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Virions produced from cell culture is the primary source for production of formalin-inactivated whole virus vaccines for enteroviruses. EV-A71 particles produced from culture system comprise two major types, the immature/empty (E)-particle and the mature/full (F)-particle, which both exhibit low isoelectric point (pI) values but have distinct differences in infectivity and immunogenicity. Although EV-A71 particles can conventionally be separated into E-particle and F-particle using sucrose gradient ultracentrifugation, this procedure is cumbersome and difficult to put into practice for vaccine production. Methods based on ion-exchange chromatography have been exploited to improve the purification efficacy; however, none of them are capable of separating the E- and F-particles efficiently. In this study, we aimed to develop an approach to isolate and purify the highly immunogenic mature EV-A71 particles. By applying a step gradient elution procedure, we successfully isolated the viral structure protein VP0-cleaved particles of EV-A71 from a mixture of cultured viral solution using the Q-membrane anion-exchange chromatography. The elution started with 0.1x phosphate buffered saline (PBS) solution while increasing the percentage of 1x PBS containing 1M NaCl in sequential steps. By this procedure, the VP0-cleaved mature particles and VP0-uncleaved immature particles of EV-A71 could be separated into different fractions in Q-membrane with gradually increased NaCl concentration in elution buffer. The purified VP0-cleaved particles were shown to have characteristics equivalent to those of the highly infectious F-particles of EV-A71. The overall recovery rate for the mature EV-A71 particles by Q-membrane is 56% and its purity was shown to be equivalent to those isolated by the sucrose gradient ultracentrifugation. Our approach provides a simple and efficient purification method for recovering mature, highly infectious virus particles from the EV-A71 culture bulk.

Hand, foot, and mouth disease (HFMD) poses a great threat to young children in the Asia-Pacific region. HFMD is usually caused by enterovirus A, and infection with enterovirus A71 (EV-A71) is particularly associated with severe complications. However, coxsackievirus CV-A16, CV-A6, and CV-A10 pandemics have been observed in recent HFMD outbreaks. Inactivated monovalent EV-A71 vaccines are available to prevent EV-A71 infection; however, they cannot prevent infections by non-EV-A71 enteroviruses. Anti-enteroviral drugs are still in the developmental stage. Application of novel strategies will facilitate the development of new therapies against these emerging HFMD-associated enteroviruses.The authors highlight the current approaches for anti-enterovirus therapeutic development and discuss the application of these novel strategies for the discovery of vaccines and antiviral drugs for enteroviruses.The maturation of DNA/RNA vaccine technology could be applied for rapid and robust development of multivalent enterovirus vaccines. Structure biology and neutralization antibody studies decipher the immunodominant sites of enteroviruses for vaccine design. Nucleotide aptamer library screening is a novel, fast, and cost-effective strategy for the development of antiviral agents. Animal models carrying viral receptors and attachment factors are required for enterovirus study and vaccine/antiviral development. Currently developed antivirals require effectiveness evaluation in clinical trials.

The novel H7N9 avian influenza A virus has caused human infections in China since 2013; some isolates from the fifth wave of infections have emerged as highly pathogenic avian influenza viruses. Recombinant hemagglutinin proteins of H7N9 viruses can be rapidly and efficiently produced with low-level biocontainment facilities. In this study, recombinant H7 antigen was obtained from engineered stable clones of Chinese Hamster Ovary (CHO) cells for subsequent large-scale production. The stable CHO cell clones were also adapted to grow in serum-free suspension cultures. To improve the immunogenicity of the recombinant H7 antigens, we evaluated the use of a novel combination adjuvant of PELC and CpG (PELC/CpG) to augment the anti-H7N9 immune responses in mice. We compared the effects with other adjuvants such as alum, AddaVax (MF59-like), and several Toll-like receptor ligands such as R848, CpG, and poly (I:C). With the PELC/CpG combination adjuvant, CHO cell-expressed rH7 antigens containing terminally sialylated complex type N-glycans were able to induce high titers of neutralizing antibodies in sera and conferred protection following live virus challenges. These data indicate that the CHO cell-expressed recombinant H7 antigens and a PELC/CpG combination adjuvant can be used for H7N9 subunit vaccine development.

Virus-like particle (VLP) technology is an attractive platform for the development of seasonal and pandemic influenza vaccines. Influenza VLPs can be obtained by the overexpression of HA, M1, NA, and/or M2 viral proteins in insect, mammalian, or plant cells. In this study, we reported to obtain highly immunogenic influenza VLPs by molecular incorporation with B-cell-activating factor (BAFF) or proliferation-inducing ligand (APRIL). Since BAFF and APRIL act as homotrimers to interact with their receptors, we engineered the VLPs by direct fusion of BAFF or APRIL to the transmembrane anchored domain of H5HA gene. Results showed that immunizations with the HA-transmembrane anchored BAFF- or APRIL-VLPs only formulated in alum but not MPL adjuvant elicited significantly higher IgG titers in sera. However, only the BAFF-VLPs formulated in alum adjuvant elicited more broadly neutralizing antibodies against the homologous and two heterologous H5N1 clade/subclade viruses and conferred protective immunity against live virus challenges. As the multi-subtype influenza vaccines containing a variety of HA subtypes can confer broader protective immunity, we also obtained multi-subtype H5H7 BAFF-VLPs and H1H5H7 BAFF-VLPs and demonstrated that these multi-subtype BAFF-VLPs were able to induce the production of neutralizing antibodies against multiple HA subtypes. Our findings provided useful information for the development of highly immunogenic, multi-subtype influenza VLP vaccines.

