Finally, flavivirus replicon RNA produced in cells as a result of its self-
amplification either after RNA transfection (RNA-based vector) or after
plasmid
DNA transfection (DNA-based vectors) can be packageed into the secreted virus-
like particles by providing KUN structural proteins from a second vector. VLPs
can then be used to deliver the encapsidated replicon RNA into cells by
infection.
In one example of the invention the DNA-based replicon vector is derived from
KUN virus and contains a eucaryotic promoter sequence (such as CMV or hybrid
CMV enhancer-chicken ~i-actin promoter [GAG]) upstream of the KUN 5'UTR and
a hepatitis delta virus ribozyme sequence followed by an SV40, bovine growth
hormone, or rabbit a-globin transcription terminator sequences downstream of
the
KUN 3'UTR. Transfection of the resulting plasmid DNA in cells will ensure
production of a KUN replicon RNA transcript with the authentic 5'-end by
cellular
RNA polymerise II and with the authentic 3'-end cleaved by hepatitis delta
virus
ribozyme, which is preferred for its efficient replication.
It will be appreciated that the nucleotide sequence inserted into the replicon
may
encode part or all of any natural or recombinant protein except for the
structural
protein sequence into which or in place of which the nucleotide sequence is
inserted. For example, the nucleotide sequence may encode a single
polypeptide sequence or a plurality of sequences linked together in such a way
that each of the sequences retains their identity when expressed as an amino
acid sequence. Where the nucleotide sequence encodes a plurality of peptides,
the peptides should be linked together in such a way that each retains its
identity
when expressed. Such polypeptides may be produced as a fusion protein or
engineered in such a manner to result in separate polypeptide or peptide
sequences.
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Where the vector is used to deliver nucleotide sequences to a host cell to
enable
host cell expression of immunogenic polypeptides, the nucleotide sequence may
encode one or more immunogenic polypeptides in association with a range of
epitopes which contribute to T-cell activity. In such circumstances the
heterologous nucleotide sequence preferably encodes epitopes capable of
eliciting either a T helper cell response or a cytotoxic T-cell (CTL) response
or
both.
The replicon described herein may also be engineered to express multiple
nucleotide sequences allowing co-expression of several proteins such as a
plurality of antigens together with cytokines or other immunomodulators to
enhance the generation of an immune response. Such a replicon might be
particularly useful for example in the production of various proteins at the
same
time or in gene therapy applications.
By way of example only the nucleotide sequence may encode the cDNA
sequence of one or more of the following: malarial surface antigens; beta-
galactosidase; any major antigenic viral antigen eg Haemagglutinin from
influenza virus or a human immunodeficiency virus (HIV) protein such as HIV gp
120 and
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HIV gag protein or part thereof; any eukaryotic polypeptide such as, for
example,
a mammalian polypeptide such as an enzyme, e.g. chymosin or gastric lipase; an
enzyme inhibitor, e.g. tissue inhibitor of metalloproteinase (TIMP); a
hormone,
e.g. growth hormone; a lymphokine, e.g. an interferon; a cytokine, e.g an
interleukin (eg IL-2, IL-4, IL-6 etc); a chemokine eg macrophage inflammatory
protein-2; a plasminogen activator, e.g. tissue plasminogen activator (tPA) or
prourokinase; or a natural, modified or chimeric immunoglobulin or a fragment
thereof including chimeric immunoglobulins having dual activity such as
antibody-
enzyme or antibody-toxin chimeras.
The nucleotide sequence may also code for one or more amino acid sequences
that serve to enhance the effect of the protein being expressed. For example,
ubiquitination of viral proteins expressed from DNA vectors results in
enhancement of cytotoxic T-lymphocyte induction and antiviral protection after
immunization. Thus, in a preferred embodiment of the invention the replicon
may
encode ubiquitin in association with the protein to be expressed thus
targeting the
resulting fusion protein to proteosomes for efficient processing and uptake by
the
MHC class I complexes.
In frame fusion of proteins other than flavivirus replicon encoded proteins to
the
C-terminus of ubiquitin also results in the efficient cleavage of such fusion
protein
after the last C-terminal residue of ubiquitin thus releasing free protein of
interest.
Preferably a ubiquitin sequence is inserted into the replicon vector. By way
of
example only the ubiquitin sequence is preferably inserted either prior to the
5'
end of the heterologous genetic sequence or at the 3' end of the heterologous
genetic sequence.
The second vector that contains the flavivirus structural genes) should be
engineered to prevent recombination with the self-replicating expression
vector.
One means for achieving this end is to prepare the second vector from genetic
material that is heterologous in origin to the origin of the self-replicating
expression vector. For example, the second vector might be prepared from SFV
when the replicon is prepared from KUN virus.
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To optimise expression of the flavivirus structural genes, the second vector
might
include such sequences as: sequences to promote expression of the genes of
interest, including appropriate transcription initiation, termination, and
enhancer
sequences; as well as sequences that enhance translation efficiency, such as
the
Kozak consensus sequence. Preferably, the second vector contains separate
regulatory elements associated with each of the different structural genes
expressed by the vector. Most preferably, the flavivirus C gene and the prME
genes are placed under the control of separate regulatory elements in the
vector.
The processing of flavivirus structural proteins during virus replication in
cells is
complex and requires a number of post-translational cleavages by host and
viral
proteases. Numerous in vitro and in vivo studies on processing of the C-prM
region have established two cleavage events: cleavage at a dibasic cleavage
site
by viral NS2B-NS3 protease generating the carboxy terminus of mature virion C
protein, which appears to be a prerequisite for the efficient cleavage at the
NH2
terminus of prM by cellular signalase. While viral proteases are expressed by
the
replicon during expression of the genes forming the nonstructural region of a
flavivirus, it will be appreciated that the second vector may also be adapted
to
include genes encoding viral NS2B-NS3 protease.
Further C-prM-E genes can be expressed as a single cassette only if C and prM
genes separated by a self-cleaved peptide like for example 2A autoprotease of
foot-and-mouth disease virus in order to ensure proper processing of C-pM
region
in the absence of KUN virus encoded NS2B-NS3 protease.
The present invention also provides stable cell lines capable of persistently
producing replicon RNAs. To prepare such cell lines, the described vectors are
preferably constructed in selectable form by inserting an 1RES-Neo or IRES-pac
cassette into the 3'UTR.
Host cell lines contemplated to be useful in the method of the invention
include
any eukaryotic cell lines that can be immortalised, ie., are viable for
multiple
passages, (eg., greater than 50 generations), without significant reduction in
growth rate or protein production. Useful cell line should also be easy to
transfect, be capable of stably maintaining foreign RNA with an unarranged
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sequence, and have the necessary cellular components for efficient
transcription,
translation, post-translation modification, and secretion of the protein.
Currently
preferred cells are those having simple media component requirements, and
which can be adapted for suspension culturing. Most preferred are mammalian
cell lines that can be adapted to growth in low serum or serum-free medium.
Representative host cell lines include BHK (baby hamster kidney), VERO, C6-36,
COS, CHO (Chinese hamster ovary), myeloma, HeLa, fibroblast, embryonic and
various tissue cells, eg., kidney, liver, lung and the like and the like.
Desirably a
cell line is selected from one of the following: BHK21 (hamster), SK6 (swine),
VERO (monkey), L292 (mouse), HeLa (human), HEK (human), 2fTGH cells,
HepG2 (human). Useful cells can be obtained from the American Type Culture
Collection (ATCC), Rockville, Md. or from the European Collection of Animal
Cell
Cultures, Porton Down, Salisbury SP40JG, U.K.
With respect to the transfection process used in the practice of the
invention, all
means for introducing nucleic acids into a cell are contemplated including,
without
limitation, CaPO<sub>4</sub> co-precipitation, electroporation, DEAF-dextran
mediated
uptake, protoplast fusion, microinjection and lipofusion. Moreover, the
invention
contemplates either simultaneous or sequential transfection of the host cell
with
vectors containing the RNA sequences. In one preferred embodiment, host cells
are sequentially transfected with at least two unlinked vectors, one of which
contains flavivirus replicon expressing heterologous gene, and the other of
which
contains the structural genes.
The present invention also provides virus like particles containing flavivirus
replicons and a method for producing such particles. It will be appreciated by
those skill in the art that virus like particles that contain flavivirus
derived replicons
can be used to deliver any nucleotide sequence to a cell. Further, the
replicons
may be of either DNA or RNA in structure. One particular use for such
particles is
to deliver nucleotide sequences coding for polypeptides that stimulate an
immune
response. Such particles may be employed as a therapeutic or in circumstances
where the nucleotide sequence encodes peptides that are capable of eliciting a
protective immune response they may be used as a vaccine. Another use for
such particles is to introduce into a subject a nucleotide sequence coding for
a
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protein that is either deficient or is being produced in insufficient amounts
in a
cell.
The replicon containing flavivirus like particles that contain nucleotide
coding
sequence for immunogenic polypeptide(s) as active ingredients may be prepared
as injectables, either as liquid solutions or suspensions; solid forms
suitable for
solution in, or suspension in, liquid prior to injection may also be prepared.
The
flavivirus replicon therapeutics) may also be mixed with excipients that are
pharmaceutically acceptable and compatible with the replicon encapsulated
viral
particle. Suitable excipients are, for example, water, saline, dextrose
glycerol,
ethanol, or the like and combinations thereof. In addition, if desired, the
therapeutic may contain minor amounts of auxiliary substances such as wetting
or
emulsifying agents, pH buffering agents, and/or adjuvant which enhance the
effectiveness of the therapeutic.
The repiicon containing flavivirus like particles may be conventionally
administered parenterally, by injection, for example, either subcutaneously or
intramuscularly. Additional formulations which are suitable for other modes of
administration include suppositories and, in some cases, oral formulations.
For
suppositories, traditional binders and carriers may include, for example,
polyalkylene glycols or triglycerides; such suppositories may be formed from
mixtures containing the active ingredient in the range of 0.5% to 10%,
preferably
1 %-2%.
Oral formulations include such normally employed excipients as, for example,
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate and the like, These compositions
take the form of solutions, suspensions, tablets, pills, capsules, sustained
release
formulations or powders and contain 10%-95% of virus like particles,
preferably
25-70%.
The flavivirus like particles may be formulated into the vaccine as neutral or
salt
forms. Pharmaceutically acceptable salts include the acid addition salts
(formed
with free amino groups of the peptide) and which are formed with inorganic
acids
such an, for example, hydrochloric or phosphoric acids, or such organic acids
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such as acetic, oxalic, tartaric, malefic, and the like. Salts formed with the
free
carboxyl groups may also be derived from in- organic bases such as, for
example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such
organic bases as isopropylamins, trimethylamine, 2-ethylamino ethanol,
histidino,
procaine, and the like.
The flavivirus like particles may be administered in a manner compatible with
the
dosage formulation and in such amount as will be prophylactically andlor
therapeutically effective. The dose of viral particles to be administered
depends
on the subject to be treated, the type of nucleotide sequence that is being
administered and the type of expression efficiency of that sequence and in the
case where the nucleotide sequence encodes immunogenic peptide/polypeptides
the degree of protection desired. Precise amounts of active ingredient
required
to be administered may depend on the judgment of the practitioner and may be
peculiar to each subject.
