U.S. patent application number 10/238786 was filed with the patent office on 2003-08-07 for infectious clones.
Invention is credited to Sanchez, Luis Enjuanes.
Application Number | 20030148325 10/238786 |
Document ID | / |
Family ID | 8310818 |
Filed Date | 2003-08-07 |
United States Patent
Application |
20030148325 |
Kind Code |
A1 |
Sanchez, Luis Enjuanes |
August 7, 2003 |
Infectious clones
Abstract
The present invention relates to methods of preparing a DNA
comprising steps, wherein (a) a DNA comprising a full length copy
of the genomic RNA (gRNA) or an RNA virus; or (b) a DNA comprising
one or several fragments of a gRNA of an RNA virus, which fragments
code for an RNA dependent RNA polymerase and at least one
structural or non-structural protein; or (c) a DNA having a
homology of at least 60% to the sequences of (a) or (b); is cloned
into a bacterial artificial chromosome (BAC). Additionally, DNAs
are provided, which comprise sequences derived from the genomic RNA
(gRNA) of a coronavirus which sequences have a homology of at least
60% to the natural sequence of the virus and code for an RNA
dependent RNA polymerase and at least one structural or
no-structural protein, wherein a fragment of said DNA is capable of
being transcribed into RNA which RNA can he assembled to a virion.
Further, the use of these nucleic acids for preparation of viral
RNA or virions as well as pharmaceutical preparations comprising
these DNAs, viral RNAs or virions is disclosed.
Inventors: |
Sanchez, Luis Enjuanes;
(Madrid, ES) |
Correspondence
Address: |
Sharon E. Crane, Ph.D.
BURNS, DOANE, SWECKER & MATHIS, L.L.P.
P.O. Box 1404
Alexandria
VA
22313-1404
US
|
Family ID: |
8310818 |
Appl. No.: |
10/238786 |
Filed: |
September 11, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10238786 |
Sep 11, 2002 |
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10148669 |
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10148669 |
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PCT/EP00/12063 |
Nov 30, 2000 |
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Current U.S.
Class: |
435/6.12 ;
435/6.13; 435/69.1; 435/91.1; 435/91.33 |
Current CPC
Class: |
C12N 2770/20034
20130101; C12N 2800/204 20130101; A61P 31/00 20180101; A61P 1/00
20180101; C12N 2770/20043 20130101; C12N 2770/20062 20130101; C12N
15/86 20130101; A61K 39/12 20130101; A61K 39/225 20130101; A61K
2039/53 20130101; C12N 2800/80 20130101; A61K 2039/525 20130101;
A61P 31/12 20180101; A61K 2039/5256 20130101; C12N 7/00 20130101;
C12N 15/70 20130101; C07K 14/005 20130101; A61P 43/00 20180101;
A61K 2039/541 20130101; A61P 11/00 20180101; A61P 29/00 20180101;
A61P 37/04 20180101 |
Class at
Publication: |
435/6 ; 435/91.1;
435/91.33; 435/69.1 |
International
Class: |
C12Q 001/68; C12P
021/06; C12P 019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 3, 1999 |
ES |
9902673 |
Claims
1. Method of preparing a DNA comprising steps, wherein (a) a DNA
comprising a full length copy of the genomic RNA (gRNA) of an RNA
virus; or (b) a DNA comprising one or several fragments of a gRNA
of an RNA virus, which fragments code for an RNA dependent RNA
polymerase and at least one structural or non-structural protein;
or (c) a DNA having a homology of at least 60% to the sequences of
(a) or (b); is cloned into a bacterial artificial chromosome
(BAC).
2. Method according to claim 1, wherein the DNA cloned into the BAC
further comprises sequences coding for several or all except one of
the structural or non-structural proteins of a virus.
3. Method according to claim 1 or 2, wherein the DNA cloned into
the BAC further comprises sequences encoding one or several
heterologous gene.
4. Method according to claim 3, wherein the heterologous gene
encodes at least one antigen suitable for inducing an immune
response against an infectious agent, at least one molecule
interfering with the replication of an infectious agent, an
antibody providing protection against an infectious agent, an
immune modulator, a cytokine, an immoenhancer or an
anti-inflammatory compound.
5. Method according to one of claims 1 to 4, wherein the DNA cloned
into the BAC has a size of at least 5 Kb.
6. Method according to one of claims 1 to 5, wherein the DNA cloned
into the BAC has a size of at least 15 Kb.
7. Method according to one of claims 1 to 6, wherein the DNA cloned
into the BAC has a size of at least 25 Kb.
8. Method according to one of claims 1 to 7, wherein the BAC
comprises a sequence controlling the transcription of the DNA
cloned into the BAC.
9. Method according to one of claims 1 to 8, wherein one of the
genes of the virus has been modified by substituting, deleting or
adding nucleotides.
10. Method according to claim 9, wherein the gene controlling
tropism of the virus has been modified.
11. Method according to one of claims 1 to 10, wherein the gene
controlling tropism of the virus has been substituted with the
respective gene of another virus.
12. Method according to one of claims 1 to 11, wherein the DNA
cloned into the BAC is capable of being transcribed into RNA which
RNA can be assembled to a virion.
13. Method according to of claim 12, wherein the virion is an
infectious, attenuated, replication defective or inactivated
virus.
14. Method according to one of claims 1 to 13, wherein the virus
naturally has a plus strand genome.
15. Method according to one of claims 1 to 14, wherein the virus is
a picornavirus, flavivirus, togavirus, coronavirus, torovirus,
arterivurs, calcivirus, rhabdovirus, paramixovirus, filovirus,
bornavirus, orthomyxovirus, bunyavirus, arenavirus or reovirus.
16. Method of preparing a viral RNA comprising steps, wherein a DNA
is prepared according to one of claims 1 to 15, the DNA is
expressed in a suitable host cell or the DNA is mixed with
chemicals, biological reagents and/or cell extracts under
conditions allowing the transcription of the DNA and the viral RNA
is isolated.
17. Method of preparing a virion comprising steps, wherein a DNA is
prepared according to one of claims 1 to 15, the DNA is expressed
in a suitable host cell or the DNA is mixed with chemicals,
biological reagents and/or cell extracts under conditions allowing
the transcription and translation of the DNA and the virion is
isolated.
18. Method according to claim 17, wherein the virion is
subsequently inactivated or killed.
19. Method for preparing a pharmaceutical composition comprising
steps, wherein a DNA is prepared according to one of claims 1 to
15, a viral RNA is prepared according to claim 16 or a virion is
prepared according to claim 17 or 18 and is subsequently mixed with
a pharmaceutically acceptable adjuvans or carrier.
20. Method according to claim 19, wherein the pharmaceutical is a
vaccine for protecting humans or animals against an infectious
disease.
21. Method according to claim 19, wherein the pharmaceutical is
used for gene therapy of humans or animals.
22. DNA comprising sequences derived from the genomic RNA (gRNA) of
a coronavirus which sequences have a homology of at least 60% to
the natural sequence of the virus and code for an RNA dependent RNA
polymerase and at least one structural or non-structural protein,
wherein a fragment of said DNA is capable of being transcribed into
RNA which RNA can be assembled to a virion.
23. DNA according to claim 22, further comprising a sequence
encoding a heterologous gene.
24. DNA according to claim 23, wherein the heterologous gene
encodes at least one antigen suitable for inducing an immune
response against an infectious agent, at least one molecule
interfering with the replication of an infectious agent, an
antibody providing protection against an infectious agent, an
immune modulator, a cytokine, an immonenhancer or an
anti-inflammatory compound.
25. DNA according to claim 22 or 24, wherein said fragment has a
size of at least 25 Kb.
26. DNA according to one of claims 22 to 25, which further
comprises sequences derived from a coronavirus coding for several
or all except one of the structural or non-structural proteins of a
virus.
27. DNA according to one of claims 22 to 26, which further
comprises sequences derived from a coronavirus coding for all of
the structural or non-structural proteins of a coronavirus.
28. DNA according to one of claims 22 to 27, further comprising a
sequence controlling the transcription of the viral gRNA.
29. DNA according to one of claims 22 to 28, wherein the sequence
controlling transcription of the viral gRNA is the immediately
early (IE) promoter of cytomegalovirus (CMV).
30. DNA according to one of claims 22 to 29, wherein the sequence
is flanked at the 3'-end by a poly(A)tail, the ribozyme of the
hepatitis 8 virus (HDV) and the termination and polyadenylation
sequences of bovine growth hormone (BGH).
31. DNA according to one of claims 22 to 30, wherein the viral
sequences are derived from an isolate of the porcine transmissible
gastroenteritis virus (TGEV), murine hepatitits virus (MHV),
infectious bronchitis virus (IBV), bovine coronavirus (BoCV),
canine coronavirus (CCoV), feline virus (FCoV), human coronavirus
(HCoV), toroviruses or arterivurses.
32. DNA according to one of claims 22 to 31, wherein the virion is
an infectious, non-infectious or replication deficient virus.
33. DNA according to one of claims 22 to 32, wherein the sequence
of the a structural or non-structural gene derived from the
coronavirus has been modified by substituting, deleting or adding
one or several nucleotides of the natural gene sequence.
34. DNA according to one of claims 22 to 33, wherein the sequence
of the S, N or M gene has been modified.
35. DNA according to one of claims 22 to 34, wherein the sequence
of the S gene derived from a coronavirus has been modified to
obtain an attenuated virion.
36. DNA according to one of claims 22 to 35, wherein the sequence
of the S gene derived from a coronavirus has been modified to
obtain a virion with a tropism differing from the tropism of the
coronavirus.
37. Vector comprising a nucleic acid according to one of claims 22
to 36.
38. Vector according to claim 37, wherein the vector is a plasmid
or bacterial artificial chromosome (BAC).
39. Host cell comprising a nucleic acid according to one of claims
22 to 38.
40. E. coli deposited under CECT 5265 at the Spanish Collection of
Type Cultures.
41. Method for producing a recombinant virion or a recombinant
viral RNA comprising steps, wherein a DNA according to one of
claims 22 to 38 is introduced into a host cell, host cells
containing the DNA are cultivated under conditions allowing the
expression thereof and the recombinant virion or viral RNA is
recovered.
42. Method for producing a recombinant virion or a recombinant
viral RNA, wherein a DNA according to one of claims 22 to 38 is
mixed with chemicals, biological reagents and/or cell extracts
under conditions allowing the transcription of the DNA and the
recombinant virion or viral RNA is recovered.
43. Method according to claim 41 or 42, wherein the DNA is a DNA
according to one of claims 23 to 38.
44. Virion obtainable by a method according to one of claims 41 to
43.
45. Virion according to claim 44, wherein the virion is an
infectious, attenuated, replication defective or inactivated
virus.
46. Virion according to claim 44 or 45, wherein the virion
comprises a modified S, M or N gene.
47. Virion according to claim 46, wherein modification of the S
gene gives raise to an attenuated virus.
48. Virion according to claim 46, wherein modification of the S
gene gives raise to a virion with altered tropism.
49. Viral RNA obtainable by a method according to claim 41 to
43.
50. Pharmaceutical preparation comprising a nucleic acid according
to one of claims 22 to 38, a host cell according to claim 39 or 40,
a virion according to one of claims 41 to 48 or a viral RNA
according to claim 49.