Virus-like particle (VLP) technology is an alternative platform for developing vaccines to combat seasonal and pandemic influenza. Influenza VLPs are non-infectious nanoparticles that can elicit effective vaccine immunogenicity in hosts. B-cell-activating factor (BAFF, or BLyS) and a proliferation-inducing ligand (APRIL) are members of the tumor necrosis factor (TNF) superfamily of cytokines. Both BAFF and APRIL are homotrimers that interact with homotrimeric receptors. Here, we report a method of the production of influenza VLPs by molecular incorporation with BAFF or APRIL homotrimers to interact with their receptors. We engineered the VLPs by direct fusion of BAFF or APRIL to the transmembrane anchored domain of the hemagglutinin (HA) gene. We also describe procedures for the production of BAFF-VLPs containing H5H7 and H1H5H7 for multi-subtype vaccine development.

We evaluated the efficacy of a recombinant adenovirus that expresses a membrane-truncated respiratory syncytial virus (RSV) fusion protein (Ad-F0ΔTM) in newborns via maternal immunization (MI) of pregnant cotton rats. Intranasal Ad-F0ΔTM immunization was given to pregnant female rats, and MI-newborn rats were then challenged intranasally with RSV. Anti-RSV IgGs were observed in the serum of MI-newborn rats after birth. The pulmonary viral loads in Ad-F0ΔTM vs. control vector, Ad-LacZ, and MI-newborns on day 3 post-challenge were reduced by 4 log 10 /g lung. The neutralizing antibody remained for up to 3 weeks in the serum of MI-newborns, which is when weaning began. Ad-F0ΔTM protected MI-newborns from RSV challenge for 1 week. Vertical-transferred protective antibodies were examined in the breast milk and placenta as well. Finally, anti-RSV immunity was not boosted but was only primed during the next RSV exposure in Ad-F0ΔTM-MI-newborns. Maternal Ad-F0ΔTM immunization provides acute protection against RSV infection in neonates.

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.

Enterovirus 71 (EV71) is a major cause of hand, foot and mouth disease (HFMD). The current EV71 propagating in Vero (EV-V) or sub-passaged in RD (EV-R) cells was used as a pathogen. Interestingly, EV-R exhibited differential virulence; challenging human scavenger receptor class B2-expressing (hSCARB2-Tg) mice with EV71 revealed that EV-V was more virulent than EV-R: 100% of mice that received lethal amounts of EV-V died, while all the mice that received EV-R survived. Severe pathogenesis correlated with viral burdens and proinflammatory cytokine levels were observed in EV-V-challenged mice, but controversy in EV-R-challenged mice. Consensus sequence analysis revealed EV-R rapidly acquired complete mutations at E145G and S241L and partial mutations at V146I of VP1, and acquired a T to C substitution at nucleotide 494 of the 5'-UTR. EV-R exhibited higher binding affinity for another EV71 receptor, human P-selectin glycoprotein ligand-1 (hPSGL-1), than EV-V. Both EV71s exhibited no significant difference in binding to hSCARB2. The molecular modelling indicate that these mutations might influence EV71 engagement with PSGL-1 and in vivo virulence.

Enterovirus 71 (EV71) is a major cause of hand, foot and mouth disease (HFMD). The current EV71 propagating in Vero (EV-V) or sub-passaged in RD (EV-R) cells was used as a pathogen. Interestingly, EV-R exhibited differential virulence; challenging human scavenger receptor class B2-expressing (hSCARB2-Tg) mice with EV71 revealed that EV-V was more virulent than EV-R: 100% of mice that received lethal amounts of EV-V died, while all the mice that received EV-R survived. Severe pathogenesis correlated with viral burdens and proinflammatory cytokine levels were observed in EV-V-challenged mice, but controversy in EV-R-challenged mice. Consensus sequence analysis revealed EV-R rapidly acquired complete mutations at E145G and S241L and partial mutations at V146I of VP1, and acquired a T to C substitution at nucleotide 494 of the 5′-UTR. EV-R exhibited higher binding affinity for another EV71 receptor, human P-selectin glycoprotein ligand-1 (hPSGL-1), than EV-V. Both EV71s exhibited no significant difference in binding to hSCARB2. The molecular modelling indicate that these mutations might influence EV71 engagement with PSGL-1 and in vivo virulence.

