Development of an Enterovirus 71 Vaccine Efficacy Test Using Human Scavenger Receptor B2 Transgenic Mice

The development of new vaccines for EV71 relies on the availability of small animal models suitable for in vivo efficacy testing. Monkeys and neonatal mice have been used, but the use of these animals has several drawbacks, including high costs, limited susceptibility, and poor experimental reproducibility. In addition, the related ethical issues are considerable. The new efficacy test based on hSCARB2 Tg mice and virulent EV71 strains propagated in genetically modified cell lines presented here can overcome these disadvantages and is expected to accelerate the development of new EV71 vaccines.

ABSTRACT

Enterovirus 71 (EV71) is a causative agent of hand-foot-mouth disease, and it sometimes causes severe neurological disease. Development of effective vaccines and animal models to evaluate vaccine candidates are needed. However, the animal models currently used for vaccine efficacy testing, monkeys and neonatal mice, have economic, ethical, and practical drawbacks. In addition, EV71 strains prepared for lethal challenge often develop decreased virulence during propagation in cell culture. To overcome these problems, we used a mouse model expressing human scavenger receptor B2 (hSCARB2) that showed lifelong susceptibility to EV71. We selected virulent EV71 strains belonging to the subgenogroups B4, B5, C1, C2, and C4 and propagated them using a culture method for EV71 without an apparent reduction in virulence. Here, we describe a novel EV71 vaccine efficacy test based on these hSCARB2 transgenic (Tg) mice and these virulent viruses. Adult Tg mice were immunized subcutaneously with formalin-inactivated EV71. The vaccine elicited sufficient levels of neutralizing antibodies in the immunized mice. The mice were subjected to lethal challenge with virulent viruses via intravenous injection. Survival, clinical signs, and body weight changes were observed for 2 weeks. Most immunized mice survived without clinical signs or histopathological lesions. The viral replication in immunized mice was much lower than that in nonimmunized mice. Mice immunized with the EV71 vaccine were only partially protected against lethal challenge with coxsackievirus A16. These results indicate that this new model is useful for in vivo EV71 vaccine efficacy testing.

IMPORTANCE The development of new vaccines for EV71 relies on the availability of small animal models suitable for in vivo efficacy testing. Monkeys and neonatal mice have been used, but the use of these animals has several drawbacks, including high costs, limited susceptibility, and poor experimental reproducibility. In addition, the related ethical issues are considerable. The new efficacy test based on hSCARB2 Tg mice and virulent EV71 strains propagated in genetically modified cell lines presented here can overcome these disadvantages and is expected to accelerate the development of new EV71 vaccines.

INTRODUCTION

Enterovirus 71 (EV71) is one of the major causative pathogens of hand-foot-mouth disease (HFMD). Although HFMD is a mild and self-limiting disease, EV71 infection can result in severe neurological complications. To prevent these issues, new vaccines are required (1). To date, formalin-inactivated vaccines have been developed by three companies (Sinovac Biotech, Ltd.; Beijing Vigoo Biological Co., Ltd.; and the Institute of Medical Chinese Academy of Medical Sciences); all have been approved for marketing in China (2–4). Similar formalin-inactivated vaccines are being developed by the National Health Research Institutes, Taiwan, (5) and by Takeda Pharmaceuticals (6, 7). In addition, different types of vaccines are under development; these types include EV71 virus-like particles (VLPs) (8–12), recombinant VLPs based on other viruses displaying EV71-neutralizing epitopes (13–15), recombinant VP1 proteins (16–19), DNA vaccines (20), and live attenuated vaccines (21). The formalin-inactivated vaccines have been shown to be effective (2–4). However, the viral yield of EV71 is low, resulting in a poor production efficiency for vaccines. The development of new vaccines that are effective and have high production efficiency is still needed. Therefore, reliable and convenient efficacy tests of vaccines are also needed to accelerate the development of new vaccines.

