U.S. patent application number 11/523004 was filed with the patent office on 2007-03-29 for recombinant mva virus, and the use thereof.
This patent application is currently assigned to GSF-Forschungszentrum Fur Umwelt und Gesundheit GmbH. Invention is credited to Volker Erfle, Marion Ohlmann, Gerd Sutter.
Application Number | 20070071769 11/523004 |
Document ID | / |
Family ID | 8097499 |
Filed Date | 2007-03-29 |
United States Patent
Application |
20070071769 |
Kind Code |
A1 |
Sutter; Gerd ; et
al. |
March 29, 2007 |
Recombinant MVA virus, and the use thereof
Abstract
The present invention relates to recombinant vaccinia viruses
derived from the modified vaccinia virus Ankara (MVA) and
containing and capable of expressing foreign genes which are
inserted at the site of a naturally occurring deletion in the MVA
genome, and the use of such recombinant MVA viruses for the
production of polypeptides, e.g. antigens or therapeutic agents, or
viral vectors for gene therapy, and the use of such recombinant MVA
viruses encoding antigens as vaccines.
Inventors: |
Sutter; Gerd; (Munich,
DE) ; Ohlmann; Marion; (Munich, DE) ; Erfle;
Volker; (Munich, DE) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
GSF-Forschungszentrum Fur Umwelt
und Gesundheit GmbH
Oberschleissheim
DE
|
Family ID: |
8097499 |
Appl. No.: |
11/523004 |
Filed: |
September 19, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10147284 |
May 15, 2002 |
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11523004 |
Sep 19, 2006 |
|
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09002443 |
Jan 2, 1998 |
6440422 |
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10147284 |
May 15, 2002 |
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PCT/EP96/02926 |
Jul 3, 1996 |
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09002443 |
Jan 2, 1998 |
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Current U.S.
Class: |
424/199.1 ;
435/235.1; 435/325; 435/456; 435/69.3; 536/23.72; 977/802 |
Current CPC
Class: |
C12N 9/1247 20130101;
C12N 7/00 20130101; A61K 48/0091 20130101; A61P 31/16 20180101;
A61P 37/04 20180101; A61P 35/00 20180101; C12N 15/86 20130101; C12N
9/0059 20130101; Y02A 50/30 20180101; C12N 2710/24143 20130101;
A61P 31/12 20180101; C07K 14/005 20130101; A61P 31/18 20180101;
A61P 31/22 20180101; C12N 2740/16322 20130101; A61P 31/00 20180101;
Y02A 50/412 20180101; A61K 2039/5256 20130101 |
Class at
Publication: |
424/199.1 ;
435/069.3; 435/456; 435/325; 435/235.1; 536/023.72; 977/802 |
International
Class: |
A61K 39/12 20060101
A61K039/12; C07H 21/02 20060101 C07H021/02; C12N 7/00 20060101
C12N007/00; C12N 15/86 20060101 C12N015/86 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 4, 1995 |
DK |
DK 0782/95 |
Claims
1-30. (canceled)
31. A method of inducing an immune response against human
tyrosinase in a melanoma patient comprising: introducing a Modified
Vaccinia Virus Ankara (MVA) containing a foreign gene encoding
human tyrosinase into cells from the patient in vitro, such that
the cells express human tyrosinase; and transferring the cells
expressing tyrosinase back into to the patient to induce an immune
response against human tyrosinase.
32. The method of claim 31, wherein the foreign gene encoding human
tyrosinase is inserted at a site of a naturally occurring deletion
within an MVA genome selected from the group consisting of deletion
site I, site II, site IV, site V, and site VI, and site VI.
33. The method of claim 32, wherein the site of a naturally
occurring deletion within an MVA genome is deletion site I.
34. The method of claim 32, wherein the site of a naturally
occurring deletion within an MVA genome is deletion site II.
35. The method of claim 32, wherein the site of a naturally
occurring deletion within an MVA genome is deletion site IV.
36. The method of claim 32, wherein the site of a naturally
occurring deletion within an MVA genome is deletion site V.
37. The method of claim 32, wherein the site of a naturally
occurring deletion within an MVA genome is deletion site VI.
38. The method of claim 31, wherein the foreign gene is under the
control of a T7 RNA polymerase promoter.
39. The method of claim 31, wherein the foreign gene is under the
control of the vaccinia virus early/late promoter p7.5.
Description
RELATED APPLICATION(S)
[0001] This application is a division of U.S. application Ser. No.
10/147,284, filed May 15, 2002, which is a continuation of U.S.
application Ser. No. 09/002,443, filed Jan. 2, 1998, which is a
continuation of International Application No. PCT/EP96/02926, which
designated the United States and was filed on Jul. 3, 1996,
published in English, which claims the benefit of Danish Patent
Application No. DK 0782/95, filed Jul. 4, 1995. The entire
teachings of the above application(s) are incorporated herein by
reference
BACKGROUND OF THE INVENTION
[0002] Vaccinia virus, a member of the genus Orthopoxvirus in the
family of Poxviridae, was used as live vaccine to immunize against
the human smallpox disease. Successful world-wide vaccination with
vaccinia virus culminated in the eradication of variola virus, the
causative agent of the smallpox (The global eradication of
smallpox. Final report of the global commission for the
certification of smallpox eradication. History of Public Health,
No. 4, Geneva: World Health Organization, 1980). Since that WHO
declaration, vaccination has been universally discontinued except
for people at high risk of poxvirus infections (e.g. laboratory
workers).
[0003] More recently, vaccinia viruses have also been used to
engineer viral vectors for recombinant gene expression and for the
potential use as recombinant live vaccines (Mackett, M. et al.,
P.N.A.S. USA, 79:7415-7419 (1982); Smith, G. L et al., Biotech. and
Genetic Engineering Reviews 2:383-407, (1984)). This entails DNA
sequences (genes) which code for foreign antigens being introduced,
with the aid of DNA recombination techniques, into the genome of
the vaccinia viruses. If the gene is integrated at a site in the
viral DNA which is non-essential for the life cycle of the virus,
it is possible for the newly produced recombinant vaccinia virus to
be infectious, that is to say able to infect foreign cells and thus
to express the integrated DNA sequence (EP Patent Applications No.
83,286 and No. 110,385)). The recombinant vaccinia viruses prepared
in this way can be used, on the one hand, as live vaccines for the
prophylaxis of infectious diseases, on the other hand, for the
preparation of heterologous proteins in eukaryotic cells.
[0004] Recombinant vaccinia virus expressing the bacteriophage T7
RNA polymerase gene allowed the establishment of widely applicable
expression systems for the synthesis of recombinant proteins in
mammalian cells (Moss, B., et al., Nature, 348:91-92 (1990)). In
all protocols, recombinant gene expression relies on the synthesis
of the T7 RNA polymerase in the cytoplasm of eukaryotic cells. Most
popular became a protocol for transient-expression (Fuerst, T. R.,
et al., Proc. Natl. Acad. Sci. USA, 83:8122-8126 (1986) and U.S.
patent application 7,648,971)). First, a foreign gene of interest
is inserted into a plasmid under the control of the T7 RNA
polymerase promoter. In the following, this plasmid is introduced
into the cytoplasm of cells infected with a recombinant vaccinia
virus producing T7 RNA polymerase using standard transfection
procedures.
