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Efficient expression of enterovirus 71 based on virus-like particles vaccine.

ABSTRACT: Enterovirus (EV) 71 is the main pathogen associated with hand-foot-mouth disease (HFMD) and can lead to the disease with severe mortality in children. Since 2009, in the Republic of Korea, an outbreak of EV71 C4a infection with neurologic involvement emerged, where in HFMD involvement was identified and central nervous system complications were reported. In this study, EV71 C4a virus-like particles (VLPs) produced by recombinant technology were generated in a baculovirus expression system. To improve the production yield, EV71 VLP was constructed using the dual promoter system baculovirus P1 and 3CD (baculo-P1-3CD), which harbored both the structural protein-encoding P1 region under the control of the polyhedron promoter and the 3CD protease gene under the regulation of the CMV-IE, lef3, gp41, or chitinase promoters to augment the level of gene transcription. Efficient VLP expression was demonstrated through optimization of incubation time and insect cell type. In addition, to evaluate the potential of VLP as a vaccine candidate, we tested the neutralizing antibodies and total anti-EV71 IgG from the purified EV71 C4a VLP serum. The recombinant EV71 VLP exhibited the morphology of self-assembled VLP, as determined by electron microscopy. Use of baculo-P1-3CD-gp41 led to a high yield (11.3mg/L < 40kDa) of VLPs in High-FiveTM cells at 3 days post-infection. Furthermore, the potential of VLP as a vaccine was evaluated through the neutralizing ability elicited by the purified EV71 VLP after immunization of BALB/c mice, which was shown to induce potent and long-lasting humoral immune responses as evidenced by the cross-neutralization titer. Our results could be used to expedite the developmental process for vaccines under clinical trials and to ensure manufacturing consistency for licensing requirements.

SUBMITTER: Kim HJ

PROVIDER: S-EPMC6405078 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Efficient expression of enterovirus 71 based on virus-like particles vaccine.

Kim Hye-Jin HJ Son Ho Sun HS Lee Sang Won SW Yoon Youngsil Y Hyeon Ji-Yeon JY Chung Gyung Tae GT Lee June-Woo JW Yoo Jung Sik JS

PloS one 20190307 3

Enterovirus (EV) 71 is the main pathogen associated with hand-foot-mouth disease (HFMD) and can lead to the disease with severe mortality in children. Since 2009, in the Republic of Korea, an outbreak of EV71 C4a infection with neurologic involvement emerged, where in HFMD involvement was identified and central nervous system complications were reported. In this study, EV71 C4a virus-like particles (VLPs) produced by recombinant technology were generated in a baculovirus expression system. To im ...[more]

PMID: 30845175

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Project description:Enterovirus 71 (EV71) and coxsackieviruses (CV) are the major causative agents of hand, foot and mouth disease (HFMD). There is not currently a vaccine available against HFMD, even though a newly developed formalin-inactivated EV71 (FI-EV71) vaccine has been tested in clinical trial and has shown efficacy against EV71. We have designed and genetically engineered a recombinant adenovirus Ad-EVVLP with the EV71 P1 and 3CD genes inserted into the E1/E3-deleted adenoviral genome. Ad-EVVLP were produced in HEK-293A cells. In addition to Ad-EVVLP particles, virus-like particles (VLPs) formed from the physical association of EV71 capsid proteins, VP0, VP1, and VP3 expressed from P1 gene products. They were digested by 3CD protease and confirmed to be produced by Ad-EVVLP-producing cells, as determined using transmission electron microscopy and western blotting. Mouse immunogenicity studies showed that Ad-EVVLP-immunized antisera neutralized the EV71 B4 and C2 genotypes. Activation of VLP-specific CD4+ and CD8+/IFN-γ T cells associated with Th1/Th2-balanced IFN-ɣ, IL-17, IL-4, and IL-13 was induced; in contrast, FI-EV71 induced only Th2-mediated neutralizing antibody against EV71 and low VLP-specific CD4+ and CD8+ T cell responses. The antiviral immunity against EV71 was clearly demonstrated in mice vaccinated with Ad-EVVLP in a hSCARB2 transgenic (hSCARB2-Tg) mouse challenge model. Ad-EVVLP-vaccinated mice were 100% protected and demonstrated reduced viral load in both the CNS and muscle tissues. Ad-EVVLP successfully induced anti-CVA16 immunities. Although antisera had no neutralizing activity against CVA16, the 3C-specific CD4+ and CD8+/IFN-γ T cells were identified, which could mediate protection against CVA16 challenge. FI-EV71 did not induce 3C-mediated immunity and had no efficacy against the CVA16 challenge. These results suggest that Ad-EVVLP can enhance neutralizing antibody and protective cellular immune responses to prevent EV71 infection and cellular immune responses against CV infection.

