Is a multivalent hand, foot and mouth disease vaccine feasible? Michel Klein1 and Pele Chong2,3
1VaxioBio Inc, Toronto, Ontario, Canada.
2Vaccine R&D Center, National Health Research Institutes, Zhunan Town, Miaoli County 350, Taiwan.
3Graduate Institute of Immunology, China Medical University, Taichung, Taiwan.
Correspondence:
Michel H. Klein, MD., 54 Strathgowan Avenue, Toronto, Ontario, Canada, M4N 1B9. Tel: 1 416 481 3549, E-mail: [email protected].
Pele Chong, Vaccine R&D Center, National Health Research Institutes, Zhunan Town, Miaoli County 350, Taiwan. Tel: 886-37-246166 ext 37710, Fax: 886-37-583009. E-mail: [email protected]
Key Words: Human Enterovirus A (HEV-A); Hand, Foot and Mouth Diseases; Enterovirus A71; Coxsackie viruses A16; coxsackieviruses B3 and B5; echovirus 30; epidemiology; monovalent, bivalent and multivalent vaccines; inactivated whole virion vaccines.
Running Title: Is a multivalent hand, foot and mouth disease vaccine feasible?
Enterovirus A infections are the primary cause of hand, foot and mouth disease (HFMD) in infants and young children. Although enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) are the predominant causes of HFMD epidemics worldwide, EV71EV-A71 has emerged as a major neurovirulent virus responsible for severe neurological complications and fatal outcomes. HFMD is a serious health threat and economic burden across the Asia-Pacific region. Inactivated EV71EV-A71 vaccines have elicited protection against EV71EV-A71 but not against CA16CV-A16 infections in large efficacy trials. The current development of a bivalent inactivated EV71EV-A71/CA16CV-A16 vaccine is the next step towards that of multivalent HFMD vaccines. These vaccines should ultimately include other prevalent pathogenic coxsackieviruses A (CA6CV-A6 and CA10CV-A10), coxsackieviruses B (CV-B3 and CV-B5) and echovirus 30 that often co-circulate during HFMD epidemics and can cause severe HFMD, aseptic meningitis and acute viral myocarditis. The prospect and challenges for the development of such multivalent vaccines are discussed.
Introduction
Human eEnteroviruses (HEVs) have been classified into four species HEV-A, HEV-B, HEV-C and HEV-D [1]. HEVs are positive-sense, single-stranded RNA viruses within the Picornaviridae family. They are responsible for a spectrum of various clinical manifestations, including severe neurological complications, and cardiopulmonary diseases in young children [2-7]. More than 100 HEV serotypes have been identified including polioviruses, coxsackieviruses A (CV-A), coxsackieviruses B (CV-B), echoviruses (E) and numbered enterovirus serotypes (EV). In the past two decades, enterovirus -A infections have become the primary cause of an increase in the incidence and severity of hand, foot and mouth disease (HFMD) in infants and young children. Both coxsackievirus A16 (CA-CV-A16) and enterovirus 71 (EV71EV-A71) have been the predominant etiologic agents of herpangina (HA) and HFMD epidemics [5-9 and Table I]. Several other enterovirus serotypes usually detected in sporadic cases or outbreaks of HFMD frequently co-circulate with EV71EV-A71 and CA16CV-A16 in large epidemics. These enteroviruses include coxsackieviruses A CV-A2, CV-A3, CA4CV-A4, CA5CV-A5, CA6CV-A6, CV-A8, CV-A9, CA10CV-A10, CV-A12, CV-A14, coxsackieviruses B CV-B1 to CV-B6 and echoviruses E-4, E-5, E-6, E-7, E-9, E-11, E-18, E-25, E30E-30 [910-69 and Table I]. HFMD has become a major health issue and a substantial economic burden throughout the Asia Pacific region [5-7]. Following the near complete eradication of poliovirus, EV71EV-A71 has emerged as a major neurotropic virus responsible for severe neurological complications and fatal outcomes. Besides EV71EV-A71, other co-circulating life-threatening enteroviruses such as CB3CV-B3, CB5CV-B5 and E30E-30 expose children to aseptic meningitis and acute myocarditis [2-4]. The recurrence of outbreaks associated with high morbidity and mortality has prompted the World Health Organization in 2009 to declare HFMD a rising menace in Asia [70]. The largest population-based HFMD epidemiological survey has recently revealed that the case-severity rate for patients with cardiopulmonary and neurological complications was 1.1% and that the fatality rate for patients with severe disease was 0.03% [9]. In the absence of approved
antiviral treatment [71], a multivalent prophylactic vaccine against HFMD is urgently needed and the development of an efficacious EV71EV-A71 vaccine in particular has been a national health priority in some Asian countries [72].
Clinical presentation
Human eEnteroviruses A predominantly infect infants and young children below 5 years of age. Most EV71EV-A71 infectees (71%) remain asymptomatic [5,7]. Following a 2-5 day incubation period with a prodrome of fever, malaise, abdominal pain and myalgia, HFMD is typically characterized by a papulovesicular or maculopapular rash, blisters of the hands, soles, and buttocks associated with painful ulcerative lesions of the mouth. HFMD is usually a self-limiting infection and most infected children recover within two weeks in the absence of secondary cutaneous infection. However, the virus may be present in the faeces for several weeks after recovery. HFMD is a highly contagious illness which is efficiently propagated to household, day care centre and kindergarten contacts by oro-pharyngeal secretions or fecal-oral transmission. EV71EV-A71 outbreaks have been responsible for severe neurological complications including aseptic meningitis, cerebella ataxia, poliomyelitis-like paralysis, Guillain-Barré syndrome, acute brainstem encephalitis, and fulminant neurogenic pulmonary edema/hemorrhage associated with high mortality [73]. The annual fatality rate in Taiwan over the last decade has been between 0 to 25%, with an average of 13% [74]. Survivors from brainstem encephalitis often suffer from neurological sequelae including long-term motor deficits and cognitive impairment [75]. EV71EV-A71 elicits humoral responses, but there is no correlation between neutralizing antibody levels and disease severity indicating that altered cellular responses such as an imbalance in Th1/Th2 and Th17/Treg subset ratios play a significant role in disease outcome and may have potential prognostic value [76-78] However, the presence of EV71EV-A71 neutralizing antibodies was found to be inversely correlated with the number of severe HFMD and the loss of maternal antibodies to be responsible for an
increase in severe cases in the 1-2 year age group [7934]. The presence of EV71EV-A71 neutralizing antibodies did not reduce the incidence of infections caused by other non-A71 enteroviruses [3479].
Coxsackievirus A16 is the other major cause of herpangina and mild HFMD. However, a small number of patients develop neurological complications such as aseptic meningitis, encephalitis, even fatal pneumonia and acute viral myocarditis [8]. Coxsackieviruses CA6CV-A6 and CA10CV-A10 have been mainly associated with HA outbreaks and more recently with HFMD epidemics. CA6CV-A6 tends to be a virulent strain which unusually affects both pediatric and adult populations. The virus has been responsible for severe atypical cases of HFMD often requiring hospitalization, characterized by extensive vesiculobullous and erosive cutaneous eruptions, eczema, purpuric lesions and onychomadesis with nail shedding [80,81]. CA6CV-A6 infections have also led to fatal encephalitis/encephalopathy and myocarditis [82,83]. CA10CV-A10-associated HFMD cases are characterized by high fever, vesicular rashes and oral ulcers [63] with occasional meningoencephalitic complications [3736].
