A Neonatal Mouse Model of Coxsackievirus A16 for Vaccine Evaluation

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1 JVI Accepts, published online ahead of print on 5 September 2012 J. Virol. doi: /jvi Copyright 2012, American Society for Microbiology. All Rights Reserved A Neonatal Mouse Model of Coxsackievirus A16 for Vaccine Evaluation Qunying Mao 1, a, Yiping Wang 1, a, Rong Gao 2, Jie Shao 1, Xin Yao 1, Shuhui Lang 1, Chao Wang 3, Panyong Mao 2, Zhenglun Liang 1 *, Junzhi Wang 1 * 1: National Institutes for Food and Drug Control, Beijing, , China; 2: 302 Military Hospital of China, Beijing, , China; 3: Hualan Biological Engineering Inc, Henan, , China. Running Title: A Neonatal Mouse Model of Coxsackievirus A16 for Vaccine Evaluation Word Count Abstract: 236 Word Count Article: 6,235 a :These authors contributed equally to this work. * Corresponding author at: National Institutes for Food and DrugControl, , No. 2, Tiantan Xili, Beijing, PR China. Tel.: , fax: , address: wangjz@nicpbp.org.cn(j. Wang) ;Tel.: , fax: , address: 21 lzlun@yahoo.com(z. Liang). 1

2 Abstract To evaluate vaccine efficacy in protecting against coxsackievirus A16 (CA16), which causes human hand, foot, and mouth disease (HFMD), we established the first neonatal mouse model. In this article we report data concerning CA16-induced pathological changes, and demonstrate that anti-ca16 antibody can protect mice against lethal challenge and that the neonatal mouse model could be used to evaluate vaccine efficacy. To establish a mouse model, a BJCA08/CA16 strain (260LD 50 ) was isolated from a patient and used to intracerebrally (i.c.) inoculate neonatal mice. The infection resulted in wasting, hind-limb paralysis, and even death. Pathological examination and immunohistochemistry (IHC) staining indicated that BJCA08 had a strong tropism to muscle and caused severe necrosis in skeletal and cardiac muscles. We then found that BJCA08 pretreated with goat anti-g10/ca16 serum could significantly lose its lethal effect in neonatal mice. When the anti-g10 serum was intraperitoneally (i.p.) injected into the neonatal mice and, within one hour, the same mice were intracerebrally inoculated with BJCA08, there was significant passive immunization protection. In a separate experiment, female mice were immunized with formaldehyde-inactivated G10/CA16 and BJCA08/CA16, and then allowed to mate one hour after the first immunization. We found that there was significant protection against BJCA08 for neonatal mice born to the immunized dams. These data demonstrated that anti-ca16 antibody may block virus invasion and protect mice against lethal challenge, and that the neonatal mouse model was a viable tool for evaluating vaccine efficacy. Key Words: Coxsackievirus A16 (CA16), neonatal mouse model, vaccine 2

3 Introduction Coxsackievirus A16 (CA16) belongs to the Enterovirus genus of the Picornaviridae family and is one of the major pathogens associated with human hand, foot, and mouth disease (HFMD) (3, 10, 13). CA16 was first isolated in 1951 (36). It is a single positive-stranded RNA virus and an icosahedral symmetry structure. Its genome has approximately 7410 nucleotides with one predominant serotype. Based on the VP4 nucleotide sequences, CA16 is classified into three distinct genetic lineages: A, B, and C. Before the 1990 s, lineages A and B were the major epidemic strains in Asia (predominantly the B strain). After that, the CA16 gene gradually mutated to form lineage C, which replaced the B strain as the predominant epidemic strain (17). Epidemics of HFMD have been reported in England, Australia, Japan, Germany, Malaysia, Singapore, Mainland China, and Taiwan (2-3, 6, 13, 18, 21, 26, 31, 34, 41). Recently, HFMD was highly epidemical in the West Pacific region resulting in severe illness and fatalities (9, 18). The HFMD epidemics were mainly caused by CA16 and human enterovirus 71 (EV71), which alternatively or cocirculated in the epidemic area (13, 17-19, 30). Because the most severe or fatal cases were caused by EV71, studies have mainly focused on EV71 but not CA16. Yet, in England, the largest HFMD outbreak (in 1994) was caused by CA16 (2). Similarly, in Taiwan the leading cause of HFMD from 1999 to 2006 was also CA16 (2579 cases), followed by EV71 (1760 cases) ( From 2001 to 2007, surveillance data in Singapore showed that the predominantly-circulating virus was CA16 for three epidemical years (2002, 2005, and 2007), and was EV71 for only one year (2006) (18). Recently, in Mainland China the predominant circulating virus strain was also CA16 (22, 35). While most CA16-associated HFMD infections present only mild symptoms, many recent reports show that CA16 infections might lead to severe health issues, such as aseptic meningitis, rhombencephalitis, cardiac and pericardial disease, pulmonary complications, spontaneous abortion, and even lethal myocarditis and pneumonia (15-16, 18, 33, 37-39, 43). The co-infection of EV71 and CA16 makes it more complex and difficult to control epidemical HFMD (46). Also, the co-infections of EV71 and CA16 can easily cause serious complications in the central nervous system (CNS) with worse 3

