Effective expression and characterization of the receptor binding domains in SARS-CoV-2 Spike proteins from original strain and variants of concern using Bombyx mori nucleopolyhedrovirus in silkworm.

A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the global pandemic of COVID-19 in 2020. Through structural analysis, it was found that several amino acid residues in the human angiotensin-converting enzyme-2 (hACE2) receptor directly interact with those in the receptor binding domain (RBD) of the spike glycoprotein (S-protein). Various cell lines, including HEK293, HeLa cells, and the baculovirus expression vector system (BEVS) with the insect cell line Sf9, have been utilized to produce the RBD. In this study, we investigated the use of Bombyx mori nucleopolyhedrovirus (BmNPV) and BEVS. For efficient production of a highly pure recombinant RBD protein, we designed it with two tags (His tag and STREP tag) at the C-terminus and a solubilizing tag (SUMO) at the N-terminus. After expressing the protein using BmNPV and silkworm and purifying it with a HisTrap excel column, the eluted protein was digested with SUMO protease and further purified using a Strep-Tactin Superflow column. As a result, we obtained the RBD as a monomer with a yield of 2.6 mg/10 mL serum (equivalent to 30 silkworms). The RBD showed an affinity for the hACE2 receptor. Additionally, the RBDs from the Alpha, Beta, Gamma, Delta, and Omicron variants were expressed and purified using the same protocol. It was found that the RBD from the Alpha, Beta, Gamma, and Delta variants could be obtained with yields of 1.4-2.6 mg/10 mL serum and had an affinity to the hACE2 receptor.

Comprehensive Transcriptome Analysis in the Testis of the Silkworm, Bombyx mori.

Spermatogenesis is an important process in reproduction and is conserved across species, but in Bombyx mori, it shows peculiarities, such as the maintenance of spermatogonia by apical cells and fertilization by dimorphic spermatozoa. In this study, we attempted to characterize the genes expressed in the testis of B. mori, focusing on aspects of expression patterns and gene function by transcriptome comparisons between different tissues, internal testis regions, and Drosophila melanogaster. The transcriptome analysis of 12 tissues of B. mori, including those of testis, revealed the widespread gene expression of 20,962 genes and 1705 testis-specific genes. A comparative analysis of the stem region (SR) and differentiated regions (DR) of the testis revealed 4554 and 3980 specific-enriched genes, respectively. In addition, comparisons with D. melanogaster testis transcriptome revealed homologs of 1204 SR and 389 DR specific-enriched genes that were similarly expressed in equivalent regions of Drosophila testis. Moreover, gene ontology (GO) enrichment analysis was performed for SR-specific enriched genes and DR-specific enriched genes, and the GO terms of several biological processes were enriched, confirming previous findings. This study advances our understanding of spermatogenesis in B. mori and provides an important basis for future research, filling a knowledge gap between fly and mammalian studies.

The biological role of core 1ß1-3galactosyltransferase (T-synthase) in mucin-type O-glycosylation in Silkworm, Bombyx mori.

O-glycosylation of secreted and membrane-bound proteins is an important post-translational modification that affects recognition of cell surface receptors, protein folding, and stability. However, despite the importance of O-linked glycans, their biological functions have not yet been fully elucidated and the synthetic pathway of O-glycosylation has not been investigated in detail, especially in the silkworm. In this study, we aimed to investigate O-glycosylation in silkworms by analyzing the overall structural profiles of mucin-type O-glycans using LC-MS. We found GalNAc or GlcNAc monosaccharide and core 1 disaccharide (Galß1-3-GalNAcα1-Ser/Thr) were major components of the O-glycan attached to secreted proteins produced in silkworms. Furthermore, we characterized the 1 b1,3-galactosyltransferase (T-synthase) required for synthesis of the core 1 structure, common to many animals. Five transcriptional variants and four protein isoforms were identified in silkworms, and the biological functions of these isoforms were investigated. We found that BmT-synthase isoforms 1 and 2 were localized in the Golgi apparatus in cultured BmN4 cells and functioned both in cultured cells and silkworms. Additionally, a specific functional domain of T-synthase, called the stem domain, was found to be essential for activity and is presumed to be needed for dimer formation and galactosyltransferase activity. Altogether, our results elucidated the O-glycan profile and function of T-synthase in the silkworm. Our findings allow the practical comprehension of O-glycosylation required for employing silkworms as a productive expression system.

