The Insect Cell-Baculovirus Expression Vector System (IC-BEVS) is a widely used eukaryotic expression system that not only achieves high protein expression but also possesses post-translational modification processing and foreign protein transfer capabilities similar to most higher eukaryotes. Additionally, due to the absence of any animal virus fragments potentially threatening to mammals, the baculovirus system has become one of the most universal and powerful eukaryotic vector systems for recombinant protein expression since the introduction of BEVS technology in 1983^[1].

1. Classification, Structural Phenotype, and Infection Modes of Baculoviruses

Baculoviruses possess large circular double-stranded DNA genomes, ranging from approximately 80 to 180 kbp. The Baculoviridae family is further divided into four genera: Alphabaculovirus, Betabaculovirus, Deltabaculovirus, and Gammabaculovirus^[2], as shown in Figure 1.

Fig 1. 91 baculovirus whole genomes queried from NCBI

Among the 91 known whole genomes of baculoviruses, the genus Alphabaculovirus accounts for 61 species, making it the most extensively studied. This includes viruses such as Autographa californica multiple nucleopolyhedrovirus (AcMNPV), Bombyx mori nucleopolyhedrovirus (BmNPV), Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV), and Helicoverpa armigera nucleopolyhedrovirus (HearNPV). The Autographa californica multiple nucleopolyhedrovirus (AcMNPV), isolated from the alfalfa looper moth, is currently one of the most widely studied baculoviruses^[3].

A notable feature of the typical baculovirus infection cycle is the production of two physically distinct types of virus particles. One viral phenotype is known as the occlusion-derived virus (ODV), while the other is called the budded virus (BV)^[4], as shown in Figure 2. The formation times of the two virus types are different. BV begins to form early in virus replication and contains a single nucleocapsid enveloped by a membrane with GP64 protein, allowing for cell-to-cell transmission within the insect. ODV starts forming late in virus replication and contains one or more nucleocapsids surrounded by a protein matrix.

Fig 2. Morphological Structure of Virus Particles: BV and ODV

In the ODV particle form, the nucleocapsid acquires an envelope within the cell nucleus, after which the virus particles are enclosed or embedded in a crystalline protein matrix, forming an occlusion body (OB). ODV particles spread infection between insects, particularly infecting the epithelial cells of the insect midgut. After ODV infects the midgut cells, BV particles bud from the basal surface of infected midgut epithelial cells into the hemocoel, systematically spreading infection from cell to cell and from tissue to tissue within the infected animal. For viruses such as AcMNPV, most tissues in the hemocoel (tracheal epithelium, hemocytes, epidermis, muscles, etc.) become infected and produce additional BV, further spreading the infection within the animal. BV contains a biologically prominent envelope glycoprotein, Gp64, which is acquired when the baculovirus buds through the plasma membrane. Without this protein, cell-to-cell infection cannot be completed^[5].

Figure 3 illustrates the baculovirus particle phenotypes and their roles in host tissue infection, explaining the primary and secondary stages of infection. The primary stage of infection occurs when occlusion bodies (OBs) dissolve at high pH and release occlusion-derived virus (ODV) particles into the midgut lumen. After passing through the peritrophic membrane, ODV particles bind to and fuse with microvilli of polarized epithelial cells of the midgut, releasing nucleocapsids into the cytoplasm. Following viral replication and nucleocapsid assembly in the nucleus (or direct transit), nucleocapsids are transported to the basal plasma membrane, where they bud to produce the budded virus (BV) phenotype. BV particles may directly infect some cells (tracheal cells and hemocytes) or circulate in the hemolymph, infecting other tissues such as fat body and muscle. In the secondary stage of infection, infection of other tissues results in the production of additional BV, further spreading the infection within the animal. ODV and OBs are produced in all cell types and are subsequently released upon cell lysis and animal dissolution or liquefaction^[4]. OBs are released from infected cells during cell lysis late in the animal infection, typically as infected insects liquefy. This is a process mediated by at least two virus-encoded enzymes (chitinase and cathepsin) that catalyze the breakdown of the insect exoskeleton, releasing OBs into the environment and completing the infection cycle in nature.