Project description:Human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the two major causative agents for hand-foot-and-mouth disease (HFMD). Previously, we demonstrated that a virus-like particle (VLP) for EV71 produced from Saccharomyces cerevisiae is a potential vaccine candidate against EV71 infection, and an EV71/CVA16 chimeric VLP can elicit protective immune responses against both virus infections. Here, we presented the crystal structures of both VLPs, showing that both the linear and conformational neutralization epitopes identified in EV71 are mostly preserved on both VLPs. The replacement of only 4 residues in the VP1 GH loop converted strongly negatively charged surface patches formed by portions of the SP70 epitope in EV71 VLP into a relatively neutral surface in the chimeric VLP, which likely accounted for the additional neutralization capability of the chimeric VLP against CVA16 infection. Such local variations in the amino acid sequences and the surface charge potential are also present in different types of polioviruses. In comparison to EV71 VLP, the chimeric VLP exhibits structural changes at the local site of amino acid replacement and the surface loops of all capsid proteins. This is consistent with the observation that the VP1 GH loop located near the pseudo-3-fold junction is involved in extensive interactions with other capsid regions. Furthermore, portions of VP0 and VP1 in EV71 VLP are at least transiently exposed, revealing the structural flexibility of the VLP. Together, our structural analysis provided insights into the structural basis of enterovirus neutralization and novel vaccine design against HFMD and other enterovirus-associated diseases.Our previous studies demonstrated that the enterovirus 71 (EV71) virus-like particle (VLP) produced from yeast is a vaccine candidate against EV71 infection and that a chimeric EV71/coxsackievirus A16 (CVA16) VLP with the replacement of 4 amino acids in the VP1 GH loop can confer protection against both EV71 and CVA16 infections. This study reported the crystal structures of both the EV71 VLP and the chimeric EV71/CVA16 VLP and revealed that the major neutralization epitopes of EV71 are mostly preserved in both VLPs. In addition, the mutated VP1 GH loop in the chimeric VLP is well exposed on the particle surface and exhibits a surface charge potential different from that contributed by the original VP1 GH loop in EV71 VLP. Together, this study provided insights into the structural basis of enterovirus neutralization and evidence that the yeast-produced VLPs can be developed into novel vaccines against hand-foot-and-mouth disease (HFMD) and other enterovirus-associated diseases.

Project description:Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two most common etiological agents responsible for the epidemics of hand, foot, and mouth disease (HFMD), a childhood illness with occasional severe neurological complications. A number of vaccine candidates against EV71 or CA16 have been reported; however, no vaccine is currently available for clinical use. Here, we generated a secreted version of EV71 and CA16 virus-like particles (VLPs) using a baculovirus-insect cell expression system and reconstructed the three-dimensional (3D) structures of both VLPs by cryo-electron microscopy (cryo-EM) single-particle analysis at 5.2-? and 5.5-? resolutions, respectively. The reconstruction results showed that the cryo-EM structures of EV71 and CA16 VLPs highly resemble the recently published crystal structures for EV71 natural empty particles and CA16 135S-like expanded particles, respectively. Our cryo-EM analysis also revealed that the majority of previously identified linear neutralizing epitopes are well preserved on the surface of EV71 and CA16 VLPs. In addition, both VLPs were able to induce efficiently neutralizing antibodies against various strains of EV71 and CA16 viruses in mouse immunization. These studies provide a structural basis for the development of insect cell-expressed VLP vaccines and for a potential bivalent VLP vaccine against both EV71- and CA16-associated HFMD.The recent outbreaks of hand, foot, and mouth disease (HFMD) in the Asia Pacific region spurred the search for effective vaccines against EV71 and CA16 viruses, the two most common etiological agents responsible for HFMD. In this paper, we show that secreted versions of EV71 and CA16 VLPs generated in the baculovirus-insect cell expression system highly resemble the crystal structures of their viral conterparts and that the majority of previously identified linear neutralizing epitopes are well preserved on the VLP surfaces. In addition, the generated VLPs can efficiently induce neutralizing antibodies against various strains of EV71 and CA16 viruses in mouse immunization. These studies provide a structural basis for the development of insect cell-expressed VLP vaccines and for a potential bivalent VLP vaccine against both EV71- and CA16-associated HFMD.

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Efficient expression of enterovirus 71 based on virus-like particles vaccine.

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Efficient expression of enterovirus 71 based on virus-like particles vaccine.

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