High yield production of norovirus GII.4 virus-like particles using silkworm pupae and evaluation of their protective immunogenicity.

Noroviruses (NoVs) are one of the major causes of acute viral gastroenteritis in humans. Virus-like particles (VLPs) without genomes that mimic the capsid structure of viruses are promising vaccine candidates for the prevention of NoVs infection. To produce large amounts of recombinant protein, including VLPs, the silkworm-expression vector system (silkworm-BEVS) is an efficient and powerful tool. In this study, we constructed a recombinant baculovirus that expresses VP1 protein, the major structural protein of NoV GII.4. Expression analysis showed that the baculovirus-infected silkworm pupae expressed NoV VP1 protein more efficiently than silkworm larval fat bodies. We obtained about 4.9 mg of purified NoV VP1 protein from only five silkworm pupae. The purified VP1 protein was confirmed by dynamic light scattering and electron microscopy to form VLPs of approximately 40 nm in diameter. Antisera from mice immunized with the antigen blocked NoV VLPs binding to histo-blood group antigens of pig gastric mucin and also blocked NoV infection in intestinal epithelial cells derived from human induced pluripotent stem (iPS) cells. Our findings demonstrated that NoV VLP eliciting protective antibodies could be obtained in milligram quantities from a few silkworm pupae using the silkworm-BEVS.

Construction of gene co-expression networks in cultured silkworm cells and identification of previously uncharacterized lepidopteran-specific genes required for chromosome dynamics.

Advances in sequencing technology and bioinformatics have accelerated gene discovery and homology-based functional annotation in many species, and numerous targeted gene studies have greatly expanded the understanding of gene functions. Nevertheless, there are still many genes that lack homology with genes in other evolutionary lineages and are left as genes with unknown functions. We constructed a gene co-expression network from the Bombyx mori ovary-derived cell line, BmN4, and attempted to infer the biological roles of uncharacterized genes based on the correlation between the function-known and unknown genes. Within this network, we focused on the co-expression modules involved in chromosome architecture, dynamics, and integrity, and selected the uncharacterized genes for subsequent RNAi-based phenotypic screening. This approach enabled the identification of 5 genes whose knockdown led to abnormalities in chromosome dynamics and spindle morphology in mitosis. One of them was a recently characterized gene, BmCenp-T, which plays a central role in building the kinetochore protein complex on the silkworm holocentric chromosomes. In this study, we suggest a method for constructing the gene co-expression network and selecting candidate genes for small-scale RNAi screening. This approach is complementary to homology-based annotation and may be useful for the analysis of lineage-specific uncharacterized genes such as orphan genes.

Characterization of a Novel Heterochromatin Protein 1 Homolog "HP1c" in the Silkworm, Bombyx mori.

Heterochromatin protein 1 plays an important role in chromatin structure and gene expression regulation. Three HP1 genes have been found in Homo sapiens, and five HP1 genes have been reported in Drosophila melanogaster. On the other hand, in Bombyx mori, only two HP1 genes, BmHP1a and BmHP1b, were reported. In this research, we have reported the molecular and functional characterization of a novel Bombyx mori HP1 gene (BmHP1c), which had stronger transcriptional repression activity than BmHP1a. BmHP1a and BmHP1b is reported to form homo- and heterodimers, but in co-immunoprecipitation experiments, no homo- or hetero-dimer formation of BmHP1c with the other silkworm HP1s is detected. The intracellular localization of BmHP1c is not only in the nucleus but also in the cytoplasm like mammalian HP1γ. In contrast to human HP1a and b, all three BmHP1s were localized preferentially in the regions poorly stained with DAPI. Interestingly, the double knockdown of BmHP1a and b, but not BmHP1c with a or b, arrested the cell cycle at the G2/M phase. These results suggest that BmHP1c is not essential for cell progression and plays a different role than BmHP1a and BmHP1b.