Refocusing of B-cell responses can be achieved by preserving the overall fold of the antigen structure but selectively mutating the undesired antigenic sites with additional N-linked glycosylation motifs for glycan masking the vaccine antigen. We previously reported that glycan-masking recombinant H5 hemagglutinin (rH5HA) antigens on residues 83, 127, and 138 (g127 + g138 or g83 + g127 + 138 rH5HA) elicited broader neutralizing antibodies and protection against heterologous clades/subclades of high pathogenic avian influenza H5N1 viruses. In this study, we engineered the stably expressing Chinese hamster ovary (CHO) cell clones for producing the glycan-masking g127 + g138 and g83 + g127 + g138 rH5HA antigens. All of these glycan-masking rH5HA antigens produced in stable CHO cell clones were found to be mostly oligomeric structures. Only the immunization with the glycan-masking g127 + g138 but not g83 + g127 + g138 rH5HA antigens elicited more potent neutralizing antibody titers against four out of five heterologous clades/subclades of H5N1 viral strains. The increased neutralizing antibody titers against these heterologous viral strains were correlated with the increased amounts of stem-binding antibodies, only the glycan-masking g127 + g138 rH5HA antigens can translate into more protection against live viral challenges. The stable CHO cell line-produced glycan-masking g127 + g138 rH5HA can be used for H5N1 subunit vaccine development.

Enterovirus 71 (EV71), one of the major causative agents of hand, foot, and mouth disease (HFMD), is now recognized as an emerging neurotropic virus in Asia and may cause severe neurologic complications and mortalities. Laboratory diagnosis of EV71 infection must be efficient and accurate, which could be accomplished by various immunoassays. In this study we use a live EV71 isolate, Tainan/4643/98, with genotype C2 as an immunogen to sensitize BALB/c (H-2(d)) mice and then generate the EV71-specific murine monoclonal antibodies. Five hybridoma clones were established and their monoclonal antibodies were characterized. All five clones are applicable in immunofluorescence staining but with different sensitivities-that is, MAbs 22, 24, and 27 were sensitive in IFA detection, and MAbs 22 and 24 were also confirmed in flow cytometry. None of these cross-reacted with coxsackievirus A16 (CVA16) or Echovirus type 6 (ECHO6), but each varied in binding to different EV71 subgenogroups (B1, B4, B5, C2, and C4). Western blot analysis revealed that all of these MAbs reacted with EV71 VP1 capsid proteins, and in addition MAbs 22 and 24 exhibited potent neutralizing activities against EV71 and protected cells from infection. Further, mapping the epitopes for each MAb revealed that only MAb 27 showed positive for the linear epitope DVIESSIGDSVSRAL, which was located at the N-terminus (a.a. 6-20) of EV71 VP1 and highly conserved among all EV71 subgenotypes. Thus, these MAbs may provide valuable tools for the laboratory diagnosis of EV71 infection and for vaccine development.

Enterovirus 71 (EV71) is now recognized as an emerging neurotropic virus in Asia and with Coxsackie virus (CV) it is the other major causative agent of hand-foot-mouth diseases (HFMD). Effective medications and/or prophylactic vaccines against HFMD are urgently needed. From a scientific (the feasibility of bioprocess, immunological responses and potency in animal challenge model) and business development (cost of goods) points of view, we in this review address and discuss the pros and cons of different EV71 vaccine candidates that have been produced and evaluated in animal models. Epitope-based synthetic peptide vaccine candidates containing residues 211–225 of VP1 formulated with Freund’s adjuvant (CFA/IFA) elicited low EV71 virus neutralizing antibody responses, but were protective in the suckling mouse challenge model. Among recombinant EV71 subunits (rVP1, rVP2 and rVP3) expressed in E. coli, purified and formulated with CFA/IFA, only VP1 elicited mouse antibody responses with measurable EV71-specific virus neutralization titers. Immunization of mice with either a DNA plasmid containing VP1 gene or VP1 expressed in Salmonella typhimurium also generated neutralizing antibody responses and protected animals against a live EV71 challenge. Recombinant EV71 virus-like particles (rVLP) produced from baculovirus formulated either with CFA/IFA or alum elicited good virus neutralization titers in both mice and non-human primates, and were found to be protective in the suckling mouse EV71 challenge model. Synthetic peptides or recombinant EV71 subunit vaccines (rVP1 and rVLP) formulated in alum were found to be poorly immunogenic in rabbits. Only formalin-inactivated (FI) EV71 virions formulated in alum elicited cross-neutralizing antibodies against different EV71 genotypes in mice, rabbits and non-human primates but induced weak neutralizing responses against CAV16. From a regulatory, economic and market acceptability standpoint, FI-EV71 virion vaccines are the most promising candidates and are currently being evaluated in human clinical trials. We further describe and analyze some new bioprocesses technologies that have great potential applications in EV71 vaccine development. This review also demonstrates the opportunities and challenges that the Asian vaccine industry faces today.

