Dual display hemagglutinin 1 and 5 on the surface of enveloped virus-like particles in silkworm expression system.

Rous sarcoma virus-like particles (RSV-LPs) displaying hemagglutinins of H1N1 (A/New Caledonia/20/99) (H1) and H5N1 (A/Vietnam/1194/2004) (H5) of the influenza A virus were produced. The H1 has its transmembrane domain, but the H5 was fused with the transmembrane domain of glycoprotein 64 (BmGP64) from Bombyx mori nucleopolyhedrovirus (BmNPV). H1 and RSV Gag protein were coexpressed in the hemolymph of silkworm larvae, copurified, and confirmed RSV-LP displaying H1 (VLP/H1). Similarly, the RSV-LP displaying H5 (VLP/H5) production was also achieved. Using fetuin agarose column chromatography, RSV Gag protein-coexpressed H1 and H5 in silkworms were copurified from the hemolymph. By immuno-TEM, H1 and H5 were observed on the surface of an RSV-LP, indicating the formation of bivalent RSV-LP displaying two HAs (VLP/BivHA) in the hemolymph of silkworm larvae. VLP/H1 induced the hemagglutination of red blood cells (RBCs) of chicken and rabbit but not sheep, while VLP/H5 induced the hemagglutination of RBCs of chicken and sheep but not rabbit. Additionally, VLP/BivHA allowed the hemagglutination of RBCs of all three animals. Silkworm larvae can produce RSV-LPs displaying two HAs and is a promising tool to produce the bivalent enveloped VLPs for the vaccine platform.

Development of Rous sarcoma Virus-like Particles Displaying hCC49 scFv for Specific Targeted Drug Delivery to Human Colon Carcinoma Cells.

PURPOSE: Virus-like particles (VLPs) have been used as drug carriers for drug delivery systems. In this study, hCC49 single chain fragment variable (scFv)-displaying Rous sarcoma virus-like particles (RSV VLPs) were produced in silkworm larvae to be a specific carrier of an anti-cancer drug. METHOD: RSV VLPs displaying hCC49 scFv were created by the fusion of the transmembrane and cytoplasmic domains of hemagglutinin from influenza A (H1N1) virus and produced in silkworm larvae. The display of hCC49 scFv on the surface of RSV VLPs was confirmed by enzyme-linked immunosorbent assay using tumor-associated glycoprotein-72 (TAG-72), fluorescent microscopy, and immunoelectron microscopy. Fluorescein isothiocyanate (FITC) or doxorubicin (DOX) was incorporated into hCC49 scFv-displaying RSV VLPs by electroporation and specific targeting of these VLPs was investigated by fluorescent microscopy and cytotoxicity assay using LS174T cells. RESULTS: FITC was delivered to LS174T human colon adenocarcinoma cells by hCC49 scFv-displaying RSV VLPs, but not by RSV VLPs. This indicated that hCC49 scFv allowed FITC-loaded RSV VLPs to be delivered to LS174T cells. DOX, which is an anti-cancer drug with intrinsic red fluorescence, was also loaded into hCC49 scFv-displaying RSV VLPs by electroporation; the DOX-loaded hCC49 scFv-displaying RSV VLPs killed LS174T cells via the specific delivery of DOX that was mediated by hCC49 scFv. HEK293 cells were alive even though in the presence of DOX-loaded hCC49 scFv-displaying RSV VLPs. CONCLUSION: These results showed that hCC49 scFv-displaying RSV VLPs from silkworm larvae offered specific drug delivery to colon carcinoma cells in vitro. This scFv-displaying enveloped VLP system could be applied to drug and gene delivery to other target cells.

A model for targeting colon carcinoma cells using single-chain variable fragments anchored on virus-like particles via glycosyl phosphatidylinositol anchor.