Silkworm FoxL21 plays important roles as a regulator of ovarian development in both oogenesis and ovariole development.

The ovary is an important organ in reproduction. In insects, especially lepidopteran insects, the oocytes and reproductive organs develop rapidly during the pupal stage. Despite their drastic morphological changes, the molecular mechanisms of ovary development are not fully understood. In this study, it is found that forkhead box transcription factor L2, member 1 (FoxL21), which is known to be involved in ovarian differentiation and maintenance in vertebrates, is required for the development of the ovary in the silkworm, Bombyx mori. FoxL21 was expressed in the ovary and ovariole during the larval and pupal stage, respectively. In silkworms in which FoxL21 was knocked out by genome editing, multiple ovarian dysfunctions, such as, abnormal egg formation, thinning of the ovariole sheaths, and defective connection of the oviductus geminus with the ovariole were observed. Finally, ovarian transplantation experiments using the knockout silkworms revealed that FoxL21 functions in the ovariole, but not in the oviductus geminus.

Leptin Is an Important Endocrine Player That Directly Activates Gonadotropic Cells in Teleost Fish, Chub Mackerel.

Leptin, secreted by adipocytes, directly influences the onset of puberty in mammals. Our previous study showed that leptin stimulation could promote the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from pituitary cells in primary culture and ovarian development in chub mackerel. This study aimed to elucidate the detailed mechanism of leptin-induced effects on gonadotropin hormone-producing cells. We produced recombinant leptin using silkworm pupae and investigated the effects of leptin on FSH and LH secretion and gene expression in the primary culture of pituitary cells from chub mackerel. The presence or absence of co-expression of lepr mRNA, FSH and LH b-subunit mRNA in gonadotropic cells was examined by double-labeled in situ hybridization. The addition of leptin significantly increased the secretion and gene expression of FSH and LH from male and female pituitary cells in primary culture. In contrast, gonadotropin-releasing hormone 1 affected neither FSH secretion in cells from females nor fshb and lhb expression in cells from males and females. The expression of lepr was observed in FSH- and LH-producing cells of both males and females. The results indicate that leptin directly regulates gonadotropin synthesis and secretion and plays an important role in the induction of puberty in teleost fish.

Stable trimer formation of spike protein from porcine epidemic diarrhea virus improves the efficiency of secretory production in silkworms and induces neutralizing antibodies in mice.

Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen of watery diarrhea that causes serious economic loss to the swine industry worldwide. Especially because of the high mortality rate in neonatal piglets, a vaccine with less production cost and high protective effect against PEDV is desired. The intrinsically assembled homotrimer of spike (S) protein on the PEDV viral membrane contributing to the host cell entry is a target of vaccine development. In this study, we designed trimerized PEDV S protein for efficient production in the silkworm-baculovirus expression vector system (silkworm-BEVS) and evaluated its immunogenicity in the mouse. The genetic fusion of the trimeric motif improved the expression of S protein in silkworm-BEVS. A small-scale screening of silkworm strains to further improve the S protein productivity finally achieved the yield of about 2 mg from the 10 mL larval serum. Mouse immunization study demonstrated that the trimerized S protein could elicit strong humoral immunity, including the S protein-specific IgG in the serum. These sera contained neutralizing antibodies that can protect Vero cells from PEDV infection. These results demonstrated that silkworm-BEVS provides a platform for the production of trimeric S proteins, which are promising subunit vaccines against coronaviruses such as PEDV.

Active Human and Murine Tumor Necrosis Factor α Cytokines Produced from Silkworm Baculovirus Expression System.

The tumor necrosis factor α (TNFα) has been employed as a promising reagent in treating autoimmunity and cancer diseases. To meet the substantial requirement of TNFα proteins, we report in this study that mature types of recombinant human and murine TNFα proteins are successfully expressed in the baculovirus expression system using silkworm larvae as hosts. The biological activities of purified products were verified in culture murine L929 cells, showing better performance over a commercial Escherichia coli-derived murine TNFα. By comparing the activity of purified TNFα with or without the tag removal, it is also concluded that the overall activity of purified TNFα cytokines could be further improved by the complete removal of C-terminal fusion tags. Collectively, our current attempt demonstrates an alternative platform for supplying high-quality TNFα products with excellent activities for further pharmaceutical and clinical trials.

