Enterovirus 71 Vaccine Development Research Articles

Development of a high-growth enterovirus 71 vaccine candidate inducing cross-reactive neutralizing antibody responses

Although Enterovirus 71 (EV71) has only one serotype based on serum neutralization tests using hyperimmune animal antisera, three major genogroups (A, B and C) including eleven genotypes (A, B1-B2, and C1-C5) can be well classified based on phylogenetic analysis. Since 1997, large-scale EV71 epidemics occurred cyclically with different genotypes in the Asia-Pacific region. Therefore, development of EV71 vaccines is a national priority in several Asian countries. Currently, five vaccine candidates have been evaluated in clinical trials in China (three C4 candidates), Singapore (one B2 candidate), and Taiwan (one B4 candidate). Overall, the peak viral titers of these 5 vaccine candidates could only reach about 107 TCID50/mL. Moreover, genotypes of these 5 candidates are different from the current predominant genotype B5 in Taiwan and South-Eastern Asia. We adapted a high-growth EV71 genotype B5 (HG-B5) virus after multiple passages and plaque selections in Vero cells and the HG-B5 virus could reach high titers (>108 TCID50/mL) in a microcarrier-based cell culture system. The viral particles were further purified and formulated with alum adjuvant. After two doses of intramuscular immunization in rabbits, the HG-B5 vaccine candidate could induce cross-reactive neutralizing antibodies against the three major EV71 genogroups. In conclusion, a high-growth EV71 virus was successfully adapted in Vero cells and could induce broad spectrum neutralizing antibody titers against three (A, B5, and C4) genotypes in rabbits.

Epidemiologic Features of Enterovirus 71-Associated Hand-Foot-and-Mouth Disease from 2009 to 2013 in Zhejiang, China.

Enterovirus 71 (EV71) usually causes hand-foot-and-mouth disease (HFMD) with severe clinical symptoms and even deaths in China. There is no efficient antiviral drug to protect against severe EV71-associated HFMD, making the development of EV71 vaccines therefore a priority. However, the potential target subject population(s) to be immunized with EV71 vaccine are not well understood. In this study, we characterized the epidemiology regarding EV71-associated HFMD on the basis of provincial-level surveillance. We extracted data on EV71-associated HFMD from the National Notifiable Disease Reporting System in Zhejiang Province, China between 1 January 2009 and 31 December 2013 (n = 7650). The higher incidence rate of EV71 cases occurred in those children aged 12–23 months, with boys being predominant. Interestingly, different peaks activities of EV71 infection was observed in different calendar year, with one peak in 2009 and 2013 and two peaks in 2010–2012. However, EV71 infection seemed to predominately occur in warm season and a distinguished cyclic peak that seemed to be of about 12 months. Children aged 12–23 months are thus identified as an important target population for public health intervention, for example, it is recommended that these key subjects immunized with EV71 vaccine. In addition, an enhanced surveillance system for EV71-associated with HFMD needs to focus on generic and phylogenetic analysis.

The Current Status of the Disease Caused by Enterovirus 71 Infections: Epidemiology, Pathogenesis, Molecular Epidemiology, and Vaccine Development.

Enterovirus 71 (EV71) infections have a major public health impact in the Asia-Pacific region. We reviewed the epidemiology, pathogenesis, and molecular epidemiology of EV71 infection as well as EV71 vaccine development. Previous studies were found using the search terms “enterovirus 71” and “epidemiology” or “pathogenesis” or “molecular epidemiology” or “vaccine” in Medline and PubMed. Articles that were not published in the English language, manuscripts without an abstract, and opinion articles were excluded from the review. The reported epidemiology of cases caused by EV71 infection varied from country to country; seasonal variations in incidence were observed. Most cases of EV71 infection that resulted in hospitalization for complications occurred in children less than five years old. The brainstem was the most likely major target of EV71 infection. The emergence of the EV71 epidemic in the Asia-Pacific region has been associated with the circulation of different genetic lineages (genotypes B3, B4, C1, C2, and C4) that appear to be undergoing rapid evolutionary changes. The relationship between the gene structure of the EV71 virus and the factors that ensure its survival, circulation, and evasion of immunity is still unknown. EV71 infection has emerged as an important global public health problem. Vaccine development, including the development of inactivated whole-virus live attenuated, subviral particles, and DNA vaccines, has been progressing.