The flavivirus like particles may be given to a subject in a single delivery
schedule, or preferably in a multiple delivery schedule. A multiple delivery
schedule is one in which a primary course of delivery may be with 1-10
separate
doses, followed by other doses given at subsequent time intervals required to
maintain and or re-enforce the effect sought and if needed, a subsequent
doses)
after several months. The delivery regimen will also, at least in part, be
determined by the need of the individual and be dependent upon the judgment of
the practitioner.
BRIEF DESCRIPTION OF THE DRAWINGS
Further features of the present invention are more fully described in the
following
Figures and Examples. In the figures:
Figure 1: illustrates the construction and specific infectivity of the full-
length KUN cDNA clones, and the structure of KUN replicon RNAs.
Schematic representations of the full-length and deleted (replicon)
constructs show consecutive substitutions of the cDNA fragments in AKUN
clone (textured boxes} with analogous fragments obtained by RT-PCR
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from KUN virion RNA (shaded boxes). PFU titers on the right hand side of
the figure represent an average (from three experiments) obtained after
electroporation of the transcribed RNAs into BHK21 cells and determined
by plaque assay; the titer of purified wild type KUN RNA was 105-106
PFU/pg RNA. Bgl(89), Sac(1481), Sph(2467), Dra(8376), Xho(11021)
show restriction enzyme sites used in replacement cloning with the
numbers in brackets representing nucleotide numbers in the KUN
sequence. An Expand High Fidelity PCR kit (Boehringer Mannheim) was
used to obtain the indicated cDNA fragment of 6895 nucleotides in the
FLSD and FLSDX constructs, and "Pfu PCR" in FLSDX indicates that this
cDNA fragment of 2645 nucleotides was obtained using Pfu DNA
polymerase (Stratagene). C20DXrep and C20DXrepNeo constructs were
prepared as described below in Example 1 (C20DXrep) and in Example 4
(C20DXrepNeo). Open boxes represent the deleted part of the genome;
Ires - internal ribosomal entry site of encephalomyelitis virus RNA; Neo -
neomycin transferase gene.
Figure 2 illustrates a schematic representation of the recombinant SFV
constructs. The solid line in all constructs represents the segment of the
SFV replicon genome flanking the multiple cloning site, open boxes show
the inserted KUN structural genes C, prM, and E as indicated, 2fiS shows
the position of the subgenomic SFV promoter, the filled and partially filled
boxes in the KUN prM and E genes represent hydrophobic signal and
anchor sequences, respectively. Capital letters in the nucleotide
sequences show authentic KUN nucleotides, small letters show
nucleotides derived from the pSFV1 vector or encoded in the primers used
for PCR amplification of KUN genes. Bold and italicised letters show
initiation (ATG) and termination (taa, tag) codons. Numbers with arrows
represent amino acid positions in the KUN polyprotein. Msc, Sma, Spe,
Bam, and Bgl represent specific restriction sites. Asterisks indicate that
these restriction sites were destroyed during the cloning procedure.
Figure 3 illustrates expression of KUN C protein by recombinant SFV-C
replicon. A) Immunofluorescence analysis of BHK21 cells at 18h after
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transfection with SFV-C RNA (SFV-C, panels 1, 3, and 5) using KUN anti-
c antibodies. SFV1 (panels 2, 4, and 6) represents IF of cells transfected
with the control SFV1 RNA prepared from pSFV1 vector. Cells in panels 1
and 2 were photographed at lower magnification then in panels 3 to 6. Ace
is an abbreviation for acetone fixation, F+Me is an abbreviation for
formaldehyde-methanol fixation. B) Metabolic labelling with 35S-
methionine/cysteine and radioimmunoprecipitation analysis with antibodies
to C protein (+anti-C) of SFV-C and SFV1 transfected BHK21 cells. BHK21
cells in 60mm culture dishes at 18h after transfection were continuously
labelled with 50 ~Ci/ml of 35S-methionine/cysteine for 4h. Labelled cell
lysates and radioimmunoprecipitates were prepared and samples were
electrophoresed in a 7 5% polyacrylamide gel. Sample volumes were 1 ~I
of 500 ~I in SFV-C, 0.5 p.1 of 300 ~.I in SFV1, 10 ~I of 30 ~I
radioimmunoprecipitate from 160 w1 of both SFV-C and SFV1 (+anti-C) cell
lysates. Dots indicate the location of KUN proteins NSS, NS3, E, NS4B,
prM, NS2A, C, and NS4A/NS2B (from top to bottom) in the radiolabeled
KUN infected cell lysate. The arrow shows position of KUN C protein.
Numbers represent molecular weights of low range pre-stained Bio-Rad
protein standards. This and following figures were prepared by scanning all
the original data (slides, autoradiograms, etc.) on the Arcus II scanner
{Agfa) using FotoLook software (Agfa) for Macintosh at 150 Ipi resolution,
followed by assembling of the montages using Microsoft PowerPoint 97
software and printing on Epson Stylus Color 800 printer at 720-1440 dpi
resolution using Epson photo quality ink jet paper.
Figure 4 illustrates expression of KUN prME genes by recombinant SFV
replicon. A) IF analysis of SFV-prME and SFV1 transfected BHK21 cells at
18h after transfection using KUN monoclonal anti-E antibodies. (B) and (C)
show the results of pulse-chase labelling and radioimmunoprecipitation
analysis with KUN monoclonal anti-E antibodies, respectively, of SFV-
prME transfected BHK21 cells, where CF {culture fluid) and C (cells)
represent samples collected during chase periods. Lanes 1 to 9 in (B) and
(C) represent the same samples either directly electrophoresed in 12.5%
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SDS-polyacrylamide gel (B), or radioimmunoprecipitated with anti-E
antibodies followed by electrophoresis in a 12.5% SDS-polyacrylamide gel
(C). Lanes 2 and 9 show samples collected after a 4h-chase period from
culture fluid and cells, respectively, after transfection with the control
SFV1
RNA. Lanes 3, 4, and 5 show culture fluid samples collected at 1 h, 4h, and
6h of chase periods, respectively, and lanes 6, 7, and 8 show the
corresponding chase samples from the cells. fn (B) 10p1 of total 700 ~I of
culture fluid and 5 p1 of total 300 p.1 of cell lysates samples were used for
electrophoresis. In (C) 10 p1 of total 30 p.1 of immunoprecipitate prepared
either from 150 p1 of the cell lysate or from 350 w1 of the culture fluid were
used for electrophoresis. The exposure time of the dried gel for cell lysates
was 1 day, and 5 days for culture fluids. Dots in lane 1 of (B) and (C)
indicate KUN proteins in the radiolabeled KUN cell lysates, as in Fig. 2B.
Numbers represent molecular weights in the low range pre-stained Bio
Rad protein standards.
Figure 5 illustrates expression of all three KUN structural proteins by the
recombinant SFV-prME-C replicon. A) Double IF analysis of the same field
in BHK21 cells at 18h after transfection with SFV-prME RNA using KUN
anti-C (panel 1 ) and anti-E (panel 2) antibodies, with Texas Red (TR) and
F1TC conjugated secondary antibodies, respectively. In (B) and (C), cells at
18h after transfection with SFV-prME-C RNA were pulsed with 35S-
methionine/cysteine for 1 h; subsequently, 300 ~I (from total of 600p1) of
cell lysates ("C" in [B] and in [C]) and 1 ml (from total of 2m1) of culture
fluids ("CF" in [B]) collected at different chase intervals (1 h, 6h, and 9h),
were immunoprecipitated either with KUN monoclonal anti-E antibodies
(B), or with KUN anti-C antibodies (C). Ten ~I (from total of 30p1) of
immunoprecipitated samples were electrophoresed in 12.5% (B) and 15%
{C) SDS-polyacrylamide gels. Dots in (B) indicate KUN proteins in the
labelled KUN cell lysates as in Fig. 2B. Dots in (C) represent KUN proteins
prM, NS2A, C, and NS4A/NS2B (from top to bottom) in the radiolabeled
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KUN infected cell lysate. Numbers represent molecular weights of the low
range pre-stained Bio-Rad protein standards.
Figure 6 illustrates packaging of KUN replicon RNA by KUN structural
proteins expressed from the recombinant SFV replicons. {A) IF analysis
with KUN anti-NS3 antibodies of BHK21 cells infected with the culture fluid
collected from BHK21 cells at 26h after transfection first with C20DXrep
RNA and 26h later with SFV-prME-C RNA (panel 1 ), or with SFV-prME and
SFV-C RNAs (panel 2), or with SFV-prME RNA (panel 3). {B) and (C) show
Northern blot analysis of RNAs isolated from BHK21 cells infected as
described in (A), using labelled KUN-specific (B) and SFV-specific (C)
cDNA probes. Lane 1 in (B) and lane 2 in (C) correspond to the cells in
panel 1 in (A). Lane 2 in (B) and lane 3 in (C) correspond to the cells in
panel 2 in (A). Lane 1 in (C) represents in vitro synthesized SFV-prME-C
RNA. Arrows in (B) and (C) indicate the positions of RNAs of about 8.8 kb
for KUN replicon RNA and about 10.8 kb for SFV-prME-C RNA determined
relative to migration in the same gel of ethidium bromide-stained 7~ DNA
digested with BstEll (New England Biolab).
Figure 7 illustrates optimisation of conditions for packaging of KUN
replicon RNA. Northern blot analysis of BHK21 cells infected with filtered
and RNase-treated culture fluid samples. In (A), samples were collected at
a fixed time (24h) after second transfection (with SFV-prME-C RNA) and
using different time intervals as shown between transfections of C20DXrep
and SFV-prME-C RNAs. In (B), samples were collected at different times
as shown after the second transfection (with SFV-prME-C RNA) which
occurs at a fixed time (30h) after the first transfection (with C20DXrep
RNA). The probe in both (A) and (B) was a radiolabeled cDNA fragment
representing the last 761 nucleotides of the KUN genome. Titers in (A)
shown under the lanes in the Northern blot represent the amounts of
infectious units (1U) contained in the corresponding samples of culture
fluids used for infections and determined by IF analysis with anti-NS3
antibodies and counting of IF positive cells.
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Figure 8 illustrates characterisation of infectious particles. (A) Inhibition
of
infection with encapsidated particles, released from cells transfected
sequentially with C20DXrep and SFV-prME-C RNAs (as in Fig. 6), by
incubation with KUN anti-E monoclonal antibodies. Panel 1 represents IF
with anti-NS3 antibodies of cells infected with culture fluid collected after
the transfections and incubated with anti-E monoclonal antibodies for 1 h at
37°C. Panel 2 represents IF with anti-NS3 antibodies of cells infected
with
the same sample of culture fluid incubated under similar conditions in the
absence of anti-E antibodies. (B) shows IF analysis with anti-N3 antibodies
of cells infected with equal proportions of resuspended pellet (panel 1; 2p,1
from 50w1 of total volume) or supernatant fluid (panel 2; 200p.1 from 5mf of
total volume) from the culture fluid collected from cells transfected with
C20DXrep and SFV-prME-C RNAs and subjected to ultracentrifugation. (C)
Radioimmunoprecipitation analysis with anti-E antibodies of culture fluids
from cells transfected with SFV-prME-C RNA (lane 2), sequentially
transfected with C20DXrep and SFV-prME-C RNAs (lane 1 ), and infected
with KUN virus (lane 3). Dots show faint bands corresponding to C and
prM visible (in the original autoradiogram) in lane 1, but only a faint band
for prM in lane 2. (D) RT-PCR analysis with KUN-specific primers of RNAs
extracted from the anti-E-immunoprecipitates of culture fluid samples
collected after transfection sequentially with C20DXrep and SFV-prME-C
RNAs (lane 2), or after transfection only with SFV-prME-C RNA (lane 3), or
after infection with KUN virus (lane 4). Lane 1 represents PhiX174 RF DNA
digested with Haelll (New England Biolab).