51. Vaccine capable of protecting an animal or a human against
deseases caused by an infectious agent comprising a nucleic acid
according to one of claims 22 to 38, a host cell according to claim
39 or 40, a virion according to one of claims 41 to 48 or a viral
RNA according to claim 49.
52. Vaccine according to claim 51, wherein the nucleic acid
comprises sequences encoding least one antigen suitable for
inducing an immune response against the infectious agent, at least
one gene interfering with the replication of the infectious agent
or an antibody providing protection against said infectious
agent.
53. Vaccine according to claim 51 or 52, wherein said virion vector
expresses at least one replication interfering molecule, an antigen
capable of inducing a systemic immune response and/or an immune
response in mucous membranes against different infectious agents
that propagate in respiratory or intestinal mucous membranes or in
other tissues.
54. Multivalent vaccine capable of protecting an animal or a human
against the infection caused by more than one infectious agent,
that comprises more than one nucleic acid according to one of
claims 22 to 38, a host cell according to claim 39 or 40, a virion
according to one of claims 41 to 48 or a viral RNA according to
claim 49, each of which expresses an antigen adequate for inducing
an immune response against each of said infectious agents, an
interfering molecule or an antibody providing protection against
each of said infectious agents.
55. Vaccine according to one of claims 51 to 54 further comprising
a pharmaceutically acceptable carrier or diluent.
56. Method of preparing a DNA according to one of claims 22 to 38
comprising steps, wherein an interfering defective genome derived
from a coronavirus is cloned under the expression of a promotor
into a BAC vector and the deleted sequences within the defective
genome are re-inserted.
57. Method of preparing a DNA according to claim 56, wherein toxic
sequences within the viral genome are identified before
re-insertion into the DNA.
58. Method of preparing a DNA according to claim 56 or 57, wherein
the toxic sequences within the viral genome are the last sequences
to be re-inserted when completing the genome.
59. An infective clone derived from a coronavirus that comprises a
full-length copy of complementary DNA (cDNA) to the genomic RNA
(gRNA) of a coronavirus, cloned under a transcription-regulatory
sequence.
60. Infective clone according to claim 59, in which said
coronavirus is an isolate of the porcine transmissible
gastroenteritis virus (TGEV).
61. Infective clone according to claim 59 or 60, in which said
promoter is the immediately early (IE) promoter of expression of
cytomegalovirus (CMV).
62. Infective clone according to one of claims 59 to 61, wherein
said full-length cDNA is flanked at the 3'-end by a poly(A) tail,
the ribozyme of the hepatitis delta virus (HDV), and the
termination and polyadenylation sequences of bovine growth hormone
(BGH).
63. Infective clone according to one of claim 59 to 62, wherein
said infective cDNA has been cloned in a bacterial artificial
chromosome (BAC).
64. A procedure for obtaining of an infective clone according to
any of claims 59 to 63, which comprises constructing the
full-length cDNA from the gRNA of a coronavirus and to assembly the
transcription-regulatory elements.
65. Procedure according to claim 64, in which the construction of
the full-length cDNA of the gRNA of a coronavirus comprises: (i)
cloning an interfering defective genome derived from said
coronavirus under a promoter of expression in a BAC; (ii)
completing the deletions of said interfering defective genome and
regenerating the deleted sequences with respect to the infective
gRNA; (iii) identifying the toxic sequences for the bacteria in
which it is going to be cloned, removing the toxic sequences, and
inserting said toxic sequences just before effecting the
transfection in eukaryotic cells to obtain the cDNA clone,
corresponding to the gRNA of the coronavirus.
66. A recombinant viral vector that comprises an infective clone
according to any of claims 59 to 63, or obtainable according to the
procedure of either of claims 58 or 59, modified to contain a
heterologous nucleic acid inserted into said infective clone under
conditions that allow said heterologous nucleic acid-to be
expressed.
67. Vector according to claim 60, in which said heterologous
nucleic acid is selected between a gene and a gene fragment that
codes a gene product of interest.
68. A method for producing a product of interest that comprises
cultivating a host cell that contains a viral vector according to
either of claims 60 or 61 under conditions that allow the
heterologous nucleic acid to be expressed and the product of
interest to be recovered.
69. A method for producing a modified recombinant coronavirus that
contains a heterologous nucleic acid in a sequence of cDNA
corresponding to the genome of a coronavirus, which comprises
introducing a viral vector according to either of claims 60 or 61
into a host cell, cultivating said host cell containing said viral
vector under conditions that allow the viral vector to be expressed
and replicated, and the virions obtained from the modified
recombinant coronavirus to be recovered.
70. A vaccine capable of protecting an animal against the infection
caused by an infectious agent that comprises (i) at least one viral
vector according to claim 60 or 61 that expresses at least one
antigen suitable for inducing an immune response against said
infectious agent, or an antibody that provides protection against
said infectious agent, along with, optionally, (ii) a
pharmaceutically acceptable excipient.
71. Vaccine according to claim 64, in which said viral vector
expresses at least one antigen capable of inducing a systemic
immune response and/or an immune response in mucous membranes
against different infectious agents that propagate in respiratory
or intestinal mucous membranes.
72. A multivalent vaccine capable of protecting an animal against
the infection caused by more than one infectious agent,
that,comprises (i) a viral vector according to claim 60 or 61, that
expresses an antigen adequate for inducing an immune response
against said infectious agents, or antibodies that provide
protection against said infectious agents, along with, optionally,
(ii) a pharmaceutically acceptable excipient.
73. A multivalent vaccine capable of protecting an animal against
the infection caused by more than one infectious agent, which
comprises (i) more than one viral vector according to claim 60 or
61, each one of which expresses an antigen adequate for inducing an
immune response against each one of said infectious agents, or
antibodies that provide protection against each one of said
infectious agents, along with, optionally, (ii) a pharmaceutically
acceptable excipient.
74. A method for producing a recombinant coronavirus that comprises
introducing an infective clone according to any of claims 59 to 63,
or obtainable according to the procedure of either of claims 64 or
65 into a host cell, cultivating said host cell that contains the
infective clone under conditions that allow the infective clone to
be expressed and replicated, and recovering virions obtained from
the recombinant coronavirus containing the complete genome of the
coronavirus.
Description
FIELD OF THE INVENTION
[0001] This invention relates to methods of preparing a DNA or an
RNA, nucleic acids obtainable by this method and their use as
vaccines and for gene therapy.
BACKGROUND OF THE INVENTION
[0002] Advances in recombinant DNA technology have led to progress
in the development of gene transfer between organisms. At this
time, numerous efforts are being made to produce chemical,
pharmaceutical, and biological products of economic and commercial
interest through the use of gene transfer techniques.
[0003] One of the key elements in genetic manipulation of both
prokaryotic and eukaryotic cells is the development of vectors and
vector-host systems. In general, a vector is a nucleic acid
molecule capable of replicating or expressing in a host cell. A
vector-host system can be defined as a host cell that bears a
vector and allows the genetic information it contains to be
replicated and expressed.
[0004] Vectors have been developed from viruses with both DNA and
RNA genomes. Viral vectors derived from DNA viruses that replicate
in the nucleus of the host cell have the drawback of being able to
integrate into the genome of said cell, so they are generally not
very safe. In contrast, viral vectors derived from RNA viruses,
which replicate in the cytoplasm of the host cell, are safer than
those based on DNA viruses, since the replication occurs through
RNA outside the nucleus. These vectors are thus very unlikely to
integrate into the host cell's genome.
[0005] cDNA clones have been obtained from single-chain RNA viruses
with positive-polarity [ssRNA(+)], for example, picornavirus
(Racaniello & Baltimore, 1981); bromovirus (Ahlquist et al.,
1984); alphavirus, a genus that includes the Sindbis virus; Semliki
Forest virus (SFV) and the Venezuelan equine encephalitis virus
(VEE) (Rice et al., 1987; Liljestrom and Garoff, 1991; Frolov et
al., 1996; Smerdou and Liljestrom, 1999); flavivirus and pestivirus
(Rice and Strauss, 1981; Lai et al., 1991; Rice et al., 1989); and
viruses of the Astroviridae family (Geigenmuller et al., 1997).
Likewise, vectors for the expression of heterologous genes have
been developed from clones of DNA complementary to the genome of
ssRNA(+) virus, for example alphavirus, including the Sindbis
virus, Semliki Forest virus (SFV), and the Venezuelan equine
encephalitis (VEE) virus (Frolov et al., 1996; Liljestrom, 1994;
Pushko et al., 1997). However, all methods of preparing recombinant
viruses starting from RNA viruses are still complicated by the fact
that most of the viruses comprise sequences which are toxic for
bacteria. Preparing a cDNA of the viral RNA and subcloning of the
cDNA in bacteria therefore often leads to deletion or rearangement
of the DNA sequences in the bacterial host. For this purpose most
of the commonly used subcloning and expression vectors cannot be
used for preparation of large DNA sections derived from recombinant
RNA viruses. However, obtaining vectors, which can carry long
foreign DNA sequences is required for a number of aspects in the
development of pharmaceuticals, specifically vaccines.
[0006] The coronaviruses are ssRNA(+) viruses that present the
largest known genome for an RNA virus, with a length comprised
between about 25 and 31 kilobases (kb) (Siddell, 1995; Lai &
Cavanagh, 1997; Enjuanes et al., 1998). During infection by
coronavirus, the genomic RNA (gRNA) replicates and a set of
subgenomic RNAs (sgRNA) of positive and negative polarity is
synthesized (Sethna et al., 1989; Sawicki and Sawicki, 1990; van
der Most & Spaan, 1995). The synthesis of the sgRNAs is an
RNA-dependent process that occurs in the cytoplasm of the infected
cell, although its precise mechanism is still not exactly
known.
[0007] The construction of cDNAs that code defective interfering
(DI) genomes (deletion mutants that require the presence of a
complementing virus for their replication and transcription) of
some coronaviruses, such as the murine hepatitis virus (MHV),
infectious bronchitis virus (IBV), bovine coronavirus (BCV) (Chang
et al., 1994), and porcine gastroenteritis virus (TGEV) (Spanish
Patent Application P9600620; Mndez et al., 1996; Izeta et al.,
1999; Snchez et al., 1999) has been described. However, the
construction of a cDNA clone that codes a complete genome of a
coronavirus has not been possible due to the large size of and the
toxic sequences within the coronavirus genome.
[0008] In summary, although a large number of viral vectors have
been developed to replicate and express heterologous nucleic acids
in host cells, the majority of the known vectors for expression of
heterologous genes are not well suited for subcloning of RNA
viruses. Further, the viral vectors so obtained present drawbacks
due to lack of species specificity and target organ or tissue
limitation and to their limited capacity for cloning, which
restricts the possibilities of use in both basic and applied
research.
[0009] Hence there is a need for methods to develop new vectors for
expression of heterologous genes that can overcome the aforesaid
problems. In particular, it would be advantagous to have large
vectors for expression of heterologous genes with a high level of
safety and cloning capacity, which can be designed so that their
species specificity and tropism can be controlled.