Determinants of EV71 immunogenicity and protection against lethal challenge in a mouse model.

Circulating enterovirus 71 (EV71)-associated hand, foot, and mouth disease (HFMD) is a major public health problem in the Asian-Pacific region. An EV71 vaccine for HFMD prevention is currently being developed. However, viral determinants that could influence the vaccine's efficacy have not been well characterized. In this study, we isolated and characterized several EV71 strains that are currently circulating in northern and southern China. We determined that VP1 variation is a major determinant of EV71 immunogenicity. A single amino acid variation in VP1 can lead to significant differences in the breadth and potency of immune responses against primary EV71 isolates as well as the sensitivity of EV71 to heterologous neutralizing antibody responses. We also identified EV71 strains that could induce potent immunogenic and cross-neutralizing antibody responses against diverse EV71 strains. Furthermore, these neutralizing antibodies could protect neonatal mice from lethal dose challenge with various circulating EV71 viruses. Our study provides useful information for EV71 vaccine development and evaluation.

Recombinant tandem multi-linear neutralizing epitopes of human enterovirus 71 elicited protective immunity in mice.

BackgroundHuman Enterovirus 71 (EV71) has emerged as the leading cause of viral encephalitis in children, especially in the Asia-Pacific regions. EV71 vaccine development is of high priority at present, and neutralization antibodies have been documented to play critical roles during in vitro and in vivo protection against EV71 infection.ResultsIn this study, a novel strategy to produce EV71 vaccine candidate based on recombinant multiple tandem linear neutralizing epitopes (mTLNE) was proposed. The three well identified EV71 linear neutralizing epitopes in capsid proteins, VP1-SP55, VP1-SP70 and VP2-SP28, were sequentially linked by a Gly-Ser linker ((G4S)3), and expressed in E.coli in fusion with the Trx and His tag at either terminal. The recombinant protein mTLNE was soluble and could be purified by standard affinity chromatography. Following three dosage of immunization in adult mice, EV71-specific IgG and neutralization antibodies were readily induced by recombinant mTLNE. IgG subtyping demonstrated that lgG1 antibodies dominated the mTLNE-induced humoral immune response. Especially, cytokine profiling in spleen cells from the mTLNE-immunized mice revealed high production of IL-4 and IL-6. Finally, in vivo challenge experiments showed that passive transfer with anti-mTLNE sera conferred full protection against lethal EV71 challenge in neonatal mice.ConclusionOur results demonstrated that this rational designed recombinant mTLNE might have the potential to be further developed as an EV71 vaccine in the future.

Neutralizing Antibodies Induced by Recombinant Virus-Like Particles of Enterovirus 71 Genotype C4 Inhibit Infection at Pre- and Post-attachment Steps

BackgroundEnterovirus 71 (EV71) is a major causative agent of hand, foot and mouth disease, which has been prevalent in Asia–Pacific regions, causing significant morbidity and mortality in young children. Antibodies elicited by experimental EV71 vaccines could neutralize infection in vitro and passively protect animal models from lethal challenge, indicating that neutralizing antibodies play an essential role in protection. However, how neutralizing antibodies inhibit infection in vitro remains unclear.Methods/FindingsIn the present study, we explored the mechanisms of neutralization by antibodies against EV71 virus-like particles (VLPs). Recombinant VLPs of EV71 genotype C4 were produced in insect cells using baculovirus vectors. Immunization with the VLPs elicited a high-titer, EV71-specific antibody response in mice. Anti-VLP mouse sera potently neutralized EV71 infection in vitro. The neutralizing antibodies in the anti-VLP mouse sera were found to target mainly an extremely conserved epitope (FGEHKQEKDLEYGAC) located at the GH loop of the VP1 protein. The neutralizing anti-VLP antisera were able to inhibit virus binding to target cells efficiently. In addition, post-attachment treatment of virus-bound cells with the anti-VLP antisera also neutralized virus infection, although the antibody concentration required was higher than that of the pre-attachment treatment.ConclusionsCollectively, our findings represent a valuable addition to the understanding of mechanisms of EV71 neutralization and have strong implications for EV71 vaccine development.

