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

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 VLPΔVP4) and investigated its structure, antigenicity, and vaccine potential. The cryo-electron microscopy (cryo-EM) structure of VLPΔVP4 was reconstructed to 3.71-Å resolution. Results from structural and biochemical analyses revealed that VLPΔVP4 resembles the end product of the viral uncoating process, the 80S empty capsid. VLPΔVP4 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-VLPΔVP4 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.IMPORTANCE 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 VLPΔVP4, structurally mimics the 80S empty capsid, which is the end stage of EV71 uncoating. We further show that VLPΔVP4 exhibits desirable immunogenicity and protective efficacy in proof-of-concept studies. In addition, the inhibitory mechanisms of the VLPΔVP4-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 VLPΔVP4 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.

Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody.

Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection.

An Ebola Virus-Like Particle-Based Reporter System Enables Evaluation of Antiviral Drugs In Vivo under Non-Biosafety Level 4 Conditions.

UNLABELLED: Ebola virus (EBOV) is a highly contagious lethal pathogen. As a biosafety level 4 (BSL-4) agent, however, EBOV is restricted to costly BSL-4 laboratories for experimentation, thus significantly impeding the evaluation of EBOV vaccines and drugs. Here, we report an EBOV-like particle (EBOVLP)-based luciferase reporter system that enables the evaluation of anti-EBOV agents in vitro and in vivo outside BSL-4 facilities. Cotransfection of HEK293T cells with four plasmids encoding the proteins VP40, NP, and GP of EBOV and firefly luciferase (Fluc) resulted in the production of Fluc-containing filamentous particles that morphologically resemble authentic EBOV. The reporter EBOVLP was capable of delivering Fluc into various cultured cells in a GP-dependent manner and was recognized by a conformation-dependent anti-EBOV monoclonal antibody (MAb). Significantly, inoculation of mice with the reporter EBOVLP led to the delivery of Fluc protein into target cells and rapid generation of intense bioluminescence signals that could be blocked by the administration of EBOV neutralizing MAbs. This BSL-4-free reporter system should facilitate high-throughput screening for anti-EBOV drugs targeting viral entry and efficacy testing of candidate vaccines. IMPORTANCE: Ebola virus (EBOV) researches have been limited to costly biosafety level 4 (BSL-4) facilities due to the lack of animal models independent of BSL-4 laboratories. In this study, we reveal that a firefly luciferase-bearing EBOV-like particle (EBOVLP) with typical filamentous EBOV morphology is capable of delivering the reporter protein into murine target cells both in vitro and in vivo Moreover, we demonstrate that the reporter delivery can be inhibited both in vitro and in vivo by a known anti-EBOV protective monoclonal antibody, 13C6. Our work provides a BSL-4-free system that can facilitate the in vivo evaluation of anti-EBOV antibodies, drugs, and vaccines. The system may also be useful for mechanistic study of the viral entry process.

Single Neutralizing Monoclonal Antibodies Targeting the VP1 GH Loop of Enterovirus 71 Inhibit both Virus Attachment and Internalization during Viral Entry.

UNLABELLED: Antibodies play a critical role in immunity against enterovirus 71 (EV71). However, how EV71-specific antibodies neutralize infections remains poorly understood. Here we report the working mechanism for a group of three monoclonal antibodies (MAbs) that potently neutralize EV71. We found that these three MAbs (termed D5, H7, and C4, respectively) recognize the same conserved neutralizing epitope within the VP1 GH loop of EV71. Single MAbs in this group, exemplified by D5, could inhibit EV71 infection in cell cultures at both the pre- and postattachment stages in a cell type-independent manner. Specifically, MAb treatment resulted in the blockade of multiple steps of EV71 entry, including virus attachment, internalization, and subsequent uncoating and RNA release. Furthermore, we show that the D5 and C4 antibodies can interfere with EV71 binding to its key receptors, including heparan sulfate, SCARB2, and PSGL-1, thus providing a possible explanation for the observed multi-inhibitory function of the MAbs. Collectively, our study unravels the mechanism of neutralization by a unique group of anti-EV71 MAbs targeting the conserved VP1 GH loop. The findings should enhance our understanding of MAb-mediated immunity against enterovirus infections and accelerate the development of MAb-based anti-EV71 therapeutic drugs. IMPORTANCE: Enterovirus 71 (EV71) is a major causative agent of hand, foot, and mouth disease (HFMD), which has caused significant morbidities and mortalities in young children. Neither a vaccine nor an antiviral drug is available. Neutralizing antibodies are major protective components in EV71 immunity. Here, we unraveled an unusual mechanism of EV71 neutralization by a group of three neutralizing monoclonal antibodies (MAbs). All of these MAbs bound the same conserved epitope located at the VP1 GH loop of EV71. Interestingly, mechanistic studies showed that single antibodies in this MAb group could block EV71 attachment and internalization during the viral entry process and interfere with EV71 binding to heparan sulfate, SCARB2, and PSGL-1 molecules, which are key receptors involved in different steps of EV71 entry. Our findings greatly enhance the understanding of the interplays among EV71, neutralizing antibodies, and host receptors, which in turn should facilitate the development of an MAb-based anti-EV71 therapy.

