Crystal Structures of Yeast-Produced Enterovirus 71 and Enterovirus 71/Coxsackievirus A16 Chimeric Virus-Like Particles Provide the Structural Basis for Novel Vaccine Design against Hand-Foot-and-Mouth Disease

Lyu, Ke; He, Ya-Ling; Li, Hao-Yang; Chen, Rong

doi:10.1128/jvi.00422-15

Cited by 20 publications

(28 citation statements)

References 34 publications

(42 reference statements)

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“…In this work, evidence is presented that EV71 VLPs of the C4 strain produced from the BEVS and purified through our multiple chromatography process retained key molecular and structural features for the first time, compared with those reported for native empty virions [24] and other EV71 VLPs [23, 35, 36]. The sedimentation coefficient of purified EV71 VLPs in this study (78S) was different from that of EV71 VLPs purified by CsCl (85S) as previously reported by Gong et al [23] using AUC analysis, and SDS-PAGE results showed that our EV71 VLPs consisted of VP0 (35 KDa), VP1 (33 KDa) and VP3 (26 KDa) capsid proteins were also different from EV71 VLPs consisted mostly of VP0 (35KDa), VP1(33 and 30KDa), VP3(27kDa), and a presumably incompletely processed band VP0-VP3(62kDa) purified by Gong et al As different strains, culture conditions, and storage conditions may influence on the final production[24, 35, 37], we speculate that these difference may be caused by our different production conditions.…”

Section: Discussionmentioning

confidence: 86%

“…The sedimentation coefficient of purified EV71 VLPs in this study (78S) was different from that of EV71 VLPs purified by CsCl (85S) as previously reported by Gong et al [23] using AUC analysis, and SDS-PAGE results showed that our EV71 VLPs consisted of VP0 (35 KDa), VP1 (33 KDa) and VP3 (26 KDa) capsid proteins were also different from EV71 VLPs consisted mostly of VP0 (35KDa), VP1(33 and 30KDa), VP3(27kDa), and a presumably incompletely processed band VP0-VP3(62kDa) purified by Gong et al As different strains, culture conditions, and storage conditions may influence on the final production[24, 35, 37], we speculate that these difference may be caused by our different production conditions. Although they were both BEVS-produced, the purified EV71 VLPs in this study were shown to be mainly located in the cytoplasm.…”

Section: Discussionmentioning

confidence: 99%

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Characterization of human enterovirus71 virus-like particles used for vaccine antigens

Zhao

Sun

et al. 2017

PLoS ONE

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Human enterovirus 71 (EV71) is a major causative pathogen of hand, foot and mouth disease (HFMD) and has caused outbreaks with significant mortality among young children in the Asia-Pacific region in recent years. Towards developing a vaccine for this disease, we have expressed and purified EV71 virus-like particles (VLPs), which resemble the authentic virus in appearance, capsid structure and protein sequence, from insect cells (Sf9) using a multistep chromatography process. We demonstrated intracellular localization of the VLPs in host cells by in situ immunogold detection, electron microscopy and immunofluorescence. Characteristics of these EV71 VLPs were studied using a variety of immunological and physicochemical techniques, which aimed to reveal that the purified EV71 VLPs have good morphology and structure consistent with natural EV71 empty capsids. Results of the amino acid analysis, SDS-PAGE, Western blotting and high-performance liquid chromatography confirmed the high purity of the EV71 VLPs. However the sedimentation coefficient of the VLPs showed that they were smaller than that of secreted EV71 VLPs purified by discontinuous cesium chloride density gradients, they were similar to the empty capsids of natural EV71 virions reported previously. Combined with the previous study that EV71 VLPs purified by a multistep chromatography process were able to elicit strong humoral immune responses in mice, our results further supported the conclusion that our EV71 VLPs had well-preserved molecular and structural characteristics. The EV71 VLPs produced from the baculovirus expression system and purified by a multistep chromatography process displayed key structural and immunological features, which would contribute to their efficacy as a HFMD vaccine.

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Section: Discussionmentioning

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Characterization of human enterovirus71 virus-like particles used for vaccine antigens

Zhao

Sun

et al. 2017

PLoS ONE

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“…Coexpression of full-length P1 and 3CD genes of EV71 in recombinant systems resulted in the cleavage of P1 into full-length VP0, VP1, and VP3 subunits; together, they assemble into full-length VLPs (termed VLP full here) (18)(19)(20)(21). VLP full resembles the naturally occurring NEP in protein composition and overall structure (19,22). Although VLP full is highly immunogenic and protective in animal models (20,23,24), autocleavage of VP0 has been observed for unknown reasons in studies of EV71 (20,24) and the related virus coxsackievirus A16 (CVA16) (25,26).…”

mentioning

confidence: 99%

Structure, Immunogenicity, and Protective Mechanism of an Engineered Enterovirus 71-Like Particle Vaccine Mimicking 80S Empty Capsid