[0005] This transfection protocol is simple because no new
recombinant viruses need to be made and very efficient with greater
than 80% of the cells expressing the gene of interest (Elroy-Stein,
O. and Moss, B., Proc. Natl. Acad. Sci. USA, 87:6743-6747 (1990)).
The advantage of the vaccinia virus/T7 RNA polymerase hybrid system
over other transient expression systems is very likely its
independence on the transport of plasmids to the cellular nucleus.
In the past, the system has been extremely useful for analytical
purposes in virology and cell biology (Buonocore, L. and Rose, J.
K, Nature, 345:625-628, (1990); Pattnaik, A. K and Wertz, G. W.,
Proc. Natl. Acad. Sci. USA, 88:1379-1383 (1991); Karschin, A.
etal., FEBS Lett. 278: 229-233 (1991), Ho, B. Y. et al., FEBS
Lett., 301:303-306 (1992); Buchholz, C. J. et al., Virology,
204:770-776 (1994)). However, important future applications of the
vaccinia virus/T7 RNA polymerase hybrid system, as e.g. to generate
recombinant proteins or recombinant viral particles for novel
therapeutic or prophylactic approaches in humans, might be hindered
by the productive replication of the recombinant vaccinia
vector.
[0006] Vaccinia virus is infectious for humans and upon vaccination
during the smallpox eradication campaign occasional serious
complications were observed. The best overview about the incidence
of complications is given by a national survey in the United States
monitoring vaccination of about 12 million people with a vaccine
based on the New York City Board of Health strain of vaccinia virus
(Lane, J. et al. New Engl. J Med., 281:1201-1208,(1969)). Therefore
the most exciting possibility to use vaccinia virus as vector for
the development of recombinant live vaccines has been affected by
safety concerns and regulations. Furthermore, most of the
recombinant vaccinia viruses described in the literature are based
on the Western Reserve strain of vaccinia virus. On the other hand,
it is known that this strain has a high neurovirulence and is thus
poorly suited for use in humans and animals (Morita et al.,
Vaccine, 5:65-70 (1987)).
[0007] For vector applications health risks would be lessened by
the use of a highly attenuated vaccinia virus strain. Several such
strains of vaccinia virus were especially developed to avoid
undesired side effects of smallpox vaccination. Thus, the modified
vaccinia virus Ankara (MVA) has been generated by long-term serial
passages of the Ankara strain of vaccinia virus (CVA) on chicken
embryo fibroblasts (for review see Mayr, A., et al., Infection,
3:6-14 (1975); Swiss Patent No. 568,392)). The MVA virus was
deposited in compliance with the requirements of the Budapest
Treaty at CNCM (Institut Pasteur, Collection Nationale de Cultures
Microorganisms, 25, rue du Docteur Roux, 75724 Paris Cedex 15) on
Dec. 15, 1987 under Depositary No. 1-721. MVA is distinguished by
its great attenuation, that is to say by diminished virulence or
infectiosity while maintaining good immunogenicity. The MVA virus
has been analyzed to determine alterations in the genome relative
to the wild CVA strain. Six major deletions of genomic DNA
(deletion I, II, III, IV, V, and VI) totaling 31,000 base pairs
have been identified (Meyer, H., et al., J. Gen. Virol.
72:1031-1038 (1991)). The resulting MVA virus became severely host
cell restricted to avian cells.
[0008] Furthermore, MVA is characterized by its extreme
attenuation. When tested in a variety of animal models, MVA was
proven to be a virulent even in immunosuppressed animals. More
importantly, the excellent properties of the MVA strain have been
demonstrated in extensive clinical trials (Mayr et al., Zbl. Bakt.
Hyg. I, Abt. Org. B 167:375-390 (1987), Stickl et al., Dtsch. med.
Wschr. 99:2386-2392 (1974)). During these studies in over 120,000
humans, including high risk patients, no side effects were
associated with the use of MVA vaccine.
[0009] MVA replication in human cells was found to be blocked late
in infection preventing the assembly to mature infectious virions.
Nevertheless, MVA was able to express viral and recombinant genes
at high levels even in non-permissive cells and was proposed to
serve as an efficient and exceptionally safe gene expression vector
(Sutter, G. and Moss, B., Proc. Natl. Acad. Sci. USA 89:10847-10851
(1992)). Recently, novel vaccinia vector systems were established
on the basis of MVA, having foreign DNA sequences inserted at the
site of deletion III within the MVA genome or within the TK gene
(Sutter, G. and Moss, B. Dev. Biol. Stand. Basel, Karger 84:195-200
(1995) and U.S. Pat. No. 5,185,146)).
[0010] To further exploit the use of MVA a novel possible way to
introduce foreign genes by DNA recombination into the MVA strain of
vaccinia virus has been sought. Since the intention was not to
alter the genome of the MVA virus, it was necessary to use a method
which complied with this requirement. According to the present
invention a foreign DNA sequence was recombined into the viral DNA
precisely at the site of a naturally occurring deletion in the MVA
genome.
