A virus-like particle-based tetravalent vaccine for hand, foot, and mouth disease elicits broad and balanced protective immunity.
ABSTRACT: 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.
Project description:Hand, foot, and mouth disease (HFMD) has recently emerged as a major public health concern across the Asian-Pacific region. Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are the primary causative agents of HFMD, but other members of the Enterovirus A species, including Coxsackievirus A6 (CVA6), can cause disease. The lack of small animal models for these viruses have hampered the development of a licensed HFMD vaccine or antivirals. We have previously reported on the development of a mouse model for EV71 and demonstrated the protective efficacy of an inactivated EV71 vaccine candidate. Here, mouse-adapted strains of CVA16 and CVA6 were produced by sequential passage of the viruses through mice deficient in interferon (IFN) ?/? (A129) and ?/? and ? (AG129) receptors. Adapted viruses were capable of infecting 3 week-old A129 (CVA6) and 12 week-old AG129 (CVA16) mice. Accordingly, these models were used in active and passive immunization studies to test the efficacy of a trivalent vaccine candidate containing inactivated EV71, CVA16, and CVA6. Full protection from lethal challenge against EV71 and CVA16 was observed in trivalent vaccinated groups. In contrast, monovalent vaccinated groups with non-homologous challenges failed to cross protect. Protection from CVA6 challenge was accomplished through a passive transfer study involving serum raised against the trivalent vaccine. These animal models will be useful for future studies on HFMD related pathogenesis and the efficacy of vaccine candidates.
Project description: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 Enterovirus 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.IMPORTANCE 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.
Project description:Human enterovirus species A (HEV-A) consists of at least 16 members of different serotypes that are known to be the causative agents of hand, foot, and mouth disease (HFMD), herpangina, and other diseases, such as respiratory disease and polio-like flaccid paralysis. Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the major causative agents of HFMD. CVA5, CVA6, CVA10, and CVA12 mainly cause herpangina or are occasionally involved with sporadic cases of HFMD. We have previously shown that human scavenger receptor class B, member 2 (SCARB2) is a cellular receptor for EV71 and CVA16. Using a large number of clinical isolates of HEV-A, we explored whether all clinical isolates of EV71 and other serotypes of HEV-A infected cells via SCARB2. We tested this possibility by infecting L-SCARB2 cells, which are L929 cells expressing human SCARB2, by infecting human RD cells that had been treated with small interfering RNAs for SCARB2 and by directly binding the viruses to a soluble SCARB2 protein. We showed that all 162 clinical isolates of EV71 propagated in L-SCARB2 cells, suggesting that SCARB2 is the critical receptor common to all EV71 strains. In addition, CVA7, CVA14, and CVA16, which are most closely related to each other, also utilized SCARB2 for infection. EV71, CVA14, and CVA16 are highly associated with HFMD, and EV71 and CVA7 are occasionally associated with neurological diseases, suggesting that SCARB2 plays important roles in the development of these diseases. In contrast, another group of viruses, such as CVA2, CVA3, CVA4, CVA5, CVA6, CVA8, CVA10, and CVA12, which are relatively distant from the EV71 group, is associated mainly with herpangina. None of these clinical isolates infected via the SCARB2-dependent pathway. HEV-A viruses can be divided into at least two groups depending on the use of SCARB2, and the receptor usage plays an important role in developing the specific diseases for each group.
Project description:Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) have caused severe epidemics of hand, foot and mouth disease (HFMD) in the Asia Pacific in recent years, particularly in infants and young children. This disease has become a serious public health problem, as no vaccines or antiviral drugs have been approved for EV71 and CA16 infections. In this study, we compared four monovalent vaccines, including formalin-inactivated EV71 virus (iEV71), EV71 virus-like particles (VLPs) (vEV71), formalin-inactivated CVA16 virus (iCVA16) and CVA16 VLPs (vCVA16), along with two bivalent vaccines, including equivalent doses of formalin-inactivated EV71+CVA16 virus (iEV71+iCVA16) and EV71+CVA16 VLPs (vEV71+vCVA16). The IgG titers and neutralization antibodies titers demonstrated that there are no immune interference exists between the two immunogens of EV71 and CVA16. IgG subclass isotyping revealed that IgG1 and IgG2b were induced primarily in all vaccine groups. Furthermore, cross-neutralization antibodies were elicited in mouse sera against other sub-genotypes of EV71 and CVA16. In vivo challenge experiments showed that the immune sera from vaccinated animals could confer passive protection to newborn mice against lethal challenge with 14 LD50 of EV71 and 50 LD50 of CVA16. Our results indicated that bivalent vaccination is promising for HFMD vaccine development. With the advantage of having a better safety profile than inactivated virus vaccines, VLPs should be used to combine both EV71 and CVA16 antigens as a candidate vaccine for prevention of HFMD virus transmission.
