Appl Microbiol Biotechnol (2014) 98:1963–1970 DOI 10.1007/s00253-013-5474-9

MINI-REVIEW

Suitability and perspectives on using recombinant insect cells for the production of virus-like particles Hideki Yamaji

Received: 3 October 2013 / Revised: 12 December 2013 / Accepted: 14 December 2013 / Published online: 10 January 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Virus-like particles (VLPs) can be produced in recombinant protein production systems by expressing viral surface proteins that spontaneously assemble into particulate structures similar to authentic viral or subviral particles. VLPs serve as excellent platforms for the development of safe and effective vaccines and diagnostic antigens. Among various recombinant protein production systems, the baculovirus–insect cell system has been used extensively for the production of a wide variety of VLPs. This system is already employed for the manufacture of a licensed human papillomavirus-like particle vaccine. However, the baculovirus–insect cell system has several inherent limitations including contamination of VLPs with progeny baculovirus particles. Stably transformed insect cells have emerged as attractive alternatives to the baculovirus–insect cell system. Different types of VLPs, with or without an envelope and composed of either single or multiple structural proteins, have been produced in stably transformed insect cells. VLPs produced by stably transformed insect cells have successfully elicited immune responses in vivo. In some cases, the yield of VLPs attained with recombinant insect cells was comparable to, or higher than, that obtained by baculovirus-infected insect cells. Recombinant insect cells offer a promising approach to the development and production of VLPs.

Keywords Insect cells . Recombinant protein production . Virus-like particles . Vaccine . Stably transformed cells . Drosophila S2 cells . High Five cells

H. Yamaji (*) Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan e-mail: [email protected]

Introduction Vaccines have been widely used as one of the most effective means of controlling and preventing infectious diseases. The majority of currently available vaccines are derived from inactivated or live-attenuated infectious pathogens (Cox 2012). Vaccines against viral infectious diseases are produced by propagating viruses in large quantities in the living cells of susceptible organisms, since a virus cannot reproduce itself autonomously. For example, influenza vaccines and Japanese encephalitis (JE) vaccines are manufactured traditionally using embryonated chicken eggs and mouse brain tissue as the substrates, respectively, for virus propagation. Recently, however, these vaccines have been licensed for production using cultured mammalian cells such as MDCK and Vero cells for virus propagation (Cox and Hashimoto 2011; Feng et al. 2011; Kikukawa et al. 2012). Compared with timeconsuming, labor-intensive egg-based and in vivo production, cell culture-based virus propagation systems allow a more rapid and large-scale production of vaccines. However, the vaccines continue to be manufactured from infectious pathogens, which poses potential safety concerns. Recombinant protein production systems can provide the next alternatives by synthesizing viral immunodominant components in vitro. Subunit vaccines composed of a single recombinant viral protein or peptide are safe because they do not contain the genetic material of the virus. However, such subunit vaccines often suffer from poor immunogenicity probably due to incorrect folding or modification of the target virus protein (Noad and Roy 2003; Roy and Noad 2008). Structural proteins of a number of viruses, such as envelope and capsid proteins, spontaneously assemble into particulate structures that are similar to authentic virus particles or naturally occurring subviral particles. Based on this inherent property, viruslike particles (VLPs) can be produced by expressing such viral surface proteins in heterologous systems using recombinant