Figure. 9. Sedimentation and electron microscopy analyses of KUN
replicon and virion particles. (A) Sedimentation profiles of virions and
replicon particles in parallel sucrose density gradients. Particles were
collected from culture fluids of BHK cells either at 35h after sequential
transfections with C20DXrep and SFV-prME-C107 RNAs, or at 24h after
infection with KUN virus, and were concentrated by ultracentrifugation as
described in Materials and Methods. The pelleted particles were
resuspended in 300 ~I of PBS-0.1 %BSA overnight at 4°C, and clarified
by
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centrifugation at 16,OOOg in the microcentrifuge for 10 min. The
supernatant was overlaid on the top of a 12 ml 5-25% sucrose density
gradient which was centrifuged at 38,000 rpm for 70 min at 20°C in an
SW41 rotor. 0.5 ml fractions were collected from the bottom of the gradient
and diluted 1:2 (replicon particles) or 1:100 (KUN virions) for infectivity
assays by IF on cover slip cultures of BHK cells at 24h (replicon particles)
or at 18h (KUN virions) after infection, using anti-E antibodies; titers of
infectious particles were determined as described earlier (see . (B) Electron
micrographs of virions (left panel) and encapsidated replicon particles (right
panel) stained with uranyl acetate. Fractions 5-7 of replicon particles in
(A),
and fractions 2-4 of KUN virions, were pooled and incubated with 1/20
dilution of anti-E antibodies for 1 h at 20°C, followed by 2h
incubation at
4°C with constant rotation. Particles were then again concentrated by
ultracentrifugation as described above, and pellets were resuspended in
175 p.1 of PBS-0.1 %BSA overnight at 4°C. Resuspended particles were
then sonicated in the Transsonic 700/h sonicating water bath (CAMLAB,
Germany) for 1 min and pelleted onto a carbon coated formvar grid by
centrifugation in an 18° fixed angle A-100 rotor in a Beckman Airfuge
for 1 h
at 80,000 rpm. Grids were stained with 4% uranyl acetate and particles
were visualized by electron microscopy. The bar represents 200nm.
Figure. 10. Schematic representation of the Kunjin replicon expression
vectors and recombinant constructs. (A) shows C20DX2Arep(Neo)
vectors) and its derivatives. SP6 shows the position of the SP6 promoter.
5'UTR and 3'UTR represent 5' and 3' untranslated regions, respectively.
C20 corresponds to the first twenty amino acids of KUN Core protein. 22E
corresponds to the last twenty two amino acids of KUN E protein. NS1-
NS5 correspond to the sequence coding for KUN nonstructural proteins.
2A indicates sequence coding for 2A autoprotease of foot-and-mouth
disease virus (FMDV) with its cleavage site indicated. IRESNeo
represents a sequence of an internal ribosomal entry site (IRES) of
encephalomyocarditis virus (EMCV) RNA followed by a sequence coding
for the neomycin transferase gene (Neo). This cassette was inserted at
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the indicated position in the 3'UTR to obtain C20DX2ArepNeo vector for
stable selection of replicon expressing cells (similar to OME/76Neo,
Khromykh and Westaway, J. Virol.,1997, 71:1497-1505). Spel shows a
unique restriction site for cloning of heterologous genes. (B) shows a list
of KUN replicons with heterologous genes inserted into the Spel site of
C20DX2Arep vector. hcv-trCore and hcv-flCore - sequences coding for
the first 160 and 191 amino acids of hepatitis C virus Core protein,
respectively; CAT - chloramphenicol acetyltransferase; GFP - green
fluorescent protein, hcv-NS3 - sequence coding for amino acids 183 to
617 of hepatitis C virus NS3 protein; VSV-G - glycoprotein G of vesicular
stomatitis virus; ~i-GAL - Escherichia coli ~i-galactosidase. +IRESNeo
signs opposite to CAT and GFP indicate that these genes were also
cloned into C20DX2ArepNeo vector. (C) Dicistronic C20DXIRESrep
vector and its derivative construct C20DX/CAT/IRESrep. Ascl-Stop shows
the position of a unique site for cloning of heterologous genes followed by
the translation termination codon (Stop). The other abbreviations are as in
(A) .
Figure. 11. Expression of heterologous genes in BHK21 cells
electroporated with recombinant RNAs. (A) and (C) show IF analysis of
BHK21 cells at 24 to 40 hours after transfection with the recombinant KUN
replicon RNAs expressing different heterologous genes (indicated under
each panel) using corresponding antibodies. Dilutions of antibodies were
as follows: 1/100 for rabbit anti-CAT polyclonal antibodies (panels 1 and 2
in A); 1/150 for rabbit anti-VSV-G pofyclonal antibodies (panels 3 and 4 in
A); 1/40 for human anti-HCV polyclonal serum (panels 1-4 in C). Mock
show parallel IF analyses of untransfected BHK21 cells. (B) GFP panel
shows fluorescence of live unfixed BHK21 cells at 24 h after transfection
with C20DX/GFP/2Arep RNA. ~3-Gal panel represents X-gal staining of
BHK21 cells at 46 h after transfection with C20DX/~-GAU2Arep RNA
performed as described in the examples.
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Figure. 12. Time course analyses of the CAT and [i-GAL expression in
cells transfected with corresponding recombinant KUN replicon RNAs. (A)
Comparative analysis of CAT expression in BHK21 cells at different times
after transfection with the same amounts (~10 pg) of KUN replicon
(C20DX/CAT/2Arep) or Sindbis replicon (TRCAT) RNAs. CAT activity is
expressed in cpm/min of radioactive acetylated chloramphenicol
determined by LSC CAT assay as described in the examples. Because of
a severe cytopathic effect, incubation of cells transfected with TRCAT RNA
was aborted after 24h post transfection. (B) Comparative analysis of (i-
galactosidase expression in BHK21 cells after transfection with the same
amounts (--5 wg) of C20DX/[3-GAL/2Arep or SFV3/LacZ RNAs. Expression
of [i-galactosidase (fig per 106cells) was calculated from the comparison of
the results of [i-galactosidase assay of the transfected cell iysates and ~-
galactosidase enzyme standard using [i-GAL Enzyme Assay System Kit
(Promega, Madison, WI, USA) essentially as described by the manufacture
(see the examples).
Figure. 13. Processing of polyproteins translated from the electroporated
recombinant KUN replicon RNAs. (A) Radioimmunoprecipitation (RIP)
analysis of radiolabelled BHK21 cells transfected with C20DX/CAT/2Arep
(lane 1 ), C20DXCAT/IRESrep (lane 2), and C20DX2Arep (lane 3) RNAs
using anti-CAT antibodies. 60 mm-diameter tissue culture dishes of BHK21
cells at 46h after electroporation with corresponding RNAs were labeled
with 100 pCi of [35S]-methionine-cysteine for 5 h and RIP analysis of cell
lysates was performed using 1/100 dilution of anti-CAT antibodies.
Samples recovered after RIP analysis were electrophoresed on SDS-
12.5% polyacrylamide gel. Arrows show the positions of corresponding
CAT fusion protein products. (B) RIP analysis with rabbit anti-VSV-G
antibodies {1/100 dilution) of BHK21 cells electroporated with C20DX/VSV-
G/2Arep (lanes 1 and 2) and C20DX2Arep (lane 3) RNAs. 60 mm-diameter
tissue culture dishes of BHK21 cells at 33h after electropration were
labeled with ~50 NCi of [35S]-methionine-cysteine for 8 h. One half (10 p,1)
of C20DX/VSV-G/2Arep RIP sample was treated with endoglycosidase F
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(endo F) as described elsewhere and both endo F-treated and untreated
samples were electrophoresed on SDS-10% polyacrylamide gel. Arrows
show the positions of glycosylated (gVSV-G) and nonglycosylated (VSV-G)
proteins.
Figure. 14. Packaging of the recombinant KUN replicon RNAs. (A) GFP
fluorescence and IF analysis of BHK21 and Vero cells at 35h after infection
with culture fluid collected from BHK21 cells sequentially transfected with
recombinant KUN replicon RNAs and SFV-prME-C105 RNA using
corresponding antibodies as indicated. Time intervals between
transfections were 30 h for C20DX/GFP/2Arep, 34 h for C20DXIVSV-
G/2Arep, and 42 h for C20DX/hcv-NS3/2Arep RNAs. Time intervals for
harvesting culture fluid after second transfections with SFV-prME-C105
RNA were 24h, 37h, and 38h, respectively. (B) Autoradiogram of the CAT
assay of the lysates from BHK21 cells (BHK mock) or BHK21 cells at 30 h
after infection with the culture fluid collected from BHK21 cells at 26 h
after
transfection with C20DX/CAT/2Arep RNA and 42 h after transfection with
SFV-prME-C105 RNA. CAT assay in was performed as described the
examples.
Figure. 15. Stable BHK cell lines expressing GFP (repGFP-BHK) and CAT
(repCAT-BHK). Cell lines were established by selection of BHK21 cells
transfected with C20DX/GFP/2Arep and C20DX/CAT/2Arep RNAs,
respectively, in the medium containing 1 mg per ml of 6418 {Geneticin). (A)
GFP fluorescence of passage 5 of repGFP-BHK cells. {B) Autoradiogram
of the CAT assay of the lysates from repCAT-BHK cells at passages 6 and
17.
Figure. 16. (A) Schematic representation of KUN replicon expression
vector containing ubiquitin gene (C20DXUb2Arep). Ub shows ubiquitin
gene, all the other abbreviations as in Fig. 10. (B) IF analysis of BHK cells
at 24h after transfection with C20DXrep and C20DXUb2Arep RNAs using
anti-NS3 antibodies.
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Figure. 17. (A). illustrates the construction of full-length
C20DXUb2A_HDVrep vector (Fig. 17A). (B) illustrates efficient replication
of C20DXUb2A HDVrep RNA in 100% BHK21 cells compared to ~60%
positive cells obtained after transfection with the same amounts of parental
C20DXUb2Arep RNA (Fig. 17B).
Figure. 18. (A). illustrates the construction of DNA-based pKUNRep1
vector (Fig. 18A). (B) shows successful detection of expression of the
KUN NS3 protein (indicator of the replicating KUN replicon RNA) at 42 h
post transfection with pKUNRep1 plasmid DNA (Fig. 18B).
