U.S. patent application number 12/148711 was filed with the patent office on 2009-03-05 for recombinant negative strand rna virus expression systems and vaccines.
Invention is credited to George G. Brownlee, Ervin Fodor, Adolfo Garcia-Sastre, Peter Palese.
Application Number | 20090061521 12/148711 |
Document ID | / |
Family ID | 29424406 |
Filed Date | 2009-03-05 |
United States Patent
Application |
20090061521 |
Kind Code |
A1 |
Palese; Peter ; et
al. |
March 5, 2009 |
Recombinant negative strand RNA virus expression systems and
vaccines
Abstract
The present invention relates methods of generating infectious
negative-strand virus in host cells by an entirely vector-based
system without the aid of a helper virus. In particular, the
present invention relates methods of generating infectious
recombinant negative-strand RNA viruses intracellularly in the
absence of helper virus from expression vectors comprising cDNAs
encoding the viral proteins necessary to form ribonucleoprotein
complexes (RNPs) and expression vectors comprising cDNA for genomic
viral RNA(s) (vRNAs) or the corresponding cRNA(s). The present
invention also relates to methods of generating infectious
recombinant negative-strand RNA viruses which have mutations in
viral genes and/or which express, package and/or present peptides
or polypeptides encoded by heterologous nucleic acid sequences. The
present invention further relates the use of the recombinant
negative-strand RNA viruses or chimeric negative-strand RNA viruses
of the invention in vaccine formulations and pharmaceutical
compositions.
Inventors: |
Palese; Peter; (Leonia,
NJ) ; Garcia-Sastre; Adolfo; (New York, NY) ;
Brownlee; George G.; (Oxford, GB) ; Fodor; Ervin;
(Oxford, GB) |
Correspondence
Address: |
PENNIE & EDMONDS LLP
1155 Avenue of the Americas
New York
NY
10036-2711
US
|
Family ID: |
29424406 |
Appl. No.: |
12/148711 |
Filed: |
April 22, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10652912 |
Aug 28, 2003 |
7384774 |
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12148711 |
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09724412 |
Nov 28, 2000 |
6649372 |
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10652912 |
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09616527 |
Jul 14, 2000 |
6544785 |
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09724412 |
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09152845 |
Sep 14, 1998 |
6146642 |
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09616527 |
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60143645 |
Jul 14, 1999 |
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Current U.S.
Class: |
435/455 ;
435/235.1; 435/236 |
Current CPC
Class: |
C12N 2760/18151
20130101; A61K 39/00 20130101; A61K 2039/5256 20130101; C12N 15/86
20130101; A61K 2039/523 20130101; A61K 48/00 20130101; C12N
2760/18152 20130101; C07K 14/005 20130101; C12N 2760/16143
20130101; C12N 2760/18143 20130101; C12N 2840/44 20130101; C12N
7/00 20130101; Y02A 50/30 20180101; Y02A 50/466 20180101; C12N
2760/18122 20130101; A61K 2039/525 20130101 |
Class at
Publication: |
435/455 ;
435/235.1; 435/236 |
International
Class: |
C12N 15/87 20060101
C12N015/87; C12N 7/00 20060101 C12N007/00 |
Claims
1.-30. (canceled)
31. A method for producing influenza virus particles for
preparation of a vaccine, comprising: growing reassortant influenza
virus to produce virus particles and recovering influenza virus
particles produced by said growing for preparation of a vaccine,
said reassortant influenza virus being a recovered reassortant
influenza virus that was produced in cultured cells by introducing
into cultured cells expression vectors which direct the expression
of the genomic vRNA or antigenomic vRNA (cRNA) segments of said
reassortant influenza virus, said cells providing a nucleoprotein,
and an RNA dependent polymerase so that RNP complexes containing
the genomic vRNA segments of said reassortant influenza virus can
be formed and wherein said reassortant influenza virus can be
assembled within said cells in the absence of helper virus and
wherein said cells were cultured so that said reassortant influenza
virus was produced.
32. The method of claim 31 wherein one or more further expression
vectors were employed in said cells to express one or more proteins
selected from said nucleoprotein and the subunits of said
RNA-dependent RNA polymerase.
33. The method of claim 31 wherein a cell line was employed which
was capable of expressing one or more of said nucleoprotein and the
subunits of said RNA-dependent RNA polymerase.
34. The method of claim 31 wherein said virus is an influenza virus
of type A, B or C.
35. The method of claim 31 wherein said cells were selected from
Vero cells and other cells which are deficient in interferon
activity and capable of supporting growth of said virus.
36. The method of claim 31 wherein said expression vectors were
capable of directly expressing genomic vRNA segments of said
virus.
37. The method of claim 31 wherein said growing of reassortant
influenza virus to produce virus particles occurs in an egg.
38. The method of claim 31 further comprising a viral attenuation
step.
39. The method of claim 31 further comprising a viral killing
step.
40. The method of claim 31 wherein all the required expression
vectors were cotransfected into said cells by use of a liposomal
transfection reagent, by calcium phosphate precipitation, or by
electroporation.
41. The method of claim 31 wherein said expression vectors were all
plasmids.
42. The method of claim 31 wherein each VRNA segment of said virus
or the corresponding cRNAs was present in a separate expression
vector.
43. The method of claim 31 wherein the expression of each vRNA
segment or cRNA was under the control of a promoter sequence
derived from a mammalian Pol I promoter.
44. The method of claim 43 wherein said promoter sequence was a
truncated human Pol I promoter sequence consisting of nucleotides
-250 to -1 of the corresponding native promoter or a functional
derivative thereof.
45. The method of claim 31 wherein the coding sequence for each
vRNA segment or cRNA in said expression vectors was followed by a
ribozyme sequence or transcription terminator to ensure a correct
3' end of each said RNA.
46. The method of claim 32 wherein expression of one or more viral
proteins from said further expression vectors was under the control
of a regulatory sequence selected from the adenovirus 2 major late
promoter linked to the spliced tripartite leader sequence of human
adenovirus type 2 or the human cytomegalovirus immediate-early
promoter, or a functional derivative of said regulatory sequence
wherein said cell in (a) is a Vero cell.
47. The method of claim 33 wherein said component was a viral
nucleoprotein.
48. The method of claim 47 wherein said cell was EcR-293NP.
49. A method for producing an influenza virus comprising: (a)
producing in a cell all of the genomic RNA of an influenza virus by
introducing into the cell DNA encoding at least one of said genomic
RNA or cRNA corresponding to said genomic RNA of the virus, (b)
producing in the cell RNA dependent polymerase and nucleoprotein
and assembling in the cell the influenza virus, said cell being
free of helper virus.
Description
[0001] This application is a continuation-in-part of application
Ser. No. 60/143,645, filed Jul. 14, 1999 and application Ser. No.
09/152,845, filed Sep. 14, 1998, each of which is incorporated
herein by reference in its entirety.
1. INTRODUCTION
[0002] The present invention relates to methods of generating
infectious recombinant negative-strand RNA viruses in mammalian
cells from expression vectors in the absence of helper virus. The
present invention also relates to methods of generating infectious
recombinant negative-strand RNA viruses which have mutations in
viral genes and/or which express, package and/or present peptides
or polypeptides encoded by heterologous nucleic acid sequences. The
present invention further relates the use of the recombinant
negative-strand RNA viruses or chimeric negative-strand RNA viruses
of the invention in vaccine formulations and pharmaceutical
compositions.
2. BACKGROUND OF THE INVENTION
[0003] A number of DNA viruses have been genetically engineered to
direct the expression of heterologous proteins in host cell systems
(e.g., vaccinia virus, baculovirus, etc.). Recently, similar
advances have been made with positive-strand RNA viruses (e.g.,
poliovirus). The expression products of these constructs, i.e., the
heterologous gene product or the chimeric virus which expresses the
heterologous gene product, are thought to be potentially useful in
vaccine formulations (either subunit or whole virus vaccines). One
drawback to the use of viruses such as vaccinia for constructing
recombinant or chimeric viruses for use in vaccines is the lack of
variation in its major epitopes. This lack of variability in the
viral strains places strict limitations on the repeated use of
chimeric vaccinia, in that multiple vaccinations will generate
host-resistance to the strain so that the inoculated virus cannot
infect the host. Inoculation of a resistant individual with
chimeric vaccinia will, therefore, not induce immune
stimulation.
[0004] By contrast, the negative-strand RNA viruses, would be
attractive candidates for constructing chimeric viruses for use in
vaccines. The negative-strand RNA virus, influenza, for example is
desirable because its wide genetic variability allows for the
construction of a vast repertoire of vaccine formulations which
stimulate immunity without risk of developing a tolerance.
2.1. ENGINEERING NEGATIVE STRAND RNA VIRUSES
[0005] The RNA-directed RNA polymerases of animal viruses have been
extensively studied with regard to many aspects of protein
structure and reaction conditions. However, the elements of the
template RNA which promote optimal expression by the polymerase
could only be studied by inference using existing viral RNA
sequences. This promoter analysis is of interest since it is
unknown how a viral polymerase recognizes specific viral RNAs from
among the many host-encoded RNAs found in an infected cell.
[0006] Animal viruses containing plus-sense genome RNA can be
replicated when plasmid-derived RNA is introduced into cells by
transfection (for example, Racaniello et al., 1981, Science
214:916-919; and Levis, et al., 1986, Cell 44:137-145). In the case
of poliovirus, the purified polymerase will replicate a genome RNA
in in vitro reactions and when this preparation is transfected into
cells it is infectious (Kaplan, et al., 1985, Proc. Natl. Acad.
Sci. USA 82:8424-8428). However, the template elements which serve
as transcription promoter for the poliovirus-encoded polymerase are
unknown since even RNA homopolymers can be copied (Ward, et al.,
1988, J. Virol. 62:558-562). SP6 transcripts have also been used to
produce model defective interfering (DI) RNAs for the Sindbis viral
genome. When the RNA is introduced into infected cells, it is
replicated and packaged. The RNA sequences which were responsible
for both recognition by the Sindbis viral polymerase and packaging
of the genome into virus particles were shown to be within 162
nucleotides (nt) of the 5' terminus and 19 nt of the 3' terminus of
the genome (Levis, et al., 1986, Cell 44:137-145). In the case of
brome mosaic virus (BMV), a positive strand RNA plant virus, SP6
transcripts have been used to identify the promoter as a 134 nt
tRNA-like 3' terminus (Dreher, and Hall, 1988, J. Mol. Biol.
201:31-40). Polymerase recognition and synthesis were shown to be
dependent on both sequence and secondary structural features
(Dreher, et al., 1984, Nature 311:171-175).
[0007] The negative-sense RNA viruses have been refractory to study
of the sequence requirements of the replicase. The purified
polymerase of vesicular stomatitis virus is only active in
transcription when virus-derived ribonucleoprotein complexes (RNPs)
are included as template (De and Banerjee, 1985, Biochem. Biophys.
Res. Commun. 126:40-49; Emerson and Yu, 1975, J. Virol.
15:1348-1356; Naito, and Ishihama, 1976, J. Biol. Chem.
251:4307-4314). With regard to influenza viruses, it was reported
that naked RNA purified from virus was used to reconstitute RNPs.
The viral nucleocapsid and polymerase proteins were gel-purified
and renatured on the viral RNA using thioredoxin (Szewczyk, et al.,
1988, Proc. Natl. Acad. Sci. USA 85:7907-7911). However, these
authors did not show that the activity of the preparation was
specific for influenza viral RNA, nor did they analyze the signals
which promote transcription.
[0008] Only recently has it been possible to recover negative
strand RNA viruses using recombinant reverse genetic techniques
(see, e.g., U.S. Pat. No. 5,166,087, which is incorporated herein
by reference in its entirety). In one embodiment of the reverse
genetic technique, ribonucleoprotein complexes (RNPs) are
reconstituted in vitro from RNA transcribed from plasmid DNA in the
presence of influenza virus polymerase proteins (PB1, PB2 and PA)
and nucleoprotein (NP) isolated from purified influenza virus
(Enami et al., 1990, Proc. Natl. Acad. Sci. USA 87:3802-3805; Enami
and Palese, 1991, J. Virol. 65:2711-2713; and Muster and
Garcia-Sastre, Genetic manipulation of influenza viruses in
Textbook of influenza (1998), ch. 9, eds. Nicholson et al.). The in
vitro reconstituted RNPs are transfected into cells infected with a
helper influenza virus, which provides the remaining required viral
proteins and RNA segments to generate transfectant viruses. In
another embodiment of the reverse genetic technique, RNPs are
reconstituted intracellularly from plasmids expressing influenza
virus polymerase proteins, nucleoprotein, and an influenza-like
vRNA segment (Neumann et al., 1994, Virology 202:477-479; Zhang et
al., 1994, Biochem. Biophys. Res. Comm. 200:95-101; and Pleschka et
al., J. Virol., 1996, 70:4188-4192). The RNPs are packaged into
transfectant viruses upon infection with helper influenza
virus.
2.2. INFLUENZA VIRUS
[0009] Virus families containing enveloped single-stranded RNA of
the negative-sense genome are classified into groups having
non-segmented genomes (Paramyxoviridae, Rhabdoviridae, Filoviridae
and Borna Disease Virus) or those having segmented genomes
(Orthomyxoviridae, Bunyaviridae and Arenaviridae). The
Orthomyxoviridae family, described in detail below, and used in the
examples herein, includes the viruses of influenza, types A, B and
C viruses, as well as Thogoto and Dhori viruses and infectious
salmon anemia virus.
[0010] The influenza virions consist of an internal
ribonucleoprotein core (a helical nucleocapsid) containing the
single-stranded RNA genome, and an outer lipoprotein envelope lined
inside by a matrix protein (Ml). The segmented genome of influenza
A virus consists of eight molecules (seven for influenza C) of
linear, negative polarity, single-stranded RNAs which encode ten
polypeptides, including: the RNA-dependent RNA polymerase proteins
(PB2, PB1 and PA) and nucleoprotein (NP) which form the
nucleocapsid; the matrix membrane proteins (M1, M2); two surface
glycoproteins which project from the lipid containing envelope:
hemagglutinin (HA) and neuraminidase (NA); the nonstructural
protein (NS1) and nuclear export protein (NEP). Transcription and
replication of the genome takes place in the nucleus and assembly
occurs via budding on the plasma membrane. The viruses can reassort
genes during mixed infections.
[0011] Influenza virus adsorbs via HA to sialyloligosaccharides in
cell membrane glycoproteins and glycolipids. Following endocytosis
of the virion, a conformational change in the HA molecule occurs
within the cellular endosome which facilitates membrane fusion,
thus triggering uncoating. The nucleocapsid migrates to the nucleus
where viral mRNA is transcribed. Viral mRNA is transcribed by a
unique mechanism in which viral endonuclease cleaves the capped
5'-terminus from cellular heterologous mRNAs which then serve as
primers for transcription of viral RNA templates by the viral
transcriptase. Transcripts terminate at sites 15 to 22 bases from
the ends of their templates, where oligo(U) sequences act as
signals for the addition of poly(A) tracts. Of the eight viral RNA
molecules so produced, six are monocistronic messages that are
translated directly into the proteins representing HA, NA, NP and
the viral polymerase proteins, PB2, PB1 and PA. The other two
transcripts undergo splicing, each yielding two mRNAs which are
translated in different reading frames to produce M1, M2, NS1 and
NEP. In other words, the eight viral RNA segments code for ten
proteins: nine structural and one nonstructural. A summary of the
genes of the influenza virus and their protein products is shown in
Table 1 below.
TABLE-US-00001 TABLE 1 INFLUENZA VIRUS GENOME RNA SEGMENTS AND
CODING ASSIGNMENTS.sup.a Length.sup.b Encoded.sup.c Length.sup.d
Molecules Segment (Nucleotides) Polypeptide (Amino Acids) Per
Virion Comments 1 2341 PB2 759 30-60 RNA transcriptase component;
host cell RNA cap binding 2 2341 PB1 757 30-60 RNA transcriptase
component; initiation of transcription 3 2233 PA 716 30-60 RNA
transcriptase component 4 1778 HA 566 500 Hemagglutinin; trimer;
envelope glycoprotein; mediates attachment to cells 5 1565 NP 498
1000 Nucleoprotein; associated with RNA; structural component of
RNA transcriptase 6 1413 NA 454 100 Neuraminidase; tetramer;
envelope glycoprotein 7 1027 M.sub.1 252 3000 Matrix protein; lines
inside of envelope M.sub.2 96 ? Structural protein in plasma
membrane; spliced mRNA 8 890 NS.sub.1 230 Nonstructural protein;
function unknown NEP 121 ? Nuclear export protein; spliced mRNA
.sup.aAdapted from R. A. Lamb and P. W. Choppin (1983), Annual
Review of Biochemistry, Volume 52, 467-506. .sup.bFor A/PR/8/34
strain .sup.cDetermined by biochemical and genetic approaches
.sup.dDetermined by nucleotide sequence analysis and protein
sequencing
[0012] The influenza A virus genome contains eight segments of
single-stranded RNA of negative polarity, coding for one
nonstructural and nine structural proteins. The nonstructural
protein NS1 is abundant in influenza virus infected cells, but has
not been detected in virions. NS1 is a phosphoprotein found in the
nucleus early during infection and also in the cytoplasm at later
times of the viral cycle (King et al., 1975, Virology 64:378).
