Characterization of an Sf-rhabdovirus-negative Spodoptera frugiperda cell line as an alternative host for recombinant protein production in the baculovirus-insect cell system

Abstract

Cell lines derived from the fall armyworm, Spodoptera frugiperda (Sf), are widely used as hosts for recombinant protein production in the baculovirus-insect cell system (BICS). However, it was recently discovered that these cell lines are contaminated with a virus, now known as Sf-rhabdovirus [1]. The detection of this adventitious agent raised a potential safety issue that could adversely impact the BICS as a commercial recombinant protein production platform. Thus, we examined the properties of Sf-RVN, an Sf-rhabdovirus-negative Sf cell line, as a potential alternative host. Nested RT-PCR assays showed Sf-RVN cells had no detectable Sf-rhabdovirus over the course of 60 passages in continuous culture. The general properties of Sf-RVN cells, including their average growth rates, diameters, morphologies, and viabilities after baculovirus infection, were virtually identical to those of Sf9 cells. Baculovirus-infected Sf-RVN and Sf9 cells produced equivalent levels of three recombinant proteins, including an intracellular prokaryotic protein and two secreted eukaryotic glycoproteins, and provided similar N-glycosylation patterns. In fact, except for the absence of Sf-rhabdovirus, the only difference between Sf-RVN and Sf9 cells was SF-RVN produced higher levels of infectious baculovirus progeny. These results show Sf-RVN cells can be used as improved, alternative hosts to circumvent the potential safety hazard associated with the use of Sf-rhabdovirus-contaminated Sf cells for recombinant protein manufacturing with the BICS.

Infectivity of Sf-rhabdovirus variants in insect and mammalian cell lines

“…Our basic approach was to use cell-free media filtrates from our Sf9 and Sf21 cultures, which were known to be contaminated with the Sf-rhabdoviral variants described above, to inoculate Sf-RVN, a novel Sf line that has no Sf-rhabdovirus (Maghodia et al, 2016). One day after inoculation, the Sf-RVN cells were extensively washed, returned to the incubator, and then samples were harvested and used to prepare cell lysates at various times post-inoculation.…”

“…Our sequence analyses revealed multiple single nucleotide polymorphisms that can be used to distinguish the Sf-rhabdovirus variants harbored by different Sf cell lines. Interestingly, PCR analysis of genomic DNAs isolated from our Sf9, Sf21, and Sf-rhabdovirus-negative (Sf-RVN; (Maghodia et al, 2016) cells revealed none of the integrated Sf-rhabdoviral sequences detected by Hashimoto and coworkers (2017) in the Sf9 L5814 cell genome. Subsequently, we found the Sf-rhabdoviral variants produced by our Sf9 and Sf21 cells were both able to establish persistent, productive infections in Sf-RVN cells, which is the first direct evidence Sf-rhabdoviruses are infectious for Sf cells.…”

“…The primary PCR products were analyzed by agarose gel electrophoresis with ethidium bromide staining and amplimers of interest were then gel purified using HiBind DNA Mini Columns (Omega Bio-Tek, Inc., Norcross, GA), according to the manufacturer’s protocol, directly sequenced (Genewiz, South Plainfield, NJ), and the sequences were assembled and aligned using Vector NTI 10.3.1 (Invitrogen). For some experiments, the RT-PCR assay described above was extended to include a secondary, nested PCR step, essentially as described previously (Maghodia et al, 2016), except we used the N-specific primer pairs NSP and NASP and NSeq1 and NSeq2 (Table 1) for the primary and secondary PCRs, respectively.…”

“…1). Since the original discovery of this agent, other investigators have confirmed the presence of Sf-rhabdoviral RNA sequences in Sf9 (Haynes, 2015; Maghodia et al, 2016) and Sf9 L5814 (Hashimoto et al, 2017) cells. Thus, Sf-rhabdovirus is now widely recognized as a common contaminant of at least some Sf cell lines used as hosts for baculovirus-mediated recombinant protein production.…”

Synthetic gene design—The rationale for codon optimization and implications for molecular pharming in plants

“…marginale (Pierlé et al 2013), R. pomeroyi (Green et al 2013), L. acidophilus (Askelson et al 2014), C. reinhardtir (Erpel et al 2016), Y. lipolytica (Matthaus et al 2014), S. elongates (van der Woude et al 2016) & baculovirus (Maghodia et al 2016). While it is encouraging that codon optimization software programs are used for a variety of species and purposes, most of the papers…”