Coxsackievirus A 16 infection does not interfere with the specific immune response induced by an enterovirus 71 inactivated vaccine in rhesus monkeys

Hand, foot and mouth disease is usually caused by enterovirus 71 (EV71) and coxsackievirus A 16 (CA16), which are members of the Picornaviridae family. In the present study, the characteristics of the immune response induced by an EV71 inactivated vaccine (made from human diploid cells) were explored in the presence of CA16 infection, based on the previously established neonatal rhesus monkey model. The typical clinical manifestations, including body temperature, viral viremia and virus shedding in the mouth, pharynx and feces, were characterized. A specific neutralizing antibody assay showed that the specific immune response induced by the EV71 inactivated vaccine was active against EV71 but not against CA16. No remarkable fluctuation in proinflammatory cytokine release was identified in the serum of immunized monkeys with EV71 vaccine and CA16 infections subsequently. The results showed that the specific immune response induced by the EV71 inactivated vaccine is effective against EV71 infection but is not affected by CA16 infection.     

Enterovirus type 71 neutralizing antibodies in the serum of macaque monkeys immunized with EV71 virus-like particles

Enterovirus type 71 (EV71) is a virulent form of enteroviruses causing hospitalizations for children less than three years of age. Currently there are no anti-viral therapies or vaccines available for EV71. Due to the high risk of poliomyelitis-like paralysis and fatal encephalitis, an effective vaccine to EV71 could potentially prevent virus-induced morbidity and mortality. In this study, we first tested a potential EV71 vaccine candidate based on virus-like particles (VLP). We vaccinated macaque monkeys to validate the immunogenicity of the VLP vaccine to EV71. We detected the VLP or EV71-specific antibodies, neutralization titers, ELISPOT, and T cell response to find their immune responses to EV71. When the VLP vaccine adjuvanted with alum was given to macaque monkeys, these monkeys developed both specific humoral and cellular immune responses to EV71. Despite lower neutralizing antibodies to EV71 were found in sera of VLP-immunized monkeys than monkeys vaccinated with inactivated EV71, VLP-based vaccine generated a memory immune response to EV71. Hence, VLP-based EV71 vaccine is a potential vaccine against EV71 infection.     

Preclinical evaluation of recombinant HFMD vaccine based on enterovirus 71 (EV71) virus-like particles (VLP): Immunogenicity,efficacy and toxicology

Enterovirus 71 (EV71) is one of the major causative agents for hand, foot and mouth disease (HFMD) in children. Currently, three inactivated EV71 vaccines have been approved by Chinese government. We previously demonstrated that recombinant EV71 virus-like particles (VLP) produced in Pichia pastoris can be produced at a high yield with a simple manufacturing process, and the candidate vaccine elicited protective humoral immune responses in mice. In present study, the nonclinical immunogenicity, efficacy and toxicity of the EV71 vaccine was comprehensively evaluated in rodents and non-human primates. The immunogenicity assessment showed that EV71 VLPs vaccine elicited high and persistent neutralizing antibody responses, which could be comparable with a licensed inactivated vaccine in animals. The immune sera of vaccinated mice also exhibited cross-neutralization activities to the heterologous subtypes of EV71. Both passive and maternal antigen specific antibodies protected the neonatal mice against the lethal EV71 challenge. Furthermore, nonclinical safety assessment of EV71 VLP vaccine showed no signs of systemic toxicity in animals. Therefore, the excellent immunogenicity, efficacy and toxicology data supported further evaluation of the VLP-based EV71 vaccine in humans.     

Intradermal injection of a fractional dose of an inactivated HFMD vaccine elicits similar protective immunity to intramuscular inoculation of a full dose of an Al(OH)3-adjuvanted vaccine

Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are the two major causative agents of hand, foot and mouth disease (HFMD), which erupts in the Asia-Pacific regions. A bivalent vaccine against both EV71 and CVA16 is highly desirable. In the present study, on the bases that an experimental bivalent vaccine comprising of inactivated EV71 and CVA16 induces a balanced protective immunity against both EV71 and CVA16, we compare the immunogenicity and reactogenicity of one fourth of a full dose of an intradermal vaccine administered by needle-free liquid jet injector with a full dose of an intramuscular vaccine administered by needle-syringe in monkeys. The results suggest that intradermal injection of a fractional dose of an inactivated HFMD vaccine elicits similar immunogenicity and reactogenicity to intramuscular inoculation of a full dose of an Al(OH)₃-adjuvanted vaccine, regardless of whether monovalent or bivalent vaccines were used. Our results support the use of an intradermal bivalent vaccine strategy for HFMD vaccination in order to satisfy the requirements and reduce the costs.     

