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Update of enterovirus 71 infection: epidemiology, pathogenesis and vaccine Expert Rev. Anti Infect. Ther. 12(4), 447–456 (2014)

Shih-Min Wang*1,2 and Ching-Chuan Liu2,3 1 Department of Emergency Medicine, College of Medicine, National Cheng Kung University and Hospital, Tainan, Taiwan 2 Center of Infectious Disease and Signaling Research, National Cheng Kung University, 138 Sheng Li Road, Tainan, 70403, Taiwan 3 Department of Pediatrics, College of Medicine, National Cheng Kung University and Hospital, Tainan, Taiwan *Author for correspondence: Tel.: +88 662 766 120 Fax: +88 662 359 562 [email protected]

Enterovirus 71 (EV71) is a neurotropic human pathogen that is the causative agent of hand foot and mouth disease (HFMD), herpangina and brain stem encephalitis. Recurrent EV71 epidemics of various scales have occurred in the Asia-Pacific region. Several specific cell surface molecules serve as the receptors for EV71. Identification of the receptors is an important step to understand EV71 disease. Cytokines, lymphocytes and monocytes contribute significantly to EV71 pathogenesis. The interaction of EV71 and receptors may be associated with the cytokines immunopathogenesis. Some animal models have been established and aim to explore the pathogenesis of EV71 infections. EV71 antibodies can neutralize or enhance infection at subneutralizing levels. These results are important for EV71 vaccine and therapeutics design. Several clinical trials of human inactivated EV71 vaccine have recently been completed. The purpose of this review is to summarize recent discoveries about the epidemiology and pathogenesis of EV71 and provide insights into human vaccine development. KEYWORDS: brain stem encephalitis • enterovirus 71 • hand foot and mouth disease • receptor • vaccine

Enterovirus 71 (EV71) is in its neurotropism to cause neurological complications, aseptic meningitis, acute flaccid paralysis and brainstem encephalitis. It was first identified in 1969 in California and described by Schmidt et al. in 1974 [1]. EV71 belongs to human EV species A of the genus EV, family Picornaviridae. EV71 distributed worldwide, a cause for great concern of public health. Recent outbreaks of severe and fatal EV71 disease are across Southeast Asia and Asia Pacific areas [2–5]. Hand foot and mouth disease (HFMD) and herpangina in infants and children are the major clinical features of EV71 infection. Whereas a minority of children, approximately 2 in 10,000 children [6], infected with EV71 may develop severe disease manifestations. In 2011, WHO Regional Office for the Western Pacific and the Regional Emerging Diseases Intervention center documented guide for clinical management on hand, foot and mouth disease [7], has proposed the simple clinical stages of disease manifestation to describe the disease severity as suggested previously [3]. The EV71 brain stem encephalitis (BE) was categorized into three important

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10.1586/14787210.2014.895666

critical stages by disease severity, including BE, autonomic nervous system (ANS) dysregulation and pulmonary edema (PE) (FIGURE 1), which resulted in high mortality rates [8–10] or longterm neurological sequelae in survivors [11]. Epidemiology

EV71 can be divided into four genotypes (A, B, C and D) [12,13] among which virus isolates share more than 92% nucleotide sequence identity in VP1 compared with 78–83% identity between genotypes. Genotypes A and D are represented by single virus strains; the prototype BrCr for genotype A and a clinical isolate from India for genotype D. Genotypes B and C each have five known subgenotypes, of which B3–B5 and C3–C5 have recently evolved in Southeast Asia. The EV of different serotypes has been cocirculated in Taiwan year by year. The leading serotypes of EV after 1998 in our hospital were depicted in TABLE 1. EV71 plays a cardinal role among the EV. Two recent major outbreaks of EV71 infection in Taiwan were in 2008 and in 2012. The dominant genotypes had changed twice, from B to C and from C


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Cardiovascular collapse, pulmonary edema

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Autonomic nerve system dysregulation Aseptic meningitis, brainstem encephalitis

HFMD, herpangina

Figure 1. Stages of enterovirus 71 infection by disease severity. HFMD: Hand foot and mouth disease.