The efficacy of EV71 vaccines has been examined experimentally in animal models. The most simple and basic experiment is to immunize nonsusceptible animals, such as rabbits or rats, with vaccine candidates to confirm the ability of the vaccine to induce the production of neutralizing antibodies (NAbs). The NAb titer in the serum is measured using in vitro neutralization tests. However, in vivo protection assays are more reliable because they measure the efficacy of the vaccines directly in immunized susceptible animals subjected to challenge with a virulent virus. However, these assays for EV71 vaccines are difficult to perform due to the limited host range of EV71. Humans are the natural host, although monkeys can be infected experimentally. Several studies on the efficacy of EV71 vaccines have been performed in monkeys (9, 22–26); however, primates have major disadvantages. These issues include requirements for large facilities with a large staff, high costs, genetic heterogeneity, and obvious ethical issues. In monkey tests, the induction of humoral and cellular immune responses has been observed in immunized animals. Protection against viral challenge has been assessed mainly by evaluating the inhibition of viral replication in the organs and by performing pathological analyses. Lethal challenge experiments have not been performed. Neonatal mice, which are susceptible to infection by EV71 strains, have been widely used for efficacy testing of EV71 vaccines (10–13, 15–18, 27); these tests involve subjecting neonatal mice harboring maternally transferred antibodies to lethal challenge with a virulent virus. However, neonatal mice lose their susceptibility to infection within a few weeks after birth, which makes it difficult to set up a desired immunization schedule and quantitatively evaluate the direct correlation between NAbs elicited by vaccination and protection effects. To extend the window of susceptibility, some studies have been performed using genetically modified mice, such as AG129 mice, which are defective in type 1 and type 2 interferon responses (28); however, the window of susceptibility is extended by only 1 to 2 weeks. Caine et al. (29) selected adapted viruses that caused disease in young AG129 mice up to 10 weeks old. The combined use of AG129 mice and age-adapted strains of the EV71 subgenogroup B2 permitted a lengthened immunization schedule for vaccine testing. However, this method is applicable only to adapted viruses, and much effort has to be expended to achieve adaptation.

To overcome these problems, we established a transgenic (Tg) mouse model expressing human scavenger receptor B2 (hSCARB2) (30), the main receptor that supports EV71 cell surface attachment, internalization, and initiation of uncoating; this receptor is used by all strains of EV71, as well as by coxsackievirus A16 (CVA16), CVA14, and CVA7 (31–33). Unlike wild-type neonatal mice and immunologically modified mice, hSCARB2 Tg mice show lifelong susceptibility to infection by EV71 (30), which makes it possible to establish a suitable efficacy testing protocol for EV71 vaccines. hSCARB2 Tg mice are susceptible to all EV71 strains, as long as the viruses are virulent enough for use in lethal challenge experiments.

In vivo challenge with EV71 presents another problem, namely, that EV71 is genetically unstable during passage in cultured cells. When viruses are propagated in cultured cells, amino acid mutations in the capsid protein (associated with the heparan sulfate [HS]-binding phenotype) occur, resulting in positive selection of mutated viruses (K. Kobayashi, K. Mizuta, and S. Koike, submitted for publication). These mutated EV71 strains show an attenuated phenotype in vivo (34, 35). Therefore, it is difficult to consistently prepare virulent strains because virulence varies from preparation to preparation. To avoid this problem, we selected several virulent strains of EV71 as candidates for lethal challenge; these viruses were prepared using a novel cell line lacking HS expression but overexpressing hSCARB2 (Kobayashi et al., submitted for publication). Virus stocks prepared using this method can be used without changing the lethal challenge conditions. Using a combination of these techniques makes it possible to easily and reproducibly test the efficacy of vaccines.

RESULTS

Selection and preparation of challenge viruses.

To develop a vaccine efficacy test, we needed virulent viruses suitable for lethal challenge. To screen for virulent viruses, hSCARB2 Tg mice were inoculated intraperitoneally with 26 clinical isolates of EV71 (isolated in the Miyagi and Yamagata prefectures of Japan) belonging to the subgenogroups B4, B5, C1, and C4; all viruses were used at a median tissue culture infectious dose (TCID₅₀) of 10⁶ per mouse. The mice were monitored for clinical signs and survival for 2 weeks, and the virulence of each isolate was assessed by calculating the mortality rate. Three strains (2716-Yamagata-2003 [2716-Ymg-03], Y90-3896, and N772-Sendai-2006 [N772-Snd-06]) belonging to subgenogroups B5, C1, and C4, respectively, showed more than 80% mortality (data not shown). We constructed full-length infectious cDNA clones of these virulent viruses. In addition, we had previously obtained cDNA clones for C7/Osaka/Japan/97 (C7/Osaka; subgenogroup B4) and SI/Isehara/Japan/99 (Isehara; subgenogroup C2), which show a level of virulence similar to that of the selected viruses described above (35). We found that the propagation of virulent viruses in cultured cells such as RD-A cells resulted in amino acid changes associated with attenuation, whereas propagation in RD-ΔEXT1+hSCARB2 cells, which lack HS expression but overexpress hSCARB2, minimized this effect (Kobayashi et al., submitted for publication). Therefore, viruses were recovered from cDNA by transfecting RD-ΔEXT1+hSCARB2 cells with in vitro-transcribed RNA. The recovered viruses were propagated only once in RD-ΔEXT1+hSCARB2 cells. The virulence of the challenge viruses was tested by inoculating hSCARB2 Tg mice intravenously with 10⁵ or 10⁶ TCID₅₀ of each virus (Table 1). Upon inoculation with 10⁵ TCID₅₀ of virus, 30 to 100% of the mice died. All mice inoculated with 10⁶ TCID₅₀ of virus died within 2 weeks (most died during the first week) postinoculation (data not shown). From these data, we estimated that the median lethal dose (LD₅₀) for these viruses was approximately 10⁵ TCID₅₀ or lower and that a dose of 10⁶ TCID₅₀ corresponded to 10 to 100 times the LD₅₀ value.