A combination vaccine comprising of inactivated enterovirus 71 and coxsackievirus A16 elicits balanced protective immunity against both viruses

Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two major causative agents of hand, foot and mouth disease (HFMD), which is an infectious disease frequently occurring in children. A bivalent vaccine against both EV71 and CA16 is highly desirable. In the present study, we compare monovalent inactivated EV71, monovalent inactivated CA16, and a combination vaccine candidate comprising of both inactivated EV71 and CA16, for their immunogenicity and in vivo protective efficacy. The two monovalent vaccines were found to elicit serum antibodies that potently neutralized the homologous virus but had no or weak neutralization activity against the heterologous one; in contrast, the bivalent vaccine immunized sera efficiently neutralized both EV71 and CA16. More importantly, passive immunization with the bivalent vaccine protected mice against either EV71 or CA16 lethal infections, whereas the monovalent vaccines only prevented the homologous but not the heterologous challenges. Together, our results demonstrate that the experimental bivalent vaccine comprising of inactivated EV71 and CA16 induces a balanced protective immunity against both EV71 and CA16, and thus provide proof-of-concept for further development of multivalent vaccines for broad protection against HFMD.     

Formaldehyde-inactivated human enterovirus 71 vaccine is compatible for co-immunization with a commercial pentavalent vaccine

In this study we tested the effectiveness of a formaldehyde-inactivated EV71 vaccine and its compatibility for co-immunization with a pentavalent vaccine that contained inactivated poliovirus (PV) vaccine. The inactivated EV71 vaccine (C2 genogroup) elicited an antibody response which broadly neutralized homologous and heterologous genogroups, including B4, C4, and B5. Pups from vaccinated dams were resistant to the EV71 challenge and had a high survival rate and a low tissue viral burden when compared to those from non-vaccinated counterparts. Co-immunization with pentavalent and inactivated EV71 vaccines elicited antibodies against the major components of the pentavalent vaccine including the PV, Bordetella pertussis, Haemophilus influenzae type b, diphtheria toxoid, and tetanus toxoid at the same levels as in mice immunized with pentavalent vaccine alone. Likewise, EV71 neutralizing antibody titers were comparable between EV71-vaccinated mice and mice co-immunized with the two vaccines. These results indicate that formaldehyde-inactivated whole virus EV71 vaccine is feasible for designing multivalent vaccines.     

Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine

Enterovirus type 71 (EV71) and coxsackievirus A 16 (CA16) are recognized as the major pathogens responsible for human hand-foot-mouth disease. To develop a bivalent EV71-CA16 vaccine, rhesus macaques immunized with two doses of this vaccine via the intradermal route were challenged with EV71 or CA16, and their clinical symptoms, viral shedding, neutralizing antibodies, IFN-γ-specific ELISpots, and tissue viral load were examined longitudinally. Specific immunity against EV71 and CA16 was observed in the macaques, which exhibited controlled proliferation of the EV71 and CA16 viruses and upregulated expression of immune-related genes compared with the controls. Furthermore, broad protection against EV71 and CA16 challenge without immunopathological effects was observed in all the immunized macaques. These studies suggest that the bivalent EV71-CA16 inactivated vaccine was effective against wild-type EV71 or CA16 viral challenge in rhesus macaques.     

Comparison of live and inactivated tick-borne encephalitis virus vaccines for safety, immunogenicity and efficacy in rhesus monkeys