SUMMARY OF THE INVENTION
[0011] The present invention thus, inter alia, comprises the
following, alone or in combination: [0012] A recombinant MVA virus
containing and capable of expressing at least one foreign gene
inserted at the site of a naturally occurring deletion within the
MVA genome; [0013] a recombinant MVA virus as above containing and
capable of expressing at least one foreign gene inserted at the
site of deletion II within the MVA genome; [0014] a recombinant MVA
virus as above wherein the foreign gene codes for a marker, a
therapeutic gene or an antigenic determinant; [0015] a recombinant
MVA virus as above wherein the foreign gene codes for an antigenic
determinant from a pathogenic virus, a bacteria, or other
microorganism, or from a parasite, or a tumor cell; [0016] a
recombinant MVA virus as above wherein the foreign gene codes for
an antigenic determinant from Plasmodium Falciparum, Mycobacteria,
Herpes virus, influenza virus, hepatitis, or human immunodeficiency
viruses. [0017] a recombinant MVA virus as above wherein the
antigenic determinant is HIV nef or human tyrosinase; [0018] a
recombinant MVA virus as above which is MVA-LAInef or MVA-hTYR;
[0019] a recombinant MVA virus as above wherein the foreign gene
codes for T7 RNA polymerase; [0020] a recombinant MVA virus as
above which is MVA-T7 pol; [0021] a recombinant MVA virus as above
wherein the foreign gene is under transcriptional control of the
vaccinia virus early/late promoter P7.5; [0022] recombinant MVA
viruses as above essentially free from viruses being able to
replicate in human cells; [0023] the use of a recombinant MVA virus
as above for the transcription of DNA sequences under
transcriptional control of a T7 RNA polymerase promoter; [0024] a
eukaryotic cell infected by a recombinant MVA virus as any above;
[0025] a cell infected by a recombinant MVA virus as above wherein
the foreign gene code for T7 RNA polymerase; [0026] a cell infected
by a recombinant MVA virus as above wherein the foreign gene code
for T7 RNA polymerase, additionally containing one or more
expression vectors carrying one or more foreign genes under
transcriptional control of a T7 RNA polymerase promoter; [0027] the
use of cells as above for the production of the polypeptides
encoded by said foreign genes comprising: [0028] a) culturing said
cells under suitable conditions, and [0029] b) isolating the
polypeptides encoded by said foreign genes. [0030] a cell infected
by a recombinant MVA virus as above wherein the foreign gene code
for T7 RNA polymerase, additionally containing expression vectors
carrying viral genes, and/or a viral vector construct encoding the
genome of a viral vector under transcriptional control of a T7 RNA
polymerase promoter; [0031] the use of a cells as above for the
production viral particles comprising: [0032] a) culturing said
cells under suitable conditions, and [0033] b) isolating the viral
particles; [0034] a cell infected by a recombinant MVA virus as
above wherein the foreign gene code for T7 RNA polymerase,
additionally containing [0035] a) an expression vector carrying a
retroviral vector construct capable of infecting and directing the
expression in target cells of one or more foreign genes carried by
said retroviral vector construct, and [0036] b) one or more
expression vectors carrying the genes encoding the polypeptides
required for the genome of said retroviral vector construct to be
packaged under transcriptional control of a T7 RNA polymerase
promoter; [0037] the use of cells as above for the production of
retroviral particles comprising [0038] a) culturing said cells
under suitable conditions, and [0039] b) isolating the retroviral
particles; [0040] a vaccine containing a recombinant MVA virus as
above wherein the foreign gene code for an antigenic determinant in
a physiologically acceptable carrier; [0041] the use of a
recombinant MVA virus as above wherein the foreign gene code for an
antigenic determinant preparation of a vaccine; [0042] the use of a
vaccine as above for the immunization of a living animal body,
including a human; [0043] the use of a vaccine as above containing
MVA-LAInef for the prevention or treatment of HIV infection or
AIDS; [0044] the use of a vaccine as above containing MVA-hTYR for
the prevention or treatment of melanomas; [0045] a vaccine
comprising as a first component, a recombinant MVA virus as above
wherein the foreign gene code for T7 RNA polymerase in a
physiologically acceptable carrier, and as a second component a DNA
sequence carrying an antigenic determinant under transcriptional
control of a T7 RNA polymerase promoter in a physiologically
acceptable carrier, the two components being contained together or
separate; [0046] the use of a vaccine as above for the immunization
of a living animal body, including a human, comprising inoculation
of said living animal body, including a human, with the first and
second component of the vaccine either simultaneously or with a
timelag using the same inoculation site; and [0047] The term "gene"
means any DNA sequence which codes for a protein or peptide.
[0048] The term "foreign gene" means a gene inserted in a DNA
sequence in which it is not normally found.
[0049] The foreign gene can be a marker gene, a therapeutic gene, a
gene encoding an antigenic determinant, or a viral gene, for
example. Such genes are well known in the art.
BRIEF DESCRIPTION OF THE FIGURES
[0050] FIG. 1 is a schematic map of the genome of MVA and plasmid
for insertion of foreign DNA by homologous recombination: HindIII
restriction sites within the genome of MVA are indicated at the
top; the 900-bp HindIII-HindIII N fragment that overlaps the
junction of deletion II within the MVA genome is shown; MVA DNA
sequences adjacent to deletion II (flank 1 and flank 2) were
amplified by PCR and used for the construction of insertion plasmid
pUC II LZ.
[0051] FIG. 2 is a schematic map of pUC II LZ P7.5: MVA vector
plasmid for insertion into deletion II containing P1l-LacZ
expression cassette and the vaccinia virus early/late promoter P7.5
to express genes of interest that can be cloned into the SmaI site
of the plasmid.
[0052] FIG. 3 is a schematic map of pUCII LZdel P7.5: MVA vector
plasmid for insertion of foreign genes at the site of deletion II
in the MVA genome, containing a self-deleting P1l-LacZ expression
cassette and the vaccinia virus early/late promoter P7.5 to express
genes of interest that can be cloned into the SmaI/Notl cloning
site of the plasmid.
[0053] FIG. 4 is a schematic map of the construction of recombinant
virus MVA-T7pol: schematic maps of the MVA genome (HindIII
restriction endonuclease sites) and the vector plasmid pUC II LZ
T7pol that allows insertion of the T7 RNA polymerase gene at the
site of deletion II within the HindIII N fragment of the MVA
genome.
[0054] FIG. 5 is a schematic map of the construction of MVA-LAInef:
schematic maps of the MVA genome (HindIII restriction endonuclease
sites) and the vector plasmid pUC II LZdel P7.5-LAInef that allows
insertion of the nef gene of HIV-1 LAI at the site of deletion II
within the HindIII N fragment of the MVA genome.
[0055] FIG. 6 is a schematic map of the construction of MVA-hTYR:
schematic maps of the MVA genome (HindIII restriction endonuclease
sites) and the vector plasmid pUC II LZdel P7.5-TYR that allows
insertion of the human tyrosinase gene at the site of deletion II
within the HindIII N fragment of the MVA genome.
DETAILED DESCRIPTION OF THE INVENTION
[0056] It is an object of the present invention to provide a
recombinant MVA virus which can serve as an efficient and
exceptionally safe expression vector.
[0057] Another object of the present invention is to provide a
simple, efficient and safe method for the production of
polypeptides, e.g. antigens or therapeutic agents, recombinant
viruses for vaccines and viral vectors for gene therapy.
[0058] Still another object of the present invention is to provide
an expression system based on a recombinant MVA virus expressing T7
RNA polymerase, and methods for the production of polypeptides,
e.g. antigens or therapeutic agents, or for generating viral
vectors for gene therapy or vaccines, based on this expression
system.
The Present Invention
[0059] Modified vaccinia virus Ankara (MVA), a host range
restricted and highly attenuated vaccinia virus strain, is unable
to multiply in human and most other mammalian cell lines tested.
But since viral gene expression is unimpaired in non-permissive
cells the recombinant MVA viruses according to the invention may be
used as exceptionally safe and efficient expression vectors.
Recombinant MVA Viruses Encoding an Antigenic Determinant
[0060] In one embodiment, the present invention relates to
recombinant MVA vaccinia viruses which contain a gene which codes
for a foreign antigen, preferably of a pathogenic agent, and
vaccines containing such a virus in a physiologically acceptable
form. The invention also relates to methods for the preparation of
such recombinant MVA vaccinia viruses or vaccines, and to the use
of these vaccines for the prophylaxis of infections caused by such
pathogenic agents.
[0061] In a preferred embodiment of the invention, the foreign gene
inserted in the MVA virus is a gene encoding HIV nef.