to B, between 2009 and 2012. The subgenotype B5 (B5b cluster) was dominant in 2008–2009 but was replaced by subgenotype C4 in 2010–2011. The re-emerging subgenotype B5 (B5c cluster) was identified from the end of 2011–2012 [14,15]. The EV71 subgenotype B5 epidemic was likely to introduce to Taiwan from Southeast Asia because of phylogenetically close to B5 EV71 circulating in Southeast Asia [16]. The subgenotype B5 was first isolated in 1999 in Malaysia and Brunei [17]. In the 2008 Singapore outbreak, subgenotype B5 accounted for about 80% of EV71-positive samples, relegating it to a major role [18]. B5 also dominated the outbreak of HFMD in Denmark 2007, has not previously been reported in Europe [19]. Huang et al. showed the re-emerging EV71 subgenotype B5 strains formed by a separate cluster that was antigenically distinct from the B4 and C genotypes [20]. Dynamics of genetic and antigenic evolution of EV71 in the recent decade showed genotype shift with antigenic property changes and genome recombination. In China, large-scale outbreak of HFMD associated with EV71 has emerged since 2007 [21]. The nationwide epidemics of EV71 started in 2008. It caused nearly 500 fatalities in 2008 and 2009 [22]. In 2012, febrile illness with neurological complication in Cambodia was caused by EV71 and associated with more than 50 deaths [23]. EV71 outbreak was reported in South Korea during 2009. The predominant genotype was C4, particularly C4a, which associated with a relatively low severity and case-fatality rate [24]. However, Zhang and coworkers reported that the mean evolution rate of C4a EV71 was faster than all other EV71 genotypes [25]. Evolutionary branch C4a has some crucial nucleotide or amino acid mutations relative to branch C4b, and these changes may be responsible for its increased neurovirulence and the epidemic of large-scale outbreaks of HFMD in China [25]. EV71 genotype C4a viruses also spread from China to Vietnam and caused a large-scale epidemic in Ho Chi Minh City and southern Vietnam in 2011, through genetic and antigenic analysis [26]. Wu and coworkers conducted a comprehensive evolutionary dynamic study 448

of EV71 during 1994–2013 in Asia Pacific areas, based on phylogenetic analyses of the VP1 sequences. They showed C4, C1, C2 and B4 are the predominant strains and the polymorphisms and divergence and VP1 gene of EV71 strains with very slow evolution rate, which may be one of the reasons for periodic outbreaks in this area [27]. An epidemiology study of EV71 infections in France during 2000–2009 showed that C1 isolates were predominant between 1994 and 2005, and C2 strains have been predominant since 2007 [28]. Another epidemiological and genetic data on EV71 circulation in the Netherlands from 1963 to 2008 were analyzed. Infections were caused by three different and successive lineages belonging to subgenotype B0, B1 and B2 from 1963 to 1986. After 1987, the B genotype was replaced by genotype C strains of lineages C1 and C2. The epidemiology of EV71 subgenotypes B1, B2, C1 and C2 appeared to have a global nature [29]. A large-scale genetic analysis of isolates was collected from 19 countries worldwide over a 40-year period. A series of recombination events occurred over the study period, which had been identified through incongruities in sequence grouping between the VP1 and 3Dpol regions. The likelihood of recombination increased with VP1 sequence divergence and recombination events occurred as an initiation of most subgenotypes immediately preceding their lineage expansion and global emergence [30]. Chang et al. examined the relationship between meteorological parameters and EV71 infection. They found that the incidence of EV71 infections reflected significant summer seasonality from April to June in Taiwan. The incidence of EV71 infections began to rise at temperatures above 13˚C; at temperatures higher than approximately 26˚C, the incidence began to decline, producing an inverted V-shaped relationship. This study indicated that warmer temperature and elevated humidity would lead to an increased rate of EV 71 infection in Taiwan [31]. The basic reproductive number has a cardinal role in infectious disease epidemiology. It is defined as the average number of secondary cases from an index case introduced into a very susceptible population [32]. The median basic reproductive number of EV71 was 5.48 higher than that of Coxsackievirus A16 (CA16), 2.50. The transmissibility of EV71 is more efficiently among children than that of CA16 [33]. EV71 can be transmitted via contaminated hands by directly from vesicles on the hands or secretions from oropharynx and feces. Ruan et al. reported a strong protective effect from better hand-washing habits during an outbreak of HFMD and herpangina [34]. A reduction in risk of more than 95% was supported by a consistently increasing dose–response effect. Pathogenesis Receptor for EV71