Progress on the research and development of human enterovirus 71 (EV71) vaccines

Enterovirus 71 (EV71) infections, which can cause severe complications, have become one of the serious public health issues in the Western Pacific region and China. To date, a number of pharmaceutical companies and institutes have initiated the research and development of EV71 vaccines as a countermeasure. As is the case with innovative vaccine development, there are several critical bottlenecks in EV71 vaccine development that must be overcome before the clinical trials, including the selection of vaccine strain, standardization of the procedure for quantifying neutralizing antibody (NTAb) and antigen, establishment and application of a reference standard and biological standards, development of animal models for the evaluation of protective efficacy, and identification of the target patient population. To tackle these technical obstacles, researchers in Mainland of China have conducted a series of studies concerning the screening of vaccine strains and the establishment of criteria, biological standards and detection methods, thereby advancing EV71 vaccine development. This review summarizes recent worldwide progress on the quality control and evaluation of EV71 vaccines.

Antigenic analysis of divergent genotypes human Enterovirus 71 viruses by a panel of neutralizing monoclonal antibodies: Current genotyping of EV71 does not reflect their antigenicity

In recent year, Enterovirus 71 (EV71)-associated hand, foot and mouth disease (HFMD) has become an important public health issue in China. EV71 has been classified into genotypes A, B1–B5 and C1–C5. With such genetic diversity, whether the convalescent or recovery antibody responses can cross-protect infections from other genotypes remains a question. Understanding of the antigenicity of such diverse genetic EV71 isolates is crucial for the EV71 vaccine development. Here, a total of 186 clones anti-EV71 MAbs was generated and characterized with Western blot and cell-based neutralization assay. Forty neutralizing anti-EV71 MAbs were further used to analyze the antigenic properties of 18 recent EV71 isolates representing seven genotypes in neutralization assay. We found that most neutralizing anti-EV71 MAbs are specific to conformational epitopes. We also classified the 40 neutralizing anti-EV71 MAbs into two classes according to their reactivity patterns with 18 EV71 isolates. Class I MAb can neutralize all isolates, suggesting conserved epitopes are present among EV71. Class II MAb includes four subclasses (IIa–IId) and neutralizes only subgroups of EV71 strains. Conversely, 18 EV71 strains were grouped into antigenic types 1 and four antigenic subtypes (2.1–2.4). These results suggest that the current genotyping of EV71 does not reflect their antigenicity which may be important in the selection of EV71 vaccine strains. This panel of neutralizing anti-EV71 MAbs may be useful for the recognition of emerging antigenic variants of EV71 and vaccine development.

Selection and characterization of vaccine strain for Enterovirus 71 vaccine development

Enterovirus 71 (EV71) has recently emerged as an important neurotropic virus in Asia because effective medications and prophylactic vaccine against EV71 infection are not available. Based on the success of inactivated poliovirus vaccine, the Vero cell-based chemically inactivated EV71 vaccine candidate could be developed. Identification of EV71 vaccine strain which can grow to high titer in Vero cell and induce cross-genotype virus neutralizing antibody responses represents the first step in vaccine development. In this report we describe the characterization and validation of a clinical isolate E59 belonging to B4 sub-genotype based on VP1 genetic analysis. Before selected as the vaccine strain, the genetic stability of E59 in passage had been analyzed based on the nucleotide sequences obtained from the Master Virus Seed, Working Seed banks and the virus harvested from the production lots, and found to be identical to those found in the original isolate. These results indicate that E59 vaccine strain has strong genetic stability in passage. Using this vaccine strain the prototype EV71 vaccine candidate was produced from 20L of Vero cell grown in serum-containing medium. The production processes were investigated, characterized and quantified to establish the potential vaccine manufacturing process including the time for virus harvest, the membrane for diafiltration and concentration, the gel-filtration chromatography for the down-stream virus purification, and the methods for viral inactivation. Finally, the inactivated virion vaccine candidate containing sub-microgram of viral proteins formulated with alum adjuvant was found to induce strong virus neutralizing antibody responses in mice and rabbits. Therefore, these results provide valuable information for cell-based EV71 vaccine development.

