Broad protection with an inactivated vaccine against primary-isolated lethal enterovirus 71 infection in newborn mice
© Chang et al. 2015
Received: 24 February 2015
Accepted: 26 June 2015
Published: 15 July 2015
Circulating enterovirus 71 (EV-A71)-associated hand, foot, and mouth disease is on the rise in the Asian-Pacific region. Although animal models have been developed using mouse-adapted EV-A71 strains, mouse models using primary EV-A71 isolates are scarce. Lethal animal models with circulating EV-A71 infection would contribute to studies of pathogenesis as well as vaccine development and evaluation.
In this study, we established a lethal mouse model using primary EV-A71 isolates from patients infected with serotypes that are currently circulating in humans. We also characterized the dose-dependent virulence and pathologic changes of circulating EV-A71 in this mouse model. Most importantly, we have established this mouse model as a suitable system for EV-A71 vaccine evaluation. An inactivated EV-A71 vaccine candidate offered complete protection from death induced by various circulating EV-A71 viruses to neonatal mice that were born to immunized female mice. The sera of the immunized dams and their pups showed higher neutralization titers against multiple circulating EV-A71 viruses.
Thus, our newly established animal model using primary EV-A71 isolates is helpful for future studies on viral pathogenesis and vaccine and drug development.
Human enterovirus 71 (EV-A71) is a non-enveloped, single-stranded positive-sense RNA virus that belongs to the Enterovirus species A genogroup in the Picornaviridae family. It began circulating in the Netherlands as early as 1963 and was first described in the USA in 1969 [1, 2]. EV-A71 and Coxsackievirus A16 (CV-A16) are the two major etiological agents that cause hand, foot, and mouth disease (HFMD); periodic large epidemics have occurred in recent decades, and it has become a severe public health problem [3–9].
Previous studies have shown that EV-A71 usually causes HFMD with severe neurological complications, including aseptic meningitis, brainstem encephalitis, poliomyelitis, encephalomyelitis, and even death [10–20]. In 1997, a large outbreak of HFMD caused by highly neurovirulent EV-A71 emerged in Malaysia and led to 41 deaths among young children . In 1998, a large outbreak of enterovirus infection occurred in Taiwan that resulted in 405 severe cases in children and 78 deaths. Of the 78 children who died, 71 (91 %) were under 5 years of age . In 2011, the largest recorded outbreak of EV-A71-associated HFMD occurred in mainland China, comprising >1.7 million cases and including 27,000 patients who exhibited severe neurological complications and 905 deaths .
EV-A71 has one serotype and can be classified into three genotypes (A, B, and C) and many subtypes (A, B0, B1-B5, and C1-C5). In Taiwan, the major subtypes of EV-A71 were C2 in 1998, B4 in the 2002 epidemic, C4 in the 2004-2005 epidemic, C5 in the 2006-2007 epidemic, B5 in the 2008-2009 epidemic, C4 in the 2010 epidemic, and B5 in the 2011-2012 epidemic [24, 25]. The predominant EV-A71 genotypes detected in Singapore were B3 in 1997-1999, B4 in 2000-2003, C1 in 2002, and B5 in 2006-2008. In mainland China in 1998-2011, all the strains were clustered in the C4 subgenotype of EV-A71.
Most research has been focused on developing vaccines against EV-A71 [26–35]. Given the successful experience in the development of inactivated whole viruses for poliovirus, influenza virus, and rabies virus, inactivated EV-A71 whole-virus vaccines have been produced by five manufacturers in mainland China, Taiwan, and Singapore. These vaccines have completed Phase III (mainland China) and Phase I (Taiwan and Singapore), respectively . In mainland China, Beijing Vigoo Biological Co., Ltd (Vigoo), Sinovac Biotec Co., Ltd (Sinovac), and the Chinese Academy of Medical Science (CAMS) have used EV-A71 subgenotype C4 as a virus seed because it is the prevalent genotype in mainland China; however, Vigoo and Sinovac chose distinct strains, FY and H07, respectively, which were all isolated from Anhui province in South China [36, 37]. Thus far, no vaccine has effectively prevented EV-A71 infection in HFMD patients is available.
