Background

Hand, foot and mouth diseases (HFMD) are common self-limiting illness in infants and young children, characterized by ulcerating vesicles in the mouth and lesions on the hands and feet. Small outbreaks of mild HFMD have occurred periodically throughout the world for a long time. Two closely related viruses, coxsackievirus 16 (CA16) and enterovirus 71 (EV71) have been identified as the most frequent pathogens of HFMD, and other enteroviruses, including CA5 and CA10, can also cause HFMD. While since 1997, large outbreaks of HFMD associated with severe neurological complications and a high case-fatality rate have been reported in Malaysia [1], Taiwan [2], Singapore [3], Japan [4] and other Asian-Pacific areas. In mainland China, large outbreaks of HFMD have been reported since 2008, resulting in millions of cases and hundreds of deaths in children [10], and large outbreaks are probably associated with genotype replacement [8]. A recent report has shown that repeated EV71 infection is common during large outbreaks in China [29]. This is particularly important considering the fact that most primary EV71 infections are asymptomatic.

The target cells of EV71 in human tissues and organs have not been identified. Previous experiments showed that coxsackieviruses could replicate in lymphocytes and monocytes [17, 30]. Human monocytic cell line THP-1 has Fc and complement receptor that is used for in vitro ADE assay in this study. The mechanism for ADE of EV71 infection is still unknown. Different mechanisms for ADE has been hypothesized, in which Fc receptor, complement receptor, β2-microglobulin have been reported to play a role. Wang et al recently demonstrated that Fcγ-receptor may participate in EV71-induced ADE based on results from THP-1 cells [19]. Arita et al also reported high-affinity Fc receptor mediated enhancement of poliovirus infection [31]. Further, EV71 encodes 4 structural proteins (VP1-4), and the viral antigenic determinants associated with ADE of EV71 are also critical for future investigation. Recently, Chehadeh et al. [32] demonstrated that VP4 protein is the major target of enhancing antibodies in the CVB4 and CVB3 ADE model. By using monoclonal antibodies screening, the prM protein of dengue virus was recently identified as the critical ADE determinants [33]. We have produced a panel of EV71 mAbs and such efforts are currently underway. Additionally, ADE infection of non-enveloped virus requires binding of virus-antibody complexes both to Fcγ-receptor and to the specific viral receptor [34]. The role of PSGL-1 [35] and SCARB2 [36], two EV71 receptors, during ADE of EV71 infection deserves further research.

IVIG is a pharmaceutically preparation of human IgG that has immunoregulatory and anti-inflammatory properties. Clinically administration during EV71 outbreaks in Taiwan [37, 38] had achieved an improvement in clinical outcome. The Ministry of Health of China has recommended high dose (total 2 g/kg in 2-5 days) of IVIG for treatment of severe EV71 infection. However, considering the fact that IVIG contains anti-EV71 antibodies [22], the potential risk of IVIG induced ADE of EV71 infection, especially the dose, must be carefully considered in future clinical application. The enhancing concentrations of IVIG may vary. Our experiments showed 50 μg/ml of IVIG enhanced EV71 infection in vitro, while 500 μg/ml in vivo. Wang et al showed that 0.5-1 or 8-16 mg/ml of IVIG enhanced EV71 infection in THP-1 cells. The difference may results from the different sources of IVIG and the methodology applied [19, 22].

Given the public health significance of EV71, a wide range of experimental EV71 vaccine approaches have been studied, and neutralizing antibodies have been suggested as one of the most important factors in limiting the severity of infection. But considering the possible impact of ADE, vaccines should be carefully designed to avoid the induction of known enhancing antibodies. Antibody responses induced by inactivated or subunit vaccines tend to be associated with severe disease by respiratory syncytial virus and retroviruses [39, 40]. Induction of cellular immunity, rather than antibodies, may represent an alternative strategy for future EV71 vaccine development [41]. Live attenuated vaccine other than inactivated vaccine will be a good candidate.

Taken together, the present findings from laboratory and animal experiments demonstrate that the presence of sub-neutralizing antibodies would enhance EV71 infection, and primary EV71 infection might represent a risk factor of severe diseases during sequential EV71 infection. Controlled clinical trials are critically needed to confirm the correlation between disease severity and ADE. Also, much effort is currently underway to determine the cellular and viral determinants of EV71-induced ADE. The results of this and future studies thus contribute to a better understanding of the pathogenesis of EV71 infection, as well as an improved ability to evaluate vaccines and therapeutic antibodies.