4 condition, longer duration and even higher critical illness transfer rates (49). Co-infection increases the chance of gene recombination of the RNA viruses. The rates of co-infection in different areas of Mainland China, vary from 0.62% (during , Hu Nan) (11) to 14.3% (2009, Hang Zhou) (45), to 7.4% (2010, Beijing) (14) to 9.3% (2010, Fo Shan) (47). Therefore, epidemical HFMD could not be controlled by solely relying on an EV71 specific vaccine. And, because the clinical symptoms of EV71 and CA16 infections are difficult to differentiate, there are further restrictions on the application of the EV71 vaccine. For these reasons, it is urgent to develop CA16 specific or combined CA16/EV71 vaccines. Because most severe cases of HFMD were caused by EV71, studies on EV71 vaccine have progressed rapidly in recent years. Among them, the EV71 inactivated vaccines developed by Mainland China and Taiwan have, respectively, entered phase III and phase I trials (Clinical trials. govid: NCT , NCT , NCT , NCT ). But the research work on CA16 has been neglected in the past. Recently, diseases induced by CA16 and the development of CA16 vaccine have received increasing attention and several companies in Mainland China have begun to launch CA16 vaccine development projects. The successful development of an EV71 vaccine benefits not only from other well-developed inactivated enterovirus vaccines, but also from the establishment and application of a neonatal mice model and a macaque model in evaluating the clinical or pre-clinical protection effects (1, 5, 27, 29). For CA16, a study using a CA16 animal experiment was first reported in 1951 (36). It indicated that CA16 might cause paralysis and even death in newborn mice. However, until now no CA16 animal model has been established to further evaluate the efficacy of a potential vaccine. In the current study, we established a neonatal mice model by applying a particular clinically isolated CA16 strain, BJCA08, and evaluated the efficacy of an inactivated CA16 vaccine to provide an useful tool for the R & D of CA16 vaccines. Materials and Methods Cells and virus. Vero cells (American Type Culture Collection, Manassas, VA) were maintained in Eagle s minimum essential medium (MEM) containing 2% or 10% fetal bovine serum (FBS) plus 2 mm 4

5 L-glutamine, 100 IU of penicillin, and 100 μg of streptomycin per ml. A local isolate, BJCA08/CA16 (GenBank accession No.JX481738), was taken from a throat swab sample of a 3-year-old boy with hand, foot, and mouth disease (HFMD) in Beijing, Viral RNA was extracted from the BJCA08/CA16 and RT-PCR was used to obtain the sequence (44). Phylogenetic trees established from alignment of the VP1 and the VP4 regions indicated that the BJCA08 belonged to a C1 subtype. A titer of BJCA08 ( PFU/ml) was determined using a plaque assay on a Vero cell. The Institute of Medical Biology, Chinese Academy of Medical Science kindly provided G10/CA16, the first prototype of a CA16 strain, (South Africa/51, GeneBank accession No.U05876). G10 belongs to the A subtype of CA16 and was grown in Vero cells with a titer of PFU/ml. All stock viruses were grown in Vero cells, subjected to three freeze-thaw cycles, clarified by centrifugation at 3900g for 10 minutes at 4 o C, filtered through a syringe filter (0.2μm, Pall Corporation, Germany), and stored at -80 o C. Mouse infectious experiments. Outbred, specific pathogen-free ICR mice (Vital River Lab Animal Technology Co., Ltd, Beijing, China) were used to develop an animal model. All institutional (National Institute for Food and Drug Control) guidelines for animal care and use were strictly followed. The mice in an age-dependent experiment (at 1, 3, 5, 7, and 14 days of age) were selected (n=8~10, per age) and intracerebrally inoculated with BJCA08 ( PFU/mouse). For the dose-dependent experiment and the 50% lethal dose (LD 50 ) study, one-day-old ICR mice (n=8-10 per group) were intracerebrally inoculated with 8-fold serial dilutions of BJCA08 (0.3, 2.4, , , and PFU/mouse). The control mice were given an uninfected culture medium and kept in a separate cage from the infected mice. Mice were observed daily for clinical illness and death until 21 days post inoculation (dpi). The grade of clinical disease was scored as follows: 0, healthy; 1, lethargy and inactivity; 2, wasting; 3, limbshake weakness; 4, hind-limb paralysis; and 5, moribund and death (Table 1) (20). The control mice were healthy throughout the experiments. LD 50 was calculated as described by Reed and Muench (32). Histopathologic and immunohistochemical staining. Five days after intracerebral inoculation with BJCA08 (300PFU/mouse) or uninfected culture medium, 12 experimental animals (grades 3 to 5) and six control animals (grade 0) were subjected to histopathologic and immunohistochemical 5