For virus to infect cells, the interaction between virus and receptors is considered to play important roles in the early steps of infection. Virus attachment and entry into host cells are generally a complex multistep processes involving sequential of one receptor and/or simultaneous recognition of multiple Expert Rev. Anti Infect. Ther. 12(4), (2014)

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CA16 CB3 CB3 CB5

Data from virology laboratory at National Cheng Kung University Hospital. CA: Coxsackievirus A; CB: Coxsackievirus B; EV: Enterovirus.

CA10

CA2

CB4

CA5

CB1

CA16

CB4

CA10

CA5

EV71

CA4

CA10

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†

CB4 CA2 CA9 CA4 CB1 CA2 CA6 CA4 CA16 CA4 Echo11 CA4 Echo6 CB2 CA16

CA16

CA6 EV71 CA10 CA16 CA6 EV71 CA10 Echo18 CB3 CA10 CA16 CB5 Echo30 CA10 EV71 Major serotypes of EV isolates

EV71

2 305 99 21 6 371 1 0 34 53 10 25 116 5 132 No. of EV71 isolates

195

706 879 724 973 579 885 585 469 844 823 483 542 1301 687 384 No. of EV isolates

1061

2013 2012 2011 2010 2009 2008 2007 2006 2005 2004 2003 2002 2001 2000 1999 1998 Year

cell-surface receptors. Virus receptors, usually membrane form, are host-cell molecules, which bind virus attachment proteins and are required for entry [35]. EV71 receptor on leukocytes is the first major step in elucidating EV71 pathogenesis at the molecular level. Human P-selectin glycoprotein ligand-1 (PSGL-1; CD162), a sialomucin membrane protein expressed on leukocytes that has a major role in early stages of inflammation, tethering and rolling of leukocytes on vascular endothelium, was proved as a functional receptor for EV71 infection by Nishimura and colleagues [36]. Because of patients with complicated EV71 BE, ANS dysregulation and PE have high concentrations of proinflammatory cytokines in the plasma and cerebrospinal fluid (CSF) [37]. The interaction of EV71 with PSGL-1 on lymphocytes may traffic to the CNS, allow the virus to spread and induce production of the inflammatory cytokines involve in BE with PE. The N-terminal region of PSGL-1 binds specifically to EV71. A post-translational modification, tyrosine sulfation, but not O-glycosylation, in the N-terminal region of PSGL-1 may facilitate virus entry and replication of EV71 in leukocytes [38]. A single amino acid residue within the capsid protein VP1 determines whether EV71 binds to PSGL-1. The conserved lysine residues on the virus surface are responsible for interaction with sulfated tyrosine residues at the PSGL-1 N-terminus, and VP1–145 acts as a switch, controlling PSGL-1 binding by modulating the exposure of VP1–244K [39]. EV71 replicated in nonleukocyte cell lines in a PSGL-1independent manner, indicating the presence of alternative receptor(s) for EV71. Yamayoshi et al. showed that human scavenger receptor class B, member 2 (hSCARB2), also known as lysosomal integral membrane protein II or CD36b like-2, serves as a receptor for EV71 infection with HFMD or with encephalitis [40]. EV71 binds soluble SCARB2 or cells expressed SCARB2, and the binding is inhibited by an antibody to SCARB2. Because SCARB2 is expressed ubiquitously, it is possible that SCARB2 may be involved in systemic EV71 infections. Furthermore, subgenotypes B2, B4, B5, C1, C2 and C4 isolates of EV71 infected cells via a SCARB2-dependent pathway. They indicated that EV-A viruses are divided into at least two groups: viruses whose infection is dependent on SCARB2 and viruses not dependent on SCARB2. CA7, CA14, CA16 and EV71 share the same receptor, SCARB2, and are frequently associated with HFMD and occasional neurological diseases [41]. Their preliminary experiments showed that SCARB2 is expressed in neurons in the CNS in humans, monkeys and transgenic (Tg) mice-expressing human SCARB2 and that the adult Tg mice showed encephalitis after infection with EV71. The underlying mechanism of PSGL-1- and hSCARB2mediated EV71 entry was investigated by Lin et al. Jurkat T and mouse L929 cells constitutively expressed human PSGL-1 (PSGL-1-L929), and human rhabdomyosarcoma (RD) cells displayed high SCARB2 but not PSGL-1. Disruption of clathrin-dependent endocytosis prevented EV71 infection in RD cells, while there was no influence in Jurkat T and

Table 1. Serotype distribution of enterovirus at National Cheng Kung University Hospital, Tainan, Taiwan, 1998–2013†.