Studies with temperature-sensitive (ts) influenza mutants carrying
lesions in the NS gene suggested that the NS1 protein is a
transcriptional and post-transcriptional regulator of mechanisms by
which the virus is able to inhibit host cell gene expression and to
stimulate viral protein synthesis. Like many other proteins that
regulate post-transcriptional processes, the NS1 protein interacts
with specific RNA sequences and structures. The NS1 protein has
been reported to bind to different RNA species including: vRNA,
poly-A, U6 snRNA, 5' untranslated region as of viral mRNAs and ds
RNA (Qiu et al., 1995, RNA 1:304; Qiu et al., 1994, J. Virol.
68:425; and Hatada Fukuda 1992, J. Gen. Virol. 73:3325-9).
Expression of the NS1 protein from cDNA in transfected cells has
been associated with several effects: inhibition of
nucleo-cytoplasmic transport of mRNA, inhibition of pre-mRNA
splicing, inhibition of host mRNA polyadenylation and stimulation
of translation of viral mRNA (Fortes et al., 1994, EMBO J. 13: 704;
Enami et al, 1994, J. Virol. 68:1432; de la Luna et al., 1995, J.
Virol. 69:2427; Lu et al., 1994, Genes Dev. 8:1817; Park et al.,
1995, J. Biol. Chem. 270:28433; Nemeroff et al., 1998, Mol. Cell.
1:991; and Chen et al., 1994, EMBO J. 18:2273-83).
[0013] Influenza remains a constant worldwide threat to human
health and hence there is a particular need for a ready method of
generating modified influenza viruses with known mutations in any
of the genomic viral RNA (vRNA) segments. Engineering influenza
vRNA segments for expression of heterologous sequences is also of
much interest, for example, in the development of new vaccines
effective against influenza virus and a second pathogenic
agent.
2.3. THE NEWCASTLE DISEASE VIRUS
[0014] The Paramyxoviridae family, described in detail below, and
used in the examples herein, contain the viruses of Newcastle
disease virus (NDV), parainfluenza virus, Sendai virus, simian
virus 5, and mumps virus. The Newcastle disease virus is an
enveloped virus containing a linear, single-strand, nonsegmented,
negative sense RNA genome. The genomic RNA contains genes in the
order of 3'-NP-P-M-F-HN-L, described in further detail below. The
genomic RNA also contains a leader sequence at the 3' end.
[0015] The structural elements of the virion include the virus
envelope which is a lipid bilayer derived from the cell plasma
membrane. The glycoprotein, hemagglutinin-neuraminidase (HN)
protrude from the envelope allowing the virus to contain both
hemagglutinin and neuraminidase activities. The fusion glycoprotein
(F), which also interacts with the viral membrane, is first
produced as an inactive precursor, then cleaved
post-translationally to produce two disulfide linked polypeptides.
The active F protein is involved in penetration of NDV into host
cells by facilitating fusion of the viral envelope with the host
cell plasma membrane. The matrix protein (M), is involved with
viral assembly, and interacts with both the viral membrane as well
as the nucleocapsid proteins.
[0016] The main protein subunit of the nucleocapsid is the
nucleocapsid protein (NP) which confers helical symmetry on the
capsid. In association with the nucleocapsid are the P and L
proteins. The phosphoprotein (P), which is subject to
phosphorylation, is thought to play a regulatory role in
transcription, and may also be involved in methylation,
phosphorylation and polyadenylation. The L gene, which encodes an
RNA-dependent RNA polymerase, is required for viral RNA synthesis
together with the P protein. The L protein, which takes up nearly
half of the coding capacity of the viral genome is the largest of
the viral proteins, and plays an important role in both
transcription and replication.
[0017] The replication of all negative-strand RNA viruses,
including NDV, is complicated by the absence of cellular machinery
required to replicate RNA. Additionally, the negative-strand genome
can not be translated directly into protein, but must first be
transcribed into a positive-strand (mRNA) copy. Therefore, upon
entry into a host cell, the virus can not synthesize the required
RNA-dependent RNA polymerase. The L, P and NP proteins must enter
the cell along with the genome on infection.
[0018] It is hypothesized that most or all of the viral proteins
that transcribe NDV mRNA also carry out their replication. The
mechanism that regulates the alternative uses (i.e., transcription
or replication) of the same complement of proteins has not been
clearly identified but appears to involve the abundance of free
forms of one or more of the nucleocapsid proteins, in particular,
the NP. Directly following penetration of the virus, transcription
is initiated by the L protein using the negative-sense RNA in the
nucleocapsid as a template. Viral RNA synthesis is regulated such
that it produces monocistronic mRNAs during transcription.
[0019] Following transcription, virus genome replication is the
second essential event in infection by negative-strand RNA viruses.
As with other negative strand RNA viruses, virus genome replication
in Newcastle disease virus (NDV) is mediated by virus-specified
proteins. The first products of replicative RNA synthesis are
complementary copies (i.e., plus-polarity) of NDV genome RNA
(cRNA). These plus-stranded copies (anti-genomes) differ from the
plus-strand mRNA transcripts in the structure of their termini.
Unlike the mRNA transcripts, the anti-genomic cRNAs are not capped
and methylated at the 5' termini, and are not truncated and
polyadenylated at the 3' termini. The cRNAs are coterminal with
their negative strand templates and contain all the genetic
information in each genomic RNA segment in the complementary form.
The cRNAs serve as templates for the synthesis of NDV
negative-strand viral genomes (vRNAs).
[0020] Both the NDV negative strand genomes (vRNAs) and antigenomes
(cRNAs) are encapsidated by nucleocapsid proteins; the only
unencapsidated RNA species are virus mRNAs. For NDV, the cytoplasm
is the site of virus RNA replication, just as it is the site for
transcription. Assembly of the viral components appears to take
place at the host cell plasma membrane and mature virus is released
by budding.
3. SUMMARY OF THE INVENTION
[0021] The present invention provides methods of generating
infectious recombinant negative-strand RNA viruses intracellularly
in the absence of helper virus from expression vectors comprising
cDNAs encoding the viral proteins necessary to form
ribonucleoprotein complexes (RNPs) and expression vectors
comprising cDNA for genomic viral RNA(s) (vRNAs) or the
corresponding cRNA(s). In particular, the present invention
provides methods of generating infectious recombinant
negative-strand RNA viruses in 293T cells in the absence of helper
virus from expression vectors comprising cDNAs encoding the viral
proteins necessary to form RNPs and expression vectors comprising
cDNA for vRNA(s) or the corresponding cRNA(s). The infectious
recombinant negative-strand RNA viruses of the invention may or may
not be capable of replicating and producing progeny. The present
invention encompasses methods of generating infectious recombinant
negative-strand RNA viruses having segmented or non-segmented
genomes.
[0022] In one embodiment, an infectious recombinant negative-strand
RNA virus having a segmented or non-segmented genome is rescued in
a method comprising introducing into a 293T cell expression vectors
capable of expressing the genomic or antigenomic RNA segments, and
a nucleoprotein, and a RNA-dependent polymerase, whereby
ribonucleoprotein complexes are formed and the recombinant
negative-strand RNA virus is produced in the absence of helper
virus. In accordance with this embodiment, the expression of the
genomic vRNA(s) or the corresponding cRNA(s) and/or the expression
of the nucleoprotein and RNA-dependent RNA polymerase may be
constitutive or inducible. For example, the expression of the
vRNA(s) or cRNA(s) under the control of a DNA-dependent RNA
polymerase promoter such as the bacteriophage T7 promoter may be
induced by inducing the expression of a DNA-dependent RNA
polymerase such as T7.
[0023] In another embodiment, an infectious recombinant
negative-strand RNA virus having a segmented or non-segmented
genome is generated in 293T cells by a method comprising: (a)
introducing expression vectors capable of expressing in said cells
genomic vRNA(s) or the corresponding cRNA(s); (b) introducing
expression vectors capable of expressing in said cells a
nucleoprotein and an RNA-dependent RNA polymerase; and (c)
culturing said cells such that RNPs are formed and the recombinant
negative-strand RNA virus is produced in the absence of helper
virus. In accordance with the present invention, the expression
vector may be engineered to express the genomic RNA segments, the
nucleoprotein and the RNA-dependent polymerase, or any combination
thereof. In another embodiment, each component may be provided to
the cell in individual expression vectors.
[0024] In another yet another embodiment, infectious recombinant
negative-strand RNA viruses are rescued in 293T cells by a method
comprising introducing expression vectors capable of expressing in
said cells genomic RNAs or antigenomic RNAs in cells which express
a nucleoprotein and an RNA dependent polymerase and culturing said
cells such that RNP's are formed and the virus is produced in the
absence of helper virus.
[0025] The present invention also provides methods of generating an
infectious recombinant negative-strand RNA viruses having greater
than 3 genomic vRNA segments in host cells, said methods
comprising: (a) expressing genomic vRNA segments or the
corresponding cRNAs from a first set of expression vectors in said
cells; and (b) expressing a nucleoprotein and an RNA-dependent RNA
polymerase from a second set of recombinant expression vectors in
said cells, whereby ribonucleoprotein complexes are formed and the
infectious recombinant negative-strand RNA viruses are produced in
the absence of helper virus. Preferably, the infectious recombinant
negative-strand RNA virus generated is a member of the
Orthomyxoviridae family and most preferably the infectious
recombinant negative-strand RNA virus generated is an influenza
virus.
[0026] In one embodiment, an infectious recombinant negative-strand
RNA virus having greater than 3 genomic vRNA segments is generated
in host cells by a method comprising: (a) introducing a first set
of expression vectors capable of expressing in said cells genomic
vRNA segments or the corresponding cRNAs; (b) introducing a second
set of expression vectors capable of expressing in said cells a
nucleoprotein and an RNA-dependent RNA polymerase; and (c)
culturing said cells such that RNPs are formed and the infectious
recombinant negative-strand RNA virus is produced in the absence of
helper virus.
[0027] In another embodiment, an infectious recombinant
negative-strand RNA virus having greater than 3 genomic vRNA
segments is generated in a host cell line expressing a
nucleoprotein and an RNA-dependent RNA polymerase by a method
comprising: (a) introducing expression vectors capable of
expressing in said cell line genomic vRNA segments or the
corresponding cRNAs; and (b) culturing said cells such that RNPs
are formed and the infectious recombinant negative-strand RNA virus
is produced in the absence helper virus. In another embodiment, an
infectious recombinant negative-strand RNA virus having greater
than 3 genomic vRNA segments is generated in a mammalian cell line
expressing genomic vRNA segments or the corresponding cRNAs by a
method comprising: (a) introducing expression vectors capable of
expressing a nucleoprotein and an RNA-dependent RNA polymerase; and
(b) culturing said cells such that RNPs are formed and the
infectious recombinant negative-strand RNA virus is produced in the
absence of helper virus.
[0028] The present invention is based, in part, on Applicants'
identification of the correct nucleotide sequence of the 5' and 3'
termini of the negative-sense genomes RNA of NDV. The nucleotide
sequence of the 3' termini of the NDV negative-sense genome RNA of
the present invention differs significantly from the NDV 3' termini
sequence previously disclosed by Collins et al. in Fundamental
Virology 3rd Ed. 1996 by Lippincott-Raven Publishers as shown in
FIG. 6. The identification of the correct nucleotide sequence of
the NDV 3' termini allows for the first time the engineering of
recombinant NDV RNA templates, the expression of the recombinant
RNA templates and the rescue of recombinant NDV particles.
Accordingly, the present invention provides methods of generating
an infectious, replicating recombinant Newcastle disease virus
(NDV) in mammalian cells, said methods comprising: (a) expressing a
genomic vRNA or the corresponding cRNA from an expression vector in
said cells; and (b) expressing a nucleoprotein and an RNA-dependent
RNA polymerase from a set of expression vectors in said cells,
whereby ribonucleoprotein complexes are formed and the recombinant
NDV is produced in the absence of helper virus.
[0029] In one embodiment, an infectious recombinant NDV is
generated in host cells by a method comprising: (a) introducing an
expression vector capable of expressing in said cells genomic vRNA
or the corresponding cRNA; (b) introducing a set of expression
vectors capable of expressing in said cells a nucleoprotein and an
RNA-dependent RNA polymerase; and (c) culturing said cells such
that RNPs are formed and recombinant NDV is produced in the absence
of helper virus.
[0030] In another embodiment, an infectious recombinant NDV is
generated in a host cell line expressing a nucleoprotein and an
RNA-dependent RNA polymerase by a method comprising: (a)
introducing expression vectors capable of expressing in said cell
line a genomic vRNA or the corresponding cRNA; and (b) culturing
said cell line such that RNPs are formed and recombinant NDV is
produced in the absence helper virus. In another embodiment, an
infectious recombinant NDV is generated in a host cell line
expressing a genomic vRNA segment or the corresponding cRNA by a
method comprising: (a) introducing expression vectors capable of
expressing in said cell line a nucleoprotein and an RNA-dependent
RNA polymerase; and (b) culturing said cell line such that RNPs are
formed and recombinant NDV is produced in the absence of helper
virus.
[0031] The ability to reconstitute negative-strand RNA viruses
intracellularly allows the design of novel recombinant viruses
(i.e., chimeric viruses) which express heterologous nucleic acid
sequences or which express mutant viral genes. The heterologous
sequences may encode, for example, epitopes or antigens of
pathogens or tumors. The ability to reconstitute negative-strand
RNA viruses intracellularly also allows the design of novel
recombinant viruses (i.e., chimeric viruses) which express genes
from different strains of viruses. Thus, the present invention
provides methods of generating chimeric viruses which express
heterologous nucleic acid sequences, mutant viral genes, or viral
genes from different strains of virus intracellularly from
expression vectors.
[0032] The present invention provides for the use of the
recombinant negative-strand RNA viruses or chimeric viruses of the
invention to formulate vaccines against a broad range of viruses
and/or antigens including tumor antigens. The recombinant
negative-strand RNA viruses or chimeric viruses of the present
invention may be used to modulate a subject's immune system by
stimulating a humoral immune response, a cellular immune response
or by stimulating tolerance to an antigen. When delivering, tumor
antigens, the invention may be used to treat subjects having a
disease amenable to immunity mediated rejection, such as non-solid
tumors or solid tumors of small size. It is also contemplated that
delivery of tumor antigens by the recombinant negative-strand RNA
viruses or chimeric viruses described herein will be useful for
treatment subsequent to removal of large solid tumors. The
recombinant negative-strand RNA viruses or chimeric viruses of the
invention may also be used to treat subjects who are suspected of
having cancer.
[0033] The present invention also provides for the use of the
recombinant negative-strand RNA viruses or chimeric viruses of the
invention in pharmaceutical compositions for the administration of
peptides or polypeptides to a subject.
3.1. DEFINITIONS
[0034] As used herein, the following terms will have the meanings
indicated: [0035] cRNA=anti-genomic RNA [0036] HIV=human
immunodefiency virus [0037] L=large protein [0038] M=matrix protein
(lines inside of envelope) [0039] MDCK=Madin Darby canine kidney
cells [0040] MDBK=Madin Darby bovine kidney cells [0041]
MLP=adenovirus type 2 major late promoter linked to a synthetic
sequence comprising the spliced tripartite leader sequence of human
adenovirus type 2 [0042] moi=multiplicity of infection [0043]
NA=neuraminidase (envelope glycoprotein) [0044] NDV=Newcastle
disease Virus [0045] NP=nucleoprotein (associated with RNA and
required for polymerase activity) [0046] NS=nonstructural protein
(function unknown) [0047] nt=nucleotide [0048] PA, PB1,
PB2=RNA-directed RNA polymerase components [0049]
pA=polyadenylation sequence from SV40 [0050] POL I=truncated human
RNA polymerase I promoter [0051] R=genomic hepatitis virus ribozyme
[0052] RNP=ribonucleoprotein (RNA, PB2, PB1, PA and NP) [0053]
rRNP=recombinant RNP [0054] vRNA=virus RNA [0055] viral polymerase
complex=PA, PB1, PB2 and NP [0056] WSN=influenza A/WSN/33 virus
[0057] WSN-HK virus=reassortment virus containing seven genes from
WSN virus and the NA gene from influenza A/HK/8/68 virus
[0058] The term "expression vectors" as used herein refers to
plasmids, viral vectors, recombinant nucleic acids and cDNA.
Preferably, the term "expression vectors" refers to plasmids.
[0059] The term "helper virus" as used herein refers to a virus
homologous to the virus being rescued. The helper virus generally
supplies one or more of the viral proteins which are required for
the production of infectious recombinant negative-strand RNA
viruses.
4. DESCRIPTION OF THE FIGURES
[0060] FIG. 1. Schematic representation of a method of generating
recombinant influenza virus. Eight transcription plasmids encoding
the vRNA segments of an influenza A virus and four protein
expression plasmids encoding influenza A virus nucleoprotein and
RNA-dependent RNA polymerase subunits are cotransfected into
cultured Vero cells (African green monkey kidney cells). Then, MDBK
(Madin-Darby bovine kidney) cells are employed for plaque assay and
amplification of rescued viral particles.