6 examination. After anesthetization, samples were taken of blood, brain, spine, heart, lung, thymus, liver, spleen, kidney, intestines, hind-limb, and spinal skeletal muscles. The mouse tissues were fixed by immersion in 10% neutral-buffered formalin for at least 2 days. Then tissues were bisected and embedded in paraffin. For histopathologic examination, tissue sections were stained with hematoxylin and eosin. For immunohistochemical testing, tissue sections were dewaxed, dehydrated, and microwaved for 14 minutes at 92 o C~99 o C in a citrate buffer. Monoclonal mice anti-ca16 VP1 antibody (MAb) T26 (1:32000 dilution; a gift from Beijing Wantai Biological Pharmacy Enterprise Co., Ltd., Beijing, China) was applied for 1 hour at 37 o C. A peroxidase-conjugated secondary Ab (1:2 dilution; Fuzhou Maixin Biotechnology Development Co., Ltd., Fuzhou, China) was added for 30 mins at room temperature, followed by 3, 3 -diaminobenzidine tetrahydrochloride (Fuzhou Maixin Biotechnology Development Co., Ltd., Fuzhou, China) chromogen. Tissues were counterstained with hematoxylin. Control sections were incubated with normal goat serum instead of anti-ca16 MAb. Virus loads in infant mouse tissues post-challenge. After intracerebral inoculation with BJCA08 (300PFU/mouse) or uninfected culture medium, 24 experimental mice and three control mice were subjected to virus load assays. After the heart was punctured, blood samples were collected and stored at -80 C. Tissue samples (from the brain, heart, lung, liver, spleen, kidney, intestines, and hind-limb skeletal muscles) were aseptically removed, weighed, and stored at -80 C. Tissues and blood samples from experimental mice (n=3, per time point) were collected at 1, 6, 12, 24, 48, 72, 96, and 120 h post infection. Samples from control mice (n=3) were collected at 0 h post inoculation. The tissue samples were homogenized in sterile phosphate-buffered saline (10%, wt/vol), disrupted by three freeze-thaw cycles, and centrifuged. Virus loads in clarified supernatants and blood were determined by real-time quantitative reverse transcriptase PCR (qrt-pcr) and expressed as log 10 copies/mg of tissue or log 10 copies/ml of blood. qrt-pcr. At selected intervals post-infection, the mice in the experimental and negative control groups were sacrificed. Blood and homogenized tissues were harvested for RNA by Mag Max TM viral isolation kit (AMBION Inc., Austin, TX, USA) following the manufacturer's recommended protocols. 6

7 According to the instructions of Ag path-id TM One-step RT-PCR kit (AMBION Inc., Austin, TX, USA), cdna was synthesized from RNA by reverse transcription for 30 min at 42 C and subsequently amplified for 40 cycles at 95 C for 5 s, 55 C for 35 s, and 72 C for 2 min. RNA from the negative control group was run simultaneously in each qrt-pcr reaction. Protective efficacy of neutralization antibody. To confirm the protective role of the humoral immune response, three experiments were carried out. First, by using goat anti-g10/ca16 serum (NT titer 1280, a gift from the Institute of Medical Biology, Chinese Academy of Medical Science), a passive immunization to protect pups against a CA16 challenge was studied in vivo. One-day-old ICR mice (n=8-10 per group) were, respectively, intraperitoneally inoculated with 50ul of 10-fold serially diluted goat anti-g10 serum (10- to fold dilutions) and medium. Within one hour after inoculation, each mouse was intracerebrally challenged with 300PFU of BJCA08. The mortality and clinical symptoms were then monitored and recorded daily after infection until 21 days after inoculation. ED 50 Ⅰwas calculated as described by Reed and Muench (32). In the second experiment, BJCA08 was first neutralized by serially diluted anti-g10 serum in vitro, and then intracerebrally inoculated into the pups to test the neutralizing effect of anti-serum. Serially diluted anti-g10/ca16 serum (10- to fold dilutions) and medium were, respectively, incubated with 300 PFU of BJCA08 at 37 C for an hour. One-day-old ICR mice (n=8-10 per group) were intracerebrally inoculated with the mixture described above. All mice were monitored daily for clinical symptoms and death until 21 days after inoculation, and ED 50Ⅱ was calculated as described by Reed and Muench (32). Lastly, maternal antibody protection was studied. BJCA08 ( PFU/ml) and G10 ( PFU/ml) were inactivated by adding 37% formaldehyde (Sinopharm Group, Beijing, China) to the suspensions of virus with a final formaldehyde concentration of 1/4,000. The viral suspension was then incubated at 37 C for 3 days (25). The inactivated suspensions were filtered as described above and stored at -80 o C. No live viruses were detected after repeated development of Vero cell cultures for a 7