Figure. 19. illustrates Expression of GFP in mouse lung epithelium after
intranasal immunization with recombinant KUN VLPs containing
encapsidated C20DX/GFP/2Arep RNA.
BEST MODES) FOR CARRYING OUT THE INVENTION
Further features of the present invention are more fully described in the
following
Examples. It is to be understood that the following Examples are included
solely
for the purposes of exemplifying the invention, and should not be understood
in
any way as a restriction on the broad description as set out above.
EXAMPLE 1
Cells.
BHK21 cells were grown in Dulbecco's modification of minimal essential medium
(Gibco BRL) supplemented with 10% foetal bovine serum at 37°C in a C02
incubator.
Construction of the replicons and vectors.
(i) C20rep: All deletion constructs were prepared from the cDNA clones used in
the construction of the plasmid pAKUN for generation of the infectious KUN RNA
(Khromykh and Westaway, ,f.Virol., 1994, 68:4580-4588) by PCR-directed
mutagenesis using appropriate primers and conventional cloning. dME cDNA
and its derivatives were deleted from nucleotides 417 to 2404, which represent
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loss of the signal sequence at the carboxy terminus of C now reduced to 107
amino acids, deletion of prM and E, with the open reading frame resumed at
codon 479 in E, preceding the signal sequence for NS1. C20 rep and C2rep
cDNAs represent progressive in frame deletions in coding sequence of C leaving
only 20 or 2 amino acids of C, respectively, with the open reading frame
continued at codon 479 in E, as in dME.
(ii) FLSDX: All RT reactions were performed with Superscript II RNase H-
reverse transcriptase (Gibco BRL) essentially as described by the manufacturer
using100-200ng of purified KUN virion RNA, or 1~g of total cell RNA and
appropriate primers. PCR amplification after RT of a 6895bp DNA fragment was
performed with the Expand High Fidelity PCR kit (Boehringer Mannheim) using
1 /25 volume of RT reaction and appropriate primers as follows. The PCR
reaction
mixture (50 ~,I) containing all necessary components except the enzyme mixture
(3 parts of Taq polymerasse and 1 part of Pwo polymerase) was preheated at
95°C for 5 min, then the enzyme mixture was added and the following
cycles
were performed: 10 cycles of 95°C for 15sec and 72°C for 6min,
followed by 6
cycles of 95°C for 15sec and 72°C for 6min with an automatic
increase of
extension time at 72°C for 20sec in each following cycle. All PCR
reactions with
Pfu DNA polymerasse (Stratagene) were performed essentially as described by
the manufacturer using 1/25-1/10 volumes of RT reactions and appropriate
primers.
All plasmids shown in Fig. 1 were obtained from the previously described
stable
KUN full-length cDNA clone pAKUN (Khromykh and Westaway, J.Virol., 1994,
68:4580-4588) by substitution of the original cDNA fragments with those
obtained
by RT and PCR amplification of purified KUN RNA using existing unique
restriction sites which were also incorporated into the primers for PCR
amplification.
Initially the Sacll'48'-DraIl183'6 (6895 bp) fragment in pAKUN clone (Fig. 1)
was
replaced with the fragment amplified using Expand High Fidelity PCR kit from
the
cDNA obtained by reverse transcription of purified KUN virion RNA using
appropriate primers. RNA transcribed from the resulting cDNA clone (FLSD) had
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a specific infectivity of 2X103 PFU per 1 p.g, compared to only 1-5 PFU per 10
pg
for AKUN RNA (Fig.1). We then commenced replacing the rest of the genome
using PCR with the high fidelity Pfu DNA polymerase (Stratagene). Thus a 2645
nts Drallla3's- Xhol"°2' fragment covering most of the NS5 gene and the
entire
3'UTR was inserted in FLSD cDNA to produce FLSDX (Fig. 1), resulting in a
total
104-105-fold improvement of the original specific infectivity, now equivalent
to 104
PFU/wg RNA (Fig. 1 ). Further replacement of the 1392 nts Bglll89-Sacll'48'
fragment covering C, prM and part of E sequence did not noticeably improve the
specific infectivity of the resulting FLBSDX RNA (data not shown). The most
infectious FLSDX clone was therefore used in all further experiments.
(iii) C20DXrep. KUN replicon cDNA construct C20DXrep was constructed from
described above C20rep by replacing an Sphl24s~- Xhol"°2' fragment
representing the sequence coding far the entire nonstructural region and the
3'UTR with the corresponding fragment from a stable full-length KUN cDNA clone
FLSDX. Transfection of BHK cells with 5-10 p.g of C20DXrep RNA resulted in
detection of ~80% replicon-positive cells compare to only ~10% positive after
transfection with the same amount of C20rep RNA.
(iv) SFV-C. An SFV replicon construct expressing KUN core (C) gene was
obtained by cloning of the Bglll-BamHl fragment, representing the sequence of
the last 7 nucleotides of the KUN 5'UTR and the sequence coding for the first
107
of the 123 amino acids of KUN C protein, from the plasmid pCINeoC107
(Khromykh, A. A. and E. G. Westaway. Arch. Virol., 1996, 141:fi85-699) into
the
BamHl site of the SFV replicon expression vector pSFV1 (Gibco BRL; Fig. 2).
(v) SFV-prME. KUN prME sequence was PCR amplified from another highly
efficient full-length KUN cDNA clone FLBSDX modified from FLSDX (which will be
described elsewhere), using appropriate primers with incorporated Bglll sites.
The
amplified fragment was digested with Bglll and cloned into the BamHl site of
the
SFV replicon expression vector pSFV1 to obtain the SFV-prME construct (Fig.
2).
(vi) SFV-prME-C. SFV replicon construct expressing both KUN prME and KUN C
genes was obtained by cloning a Mscl-Spel fragment from the SFV-C plasmid
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containing the SFV 26S subgenomic promoter, KUN C sequence and SFV 3'UTR
into the SFV-prME vector digested with Smal and Spel (Fig. 2). Thus the final
double subgenomic construct SFV-prME-C should produce SFV replicon RNA
which upon transfection into BHK cells will direct synthesis of two subgenomic
RNAs expressing KUN prME and KUN C genes.
RNA transcription and transfection.
RNA transcripts were prepared from C20DXrep plasmid DNA linearized with Xhol,
and from SFV plasmids linearised with Spel using SP6 RNA polymerase.
Electroporation of RNAs into BHK21 cells was performed. Briefly, 10-20 p,g of
in
vitro transcribed RNAs were electroporated into 2x106 BHK21 cells in 400 p1 in
a
0.2-cm cuvette (Bio-Rad) using the Bio-Rad Gene Pulser apparatus.
Immunofluorescence analysis.
Replication of KUN replicon RNA C20DXrep after initial electroporation, and
after
infection of BHK cells in packaging experiments, was monitored by
immunofluorescence (IF) analysis with antibodies to KUN NS3 protein.
Expression of KUN E protein after electroporation with SFV-prME and SFV-prME-
C RNAs was detected by IF with a cocktail of mouse monoclonal antibodies to
KUN E protein. These antibodies designated 3.91 D, 10A1, and 3.676 were
generously provided by Roy Hall, University of Queensland, Brisbane,
Australia.
All three antibodies were mixed in equal amounts and a 1/10 dilution of this
mixture was used in IF analysis. Expression and nuclear localisation of KUN C
protein after electroporation with SFV-C and SFV-prME-C RNAs was monitored
by IF analysis with rabbit polyclonal antibodies to KUN C protein. .
Metabolic labelinct and radioimmunoprecipitation analysis.
Metabolic labeling with 35S-methionine/cysteine of electroporated BHK cells
was
performed essentially as described in the SFV Gene Expression System Manual
with some minor modifications. Briefly, cells at 18 h after the
electroporation with
SFV RNAs (with or without prior electroporation with KUN replicon RNA), were
pulse labeled with 35S-methionine/cysteine for 4h, or for 1-2h followed by
different
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periods of incubation (chase) in medium with an excess of unlabeled
methionine/cysteine. Cell culture fluid was collected for analysis of secreted
proteins by electrophoresis and radioimmunoprecipitation (RIP). Labeled cells
were lysed in buffer containing 1 % Nonidet P40 (NP40), 50 mM Tris-HCI (pH
7.6),
150 mM NaCI, and 2mM EDTA, the nuclei removed by low speed centrifugation
and the lysate supernatant was used for parallel analysis with the culture
fluid.
For RIP analysis, labeled cell culture fluids were first filtered through 0.45
pm filter
(Sartorius AG, Gottingen, Germany) and digested with RNase A (20 pg per ml)
for
30 min at 37°C to ensure the removal of membrane particulate material
and
naked RNA. Filtered and RNase treated culture fluids, or untreated cell
lysates,
were then mixed with 1/20 volume of the pooled anti-E monoclonal antibodies
(see above) or with rabbit anti-C antibodies, and incubated overnight at
4°C with
constant rotation in microcentrifuge tubes. Protein A-Sepharose beads were
then
added to a final concentration of about 1 %, and incubation was continued for
another 1 h at 4°C. After three washes with RIPA buffer (50 mM Tris-
HCI, pH 7.6;
150 mM NaCI; 1% NP40; 0.5% deoxycholic acid sodium salt (DOC]; 0.1% sodium
dodecyl sulfate [SDS]) and one wash with phosphate buffered saline (PBS),
beads were resuspended in the SDS-gel sample buffer, boiled for 5 min and
subjected to electrophoresis in an SDS-polyacrylamide gel. After
electrophoresis
gels were dried and exposed to X-ray film.
Northern blot hybridisation.
Five pg total RNA, isolated using Trizol reagent (Gibco BRL) from BHK21 cells
infected with culture fluid collected from cells doubly transfected with
C20DXrep
RNA and SFV RNAs expressing KUN structural proteins, was electrophoresed for
Northern blotting. The hybridisation probes were [32P]-labelled cDNA fragments
representing the 3'-terminal 761 nucleotides of the KUN genome including the
3'UTR region (see Fig. 6B and Fig. 7), and 446 nucleotides of the SFV NSP2
region (see Fig. 6C).
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Expression of KUN C Gene by the recombinant SFV-C replicon.
For the expression of KUN C gene in the pSFV1 vector the Bglll-BamHl fragment
from plasmid pCINeoC107 was subcloned into the BamHl site of pSFV1 (Fig. 2).
This fragment represents the sequence coding for the first 107 amino acids of
KUN C protein, equivalent to the mature form of C, from which the carboxy
terminal hydrophobic sequence has been removed. The SFV-C construct also
contains a native KUN initiation codon with an extra 7 nucleotides of the KUN
5'UTR derived from the pCINeoC107 plasmid and four extra codons at the
carboxy-terminus derived from the SFV vector sequence (Fig. 2).
