Construction of SARS-CoV-2 virus-like particles in plant.

The pandemic of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused a public health emergency, and research on the development of various types of vaccines is rapidly progressing at an unprecedented development speed internationally. Some vaccines have already been approved for emergency use and are being supplied to people around the world, but there are still many ongoing efforts to create new vaccines. Virus-like particles (VLPs) enable the construction of promising platforms in the field of vaccine development. Here, we demonstrate that non-infectious SARS-CoV-2 VLPs can be successfully assembled by co-expressing three important viral proteins membrane (M), envelop (E) and nucleocapsid (N) in plants. Plant-derived VLPs were purified by sedimentation through a sucrose cushion. The shape and size of plant-derived VLPs are similar to native SARS-CoV-2 VLPs without spike. Although the assembled VLPs do not have S protein spikes, they could be developed as formulations that can improve the immunogenicity of vaccines including S antigens, and further could be used as platforms that can carry S antigens of concern for various mutations.

Covalent protein display on Hepatitis B core-like particles in plants through the in vivo use of the SpyTag/SpyCatcher system.

Virus-like particles (VLPs) can be used as nano-carriers and antigen-display systems in vaccine development and therapeutic applications. Conjugation of peptides or whole proteins to VLPs can be achieved using different methods such as the SpyTag/SpyCatcher system. Here we investigate the conjugation of tandem Hepatitis B core (tHBcAg) VLPs and the model antigen GFP in vivo in Nicotiana benthamiana. We show that tHBcAg VLPs could be successfully conjugated with GFP in the cytosol and ER without altering VLP formation or GFP fluorescence. Conjugation in the cytosol was more efficient when SpyCatcher was displayed on tHBcAg VLPs instead of being fused to GFP. This effect was even more obvious in the ER, showing that it is optimal to display SpyCatcher on the tHBcAg VLPs and SpyTag on the binding partner. To test transferability of the GFP results to other antigens, we successfully conjugated tHBcAg VLPs to the HIV capsid protein P24 in the cytosol. This work presents an efficient strategy which can lead to time and cost saving post-translational, covalent conjugation of recombinant proteins in plants.

Adaptive laboratory evolution of stable insect cell lines for improved HIV-Gag VLPs production.

Adaptive laboratory evolution (ALE) has been extensively used to modulate the phenotype of industrial model organisms (e.g. Escherichia. coli and Saccharomyces cerevisae) towards a specific trait. Nevertheless, its application to animal cells, and in particular to insect cell lines, has been very limited. In this study, we describe employing an ALE method to improve the production of HIV-Gag virus-like particles (VLPs) in stable Sf-9 and High Five cell lines. Serial batch transfer was used for evolution experiments. During the ALE process, cells were cultured under controlled hypothermic conditions (22 °C instead of standard 27 °C) for a prolonged period of time (over 3 months), which allowed the selection of a population of cells with improved phenotype. Adapted cells expressed up to 26-fold (Sf-9 cells) and 10-fold (High Five cells) more Gag-VLPs than non-adapted cells cultured at standard conditions. The production of HIV Gag-VLPs in adapted, stable insect Sf-9 cell lines was successfully demonstrated at bioreactor scale. The Gag-VLPs produced at 22 °C and 27 °C were comparable, both in size and morphology, thus confirming the null impact of adaptation process and hypothermic culture conditions on VLP's quality. This work demonstrates the suitability of ALE as a powerful method for improving yields in stable insect cell lines producing VLPs.

Preparation of virus-like particle mimetic nanovesicles displaying the S protein of Middle East respiratory syndrome coronavirus using insect cells.