Chimeric virus-like particle vaccines displaying conserved enterovirus 71 epitopes elicit protective neutralizing antibodies in mice through divergent mechanisms.

Enterovirus 71 (EV71) is a major causative agent of hand, food, and mouth disease, which frequently occurs in young children. Since there are 11 subgenotypes (A, B1 to B5, and C1 to C5) within EV71, an EV71 vaccine capable of protecting against all of these subgenotypes is desirable. We report here the vaccine potential and protective mechanism of two chimeric virus-like particles (VLPs) presenting conserved neutralizing epitopes of EV71. We show that fusions of hepatitis B core antigen (HBc) with the SP55 or SP70 epitope of EV71, designated HBcSP55 and HBcSP70, respectively, can be rapidly generated and self-assembled into VLPs with the epitopes displayed on the surface. Immunization with the chimeric VLPs induced carrier- and epitope-specific antibody responses in mice. Anti-HBcSP55 and anti-HBcSP70 sera, but not anti-HBc sera, were able to neutralize in vitro multiple genotypes and strains of EV71. Importantly, passive immunization with anti-HBcSP55 or anti-HBcSP70 sera protected neonatal mice against lethal EV71 infections. Interestingly, anti-HBcSP70 sera could inhibit EV71 attachment to susceptible cells, whereas anti-HBcSP55 sera could not. However, both antisera were able to neutralize EV71 infection in vitro at the postattachment stage. The divergent mechanism of neutralization and protection conferred by anti-SP70 and anti-SP55 sera is in part attributed to their respective ability to bind authentic viral particles. Collectively, our study not only demonstrates that chimeric VLPs displaying the SP55 and SP70 epitopes are promising candidates for a broad-spectrum EV71 vaccine but also reveals distinct mechanisms of neutralization by the SP55- and SP70-targeted antibodies.

The molecule of DC-SIGN captures enterovirus 71 and confers dendritic cell-mediated viral trans-infection.

BACKGROUND: Enterovirus 71 (EV71) is the main causative agent of hand, foot and mouth disease that occurs in young children. Neither antiviral agents nor vaccines are available for efficiently combating viral infection. Study of EV71-host interplay is important for understanding viral infection and developing strategies for prevention and therapy. Here the interactions of EV71 with human dendritic cells were analyzed. METHODS: EV71 capture, endocytosis, infection, and degradation in monocyte-derived dendritic cells (MDDCs) were detected by Flow cytometry or real-time (RT-) PCR, and MDDCs-mediated EV71 trans-infection of RD cells was determined via coculture system. Cell morphology or viability was monitored with microscopy or flow cytometry. SiRNA interference was used to knock down gene expression. RESULTS: MDDCs can bind EV71, but these loaded-EV71 particles in MDDCs underwent a rapid degradation in the absence of efficient replication; once the captured EV71 encountered susceptible cells, MDDCs efficiently transferred surface-bound viruses to target cells. The molecule of DC-SIGN (DC-specific intercellular adhesion molecule-3 grabbing nonintegrin) mediated viral binding and transfer, because interference of DC-SIGN expression with specific siRNAs reduced EV71 binding and impaired MDDC-mediated viral trans-infection, and exogenous expression of DC-SIGN molecule on Raji cell initiated viral binding and subsequent transmission. CONCLUSION: MDDCs could bind efficiently EV71 viruses through viral binding to DC-SIGN molecule, and these captured-viruses could be transferred to susceptible cells for robust infection. The novel finding of DC-mediated EV71 dissemination might facilitate elucidation of EV71 primary infection and benefit searching for new clues for preventing viruses from initial infection.