Previously, lethal mouse model in EV-A71 infection has been a pivotal evaluation role in the development of EV-A71 vaccines [27, 29, 33, 35]. However, EV-A71 viral isolates from HFMD patients in northeastern China  have not been previously studied in a mouse model or for vaccine development. Our group has isolated and identified several circulating EV-A71 strains from hospitalized HFMD children in northeastern China who had either severe or mild disease. We determined that these strains are complex recombinant viruses involving multiple type A human enterovirus (HEV) . In the present study, we examined and compared the virulence, pathological changes, and progression induced by the circulating EV-A71 viruses, including Changchun (CC, Northeast China) and Fuyang (FY, South China) strains, in a neonatal mouse model. These strains showed different virulence, and a series of lethal strains could be used as a tool for vaccine evaluation. Furthermore, the EV-A71 vaccine candidate CC063 strain with the highest virulence also presented a broadly cross-neutralizing capacity and protection to neonatal mice from lethal-dose infect with various EV-A71 viruses. At the same time, the sera of the immunized dams and their pups showed higher neutralization titers against various EV-A71 viruses. The lethal challenge and protection in mouse model from circulating primary EV-A71 strains and the select vaccine candidates can be very helpful for vaccine development and evaluation.
Cells and viruses
African green monkey kidney epithelial cell line, Vero cells (Cat no. CCL-81; ATCC) were cultured in modified Eagle’s medium (MEM) (Gibco, Invitrogen, USA) containing 10 % fetal bovine serum (FBS) (Gibco, Invitrogen, USA) and 3 % L-glutamine at 37 °C with 5 % CO2. The CC strains of EV-A71 viruses were isolated from throat swabs of HFMD patients in Changchun, China in 2010. Viruses were continuously subcultured to the tenth passage in order to ensure stable titers and genetic features. FY0805 (GenBank accession No. HQ882182.1) and SHZH98 (GenBank accession No. AF302996.1) viruses were gifts from Dr. Qi Jin (Institute of Pathogen Biology, Chinese Academy of Medical Science). BrCr (GenBank accession No. U00871.1) viruses were received from Dr. PY. Mao (302 Military Hospital of China). CV-A16-CC024 is preserved in our laboratory and was isolated from a HFMD patient in Changchun, China (GenBank accession No. KF055238). Viruses were harvested when the cytopathogenic effect (CPE) reached 90 %, and the viral titers were determined in Vero cells by the microplate CPE method and calculated by the Reed–Muench method .
Neonatal mouse infection model
One-day-old specific-pathogen-free (SPF) ICR neonatal mice (Experimental Animal Center, Jilin University) were used to establish the animal model of viral infection. All welfare and experimental procedures were carried out strictly in accordance with the guide for the care and use of laboratory animals and the related ethical regulations of the First Hospital of Jilin University. All efforts were made to minimize animal’s suffering.
The neonatal mice were divided into different groups, randomly and each group contains three litters (n = 8 ~ 10 per litter), and inoculated intracerebrally with EV-A71 viruses or MEM (10 μl/mouse), respectively. The survival rates and mean clinical symptoms were monitored daily for 21 days post-infection. The mean clinical symptoms were scored as follows: 0, healthy; 1, lethargy or weakness; 2, wasting; 3, limb shake; 4, paralysis in hind limb; 5, moribund or dead. The control mice were healthy throughout the experiments. The median lethal dose (LD50) was calculated as described by the Reed–Muench method .
Histopathological and immunohistochemical analysis (IHC)
Three mice with grade 1 to 5 of clinical disease from each of three experimental groups, EV-A71CC063 (106.5 CCID50/ml), BrCr (106.5 CCID50/ml), EV-A71CC072 (106.5 CCID50/ml), and three mice of grade 0 to 1 from the control group were subjected to histopathological and IHC analysis at 5 days post-infection. After the mice were anesthetized, tissues and organs, including lung, intestine, liver, kidney, hind limb muscle, spleen, heart, spinal muscle and brain, were harvested and immersion-fixed with 10 % formaldehyde solution for 5 days. Then, all the samples were dehydrated via an ethanol gradient, clarified through dimethylbenzene, and embedded in paraffin, and 4-μm sections were obtained for hematoxylin and eosin (HE) staining. For IHC examination, 4-μm sections of tissue samples were dewaxed and hydrated through an ethanol gradient. Antigens were then restored by microwaving for 15 min at 95 °C in citrate buffer. Used hydrogen peroxide (2.5 %) treatment to inhibit endogenous peroxidase activity of the samples. EV-A71 viral antigens were detected by anti-EV-A71 polyclonal antibody of rabbit (developed in our laboratory) and streptavidin-peroxidase anti-rabbit IgG kit (Fuzhou Maixin Biotechnology Development Co., Ltd, Fuzhou, China).