Middle East respiratory syndrome coronavirus (MERS-CoV) first emerged in 2012, and over 2000 infections and 800 deaths have been confirmed in 27 countries. However, to date, no commercial vaccine is available. In this study, structural proteins of MERS-CoV were expressed in silkworm larvae and Bm5 cells for the development of vaccine candidates against MERS-CoV and diagnostic methods. The spike (S) protein of MERS-CoV lacking its transmembrane and cytoplasmic domains (SΔTM) was secreted into the hemolymph of silkworm larvae using a bombyxin signal peptide and purified using affinity chromatography. The purified SΔTM forms small nanoparticles as well as the full-length S protein and has the ability to bind human dipeptidyl peptidase 4 (DPP4), which is a receptor of MERS-CoV. These results indicate that bioactive SΔTM was expressed in silkworm larvae. To produce MERS-CoV-like particles (MERS-CoV-LPs), the coexpression of spike proteins was performed in Bm5 cells and envelope (E) and membrane (M) proteins secreted E and M proteins extracellularly, suggesting that MERS-CoV-LPs may be formed. However, this S protein was not displayed on virus-like particles (VLPs) even though E and M proteins were secreted into the culture supernatant. By surfactant treatment and mechanical extrusion using S protein- or three structural protein-expressing Bm5 cells, S protein-displaying nanovesicles with diameters of approximately 100-200 nm were prepared and confirmed by immuno-TEM. The mechanical extrusion method is favorable for obtaining uniform recombinant protein-displaying nanovesicles from cultured cells. The purified SΔTM from silkworm larvae and S protein-displaying nanovesicles from Bm5 cells may lead to the development of nanoparticle-based vaccines against MERS-CoV and the diagnostic detection of MERS-CoV.

Bioprocess optimization for purification of chimeric VLP displaying BVDV E2 antigens produced in yeast Hansenula polymorpha.

Chimeric virus-like particles (VLP) are known as promising tools in the development of safe and effective subunit vaccines. Recently, a technology platform to produce VLP based on the small surface protein (dS) of the duck hepatitis B virus was established. In this study, chimeric VLP were investigated displaying the 195 N-terminal amino acids derived from the glycoprotein E2 of the bovine viral diarrhea virus (BVDV) on their surface. Isolation of the VLP from methylotrophic yeast Hansenula polymorpha was allowed upon co-expression of wild-type dS and a fusion protein composed of the BVDV-derived antigen N-terminally fused to the dS. It was shown the VLP could be purified by a process adapted from the production of a recombinant hepatitis B VLP vaccine. However, the process essentially depended on costly ultracentrifugation which is critical for low cost production. In novel process variants, this step was avoided after modification of the initial batch capture step, the introduction of a precipitation step and adjusting the ion exchange chromatography. The product yield could be improved by almost factor 8 to 93 ± 12 mg VLP protein per 100 g dry cell weight while keeping similar product purity and antigenicity. This allows scalable and cost efficient VLP production.

Pichia pastoris-expressed Zika virus envelope domain III on a virus-like particle platform: design, production and immunological evaluation.

Zika virus (ZIKV) is an arbovirus which shares antigenic similarity and the mosquito vector with dengue viruses (DENVs). ZIKV is a neurotropic virus capable of causing congenital neurodevelopmental birth defects. As ZIKV antibodies (Abs) can potentially enhance infection by DENVs, a preventive ZIKV vaccine must be designed to eliminate antibody dependent enhancement of infection. We developed a Zika Subunit Vaccine (ZSV) consisting of two proteins, ZS and S, in a genetically pre-determined ratio of 1:4, using the methylotrophic yeast Pichia pastoris. ZS is an in-frame fusion of ZIKV envelope domain III with the Hepatitis B virus (HBV) surface antigen, and S is the un-fused HBV surface antigen. Using specific monoclonal Abs we showed the presence of ZS and S in the co-purified material which were found to co-assemble into virus-like particles (VLPs), based on dynamic light scattering and electron microscopic analyses. These VLPs were immunogenic in BALB/c mice, eliciting Abs capable of neutralizing ZIKV reporter virus particles. Further, the VLP-induced Abs did not enhance a sub-lethal DENV-2 challenge in AG129 mice. This important safety feature, coupled to the well-documented advantage of P. pastoris expression system, warrants further exploration of ZSV VLP as a possible vaccine candidate.

Comparative purification and characterization of hepatitis B virus-like particles produced by recombinant vaccinia viruses in human hepatoma cells and human primary hepatocytes.