Materials and methods

Viruses and cells

Human rhabdomyosarcoma (RD) cells were cultured with DMEM supplemented with 2% fetal bovine serum (FBS), 100 IU of penicillin, and 100 μg of streptomycin per ml at 37°C in the presence of 5% CO2. Human monocytic cells (THP-1) were used for in vitro ADE assay and maintained in RPMI-1640 media at 37°C in 5% CO2. EV71 strain AH08/06 was isolated from the throat swab sample of an HFMD case during an outbreak in 2008 in Anhui, China [39]. EV71 strain HN08/08 was isolated from the stool sample of an HFMD case during an outbreak in 2008 in Henan, China. All the viruses were propagated and titrated in RD cells and the stock was stored in aliquots at -80°C in our laboratory. The titer of EV71 was expressed as 50% tissue culture infection dose (TCID50), plaque-forming units (PFU) or 50% lethal dose (LD50) according to the Reed-Muench method [42].

Microneutralizing assay

The titer of neutralizing antibodies against EV71 was determined in RD cells according to standard protocol [43]. Briefly, 50 μL of sample dilutions and 50 μL of virus stock containing 100 TCID50 EV71 were mixed and incubated onto the microtiter plates with RD cells at 37°C for 6 days. All the samples were tested at an initial dilution of 1: 8, and cell and virus controls were run simultaneously. The neutralizing antibody titer was defined as the highest dilution of serum that could prevent the occurrence of cytopathic effect.

Human Intravenous Immunoglobulin (IVIG) preparations

Human 5% liquid IVIG preparations (5 g/100 ml, Tonrol Bio-Pharmaceutical) manufactured from Chinese plasma donors, consisting more than 99% of IgG and a very small quantities of IgA and IgM, were used as stock dilutions. The titer of neutralizing antibodies of IVIG was calculated to 1:128 by microneutralzing assay as previously described.

ADE infection assay in vitro

Stock IVIG preparation (100 dilution) was 10-fold serially diluted to 10-4 with PBS and each dilution was then incubated with EV71 suspensions at 37°C for 1 h to form virus-antibody complexes, respectively. The EV71-antibody complexes were then added to cultured THP-1 cells at a multiplicity of infection (MOI) of 1 and incubated for 1 h at 37°C. Then, THP-1 cells were washed for three times with PBS and then incubated with fresh RPMI-1640 media for another 24 h. Finally, the infected cells and supernatants were harvested and the virus yield was determined by the plaque assays and real-time RT-PCR assays.

Plaque forming assay

Monolayer RD cells were cultured in 12-well plates and serial 10-fold dilutions of viral suspensions were added for adsorption for 1 h. Then, the virus suspension was replaced with DMEM containing 2% FBS and low melting point agarose. Followed by incubation for 72 h, the cells were fixed with 10% formaldehyde and subsequently stained with 1% crystal violet solution. The titer of EV71 was calculated and expressed as plaque-forming units (PFU)/ml.

Real-time RT-PCR assay

Viral RNA in EV71-infected THP-1 cells was assayed by real-time RT-PCR as previously described [44]. Viral RNA was extracted by the QIAmp Viral RNA Mini kit (Qiagen) and one-step RT-PCR was performed by using the PrimeScript One-Step RT-PCR kit (Takara) according to the manufacturer's instructions in Roche LightCycler 2.0 systems.

In vivo neutralization assay of EV71

Stock IVIG preparation was 10-fold serially diluted mixed with EV71 suspensions as previous described. Four groups of 1-day-old Kunming mice (n = 11) were injected intraperitoneally with these EV71-IVIG or EV71-PBS mixtures, respectively. The mortality and neurological symptoms were monitored for 15 days.

Secondary EV71 infection model

We use two EV71 strains to establish the secondary EV71 infection model: a virulent strain AH08/06 which can result in death in 14-day-old suckling mice and an avirulent strain HN08/08. Briefly, groups of suckling mice of 1-day-old were primary injected subcutaneously with 50 μl of different doses (105, 106 and 107 PFU, respectively) of HN08/08 or PBS. Then 14 days after the primary infection, all the mice were subsequently infected with 1 LD50 (50 μl) of AH08/06 by intraperitoneally injection. Before the secondary infection, 100 μl of blood was obtained via tail vein bleeding and processed for microneutralizing assay. The mortality and neurological symptoms were followed for another 14 days post the secondary infection. All the animal experiments were approved and performed according to the guideline of Animal Experiment Committee of State Key Laboratory of Pathogen and Biosecurity.

Statistical analysis

Kaplan-Meier survival curves were used to display mortality data, and log rank analyses were performed to determine statistical significance between different groups.