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PSGL-1-L929 cells. Disturbing caveolar endocytosis by specific inhibitor or caveolin-1 siRNA in Jurkat T and PSGL-1-L929 cells considerably blocked EV71 infection, whereas it had no effect on EV71 infection in RD cells. PSGL-1 initiates caveola-dependent endocytosis and hSCARB2 activates clathrindependent endocytosis [42]. Recently, a functional comparison of SCARB2 and PSGL-1 receptors for EV71 infection was also studied. Yamayoshi et al. reported that the uncoating of EV71 requires both SCARB2 and an acidic environment and occurs after the internalization of the virus–receptor complex into endosomes. SCARB2 is capable of viral binding, viral internalization and viral uncoating, and that the low infection efficiency of L-PSGL-1 cells is due to the inability of PSGL-1 to induce viral uncoating [43]. However, PSGL-1 and SCARB2 also serve as a receptor for CVA16, which is rarely to cause severe neurological complications. PSGL-1 is not expressed on neurons, the neurotropism of EV71 may have other factors involvement, which are independent of these two receptors. Annexin II (Anx2) is a member of the annexin gene family. It can interact with multiple cellular factors and is involved in regulating cellular functions including endocytotic and exocytotic pathways, calciumdependent F-actin filament bundling and the profibrinolytic generation of plasmin [44]. Anx2 expressed on the cell surface as a cellular adherent factor can interact with EV71 via VP1 binding and enhance its infectivity. However, HepG2 cells do not express Anx2 remained permissive to EV71 infection. EV71 also utilized cell surface heparan sulfate as an attachment receptor. Neutralization of the cell surface anionic charge by polycationic poly-D-lysine and blockage of heparan sulfate by an anti-heparan sulfate peptide inhibited EV71 infection. The exact mechanism of interaction of EV71 particles with negatively charged heparan sulfate on the cell surface remains under studies [45]. Su et al. found sialylation of cell surface glycoproteins assist in the attachment of EV71 to host cells. The attachment of EV71 to RD and SK-N-SH cells has diminished after the removal of cell surface sialic acids by neuraminidase [46]. In an orally infected EV71 murine model, EV71 was suggested to use the active retrograde axonal transport system to enter the CNS [47]. Multiple axonal transport routes, receptor dependent and receptor independent for poliovirus are present in Tg mice [48]. Therefore, EV71 may also be disseminated through other distinct receptor-independent pathways and mechanisms. Different receptors may play different roles on different cells at different infectious stages in the EV71 infection. Not a single receptor interacts with EV71 is sufficient enough to elicit EV71 BE. Immune responses to EV71 infection

Inflammatory mediators, cytokines and chemokines, play an important role in the pathogenesis of EV71 BE. Both the CNS and the systemic inflammatory responses to infection play distinctly different roles in the pathogenesis of EV71 PE. In addition to IL-6, TNF-a, IL-1, IL-10, IL-13 and IFN-g are associated with BE that is complicated by PE [3]. A significant 450