Viral loads in newborn infected mouse tissues
Injected intracerebrally with EV-A71CC063 (106.5CCID50/ml) or control medium, 12 viral challenged mice and 3 negative control mice were then used to detecting viral loads.
All samples including blood, lung, intestine, liver, kidney, hind limb muscle, spleen, heart, spinal muscle and brain were harvested from the experimental group (n = 3 per time point) on days 2, 4, and 6 post-infection and from the control group (n = 3) with no challenge on day 1. All the collected tissue samples were weighed individually and stored at -80 °C for further viral detection. The collected tissue samples were disrupted and homogenized in sterile phosphate buffer with the freeze-thaw/grinding method, then centrifuged. The clarified supernatants were collected, and viral loads in the tissue supernatant and blood were determined by real-time fluorescence quantitative reverse transcriptase PCR (qRT-PCR) and the results were expressed in log10 copies/ml of blood or log10 copies/mg of tissue. The 95 % confidence interval of the negative control values determined in various organs and tissues were regarded as the reference value for methodological sensitivity (0 copies/mg or ml).
Inactivation, purification, and immunogenicity of EV-A71 virus candidate vaccine
The Vero cells infected by the seven strains of EV-A71 candidate virus were collected and centrifuged at 4500 × g for 30 min; the collected supernatants were inactived with formalin at 4 °C for 72 h. The effect of viral inactivation was tested by CPE, The inactived viruses were concentrated by ultrafiltration, following purified on Sepharose 4 Fast Flow gel. The protein concentration of the final virus elution was measured with a BCA kit (Thermo Scientific, Inc.), and the viral purity was determined by silver stain plus reagent (Bio-Rad, Inc.). The purified, inactivated EV-A71 derived viral proteins and negative control antigens prepared in similar fashion were mixed with alum and 0.5 ml (10 μg/ml) of the alum adjuvant vaccine was used for each immunization via intraperitoneal injection (I.p.).
Seven groups (n = 8 per group) of female adult ICR mice were immunized i.p. with a different EV-A71 vaccine candidate, CC063, CC072, CC077, CC080, CC085, SHZH98, or FY0805 two times at 2-week intervals. Serum was collected on the fourth week after immunization, and neutralization titers against the various EV-A71 viruses were measured by TCID50 assay in Vero cells.
Serum neutralization test
Serum neutralization titers (NTAb) were determined by the TCID50 reduction assay in Vero cells. Isopycnic mixing (50 μl/well, respectively) of the serially diluted sera was carried out with the working concentration (100 TCID50/ml) variously circulating EV-A71 strain stocks; the mixtures were incubated at 37 °C for 2 h in 96-well plates. Subsequently, the Vero cells (2 × 105/ml) were seeded onto 96-well plates (100 μl/well) for infection at 35 °C with 5 % CO2 and cultured for 7 days. The CPE of the Vero cells was observed by light microscopy. The highest serum dilution that could inhibit CPE in >50 % of the wells were determined as serum neutralization titers.
Protective efficacy of vaccine-induced maternal antibody for neonatal mice
Eight-week-old female ICR mice (n = 3, each group) were injected i.p. with inactivated EV-A71CC063 vaccine or negative control phosphate-buffered saline (PBS) on weeks 0 and 2, respectively. The inactivated vaccine contained 10 μg of EV-A71CC063 virus. The first injection was 1 h after mate. After delivery, two experiments were carried out to confirm the protective efficacy of the humoral immune response of the EV-A71 vaccine candidate. First, three immunized maternal mice and their pups in the vaccine and negative control groups were euthanized, sera were collected for neutralizing antibodies assays against various EV-A71 strains. Second, four groups of pups (n = 3 litters per group, and n = 8 ~ 10 per litter) were intracerebrally challenged with different lethal doses of EV-A71 strains on day 1. Challenged mice were monitored for 21 days.