[0061] FIG. 2. Schematic representation of the NDV minigenome. Top
illustration depicts the PNDVCAT plasmid including the T7 promoter;
the 5' terminal sequence (5' end of genomic RNA, 191 nt); the
inserted nucleotides (CTTAA); 667 nt of CAT ORF; the 3'-terminal
sequence (3' end of genomic RNA, 121 nt) the BbS1 and nuclease
sites. Lower illustration depicts the chimeric NDV-CAT RNA
resulting from in vitro transcription.
[0062] FIGS. 3A-3C. Schematic representation of the PTMI expression
vectors.
[0063] PTM1-NP encodes the NDV NP protein.
[0064] PTM1-P encodes the NDV P protein.
[0065] PTM1-L encodes the NDV L protein.
[0066] FIG. 4. RNA sequence of NDV 5' and 3' non-coding terminal
regions (plus-sense). Sequences 5' to the CAT gene represent 121 nt
of the 5' non-coding terminal region of NDV plus sense genome
comprising 65 nt of the leader sequence (in bold) followed by 56 nt
of the NP gene UTR. Sequences 3' to the CAT gene represent inserted
nucleotides cuuaa (in lower case) and 191 nt of the non-coding
terminal region of NDV plus sense genome comprising 127 nt of the
UTR of the L gene followed by 64 nt of the trailer region (in
bold).
[0067] FIGS. 5A-5B. Schematic representation of a stricture of
recombinant NDV clones. FIG. 4B, representation of infectious NDV
expressing HIV Env and Gag. Top panel, HIV Env and Gag are between
the M and L genes. Lower panel, HIV Env and Gag are 3' to the NP
gene.
[0068] FIG. 6. Schematic representation of the 3' termini of NDV as
aligned with sequence of Collins et al. Parainfluenza viruses, in
Field's Virology, 3rd ed. B. N. Fields, D. M. Knipe, p. m. Howley
et al, eds., Lippincott-Raven Publishing, Philadelphia, 1996.
5. DETAILED DESCRIPTION OF THE INVENTION
[0069] The present invention provides methods of generating
infectious negative-strand RNA viruses intracellularly from
recombinant nucleic acid molecules. In particular, the present
invention provides methods of generating an infectious
negative-strand RNA virus in 293T cells, said methods comprising
providing expression vectors capable of expressing genomic or
antigenomic viral RNA segments, and nucleoproteins, and RNA
dependent RNA polymerase, whereby RNPs are formed in said cells and
infectious recombinant negative-strand RNA is produced in the
absence of helper virus. The present invention encompasses methods
of infectious recombinant negative-strand RNA virus having a
segmented or non-segmented genome.
[0070] The present invention provides methods of generating
infectious, replicating recombinant negative-strand RNA virus in
the absence of helper virus by transiently transfecting 293T cells
with expression vectors providing the genomic vRNA(s) or the
corresponding cRNA(s) and the required viral proteins. In one
embodiment, an infectious, replicating negative-strand RNA virus is
generated in 293T cells by a method comprising: (a) introducing
expression vectors which direct the expression of each required
genomic vRNA segment or the corresponding cRNA into said cells; (b)
introducing expression vectors which express a nucleoprotein and
RNA-dependent RNA polymerase subunits or one or more additional
viral proteins in said cells; and (c) culturing said cells such
that RNPs are formed and the infectious, replicating recombinant
negative-strand RNA virus is produced in the absence of helper
virus. In accordance with these embodiments, each set of expression
vectors may each comprise one or more vectors and each set of
expression vectors may be introduced by transfection methods
described herein or known to those of skill in the art.
[0071] The present invention also provides methods of generating
infectious, replicating recombinant negative-strand RNA virus in
the absence of helper virus by transfecting 293T cell lines
expressing one or more genomic vRNAs or the corresponding cRNAs
with expression vectors directing the expression of the required
viral proteins. In a specific embodiment, an infectious,
replicating recombinant negative-strand RNA virus is generated in a
293T cell line expressing genomic vRNA(s) or the corresponding
cRNA(s) by a method comprising: (a) introducing expression vectors
which express in said cells a nucleoprotein and RNA-dependent RNA
polymerase subunits; and (b) culturing said cells such that RNPs
are formed and the infectious, replicating virus is produced in the
absence of helper virus.
[0072] The present invention also provides methods of generating
infectious, replicating recombinant negative-strand RNA virus in
the absence of helper virus in a 293T cell line that expresses one
or more viral proteins required to form RNPs (i.e., nucleoprotein
and RNA-dependent RNA polymerase subunits), said methods
comprising: (a) introducing one or more expression vectors
directing the expression of genomic vRNA(s) or the corresponding
cRNA(s) in said cell line; (b) introducing one or more expression
vectors that direct the expression of any viral proteins required
to form RNPs which are not expressed by the 293T cell line; and (c)
culturing said cell lines such that RNPs are formed and the
infectious recombinant virus is produced. In accordance with this
embodiment, each set of expression vectors may each comprise one or
more vectors. For example, in the generation of an infectious,
replicating negative-strand RNA virus with a nonsegmented genome,
the first set of expression vectors would comprise one expression
vector.
[0073] In accordance with the present invention, the 293 T cells,
or any other host cell used in the methods of the invention, may be
modified in many ways in order to facilitate rescue of a
recombinant negative strand RNA virus in the absence of helper
virus. In particular, the host cell may be modified or engineered
to express viral proteins required for replication or packaging,
either constitutively or inducibly. In either event, expression of
the viral proteins is regulated by either a constitutive or
inducible promoter as described herein or known to those of skill
in the art. In such an embodiment, the host cell may be engineered
to express viral proteins required to form RNPs or viral structured
proteins. In another embodiment, the host cell may be modified to
constitutively or inducibly expresses RNA-dependent RNA
polymerases, or subunits thereof.
[0074] The present invention also provides methods of generating
infectious, non-replicating or attenuated negative-strand RNA virus
in 293T cells in the absence of helper virus, wherein method
comprises introducing expression vectors which do not encode all of
the genomic viral sequences required to form viral particles; or
introducing expression vectors which provide the genomic vRNA(s) or
corresponding cRNA(s) which contain a mutation, deletion or
insertion which result in a recombinant virus with an attenuated
phenotype. Further, the expression vectors may be introduced by
transfection methods described herein or known to those of skill in
the art.
[0075] The present invention also provides methods of generating
infectious negative strand RNA virus in 293T cells infected by a
helper virus, said methods comprising: (a) introducing expression
vectors directing the expression of one or more vRNAs or the
corresponding cRNAs in said cells; (b) introducing expression
vectors directing the expression of one or more viral proteins in
said cells; and (c) culturing the cells such that the RNPs are
formed and the infectious, replicating negative-strand RNA virus is
produced. In one embodiment, the helper virus provides viral
proteins required to form the RNPs. In a preferred embodiment, the
helper virus provides a DNA-dependent RNA polymerase such as, for
example, bacteriophage T7, T3 or the SP6 polymerase. Preferably,
the helper virus is not a negative-strand RNA virus and more
preferably the helper virus is a DNA virus such as vaccinia.
[0076] The present invention also provides methods of generating
infectious negative strand RNA virus in a 293T cell line infected
by helper virus by introducing one or more expression vectors into
said cell line. Accordingly, the 293T cell lines are transfected
with expression vectors that direct the expression of vRNA(s) or
the corresponding cRNA(s) and expression vectors that direct the
expression of the viral proteins required for the formation of RNPs
which are not provided by the helper virus.
[0077] The present invention also provides methods of generating an
infectious recombinant negative-strand RNA viruses having greater
than 3 genomic vRNA segments in mammalian cells, said methods
comprising: (a) expressing genomic vRNA segments or the
corresponding cRNAs from a first set of expression vectors in said
cells; and (b) expressing a nucleoprotein and an RNA-dependent RNA
polymerase from a second set of recombinant expression vectors in
said cells, whereby ribonucleoprotein complexes are formed and the
infectious recombinant negative-strand RNA viruses are produced in
the absence of helper virus. Preferably, the infectious recombinant
negative-strand RNA virus is a member of the Orthomyxoviridae
family and most preferably the infectious-recombinant
negative-strand RNA virus an influenza virus.
[0078] The present invention encompasses the generation of
infectious recombinant negative-strand RNA viruses having greater
than 3 genomic segments which are capable of replicating and
producing progeny. The invention also encompasses the infectious
recombinant negative-strand RNA viruses having greater than 3
genomic segments which are not capable of replicating and producing
progeny.
[0079] In one embodiment, an infectious recombinant negative-strand
RNA virus having greater than 3 genomic vRNA segments is generated
in mammalian cells by a method comprising: (a) introducing a first
set of expression vectors capable of expressing in said cells
genomic vRNA segments or the corresponding cRNAs; (b) introducing a
second set of expression vectors capable of expressing in said
cells a nucleoprotein and RNA-dependent RNA polymerase; and (c)
culturing said cells such that RNPs are formed and the infectious
recombinant negative-strand RNA virus is produced in the absence of
helper virus.
[0080] In another embodiment, an infectious recombinant
negative-strand RNA virus having greater than 3 genomic vRNA
segments is generated in a mammalian cell line expressing a
nucleoprotein and an RNA-dependent RNA polymerase by a method
comprising: (a) introducing expression vectors capable of
expressing genomic vRNA segments or the corresponding cRNAs; and
(b) culturing said cells such that RNPs are formed and the
infectious recombinant negative-strand RNA virus is produced in the
absence helper virus. In another embodiment, an infectious
recombinant negative-strand RNA virus having greater than 3 genomic
vRNA segments is generated in a mammalian cell line expressing
genomic vRNA segments or the corresponding cRNAs by a method
comprising: (a) introducing expression vectors capable of
expressing a nucleoprotein and an RNA-dependent RNA polymerase; and
(b) culturing said cells such that RNPs are formed and the
infectious recombinant negative-strand RNA virus is produced in the
absence of helper virus.
[0081] The present invention also provides methods of generating
infectious recombinant negative-strand RNA viruses having greater
than 3 vRNA segments in the presence of helper virus by introducing
into host cells expression vectors. The expression vectors
introduced into the host cells comprise vectors directing the
expression of greater than 3 vRNA segments or the corresponding
cRNAs. Further, the expression vectors introduced into the host
cells may comprise cDNA encoding one or more viral proteins,
particularly one or more viral proteins required to form the
RNPs.
[0082] The present invention provides methods of generating an
infectious, replicating recombinant Newcastle disease virus (NDV)
in mammalian cells, said methods comprising: (a) expressing genomic
vRNA or the corresponding cRNA from an expression vector in said
cells; and (b) expressing a nucleoprotein and an RNA-dependent RNA
polymerase from a set of expression vectors in said cells, whereby
ribonucleoprotein complexes are formed and the recombinant NDV is
produced in the absence of helper virus. The present invention
provides methods of generating an infectious, non-replicating
recombinant Newcastle disease virus (NDV) in mammalian cells, said
methods comprising: (a) expressing a vRNA or the corresponding cRNA
from an expression vector in said cells, wherein said vRNA or the
corresponding cRNA do not encode of the genomic viral proteins
necessary for replicating; and (b) expressing a nucleoprotein and
an RNA-dependent RNA polymerase from a set of expression vectors in
said cells, whereby ribonucleoprotein complexes are formed and the
non-replicating recombinant NDV is produced in the absence of
helper virus.
[0083] In one embodiment, an infectious recombinant NDV is
generated in mammalian cells by a method comprising: (a)
introducing an expression vectors capable of expressing in said
cells a genomic vRNA or the corresponding cRNA; (b) introducing a
set of expression vectors capable of expressing in said cells a
nucleoprotein and RNA-dependent RNA polymerase; and (c) culturing
said cells such that RNPs are formed and recombinant NDV is
produced in the absence of helper virus.
[0084] In another embodiment, an infectious recombinant NDV is
generated in a host cell line expressing a nucleoprotein and an
RNA-dependent RNA polymerase by a method comprising: (a)
introducing expression vectors capable of expressing in said cell
line a genomic vRNA segment or the corresponding cRNA; and (b)
culturing said cell line such that RNPs are formed and recombinant
NDV is produced in the absence helper virus. In another embodiment,
an infectious recombinant NDV is generated in a host cell line
expressing a genomic vRNA or the corresponding cRNA by a method
comprising: (a) introducing expression vectors capable of
expressing in said cell line a nucleoprotein and an RNA-dependent
RNA polymerase; and (b) culturing said cell line such that RNPs are
formed and recombinant NDV is produced in the absence of helper
virus.
[0085] The present invention also encompasses methods of generating
NDV in the presence of helper virus by introducing expression
vectors. The expression vectors directing the expression of genomic
vRNA or cRNA and/or one or more viral proteins.
[0086] The ability to reconstitute negative-strand RNA viruses
intracellularly in mammalian cells allows for the design of
recombinant viruses (i.e., chimeric viruses) which express
heterologous nucleic acid sequences or mutant viral genes. The
heterologous sequences may encode, for example, epitopes or
antigens of pathogens or tumors. The ability to reconstitute
negative-strand RNA viruses intracellularly also allows the design
of novel recombinant viruses (i.e., chimeric viruses) which express
genes from different strains of viruses. Thus, the present
invention provides methods of generating chimeric viruses which
express heterologous nucleic acid sequences, mutant viral genes, or
viral genes from different strains of virus intracellularly from
expression vectors in the absence or presence of helper virus.
[0087] The present invention encompasses the cells and cell lines
produced in the process of generating infectious negative-strand
RNA viruses.
[0088] The infectiousness of a recombinant or chimeric
negative-strand RNA virus of the present invention will vary
depending upon the strain of virus from which the nucleic acid
sequences encoding structural proteins such as influenza virus HA
or NA are derived. Additionally, the infectiousness of a
recombinant or chimeric negative-strand RNA virus of the invention
will vary depending upon whether or not mutations have been
introduced into the nucleic acid sequences encoding structural
proteins. For example, a recombinant influenza virus of the
invention with a mutation in HA may not be as infectious as another
recombinant influenza virus expressing identical viral proteins
without a mutation in HA.
[0089] The infectious recombinant or chimeric viruses of the
present invention may or may not be capable of replicating and
producing progeny. In a specific embodiment, an infectious
recombinant negative-strand RNA virus of the invention is capable
of replicating and producing progeny. The replication of an
infectious recombinant or chimeric negative-strand RNA virus of the
invention will vary depending upon the strain of virus from which
the genomic vRNA(s) or the corresponding cRNA(s) were derived.
Further, the replication of an infectious recombinant or chimeric
negative-strand RNA virus of the invention will vary depending upon
whether or not mutations have been introduced into the genomic
vRNA(s) or the corresponding cRNA(s). For example, an infectious
recombinant influenza virus expressing a truncated NS1 protein may
replicate better than an infectious recombinant influenza virus
expressing identical viral proteins except that it expresses a
full-length NS1 protein.
[0090] The present invention provides for the use of the
recombinant negative-strand RNA viruses or chimeric viruses of the
invention to formulate vaccines against a broad range of viruses
and/or antigens including tumor antigens. The recombinant
negative-strand RNA viruses or chimeric viruses of the present
invention may be used to modulate a subject's immune system by
stimulating a humoral immune response, a cellular immune response
or by stimulating tolerance to an antigen. When delivering, tumor
antigens, the invention may be used to treat subjects having a
disease amenable to immunity mediated rejection, such as non-solid
tumors or solid tumors of small size. It is also contemplated that
delivery of tumor antigens by the recombinant negative-strand RNA
viruses or chimeric viruses described herein will be useful for
treatment subsequent to removal of large solid tumors. The
recombinant negative-strand RNA viruses or chimeric viruses of the
invention may also be used to treat subjects who are suspected of
having cancer.
[0091] The present invention also provides for the use of the
recombinant negative-strand RNA viruses or chimeric viruses of the
invention in pharmaceutical compositions for the administration of
one or more peptides or polypeptides of interest.
5.1. EXPRESSION VECTORS FOR VRNA
[0092] Expression vectors comprising cDNA for viral RNA(s) or
corresponding cRNA(s) will preferably be under the control of a
DNA-dependent RNA polymerase promoter sequence. Examples of
DNA-dependent RNA polymerase promoters include but are not limited
to, bacterial promoters, viral promoters such as T7, T3 or SP3, and
cellular promoters such as a mammalian RNA polymerase I promoter.
Preferably, the cDNA for the viral RNA(s) or corresponding cRNA(s)
is derived from a mammalian RNA polymerase I (RNA Pol I) promoter.
Particularly preferred for this purpose is the truncated human RNA
Pol I promoter consisting of nucleotides -250 to -1 of the
corresponding native promoter or a functional derivative thereof
(Jones et al., 1988, Proc. Natl. Acad. Sci. USA 85:669-673). In yet
another embodiment, the vRNA(s) or corresponding cRNA(s) may be
under the control of a mammalian RNA polymerase II promoter or RNA
polymerase III promoter (see, e.g. Legin in Genes, Oxford
University Press, New York (1977), pp. 819-22). To ensure the
correct 3' end of each expressed vRNA or cRNA, each vRNA or cRNA
expression vector will incorporate a ribozyme sequence or
appropriate terminator sequence downstream of the RNA coding
sequence. This may be, for example, the hepatitis delta virus
genomic ribozyme sequence or a functional derivative thereof, or
the murine rDNA terminator sequence (Genbank Accession Number
M12074). Alternatively, for example, a Pol I terminator may be
employed (Neumann et al., 1994, Virology 202:477-479). The RNA
expression vectors may be constructed in the same manner as the
vRNA expression vectors described in Pleschka et al., 1996, J.