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 A71 (EV-A71) is responsible for epidemics of hand, foot and mouth disease (HFMD) in young children. To circumvent difficulties in obtaining clinical enterovirus isolates that might be contaminated with other viruses, a platform technology was developed to quickly generate vaccine virus strains based on the published enterovirus genomic sequences. A recombinant plasmid containing the full-length infectious cDNA clone of EV-A71 vaccine strain E59 was directly generated after transfecting the recombinant plasmid into Vero, RD or HEK293A cells, and phenotypic characteristics similar to the parental strain were observed. The cDNA-derived infectious EV-A71 virus grown in Vero cells produced relatively stable virus titers in both T-flasks and microcarrier culture systems. To evaluate the genetic stability of the cDNA-derived EV-A71 viruses, the immunodominant structural proteins, VP1 and VP2, of the recombinant EV-A71 viruses were sequenced and analyzed. The cDNA-derived EV-A71 virus showed weak pathogenicity in a human SCARB2 mouse model. These results show the successful generation of a recombinant virus derived from a published viral genomic sequence that demonstrated good genetic stability and viral yields, which could represent an efficient and safe vaccine strain for cGMP-grade manufacturing.

Virus-like particle (VLP) technology is an attractive platform for seasonal and pandemic influenza vaccine development. We previously showed that influenza VLPs can be modified using M2 fusion with molecular adjuvants such as Salmonella typhimurium flagellin (FliC) to enhance VLP immunogenicity. For this study, three types of chimeric VLPs were incorporated with FliC, granulocyte-macrophage colony-stimulating factor (GM-CSF), or both GM-CSF and FliC (GM-CSF/FliC) to enhance anti-influenza immunogenicity. Our results indicate that immunizations with the chimeric FliC VLPs and GM-CSF/FliC H5N1 VLPs elicited more potent and broadly neutralizing antibodies and neuraminidase-inhibiting antibodies in sera, and induced higher numbers of hemagglutinin-specific antibody-secreting cells and germinal center B cell subsets in splenoctyes. Immunization with the chimeric GM-CSF H5N1 VLPs induced stronger Th1 and Th2 cellular responses. The chimeric GM-CSF/FliC H5N1 VLP constructs were further obtained to include H7 or H1H7 bi- or tri-subtype. It is our hope that these findings provide useful information for developing multi-subtype influenza vaccines.

We have developed an efficient cell culture process to scale up the production of a recombinant adenovirus that expresses the membrane-trunked fusion protein of respiratory syncytial virus (RSV; Ad-F0ΔTM). Adherent cells of human embryonic kidney (HEK) 293-derived cell, 293A, which supports the production of E1/E3-deleted Ad-F0ΔTM when cultured in the presence of fetal bovine serum (FBS), were adapted to suspension growth under serum-free medium. In doing so, we studied the immunogenicity of Ad-F0ΔTMsus, which propagated in a bioreactor that was cultured with serum-free suspension of 293A, in comparison with Ad-F0ΔTMadh, which was produced from parental 293A cells that were adherently cultured in medium containing FBS. The size and morphology of Ad-F0ΔTMsus and Ad-F0ΔTMadh virions were identical upon inspection with electron microscopy. The results showed that anti-F IgG and RSV-neutralizing titer were raised in the serum of both mice that were intranasally immunized twice with Ad-F0ΔTMsus or Ad-F0ΔTMadh at two-week injection intervals. Furthermore, the immune responses persisted for six months after vaccination. Activation of F protein-specific CD8(+) T cell's epitope associated IFN-ɣ and IL-4 was induced in both Ad-F0ΔTMsus- and Ad-F0ΔTMadh, but not in Ad-LacZsus, -immunized mouse splenocytes. No vaccine-enhanced lung inflammation, airway mucus occlusion or eosinophils infiltration were observed in Ad-immunized mice followed by RSV challenge; however, these symptoms were observed following immunization with formalin-inactivated RSV vaccine. These results indicate that the safety and potency of Ad-F0ΔTM produced from either adherent cells or suspension and serum-free cells are the same.

A live enterovirus 71 (EV71) isolate designated, EV71/E59, with genotype B4 produced in Vero cells and purified over a sucrose gradient was used as the immunogen to generate EV71-specific murine monoclonal antibodies. Four hybridoma clones derived from the fusion of splenocytes of EV71/E59-preimmunized BALB/c (H-2(d)) mice and the NS-1 myeloma cells that exhibit stable growth were selected for detailed characterization. The proof that the hybridomas produced are indeed true independent clones was based on the obervations that they expressed different complementarity-determining regions (CDRs) in their κ light chain genes. Purified ascitic fluids produced by the individual clones reacted against the viral capsid protein, VP1, in Western blot; and recognized distinct sites of a common epitope localized at the C-terminal half of VP1. Each of the monoclonal antibodies exhibited potent neutralizing activities against the immunizing virus strain, as well as two other isolates namely, N0781-TW-01, and N2838, of subgenogroups B4 and B5, respectively, that were found commonly in recent outbreaks in Taiwan. It was also observed the monoclonal antibodies acted cooperatively in neutralizing the EV71/E59 virus.