[0062] We have constructed recombinant MVA viruses that allow
expression of the HIV-1 nef gene under the control of the vaccinia
virus early/late promoter P7.5. The regulatory Nef protein of
primate lentiviruses is synthesized early in the viral replication
cycle and has been shown to be essential for high titer virus
replication and disease induction in vivo. This suggests that HIV
Nef might play a crucial role in AIDS pathogenesis. The molecular
mechanism(s) by which Nef contributes to increased viral
infectivity and to HIV pathogenicity need to be further elucidated.
However, Nef is immunogenic and Nef-specific antigen can be used as
a vaccine against HIV infection and AIDS.
[0063] In this context, the recombinant MVA virus expressing the
HIV nef gene can be used for immunization of human beings, on one
hand, as a prophylactic vaccine against human HIV, and on the other
hand, for immunotherapy of HIV infected or AIDS patients.
Furthermore, the recombinant MVA virus expressing the HIV nef gene
can be used for the production of recombinant HIV Nef protein.
[0064] In another preferred embodiment of the invention the foreign
gene inserted in the MVA virus is a gene encoding human
tyrosinase.
[0065] We have constructed recombinant MVA viruses that allow
expression of the human tyrosinase gene under the control of the
vaccinia virus early/late promoter P7.5. Recently, human tyrosinase
was identified as a melanoma-specific tumor antigen that allows
generation of anti-tumor cytolytic T-lymphocytes (Beichard, V., et
al., J. Exp. Med., 178:489-495 (1993)). Since among normal cells,
only melanocytes appear to express the tyrosinase gene, tyrosinase
is a useful target antigen for immunotherapy of melanomas.
Therefore, the recombinant MVA virus expressing the human
tyrosinase gene can be used in melanoma patients to induce immune
responses that provoke tumor rejection or prevent metastasis.
Recombinant MVA virus expressing the human tyrosinase gene can be
used directly as an anti-melanoma vaccine, or the virus can be used
to prepare anti-melanoma vaccines. In one example, the recombinant
MVA virus expressing the human tyrosinase gene can be used for the
production of recombinant tyrosinase protein which is used as
antigen in vaccine preparations. In another example, using the
recombinant MVA virus expressing the human tyrosinase gene as
expression vector, cells derived from a tumor patient can be
modified in vitro to express tyrosinase and then transferred back
to the patient to induce anti-tumor immune responses. A vaccine
prepared on the basis of recombinant MVA expressing the human
tyrosinase gene can be used either parenterally or locally at the
site of the tumor. To prevent tumor metastasis or to phenotypically
change the tumor e.g. in size, shape, consistency, vascularization
or other features. A vaccine prepared on the basis of recombinant
MVA expressing the human tyrosinase gene can be used before,
during, or after surgical extirpation of the tumor.
[0066] For the preparation of vaccines, the MVA vaccinia viruses
according to the invention are converted into a physiologically
acceptable form. This can be done based on the experience in the
preparation of MVA vaccines used for vaccination against smallpox
(as described by Stickl, H. et al., Dtsch. med. Wschr. 99:2386-2392
(1974)). Typically, about 10.sup.6-10.sup.8 particles of the
recombinant MVA are freeze-dried in 100 ml of phosphate-buffered
saline (PBS) in the presence of 2% peptone and 1% human albumin in
an ampoule, preferably a glass ampoule. The lyophilisate can
contain extenders (such as mannitol, dextran, sugar, glycine,
lactose or polyvinylpyrrolidone) or other aids (such as
antioxidants, stabilizers, etc.) suitable for parenteral
administration. The glass ampoule is then sealed and can be stored,
preferably at temperatures below -20.degree. C., for several
months.
[0067] For vaccination or therapy the lyophilisate can be dissolved
in 0.1 to 0.5 ml of an aqueous solution, preferably physiological
saline, and administered either parenterally, for example by
intramuscular inoculation or locally, for example by inoculation
into a tumor or at the site of a tumor. Vaccines or therapeutics
according to the invention are preferably injected intramuscularly
(Mayr, A. et al., Zbl. Bakt. Hyg., I. Abt. Orig. B 167:375-390
(1978)). The mode of administration, the dose and the number of
administrations can be optimized by those skilled in the art in a
known manner. It is expedient where appropriate to administer the
vaccine several times over a lengthy period in order to obtain
appropriate immune responses against the foreign antigen.
The Use of Recombinant MVA Viruses for the Production of
Heterologous Polypeptides
[0068] The recombinant MVA vaccinia viruses according to the
invention can also be used to prepare heterologous polypeptides in
eukaryotic cells. This entails cells being infected with the
recombinant vaccinia viruses. The gene which codes for the foreign
polypeptide is expressed in the cells, and the expressed
heterologous polypeptide is isolated. The methods to be used for
the production of such heterologous polypeptides are generally
known to those skilled in the art (EP-A-206,920 and EP-A-205,939).
The polypeptides produced with the aid of the recombinant MVA
viruses are, by reason of the special properties of the MVA
viruses, more suitable for use as medicaments in humans and
animals.
Recombinant MVA Viruses Encoding T7 RNA Polymerase and the Use
Thereof for the Expression of DNA Sequences Under Transcriptional
Control of a T7 RNA Polymerase Promoter
[0069] In a further embodiment of the present invention we have
constructed recombinant MVA viruses that allow expression of the
bacteriophage T7 RNA polymerase gene under the control of the
vaccinia virus early/late promoter P7.5. The usefulness of
MVA-T7pol recombinant viruses as expression system has been tested
in transient transfection assays to induce expression of
recombinant genes under the control of a T7 RNA polymerase
promoter. Using the E. coli chloramphenicol acetyltransferase (CAT)
gene as a reporter gene we found that MVA-T7pol induced CAT gene
expression as effectively as a vaccinia/T7pol recombinant virus
derived from the replication-competent WR strain of vaccinia
virus.
[0070] The MVA/T7 polymerase hybrid system according to the
invention can thus be used as a simple, efficient and safe
mammalian expression system for production of polypeptides in the
absence of productive vaccinia virus replication.
[0071] This expression system can also be used for generating
recombinant viral particles for vaccination or gene therapy by
transformation of cell lines infected with recombinant MVA
expressing T7 RNA polymerase, with DNA-constructs containing all or
some of the genes, and the genome or recombinant genome necessary
for generating viral particles, e.g MVA particles or retroviral
particles, under transcriptional control of a T7 RNA polymerase
promoter.
[0072] Retroviral vector systems consist of two components: [0073]
1) the retroviral vector itself is a modified retrovirus (vector
plasmid) in which the genes encoding for the viral proteins have
been replaced by therapeutic genes and marker genes to be
transferred to the target cell. Since the replacement of the genes
encoding for the viral proteins effectively cripples the virus it
must be rescued by the second component in the system which
provides the missing viral proteins to the modified retrovirus.
[0074] The second component is: [0075] 2) a cell line that produces
large quantities of the viral proteins, however lacks the ability
to produce replication competent virus. This cell line is known as
the packaging cell line and consists of a cell line transfected
with one or more plasmids carrying the genes (genes encoding the
gag, pol and env polypeptides) enabling the modified retroviral
vector to be packaged.
