Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant virus encoding one or more heterologous proteins...
Reexamination Certificate
2001-12-26
2004-01-27
Mosher, Mary E. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Recombinant virus encoding one or more heterologous proteins...
C536S023720, C424S232100, C435S235100, C435S320100, C435S325000, C435S456000, C435S471000
Reexamination Certificate
active
06682742
ABSTRACT:
The present invention provides a new DNA vector comprising a nucleic acid sequence useful for inserting heterologous sequences into the genome of poxviruses by homologous recombination. The present invention relates also, inter alia, to recombinant poxviruses carrying heterologous coding sequences transferred by the vector according to the present invention.
BACKGROUND OF THE INVENTION
The successful worldwide eradication of smallpox via vaccination with live Orthopoxvirus, such as Vaccinia virus strain Western Reserve, Copenhagen or Ankara, stimulated in the early 80′ research to study poxviruses in closer detail. Subsequently, said poxviruses were developed to well understood and easy-to-handle virus vectors or research tools, respectively (Moss, 1996). Today poxvirus vectors are used in various fields e.g. as expression vector or for the development of vaccines and therapeutic substances. The main reasons for the high acceptance of poxvirus vectors are the following promising features: Firstly, the vector viruses are easy to manipulate, are highly stable and cheap to manufacture. Secondly, said vector virus can accommodate large amounts of heterologous DNA and proofed to be a versatile expression vector. Thirdly, said vector virus is easily administered in vivo and succeeded in stimulating humoral and cellular immune responses. Accordingly, its use as a recombinant vaccine for protective immunisation against infectious disease or cancer made poxvirus vectors particularly attractive. Especially, Vaccinia virus, the best-known member of the Orthopoxvirus family, has been successfully used as recombinant vaccine to protect against diseases in a large variety of animal models (Carroll et al., 1997; Sutter et al., 1994a).
To develop and establish recombinant vaccinia viruses several insertion sites have been used. The most prominent insertion site of the vaccinia genome is the locus of the viral thymidine-kinase (tk) gene (Mackett et al., 1982). However, also other non-essential genes, such as the viral hemagglutinin and ribonucleotide reductase genes (Shida et al. 1987, Howley et al. 1996) or the naturally occurring deletion site II or III have been used to insert heterologous DNA sequences into the genome of vaccinia virus (Sutter et al., 1994a). Construction of recombinant vector viruses carrying several heterologous genes or several immunogenic epitopes becomes more and more of general interest. Accordingly, there is a high need to identify further sites in the virus genome, which are suitable to insert further heterologous DNA sequences.
Insertion of heterologous DNA sequences into a poxviral genome bears the risk to destroy regions essential for the virus propagation due to a lack of complete understanding of the poxviral lifecycle. Although the sequence information of several poxvirus genomes (Goebel et al. 1990; Antoine et al. 1998) is available the function of most proteins encoded by the identified open reading frames is not known. Accordingly, it is still a complicated challenge to identify sites in the genome, which are suitable to stably take up heterologous DNA without destroying any sequences essential for viral replication and propagation.
OBJECT OF THE INVENTION
It is thus an object of the present invention to identify a new insertion site in the poxviruses genome and provide vectors suitable to direct the integration of heterologous DNA sequences into said insertion site.
DESCRIPTION OF THE INVENTION
To achieve the foregoing and other objects, the present invention provides a vector comprising a nucleic acid sequence according to SeqID No. 1 or its complementary strand. The nucleic acid sequence according to SeqID No. 1 is highly homologous with parts of the genomic sequences of a poxvirus genome. Due to this homology the nucleic acid sequence according to the present invention is capable to initiate homologous recombination between said sequence and the corresponding genomic sequences of poxviruses. Thus, the present invention provides a mean useful to direct integration of DNA sequences into the genome of different orthopoxviruses, preferably into the genome of modified vaccinia virus Ankara (MVA), but also of further related orthopoxviruses such as, e.g., Vaccinia virus strain Western Reserve or Copenhagen.
According to a preferred embodiment the nucleic acid sequence of the present invention is derived from modified vaccinia Ankara virus (MVA), especially from MVA, which has been isolated and deposited on January 27
th
, 1994 according to the Budapest Treaty at the European Collection of Animal Cell Cultures (Salisbury, UK) under. Deposit No.: V94012707.
The present invention further provides a vector comprising nucleic acid sequences, which hybridise under stringent conditions to the sequences according to SeqIDNo: 1 or its complementary strand. In the context of this invention the term “vector” is understood as DNA vehicles of circular structure, such as plasmids, cosmids or artificial chromosomes. Said vector comprises in addition to the desired nucleic acid sequence regulatory sequences, selective marker genes and replicons enabling the autonomous replication of the vector. Hence, the vector according to the present invention can easily be amplified in and isolated from unicellular host organism. Furthermore, the term “under stringent conditions” defines parameters according to standard protocols (Sambrook et al., 1989), such as reaction temperature, formamide content or salt concentrations, which allow hybridisation of DNA—DNA sequences with a homology about and above 70%. As described above, also these sequences hybridising to the corresponding sequence of the poxvirus genome and are thus, particularly, useful to integrate heterologous sequences into a genome of orthopoxviruses.
Additionally, the present invention provides a vector comprising fragments of the above-mentioned nucleic acid sequence. These fragments comprise consecutive basepairs of said nucleic acid sequence and are also useful to integrate into the poxviral genome by homologous recombination. The length of said fragments is variable and fragments with only 30 basepairs being homologous to corresponding parts of the poxvirus genome are already sufficient to initiate recombination events. However, to increase the efficiency of homologous recombination between the poxvirus genome and the fragments as used in the present invention, said fragments are preferably about and above 200 basepairs in length, more preferably about and above 300 or 500 basepairs in length.
To initiate homologous recombination the vector according to the present invention and a wildtype poxvirus is introduced into a host cell. During replication of the poxvirus genome homologous recombination between the nucleic acid sequence inserted into the vector and the corresponding sequences of the poxvirus genome occurs. Since homologous recombination events occur only with a statistical probability of 1:10
3
to 1:10
4
any resulting recombinant poxvirus needs to be isolated. For this, e.g. a marker gene with functionally associated regulatory element is inserted into a cloning site of the, nucleic acid sequence included in the vector. After homologous recombination the resulting recombinant poxviruses are isolated by screening for expression of said marker gene or by selection for the expression of a dominant-selection marker gene, respectively.
According to a further embodiment the vector of the present invention is particularly useful for insertion of a desired heterologous coding sequence into a poxviral genome. The term “heterologous” is used in the context of this invention for any combination of nucleic acid sequences that is not normally found intimately associated in nature. The heterologous genes according to the present invention are preferably selected from the group of marker genes, therapeutic genes, such as anti-viral genes, anti-tumour genes, cytokine or chemokine genes, suicide genes, but also from host range genes or immunogenic epitopes. For insertion and/or expression of a desired heterologous cod
Baier Robert
Erfle Volker
Ohlmann Marion
Sutter Gerd
Wintersperger Stefan
Dubno Herbert
GSF Forschungszentrum fur Unwelt und Gesundheit GmbH
Mosher Mary E.
Myers Jonathan
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