Electroporation of SFV-C RNA into BHK21 cells resulted in expression of KUN C
protein in almost 100% of cells as judged by IF with antibodies to KUN C
protein
(Fig.3A, panel 1 ). KUN C protein expressed in SFV-C RNA transfected cells was
localised in the cytoplasm (Fig. 3A, panel 3; acetone fixation) and also in
the
nuclei (Fig. 3A, panel 5; formaldehyde-methanol fixation). Because of
difficulties
in identification of KUN C protein in radiolabeled lysates of SFV-C
transfected
cells (Fig 3B), immunoprecipitation of the radiolabelled lysates with anti-C
antibodies was carried out. A labelled band coincident in migration with KUN C
protein was apparent in the lysates of SFV-C but not in those of SFV1
transfected
cells (compare SFV-C and SFV1 in Fig. 3B).
Expression of KUN prME Genes by the recombinant SFV-prME replicon.
The full-length prME sequence plus the preceding signal sequence in our SFV-
prME construct (see Fig. 2) was included in the replicon. As a source of cDNA
for
prME genes, full-length KUN cDNA clone FLBSDX were used. An initiation and a
termination codon, as well as Bglll sites for conventional cloning, were
incorporated into the primers for PCR amplification (see Fig. 2). To minimise
the
amount of possible mismatches which could occur during PCR amplification high
fidelity Pfu DNA polymerase (Stratagene) was used in all our PCR reactions.
When SFV-prME RNA was electroporated into BHK21 cells, nearly 100% of cells
were found to be positive in IF analysis with monoclonal antibodies to KUN E
protein at 12-18h after electroporation (Fig. 4A, panel 1 ). To confirm
expression
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of KUN prM and E proteins in transfected cells and to detect secretion of prME
into the tissue culture fluid transfected cells were labelled with 35S-
methionine/cysteine for 1 h, followed by incubation for increasing chase
periods.
A strongly labelled band corresponding to KUN E protein was apparent in both
culture fluid and cell lysates of SFV-prME transfected cells at all times (see
culture fluid and cells in Fig. 4B). A labelled band corresponding to KUN prM
protein was detected only in cell lysates (cells in Fig. 4B). A labelled band
corresponding in migration to the predicted molecular weight of KUN pr protein
was detected in the culture fluid only of transfected cells (culture fluid in
Fig. 4B).
An apparent increase in the intensity of labelling of E and possibly pr
proteins in
the culture fluid (Fig. 4B, culture fluid) and a concomitant decrease in the
intensity
of labelling of E and prM proteins in the cell lysates (Fig. 4B, cells) were
observed
during the chase period. The efficiency of the secretion of E and pr proteins
was
low, since the lanes showing labelled culture fluid were exposed to X-ray film
for
about 5 times longer than the lanes showing cell lysates (see legend to Fig.
4).
When samples from the pulse-chase labelling experiment with SFV-prME replicon
were immunoprecipitated with KUN anti-E monoclonal antibodies, E and prM
were coprecipitated from the transfected cell lysates (Fig. 4C, lanes 6-9). E
protein (Fig. 4C, lanes 3-5) and in some experiments trace amounts of prM
protein (results not shown) were precipitated also from culture fluid of
transfected
cells. Because of its low molecular weight, M protein probably ran off the gel
during electrophoresis and therefore could not be detected. A gradual increase
in
the amount of immunoprecipitated labelled E protein in the culture fluid of
transfected cells was observed throughout the chase period (Fig. 4C, lanes 3-
5),
confirming the ongoing secretion of E protein. An absence of correlation
between
the increase of immunoprecipitated labelled E protein in the culture fluid,
and an
expected decrease of labelled E and prM proteins immunoprecipitated from the
cell lysate (compare lanes 3-5 in Fig. 4C with the corresponding culture fluid
lanes
in Fig. 4B, and lanes 6-9 in Fig. 4C with the corresponding cell lanes in Fig.
4B),
can probably be explained by inadequate amounts of antibodies used for
immunoprecipitation of a large excess of expressed proteins retained in the
cells
during the relatively short chase periods. Taken together, results of the
direct
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pulse-chase labelling and RIP analyses confirmed both the correct processing
of
prME polyprotein in cells and the secretion of E, and possibly pr and M
proteins,
into the culture fluid after transfection of SFV-prME RNA into BHK21 cells.
Expression of all three KUN structural proteins by the recombinant SFV-
prME-C replicon.
Although KUN replicon was packaged using transfection with two SFV RNAs
expressing prME and C genes separately (see results in the next example), the
efficiency of packaging was low. To increase the efficiency of packaging and
to
simplify the procedure a single SFV replicon construct was prepared expressing
prME genes and C gene simultaneously. Because of the difficulties experienced
with cloning of the entire C-prM-E region into the pSFV1 vector (see the first
section of the Results) and also in order to avoid possible uncertainty
regarding
cleavage at the carboxy terminus of C in the absence of flavivirus protease
NS2B
NS3, an SFV replicon expressing prME and C genes under the control of two
separate 26S promoters was prepared (see SFV-prME-C in Fig. 2).
IF analysis of SFV-prME-C electroporated BHK cells with anti-E and anti-C
antibodies showed expression of both E and C proteins in nearly 100% of cells
by
18h after transfection (results not shown). Both E and C proteins were
expressed
in the same cell (compare dual labelling by anti-C and anti-E antibodies in
Fig.
5A). When transfected cells were pulse-chased with 35S-methionine/cysteine and
the lysates were immunoprecipitated with KUN anti-E monoclonal antibodies,
both E and prM proteins were coprecipitated, as was observed after
transfection
of SFV-prME RNA (compare Fig. 4B and Fig. 5B). A gradual increase of
immunoprecipitated labelled E protein in culture fluids, and a corresponding
decrease of immunoprecipitated labelled E and prM proteins in the cell lysates
were observed during the chase period (Fig. 5B). Immunoprecipitation of the
labelled cell lysates with anti-C antibodies confirmed expression of C protein
in
transfected cells and showed a gradual decrease of the amount of precipitated
C
during the chase period (Fig. 5C}. The results of RIP analysis of culture
fluid, not
treated with detergents, using anti-C antibodies were negative (results not
shown), indicating that no free C protein was secreted into culture fluid of
SFV-
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prME-C transfected cells. In a later experiment (see Fig. 8C, lane 2),
particles
secreted from cells transfected only with SFV-prME-C RNA were purified and
precipitated with anti-E antibodies; again no secreted C was detected.
Overall, the immunofluorescence and labelling patterns in cells transfected
with
SFV-prME-C RNA were very similar to those observed in cells transfected with
two different RNAs expressing prME and C proteins separately (compare Fig. 5
with Fig. 3 and Fig. 4), suggesting proper processing and maturation of all
three
KUN structural proteins when expressed from the recombinant SFV replicon.
EXAMPLE 2
Preparation of encapsidated particles and determination of their titer.
For all infections with encapsidated particles, cell culture fluid was
filtered through
a 0.45 ~.m filter (Sartorius AG, Gottingen, Germany) and treated with RNase A
{20pg per ml) for 0.5h at room temperature (followed by 1.5h incubation at
37°C
during infection of cells). To prepare partially purified particles, filtered
and RNase
A treated culture fluids from transfected cells were clarified by
centrifugation at
16,OOOg in the microcentrifuge for 15 min at 4°C, and the particles
were pelleted
from the resulting supernatant fluid by ultracentrifugation at 40,000 rpm for
2h at
4°C in the AH650 rotor of the Sorvall OTD55B centrifuge. The pellets
were
resuspended in 50 p1 PBS supplemented with RNAse A (20p.g per ml), left to
dissolve overnight at 4°C, and then used for infection of BHK21 cells
or for RT-
PCR analysis. To determine the titer of encapsidated particles, BHK21 cells on
1.3 cm2 coverslips were infected with 100-200 ~I of serial 10-fold dilutions
of cell
culture fluid or of pelleted material for 1.5h at 37°C. The fluid was
then replaced
with 1 ml of DMEM medium supplemented with 2% FBS; cells were incubated for
24h at 37°C in the C02 incubator and then subjected to IF analysis with
anti-NS3
antibodies as described above. The infectious titer of packaged particles was
calculated using the following formula:
Titer (1U) per 2x106 of initially transfected cells = Nx(SW:SIA)x10"X(V: VI),
where N is the average number of anti-NS3 positive cells in the image area,
calculated from 5 image areas in different parts of the coverslip; SW is the
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surface of the well in a 24-well plate (200 mm2); SIA is the surface of the
image
area (1.25 mm2 using defined magnification parameters, calculated according to
the manual for the Wild MPS46/52 photoautomat [Wild Leitz, Heerburg,
Germany]); V is the total volume of the culture fluid (usually 3-5 ml per 60
mm
dish) collected from the population of 2x106 initially electroporated BHK21
cells;
VI is the volume used for infection of the cover slips (usually 100-200 ~,I);
and 10"
is the dilution factor.
Packaaina of the KUN replicon RNA into transmissible "infectious" particles
by the KUN structural proteins expressed from the recombinant SFV
replicons.
Because the KUN replicon construct C20rep was able to successfully transfect
only 10-20% of cells a KUN replicon of greater transfection efficiency was
used
for attempted packaging in doubly transfected cells (i.e. KUN replicon, and
recombinant SFV replicons expressing KUN structural proteins). This
significantly
improved the efficiency of transfection in BHK21 cells to about 80% using the
replicon construct C20DXrep, which was used in all packaging experiments. As
noted above, all cell culture fluids from packaging experiments were filtered
to
remove large membrane fragments and treated with RNase A to remove naked
RNA.
Initial cotransfection experiments showed that simultaneous transfection of
C20DXrep RNA and SFV RNAs expressing KUN structural proteins did not result
in the detection of infectious particles. Therefore a delay period of 12h or
longer
between electroporations was used in subsequent experiments to allow KUN
replicon RNA to accumulate before electroporation of SFV RNAs. IF and
Northern blot analyses of BHK cells infected with the tissue culture fluid
collected
at 27h after the first electroporation with C20DXrep RNA, and at 26h after the
second electroporation with recombinant SFV RNAs, showed higher efficiency of
packaging when the single SFV-prME-C RNA was used compared to that
obtained with two SFV RNAs, SFV-prME and SFV-C (compare panels 1 and 2 in
Fig. 6A, and lanes 1 and 2 in Fig. 6B, respectively). Significantly, IF and
Northern
blot analysis showed that no released infectious particles containing
replication
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competent SFV RNA were produced when SFV-prME-C RNA alone, or SFV-
prME and SFV-C RNAs together, were transfected with (Fig. 6C, lanes 2 and 3)
or without previous transfection of KUN replicon RNA. These results clearly
demonstrated that the recombinant SFV replicon RNAs containing inserted KUN
structural genes could not be packaged by the KUN structural proteins, hence
the
packaging was specific for KUN RNA. Also, no infectious particles containing
packaged KUN replicon RNA were detected when only SFV-prME RNA was used
in packaging experiments with C20DXrep RNA (panel 3 in Fig. 6A),
demonstrating that coexpression of C protein is absolutely essential for the
formation of the secreted infectious particles.