[0076] To generate the packaged vector, the vector plasmid is
transfected into the packaging cell line. Under these conditions
the modified retroviral genome including the inserted therapeutic
and marker genes is transcribed from the vector plasmid and
packaged into the modified retroviral particles (recombinant viral
particles). This recombinant virus is then used to infect target
cells in which the vector genome and any carried marker or
therapeutic genes becomes integrated into the target cell's DNA. A
cell infected with such a recombinant viral particle cannot produce
new vector virus since no viral proteins are present in these
cells. However, the DNA of the vector carrying the therapeutic and
marker genes is integrated in the cell's DNA and can now be
expressed in the infected cell.
[0077] The recombinant MVA virus according to the invention
expressing T7 RNA polymerase can be used to produce the proteins
required for packaging retroviral vectors. To do this the gag, pol
and env genes of a retrovirus (e.g. the Murine Leukemia Virus
(MLV)) are placed under transcriptional control of a T7 RNA
polymerase promoter in one or more expression vectors (e.g.
plasmids). The expression vectors are then introduced into cells
infected with the recombinant MVA virus expressing T7 RNA
polymerase, together with an expression vector carrying a
retroviral vector construct, possibly under transcriptional control
of a T7 RNA polymerase promoter.
[0078] WO 94/2943 7, WO 89/11539 and WO 96/7748 describes different
types of retroviral vector which can be packaged using the
packaging system described above.
[0079] A further use of the recombinant MVA virus expressing T7 RNA
polymerase is to generate recombinant proteins, noninfectious virus
particles, or infectious mutant virus particles for the production
of vaccines or therapeutics (Buchholz et al., Virology, 204:770-776
(1994) and EP-B1-1356695)). To do this viral genes (e.g. the
gag-pol and env genes of HIV-1) are placed under transcriptional
control of the T7 promoter in an expression vector (e.g. plasmid or
another recombinant MVA virus). This construct is then introduced
into cells infected with the recombinant MVA virus expressing T7
RNA polymerase. The recombinant viral genes are transcribed with
high efficiency, recombinant proteins are made in high amounts and
can be purified. Additionally, expressed recombinant viral proteins
(e.g., HIV-1 env, gag) may assemble to viral pseudo-particles that
budd from the cells and can be isolated from the tissue culture
medium. In another embodiment, viral proteins (from e.g. HIV, SIV,
Measles virus) expressed by the MVA-T7 pol system may rescue an
additionally introduced mutant virus (derived from e.g. HIV, SIV,
Measles virus) by overcoming a defect in attachment and infection,
uncoating, nucleic acid replication, viral gene expression,
assembly, budding or another step in viral multiplication to allow
production and purification of the mentioned mutant virus.
[0080] MVA-T7pol can also be used together with DNA sequences
carrying the gene of an antigen of interest (e.g. the gene of HIV,
nef, tat, gag, pol, or env or others) for immunization. First, a
coding sequence of a given antigen (e.g HIV, HCV, HPV, HSV, measles
virus, influenza virus or other) are cloned under control of a T7
RNA polymerase promoter preferably in a plasmid vector and the
resulting DNA construct is amplified and purified using standard
laboratory procedures. Secondly, the vector DNA is inoculated
simultaneously or with appropriate limelags together with
MVA-T7pol. At the site of inoculation the recombinant gene of
interest is expressed transiently in cells containing both the
vector DNA and MVA-T7 pol and the corresponding antigen is
presented to the host immune system stimulating an antigen-specific
immune response. This protocol using the non-replication vaccinia
with MVA-T7 pol represents a promising novel approach to nucleic
acid vaccination allowing efficient transient expression of a given
antigen, but avoiding the potential risk of constitutive gene
expression.
The Recombinant MVA Vaccinia Viruses can be Prepared as Set Out
Hereinafter
[0081] A DNA-construct which contains a DNA-sequence which codes
for a foreign polypeptide flanked by MVA DNA sequences adjacent to
a naturally occurring deletion, e.g. deletion II, within the MVA
genome, is introduced into cells infected with MVA, to allow
homologous recombination.
[0082] Once the DNA-construct has been introduced into the
eukaryotic cell and the foreign DNA has recombined with the viral
DNA, it is possible to isolate the desired recombinant vaccinia
virus in a manner known per se, preferably with the aid of a marker
(compare Nakano et al., Proc. Natl. Acad. Sci. USA, 79:1593-1596
(1982); Franke et al., Mol. Cell. Biol, 1918-1924 (1985);
Chakrabarfi et al., Mol. Cell. Biol., 3403-3409 (1985); Fathi et
al., Virology 97-105 (1986)).
[0083] The DNA-construct to be inserted can be linear or circular.
A circular DNA is preferred, especially a plasmid. The
DNA-construct contains sequences flanking the left and the right
side of a naturally occurring deletion, e.g. deletion II, within
the MVA genome (Altenburger, W., Suter, C. P. and Altenburger J.,
Arch. Virol., 105:15-27 (1989)). The foreign DNA sequence is
inserted between the sequences flanking the naturally occurring
deletion. The foreign DNA sequence can be a gene coding for a
therapeutic polypeptide, e.g. t-PA or interferon, or an antigenic
determinant from a pathogenic agent. Pathogenic agents can be
viruses, bacteria and parasites which may cause a disease, as well
as tumor cells which multiply unrestrictedly in an organism and may
thus lead to pathological growths. Examples of such pathogenic
agents are described in Davis, B. D. et al., (Microbiology, 3rd
ed., Harper international Edition). Preferred antigens of
pathogenic agents are those of human immunodeficiency viruses (e.g.
HIV-1 and HIV-2), of mycobacteria causing tuberculosis, of the
parasite Plasmodium Falciparum, and of melanoma cells.
[0084] For the expression of a DNA sequence or gene, it is
necessary for regulatory sequences, which are required for the
transcription of the gene, to be present on the DNA. Such
regulatory sequences (called promoters) are known to those skilled
in the art, and includes for example those of the vaccinia 11 kDa
gene as are described in EP-A-198,328, and those of the 7.5 kDa
gene (EP-A-110,385).
[0085] The DNA-construct can be introduced into the MVA infected
cells by transfection, for example by means of calcium phosphate
precipitation (Graham et al., Virol., 52:456-467 (1973); Wigler et
al., Cell 777-785 (1979)) by means of electroporation (Neumann et
al., EMBO J., 1:841-845 (1982)), by microinjection (Graessmann et
al., Meth. Enzymol. 101:482-492 (1983)), by means of liposomes
(Straubinger et al., Methods in Enzymology, 101:512-527 (1983)), by
means of spheroplasts (Schaffner, Proc. Natl. Acad. Sci. USA,
77:2163-2167 (1980)) or by other methods known to those skilled in
the art. Transfection by means of calcium phosphate precipitation
is preferred.
[0086] The detailed examples which follow are intended to
contribute to a better understanding of the present invention.
However, it is not intended to give the impression that the
invention is confined to the subject-matter of the examples.