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DNA technology (Fig. 1) (Grgacic and Anderson 2006; Noad and Roy 2003; Roy and Noad 2008). VLPs are non-infectious and non-replicating because they assemble without the incorporation of the DNA or RNA of the virus. VLPs are a highly effective type of subunit vaccine that can elicit strong humoral and cellular immune responses because of their densely repeated display of viral antigens in an authentic conformation (Grgacic and Anderson 2006; Noad and Roy 2003; Roy and Noad 2008). Therefore, VLPs afford great opportunities for the development of safe and effective vaccines and diagnostic antigens. In addition to being suitable as a vaccine against the corresponding virus from which they were derived, VLPs can also be used as platforms for presenting foreign antigens by genetic fusion or chemical conjugation (Jennings and Bachmann 2008). Furthermore, applications of VLPs to carriers for gene and drug delivery have been explored (Yoo et al. 2011). A wide variety of VLPs have been produced in different recombinant protein production systems including bacterial, yeast, insect, mammalian, and plant cell systems and in vitro cell-free systems (Kushnir et al. 2012; Zeltins 2013). Among these systems, the baculovirus–insect cell system has been one of the most widely used systems for the production of VLPs (Fernandes et al. 2013; Kost et al. 2005; Kushnir et al. 2012; Metz and Pijlman 2011; van Oers 2006; Vicente et al. 2011; Zeltins 2013). This system is employed for the industrial-scale manufacture of a human papillomavirus-like particle vaccine, Cervarix, which has been approved for the prevention of cervical cancers (Cox 2012; Schiller et al. 2012; Vicente et al. 2011). Moreover, in 2013, a seasonal influenza vaccine, Flublok, which consists of recombinant hemagglutinin produced by the baculovirus–insect cell system, was approved in the USA (Cox 2012; Cox and Hashimoto 2011; Kushnir et al. 2012). Although Flublok is not a VLP-based vaccine, these examples demonstrate that insect cells are a practical platform for the production of the next generation of recombinant protein vaccines. The baculovirus–insect cell system is the most commonly used insect cell-based expression system. In this system, upon infection with a recombinant nucleopolyhedrovirus (NPV) carrying the foreign gene of interest, lepidopteran insect cells often express large quantities of the foreign protein under the control of the very strong promoter of the NPV polyhedrin or p10 gene during the very late stage of infection. Host insect cells perform most of the post-translational processing and modifications of higher eukaryotes, including phosphorylation, glycosylation, correct signal peptide cleavage, proteolytic processing, and fatty acid acylation, and thereby express recombinant proteins that are soluble and antigenically, immunogenically, and functionally similar to their counterparts (Luckow 1995). The baculovirus–insect cell system directs the transient expression of recombinant proteins, which has both advantages and disadvantages. This system allows attractive “plug and play” production where a single

Appl Microbiol Biotechnol (2014) 98:1963–1970

well-characterized cell line can be used for the production of different proteins (Cox 2012). However, the yields of secreted and membrane-bound proteins obtained with this system are often very low, probably because of the adverse effects of baculovirus infection on the host insect cell secretory machinery (Harrison and Jarvis 2007). Continuous protein production is virtually impossible because host insect cells are lysed during the baculovirus infection cycle. Release of intracellular proteins from lysed cells may result in protein degradation by proteases and may also complicate the downstream processing and purification of products. The baculovirus–insect cell system also requires routine maintenance of baculovirus stocks. Furthermore, in the case of VLP production, removal or inactivation of progeny baculoviruses released by budding off from infected insect cells may become a critical problem (Fernandes et al. 2013; Kushnir et al. 2012; Vicente et al. 2011), though baculoviruses are non-pathogenic to vertebrates. Stably transformed insect cells can be employed as attractive alternative platforms to the baculovirus–insect cell system (Douris et al. 2006; Farrell et al. 1998, 1999; Harrison and Jarvis 2007; Keith et al. 1999; Sonoda et al. 2012; Yamaji 2011; Yamaji et al. 2008). In stably transformed insect cell systems, host insect cells are transfected with a plasmid vector into which the heterologous gene of interest is cloned under the control of an appropriate promoter. If the introduced vector integrates into the chromosomal DNA of the host cell, the foreign protein can be synthesized either constitutively or upon induction. To enable the selection of transformed cells, a constitutively expressible antibiotic resistance marker is introduced into host cells with the heterologous gene of interest (Fig. 1). The antibiotic resistance marker is placed either on the same plasmid as the gene of interest or on a separate plasmid. This system is particularly useful for the production of complex secreted and membrane-bound proteins, because the protein synthesis and processing machinery of the host insect cell is not damaged by baculovirus infection. Attention has been recently directed toward the production of VLPs by stably transformed insect cells, and successful expressions have been reported. This mini-review focuses on VLP production in recombinant insect cells and is an overview of recent advances.

