Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2002-10-01
2004-01-06
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S005000, C435S006120, C435S325000, C424S192100, C424S199100
Reexamination Certificate
active
06673572
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to improved methods for producing nonsegmented, negative-sense, single-stranded RNA viruses of the Order designated Mononegavirales virus. Preferred embodiments relate to methods of producing such viruses as attenuated and/or infectious viruses, such as Measles virus (MV) respiratory syncytial virus (RSV) and Human parainfluenza virus (PIV). The recombinant viruses can be prepared from cDNA clones, and, accordingly, viruses having defined changes in the genome can be obtained.
BACKGROUND OF THE INVENTION
Enveloped, negative-sense, single stranded RNA viruses are uniquely organized and expressed. The genomic RNA of negative-sense, single stranded viruses serves two template functions in the context of a nucleocapsid: as a template for the synthesis of messenger RNAs (mRNAs) and as a template for the synthesis of the antigenome (+) strand. Negative-sense, single stranded RNA viruses encode and package their own RNA-dependent RNA Polymerase. Messenger RNAs are only synthesized once the virus has entered the cytoplasm of the infected cell. Viral replication occurs after synthesis of the mRNAs and requires the continuous synthesis of viral proteins. The newly synthesized antigenome (+) strand serves as the template for generating further copies of the (−) strand genomic RNA.
The polymerase complex actuates and achieves transcription and replication by engaging the cis-acting signals at the 3′ end of the genome, in particular, the promoter region. Viral genes are then transcribed from the genome template unidirectionally from its 3′ to its 5′ end. There is always less mRNA made from the downstream genes (e.g., the polymerase gene (L)) relative to their upstream neighbors (i.e., the nucleoprotein gene (N)). Therefore, there is always a gradient of mRNA abundance according to the position of the genes relative to the 3′-end of the genome.
Molecular genetic analysis of such nonsegmented RNA viruses has proved difficult until recently because naked genomic RNA or RNA produced intracellularly from a transfected plasmid is not infectious (Boyer and Haenni, 1994). This technical problem has been overcome through development of clever cDNA rescue technology that permits isolation of recombinant nonsegmented, negative-strand RNA viruses (Pattnaik et al., 1992; Schnell, Mebatsion, and Conzelmann, 1994). The techniques for rescue of these different negative-strand viruses follows a common theme, each having distinguishing requisite components for successful rescue (Baron and Barrett, 1997; Collins et al., 1995; Garcin et al., 1995; Hoffman and Banerjee, 1997; Lawson et al., 1995; Radecke et al., 1995; Schneider et al., 1997; He et al, 1997; Schnell, Mebatsion and Conzelmann, 1994; Whelan et al., 1995). After transfection of a genomic cDNA plasmid, an exact copy of genome RNA is produced by the combined action of phage T7 RNA polymerase and a vector-encoded ribozyme sequence that cleaves the RNA to form the 3′ termini. This RNA is packaged and replicated by viral proteins initially supplied by co-transfected expression plasmids. In the case of the Measles virus (MV) rescue system (Radecke et al., 1995), a stable cell line was prepared that expresses T7 RNA polymerase and the MV proteins N (nucleocapsid protein) and P (phosphoprotein polymerase subunit). Thus, MV rescue can be achieved by co-transfecting this cell line with an MV genomic cDNA clone containing an appropriately positioned T7 polymerase promoter and an expression plasmid that contains the MV polymerase gene (L).
Successful measles virus cDNA rescue apparently requires numerous molecular events to occur after transfection including: 1) accurate, full-length synthesis of genome RNA by T7 RNA polymerase and 3′ end processing by the ribozyme sequence; 2) synthesis of viral N, P, and L proteins at levels appropriate to initiate replication; 3) the de novo packaging of genomic RNA into transcriptionally-active and replication-competent nucleocapsid structures; and 4) expression of viral genes from newly-formed nucleocapsids at levels sufficient for replication to progress. Exactly what steps may be rate-limiting in successful rescue has not been determined, but the efficiency of rescue potentially may be improved by stimulating any one of the steps mentioned above.
The present invention seeks to improve the ability to recover the desired recombinant RNA viruses, such as MV. It is submitted that the ability to obtain replicating virus from rescue may diminish as the polynucleotide, which encodes the native genome and antigenome of a desired virus, is increasingly modified. Accordingly, the present invention seeks to overcome such an obstacle since these methods can substantially improve the likelihood of obtaining a desired recombinant virus from a rescue procedure.
SUMMARY OF THE INVENTION
The present invention provides for a method for producing a recombinant virus from the Order Mononegavirales comprising; (a) in at least one host cell, conducting transfection of a rescue composition which comprises (i) a transcription vector comprising an isolated nucleic acid molecule which comprises a polynucleotide sequence encoding a genome or antigenome of a nonsegmented, negative-sense, single stranded RNA virus of the Order Mononegavirales and (ii) at least one expression vector which comprises at least one isolated nucleic acid molecule encoding the trans-acting proteins necessary for encapsidation, transcription and replication; in a host cell under conditions sufficient to permit the co-expression of these vectors and the production of the recombinant virus; (b) heating the transfected rescue composition to an effective heat shock temperature under conditions sufficient to increase the recovery of the recombinant virus; and optionally, (c) harvesting the resulting recombinant virus.
An additional method relates to producing a recombinant Mononegavirales virus comprising; a) in at least one host cell, conducting transfection of a rescue composition which comprises (i) a transcription vector comprising an isolated nucleic acid molecule encoding a genome or antigenome of a nonsegmented, negative-sense, single stranded RNA virus of the Order Mononegavirales and (ii) at least one expression vector which comprises at least one isolated nucleic acid molecule which comprises a polynucleotide sequence encoding the trans-acting proteins necessary for encapsidation, transcription and replication under conditions sufficient to permit the co-expression of said vectors and the production of the recombinant virus; b) transferring the transfected rescue composition onto at least one layer of Vero cells; and optionally, harvesting the recombinant virus.
Further aspects of the present invention relate to methods combining the non-overlapping steps of the above methods, along with preferred embodiments, to create further improved methods.
In alternative embodiments, this invention provides a method for making RNA viruses of the Order Mononegavirales which are attenuated, infectious or both. Additional embodiments relate to the viruses produced from the methods of this invention, as well as vaccines containing such viruses. It is noted that such viruses may be human or non-human, such as murine or bovine.
The above-identified embodiments and additional embodiments, which are discussed in detail herein, represent the objects of this invention.
REFERENCES:
References cited in the parent application, 09/701,671.
Kovacs Gerald R.
Parks Christopher L.
Sidhu Mohinderjit S.
Udem Stephen A.
Gordon Alan M.
Park Hankyel T.
Webster Darryl L.
Wyeth Holdings Corporation
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