This study describes the comparative expression and purification of hepatitis B surface antigen (HBsAg) particles produced upon infection of human primary hepatocytes and human hepatoma cell lines (HuH-7 and HepG2) with recombinant vaccinia viruses. The highest levels of HBsAg expression were found in HuH-7 hepatoma cells following infection with recombinant vaccinia viruses, which contain the S gene under control of a 7.5 k-promoter. Four different methods for purification of the HBsAg particles were examined: isopycnic ultracentrifugation, sucrose cushion sedimentation, isocratic column gel filtration, and binding to anti-HBs-coated microparticles. The highest degree of purity of HBsAg particles was reached by the method based on anti-HBs-coated microparticles. The resulting product was >98% pure. Biochemical analysis and characterization of purified HBsAg particles were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and electron microscopy. The HBsAg, purified from human hepatoma cell lines and from human primary hepatocytes, consisted of both the non-glycosylated (p25) and the glycosylated (gp27) form and assembled into typical 22-nm particles, and thus may be of great interest and importance for research, diagnostics, and medical treatments.

Targeted Delivery of Cell Penetrating Peptide Virus-like Nanoparticles to Skin Cancer Cells.

Skin cancer or cutaneous carcinoma, is a pre-eminent global public health problem with no signs of plateauing in its incidence. As the most common treatments for skin cancer, surgical resection inevitably damages a patient's appearance, and chemotherapy has many side effects. Thus, the main aim of this study was to screen for a cell penetrating peptide (CPP) for the development of a targeting vector for skin cancer. In this study, we identified a CPP with the sequence NRPDSAQFWLHH from a phage displayed peptide library. This CPP targeted the human squamous carcinoma A431 cells through an interaction with the epidermal growth factor receptor (EGFr). Methyl-ß-cyclodextrin (MßCD) and chlorpromazine hydrochloride (CPZ) inhibited the internalisation of the CPP into the A431 cells, suggesting the peptide entered the cells via clathrin-dependent endocytosis. The CPP displayed on hepatitis B virus-like nanoparticles (VLNPs) via the nanoglue successfully delivered the nanoparticles into A431 cells. The present study demonstrated that the novel CPP can serve as a ligand to target and deliver VLNPs into skin cancer cells.

Plant-based chimeric HPV-virus-like particles bearing amyloid-ß epitopes elicit antibodies able to recognize amyloid plaques in APP-tg mouse and Alzheimer's disease brains.

The main amyloid-beta (Aß) variants detected in the human brain are full-length Aß1-40 and Aß1-42 peptides; however, a significant proportion of AD brain Aß consists also of N-terminal truncated/modified species. The majority of the previous immunotherapeutic strategies targeted the N-terminal immunodominant epitope of the full-length Aß; however, most of the pathological N-truncated forms of Aß lack this critical B cell epitope. Recently, virus-like particles (VLPs), self-assembled structures with highly ordered repetitive patterns on their surface and capable of inducing robust immune responses, were applied as a promising platform for various antigen expressions. In this study, we expressed in plants two chimeric HPV16 L1 capsid proteins obtained by introduction of the ß-amyloid 11-28 epitope (Aß 11-28) into the h4 helix or into the coil regions of the L1 protein. The Aß 11-28 epitope was chosen because it is present in the full-length Aß 1-42 as well as in the truncated/modified amyloid peptide species. After expression, we assembled the chimerical L1/Aß 11-28 into a VLP in which the Aß 11-28 epitope is exposed at very high density (360 times) on the surface of the VLP. The chimeric VLPs elicited in mice Aß-specific antibodies binding to ß-amyloid plaques in APP-tg mouse and AD brains. Our study is the first to demonstrate a successful production in plants and immunogenic properties in mice of chimeric HPV16 L1 VLPs bearing Aß epitope that may be of potential relevance for the development of multivalent vaccines for a multifactorial disease such as AD.

Recombinant expression of beak and feather disease virus capsid protein and assembly of virus-like particles in Nicotiana benthamiana.

