Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2002-04-17
2004-04-20
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Vector, per se
C435S005000, C435S006120, C435S069700, C435S325000, C536S023100, C536S023720
Reexamination Certificate
active
06723559
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to recombinant negative strand RNA molecules which may be used to express heterologous proteins in animal cells and/or to construct recombinant viruses able to express heterologous proteins during their multiplication in host animal cells.
2. Discussion of the Background
Despite the segmented nature of the influenza virus genome, several approaches have already been described to achieve the construction of stable recombinant influenza viruses, able to express heterologous protein sequences of interest. These approaches have been made possible after the development of reverse genetics techniques for the influenza viruses.
Short foreign polypeptides can be expressed by inserting the foreign sequence into an essential viral gene (see, for example, Castrucci, M. R., S. Hou, P. C. Doherty, and Y. Kawaoka 1994. Protection against lethal lymphocytic choriomeningitis virus (LCMV) infection by immunization of mice with an influenza virus containing an LCMV epitope recognized by cytotoxic T lymphocytes
Journal of Virology.
68:3486-90). This strategy is limited to the use of small size epitope inserts and the insertion of a foreign epitope into the sequence of a viral protein can modify the protein in a way which would prevent the generation of viable recombinant viruses
Longer polypeptides can be expressed by the use of a fusion protein containing a protease sensitive peptide sequence between an essential viral gene product and a foreign polypeptide (Percy, N., W. S. Barclay, A. Garcia Sastre, and P. Palese 1994. Expression of a foreign protein by influenza A virus
Journal of Virology.
68:4486-4492), but this approach results in the expression of altered viral and/or foreign proteins due to the presence of the specific protease signal in the cleaved protein products.
A foreign polypetide can also be expressed via the use of recombinant RNA segment which were made functionally dicistronic or tricistronic at the level of translation by the insertion of the IRES (Internal Ribosome Entry site) of the human BiP RNA (Garcia-Sastre, A., T. Muster, W. S. Barclay, N. Percy, and P. Palese 1994. Use of a mammalian internal ribosomal entry site element for expression of a foreign protein by a transfectant influenza virus
Journal of Virology.
68:6254-6261). In this case, the bicistronic mRNA (which is transcribed during viral replication) permits internal initiation of translation of viral sequences (via internal binding of the ribosome to the IRES sequence) and allows for the expression of foreign protein coding sequences via cap-dependant initiation of translation. A potential drawback of this approach could rely in the fact that the synthesis of the NA gene, which is under the dependance of the IRES sequence is not regulated to the same level and/or at the same time point as it is in the wild type virus ; this could modify the phenotypic characteristics of the virus as it has been shown, for example, that the amount of NA incorporated in the influenza virions affects the infectivity of the viral particles.
Garcia-Sastre et al. (Garcia-Sastre, A., N. Percy, W. Barclay, and P. Palese 1994. Introduction of foreign sequences into the genome of influenza A virus
Developments in Biological Standardization.
82:237-246) reported the design of a similar dicistronic RNA molecule in which the corresponding mRNA contains the viral sequence in a proximal position which allows translation from the regular terminal open reading frame, while the translation of the foreign sequence would be initiated from an internal site. In this publication, the IRES of the EMCV virus was used but turned out to be non functional in influenza virus infected cells, since no expression of the foreign sequence could be detected. What is noticeable here is the presence of a duplication of the 39 last nucleotides of NA ORF, which extended the 5′ terminus of the recombinant vRNA molecule from the 28 nt of the 5′ non coding region itself to the 67 first nucleotides of the 5′ terminus of the original NA segment of virus WSN. Nevertheless, this duplication seemed to arise from a facility in the genetic construction of the RNA molecule, since any data neither demonstrates nor shows the role of these additional 39 nt at the 5′ end of the recombinant NA segment encoded by the pT3NA/EMC and pT3NA/EMC-NS 1 plasmids.
More recently, Flick and Hobom (Flick, R., and G. Hobom 1999. Transient bicistronic vRNA segments for indirect selection of recombinant influenza viruses
Virology.
262:93-10) introduced another principle of constructing dicistronic vRNA molecules for the transient expression of foreign sequences and/or selection genes. They showed that a recombinant vRNA-like molecule can be made dicistronic by the duplication of its 3′-non coding flanking sequence : in this case, the recombinant RNA molecule is functionally bicistronic for its replication and transcription, in the sense that it can be transcribed and replicated in (a) a genomic mode after interaction of the single 5′ non coding sequence with the external 3′ non coding sequence and in (b) a subgenomic mode after interaction of the 5′ non coding sequence with the internal 3′ non coding sequence. Two types of mRNA molecules are produced, genomic mRNA allows translation of the first cistron, whereas the shorter subgenomic mRNA drives the translation of the second cistron. They showed that the recombinant dicistronic RNA molecules can be propagated as an additional ninth segment during a few rounds of viral multiplication, but that next, the distal segment is spontaneously lost through vRNA-internal initiation events, giving rise to a monocistronic ninth segment. Moreover, it can be anticipated that this resulting virus will be unstable since additional, independant vRNA segments coding for foreign genes are lost during virus growth in the absence of continuing selective pressure as it has been reported by others and the authors themselves in another publication (Neumann, G., and G. Hobom 1995. Mutational analysis of influenza virus promoter elements in vivo
Journal of General Virology.
76:1709-17).
Based on the disadvantages of the prior studies, the present inventors sought to design dicistronic influenza genomic segments, in which the first cistron would drive the translation of an essential viral gene and the second cistron would permit the translation of foreign sequences. In completing the invention the present inventors demonstrate that such dicistronic viral segment permitted the rescue of stable recombinant influenza viruses, able to express foreign sequences of interest. The inventors further demonstrate that such recombinant influenza viruses may be used to express heterologous proteins in an animal host. In particular, when inoculated in an animal recipient, these recombinant viruses are able to induce an immune response against the encoded foreign protein. It should be underlined that Flick and Hobom neither reported nor suggested the construction of such dicistronic genomic segments or related recombinant viruses.
The present inventors, for the first time, demonstrate the effectiveness of using recombinant RNA molecules, which were made functionally dicistronic or multicistronic at the level of RNA replication and transcription.
SUMMARY OF THE INVENTION
Objects of the Present Invention are as Follows:
A recombinant RNA molecule comprising, from the 3′ end towards the 5′ end:
a) At least two units, each of them composed of a wild-type truncated or mutated 3 ′-non coding flanking sequence of a genomic RNA segment of a segmented negative strand RNA virus, optionaly a given spacer sequence of a size chosen from 0 nucleotide to 500 nucleotides, the reverse complement of an mRNA coding sequence or of a fragment of an mRNA coding sequence linked in frame to an initiating AUG and termination codon, a second spacer sequence of a size choosen from 0 nucleotide to 500 nucleotides.
b) A wild-type, truncated or mutate
Escriou Nicolas Robert Xavier
Naffakh Nadia
Van Der Werf Sylvie
Vieira-Machado Alexandre
Institute Pasteur
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Park Hankyel T.
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