The Fe-B/FePt-type nanocomposite ribbons with strong exchange-coupling and high permanent magnetic properties have been developed by the melt-spinning method. For ternary (Fe 0.5+y Pt 0.5-y ) z B 100-z (y=0–0.2; z=82 and 84), multiple phases, namely γ 1 , Fe 2 B and Fe 3 B, with nanoscaled grain sizes (25–35 nm) can readily form in ribbons after an isothermal annealing at 500 °C. Strong exchange-coupling between grains was found in these ribbons, giving rise to an enhancement of not only the remanence but also the maximum energy product. An i H c of more than 12 kOe has been obtained in (Fe 0.6 Pt 0.4 ) 82 B 18 (B r = 6.6 kG, (BH) max =8.9 MGOe), and a B r of 9.4 kG, ft of 7.5 kOe and (BH) max of 14.0 MGOe can be achieved in (Fe 0.675 Pt 0.325 ) 84 B 16 . Magnetically soft Fe 2 B and Fe 3 B were found to present in the matrix, as evidenced by high resolution transmission electron microscopy. Because of the higher saturation magnetization of Fe 3 B than Fe 2 B, the coexistence of Fe 3 B with Fe 2 B and γ 1 phases was believed the main cause to result in the improved remanence and magnetic energy product of the (Fe 0.675 Pt 0.325 ) 84 B 16 ribbons.

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.

Enterovirus 71 (EV71) and coxsackieviruses are the major causative agents of hand, foot, and mouth disease (HFMD) outbreaks worldwide and have a significant socioeconomic impact, particularly in Asia. Formalin-inactivated (FI) EV71 vaccines evaluated in human clinical trials in China, Taiwan, and Singapore were found to be safe and to elicit strong neutralizing antibody responses against EV71 currently circulating in Asia. The results from 3 different phase 3 clinical trials performed in young children (6-60 months) indicate that the efficacy of FI-EV71 vaccines is >90% against EV71-related HFMDs and >80% against EV71-associated serious diseases, but the vaccines did not protect against coxsackievirus A16 infections. Here we discuss the critical factors affecting EV71 vaccine product registration, including clinical epidemiology, antigenic shift issues in cross-protection and vaccine strain selection, standardized animal models for potency testing, and cost-effective manufacturing processes for potential incorporation of FI-EV71 vaccine into Expanded Programme on Immunization vaccines.

Enterovirus 71 (EV71) and coxsackieviruses (CV) are the major causative agents of hand, foot and mouth disease (HFMD). There is not currently a vaccine available against HFMD, even though a newly developed formalin-inactivated EV71 (FI-EV71) vaccine has been tested in clinical trial and has shown efficacy against EV71. We have designed and genetically engineered a recombinant adenovirus Ad-EVVLP with the EV71 P1 and 3CD genes inserted into the E1/E3-deleted adenoviral genome. Ad-EVVLP were produced in HEK-293A cells. In addition to Ad-EVVLP particles, virus-like particles (VLPs) formed from the physical association of EV71 capsid proteins, VP0, VP1, and VP3 expressed from P1 gene products. They were digested by 3CD protease and confirmed to be produced by Ad-EVVLP-producing cells, as determined using transmission electron microscopy and western blotting. Mouse immunogenicity studies showed that Ad-EVVLP-immunized antisera neutralized the EV71 B4 and C2 genotypes. Activation of VLP-specific CD4+ and CD8+/IFN-γ T cells associated with Th1/Th2-balanced IFN-ɣ, IL-17, IL-4, and IL-13 was induced; in contrast, FI-EV71 induced only Th2-mediated neutralizing antibody against EV71 and low VLP-specific CD4+ and CD8+ T cell responses. The antiviral immunity against EV71 was clearly demonstrated in mice vaccinated with Ad-EVVLP in a hSCARB2 transgenic (hSCARB2-Tg) mouse challenge model. Ad-EVVLP-vaccinated mice were 100% protected and demonstrated reduced viral load in both the CNS and muscle tissues. Ad-EVVLP successfully induced anti-CVA16 immunities. Although antisera had no neutralizing activity against CVA16, the 3C-specific CD4+ and CD8+/IFN-γ T cells were identified, which could mediate protection against CVA16 challenge. FI-EV71 did not induce 3C-mediated immunity and had no efficacy against the CVA16 challenge. These results suggest that Ad-EVVLP can enhance neutralizing antibody and protective cellular immune responses to prevent EV71 infection and cellular immune responses against CV infection., Author Summary The spread of enterovirus-induced HFMD could be controlled through a robust vaccination program. Formalin-inactivated EV71 (FI-EV71) vaccines have been evaluated in human clinical trials in China, Taiwan and Singapore and were found to be safe and to elicit strong neutralizing antibody responses against EV71, which is currently circulating in Asia. However, the results from recent three phase III clinical trials performed in young children indicate that the lack of efficacy against CVA16 infections is a major challenge to the current EV71 vaccine and future HFMD vaccine development. In this study, we developed an adenovirus-based vaccine, Ad-EVVLP with the EV71 P1 and 3CD genes to express VLPs. Ad-EVVLP immunization induced EV71-specific neutralizing antibodies and Th1/Th2-balanced cellular responses in mice. Ad-EVVLP provided protection against both EV71 and CVA16 challenges in the hSCARB2-Tg mice model, whereas FI-EV71 vaccine activated only Th2-mediated EV71 neutralizing antibody responses to protect against EV71 challenge. Because Ad-EVVLP vaccination-induced antibodies had no virus neutralizing activities against CVA16, the cross-protective immunity against CVA16 was mediated by conserved 3CD-specific cellular immunity activation. These results indicate that Ad-EVVLP meets a medical need as a universal HFMD vaccine against both EV71 and CV infections.