BACKGROUND: Beak and feather disease virus (BFDV) is an important disease causing agent affecting psittacines. BFDV is highly infectious and can present as acute, chronic or subclinical disease. The virus causes immunodeficiency and is often associated with secondary infections. No commercial vaccine is available and yields of recombinant BFDV capsid protein (CP) expressed in insect cells and bacteria are yet to be seen as commercially viable, although both systems produced BFDV CP that could successfully assemble into virus-like particles (VLPs). Plants as expression systems are increasingly becoming favourable for the production of region-specific and niche market products. The aim of this study was to investigate the formation and potential for purification of BFDV VLPs in Nicotiana benthamiana. METHODS: The BFDV CP was transiently expressed in N. benthamiana using an Agrobacterium-mediated system and plant expression vectors that included a bean yellow dwarf virus (BeYDV)-based replicating DNA vector. Plant-produced BFDV CP was detected using immunoblotting. VLPs were purified using sucrose cushion and CsCl density gradient centrifugation and visualised using transmission electron microscopy. RESULTS: In this study we demonstrate that the BFDV CP can be successfully expressed in N. benthamiana, albeit at relatively low yield. Using a purification strategy based on centrifugation we demonstrated that the expressed CP can self-assemble into VLPs that can be detected using electron microscopy. These plant-produced BFDV VLPs resemble those produced in established recombinant expression systems and infectious virions. It is possible that the VLPs are spontaneously incorporating amplicon DNA produced from the replicating BeYDV plant vector. CONCLUSIONS: This is the first report of plant-made full-length BFDV CP assembling into VLPs. The putative pseudovirions could be used to further the efficacy of vaccines against BFDV.

Integrated molecular and bioprocess engineering for bacterially produced immunogenic modular virus-like particle vaccine displaying 18 kDa rotavirus antigen.

A high global burden of rotavirus disease and the unresolved challenges with the marketed rotavirus vaccines, particularly in the developing world, have ignited efforts to develop virus-like particle (VLP) vaccines for rotavirus. While rotavirus-like particles comprising multiple viral proteins can be difficult to process, modular VLPs presenting rotavirus antigenic modules are promising alternatives in reducing process complexity and cost. In this study, integrated molecular and bioprocess engineering approaches were used to simplify the production of modular murine polyomavirus capsomeres and VLPs presenting a rotavirus 18 kDa VP8* antigen. A single construct was generated for dual expression of non-tagged murine polyomavirus capsid protein VP1 and modular VP1 inserted with VP8*, for co-expression in Escherichia coli. Co-expressed proteins assembled into pentameric capsomeres in E. coli. A selective salting-out precipitation and a polishing size exclusion chromatography step allowed the recovery of stable modular capsomeres from cell lysates at high purity, and modular capsomeres were successfully translated into modular VLPs when assembled in vitro. Immunogenicity study in mice showed that modular capsomeres and VLPs induced high levels of VP8*-specific antibodies. Our results demonstrate that a multipronged synthetic biology approach combining molecular and bioprocess engineering enabled simple and low-cost production of highly immunogenic modular capsomeres and VLPs presenting conformational VP8* antigenic modules. This strategy potentially provides a cost-effective production route for modular capsomere and VLP vaccines against rotavirus, highly suitable to manufacturing economics for the developing world. Biotechnol. Bioeng. 2017;114: 397-406. © 2016 Wiley Periodicals, Inc.

Production of Chikungunya Virus-Like Particles and Subunit Vaccines in Insect Cells.

Chikungunya virus is a reemerging human pathogen that causes debilitating arthritic disease in humans. Like dengue and Zika virus, CHIKV is transmitted by Aedes mosquitoes in an epidemic urban cycle, and is now rapidly spreading through the Americas since its introduction in the Caribbean in late 2013. There are no licensed vaccines or antiviral drugs available, and only a few vaccine candidates have passed Phase I human clinical trials. Using recombinant baculovirus expression technology, we have generated CHIKV glycoprotein subunit and virus-like particle (VLP) vaccines that are amenable to large scale production in insect cells. These vaccines, in particular the VLPs, have shown high immunogenicity and protection against CHIKV infection in different animal models of CHIKV-induced disease. Here, we describe the production, purification, and characterization of these potent CHIKV vaccine candidates.

Self-assembly of virus-like particles of rabbit hemorrhagic disease virus capsid protein expressed in Escherichia coli and their immunogenicity in rabbits.