Modified Theoretical Model Predicts Radial Support Capacity of Polymer Braided Stents.

BACKGROUND AND OBJECTIVE: Self-expanding polymer braided stents are expected to replace metallic stents in the treatment of Peripheral Arterial Disease, which seriously endangers human health. To restore the patency of blocked peripheral arteries with different properties and functions, the radial supporting capacity of the stent should be considered corresponding to the vessel. A theoretical model can be established as an effective method to study the radial supporting capacity of the stent which can shorten the stent design cycle and realize the customization of the stent according to lesion site. However, the classical model developed by Jedwab and Clerc of radial force is only limited to metallic braided stents, and the predictions for polymer braided stents are deviated. METHODS: In this paper, based on the limitation of the J&C model for polymer braided stents, a modified radial force model for polymer braided stents was proposed, which considered the friction between monofilaments and the torsion of the monofilaments. And the modified model was verified by radial force tests of polymer braided stents with different structures and monofilaments. RESULTS: Compared with the J&C model, the proposed modified model has better predictability for the radial force of polymer braided stents that prepared with different braided structure and polymer monofilaments. The root mean squared error of modified model is 0.041±0.026, while that of the J&C model is 0.246±0.111. CONCLUSIONS: For polymer braided stents, the friction between the polymer monofilaments and the torsion of the monofilaments during the radial compression cannot be ignored. The radial force prediction accuracy of the modified model considering these factors was significantly improved. This work provides a research basis on the theoretical model of polymer braided stents, and improves the feasibility of rapid personalized customization of polymer braided stents.

Evaluation of resistance to radial cyclic loads of poly(L-lactic acid) braided stents with different braiding angles.

Poly(L-lactic acid) (PLLA) braided stents have superior biocompatibility and flexibility, substituting metal stents in peripheral blood vessels. However, the radial supporting capacity of PLLA braided stent should be improved to bear the dynamic load from the peripheral artery. This paper evaluated the radial support performance of PLLA braided stents with different braiding angles after the radial cyclic loads test. The results indicate that braiding angle of stents is an important parameter affecting its ability to resist radial cyclic loads. The stent with a smaller braiding angle has better initial radial support but insufficient durability, while the stent with a larger braiding angle could maintain adequate radial support and suitable ability to resist radial cyclic loads. The theoretical analysis, verified by observing the surface morphology of filament crossover points, found that filaments of the stents with smaller braiding angles have more significant axial displacement and axial rotation angle during radial compression, which made the friction phenomenon more intense and led to insufficient ability to resist radial cyclic loads. This study could provide a meaningful idea for preparing biodegradable braided stents with suitable ability to resist radial cyclic loads.

Molecular mechanism of antibody neutralization of coxsackievirus A16.

Coxsackievirus A16 (CVA16) causes hand, foot and mouth disease in infants and young children. However, no vaccine or anti-viral agent is currently available for CVA16. Here, the functions and working mechanisms of two CVA16-specific neutralizing monoclonal antibodies (MAbs), 9B5 and 8C4, are comprehensively investigated. Both 9B5 and 8C4 display potent neutralization in vitro and prophylactic and therapeutic efficacy in a mouse model of CVA16 infection. Mechanistically, 9B5 exerts neutralization primarily through inhibiting CVA16 attachment to cell surface via blockade of CVA16 binding to its attachment receptor, heparan sulfate, whereas 8C4 functions mainly at the post-attachment stage of CVA16 entry by interfering with the interaction between CVA16 and its uncoating receptor SCARB2. Cryo-EM studies show that 9B5 and 8C4 target distinct epitopes located at the 5-fold and 3-fold protrusions of CVA16 capsids, respectively, and exhibit differential binding preference to three forms of naturally occurring CVA16 particles. Moreover, 9B5 and 8C4 are compatible in formulating an antibody cocktail which displays the ability to prevent virus escape seen with individual MAbs. Together, our work elucidates the functional and structural basis of CVA16 antibody-mediated neutralization and protection, providing important information for design and development of effective CVA16 vaccines and antibody therapies.

Construction and characterization of an infectious clone of coxsackievirus A16.