Coxsackieviruses CB3CV-B3 and CB5CV-B5 are both neurotropic and cardiovirulent viruses (2-4). When they co-circulate in HFMD outbreaks, they potentially pose significant health threats to neonates and young infants by exposing them to the risk of acute myocarditis and dilated cardiomyopathy, aseptic meningoencephalitis, interstitial pneumonitis, disseminated intravascular coagulopathy, sequelae and fatal outcomes [2,4]. CB3CV-B3 infections are the major cause of myocarditis which can lead to dilated cardiomyopathy, long-term sequelae and fatal outcome [84-,86].
Echovirus E30E-30 is a major neurotropic pathogen responsible for worldwide outbreaks of aseptic meningitis and encephalitis in [2,87]. Neonates and infants are specifically at risk for disseminated CB3CV-B3, CB5CV-B5 and E30E-30 infections and these three enteroviruses have been directly implicated in the pathogenesis of type I diabetes as a result of direct cell damage and autoimmune mimicry [2,88]. Herpangina is the most frequent clinical manifestation of CA4CV-A4 and CA5CV-A5 infections [86, 89-9291].
Virology of HFMD-associated enteroviruses
Enteroviruses are non-enveloped particles containing a non-segmented, single-stranded, positive sense genomic RNA [1]. Their capsids are composed of 60 copies of four structural proteins, VP1, VP2, VP3 and VP4. After infection, viral RNA acts as mRNA and its open reading frame is translated into a polyprotein further cleaved by proteolysis into four structural proteins (VP). The VP1, VP2 and VP3 are displayed on the surface of the virion surface as shown in the EV71EV-A71 and CB3CV-B3 crystal structures [9392-9594]. These subunits expose linear and conformational neutralization epitopes and are responsible for immune responses and host-receptor binding. VP1 contains major neutralization
determinants and is used in viral identification and evolutionary analyses. The 5’untranslated region (5’ UTR) RNA contains a type I internal ribosomal site (IRES) that is poorly efficient at initiating viral translation. The efficiency of viral replication and translation depends on the complex interplay of 12 trans-acting host factors (ITAFs) with EV71EV-A71 IRES [9695]. Like poliovirus, enteroviruses produce empty (E) and full (F) particles in cell culture systems that can be separated and purified using continuous sucrose gradient ultracentrifugation [9796]. The F-particles like the poliovirus D-antigen have a high content of viral RNA and a full particle structure. In contrast, defective E-particles like the
poliovirus C antigen are empty structures virtually devoid of infectious RNA which explains their low infectivity.
Enteroviruses cell and tissue tropism
Enteroviruses use a wide array of cell-surface receptors and cell entry mechanisms. Cell tropism and pathogenicity depend on these receptors and on cellular trans-acting factors [9897, 998]. EV71EV-A71 and CA16CV-A16 but not CA6CV-A6 and CA10CV-A10, use the ubiquitous human scavenger receptor class B member 2 (hSCARB2) and the leukocyte P-selectin glycoprotein ligand 1 (PSGL-1) to infect a wide variety of host cells [10099,101100]. hSCARB2 is capable of viral binding, uncoating and internalization. The inefficiency of L-PSGL1-expressing cells is due to the inability of this receptor to
induce viral uncoating [102101]. hSCARB2 is ubiquitous and likely involved in EV71EV-A71 and CA16CV-A16 systemic and neural cell infections. Annexin-2, sialylated glycans, heparin sulfate and DC-SIGN receptors also play a role in EV71EV-A71 pathogenesis [103102-106105]. Both CB3CV-B3 and CB5CV-B5 use the decay accelerating factor (DAF/CD55) for primary attachment and the coxsackievirus and adenovirus receptor (CAR) as an internalization receptor [9998,107106]. CAR is an integral membrane protein localized to tight junctions highly expressed in developing brain and heart, and mediates CB3CV-B3 and CB5CV-B5 infection of cardiac myocytes as well as neural progenitor and stem cells leading to acute myocarditis and CNS involvement [107106-,1098]. Echovirus 30, the most common cause of aseptic meningitis in young infants, binds to the heterodimeric vitronectin v3 receptor controlling neural cell differentiation to induce productive infection of target cells and neuronal cell death through the activation of the TRIO-RhoA signaling pathway [110109,111110].
Epidemiology
Following the isolation of CA16CV-A16 in South Africa in 1951 [112111], a CA16CV-A16 outbreak occurred in 1957 in Toronto [113112] and the new illness was later described in 1960 by Aslop as "hand-foot-and-mouth" disease in 1960 [114113]. EV71EV-A71 was isolated in California in 1969 from a patient with CNS diseaseencephalitis [115114]. Since their discoveries, both viruses have been the cause of large life-threatening epidemics throughout the world, in North America, Europe, Australia, and Asia [5-79]. In the past decade, cyclic HFMD outbreaks of co-circulating or alternating EV71EV-A71 and CA16CV-A16 infections have become a major health problem in particular in the Asia-Pacific region [Table 1]. Severe EV71EV-A71 outbreaks were earlier reported in the USA, France, Hungary, Greece, Netherlands, Norway, and UK [5-7]. Large EV71EV-A71 epidemics in Malaysia (1997, 2005 and 2008), Singapore (2006, 2008), Taiwan (1998, 2008), southern Vietnam (2005), Ho Chi Minh City (2011) and Cambodia (2012) were associated with severe neurological complications and fatal outcomes [48-50,53,54,59,60,116 115 and Table I]. In the last 5 years, a significant increase in HFMD outbreaks
occurred in several provinces of Mainland China [12,117116]. In response, HFMD was declared a class C notifiable infectious disease by the Ministry of Health of China and a national surveillance system was established in 2008 [118117]. Shanghai experienced a major HFMD outbreak in 2010 [34]. EV71EV-A71 (54.1%) was the principal causative agent responsible for 86% of severe complications and 100% of fatalities. Several epidemics in Beijing (2013), Shangdong (2009), Shanghai (2011), Shenzhen (2013), Japan (2005-2009), Singapore (2004, 2006, 2008), Taiwan (2005-2008) and Thailand (2012) involved multiple enterovirus serotypes [15,31,36,40,42,4847,49,51,52,5657]. The most prevalent serotypes isolated in Japan between 2005 and 2009 were CV-A2, CA4CV-A4, CA6CV-A6, CA16CV-A16, CB3CV-B3, CB5CV-B5, E30E-30 and EV71EV-A71 [42] whereas in Taiwan the predominant HFMD-associated strains between 2004 and 2010 were CV-A2, CA4CV-A4, CA5CV-A5, CA6CV-A6, CA10CV-A10, CA16CV-A16, CV-B1, CV-B4, EV71EV-A71, E-6, and E-18 [52]. The dominant serotype varied depending on the outbreak and the country but EV71EV-A71 has always been the pathogen responsible for the majority of severe cases of neurological HFMD and fatal outcomes. Between 2008 and 2014, a total of 10,714,237 HFMD cases of HFMD were caused by EV71EV-A71 (43.4%) and CA16CV-A16 (34.4%). The average morbidity increased from 37.6/100000 in 2008 to 139.6/100000 in 2013. EV71EV-A71 and CA16CV-A16 were responsible for 90.2% and 8.7% of fatal outcomes, respectively [9].