Enterovirus 71 (EV71), an emerging neurotropic virus and coxsackieviruses (CV) are the major causative agents of hand, foot and mouth diseases (HFMD). These viruses have become a serious public health threat in the Asia Pacific region. Formalin-inactivated EV71 (FI-EV71) vaccines have been developed, evaluated in human clinical trials and were found to elicit full protection against EV71. Their failure to prevent CVA16 infections could compromise the acceptability of monovalent EV71 vaccines. Bivalent FI-EV71/FI-CVA16 vaccines have been found to elicit strong neutralizing antibody responses against both viruses in animal models but did not protect against CVA6 and CVA10 viral infections in cell culture neutralization assay. In this review, we discuss the critical bottlenecks in the development of multivalent HFMD vaccines, including the selection of vaccine strains, animal models to assess vaccine potency, the definition of end-points for efficacy trials, and the need for improved manufacturing processes to produce affordable vaccines.

Enterovirus71 (EV71) is now recognized as an emerging neurotropic virus in Asia and one major causative agent of hand-foot-mouth diseases (HFMD). However potential animal models for vaccine development are limited to young mice. In this study, we used an adeno-associated virus (AAV) vector to introduce the human EV71 receptors P-selectin glycoprotein ligand-1 (hPSGL1) or a scavenger receptor class-B member-2 (hSCARB2) into adult ICR mice to change their susceptibility to EV71 infection. Mice were administered AAV-hSCARB2 or AAV-hPSGL1 through intravenous and oral routes. After three weeks, expression of human SCARB2 and PSGL1 was detected in various organs. After infection with EV71, we found that the EV71 viral load in AAV-hSCARB2- or AAV-hPSGL1-transduced mice was higher than that of the control mice in both the brain and intestines. The presence of EV71 viral particles in tissues was confirmed using immunohistochemistry analysis. Moreover, inflammatory cytokines were induced in the brain and intestines of AAV-hSCARB2- or AAV-hPSGL1-transduced mice after EV71 infection but not in wild-type mice. However, neurological disease was not observed in these animals. Taken together, we successfully infected adult mice with live EV71 and induced local inflammation using an AAV delivery system.

Enterovirus 71 (EV71) has caused epidemics of hand, foot and mouth diseases in Asia during the past decades and no vaccine is available. A formalin-inactivated EV71 candidate vaccine (EV71vac) based on B4 subgenotype has previously been developed and found to elicit strong neutralizing antibody responses in mice and humans. In this study, we evaluated the long-term immunogenicity and safety of this EV71vac in a non-human primate model. Juvenile macaques were immunized at 0, 3 and 6 weeks either with 10 or 5 µg doses of EV71vac formulated with AlPO4 adjuvant, or PBS as control. During the 56 weeks of studies, no fever nor local redness and swelling at sites of injections was observed in the immunized macaques. After single immunization, 100% seroconversion based on 4-fold increased in neutralization titer (Nt) was detected in EV71vac immunized monkeys but not PBS controls. A dose-dependent IgG antibody response was observed in monkeys receiving EV71vac immunization. The Nt of EV71vac immunized macaques had reached the peak after 3 vaccinations, then decreased gradually; however, the GMT of neutralizing antibody in the EV71vac immunized macaques were still above 100 at the end of the study. Correspondingly, both dose- and time-dependent interferon-γ and CD4+ T cell responses were detected in monkeys receiving EV71vac. Interestingly, similar to human responses, the dominant T cell epitopes of macaques were identified mainly in VP2 and VP3 regions. In addition, strong cross-neutralizing antibodies against most EV71 subgenotypes except some C2 and C4b strains, and Coxsackievirus A16 were observed. In summary, our results indicate that EV71vac elicits dose-dependent T-cell and antibody responses in macaques that could be a good animal model for evaluating the long-term immune responses elicited by EV71 vaccines.