BACKGROUND: Coxsackievirus A16 (CVA16) is a member of the Enterovirus genus of the Picornaviridae family and it is a major etiological agent of hand, foot, and mouth disease (HFMD), which is a common illness affecting children. CVA16 possesses a single-stranded positive-sense RNA genome containing approximately 7410 bases. Current understanding of the replication, structure and virulence determinants of CVA16 is very limited, partly due to difficulties in directly manipulating its RNA genome. RESULTS: Two overlapping cDNA fragments were amplified by RT-PCR from the genome of the shzh05-1 strain of CVA16, encompassing the nucleotide regions 1-4392 and 4381-7410, respectively. These two fragments were then joined via a native XbaI site to yield a full-length cDNA. A T7 promoter and poly(A) tail were added to the 5' and 3' ends, respectively, forming a full CVA16 cDNA clone. Transfection of RD cells in vitro with RNA transcribed directly from the cDNA clone allowed the recovery of infectious virus in culture. The CVA16 virus recovered from these cultures was functionally and genetically identical to its parent strain. CONCLUSIONS: We report the first construction and characterization of an infectious cDNA clone of CVA16. The availability of this infectious clone will greatly enhance future virological investigations and vaccine development for CVA16.

Coxsackievirus A10 atomic structure facilitating the discovery of a broad-spectrum inhibitor against human enteroviruses.

Coxsackievirus A10 (CV-A10) belongs to the Enterovirus species A and is a causative agent of hand, foot, and mouth disease. Here we present cryo-EM structures of CV-A10 mature virion and native empty particle (NEP) at 2.84 and 3.12 Å, respectively. Our CV-A10 mature virion structure reveals a density corresponding to a lipidic pocket factor of 18 carbon atoms in the hydrophobic pocket formed within viral protein 1. By structure-guided high-throughput drug screening and subsequent verification in cell-based infection-inhibition assays, we identified four compounds that inhibited CV-A10 infection in vitro. These compounds represent a new class of anti-enteroviral drug leads. Notably, one of the compounds, ICA135, also exerted broad-spectrum inhibitory effects on a number of representative viruses from all four species (A-D) of human enteroviruses. Our findings should facilitate the development of broadly effective drugs and vaccines for enterovirus infections.

A virus-like particle vaccine protects mice against coxsackievirus A10 lethal infection.

Coxsackievirus A10 (CVA10) has emerged worldwide as one of the main pathogens of hand, foot, and mouth disease (HFMD) in recent years. However, there is currently no commercial vaccine available to prevent CVA10 infection. Here we report the development of a recombinant virus-like particle (VLP) based candidate vaccine for CVA10. Co-expression of the capsid protein precursor P1 and the protease 3CD of CVA10 in Pichia pastoris resulted in cleavage of P1 into three capsid subunit proteins VP0, VP1, and VP3. These three subunit proteins co-assembled into CVA10 VLPs, which were visualized as spherical particles with a diameter of ∼30 nm under electron microscope. Immunization studies showed that CVA10 VLP could efficiently induce antigen-specific serum antibodies in mice. The anti-VLP sera were able to potently neutralize homologous and heterologous CVA10 strains. Importantly, passively transferred anti-VLP sera fully protected recipient neonatal mice from lethal CVA10 infection. In addition, neonatal mice born to the VLP-immunized dams were also completely protected from CVA10 lethal challenge. Collectively, these data show that CVA10 VLP represents a promising CVA10 vaccine candidate.

A virus-like particle-based tetravalent vaccine for hand, foot, and mouth disease elicits broad and balanced protective immunity.

Hand, foot, and mouth disease (HFMD) is an infectious disease that mainly affects infants and children, causing considerable morbidity and mortality worldwide. HFMD is commonly caused by enterovirus 71 (EV71) and coxsackieviruses A16 (CVA16), A6 (CVA6), and A10 (CVA10). Formalin-inactivated EV71 vaccines are currently available in China; however, these vaccines fail to confer cross-protection against infections by other HFMD-causing enteroviruses, highlighting the necessity of developing a multivalent HFMD vaccine. Our previous studies demonstrated that recombinant virus-like particles (VLP) of EV71, CVA16, and CVA6 are capable of inducing protective immunity against homologous virus challenges in mice. In this study, we generated CVA10-VLP using a baculovirus-insect cell expression system and then combined CVA10-VLP with EV71-VLP, CVA16-VLP, and CVA6-VLP to formulate a tetravalent VLP vaccine. Immunogenicity and protective efficacy of tetravalent VLP vaccine was compared with that of monovalent VLP vaccines. Mouse immunization studies revealed that the tetravalent vaccine elicited antigen-specific and long-lasting serum antibody responses comparable to those elicited by its corresponding monovalent vaccines. Moreover, tetravalent vaccine immune sera strongly neutralized EV71, CVA16, CVA10, and CVA6 strains with neutralization titers similar to those of their monovalent counterparts, indicating a good compatibility among the four antigens in the combination vaccine. Importantly, passively transferred tetravalent vaccine-immunized sera conferred efficient protection against single or mixed infections with EV71, CVA16, CVA10, and CVA6 viruses in mice, whereas the monovalent vaccines could only protect mice against homotypic virus infections but not heterotypic challenges. These results demonstrate that the tetravalent VLP vaccine represents a promising broad-spectrum HFMD vaccine candidate.