However, CA16CV-A16 has beenwas the prevalent HFMD-associated enterovirus between EV71EV-A71 epidemics in Changchun (2008), Hebei (2012), Shangdong (2011) provinces in China, India (2009-2010), Japan (2009), Singapore (2004, 2007), Thailand (2010) and Spain (2010) [17,,24,29,31,39,40,41,44,47,49,58,67]. Although usually mild, CA16CV-A16 infection may lead to neural and muscle cells apoptosis [119118] and has occasionally been the cause of rhombencephalitis, brain stem encephalitis and acute flaccid paralysis, fatal pneumonitis and fulminant myocarditis with intractable shock [8].
Since 2004, the incidence of CA6CV-A6- and CA10CV-A10-associated HFMD epidemics has markedly increased worldwide [Table I]. Both viruses cause herpangina and occasionally meningitis, encephalitis and pleurodynia [2]. CA6CV-A6 was the first or second most common pathogen in large outbreaks in Cuba [10], China [15,17,21,37,38], India [40], Israel [80], Japan [42,45,46], Singapore [49,50], Taiwan [52.55], Thailand [56-58], Spain [67], UK [69] and USA [11]. CA6CV-A6 tends to be a virulent strain which unusually affects both pediatric and adult populations. It has been reported that 8.3% of CA6CV-A6-infected patients had high fever and developed meningoencephalitis [37,57]. In recent years, CA6CV-A6 and CA10CV-A10 have regularly co-circulated and have been independently high risk factors in HFMD outbreaks in China [13]. Dual outbreaks of CA6CV-A6 and CA10CV-A10 occurred in the Shenzhen province of in China (2013) [37], Finland (2008) [62], France (2010) [63] and Spain in 2008 and 2011 [65,66]. CA6CV-A6 was the prevalent serotype in Finland (71%) and in the 2011 outbreak in Spain (90%) whereas CA10CV-A10 was the predominant pathogen in France (39.9%), Spain in 2008 (45%) and during the onychomadesis outbreak in Valencia (50%) [66] [Table I].
Coxsackievirus CB3CV-B3, coxsackievirus CB5CV-B5 and echovirus 30 to a lesser extent have been found to frequently co-circulate with EV71EV-A71 and CA16CV-A16 during HFMD outbreaks around the world [Table 1]. However, they have rarely been detected together in the same epidemic. Their incidence in multi-serotype HFMD epidemics ranged from 0.3% to 14.7%, 0.7%to 19.0% and 0.6% to19% for CB3CV-B3, CB5CV-B5, and E30E-30, respectively. During the 2009 Shangdong epidemic, a significant proportion (78.6%) of patients with CB5CV-B5-associated HFMD suffered from neurological complications [30].
Although often detected during HFMD epidemics, coxsackieviruses CA4CV-A4 and CA5CV-A5 are predominantly associated withresponsible for herpangina outbreaks [86, 89-92]. However, CA4CV-A4 was the principal pathogen in the 2004 and 2006 HFMD epidemics in Taiwan [52].
Molecular epidemiology
There is only one EV71EV-A71 serotype. Based on VP1 gene phylogenetic studies, EV71EV-A71 has now been classified into six genotypes, A to F [72,120119]. Genotype A contains only the prototypic strain BrCr/1970. Genotypes B and C have been further divided into 6 and 5 sub-genotypes, B0-B5 and C1-C5, respectively. The C4 genotype has been further classified into C4a and C4b lineages [5-7]. More recently, additional genotypes including the Indian D genotype and two African ones (E and F) were identified, illustrating the wide genetic diversity of EV71EV-A71 [120119]. No association could be established between genotype and disease severity [7]. EV71EV-A71 epidemics occur throughout the year but usually peak in summer months. However, the seasonal distribution and cyclical patterns (every 2-4 years) of outbreaks vary depending on the year and the country [7]. Most countries use different case definitions, sample collections, data analysis and laboratory testing procedures to report HFMD cases, therefore disease burdens are likely underestimated.
Genotype A transiently re-emerged in China in 2008 [118117]. In contrast, genotypes B and C have continued to circulate and co-exist around the world since the 1970’s, causing outbreaks with CNS complications and fatal outcomes [7, 9, and Fig.1]. The first major EV71EV-A71 outbreak occurred in 1997 in Malaysia where co-circulating neurovirulent B3, B4, C1 and C2 genotypes were responsible for 41 deaths among young children [48]. In Singapore, sub-genotypes B5 and C2 caused the largest epidemic in 2008 [50]. Subgenogroups C2, C4 and B5 with genomic variations have repeatedly appeared during outbreaks in Japan between 1990 and 2013 [121120]. EV71EV-A71 has caused nationwide epidemics in Taiwan with different circulating genotypes and subgenotypes, C2 in 1998, B4 in 200-2001, C4 in 2005, and B5 in 2008 and 2012 [122121]. The C4 subgenotype emerged in 1998 in China with a predominance of the C4b lineage until 2009 followed by the exclusive occurrence of the C4a lineage until now. Between 2008 and 2014, C4 epidemics have been responsible for more than 10 million HFMD cases with a case-severity and case-fatality rates per year of 1.1% and 0.03-0.04%, respectively
[9,118117]. Recently, EV71EV-A71 sub-genotype C5 was observed in severe pandemics associated with high mortality rates that spread through Vietnam in 2005 and 2011, then to Cambodia in 2012 [59,60,116115].
Two CA16CV-A16 genotypes (A and B) have been identified [8]. The prototypic CA16CV-A16 genotype G-10 is the sole member of genotype A. Genotype B contains two subgenotypes B1 and B2 further divided into B1a, B1b, B1c, B2a, B2b and B2c. Genotypes B1a and B1b have been the predominant subgenotypes circulating in Australia and several provinces in China. However, subgenotypes B2a and B2b were identified in Shenzhen from 2005 to 2009 [123122]. A comparative study of the biological properties of two clinical isolates revealed that CA16CV-A16 strains of the B genotype may have different pathogenicity [124123].
