1. Field of the Invention
The present invention relates generally to the study of alphaviruses such as Sindbis virus. More specifically, the present invention relates to an alphavirus packaging method useful in large scale production of recombinant vaccines or recombinant proteins.
2. Description of the Related Art
Progress in understanding the molecular mechanisms of alphavirus replication and pathogenesis has made possible their use as gene delivery and expression systems. Sindbis virus is a prototype and the least pathogenic member of the alphavirus genus. Sindbis virus can establish productive replication in a wide variety of cell lines of vertebrate and invertebrate origin. The biology of Sindbis virus has been better studied than that of any other alphavirus, and this fact favors its application as a vector for development of recombinant vaccines.
Sindbis virions are formed by three structural proteins: capsid protein and glycoproteins E1 and E2. A total of 240 molecules of the capsid protein assemble into an icosahedral nucleocapsid that packages the viral genomic RNA. The nucleocapsid is surrounded by a lipid envelope holding glycoprotein spikes. The spikes are present on the virion surface in an icosahedral lattice, and each contains three E1-E2 heterodimers.
The Sindbis virus genome is a single RNA molecule of positive polarity of almost 12 kb in size (FIG. 1). It mimics the structure of cellular mRNAs in that it contains both a 5′ methylguanylate cap and a 3′ polyadenylate tail. These features allow translation of viral proteins by host cell machinery immediately after release of the genome RNAs from the nucleocapsids. The 5′ two-thirds of the genome is translated into the nonstructural proteins (nsPs) that comprise the viral components of the RNA-dependent RNA polymerase (RdRp) required for replication of the viral genome and transcription of the subgenomic RNA which is encoded by the 3′ third of the genome. The subgenomic RNA is transcribed from the subgenomic promoter and translated into the viral structural proteins that form viral particles.
Alphaviruses as Vectors
The creation of infectious cDNA copies of several alphavirus genomes, identification of the cis-acting elements required for RNA replication and packaging, and the development of efficient methods for RNA transfection made possible use of alphaviruses as a gene delivery and expression system. It has been shown that the alphavirus genome can be divided into separate RNA fragments that complement each other's functions during intracellular replication (Weiss & Schlesinger, 1991).
A method of packaging Sindbis virus into infectious viral particle is shown in FIG. 2. Self-replicating Sindbis-specific RNAs (replicons) and helper RNAs (DH RNAs) are co-transfected into cells by electroporation. Replicons supply the replicative enzymes for their own replication and for that of helper genomes and for transcription of helper subgenomic RNA. Translation of the latter leads to production of viral structural proteins required for formation of viral particles. These particles contain predominantly replicon genomes, as helper RNAs lacking the packaging signals are packaged inefficiently. The average electroporation resulted in the production of 1-5×109 infectious particles with the usual titers of 5-10×108 Inf.u./ml.
On the next passage, particles deliver replicons into naive cells where replication begins. Initially, replicons serve as usual cellular messenger RNAs and express the nonstructural proteins forming the replicative complex. Next, replicon RNAs are used as templates for synthesis of a full-length minus-strand intermediate. The minus-strand intermediates then serve as templates for production of large quantities of positive-strand replicon RNAs. Within a few hours post infection, the replicative complexes perform ˜105 fold amplification of the replicon genome. The subgenomic RNA coding the heterologous sequences is normally produced in a 10 fold excess to genome RNAs and becomes the main mRNA translated in the infected cells.
The infection does not spread to other cells because the alphavirus structural proteins required for virus particles formation are not expressed in the cells infected only by replicons. This made Sindbis virus-based and other alphavirus-based replicons a very attractive system for large-scale production of heterologous proteins. The expression systems were designed for Sindbis virus, Semliki Forest virus (SFV) and Venezuelan equine encephalitis virus (VEE). However, the use of Sindbis virus-based constructs instead of Venezuelan equine encephalitis-based or Semliki Forest virus-based replicons and packaging systems is advantageous because all of the experiments can be performed according to BSL2 regulations that dramatically increase the safety and efficiency of all the procedures.
However, initial experiments employing replicons and packaging systems revealed some phenomena that impaired application of alphavirus-based expression systems. It was noticed that the level of expression of the heterologous proteins by Sindbis virus replicons was lower than expected based on the level of accumulation of the subgenomic RNA. Replicons and helper RNAs also have a very high level of recombination that leads to formation of replication-competent viruses. Moreover, large-scale production of packaged replicons for vaccine applications was limited by the number of electroporations that could be performed.
It is an objective of the present invention to overcome these problems by developing new Sindbis virus vectors and packaging systems which are significantly different from the previously described ones. These new vectors can become prototypes for designing and manufacturing of recombinant vaccines against various pathogens.