Coxsackievirus A16 utilizes cell surface heparan sulfate glycosaminoglycans as its attachment receptor.

Coxsackievirus A16 (CVA16) is one of the major pathogens responsible for hand, foot and mouth disease, which affects more than two million children in the Asian-Pacific region annually. Previous studies have shown that scavenger receptor B2 is a functional receptor for CVA16 that facilitates the uncoating process. However, it remains unclear whether other receptors are required for efficient CVA16 infection. In this study, by using a variety of assays we demonstrated that CVA16 utilizes surface heparan sulfate glycosaminoglycans as its attachment receptor. We further showed that five surface-exposed positively charged residues located in a cluster at the five-fold vertex of the virion are critical to heparan sulfate binding and cellular attachment of CVA16. Among the five residues, the arginine at position 166 (R166) of VP1 capsid protein appeared to be the most important for the interaction between CVA16 and heparan sulfate. Alanine substitution at this site (R166A) almost completely abolished heparan sulfate binding and cellular attachment of the virus. Our work achieves insight into the early events of CVA16 infection, thereby providing information that may facilitate the rational design of antiviral drugs and vaccines against CVA16 infection.

Yeast-produced recombinant virus-like particles of coxsackievirus A6 elicited protective antibodies in mice.

Coxsackievirus A6 (CA6) has recently emerged as the predominant pathogen of hand, foot and mouth disease (HFMD), causing significant morbidity in children and adults. The increasing prevalence of CA6 infection and its associated disease burden underscore the need for effective CA6 vaccines. However, CA6 grows poorly in cultured cells, making it difficult to develop inactivated whole-virus or live attenuated vaccines. Here we report the development of a recombinant virus-like particle (VLP) based CA6 vaccine. CA6 VLPs were produced in Pichia pastoris yeast transformed with a vector encoding both P1 and 3CD proteins of CA6. Immunization with CA6 VLPs elicited CA6-specific serum antibodies in mice. Passive transfer of anti-VLP antisera protected recipient mice against lethal CA6 challenge. Collectively, these results demonstrate that CA6 VLPs represent a viable CA6 vaccine candidate which warrants further preclinical and clinical development.

Virus-like particle-based vaccine against coxsackievirus A6 protects mice against lethal infections.

Coxsackievirus A6 (CA6) is emerging as one of the major causative agents of hand, foot, and mouth disease (HFMD) worldwide. However, no vaccine is currently available for preventing CA6 infection. Here, we report the development of a virus-like particle (VLP)-based recombinant vaccine for CA6. We produced CA6 VLPs in insect cells by infecting the cells with a baculovirus coexpressing the genes encoding CA6 P1 and 3CD. Biochemical analyses showed that the produced VLPs consisted of VP0, VP1, and VP3 capsid subunit proteins generated by the cleavage of P1 by 3CD. Mice immunized with these VLPs produced CA6-specific serum antibodies. Passive transfer of antisera from CA6 VLP-immunized mice protected recipient mice from lethal infections caused by homologous and heterologous CA6 strains. Moreover, active immunization of mice with CA6 VLPs efficiently conferred protection against both homologous and heterologous CA6 infections. These results suggested that CA6 VLP-based recombinant vaccine is a promising candidate vaccine for preventing CA6 infection and can be incorporated into a multivalent HFMD vaccine formulation to achieve broad-spectrum and effective prevention of this disease.

Inactivated coxsackievirus A10 experimental vaccines protect mice against lethal viral challenge.