CA6CV-A6 strains from the Shenzhen epidemic (2008-2012) were classified into seven clusters, A to F [36]. The predominant strain belonged to genogroup D whereas genogroup C prevailed in other areas of China. A majority of CA6CV-A6 strains isolated in the Shenzhen province were closely related to those detected during the outbreaks in Finland [62], Spain [65], France [63], Spain [65], and Japan [46]. CA10CV-A10 phylogenetic trees vary depending on the geography of the epidemic. Co-circulating CA10CV-A10 strains isolated during the epidemic in the Hebei province between 2008 and 2012 were found to segregate into four clusters (A-D), the C genotype being further divided into 4 lineages [24]. All Chinese CA10CV-A10 segregated into the B and C genotypes, genotype B in Shangdong and genotype C in the other provinces. Another phylogenetic analysis of CA10CV-A10 strains indicated that they cluster into seven genotypes (A to G) [36]. The predominant strain associated with HFMD cases in the Shenzhen outbreak between 2008 and 20120 belonged to genogroup C whereas CA10CV-A10 strains isolated during epidemics in the Shangdong and Jiangsu provinces (2008-2012), Spain (2008) and France (2010) segregated into genotype C [36]. CA6CV-A6 replaced CA16CV-A16 and toppled EV71EV-A71 to become the predominant pathogen in Shenzhen epidemics from 2008 to 2009 [37].
Five (A-E) genotypes (A-E) were assigned by phylogenetic analyses to coxsackieviruses CB3CV-B3 and CB5CV-B5 [24,125124]. All Chinese CB3CV-B3 isolates segregated into genotype E but the presence of two divergent circulating genotype E strains in Shijiazhuang City (2010-2012) suggests the existence of two different lineages. Chinese CB3CV-B3 strains clustered in a genogroup totally different from CB3CV-B3 isolates from other countries [24]. The severity of the Linyi epidemic in 2009 was attributed to the introduction of an unusual and distinct CB5CV-B5 lineage [30].
Echovirus 30 has been classified into eight clusters (A-H) [23] and the E30E-30-associated HFMD outbreak in Guanxi (2010) has been attributed to a strain belonging to the H lineage [23].
Genotype switching, co-circulation, co-infection and genetic recombination
EHuman enteroviruses have high mutation rates due to evolutionary pressure and frequent recombination. The HEV genome evolves at a rate of 1% to 2% mutation per year contributing to strain diversification [126125].
EV71EV-A71 epidemics occur cyclically every 2-4 years with changes in the predominant genotype and subgenotype. These epidemics can be the result of infection by a single genotype/subgenotype, the co-circulation of divergent EV71EV-A71 isolates, the emergence of variants or or the unpredictable switching of genotypes and sub-genotypes [126] [FidFig. 1]. Multiple serotypes, genotypes and subgenotypes frequently co-circulate during HFMD epidemics, thus facilitating co-infection and genetic recombination that may lead to the generation of new variants with altered tropism, virulence and fitness. Indeed, several mixed EV71EV-A71/CA16CV-A16, EV71EV-A71/CB3CV-B3, CA16CV-A16/CV-A10, CA16CV-A16/CB3CV-B3, CA16CV-A16/CB5CV-B5, CA16CV-A16/CA6CV-A6, CA10CV-A10/CA5CV-A5, CA10CV-A10/CA6CV-A6, CA10CV-A10/CV-B1, CA10CV-A10/E-9 infections9 infections have been well documented [29,30,32-34,128127,129128]. The EV71EV-A71 C4 genotype has persisted with progressive drift through time in China [9,118117,127126]. Intra-genotype EV71EV-A71 B shifts from B3 to B4 (1997-2000) and B4 to B5 (2000-2003) have occurred in Malaysia. Sequential inter-genotype shifts from C2 to B4 and subsequently from C4 to B5 were observed in Taiwan
[7,122121,127126]. The co-circulation of several EV71EV-A71 genotypes and CAV16 CV-A16 during HFMD epidemics has been responsible for intra-typic genetic recombination between EV71EV-A71 B and C genotypes in Taiwan and inter-typic recombination between EV71EV-A71 and CVA16 in China [5,7,14,19,127126,129129]. Another inter-serotypic recombination happened between EV71EV-A71 genotype C2 and CVA8 to create genotype B4, responsible for outbreaks in Japan and Taiwan in 1998 [7,127126]. The predominant C4a genotype may be a double recombinant virus among EV71EV-A71 genotypes B, C and CVA16 [7,127126]. Almost each major HFMD outbreak was correlated to genetic variations caused by EV71EV-A71 switches [7].Phylogenetic analyses have revealed that CA16CV-A16 B1a and B1b strains circulating in China were complex recombinant forms containing sequences from multiple HEV-A donors [130129,131130]. These co-infection and recombination events have been associated with disease severity. One example is the inter-typic recombination that led to the emergence of new B3/B5 variants responsible for acute myocarditis in children with HFMD [132131]..
Thus, a continuous monitoring of antigenic variation and genetic evolution of HFMD-associated enteroviruses is critical to determine the mosaic composition of an epidemic, design vaccines and plan efficacy trials. In this regard, an enhancement of national physician-based sentinel surveillance clinics and the creation of a global surveillance network for enterovirus outbreaks similar to the WHO surveillance system for influenza are urgently needed. An automated alert and response system evaluated in China has shown good sensitivity and specificity in the detection of HFMD outbreaks [133132] and improved assays to identify multiple pathogens simultaneously are currently being developed [26,133].. Hand, foot and mouth disease vaccines
Clinical and molecular epidemiology data confirm that in the last decade [Table 1], CA16CV-A16 and EV71EV-A71 were the most prevalent aetiological agents of HFMD and that EV71EV-A71 was the most neurovirulent serotype. CA6CV-A6 and CA10CV-A10 co-circulated in 85% of all epidemics. and HFMD-associated coxsackieviruses CB3CV-B3, CB5CV-B5 and Echovirus E30E-30 that were globally detected in a third of the outbreaks remain potential serious threats to neonates and young infants due to
their neurotropism and cardiovirulence. Although CA4CV-A4 and CA5CV-A5 were identified in 45% of multiple-serotype epidemics, they are usually associated with HA outbreaks. Thus, an heptavalent vaccine including EV71EV-A71, CA16CV-A16, CA6CV-A6, CA10CV-A10, CB3CV-B3, CB5CV-B5 and E30E-30 immunogens could be designed to protect against the vast majority of pathogenic HFMD-associated HEV serotypes.
1. EV71EV-A71 vaccines: The first step towards multivalent HFMD vaccines.
The development of a vaccine against EV71EV-A71 has been a health priority because of its neurovirulence. It is also the first step towards the development of a multivalent HFMD vaccines [135134].
i. Recent EV71EV-A71 vaccine development
Several EV71EV-A71 candidate vaccines are still at the pre-clinical stage [72,136135,137136]. Synthetic vaccines based on immunodominant linear neutralization epitopes are safe, cost-effective, but poorly immunogenic even when formulated with Freund’s adjuvants. Multi-linear tandem neutralization epitopes expressed in E. coli might be more promising [138137]. Among EV71EV-A71 subunits, recombinant VP1 subunits produced in different expression systems including Pichia pastoris [139138] were capable of eliciting good antibody responses and protection in suckling mice when formulated with strong adjuvants [140139]. VP1 anchored on the surface of baculovirus via a transmembrane domain induced cross-neutralization responses in mice and conferred protection in passive immunization studies [141140,142141]. A plasmid DNA vaccine expressing VP1 was only moderately immunogenic. DNA vaccines do not elicit strong antibody responses [143142] and have never been commercialized as human vaccines. Passive transfer of immune sera from mice vaccinated with an adenovirus vector exposing an EV71EV-A71 neutralization epitope on its surface protected suckling mice from live viral challenge but not as efficiently as an inactivated EV71EV-A71 vaccine [144143].