SUMMARY OF THE INVENTION
[0010] According to the present invention the above problems are
solved by a method of preparing a DNA comprising steps, wherein
[0011] (a) a DNA comprising a full length copy of the genomic RNA
(gRNA) of an RNA virus; or
[0012] (b) a DNA comprising one or several fragments of a gRNA of
an RNA virus, which fragments code for an RNA dependent RNA
polymerase and at least one structural or non-structural protein;
or
[0013] (c) a DNA having a homology of at least 60% to the sequences
of (a) or (b); is cloned into a bacterial artificial chromosome
(BAC).
[0014] Surprisingly, the present inventors found that the problems
encountered by the prior art methods to subclone and express large
DNA sequences derived from viral gRNA can be overcome by using BACs
as a cloning vector. The use of BACs has the particular advantage
that these vectors are present in bacteria in a number of one or
two copies per cell, which considerably limits the toxicity and
reduces the possibilities of interplasmid recombinantion.
[0015] The invention further provides methods of preparing a viral
RNA or a virion comprising steps, wherein a DNA is prepared
according to one of the above methods, the DNA is expressed and the
viral RNA or the virion is isolated. Further, methods of preparing
pharmaceuticals, specifically vaccines comprising the steps of the
above methods to prepare a DNA are disclosed.
[0016] According to another aspect of the present invention
provides a DNA comprising sequences derived from the genomic RNA
(gRNA) of a coronavirus which sequences have a homology of at least
60% to the natural sequence of the coronavirus and code for an RNA
dependent RNA polymerase and at least one structural or
non-structural proteins wherein a fragment of said DNA is capable
of being transcribed into RNA and which RNA can be assembled to a
virion. The present invention also encompasses methods of preparing
respective DNAs.
[0017] The present invention further provides vectors, more
specifically bacterial artificial chromosomes (BACS) comprising
respective nucleic acids. According to a further embodiment the
present invention is directed to host cells and infectious,
attenuated or inactivated viruses comprising the DNAs or RNAs of
the present invention.
[0018] The invention also provides pharmaceutical preparations,
such as mono- or multivalent vaccines comprising nucleic acids,
vectors, host cells or virions of the present invention.
[0019] Finally, the present invention provides methods for
producing a virion or a viral RNA comprising steps, wherein a DNA
according to the present invention is transcribed and the virions
or viral RNAs are recovered, as well as viral RNAs obtainable by
this method.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 shows the construction of a cDNA clone that codes an
infective RNA of TGEV. FIG. 1A shows the genetic structure of the
TGEV, with the names of the genes indicated by letters and numbers
(1a, 1b, S, 3a, 3b, E, M, N, and 7). FIG. 1B shows the cDNA-cloning
strategy, which consisted in completing the DI-C genome. Deletions
.DELTA.1, .DELTA.2, and .DELTA.3 that have been completed to
reestablish the full length of the cDNA are indicated. The numbers
located beneath the structure of the DI-C genome indicate the
nucleotides that flank each deletion in said DI-C genome. FIG. 1C
shows the four cDNA fragments constructed to complete deletion
.DELTA.1 and the position of the principal restriction sites used
during joining. The insertion of fragment .DELTA.1 produced an
increase in the toxicity of the cDNA.
[0021] FIG. 2 shows the structure of the pBeloBAC plasmid (Wang et
al., 1997) used in cloning the infective cDNA of TGEV. The pBeloBAC
plasmid was provided by H. Shizuya and M. Simon (California
Institute of Technology) and includes 7,507 base pairs (bp) that
contain the replication origin of the F factor of E. coli (oriS),
the genes necessary to keep one single copy of the plasmid per cell
(parA, parB, parC, and repE), and the chloramphenicol-resistance
gene (CM.sup.r). The positions of the T7 and SP6 promoters and of
the unique restriction sites are indicated. CosN: site cosN of
lambda to facilitate the construction of the pBAC plasmid; lac Z:
.beta.-galactosidase gene. Sequence loxP used during the generation
of the plasmid is also indicated.
[0022] FIG. 3 shows the structure of the basic plasmids used in the
construction of TGEV cDNA. The pBAC-TcDNA.sup..DELTA.ClaI plasmid
contains all the information of the TGEV RNA except for one
ClaI-ClaI fragment of 5,198 bp. The cDNA was cloned under the
immediately early (IE) promoter of expression of cytomegalovirus
(CMV) and is flanked at the 3'-end by a poly(A) tail with 24
residues of A, the ribozyme of the hepatitis delta virus (HDV), and
the termination and polyadenylation sequences of bovine growth
hormone (BGH). The pBAC-B+C+D5' plasmid contains the ClaI-ClaI
fragment required to complete the pBAC-TcDNA.sup..DELTA.ClaI until
the cDNA is full length. The pBAC-TcDNA.sup.FL plasmid contains the
full-length cDNA of TGEV. SAP: shrimp alkaline phosphatase.
[0023] FIG. 4 shows the differences in the nucleotide sequence of
the S gene of the clones of TGEV PUR46-MAD (MAD) and C11. The
numbers indicate the positions of the substituted nucleotides,
considering as nucleotide one of each gene the A of the initiating
codon. The letters within the bars indicate the corresponding
nucleotide in the position indicated. The letters located beneath
the bars indicate the amino acid (aa) substitutions coded by the
nucleotides that are around the indicated position. .DELTA.6 nt
indicates a 6-nucleotide deletion. The arrow indicates the position
of the termination codon of the S gene.
[0024] FIG. 5 shows the strategy followed to rescue the infective
TGEV from the full-length TGEV cDNA. The pBAC-TcDNA.sup.FL plasmid
was transfected to ST cells (pig testicle cells), and 48 h after
transfection, the supernatant was used to infect new ST cells. The
virus was passed at the times indicated. At each passage, aliquots
of supernatant and of cellular monolayer were collected for virus
titration and isolation of RNA for RT-PCR analysis, respectively.
vgRNA: full-length viral RNA.
[0025] FIG. 6 shows the cytopathic effect (CPE) produced by the
TGEV cDNA in the transfected ST cells. The absence of CPE in
non-transfected (control) ST cells (FIG. 6A) and the CPE observed
14 and 20 h after transfection with pBAC-TcDNA.sup.FL in ST cells
are shown (FIGS. 6B and 6C, respectively).
[0026] FIG. 7 shows the evolution of the viral titer with the
passage. A graph representing the viral titer in the supernatant of
two series of cellular monolayers (1 and 2) at different passages
after transfection with pBAC-TcDNA.sup.FL is shown. Mock 1 and 2
refer to nontransfected ST cells. TcDNA 1 and 2 refer to ST cells
transfected with pBAC-TcDNA.sup.FL.
[0027] FIG. 8 shows the results of the analysis of the sequence of
the virus recovered after transfecting ST cells with
pBAC-TcDNA.sup.FL. The structure of the TGEV genome is indicated at
the top of the figure. Likewise, the differences in the sequence of
nucleotides (genetic markers) between the virus recovered from the
pBAC-TcDNA.sup.FL (TcDNA) plasmid, and TGEV clones C8 and C11 are
indicated. The positions of the differences between the nucleotides
are indicated by the numbers located over the bar. The cDNA
sequences of the TcDNA virus and of clone C11 were determined by
sequencing the fragments obtained by RT-PCR (reverse-transcription
and polymerase chain reaction). The sequence of clone C8 is being
sent for publication (Penzes et al., 1999) and is included at the
end of this patent. The restriction patterns are shown with ClaI
and DraIII of the fragments obtained by RT-PCR that include
nucleotides 18,997 and 20,990 of the TcDNA and C8 viruses. The
restriction patterns show the presence or absence of ClaI and
DraIII sites in the cDNA of these viruses. The result of this
analysis indicated that the TcDNA virus recovered had the S-gene
sequence expected for isolate C11. MWM: molecular weight
markers.
[0028] FIG. 9 shows the results of the RT-PCR analysis of the virus
recovered. The viral RNA was expressed under the control of the CMV
promoter recognized by the cellular polymerase pol II. In
principle, this RNA could undergo splicing during its transport to
the cytoplasm. To study whether this was the case, the sites of the
RNA with a high probability of splicing were determined using a
program for predicting splicing sites in sequences of human DNA
(Version 2.1.5.94, Department of Cell Biology, Baylor College of
Medicine) (Solovyev et al., 1994). The potential splicing site with
maximum probability of cut had the donor site at nt 7,243 and the
receiver at nt 7,570 (FIG. 9A). To study whether this domain had
undergone splicing, a RT-PCR fragment flanked by nt 7,078 and nt
7,802 (FIG. 9B) was prepared from RNA of passages 0 and 2 of
nontransfected cultures (control), or from ST cells transfected
with TcDNA with the ClaI fragment in reverse orientation
(TcDNA.sup.FL(-.DELTA.ClaI)RS, or in the correct orientation
(TcDNA.sup.FL), and the products resulting from the RT-PCR were
analyzed in agarose gels. The results obtained are shown in FIGS.
9C (passage 0) and 9D (passage 2).
[0029] FIG. 10 shows the results of the immunofluorescence analysis
of the virus produced in cultures of ST cells transfected with
TcDNA. Staining for immunofluorescence was done with antibodies
specific for the TGEV PUR46-MAD isolate, and for the virus
recovered after transfection with the pEAC-TcDNA.sup.FL plasmid.
For this, TGEV-specific monoclonal antibodies were used which bind
to both isolates or only to PUR46-MAD (Snchez et al., 1990). The
result confirmed that the TcDNA virus had the expected
antigenicity. The specific polyclonal antiserum for TGEV bound to
both viruses, but not to the uninfected cultures, and only the
expected monoclonal antibodies specific for the S (ID.B12 and
6A.C3), M (3B.B3), and N (3B.D8) proteins bound to the TcDNA virus
(Snchez et al., 1999).
[0030] FIG. 11 shows the expression of GUS under different
transcription-regulatory sequences (TRSS) that vary flanking region
5' of the intergenic (IG) sequence. Minigenome M39 was cloned under
the control of the CMV promoter. Inserted into this minigenome was
a multiple cloning sequence (PL1, 5'-CCTAGGATTTAAATCCTAAGG-3'; SEQ
ID NO: 2) and the transcription unit formed by the selected
transcription-regulating sequences (TRS), another multiple cloning
sequence (PL2, 5'-GCGGCCGCGCCGGCGAGGCCTGTCGAC-3'; SEQ ID NO: 3; or
PL3, 5'-GTCGAC-3'; SEQ ID NO: 4), sequences with the structure of a
Kozak (Kz) domain, the .beta.-glucuronidase (GUS) gene, and another
multiple cloning site (PL4, 5'-GCTAGCCCAGGCGCGCGGTACC-3'; SEQ ID
NO: 5). These sequences .sup.1were flanked at the 3'-end by the
3'-sequence of minigenome M39, the HDV ribozyme, and the
termination and polyadenylation sequences of BGH. The TRSs had a
different number (0, -3, -8, and -88) of nucleotides at the 5'-end
of the IG sequence (CUAAAC).sup.1, and came from the N, S, or M
genes, as indicated. ST cells were transfected with the different
plasmids, were infected with the complementing virus (PUR46-MAD),
and the supernatants were passed 6 times. The GUS activity in the
infected cells was determined by means of the protocol described by
Izeta (Izeta et al., 1999). The results obtained by relating the
GUS activity to the passage number are collected in FIG. 11B.