TABLE 1.

Virulence of the challenge viruses

Virus strain	No. of dead mice/no. of inoculated mice (% dead) at a TCID50 of:
	105	106
C7/Osaka-97 (B4)	5/10 (50)	10/10 (100)
2716-Ymg-03 (B5)	8/10 (80)	10/10 (100)
Y90-3896 (C1)	6/10 (60)	10/10 (100)
SI/Isehara/99 (C2)	10/10 (100)	10/10 (100)
N772-Snd-06 (C4)	3/10 (30)	10/10 (100)

Schedule for immunization with formalin-inactivated EV71.

We propagated the EV71 SK-EV006/Malaysia/97 (SK-EV006) strain (belonging to the subgenogroup B2) in RD-A cells. This strain was chosen because it grows to a high titer in RD-A cells. The purified virus was inactivated with formalin. Mice were immunized subcutaneously once or twice with formalin-inactivated EV71 (0.3 μg). The NAb titers in serum samples collected at the indicated time points were measured in a plaque reduction neutralization test (PRNT), using the homologous SK-EV006 strain as the challenge virus (Fig. 1A). NAbs were elicited at least 4 weeks after the first immunization. The NAb titer reached a plateau at approximately 6 to 8 weeks after the first immunization (at an age of 10 or 12 weeks). The geometric mean NAb titers of the serum samples from immunized mice ranged from 1:64 to 1:1,024. The NAb titers in serum samples from mice immunized twice were significantly higher than those in serum samples from mice immunized only once (P = 0.004; two-way analysis of variance [ANOVA]). Accordingly, we designed the immunization and challenge schedule shown in Fig. 1B. Mice were inoculated subcutaneously twice (once at 4 weeks old and again at 8 weeks old). A challenge virus was administered when the mice reached 10 weeks old.

FIG 1.

(A) Induction of NAbs in hSCARB2 Tg mice. Ten hSCARB2 Tg mice received one (prime only) or two (prime plus boost) subcutaneous injections of formalin-inactivated EV71 SK-EV006 (0.3 μg) formulated with alum. Serum was collected at 8, 12, 16, and 21 weeks old, and NAb titers were determined. The geometric mean NAb titer is shown (open and closed circles) with the standard deviation. The NAb titer of the group immunized twice was significantly higher than that of the group immunized once (P = 0.004; two-way analysis of variance [ANOVA]). (B) Immunization and challenge schedule. The EV71 vaccine was administered at 4 and 8 weeks old. Lethal challenge with virulent strains was performed at 10 weeks old. Serum was collected 1 day before lethal challenge, and the NAb titer was determined. Survival and body weight changes were monitored daily for 2 weeks. The brain and spinal cord of paralyzed and surviving mice were examined for histopathological changes.

Immunized mice are protected from lethal challenge with an EV71 strain.

First, we immunized mice with different doses (0.003, 0.03, or 0.3 μg) of the formalin-inactivated vaccine, followed by challenge with the virulent strain N772-Snd-06 (belonging to the subgenogroup C4); this strain was chosen because the vaccine seed strain SK-EV006 was not sufficiently virulent for use as an in vivo challenge virus (35).