Production of VLPs by recombinant Drosophila cells In stably transformed insect cell systems, a cell line derived from a dipteran insect, the Drosophila melanogaster Schneider 2 (S2) cell line, has been widely used. Expression vectors containing either a constitutive promoter such as the Drosophila actin promoter or an inducible promoter such as the Drosophila metallothionein promoter are available for heterologous gene expression in Drosophila S2 cells

Appl Microbiol Biotechnol (2014) 98:1963–1970 Fig. 1 Schematic representation of flavivirus virion formation in infected cells (a) and production of virus-like particles (VLPs) in recombinant insect cells (b). a The virion of flaviviruses such as Japanese encephalitis virus (JEV) consists of a nucleocapsid structure surrounded by a lipid bilayer containing an envelope glycoprotein (E) and a membrane protein (M). The M protein is synthesized as the precursor membrane protein (prM), which is then cleaved to M by a cellular protease during virion maturation. This cleavage causes the rearrangement of E proteins from a prM/E heterodimer to an E homodimer on virus particles, leading to the formation of mature virions that can show toxic cell-fusion activity. Nucleocapsid-free subviral particles, slowly sedimenting hemagglutinin (SHA) particles, are natural by-products of flavivirus assembly and are released from infected cells. b VLPs similar to SHA particles can be produced by co-expression of the prM and E proteins in insect cells. The use of the mutated prM gene at a pr/M cleavage site is effective in suppressing the toxic cell-fusion activity of VLPs

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a

b

Virus infection

Structural protein Genome 5 RNA

Non-structural protein

Signal 3

C E NS1 NS3 NS5 prM NS2A/B NS4A/B Polyprotein synthesis Viral genome replication Virus assembly Lipid bilayer Nucleocapsid (C) E prM

cDNA E prM

prM/E gene with a pr/M cleavage site mutation Antibiotic resistance gene Plasmid vector Transfection

Genome RNA Insect cells Immature virion Proteolytic cleavage of prM

Selection of antibioticresistant clones

E dimer M

Mature virion

(Kirkpatrick and Shatzman 1999). Expression from the metallothionein promoter is induced by the addition of CuSO4. Inducible promoters may be useful for the production of toxic viral proteins because recombinant protein expression can be induced after cells have grown to a high cell density. Hundreds of copies of the expression plasmid stably integrate into the genome of Drosophila S2 cells in a single transfectionselection event (Johansen et al. 1989; Kirkpatrick and Shatzman 1999), which may result in high levels of heterologous protein expression. Drosophila S2 cell lines secreting high yields of the small surface antigen of hepatitis B virus (HBV) were generated by transfecting cells with a plasmid vector containing the S gene of HBV under the control of the inducible Drosophila metallothionein promoter (Deml et al. 1999a) (Table 1). The expression of HBV surface antigen (HBsAg) was compared between two vector systems. One system was based on the co-transfection of an expression vector for the HBV S gene

SHA particle

VLP

and a plasmid carrying a selectable marker dihydrofolate reductase (dhfr) gene under the control of the constitutive Drosophila actin 5C promoter. The other system was based on the transfection of a single plasmid containing both expression units. Reportedly, both vector systems were suitable for the generation of stably transfected cell lines secreting high levels of HBsAg, and the transfected S genes were integrated stably into the genome of Drosophila S2 cells at high copy numbers between 10 and 240. The yield of secreted HBsAg reached 7 mg/L under serum-free culture conditions, which was comparable to that obtained with the baculovirus–insect cell system. HBsAg secreted by stably transfected Drosophila S2 cells had a particulate structure similar to 22-nm subviral HBsAg particles derived from a human hepatoma cell line (Deml et al. 1999b). Immunization of mice with purified HBsAg particles elicited humoral antibody and cytotoxic T lymphocyte (CTL) responses. HBsAg was also efficiently produced in culture medium by Drosophila S2 cells stably