Wang

Xiang

et al. 2018

J Virol

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Enterovirus 71 (EV71) is the major causative agent of severe hand, foot, and mouth disease, which affects millions of young children in the Asia-Pacific region annually. In this study, we engineered a novel EV71 virus-like particle (VLP) that lacks VP4 (therefore designated VLP) and investigated its structure, antigenicity, and vaccine potential. The cryo-electron microscopy (cryo-EM) structure of VLP was reconstructed to 3.71-Å resolution. Results from structural and biochemical analyses revealed that VLP resembles the end product of the viral uncoating process, the 80S empty capsid. VLP is able to elicit high-titer neutralizing antibodies and to fully protect mice against lethal viral challenge. Mechanistic studies showed that, at the cellular level, the anti-VLP sera exert neutralization effects at both pre- and postattachment stages by inhibiting both virus attachment and internalization, and at the molecular level, the antisera can block multiple interactions between EV71 and its key receptors. Our study gives a better understanding of EV71 capsid assembly and provides important information for the design and development of new-generation vaccines for EV71, and perhaps for other enteroviruses, as well. Enterovirus 71 (EV71) infection may lead to severe hand, foot, and mouth disease, with significant morbidity and mortality. Knowledge regarding EV71 particle assembly remains limited. Here, we report the generation and characterization of a novel EV71 virus-like particle that lacks the VP4 capsid subunit protein. This particle, termed VLP, structurally mimics the 80S empty capsid, which is the end stage of EV71 uncoating. We further show that VLP exhibits desirable immunogenicity and protective efficacy in proof-of-concept studies. In addition, the inhibitory mechanisms of the VLP-induced antibodies are unraveled at both the cellular and molecular levels. Our work provides the first evidence of picornaviral particle assembly in the complete absence of VP4 and identifies VLP as an improved EV71 vaccine candidate with desirable traits. These findings not only enhance our understanding of particle assembly and uncoating of picornaviruses, but also provide important information for structure-guided vaccine design for EV71 and other enteroviruses.

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“…The radius of the CVA6 VLP is 161 Å ( Fig. 2A), which is similar to those of the expanded particles of EV71 and CVA16 (e.g., 159 Å for the EV71 procapsid, 161 Å for the EV71 VLP, and 162 Å for the CVA16 135S particle) and is slightly larger than those of the compact particles of EV71 and CVA16 (e.g., 155 Å for the EV71 mature virion, 156 Å for the CVA16 mature virion, and 156 Å for the CVA16 VLP) (23,(25)(26)(27), suggesting that the CVA6 VLP is likely in an expanded state. However, since no CVA6 mature virus structure is currently available, it is impossible to directly compare the radius of the CVA6 VLP with that of the CVA6 mature virion.…”

Section: Resultsmentioning

confidence: 68%

A 3.0-Angstrom Resolution Cryo-Electron Microscopy Structure and Antigenic Sites of Coxsackievirus A6-Like Particles

Chen

Zhang

Zhou

et al. 2018

J Virol

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Coxsackievirus A6 (CVA6) has recently emerged as one of the predominant causative agents of hand, foot, and mouth disease (HFMD). The structure of the CVA6 mature viral particle has not been solved thus far. Our previous work shows that recombinant virus-like particles (VLPs) of CVA6 represent a promising CVA6 vaccine candidate. Here, we report the first cryo-electron microscopy (cryo-EM) structure of the CVA6 VLP at 3.0-Å resolution. The CVA6 VLP exhibits the characteristic features of enteroviruses but presents an open channel at the 2-fold axis and an empty, collapsed VP1 pocket, which is broadly similar to the structures of the enterovirus 71 (EV71) VLP and coxsackievirus A16 (CVA16) 135S expanded particle, indicating that the CVA6 VLP is in an expanded conformation. Structural comparisons reveal that two common salt bridges within protomers are maintained in the CVA6 VLP and other viruses of the genus, implying that these salt bridges may play a critical role in enteroviral protomer assembly. However, there are apparent structural differences among the CVA6 VLP, EV71 VLP, and CVA16 135S particle in the surface-exposed loops and C termini of subunit proteins, which are often antigenic sites for enteroviruses. By immunological assays, we identified two CVA6-specific linear B-cell epitopes (designated P42 and P59) located at the GH loop and the C-terminal region of VP1, respectively, in agreement with the structure-based prediction of antigenic sites. Our findings elucidate the structural basis and important antigenic sites of the CVA6 VLP as a strong vaccine candidate and also provide insight into enteroviral protomer assembly. Coxsackievirus A6 (CVA6) is becoming one of the major pathogens causing hand, foot, and mouth disease (HFMD), leading to significant morbidity and mortality in children and adults. However, no vaccine is currently available to prevent CVA6 infection. Our previous work shows that recombinant virus-like particles (VLPs) of CVA6 are a promising CVA6 vaccine candidate. Here, we present a 3.0-Å structure of the CVA6 VLP determined by cryo-electron microscopy. The overall architecture of the CVA6 VLP is similar to those of the expanded structures of enterovirus 71 (EV71) and coxsackievirus A16 (CVA16), but careful structural comparisons reveal significant differences in the surface-exposed loops and C termini of each capsid protein of these particles. In addition, we identified two CVA6-specific linear B-cell epitopes and mapped them to the GH loop and the C-terminal region of VP1, respectively. Collectively, our findings provide a structural basis and important antigenic information for CVA6 VLP vaccine development.

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Crystal Structures of Yeast-Produced Enterovirus 71 and Enterovirus 71/Coxsackievirus A16 Chimeric Virus-Like Particles Provide the Structural Basis for Novel Vaccine Design against Hand-Foot-and-Mouth Disease

Cited by 20 publications

References 34 publications

Characterization of human enterovirus71 virus-like particles used for vaccine antigens

Characterization of human enterovirus71 virus-like particles used for vaccine antigens

Structure, Immunogenicity, and Protective Mechanism of an Engineered Enterovirus 71-Like Particle Vaccine Mimicking 80S Empty Capsid

A 3.0-Angstrom Resolution Cryo-Electron Microscopy Structure and Antigenic Sites of Coxsackievirus A6-Like Particles

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