Coxsackievirus A10 (CVA10) has become one of the major causative agents of hand, foot and mouth disease (HFMD). It is now recognized that CVA10 should be targeted for vaccine development. We report here that ß-propiolactone inactivated whole-virus based CVA10 vaccines can elicit protective immunity in mice. We prepared two inactivated CVA10 experimental vaccines derived from the prototype strain CVA10/Kowalik and from a clinical isolate CVA10/S0148b, respectively. Immunization with the experimental vaccines elicited CVA10-specific serum antibodies in mice. The antisera from vaccinated mice could potently neutralize in vitro infection with either homologous or heterologous CVA10 strains. Importantly, passive transfer of the anti-CVA10 sera protected recipient mice against CVA10/Kowalik or CVA10/S0148b infections. Moreover, active immunization with the inactivated vaccines also conferred protection against homologous and heterologous infections in mice. Collectively, our results demonstrate the proof-of-concept for inactivated whole-virus based CVA10 vaccines.

Transcutaneous immunization via rapidly dissolvable microneedles protects against hand-foot-and-mouth disease caused by enterovirus 71.

Recent large outbreaks of hand-foot-and-mouth disease (HFMD) have seriously affected the health of young children. Enterovirus 71 (EV71) is the main causative agent of HFMD. Herein, for the first time, rapidly dissolvable microneedles (MNs) loaded with EV71 virus-like particles (VLPs) were evaluated whether they could induce robust immune responses that confer protection against EV71 infection. The characteristics of prepared MNs including hygroscopy, mechanical strength, insertion capacity, dissolution profile, skin irritation and storage stability were comprehensively assessed. EV71 VLPs remained morphologically stable during fabrication. The MNs made of sodium hyaluronate maintained their insertion ability for at least 3h even at a high relative humidity of 75%. With the aid of spring-operated applicator, EV71 MNs (approximately 500µm length) could be readily penetrated into the mouse skin in vivo, and then rapidly dissolved to release encapsulated antigen within 2min. Additionally, MNs induced slight erythema that disappeared within a few hours. More importantly, mouse immunization and virus challenge studies demonstrated that MNs immunization induced high level of antibody responses conferring full protection against lethal EV71 virus challenge that were comparable to conventional intramuscular injection, but with only 1/10th of the delivered antigen (dose sparing). Consequently, our rapidly dissolving MNs may present as an effective and promising transcutaneous immunization device for HFMD prophylaxis among children.

Towards broadly protective polyvalent vaccines against hand, foot and mouth disease.

Hand, foot, and mouth disease (HFMD) caused by multiple enterovirus infections is a serious health threat to children in the Asia-Pacific region. This article reviews progresses in the development of vaccines for HFMD and discusses the need for polyvalent HFMD vaccines for conferring broad-spectrum protection.

A bivalent virus-like particle based vaccine induces a balanced antibody response against both enterovirus 71 and norovirus in mice.

Noroviruses are the main cause of severe viral gastroenteritis, which results in estimated 200,000 deaths each year, primarily in children in the developing world. Genogroup II.4 (GII.4) strains are responsible for the majority of norovirus outbreaks. Enterovirus 71 (EV71), the leading causative agent of hand, foot and mouth disease, has recently been prevalent in Asia-Pacific regions, resulting in significant morbidity and mortality in young children. However, no vaccine is commercially available for either norovirus GII.4 or EV71. Recombinant virus-like particles (VLPs) derived from either GII.4 or EV71 have been shown to be promising monovalent vaccine candidates. In this study, we investigate the possibility to formulate a VLP-based bivalent vaccine for both norovirus GII.4 and EV71. The GII.4- and EV71-VLPs were produced in a baculovirus-insect cell expression system. A bivalent combination vaccine comprised of GII.4 and EV71 VLPs was formulated and compared with monovalent GII.4- and EV71-VLPs for their immunogenicity in mice. We found that the bivalent vaccine elicited durable antibody responses toward both GII.4 and EV71, and the antibody titers were comparable to that induced by the monovalent vaccines, indicating there is no immunological interference between the two antigens in the combination vaccine. More significantly, the bivalent vaccine-immunized mouse sera could efficiently neutralize EV71 infection and block GII.4-VLP binding to mucin. Together, our results demonstrate that the experimental combination vaccine comprised of GII.4 and EV71-VLPs is able to induce a balanced protective antibody response, and therefore strongly support further preclinical and clinical development of such a bivalent VLP vaccine targeting both norovirus GII.4 and EV71.