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:Hand, foot and mouth disease (HFMD) is responsible for a heavy economic and social burden in the Asia-Pacific region. Previous studies have shown that coxsackievirus A6 (CVA6) and coxsackievirus A10 (CVA10) have become the predominant agents of HFMD in mainland China in recent years, replacing enterovirus 71 (EV71) and coxsackievirus A16 (CVA16), although it is unclear if this is consistent throughout China. In this study, samples from 253 HFMD cases were collected in Shenzhen, China, from May 2013 through April 2014 to identify the etiological agent of HFMD. In total, 64.8% (164/253) of HFMD cases were enterovirus positive, in which 81.1% (133/164) were determined to be CVA6. The phylogenetic tree of the partial viral protein 1 sequence showed that the CVA6 isolates were divided into four clusters (Clusters A to D), and cluster D was further divided into four sub-clusters (Clusters D1 to D4). The 133 CVA6 samples isolated in our study were classified into cluster D4, in which the first identified sequence was isolated in Shenzhen in 2008. This study demonstrated that the CVA6 cluster D4, which is predominantly circulating in HFMD in mainland China, may have originated from a local strain identified in 2008 in Shenzhen.
Project description:In this study, we investigated the epidemiology and molecular characteristics of enteroviruses associated with severe hand, foot and mouth disease (HFMD) in Shenzhen, China, during 2014-2018. A total of 137 fecal specimens from patients with severe HFMD were collected. Enterovirus (EV) types were determined using real-time reverse transcription polymerase chain reaction (RT-PCR), RT nested PCR, and sequencing. Sequences were analyzed using bioinformatics programs. Of 137 specimens tested, 97 (70.8%), 12 (8.8%), and 10 (7.3%) were positive for EV-A71, coxsackievirus A6 (CVA6), and CVA16, respectively. Other pathogens detected included CVA2 (2.9%, 4/137), CVA10 (2.9%, 4/137), CVA5 (0.7%, 1/137), echovirus 6 (E6) (0.7%, 1/137) and E18 (0.7%, 1/137). The most frequent complication in patients with proven EV infections was myoclonic jerk, followed by aseptic encephalitis, tachypnea, and vomiting. The frequencies of vomiting and abnormal eye movements were higher in EV-A71-infected patients than that in CVA6-infected or CVA16-infected patients. Molecular phylogeny based on the complete VP1 gene revealed no association between the subgenotype of the virus and disease severity. Nevertheless, 12 significant mutations that were likely to be associated with virulence or the clinical phenotype were observed in the 5'UTR, 2A<sup>pro</sup>, 2C, 3A, 3D<sup>pol</sup> and 3'UTR of CVA6. Eight significant mutations were observed in the 5'UTR, 2B, 3A, 3D<sup>pol</sup> and 3'UTR of CVA16, and 10 significant mutations were observed in the 5'UTR, VP1, 3A and 3C<sup>pro</sup> of CVA10. In conclusion, EV-A71 is still the main pathogen causing severe HFMD, although other EV types can also cause severe complications. Potential virulence or phenotype-associated sites were identified in the genomes of CVA6, CVA16, and CVA10.