Purification and Characterization of Enterovirus 71 Viral Particles Produced from Vero Cells Grown in a Serum-Free Microcarrier Bioreactor System

BackgroundEnterovirus 71 (EV71) infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD) and can cause neurological disease during acute infection.Principal FindingIn this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g/L Cytodex 1 microcarrier. The viral titer was >106 TCID50/mL by 6 days post infection when a MOI of 10−5 was used at the initial infection. Two EV71 virus fractions were separated and detected when the harvested EV71 virus concentrate was purified by sucrose gradient zonal ultracentrifugation. The EV71 viral particles detected in the 24–28% sucrose fractions had an icosahedral structure 30–31 nm in diameter and had low viral infectivity and RNA content. Three major viral proteins (VP0, VP1 and VP3) were observed by SDS-PAGE. The EV71 viral particles detected in the fractions containing 35–38% sucrose were 33–35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. The two virus fractions were formalin-inactivated and induced high virus neutralizing antibody responses in mouse immunogenicity studies. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211–225).ConclusionThese results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification.

Identification and characterization of a cross-neutralization epitope of Enterovirus 71

Enterovirus 71 (EV71) infections in children manifest as exanthema and are most commonly known as hand-foot-and-mouth disease (HFMD). Because it can cause severe neurological complications like poliomyelitis, EV71 has now emerged as an important neurotropic virus in Asia. EV71 virus has been shown to consist of 3 (A, B and C) genotypes and many subgenotypes. Although EV71 vaccine development has recently yielded promising preclinical results, yet the correlation between the content of antigen(s) in vaccine candidates and the level of protective antibody responses is not established. The neutralization epitope(s) of EV71 antigens could be used as the surrogate biomarker of vaccine potency. Using peptide ELISA, antisera generated from animals immunized with formalin-inactivated EV71 virion vaccine formulated in alum, EV71-specific neutralizing monoclonal antibody (nMAb) and a panel of 153 overlapping synthetic peptides covering the entire sequences of VP1, VP2 and VP3 of EV71, we screened for immunodominant linear neutralization epitope(s). Synthetic peptide VP2-28, corresponding to residues 136–150 of VP2, was found to bind to and inhibit the binding to EV71 of nMAb MAB979 that was found to have cross-neutralizing activity against different genotypes of EV71 virus. In addition, VP2-28 was found to be recognized only by neutralizing antisera generated from rabbits immunized with the formalin-inactivated whole EV71 virion vaccine but not by antisera from immunized mice and rats. During the epitope mapping, a murine EV71 genotype- and strain-specific linear neutralization epitope VP1–43 was identified within residues 211–220 of VP1. Furthermore, based on sequence alignment and structure prediction analysis using poliovirus as the template for molecular modeling, the VP1-43 and VP2-28 epitopes were shown to run in parallel within 0.1nm and form a rim of the canyon at the junction site of VP1 and VP2 in the viral capsid. In mouse, rat and rabbit immunogenicity studies, a dose-dependent relationship between the number of VP2-28 epitope units measured by a quantitative assay in vaccine preparations and the magnitude of neutralizing titers was demonstrated. VP2-28 has amino acid sequences that are highly conserved among EV71 genotypes, is not affected by formalin-treatment and long-term storage. Thus, VP2-28 could be used as the surrogate biomarker in the potency testing of candidate EV71 vaccines.

The VP1 structural protein of enterovirus 71 interacts with human ornithine decarboxylase and gene trap ankyrin repeat

Enterovirus 71 (EV71) is a major etiological agent of hand, foot and mouth disease (HFMD). Several outbreaks in East Asia were associated with neurological complications and numerous deaths. EV71 possesses four structural proteins VP1–VP4 that are necessary in the formation of the pentameric icosahedral capsid. The viral capsid contributes to virulence, and VP1 is a prime target for EV71 vaccine development. Using yeast two-hybrid analysis, we demonstrated binding affinity between VP1 and three human proteins, i.e. ornithine decarboxylase (ODC1), gene trap ankyrin repeat (GTAR), and KIAA0697 expressed in brain tissue. These interactions were authenticated by co-immunoprecipitation experiments, and by indirect immunofluorescent confocal microscopy of transfected and EV71-infected Vero cells. The significant interaction between VP1 and ODC1 may compromise the latter's activity, and interfere with polyamine biosynthesis, growth and proliferation of EV71-infected cells. The interaction between VP1 and GTAR is noteworthy, since ankyrin proteins are associated with certain neural cell adhesion molecules and with the CRASH neurological syndrome. Given that VP1 is synthesized in large amounts during productive infection, these viral–host protein interactions may provide insights into the role of VP1 in the pathogenesis of EV71 disease and its neurological complications such as acute flaccid paralysis and encephalitis.