8 period of up to 3 weeks. Eight-week-old female ICR mice (n=2 per group) were, respectively, intraperitoneally injected twice at 2-week intervals with 0.5ml formaldehyde-inactivated G10, formaldehyde-inactivated BJCA08 or medium. The mice were allowed to mate one hour after the first injection. After delivery (about 5-10 days after the boost), pups were intracerebrally challenged with BJCA08 (300PFU/mouse) on postnatal day 1. Body weight, clinical symptoms, and death of the challenged suckling mice were monitored as described above. Dams were euthanized and sera were collected and stored at -80 C until use. Statistical analysis. The clinical scores and virus load values were, respectively, analyzed by the nonparametric one-way ANOVA and unpaired t test. Survival rates were evaluated by the Mantel Cox Log-rank test. The body weight was compared using Dunn's Multiple Comparison Test. The results were expressed as means and standard deviation (SD) of the means. LD 50, ED 50Ⅰ, and ED 50Ⅱ were calculated as described by Reed and Muench (32). A P value of < 0.05 was considered statistically significant. Results Intracerebral inoculation of BJCA08/CA16 results in age-related disease and mortality. To examine the sensitivities of BJCA08/CA16 on neonatal mice with different ages (in days), neonatal mice at ages 1-14 days old were intracerebrally inoculated with BJCA08 ( PFU/mouse). The results showed that varying grades of clinical diseases, such as wasting, hind-limb paralysis, and even death occurred in the neonatal mice (inoculated at ages 1, 3, 5, and 7 days), but all mice inoculated at 14 days survived (Fig.1). The severity of clinical symptoms from mild to severe was scored as five grades (Table 1). Fig.1 shows that clinical diseases of one-day-old mice first occurred at 4 days post infection (d.p.i.) with a mean clinical score grade 3 and all were dead at 6 d.p.i. Both three-day-old mice and five-day-old mice began to get sick at 5 d.p.i. at a grade 3, and these mice were dead at the 13th and 11th day post infection, respectively. Seven-day-old mice started to exhibit clinical symptoms at 5 d.p.i. at a grade 2, and five of them gradually recovered starting at 10 d.p.i., with a reduced mortality rate of 55.6%. No clinical incidents or mortality were observed among 14-day-old mice. These data indicated that one-day- 8

9 old neonatal mice are the most sensitive to BJCA08 infection. The symptoms and mortality rates steadily decreased as age increased. The Mantel-Cox log-rank test found that there was a statistically significant difference in survival rate between one-day-old mice and other age groups (p<0.0001). Therefore, neonatal one-day-old mice were selected as the target animal in the current study. Intracerebral inoculation of BJCA08/CA16 results in dose-related disease and mortality. To determine the dose-response effects and LD 50, one-day-old mice were inoculated intracerebrally with serially diluted BJCA08. Fig. 2 shows that the mice that were i.c. inoculated with doses of and PFU/mouse became sick at 4 d.p.i. at grade 3, and mice infected with a dose of PFU/mouse became sick 7 d.p.i. at grade 4. These three infective doses mentioned above ( , and PFU/mouse) caused all mice to die at different days post infection (6, 9, and 12 d.p.i., respectively). With infection doses of 2.4 and 0.3 PFU/mouse, the mortality rates were, respectively, 62.5% and 25%, with some mice progressively recovering after infection. These results demonstrated significant dose-response effects between BJCA08 infective dosages and the mortality rates of neonatal mice (the calculated LD 50 was 1.2 PFU/mouse). To guarantee the reproducibility of the animal model, a dose of PFU/mouse (i.e. 260LD 50 ) was given as a lethal challenge. Repetitive experiments showed that under this dosage all one-day-old mice became sick within 4-5 d.p.i. and all were dead between 6 to 10 d.p.i. The morbidity time and mortality rates were stable and had good reproducibility (Supplemental Table 1). Pathological changes in infected mice after intracerebral challenge with a lethal dose of BJCA08/CA16. Although mice with different challenge ages have different onset time of diseases (Fig.1), the typical clinical signs of them were same, i.e. they all exhibited wasting, hind-limb paralysis and so on. Therefore, in this study we selected the representative endpoints including wasting, hind-limb paralysis which were from one-day-old mice infected by BJCA08 (260LD 50 ) at 8 d.p.i (Fig.3 a and b). To understand the distribution of virus and pathological changes, one-day-old mice infected by BJCA08 at grade levels 3-5 were sacrificed for pathological and immunohistochemistry (IHC) examinations. The results showed that the hind-limb skeletal muscle fibers exhibited severe necrosis (Fig. 3 c); IHC staining 9