.sup.1 It should be noted that CTAAAC and CUAAC have the same
meaning for the purpose of this patent. The first represents the
sequence of the DNA and the second that of the corresponding
RNA.
[0031] FIG. 12 shows the expression of GUS under different TRSs
that vary in the 3'-flanking region of the IG sequence (see FIG.
11A). Using this transcription unit with the 5'-flanking region
corresponding to the -88 nt of the N gene of TGEV plus the IG
sequence (CUAAAC), the 3'-flanking sequences were modified. These
sequences corresponded to those of the different TGEV genes (S, 3a,
3b, E, M, N, and 7), as is indicated in FIG. 12A. In two cases,
3'-sequences were replaced by others that contained a restriction
site (SalI) and an optimized Kozak sequence (Kz), or by a sequence
identical to the one that follows the first IG sequence located
following the leader of the viral genome. The activity of GUS in
the infected cells was determined by means of the protocol
described above (Izeta et al., 1999). cL12 indicates a sequence of
12 nucleotides identical to that of 3'-end of the "leader" sequence
of the TGEV genome (see the virus sequence indicated at the end).
The results obtained by relating the expression of GUS to the
passage number are collected in FIG. 12B.
[0032] FIG. 13 shows the effect of the site of insertion of the
module of expression in the minigenome over the levels of GUS
expression. The GUS transcription unit, containing -88 nt of the N
gene flanking the 5'-end of the IG sequence (CUAAAC), and the Kz
sequences flanking the 3'-end (see FIG. 12A), was inserted into
four single restriction sites in minigenome M39 (FIG. 13A) to
determine if all these sites were equally permissive for the
expression of the heterologous gene. ST cells were transfected with
these plasmids and infected with the complementing virus
(PUR46-MAD). The GUS activity in the infected cells was determined
at passage 0 (P0) following the protocol described above (Izeta et
al., 1999). The results obtained are collected in FIG. 13B.
DETAILED DESCRIPTION OF THE INVENTION
[0033] According to the present invention methods of preparing a
DNA are provided, which comprise steps, wherein
[0034] (a) a DNA comprising a full length copy of the genomic RNA
(gRNA) of an RNA virus; or
[0035] (b) a DNA comprising one or several fragments of a gRNA of
an RNA virus, which fragments code for an RNA dependent RNA
polymerase and at least one structural or non-structural protein;
or
[0036] (c) a DNA having a homology of at least 60% to the sequences
of (a) or (b);
[0037] is cloned into a bacterial artificial chromosome (BAC).
[0038] According to the present application a "bacterial artificial
chromosome" is a DNA sequence which comprises the sequence of the F
factor. Plasmids containing this sequences, so-called F plasmids,
are capable of stably maintaining heterologous sequences longer
than 300 Kb in low copy number (one or two copies per cell).
Respective BACs are known in the art (Shizuya et al., 1992).
[0039] According to the present invention the DNA cloned into the
BAC has a homology of at least 60%, preferably 75% and more
preferably 85 or 95%, to a natural sequence of an RNA virus.
Sequence homology is preferably determined using the Clustal
computer program available from the European Bioinformatics
Institute (EBI).
[0040] According to the methods of the present invention the DNA
cloned into the BAC may further comprise sequences coding for
several or all except one of the structural or non-structural
proteins of the virus.
[0041] In a preferred embodiment of the present invention the DNA
cloned into the BAC further comprises sequences encoding one or
several heterologous gene. According to the present application a
gene is characterized as a "heterologous gene" if it is not derived
from the virus which was used as a source for the genes encoding
the RNA dependent RNA polymerase and the structural or
non-structural protein. A "heterologous gene" thus also refers to
genes derived from one type of virus and expressed in a vector
comprising sequences derived from another type of virus. Any
heterologous gene of interest can be inserted into the nucleic
acids of the present invention. The insertion of genes encoding one
or several peptides or proteins which are recognised as an antigen
from an infectious agent by the immune system of a mammal is
especially preferred. Alternatively, the method of the present
invention may be performed using heterologous genes encoding at
least one molecule interfering with the replication of an
infectious agent or an antibody providing protection against an
infectious agent. The heterologous sequences may contain sequences
encoding an immune modulator, a cytokine, an immonenhancer and/or
an anti-inflammatory compound.
[0042] The method of the present invention may be performed using a
DNA for cloning into a BAC that has any size. However, specific
advantages over the known methods to prepare subcloned DNA from
viral are obtained, if large sequences are used. The DNA cloned
into the BAC may thus comprise a length of at least 5 Kb, wherein
DNA with a size of at least 15, 25 or 30 Kb is specifically
preferred.
[0043] According to specifically preferred embodiments of the
present invention methods are provided, wherein the BAC comprises a
sequence controlling the transcription of the DNA cloned into the
BAC. This will allow transcription of the viral RNA and thus enable
expression of the virus. Any sequence controlling transcription
known in the art may be used for this purpose, including sequences
driving the expression of genes derived from DNA or RNA genomes.
The use of the immediately early (IE) promoter of cytomegalovirus
(CGV) is preferred.
[0044] The DNA cloned into the BAC may also be flanked at the
3'-end by a poly(A)tail. The nucleic acid may comprise termination
and/or polyadenylation sequences of bovine growth hormone (BGH).
Additionally or alternatively, the nucleic acids may comprise
sequences encoding a ribozyme, for example the ribozyme of the
hepatitis .delta. virus (HDV).
[0045] Additional advantages may be achieved if at least one of the
genes of the virus has been modified by substituting, deleting or
adding nucleotides. For example the gene controlling tropism of the
virus may be modified to obtain viruses with altered tropism.
Alternativly, the gene controlling tropism of the virus has been
substituted with the respective gene of another virus. The
modification is preferably performed in the S, M and/or N genes of
the virus.
[0046] In a preferred embodiment of the present invention a method
is provided, wherein the DNA cloned into the BAC is capable of
being transcribed into RNA which RNA can be assembled to an virion.
The virion may be an infectious, attenuated, replication defective
or inactivated virus.
[0047] Any RNA virus may be used in the methods of the invention.
The virus can for example be a picornavirus, flavivirus, togavirus,
coronavirus, toroviruses, arterivurses, calcivirus, rhabdovirus,
paramixovirus, filovirus, bornavirus, orthomyxovirus, bunyavirus,
arenavirus or reovirus. The use of viruses naturally having a plus
strand genome is preferred.
[0048] Additionally, the present invention provides methods of
preparing a viral RNA or a virion comprising steps, wherein a DNA
is prepared according to one of above methods, the DNA is expressed
in a suitable host cell and the viral RNA or the virion is isolated
from that host cell. Any of methods for isolating viruses from the
cell culture known in the art may be used. Alternatively, methods
of preparing a viral RNA or a virion are disclosed, wherein the DNA
of the present invention is transcribed or translated using
chemicals, biological reagents and/or cell extracts and the viral
RNA or the virion is subsequently isolated. For certain
embodiments, the virus may subsequently be inactivated or
killed.
[0049] The invention also provides methods for preparing a
pharmaceutical composition comprising steps, wherein a DNA, a viral
RNA or a virion is prepared according to one of the above methods
and is subsequently mixed with a pharmaceutically acceptable
adjuvans and/or carrier. A large number of adjuvans and carriers
and diluents are known in the prior art and may be used in
accordance with the present invention. The pharmaceutical is
preferably a vaccine for protecting humans or animals against an
infectious disease. The pharmaceutical can advantageously also be
used for gene therapy.
[0050] The present invention further provides for the first time a
DNA comprising sequences derived from the genomic RNA (gRNA) of a
coronavirus which sequences have a homology of at least 60% to the
natural sequence of the coronavirus and code for an RNA dependent
RNA polymerase and at least one structural or non-structural
protein, wherein a fragment of said DNA is capable of being
transcribed into RNA which can be assembled to a virion.
[0051] According to the present invention the term "sequence
derived from a coronavirus" is used to refer to a nucleic acid
sequence which has a homology of at least 60%, preferably 75% and
more preferably 85 or 95%, to a natural sequence of a coronavirus.
Sequence homology can be determined using the Clustal computer
program available from the European Bioinformatics Institute
(EBI).
[0052] The term "coronavirus" is used according to the present
invention to refer to a group of viruses having a single molecule
of linear, positive sense, ssRNA of 25 to 33 Kb. These viruses
usually contain 7 to 10 structural genes, i.e. genes encoding
proteins that determine the viral structure. These genes are
typically arranged in the viral genome in the order of 5'
repli-case-(hemagglutinin-esterase)-spike-envelope-mem-
brane-nucleoprotein-3'. Additionally the viral genome may comprise
a number of non-structural genes which encode a nested set of mRNAs
with a common 3' end and are largely non-essential.
[0053] The term "capable of being transcribed into RNA which can be
assembled into a virion" is used to characterize a DNA sequence,
which--once introduced into a suitable host cell--will be
transcribed into RNA and generate virions. The virions are
preferably infectious viruses, but may also be inactivated,
attenuated or replication defective viruses comprising said RNA.
Preferably all the information necessary for expression of the
virion is encoded by the DNA sequence of the present invention.
[0054] The nucleic acids of the present invention may further
comprise a sequence encoding one or several heterologous genes of
interest. According to the present invention a gene is
characterized as a "heterologous gene" if it is not derived from
the coronavirus which was used as a source for the genes encoding
the RNA dependent RNA polymerase and the structural or
non-structural protein. A "heterologous gene" thus also refers to
genes derived from one type of coronavirus and expressed in a
vector comprising sequences derived from another type of
coronavirus. Any heterologous gene of interest can be inserted into
the nucleic acids of the present invention. The insertion of genes
encoding peptides or proteins which are recognised as an antigen
from an infectious agent by the immune system of a mammal is
especially preferred. The heterologous gene may thus encode at
least one antigen suitable for inducing an immune response against
an infectious agent, at least one molecule interfering with the
replication of an infectious agent or an antibody providing
protection against an infectious agent. Alternatively or
additionally, the heterologous gene may encode an immune modulator,
a cytokine, an immonenhancer or an anti-inflammatory compound.
[0055] The fragment of the DNA according to the present invention
which is transcribed into RNA preferably has a size of at least 25
Kb. Fragments with a size of at least 30 Kb are especially
preferred.
[0056] According to a preferred embodiment of the present invention
the DNA further comprises sequences derived from a coronavirus
coding for several or all except one of the structural or
non-structural proteins of a coronavirus. Alternatively, the DNA of
the present invention further comprises sequences coding for all of
the structural or non-structural proteins of a coronavirus.
[0057] According to a further embodiment, the nucleic acids of the
present invention comprise a sequence controlling the transcription
of a sequence derived from a coronavirus gRNA. Any sequence
controlling transcription known in the art may be used for this
purpose, including sequences driving the expression of genes
derived from DNA or RNA genomes. The use of the immediately early
(IE) promoter of cytomegalovirus (CKV) is preferred.