To optimize the conditions for efficient packaging of C20DXrep RNA in
cotransfection experiments with SFV-prME-C RNA, variable time points between
electroporations (Fig. 7A), and between the second electroporation and
harvesting of the infectious particles (Fig. 7B), were examined. Initially
optimisation of the time between the two electroporations was studied with a
fixed
time for collection of the infectious particles. Equal amounts of cells were
seeded
onto cell culture dishes after the first electroporation with C20DXrep RNA,
and
cells were subsequently electroporated with SFV-prME-C RNA at 12h, 18h, 24h,
or 30h incubation intervals. Culture fluid was then harvested from each dish
at
24h after the second electroporation and serial dilutions were used to infect
BHK21 cells. IF analysis of these cells with anti-NS3 antibodies indicated a
gradual accumulation of infectious particles from 12h to 24h between
electroporations, the highest titer reaching 3.8x106 infectious particles at
24h from
2x 106 of initially transfected cells (Fig. 7A). Northern blot analysis of
total RNA
from infected cells with a labelled KUN-specific cDNA probe showed that the
optimal time interval between the electroporation of KUN replicon RNA and of
SFV-prME-C RNA was between 18h and 30h (Fig. 7A). When the interval
between electroporations was extended to 36h and 48h the yield of produced
infectious particles was reduced.
In a separate study BHK cells were electroporated with SFV-prME-C RNA at 30h
after electroporation with C20DXrep RNA and seeded into one 60 mm culture
dish. Single aliquot's of the culture fluid (1m1 of total 3m1) were then
collected at
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24h, 30h, and 42h after the second electroporation. The volume of the
remaining
culture fluid after removal of each aliquot was adjusted to the original
volume by
adding fresh media, and cells were re-incubated. Collected aliquots were then
used to infect BHK cells and total cell RNA recovered from these infected
cells at
24h was then analysed for relative amounts of amplified KUN replicon RNA using
IF analysis with anti-NS3 antibodies and Northern blot hybridization with a
labelled KUN-specific cDNA probe. The gradual increase in amplified KUN
replicon RNA from 24h to 42h after the second electroporation with SFV-prME-C
RNA detected by Northern blot analysis (Fig. 7B) was in accord with an
increase
in released infectious particles as shown by IF analysis of newly infected
cells
with anti-NS3 antibodies.
In a separate experiments we compared efficiency of packaging using SFV
replicon RNA SFV-prME-C105 expressing prME genes and a C gene coding
precisely for the first 105 amino acids of KUN core protein (representing an
exact
copy of mature KUN core protein found in infectious virions) with that of SFV-
prME-C RNA expressing prME genes and a C gene coding for the first 107 amino
acids of KUN core protein plus an extra 4 amino acids derived from the vector
(see Fig. 2). The same yields of encapsidated KUN replicon particles were
obtained using either of these two RNAs (data not shown) indicating that extra
6
amino acids present in the core protein expressed from SFV-prME-C RNA (see
Fig. 2) did not interfere with its packaging efficiency. Therefore both RNAs
can be
used for efficient production of encapsidated particles.
Characterisation of the infectious particles.
To prove that infectious particles secreted into the culture fluid of cells
transfected
with C20DXrep and SFV-prME-C105 RNAs were in fact virus-like particles
incorporating KUN structural proteins, a virus neutralisation test was
performed.
An hour incubation of this tissue culture fluid at 37°C with a 1/10
dilution of the
cocktail of monoclonal antibodies to KUN E protein with known neutralising
activity resulted in almost complete loss of infectivity (panel 1 in Fig. 8A),
while no
loss of infectivity was observed in the control sample incubated under similar
conditions in the absence of antibodies (panel 2 in Fig. 8A). Similar results
were
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obtained when incubations with antibodies were performed at room temperature
or at 4°C.
To show that the infectious particles can be concentrated by pelleting, a
clarified
culture fluid of cotransfected cells was subjected to ultracentrifugation.
When
pellet and supernatant after ultracentrifugation were used to infect BHK cells
which were later (at 24h) analysed by IF with anti-NS3 antibodies, virtually
all the
infectious particles were present in the pelleted material (compare panels 1
and 2
in Fig. 8B).
To identify the proteins and to detect the presence of KUN replicon RNA in the
recombinant infectious particles, they were immunoprecipitated in the absence
of
detergents from the culture fluid of cotransfected and radiolabeled cells
using
anti-E antibodies. Half of the immunoprecipitated sample was used for
separation in the SDS-polyacrylamide gel, and the other half was used to
extract
RNA by proteinase K digestion. Radioautography of the polyacrylamide gel
showed the presence of E, prM, and C proteins in the immunoprecipitates of
culture fluid collected from cells either sequentially transfected with
C20DXrep
and SFV-prME-C RNAs or infected with KUN virus (Fig. 8C, lanes 1 and 3,
respectively). E and prM proteins, but no C protein was immunoprecipitated
from
culture fluid of cells transfected only with SFV-prME-C RNA (Fig. 8C, lane 2),
suggesting that specific interaction between KUN replicon RNA and KUN C
protein was required for assembly of secreted infectious particles. Note that
secreted flaviviruses often contain significant amounts of uncleaved prM as
observed in Fig. 8C.
RNA extracted from the immunoprecipitates was reverse transcribed and PCR
amplified using KUN-specific primers. A DNA fragment of predicted size 0700
bp, NS2A region) was observed in the RT-PCR reactions of RNAs extracted from
the immunoprecipitates of the culture fluid collected from cells either
transfected
sequentially as in Fig. 6 with both C20DXrep and SFV-prME-C RNAs (Fig. 8D,
lane 2) or infected with KUN virus (Fig. 8D, lane 4). No RT-PCR product was
obtained from RNA extracted from the immunoprecipitate of the culture fluid
collected from cells transfected with SFV-prME-C RNA alone (Fig. 8D, lane 3).
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These analyses established that the infectious RNA recovered from packaging
experiments was demonstrably packaged in particles encapsidated by the KUN
structural proteins.
Further characterization of the packaged particles containing replicon RNA was
performed by sedimentation analysis. In parallel with KUN virions (both
concentrated by ultracentrifugation) they were sedimented through 5-25%
sucrose density gradients. 0.5 ml fractions were collected, diluted and
assayed for
infectivity by IF assay using anti-NS3 antibodies at 18h for KUN virions or at
24h
for replicon particles (see legend to Fig. 9A). The maximum infectivity for
repiicon
particles was concentrated in fractions 5-7 with a peak titer of 1.3x105 IU/ml
(fraction 6), while infectious KUN virions were mostly concentrated in
fractions 2-4
with a peak titer of 2.8x10' IU/ml (fraction 3; Fig. 9A). These three
fractions from
each gradient were combined, incubated with anti-E antibodies to aggregate
virions and encapsidated particles, and concentrated by ultracentrifugation
for
electron microscopy (for experimental details see legend to Fig. 9B). As might
be
expected from the gradient sedimentation results (Fig. 9A), particles
containing
encapsidated replicon RNA were smaller than KUN virions, ~35nm diameter
compared to ~50nm diameter of virion particles (Fig. 9B). Both replicon and
virion
particles appeared to be spherical and uniform in size; surface details were
not
resolved, probably because of attachment of some anti-E antibody molecules
(Fig. 9B).
EXAMPLE 3
Construction of modified KUN replicon vectors and expression of
heterologous genes.
Cells.
BHK21 cells were grown in Dulbecco's modification of minimal essential medium
(DMEM, Gibco BRL) supplemented with 10% of fetal bovine serum (FBS). Vero
cells were grown in M199 medium (Gibco BRL) supplemented with 5% FBS.
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Construction of the plasmids.
(I) C20DXrspNeo: The dicistronic replicon construct C20DXrepNeo used for
generation of replicon-expressing BHK cells (repBHK) was prepared from
C20DXrep by cloning an Ices-Neo cassette into the 3'UTR 25 nucleotides
downstream of the polyprotein termination codon. An Xmal-Xhol fragment from
oME/76Neo plasmid (Khromykh and Westaway, J.Virol.1997, 71:1497-1505)
representing nucleotides 10260 -10422 of KUN sequence, followed by the IRES-
Neo cassette and the last 522 nucleotides of KUN sequence was used to
substitute Xmal'o2so Xhol"°2' fragment in C20DXrep construct. Note,
that IRES-
Neo cassette was initially derived from the mammalian expression vector
plresNeo1 (a derivative of pCIN1, provided by S. Rees (Rees et al.,
BioTechniques, 1996, 20:102-110)). The nucleotide sequence at the C-terminus
of IRES element in this IRES-Neo cassette was modified by authors in order to
decrease the level of Neo expression thus forcing selection of cells
expressing
only high levels of inserted genes when using this (plresNeo1) vector and high
concentrations of antibiotic 6418.
(i) C20DX2Arep and C20DX2ArepNeo. To ensure cytosolic cleavage of
heterologous genes expressed from the KUN replicon vectors, the C20Dxrep,
C20DXrepNeo constructs were modified by inserting sequence coding for 2A
autoprotease of the food-and-mouth disease virus (FMDV-2A) between the first
twenty amino acids of KUN C and the last twenty two amino acids of KUN E
proteins in each plasmid preserving the KUN polyprotein open reading frame.
(C20DX2Arep, Fig. 10A). FMDV-2A peptide represents a specific sequence of 19
amino acids which cleaves itself at the C-terminus between the glycine-proline
dipeptide and has been used to mediate cleavage of artificial polyproteins.
The
KUN replicon cDNA constructs C20DX2Arep and C20DX2ArepNeo (Fig. 10A)
were prepared by cloning FMDV-2A sequence PCR amplified from the plasmid
pT3CAT2A/NAmodll (Percy et al, J.Virol.,1994, 68:4486-4492, obtained from
Peter Palese) using forward primer with incorporated Mlul-Spel restriction
sites
and reverse primer with incorporated Eagl-Mlul restriction sites, into Ascl
site of
the previously described C20DXrep and C20DXrepNeo plasmids, respectively
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(Fig. 10A). High-fidelity Pfu DNA polymerise (Stratagene) was used for all PCR
reactions.
Two unique sites for cloning of foreign genes were also incorporated into
these
vectors: (1.) a Spel site between the first 20 amino acids of C protein and
the 2A
sequence, and (2.) a Eagl site between the 2A sequence and the rest of the KUN
replicon sequence. Cloning into Spel site ensures the correct cleavage of C20-
FG-2A fusion protein from the rest of the KUN polyprotein sequence. Cloning
into
the Eagl site permits correct N-terminus cleavage, but it will have its C-
terminus
fused to the 22aa of E protein.
(iii) C20DXICATI2Arep, and C20DXICATI2ArepNeo. The FMDV-2A-CAT
sequence was PCR amplified from the plasmid pT3CAT2A/NAmodll (Percy et al.,
J.Virol. 1994, 68:4486-4492), by using the same as for FMDV-2A amplification
reverse primer and a forward primer with incorporated Mlul restriction site,
and
cloned into the Ascl site of the C20DXrep and C20DXrepNeo plasmids to obtain
C20DXICATI2Arep, and C20DXICATI2ArepNeo constructs, respectively (Fig.
10B).
(iv) C20DXIRESrep and C20DX/CATIIRESrep. C20DXIRESrep was constructed
by cloning EMCV IRES sequence PCR amplified from OME/76Neo plasmid
(Khromykh and Westaway, J.Virol., 1997, 71:1497-1505) using the appropriate
primers with incorporated Ascl (forward primer) and Mlul (reverse primer)
restriction sites into the Ascl site of the C20DXrep plasmid.