Figure 2A shows the NAb titers in serum sample collected 1 day before challenge with SK-EV006. Figure 2B shows a stacked bar chart representing the percentages of mice with different outcomes after immunization with different doses of the vaccine. Changes in body weight are shown in Fig. 2C to F. No NAbs were induced in the nonimmunized mouse group (Fig. 2A). Body weight decreased daily; all mice showed neurological signs or severe limb paralysis, and all died before 9 days postinfection (dpi) (Fig. 2B and C). In the group immunized with 0.003 μg of vaccine, half of the mice showed an NAb titer ranging from 1:64 to 1:1,024; NAbs were undetectable (<1:16) in the other half of the mice (Fig. 2A). Eight mice died; however, one survived with transient body weight loss and clinical signs that included wobbling, limb weakness, or mild paralysis (blue circles in Fig. 2D), and another survived without clinical signs (red circles in Fig. 2D). In the group immunized with 0.03 μg of vaccine, the NAb titers ranged from 1:64 to 1:1,024 (Fig. 2A). Three mice died, two showed mild paralysis and body weight loss, and five survived without clinical signs (Fig. 2B and E). Some of the mice with mild paralysis recovered during the course of the experiments. In the group immunized with 0.3 μg of vaccine, the NAb titers ranged from 1:256 to 1:4,096 (Fig. 2A). All mice survived without clinical signs (Fig. 2B and F). The results show that the induction of NAbs and subsequent protection are dependent on the dose of the inoculated vaccine. Two immunizations with 0.03 μg or 0.3 μg of formalin-inactivated SK-EV006 were sufficient for 100% seroconversion and 100% survival in hSCARB2 Tg mice, respectively. Mice with higher NAb titers tended to have a better outcome, although the value of the NAb titer for each mouse did not correlate strictly with the outcome. The survival rates of the mice with NAb titers no lower than 1:64, 1:256, or 1:1,024 were 76%, 81%, and 87%, respectively. No mouse with an NAb titer lower than 1:16 survived.

FIG 2.

Outcomes, NAb titers, and body weight changes in challenged mice. A group of ten mice were immunized with a mock vaccine or 0.003, 0.03, or 0.3 μg of vaccine, followed by a lethal challenge with 10⁶ TCID₅₀ of N772-Snd-06. (A) The NAb titers in serum samples collected 1 day before challenge were measured. The NAb titer for each group is shown in the box plot. The value for each mouse is indicated by a circle. The outcome of each mouse is denoted by the circle color. Black, blue, and red circles indicate mice that died, survived with clinical signs, or survived without clinical signs, respectively. The closed red circles denote mice that survived without clinical signs but with mild inflammation in the central nervous system (CNS) upon histopathological examination. * and ** denote P < 0.05 and P < 0.01, respectively (one-way ANOVA). (B) The percentages of mice that died, survived with clinical signs, or survived without clinical signs with or without histopathological changes are indicated by solid black, solid blue, solid red, and open red bars, respectively. (C to F) The body weight changes of mice immunized with the mock vaccine (C) or 0.003 (D), 0.03 (E), or 0.3 μg (F) of vaccine after challenge are shown. The color of the circles indicates the outcome (as in panel A). The numbers indicate individual mice that exhibited the typical pathological lesions shown in Fig. 4. The survival of mice immunized with 0.03 (E) or 0.3 μg (F) of vaccine was significantly longer than that of nonimmunized mice (P = 0.002 and 0.000, respectively; log-rank test).

Histopathological examination of immunized mice.

Thin sections of the brain and spinal cord from dead and surviving mice were stained with hematoxylin and eosin (H&E) to observe histopathological changes such as cellular infiltration and neuronal damage (Fig. 3). Sections were also stained with an anti-EV71 antibody. All mice in the phosphate-buffered saline (PBS)-inoculated group showed evidence of severe neuronal damage, including degenerated neurons, neuronophagia, cellular infiltration, and perivascular cuffing in the spinal cord (Fig. 3A and B), brainstem, hypothalamus, thalamus, cerebellum, cerebrum, and dorsal root ganglia (data not shown). The degenerated neurons were EV71 antigen positive (Fig. 3C). In contrast, almost none of the mice that survived without clinical signs (open red circles in Fig. 2A and D to F) showed pathological lesions in the central nervous system (CNS) (Fig. 3D and E). A few of the mice that survived without clinical signs showed mild lesions in the CNS (Fig. 3F and G and closed red circles in Fig. 2A and F). The mice that survived but exhibited clinical signs (blue circles in Fig. 2A, D, and E) showed mild inflammatory reactions (e.g., cellular infiltration and gliosis; Fig. 3H and I). Thus, the severity of histopathological lesions correlated well with the presentation of clinical signs.

FIG 3.