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Appl Microbiol Biotechnol (2014) 98:1963–1970

Table 1 Virus-like particles produced in stably transformed insect cells Virus

Expressed protein(s)

Cell line Note

Reference

Hepatitis B virus (HBV) Enveloped

S

S2

Deml et al. (1999a, b)

Japanese encephalitis virus (JEV) Mouse polyomavirus

Enveloped

prM, E

S2

Non-enveloped

VP1

S2

Non-enveloped, doublelayered Enveloped

VP2, VP6

S2

Drosophila metallothionein promoter, 7 mg HBsAg/L (comparable to the baculovirus–insect cell system) Drosophila actin 5C promoter (pAc5.1/V5-His; Life Technologies), prM signal sequence Drosophila metallothionein promoter, 2–4 mg VP1/L (lower than that from the baculovirus system), VP1 fused to the Drosophila BiP signal peptide (pMT/BiP/V5; Life Technologies) was efficiently secreted Drosophila metallothionein promoter and Drosophila BiP signal sequence (pMT/BiP/V5), bicistronic expression using EMCV-derived IRES element Drosophila metallothionein and actin 5C promoters (pMT/BiP/V5, pMT/V5-His [Life Technologies], pAc5.1/V5-His), co-transfection of four plasmids, 7.5 mg gp120/L and 9.5 Gag mg/L (comparable to the baculovirus system) BmNPV IE-1 transactivator, BmNPV HR3 enhancer, and Bombyx mori actin promoter, honeybee melittin signal sequence, 1.7 mg L/L AcNPV HR5 enhancer and AcNPV IE-1 promoter (pBiEx-3; Merck Chemicals), 1–3 mg Gag (secreted)/L AcNPV IE-1 promoter, piggyBac transposonmediated mutagenesis, low yield compared to the baculovirus system AcNPV HR5 enhancer and AcNPV IE-1 promoter (pBiEx-3), honeybee melittin signal sequence for gp140, double transformation OpNPV IE-2 promoter (pIB/V5-His-Dest; Life Technologies) BmNPV IE-1 transactivator, BmNPV HR3 enhancer, and B. mori actin promoter, prM gene with a pr/M cleavage site mutation, 30 mg E/L (higher than the baculovirus system) AcNPV HR5 enhancer and AcNPV IE-1 promoter (pBiEx-3)

Yamaji et al. (2013)

Human rotavirus

Human immunodeficiency virus type 1 (HIV-1)

VLP type

gp160, Rev, S2 Pr55 Gag

HBV

Enveloped

L

High Five

HIV-1

Enveloped

Pr55 Gag

High Five

HIV-1

Enveloped

Pr55 Gag

Sf9

HIV-1

Enveloped

Rous sarcoma virus

Enveloped

JEV

Enveloped

Pr55 Gag, High gp140 Five (chimeric) Gag lacking High protease Five region prM, E High Five

Bovine rotavirus

Non-enveloped core-like particle

VP2

High Five

transfected with a plasmid vector containing the HBV S gene under the control of the constitutive Drosophila actin 5C promoter (Jorge et al. 2008). Stably transformed Drosophila S2 cells secreting JE VLPs were generated by transfecting cells with a plasmid vector containing the coding sequence of the JE virus (JEV) prM signal peptide, the precursor (prM) of the viral membrane protein (M), and the envelope glycoprotein (E) (Fig. 1) under the control of the constitutive Drosophila actin 5C promoter (Zhang et al. 2007). The secreted E proteins were in a particulate form, and mice immunized with purified E protein particles developed specific antibodies. Mouse polyomavirus major coat protein VP1 was expressed under the control of the Drosophila metallothionein promoter in stably transformed Drosophila S2 cells (Ng et al. 2007). VP1 was expressed from recombinant Drosophila S2