EXAMPLES
1. Growing and Purification of the Viruses
1.1 Growing of the MVA Virus
[0087] The MVA virus is a highly attenuated vaccinia virus derived
from the vaccinia virus strain Ankara (CVA) by long-term serial
passages on primary chicken embryo fibroblast (CEF) cultures. For a
general rewiew of the history of the production, the properties and
the use of MVA strain, reference may be made to the summary
published by Mayr et al., in Infection, 3:6-14 (1975). Due to the
attenuation in CEF, the MVA virus replicates to high titers in this
avain host cell. In mammalian cells, however, MVA is severely
growth restricted, and typical plaque formation by the virus is not
detectable. Therefore, MVA virus was grown on CEF cells. To prepare
CEF cells, 1-day-old embryos were isolated from incubated chicken
eggs, the extremities are removed, and the embryos are minced and
dissociated in a solution composed of 0.25% trypsin at 37.degree.
C. for 20 minutes. The resulting cell suspension was filtered and
cells were sedimented by centrifugation at 2000 rpm in a Sorvall
RC-3B centrifuge at room temperature for 5 minutes, resuspended in
10 volumes of medium A (MEM Eagle, for example obtainable from Life
Technologies GmbH, Eggenstein, Germany), and sedimented again by
centrifugation at 2000 rpm in a Sorvall RC-3B centrifuge at room
temperature for 5 minutes. The cell pellet was reconstituted in
medium A containing 10% fetal calf serum (FCS), penicillin (100
units/mi), streptomycin (100 mg/ml) and 2 mM glutamine to obtain a
cell suspension containing 500,000 cells/ml. CEF cells obtained in
this way were spread on cell culture dishes. They were left to grow
in medium A in a CO.sub.2 incubator at 37.degree. C. for 1-2 days,
depending on the desired cell density, and were used for infection
either directly or after one further cell passage. A detailed
description of the preparation of primary cultures can be found in
the book by R. I. Freshney, "Culture of animal cell, Alan R. Liss
Verlag, New York (1983) Chapter 11, page 99 et seq.
[0088] MVA viruses were used for infection as follows. CEF cells
were cultured in 175 cm.sup.2 cell culture bottles. At 90-100%
confluence, the medium was removed and the cells were incubated for
one hour with an MVA virus suspension (0.01 infectious units (IU)
per cell, 0.02 ml/cm.sup.2) in medium A. Then more medium A was
added (0.2 ml/cm.sup.2) and the bottles were incubated at
37.degree. C. for 2-3 days (until about 90% of the cells show
cytopathogenic effect). Crude virus stocks were prepared by
scraping cell monolayers into the medium and pelleting the cell
material by centrifugation at 3000 rpm in a Sorvall RC-3B
centrifuge at 4.degree. C. for 5 minutes. The crude virus
preparation was stored at -20.degree. C. before processing (e.g.,
virus purification).
1.2 Purification of the Viruses
[0089] The purification steps undertaken to obtain a virus
preparation which was as pure as possible and free from components
specific to the host cell were similar to those described by
Joklik, Virology, 18:9-18 (1962)). Crude virus stocks which had
been stored at -20.degree. C. were thawed and suspended once in PBS
(10-20 times the volume of the sediment), and the suspension was
centrifuged as above. The new sediment was suspended in 10 times
the volume of Tris buffer 1 (10 mM Tris-HCl pH 9.0,), and the
suspension was briefly treated with ultrasound (Labsonic L, B.Braun
Biotech International, Melsungen Germany; 2.times.10 seconds at 60
watts and room temperature) in order to further disintegrate cell
debris and to liberate the virus particles from the cellular
material. The cell nuclei and the larger cell debris were removed
in the subsequent brief centrifugation of the suspension (Sorvall
GSA rotor obtainable from DuPont Co., D-6353 Bad Nauheim, FRG; 3
minutes at 3000 rpm and 10.degree. C.). The sediment was once again
suspended in Tris buffer 1, treated with ultrasound and
centrifuged, as described above. The collected supernatants
containing the free virus particles were combined and layered over
a cushion of 10 ml of 36% sucrose in 10 mM Tris-HCl, pH 9.0, and
centrifuged in a Beckman SW 27/SW 28 rotor for 80 minutes with
13,500 rpm at 40.degree. C. The supernatant was discarded, and the
sediment containing the virus particles was taken up in 10 ml of 1
mM Tris-HCl, pH 9.0, homogenized by brief treatment with ultrasound
(2.times.10 seconds at room temperature, apparatus as described
above), and applied to a 20-40% sucrose gradient (sucrose in 1 mM
Tris-HCl, pH 9.0) for further purification. The gradient was
centrifuged in Beckmann SW41 rotor at 13,000 rpm for 50 minutes at
4.degree. C. After centrifugation, discrete bands containing virus
particles were harvested by pipetting after aspirating volume above
band. The obtained sucrose solution was diluted with three volumes
PBS and the virus particles were sedimented again by centrifugation
(Beckmann SW 27/28, 60 minutes at 13,500 rpm, 4.degree. C.). The
pellet, which now consisted mostly of pure virus particles, was
resuspended in PBS and equilibrated to virus concentrations
corresponding on average to 1-5 .times.109 IU/ml. The purified
virus stock solution was stored at -80.degree. C. and used either
directly or diluted with PBS for subsequent experiments.
1.3 Cloning of MVA Virus
[0090] To generate homogeneous stock virus preparations MVA virus
obtained from Prof. Anton Mayr was cloned by limiting dilution
during three consecutive passages in CEF cultured on 96-well tissue
culture plates. The MVA clone F6 was selected and amplified in CEF
to obtain working stocks of virus that served as starting material
for the generation of recombinant MVA viruses described in this
patent application as well as for the generation of recombinant MVA
viruses described previously (Sutter, G. and Moss, B., Proc. Natl.
Acad. Sci. USA, 89:10847-10851 (1992); Sutter, G. et al., Vaccine,
12:1032-1040 (1994); Hirsch, V. et al., J. Virol., 70:3741-3752
(1996)).
2. Construction and Characterization of Recombinant MVA Viruses
2.1. Construction of Vector Plasmids
[0091] To allow the generation of recombinant MVA viruses novel
vector plasmids were constructed. Insertion of foreign genes into
the MVA genome was targeted precisely to the site of the naturally
occurring deletion II in the MVA genome. Sequences of MVA DNA
flanking the site of a 2500-bp deletion in the HindIII N fragment
of the MVA genome (Altenburger, W. et al., J. Arch. Virol.,
105:15-27 (1989)) were amplified by PCR and cloned into the
multiple cloning site of plasmid pUC18. The primers for the left
600-bp DNA flank were 5'-CAG CAG GGT ACC CTC ATC GTA CAG GAC GTT
CTC-3' (SEQ ID NO: 1) and 5'-CAG CAG CCC GGG TAT TCG ATG ATT ATT
TTT AAC AAA ATA ACA-3' (SEQ ID NO: 2) (sites for restriction
enzymes Kpnl and Smal are underlined).