10 indicated that widespread CA16 antigen was observed in the corresponding areas of lesion (Fig. 4 a). Moreover, severe necrosis and CA16 antigen were also observed in the juxtaspinal skeletal muscles fibers (Fig. 3 d and Fig. 4 c), while on the same IHC section, intramedullary nerve cells surrounded by necrotic skeletal muscles were normal with no viral antigen (Fig. 4 d). This indicated that BJCA08 had a strong tropism to muscle rather than to nervous tissues, and also indicated how it differed from EV71. Within the encephalocoele (the place of inoculation), limited CA16 antigen was only observed in the ependymal epithelium and the choroid plexus epithelium of lateral ventricle (Fig 4. g), but no obviously pathological change was found (Fig 3. g). Also, on the same IHC section, no obviously positive reaction to viral antigen was found in the neurocytes of the brain (Fig 4. h). In addition, for moribund neonatal mice (grade 5), necrosis in muscle fiber was observed in focal cardiac tissues (Fig 3.e), and the relevant IHC staining showed viral antigen (Fig 4. e). No viral antigen and pathological change were detected in lymphoid organs including the spleen and thymus of the BJCA08 infected neonatal mice. In the control group, treated with the culture medium, all detected tissues had no observable pathological change, and the IHC staining also showed negative reactions (Fig. 3 f, h and Fig. 4 b, f). Tissue viral loads in BJCA08/CA16-infected mice. Detection of tissue viral loads may indicate the characteristics of virus distribution in infected mice at different time points after inoculation. Fig. 5 shows that the virus was first detected in brain ( copies/mg) and blood ( copies/ml) tissues at 1 h after inoculation. At different time points post infection (6 h, 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h), the quantity of virus was at a stable level in the blood ( , , , , , , and copies/ml, respectively). Twenty-four hours after inoculation, the virus was detected in the brain, hindlimb skeletal muscle, and cardiac tissues ( , and copies/mg, respectively). Seventy-two hours later, the virus was ubiquitous in all tissues, such as intestine ( copies/mg), lung ( copies/mg), liver ( copies/mg), muscle ( copies/mg), brain ( copies/mg), spleen ( copies/mg), kidney ( copies/mg), and heart ( copies/mg), suggesting that the virus had already spread throughout the entire the body by means of the blood stream. The viral loads in the hind- limb skeletal muscles steadily increased with time (from copies/mg at 24 h to copies/mg at 10

11 h). At 120 h, the viral load in the hind-limb skeletal muscles was 4 log values higher than that in the blood ( copies/ml), and 2-3 log values higher than that in other tissues. For control mice, no viral loads were detected in the intestine, lung, liver, muscle, brain, spleen, kidney, heart, and blood. This observation was consistent with the results of pathological change and IHC staining, indicating BJCA08 has strong tropism to muscle tissues, which may be the major location for viral duplication. Passive immunization with anti-g10/ca16 immune serum protected pups against CA16 challenge in vivo and ED 50Ⅰ. The anti-g10/ca16 serum (neutralization titer 1280) was serially diluted and separately i.p. was injected into neonatal mice. Within one hour, the same mice were i.c. inoculated with the BJCA08 at a dosage of 260LD 50. The results (Fig. 6) showed that the control mice (i.p. injection culture medium) got sick at 6 d.p.i. (grade 3), started to die at 7 d.p.i., and all were dead by 11 d.p.i. Mice i.p. inoculated with a 1:10 dilution antiserum had 100% survival with no clinical symptoms. When i.p. inoculated with antiserum at 1:100 or 1:1000 dilutions, the experimental mice began to die at the 9th or 6th day post infection, respectively. Some mice in both groups suffered from mild symptoms and gradually recovered. The protection rates of the 1:100 and the 1:1000 dilution groups were, respectively, 25% and 22% at the end of the 21-day observation period. All mice i.p. inoculated with antiserum at a dilution of 1:10000 were dead at 11 d.p.i. The Mantel-Cox log-rank tests suggested that the survival rates in mice treated with an antiserum of 1:10 or 1:100 dilution were statistically different from the control group (P<0.0001). According to the survival rates for different serum-protective groups and the NT titer (1280) of anti-g10 serum, ED 50 Ⅰwas calculated using the Reed and Muench method (32). A significant dose-response effect was observed between the antibody concentration and the protection rates. Anti-G10 serum at the 85 times diluted concentration may protect half of the experimental animals in the in-vivo blocking test (i.e.ed 50 Ⅰ= 15) BJCA08/CA16 in vitro pretreated with anti-g10/ca16 serum could significantly lose its lethal effect in neonatal mice and ED 50Ⅱ. The In-vivo neutralizing study showed that mice in the control group 11

