Yao-Chi Chung

Immunization with virus-like particles of enterovirus 71 elicits potent immune responses and protects mice against lethal challenge.

Enterovirus 71 (EV71) is an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes outbreaks with significant mortality among young children. To develop the vaccine, we have produced and purified the EV71 virus-like particle (VLP) that resembles the authentic virus in appearance, capsid structure and protein composition. In this study, we further evaluated the potential of VLP as a vaccine by comparing the humoral and cellular immune responses elicited by the purified VLP, denatured VLP and heat-inactivated EV71 virus. After immunization of BALB/c mice, EV71 VLP induced potent and long-lasting humoral immune responses as evidenced by the high total IgG titer and neutralization titer. The splenocytes collected from the VLP-immunized mice exhibited significant cell proliferation and produced high levels of IFN-gamma, IL-2 and IL-4 after stimulation, indicating the induction of Th1 and Th2 immune responses by VLP immunization. More importantly, the VLP immunization of mother mice conferred protection (survival rate up to 89%) to neonatal mice against the lethal (1000 LD(50)) viral challenge. Compared with the VLP immunization, immunization with denatured VLP and heat-inactivated EV71 elicited lower neutralization titers and conferred less effective protection to newborn mice, although they induced comparable levels of total IgG and cellular immune responses. These data collectively indicate the importance of the preservation of VLP structure and implicate the potential of VLP as a vaccine to prevent EV71 infection.

Transgene expression and differentiation of baculovirus-transduced human mesenchymal stem cells

Mesenchymal stem cells (MSCs) have drawn considerable attention as vehicles for cell‐ or gene‐based therapies, yet various problems still exist for current gene delivery vectors. On the other hand, baculovirus has emerged as a novel gene therapy vector, but its transduction of stem cells has not been reported.

Enterovirus 71 virus-like particle vaccine: Improved production conditions for enhanced yield

To develop the enterovirus 71 (EV71) vaccine, we previously constructed a recombinant baculovirus (Bac-P1-3CD) co-expressing EV71 P1 (under polyhedrin promoter) and 3CD (under p10 promoter) proteins, which caused P1 cleavage by 3CD protease and self-assembly of virus-like particles (VLPs) in Sf-9 cells. Assuming that reducing the 3CD expression can alleviate the competition with P1 expression and elevate the VLPs yield, hereby we constructed Bac-P1-C3CD and Bac-P1-I3CD expressing 3CD under weaker CMV and IE-1 promoters, respectively. Western blot and ELISA analyses revealed that Bac-P1-C3CD and Bac-P1-I3CD led to the VLPs release into the supernatant and enhanced the extracellular VLPs yield in Sf-9 cells, but gave poor VLPs production in High Five™ (Hi-5) cells. By optimizing the process parameters including host cells, cell density, culture mode and dissolved oxygen (DO), the best extracellular VLPs yield was achieved by infecting Sf-9 cells (4 × 10(6)cells/mL) cultured in the bioreactor (DO=30%) with Bac-P1-C3CD, which approached ≈64.3mg/L and represented a ≈43-fold increase over the yield (1.5mg/L) attained using the old process (Bac-P1-3CD infection of Sf-9 cells in the spinner flasks). The resultant VLPs not only resembled the VLPs produced from Bac-P1-3CD infection in density, size and shape, but also induced potent antibody responses in mouse models. The antibodies neutralized EV71 strains of homologous and heterologous genogroups, implicating the potential of the VLPs to confer cross-protection for the prevention of future epidemics. Altogether, Bac-P1-C3CD and the bioprocess render mass production more economical, obviate the need for cell lysis and hold promise for future industrial vaccine production.

Generation of chimeric baculovirus with histidine-tags displayed on the envelope and its purification using immobilized metal affinity chromatography

Abstract Baculovirus/insect cell system has been widely used for recombinant protein production. Since the finding that baculovirus is capable of transducing hepatocytes in 1995, success to utilize baculovirus as a gene delivery vehicle into mammalian cells has been reported. Due to the high transduction efficiency and the non-replication nature in mammalian cells, baculovirus has emerged as a vector for in vivo gene therapy. For gene therapy studies and future clinical needs, a simple and efficient purification scheme for baculovirus is necessary. In this study, a recombinant baculovirus with a hexahistidine (His6) tag fused on the viral envelope protein, gp64, was constructed. The presence of the His6-tagged gp64 was confirmed by Western blot and its display on the virus surface was visualized by immunogold electron microscopy. The His6 tag displayed on the virus surface enabled the virus purification by a simple immobilized metal affinity chromatography (IMAC) with high purity (≈87%), thus obviating the need for successive ultracentrifugation steps. The recovery yield was higher than that obtained in conventional gradient ultracentrifugation but necessitated further process improvement. This study demonstrates the feasibility of combining the baculoviral display technology and IMAC for viral vector purification.