C20DXICAT/IRESrep
construct was prepared by cloning CAT gene PCR amplified from the plasmid
pT3CAT2AINAmodll (Percy et al., J.Virol. 1994, 68:4486-4492) using primers
with
incorporated Mlul restriction sites into the Ascl site of C20DXIRESrep plasmid
(Fig.10C).
(v) C20DXIGFPl2Arep, C20DXIGFPI2ArepNeo, C20DXIhcvCORE16012Arep,
C20DXIhcvCORE19112Arep, C20DXIhcvNS312Arep, C20DXlVSV-GI2Arep, and
C20DXI(3-GAtJ2Arep. All these constructs (Fig. 10B) were prepared in a similar
way as follows. The heterologous genes were PCR amplified from corresponding
plasmids using primers with incorporated Spel and/or Xbal restriction sites
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(sequences of the primers may be obtained from the corresponding author), and
cloned into the Spel site of the C20DX2Arep or C20DX2ArepNeo (Fig. 10A).
Plasmids for PCR amplifications of the above genes were: GFP - pEGFP
(Clontech), hcv Core - pcDNA3/HCV-Core (obtained from Eric Gowans, Sir Albert
Sakzewski Virus Research Center, Brisbane), hcvNS3 - p3B-271 (obtained from
Eric Gowans), VSV-G - pHCMV19 (obtained from Michael Bruns, Heinrich-Pette-
Institute, University of Hamburg), ~i-GAL - pSFV3/LacZ (Gibco BRL).
RNA transcription and electroporation.
Recombinant KUN replicon RNA transcripts were prepared using SP6 RNA
polymerase from the corresponding recombinant KUN replicon plasmid DNAs
linearized with Xhol or from the SFV-prME-C105 plasmid linearized with Spel.
Eiectroporation of RNAs into BHK21 cells was performed according to the
method described in Example 1.
Immunofluorescence analysis.
Immunofluorescence (IF) analysis of electroporated or infected cells was
performed as described using antibodies specific to expressed proteins or KUN
anti-NS3 antibodies. Rabbit polyclonal anti-CAT antibodies were purchased from
5 Prime --~ 3 Prime (Boulder, CO, USA), rabbit polyclonal anti-VSV-G
antibodies
were obtained from Michael Bruns (Heinrich-Pette-Institut, Hamburg, Germany),
human anti-HCV polyclonal serum was provided by Eric Gowans (Sir Albert
Sakzewski Virus Research Centre, Brisbane, Australia). Preparation and
characterization of KUN anti-NS3 antibodies were described previously
(Westaway et al., J.Virol., 1997, 71:6650-6661).
In Situ Q-Galactosidase staining and ii-Galactosidase assay.
X-gal staining of BHK21 cells either electroporated with C20DX/~-GAU2Arep
RNA or infected with VLP containing encapsidated C20DX/~i-GAU2Arep RNA
and determination of ~i-galactosidase activity in the cell lysates was
performed
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using commercial ø-GAL Enzyme Assay System Kit (Promega, Madison, WI,
USA) essentially as described by the manufacture.
CAT assay.
CAT activity in lysates of BHK21 cells either electroporated with TRCAT and
C20DX/CAT/2Arep RNAs, or after infection with VLPs containing encapsidated
C20DX/CAT/2Arep RNA, or in stable cell line expressing C20DX/CATI2ArepNeo
RNA was determined using TLS or LSC assays as described previously
(Khromykh and Westaway, J.Virol., 1997, 71:1497-1505).
Preparation of encapsidated particles and determination of their titer.
Preparation of the recombinant VLPs expressing CAT, GFP, and VSV-G proteins
and determination of their titers was performed as described in Example 1.
Optimal time of expression of heterologous products: In order to estimate the
level and the optimal time of expression of heterologous products using this
system, as well as to evaluate possible effects of the size of inserted
sequences
on the replication and packaging efficiency of resulting recombinant KUN
replicon
RNAs, KUN replicons expressing CAT (218 amino acids), GFP (237 amino
acids), and ø-Gal (1017 aa) genes were prepared in C20DX2Arep vector (Fig.
10B). In addition, CAT gene was also inserted into C20DXIRESrep vector
producing C20DXICAT/IRESrep RNA (Fig. 10C). To demonstrate proper
glycosylation of expressed glycoproteins in our system a C20DXNSV-G/2Arep
construct expressing VSV G glycoprotein (Fig. 10B) was prepared. The
expression of these genes in electroporated BHK21 cells was initially
demonstrated by IF analysis with specific antibodies for CAT and VSV-G
proteins
(Fig. 11A), by fluorescence analysis of live unfixed cells for GFP protein
(panel 1
l
in Fig. 11 B), and by X-gal staining for ø-Gal protein (panel 2 in Fig. 11 B).
The
percentage of expressing cells in these experiments varied amongst the
f
constructs from ~10% for C20DX/CAT/IRESrep RNA, ~20% for C20DXIø
Gal/2Arep, C20DXNSV-G/2Arep, and C20DX/CAT/2Arep RNAs to ~50% for
E
C20DX/GFPI2A RNA at 24-48 after electroporation (data not shown). In a
c
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separate experiment ~80-90% cells were transfected with C20DX/(i-Gal/2Arep
RNA.
Expression of HCV proteins.
To express HCV proteins using the KUN replicon system, Core and NS3 genes of
an Australian isolate of HCV (Trowbridge and Gowans, Arch.Virol., 1998,
143:501-511) were expressed using the replicon vector C20DX2Arep. A truncated
form of HCV NS3 gene (coding for amino acids 183 to fi17), containing most of
the HCV NS3 cytotoxic T cell epitopes was cloned into C20DX2Arep vector.
Transfection of the recombinant C20DX/hcvNS3/2Arep RNA into BHK21 cells
resulted in detection of expression of HCV NS3 gene in ~20-30% of transfected
cells (panel 2 in Fig. 11 C). HCV Core gene was expressed in two forms: as a
full
length gene (coding for 191 amino acids, C20DX/hcv-flCore/2Arep RNA, Fig.
10B), and as a truncated gene (coding for the first 160 amino acids, C20DX/hcv-
trCore/2Arep RNA, Fig. 10B). Electroporation of both RNAs into BHK21 cells
resulted in expression of HCV Core protein in ~60-70% of transfected cells
(data
not shown), and at a similar levels, as judged by the intensity of IF with
human
anti-HCV antiserum (Fig. 11 C, panels 3 and 4). Significantly, similar to the
reports
on expression of different forms of HCV Core in other systems, truncated HCV
Core expressed from KUN replicon vector was localized in the nuclei, while
full
length Core was not (data not shown).
Kinetics of expression, processinc and alycosylation of heterolocous
proteins using KUN replicon vectors.
The kinetics of expression of two different size reporter genes CAT {218 amino
acids) and ~3-Gal (1017 amino acids) after electroporation of corresponding
recombinant replicon RNAs into BHK21 cells were compared by appropriate
reporter gene assays. Similar to previous results with detection of
replicating KUN
replicon RNA, a delay of about 10-16h in detectable expression of both
reporter
genes was observed (Fig. 12). Further incubation of electroporated cells
resulted
in a rapid accumulation of CAT protein, reaching maximum at ~30h after
transfection (Fig. 12A), while accumulation of (i-Gal protein was slightly
delayed,
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reaching maximum at ~36-40h after transfection (Fig. 12B). In parellel
experiments, expression of CAT gene from SIN replicon {TRCAT in Fig. 12A) and
~i-GAL gene from SFV replicon (SFV3/LacZ in Fig. 12B) reached maximum level
earlier after transfection (6-8h) and remained approximately at the same level
during the experiment. The maximum levels of expression of the CAT and ~i-GAL
genes in cells electroporated with the same amounts of alphavirus replicon
RNAs
and corresponding KUN replicon RNAs were similar (Fig. 12 A and B).
Quantitative analysis of ~3-GAL expression showed that ~6-7 pg and ~7-8 p,g of
~3-
GAL protein per 106 initially transfected cells was produced from ~5 ~.g of
electroporated C20DX/(i-GAL/2Arep and SFV3/LacZ RNAs, respectively (Fig.
12B). Importantly, in contrast to the massive destruction of cells expressing
(3-
GAL from SFV replicon RNA (data not shown), cells expressing ~i-GAL from KUN
replicon looked quite healthy (see for example Fig. 11 B).
To examine whether proper proteolytic cleavage mediated by FMDV-2A protease
occurred during translation of recombinant KUN replicon RNAs in electroporated
cells, the sizes of the radiolabelled protein products expressed from
C20DX/CAT/2Arep RNA were examined using radioimmunoprecipitation (RIP)
analysis with anti-CAT antibodies. Strong radiolabelled band of ~30 kDa,
corresponding to a predicted size of C20/CAT/2A fusion protein (257 amino
acids) was observed (lane 1, Fig. 13A), suggesting that FMDV-2A cleavage
indeed occurred. The presence of a very weak band of ~33 kDa, corresponding to
the predicted size of C20/CAT/2A/22E fusion protein (286 amino acids) was also
observed (lane 1, Fig. 13A), indicating that the cleavage by FMDV-2A protease
was not complete. However, comparative analysis of the relative intensities of
these two bands clearly demonstrated that most of the fusion protein (~95-98%)
was efficiently cleaved. Note that the cleavage between 22E and NS1 (Fig. 10A)
is mediated by cellular signal peptidase.
Expression and proper processing of heterologous genes from the dicistronic
KUN replicon vector C20DXIRESrep was demonstrated by detection of 27.5
kDa radiolabelled band corresponding to a predicted size of C20CAT protein
(240
amino acids) in the anti-CAT immunoprecipitate from the lysate of BHK21 cells
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transfected with C20DX/CAT/IRESrep RNA (lane 2, Fig. 13A). Gfycosylation of
the VSV-G glycoprotein expressed from KUN replicon was demonstrated by the
observed reduction in size of the endoglycosidase F treated VSV-G protein
immunoprecipitated from the radiolabbeled lysates of BHK21 cells transfected
with C20DXIVSV-G/2Arep RNA (compare lanes 1 and 2 in Fig. 13B).
Packa~ina of recombinant KUN replicon RNAs into pseudoinfectious virus-
like particles.
Although relatively high level of expression of heterologous genes was
achieved
in BHK21 cells after electroporation of recombinant KUN replicon RNAs, it is
well
known that the efficiencies of transfection of different cell lines varies
tremendously. Therefore it was desirable to prepare a stocks of virus-like
particles
(VLP) containing encapsidated recombinant replicon RNAs in order to broaden
the spectrum of cells which could be used for expression. According to the
present invention a heterologous packaging system has been developed allowing
production of VLPs containing KUN replicon RNA encapsidated by the KUN
structural proteins using consecutive transfections with KUN replicon RNA and
SFV replicon RNA SFV-prME-C105 expressing KUN structural genes. The
highest titer of VLPs was achieved when the second electroporation with SFV-
prME-C105 RNA was performed at the time of the maximum replication of KUN
replicon RNA (delay of ~24-27h). Therefore in packaging experiments with
recombinant KUN replicon RNAs, second electroporation with SFV-prME-C105
RNA was performed at the estimated time of maximum replication of recombinant
KUN replicon RNAs (for time intervals see legend to Fig. 14).