PURPOSE: VLPs displaying tumor targeting single-chain variable fragments (VLP-rscFvs) which targets tumor-associated glycoprotein-72 (TAG-72) marker protein have a potential for immunotherapy against colon carcinoma tumors. In this study, scFvs anchored on VLPs using glycosylphosphatidylinositol (GPI) were prepared to target colon carcinoma spheroids in vitro. METHODS: VLPs-rscFvs were produced by co-injecting two types of Bombyx mori nucleopolyhedrovirus (BmNPV) bacmids, encoding RSV-gag and rscFvs cDNA into silkworm larvae. Large unilamellar vesicles (LUVs) of 100 nm in diameter were made using 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and packaged with Sulforhodamine B (SRB). LUV-SRB was used to associate with VLP-rscFvs assisted by GP64 present on VLP-rscFvs to produce VLP-rscFv associated SRB (VLP-rscFvs-SRB) at pH 7.5. RESULTS: The antigenicity of the purified VLPs-rScFvs was confirmed by enzyme-linked immunosorbent assay (ELISA) using TAG-72 as antigen. LUV-SRB made of DOPC was used to associate with 100 µg of VLP-rscFvs to produce VLP-rscFv-SRB. Specific delivery and penetration of SRB up to 100 µm into the spheroids shows the potential of the new model. CONCLUSIONS: The current study demonstrated the display, expression and purification of VLP-rscFvs efficiently. As a test model VLP-rscFv-SRB were prepared which can be used for immunotherapy. rscFvs provide the specificity needed to target tumors and VLPs serve as carrier transporting the dye to target.

A new compound from the mushroom Tricholoma flavovirens.

A novel compound (1) and a known one (2) were isolated from the fruiting bodies of Tricholoma flavovirens. Their structures were determined by the interpretation of spectroscopic data. Both compounds showed inhibition effects on the growth of hypocotyl of lettuce with significant differences. In addition, compound 1 showed a promotion effect on the growth of root with significant differences and 2 had the similar tendency to promote the growth.

Sero-diagnostic potential of Plasmodium falciparum recombinant merozoite surface protein (MSP)-3 expressed in silkworm.

Plasmodium falciparum is a blood protozoan parasite, transmitted by Anopheles mosquitoes vectors, that can cause morbidity and even leads to mortality in tropical countries. Strategies are directed to combat malaria including development of diagnostic tools, serological markers and vaccinations. A target under intensive studies is Merozoite Surface Protein (MSP)-3. The aim of this study is to express and purify recombinant MSP3 of P. falciparum (rPfMSP3) using silkworm expression system as a host for its large-scale production and to investigate its potential effectiveness for sero-diagnosis. The rPfMSP3 formed oligomers in a blue-native PAGE and its N-glycosylation was confirmed by periodic acid-Schiff staining and PNGase F treatment. The amyloid-like morphology of the rPfMSP3 oligomers was observed. Enzyme-linked immunosorbent assay showed that 60-70% of human samples from subjects living in malaria endemic areas in Indonesia detected the rPfMSP3. Western blot results showed that the rPfMSP3 was recognized by a malaria infected human serum but not by an uninfected human serum. The rPfMSP3 was successfully expressed in silkworm as a soluble protein and has the potential to be used in serological measurement for detecting PfMSP3-specific antibodies in sera from individuals living in endemic areas.

Single-step detection of norovirus tuning localized surface plasmon resonance-induced optical signal between gold nanoparticles and quantum dots.

A new method of label free sensing approach with superior selectivity and sensitivity towards virlabel-freeon is presented here, employing the localized surface plasmon resonance (LSPR) behavior of gold nanoparticles (AuNPs) and fluorescent CdSeTeS quantum dots (QDs). Inorganic quaternary alloyed CdSeTeS QDs were capped with L-cysteine via a ligand exchange reaction. Alternatively, citrate stabilized AuNPs were functionalized with 11-mercaptoundecanoic acid to generate carboxylic group on the gold surface. The carboxylic group on the AuNPs was subjected to bind covalently with the amine group of L-cysteine capped CdSeTeS QDs to form CdSeTeS QDs/AuNPs nanocomposites. The fluorescence of CdSeTeS QDs/AuNPs nanocomposite shows quenched spectrum of CdSeTeS QDs at 640 nm due to the close interaction with AuNPs. However, after successive addition of norovirus-like particles (NoV-LPs), steric hindrance-induced LSPR signal from the adjacent AuNPs triggered the fluorescence enhancement of QDs in proportion to the concentration of the target NoV-LPs. A linear range of 10-14 to 10-9 g mL-1 NoV-LPs with a detection limit of 12.1 × 10-15 g mL-1 was obtained. This method was further applied on clinically isolated norovirus detection, in the range of 102-105 copies mL-1 with a detection limit of 95.0 copies mL-1, which is 100-fold higher than commercial ELISA kit. The superiority of the proposed sensor over other conventional sensors is found in its ultrasensitive detectability at low virus concentration even in clinically isolated samples. This proposed detection method can pave an avenue for the development of high performance and robust sensing probes for detection of virus in biomedical applications.