[0058] The nucleic acid according to the present invention may also
be flanked at the 3'-end by a poly(A)tail. The nucleic acid may
comprise termination and/or polyadenylation sequences of bovine
growth hormone (BGH). Additionally or alternatively, the nucleic
acids may comprise sequences encoding a ribozyme, for example the
ribozyme of the hepatitis .delta. virus (HDV).
[0059] The nucleic acids of the present invention may comprise
sequences derived from any coronavirus, for example derived from an
isolate of the porcine transmissible gastroenteritis virus (TGEV),
murine hepatitits virus (MHV), infectious bronchitis virus (IBV),
bovine coronavirus (BoCV), canine coronavirus (CCoV), feline
coronavirus (FcoV) or human coronavirus. According to a preferred
embodiment the nucleic acid is derived from a transmissable
gastroenteritis virus.
[0060] According to a further embodiment of the present invention,
the DNAs of the present invention are part of a plasmid, preferably
part of a bacterial artificial chromosome (BAC).
[0061] The present invention further provides host cells comprising
respective nucleic acids or vectors. The host cells may be
eucaryotes or procaryotes. Alternatively, the present invention
provides virions comprising a nucleic acid according the present
invention. Respective virions may for example be isolated from cell
cultures transfected or infected with the nucleic acids of the
present invention.
[0062] According to a further embodiment, the present invention
provides methods for producing a virion or a viral RNA comprising
steps, wherein a DNA of the present invention is introduced into a
host cell, host cells containing the DNA are cultivated under
conditions allowing the expression thereof and the virion or viral
RNA is recovered. Additionally, methods for producing a virion or a
viral RNA are provided, wherein a DNA of the present invention is
mixed in vitro with chemicals, biological reagents and/or cell
extracts under conditions allowing the expression of the DNA and
the virion or viral RNA is recovered. The present invention also
encompasses the virions and viral RNAs obtainable by either of the
above methods. RNAs and virions carrying a heterologous gene are
preferred. The viruses so obtained may have the form of an
infectious, attenuated, replication defective or inactivated
virus.
[0063] The virus may comprise modified genes, for example a
modified S, N or M gene. In a specific embodiment of the present
invention the modification of the S, N or M gene gives raise to an
attenuated virus or a virus with altered tropism.
[0064] According to a further embodiment the invention provides a
pharmaceutical preparation comprising nucleic acids, host cells or
virions according to the present invention. According to a
preferred embodiment the pharmaceutical preparation is a vaccine
capable of protecting an animal against deseases caused by an
infectious agent. The vaccine may for example comprise sequences of
at least one antigen suitable for inducing an immune response
against the infectious agent or an antibody providing protection
against said infectious agent. The vaccine may comprise a DNA
expressing at least one molecule interfering with the replication
of the infectious agent. Alternatively the vaccine may comprise a
vector expressing at least one antigen capable of inducing a
systemic immune response and/or an immune response in mucous
membranes against different infectious agents that propagate in
respiratory, intestinal mucous membranes or in other tissues. The
vaccine may also be a multivalent vaccine capable of protecting an
animal against the infection caused by more than one infectious
agent, that comprises more than one nucleic acid of the present
invention each of which expresses an antigen capable of inducing an
immune response against each of said infectious agents, or
antibodies that provide protection against each one of said
infectious agents or other molecules that interfere with the
replication of any infectious agent.
[0065] The vaccines of the present invention may further comprise
any of the pharmaceutically acceptable carriers or diluents known
in the state of the art.
[0066] The present invention further provides methods for preparing
a DNA of the present invention comprising steps, wherein an
interfering defective genome derived from a coronavirus is cloned
under the expression of a promotor into a BAC vector and the
deletions within the defective genome are re-inserted. The method
may further comprise steps, wherein toxic sequences within the
viral genome are identified before re-insertion into the remaining
genomic DNA. Preferably, the toxic sequences within the viral
genome are the last sequences to be re-inserted before completing
the genome. According to the present invention this method is
suitable to yield infectious clones of coronaviruses which are
stable in bacteria for at least 80 generations and thus provides a
very efficient cloning vector.
[0067] The present invention provides the development of infective
clones of cDNA derived from coronavirus (Almazan et al., 2000), as
well as vectors constructed from said infective clones that also
include heterologous nucleic acid sequences inserted into said
clones. The infective clones and vectors provided by this invention
have numerous applications in both basic and applied research, as
well as a high cloning capacity, and can be designed in such a way
that their species specificity and tropism can be easily
controlled.
[0068] This patent describes the development of a method that makes
it possible to obtain, for the first time in the history of
coronavirus, a full-length infective cDNA clone that codes the
genome of a coronavirus (Almazan et al., 2000).
[0069] A new vector or system of expression of heterologous nucleic
acids based on a coronavirus generated from an infective cDNA clone
that codes the genomic RNA (gRNA) of a coronavirus has been
developed. In one particular realization of this invention, the
coronavirus is the porcine transmissible gastroenteritis virus
(TGEV).
[0070] The new system of expression can be used in basic or applied
research, for example, to obtain products of interest (proteins,
enzymes, antibodies, etc.), as a vaccinal vector, or in gene
therapy in both humans and animals. The infective coronavirus
obtained from the infective cDNA clone can be manipulated by
conventional genetic engineering techniques so that new genes can
be introduced into the genome of the coronavirus, and so that these
genes can be expressed in a tissue- and species-specific manner to
induce an immune response or for gene therapy. In addition, the
expression has been optimized by the selection of new
transcription-regulating sequences (TRS) that make it possible to
increase the levels of expression more than a hundredfold.
[0071] The vectors derived from coronavirus, particularly TGEV,
present several advantages for the induction of immunity in mucous
membranes with respect to other systems of expression that do not
replicate in them: (i) TGEV infects intestinal and respiratory
mucous membranes (Enjuanes and Van der Zeijst, 1995), that is, the
best sites for induction of secretory immunity; (ii) its tropism
can be controlled by modifying the S (spike) gene (Ballesteros et
al., 1997); (iii) there are nonpathogenic strains for the
development of systems of expression that depend on complementing
virus (Sanchez et al., 1992); and (iv) coronaviruses are
cytoplasmic RNA viruses that replicate without passing through an
intermediate DNA stage (Lai and Cavanagh, 1997), making its
integration into the cellular chromosome practically
impossible.
[0072] The procedure that has made it possible to recover an
infective coronavirus from a cDNA that codes the gRNA of a
coronavirus includes the following strategies:
[0073] (i) expression of the RNA of the coronavirus under the
control of an appropriate promoter;
[0074] (ii) cloning of the genome of the coronavirus in bacterial
artificial chromosomes (BACs);
[0075] (iii) identification of the sequences of cDNA of the
coronavirus that are directly or indirectly toxic to bacteria;
[0076] (iv) identification of the precise order of joining of the
components of the cDNA that codes an infective RNA of coronavirus
(promoters, transcription-termination sequences, polyadenylation
sequences, ribozymes, etc.); and
[0077] (v) identification of a group of technologies and processes
(conditions for the growth of BACs, modifications to the
purification process of BAC DNA, transformation techniques, etc.)
that, in combination, allow the efficient rescue of an infective
coronavirus of a cDNA.
[0078] The promoter plays an important role in increasing the
expression of viral RNA in the nucleus, where it is synthesized, to
be transported to the cytoplasm later on.
[0079] The use of BACs constitutes one of the key points of the
procedure of the invention. As is known, cloning of eukaryotic
sequences in bacterial plasmids is often impossible due to the
toxicity of the exogenous sequences for bacteria. In these cases,
the bacteria usually eliminate toxicity by modifying the introduced
sequences. Nevertheless, in the strategy followed in this case, to
avoid the possible toxicity of these viral sequences, the necessary
clonings were carried out to obtain a complete cDNA of the
coronavirus in BACs. These plasmids appear in only one copy or a
maximum of two per cell, considerably limiting their toxicity and
reducing the possibilities of interplasmid recombination.
[0080] Through the identification of the bacteriotoxic cDNA
sequences of the coronavirus, the construction of the cDNA that
codes the complete genome of a coronavirus can be completed, with
the exception of the toxic sequences, which are added in the last
step of construction of the complete genome, that is, just before
transfection in eukaryotic cells, avoiding their modification by
the bacteria.
[0081] One object of the present invention therefore consists in an
infective double-chain cDNA clone that codes the gRNA of a
coronavirus, as well as the procedure for obtaining it.
[0082] An additional object of this invention consists in a set of
recombinant viral vectors that comprises said infective clone and
sequences of heterologous nucleic acids inserted into said
infective clone.
[0083] An additional object of this invention consists in a method
for producing a recombinant coronavirus that comprises the
introduction of said infective clone into a host cell, the culture
of the transformed cell in conditions that allow the replication of
the infective clone and production of the recombinant coronavirus,
and recovering the recombinant coronavirus from the culture.
[0084] Another object of this invention consists in a method for
producing a modified recombinant coronavirus that comprises
introducing the recombinant viral vector into a host cell,
cultivating it in conditions that allow the viral vector to
replicate and the modified recombinant coronavirus to be produced,
and recovering the modified recombinant coronavirus from the
culture. Another object of this invention consists in a method for
producing a product of interest that comprises cultivating a host
cell that contains said recombinant viral vector in conditions that
allow the expression of the sequence of heterologous DNA.
[0085] Cells containing the aforementioned infective clones or
recombinant viral vector constitute another object of the present
invention.
[0086] Another object of this invention consists in a set of
vaccines that protect animals against infections caused by
infectious agents. These vaccines comprise infective vectors that
express at least one antigen adequate for inducing an immune
response against each infective agent, or at least one antibody
that provides protection against said infective agent, along with a
pharmaceutically acceptable excipient. The vaccines can be mono- or
multivalent, depending on whether the vectors express one or more
antigens capable of inducing an immune response to one or more
infectious agents, or, alternatively, one or more antibodies that
provide protection against one or more infectious agents.
[0087] Another object provided by this invention comprises a method
of animal immunization that consists in the administration of said
vaccine.
[0088] The invention provides a cDNA clone that codes the infective
RNA of a coronavirus, henceforth the infective clone of the
invention, which comprises: (1) a copy of the complementary DNA
(cDNA) to the infective genomic RNA (gRNA) of a coronavirus or the
viral RNA itself; and (2) an expression module for an additional
gene, which includes optimized transcription-promoting
sequences.
[0089] In one particular realization of this invention, the
coronavirus is a TGEV isolate, in particular, the PUR46-MAD isolate
(Snchez et al., 1990), modified by the replacement of the S gene of
this virus by the S gene of the clone C11 TGEV isolate or the S
gene of a canine or human coronavirus.
[0090] The transcription-promoting sequence, or promoter, is an RNA
sequence located at the 5'-terminal end of each messenger RNA
(mRNA) of coronavirus, to which the viral polymerase RNA binds to
begin the transcription of the messenger RNA (mRNA). In a
particular and preferred embodiment the viral genome is expressed
from a cDNA using the IE promoter of CMV, due to the high level of
expression obtained using this promoter (Dubensky et al., 1996),
and to previous results obtained in our laboratory that indicated
that large defective genomes (9.7 kb and 15 kb) derived from the
TGEV coronavirus expressed RNAs that did not undergo splicing
during their transport from the nucleus, where they are
synthesized, to the cytoplasm.