In this study, virus-like particles (VLPs) derived from rabbit hemorrhagic disease virus (RHDV) were evaluated for the development of a vaccine against RHDV infection. The VP60 gene was cloned and inserted into a pSMK expression vector containing a small ubiquitin-like modifier (SUMO) tag that can promote the soluble expression of heterologous proteins in Escherichia coli cells. After expression and purification of His-SUMO-VP60 and cleavage of the SUMO tag, we found that the RHDV VP60 protein had self-assembled into VLPs with a similar shape and smaller size compared with authentic RHDV capsid. Next, the antigenicity and immunogenicity of the VLPs were examined. The results showed that RHDV-specific responses were clearly induced in rabbits and that all rabbits in the VLP group survived while those in the negative control group died within 72 h post-infection. These results suggest that VLP-based RHDV could be a promising RHDV vaccine candidate.

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.

Assembly and immunogenicity of baculovirus-derived infectious bronchitis virus-like particles carrying membrane, envelope and the recombinant spike proteins.

OBJECTIVE: To assemble infectious bronchitis virus (IBV)-like particles bearing the recombinant spike protein and investigate the humoral immune responses in chickens. RESULTS: IBV virus-like particles (VLPs) were generated through the co-infection with three recombinant baculoviruses separately encoding M, E or the recombinant S genes. The recombinant S protein was sufficiently flexible to retain the ability to self-assemble into VLPs. The size and morphology of the VLPs were similar to authentic IBV particles. In addition, the immunogenicity of IBV VLPs had been investigated. The results demonstrated that the efficiency of the newly generated VLPs was comparable to that of the inactivated M41 viruses in eliciting IBV-specific antibodies and neutralizing antibodies in chickens via subcutaneous inoculation. CONCLUSIONS: This work provides basic information for the mechanism of IBV VLP formation and develops a platform for further designing IBV VLP-based vaccines against IBV or other viruses.

Synthesis of empty african horse sickness virus particles.

As a means to develop African horse sickness (AHS) vaccines that are safe and DIVA compliant, we investigated the synthesis of empty African horse sickness virus (AHSV) particles. The emphasis of this study was on the assembly of the major viral core (VP3 and VP7) and outer capsid proteins (VP2 and VP5) into architecturally complex, heteromultimeric nanosized particles. The production of fully assembled core-like particles (CLPs) was accomplished in vivo by baculovirus-mediated co-synthesis of VP3 and VP7. The two different outer capsid proteins were capable of associating independently of each other with preformed cores to yield partial virus-like particles (VLPs). Complete VLPs were synthesized, albeit with a low yield. Crystalline formation of AHSV VP7 trimers is thought to impede high-level CLP production. Consequently, we engineered and co-synthesized VP3 with a more hydrophilic mutant VP7, resulting in an increase in the turnover of CLPs.

Ebolavirus Vaccines: Progress in the Fight Against Ebola Virus Disease.

Ebolaviruses are highly infectious pathogens that cause lethal Ebola virus disease (EVD) in humans and non-human primates (NHPs). Due to their high pathogenicity and transmissibility, as well as the potential to be misused as a bioterrorism agent, ebolaviruses would threaten the health of global populations if not controlled. In this review, we describe the origin and structure of ebolaviruses and the development of vaccines from the beginning of the 1980s, including conventional ebolavirus vaccines, DNA vaccines, Ebola virus-like particles (VLPs), vaccinia virus-based vaccines, Venezuelan equine encephalitis virus (VEEV)-like replicon particles, Kunjin virus-based vaccine, recombinant Zaire Ebolavirusx2206;VP30, recombinant cytomegalovirus (CMV)-based vaccines, recombinant rabies virus (RABV)-based vaccines, recombinant paramyxovirus-based vaccines, adenovirus-based vaccines and vesicular stomatitis virus (VSV)-based vaccines. No licensed vaccine or specific treatment is currently available to counteract ebolavirus infection, although DNA plasmids and several viral vector approaches have been evaluated as promising vaccine platforms. These vaccine candidates have been confirmed to be successful in protecting NHPs against lethal infection. Moreover, these vaccine candidates were successfully advanced to clinical trials. The present review provides an update of the current research on Ebola vaccines, with the aim of providing an overview on current prospects in the fight against EVD.

Assembly and Purification of Polyomavirus-Like Particles from Plants.