Histopathological changes in the spinal cord of immunized hSCARB2 Tg mice after lethal challenge with a virulent virus. Representative images of the spinal cord of nonimmunized and immunized mice after EV71 inoculation. Massively damaged neurons with cellular infiltration associated with EV71 infection in the spinal cord of a nonimmunized mouse (no. 7702) that died at 4 dpi (A to C). No histopathological changes in the spinal cord of a mouse (no. 7684) that survived without clinical signs (D and E). Slight cellular infiltration in the spinal cord of a mouse (no. 7704) that survived with no clinical signs (F and G) and a mouse (no. 7676) that survived with clinical signs (H and I). (A, B, and D to I) HE staining. (C) Immunohistochemical analysis using an anti-EV71 antibody. The black, gray, and striped arrows indicate neuronophagia, gliosis, and perivascular cuffing, respectively. The open arrows indicate EV71 antigen-positive neurons. Panels B, E, G, and I show high-magnification views of the areas bordered by the boxes in panels A, D, F, and H, respectively.

Inhibition of viral replication in immunized mice.

Next, we compared viral replication in immunized mice with that in nonimmunized mice. A group of 18 mice was immunized with or without a dose of 0.3 μg and challenged as shown in Fig. 1B. Six mice in each group were sacrificed at 1, 2, and 3 dpi, and the brain, spinal cord, plasma, liver, heart, lungs, and kidneys were collected for virus titration experiments. The viral load in the CNS tissues of the nonimmunized mice increased daily; however, the load in the CNS of the immunized mice was significantly lower than that in the CNS of the nonimmunized mice (below the detection threshold in some cases; Fig. 4A and B). The virus was detected in the plasma samples from the nonimmunized mice at 1 dpi, but viral levels decreased gradually thereafter. The virus was almost undetectable in the plasma samples from the immunized mice (Fig. 4C). In other nonneural tissues, the viral load in the immunized mice was always much lower than that in the nonimmunized mice (Fig. 4D to H). Thus, viral replication is strongly inhibited in immunized mice.

FIG 4.

Inhibition of viral replication in immunized mice. hSCARB2 Tg mice were immunized twice with 0.3 μg of SK-EV006 vaccine and then infected with 10⁶ TCID₅₀ of N772-Snd-06. Six mice were sacrificed at 1, 2, and 3 dpi; the brain (A), spinal cord (B), plasma (C), liver (D), heart (E), lungs (F), kidneys (G), and spleen (H) were collected; and virus titers were determined. The virus titers in immunized and nonimmunized mice are denoted by red circles and black squares, respectively. The horizontal bars indicate the average value. * and ** denote P < 0.05 and 0.01, respectively (Student’s t test). The gray areas indicate values below the limit of detection. All experiments were repeated twice and produced similar results.

Immunized mice are protected from lethal challenge with EV71 strains belonging to different subgenogroups.

The results reported above suggest that immunization with SK-EV006 (subgenogroup B2) at a dose of 0.3 μg provides effective protection from lethal challenge with the virulent C4 strain N772-Snd-06. To examine whether this vaccine is effective against lethal challenge with EV71 strains belonging to other subgenogroups, we injected immunized mice with one of the following four different virulent EV71 strains: C7/Osaka (B4), 2716-Ymg-03 (B5), Y90-3896 (C1), or Isehara (C2). The NAb titers in serum samples collected 1 day before challenge were measured with the homologous SK-EV006 and heterologous virulent strains (Fig. 5A). The NAb titer measured using virulent strains was much lower than that measured using SK-EV006 and was below the detection limit in some cases (Fig. 5A). However, all mice survived lethal challenge with the virulent strains (Fig. 5B to F). Only a few mice showed mild pathological lesions in the CNS. These results suggest that the formalin-inactivated vaccine belonging to the subgenogroup B2 protects mice from lethal challenge with virulent viruses belonging to other subgenogroups, although the cross-reactivity measured by the in vitro PRNT was not very high.

FIG 5.

Protection of immunized mice challenged with the C7/Osaka, 2716-Ymg-03, Y90-3896, or Isehara strains. (A) NAb titers measured using homologous and heterologous virus strains. The PRNT was performed using serum samples from mice immunized with formalin-inactivated SK-EV006 and challenged with SK-EV006 (left) and the virulent virus strains (right). The NAb titer for each mouse is shown in the box plot. Serum samples from nonimmunized mice did not show neutralizing activity (data not shown). (B) Outcomes of challenged mice. The percentages of mice that died and those that survived without clinical signs with or without histopathological changes are indicated by solid black, solid red, and open red bars, respectively. Body weight changes of mice challenged with C7/Osaka (C), 2716-Ymg-03 (D), Y90-3896 (E), or Isehara (F) strains. The values of nonimmunized and immunized mice are indicated by triangles and circles, respectively. Black and red indicate mice that died or survived without clinical signs, respectively. The closed red circles indicate the mice that survived without clinical signs but with mild inflammation in the CNS that was observed upon histopathological examination.