Recombinant virus-like particles (VLPs) mimic the conformation of authentic native viruses and are safe because devoid of viral genome. Prophylactic VLP-based vaccines against hepatitis B virus and human papillomavirus are currently commercially available. EV71EV-A71 virus-like particles produced in the baculovirus system [139138] and in Saccharomyces cerevisiae [145144] induced robust neutralization responses in mice as well as potent cellular responses and immune sera conferred protection in neonatal mice against lethal EV71EV-A71 challenge. Their safety, immunogenicity and high-yield production make them attractive candidates for future combination vaccines. However, EV71EV-A71 VLPs elicited only low neutralizing titers in macaques [146145]. Several immunogens have been evaluated in oral immunization studies. Sera from mice fed with transgenic tomatoes expressing VP1 exhibited neutralizing activity in vitro [147146]. Oral immunization of maternal mice with VP1 formulated with chitosan [148147], recombinant baculovirus displaying VP1 mixed with bilosomes [149148], Salmonella
Typhimurium and Bifidobacterium longum vectors expressing VP1 [150149,151150] conferred protection to neonatal mice. Although a safe live attenuated EV71EV-A71 would be an ideal and low-cost vaccine, such vaccine exposes to the risk of genetic instability and the possibility of reversion to virulence. An engineered, temperature-sensitive EV71EV-A71 BrCr mutant was shown to be less neurovirulent and to elicit cross-neutralizing antibody responses but it caused mild tremor in cynomolgus monkeys [152151]. However, the possibility to engineer attenuated high-fidelity-variants of EV71EV-A71 with low pathogenicity could be a promising approach for future live vaccines [153152].
In a comparative study of prototyptic EV71EV-A71 vaccines produced by different technologies, formalin-inactivated EV71EV-A71 virions adjuvanted in alum were found to be very immunogenic, to elicit strong cross-neutralization titers against different EV71EV-A71 genotypes and subgenotypes in mice and non-human primates and to be the most potent and promising immunogens [72,140139,154153,155154].
ii. Formalin-inactivated EV71EV-A71 vaccines and efficacy trials
Based on the promising results of pre-clinical studies and the efficacy of the inactivated poliovirus vaccine (IPV), for regulatory, economic and market acceptability reasons formalin-inactivated A71 virions were selected for the clinical development of stable and cost-effective monovalent EV71EV-A71 vaccines [72]. Five inactivated EV71EV-EV71EV-A71 vaccines have been rapidly developed in the past few years [72,137136] The Vaccine R&D Center of the National Health Research Institutes (NHRI) of Taiwan produced a B4-based FI-EV71EV-A71 vaccine (EV71EV-A71vac) and launched the first human Phase I clinical trial in adults in 2010. A single vaccine dose of 5 µg or 10 µg was safe and highly immunogenic [156155]. It elicited 100% seroconversion in naïve volunteers and strong virus neutralizing antibody (VNA) responses (geometric mean titer (GMT) = 210) against the vaccine strain and the B1, B5 and C4a subgenotypes in 85% of the vaccinees [157156]. In contrast, neutralizing responses against C4b and CVA16 were weak in 20% of the subjects and 90% of the vaccinees did not develop any VNA against an atypical C2 strain. Inviragen (Takeda Pharmaceuticals Co. Ltd) reports the results of a Phase I trial in adults with an inactivated EV71EV-A71 B2 vaccine. All subjects who received 0.6 ug or 3 ug of vaccine at days 0 and 28 seroconverted and developed VNA GMTs of 323 and 452, respectively [138136].
Inactivated EV71EV-A71 vaccines based on different C4 isolates were independently developed and evaluated by three different Chinese companies, Vigoo Biological, Sinovac Biotech, and the Institute of Medical Biology, Chinese Academy of Medical Sciences (CAMS) [158157-161160]. The clinical efficacy of these vaccines formulated in alum was assessed in large Phase III trials involving more than 30,000 healthy infants and young children (6 to 35 months of age) who received two doses of vaccine 28 days apart or a placebo control. All three vaccines were found to be safe and well tolerated. The most common side-effects observed were indurations, erythema and pain at the injection site that resolved within 24-72 hours and as well as a grade-3 fever. The rate of seldom-reported serious adverse events
(SAEs) in vaccinees was not different from that observed in the control groups and were not causally related to vaccination. The Vigoo’s vaccine [159158] was >90% efficacious against EV71EV-A71-related HFMD and >80% protective against EV71EV-A71-associated serious diseases including herpangina. In the Sinovac’s trial, the incidence rate of EV71EV-A71-associated disease was 0.3% vs 2.1% in the control group, corresponding to an 89.3% efficacy [160159]. In the CAMS’ study [161160], the seroconversion rate was 100% after two vaccinations, with a VNA GMT of 170.6. The vaccine was 97.4% efficacious against EV71EV-A71-related diseases. All C4-based vaccines prevented herpangina and EV71EV-A71-associated hospitalizations. Immune sera from subjects immunized with the Vigoo’s and Sinovac’s vaccines cross-neutralized the circulating EV71EV-A71 genotypes and subgenotypes (B4, B5, C2, C5) associated with epidemics in recent years [162161]. Furthermore, pre-existing antibodies due to stealth infections of young children did not interfere with vaccine efficacy against different EV71EV-A71 genotypes [162161]. However, the vaccines did not protect against CAV16 [158157,159158] and conversely, CA16CV-A16 infection does not interfere with EV71EV-A71 vaccination [163162]. Interestingly, the VNA titers decreased by half after 6 months but this waning did not affect vaccine efficacy [158157]. Most importantly, Phase III results suggest that a VNA titer of 1/16 can serve as a correlate of protection against EV71EV-A71-related HFMD [158157,159158]. In spite of differences in vaccine strains and manufacturing processes, C4-based vaccines have shown batch consistency and efficacy [164163] which should facilitate their licensure and market entry in China if there were no issues regarding vaccine stability, manufacturing capacity and production cost for which information is yet not available.
2. Development of a bivalent EV71EV-A71/CA16CV-A16 vaccine: The next critical step.
Except for the rare instances when EV71EV-A71 is virtually the only causative agent of HFMD, monovalent EV71EV-A71 vaccines will only protect against a fraction of HFMD cases, in particular when if the aetiologyetiology of HFMD changes over a short period of time [29] and CA16CV-A16
infections become predominant (50%-72%) [17,24,29.31,40,41,49,58 and Table 1], thus raising the issue of public acceptance of an EV71EV-A71 vaccine. The availability of a bivalent EV71EV-A71/CA16CV-A16 would critically enhance the breadth of protection against HFMD and a combination vaccine is highly desirable. Six conserved, KLH-conjugated VP1 peptides formulated in complete Freund’s adjuvants induced neutralizing antibodies against both homologous and heterologous CA16CV-A16 strains [165164]. The development of chemically-inactivated monovalent CA16CV-A16 vaccines produced in Vero or KMB cells and adjuvanted with alum paved the way towards that of a bivalent vaccine [166165-169168]. Monovalent CA16CV-A16 vaccines have induced neutralizing antibody responses against the vaccine strain [166] and against heterologous CA16CV-A16 isolates [167166,168167]. Furthermore, vaccination conferred full protection to mice lethally challenged with the mouse-adapted strain CA16CV-A16-MAV [1667] and maternal immunization protected neonatal mice from challenges with a series of circulating CA16CV-A16 isolates [169168]. Virus-like particles (VLP) produced in Saccharomyces cervisiae have also elicited potent neutralizing responses and passive transfer of immune sera protected neonate mice against lethal CA16CV-A16 challenge [170169].