Production of scFv, Fab, and IgG of CR3022 Antibodies Against SARS-CoV-2 Using Silkworm-Baculovirus Expression System.

COVID-19, caused by SARS-CoV-2, is currently spreading around the world and causing many casualties. Antibodies against such emerging infectious diseases are one of the important tools for basic viral research and the development of diagnostic and therapeutic agents. CR3022 is a monoclonal antibody against the receptor binding domain (RBD) of the spike protein (S protein) of SARS-CoV found in SARS patients, but it was also shown to have strong affinity for that of SARS-CoV-2. In this study, we produced large amounts of three formats of CR3022 antibodies (scFv, Fab and IgG) with high purity using a silkworm-baculovirus expression vector system. Furthermore, SPR measurements showed that the affinity of those silkworm-produced IgG antibodies to S protein was almost the same as that produced in mammalian expression system. These results indicate that the silkworm-baculovirus expression system is an excellent expression system for emerging infectious diseases that require urgent demand for diagnostic agents and therapeutic agents.

Quick and Easy Assembly of a One-Step qRT-PCR Kit for COVID-19 Diagnostics Using In-House Enzymes.

One-step reverse-transcription quantitative polymerase chain reaction (qRT-PCR) is the most widely applied method for COVID-19 diagnostics. Notwithstanding the facts that one-step qRT-PCR is well suited for the diagnosis of COVID-19 and that there are many commercially available one-step qRT-PCR kits in the market, their high cost and unavailability due to airport closures and shipment restriction became a major bottleneck that had driven the desire to produce the key components of such kits locally. Here, we provide a simple, economical, and powerful one-step qRT-PCR kit based on patent-free, specifically tailored versions of Moloney murine leukemia virus reverse transcriptase and Thermus aquaticus DNA polymerase and termed R3T (Rapid Research Response Team) one-step qRT-PCR. We also demonstrate the robustness of our enzyme production strategies and provide the optimal reaction conditions for their efficient augmentation in a one-step approach. Our kit was routinely able to reliably detect as low as 10 copies of the synthetic RNAs of SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity to that of the Invitrogen SuperScript III Platinum One-step qRT-PCR and TaqPath one-step RT-qPCR kits. Overall, our kit has shown robust performance in both laboratory settings and the Saudi Ministry of Health-approved testing facility.

Optimization of SARS-CoV-2 Spike Protein Expression in the Silkworm and Induction of Efficient Protective Immunity by Inoculation With Alum Adjuvants.

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing a spread of coronavirus disease 2019 (COVID-19) globally. In order to end the COVID-19 pandemic, an effective vaccine against SARS-CoV-2 must be produced at low cost and disseminated worldwide. The spike (S) protein of coronaviruses plays a pivotal role in the infection to host cells. Therefore, targeting the S protein is one of the most rational approaches in developing vaccines and therapeutic agents. In this study, we optimized the expression of secreted trimerized S protein of SARS-CoV-2 using a silkworm-baculovirus expression vector system and evaluated its immunogenicity in mice. The results showed that the S protein forming the trimeric structure was the most stable when the chicken cartilage matrix protein was used as the trimeric motif and could be purified in large amounts from the serum of silkworm larvae. The purified S protein efficiently induced antigen-specific antibodies in mouse serum without adjuvant, but its ability to induce neutralizing antibodies was low. After examining several adjuvants, the use of Alum adjuvant was the most effective in inducing strong neutralizing antibody induction. We also examined the adjuvant effect of paramylon from Euglena gracilis when administered with the S protein. Our results highlight the effectiveness and suitable construct design of the S protein produced in silkworms for the subunit vaccine development against SARS-CoV-2.

Efficient production of recombinant SARS-CoV-2 spike protein using the baculovirus-silkworm system.