Project description:Hand, foot and mouth disease (HFMD), caused by a group of enteric viruses such as Enterovirus 71 (EV71), Coxsackievirus A16 (CVA16) and Coxsackievirus A10 (CVA10), is heavily epidemic in East Asia. This research focused on investigating the occurrence of HFMD pathogens in domestic sewage and secondary effluent before disinfection in a wastewater treatment plant (WWTP) in Xi'an, the largest megacity in northwest China. In order to simultaneously detect all three HFMD pathogens, a semi-nested RT-PCR assay was constructed with a newly designed primer set targeting conservative gene regions from the 5' untranslated region (UTR) to VP2. As a result, 86% of raw sewage samples and 29% of the secondary effluent samples were positive for the HFMD viral gene, indicating that HFMD pathogens were highly prevalent in domestic wastewater and that they could also persist, even with lower probability, in the secondary effluent before disinfection. Of the three HFMD pathogens, CVA10 was positive in 48% of the total samples, while the occurrences of CVA16 and EV71 were 12% and 2%, respectively. It could thus be stated that CVA10 is the main HFMD pathogen prevailing in the study area, at least during the investigation period. High genetic diversity in the conservative gene region among the same serotype of the HFMD pathogen was identified by phylogenetic analysis, implying that this HFMD pathogen replicates frequently among the population excreting the domestic sewage.
Project description:OBJECTIVES:Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) were responsible for 43.3% (235 123/543 243) and 24.8% (134 607/543 243) of all laboratory-confirmed hand, foot and mouth disease (HFMD) cases during 2010-2015 in China. Three monovalent EV71 vaccines have been licensed in China while bivalent EV71/CA16 vaccines are under development. A comparative cost-effectiveness analysis of bivalent EV71/CA16 versus monovalent EV71 vaccination would be useful for informing the additional value of bivalent HFMD vaccines in China. METHODS:We used a static model parameterized with the national HFMD surveillance data during 2010-2013, virological HFMD surveillance records from all 31 provinces in mainland China during 2010-2013 and caregiver survey data of costs and health quality of life during 2012-2013. We estimated the threshold vaccine cost (TVC), defined as the maximum additional cost that could be paid for a cost-effective bivalent EV71/CA16 vaccine over a monovalent EV71 vaccine, as the outcome. The base case analysis was performed from a societal perspective. Several sensitivity analyses were conducted by varying assumptions governing HFMD risk, costs, discounting and vaccine efficacy. RESULTS:In the base case, choosing the bivalent EV71/CA16 over monovalent EV71 vaccination would be cost-effective only if the additional cost of the bivalent EV71/CA16 compared with the monovalent EV71 vaccine is less than €4.7 (95% CI 4.2-5.2). Compared with the TVC in the base case, TVC increased by up to €8.9 if all the test-negative cases were CA16-HFMD; decreased by €1.1 with an annual discount rate of 6% and exclusion of the productivity loss; and increased by €0.14 and €0.3 with every 1% increase in bivalent vaccine efficacy against CA16-HFMD and differential vaccine efficacy against EV71-HFMD, respectively. CONCLUSIONS:Bivalent EV71/CA16 vaccines can be cost-effective compared with monovalent EV71 vaccines, if suitably priced. Our study provides further evidence for determining the optimal use of HFMD vaccines in routine paediatric vaccination programme in China.
Project description:Hand, foot, and mouth disease (HFMD), an enteroviral disease has emerged as a major emerging infection in India. This is caused most commonly by enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) but can also be due to CVA4-10, CVA24, CVB2-5, and echovirus 18 (Echo18). Virological analysis of the cases of HFMD has been infrequently done in India. West Bengal is one of the worst affected states in India.To document the clinical and etiological aspect, the changing patterns and clinic-virological correlation. Method: a total of 62 samples of throat swab were collected from affected children over 3 successive years in Kolkata, West Bengal, India.Five cases had a previous history of HFMD during the last 1-5 years. Fever was usually of mild degree (highest 102°C). There was no apparent correlation between fever of >100°C and a positive test. There was no correlation of viral strain and clinical severity. A test positive for the Viral RNA was noted among 64.51% (40/62) cases. Multiple strains were characteristically present in each year. CVA6, EV71 were found in 2013, CVA6, EV71 in 2014, and CVA6, CVA16 in 2015.Presence of multiple strains explained the frequent occurrence of relapses. We expect this small study will serve as an important document for all future studies on HFMD.