[0092] The primers for the right 550-bp DNA flank were 5'-CAG CAG
CTG CAG GAA TCA TCC ATT CCA CTG AAT AGC-3' (SEQ ID NO: 3); and
5'-CAG CAG GCA TGC CGA CGA ACA AGG AAC TGT AGC AGA-3' (SEQ ID NO:
4)(sites for restriction enzymes Pstl and Sphl are underlined).
Between these flanks of MVA DNA inserted in pUC18, the Escherichia
coli LacZ gene under control of the vaccinia virus late promoter
P11 (prepared by restriction digest from pIII LZ, Sutter, G. and
Moss, B., PNAS USA 89:10847-10851(1992)) was inserted, using the
BamHI site, to generate the MVA insertion vector pUCII LZ (FIG. 1).
In the following, a 289 bp fragment containing the vaccinia virus
early-late promoter P7.5 together with a Smal site for cloning
(prepared by restriction digest with EcoRI and Xbal from the
plasmid vector pSC11 (Chakrabarbti et al., Mole. Cell. Biol.,
5:3403-3409 (1985)) was inserted into the Smal site of pUCII LZ to
give the MVA vector pUC II LZ P7.5 [FIG. 2]. To construct a vector
plasmid that allows isolation of recombinant MVA viruses via
transient synthesis of the reporter enzyme .beta.-galactosidase a
330 bp DNA fragment from the 3'-end of the E. coli LacZ open
reading frame was amplified by PCR (primers were 5'-CAG CAG GTC GAC
CCC GAC CGC CTT ACT GCC GCC-3' (SEQ ID NO: 5) and 5'-GGG GGG CTG
CAG ATG GTA GCG ACC GGC GCT CAG-3' (SEQ ID NO: 6)) and cloned into
the SalL and Pstl sites of pUC II LZ P7.5 to obtain the MVA vector
pUC II LZdel P7.5 (FIG. 3). Using the Smal site, this vector
plasmid can be used to insert DNA sequences encoding a foreign gene
under transcriptional control of the vaccinia virus promoter P7.5
into the MVA genome. After the desired recombinant virus has been
isolated by screening for expression of .beta.-galactosidase
activity further propagation of the recombinant virus leads to the
self-deletion of the reengineered P11-LacZ expression cassette by
homologous recombination.
2.2. Construction and Characterization of Recombinant Virus MVA
T7pol
[0093] A 3.1 kbp DNA fragment containing the entire gene of
bacteriophage T7 RNA polymerase under control of the vaccinia virus
early/late promoter P7.5 was excised with EcoRI from plasmid pTF7-3
(Fuerst, T.R. et al., P.N.A.S. USA, 83:8122-8126 (1986), modified
by incubation with Klenow DNA polymerase to generate blunt ends,
and cloned into a unique Smal restriction site of pUCII LZ to make
the plasmid transfer vector pUCII LZ T7pol (FIG. 4). As
transcriptional regulator for the expression of the T7 RNA
polymerase gene the vaccinia virus early/late promoter P7.5 was
chosen. Contrary to stronger vaccinia virus late promoters (e.g.
P11) this promoter system allows expression of recombinant genes
immediately after the infection of target calls. The plasmid pUCII
LZ T7pol that directs the insertion of the foreign-genes into the
site of deletion II of the MVA genome was used to generate the
recombinant virus MVA T7pol.
[0094] CEF cells infected with MVA at a multiplicity of 0.05
TCID.sub.50 per cell were transfected with DNA of plasmid pUCII LZ
T7pol as described previously (Sutter, G, et al., Vaccine,
12:1032-1040 (1994)). Recombinant MVA virus expressing the T7 RNA
polymerase and co-expressing .beta.-D-galactosidase (MVA
P7.5-T7pol) was selected by five consecutive rounds of plaque
purification in CEF cells stained with 5-bromo-4-chloro-3-indolyl
.beta.-D-galactoside (300 .mu.g/ml). Subsequently, recombinant
viruses were amplified by infection of CEF monolayers, and the DNA
was analyzed by PCR to confirm genetic homogeneity of the virus
stock. Southern blot analysis of MVA-T7pol viral DNA demonstrated
stable integration of the recombinant genes at the site of deletion
II within the MVA genome.
[0095] To monitor expression of T7 RNA polymerase by recombinant
MVA T7pol [.sup.35S]-labeled polypeptides from virus infected
tissue culture were analyzed. Monolayers of the monkey kidney cell
line CV-1 grown in 12-well plates were infected with virus at a
multiplicity of 20 TCID.sub.50 per cell. At 3 to 5 hours after
infection, the medium was removed, and the cultures were washed
once with 1 ml of methionine free medium. To each well, 0.2 ml of
methionine-free medium supplemented with 50 .mu.Ci of
[.sup.35S]methionine was added and incubated for 30 minutes at
37.degree. C. Cytoplasmic extracts of infected cells were prepared
by incubating each well in 0.2 ml of 0.5% Nonidet P-40 lysis buffer
for 10 mm at 37.degree. C. and samples were analyzed by SDS-PAGE.
The metabolic labeling of the CV-1 cells with MVA T7pol revealed
the synthesis of two additional polypeptides (i) a protein of about
116,000 Da representing the E. coli .beta.-galactosidase
co-expressed to allow the screening for recombinant virus and (ii)
a 98,000 Da protein with the expected size of the bacteriophage T7
RNA polymerase. The large amount of .beta.-galactocidase made by
MVA T7pol is remarkable. The results from the in vivo labeling
experiments demonstrate a very strong expression of the P11-LacZ
gene construct when inserted into the MVA genome at the site of
deletion II indicating that recombinant genes in MVA vector viruses
might be expressed more efficiently when inserted into this locus
of the MVA genome.
[0096] The usefulness of MVA-T7pol recombinant viruses as
expression system in comparison to the WR-T7pol recombinant virus
vTF7-3 (Fuerst et al. 1986) was tested by the co-transfection of
DNA of a plasmid vector that is derived from pTM1 (Moss, B., et
al., Nature, 348:91-92 (1990)) and contains (cloned into the Ncol
and BamHI sites of the pTM 1 multiple cloning site) the E. coli
chloramphenicol acetyltranferase (CAT) gene under the control of a
T7 RNA polymerase promoter (PT.sub.7). Transfected and infected
CV-1 cells were suspended in 0.2 ml of 0.25 M Tris-HCl (pH 7.5).
After three freeze-thaw cycles, the lysates were cleared by
centrifugation, the protein content of the supernatants was
determined, and samples containing 0.5, 0.25, 0.1 .mu.g total
protein were assayed for enzyme activity as described by Mackett,
M., et al., J. Virol., 49:857-864 (1984). After autoradiography,
labeled spots were quantitated using the Fuji imaging analysis
system.
[0097] The results demonstrate that by using the highly attenuated
vaccinia vector MVA it is possible to exploit the vaccinia virus-T7
RNA polymerase system as efficiently as by using a fully
replication-competent vaccinia virus recombinant.
