Adventitious viruses in insect cell lines used for recombinant protein expression
Abstract
Insect cells are widely used for recombinant protein expression, typically as hosts for recombinant baculovirus vectors, but also for plasmid-mediated transient transfection or stable genetic transformation. Insect cells are used to express proteins for research, as well as to manufacture biologicals for human and veterinary medicine. Recently, several insect cell lines used for recombinant protein expression were found to be persistently infected with adventitious viruses. This has raised questions about how these infections might affect research performed using those cell lines. Furthermore, these findings raised serious concerns about the safety of biologicals produced using those cell lines. In response, new insect cell lines lacking adventitious viruses have been isolated for use as improved research tools and safer biological manufacturing platforms. Here, we review the scientific and patent literature on adventitious viruses found in insect cell lines, affected cell lines, and new virus-free cell lines.
Introduction
The first immortalized insect cell lines were established in the 1960s [1], Since then, hundreds of insect cell lines have been established from insects in many different Orders, with 940 different cell lines listed in the ExPASy Cellosaurus database at the time of this writing (http://web.expasy.org/cellosaurus/). Several of these cell lines are routinely used to express recombinant proteins for basic research. In addition, some insect cell lines are now used to manufacture biologicals approved for use in human or veterinary medicine (see Ref. [2] for a recent list). The insect cell lines most commonly used for recombinant protein expression are derived from the Orders Lepidoptera (moths and butterflies) and Diptera (flies).
Lepidopteran cell lines are typically used as hosts for recombinant baculoviruses encoding the protein(s) of interest (recently reviewed by Ref. 13]). Alternatively, they can express recombinant proteins following transient transfection or stable genetic transformation with insect-specific expression plasmids encoding the protein(s) of interest (reviewed by Refs. [4], [5]). Lepidopteran cell lines used for recombinant protein expression include lines derived from Trichoplusia ni (Tn), such as TN-368 [6], BTI-Tn5B 1-4 (commercialized as High Five™; [7]), and Tni PRO™ (Expression Systems, LLC), lines derived from Spodoptera frugiperda (Sf), such as IPLB-Sf21 AE (Sf21; [8]), Sfe [9], and SI900+ (commercialized as expresSF+™; [10]), and a few lines derived from Bombyx mori (Bm),suchasBm-N[ll].
Dipteran insect cell lines are typically used for plasmid-mediated recombinant protein expression [4], [5], Like mammalian cells, they are not susceptible to baculovirus infection, but can be transduced with baculovirus vectors 112], 113). The dipteran cell lines most commonly used for recombinant protein expression are S2 and S2R+, both derived from Drosophila melanogaster (Dm) [14], [15],
A problem with insect cell-based recombinant protein expression is most of the relevant cell lines are persistently infected with various adventitious viruses (see below). The absence of obvious cytopathic effects (CPEs), such as syncytia formation, nuclear hypertrophy, apoptosis, or inclusion body formation allowed these infections to go undetected for decades. The specific questions raised by the presence of adventitious viral contaminants in these cell lines focus on the validity of conclusions obtained using these lines for basic research and the potential biosafety hazards associated with their use as substrates for biologicals manufacturing.
In this review, we have compiled information available on persistent viral infections in insect cell lines used to produce recombinant proteins, which is scattered throughout the scientific and patent literature. In addition, we discuss new insect cell lines that are not contaminated with adventitious viruses. Finally, we briefly discuss measures to prevent contaminating virus-free cell lines.
Section snippets
Discovery and characteristics
Sf-rhabdovirus was independently discovered in various Sf cell lines at about the same time by three different groups. One group at the FDA`s Center for Biologicals Evaluation and Research (CBER) discovered Sf-rhabdovirus in Sf9 and Sf21 cells by using a combination of degenerate PCR and massively parallel sequencing [ 16]. A separate group atTakeda Vaccines discovered Sf-rhabdovirus in a noroviral vaccine candidate produced by Sf9 cells infected with a recombinant baculovirus encoding a...
Discovery and characteristics
Trichoplusia ni cell line virus, or TnCLV, was first described in 2007 by a group at the National Institute of Infectious Diseases in Japan [29]. Much like the discovery of Sf-rhabdovirus at Takeda, this group serendipitously discovered TnCLV when they infected High Five™ cells (a Tn cell line) with a recombinant baculovirus encoding the hepatitis E capsid protein. They found these cells produced not only the expected baculovirus and hepatitis E virus-like particles, but also...
Discovery and characteristics
Bombyx mori macula-like virus (BmMLV) was serendipitously discovered in both Bm-N cells and silkworms [51]. BmMLV has a single linear 6.5 kb genomic RNA encoding 3 putative protein ORFs, and is classified in the Family tymoviridae, genus maculavirus. BmMLV can be inactivated by heat treatment (1 h at 75 °C), irradiation (10 kGy), and UV light (140 mJ/cm2 at 254 nm) [52].
Contaminated and susceptible cell lines
There has been comparatively little research on insect cell line contamination with and susceptibility to BmMLV. This is
Arboviruses (Flaviviridae; Flavivirus)
Arboviruses are of special interest, as many are vertebrate pathogens that can infect and cause serious disease in humans. Several arboviruses can stably and persistently infect cell lines derived from mosquitoes, which are naturally permissive hosts. However, it also has been shown arboviruses including St. Louis encephalitis, Dengue and Japanese B encephalitis virus can persistently infect lepidopteran cell lines used for recombinant protein production. As outlined below, the only examples of
Persistent baculovirus infection (Baculoviridae)
Baculoviruses are strictly arthropod pathogens and are not known to cause disease in any vertebrate [63], [64]. Thus, they are of relatively low concern in terms of biosafety.
Typically, infecting susceptible lepidopteran cell lines with baculovirus causes obvious CPEs and cell lysis followed by cell culture death within days. However, infection with some baculovirus mutants can produce a persistent infection [65]. Also, persistently infected lepidopteran cell cultures have been obtained using
Drosophila cell line viruses
Drosophila cells are used for recombinant protein expression for both basic research [77] as well as therapeutic [78] applications (Drosophila expression system, DES). Typically, the DES employs Drosophila S2 cells [14], which can be persistently infected by several adventitious viruses, as outlined below.
Viruses are associated with most, if not all natural insects populations
Recent studies indicate previously unknown viruses are associated with most, if not all natural insect populations [84], [85], [86]. Discovery of these diverse and ubiquitous viruses has only recently become possible by the emergence of massively parallel sequencing technologies. These viruses are well adapted, as they do not appear to cause pathology, infected populations are thriving, and they are often transmitted vertically [26]. Thus, it should be expected, a priori, that cell lines newly
Summary
As demonstrated by the many examples above, insect cell lines are often contaminated with adventitious viruses. In fact, one or more adventitious viruses have been found to be associated with all cell lines routinely used for recombinant protein expression. Considering this current state of the art, insect cell lines should only be considered free of adventitious viruses following rigorous experimentation including appropriate positive and negative controls over a substantial period of time.
The Acknowledgements
This work was supported by Awards R43 GM102982 and R43 AI112118 from the National Institutes of Health, Institutes of General Medical Sciences and Allergy and Infectious Diseases, respectively. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, National Institute of General Medical Sciences or National Institute of Allergy and Infectious Diseases.