Construction of hybrib Autographa californica nuclear polyhedrosis bacmid by modification of p143 helicase.

We developed a new hybrid nuclear polyhedrosis virus (NPV) bacmid capable of infecting Spodoptera frugiperda, Tricoplusia ni, and Bombyx mori, and B. mori cell lines for producing hybrid recombinant baculovirus that can carry a gene of interest and express it in a broad range of hosts. A GFP(uv)-beta1,3-N-acetylglucosaminyltransferase 2 fusion gene was expressed successfully in silkworm larvae using this hybrid bacmid. The hybrid NPV bacmid provides an altogether simple and realistically feasible method for large-scale applications using silkworm larvae. It can be easily managed in E. coli, which has no biohazard safety concerns, in addition to the baculovirus-based expression system.

Virus-Like Particles Displaying Recombinant Short-Chain Fragment Region and Interleukin 2 for Targeting Colon Cancer Tumors and Attracting Macrophages.

Functionalized virus-like particles (VLPs) can target with specificity as drug delivery systems and can attract macrophages for the destruction of cancer cells. Here, the group antigen capsid protein from the Rous sarcoma virus was used to prepare VLPs, functionalized by displaying glycol-inositol phosphate-anchored recombinant single chain fragment variable (rscFv) and hemagglutinin transmembrane region anchored recombinant human interleukin-2 (rhIL2) (designated as VLP-rscFv-rhIL2s) in silkworms. The rscFv specifically binds the tumor-associated glycoprotein 72 that is expressed at the surface of colon cancer cells. VLP-rscFv-rhIL2 was affinity purified and had a smooth particle size with a diameter of 50 nm. Calcein-AM-packaged VLP-rscFv-rhIL2s successfully targeted cancer cells as a model for drug delivery system. VLP-rscFv-rhIL2 bound with colon cancer cells that attracted macrophages (human monocytic cell line-1 cells) in chemotaxis chamber assays compared with negative controls. The macrophages secreted tumor necrosis factor-α, a cytokine that is necessary to destroy cancer cells. These results demonstrate the potential of VLP-rscFv-rhIL2 as an intelligent nano biomaterial that is capable of attracting macrophages.

Display of Neospora caninum surface protein related sequence 2 on Rous sarcoma virus-derived gag protein virus-like particles.

Virus-like particles (VLPs) displaying antigen have been increasingly recognized as a potential vaccine in the livestock industry. In this study, Neospora caninum surface protein related sequence (NcSRS)2 was displayed on the surface of Rous sarcoma virus group-antigen protein (RSV-gag) VLPs. Two types of Bombyx mori nucleopolyhedrovirus (BmNPV) bacmids, encoding RSV-gag and NcSRS2 genes, were co-injected into silkworm larvae to produce VLPs-NcSRS2. At 7 days post-injection, VLPs-NcSRS2 were collected from hemolymph and purified. The antigenicity of the purified protein was confirmed by enzyme-linked immunosorbent assay (ELISA) using neosporosis-positive bovine serum. ELISA revealed that ~0.16µg rNcSRS2 was displayed per 1µg VLPs-NcSRS2. To develop an antibody specific for VLPs-NcSRS2, purified VLPs-NcSRS2 were used to immunize mice in a three-dose regimen without adjuvant and the production of antibodies was confirmed in serum samples. By using a silkworm expression system, we demonstrated the display, expression and immunization of neosporosis-targeting membrane proteins, which are vaccine candidates for neosporosis.