The EV71 vaccine does not provide complete protection against CVA16.

CVA16 is the serotype most closely related to EV71. Some neutralizing monoclonal antibodies against EV71, including MAB979, cross-react with CVA16 (32). However, polyclonal antibodies against EV71 neutralize CVA16 poorly and vice versa (36). Therefore, we asked whether the EV71 vaccine protects against infection by CVA16. A virulent CVA16 strain, CVA16 2065-Yamagata-2004 (2065-Ymg-04), was chosen from six clinical isolates (data not shown) and propagated in RD-A cells. The EV71 NAb titer in immunized mice ranged from 1:256 to 1:4,096 (Fig. 6A and B), which is sufficient to protect against lethal challenge with virulent EV71 strains. However, the neutralizing activity against CVA16 was below the detection limit (<1:8). Next, immunized mice were challenged with a moderate (10⁶ TCID₅₀) or high (10⁷ TCID₅₀) dose of CVA16 2065-Ymg-04. We found that 90% of nonimmunized mice died after receiving the moderate dose, and all died after receiving the high dose (Fig. 6B and C). Of 10 immunized mice challenged with the moderate dose, two died, seven survived with transient body weight loss and clinical signs, and one survived without clinical signs (Fig. 6B and D). In contrast, all immunized mice died after challenge with the high dose of CVA16 (Fig. 6B and F). The results suggest that the EV71 vaccine provides partial protection against CVA16, although this protection is very weak.

FIG 6.

Protection of EV71-immunized mice challenged with virulent CVA16. NAb titers (A) after challenge with EV71 SK-EV006 (left) or CVA16 N0722-Snd-07 (right). Outcomes (B) and body weight changes of mice (C to F). Nonimmunized mice challenged with a moderate (C) or high dose of (E) CVA16. Mice immunized with the EV71 vaccine followed by challenge with a moderate (D) or high (E) dose of CVA16. The survival of immunized mice (D) challenged with a moderate dose was significantly longer than that of nonimmunized mice (C; P = 0.015; log-rank test).

DISCUSSION

Previously, we developed a mouse model expressing human poliovirus receptor (hPVR) susceptible to infection by poliovirus (PV) (37, 38). These hPVR Tg mice are useful for studies on PV pathogenesis (39–41) and for safety testing of live attenuated vaccines (42, 43). Next, we developed a new model for EV71 vaccine efficacy testing based on hSCARB2 Tg mice and a cell culture system that can propagate virulent viruses with minimum alterations in virulence. As expected, this model overcomes problems associated with conventional animal models. We examined the NAb titer elicited by the vaccine, clinical signs, body weight changes, and the mortality rate after lethal challenge. All of these parameters correlated well with each other. Therefore, it is possible to assess the efficacy of vaccine candidates simply by calculating the mortality rate of these mice. hSCARB2 mice are bred in a standard mouse facility, and infection experiments are performed in a standard mouse ABSL2 facility. Here, we used 10 mice per experimental group, which was sufficient to draw simple conclusions about whether a vaccine candidate is effective. If more detailed analyses are required, it is possible to conduct experiments on a larger scale. We can use age-matched mice with a uniform genetic background. These advantages make it possible to obtain accurate and reproducible results. Additionally, unlike the neonatal mouse model, there is no need to transfer NAbs. Thus, this model is convenient and allows us to test a variety of experimental conditions. Although we used the immunization and challenge schedule shown in Fig. 1B, alternative schedules are possible because hSCARB2 Tg mice show lifelong susceptibility to EV71. Here, lethal challenge was performed 2 weeks after the last immunization. However, lethal challenge can be performed several months or 1 year after immunization if one’s aim is to investigate the period over which a vaccine is effective.

Similar EV71 vaccine efficacy tests using mouse models expressing hSCARB2 have been reported. Lin et al. (44) and Tsou et al. (14) reported EV71 vaccine efficacy tests based on another hSCARB2 Tg mouse model in which hSCARB2 cDNA was expressed using the PEF-1α promoter. In these studies, immunization was performed at 1 and 7 days old, and lethal challenge was performed at 14 days old. This immunization schedule might have been chosen because the adult hSCARB2 Tg mice generated by Lin et al. (44) appeared to lose susceptibility to infection. Zhou et al. (45) also reported an EV71 vaccine efficacy test using hSCARB2 knock-in mice in which hSCARB2 cDNA expression is driven by the CAG promoter and a recombinant EV71-harboring luciferase. They assessed the protective effect of a vaccine by monitoring the inhibition of luciferase activity after infection of immunized mice with recombinant EV71. They reported that hSCARB2 knock-in mice did not die after EV71 infection. A lethal challenge experiment might not be possible in this model. These differences in susceptibility to EV71 among mouse models are possibly due to characteristics derived from the promoter used and the hSCARB2 expression profile.