Combination of inactivated EV71EV-A71/CA16CV-A16 vaccines formulated in alum [171170] or with PELC/CpG [172171] elicited balanced neutralizing responses against both viruses whereas monovalent CA16CV-A16 vaccines did not protect against EV71EV-A71 infection. Furthermore, maternal immunization of mice with the bivalent vaccine protected neonates challenged with the mouse-adapted EV71EV-A71/MVA-N strain and the clinical isolate CA16CV-A16/G08 [171170]. Maternal immunization with a VLP-based bivalent vaccine produced in the baculovirus system and adjuvanted with alum conferred full protection to newborns against lethal challenge either with EV71EV-A71 or CA16CV-A16 [173172]. Crystallographic studies revealed that the EV71EV-A71 BC loop could serve as an ideal insertion site for the display of foreign neutralization epitopes without perturbing the capsid structure [9493], thus providing the mean to engineer potential EV71EV-A71/CA16CV-A16 hybrid immunogens. Along a similar concept, antisera raised in mice vaccinated with a novel chimeric
EV71EV-A71-based VLP in which the autologous neutralization epitope SP70 had been replaced by that of CA16CV-A16 conferred protection in neonates against lethal challenge in a passive transfer experiment [174173]. EV71EV-A71 and CA16CV-A16 vaccines produced both as inactivated virions and VLPs were compared for their immunogenicity and protective ability when administered either alone or in combination. Monovalent and bivalent vaccines adjuvanted with alum induced the same level of strain-specific neutralizing antibodies confirming that there is no interference between immunogens in the bivalent vaccine. All bivalent vaccines elicited cross-neutralizing antibodies against 12 EV71EV-A71 and 6 CA16CV-A16 sub-genotypes, respectively. Passive transfer of immune sera conferred protection in newborn mice against lethal challenge with both viruses although bivalent VLPs vaccines were more potent than individual VLPs formulations [175174].
3. Multivalent HFMD vaccines: The ultimate need, the ultimate goal..
Results obtained with bivalent EV71EV-A71/CA16CV-A16 vaccines serve as proof of concept for a two-step development of a multivalent vaccine necessary for broad protection against HFMD. Based on epidemiological data, we propose that the first step should be to generate a tetravalent vaccine containing EV71EV-A71, CA16CV-A16, CA6CV-A6 and CA10CV-A10 [135134,176175] to cover the most prevalent HFMD pathogens, then to further incorporate CB3CV-B3, CB5CV-B5 and E30E-30 immunogens in a heptavalent vaccine to prevent the risks of aseptic meningitis and acute carditis associated with these viruses in the course of multi-serotype HFMD epidemics [135134]. The addition of CA4CV-A4 and CA5CV-A5 components components should not be considered at this stage since they are essentially responsible for HA outbreaks..
There is little information on CA6CV-A6 and CA10CV-A10 immunological properties. A bivalent EV71EV-A71/CA16CV-A16 vaccine induced strong humoral responses in mice and rabbits, but immune sera did not neutralize CA6CV-A6 nor CA10CV-A10 in an in vitro assay [176175]. CA6CV-A6 and CA10CV-A10 VLPs were found to be highly immunogenic in mice. but only anti-CA10CV-A10 antisera
were tested and shown to neutralize CA10CV-A10 infection in vitro. The protective ability of CA6CV-A6 and CA10CV-A10 VLPs need to be assessed in an appropriate animal model. However, an experimental tetravalent vaccine combining inactivated EV-A71, CV-A16, CV-A6 and CV-A10 was recently found to elicit neutralizing antibody responses in mice against all four viruses, indicating that producing such a vaccine is highly feasible (Dr CC. Liu, personal communication).
Since the early attempts to develop a live attenuated temperature-sensitive CB3CV-B3 mutant vaccine against myocarditis in 1997, several recent approaches have been evaluated for their potential to protect against CB3CV-B3 infection. Previous studies have highlighted the importance of both humoral and cellular immunity in preventing CB3CV-B3-induced disease [177176]. A -propionolactone-inactivated CB3CV-B3 strain formulated with Quil A matrix or ISCOMs induced neutralizing antibodies and protected mice against myocarditis but was abandoned for technical issues and the lack of interest from the industry [177176,178177]. Several types of either natural or engineered attenuated vaccines have induced protection against experimental myocarditis and pancreatitis but they are prone to antibody-dependent enhancement of disease, reversion to cardiovirulence, and persistent infection in the target tissues [177176]. Very recently, an attenuated CB3CV-B3 Sabin3-like strain administered orally induced a protective immune response in mice but a limited amount of pancreatic inflammation was still detected in some challenged animals [179178]. Priming with a DNA vector expressing CB3CV-B3 VP1 followed by two VP1 subunit boosts induced neutralizing antibodies and cytotoxic T cells but was only partially protective against live CB3CV-B3 challenge challenge [180179]. Intranasal co-administration of a encapsulated plasmid DNA vector expressing VP1 (chito-pDNA-VP1) and a second chitosan-DNA plasmid producing the high mobility group box 1 protein as an immunostimulant induced both systemic and mucosal immune responses and reduced the viral load and the severity of CB3CV-B3-induced myocarditis [181180]. Vaccination with CB3CV-B3 VLPs produced in the baculovirus expression system and formulated in complete or incomplete Freund’s adjuvant have induced neutralizing antibody titers of 1/320 that conferred incomplete protection upon passive immune serum
transfer to mice challenged with a cadiovirulent virus [182181]. Chromatographically-purified VLPs elicited higher neutralizing antibody titers (1/1100) and an increase in effector-memory T cells. However, VLPs were less immunogenic than a formalin-inactivated CB3CV-B3 vaccine used as positive control and challenge experiments were not performed [183182]. The immunoprotective ability of a recombinant vesicular. stomatitis virus (VSV) vector expressing CB3CV-B3 VP1 was compared to that of a chitosan-pDNA-VP1 vaccine following intranasal administration. The VSV-VP1 vaccine induced significantly higher levels of antigen-specific, systemic and mucosal antibody responses than chitosan-pDNA-VP1 as well as strong polyfunctional T-cell responses and dendritic cell maturation, but was not fully protective against a 50% lethal dose of live CB3CV-B3 [184183]. There is no information available on CB5CV-B5 and E30E-30 vaccine research activities.