[0091] The infective clone of the invention also contains a
transcription termination sequence and a polyadenylation signal
such as that coming from the BGH gene. These termination sequences
have to be placed at the 3'-end of the poly(A) tail. In one
particular realization, the infective clone of the invention
contains a poly(A) tail of 24 residues of A and the termination and
polyadenylation sequences of the BGH separated from the poly(A)
tail by the sequence of the HDV ribozyme.
[0092] The plasmid into which the infective cDNA of the virus has
been inserted is a DNA molecule that possesses a replication
origin, and is therefore potentially capable of replicating in a
suitable cell. The plasmid used is a replicon adequate for
maintaining and amplifying the infective clone of the invention in
an adequate host cell such as a bacterium, for example, Escherichia
coli. The replicon generally carries a gene of resistance to
antibiotics that allows the selection of the cells that carry it
(for example, cat).
[0093] In Example 1, the construction of an infective clone of TGEV
under the control of the IE promoter of CMV is described. The
3'-end of the cDNA appears flanked by a 24 nt poly(A) sequence, the
HDV ribozyme, and the transcription termination sequence of the
BGH.
[0094] The procedure for obtaining the infective clone of the
invention comprises constructing the full-length cDNA from the gRNA
of a coronavirus and joining the transcription-regulating
elements.
[0095] The cDNA that codes the infective gRNA of a coronavirus was
obtained from a DI genome derived from a coronavirus cloned as a
cDNA under the control of an appropriate promoter in a BAC, for the
purpose of increasing the cDNA's stability. Then the bacteriotoxic
sequences were identified and, for the purpose of eliminating that
toxicity, said toxic sequences were removed and inserted at the end
of the construction of the complete genome, just before
transfection in eukaryotic cells. The viral progeny can be
reconstituted by means of transfection of the BAC plasmid that
contains the coronavirus genome in eukaryotic cells that support
viral replication.
[0096] The transcription-regulating elements are joined by means of
conventional techniques (Maniatis et al., 1989).
[0097] The infective clone of the invention can be manipulated by
conventional genetic engineering techniques to insert at least one
sequence of a heterologous nucleic acid that codes a determined
activity, under the control of the promoter that is present in the
infective clone and of the regulating sequences contained in the
resulting expression vector.
[0098] The infective clone of the invention presents numerous
applications; for example, it can be used both in basic research,
for example, to study the mechanism of replication and
transcription of coronaviruses, and in applied research, for
example, in the development of efficient systems of expression of
products of interest (proteins, enzymes, antibodies, etc.).
[0099] Appropriate cells can be transformed from the infective cDNA
clone of the invention, and the virions obtained containing the
complete genome of the coronavirus can be recovered. Therefore, the
invention moreover provides a method for producing a recombinant
coronavirus that comprises the introduction of an infective cDNA of
the invention into a host cell, the culture of said cell under
conditions that allow the expression and replication of the
infective clone and the recovery of the virions obtained from the
recombinant coronavirus, which contain the infective genome of the
coronavirus. The infective clone of the invention can be introduced
into the host cell in various ways, for example by transfection of
the host cell with an RNA transcribed in vitro from an infective
clone of the invention, or by infecting the host cell with the
infective cDNA clone of the invention. Said host cells that contain
the infective clone of the invention constitute an additional
object of the present invention.
[0100] The invention also provides a set of recombinant viral
vectors derived from an infective clone of the invention,
henceforth viral vectors of the invention. The viral vectors of the
invention comprise an infective cDNA clone of the invention
modified to contain a heterologous nucleic acid inserted into said
infective clone under conditions that allow said heterologous
nucleic acid to be expressed.
[0101] The term "nucleic acid," as it is used in this description,
includes genes or gene fragments as well as, in general, any
molecule of DNA or RNA.
[0102] In the sense used in this description, the term
"heterologous" applied to a nucleic acid refers to a nucleic acid
sequence that is not normally present in the vector used to
introduce the heterologous nucleic acid into a host cell.
[0103] The heterologous nucleic acid that can contain the viral
vector of the invention can be a gene or fragment that codes a
protein, a peptide, an epitope, or any gene product of interest
(such as antibodies, enzymes, etc.). The heterologous nucleic acid
can be inserted into the infective clone of the invention by means
of conventional genetic engineering techniques in any appropriate
region of the cDNA, for example, after ORF 1b or between genes N
and 7, following the initiator codon (AUG), and in reading frame
with that gene; or, alternatively, in the zones corresponding to
other ORFs. In the construction of the viral vector of the
invention, it is essential that the insertion of the heterologous
nucleic acid not interfere with any of the basic viral
functions.
[0104] The viral vector of the invention can express one or more
activities. In this latter case, the viral vector will include as
many sequences of heterologous nucleic acid as activities to be
expressed, preceded by one or several promoters, or by a promoter
and various ribosome recognition sites (IRES, internal ribosome
entry sites), or by various promoters and one ribosome recognition
site.
[0105] Therefore, the invention provides a method for producing a
product of interest that comprises cultivating a host cell that
contains a viral vector of the invention under conditions that
allow the heterologous nucleic acid to be expressed and the product
of interest to be recovered. Said host cells that contain the viral
vector of the invention constitute an additional object of the
present invention.
[0106] The viral vector of the invention can be designed so that
its species specificity and tropism can be easily controlled. Due
to these characteristics, a very interesting application of the
viral vectors of the invention is their use in gene therapy as a
vector of the gene of interest, or as a vaccinal vector to induce
immune responses against different pathogens.
[0107] The invention furthermore provides vaccines, capable of
protecting an animal against the infection caused by an infectious
agent, that comprise (i) at least one viral vector of the invention
that expresses at least one antigen suitable for inducing an immune
response against said infectious agent, or an antibody that
provides protection against said infectious agent, along with,
optionally, (ii) a pharmaceutically acceptable excipient.
[0108] In the sense used in this description, "inducing protection"
should be understood as the immune response of the receiving
organism (animal to be immunized) induced by the viral vector of
the invention, through suitable mechanisms such as that induced by
substances that potentiate cellular response (interleukins,
interferons, etc.), cellular necrosis factors, and similar
substances that protect the animal from infections caused by
infectious agents.
[0109] Included under the term "animal" are all animals of any
species, preferably mammals, including man.
[0110] The term "infectious agent" in the sense used in this
description includes any viral, bacterial, fungal, parasitic, or
other infective agent that can infect an animal and cause it a
pathology.
[0111] In one particular realization, the vaccine provided by this
invention comprises at least one viral vector of the invention that
expresses at least one antigen capable of inducing a systemic
immune response and/or an immune response in mucous membranes
against different infectious agents that propagate in respiratory
or intestinal mucous membranes. The vectors of the invention are
quite suitable to induce immunity in mucous membranes as well as
lactogenic immunity, which is of special interest in protecting
newborns against intestinal tract infections.
[0112] In another particular realization, the vaccine provided by
this invention comprises at least one viral vector of the invention
that expresses at least one gene that codes for the light and heavy
chains of an antibody of any isotype (for example, IgG.sub.1, IgA,
etc.) that protects against an infectious agent.
[0113] Species specificity can be controlled so that the viral
vector may express the S protein of the envelope of a coronavirus
that infects the desired species (man, dog, cat, pig, etc.),
suitable to be recognized by the cellular receptors of the
corresponding species.
[0114] The vaccines provided by this invention can be monovalent or
multivalent, depending on whether the viral vectors of the
invention express one or more antigens capable of inducing an
immune response to one or more infectious agents, or one or more
antibodies that provide protection against one or more infectious
agents.
[0115] In a particular realization of this invention, monovalent
vaccines capable of protecting man, pigs, dogs and cats against
different infectious human, porcine, canine, and feline agents are
provided, and tropism is controlled by expressing the S
glycoprotein of the coronavirus with the desired species
specificity.
[0116] The monovalent vaccines against porcine infectious agents
can contain a vector that expresses an antigen selected from the
group consisting essentially of antigens of the following porcine
pathogens: Actinobacillus pleuropneumoniae, Actinobacillus suis,
Haemophilus parasuis, porcine parvovirus, Leptospira, Escherichia
coli, Erysipelotrix rhusiopathiae, Pasteurella multocida,
Bordetella bronchiseptica, Clostridium sp., Serpulina
hydiosenteriae, Mycoplasma hyopneumoniae, porcine epidemic diarrhea
virus (PEDV), porcine respiratory coronavirus, rotavirus, or
against the pathogens that cause porcine respiratory and
reproductive syndrome, Aujeszky's disease (pseudorabies), swine
influenza, or transmissible gastroenteritis, and the etiological
agent of atrophic rhinitis and proliferative ileitis. The
monovalent vaccines against canine infectious agents can contain an
expression vector that expresses an antigen selected from the group
essentially consisting of antigens of the following canine
pathogens: canine herpes viruses, types 1 and 2 canine adenovirus,
types 1 and 2 canine parvovirus, canine reovirus, canine rotavirus,
canine coronavirus, canine parainfluenza virus, canine influenza
virus, distemper virus, rabies virus, retrovirus, and canine
calicivirus.
[0117] The monovalent vaccines against feline infectious agents can
contain an expression vector that expresses an antigen selected
from the group essentially consisting of antigens of the following
feline pathogens: cat calicivirus, feline immunodeficiency virus,
feline herpes viruses, feline panleukopenia virus, feline reovirus,
feline rotavirus, feline coronavirus, cat infectious peritonitis
virus, rabies virus, feline Chlamydia psittaci, and feline leukemia
virus.
[0118] The vectors can express an antibody that provides protection
against an infectious agent, for example, a porcine, canine or
feline infectious agent such as those cited above. In one
particular realization, the vector expresses the recombinant
monoclonal antibody identified as 6A.C3, which neutralizes TGEV,
expressed with isotypes IgG.sub.1 or IgA, in which the constant
part of the immunoglobulin is of porcine origin, or neutralizing
antibodies for human and porcine rotaviruses.
[0119] As the excipient, a diluent such as physiological saline or
other similar saline solutions can be used. Likewise, these
vaccines can also contain an adjuvant from those usually used in
the formulation of both aqueous vaccines, such as aluminum
hydroxide, QuilA, suspensions of alumina gels and the like, and
oily vaccines based on mineral oils, glycerides, fatty acid
derivatives, and their mixtures.
[0120] The vaccines of the present invention can also contain
cell-response-potentiating (CRP) substances, that is, substances
that potentiate subpopulations of helper T-cells (Th.sub.1 and
Th.sub.2) such as interleukin-1 (IL-1), IL-2, IL-4, IL-5, IL-6,
IL-12, gamma-IFN (gamma-interferon), cellular necrosis factor, and
similar substances that could theoretically provoke cellular
immunity in vaccinated animals. These CRP substances could be used
in vaccine formulations with aqueous or oily adjuvants. Another
type of adjuvants that modulate and immunostimulate cellular
response can also be used, such as MDP (muramyl dipeptide), ISCOM
(Immunostimulant Complex), or liposomes.