Production of Rous sarcoma virus-like particles displaying human transmembrane protein in silkworm larvae and its application to ligand-receptor binding assay.

Two types of Rous sarcoma virus (RSV) group-antigen protein (Gag) virus like particles (VLPs), full-length Gag (Gag701) and RSV protease domain (PR)-deleted mutant (Gag577) were expressed in silkworm larvae. Gag577 was secreted into hemolymph efficiently using wild type bacmid (WT), cysteine protease-deficient bacmid (CP(-)), cysteine protease and chitinase-deficient bacmid (CP(-)Chi(-)) bacmids, but comparatively Gag701 secretion levels were low. VLPs were purified on 10-60% (v/v) sucrose density gradient by ultracentrifugation and their structures confirmed under electron microscope. When hPRR and RSV Gag577 were co-expressed in silkworm larvae, human prorenin receptor (hPRR) was displayed on the surface of RSV VLPs, which was detected by Western blotting and immunoelectron microscopy. Moreover, binding of hPRR localized on the surface of VLPs to human prorenin was confirmed by ELISA. These results indicate that active hPRR was displayed on the surface of RSV VLPs, which can be utilized for drug discovery of hPRR blockers to prevent nephropathy. Moreover, this transmembrane protein display system using RSV Gag in silkworm larvae is applicable to expression of intact transmembrane proteins and binding assay of transmembrane proteins to its ligands, especially for transmembrane proteins which cannot be purified from membrane fractions in active states.

Expression of an RSV-gag virus-like particle in insect cell lines and silkworm larvae.

Rous sarcoma virus group antigen protein-based virus-like particles (VLPs) are well known for their structural integrity and ease of handling. VLPs play an important role in drug delivery systems because they can be manipulated with ease. In this study, a new method was established for expressing Rous sarcoma virus group antigen protein based VLPs in silkworm larvae and establishing stably expressing insect cell lines. These VLPs have been isolated by ultracentrifugation using a sucrose step gradient of 10-60% (v/v), and their spherical structure has been confirmed using transmission electron microscopy (TEM). The spherical morphology is similar in both the silkworm larvae and in stably expressing cell lines. Silkworm larvae are better suited for producing Rous sarcoma virus group antigen protein-based VLPs on a large scale; yields from silkworm larvae were approximately 8.2-fold higher than yields from stable cell lines. These VLPs provide a new method for large-scale application in vaccine development and drug delivery systems.

Multiple co-transfection and co-expression of human beta-1,3-N-acetylglucosaminyltransferase with human calreticulin chaperone cDNA in a single step in insect cells.

Human beta-1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) is indispensable for the conversion of lacto-N-triose II into lacto-N-tetraose and lacto-N-neotetraose. In this paper, we report multiple co-transfection in a single step using two different human cDNAs in an insect cell, beta3GnT2 and calreticulin chaperone respectively. This minimized the time required to isolate stably expressing cell line from 12 weeks to 4 weeks and simplified the isolation technique to a one-step process. We tried to insert as much cDNA as possible and used various concentrations of two antibiotics, Blasticidin and Geneticin, at 25-1500 microg/ml respectively during co-transfection for the selection of an efficiently expressing stable cell line with no adverse effects. A stably expressing cell line was isolated which expressed beta3GnT2 and chaperone simultaneously, which gave an activity of 10.1 m-units/ml compared with 6.7 m-units/ml by a cell only carrying beta3GnT2. In this study we correlated the activity of beta3GnT2 with the amount of beta3GnT2 and human calreticulin cDNA in a stably expressing insect cell line simultaneously expressing calreticulin chaperone. When the amounts of chaperone and beta3GnT2 cDNA were in a rough ratio of 1:1, the beta3GnT2 activity was at a high level. In order to achieve better expression levels of beta3GnT2 with less cost and time, efficient ways have to be devised.