elevation of serum IL-1 receptor antagonist and granulocyte colony-stimulating factor (G-CSF) levels are observed in patients with PE. A serum G-CSF to IL-5 ratio of >100 at admission was suggested to be a prognostic marker for death [49]. The CSF level of IL-6 was significantly high and correlated with disease severity during the first or second day of CNS involvement in EV71 infection [50]. Khong et al. showed that sustained high levels of IL-6 led to severe tissue damage and eventually death in the EV71-infected neonate mouse model [51]. Administration of anti-IL-6 neutralizing antibodies increased the survival rates and clinical scores and displayed reduced tissue damage, absence of splenic atrophy, increased immune cell activation and markedly elevated systemic levels of IL-10. They suggested that anti-IL-6 antibody treatment represents a potential therapeutic approach to provide protection from severe EV71 complications. These findings are justified in treating EV71-infected patients complicated with ANS dysregulation with intravenous immunoglobulin (IVIG) [52]. Plasma levels of IFN-g-induced protein (IP)-10 was significantly high in patients with PE [53]. Shen and coworkers investigated the induction of IP-10 by EV71 infection in a murine model. They found that IP-10 deficiency significantly reduced levels of monokine induced by IFN-g (MIG) in serum, and levels of IFN-g and the number of CD8+ T cells in the mouse brain. Absence of IP-10 significantly increased the mortality of infected mice by 45% and slowed down virus clearance in several vital tissues [54]. Chen and coworkers reported high expression of acid-related orphan nuclear receptor gamma t (ROR t) in peripheral blood monnuclear cells and elevated concentrations of IL-17 and IL-23 in sera of EV71 infections. Further, the frequencies of Th17 cells in blood from EV71-infected children were significantly higher in comparison with controls [55]. Polymorphism of some inflammatory mediators associated with the complications and disease severity. Yang et al. reported that IFN-g + 874 A allele and IL-10-1082 A allele was observed with significantly higher frequency in patients with EV71 encephalitis compared with HFMD patients without complications [56]. Upregulation of toll-like receptor (TLR) 2, TLR7 and TLR8 mRNA expression were found at different time points in EV71-infected human primary monocyte-derived macrophages in vitro. These results indicated that the cytokine productions in EV71-infected monocyte-derived macrophages may be partly through the activation of TLR2, TLR7 and TLR8 [57]. A proteomic analysis study aimed to detect the related protein biomarkers from five extremely severe HFMD children of EV71 infection. It showed an apparent downregulation of complement C3(C3), inter-a (globulin) inhibitor H2, serum albumin, plasminogen and retinol-binding protein 4; but increased peroxiredoxin-2 in extremely severe HFMD patient. These proteins may be used as potential molecular markers to monitor the progression of severe EV71 infection [58]. Previous study demonstrated that patients with PE had lower circulating CD4+ T cells, CD8+ T cells and natural killer cells [59]. However, CD19+ B cells and CD20+ B cells expression Expert Rev. Anti Infect. Ther. 12(4), (2014)

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Update of EV71 infection

frequency and absolute cell number did not change significantly in EV71 infection by the disease severity [WANG SM, + + + UNPUBLISHED DATA]. CD20 B cells, CD4 T cells and CD8 T cells infiltration were detected in the spinal cord by immunohistochemical staining in a fatal EV71-infected patient. Lin et al. showed that the disease severity, mortality and tissue viral loads of mice deficient in CD19+ B, CD4+ T cells or CD8+ T cells were significantly higher than those of wild-type mice. Treatment with EV71-specific antibody before or after infection considerably reduced the disease severity, mortality and tissue viral loads in B cells deficient mice. They indicated both lymphocyte and antibody responses protect mice from EV71 infection [60]. Marked elevations of absolute counts and percentages of CD19+ B cells were observed clinically in the cases with neurological involvement compared with the mild cases of EV71 infection. Xie et al. showed a significant elevation of plasma immunoglobulin levels in patients with the CNS complications [61]. Dendritic cells (DCs) play a crucial role in antiviral immunity by functioning as professional antigen-presenting cells to prime T cells and by secreting cytokines to modulate immune responses. EV71 productively infected human immature DCs and expressed viral antigen in DCs. EV71 entry into DCs was partially mediated by DC-SIGN. Further analysis revealed that EV71 increased the viability, activation and release of cytokines, IL-6, IL-12 and TNF-a in DCs [62]. Animal model