[0121] The invention provides multivalent vaccines capable of
preventing and protecting animals from infections caused by
different infectious agents. These multivalent vaccines can be
prepared from viral vectors of the invention into which the
different sequences that code the corresponding antigens have been
inserted in the same recombinant vector, or by constructing
independent recombinant vectors that would later be mixed for joint
inoculation. Therefore, these multivalent vaccines comprise a viral
vector that contains more than one sequence of heterologous nucleic
acids that code for more than one antigen or, alternatively,
different viral vectors, each of which expresses at least one
different antigen.
[0122] Analogously, multivalent vaccines that comprise multivalent
vectors can be prepared using sequences that code antibodies that
protect against infectious agents, instead of sequences that code
the antigens.
[0123] In one particular realization of this invention, vaccines
capable of immunizing humans, pigs, dogs, and cats against
different porcine, canine and feline infectious agents,
respectively, are provided. For this, the viral vectors contained
in the vaccine must express different antigens of the human,
porcine, canine or feline pathogens mentioned above or others.
[0124] The vaccines of this invention can be presented in liquid or
lyophilized form and can be prepared by suspending the recombinant
systems in the excipient. If said systems were in lyophilized form,
the excipient itself could be the reconstituting substance.
[0125] Alternatively, the vaccines provided by this invention can
be used in combination with other conventional vaccines, either
forming part of them or as a diluent or lyophilized fraction to be
diluted with other conventional or recombinant vaccines.
[0126] The vaccines provided by this invention can be administered
to the animal orally, nasally, subcutaneously, intradermally,
intraperitoneally, intramuscularly, or by aerosol.
[0127] The invention also provides a method for the immunization of
animals, in particular pigs, dogs and cats, against one or various
infectious agents simultaneously, that comprises the oral, nasal,
subcutaneous, intradermal, intraperitoneal, intramuscular, or
aerosol administration (or combinations thereof) of a vaccine that
contains an immunologically efficacious quantity of a recombinant
system provided by this invention.
[0128] In addition, the invention also provides a method for
protecting newborn animals against infectious agents that infect
said animals, consisting in the oral, nasal, subcutaneous,
intradermal, intraperitoneal, intramuscular, or aerosol
administration (or combinations thereof) of a vaccine of those
provided by this invention to mothers before or during the
gestation period, or to their offspring.
[0129] The invention is illustrated by the following examples,
which describe in detail the obtainment of infective clones and the
construction of the viral vectors of the invention. These examples
should not be considered as limiting the scope of the invention,
but as illustrating it. In said example, the transformation and
growth of bacteria, DNA purification, sequence analysis, and the
assay to evaluate the stability of the plasmids were carried out
according to the methodology described below.
[0130] Transformation of Bacteria
[0131] All of the plasmids were electroporated in the E. coli DH10B
strain (Gibco BRL), introducing slight modifications to previously
described protocols (Shizuya et al., 1992). For each
transformation, 2 .mu.L of the ligation and 50 .mu.L of competent
bacteria were mixed in 0.2-cm dishes (BioRad) and electroporated at
200 .OMEGA., 2.5 kV, and 25 .mu.F. Then, 1 mL of SOC medium
(Maniatis et al., 1989) was added at each transformation, the cells
were incubated a 37.degree. C. for 45 min, and finally, the
recombinant colonies were detected on plates of LB SOC media
(Maniatis et al., 1989) with 12.5 .mu.g/mL of chloramphenicol.
[0132] Growth Conditions of the Bacteria
[0133] The bacteria containing the original plasmids, in which the
incomplete genome of TGEV was cloned (FIG. 3), were grown at
37.degree. C., showing normal growth kinetics. On the other hand,
the BAC that contained the complete cDNA was grown at 30.degree. C.
for the purpose of minimizing instability as much as possible. Even
so, the size of the colonies was reduced and incubation periods of
up to 24 h were necessary to achieve normal colony sizes.
[0134] Purification of DNA
[0135] The protocol described by Woo (Woo et al., 1994) was
followed, with slight modifications. From a single colony, 4 L of
LB were inoculated with chloramphenicol (12.5 gg/ml). After an
incubation period of 18 h at 30.degree. C., the bacteria were
collected by centrifugation at 6,000 G, and the plasmid was
purified using the Qiagen Plasmid Maxipreparations kit according to
the manufacturer's recommendations. By means of this procedure, it
was observed that the plasmid DNA obtained was contaminated with
bacterial DNA. To eliminate the contaminating bacterial DNA, the
plasmidic DNA was purified by means of centrifugation at 55,000 rpm
for 16 h on a CsCl gradient. The yield obtained was between 15 and
30 .mu.g/L, depending on the size of the plasmid.
[0136] Sequence Analysis
[0137] The DNA was sequenced in an automatic sequencer (373 DNA
Sequencer, Applied Biosystems) using dideoxynucleotides marked with
fluorochromes and a temperature-resistant polymerase (Perkin
Elmer). The reagents were obtained by way of a kit (ABI PRISM Dye
Terminator Cycle Sequencing Ready Reaction Kit) from the Applied
Biosystems company. The thermocycler used to perform the sequencing
reactions was a "GeneAmpPCR System 9600" (Perkin Elmer).
[0138] The joining of the sequences and their comparison with the
consensus sequence of the TGEV were carried out using the SeqMan II
and Align (DNASTAR) programs, respectively. No differences in
relation to the consent sequence were detected.
[0139] Stability of the Plasmids
[0140] From the original glycerolates, the bacteria that contained
recombinant pBeloBAC11 plasmids were grown in 20 mL of LB with
chloramphenicol (12.5 .mu.g/mL) for 16 h at 30.degree. C. and
37.degree. C. This material was considered passage 0. The bacteria
were diluted 10.sup.6 times and grown at 30.degree. C. and
37.degree. C. for 16 h. Serial passages were realized during eight
consecutive days (each passage represents approximately 20
generations). The plasmid DNA was purified by Miniprep at passages
0 and 8 (160 generations) and analyzed with restriction
endonucleases. The two plasmids that contained part of the genome
of TGEV were highly stable, whereas the plasmid that contained the
complete genome of TGEV showed a certain instability after 40
generations (at this point approximately 80% of the DNA presented
the correct restriction pattern).
EXAMPLE 1
[0141] Construction of a Recombinanat Vector Based on a Clone of
Infective cDNA Derived from TGEV
[0142] 1.1 Generation of an Infective cDNA of TGEV
[0143] For the purpose of obtaining a cDNA that coded for the
complete TGEV genome, we originally started with a cDNA that coded
for the defective DI-C genome (Mendez et al., 1996). This cDNA,
with an approximate length of one third of the TGEV genome, was
cloned in the low-copy pACNR1180 plasmid (Ruggli et al., 1996) and
its sequence was determined. The cDNA that coded the defective
genome was efficiently rescued (replicated and packaged) with the
help of a complementing virus (Mendez et al., 1996; Izeta et al.,
1999).
[0144] The DI-C genome presents three deletions (.DELTA.1,
.DELTA.2, and .DELTA.3) of approximately 10, 1 and 8 kilobases
(kb), at ORFs 1a, 1b, and between genes S and 7, respectively (see
FIG. 1).
[0145] The strategy followed to complete the sequence of a cDNA
that would code for an infective TGEV genome was to incorporate,
step by step, the sequences deleted in the DI-C genome, analyzing
the bacteriotoxicity of the new generated constructions. This
aspect is very important, since it is widely documented in the
scientific literature that recombinant plasmids presenting cDNAs of
RNA virus generally grew poorly and were unstable (Boyer and
Haenni, 1994; Rice et al., 1989; Mandl et al., 1997).
[0146] The first deletion to be completed was deletion .DELTA.2, of
1 kb, of ORF 1b, yielding a stable recombinant plasmid. The
sequence that lacked ORF 1a was introduced by cloning cDNA
fragments A, B, C, and D (FIG. 1) (Almazan et al., 2000) in such a
way that all the information required for the gene of the replicase
would be complete. The recombinant plasmid obtained was unstable in
the bacteria, generating new plasmids that had incorporated
additions and deletions in fragment B (Almazan et al., 2000).
Interestingly, the elimination of a 5,198 bp ClaI-ClaI restriction
fragment that encompassed the region of the genome comprised
between nucleotides 4,417 and 9,615 (Penzes et al., 1999) yielded a
relatively stable plasmid in the E. coli DH10B strain. Later, the
sequence of deletion .DELTA.3 was added by cloning all the genetic
information for the structural and nonstructural proteins of the
3'-end of the TGEV genome (FIG. 1).
[0147] For the purpose of incrementing the stability of the TGEV
cDNA, it was decided that it would be subcloned in BAC using the
pBeloBAC11 plasmid (Kim et al., 1992) (see FIG. 2). The pBeloBAC11
plasmid was a generous gift from H. Shizuya and M. Simon
(California Institute of Technology). The plasmid, 7,507 bp in
size, includes the replication origin of the F factor from parB,
parC, E. coil (oriS) and the genes necessary to keep a single copy
of the plasmid per cell (parA, and repE). The plasmid also presents
the gene of resistance to chloramphenicol (cat) as a selection
marker. The cDNA was cloned under the control of the IE promoter of
CMV, due to the high level of expression obtained using this
promoter (Dubensky et al., 1996) and to previous results obtained
in our laboratory, indicating that large (9.7 kb and 15 kb)
defective genomes derived from TGEV expressed RNAs that did not
undergo splicing during transport from the nucleus, where they are
synthesized, to the cytoplasm (Izeta et al., 1999; Penzes et al.,
1999; Almazan et al., 2000). The generated TGEV cDNA
(pBAC-TcDNA-.DELTA.ClaI) contained the information for the genes of
the replicase, with the exception of the deleted 5,198 bp ClaI
fragment, and all the information of the structural and
non-structural genes. The 3'-end of the cDNA appears flanked by a
24 nt polyA sequence, the HDV ribozyme, and the transcription
termination sequence of BGH (Izeta et al., 1999). On the other
hand, the ClaI fragment necessary to generate a complete genome of
TGEV was cloned in BAC, generating the plasmid pBAC-B+C+D5', which
contained the region of the TGEV genome between 4,310 and 9,758
(see FIG. 3). Both plasmids were grown in the E. coli DH10B strain
and sequenced in their entirety. The sequence obtained was
identical to the consent sequence of the PUR46-MAD isolate of TGEV
provided at the end of this document (SEQ ID NO: 1), with the
exception of two replacements in the positions of nucleotides 6,752
(A=>G, silent) and 18,997 (T=>C, silent), and the changes in
the S gene of the PUR46-MAD that has been replaced by the D gene of
isolate C11 (these changes are indicated in FIG. 4).
[0148] Furthermore, for the purpose of generating a cDNA that would
code a virulent TGEV, the S gene of the PUR46-MAD isolate, which
replicates at highs levels in the respiratory tract (>10.sup.6
PFU/g of tissue) and at low levels in the intestinal tract
(<10.sup.3 PFU/mL), was completely replaced by the S gene of
TGEV clone 11, henceforth C11, which replicates with elevated
titers both in the respiratory tract (<10.sup.6 PFU/mL) and in
the intestinal tract (<10.sup.6 PFU/mL) (Snchez et al., 1999).