The clinical scores, viral loads, and antibody titers were analyzed with nonparametric one-way ANOVA tests. The survival rates were determined by log-rank test. All statistical analysis were expressed as means ± the standard error of the mean (SEM). P < 0.05 was considered significant.
Establishment of a neonatal mice model using primate EV-A71 viruses that produce strain-specific morbidity and mortality
Lethal circulating EV-A71 viruses produce dose-dependent morbidity and mortality
For CC077, the challenged mice given 107.5 CCID50/ml, 106.5 CCID50/ml, or 105.5 CCID50/ml began to sicken on days 4, 5, and 5, respectively, with a 0 %, 80 %, and 90 % survival rate (mean clinical score of grade 3), respectively (Fig. 2b). The challenged mice with 104.5 CCID50/ml began to sicken on day 7 (grade 1), and no deaths occurred during the period of observation (Fig. 2b).
The mice that were infected with CC080 in various titers all became sick on day 3 post-infection (grade 1); after an increased tropism in clinical grade, only the 106.5 CCID50/ml group reached grade 5 on day 12, whereas the 105.5 and 104.5 CCID50/ml groups had mean clinical scores of grade 3 during the whole test period (Fig. 2c). The challenged mice with 106.5 ~ 104.5 CCID50/ml began to die on days 5, 5, and 7, respectively (0 %, 60 %, and 80 % survival rate, respectively) (Fig. 2c). In the 103.5 CCID50/ml group, no obvious symptoms or death of the infected mice were observed (Fig. 2c).
The mice challenged with 107.5 CCID50/ml, 106.5 CCID50/ml, 105.5 CCID50/ml, or 104.5 CCID50/ml FY0805 began to sicken on days 4, 5, 5, and 6 (grade 1), with 100 %, 100 %, 60 %, and 10 % mortality rates by days 8, 9, 12, and 9, respectively. The mice that were infected with the lowest dosage of 103.5 CCID50/ml exhibited only mild symptoms (grade 1) on days 6 to 10 and no deaths (Fig. 2d).
In the aforementioned four experiments, no clinical symptoms or death were observed in the negative control group (Fig. 2a-d), indicating that the disease and death of the mice were specifically attributable to the EV-A71 viruses.
In conclusion, the symptoms and mortality rate produced by the four pathogenic virus strains increased in a dose-dependent manner. The median lethal doses (LD50) for CC063, CC077, CC080, and FY0805 were about 105.0 CCID50/ml, 107.0 CCID50/ml, 105.5 CCID50/ml, and 105.5 CCID50/ml, respectively. CC063 appeared to be the most pathogenic strain (Fig. 2). Thus, we have identified multiple circulating EV-A71 viruses that can induce strain-specific mortality and disease symptoms in newborn mice.
Pathology in the mice post-infected with a lethal dose of circulating CC063
Viral loads in selected tissues of CC063-infected mice
EV-A71CC063 vaccine candidate showed strongest immunogenicity and broader cross-neutralization capacity
Maternal antibody against the CC063 vaccine candidate can broadly protect the neonatal mice from lethal virus challenge
Animal models plays a critical role in the development and evaluation of potential EV-A71 vaccines. Since normal adult mice are insensitive to EV-A71 infection, some researchers have suggested using neonatal mice or transgenic mice challenged with single viruses to evaluate the protective efficacy of vaccines [27, 29, 33, 35]. In our study, we have identified multiple lethal circulating EV-A71 viruses in a neonatal mouse model and have analyzed the pathogenic features of the lethal and non-lethal strains. These circulating EV-A71 strains were isolated from different regions of China. Early genome analysis demonstrated that these EV-A71 strains are circulating recombinant viruses  that are distinct from the prototype EV-A71 (BrCr). Interestingly, although the prototype EV-A71 (BrCr) did not cause lethal infection in the neonatal mice, several primary EV-A71 isolates readily generated pathogenic infections in these mice. On the other hand, some primary EV-A71 isolates could not cause lethal infection, despite the fact that they are genetically closely related to the pathogenic primary EV-A71 viruses.