NAbs in the serum play an important role in protection against many EVs, including EV71. The NAb titer is a critical parameter in efficacy tests of EV71 vaccines (1). However, genetically distant EV71 strains may not cross-react properly with each other, or it may be difficult to determine the titers of NAbs specific for some virulent EV71 strains using cultured cells in vitro. According to our previous observations, resistance to NAbs depends on the amino acid at position 145 within VP1. Viruses with glutamic acid at this position are more virulent in hSCARB2 Tg mice and monkeys and more resistant to NAbs than viruses harboring glutamine or glycine at this position (34, 35). All of the challenge viruses selected in this study harbor glutamic acid at position 145. Indeed, the NAb titers measured with these viruses were much lower than the titer measured with SK-EV006, which harbors glycine at this position. In some mice, the NAb titer was below the detection limit of the PRNT (<1:8; Fig. 5A). However, lethal challenge experiments using these virulent viruses revealed that the SK-EV006 vaccine protected mice (Fig. 5B). It is likely that a vaccine belonging to a particular subgenogroup of EV71 may provide effective protection against viruses belonging to different subgenogroups of EV71, as predicted by our previous study (46). The NAb titers of all mice inoculated with 0.3 μg of vaccine and measured using SK-EV006 were greater than or equal to 1:64, and all mice challenged with any of the five different virulent strains survived (Fig. 2 and 5). The minimum protective antibody titer under these conditions was approximately 1:64. The virus strain SK-EV006, the NAb titer of which can be easily determined, might be useful as a suitable surrogate marker for in vitro neutralization tests. Our results showed that we could obtain clear evidence of protection in vivo, even when using viruses whose NAb titers could not be determined clearly in vitro. Therefore, this system provides direct and sensitive evidence of whether a vaccine candidate protects against a wide range of challenge viruses.

EV71-vaccinated hSCARB2 Tg mice were partially protected against CVA16 (Fig. 6). When challenged with a moderate dose, most mice survived, albeit with some clinical signs. When challenged with a high dose, no protection was observed. These results suggest that this system detected weak cross-protection. It is not clear whether EV71 vaccines will protect humans from infection with CVA16. However, because both EV71 and CVA16 use hSCARB2 as the main receptor, successful generation of vaccine candidates and virulent challenge viruses based on CVA16 would make it possible to extend this model to encompass CVA16 vaccine efficacy tests.

The results described above demonstrate the advantages associated with this model system. To further improve the system, it may be necessary to examine the correlation between protection in mice and protection in humans (the latter will necessitate conducting clinical trials). Taken together, the results suggest that the system described here will contribute to the development of new EV71 vaccines.

MATERIALS AND METHODS

Ethics statement.

Experiments using recombinant DNA and pathogens were approved by the Committee for Experiments using Recombinant DNA and Pathogens at the Tokyo Metropolitan Institute of Medical Science (approval numbers 15-023 and 18-033). Experiments using mice were approved by the Animal Use and Care Committee and performed in accordance with the Guidelines for the Care and Use of Animals of the Tokyo Metropolitan Institute of Medical Science, 2012 (approval numbers 15071, 16016, 17036, and 18047).

Cells and viruses.

RD-A cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Nissui, Japan) supplemented with 5% fetal calf serum (FCS). RD-ΔEXT1+hSCARB2 cells were established as described previously (Kobayashi et al., submitted for publication) and maintained in DMEM containing 5% FCS and 1 μg/ml puromycin. A total of 26 EV71 virus strains isolated from HFMD patients in the Miyagi and Yamagata prefectures of Japan were propagated in RD-SCARB2 cells and used for initial mouse virulence tests to select virulent viruses. The EV71 C7/Osaka-97 (35), 2716-Ymg-03 (47), Y90-3896 (46), Isehara (35), and N772-Snd-03 (GenBank accession numbers LC375765.1, LC375766.1, LC506514.1, LC375764.1, and LC506513.1, respectively) strains belonging to subgenogroups B4, B5, C1, C2, and C4, respectively, were selected. RNA isolated from these viruses was used to establish infectious cDNA clones, as described previously (35). RD-ΔEXT1+hSCARB2 cells were transected with RNA transcribed in vitro from full-length infectious viral cDNA. The recovered viruses were propagated once in RD-ΔEXT1+hSCARB2 cells. The CVA16 2065-Ymg-04 and N0722-Snd-07 strains (accession numbers LC506516 and LC506515, respectively) were isolated from HFMD patients in the Yamagata and Miyagi prefectures, respectively (48), using GMK cells and Vero cells. These viruses were propagated in RD-SCARB2 cells and titrated on RD-A cells. The virus titer was measured using a microplate method and is expressed as the TCID₅₀.