Challenges for a multivalent HFMD vaccine registration
HFMD epidemics will persist for a long time owing to the co-circulation of multiple pathogens, the occurrence of co-infection and recombination, the ever increasing number of travelers and migrants, and the lack of a multivalent vaccine. However, it would be overoptimistic to think that such a vaccine will be available soon because of the numerous challenges faced by its development.
i.Cross-protective ability of a multivalent vaccine and selection of potential vaccine strains.
A multivalent vaccine should ideally protect against all genotypes and subgenotypes of HFMD-associated viruses due to the unpredictability of the composition of epidemics and the emergence of potentially new variants.
With respect to the monovalent EV71EV-A71 vaccine, both C4-based and B4-based vaccines cross-neutralized the current circulating EV71EV-A71 isolates [157156-162161], but the B4 vaccine poorly neutralized an atyptical C2 strain [157156]. However, the degree of cross-neutralizing activity of immune responses induced by the C4- and B4-based vaccines against viruses from genotypes D, E and F remains to be evaluated. However, due to its good cross-immunogenicity as well as its large contribution to
endemicity and HFMD epidemics, the C4a strain emerges as the best candidate for inclusion in a multivalent vaccine. Only results from international efficacy trials conducted in regions and countries where different epidemic enterovirus A71 circulate will help assess the breadth of the cross-protective ability of a C4a-based vaccine and determine whether additional genotypes/subgenotypes need to be included in a universal EV-A71 vaccine.
FI-EV71EV-A71 vaccines failed to protect against CVA16 infections that are predominantly responsible for annual HFMD outbreaks [158157-163162]. It is likely that FI-EV71EV-A71 vaccinations may may not significantly reduce the number of clinical cases of HFMD during outbreaks. In this regard, the introduction of a protective bivalent EV71EV-A71/ CA16CV-A16 vaccines on the market should markedly reduce the number of HFMD cases [171170-175174]. CA16CV-A16 strains that have elicited both homologous and heterologous protection against genotypes A and B in pre-clinical studies are potential candidates for a multivalent HFMD vaccine [1667-169168]. The selection of CA6CV-A6, CA10CV-A10, CB3CV-B3, CB5CV-B5 and E30E-30 vaccine strains will have to be based on comprehensive epidemiological information .to identify the most prevalent circulating genotype(s) for each enterovirus serotype. Immunogenicity studies will be necessary to determine whether like for EV-A71 and CV-A16, vaccine strain candidates induce cross-neutralizing antibody responses against most or all of their respective genotypes. In the absence of broad cross-neutralizing activity, the need for more than a restricted number of vaccine strains for each EV serotype would be a serious obstacle to the production of multivalent HFMD vaccines. Because of the risk of inter-typic and intra-typic recombination and the possible emergence of new strains with increased virulence, only results from multinational efficacy trials with a multivalent vaccine will reveal if it can elicit broad protection against divergent epidemic viruses in the target age group. Severe HFMD cases would not be suitable as clinical end points due to their low frequency. Selecting herpangina or mild illness would be more appropriate. The efficacy of a multivalent vaccine against coxsakieviruses B and echovirus E30E-30 infections should also be assessed during aseptic meningitis and acute myocarditis outbreaks. In this regard, the
harmonization and standardization of virus strains, immunoassays and rapid diagnostic tools should be established at the national and international levels. A global surveillance network for enterovirus outbreaks and a rapid response system are is also urgently needed.
ii. Duration of humoral immunity, role of cellular responses and oral immunization.
Phase III trials have unambiguously revealed that humoral immunity is protective against EV71EV-A71 infection. But a significant waning of neutralizing antibody titers during the first 6 months after 2 vaccinations was noticed [158157]. Importantly, the risk of subneutralizing antibody levels exposing to antibody-dependent enhancement of disease described for EV71EV-A71, CA16CV-A16 and CB3CV-B3 should be prevented [173172,177176,185184]. In an early Phase II trial, 773 participants who had received at least 1 dose of EV71EV-A71 vaccine were enrolled to receive a vaccine booster dose [186185]. A 10-fold increase at least in neutralizing antibody responses was induced by the booster injection. The booster dose was very immunogenic and well tolerated. Phase IV clinical trials will determine whether the current schedules and vaccine doses need to be optimized and whether a third immunization at 18-24 months is necessary to ensure long-lasting protection. The development of mucosal vaccines to prevent viral entry in the gastrointestinal tract is attractive, but it may not be necessary since parenteral immunization confers protection. Although live-attenuated viruses would be the best vaccines to induce both systemic and mucosal immunity as well as immune memory, the risk of reversion to virulence remains a major obstacle to their development.
Inducing polyfunctional T-cell responses and broad T-cell memory might be particularly critical for clearing coxsackievirus B infections. The vaccine might require immunogens and adjuvants/delivery systems different from inactivated viruses formulated in alum. Prospective studies should be conducted during EV71EV-A71 and coxsackieviruses epidemics to assess the role of cellular immunity in long-term cross-protection and viral pathogenesis. In addition, longitudinal studies are necessary to evaluate the role
of multivalent vaccines in controlling antigenic shift, virus fitness and the emergence of new virus variants.
iii. Standardized animal models for vaccine potency test
Standardized animal models necessary to understand HEV-A pathogenesis and evaluate the potency and consistency of vaccine batches are not yet available [187186]. Mouse-adapted strains, neonatal suckling mice and immunodeficient animals have been widely used to evaluate the protective efficacy of EV71EV-A71 and CA16CV-A16 vaccine candidates, but they do not mimic human infections [169168,187186,188187]. In contrast, cardiac pathogenesis in Balb/c and SWR mice infected with CB3CV-B3 is very similar to that of human patients [178177]. Macaques develop antibody responses to EV71EV-A71 vaccines similar to those observed in human; however they are not suitable to study neurovirulence and pulmonary edema complications and their use is limited by ethical and economic considerations [187186]. Transgenic mice carrying the human receptor hSCARB2 [189188,190189] develop HFMD-like skin rashes upon infection with EV-A71 B4 and B5 clinical EV71 isolates and severe limb paralysis and death occurred in animals inoculated with a C2 strain [189188]. The presence of EV71EV-A71 in tissues and CNS was accompanied by the up-regulation of pro-inflammatory mediators (CXCL10, CCL3, TNF-α, and IL-6) and correlated with the recruitment of T lymphocytes and disease severity [189188]. In addition, passive administration of the monoclonal anti-EV71EV-A71 VP1 neutralizing antibody N3 reduced symptoms induced by EV71EV-A71 B5 infection and protected the transgenic mice against EV71EV-A71 C2-induced severe limb paralysis and death [191190]. Once standardized, the transgenic mouse model will be useful to assess the cross-protective ability of vaccines against coxsackieviruses A using hSCARB-2 as receptor. However, CA6CV-A6 and CA10CV-A10 do not use this receptor.
