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Childhood exanthema caused by different serotypes of coxsackievirus (CV-A) and enterovirus A71 (EV-A71) has become a serious global health problem; it is commonly known as hand, foot, and mouth disease (HFMD). Current EV-A71 vaccine clinical trials have demonstrated that human antibody responses generated by EV-A71 vaccinations do not cross-neutralize coxsackievirus A16 (CV-A16). An effective multivalent HFMD vaccine is urgently needed. From molecular epidemiological studies in Southeast Asia, CV-A6 and CV-A10 are commonly found in HFMD outbreaks. In this study, CV-A6 and CV-A10 were individually cultured in rhabdomyosarcoma (RD) cells grown in medium containing serum, harvested and concentrated. In viral downstream purification, two viral fractions were separated by sucrose gradient zonal ultracentrifugation and detected using a SDS-PAGE analysis and a virus infectivity assay. These two viral fractions were formalin-inactivated, and only the infectious particle fraction was found to be capable of inducing CV-A serotype-specific neutralizing antibody responses in animal immunogenicity studies. These mouse and rabbit antisera also failed to cross-neutralize EV-A71 and CV-A16 infections. Only a combination of formalin-inactivated EV-A71, CV-A6, CV-A10 and CV-A16 multivalent vaccine candidates elicited cross-neutralizing antibody responses in both mouse and rabbit immunogenicity studies. The current results certainly provide important information for multivalent HFMD vaccine development.

Hand, foot and mouth diseases (HFMD) are mainly caused by Enterovirus A71 (EV-A71) infections. Clinical trials in Asia conducted with formalin-inactivated EV-A71 vaccine candidates produced from serum-free Vero cell culture using either roller bottle or cell factory technology, are found to be safe and highly efficacious. To increase vaccine yields and reduce the production costs, the bioprocess improvement for EV-A71 vaccine manufacturing is currently being investigated. The parameters that could affect and enhance the production yields of EV-A71 virus growth in the microcarrier bioreactor were investigated. The medium replacement culture strategy included a multi-harvested semi-batch process and perfusion technology and was found to increase the production yields more than 7-14 folds. Based on the western blot and cryo-EM analyses of the EV-A71 virus particles produced from either the multi-harvested semi-batch (MHSBC) or perfusion cultures were found to be similar to those virus particles obtained from the single batch culture. Mouse immunogenicity studies indicate that the EV-A71 vaccine candidates produced from the perfusion culture have similar potency to those obtained from single batch bioprocess. The physical structures of the EV-A71 particles revealed by the cryo-EM analysis were found to be spherical capsid particles. These results provide feasible technical bioprocesses for increasing virus yields and the scale up of EV-A71 vaccine manufacturing using the bioreactor cell culture methods.

Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are major causative agents of hand, foot, and mouth diseases (HFMDs), and EV71 is now recognized as an emerging neurotropic virus in Asia. Effective medications and/or prophylactic vaccines against HFMD are not available. The current results from mouse immunogenicity studies using in-house standardized RD cell virus neutralization assays indicate that (1) VP1 peptide (residues 211–225) formulated with Freund’s adjuvant (CFA/IFA) elicited low virus neutralizing antibody response (1/32 titer); (2) recombinant virus-like particles produced from baculovirus formulated with CFA/IFA could elicit good virus neutralization titer (1/160); (3) individual recombinant EV71 antigens (VP1, VP2, and VP3) formulated with CFA/IFA, only VP1 elicited antibody response with 1/128 virus neutralization titer; and (4) the formalin-inactivated EV71 formulated in alum elicited antibodies that cross-neutralized different EV71 genotypes (1/640), but failed to neutralize CVA16. In contrast, rabbits antisera could cross-neutralize strongly against different genotypes of EV71 but weakly against CVA16, with average titers 1/6400 and 1/32, respectively. The VP1 amino acid sequence dissimilarity between CVA16 and EV71 could partially explain why mouse antibodies failed to cross-neutralize CVA16. Therefore, the best formulation for producing cost-effective HFMD vaccine is a combination of formalin-inactivated EV71 and CAV16 virions.

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 pep- tide 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 over- lapping 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 pre- diction 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.

The maltooligosyltrehalose trehalohydrolase (MTHase) mainly cleaves the alpha-1,4-glucosidic linkage next to the alpha-1,1-linked terminal disaccharide of maltooligosyltrehalose to produce trehalose and the maltooligosaccharide with lower molecular mass. In this study, the treZ gene encoding MTHase was PCR-cloned from Sulfolobus solfataricus ATCC 35092 and then expressed in Escherichia coli. A high yield of the active wild-type MTHase, 13300 units/g of wet cells, was obtained in the absence of IPTG induction. Wild-type MTHase was purified sequentially using heat treatment, nucleic acid precipitation, and ion-exchange chromatography. The purified wild-type MTHase showed an apparent optimal pH of 5 and an optimal temperature at 85 degrees C. The enzyme was stable at pH values ranging from 3.5 to 11, and the activity was fully retained after a 2-h incubation at 45-85 degrees C. The k(cat) values of the enzyme for hydrolysis of maltooligosyltrehaloses with degree of polymerization (DP) 4-7 were 193, 1030, 1190, and 1230 s(-1), respectively, whereas the k(cat) values for glucose formation during hydrolysis of DP 4-7 maltooligosaccharides were 5.49, 17.7, 18.2, and 6.01 s(-1), respectively. The K(M) values of the enzyme for hydrolysis of DP 4-7 maltooligosyltrehaloses and those for maltooligosaccharides are similar at the same corresponding DPs. These results suggest that this MTHase could be used to produce trehalose at high temperatures.