Fig 3. Baculovirus Particle Phenotype and its Role in Host Tissue Infection

Figure 4 illustrates the entry, replication, and egress of baculoviruses in non-midgut host cells. The figure represents infection by an alphabaculovirus BV and the subsequent viral replication and production of BV and ODV. After BV binding and entry via clathrin-mediated endocytosis, it is transported along microtubules, and upon acidification, the nucleocapsid (blue) is released. Polymerization of the P78/83 Arp2/3 complex leads to the initiation of actins, which provides the propulsive force for nucleocapsid transport in the cytoplasm and passage through nuclear pores. The release of nucleocapsids in the nucleus results in viral gene expression, DNA replication, and the assembly of progeny nucleocapsids (blue and green) in viral stroma. Within the nucleocapsids, some are designated for egress from the nucleus (blue), while others are designated for ODV production (green). Nucleocapsids leaving the nucleus are observed in cytoplasmic vesicles (transport vesicles), which release the nucleocapsids into the cytoplasm^[4].

Fig 4. Entry, replication, and egress of baculoviruses in non-midgut host cells

Furthermore, ODV viral particles have a high specificity for infecting insect midgut cells and must overcome significant physical and biological barriers. Additionally, the stability of ODV in the environment heavily relies on OB protein, which surrounds and protects ODV viral particles from desiccation and possible UV inactivation. The OB protein (called polyhedrin in most baculoviruses) forms natural crystals in OBs, where polyhedrin trimers are connected by disulfide bonds to form dodecamers. Figure 5 shows electron micrographs of AcMNPV replication and viral particle phenotypes.

Fig 5. Electron micrographs of AcMNPV replication and particle phenotypes

In cell culture systems, infection is initiated by BV particles, as ODV particles have poor infectivity in cultured cells. Infections typically occur in larvae. Therefore, ODV is not infectious in cell culture, and there is no need for the protective crystallization of polyhedrin protein in vitro^[6].

2. Insect Cell Line Culture and Characteristics

Over the past few decades, hundreds of insect cell lines have been isolated from more than 100 insect species across 6 genera^[7]. Lepidopteran cell lines are primarily used with BEVS for recombinant protein expression and baculovirus biopesticide production, particularly cell lines derived from silkworm, cabbage looper, fall armyworm, and Trichoplusia species.

Among these, Sf-9, Sf-21, Tn-368, and High Five are the most widely used cell lines in industrial applications. These cell lines and related ones are highly susceptible to infection by AcMNPVs and other baculoviruses. Figure 6 illustrates some commonly used engineered cell lines and their applicable directions^[8].

Fig 6. Commonly Used Engineered Cell Lines and Their Applicable Directions

· Sf-21 Cell Line

The first cell line widely used for research and technological applications is Sf-21, an ovarian cell line from the fall armyworm Spodoptera frugiperda, established by Vaughn and colleagues in 1977. It is also the parental line of the Sf-9 cell clone^[9]. The Sf-21 cell line can produce samples with relatively high uniformity, and after culturing for a certain number of generations, the resulting cell line achieves homogeneity, belonging to the same type of cells.

· Sf-9 Cell Line

Sf-9 cells are derived from the ovarian tissue of female fall armyworm pupae and are better suited for replicating baculovirus expression vectors. The Sf-9 cell line originates from Sf-21 and is selected from Sf-21 clones. Compared to Sf-21, Sf-9 has better tolerance to pH, osmotic pressure, and shear forces. Both Sf-21 and Sf-9 cell lines are suitable for baculovirus infection, purification, high-titer viruses production, and expression of recombinant proteins. The growth and infection characteristics of Sf-9 cells make them excel in baculovirus transfection and amplification. Additionally, Sf-9 cells are relatively small in volume, uniform in shape, and easily form monolayers and culture plates.