Primary circulating EV-A71 viruses cause many symptoms in neonatal mice, including wasting, limb-shake weakness, and hind leg paralysis. HE staining showed severe lesions in the hind limb and spinal skeletal muscles, but not in the cardiac muscles or lung tissues; these findings are very different from those in the neonatal mice infected with lethal CV-A16 viruses, which suffered from severe lung damage . We could also detected viral antigen in the hind limb, cardiac, and spinal skeletal muscles. As expected, a large amount of viral antigen was detected in the brain tissue (data not shown), a point that has been emphasized in many previous studies [11, 14, 17, 19, 42, 43]. Virus loads in these organs were consistent with our results. However, the expression of viral antigen could also be detected by IHC (data not shown) in many other tissues of the infected mice that showed tissue-specific pathogenesis.
Several viruses, such as CC072, CC085, SHZH98, and BrCr, were unable to induce neonatal death in the mice (Figs. 1 and 2). Consistent with the findings, pathological changes and viral loads could not be detected in the organs of these infected mice (data not shown), suggestnig that the absence of viral replication in the neonatal mice might be the main reason that the virus could not induce animal death. Our mouse model should be useful for future identification of viral determinants of pathogenic infection.
Widespread HFMD has raised serious public health concerns in the Asia-Pacific region [4, 8, 9]. Coxsackievirus 16 (CV-A16) and enterovirus 71 (EV-A71) can both cause HFMD, major studies are focused on the latter as EV-A71 infection often associated with severe complications, including various neurological symptoms. Interestingly, we observed that administration of only the higher dosage of the various EV-A71 strains (CC063 LD50 ≈ 105.0 CCID50/ml) could induce neonatal mouse death, as compared to circulating CV-A16 viruses (CC024 LD50 ≈ 101.65 CCID50/ml) . The pathogenesis induced by EV-A71 and CV-A16 needs to be further investigated in the future.
Our study demonstrates that primary isolates of circulating EV-A71 viruses differ greatly in their ability to elicit broad-range neutralizing antibodies. Using these viruses isolated from diverse areas, we characterized EV-A71 vaccine candidates with the highest virulence and determined that these candidates, derived from virus from Northeast China, could protect neonatal mice from challenge with diverse lethal strains. Moreover, we found that vaccine candidate CC063 had the highest virulence (LD50 ≈ 105.0 CCID50/ml) (Fig. 2a), the strongest immunogenicity (Figs. 6 and 7), and the broadest cross-protection (Fig. 8). Therefore, using a sensitive mouse model and EV-A71 viruses, we have successfully studied the viral pathogenesis and identified a potential vaccine candidate.
Since no effective antiviral agents are available, EV-A71 vaccine will play an important role in controlling EV-A71-induced HFMD in children. The selection of a vaccine candidate strain is the crucial factor, especially for the EV-A71 vaccine, because of its high mutation rate and frequent recombination . In general, the virus vaccine candidate strain should have higher virulence, better immunogenicity, broad cross-protection, and genetic stability . For vaccine evaluation, with the exception of the cellular CPE method in vitro, animal model systems can serve directly evaluation to protective efficacy and immunogenicity of candidate vaccines. Indeed, we observed high immunogenicity of the EV-A71 vaccine candidate both in vitro and in vivo. Even more important is the fact that the EV-A71 candidate vaccine in our study demonstrated broad cross-protection against lethal challenge with multiple EV-A71 viruses from the Jilin and Anhui provinces of China. Our study therefore provides important guidance for future HFMD vaccine development and evaluation which may lead to the discovery of potential vaccine candidates for the prevention and control of HFMD.
We thank Chunyan Dai, Xiuying Zhang, Qunying Mao, Zhenglun Liang, Panyong Mao, Qi Jin and Pangyong Mao for for critical reagents and Deborah McClellan for editorial assistance. This work was supported in part by funding from the Chinese Ministry of Science and Technology (2012CB911100 and 2013ZX10001-005), the Chinese Ministry of Education (IRT1016), the National Natural Science Foundation of China (No. 31270202), the Key Laboratory of Molecular Virology, Jilin Province (20102209), and the Health and Family Planning Commission of Jilin Province (2013Z066).
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