Virol. 70:4188-4192.
[0093] In a specific embodiment of the present invention, vRNA or
cRNA expression vectors for the production of infectious
recombinant NDV comprise the nucleotide sequence of the 3' termini
of the NDV negative-sense genome RNA first identified by the
Applicants'. This 3' termini of the NDV negative-sense genome RNA
differs significantly from the NDV 3' termini sequence previously
disclosed by Collins et al. in Fundamental Virology 3rd Ed. 1996 by
Lippincott-Raven Publishers as shown in FIG. 6. The identification
of the correct nucleotide sequence of the NDV 3' termini allows for
the first time the engineering of recombinant NDV RNA templates,
the expression of the recombinant RNA templates and the rescue of
recombinant NDV particles.
[0094] A DNA-dependent RNA polymerase which recognizes the promoter
sequence in the vRNA or corresponding cRNA expression vectors is
used to produce the vRNA or corresponding cRNA from the nucleic
acid sequences. Examples of DNA-dependent RNA polymerases include,
but are not limited to, viral DNA-dependent RNA polymerase such as
T7, T3 or the SP6 polymerase, bacterial DNA-dependent RNA
polymerase, and cellular DNA-dependent RNA such as mammalian RNA
polymerase I. In one embodiment, the expression vectors comprising
the cDNA directing the expression of vRNA(s) or corresponding
cRNA(s) are introduced into a host cell that does not express the
DNA-dependent RNA polymerase which recognizes the DNA-dependent RNA
polymerase promoter and one or more vectors expressing the
DNA-dependent RNA polymerase subunits are introduced into said host
cell. In accordance with this embodiment, the vectors expressing
the DNA-dependent RNA polymerase subunits may be regulated by an
inducible promoter. The expression of the DNA-dependent RNA
polymerase then regulates the expression of the vRNA(s) or
corresponding cRNAs.
[0095] The present invention provides expression vectors directing
the expressing of genomic vRNA(s) or corresponding cRNA(s) which
have one or more mutations. These mutations may result in the
attenuation of the virus. For example, the vRNA segments may be the
vRNA segments of an influenza A virus having an attenuated base
pair substitution in a pan-handle duplex promoter region, in
particular, for example, the known attenuating base pair
substitution of A for C and U for G at position 11-12' in the
duplex region of the NA-specific vRNA (Fodor et al., 1998, J.
Virol. 6923-6290). By using the methods of the invention to produce
recombinant negative-strand RNA virus, new attenuating mutations
may be identified.
[0096] Sequences heterologous to a viral genome may be engineered
into expression vectors directing the expression of vRNA(s) or
corresponding cRNA(s) and introduced into host cells along with
expression vectors directing the expression of viral proteins to
generate novel infectious recombinant negative-strand RNA viruses
or chimeric viruses. Heterologous sequences which may be engineered
into these viruses include antisense nucleic acids and nucleic acid
such as a ribozyme. Alternatively, heterologous sequences which
express a peptide or polypeptide may be engineered into these
viruses. Heterologous sequences encoding the following peptides or
polypeptides may be engineered into these viruses include: 1)
antigens that are characteristic of a pathogen; 2) antigens that
are characteristic of autoimmune disease; 3) antigens that are
characteristic of an allergen; and 4) antigens that are
characteristic of a tumor. For example, heterologous gene sequences
that can be engineered into the chimeric viruses of the invention
include, but are not limited to, epitopes of human immunodeficiency
virus (HIV) such as gp160; hepatitis B virus surface antigen
(HBsAg); the glycoproteins of herpes virus (e.g., gD, gE); VP1 of
poliovirus; and antigenic determinants of nonviral pathogens such
as bacteria and parasites to name but a few.
[0097] Antigens that are characteristic of autoimmune disease
typically will be derived from the cell surface, cytoplasm,
nucleus, mitochondria and the like of mammalian tissues, including
antigens characteristic of diabetes mellitus, multiple sclerosis,
systemic lupus erythematosus, rheumatoid arthritis, pernicious
anemia, Addison's disease, scleroderma, autoimmune atrophic
gastritis, juvenile diabetes, and discoid lupus erythromatosus.
[0098] Antigens that are allergens are generally proteins or
glycoproteins, including antigens derived from pollens, dust,
molds, spores, dander, insects and foods.
[0099] Antigens that are characteristic of tumor antigens typically
will be derived from the cell surface, cytoplasm, nucleus,
organelles and the like of cells of tumor tissue. Examples include
antigens characteristic of tumor proteins, including proteins
encoded by mutated oncogenes; viral proteins associated with
tumors; and glycoproteins. Tumors include, but are not limited to,
those derived from the types of cancer: lip, nasopharynx, pharynx
and oral cavity, esophagus, stomach, colon, rectum, liver, gall
bladder, pancreas, larynx, lung and bronchus, melanoma of skin,
breast, cervix, uterine, ovary, bladder, kidney, uterus, brain and
other parts of the nervous system, thyroid, prostate, testes,
Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma and
leukemia.
[0100] In one specific embodiment of the invention, the
heterologous sequences are derived from the genome of human
immunodeficiency virus (HIV), preferably human immunodeficiency
virus-1 or human immunodeficiency virus-2. In another embodiment of
the invention, the heterologous coding sequences may be inserted
within an negative-strand RNA virus gene coding sequence such that
a chimeric gene product is expressed which contains the
heterologous peptide sequence within the viral protein. In such an
embodiment of the invention, the heterologous sequences may also be
derived from the genome of a human immunodeficiency virus,
preferably of human immunodeficiency virus-1 or human
immunodeficiency virus-2.
[0101] In instances whereby the heterologous sequences are
HIV-derived, such sequences may include, but are not limited to
sequences derived from the env gene (i.e., sequences encoding all
or part of gp160, gp120, and/or gp41), the pol gene (i.e.,
sequences encoding all or part of reverse transcriptase,
endonuclease, protease, and/or integrase), the gag gene (i.e.,
sequences encoding all or part of p7, p6, p55, p17/18, p24/25) tat,
rev, nef, vif, vpu, vpr, and/or vpx.
[0102] One approach for constructing these hybrid molecules is to
insert the heterologous coding sequence into a DNA complement of a
negative-strand RNA virus gene so that the heterologous sequence is
flanked by the viral sequences required for viral polymerase
activity; i.e., the viral polymerase binding site/promoter,
hereinafter referred to as the viral polymerase binding site, and a
polyadenylation site. In an alternative approach, oligonucleotides
encoding the viral polymerase binding site, e.g., the complement of
the 3'-terminus or both termini of the virus genomic segments can
be ligated to the heterologous coding sequence to construct the
hybrid molecule. The placement of a foreign gene or segment of a
foreign gene within a target sequence was formerly dictated by the
presence of appropriate restriction enzyme sites within the target
sequence. However, recent advances in molecular biology have
lessened this problem greatly. Restriction enzyme sites can readily
be placed anywhere within a target sequence through the use of
site-directed mutagenesis (e.g., see, for example, the techniques
described by Kunkel, 1985, Proc. Natl. Acad. Sci. U.S.A. 82:488).
Variations in polymerase chain reaction (PCR) technology,
described, also allow for the specific insertion of sequences
(i.e., restriction enzyme sites) and allow for the facile
construction of hybrid molecules. Alternatively, PCR reactions
could be used to prepare recombinant templates without the need of
cloning. For example, PCR reactions could be used to prepare
double-stranded DNA molecules containing a DNA-directed RNA
polymerase promoter (e.g., bacteriophase T3, T7 or SP6) and the
hybrid sequence containing the heterologous gene and the polymerase
binding site. RNA templates could then be transcribed directly from
this recombinant DNA. In yet another embodiment, the recombinant
vRNAs or corresponding cRNAs may be prepared by ligating RNAs
specifying the negative polarity of the heterologous gene and the
viral polymerase binding site using an RNA ligase.
[0103] Bicistronic mRNA could be constructed to permit internal
initiation of translation of viral sequences and allow for the
expression of foreign protein coding sequences from the regular
terminal initiation site. Alternatively, a bicistronic mRNA
sequence may be constructed wherein the viral sequence is
translated from the regular terminal open reading frame, while the
foreign sequence is initiated from an internal site. Certain
internal ribosome entry site (IRES) sequences may be utilized. The
IRES sequences which are chosen should be short enough to not
interfere with Newcastle disease virus packaging limitations. Thus,
it is preferable that the IRES chosen for such a bicistronic
approach be no more than 500 nucleotides in length, with less than
250 nucleotides being preferred. Further, it is preferable that the
IRES utilized not share sequence or structural homology with
picornaviral elements. Preferred IRES elements include, but are not
limited to the mammalian BiP IRES and the hepatitis C virus
IRES.
[0104] Alternatively, a foreign protein may be expressed from an
internal transcriptional unit in which the transcriptional unit has
an initiation site and polyadenylation site. In another embodiment,
the foreign gene is inserted into a negative-strand RNA virus gene
such that the resulting expressed protein is a fusion protein.
5.2. EXPRESSION VECTORS ENCODING VIRAL PROTEINS
[0105] Expression vectors used to express viral proteins, in
particular viral proteins for RNP complex formation, will
preferably express viral proteins homologous to the desired virus.
The expression of viral proteins by these expression vectors may be
regulated by any regulatory sequence known to those of skill in the
art. The regulatory sequence may be a constitutive promoter, an
inducible promoter or a tissue-specific promoter. In a specific
embodiment, the regulatory sequence comprises the adenovirus 2
major late promoter linked to the spliced tripartite leader
sequence of human adenovirus 2, as described by Berg et al.,
BioTechniques 14:972-978.
[0106] Promoters which may be used to control the expression of
viral proteins in protein expression vectors include, but are not
limited to, the SV40 early promoter region (Bernoist and Chambon,
1981, Nature 290:304-310), the promoter contained in the 3' long
terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell
22:787-797), the herpes thymidine kinase promoter (Wagner et al.,
1981, Proc. Natl. Acad. Sci. USA 78:1441-1.445), the regulatory
sequences of the metallothionein gene (Brinster et al., 1982,
Nature 296:39-42); prokaryotic expression vectors such as the
.beta.-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl.
Acad. Sci. USA 75:3727-3731), or the tac promoter (DeBoer et al.,
1983, Proc. Natl. Acad. Sci. USA 80:21-25); see also "Useful
proteins from recombinant bacteria" in Scientific American, 1980,
242:74-94; plant expression vectors comprising the nopaline
synthetase promoter region (Herrera-Estrella et al., Nature
303:209-213) or the cauliflower mosaic virus 35S RNA promoter
(Gardner et al., 1981, Nucl. Acids Res. 9:2871), and the promoter
of the photosynthetic enzyme ribulose biphosphate carboxylase
(Herrera-Estrella et al., 1984, Nature 310:115-120); promoter
elements from yeast or other fungi such as the Gal 4 promoter, the
ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase)
promoter, alkaline phosphatase promoter, and the following animal
transcriptional control regions, which exhibit tissue specificity
and have been utilized in transgenic animals: elastase I gene
control region which is active in pancreatic acinar cells (Swift et
al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor
Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology
7:425-515); insulin gene control region which is active in
pancreatic beta cells (Hanahan, 1985, Nature 315:115-122),
immunoglobulin gene control region which is active in lymphoid
cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al.,
1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.
7:1436-1444), mouse mammary tumor virus control region which is
active in testicular, breast, lymphoid and mast cells (Leder et
al., 1986, Cell 45:485-495), albumin gene control region which is
active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276),
alpha-fetoprotein gene control region which is active in liver
(Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et
al., 1987, Science 235:53-58; alpha 1-antitrypsin gene control
region which is active in the liver (Kelsey et al., 1987, Genes and
Devel. 1:161-171), beta-globin gene control region which is active
in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias
et al., 1986, Cell 46:89-94; myelin basic protein gene control
region which is active in oligodendrocyte cells in the brain
(Readhead et al., 1987, Cell 48:703-712), myosin light chain-2 gene
control region which is active in skeletal muscle (Sani, 1985,
Nature 314:283-286), and gonadotropic releasing hormone gene
control region which is active in the hypothalamus (Mason et al.,
1986, Science 234:1372-1378).
[0107] Appropriate protein expression vectors known to those of
skill in the art can be used to express the viral proteins. For
example, the plasmid pGT-h described in Berg et al., BioTechniques
14:972-978 or pcDNA3 vectors can be used to construct expression
vectors for viral proteins.
[0108] In a specific embodiment, the protein expression vector
comprises a promoter operably linked to a nucleic acid sequence,
one or more origins of replication, and, optionally, one or more
selectable markers (e.g., an antibiotic resistance gene). In
another embodiment, a protein expression vector that is capable of
producing bicistronic mRNA may be produced by inserting bicistronic
mRNA sequence. Certain internal ribosome entry site (IRES)
sequences may be utilized. Preferred IRES elements include, but are
not limited to the mammalian BiP IRES and the hepatitis C virus
IRES.
[0109] Expression vectors containing gene inserts can be identified
by three general approaches: (a) nucleic acid hybridization; (b)
presence or absence of "marker" gene functions; and (c) expression
of inserted sequences. In the first approach, the presence of the
viral gene inserted in an expression vector(s) can be detected by
nucleic acid hybridization using probes comprising sequences that
are homologous to the inserted gene(s). In the second approach, the
recombinant vector/host system can be identified and selected based
upon the presence or absence of certain "marker" gene functions
(e.g., resistance to antibiotics or transformation phenotype)
caused by the insertion of the gene(s) in the vector(s). In the
third approach, expression vectors can be identified by assaying
the gene product expressed. Such assays can be based, for example,
on the physical or functional properties of the viral protein in in
vitro assay systems, e.g., binding of viral proteins to
antibodies.
[0110] In a specific embodiment, one or more protein expression
vectors encode and express the viral proteins necessary for the
formation of RNP complexes. In another embodiment, one or more
protein expression vectors encode and express the viral proteins
necessary to form viral particles. In yet another embodiment, one
or more protein expression vectors encode and express the all of
the viral proteins of a particular negative-strand RNA virus.
5.3. GENERATION OF RECOMBINANT NEGATIVE STRAND RNA VIRUSES
[0111] The present invention provides methods of generating
infectious recombinant negative-strand RNA virus by introducing
protein expression vectors and vRNA or corresponding cRNA
expressing expression vectors into host cells in the absence of
helper virus. The present invention also provides methods of
generating infectious recombinant negative-strand RNA virus by
introducing protein expression vectors and vRNA or corresponding
cRNA expressing expression vectors into host cells in the presence
of helper virus.
[0112] Protein expression vectors and expression vectors directing
the expression of vRNAs or corresponding cRNAs can be introduced
into host cells using techniques known to those of skill in the
art. For example, expression vectors of the invention can be
introduced into host cells by employing electroporation,
DEAE-dextran, calcium phosphate precipitation, liposomes,
microinjection, and microparticle-bombardment (see, e.g., Sambrook
et al., Molecular Cloning: A Laboratory Manual, 2 ed., 1989, Cold
Spring Harbor Press, Cold Spring Harbor, N.Y.). The expression
vectors of the invention may be introduced into host cells
simultaneously or sequentially.
[0113] In one embodiment, one or more expression vectors directing
the expression of vRNA(s) or corresponding cRNA(s) are introduced
into host cells prior to the introduction of expression vectors
directing the expression of viral proteins. In another embodiment,
one or more expression vectors directing the expression of viral
proteins are introduced into host cells prior to the introduction
of the one or more expression vectors directing the expression of
vRNA(s) or corresponding cRNA(s). In accordance with these
embodiments, the expression vectors directing the expression of the
vRNA(s) or corresponding cRNA(s) may introduced together or
separately in different transfections. Further, in accordance with
these embodiments, the expression vectors directing the expression
of the viral proteins can be introduced together or separately in
different transfections.
[0114] In another embodiment, one or more expression vectors
directing the expression of vRNA(s) or corresponding cRNA(s) and
one or more expression vectors directing the expression of viral
proteins are introduced into host cells simultaneously. Preferably,
all of the expression vectors are introduced into host cells using
liposomes. Appropriate amounts and ratios of the expression vectors
for carrying out a method of the invention may be determined by
routine experimentation. As guidance, in the case of liposomal
transfection or calcium precipitation of plasmids into the host
cells, it is envisaged that each plasmid may be employed at a few
.mu.gs, e.g., 1 to 10 .mu.g, for example, diluted to a final total
DNA concentration of about 0.1 .mu.g/ml prior to mixing with
transfection reagent in conventional manner. It may be preferred to
use vectors expressing NP and/or RNA-dependent RNA polymerase
subunits at a higher concentration than those expressing vRNA
segments. One skilled in the art will appreciate that the amounts
and ratios of the expression vectors may vary depending upon the
host cells.
