Genetic engineering of baculovirus-insect cell system to improve protein production

Abstract

The Baculovirus Expression Vector System (BEVS), a mature foreign protein expression platform, has been available for decades, and has been effectively used in vaccine production, gene therapy, and a host of other applications. To date, eleven BEVS-derived products have been approved for use, including four human vaccines [Cervarix against cervical cancer caused by human papillomavirus (HPV), Flublok and Flublok Quadrivalent against seasonal influenza, Nuvaxovid/Covovax against COVID-19], two human therapeutics [Provenge against prostate cancer and Glybera against hereditary lipoprotein lipase deficiency (LPLD)] and five veterinary vaccines (Porcilis Pesti, BAYOVAC CSF E2, Circumvent PCV, Ingelvac CircoFLEX and Porcilis PCV). The BEVS has many advantages, including high safety, ease of operation and adaptable for serum-free culture. It also produces properly folded proteins with correct post-translational modifications, and can accommodate multi-gene– or large gene insertions. However, there remain some challenges with this system, including unstable expression and reduced levels of protein glycosylation. As the demand for biotechnology increases, there has been a concomitant effort into optimizing yield, stability and protein glycosylation through genetic engineering and the manipulation of baculovirus vector and host cells. In this review, we summarize the strategies and technological advances of BEVS in recent years and explore how this will be used to inform the further development and application of this system.

Advances in CRISPR-Cas9 for the Baculovirus Vector System: A Systematic Review

“…BEVS has also provided an effective way to overcome some difficulties in the implementation of other expression systems, as it was used to overcome the poor production of recombinant adeno-associated viruses (AAVs), which are now produced in sufficient quantities of AAV vectors for large scale studies [ 13 ]. For the generation of baculoviruses, virus-like particles, recombinant proteins, and gene therapy vectors, more than 400 different types of insect cell lines have been developed but the most widely used type is Sf9, which is a clonal isolate of Sf21 cell line derived from pupal ovarian tissue of Spodoptera frugiperda [ 14 ]. Another commonly used cell line is BT1-TN5B1-4, also known as High Five, which is derived from ovarian tissue of adults from Cabbage Looper Trichoplusia ni [ 15 ].…”

“…To facilitate the use of these systems, several improvements and modifications have been applied. One improvement includes ExpiFectamine Sf (Thermo Fisher Scientific, Waltham, MA, USA), a next-generation cationic lipid based reagent for more robust transfection, and modifications include using RNAi technology or CRISPR Cas9 for engineering genome composition, which led to improved expression process and helped in overcoming some of the limitations of the BEVS [ 14 ].…”

“…However, mammalian cells extend this intermediate to complex N glycans with various glycosyltransferases, while insect cells cannot elongate this product due to a membrane-bound enzyme, N-acetylglucosaminidase, which catalyzes the removal of terminal N-acetylglucosamine residue in trimmed N-glycan processing intermediates [ 128 ]. Similarly, two Lepidopteran insect cell lines have been shown to transfer and trim nascent polypeptides to yield the same processing intermediates as mammalian cells [ 14 ]. However, the N-glycosyl intermediate is eliminated as a substrate for N-acetylglucosamine transferase II by N-acetylglucosaminidase, terminating as simple paucimannosidic N-glycans in insect cells [ 14 ].…”

“…Similarly, two Lepidopteran insect cell lines have been shown to transfer and trim nascent polypeptides to yield the same processing intermediates as mammalian cells [ 14 ]. However, the N-glycosyl intermediate is eliminated as a substrate for N-acetylglucosamine transferase II by N-acetylglucosaminidase, terminating as simple paucimannosidic N-glycans in insect cells [ 14 ]. As a solution, the fdl gene, which encodes for N-acetylglucosaminidase, was recently deleted in Sf9 cells, thus promoting further elongation toward homogenous, terminally sialylated N-glycans [ 126 ].…”

The efficacy of new oral vaccine feeds against Salmonid novirhabdovirus in rainbow trout

“…The baculovirus/insect cell expression system has been widely used for the expression of foreign genes, having a great potential for vaccine development [27] , [28] , [29] . The system is attractive because it is non-pathogenic towards vertebrates [30] . It provides for an abundant supply of the protein-of-interest due to the strong polyhedrin promoter [31] .…”