The lack of a relevant animal model has hampered the understanding of EV71 pathogenesis, in particular the occurrence of ANS dysregulation and PE in human. Monkeys and neonatal mice that are susceptible to some EV71 strains were used as study models. Zhang et al. reported the pathogenic process of systemic EV71 infection in rhesus monkeys after inoculation via intracerebral, intravenous, respiratory and digestive routes. Only intracerebral inoculation resulted in PE and hemorrhage along with impairment of neurons [63]. Exogenous IL-6, IL-13 and IFN-g treatment could induce pulmonary dysfunction and clinical symptoms, and provoked a mild PE in EV71infected mice [64]. Currently, there is no any mice model can develop PE after natural infection of EV71. Fujii et al. generated hSCARB2 Tg mouse model with an expression profile similar to that in humans. Tg mice infected with EV71 exhibited ataxia, paralysis and death clinically. Pathological findings showed that most severely affected were neuron cells in the spinal cord, brain stem, cerebellum, hypothalamus, thalamus and cerebrum [65]. Lin and coworkers also infected hSCARB2 Tg mice with clinical EV71 isolates lead to HFMD-like and neurological syndromes. EV71 viral loads were evident in the tissues and CNS accompanied the upregulated proinflammatory mediators (CXCL10, CCL3, TNF-a and IL-6), correlating to recruitment of the infiltrated T lymphocytes that result in severe diseases [66]. hSCARB2 Tg mouse model was used to evaluate the protective efficacy conferred by a previously described EV71-specific neutralizing informahealthcare.com

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antibody, N3. A single injection of N3 effectively inhibited the HFMD-like cutaneous lesions in mice preinfected with B4 genotype of EV71 or prevented severe limb paralysis and death in mice preinoculated with C2 genotype EV71. The protection associated with the reduction of the viral load in the brain, spinal cords and limb muscles and also associated with the reduction of the proinflammatory cytokines and chemokines production in tissues [67]. Khong et al. set up an animal model of 2-week-old immunodeficient AG129 mice, which lacks types I and II interferon receptors, are susceptible to infection with a nonmouse-adapted EV71 strain via both the intraperitoneal and oral routes of inoculation. The infected mice displayed progressive limb paralysis before death and indicated that limb paralysis is a consequence of EV71 neural invasion [68]. Caine and coworkers established AG129 adult mice infected model to produce systemic clinical signs of polio-like disease using adapted subgenotype B2 EV71 strain. Using this model, they showed passive immunization with rabbit EV71 immune sera and active immunization with an alum adjuvanted inactivated EV71 candidate vaccine provided full protection against EV71 disease [69]. Expert commentary Antibody-dependent enhancement

The interplay between virus and antibody may have neutralization, cross reaction or protection and antibody-dependent enhancement (ADE) of infectivity. Immunity to EV71 is mainly dependent on humoral factors, the formation of neutralizing serum antibodies. Pre-existing antibodies may play a critical role in controlling viral infection or in aggregating the disease severity. It depends on various factors including viral serotype, viral capsid, cell type, antibody concentration, class and subclass and epitope. The seropositive rates of EV71 neutralizing antibodies in the family cohorts were 65% in the mothers, 50% in the neonates and 1% in the 6-month-old infants. These maternal EV71-neutralizing antibodies declined to undetectable levels in 99% of the 6-month-old infants [70]. The highest fatality rate of complicated HFMD and herpangina was in children younger than 1 year from 1998 to 2008 in Taiwan [4,71]. Severe disease and death typically occur only in young children, which is likely due to age-dependent hyperactive innate immune responses [72]. These clinical observations have led to the widely accepted hypothesis of ADE of infectivity. ADE describes an increase in the efficiency of virus infection in the presence of subneutralizing concentrations of virusreactive antibody [73]. In this connection, antibodies may increase the efficiency of virus attachment and infection through interactions with Fcg receptors expressed on the surface of target cells [74]. EV71 can infect peripheral blood monnuclear cells, lymphocytes and monocytes. Anti-EV71 immune serum and human IVIG can enhance EV71 infection on THP-1 cells at subneutralizing concentrations. Anti-FcgRI (CD64), but not anti-FcRgII (CD32) and anti-FcgRIII (CD16), significantly inhibited immune serum-mediated infection by ADE. These 451