Polyomaviruses are small DNA viruses that have a history of use in biotechnology. The capsids of a number of species have been developed into experimental prophylactic and therapeutic virus-like particle (VLP) vaccines. In order to explore plants as a host for the expression and purification of polyomavirus-like particles, we have transiently expressed the major capsid protein, VP1, in Nicotiana benthamiana leaves. Deletion of a polybasic motif from the N-terminal region of VP1 resulted in increased expression as well as reduced necrosis of leaf tissue, which was associated with differences in subcellular localisation and reduced DNA binding by the deletion variant (ΔVP1). Self-assembled VLPs were recovered from tissue expressing both wild-type VP1 and ΔVP1 by density gradient ultracentrifugation. VLPs composed of ΔVP1 were more homogenous than wtVPLs and, unlike the latter, did not encapsidate nucleic acid. Such homogenous, empty VLPs are of great interest in biotechnology and nanotechnology. In addition, we show that both MPyV VLP variants assembled in plants can be produced with encapsidated foreign protein. Thus, this study demonstrates the utility of plant-based expression of polyomavirus-like particles and the suitability of this host for further developments in polyomavirus-based technologies.

Incorporation of a GPI-anchored engineered cytokine as a molecular adjuvant enhances the immunogenicity of HIV VLPs.

HIV vaccines should elicit immune responses at both the mucosal portals of entry to block transmission and systemic compartments to clear disseminated viruses. Co-delivery of mucosal adjuvants has been shown to be essential to induce effective mucosal immunity by non-replicating vaccines. A novel cytokine, GIFT4, engineered by fusing GM-CSF and interleukin-4, was previously found to simulate B cell proliferation and effector function. Herein a membrane-anchored form of GIFT4 was constructed by fusing a glycolipid (GPI)-anchoring sequence and incorporated into Env-enriched HIV virus-like particles (VLPs) as a molecular adjuvant. Guinea pigs were immunized with the resulting HIV VLPs through an intramuscular priming-intranasal boosting immunization route. The GIFT4-containing VLPs induced higher levels of systemic antibody responses with significantly increased binding avidity and improved neutralizing breadth and potency to a panel of selected strains, as well as higher levels of IgG and IgA at several mucosal sites. Thus, the novel GPI-GIFT4-containging VLPs have the potential to be developed into a prophylactic HIV vaccine. Incorporation of GPI-anchored GIFT4 into VLPs as a molecular adjuvant represents a novel approach to increase their immunogenicity.

Enhanced enterovirus 71 virus-like particle yield from a new baculovirus design.

Enterovirus 71 (EV71) is responsible for the outbreaks of hand-foot-and-mouth disease in the Asia-Pacific region. To produce the virus-like particle (VLP) vaccine, we previously constructed recombinant baculoviruses to co-express EV71 P1 polypeptide and 3CD protease using the Bac-to-Bac(®) vector system. The recombinant baculoviruses resulted in P1 cleavage by 3CD and subsequent VLP assembly in infected insect cells, but caused either low VLP yield or excessive VLP degradation. To tackle the problems, here we explored various expression cassette designs and flashBAC GOLD™ vector system which was deficient in v-cath and chiA genes. We found that the recombinant baculovirus constructed using the flashBAC GOLD™ system was insufficient to improve the EV71 VLP yield. Nonetheless, BacF-P1-C3CD, a recombinant baculovirus constructed using the flashBAC GOLD(TM) system to express P1 under the polh promoter and 3CD under the CMV promoter, dramatically improved the VLP yield while alleviating the VLP degradation. Infection of High Five(TM) cells with BacF-P1-C3CD enhanced the total and extracellular VLP yield to ≈268 and ≈171 mg/L, respectively, which enabled the release of abundant VLP into the supernatant and simplified the downstream purification. Intramuscular immunization of mice with 5 µg purified VLP induced cross-protective humoral responses and conferred protection against lethal virus challenge. Given the significantly improved extracellular VLP yield (≈171 mg/L) and the potent immunogenicity conferred by 5 µg VLP, one liter High Five(TM) culture produced ≈12,000 doses of purified vaccine, thus rendering the EV71 VLP vaccine economically viable and able to compete with inactivated virus vaccines.