Zhang et al. (2007) Ng et al. (2007)

Lee at al. (2011)

Yang et al. (2012)

Shishido et al. (2006)

Tagliamonte et al. (2010) Lynch et al. (2010)

Tagliamonte et al. (2011) Deo et al. (2011)

Shoja et al. (2013)

cells at a concentration of 4 mg/L with the majority of VP1 being associated with the cells, and self-assembled into VLPs intracellularly. This expression level was lower than that from the baculovirus–insect cell system. When the VP1 gene was expressed downstream of the secretion signal sequence of the Drosophila immunoglobulin heavy chain binding protein (BiP), VP1 was efficiently secreted into the culture medium at a concentration of 2 mg/L, but it was glycosylated. Nevertheless, a small fraction of the secreted VP1 assembled into VLP-like structures. Human rotavirus Wa capsid proteins, VP2 and VP6, were expressed using the Drosophila metallothionein promoter and the Drosophila BiP signal sequence in stably transformed Drosophila S2 cells (Lee at al. 2011). An encephalomyocarditis virus (EMCV)-derived internal ribosomal entry site (IRES) element was employed for bicistronic expression of

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VP2 and VP6. Stably transformed cells secreted doublelayered VLPs consisting of rotavirus VP2 and VP6 to the culture medium. Drosophila S2 cells were co-transfected with four plasmid vectors containing the genes encoding human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp160, Rev-independent precursor 55 kDa (Pr55) Gag protein, Rev protein, and an antibiotic resistance marker (Yang et al. 2012). The genes encoding the HIV-1 envelope and Rev proteins were driven by the inducible Drosophila metallothionein promoter, while the gene encoding HIV-1 Gag was placed under the control of the constitutive Drosophila actin 5C promoter. The gene encoding the HIV-1 envelope protein was in frame with the Drosophila BiP signal sequence. In stably transformed Drosophila S2 cells, HIV-1 envelope proteins were properly cleaved, glycosylated, and incorporated into VLPs with Gag. The yield of HIV-1 VLPs secreted to the culture medium was equivalent to 7.5 mg gp120/L and 9.5 mg Gag/L, which was comparable to that produced in the baculovirus– insect cell system. Mice primed with plasmids carrying the HIV-1 gp120 and Gag genes and boosted with HIV-1 VLPs produced by stably transfected cells in the presence of CpG exhibited both anti-envelope antibody responses and envelope- and Gag-specific CD8 T cell responses.

VLP production in recombinant lepidopteran insect cells Stable expression of heterologous genes in lepidopteran insect cells has also been investigated. Cell lines commonly used with Autographa californica NPV (AcNPV) in the baculovirus–insect cell system, such as Sf9 derived from Spodoptera frugiperda and BTI-TN-5B1-4 (High Five) from Trichoplusia ni, are often employed as host cells for stable expression as well. In particular, High Five cells have been shown to be an excellent host for the production of recombinant secreted proteins (Farrell et al. 1999; Keith et al. 1999; Yamaji et al. 2008). In the case of lepidopteran insect cells, the choice of a promoter to drive the heterologous gene expression is important, as the use of a weak promoter results in low recombinant protein yields (Yamaji 2011). The activity of promoters used in insect cell expression systems can be enhanced by certain cis- or trans-acting elements derived from baculoviruses (Douris et al. 2006; Harrison and Jarvis 2007). Stably transformed High Five cells expressing the large surface antigen of HBV (L protein) were established by cotransfecting cells with an expression plasmid vector and a plasmid carrying an antibiotic resistance marker (Shishido et al. 2006) (Table 1). The expression vector contained the Bombyx mori cytoplasmic actin promoter downstream of the B. mori NPV (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer, which was developed for high-level expression of foreign gene in lepidopteran insect cells (Farrell et al.