· Tn-368 Cell Line

Tn-368 and High Five are both cell lines derived from the cabbage looper (Trichoplusia ni, Tn). However, studies have found that High Five cells can be latently infected by nodavirus. Furthermore, baculovirus infection of High Five cells can stimulate the production of nodavirus particles.

· High Five Cell Line

Tn-368 and High Five are both derived from the ovarian tissue of the cabbage looper (Trichoplusia ni, Tn). In terms of recombinant protein expression, the High Five cell line shows higher expression levels, sometimes even more than 20 times that of Sf-9 cells for certain proteins. High Five cells have more complex glycosylation and phosphorylation modifications compared to Sf9, making them more suitable for expressing some secretory recombinant proteins. The virus-like particle vaccine for human papillomavirus is produced in High Five cells. The High Five cell line exhibits superior performance in expressing recombinant viruses and is frequently used for transfection and plaque purification.


Compared to Sf cell lines, High Five cell lines have demonstrated superior secretory glycoprotein production capabilities from the outset. Figure 7 summarizes some key characteristics of Sf-9 and High Five cell lines^[7].

Fig 7. Some key characteristics of Sf-9 and High Five cell lines.

Comparative findings:

(1) Sf9 cells are more suitable for recombinant virus amplification and packaging; Sf21 cells, due to their larger cell diameter, are more suitable for virus titer plaque assays; High Five cells are more suitable for secretory protein expression.

(2) Sf9 cells have stronger tolerance to osmotic pressure, shear force, and pH compared to Sf21 cells, showing excellent performance in transfection and production of high-titer baculovirus.

(3) High Five cells have a shorter proliferation time and higher expression levels compared to Sf9 cells. The proteins expressed by High Five cells have more complex glycosylation modifications than those expressed by Sf9 cells. Both Sf9 and High Five cells have the characteristics of suspension and adherent growth, therefore enabling large-scale production of recombinant proteins through shake flasks and bioreactors.


Sf cell lines are suitable for suspension culture and can be easily detached from culture surfaces with appropriate agitation without trypsinization. Tn cell lines were initially adherent-dependent, but today they have been well-adapted to suspension culture. Sf-21 is more fragile than Sf-9, with lower tolerance to osmotic, pH, and shear stress, and has a lower growth rate. Nowadays, the use of Sf-21 has decreased due to its growth and infection characteristics.

High Five is an insect cell line that typically produces more recombinant protein, up to 20 times higher compared to Sf-9. High Five cells are more resistant to shear stress and agitation than Sf-9. High Five cells are larger than Sf-9 cells, have a higher protein content, and their cell size distribution is broader than that of Sf-9 cells. However, cell size depends on media osmolarity, shear stress, and cell status (viable, apoptotic, etc.). In recent years, the superior characteristics of the express SF+ cell line, derived from SF-9 cells, have led to its use in the production of several biologics, such as the influenza vaccine FluBlok.

References

[1] Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol Cell Biol. 1983;3(12):2156-65.
[2] The interaction between baculoviruses and their insect hosts. Dev Comp Immunol.2018;83:114-123.
[3] 凌同, 余黎, 白慕群. 昆虫杆状病毒表达系统的研究进展与应用[J]. 微生物学免疫学进展,2014,42(02): 70-78.
[4] Baculovirus Entry and Egress from Insect Cells. Annu Rev Virol. 2018; 29; 5(1): 113-139.
[5] 冯敏, 吴小峰. 昆虫杆状病毒囊膜蛋白GP64与宿主细胞表面因子互作的研究综述. 蚕业科学, 2014, 40(5): 911–916.
[6] An Overview of Cell Culture Engineering for the Insect Cell-Baculovirus Expression Vector System (BEVS). Animal Cell Culture. 2015. 9: 501-515.
[7] Insect cells as factories for biomanufacturing. Biotechnol Adv. 2012;30(5):1140-57. 
[8] Genetic engineering of baculovirus-insect cell system to improve protein production. Front Bioeng Biotechnol. 2022；20; 10: 994743.
[9] The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae).In Vitro. 1977;13(4):213-7.