[0115] In one embodiment, at least 0.5 .mu.g, preferably at least 1
.mu.g, at least 2.5 .mu.g, at least 5 .mu.g, at least 8 .mu.g, at
least 10 .mu.g, at least 15 .mu.g, at least 20 .mu.g, at least 25
.mu.g or at least 50 .mu.g of one or more protein expression
vectors of the invention are introduced into host cells to generate
infectious recombinant negative-strand RNA virus. In another
embodiment, at least 0.5 .mu.g, preferably at least 1 .mu.g, at
least 2.5 .mu.g, at least 5 .mu.g, at least 8 .mu.g, at least 10
.mu.g, at least 15 .mu.g, at least 20 .mu.g, at least 25 .mu.g or
at least 50 .mu.g of one or more expression vectors of the
invention directing the expression of vRNAs or cRNAs are introduced
into host cells to generate infectious recombinant negative-strand
RNA virus.
[0116] Host cells which may be used to generate the negative-strand
RNA viruses of the invention include primary cells, cultured or
secondary cells, and transformed or immortalized cells (e.g., 293
cells, 293T cells, CHO cells, Vero cells, PK, MDBK, OMK and MDCK
cells). Host cells are preferably animal cells, more preferably
mammalian cells, and most preferably human cells. In a preferred
embodiment, infectious recombinant negative-strand RNA viruses of
the invention are generated in 293T cells.
[0117] It is known that Vero cells are deficient in interferon
expression (Diaz et al., 1998, Proc. Natl. Acad. Sci. USA
85:5259-5263), which might be a factor in attaining good viral
rescue. Hence, it is extrapolated that Vero cells and other cells
deficient in interferon activity or response which will support
growth of segmented negative-strand RNA viruses may be useful in
the practice of the invention.
[0118] In order to rescue recombinant influenza B viruses, 293 T
cells may not be the most efficient host cell to achieve rescue.
Thus, in accordance with the present invention, methods to achieve
rescue influenza B virus should utilize host cells which support
the efficient replication of influenza B, such as MDCK (canine
kidney), PK (porcine kidney) or OMK (owl monkey kidney) cells.
Alternatively, MDBK (bovine kidney) cells may be used as hots cells
to support rescue of influenza B. Despite the fact that MDBK cells
do not support the growth of influenza B, using a reverse genetics
approach this cell line supports rescue of influenza B (Barclay et
al., 1995, J. Virol. 69:1275-1279).
[0119] The present invention provides methods of generating
infectious recombinant negative-strand RNA virus in stably
transduced host cell lines. The stably transduced host cell lines
of the invention may be produced by introducing cDNA controlled by
appropriate expression control elements (e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable marker into host cells. Following the introduction
of the foreign DNA, the transduced cells may be allowed to grow for
1-2 days in an enriched media, and then are switched to a selective
media. The selectable marker confers resistance to the cells and
allows the cells to stably integrate the DNA into their
chromosomes. Transduced host cells with the DNA stably integrated
can be cloned and expanded into cell lines.
[0120] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler, et
al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes
can be employed in tk-, hgprt- or aprt-cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
dhfr, which confers resistance to methotrexate (Wigler et al.,
1980, Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc.
Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to
mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad.
Sci. USA 78:2072); neo, which confers resistance to the
aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol.
150:1); and hygro, which confers resistance to hygromycin (Santerre
et al., 1984, Gene 30:147) genes.
[0121] The infectious recombinant negative-strand RNA viruses
generated by methods of the invention which are not attenuated, may
attenuated or killed by, for example, classic methods. For example,
recombinant negative-strand RNA viruses of the invention may be
killed by heat or formalin treatment, so that the virus is not
capable of replicating. Recombinant negative-strand RNA viruses of
the invention which are not attenuated may be attenuated by, e.g.,
passage through unnatural hosts to produce progeny viruses which
are immunogenic, but not pathogenic.
[0122] Attenuated or killed viruses produced in accordance with the
invention may subsequently be incorporated into a vaccine
composition in conventional manner. Where such a virus has a
chimeric vRNA segment as discussed above which encodes a foreign
antigen, it may be formulated to achieve vaccination against more
than one pathogen simultaneously. Attenuated recombinant viruses
produced in accordance with the invention which possess a chimeric
vRNA segment may also be designed for other therapeutic uses, e.g.,
an anti-tumor agent or gene therapy tool, in which case production
of the virus will be followed by its incorporation into an
appropriate pharmaceutical composition together with a
pharmaceutically acceptable carrier or diluent.
[0123] Helper virus free rescue in accordance with the invention is
particularly favored for generation of reassortant viruses,
especially reassortant influenza viruses desired for vaccine use.
For example, by means of viral rescue in accordance with the
invention the HA and NA vRNA segments of an influenza virus, e.g.,
influenza A/PR8/34 which is recognized as suitable for human
administration, may be readily substituted with the HA and NA vRNA
segments of an influenza strain associated with an influenza
infection epidemic. Such reassortant influenza viruses may, for
example, be used for production of a killed influenza vaccine in
conventional manner.
[0124] The methods of the present invention may be modified to
incorporate aspects of methods known to those skilled in the art,
in order to improve efficiency of rescue of infectious viral
particles. For example, the reverse genetics technique involves the
preparation of synthetic recombinant viral RNAs that contain the
non-coding regions of the negative strand virus RNA which are
essential for the recognition by viral polymerases and for
packaging signals necessary to generate a mature virion. The
recombinant RNAs are synthesized from a recombinant DNA template
and reconstituted in vitro with purified viral polymerase complex
to form recombinant ribonucleoprotein (RNPs) which can be used to
transect cells. A more efficient transfection is achieved if the
viral polymerase proteins are present during transcription of the
synthetic RNAs either in vitro or in vivo. The synthetic
recombinant RNPs can be rescued into infectious virus particles.
The foregoing techniques are described in U.S. Pat. No. 5,166,057
issued Nov. 24, 1992; in U.S. Pat. No. 5,854,037 issued Dec. 29,
1998; in U.S. Pat. No. 5,789,229 issued Aug. 4, 1998; in European
Patent Publication EP 0702085A1, published Feb. 20, 1996; in U.S.
patent application Ser. No. 09/152,845; in International Patent
Publications PCR WO97/12032 published Apr. 3, 1997; WO96/34625
published Nov. 7, 1996; in European Patent Publication EP-A780475;
WO99/02657 published Jan. 21, 1999; WO98/53078 published Nov. 26,
1998; WO98/02530 published Jan. 22, 1998; WO99/15672 published Apr.
1, 1999; WO98/13501 published Apr. 2, 1998; WO97/06720 published
Feb. 20, 1997; and EPO 780 47SA1 published Jun. 25, 1997, each of
which is incorporated by reference herein in its entirety.
5.4. SEGMENTED NEGATIVE-STRAND RNA VIRUS EMBODIMENTS
[0125] The present invention provides a method for generating in
cultured cells infectious viral particles of a segmented
negative-strand RNA virus having greater than 3 genomic vRNA
segments, for example an influenza virus such as an influenza A
virus, said method comprising: (a) providing a first population of
cells capable of supporting growth of said virus and having
introduced a first set of expression vectors capable of directly
expressing in said cells genomic vRNA segments to provide the
complete genomic vRNA segments of said virus, or the corresponding
cRNAs, in the absence of a helper virus to provide any such RNA
segment, said cells also being capable of providing a nucleoprotein
and RNA-dependent RNA polymerase whereby RNP complexes containing
the genomic vRNA segments of said virus can be formed and said
viral particles can be assembled within said cells; and (b)
culturing said cells whereby said viral particles are produced.
[0126] The present invention also provides a method for generating
in cultured cells infectious viral particles of a segmented
negative-strand RNA virus, said method comprising: (i) providing a
first population of cells which are capable of supporting the
growth of said virus and which are modified so as to be capable of
providing (a) the genomic vRNAs of said virus in the absence of a
helper virus and (b) a nucleoprotein and RNA-dependent RNA
polymerase whereby RNA complexes containing said genomic vRNAs can
be formed and said viral particles can be assembled, said genomic
vRNAs being directly expressed in said cells under the control of a
human Pol I promoter or functional derivative thereof: and (ii)
culturing said cells whereby said viral particles are produced.
[0127] The present specification also provides a method for
generating in cultured cells infectious viral particles of a
segmented negative-strand RNA virus, said method comprising: (i)
providing a population of cells which are capable of supporting the
growth of said virus and which are modified so as be capable of
providing (a) the genomic vRNAs of said virus in the absence of a
helper virus and (b) a nucleoprotein and RNA-dependent RNA
polymerase whereby RNP complex or complexes containing said genomic
vRNAs can be formed and said viral particles can be assembled, said
genomic RNAs being directly expressed in said cells under the
control of a mammalian Pol I, Pol II or Pol III promoter or a
functional derivative thereof, e.g., the truncated human Pol I
promoter as previously noted above; and (ii) culturing said cells
whereby said viral particles are produced. In a specific
embodiment, an infectious recombinant negative-strand RNA virus
having at least 4, preferably at least 5, at least 6, or at least 7
genomic vRNA segments in a host cell using the methods described
herein.
[0128] In a preferred embodiment, the present invention provides
for methods of generating infectious recombinant influenza virus in
host cells using expression vectors to express the vRNA segments or
corresponding cRNAs and influenza virus proteins, in particular
PB1, PB2, PA and NA. In accordance with this embodiment, helper
virus may or may not be included to generate the infectious
recombinant influenza viruses.
[0129] The infectious recombinant influenza viruses of the
invention may or may not replicate and produce progeny. Preferably,
the infectious recombinant influenza viruses of the invention are
attenuated. Attenuated infectious recombinant influenza viruses
may, for example, have a mutation in the NS1 gene.
[0130] In a preferred embodiment, the infectious recombinant
influenza viruses of the invention express heterologous (i.e.,
non-influenza virus) sequences. In another embodiment, the
infectious recombinant influenza viruses of the invention express
influenza virus proteins from different influenza strains. In yet
another preferred embodiment, the infectious recombinant influenza
viruses of the invention express fusion proteins.
5.5. NEWCASTLE DISEASE VIRUS EMBODIMENTS
[0131] A specific embodiment of the present invention is the
Applicants' identification of the correct nucleotide sequence of
the 5' and 3' termini of the negative-sense genomes RNA of NDV. The
nucleotide sequence of the 3' termini of the NDV negative-sense
genome RNA of the present invention differs significantly from the
NDV 3' termini sequence previously disclosed by Collins et al. in
Fundamental Virology 3rd Ed. 1996 by Lippincott-Raven Publishers as
shown in FIG. 6. The identification of the correct nucleotide
sequence of the NDV 3' termini allows for the first time the
engineering of recombinant NDV RNA templates, the expression of the
recombinant RNA templates and the rescue of recombinant NDV
particles.
[0132] Heterologous gene coding sequences flanked by the complement
of the viral polymerase binding site/promoter, e.g, the complement
of 3'-NDV virus terminus of the present invention, or the
complements of both the 3'- and 5'-NDV virus termini may be
constructed using techniques known in the art. The resulting RNA
templates may be of the negative-polarity and contain appropriate
terminal sequences which enable the viral RNA-synthesizing
apparatus to recognize the template. Alternatively,
positive-polarity RNA templates which contain appropriate terminal
sequences which enable the viral RNA-synthesizing apparatus to
recognize the template, may also be used. Recombinant DNA molecules
containing these hybrid sequences can be cloned and transcribed by
a DNA-dependent RNA polymerase, such as bacteriophage T7, T3, or
the SP6 polymerase and the like, to produce in vitro and in vivo
the recombinant RNA templates which possess the appropriate viral
sequences that allow for viral polymerase recognition and
activity.
[0133] As described above, heterologous sequences can be: 1)
antigens that are characteristic of a pathogen; 2) antigens that
are characteristic of autoimmune disease; 3) antigens that are
characteristic of an allergen; and 4) antigens that are
characteristic of a tumor. The heterologous sequences can be
introduced into viral nucleic acid sequences by techniques
described herein or known to those of skill in the art.
[0134] The gene segments coding for the NDV HN, P, NP, M, F, or L
proteins may be used for the insertion of heterologous gene
products. Insertion of a foreign gene sequence into any of these
segments could be accomplished by either a complete replacement of
the viral coding region with the foreign gene or by a partial
replacement. Complete replacement would probably best be
accomplished through the use of PCR-directed mutagenesis. Briefly,
PCR-primer A would contain, from the 5' to 3' end: a unique
restriction enzyme site, such as a class IIS restriction enzyme
site (i.e., a "shifter" enzyme; that recognizes a specific sequence
but cleaves the DNA either upstream or downstream of that
sequence); a stretch of nucleotides complementary to a region of
the NDV gene; and a stretch of nucleotides complementary to the
carboxy-terminus coding portion of the foreign gene product.
PCR-primer B would contain from the 5' to 3' end: a unique
restriction enzyme site; a stretch of nucleotides complementary to
a NDV gene; and a stretch of nucleotides corresponding to the 5'
coding portion of the foreign gene. After a PCR reaction using
these primers with a cloned copy of the foreign gene, the product
may be excised and cloned using the unique restriction sites.
Digestion with the class IIS enzyme and transcription with the
purified phage polymerase would generate an RNA molecule containing
the exact untranslated ends of the NDV gene with a foreign gene
insertion. In an alternate embodiment, PCR-primed reactions could
be used to prepare double-stranded DNA containing the bacteriophage
promoter sequence, and the hybrid gene sequence so that RNA
templates can be transcribed directly without cloning.
[0135] The hemagglutinin and neuraminidase activities of NDV are
coded for by a single gene, HN. The HN protein is a major surface
glycoprotein of the virus. For a variety of viruses, such as
influenza, the hemagglutinin and neuraminidase proteins have been
demonstrated to contain a number of antigenic sites. Consequently,
this protein is a potential target for the humoral immune response
after infection. Therefore, substitution of antigenic sites within
HN with a portion of a foreign protein may provide for a vigorous
humoral response against this foreign peptide. If a sequence is
inserted within the HN molecule and it is expressed on the outside
surface of the HN it will be immunogenic. For example, a peptide
derived from gp160 of HIV could be inserted into antigenic site of
the HN protein for antigenic presentation by the chimeric virus,
resulting in the elicitation of both a humoral immune response. In
a different approach, the foreign peptide sequence may be inserted
within the antigenic site without deleting any viral sequences.
Expression products of such constructs may be useful in vaccines
against the foreign antigen, and may indeed circumvent a problem
discussed earlier, that of propagation of the recombinant virus in
the vaccinated host. An intact HN molecule with a substitution only
in antigenic sites may allow for HN function and thus allow for the
construction of a viable virus. Therefore, this virus can be grown
without the need for additional helper functions. The virus may
also be attenuated in other ways to avoid any danger of accidental
escape.
[0136] Other hybrid constructions may be made to express proteins
on the cell surface or enable them to be released from the cell. As
a surface glycoprotein, the HN has an amino-terminal cleavable
signal sequence necessary for transport to the cell surface, and a
carboxy-terminal sequence necessary for membrane anchoring. In
order to express an intact foreign protein on the cell surface it
may be necessary to use these HN signals to create a hybrid
protein. In this case, the fusion protein may be expressed as a
separate fusion protein from an additional internal promoter.
Alternatively, if only the transport signals are present and the
membrane anchoring domain is absent, the protein may be secreted
out of the cell.
[0137] The recombinant templates prepared as described above can be
used in a variety of ways to express the heterologous gene products
in appropriate host cells or to create chimeric viruses that
express the heterologous gene products. In one embodiment, the
recombinant template can be used to transect appropriate host
cells, may direct the expression of the heterologous gene product
at high levels. Host cell systems which provide for high levels of
expression include continuous cell lines that supply viral
functions such as cell lines superinfected with NDV, cell lines
engineered to complement NDV functions, etc.
[0138] In an alternate embodiment of the invention, the recombinant
templates may be used to transect cell lines that express a viral
polymerase protein in order to achieve expression of the
heterologous gene product. To this end, transformed cell lines that
express a polymerase protein such as the L protein may be utilized
as appropriate host cells. Host cells may be similarly engineered
to provide other viral functions or additional functions such as NP
or HN.
[0139] In another embodiment, a helper virus may provide the RNA
polymerase protein utilized by the cells in order to achieve
expression of the heterologous gene product.
[0140] In yet another embodiment, cells may be transfected with
vectors encoding viral proteins such as the NP, P and L proteins.
Examples of such vectors are illustrated in FIG. 2A-2C.
[0141] In order to prepare chimeric virus, containing modified NDV
virus RNAs or RNA coding for foreign proteins in the plus or minus
sense, may be used to transect cells which are also infected with a
"parent" NDV virus. Following reassortment, the novel viruses may
be isolated and their genomes be identified through hybridization
analysis. In additional approaches described herein the production
of infectious chimeric virus may be replicated in host cell systems
that express an NDV viral polymerase protein (e.g., in virus/host
cell expression systems; transformed cell lines engineered to
express a polymerase protein, etc.), so that infectious chimeric
virus are rescued. In this instance, helper virus need not be
utilized since this function is provided by the viral polymerase
proteins expressed.