12 got sick 3 d.p.i. (grade 2), and all died at 9 d.p.i. (Fig. 7). The survival rates for anti-g10 serum at dilutions of 1:10, 1:100, and 1:1000 were, respectively, 80%, 50%, and 40%, and the clinical grades for different dilutions also reduced with the increasing quantities of antiserum. No protective role was found at a dilution of 1: At 9 d.p.i., all mice in the 1:10000 group were dead. Compared to the culture medium control, the anti-g10 serum at dilutions of 1:10, 1:100, and 1:1000 neutralized with BJCA08 in vitro at 37 for an hour can significantly reduce the mortality rates of neonatal mice (P<0.001). According to the survival rates for different serum-dilution groups and the NT titer(1280) of anti-g10 serum, ED 50 Ⅱwas calculated using the Reed and Muench method (32). Anti-G10 serum at the 160 times diluted concentration can neutralize virus and protect 50% of the experimental animals (ED 50Ⅱ = 8.3). Protection role of maternal antibody. In a previous study, we found that formaldehyde-inactivated G10/CA16 could induce a moderate humoral response in adult mice with neutralization titers from 64 to 512 one week after the second inoculation (unpublished data). Whether the maternal antibody could neutralize BJCA08 and protect the neonatal mice from disease or death is still unknown. Inactivated BJCA08, inactivated G10, and culture medium were used to i.p. immunize female adult mice twice at two-week intervals. They were allowed to mate one hour after the first injection. The female mice delivered pups 5-10 days after the second inoculation. Then, BJCA08 at a dosage of 260LD 50 was used to i.c. inoculate the one-day-old mice born to the immunized dams. Fig. 8 shows that the neonatal mice in the control group started to die at 6 d.p.i., and all were dead at 10 d.p.i. One-day-old mice born to dams that were immunized by inactivated BJCA08, had a survival rate of 93% without severe clinical symptoms. One-day-old mice born to dams immunized by inactivated G10 exhibited diseases and survived with a rate of 47%. These data suggest that using inactivated BJCA08 and G10 to immunize female adult mice may significantly reduce mortality rates in the neonatal mice (Mantel-Cox log-rank test, P<0.0001). The weight gain for neonatal mice born to inactivated BJCA08 immunized dams was fastest, followed by the pups born to dams immunized with inactivated G10. Almost no weight gain was 12

13 observed for the control mice and all control mice were dead at 10 d.p.i. (Fig.8). Weight gain was significantly higher in the inactivated BJCA08 group and in the G10 immunized groups as compared with the control group (P<0.0001). Discussion The animal model is very important in evaluating immunogenicity and the protective efficacy of a vaccine (23). To develop an EV71 vaccine, animal models for the neonatal mouse, the cynomolgus monkey, and the rhesus monkey have been established and improved. These animal models confirmed the protective role of vaccine on inducing neutralizing antibody and also promoted the development of the EV71 vaccine (1, 5, 27, 29). Until our study, no animal model had been developed to evaluate the protective efficacy of the CA16 vaccine. In 1948 Dolldorf and Sickles discovered that the coxsackievirus A generally had strong pathogenicity within newborn mice (36). But the details of the infection are still unclear. In 1993, Timo Hyypiä detected the genomes of the coxsackievirus in the tissues of newborn mice that were infected by five serotypes (A2, A9, A21, B3, and B4) using in situ hybridization. The results showed that coxsackievirus A could affect skeletal and cardiac muscles, while the coxsackievirus B subgroup infected a wide range of tissues (12). To develop an animal model for evaluation of the CA16 vaccine s protective efficacy, the BJCA08 strain was used to establish the neonatal mouse model which was the first CA16 strain isolated from a throat swab sample of a 3-year-old boy with HFMD in Beijing by our lab, The isolate was confirmed by serological and molecular biological detections without contamination of mycoplasma and adventitious viruses. The complete genome was sequenced (GenBank accession No.JX481738), which indicated that it belongs to C subtype demonstrating homology in VP1 with other CA16 strains isolated from Mainland China and Taiwan (Table 2). In the mouse model, newborn mice showed typical clinical symptoms and death following the infection of virus (Supplemental Figure 2). Since C subtype was major subtype of CA16 in China mainland in recent years, we selected BJCA08 to establish the neonatal mouse model to meet the need of evaluation of CA16 vaccines in China, although the pathogenesis comparison with other CA16 strains was not carried out in newborn mice. 13