An ideal multivalent HFMD vaccine should be inexpensive, safe, compatible with large-scale production, easy to administer and acceptable to parents. There is an urgent need to improve and scale-up the current manufacturing processes for inactivated vaccines for broad approval of EV71EV-A71 vaccines by regulatory authorities. Due to intellectual property rights and proprietary technologies, information on the influence of culture medium and production systems on vaccine yields is totally missing. Both the roller-bottle and cell factory technologies used in producing current clinical lots are easy to implement and operate, although labor intensive. Developing countries could start implementing these technologies first and subsequently optimize the manufacturing processes for large-scale vaccine production. Other technologies could potentially be used in the future to increase virus yields. They include the selection for each virus of optimal cGMP-compliant cell lines, the transfection of more viral receptor genes into host cells or the removal of genes inhibiting viral replication to enhance virus production. Reverse-genetic could also be used to improve virus yields by inserting specific protease cleavage sites to increase virus infectivity [72,136135]. The development of a multivalent HFMD vaccine remains a challenging task. Although inactivated poliovirus and EV71EV-A71 vaccines have been successfully developed and inactivated CA16CV-A16 candidate vaccines are very promising, there is some indication that VLP-based bivalent vaccines are more immunogenic than a combination of inactivated EV71EV-A71/CA16CV-A16 vaccines [175174]. It is not clear at the present time whether the production yields for EV71EV-A71 and other HFMD-associated viruses will be sufficient to meet global needs. The selection process for an ideal vaccine strains needs to be addressed in the light of comprehensive epidemiological surveys, the optimal technology to efficiently produce potent and safe immunogens must be defined for each serotype, and clinical trials will have to be conducted for each individual vaccine before combining them. Large-scale production of EV71EV-A71 vaccines will require an improvement of the current manufacturing processes. The use of bioreactors, micro-carriers and perfusion technology could increase cell growth and virus yield by one order of magnitude. To lower the production cost, a simple and efficient downstream chromatographic purification step could be optimized
to co-purify immunogenic defective and infectious virus particles [9796,136135,176175]. A similar approach could be applied for the production of the other enteroviruses. If the yields of inactivated vaccines could not be improved, VLP-based vaccines are an alternative. They are good and safe immunogens that can be produced at the industrial scale and two VLP-based vaccines (HBV, HPV) have already been successfully commercialized. One challenge in the development of multivalent HFMD vaccines will be to avoid interference between their various immunogens and between potentially different adjuvants in order to ensure vaccine stability and consistent immunogenicity. However, two hexavalent diphtheria, tetanus, acellular pertussis, Haemophilus influenzae type b, poliovirus and hepatitis B (DTaP-Hib-IPV-HepB) combination vaccines that contain 10 different immunogens have been successfully licensed and commercialized and contribute to a significant increase in the infant vaccine coverage.
v. Strategy for immunization
Approximately 50% of neonates have neutralizing anti-EV71EV-A71 antibody titers that decline to be undetectable after 6 months [192191] while the disease peak occurs between one and two years of age. Ideally an HFMD vaccine should target infants before or at 6 months of age. This vaccination schedule will overlap with the administration of other pediatric (DTaP, Hib, IPV, HepB) combination vaccines, Rotavirus and Pneumococcal vaccines depending on the country. Pre-clinical studies and human trials should demonstrate that the co-administration of an inactivated multivalent HFMD vaccine with these vaccines will alter neither its immunogenicity nor the potency of the co-administered vaccine. In this regard, preliminary studies have shown that co-immunization of an inactivated EV71EV-A71 vaccine with PediacelPedicel (Sanofi Pasteur) did not alter antibody responses to individual vaccine components [193192]. Furthermore, the immunogenicity of an inactivated EV71EV-A71 vaccine was not affected by pre-existing anti-CA16CV-A16 or anti-poliovirus neutralizing antibodies nor by its co-administration with CA16CV-A16 and poliovirus 1,2,3 vaccines [194193].
vi. Economic issues
HFMD is a significant health and economic burden for the Asia Pacific region in particular for China. An accurate estimation of this burden will require an enhancement of the surveillance system, an improved training of physicians in particular in rural areas and the generalized availability of rapid diagnostic tools. It has been estimated that the average direct and indirect cost was US$129 for an outpatient, $484 for an inpatient, and $1936 for a severe case of HFMD, respectively. The annual economic burden of EV71EV-A71-associated HFMD only amounts to US$161-323 million per year [195194]. This figure is still optimistic since it does not take into account the fact that the number of annual EV71EV-A71 cases is likely underestimated, nor the number of HFMD cases caused by other enteroviruses, nor the costs linked to sequelae and fatalities. Clinical trials have revealed that the administration of 400 units (1µg) dose of EV71EV-A71 vaccine twice achieved efficacy [72,158157,161160]. A recent publication [136135] reports that a 40-liters pilot-scale production batch could yield 50,000 1µg doses of FI-EV71EV-A71 at a cost of 0.4 US dollar/dose. This translates into 200,000 doses of the lowest C4-vaccine protective dose (0.25 µg) as determined in a Phase III trial [72,158157] at 0.1 US dollar/dose. Assuming similar potency and yields for the other components of a heptavalent HFMD vaccine, one could speculate that the production cost of the vaccine would be in the range of US$ 1 per dose. It has been forecasted that routine immunization with a 70% efficacious EV71EV-A71 vaccine sold at 25 US dollars/dose would be of great economic value [196195]. Another recent study reports that immunization with an EV71EV-A71 vaccine would be cost-effective if vaccination costs were ≤ US$75 per dose for 90% efficacy [195194]. With this profit margin and the new emerging vaccine markets in the Asia-Pacific region, the global vaccine companies should become interested in manufacturing monovalent, bivalent and ultimately multivalent HFMD vaccines.
A multivalent HFMD vaccine is an unmet medical need for a life-threatening disease with huge economic burden. Although EV71EV-A71 is responsible for the vast majority of severe complications and fatalities, a monovalent EV71EV-A71 vaccine may not significantly reduce the number of HFMD cases which poses a problem of public acceptance. EV71EV-A71 vaccines will move soon to market in China and a bivalent EV71EV-A71/CA16CV-A16 vaccine should enter Phase 1 I clinical trials in the near future. Based on the current clinical and molecular epidemiology data, there is a strong rationale for including CA6CV-A6 and CA10CV-A10 immunogens in the next tetravalent vaccine. This vaccine would form the core of an ultimate heptavalent HFMD vaccine incorporating CB3CV-B3, CB5CV-B5 and E30E-30 viral components. The safety and efficacy of poliovirus and EV71EV-A71 vaccines should serve as a rationale for the stream-lined development of a multivalent vaccine based on inactivated viruses. Improvement in manufacturing processes will still be needed to produce cost-effective viral immunogens. The development of a heptavalent HFMD vaccine is feasible but it remains a major undertaking and challenge that will require the cooperation of epidemiologists, policy makers, market analysts, Research and Development Institutes, Governments, and the global vaccine industry. The implementation of EV71EV-A71 vaccination programs might change the landscape of HFMD epidemics by creating a niche for other viruses as a result of selective pressure. Thus, the establishment of a global surveillance system is urgently needed to monitor the safety and long-term immunogenicity of the EV71EV-A71 vaccines, determine whether booster doses are necessary, and detect potential changes in the composition of future HFMD epidemics. Ultimately, the combination of a multivalent HFMD vaccine with Expanded Programme on Immunization vaccines would greatly simplify immunization schedules. Such an achievement would be another "invaluable gift to children" [197196] and a new milestone in the history of combination vaccines.
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