[0142] In a particularly desirable approach, cells engineered to
express all NDV viral genes may result in the production of
infectious chimeric virus which contain the desired genotype; thus
eliminating the need for a selection system. Theoretically, one can
replace any one of the six genes or part of any one of the six
genes of NDV with a foreign sequence. However, a necessary part of
this equation is the ability to propagate the defective virus
(defective because a normal viral gene product is missing or
altered). A number of possible approaches exist to circumvent this
problem. In one approach a virus haying a mutant protein can be
grown in cell lines which are constricted to constitutively express
the wild type version of the same protein. By this way, the cell
line complements the mutation in the virus. Similar techniques may
be used to construct transformed cell lines that constitutively
express any of the NDV genes. These cell lines which are made to
express the viral protein may be used to complement the defect in
the recombinant virus and thereby propagate it. Alternatively,
certain natural host range systems may be available to propagate
recombinant virus.
[0143] A third approach to propagating the recombinant virus may
involve co-cultivation with wild-type virus. This could be done by
simply taking recombinant virus and co-infecting cells with this
and another wild-type virus (preferably a vaccine strain). The
wild-type virus should complement for the defective virus gene
product and allow growth of both the wild-type and recombinant
virus. Alternatively, a helper virus may be used to support
propagation of the recombinant virus.
[0144] In another approach, synthetic templates may be replicated
in cells co-infected with recombinant viruses that express the NDV
virus polymerase protein. In fact, this method may be used to
rescue recombinant infectious virus in accordance with the
invention. To this end, the an NDV polymerase protein may be
expressed in any expression vector/host cell system, including but
not limited to viral expression vectors (e.g., vaccinia virus,
adenovirus, baculovirus, etc.) or cell lines that express a
polymerase protein (e.g., see Krystal et al., 1986, Proc. Natl.
Acad. Sci. USA 83: 2709-2713). Moreover, infection of host cells
expressing all six NDV proteins may result in the production of
infectious chimeric virus particles. This system would eliminate
the need for a selection system, as all recombinant virus produced
would be of the desired genotype.
[0145] It should be noted that it may be possible to construct a
recombinant virus without altering virus viability. These altered
viruses would then be growth competent and would not need helper
functions to replicate. For example, alterations in the
hemagglutinin neuraminidase gene discussed, supra, may be used to
construct such viable chimeric viruses.
5.6. PURIFICATION/ISOLATION OF RECOMBINANT NEGATIVE STRAND RNA
VIRUSES
[0146] The recombinant negative strand RNA viruses of the invention
can be isolated or purified using techniques known to those of
skill in the art (see, e.g., U.S. Pat. No. 5,948,410 and R. J.
Kuchler, "Biochemical Methods in Cell Culture and Virology",
Dowden, Hutchinson and Ross, Inc., Stroudsburg, Pa. (1977)). For
example, using one isolation method, supernatant from host cells
expressing the recombinant negative-strand RNA viruses of the
invention are filtered through a depth filter with a nominal pore
size of 0.5 micron to remove the cellular debris. Subsequently, the
recombinant negative-strand RNA viruses are concentrated and
purified by ultrafiltration using a membrane with a molecular
weight cut-off. Sucrose is added to the concentrate to a final
concentration of 30% (w/v) after which formaldehyde is added to a
final concentration of 0.015% (w/v). This mixture is stirred at
2-8.degree. C. for 72 hours. Next the virus concentrate is diluted
five-fold with phosphate buffered saline and loaded onto a affinity
column containing Amicon Cellufine Sulphate. After removing
impurities by washing with phosphate buffered saline the virus is
eluted with a solution of 1.5 molar sodium chloride in phosphate
buffered saline. The eluate is concentrated and desalted by
ultrafiltration using a membrane with a molecular weight
cut-off.
[0147] In another isolation method, supernatant from host cells
expressing the recombinant negative-strand RNA viruses of the
invention is subject to centrifugation at a speed which will not
pellet the virus (e.g., 2,500 rpm for about 20 minutes). The
supernatant may then be further purified by ultrafiltration
employing a filter having a pore size that is larger than the viral
particles. Preferably, a filter of approximately 0.22 microns is
used. Following filtration, the viral particles are collected by
polyethylene glycol precipitation followed by centrifugation or,
more preferably, by high speed centrifugation at about 70,000 rpm.
The viral particles are then resuspended in a small volume of
buffer, preferably TNE (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, pH
7.4). A non-ionic detergent may optionally be added to the viral
particle suspension to dissolve any contaminants. Although the high
speed viral pellet is sufficiently pure to use as a source of viral
RNA the viral suspension may optionally be further purified by
sucrose density gradient centrifugation.
[0148] An "isolated" or "purified" recombinant negative-strand RNA
virus is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the protein is derived, and is substantially free of contaminating
viruses (e.g., helper virus). A recombinant negative-strand RNA
virus that is substantially free of cellular material includes
preparations of the recombinant negative-strand RNA virus is at
least 50%, preferably at least 60%, at least 75%, at least 85%, at
least 95%, or at least 99% free of heterologous protein (also
referred to herein as a "contaminating protein"). A recombinant
negative-strand RNA virus that is substantially free of
contaminating virus includes preparations of the recombinant
negative-strand RNA virus is at least 50%, preferably at least 60%,
at least 75%, at least 85%, at least 95%, or at least 99% free of
contaminating viruses.
5.7. ASSAYS FOR THE IDENTIFICATION OF RECOMBINANT NEGATIVE STRAND
RNA VIRUSES
[0149] The production of the recombinant negative-strand RNA
viruses of the invention may assessed using any technique known to
one of skill in the art. For example, recombinant negative-strand
RNA viruses of the invention may be assessed by cell-free reverse
transcriptase (hereinafter "RT") activity assay in the cultures and
by electron microscopy. Further, any conventional assay which
detects virus-specific proteins may be employed to detect the
production of the recombinant negative-strand RNA viruses of the
invention. Such assays include, for example, Western blots, ELISA,
radioimmunoassay, or polyacrylamide gel electrophoresis and
comparison to a virus standard.
[0150] The production of infectious, replicating recombinant
negative-strand RNA viruses of the invention may be assessed using
techniques known to those of skill in the art. In particular, the
production of infectious, replicating recombinant negative-strand
RNA viruses of the invention may be assessed by a plaque assay
using, for example, MDCK cells.
5.8. VACCINE FORMULATIONS
[0151] Virtually any heterologous gene sequence may be constructed
into the viruses of the invention for use in vaccines. Preferably,
epitopes that induce a protective immune response to any of a
variety of pathogens, or antigens that bind neutralizing antibodies
may be expressed by or as part of the viruses. For example,
heterologous gene sequences that can be constructed into the
viruses of the invention for use in vaccines include but are not
limited to epitopes of human immunodeficiency virus (HIV) such as
gp120; hepatitis B virus surface antigen (HBsAg); the glycoproteins
of herpes virus (e.g. gD, gE); VP1 of poliovirus; antigenic
determinants of non-viral pathogens such as bacteria and parasites,
to name but a few. In another embodiment, all or portions of
immunoglobulin genes may be expressed. For example, variable
regions of anti-idiotypic immunoglobulins that mimic such epitopes
may be constructed into the viruses of the invention.
[0152] Either a live recombinant viral vaccine or an inactivated
recombinant viral vaccine can be formulated. A live vaccine may be
preferred because multiplication in the host leads to a prolonged
stimulus of similar kind and magnitude to that occurring in natural
infections, and therefore, confers substantial, long-lasting
immunity. Production of such live recombinant virus vaccine
formulations may be accomplished using conventional methods
involving propagation of the virus in cell culture or in the
allantois of the chick embryo followed by purification.
[0153] Vaccine formulations may include genetically engineered
negative strand RNA viruses that have mutations in the NS1 or
analogous gene. They may also be formulated using negative strand
RNA viruses that have mutations in the NS1 or analogous gene that
are natural variants, such as the A/turkey/Ore/71 natural variant
of influenza A, or B/201, and AWBY-234, which are natural variants
of influenza B. Furthermore, vaccines can include viruses that have
mutations in the NS1 or analogous gene resulting from spontaneous
mutation events, UV irradiation, exposure to chemical mutagens, or
any other genetically-altering event.
[0154] Many methods may be used to introduce the vaccine
formulations described above, these include but are not limited to
oral, intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, and intranasal routes. It may be preferable to
introduce the virus vaccine formulation via the natural route of
infection of the pathogen for which the vaccine is designed. Where
a live virus vaccine preparation is used, it may be preferable to
introduce the formulation via the natural route of infection for
influenza virus. The ability of influenza virus to induce a
vigorous secretory and cellular immune response can be used
advantageously. For example, infection of the respiratory tract by
influenza viruses may induce a strong secretory immune response,
for example in the urogenital system, with concomitant protection
against a particular disease causing agent.
5.9. PHARMACEUTICAL COMPOSITIONS
[0155] The present invention encompasses pharmaceutical
compositions comprising recombinant viruses of the invention to be
used as anti-viral agents or anti-tumor agents. The pharmaceutical
compositions have utility as an anti-viral prophylactic and thus in
accordance may be administered to a subject when the subject has
been exposed or is expected to be exposed to a virus. For example,
in the event that a child comes home from school where he is
exposed to several classmates with the flu, a parent would
administer the anti-viral pharmaceutical composition of the
invention to herself, the child and other family members to prevent
viral infection and subsequent illness.
[0156] Various delivery systems are known and can be used to
administer the pharmaceutical composition of the invention, e.g.,
encapsulation in liposomes, microparticles, microcapsules,
recombinant cells capable of expressing the mutant viruses,
receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol.
Chem. 262:4429-4432). Methods of introduction include but are not
limited to intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, and oral routes.
The compounds may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. In addition, in a preferred embodiment in a preferred
embodiment it may be desirable to introduce the pharmaceutical
compositions of the invention into the lungs by any suitable route.
Pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing
agent.
[0157] In a specific embodiment, it may be desirable to administer
the pharmaceutical compositions of the invention locally to the
area in need of treatment; this may be achieved by, for example,
and not by way of limitation, local infusion during surgery,
topical application, e.g., in conjunction with a wound dressing
after surgery, by injection, by means of a catheter, by means of a
suppository, or by means of ari implant, said implant being of a
porous, non-porous, or gelatinous material, including membranes,
such as sialastic membranes, or fibers. In one embodiment,
administration can be by direct injection at the site (or former
site) of a malignant tumor or neoplastic or pre-neoplastic
tissue.
[0158] In another embodiment, the pharmaceutical composition can be
delivered in a vesicle, in particular a liposome (see Langer, 1990,
Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of
Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),
Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.
317-327; see generally ibid.)
[0159] In yet another embodiment, the pharmaceutical composition
can be delivered in a controlled release system. In one embodiment,
a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref.
Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; and
Saudek et al., 1989, N. Engl. J. Med. 321:574). In another
embodiment, polymeric materials can be used (see Medical
Applications of Controlled Release, Langer and Wise (eds.), CRC
Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,
Drug Product Design and Performance, Smolen and Ball (eds.), Wiley,
New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev.
Macromol. Chem. 23:61 (1983); see also Levy et al., 1985, Science
228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al.,
1989, J. Neurosurg. 71:105). In yet another embodiment, a
controlled release system can be placed in proximity of the
composition's target, i.e., the lung, thus requiring only a
fraction of the systemic dose (see, e.g., Goodson, in Medical
Applications of Controlled Release, supra, vol. 2, pp. 115-138
(1984)).
[0160] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[0161] The pharmaceutical compositions of the present invention
comprise a therapeutically effective amount of a mutant virus, and
a pharmaceutically acceptable carrier. In a specific embodiment,
the term "pharmaceutically acceptable" means approved by a
regulatory agency of the Federal or a state government or listed in
the U.S. Pharmacopeia or other generally recognized pharmacopeia
for use in animals, and more particularly in humans. The term
"carrier" refers to a diluent, adjuvant, excipient, or vehicle with
which the pharmaceutical composition is administered. Such
pharmaceutical carriers can be sterile liquids, such as water and
oils, including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. Water is a preferred carrier when the pharmaceutical
composition is administered intravenously.
[0162] Saline solutions and aqueous dextrose and glycerol solutions
can also be employed as liquid carriers, particularly for
injectable solutions. Suitable pharmaceutical excipients include
starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,
chalk, silica gel, sodium stearate, glycerol monostearate, talc,
sodium chloride, dried skim milk, glycerol, propylene, glycol,
water, ethanol and the like. The composition, if desired, can also
contain minor amounts of wetting or emulsifying agents, or pH
buffering agents. These compositions can take the form of
solutions, suspensions, emulsion, tablets, pills, capsules,
powders, sustained-release formulations and the like. The
composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. Oral formulation can
include standard carriers such as pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, etc. Examples of suitable
pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin. Such compositions will
contain a therapeutically effective amount of the Therapeutic,
preferably in purified form, together with a suitable amount of
carrier so as to provide the form for proper administration to the
patient. The formulation should suit the mode of
administration.
[0163] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0164] The pharmaceutical compositions of the invention can be
formulated as neutral or salt forms. Pharmaceutically acceptable
salts include those formed with free amino groups such as those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc., and those formed with free carboxyl groups such as
those derived from sodium, potassium, ammonium, calcium, ferric
hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,
histidine, procaine, etc.
[0165] The amount of the pharmaceutical composition of the
invention which will be effective in the treatment of a particular
disorder or condition will depend on the nature of the disorder or
condition, and can be determined by standard clinical techniques.
In addition, in vitro assays may optionally be employed to help
identify optimal dosage ranges. The precise dose to be employed in
the formulation will also depend on the route of administration,
and the seriousness of the disease or disorder, and should be
decided according to the judgment of the practitioner and each
patient's circumstances. However, suitable dosage ranges for
intravenous administration are generally about 20-500 micrograms of
active compound per kilogram body weight. Suitable dosage ranges
for intranasal administration are generally about 0.01 pg/kg body
weight to 1 mg/kg body weight. Effective doses may be extrapolated
from dose-response curves derived from in vitro or animal model
test systems.
[0166] Suppositories generally contain active ingredient in the
range of 0.5% to 10% by weight; oral formulations preferably
contain 10% to 95% active ingredient.
[0167] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration. In a preferred embodiment, the kit
contains a Therapeutic of the invention, e.g., a lats protein, or
therapeutically effective lats derivative or analog, or nucleic
acid encoding the same, and one or more chemotherapeutic
agents.
6. EXAMPLE: HELPER VIRUS FREE RESCUE OF INFLUENZA A/WSN/33
[0168] The present invention demonstrates the ability to generate
recombinant negative-strand RNA viruses in the absence of helper
virus using expression vectors.
Materials & Methods
Preparation of Plasmids Encoding the
[0169] vRNA Segments of an Influenza A Virus
[0170] Eight plasmids (pPOL1-PB2-RT, pPOL1-PB1-RT, pPOL1-PA-RT,
pPOL1-HA-RT, pPOL1-NP-RT, pPOL1-NA-RT, pPOL1-M-RT and pPOL1-NS-RT)
each expressing a different vRNA segment of influenza A/WSN/33 were
constructed. These plasmids are based on the pUC19 or pUC18
plasmids and have a structure analogous to the model vRNA segment
encoding plasmid, pPOL1-CAT-RT, described in Pleschka et al., 1996,
J. Virol. 70:4183-4192, except that the cDNA encoding the vRNA CAT
reporter gene segment (an open reading frame for chloramphenicol
acetyltransferase in negative polarity flanked by the non-coding
regions of the NS-encoding vRNA segment of influenza A/WSN/33) has
been substituted by a cDNA encoding a native vRNA segment of
influenza A/WSN/33. Each of the plasmids comprise a truncated human
RNA Pol I promoter (positions -250 to -1) fused to the end of the
vRNA segment encoding cDNA to ensure that the correct 5' end of the
transcribed vRNA. Further, each of the vRNA segment encoding
plasmids comprise the sequence of the hepatitis delta virus genomic
ribozyme to that ensure the correct 3' end of the transcribed
vRNA.
[0171] Samples of influenza A/WSN/33 for preparation of the cDNA
inserts of the above-described plasmids are obtainable, for
example, from the W.H.O. Collaborating Centre, Division of
Virology, National Institute for Medical research, London,
U.K.)
Preparation of Plasmids for Expression of the PB1, PB2, PA and NP
Proteins of Influenza A/WSN/33
[0172] Four expression plasmids, pGT-h-PB1, pGT-h-PB2, pGT-h-PA and
p-GT-h-NP, encoding the influenza PB1, PB2, PA and NP proteins,
respectively, under the control of the adenovirus 2 major late
promoter linked to a synthetic sequence comprising the spliced
tripartite leader sequence of human adenovirus type 2 were
constructed. This promoter has been reported to give high-level
expression of proteins in cells adapted to serum-free suspension
culture (Berg et al., 1993, BioTechniques 14:972-978). The pGT-h
set of protein expression plasmids was constructed by inserting the
open reading frames for the PB1, PB2, PA and NP proteins into the
Bcl I cloning site of the pGT-h plasmid (Berg et al., 1993,
ibid).