2.3. Construction and Characterization of Recombinant Virus
MVA-LAInef
[0098] A 648 bp DNA fragment containing the entire nef gene of
HIV-1 LAI was prepared by PCR from plasmid DNA (pTG1166 kindly
provided by M.-P. Kieny, Transgene S. A., Strasbourg; PCR primers
were 5'-CAG CAG GGA TCC ATG GGT GGC AAG TGG TCA AAA AGT AGT-3' (SEQ
ID NO: 7) and 5'-CAG CAG GGA TCC ATG TCA GCA GTT CTT GAA GTA CTC
CGG-3' (SEQ ID NO: 8)), digested with restriction endonuclease
BamHI, modified by incubation with Klenow DNA polymerase to
generate blunt ends, and cloned into the SmaI site of pUC II LZdel
P7.5 to make the vector pUC II LZdel P7.5-LAInef (FIG. 5). This
plasmid could be used to engineer MVA recombinant virus that
expresses the nef gene of HIV-1 LAI under control of the vaccinia
virus early/late promoter P7.5.
[0099] CEF cells infected with MVA at a multiplicity of 0.05
TCID.sub.50 per cell were transfected with DNA of plasmid pUC II
LZdel P7.5-LAInef as described previously (Sutter, G. et al.,
Vaccine, 12:1032-1040 (1994)). Recombinant MVA viruses containing
the nef gene and transiently co-expressing the E. coli LacZ marker
gene were selected by consecutive rounds of plaque purification in
CEF cells stained with 5-bromo-4-chloro-3-indolyl
.beta.-D-galactoside (300 .mu.g/ml). In the following, recombinant
MVA viruses containing the nef gene and having deleted the LacZ
marker gene were isolated by three additional consecutive rounds of
plaque purification screening for non-staining viral foci in CEF
cells in the presence of 5-bromo-4-chloro-3-indolyl
.beta.-galactoside (300 .mu.g/ml). Subsequently, recombinant
viruses were amplified by infection of CEF monolayers, and the
MVA-LAInef viral DNA was analyzed by PCR to confirm genetic
homogeneity of the virus stock. Southern blot of viral DNA
confirmed genetic stability of MVA-LAlnef and precisely
demonstrated integration of the nef gene and deletion of the E.
coli LacZ marker gene at the site of deletion II within the viral
genome.
[0100] Efficient expression of recombinant Nef protein was
confirmed by Western blot analysis of protein lysates from CEF
cells infected with MVA-LAInef using mouse monoclonal antibodies
directed against HIV-1 Nef (kindly provided by K. Krohn and used as
described by Ovod, V. et al., AIDS, 6:25-34 (1992)).
2.4. Construction and Characterization of Recombinant Virus
MVA-hTYR
[0101] A 1.9 kb DNA fragment containing the entire gene encoding
human tyrosinase (Tyrosinase c-DNA clone 123.B2 isolated from the
melanome cell line SK29-MEL of patient SK29 (AV), GenBank Acct. No.
U01873; Brichard, V. et al., J. Exp. Med., 178:489-495 (1993)) was
prepared from the plasmid pcDNAI/Amp-Tyr (Wolfel, T. et al., Eur.
J. Immunol., 24:759-764 (1994)) by EcoRI digest, modified by
incubation with Klenow DNA polymerase to generate blunt ends, and
cloned into the Smal site of pUC II LZdel P7.5 to make the vector
pUC II LZdel P7.5-TYR (FIG. 6).
[0102] This plasmid could be used to engineer MVA recombinant virus
that expresses the human tyrosinase gene under control of the
vaccinia virus early/late promoter P7.5.
[0103] CEF cells infected with MVA at a multiplicity of 0.05
TCID.sub.50 per cell were transfected with DNA of plasmid pUC II
LZdel P7.5-TYR as described previously (Sutter, G, et al., Vaccine,
12:1032-1040 (1994)). Recombinant MVA virus stably expressing the
gene for human tyrosinase and transiently co-expressing the E. coli
LacZ gene was selected by consecutive rounds of plaque purification
in CEF cells stained with 5-bromo-4-chloro-3-indolyl
.beta.-D-galactoside (300 .mu.g/ml). In the following, recombinant
MVA virus expressing the gene encoding human tyrosinase and having
deleted the LacZ marker gene was isolated by three additional
consecutive rounds of plaque purification screening for
non-staining viral foci in CEF cells in the presence of
5-bromo-4-chloro-3-indolyl .beta.-D-galactoside (300 .mu.g/ml).
Subsequently, recombinant viruses were amplified by infection of
CEF monolayers, and the MVA-hTYR viral DNA was analyzed by PCR to
confirm genetic homogeneity of the virus stock. Southern blot
analysis of viral DNA confirmed genetic stability of MVA-hTYR and
precisely demonstrated integration of the recombinant tyrosinase
gene and deletion of the E. coli LacZ marker gene at the site of
deletion II within the viral genome.
[0104] Efficient expression of recombinant human tryosinase was
confirmed by Western blot analysis of protein lysates from CEF
cells infected with MVA-hTYR using rabbit polyclonal antibodies
(kindly provided by V. Hearing and used as described by Jimenez,
M., et al., P.N.A.S. USA, 85:3830-3834 (1988)) or mouse monoclonal
antibodies (kindly provided by L. Old and used as described by
Chen, Y. et al., P.N.A.S. USA 92:8125-8129 (1995)) directed against
tyrosinase.
EQUIVALENTS
[0105] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims. Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
specifically herein. Such equivalents are intended to be
encompassed in the scope of the claims.
Sequence CWU 1
1
8 1 33 DNA Unknown Primer for left 660-bp of MVA DNA flanking the
site of a 2500-bp deletion in the HindIII N fragment of the MVA
genome 1 cagcagggta ccctcatcgt acaggacgtt ctc 33 2 42 DNA Unknown
Primer for left 660-bp of MVA DNA flanking the site of a 2500-bp
deletion in the HindIII N fragment of the MVA genome 2 cagcagcccg
ggtattcgat gattattttt aacaaaataa ca 42 3 36 DNA Unknown Primer for
right 550-bp of MVA DNA flanking the site of a 2500-bp deletion in
the HindIII N fragment of the MVA genome 3 cagcagctgc aggaatcatc
cattccactg aatagc 36 4 36 DNA Unknown Primer for right 550-bp of
MVA DNA flanking the site of a 2500-bp deletion in the HindIII N
fragment of the MVA genome 4 cagcaggcat gccgacgaac aaggaactgt
agcaga 36 5 33 DNA Unknown PCR primer for amplifying E. coli LacZ
open reading frame 5 cagcaggtcg accccgaccg ccttactgcc gcc 33 6 33
DNA Unknown PCR primer for amplifying E. coli LacZ open reading
frame 6 ggggggctgc agatggtagc gaccggcgct cag 33 7 39 DNA Unknown
Primer for amplifying a 648bp DNA fragment containing the entire
nef gene of HIV-1 LAI 7 cagcagggat ccatgggtgg caagtggtca aaaagtagt
39 8 39 DNA Unknown Primer for amplifying a 648bp DNA fragment
containing the entire nef gene of HIV-1 LAI 8 cagcagggat ccatgtcagc
agttcttgaa gtactccgg 39
* * * * *