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1998, 1999; Keith et al. 1999). The use of the IE-1 transactivator and the HR3 enhancer allowed an increase of more than 1,000-fold in the stimulation of foreign gene expression through the actin promoter (Douris et al. 2006; Lu et al. 1997). By using this expression vector with the honeybee melittin signal sequence, stably transformed High Five cells secreted particles composed of L protein to culture medium at a concentration of 1.7 mg/L. Stably transformed High Five cells expressing VLPs based on HIV-1 Pr55 Gag protein were generated by co-transfecting cells with a plasmid vector containing the HIV-1 Gag coding gene downstream of the AcNPV HR5 enhancer and the AcNPV IE-1 immediate early promoter, together with a plasmid vector carrying a selection marker (Tagliamonte et al. 2010). Yields of Pr55 Gag protein expressed in the cytoplasm of stably transformed High Five cells and those in the culture medium were 5–8 and 1–3 mg/L, respectively. Expressed Pr55 Gag proteins auto-assembled into enveloped VLPs, which were released into the culture medium. VLPs produced by recombinant High Five cells elicited a specific systemic humoral response in immunized mice. In a similar manner, stably transformed High Five cells secreting rotavirus corelike particles were established by transfecting cells with the VP2 gene of the bovine rotavirus RF strain (Shoja et al. 2013). Stably transformed High Five cells expressing HIV-1 Pr55 Gag VLPs were doubly transfected with a plasmid vector carrying the modified HIV-1 gp140 envelope gene, which included the honeybee melittin signal sequence, coding sequences for the HIV-1 gp120 and gp41 ectodomain, and the trans-membrane domain of AcNPV major glycoprotein gp64 (Tagliamonte et al. 2011). HIV-1 Gag and gp140 Env proteins were detected in the culture medium as well as in the cytoplasmic fraction of doubly transformed High Five cells. Pr55 Gag VLPs expressing trimeric forms of gp120/gp41 complexes on their surface were released by cell membrane budding into the culture medium. The Pr55 Gag/gp140 Env-VLPs produced by the doubly transformed High Five cells elicited systemic anti-Env humoral response in immunized mice. The cDNA of Rous sarcoma virus (RSV) Gag protein lacking the protease region (PR) was stably expressed under the control of the Orgyia pseudotsugata NPV (OpNPV) IE-2 promoter in High Five cells (Deo et al. 2011). RSV gag-based VLPs, which were structurally similar to native RSV particles, were secreted into the culture medium. High Five cells were stably transfected with a plasmid vector encoding the JEV prM signal sequence and the prM and E genes (Yamaji et al. 2013). DNA encoding a form of prM with a pr/M cleavage site mutation (Konishi et al. 2001) was used to suppress the toxic cell-fusion activity of VLPs (Fig. 1). A single plasmid vector was used, which contained the BmNPV IE-1 transactivator, the BmNPV HR3 enhancer, and the B. mori actin promoter for high-level expression, together with a blasticidin resistance gene for use as a

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selectable marker (Yamaji et al. 2008). High Five cells stably secreting a high concentration of E protein were efficiently developed by incubation in the presence of blasticidin. E proteins secreted from stably transformed High Five cells were in a particulate form that was similar to slowly sedimenting hemagglutinin (SHA) particles released from JEV-infected mosquito cells. VLPs recovered from the culture supernatant successfully induced neutralizing antibodies in mice. A significantly higher yield of E protein (30 mg/L) was attained using recombinant High Five cells compared with using the baculovirus–insect cell system (3 mg/L; Yamaji et al. 2012). In place of the JEV authentic prM signal sequence, the Drosophila BiP signal sequence was also available for efficient secretory production of JE VLPs in High Five cells (Yamaji and Konishi 2013). As an alternative to the random integration of an expression vector into the insect cell genome through recombination under antibiotic selection, the piggyBac transposable element was used for the generation of stably transformed Sf9 cells that expressed HIV-1 Pr55 Gag protein (Lynch et al. 2010). Stably transformed Sf9 cells secreted HIV-1 Pr55 Gag VLPs to the culture medium, but the yield of VLPs was lower than that obtained with the baculovirus–insect cell system. Immunogenicity in mice of Gag VLPs produced by stably transformed cells was lower than when VLPs were produced by baculovirus-infected cells, which may be due to a lack of baculovirus glycoprotein incorporation in the VLPs obtained from stably transformed cells. With respect to transgene integration, a recombinase-mediated cassette exchange system using flipase was successfully used in Sf9 cells for stable integration of the enhanced green fluorescent protein (EGFP) gene (Fernandes et al. 2012).