Viral Rescue
[0173] Five .mu.g of each of the polymerase protein expression
plasmids, 10 .mu.g of the NP-expressing plasmid and 3 .mu.g of each
of the 8 vRNA-encoding plasmids were diluted to a concentration of
0.1 .mu.g/.mu.l in 20 mM Hepes buffer (pH 7.5). The DNA solution
was added to diluted DOTAP liposomal transfection reagent
(Boehringer Mannheim) containing 240 .mu.l of DOTAP and 720 .mu.l
of 20 mM Hepes buffer (pH 7.5). The transfection mixture was
incubated at room temperature for 15 minutes and then mixed with
6.5 ml of Minimal Essential Medium (MEM) containing 0.5% fetal calf
serum (FCS), 0.3% bovine serum albumin (BSA), penicillin and
streptomycin. This mixture was added to near-confluent Vero cells
in 8.5 cm diameter dishes (about 10' cells covering about 90% of
the dish) washed with PBS. After 24 hours, the transfection medium
was removed from the cells and replaced with 8 ml of fresh medium
(MEM) containing 0.5% FCS, 0.3% BSA, penicillin and streptomycin.
The transfected Vero cells were cultured for at least 4 days after
transfection. Every day, the medium from the transfected cells was
collected and assayed for the presence of influenza virus by
plaquing a 0.5 ml aliquot on MDBK cells in conventional manner. The
rest of the medium was transferred into 75 cm.sup.2 flasks of
subconfluent MDBK cells for amplification of any rescued virus. The
original transfected cells were further incubated after adding 8 ml
of fresh medium.
Introduction of Genetic Tags into 2 vRNA Segments
[0174] A cDNA was constructed encoding an HA vRNA segment with a
mutation of 6 nucleotides near the 3' end of the segment.
Nucleotides 31 to 35 from the 3' end (3'-UUUUG-5') were replaced
with 3'-AAAAC-5' resulting in amino acid substitution at amino acid
4(K.fwdarw.F) and at amino acid 5 (L.fwdarw.V) near the N-terminus
of HA within the signal peptide. In addition, a silent C.fwdarw.U
mutation was created at nucleotide 40. These changes introduced
several new restriction sites, including a unique SpeI site. The
cDNA encoding the NA segment was mutated to encode an NA segment
containing two silent mutations at nucleotides 1358 and 1360 so as
to introduce a new unique SacI restriction site (Pleschka et al.,
1996, J. Virol. 70:4188-4192). These cDNA were incorporated into
the pPOL1 expression vectors described above.
[0175] Rescued transfectant virus was generated in Vero cells using
the two expression plasmids encoding 2 genetic tags in place of the
pPOL1-HA-RT and pPOL1-NA-RT plasmids described above. The rescued
virus was amplified in MDCK cells as described above. Medium from
MDBK cells infected with the rescued transfectant virus was used to
isolate vRNA. One .mu.l of the medium was treated with 5 .mu.l of
RNase-free DNase to remove any residual plasmid DNA carried over.
After 15 minutes at 37.degree. C., vRNA was isolated using the
RNeasy Mini Kit (Qiagen). Short regions of the HA and NA vRNAs
expected to contain the genetic tags were amplified by RT-PCR and
then analysed by digestion with SpeI and SacI restriction enzymes,
respectively. As a control, the same regions of the HA and NA
segments were amplified from vRNA isolated from authentic influenza
A/WSN/33 virus using the same RT-PCR primers.
Results
[0176] The pGT-h-PB1, pGT-h-PB2, pGT-h-PA and p-GT-h-NP expression
plasmids encoding the viral nucleoprotein and 3 protein subunits of
the viral RNA-dependent RNA polymerase were cotransfected into
human 293 cells or Vero cells with the expression plasmid
pPOL1-CAT-RT. In both the transfected human 293 cells and Vero
cells, CAT activity could be detected (data not shown). Vero cells
were chosen for helper virus free generation of influenza A/WSN/33
from transfected vRNA segments since they support better growth of
influenza A/WSN/33 than human 293 cells (about one log difference
in maximum viral titre).
[0177] At early stages post-transfection positive-sense mRNA from
the 4 protein expression plasmids coexists with naked
negative-sense genomic vRNA transcribed from the transcription
plasmids. Thus, double-stranded RNA may form. Formation of such
double-stranded RNA in human cells could possibly lead to the
induction of interferon-mediated antiviral responses and
consequently to suppression of the growth of any rescued virus.
However, such interferon-induction is obviated as a problem in Vero
cells since such cells are deficient in interferon expression.
[0178] To rescue transfectant influenza A/WSN/33 virus, 4
expression plasmids encoding the viral nucleoprotein and 3 protein
subunits of the viral RNA-dependent RNA polymerase (pGT-h-PB1,
pGT-h-PB2, pGT-h-PA and p-GT-h-NP) and 8 plasmids (pPOL1-PB2-RT,
pPOL1-PB1-RT, pPOL1-PA-RT, pPOL1-HA-RT, pPOL1-NP-RT, pPOL1-NA-RT,
pPOL1-M-RT and pPOL1-NS-RT) each expressing a different vRNA
segment of influenza A/WSN/33 were cotransfected into Vero cells.
The culture supernatant from the Vero cells was assessed for
rescued transfectant virus by plaque assays using MDCK cells. Four
days post-transfection infectious influenza virus was recovered.
Approximately 10-20 plaque-forming viral particles were obtained
from a 8.5 cm dish containing approximately 10.sup.7 cells. The
rescued virus showed a specific property characteristic of
influenza A/WSN/33 virus, i.e., it formed plaques on MDBK cells in
the absence of trypsin. The plaques formed by the rescued virus
were comparable in size to those formed by a control authentic
A/WSN/33 virus sample grown on the same MDBK cells.
[0179] To confirm that the viral plaques observed on the MDBK cells
treated with virus harvested from the culture medium of transfected
cells were derived from the cloned cDNAs, genetic tags were
introduced into two of the 8 vRNA segment cDNAs. Isolated vRNA from
rescued transfectant virus was used to amplify the genetic tags by
RT-PCR. The PCR products obtained from the rescued virus and the
control virus were the same size. Those PCR products originating
from the HA and NA segments of the rescued virus could be digested
with SpeI and SacI, respectively. However, the PCR products
corresponding to the control virus were, as expected, not digested
by the same enzymes. The omission of reverse transcriptase in
control RT-PCR reactions resulted in no visible PCR products.
[0180] The results described herein demonstrate that an influenza A
virus can be rescued by cotransfecting 8 transcription plasmids for
the individual vRNA segments and 4 expression plasmids encoding the
required NP, PB1, PB2 and PA proteins into Vero cells in the
absence of any helper virus.
[0181] It is noted that unlike some of the earlier studies which
emphasized the importance of using positive strand RNA for rescuing
negative strand RNA viruses, including Bunyamwera virus whose
genome is in 3 segments (Schnell et al., 1994, EMBO J.
13:4195-4203; Roberts and Rose, 1998, Virology 247:1-6; and Bridgen
and Elliot, 1996, 93:15400-15404), individual negative sense vRNA
segments were used herein to generate the recombinant influenza
virus.
7. EXAMPLE: HELPER VIRUS FREE RESCUE OF A/PR/8/34 INFLUENZA
(CAMBRIDGE VARIANT)
[0182] In order to rescue A/PR/8/34 entirely from recombinant DNA,
12 plasmids were generated. The 12 plasmids are analogous to those
described for the rescue of A/WSN/33 virus (see Example 6 above),
with a few modifications. The 8 plasmids required for the synthesis
of the 8 vRNA segments, by cellular RNA Polymerase I, have a marine
rDNA terminator sequence (GenBank Accession Number M12074) instead
of the hepatitis delta virus ribozyme to generate the exact 3' end
of the vRNA segments. The 4 protein expression plasmids for the
A/PR/8/34 polymerase subunits (PB1, PB1, PA) and the nucleoprotein
(NP) are based on the commercially available pcDNA3 (Invitrogen,
Catalogue No. V790-20), which has a cytomegalovirus (CMV) promoter
and a bovine growth hormone (BGH) poly(A) site.
Construction of the plasmid pPolISapIT
[0183] In order to allow easy cloning of the 8 vRNA segments, a new
basic cloning vector, pPolISapIT, was constructed. In this new
construct, the marine rDNA terminator sequence (positions +572 to
+715) is positioned downstream of the Pol I promoter. The Pol I
promoter and terminator sequences are separated by a 24 bp linker
sequence (5'-AGAAGAGCCAGATCTGGCTCTTCC-3'), containing SapI
restriction sites.
[0184] Plasmid pPolISapIT was derived from pPolI-CAT-RT (originally
described in Pleschka et al., J. Virol. 70, 4188-4192, 1996). A DNA
fragment containing a region of the marine rDNA terminator sequence
(positions +335 to +715, GenBank accession number M12074) was
inserted into the SalI site of pPolI-CAT-RT to generate
pPolI-CAT-T. Subsequently, by using an inverse PCR technique, the
CAT gene, the ribozyme and part of the marine rDNA terminator
sequence (positions +335 to +571) were deleted from pPolI-CAT-T. At
the same time, the 24 bp linker sequence as given above was
introduced through the PCR primers between the Pol I promoter and
the marine rDNA terminator sequence.
Construction of the vRNA Expression Vectors
[0185] cDNA was generated by RT-PCR from vRNA isolated from
influenza A/PR/8/34 virus (Cambridge variant) using PCR primers
with SapI overhangs. After SapI digestion, the PCR products were
cloned into pPolISapIT digested with SapI.
Viral Rescue
[0186] Cotransfection of the 12 plasmids into Vero cells using
DOTAP transfection reagent was performed as described in Example 6
(see also Fodor et al., 1999, J. Virol. 73:9679-9682). Plaque
assays and viral amplification were performed on MDCK cells in the
presence of 0.5 .mu.g/ml trypsin.
Results
[0187] Cotransfection of the 12 plasmids described supra resulted
in the rescue of infectious influenza A/PR/8/34 particles 4 days
post transfection. These results demonstrate that influenza
A/PR8/34 virus can be successfully rescued by the helper virus-free
method of the invention. This is of particular interest since
influenza A/PR8/34 is known to be avirulent to humans (Beare et
al., 1975, Lancet (ii):729-732), whereas influenza A/WSN/33 is
considered unsuitable for administration to humans because of its
known neurotropism in mice. It is thus proposed that influenza
A/PR8/34, in a suitably attenuated form, would be suitable as a
parent virus for live vaccine development. For example, helper
virus-free viral rescue in accordance with the invention could be
used to generate an attenuated reassortant virus starting with
expression vectors for the vRNAs of influenza A/PR8/34 apart from
substitution of the HA and NA genomic segments of A/PR8/34 virus
with the HA and NA genomic segments of an influenza strain
associated with an influenza infection epidemic.
8. EXAMPLE: IMPROVED PROTOCOLS FOR THE HELPER VIRUS FREE RESCUE OF
INFLUENZA A/WSN/33
[0188] 293T cells were cotransfected with the four protein
expression plasmids described in Example 7 and the eight vRNA
expression plasmids described in Example 7 (see also Fodor et al.,
1999, J. Virol. 73:9679-9682) using in the 3 protocols set out
below. These protocols resulted in the production of between
100-10,000 plaque-forming viral particles from 10.sup.6 cells were
obtained on day 2 posttransfection. This is at least 100 times more
influenza virus than obtained by the transfection studies reported
in Example 6.
Protocol (a): Transfection of 293T Cells Using "Lipofectamine 2000"
Transfection Reagent
[0189] One .mu.g of each of the 12 plasmids were combined and the
volume adjusted to 50 .mu.l by adding OPTIMEM medium (Gibco BRL).
In a polystyrene tube, 12 .mu.l of LipofectAMINE 2000 (Gibco BRL,
Cat. No. 11 668-027) and 238 .mu.l of OPTIMEM medium were combined
and the mixture incubated for 5 minutes at room temperature. The
DNA mixture was then added drop-wise into the diluted LipofectAMINE
2000 transfection reagent. After incubating the DNA-Lipofectamine
mixture at room temperature for about 20 minutes, the mixture was
added drop-wise into a 293T cell suspension (about 10.sup.6 cells
in 1 ml of DMEM containing 10% FCS without antibiotics). At about
16-24 hours post transfection, the transfection mixture was removed
and replaced with 1 ml of DMEM containing 0.5% FCS, 0.3% BSA,
penicillin and streptomycin. Twenty-four to forty-eight hours
later, rescued virus was screened for by plaquing 100 .mu.l of the
medium from the transfected 293T cells on MDBK cells and by
passaging the rest of the medium on a 25 cm.sup.2 semiconfluent
MDBK flask. One ml of DMEM containing 0.5% FCS, 0.3% BSA penicillin
and streptomycin was added to the transfected 293T cells and
incubation continued for another 2 to 3 days before repeating the
plaquing and amplification on MDBK cells.
Protocol (b): Transfection of 293T Cells Using Calcium Phosphate
Precipitation
[0190] For transfection using calcium phosphate precipitation, 1
.mu.g of each of the 12 plasmids was combined and the plasmid
mixture added to 250 .mu.l 2.times.HEBS buffer (40 mM Hepes, 280 mM
NaCl, 10 mM KCl, 2 mM Na.sub.2HPO.sub.4, 10 mM glucose, pH 7.05).
Then 250 .mu.l of 250 mM CaCl.sub.2 was added and the contents of
the tube mixed vigorously. After 20-30 mins at room temperature,
the precipitate was mixed with 1 ml of DMEM containing 10% FCS,
penicillin and streptomycin and added to a 293T cell suspension
(about 10.sup.6 cells in 1 ml of DMEM containing 10% FCS without
antibiotics). At about 16-24 hours post transfection, the
transfection mixture was removed and replaced with 1 ml of DMEM
containing 0.5% FCS, 0.3% BSA, penicillin and streptomycin. 24-48
hours later, rescued virus was screened for as in protocol (a)
above.
Protocol (c): Transfection of Vero Cells Using DOTAP Transfection
Reagent
[0191] One .mu.g of each of the 12 plasmids was combined and the
volume adjusted to 120 .mu.l by adding 20 mM hepes (pH 7.5) to give
a DNA concentration of about 0.1 .mu.g/.mu.l. The DNA solution was
then added to diluted DOTAP transfection reagent (Boehringer)
containing 60 .mu.l of DOTAP and 200 .mu.l of 20 mM Hepes (pH 7.5)
in a polystyrene tube. After incubation of the DNA-DOTAP mixture at
room temperature for about 15-20 minutes, the mixture was added
drop-wise into a Vero cell suspension (about 10.sup.6 cells in 1 ml
of MEM containing 10% FCS, penicillin and streptomycin). At about
16-24 hours post transfection, the transfection mixture was removed
and replaced with 1 ml of MEM containing 0.5% FCS, 0.3% BSA,
penicillin, and streptomycin. Twenty-four to forty-eight hours
later, rescued virus was screened for by plaquing 100 .mu.l of the
medium from the transfected Vero cells on MDBK cells and by
passaging the rest of the medium on a 25 cm.sup.2 semiconfluent
MDBK flask. One ml of MEM containing 0.5% FCS, 0.3% BSA,
penicillin, and streptomycin was added to the transfected Vero
cells and incubation continued for another 2 to 3 days before
repeating the plaquing and amplification on MDBK cells.
9. EXAMPLE: HELPER VIRUS FREE RESCUE OF REASSORTANT INFLUENZA
VIRUSES
[0192] Plasmid-based rescue in accordance with the invention has
been successfully used to generate reassortant influenza viruses.
The following reassortant viruses were generated:
(i) A/WSN/33 with the PA segment derived from A/PR/8/34 (ii)
A/WSN/33 with the NP segment derived from A/PR/8/34 (iii) A/WSN/33
with the M segment derived from A/PR/8/34 (iv) A/WSN/33 with the
PB2 segment derived from A/FPV/Dobson/34
[0193] These examples demonstrate the utility of the helper virus
free method for isolating reassortants. Reassortant viruses based
on A/PR8/34 (or other suitable strains) are required for the
production of conventional killed vaccines because they grow to
high titre in embryonated chicken eggs--used in the commercial
production of killed influenza vaccines. As previously indicated
above, an important application of helper virus free viral rescue
in accordance with the invention is thus seen to be easier and more
direct isolation of reassortant viruses than by the classic method
of isolating reassortants from a mixed infection of cells with two
live viruses. Importantly, using a method of the invention, the
need to screen many potential reassortants before the required one
is isolated is obviated.
10. EXAMPLE: HELPER VIRUS FREE RESCUE OF INFLUENZA A/WSN/330N
ECR-293 CELLS
[0194] Rescue of influenza A/WSN/33 has been achieved on EcR-293NP
cells, a cell line stably expressing influenza NP, by transfecting
II plasmids expressing genomic vRNA segments and RNA-dependent RNA
polymerase subunits PB1, PB2 and PA.
[0195] EcR-293NP cells were derived from the commercially available
cell line EcR-293 (Invitrogen, Catalogue No. R650-07) which
constitutively expresses the VgEcR and RXR subunits of the ecdysone
receptor. Influenza NP expression in such cells is inducible in
response to ponasterone A. The same protocol specified in Example
8(a) employing LipofectAMINE 2000 transfection reagent was used
except that pcDNA-NP was omitted, since the NP protein for the
initial encapsidation of the vRNA segments was provided by the
EcR-293NP cells.