Preparation of a formalin-inactivated vaccine.

SK-EV006 (31), which belongs to subgenogroup B3, was propagated in RD-A cells. The virus was recovered by three cycles of freezing and thawing. After removal of cell debris by centrifugation at 10,000 × g for 20 min, the virus in the supernatant was precipitated in 8% polyethylene glycol (PEG) and 0.4 M NaCl. The PEG was removed by chloroform extraction. Sodium N-lauroyl sarcosinate (final concentration, 0.5%), EDTA (2 mM), and NaCl (0.3 M) were added to the virus solution, which was then centrifuged at 141,000 × g for 3 h. The pellet was dissolved in PBS, and filled particles (F-particles) were purified by cesium chloride equilibrium density gradient centrifugation. The F-particle fraction was collected and diluted with PBS, followed by centrifugation at 141,000 × g for 3 h. The pellet was then dissolved in PBS. A total of 100 μg of purified virus was inactivated at 37°C for 7 days in 1 ml of M199 medium containing 5% glycine and 0.925% formalin (49). Sodium sulfite (final concentration, 2.64 mM) and EDTA (final concentration, 0.9 mM) were added to neutralize the formalin. Finally, the formalin-inactivated virus was mixed with an equal volume of Alhydrogel adjuvant (2%; InvivoGen, CA, USA).

hSCARB2 Tg mice and animal experiments.

Human scavenger receptor B2 (hSCARB2) Tg mice (30) were used for experiments. Mice were inoculated with formalin-inactivated EV71 when they reached 4 weeks old and then again at 8 weeks old. Blood was collected from the facial vein using a Goldenrod animal lancet (MEDIpoint, NY, USA) at the indicated times. Lethal challenge with virulent strains was performed when the mice reached 10 weeks old. The mice were monitored for 2 weeks to observe clinical signs and body weight changes. The organs of dead mice were removed, fixed in 4% paraformaldehyde, and embedded in paraffin. Surviving mice were euthanized, fixed with 4% paraformaldehyde by perfusion, and subjected to the same process at 14 dpi Thin sections were prepared and stained with H&E as described previously (39). Immunostaining with an anti-EV71 antibody was performed as described previously (30). To examine viral replication in the CNS, six mice were sacrificed at 1, 2, or 3 dpi, and the brain, spinal cord, plasma, liver, heart, lungs, and kidneys were collected. The tissues were stored at −80°C and then homogenized in 10 volumes of DMEM containing 5% FCS. The virus titer in the supernatant was determined by titration on RD-A cells.

PRNT.

The PRNT for EV71 was performed as described previously (34). Briefly, serum samples were heated at 56°C for 30 min prior to serial dilution (4-fold), starting from 1:16. These solutions were mixed with 200 to 500 PFU of challenge virus (SK-EV006 or other virulent viruses) for 3 h. The titer of each mixture was measured using RD-A cells. NAb titers are expressed as the highest serum dilution that reduced the plaque number to less than 20% of the original number. The NAb titer for CVA16 was determined using a microplate method (starting with a 1:8 dilution of serum) and the CVA16 N0722-Snd-07 strain. The capsid sequence of N0722-Snd-07 is identical to that of 2065-Ymg-04. The NAb titer for CVA16 was measured easily using the N0722-Snd-07 strain.

Statistical analyses.

Statistical analyses were performed using GraphPad Prism v.7.04. Analyses of the NAb titer (Fig. 1A to 2A), viral load (Fig. 4), and survival of mice (Fig. 2C to F and Fig. 5C to F) were performed using two-way ANOVA, one-way ANOVA, Student’s t test, or the log-rank test, as appropriate. For multiple comparisons (Fig. 2A), P values were adjusted using the original false-discovery rate method of Benjamin and Hochberg. A P value of less than 0.05 was considered significant.