14 The appropriated route of challenge is one of the important considerations for animal mode development. Because CA16 belongs to Enterovirus genus, in previous study we first compared the sensitivity of oral inoculation (p.o.), i.p. inoculation and i.c. inoculation in one-day-old mice. The results showed that one-day-old mice inoculated by p.o. with 20ul BJCA08 died earlier (4 to 6 days post infection) than those inoculated using i.p. with 50ul BJCA08 (7 to 12 days post infection), while 100ul BJCA08 given orally to one-day-old mice failed to induce 100% death (Supplemental Figure 3). This result indicated that the p.o. route was the most sensitive for the neonatal mice. Furthermore, for the oral route, newborn mice should be fasted for hours which may affect their normal growths. For the i.p route, the abdominal capacity of one-day-old mice was very limited, so the leakage often occurred which can make the challenge dose inaccurate. Given that aseptic meningitis, rhombencephalitis caused by CA16 in humans and the validity of EV71 mouse model of i.c. inoculation (4, 51-52), the i.c. inoculation was finally selected to establish this CA16 mouse model. According to the results, we found that the effects of BJCA08 were significantly correlated with the age of mice and the dosage used. On the basis of experimental data, we chose a 260LD 50 dosage of BJCA08 as the challenge dose to i.c. inoculate the neonatal mice. The infected mice exhibited wasting, hind-limb paralysis, and even death. Under this challenge dose, the average onset time of disease was about five days post infection, and all mice were dead between 6 to 10 days post infection (100% mortality rate). These data indicated good feasibility and reproducibility (Supplemental Table 1). Previous studies demonstrated that CA16 infection or immunization may induce neutralizing antibody generation (42). This is consistent with reports that the positive rate of neutralizing antibody was high in babies or infants living in epidemic areas (24, 48). Anti-CA16 serum was able to inhibit the cytopathic effect induced by CA16 in vitro (24). It is important for the development of a CA16 vaccine to verify whether anti-ca16 serum has a similarly protective function in vivo as anti-polio and anti-ev71 serum. In the current study, we intracerebrally inoculated neonatal mice with previously prepared hyperimmune anti-g10 serum to examine the serum s protective efficacy. We conducted the in-vivo neutralizing experiment. Serially diluted anti-g10 sera were, respectively, mixed with 260LD 50 of BJCA08 at the 14

15 same volume. Then the neonatal mice were i.c. inoculated with the mixture after it had been neutralized at 37 for one hour. The results showed that anti-g10 serum could neutralize BJCA08 and reduce the severity of the diseases and the mortality rate, which displayed significant dose-response effects. A 10μl anti-g10 serum with neutralization titer 8.3 may protect 50% of the neonatal mice from death. Second, a passive immunization protection test was performed to examine whether anti-g10 serum displayed similarly protective function as the in-vivo neutralizing test. We intraperitoneally injected a series of diluted anti-g10 serum into one-day-old mice. Within one hour, the mice were i.c inoculated by BJCA08 with a dosage of 260LD 50. The results showed that in a passive immunization protection test, the neutralizing antibody also had a protective efficacy with a significant dose-response effect, but required a higher quantity to achieve the same effect as the in-vivo neutralizing test. A 50μl anti-g10 serum with neutralization titer 15 may protect 50% of the neonatal mice from death. These data mentioned above suggested that sufficient anti-g10 serum was able to protect neonatal mice from a lethal challenge. To further explore the protective effect of vaccine in the neonatal mouse model, we chose maternal instead of neonatal immunization because immunizing one-day-old puppy is not only technically difficult but also not suitable for the evaluation of CA16 vaccine as the susceptibility of the animal is only within a few days. Similarly, EV71, with the very close gene homology and clinical symptoms to CA16, is also sensitive to suckling mice (1,52). Thus, the maternal antibody protection method for the evaluation of EV71 vaccines was successfully used (1,40,50,52,56-57), in which dams were immunized and specific maternal antibody vertically transferred from dams to pups demonstrated excellent protection in the pups against EV71 challenge. Moreover, the protection rates of newborn mice were dose-dependent (1). In 2012, Li et al. first reported of a phase Ⅰ clinical trial of the EV71 vaccine (Clinical trials. govid: NCT and NCT ), in which a clear dose-dependent correlation was observed (55). Referring to the EV71 maternal model, in our research BJCA08/CA16 at a dosage of 260LD 50 was used to intracerebrally challenge the newborns of the dams immunized with inactivated CA16 (G10 and BJCA08). The clinical symptoms and survival rates of infected neonatal mice were analyzed to indirectly 15