Three antigenic chimeric live attenuated tick-borne encephalitis virus (TBEV) vaccine candidates were compared for level of replication in murine and human neuroblastoma cells, for neurovirulence and neuroinvasiveness in mice, and for safety, immunogenicity and efficacy in rhesus monkeys. Two chimeric viruses were generated by replacing the membrane precursor and envelope protein genes of dengue type 4 virus (DEN4) with the corresponding genes of a Far Eastern TBEV, Sofjin strain, in the presence (TBEV/DEN4Delta30) or absence (TBEV/DEN4) of a 30 nucleotide deletion (Delta30) in the 3' noncoding region of the DEN4 part of the chimeric genome. A third chimeric TBEV vaccine candidate was based on the antigenically distant, but naturally attenuated Langat virus (LGT). Chimerization of LGT with DEN4 resulted in decreased neurovirulence and neuroinvasiveness in mice and highly restricted viremia in rhesus monkeys. Also, the LGT/DEN4 chimera was highly restricted in replication in both murine and human neuroblastoma cells. In contrast, TBEV/DEN4 and TBEV/DEN4Delta30 were neither attenuated for neurovirulence in the mice nor restricted in replication in the neuroblastoma cells. However, both were highly attenuated for neuroinvasiveness in mice. TBEV/DEN4 replicated to moderately high titer in rhesus monkeys (mean peak viremia=10(3.1)PFU/ml) indicating that the TBEV/DEN4 chimerization had only a modest, if any, attenuating effect in monkeys. However, the addition of the Delta30 mutation to TBEV/DEN4 greatly attenuated the chimeric virus for rhesus monkeys (mean peak viremia=10(0.7)PFU/ml) and induced a higher level of antibody against the TBEV than did LGT/DEN4. A single dose of either highly attenuated TBEV/DEN4Delta30 or LGT/DEN4 vaccine candidate or three doses of an inactivated TBEV vaccine were efficacious in monkeys against wild-type LGT challenge. These results indicate that both TBEV/DEN4Delta30 and LGT/DEN4 are safe and efficacious in rhesus monkeys and should be further evaluated as vaccine candidates for use in humans.     

High immunogenic enterovirus 71 strain and its production using serum-free microcarrier Vero cell culture

Developing an effective vaccine against enterovirus 71 (EV71) infection provides the best means to control the disease. We have previously reported that large-scale preparation of a low immunogenic EV71 strain can be achieved using serum free microcarrier Vero cell culture in a 2-l bioreactor [Wu SC, Liu CC, Lian WC. Optimization of microcarrier cell culture process for the inactivated enterovirus type 71 vaccine development. Vaccine 2004;22:3858-64]. This present work further investigated the virus growth and the immunogenicity of two high immunogenic strains (EV71-075 and EV71-117) prepared in serum-free microcarrier cell cultures. Our results showed that serum free culture increased cell death rate after infection, reduced the virus specific productivity, but resulted in elicitation of higher neutralizing titers in immunized mice as compared to that parallel obtained in serum-containing cultures. Therefore, serum-free microcarrier culture is a valuable process for developing inactivated EV71 vaccines.     

Similar protective immunity induced by an inactivated enterovirus 71 (EV71) vaccine in neonatal rhesus macaques and children

During the development of enterovirus 71 (EV71) inactivated vaccine for preventing human hand, foot and mouth diseases (HFMD) by EV71 infection, an effective animal model is presumed to be significant and necessary. Our previous study demonstrated that the vesicles in oral regions and limbs potentially associated with viremia, which are the typical manifestations of HFMD, and remarkable pathologic changes were identified in various tissues of neonatal rhesus macaque during EV71 infection. Although an immune response in terms of neutralizing antibody and T cell memory was observed in animals infected by the virus or stimulated by viral antigen, whether such a response could be considered as an indicator to justify the immune response in individuals vaccinated or infected in a pandemic needs to be investigated. Here, a comparative analysis of the neutralizing antibody response and IFN-γ-specific T cell response in vaccinated neonatal rhesus macaques and a human clinical trial with an EV71 inactivated vaccine was performed, and the results showed the identical tendency and increased level of neutralizing antibody and the IFN-γ-specific T cell response stimulated by the EV71 antigen peptide. Importantly, the clinical protective efficacy against virus infection by the elicited immune response in the immunized population compared with the placebo control and the up-modulated gene profile associated with immune activation were similar to those in infected macaques. Further safety verification of this vaccine in neonatal rhesus macaques and children confirmed the potential use of the macaque as a reliable model for the evaluation of an EV71 candidate vaccine.     

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.     

Fibroblast-stimulating lipopeptide-1 as a potential mucosal adjuvant enhances mucosal and systemic immune responses to enterovirus 71 vaccine