We assessed two strategies for preparing candidate vaccines against hand, foot, and mouth disease (HFMD) caused mainly by infections of enterovirus (EV) 71 and coxsackievirus (CV) A16. We firstly design and optimize the potency of adjuvant combinations of emulsion-based delivery systems, using EV71 candidate vaccine as a model. We then perform immunogenicity studies in mice of EV71/CVA16 antigen combinations formulated with PELC/CpG. A single dose of inactivated EV71 virion (0.2 μg) emulsified in submicron particles was found (i) to induce potent antigen-specific neutralizing antibody responses and (ii) consistently to elicit broad antibody responses against EV71 neutralization epitopes. A single dose immunogenicity study of bivalent activated EV71/CVA16 virion formulated with either Alum or PELC/CpG adjuvant showed that CVA16 antigen failed to elicit CVA16 neutralizing antibody responses and did not affect EV71-specific neutralizing antibody responses. A boosting dose of emulsified EV71/CVA16 bivalent vaccine candidate was found to be necessary to achieve high seroconversion of CVA16-specific neutralizing antibody responses. The current results are important for the design and development of prophylactic vaccines against HFMD and other emerging infectious diseases.

When BmN-4 and M-BmN cells were grown in shake flasks, the pH initially dropped and later increased. The increase in pH signaled a ‘metabolic switch’ that was used here as an indicator for initiating a supplemental glucose and glutamine feed. Using the pH-based fed-batch culture method described, the maximum cell densities of BmN-4 cells and M-BmN cells were increased from 30×105 cells ml−1 to 43×105 and 52×105 cells ml−1, respectively. Correspondingly, the production of polyhedra (4·5×105 OBs ml−1) and HBsAg (574 ng ml−1), from the infection of BmN-4 and M-BmN by wild-type and recombinant BmNPV viruses, respectively, were both significantly enhanced 50% and 100%, respectively. This feeding strategy was implemented with no advanced instrumentation yet facilitated significantly increased yield in shake flasks. The technique should benefit those in research laboratories employing the baculovirus expression system as a rapid and efficient production system.

Effect of annealing time on the magnetic properties and microstructure of (Fe0.675Pt0.325)84B16 ribbon has been studied. For the as-quenched ribbons (Vs=45 m/s) annealed isothermally at 500 °C, iHc increases from 0.05 kOe for the quenched ribbons to the optimal value of 7.5 kOe after 5 h annealing, but Br increases from 1.2 kG for the as-quenched ribbons to 7.5 kG after 5 h annealing at first, then decreases when the annealing time is over 6 h. From thermal magnetic analyzer (TMA) and high resolution transmission electron microscope (HRTEM), magnetically soft Fe2B and Fe3B were found to coexist with magnetically hard γ1-FePt phase in the ribbons after suitable annealing. The existence of sufficient fine Fe3B with Fe2B phases and the well exchange coupling effect between hard and soft phases are the main cause of the improved remanence and magnetic energy product of the ternary (Fe0.675Pt0.325)84B16 ribbons.

Enterovirus 71 (EV71) causes hand, foot, and mouth disease and severe neurological disorders in children. Human scavenger receptor class B member 2 (hSCARB2) and P-selectin glycoprotein ligand-1 (PSGL-1) are identified as receptors for EV71. The underling mechanism of PSGL-1-mediated EV71 entry remains unclear. The endocytosis required for EV71 entry were investigated in Jurkat T and mouse L929 cells constitutively expressing human PSGL-1 (PSGL-1-L929) or human rhabdomyosarcoma (RD) cells displaying high SCARB2 but no PSGL-1 by treatment of specific inhibitors or siRNA. We found that disruption of clathrin-dependent endocytosis prevented EV71 infection in RD cells, while there was no influence in Jurkat T and PSGL-1-L929 cells. Disturbing caveolar endocytosis by specific inhibitor or caveolin-1 siRNA in Jurkat T and PSGL-1-L929 cells significantly blocked EV71 infection, whereas it had no effect on EV71 infection in RD cells. Confocal immunofluorescence demonstrated caveola, and EV71 was directly colocalized. pH-dependent endosomal acidification and intact membrane cholesterol were important for EV71 infection, as judged by the pretreatment of inhibitors that abrogated the infection. A receptor-dominated endocytosis of EV71 infection was observed: PSGL-1 initiates caveola-dependent endocytosis and hSCARB2 activates clathrin-dependent endocytosis.

Combination vaccines can reduce the number of injections and simplify the immunization schedule required to prevent different diseases. Here we assessed the immunogenicity in a mouse model of a vaccine composition comprising inactivated influenza viruses (H5N1/H1N1), enterovirus 71 (EV71), and/or Japanese encephalitis virus (JEV) and investigated whether the vaccine formulations can overcome the immunologic interference between the individual vaccine components. We demonstrated that the antigenic competition happens between H5N1/H1N1 or H5N1/EV71 inactivated virions when the vaccine combinations either formulated with Alum suspensions or without adjuvant. In the presence of PELC emulsified particles, EV71-specific immune responses before and after incorporating H5N1 virus into EV71 vaccine were detected of no significant difference; in addition, H5N1- and EV71-specific immune responses were found at the same level when H5N1/EV71/JEV consolidating into combination vaccine. Emulsified vaccine formulation was represented as a potential tool that is found to reduce the number of injections required to prevent multiple infectious strains causing the same disease (H5N1/H1N1) and/or that protect against different diseases (H5N1/EV71). Combination vaccines can also include a third component to protect against H5N1/EV71/JEV at the same time.