In the case of a new viral disease outbreak, an immediate development of virus detection kits and vaccines is required. For COVID-19, we established a rapid production procedure for SARS-CoV-2 spike protein (S protein) by using the baculovirus-silkworm expression system. The baculovirus vector-derived S proteins were successfully secreted to silkworm serum, whereas those formed insoluble structure in the larval fat body and the pupal cells. The ectodomain of S protein with the native sequence was cleaved by the host furin-protease, resulting in less recombinant protein production. The S protein modified in furin protease-target site was efficiently secreted to silkworm serum and was purified as oligomers, which showed immunoreactivity for anti-SARS-CoV-2 S2 antibody. By using the direct transfection of recombinant bacmid to silkworms, we achieved the efficient production of SARS-CoV-2 S protein as fetal bovine serum (FBS)-free system. The resultant purified S protein would be useful tools for the development of immunodetection kits, antigen for immunization for immunoglobulin production, and vaccines.

Expression and Purification of Vaccinia Virus DNA Topoisomerase IB Produced in the Silkworm-Baculovirus Expression System.

Type IB DNA topoisomerases are enzymes to change the topological state of DNA molecules and are essential in studying replication, transcription, and recombination of nucleic acids in vitro. DNA topoisomerase IB from Vaccinia virus (vTopIB) is a 32 kDa, type I eukaryotic topoisomerase, which relaxed positively and negatively supercoiled DNAs without Mg2+ and ATP. Although vTopIB has been effectively produced in E. coli expression system, no studies remain available to explore an alternative platform to express recombinant vTopIB (rvTopIB) in a higher eukaryote, where the one can expect post-translational modifications that affect the activity of rvTopIB. Here in this study, rvTopIB with N-terminal tags was constructed and expressed in a silkworm-baculovirus expression vector system (silkworm-BEVS). We developed a simple two consecutive chromatography purification to obtain highly pure rvTopIB. The final yield of rvTopIB obtained from a baculovirus-infected silkworm larva was 83.25 µg. We also evaluated the activity and function of rvTopIB by the DNA relaxation activity assays using a negatively supercoiled pUC19 plasmid DNA as a substrate. With carefully assessing optimized conditions for the reaction buffer, we found that divalent ions, Mg2+, Mn2+, Ca2+, as well as ATP stimulate the DNA relaxation activity by rvTopIB. The functional and active form of rvTopIB, together with the yields of the protein we obtained, suggests that silkworm-BEVS would be a potential alternative platform to produce eukaryotic topoisomerases on an industrial scale.

Expression and purification of biologically active human granulocyte-macrophage colony stimulating factor (hGM-CSF) using silkworm-baculovirus expression vector system.

Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a hematopoietic growth factor. It is widely employed as a therapeutic agent targeting neutropenia in cancer patients undergoing chemotherapy and in patients with AIDS or after bone marrow transplantation. In this study, we constructed the recombinant baculoviruses for the expression of recombinant hGM-CSF (rhGM-CSF) with two small affinity tags (His-tag and Strep-tag) at the N or C-terminus. Compared to N-tagged rhGM-CSF, C-tagged rhGM-CSF was highly recovered from silkworm hemolymph. The purified rhGM-CSF proteins migrated as a diffuse band and were confirmed to hold N-glycosylations. A comparable activity was achieved when commercial hGM-CSF was tested as a control. Considering the high price of hGM-CSF in the market, our results and strategies using silkworm-baculovirus system can become a great reference for mass production of the active rhGM-CSF at a lower cost.

Identification of an insecticidal toxin produced by Enterobacter sp. strain 532 isolated from diseased Bombyx mori silkworms.