To prevent viral infection at the site of entry, mucosal vaccines are potent tools for inducing IgA secretion for defense. Because Toll-like receptor (TLR) ligands serve as strong adjuvants, two ligands that mimic the structure of mycoplasmal and bacterial lipopeptides represent interesting vaccine candidates. Pam3CSK4, a synthetic triacylated lipopeptide, interacts with TLR2/1. Because fibroblast-stimulating lipopeptide-1 (FSL-1), a synthetic diacylated lipopeptide, is recognized by TLR2/6, we targeted the potential immuno-inducibility of Pam3CSK4 and FSL-1 as adjuvants of an enterovirus 71 (EV71) mucosal vaccine. Naïve BALB/c mice were used for intranasal immunization three times over a 3-week interval, with results showing that EV71-specific IgG and IgA in serum, nasal washes, bronchoalveolar lavage fluid, and feces from the EV71 + FSL-1 group were significantly higher than levels observed in mice treated with EV71 + Pam3CSK4, EV71 alone, or the control group treated with phosphate-buffered saline. Furthermore, we observed more EV71-specific IgG and IgA-producing cells in treatments using EV71 formulated with FSL-1. Additionally, T cell-proliferative responses and interferon-γ and interleukin-17 secretion were significantly increased when inactivated EV71 was formulated using FSL-1. Moreover, serum from immunized mice was capable of neutralizing the infectivity of EV71 (C2 genotype) and was able to cross-neutralize the B4 and B5 genotypes of EV71. Our data suggested that FSL-1 could be used as an efficient adjuvant for intranasal EV71-vaccine immunization.     

Immunogenicity and protective efficacy in monkeys of purified inactivated Vero-cell SARS vaccine

BACKGROUND: In 2003, severe acute respiratory syndrome (SARS) resulted in hundreds of infections and deaths globally. We aim to assess immunogenicity and protective efficacy of purified inactivated Vero-cell SARS vaccine in monkeys. METHODS: The cultures of SARS coronavirus (SARS-CoV) BJ-01 strain infected Vero cells were inactivated with beta-propiolactone. Sequential procedures, including ultrafiltration, gel filtration and ion exchange chromatography, were performed to obtain purified inactivated SARS vaccine. The purified SARS vaccine was analyzed with electron microscope, HPLC and Western blotting. We immunized three groups of cynomolgus macaques fascicularis with adjuvant-containing purified vaccine, purified vaccine and unpurified vaccine, respectively, and a fourth group served as a control. Antibody titers were measured by plaque reduction neutralization test. The vaccinated monkeys were challenged with SARS-CoV BJ-01 strain to observe protective efficacy. Additionally, three groups of rhesus monkeys were immunized with different doses of the purified inactivated SARS vaccine (0.5, 1 and 2mug/time/monkey) on days 0 and 7, and the monkeys were challenged with SARS-CoV GZ-01 strain. We assessed the safety of the SARS vaccine and observed whether the antibody dependent enhancement (ADE) occurred under low levels of neutralizing antibody in rhesus. FINDINGS: The purity of SARS vaccine was 97.6% by HPLC identification and reacted with convalescent sera of SARS patients. The purified SARS vaccine induced high levels of neutralizing antibodies and prevented the replication of SARS-CoV in monkeys. Under low levels of neutralizing antibody, no exacerbation of clinical symptoms was observed when the immunized monkeys were challenged with SARS-CoV. In this preliminary animal trial, no side effects were detected when monkeys were immunized with purified SARS vaccine either at normal or large doses. INTERPRETATION: The purified inactivated SARS vaccine could induce high levels of neutralizing antibody, and protect the monkeys from the challenge of SARS-CoV. The SARS vaccine prepared in the study appeared to be safe in monkeys.     

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 20 L 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.     

肠道病毒71型灭活疫苗(Vero细胞)分别与乙肝、乙脑疫苗联合接种的免疫原性研究

目的 评价、探索肠道病毒71型(enterovirus 71,EV71)灭活疫苗与免疫规划疫苗中的重组乙型肝炎疫苗(hepatitis B vaccine,HepB)、乙型脑炎减毒活疫苗(live-attenuated Japanese encephalitis vaccine,LJEV)联合接种的效果及可行性,为EV...     

Optimization of microcarrier cell culture process for the inactivated enterovirus type 71 vaccine development

Enterovirus 71 (EV71) is an enterovirus that could lead to severe neurological disorders and fatalities. The inactivated vaccine is an appropriate EV71 vaccine format for meeting current needs. Large-scale preparation of the inactivated EV71vaccine depends on a scalable cell culture system for industrial mass production. In this paper, Vero cells were found to produce higher titers of EV71 than did MRC-5 and WI-38 cells. High-density microcarrier Vero cell cultures were established using 5g/L Cytodex 1 microcarriers and found to promote the release of EV71s from infected Vero cells. For the large-scale production of the inactivated vaccine antigen, the extracellular virus titers produced in the 2L bioreactor were found to be 10 times lower than the spinner flask culture but improved by 30-folds using glucose/glutamine feedings during infection. A serum-free Vero cell microcarrier culture was also established in the bioreactor, yielding a high-titer of 5.8 x 10(7) TCID50/mL for EV71 production. The immunogenicity of the inactivated virions produced in serum-free culture elicited a slightly higher level of neutralizing antibody response in immunized mice. These results constitute valuable information on the development of a large-scale microcarrier cell culture process for producing inactivated EV71 vaccine.     