Essentially all recombinant replicon RNAs were packaged into VLPs (Fig. 14),
albeit with different efficiencies. The lowest efficiency of packaging was
obtained
for replicon RNAs expressing HCV Core protein (~10~ infectious units (1U) per
ml,
results not shown), suggesting strong interference of HCV Core gene sequence
or its protein product with the encapsidation of recombinant KUN replicon RNA.
The titers of extracellular VLPs recovered in the packaging experiments with
other
recombinant RNAs were all in a range of 106-106 IU per ml depending on the
type
of cells used for infectivity assays (Vero or BHK21) and the inserted sequence
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(results not shown). In general, higher titers were obtained when infectivity
assays
were performed on Vero cells than on BHK21 cells, and when packaging was
performed with recombinant KUN replicon RNAs possessing higher initial
transfection/replication efficiency. Similar amounts of infectious VLPs were
also
recovered from the lysates of transfected cells (results not shown).
Establishment of stable cell lines exuressing CAT and GFP Genes using
C20DX2ArepNeo vector.
To demonstrate the utility of this dicistronic KUN-Neo replicon vector for the
establishment of stable cell lines expressing heterologous genes two
constructs,
C20DXICAT/2ArepNeo and C20DX/GFP/2ArepNeo were prepared by cloning
CAT and GFP sequences into the Spel site of the C20DX2ArepNeo vector (Fig.
10A and B). Transfection of the resulting RNAs into BHK21 cells and subsequent
incubation of these cells in the medium supplemented with 1 mg/ml 6418
(Geneticin) resulted in a rapid enrichment of cells expressing CAT and GFP
genes (repCAT-BHK and repGFP-BHK, respectively; Fig.15). Most of the cells in
the total cell population were producing relatively high levels of
heterologous
protein (see for example Fig. 15A). Importantly, the level of expression
remained
stable during further passaging of the cells (compare CAT expression in repCAT-
BHK cells at passages 6 and 17 in Fig. 15B).
The above examples show that noncytopathic flavivirus KUN replicon vectors can
be used for transient or stable expression of heterologous genes in mammalian
cells. They also show that recombinant KUN replicon RNAs expressing
heterologous genes can be encapsidated into pseudoinfectious virus-like
particles
by subsequent transfection with SFV repiicon RNA expressing KUN structural
genes. These virus-like particles can be used for delivery of the recombinant
self-
replicating RNAs into a wide range of cells or animals leading to a long-term
production of heterologous proteins. Importantly, because of the heterologous
nature of the developed packaging system, no recombination between KUN and
SFV RNAs producing an infectious virus can occur.
While the amounts of produced heterologous proteins using KUN replicon vectors
were lower than those reported in using alphavirus replicon vectors,
replication of
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KUN repficons in contrast to alphavirus replicons did not produce any
cytopathic
effect in cells. This noncytopathic nature and persistence of replication of
KUN
replicons allowed the development of a vector for generation of stable cell
lines
continuously expressing heterologous genes by inserting IRES-Neo cassette into
the 3'UTR of C20DX2Arep replicon. Using such a selectable vector
(C20DX2ArepNeo), a stable BHK cell lines continuously expressing GFP and
CAT genes were rapidly established by selection of transfected cells with
antibiotic 6418. The expression of these genes in the established cells lines
maintained at the same level for at least 17 passages.
EXAMPLE 4
Construction of repiicon vector containing ubiquitin gene
Mouse ubiquitin gene was PCR amplified from the plasmid pRB269 (Baker et al.,
J Biol Chem 269:25381-25386) using appropriate primers with incorporated
unique cloning sites (see Fig. 16A). Resulting PCR fragment containing also
Xbal
site at the 5'end and Spel site at the 3'end was then cloned into the Spel
site of
C20DX2Arep plasmid (see Fig. 10A), producing C20DXUb2Arep vector (Fig. 16).
Thus the gene of interest can be cloned either between C20 and ubiquitin or
between ubiquitin and FMDV 2A protease sequences (Fig. 16A). If heterologous
sequence inserted between C20 and ubiquitin, the resulting product would be a
fusion with C20 at the N-terminus and with ubiquitin at the C-terminus for
efficient
targeting to proteosomes. If heterologous sequence inserted between ubiquitin
and FMDV2A, the resulting product would have a correctly processed N-terminus
but would contain an FMDV 2A peptide fused to its C-terminus. Transfection of
C20DXUb2Arep RNA into BHK21 cells resulted in its replication with efficiency
similar to that of C20DXrep RNA (Fig 16B).
EXAMPLE 5
Modified Kunjin replicon vector with HDV antigenomic ribozyme sequence
To produce KUN replicon transcripts with authentic 3'-termini we incorporated
hepatitis delta virus (HDV) antigenomic ribozyme sequence (Perrotta and Been,
1991, Nature (London) 350:434-436) followed by the simian virus 40 (SV40)
polyadenylation signal (HDVribo/SV40polyA) immediately downstream of the last
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nucleotide of KUN replicon sequence (Fig. 17A). Delta virus ribozyme should
cleave itself off either during in vitro transcription reaction or after
transfection into
cells thus releasing precise 3'-termini of the KUN replicon RNA, which is
important for efficient initiation of KUN RNA replication. The fragment
containing
the last 1331 nucleotides of the KUN replicon sequence followed by
HDVribo/SV40polyA cassette was produced by fusion PCR reaction (Karreman,
1998, BioTechniques 24:736-742) using Pfu DNA polymerase (Stratagene),
appropriate primers and two plasmid DNAs pTMSVSA (obtained from Tom
Macnaughton, Sir Albert Sakzewski Virus Research Center, Brisbane, Australia)
and C20DXrep, as templates. Primers were: NSSdGDD-F (KUN NS5 sequence,
forward) -- 5'- CTG GTT AAC TGT GTG GTA AAG CCC TT -3'; 3'UTRHDV
(junction of KUN 3'end and HDV ribozyme) -- 5'- GAG AAC ACA GGA TCT GGG
TCG GCA TGG CAT CT -3'; SV40pA R (SV40 polyadenyiation signal, reverse)
-- 5'- GGC CTC GAG CAA TTG TTG TTG TTA ACT T- 3'
Resulting PCR product was digested with Xmal (5'end) and Xhol (3'end) and
cloned into Xmal l Xhol digested C20DXUb2Arep DNA, producing
C20DXUb2A HDVrep vector (Fig. 17A).
Electroporation of ~5-9 0 wg RNA transcribed from this construct resulted in
its
efficient replication in 100% BHK21 cells compared to ~60% positive cells
obtained after transfection with the same amounts of parental C20DXUb2Arep
RNA (Fig. 17B).
EXAMPLE 6
DNA-based Kunjin replicon expression vector
To allow in vivo transcription of the KUN replicon RNA by cellular RNA
polymerase II after transfection of the corresponding plasmid DNA we modified
existing KUN replicon vector C20DXUb2A-HDVrep by inserting cytomegalovirus
immediate-early (CMV-IE) enhancer/promoter region immediately upstream of the
KUN replicon sequence. The fragment containing CMV-IE promoter sequence
followed by 5'end of the KUN replicon sequence was produced in fusion PCR
reaction (Karreman, 1998, BioTechniques 24:736-742) using Pfu DNA
polymerase, appropriate primers and pCl (Promega) and C20DXUb2Arep
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plasmid DNAs as templates. Primers were: CMV F (CMV IE promoter, forward) --
5'- GCG CTT AAG ACA TTG ATT ATT GAC TAG TTA -3'; CMVS'UTR (junction
of CMV promoter and 5'UTR of the KUN sequence) --5'- CGT TTA GTG AAC
CGA GTA GTT CGC CTG TGT GA -3'; FMDV2AR (end of FMDV-2A
autoprotease sequence, reverse) --5'- GTG ACG CGT CGG CCG GGC CCT
GGG TTG GA -3'. Resulting PCR product was digested with Eagl (3'end) and
cloned into Nrul (blunt) l Eagl digested C20DXUb2A HDVrep plasmid, producing
pKUNRep1 vector (Fig. 18A). SV40polyA sequence was~previously incorporated
downstream of HDV antigenomic ribozyme sequence (see Fig. 17A) to ensure
termination of transcription by RNA polymerase II.
Transfection of the plasmid DNA pKUNRep1 into BHK21 cells using FuGENE 6
transfection reagent (Boehringer Mannheim) resulted in successful detection of
expression of the KUN NS3 protein (indicator of the replicating KUN replicon
RNA) at 42 h post transfection {Fig. 18B).
EXAMPLE 7
Expression of GFP in mouse lung epithelium after intranasai immunization
with recombinant KUN VLPs containing encapsidated C20DX/GFPI2Arep
RNA
Two female BALB/c mice were immunized intra-nasally with 106 IU per mouse of
the recombinant KUN VLPs expressing GFP (for details of the VLP preparation
and determination of their titre see Example 3). Mice were anaesthetized with
ketamine/xylazine (100u1 per 20g of mouse weight) via intra-peritoneal route
prior
to immunization. At days 2, and 4 after immunization mice were euthanased with
C02, their lungs were collected, rinsed in PBS and fixed in 4%
paraformaldehyde
for 2-4 hours at 4°C. Lungs were also collected from the control
nonimmunized
mouse using the same procedure. All the specimens were paraffin embedded
and microtome sectioned at ~5 micron, mounted on a microscope slide and
analyzed under ultraviolet light using FITC filter. Strong GFP fluorescence
was
observed in epithelial cells lining the airways passages of the lung sections
prepared from mice immunized with recombinant KUN VLPs but not in the lung
section prepared from the control mouse (Fig. 19). These results clearly
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demonstrate efficient delivery and expression of the heterologous gene in vivo
using recombinant KUN VLPs.
EXAMPLE $
Immunogenic properties of KUN replicon VLPs in mice
In order to evaluate immunogenic properties of KUN replicon VLPs, three BALB/C
mice were immunized intra-dermally (in the base of a tail) with 5x105 IU of
VLPs
containing packaged C20DX/GFP/2Arep RNA (see Example 3). Two weeks after
immunization their serum was analyzed on the presence of anti-GFP antibodies
by ELISA with purified GFP protein. The results of 50% end point titrations
(ELISA t5o) for each mouse were: mouse #1 - 1/200, mouse #2 - 1/130, mouse
#3 - 1/100. These results clearly demonstrate that specific humoral immune
response to the heterologous protein encoded by the KUN replicon vector can be
developed as early as at 2 weeks after only a single immunization with the
recombinant KUN VLPs. It is anticipated that the antibody response will be
greatly
enhanced after the second immunization.
It should be understood that the foregoing description of the invention
including
the principles, preferred embodiments and Examples cited above are
illustrative
of the invention and should not be regarded as being restrictive on its scope.
Variations and modifications may be made to the invention by others without
departing from the spirit of that which is described as the invention and it
is
expressly intended that all such variations and changes which fall within this
ambit are embraced thereby is intended merely to be illustrative thereof.
