Influenza epidemics and pandemics continue to claim human lives and impact the global economy. In the United States alone, influenza causes an estimated 50,000 deaths annually (Thompson et al., 2003), while global pandemics can result in millions of influenza-related deaths. A classic example is the so-called ‘Spanish influenza’, which killed an estimated 40-50 million people worldwide in 1918-1919 (Potter, 1998). The threat imposed by influenza virus has been further elevated with the recent introductions of avian influenza viruses into the human population. Avian influenza viruses were long thought not to be directly transmissible to humans and cause lethal outcomes. However, this perception changed in 1997, when 18 Hong Kong residents were infected by a wholly avian influenza virus of the H5N1 subtype, that resulted in 6 deaths (Subbarao et al., 1998; Claas et al., 1998). Over the next few years, several other cases of direct avian-to-human transmission were reported (Peiris et al., 2004; Fouchier et al., 2004; Koopsman et al., 2004), including the ongoing outbreak of highly pathogenic H5N1 influenza viruses in several Asian countries that has claimed 41 lives of 54 infected individuals as of Jan. 26, 2005 (WHO, 2004). The increasing numbers of human H5N1 virus infections, together with a high mortality rate and possible human-to-human transmission, make the development of vaccines to these viruses essential.
In the United States, two influenza vaccines are licensed for human use: an inactivated vaccine and a live attenuated vaccine virus. The production of influenza virus vaccines relies on reassortment (Gerdil, 2003), which requires coinfection of cells with a circulating wild-type strain that provides the hemagglutinin (HA) and neuraminidase (NA) segments and either A/PR/8/34 (PR8) virus (an attenuated human virus that provides high-growth properties in eggs) or a live attenuated virus that provides the attenuated phenotype. The selection of the desired “6+2” reassortants (i.e., those containing the HA and NA gene segments of the circulating wild-type strain in the genetic background of PR8 or live attenuated virus) is time-consuming and cumbersome. Moreover, the need for reassortment and selection, as well as the inability of some reassortant viruses to grow to high titers, have resulted in delays in vaccine production.
The artificial generation of influenza A and B viruses entirely from cloned cDNA in plasmid-transfected cells, the so-called “plasmid-based reverse genetics system” (Fodor et al., 1999; Neumann et al., 1999; Neumann et al., 2004; Neumann et al., 2002; Neumann et al., 1999; Hoffmann et al., 2002; Fodor et al., 1999; Hoffmann et al., 2000), represents an important advance for influenza virology. This technology has advanced both basic and applied research of influenza virus; most notably, the development of vaccine seed strains for highly pathogenic influenza viruses, including the currently circulating H5N1 viruses (Horimoto et al., 2006; Subbarao et al., 2003; Takada et al., 1999; Webby et al., 2004; Wood et al., 2004).
In one system (Neumann et al., 1999), eight plasmids encoding the eight influenza viral RNA segments under the control of the RNA polymerase I (PolI) promoter and terminator sequences are transfected into eukaryotic cells together with four RNA polymerase II (PolII)-driven plasmids for the expression of the three viral polymerase subunits and the nucleoprotein NP; these four proteins are required to initiate viral replication and transcription. An alternative system has been developed (Hoffmann et al., 2000) that relies on eight plasmids in which the viral cDNAs are flanked by an RNA polymerase I promoter on one site and an RNA polymerase II promoter on the other site, which permits the vRNA and mRNA to be derived from the same template. These systems have allowed 6+2 reassortants to be engineered without the need for reassortment and screening procedures.
Since at least eight plasmids have to be transfected into a single cell for virus generation, the limiting factor for plasmid-based reverse genetics is the transfection efficiency of the cells. In general, 293T cells, which are readily transfected with plasmids (Goto et al., 1997), have been used for plasmid-based systems (Hoffmann et al., 2000; Neumann et al., 1999). However, 293T cells cannot be used for the development of human vaccine seed strains because they are not validated for such use. African green monkey kidney (Vero) cells, which have been used for the production of rabies and polio virus vaccines (Montagnon et al., 1999), are the WHO-recommended cell line for vaccine production (Wood et al., 2004). Although these cells are not readily transfected (Kistner et al., 1998; Kistner et al., 1999a; Kistner et al., 1999b; Bruhl et al., 2000), the generation of influenza virus in Vero cells has been demonstrated (Fodor et al., 1999; Nicolson et al., 2005). Madin-Darby canine kidney (MDCK) cells (Brands et al., 1999; Palache et al., 1999; Halperin et al., 2002) are available for the production of influenza virus vaccine cells, but the cell line cannot be transfected with high efficiencies. It is, therefore, difficult to efficiently generate influenza viruses by using plasmid-based systems in influenza virus vaccine cells.