Characterization of a Vero cell-adapted virulent strain of enterovirus 71 suitable for use as a vaccine candidate

Enterovirus 71 (EV71) is a neurotrophic virus that causes seasonal morbidity and mortality in children throughout the world with increasing frequency in recent years. Because of the lack of an effective antiviral agent, primary prevention, including the development of effective vaccines, is a top priority in terms of control strategies. Poliovirus vaccine technology, both live attenuated and inactivated, killed virus vaccines, can be adopted for use with EV71 because of their relatedness. In this study, we have characterized a laboratory-adapted EV71 strain, YN3-4a, which exhibits different characteristics from those of its parent isolate, neu, in having a rapid growth rate in Vero cells, a larger plaque size, and a lower LD(50) in newborn mice. The YN3-4a can be produced at a high viral titer of up to 10(10) tissue culture infective dose (TCID(50)) when grown in Vero cells, an approved substrate for virus vaccine production. Mouse antiserum raised against YN3-4a can neutralize a broad range of strains of EV71 isolated at different times from a variety of geographic regions. On passage in Vero cells, YN3-4a remained genetically and phenotypically stable. Many of the above-described features, such as high viral yield, strong immunogenicity, broad-based antigenic coverage, and passage stability, are desirable features in a prototype virus for the development of an inactivated viral vaccine.     

Evaluation of Japanese encephalitis virus DNA vaccine candidates in rhesus monkeys [Macaca mulatta

We have previously described DNA vaccine candidates against Japanese encephalitis virus (JEV) that were immunogenic in mice. Present study was conducted to evaluate their immunogenicity in rhesus monkeys (Macaca mulatta) and compare it with the commercial mouse brain-derived, formalin-inactivated vaccine. Groups of four monkeys were immunized with either pMEa (expressing the anchored form of the envelope protein along with the pre-membrane protein of JEV) or pMEs (expressing the secretory form of the envelope protein along with pre-membrane protein of JEV) by intra-muscular (IM, using needle) or intra-dermal (ID, using gene gun) routes. Following primary immunization with 1mg plasmid DNA given IM, or 5 microg plasmid DNA given ID, the monkeys were boosted after 1 and 2 months with 0.5mg DNA given IM or 5 microg DNA given ID, and observed for a period of 6 months. After the second booster, most of the monkeys sero-converted and developed JEV neutralizing antibodies, albeit of low titer. Importantly however, following a sham challenge with the mouse brain-derived inactivated JEV vaccine given 6 months after immunization, the neutralizing antibody titers rose rapidly indicating a vigorous anamnestic response. Based on the JEV neutralizing antibody response following the vaccination and the extent of anamnestic response generated in the immunized monkeys, plasmid pMEa was superior to pMEs. This study indicates that the JEV candidate DNA vaccine is capable of generating protective levels of JEV neutralizing antibodies in rhesus monkeys and prime the immune system effectively against a subsequent exposure to JEV.     

Efficacy of an inactivated bivalent vaccine for enterovirus 71 and coxsackievirus A16 in mice immunized intradermally

Human hand, foot, and mouth disease (HFMD), an important infectious disease in children, is caused mainly by enterovirus 71 (EV71) and coxsackievirus A16 (CA16). In this study, a bivalent inactivated EV71/CA16 vaccine is developed and evaluated in immunized BALB/c mice injected through the intradermal route. Q-RT-PCR detection of the mRNA of immune signal molecules in local epithelial tissues inoculated with the vaccine indicates activation of innate immunity, which includes upregulation of immune-related chemokines, interferons and CD molecules. Further, the finding that neutralizing antibodies and specific T cellular responses were elicited in adult mice after two immunizations with the vaccine at a 28-day interval, which endowed offspring mice to defend a viral challenge, suggests the successful induction of specific protective antiviral immunity. All these data suggest that immunization with this bivalent EV71/CA16 vaccine via the intradermal route elicits effective immunity against EV71 and CA16 infection.     

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 0.1 nm 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.