The S gene of C11 presents 14 nucleotides that differ from the S
gene of the PUR46-MAD isolate, plus a 6 nt insertion at the 5'-end
of the S gene (see FIG. 4) (Snchez et al., 1999). Previous results
in our laboratory (Snchez et al., 1999) showed that mutants
generated by directed recombination, in which the S gene of the
PUR46-MAD isolate of the TGEV was replaced with the S gene of the
C11 intestinal isolate, acquired intestinal tropism and increased
virulence, unlike the natural PUR46-MAD isolate of the TGEV that
replicates very little or not at all in the intestinal tracts of
infected pigs.
[0149] A cDNA was constructed from the PUR46-MAD isolate of TGEV
with the S gene of the intestinal isolate C11, by means of cloning
of the 5,198 bp ClaI-ClaI fragment, obtained from the pBAC-B+C+D5'
plasmid, in the pBAC-TcDNA.sup..DELTA.ClaI plasmid, to generate the
pBAC-TcDNA.sup.FL plasmid that contains the cDNA that codes for the
complete TGEV genome (FIG. 3).
[0150] The stability in bacteria of the plasmids used in the
construction of the clone of infective cDNA
(pBAC-TcDNA.sup.-.DELTA.ClaI and pBAC-ClaI.sup.F), as well as the
plasmid that contains the complete cDNA (pBAC-TcDNA.sup.FL), was
analyzed after being grown in E. coli for 160 generations. The
stability was analyzed by means of digestion with restriction
enzymes of the purified DNAs. No deletions or insertions were
detected, although the presence of minor changes not detected by
the analysis technique used cannot be ruled out in the case of the
pBAC-TcDNA.sup.-.DELTA.ClaI plasmid and the pBAC-B+C+D5' plasmid.
In the case of the pBAC-TcDNA plasmid, which contains the complete
genome of TGEV, a certain instability was detected after 40
generations (at this point approximately 80% of the DNA presented
the correct restriction pattern). This slight instability, however,
does not represent an obstacle to the rescue of the infective
virus, since 20 generations (4 L of culture) of bacterial growth
are sufficient to generate a quantity of plasmid DNA that allows
the virus to be rescued.
[0151] 1.2 Rescue of an Infective TGEV from a cDNA that Codes for
the Complete Genome
[0152] ST cells were transfected with the pBAC-TcDNA.sup.FL
plasmid. At 48 h posttransfection, the supernatant of the culture
was collected and passed into ST cells six times (see FIG. 5).
Starting at passage 2, at 14 h postinfection, the cytopathic effect
became apparent, extending later, at 20 h postinfection, to
practically all of these cells that formed the monolayer (see FIG.
6). On the other hand, the titer of rescued virus increased rapidly
with the passages, reaching values on the order of 10.sup.8 PFU/mL
as of passage 3 (see FIG. 7). The experiment was repeated five
times, and in ail cases, infective virus with similar titers were
recovered, whereas, in the case of nontransfected ST cells or ST
cells transfected with a similar plasmid, where the ClaI-ClaI
fragment was found in the opposite orientation, virus was never
recovered.
[0153] For the purpose of eliminating the possibility that the
virus obtained was the product of contamination, the sequence at
positions 6,752 and 18,997 was determined by means of sequencing of
cDNA fragments amplified by RT-PCR using the genomic RNA of the
rescued virus as a template. The analysis of the sequence
determined that the nucleotides in positions 6,752 and 18,997 were
those present in the cDNA. Furthermore, the rescued virus
presented, in the cDNA sequence of the S gene, a restriction site
DraIII at position 20,990, as was expected for the S gene of C11
(FIG. 8). The presence of these three genetic markers confirmed
that the isolated virus came from the cDNA.
[0154] In a more in-depth characterization of the virus generated,
a comparative analysis was made by immunofluorescence of infected
cells with the virus recovered (TcDNA) after transfection with the
pBAC-TcDNA.sup.FL plasmid or cells infected with the PUR46-MAD
isolate of the TGEV. For this, specific polyclonal and monoclonal
antibodies that recognized both the C11 isolate and the PUR46-MAD
isolate, or only the latter, were used (see FIG. 10). The results
obtained confirmed the antigenicity expected for the new TcDNA
virus. The polyclonal antibody specific for TGEV, the expected
specific monoclonal of the S protein (ID.B12 and 6A.C3), as well as
the specific monoclonal of the M (3B.B3) and N (3B.D8) proteins
recognized both the TcDNA and the PUR46-MAD. The data obtained
indicated that the virus generated presented the M and N proteins
of the PUR46-MAD isolate and the S protein of the C11 isolate, as
had been designed in the original cDNA.
[0155] 1.3 In vivo Infectivity and Virulence
[0156] For the purpose of analyzing the in vivo infectivity of the
TcDNA virus, a group of five newborn pigs was inoculated with virus
cloned from passage 6, and mortality was analyzed. The five
inoculated pigs died 3 to 4 days postinoculation, indicating that
the TcDNA virus was virulent. In contrast, two pigs inoculated only
with the diluent of the virus and maintained in the same conditions
did not suffer alterations.
[0157] 1.4 Optimization of the Levels of Expression by Modification
of the Transcription-Regulating Sequences
[0158] RNA synthesis in coronavirus takes place by means of an
RNA-dependent process, in which the mRNAs are transcribed from
templates with negative polarity. In the TGEV, a conserved
consensus sequence, CUAAAC, appears, which is located just in front
of the majority of the genes. These sequences represent signals for
the transcription of the subgenomic mRNAs. In coronavirus, there
are between six and eight types of mRNAs with variable sizes,
depending on the type of coronavirus and of the host. The largest
corresponds to the genomic RNA, which in turn serves as mRNA for
ORFs 1a and 1b. The rest of the mRNAs correspond to subgenomic
mRNAs. These RNAs are denominated mRNA 1 to 7, in decreasing size
order. On the other hand, some mRNAs that have been discovered
after the set of originally described mRNAs have been denominated
with the name of the corresponding mRNA, a dash, and a number,
e.g., mRNA 2-1. The mRNAs present a coterminal structure in
relation to the structure of the genomic RNA. With the exception of
the smallest mRNA, the rest are structurally polycistronic, while,
in general, only the ORF located closest to 5' is translated.
[0159] The efficiency in the expression of a marker gene (GUS) has
been studied using different sequences flanking the 5'-terminal of
the minimal intergenic (IG) sequence CUAAAC (FIG. 11), different
sequences flanking the 3'-terminal of the IG sequence (FIG. 12),
and various insertion sites (FIG. 13). The results obtained (FIGS.
11 to 13) indicated that optimal expression was achieved with a TRS
consisting of: (i) the -88 nt flanking the consent sequence for the
N gene of TGEV; (ii) the IG sequence; and (iii) the 3'-flanking
sequence of the IG sequence of the S gene. Furthermore, in
agreement with the results obtained in relationship to the point of
insertion of the heterologous gene, the greatest levels of
expression were achieved when the heterologous gene was located at
the 3'-end of the genome. A TRS such as that described allows the
GUS to be expressed at levels between 2 and 8 .mu.g per 10.sup.6
cells.
[0160] 1.5 Tissue Specificity of the System of Expression
[0161] Many pathogens enter the host through the mucous membranes.
To prevent this type of infections, it is important to develop
systems of expression that allow the induction of high levels of
secretory immunity. This can be achieved fundamentally through the
administration of antigens in the lymph nodes associated with the
respiratory and intestinal tract. To achieve this goal, and in
general to direct the expression of a gene at the tissue of
interest, the molecular bases of the tropism of TGEV have been
studied. These studies have showed that the tissue specificity of
TGEV can be modified by the construction of recombinant viruses
containing the S gene of coronavirus with the desired tropism
(Ballesteros et al., 1997; Snchez et al., 1999). This information
makes it possible to construct systems of expression based on cDNA
genomes of coronavirus with respiratory or intestinal tropism.
[0162] 1.6 Expression of the Viral Antigen Coded by the ORE5 of
PRRSV Using Infective cDNA
[0163] For the purpose of optimizing the levels of expression of
heterologous genes, constructions were made from a vector of
interchangeable modules flanked by cloning sequences that
facilitate the exchange of TRSs and heterologous genes within the
vector. The construction, which included ORF 5 of the PRRSV
(Porcine respiratory and reproductive syndrome virus) flanked at
the 5'-end by the minimal IGS consensus sequence (CUAAAC) preceded
by the -88 nts flanking the gene of the viral nucleocapsid (N), and
at the 3'-end by restriction site SalI (GTCGAC) and a sequence
analogous to that of Kozak (AC)GACC, yielded an optimal expression
(about 10 .mu.g/10.sup.6 cells). In principle, these levels of
expression of the heterologous gene are more than sufficient to
induce an immune response. The heterologous gene was inserted into
the position previously occupied by genes 3a and 3b of the virus,
which are dispensable.
[0164] 1.7 Induction of an Immune Response in Swine to an Antigen
Expressed With the cDNA Derived Virus Vector
[0165] Using the same type of virus vector derived from the cDNA
and the TRSs described above, the gene encoding the green
fluorescent protein (GFP) was expressed at high levels (20 .mu.g of
protein per million of cells in swine testis, ST, cells). The
expression levels were stable for more than 20 passages in cell
culture. Furthermore, a set of swine were immunized with the live
virus vector, that was administered by the oral, intranasal and
intragastric routes and a strong humoral immune response was
detected against both the virus vector and the GFP. Interestingly,
no secondary effect was observed in the inoculated animals after
the administration of three doses of the virus vector.
[0166] 1.8 Construction of a Safe Virus Vector That Expresses the
Foreign Gene Without Leading to the Generation of an Infectious
Virus.
[0167] To design vector for humans, biosafety is a priority. To
achieve this goal, three types of safety guards are being
engineered in the vector. Two of these are based on the deletion of
two virus components, mapping at different positions of the virus
genome, essential for the replication of the virus. These
components are being provided in trans by a packaging cell line.
This cell (Baby Hamster Kidney, BHK) expresses the missing TGEV
genes encoding essential structural proteins of the virus (the
envelope E and the membrane M proteins). The third safety guard is
the relocation of the packaging signal of the virus genome, in such
a way that the recovery of an infectious virus by recombination is
prevented, leading to the generation of a suicide vector that
efficiently expresses the heterologous genes but that is unable to
propagate even to the closest neighbor cell.
[0168] With the design of the new vector for use in humans, we are
not producing a new virus that could be propagated within the human
species, since this vector can not be transmitted from cell to cell
in human beings. The vector is based on a replication defective
virus. It can only be grown in the vaccine factory by using
packaging cells complementing the deletions of the virus. These
safety guards represent novel procedures in the engineering of
coronaviruses. The recombinant virus with a new tropism will be
replication competent at least in feline cells, since these cells
replicate human, porcine, canine and feline coronaviruses.
[0169] Deposition of Microorganisms:
[0170] The bacterium derived from Escherichia coli, carrying the
plasmid with the infective clone of the invention, identified as
Escherichia coli pBAC-TcDNA.sup.FL, has been deposited with the
Spanish Collection of Type Cultures (CECT), Burjassot (Valencia),
on Nov. 24.sup.th 1999, under registration number CECT 5265.
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Sequence CWU 0
0
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