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findings support the notion that ADE augments the infectivity of EV71 and contributes to the age-dependent pathogenesis of EV71-induced disease [75]. Han and coworkers developed a secondary infection model of EV71 in which 1-day-old mice were first infected with an avirulent EV71 strain, to induce the subneutralizing antibody, and subsequently infected with a virulent EV71 strain 14 days after the primary infection. Subneutralizing antibody increased the mortality of EV71 infection in a secondary infection suckling mouse model [76]. They indicated that ADE might contribute to the pathogenesis of severe EV71 infection. Newborn mice that received anti-EV71 IgG at the subneutralizing dose exhibited higher clinical score and greater mortality than mice that received virus control. The histopathological findings revealed extensive neuronal and muscular damage and markedly increased viral titers in brain, spinal cord and lung in ADE group. Mice that received subneutralizing anti-EV71 IgG plus virus had significantly high serum levels of IFN-g and monocyte chemoattractant protein-1 (MCP-1) [77]. These findings support the concept that subneutralizing antibodies directed enhance EV71 induce ADE in newborn mice. A recent study investigated the distinct role of each IgG subclass on neutralization and enhancement of EV71 infection. Cao et al. found that the strongest neutralizing activity of human IVIG is mainly mediated by IgG1 subclass. However, IgG3 presents only enhancing activity without neutralization, whereas IgG2 has weak ADE activity in vitro [78]. The subneutralizing antibodies enhanced that EV71 infections may involve in a concentration-dependent, IgG subclass-dependent, genotypedependent and receptor-dependent manner. Vaccine development should seek high concentration and long duration of antibodies production that mediate neutralization but not ADE. Five-year view Vaccine

The need for effective vaccines to control EV71 is urgent. Many EV71 vaccine (EV71vac) candidates have been developed including inactivated virus, virus-like particles, capsid protein VP1 peptide or recombinant protein-based constructs, VP1expressing viral or bacterial vectors, VP1 DNA constructs and live attenuated EV71 [79]. According to the experiences of poliovirus vaccines development and animal studies, the inactivated whole-virus vaccines are more likely to be feasible [80]. Ong and colleagues tested the protective efficacy of formaldehyde-inactivated whole-virus vaccines in mice model of EV71 encephalomyelitis. Following immunization of mice at birth and administration of a booster dose at day 7, complete protection from disease in mice at day 14 was observed. Cross-neutralizing EV71 antibodies to strains with genotypes B3, B4 and C1–C5 generated in immunized adult mice were able to passively protect 14-day-old mice from disease [81]. A formalin-inactivated EV71vac was reported demonstrated excellent protective efficacy in a maternal vaccination-neonatal challenge mice model [82]. Although the major cause of morbidity and mortality in this mice model is skeletal, muscle 452

involvement but not the involvement of the CNS. However, the vaccine protective efficacy demonstrated that the protection from lethal challenge was transferred from immunized dams to their offspring, demonstrated that humoral immune responses are likely to be the main component of the adaptive immune response providing protection to mice against EV71 infection. Several large-scale clinical trials of EV71vac were conducted recently. A Phase I, uncontrolled, prospective, randomized, open-label, two-center clinical study of chemically inactivated EV71vac, based on an EV71 subgenotype B4 strain, was performed in adult volunteers in Taiwan. It elicits crossneutralizing antibody responses against EV71 subgenotypes B1, B4, B5 and C4A. However, only weak cross-neutralizing antibody responses against a C4b and CA16 were shown. Further, more than 90% of vaccinated volunteers did not develop crossneutralizing antibody responses against a C2 strain [83]. A double-blind, randomized controlled study was to evaluate the safety and immunogenicity of a human EV71vac in healthy adults, children and infants. The vaccine dosages were 200 and 400 U for children and adults, and 100, 200 and 400 U for infants. Adults received intramuscular injection on days 0, 14 and 28 and children and infants received on days 0, 28 and 56. For immune response, the seropositive rates of neutralizing antibody increased to 100% for all dosage groups after the second vaccination [84]. Zhu and colleagues undertook a randomized, double-blind, placebo-controlled clinical trial (Phase Ib) of inactivated EV71vac in healthy infants and children, who were divided into 6–12 and 13–60 months, and randomly allocated to receive placebo or the vaccine containing 160 U, 320 U or 640 U antigen. After two doses, all the participants receiving EV71vacs were seropositive, and the seroconversion rates were above 98.1%. In the participants with seropositive baseline, one dose induced good seroconversion rates of more than 64.3% in participants receiving EV71vacs [85]. A randomized, double-blind, placebo-controlled, Phase II trial of EV71vac was undertaken in China. More evidence for the immunogenicity, safety profile and assessed vaccine-induced neutralizing antibody geometric mean titers over 8 months of the vaccines were provided. The most common systemic adverse reactions across treatment groups were fever and diarrhea. They suggested that an alum-adjuvanted 320 U formulation is the best possible formulation for the Phase III trial [86]. Li et al. conducted a double-blind, randomized, controlled trial to evaluate the optimal dose, immunogenicity, safety and immune persistence of the EV71vac. The dose–response relationship, with the 400 U dose showing higher immunogenicity than the 100 and 200 U doses, remained until 13 months after the second vaccination, despite waning antibody levels. In this trial, 1-year antibody persistence induced by EV71vac was first evaluated [87]. The clinical efficacy of an inactivated EV71vac was proved through randomized, double-blind, placebo-controlled, Phase III trial of more than ten thousands enrolled participants. The vaccine provided significant protection against EV71-associated disease, especially EV71-associated HFMD in children aged Expert Rev. Anti Infect. Ther. 12(4), (2014)