Hand-foot-mouth diseases (HFMD) caused by enterovirus 71 (EV71) and coxsackievirus 16 (CVA16) in children have now become a severe public health issue in the Asian-Pacific region. Recently we have successfully developed transgenic mice expressing human scavenger receptor class B member 2 (hSCARB2, a receptor of EV71 and CVA16) as an animal model for evaluating the pathogenesis of enterovirus infections. In this study, hSCARB2-transgenic mice were used to investigate the efficacy conferred by a previously described EV71 neutralizing antibody, N3. A single injection of N3 effectively inhibited the HFMD-like skin scurfs in mice pre-infected with clinical isolate of EV71 E59 (B4 genotype) or prevented severe limb paralysis and death in mice pre-inoculated with 5746 (C2 genotype). This protection was correlated with remarkable reduction of viral loads in the brain, spinal cord and limb muscles. Accumulated viral loads and the associated pro-inflammatory cytokines were all reduced. The protective efficacy of N3 was not observed in animals challenged with CVA16. This could be due to dissimilarity sequences of the neutralizing epitope found in CVA16. These results indicate N3 could be useful in treating severe EV71 infections and the hSCARB2-transgenic mouse could be used to evaluate the protective efficacy of potential anti-enterovirus agent candidates.

Although several factors participating in enterovirus 71 (EV71) entry and replication had been reported, the precise mechanisms associated with these events are far from clear. In the present study, we showed that heat shock protein 90 (HSP90) is a key element associated with EV71 entry and replication in a human rhabdomyosarcoma of RD cells. Inhibition of HSP90 by pretreating host cells with HSP90β siRNA or blocking HSP90 with a HSP90-specific antibody or geldanamycin (GA), a specific inhibitor of HSP90, as well as recombinant HSP90β resulted in inhibiting viral entry and subsequent viral replication. Co-immunprecipitation of EV71 with recombinant HSP90β and colocalization of EV71-HSP90 in the cells demonstrated that HSP90 was physically associated with EV71 particles. HSP90 seems to mediate EV71 replication by preventing proteosomal degradation of the newly synthesized capsid proteins, but does not facilitate viral gene expression at transcriptional level. This was evident by post-treatment of host cells with GA, which did not affect the expression of viral transcripts but accelerated the degradation of viral capsid proteins and interfered with the formation of assembled virions. In vivo studies were carried out using human SCARB2-transgenic mice to evaluate the protection conferred by HSP90 inhibitor, 17-allyamino-17-demethoxygeldanamycin (17-AAG), an analog of geldanamycin, that elicited similar activity but with less toxicity. The results showed that the administration of 17-AAG twice conferred the resistance to hSCARB2 mice challenged with C2, C4, and B4 genotypes of EV71. Our data supports HSP90 plays an important role in EV71 infection. Targeting of HSP90 with clinically available drugs might provide a feasible therapeutic approach to treat EV71 infection.

Background Large-scale outbreaks of enterovirus 71 (EV71) infections have occurred in Asia-Pacific regions. Severe complications include encephalitis and poliomyelitis-like paralysis, cardiopulmonary collapse, and death, necessitating an effective vaccine against EV71. Methods In this randomized Phase I study, we evaluated the safety and immunogenicity of an inactivated alum-adjuvanted EV71 whole-virus vaccine produced on Vero cell cultures. Sixty healthy volunteers aged 20–60 years received two doses of vaccine, administered 21 days apart. Each dose contained either 5 μg of EV71 antigen with 150 μg of adjuvant (Group A05) or 10 μg of EV71 antigen with 300 μg of adjuvant (Group B10). Serologic analysis was performed at baseline, day 21, and day 42. Results There were no serious adverse events. Mild injection site pain and myalgia were the most common adverse events with either vaccine formulation. The immunogenicity data showed that 90% of vaccine recipients have a 4-fold or greater increase in neutralization antibody titers (NT) after the first dose, without a further increase in NT after the second dose. The seroconversion rates on day 21 and day 42 were 86.7% and 93.1% respectively, in Group A05, and 92.9% and 96.3%, respectively, in Group B10. Thus, 5 μg and 10 μg of the EV71 vaccine can induce a remarkable immune response in healthy adults after only the first vaccination. Conclusion The 5 μg and 10 μg adjuvanted EV71 vaccines are generally safe and immunogenic in healthy adults. (ClinicalTrials.gov number, NCT01268787 ).