11. EXAMPLE: HELPER VIRUS FREE RESCUE OF A RECOMBINANT INFLUENZA
VIRUS EXPRESSING A FOREIGN ANTIGEN
[0196] The plasmid pPOL1-E6N18-2A-NA which is capable of expressing
a chimeric vRNA segment based on the NA vRNA segment of influenza
A/WSN/33 virus was constructed. The modified vRNA coding sequence
was inserted between sequences corresponding to a truncated human
Pol I promoter and hepatitis delta virus ribozyme as for
preparation of the Pol I-expression plasmids described in Example
6. The resultant chimeric gene contained a long open reading frame
(ORF) encoding the first 88 amino acids of the E6 protein of human
papillomavirus 18 (HPV 18), followed by 17 amino acids
corresponding to the self-cleavage motif of the 2A protease of
foot-and-mouth-disease virus (FMDV), followed by the amino acid
sequence of the NA of influenza A/WSN/33. The coding region was
flanked by the non-coding regions of the NA gene of A/WSN/33 virus.
In this way, a chimeric influenza virus gene was generated encoding
a polyprotein that undergoes self-cleavage, resulting in the
generation of an HPV-derived polypeptide and the NA protein. A
similar strategy for the expression of foreign antigens by
influenza virus vectors generated by classical RNP-transfection has
previously been described (T. Muster and A. Garcia-Sastre, Genetic
manipulation of influenza viruses, in Textbook of Influenza, K. G.
Nicholson, R. G. Webster & A. J. Hay, eds., pp. 93-106 (1998),
Blackwell Science Ltd, Oxford, UK.)
[0197] The recombinant influenza virus vector expressing the
HPV18-derived antigen was generated by cotransfecting into 293T
cells pPOL1-E6N18-2A-NA together with 7 Poll-expression vectors
encoding wild-type viral RNAs, i.e., PB2, PB1, PA, HA, NP, M and NS
as described in Example 6 and the 4 PolI-expression vectors
encoding the PB2, PB1, PA and NP proteins as described in Example
7. The rescued virus had the correct nucleotide sequence as
confirmed by sequence analysis of its NA-specific viral RNA.
12. EXAMPLE: EXPRESSION AND PACKAGING OF A FOREIGN GENE BY
RECOMBINANT NDV
[0198] The expression of the chloramphenicol transferase gene (CAT)
using the NDV minigenome is described. The NDV minigenome was
prepared using pNDVCAT, a recombinant plasmid containing the CAT
gene. The pNDVCAT plasmid is a pUC19 plasmid containing in
sequence: the T7-promoter; the 5'-end of the NDV genomic RNA
comprising 191 nucleotides of noncoding NDV RNA sequence; 5
inserted nucleotides (3'CTTAA); the complete coding sequence of the
chloramphenicol transferase (CAT) gene in the reversed and
complemented order; the 3'-end of the NDV genomic RNA sequence
comprising 121 nucleotides of noncoding NDV RNA sequence; a BbsI
cloning site and several restriction sites allowing run-off
transcription of the template. The pNDVCAT can be transcribed using
T7 polymerase to create an RNA with Newcastle disease viral-sense
flanking sequences around a CAT gene in reversed orientation.
[0199] The length of a paramyxovirus RNA can be a major factor that
determines the level of RNA replication, with genome replication
being most efficient when the total number of nucleotides is a
multiple of six. For NDV, the question of whether this rule of six
is critical for replication was examined by generating CAT
mini-replicons of varying lengths, differing by one to five
nucleotides. Only one construct whose genome was divisible by six
was able to induce high CAT activity.
Construction of the Newcastle Disease Virus Minigenome
[0200] In order to construct an NDV minigenome, as described supra,
the following strategy was used. The 5' terminal sequence of
genomic NDV RNA was obtained by RACE (Gibco, BRL) using standard
techniques in the art. The template for the RACE reaction was
genomic RNA which was purified from NDV virions (strain:
California-11914-1944). As illustrated in FIG. 4, this terminal
sequence comprised 64 nucleotides of a trailer sequence plus 127
nucleotides of the untranslated region of the L gene. Located
adjacent to the 191 viral nucleotide sequence, a 5 nucleotide
sequence (3'CCTTAA) was inserted. A CAT gene comprised 667
nucleotides of the CAT open reading frame which was placed between
the viral 5' and 3'terminal non-coding regions. In order to obtain
the 3' terminal region of the NDV sequence, RT-PCR was used. The
template for the RT-PCR reaction was in vitro polyadenylated
genomic RNA of NDV. As illustrated in FIG. 3, the 3' terminal
region of 121 nucleotides was comprised of 56 nucleotides of the
untranslated region of the NP gene plus 65 nucleotides of a leader
sequence. The resulting construct of the NDV minigenome is shown in
FIG. 2. Nucleotide sequences of 3' and 5' non-coding terminal
region shown in FIG. 4.
Construction of the NDV NP, P & L Expression Plasmids
[0201] As described in Section 5, the transcription or replication
of a negative strand RNA genome requires several protein components
to be brought in with the virus, including the L protein, P protein
and NP protein. In order to facilitate the expression from the NDV
minigenome, the genes encoding each of the L, P and NP proteins
were cloned into pTM1 expression vectors as illustrated in FIG.
3A-C. The pTM1 expression vectors comprises a T7 promoter, several
cloning sites for insertion of the gene of interest (L, P or NP), a
T7 terminator, a pUC19 origin of replication and an ampicillin
resistance gene. In order to construct the expression plasmids,
full length DNA of NDV nucleoprotein (NP), phosphoprotein (P) and
polymerase (L) were obtained by PCR amplification. These DNAs were
cloned into T7 polymerase expression vector pTM1, respectively
(FIG. 3A-C).
RNA Transcription of the NDV Minigenome
[0202] RNA transcription from the NDV minigene plasmid was
performed with the Ribomax kit (Promega) as specified by the
manuscripts. In order to allow run-off transcription, 1 .mu.g of
NDV minigenome plasmid (pNDVCAT) was digested with Bbs I. The
linearized plasmid was then used as a template of transcription
reaction (for 2 hours at 37.degree. C.). In order to remove
template DNA, the resulting reaction mixture was treated with
RNase-free DNase (for 15 min. at 37.degree. C.) and purified by
phenol-chloroform extraction, followed by ethanol
precipitation.
Cell Transfections
[0203] Cos-1 cells, or 293T cells were grown on 35 mm dishes and
infected with the helper virus rVV T7 at a multiplicity of
infection (moi) of approximately 1 for 1 hour before transfection.
The cells were then transfected with the expression vectors
encoding the NP, P and L proteins of NDV. Specifically,
transfections were performed with DOTAP (Boehringer Mannheim).
Following helper virus infection, cells were transfected with the
pTM1-NP (1 .mu.g), pTM1-P (1 .mu.g) and pTM1-L (0.1 .mu.g) for 4
hours. Control transfections, lacking the L protein, were performed
on a parallel set of cells with pTM1-NP (1 .mu.g), pTM1-P (1 .mu.g)
and mock pTM1-L (0 .mu.g). After the 4 hour incubation period,
cells were subjected to RNA transfection with 0.5 .mu.g of the
NDV-CAT chimeric (-) RNA (see FIG. 1). Following RNA transfection,
cells were allowed to incubate for 18 hours. The cell lysates were
subsequently harvested for the CAT assay.
CAT Assays
[0204] CAT assays were done according to standard procedures,
adapted from Gorman et al., 1982, Mol. Cell. Biol. 2: 1044-1051.
The assays contained 10 .mu.l of .sup.14C chloramphenicol (0.5
.mu.Ci; 8.3 nM; NEN), 20 .mu.l of 40 mM acetyl CoA (Boehringer) and
50 .mu.l of cell extracts in 0.25 M Tris buffer (pH 7.5).
Incubation times were 16-18 hours.
Results
[0205] In each cell line transfected with the NP, P, L expression
vectors, and the chimeric NDV-CAT RNA, high levels of expression of
CAT was obtained 18 hours post-infection. In addition, control
transfected cells lacking the L protein did not express CAT.
Rescue of Infectious NDV Viruses Using RNA Derived From Specific
Recombinant DNA
[0206] The experiments described in the subsections below
demonstrate the rescue of infectious NDV using RNA which is derived
from specific recombinant DNAs. RNAs corresponding to the chimeric
NDV-CAT RNA may be used to show that the 191 nucleotides of the 5'
terminal and the 121 nucleotides of the 3' terminal nucleotides of
the viral RNAs contain all the signals necessary for transcription,
replication and packaging of model NDV RNAs. RNAs containing all
the transcriptional units of the NDV genomes can be expressed from
transfected plasmids. Thus, this technology allows the engineering
of infectious NDV viruses using cDNA clones and site-specific
mutagenesis of their genomes. Furthermore, this technology may
allow for the construction of infectious chimeric NDV viruses which
can be used as efficient vectors for gene expression in tissue
culture, animals or man.
13. EXAMPLE: RECOMBINANT NEWCASTLE DISEASE VIRUS CONTAINING AN HIV
ANTIGEN gp160EPITOPE INSERTED INTO THE NDV GENOME
[0207] In the Example presented herein, a chimeric NDV is
constructed to express a heterologous antigen derived from gp160 of
HIV. The experiments described in the subsections below demonstrate
the use of a recombinant RNA template to generate a chimeric NDV
that expresses a HIV gp160-derived peptide within the NDV genome
and, further, this chimeric NDV is used to elicit a vertebrate
humoral and cell-mediated immune response.
Construction of Plasmid
[0208] Recombinant NDV cDNA clones expressing HIV gp160 proteins
may be constructed in a number of ways known in the art. For
example, as illustrated in FIG. 4, the HIV Env and Gag proteins may
be inserted into the NDV in a number of locations. In one example,
the Env and Gag proteins are inserted between the M and L genes. In
a different example, the Env and Gag proteins are inserted 3' to
the NP gene (between the leader sequence and NP). Alternatively,
these HIV proteins will be incorporated between the NDV envelope
proteins (HN and F) at the 3' end. These proteins may also be
inserted into or between any of the NDV genes.
Generation of Infectious Chimeric Virus
[0209] Transfection of RNA derived from plasmid comprising a
recombinant NDV genome may be transfected into cells such as, for
example, COS, 293 MDBK and selection of infectious chimeric virus
may be done as previously described. See U.S. Pat. No. 5,166,057,
incorporated herein by reference in its entirety. The resulting RNA
may be transfected into cells infected with wild type virus by
using standard transfection protocol procedures. Posttransfection,
the supernatant may be collected and used at different dilutions to
infect fresh cells in the presence of NDV antiserum. The
supernatant may also be used for plaque assays in the presence of
the same antiserum. The rescued virus can then be purified and
characterized, and used, for example, in antibody production.
Hemagglutination Inhibition and Virus Neutralization Assays
[0210] Hemagglutination inhibition (HI) assays are performed as
previously described (Palmer et al., 1975, Immunol. Ser. 6:51-52).
Monoclonal antibodies (2G9, 4B2, 2F1O, 25-5) are prepared by
standard procedures with a human anti-gp120 monoclonal antibody.
Ascites fluid containing monoclonal antibodies is treated with
receptor-destroying enzyme as previously described (Palmer et al.,
1975, Immunol. Ser. 6:51-52).
[0211] For virus neutralization assay, cells in 30-mm-diameter
dishes are infected virus. After a 1 h adsorption, agar overlay
containing antibody at different dilutions is added. The cell
monolayer is then stained with 0.1% crystal violet at 72 h
postinfection.
Immunization
[0212] 6 weeks old BALB/c mice are infected either via the aerosol
route with the virus, or are immunized intraperitoneally (i.p.)
with 10 .mu.g of purified virus. For all booster immunizations, 10
.mu.g of purified virus is administered i.p. Sera is collected 7
days after each immunization.
Radioimmunoassay
[0213] The radioimmunoassay is performed as previously described
(Zaghouani, H. et al., 1991, Proc. Natl. Acad. Sci. USA
88:5645-6549). Briefly, microtiter plates are coated with 5 ug/ml
peptide-BSA conjugate, saturated with 2% BSA in phosphate-buffered
saline(PBS) and incubated with various dilution of serum. Bound
antibodies are revealed by using .sup.125I labelled antimouse kappa
monoclonal antibody.
Radioimmunoprecipitation
[0214] The H9 human T cell line is acutely infected with HIV. Four
days postinfection, 5.times.10.sup.7 infected cells are labelled
with .sup.35S-cysteine, .sup.35S-methionine, and .sup.3H-isoleucine
at 2.times.10.sup.6/ml in media containing 100 .mu.Ci of each
isotope per ml. After 20 h of metabolic labelling, the radioactive
virions are pelleted by centrifugation for 1 h at 45,000 rpm. The
pellet is then resuspended in 1.0 ml of lysis buffer containing 1%
Triton X-100 and 2 mM phenylmethylsulfonyl fluoride (PMSF).
Approximately 20 .mu.l of sera or 0.5 .mu.g of monoclonal antibody
in 20 .mu.l PBS) and 175 .mu.l of virion lysate are incubated
overnight at 4.degree. C. in 0.5 ml immunoprecipitation buffer
containing 0.5% sodium dodecyl sulfate (SDS), 1 mg/ml BSA, 2%
Triton X-100, and 50 mM sodium phosphate (pH 7.4). The
antigen-antibody complexes are bound to protein A-Sepharose beads,
and are analyzed by electrophoresis on a 10% SDS-polyacrylamide
gel.
HIV-1 Neutralization Assays
[0215] The in vitro neutralization assay are performed as described
previously (Nara, P. L. et al., 1987, AIDS Res. Hum. Retroviruses
3:283-302). Briefly, serial twofold dilutions of heat-inactivated
serum are incubated for 1 h at room temperature with 150-200
syncytium forming units of HIV virus produced in H9 cells. The
virus/serum mixture is incubated for 1 h at 37.degree. C. with
50,000 DEAE-dextran treated CEMss cells (adhered to microplate
dishes using poly-L-lysine), or 50,000H9 suspension cells. After
virus adsorption, the unbound virus is removed and 200 .mu.l of
media is added to each well. Four days postinfection, 50 .mu.l of
supernatant media is removed for viral p24.sup.gag protein
quantitation (Coulter Source, Inc.). The total number of syncytia
in CEMss cells is counted five days postinfection. The
neutralization titers are calculated by comparison with control
wells of virus only, and are expressed as the reciprocal of the
highest serum dilution which reduced syncytia numbers by more than
50% or inhibited the p24 synthesis by more than 50%.
Induction of CTL Response
[0216] BALB/c mice is immunized with 0.2 ml viral suspension
containing 10.sup.7 PFU of chimeric NDV virus. 7 days later, spleen
cells are obtained and restimulated in vitro for 5 days with
irradiated spleen cells, alone or coated with immunogenic peptides,
in the presence of 10% concanavalin A in the supernatant as
previously described (Zaghouani, H. et al., 1992, J. Immunol.
148:3604-3609).
Cytolysis Assay
[0217] The target cells coated with peptides are labeled with
Na.sup.51Cr.sub.4 (100 .mu.Ci/10.sup.6 cells) for 1 h at 37.degree.
C. After being washed twice, the cells are transferred to V-bottom
96-well plates, the effector cells are added, and incubated at
37.degree. C. in 7% C0.sub.2. Four hours later, the supernatant is
harvested and counted. The maximum chromium release is determined
by incubating the cells with 1% Nonidet P40 detergent. The
percentage of specific lysis is calculated according to the
following formula: [(cpm samples-cpm spontaneous release)/(cpm
maximum release-cpm spontaneous release)].times.1OO.
[0218] The present invention is not to be limited in scope by the
specific embodiments described which are intended as single
illustrations of individual aspects of the invention, and any
constructs, viruses or enzymes which are functionally equivalent
are within the scope of this invention. Indeed, various
modifications of the invention in addition to those shown and
described herein will become apparent to those skilled in the art
from the foregoing description and accompanying drawings. Such
modifications are intended to fall within the scope of the appended
claims.
[0219] All references cited herein are incorporated herein by
reference in the entirety for all purposes.
Sequence CWU 1
1
51121RNANewcastle Disease Virus 1accaaacaga gaauccguaa gguacguuaa
aaagcgaagg agcaauugaa gucgcacggg 60uagaaggugu gaaucucgag ugcgagcccg
aagcacaaac ucgagaaagc cuucuaccaa 120c 1212196RNANewcastle Disease
Virus 2cuuaacgaca aucacauauu aauaggcucc uuuucuggcc aauuguaucc
uuguugauuu 60aaucauacua uguuagaaaa aaguugaacu ccgacuccuu aggacucgaa
cucgaacuca 120aauaaauguc uuagaaaaag auugcgcaca guuauucuug
aguguagucu ugucauucac 180caaaucuuug uuuggu 196364DNANewcastle
Disease Virus 3tggtttgtct cttaggcatt ccatgcaatt tttcgcttcc
tcgttaactt catgcccatc 60ttcc 64467DNAArtificialIllustrative
parainfluenza comparison sequence 4tggtttgtct cttaggcatt ccatgctatt
ttccgcttcc tcgttaactt cagcatgccc 60atcttcc
67524DNAArtificialSynthetic Linker Sequence 5agaagagcca gatctggctc
ttcc 24
* * * * *