Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2001-05-31
2004-07-27
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S004000, C435S320100, C435S034000, C435S173900, C424S218100
Reexamination Certificate
active
06767699
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates generally to the purification of viruses and virus-derived vectors, including those related to alphaviruses, from biological and chemical preparations. In particular, this invention relates to methods of purification of such viruses and vectors from preparations by subjecting the preparation to chromatographic purification using an ion exchange resin or combination of an ion exchange resin step and another chromatographic process step such as size exclusion or affinity chromatography. The method provides purified viruses and vectors for use as effective vaccines and therapeutics. Moreover related methods for quantifying replicon vector preparations and verifying the replication incompetency of purified vectors are provided.
BACKGROUND OF THE INVENTION
Alphaviruses comprise a set of genetically, structurally, and serologically related arthropod-borne viruses of the Togaviridae family. Twenty-six known viruses and virus subtypes have been classified within the alphavirus genus, including, Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelan equine encephalitis virus.
Sindbis virus is the prototype member of the Alphavirus genus of the Togaviridae family. Its replication strategy has been well characterized in a variety of cultured cells and serves as a well-accepted model for other alphaviruses. Briefly, the genome of Sindbis virus (like other alphaviruses) is an approximately 12 kb single-stranded positive-sense RNA molecule which is capped and polyadenylated, and contained within a virus-encoded capsid protein shell. The nucleocapsid is further surrounded by a host-derived lipid envelope into which two viral-specific glycoproteins, E1 and E2, are inserted and anchored to the nucleocapsid. Certain alphaviruses (e.g., SFV) also maintain an additional protein, E3, which is a cleavage product of the E2 precursor protein, PE2.
After virus particle adsorption to target cells, penetration, and uncoating of the nucleocapsid to release viral genomic RNA into the cytoplasm, the replicative process occurs via four alphaviral nonstructural proteins (nsPs), translated from the 5′ two-thirds of the viral genome. Synthesis of a full-length negative strand RNA, in turn, provides template for the synthesis of additional positive strand genomic RNA and an abundantly expressed 26 S subgenomic RNA, initiated internally at the junction region promoter. The alphavirus structural proteins (sPs) are translated from the subgenomic 26S RNA, which represents the 3′ one-third of the genome, and like the nsPs, are processed post-translationally into the individual proteins.
Several members of the alphavirus genus are being developed as “replicon” expression vectors for use as vaccines and therapeutics. Replicon vectors may be utilized in several formats, including DNA, RNA, and recombinant vector particles. Such replicon vectors have been derived from alphaviruses that include, for example, Sindbis virus (Xiong et al., Science 243:1188-1191,1989; Dubensky et al., J. Virol. 70:508-519,1996; Hariharan et al., J. Virol. 72:950-958, 1988; Polo et al., PNAS 96:4598-4603, 1999), Semliki Forest virus (Liljestrom, Bio/Technology 9:1356-1361, 1991; Berglund et al., Nat. Biotech. 16:562-565, 1998), and Venezuelan equine encephalitis virus (Pushko et al., Virology 239:389-401, 1997). A wide body of literature has now demonstrated efficacy of such replicon vectors for applications such as vaccines (see for example, Dubensky et al., ibid; Berglund et al., ibid; Hariharan et al., ibid, Pushko et al., ibid; Polo et al., ibid; Davis et al., J Virol. 74:371-378, 2000; Schlesinger and Dubensky, Curr Opin. Biotechnol. 10:434-439, 1999; Berglund et al., Vaccine 17:497-507, 1999).
Because of their configuration, vector replicons do not express the alphavirus structural proteins necessary for packaging into recombinant alphavirus particles (replicon particles). Thus, to generate replicon particles, these proteins must be provided in trans. Packaging may be accomplished by a variety of methods, including transient approaches such as co-transfection of in vitro transcribed replicon and defective helper RNA(s) (Liljestrom, Bio/Technology 9:1356-1361, 1991; Bredenbeek et al., J. Virol. 67:6439-6446, 1993; Frolov et al., J. Virol. 71:2819-2829, 1997; Pushko et al., Virology 239:389-401, 1997; U.S. Pat. Nos. 5,789,245 and 5,842,723) or plasmid DNA-based replicon and defective helper constructs (Dubensky et al., J. Virol. 70:508-519, 1996), as well as introduction of alphavirus replicons into stable packaging cell lines (PCL) (Polo et al., PNAS 96:4598-4603, 1999; U.S. Pat. Nos. 5,789,245, 5,842,723, and 6,015,694; PCT publications WO 9738087 and WO 9918226).
Alphavirus replicon particles produced using any of the above methodologies subsequently are harvested in the cell culture supernatants. The replicon particles then may be concentrated and partially purified using one of several published approaches, including polyethylene glycol (PEG) precipitation, ultracentrifugation, or Cellufine sulfate™ ion exchange chromatography. Unfortunately, these methods do not remove a sufficient level of non-alphavirus derived protein contaminants, are not scalable, or are costly, and therefore are likely not amenable for commercial manufacture necessary of vaccine and therapeutic products.
The present invention provides methods of production and purification with utility for the large-scale manufacture of alphavirus replicon particles. Also disclosed are novel methods for quantitating vector particles in a preparation and determining the presence or absence of contaminating replication-competent virus in a preparation. Additional methods are provided for detecting the presence of packaged helper RNAs in a preparation of replicon particles. Alphavirus particles produced and characterized according to the methods described herein may be used for a variety of applications, including for example, vaccines and gene therapy.
SUMMARY OF THE INVENTION
Briefly stated, the present invention provides methods of production and purification for alphavirus replicon particles. Such replicon particles may be derived from a wide variety of alphaviruses (e.g., Semliki Forest virus, Ross River virus, Venezuelan equine encephalitis virus, Sindbis virus), and are designed to express a variety of heterologous proteins (e.g., antigens, immunostimulatory proteins, therapeutic proteins).
Within one aspect of the invention, a method of purifying alphavirus replicon particles is provided. Purification is achieved by first contacting a preparation containing alphavirus replicon particles with an ion exchange resin, under conditions and for a time sufficient to bind to the resin. Next, the portion of the preparation which is not bound to the ion exchange resin is removed from the ion exchange resin, and then the bound alphavirus replicon particles are eluted from the ion exchange resin and recovered. In one embodiment, the ion exchange resin is a tentacle ion exchange resin. In another embodiment, the tentacle ion exchange resin is a cationic exchange resin. In yet another embodiment, the tentacle ion exchange resin is an anionic exchange resin.
Within another aspect of the invention, a method of purification for alphavirus replicon particles is provided, comprising at least two chromatographic purification steps. The chromatographic purification steps are selected from the group consisting of ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and affinity chromatography. In one preferred embodiment, purification is performed using a first step of ion exchange chromatography and a second step of size exclusion chromatography.
Within another aspect of the invention, a method of producing alphavirus replicon particles is provided. Alphavirus packaging cells are infected with a seed stock of alphavirus replicon particles and then incubated in a bioreactor, under conditions and for a time sufficient to permit the production of alphavirus re
Dubensky, Jr. Thomas W.
Greer Catherine
Polo John M.
Blackburn Robert P.
Chen Stacy B.
Chiron Corporation
Housel James
Moran Michael J.
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