High level expression of the key antigenic protein, σC, from avian reovirus into insect cells and its purification by immobilized metal affinity chromatography

Avian reovirus (ARV) structural protein, σC, the prime candidate for vaccine against ARV, was expressed using a baculovirus/insect cell system. The expressed protein remained intracellular and reached 96 μg/106 cells. Total product yield from a 200 ml suspension culture was 19 mg. When the protein was fused with a histidine tag and an enterokinase (EK) cleavage site, purification of 94% was achieved in a single step. The histidine tag was removed by EK.

Evaluation of the stability of enterovirus 71 virus-like particle

Enterovirus 71 (EV71) is responsible for the outbreaks of hand-foot-and-mouth disease that caused significant mortality in children, but no vaccine is available yet. EV71 virus-like particle (VLP) is the empty capsid consisting of viral structural proteins but can elicit potent immune responses, rendering VLP a promising EV71 vaccine candidate. To evaluate whether VLP remains stable after long-term storage, which is crucial for advancing the VLP vaccine to the clinical setting, we evaluated the effects of NaCl concentration, buffers and temperatures on the VLP stability. We first validated the use of dynamic light scattering (DLS) for measuring the hydrodynamic diameter (≈30-35 nm) of VLP, which was close to the VLP diameter (≈25-27 nm) as measured by transmission electron microscopy (TEM). Using these techniques, we found that EV71 VLP remained stable for 5 months in sodium phosphate (NaPi) buffers with various NaCl concentrations. EV71 VLP also remained morphologically stable in NaPi, citrate and TE(+) buffers for 5 months, yet the enzyme-linked immunosorbent assay (ELISA) revealed that the VLP stored in citrate and TE(+) buffers partially lost the immunogenicity after 5 months. In contrast, the VLP stored in the NaPi buffer at 4°C remained stable macroscopically and microscopically for 5 months, as judged from the DLS, TEM and ELISA. The VLP stored at 25°C and 37°C also retained stability for 1 month, which would obviate the need of a cold chain during the shipping. These data altogether proved the stability of EV71 VLP and suggested that the VLP is amenable to bioprocessing and storage.

Update on baculovirus as an expression and/or delivery vehicle for vaccine antigens.

After three decades of development, the baculovirus/insect cell expression system is now recognized as a powerful platform for recombinant protein production. With a number of distinct advantages, the baculovirus/insect cell expression system has been extensively used for the production of various vaccine candidates, and several human and veterinary vaccine products have been commercially available. In addition to insect cells, baculovirus is capable of entering a broad range of mammalian cells, lending itself to a promising gene delivery vehicle for antigen expression and display in vivo. The use of baculovirus for antigen expression and delivery has been reviewed in 2008. Rather than a critical evaluation, this paper aims to provide an update of the applications of baculovirus as an in vitro or in vivo antigen expression/delivery vehicle, with special focuses on developments and advances after 2008.

High-density cultivation of insect cells and production of recombinant baculovirus using a novel oscillating bioreactor

A novel two-compartment bioreactor, BelloCell®, was used to cultivate insect cells and a maximum yield of 4.6 × 109 cells was attained. The cells were immobilized in a packed bed fixed in the upper chamber, and the bellow in the lower chamber was compressed and released in an alternating fashion. The motion resulted in gentle, cyclic movement of the medium that was contained in the lower chamber and consequently exposed the cells to air in an oscillatory manner, thus rendering adequate aeration and uniform cell distribution in the bed. The baculovirus yield produced in BelloCell® could amount up to 3.3 × 1017 pfu using as little as 1.1 l medium in the production run. Besides, BelloCell® was extremely easy to handle and operate. These benefits underline the potential of BelloCell® for simple, economical and high-density cell culture and protein/virus production.

Corrigendum to “Avian Influenza Virus Hemagglutinin Display on Baculovirus Envelope: Cytoplasmic Domain Affects Virus Properties and Vaccine Potential”

After the publication of this article, the authors indicated that Dr. Hung-Jen Liu should be included as the second corresponding author. His contact information is:

Expression and Purification of N and E Proteins from Severe Acute Respiratory Syndrome (SARS)-Associated Coronavirus: a Comparative Study

Histidine-tagged N (rNH) and E (rEH) proteins of Severe Acute Respiratory Syndrome (SARS)-coronovirus were expressed in the baculovirus/insect cell system and purified by immobilized metal affinity chromatography. rNH and rEH proteins differed markedly with respect to expression levels, cell death kinetics and subcellular localizations that led to different extraction and purification schemes. The features of both proteins are compared and the potential applications of purified rNH and rEH are discussed.