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6–35 months. Evidence of immunological correlates of protection against EV71-associated disease was provided [88]. Furthermore, Mao et al. indicated that EV71 vaccination of subgenotype C4 provided the broad and homogeneous protection against B4, B5, C2 and C5 subgenotypes of EV71 in health children and infants, similar to the outcome of natural infection [89]. Another study also indicates that the EV71vac was highly consistent between production lots, and it was well tolerated and highly immunogenic in children, which supports the licensing of the inactivated vaccine for marketing [90]. Chen and colleagues tested the effectiveness of a formaldehydeinactivated EV71vac and its compatibility for coimmunization with a pentavalent vaccine [91]. They displayed coimmunization with pentavalent and inactivated EV71vacs elicited antibodies against the major components of the pentavalent vaccine including poliovirus, Bordetella pertussis, Haemophilus influenzae type b, diphtheria toxoid and tetanus toxoid at the same levels as in mice immunized with pentavalent vaccine alone. These findings indicate that formaldehyde-inactivated EV71vac is feasible for designing multivalent vaccines. While interaction or

Review

immunogenicity effect on vaccines coadministered in infants still needed to assess clinically. Once the vaccination of EV71 was introduced to routine childhood vaccination schedules, EV71 will be put in the list of vaccine-preventable disease in children. Financial & competing interests disclosure

The authors thank the G.D. Hsiung Virology Laboratory at National Cheng Kung University Hospital for providing data. Our studies have been supported by grants from the National Science Council, Taiwan; the Multidisciplinary Center of Excellence for Clinical Trial and Research, Department of Health, Executive Yuan, Taiwan; and the Center of Infectious Disease and Signaling Research, National Cheng Kung University, Taiwan. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Key issues • B5 and C4 genotypes were the predominant strain of Enterovirus 71 (EV71) circulated in the Asia region recently. • P-selectin glycoprotein ligand-1, scavenger receptor class B, member 2, Annexin II and heparan sulfate are identified as receptors for EV71 infection. • Anti-IL-6 neutralizing antibodies therapy increased the survival rates and immune cell activation and reduced tissue damage in mice. It indicated the usefulness of IVIG treatment in severe EV71 infection. • T cells, B cells and dendritic cell participated in EV71 infection and associated with disease severity and cytokine production. • Several mouse models have been applied to EV71 infection by demonstrating neurological complications clinically and pathologically, however, failed to show pulmonary edema. • Antibody-dependent enhancement of EV71 infectivity is proved by clinical implication, in vitro studies and animal studies. • Phase I through Phase III clinical trials of inactivated EV71 vaccine had completed, some unresolved issues still leave behind.

J Clin Microbiol Infect Dis 2012;31: 1219-24

References Papers of special note have been highlighted as: • of interest •• of considerable interest 1.

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Test EV71-inactivated vaccine using maternal vaccination-neonatal challenge mice model.

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First study of Phase II clinical trial of EV71 vaccine.

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Large-scale study of Phase III clinical trial of EV71 vaccine.

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