Concluding remarks Insect cells are ideal host cells that efficiently and safely produce mammalian virus proteins including VLPs. Insect cells are capable of producing recombinant proteins with complex folding and post-translational processing and modifications performed in higher eukaryotes. Insect cells can grow to a high cell density in suspension culture with a serum-free or animal-derived component-free medium. In addition, insect cells do not support the growth of mammalian viruses or mycoplasmas. Lepidopteran insect cell lines, a subclone derived from High Five cells (Schiller et al. 2012) and expresSF + cells derived from the Sf9 cell line (Cox 2012; Cox and Hashimoto 2011), are employed as host cells in the baculovirus–insect cell system for the commercial manufacture of a human papillomavirus-like particle vaccine and a recombinant hemagglutinin influenza vaccine, respectively. In insect cell-based expression systems, the baculovirus–insect cell system is well developed and has been used extensively

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for the production of a wide variety of VLPs. However, this system has several inherent drawbacks including contamination of VLPs with progeny baculovirus particles. The relatively new stably transformed insect cell systems can be available as attractive alternative platforms for the baculovirus–insect cell system. Secretory production of different types of VLPs, with or without an envelope and composed of either single or multiple structural proteins, have been demonstrated in stably transformed cell lines derived from dipteran and lepidopteran insects (Table 1). VLPs produced by stably transformed insect cells have successfully induced immune responses in vivo. Cotransfection with multiple plasmid vectors and bicistronic expression using an IRES element have been developed for the production of complex VLPs composed of two or more viral structural proteins. Signal sequences of insect proteins such as Drosophila BiP and honeybee melittin efficiently functioned in stably transformed insect cells. In addition to the random integration of an expression plasmid into the insect cell genome through recombination under antibiotic selection, transposon-mediated mutagenesis was available to integrate transgenes into the insect cell genome. In some cases, the yield of VLPs attained with recombinant insect cells was comparable to, or higher than, that obtained by baculovirus-infected insect cells. This mini-review shows that recombinant insect cells offer a promising approach to the development and production of VLPs for use as next-generation vaccines and diagnostic antigens. Stable insect cell lines have been developed for a relatively short period of 1–2 months (Deml et al. 1999a; Deo et al. 2011; Douris et al. 2006; Lee et al. 2011; Ng et al. 2007; Shishido et al. 2006; Shoja et al. 2013; Tagliamonte et al. 2010, 2011; Yamaji et al. 2013; Yang et al. 2012; Zhang et al. 2007). However, further developments are needed in order to increase the productivity of VLPs. These developments include applications of genomics, proteomics, and metabolomics technologies, host cell engineering, and optimization of culture medium and feeding strategies, as developed for the manufacture of biopharmaceuticals in mammalian cell culture. Advances have been made in the development of bioprocesses for the large-scale, high-density culture of insect cells, particularly for the baculovirus–insect cell system (Drugmand et al. 2012; Ikonomou et al. 2003; Jardin et al. 2007; Marteijn et al. 2003; Meghrous et al. 2010; Shishido et al. 2007; Vicente et al. 2011; Yamaji and Konishi 2013; Yamaji et al. 2006), which also lead to increased yields of VLPs.

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