Although Enterobacter sp. 532 shows pathogenicity in Bombyx mori, the insecticidal mechanisms are unclear. Here, we identified and characterised an insecticidal protein from Enterobacter. The insecticidal protein was purified from the strain and inoculated into B. mori larvae. Intracellular proteins were prepared, purified and separated by preparative native polyacrylamide gel electrophoresis (PAGE); one protein band had insecticidal activity. Sodium dodecyl sulfate-PAGE showed the presence of several bands, indicating that the insecticidal protein formed a complex. Peptide mass fingerprinting of a prominent 255.3-kDa band revealed 64 peptides that matched one protein with 33.0% sequence coverage. This protein was a homologue of the A component of the toxin complex (Tc), and the VRP1 domain was conserved; thus, the gene was named itcA (insecticidal toxin complex A). In the itcA downstream region, B and C component gene homologues were found, and these genes were located on an 86.2-kb contig sequence. Two repA genes and 27 genes related to conjugation transfer of plasmids were located on the contig, suggesting that the contig originated from a mobilisable plasmid. Therefore, these findings suggested that the strain may have acquired the Tc genes by horizontal transfer. This is the first description of Tc produced by the genus Enterobacter.

Expression, Purification, and Characterization of Recombinant Human α1-Antitrypsin Produced Using Silkworm-Baculovirus Expression System.

Human α1-antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α1-antitrypsin is mainly purified from human plasma. There is a high demand for a stable and low-cost supply of recombinant AAT (rAAT). In this study, the baculovirus expression vector system using silkworm larvae as host was employed and a large amount of highly active AAT was recovered from the silkworm serum (~ 15 mg/10 ml) with high purity. Both the enzymatic activity and stability of purified rAAT were comparable with those of commercial product. Our results provide an alternative method for mass production of the active rAAT in pharmaceutical use.

A functional polypeptide N-acetylgalactosaminyltransferase (PGANT) initiates O-glycosylation in cultured silkworm BmN4 cells.

Mucin-type O-glycosylation is initiated by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts or PGANTs), attaching GalNAc to serine or threonine residue of a protein substrate. In the insect model from Lepidoptera, silkworm (Bombyx mori), however, O-glycosylation pathway is totally unexplored and remains largely unknown. In this study, as the first report regarding protein O-glycosylation analysis in silkworms, we verified the O-glycan profile that a common core 1 Gal (ß1-3) GalNAc disaccharide branch without terminally sialylated structure is mainly formed for a baculovirus-produced human proteoglycan 4 (PRG4) protein. Intriguingly, functional screenings in cultured silkworm BmN4 cells for nine Bmpgants reveal that Bmpgant2 is the solo functional BmPGANT for PRG4, implying that Bmpgants may have unique cell/tissue or protein substrate preferences. Furthermore, a recombinant BmPGANT2 protein was successfully purified from silkworm-BEVS and exhibited a high ability to transfer GalNAc for both peptide and protein substrates. Taken together, the present results clarified the functional BmPGANT2 in cultured silkworm cells, providing crucial fundamental insights for future studies dissecting the detailed silkworm O-glycosylation pathways and productions of glycoproteins with O-glycans.

Purification and characterization of immunogenic recombinant virus-like particles of porcine circovirus type 2 expressed in silkworm pupae.

Porcine circovirus type 2 (PCV2) is a primary causative agent of postweaningmultisystemic wasting syndrome (PMWS), which has a significant economic impact on the swine industry. The capsid protein (Cap) encoded by ORF2 of the viral genome has been used effectively as a vaccine against PCV2 infection. The Cap protein can spontaneously assemble into virus-like particles (VLPs) that are safe and highly immunogenic for vaccine applications. Several expression systems, including bacteria, yeast and insect cells, have been utilized to produce PCV2 VLPs. However, in some cases, the recombinant Cap (rCap) proteins produced in bacteria and yeast do not assemble spontaneously. In this study, we expressed rCap protein using a silkworm-baculovirus expression vector system (silkworm-BEVS) for mass production of PCV2 VLPs and established a simple three-step protocol for its purification from pupae: extraction by detergent, ammonium sulfate precipitation and anion exchange column chromatography. Size-exclusion chromatography (SEC) analysis and transmission electron microscope (TEM) observation showed that purified rCap proteins formed VLPs with a similar morphology to that of the original virus. Furthermore, the VLPs produced in silkworms were capable of inducing neutralizing antibodies against PCV2 in mice. Our results demonstrated that the silkworm system is a powerful tool for the production of PCV2 VLPs and will be useful for the development of a reliable and cost-effective PCV2 vaccine.