16 evaluate the protective effect of inactivated CA16. The results demonstrated that both inactivated G10 and BJCA08 may protect the infected neonatal mice to varying degrees, and also indicated that vertical immunity could be induced by the CA16 vaccine. The survival rates of neonatal mice born to dams immunized with inactivated G10 and BJCA08 were, respectively 47% and 93%. At the end of the observation period (21 days post infection), the serum neutralization titers of female mice immunized by G10 and BJCA08 were, respectively 28 and 14 (Supplemental Table 2). These data indicated that immunization with inactivated CA16 virus can induce maternal antibody, which in turn prevents neonatal mice from a lethal challenge of BJCA08. But an unparalleled phenomenon was observed between the protection rates for neonatal mice and the antibody levels of immunized dams. The result was consistent with the observation by Wu et al. concerning the EV71 neonatal mouse model (40). It suggested that the higher protective level offered by the inactivated virus vaccine was not corroborated by a demonstrably higher neutralization titer. This may be explained by differences between in vivo and in vitro functions of pathogen-specific immunoglobulin. It may also be associated with the great differences in the genomic sequences between G10 and BJCA08. Comparing the nucleotide sequences of G10 and BJCA08, it can be seen that they belong to the same serotype, but to a different genotype (A and C1). The homology of VP1 nucleotide sequences is only 76.3% (Supplemental Figure 1) (44). Whether they have different neutralizing epitopes or cross-protective capacity requires further investigation. Both EV71 and CA16 have a similar viral structure because they belong to the same Enterovirus genus. Studies concerning the EV71 neonatal mouse model showed that EV71 was neurotropic, and might enter the brain through the nervous system and cause CNS complications (4, 29). To understand the pathogenic mechanism of CA16 infections, we conducted a pathological examination and IHC staining on the infected neonatal mice. No pathological change or positive CA16 antigen was found in neurocytes of the brain or the spinal cord, except for in the brain ependymal epithelium. This indicated that BJCA08/CA16, differing from EV71, had no significant neurotropism in the neonatal mouse. However, the hind-limb skeletal muscles showed severe necrosis. CA16 antigen was also observed in the corresponding areas of lesion. Moreover, severe necrosis in the juxtaspinal skeletal muscles fibers 16

17 and positive CA16 antigen indicated that BJCA08 has a strong tropism to muscle tissues. These findings are consistent with the results reported by Sickles (36). In addition, focal necrosis was also observed in the cardiac tissues of the moribund mice, and positive CA16 antigen was found in the corresponding IHC staining. These findings suggest that the lethal lesions occurring in the cardiac tissues at late stages of the disease may cause death in neonatal mice. The findings were also consistent with clinical observations that some deaths resulted from lethal myocarditis (7-8, 39). Using qrt-pcr, we found that CA16 was detected in brain tissue and blood at 1 h post inoculation, while the virus in the brain may be residual due to the i.c. inoculation at this time. The viral loads in the blood were steady and increased slightly from 1 h to 120 h post infection. Twenty-four hours after inoculation, the virus was first detectable in the brain, the hind-limb skeletal muscles, and the cardiac tissues. Thereafter, the virus gradually spread to all tissues (liver, lung, and intestine at 48 h; spleen and kidney at 72 h). These data revealed that after intracerebral injection, BJCA08 may enter the blood stream by the capillary vessels of the brain, then arrive and replicate in target tissues through blood circulation, and finally spread to the whole body. Among tissues having the virus at the earliest time, only the skeletal muscles had viral loads that steadily increased. It was speculated that CA16 was continuously replicated in the muscles and released into blood circulation, which then caused lesions and necrosis in cardiac muscles, and finally led to death. The population most vulnerable to severe disease from CA16 infection is children under five yearsof-age. Clinical symptoms caused by CA16 infection are usually mild such as HFMD, fever, and herpangina. But some reports indicated that CA16 infection also can cause severe complications such as aseptic meningitis, rhombencephalitis, pulmonary complications, cardiac and pericardial disease, and even fatal myocarditis and pneumonitis (15-16, 28, 33, 37-39, 43). The disease process caused by the CA16 infection progressed very quickly when severe complications occurred. However, the potential pathogenesis as well as the mechanism underlying lethal CA16 infection in humans is still unclear. Thus, in this study a newborn mouse model of CA16 infection was established by using i.c. inoculation with the clinical isolate strain BJCA08. Although the main cause of morbidity and mortality in our animal model is 17

18 skeletal muscle myositis with no significant involvement of the CNS, a moribund mouse exhibited severe focal myocarditis that is similar to a human case of a 15-month-old boy died of myocarditis following HFMD (38). Our data clearly indicated that this model is useful for the assessment of vaccine efficacy. A few cases of EV71 and CA16 co-infections have been reported in humans (11, 14, 45, 47, 49). Clearly, the incidence, pathogenicity, and pathogenesis of the co-infections need further investigation. It would be of interest to evaluate whether this mouse model could be applied to the study of such co-infection. Experiments are ongoing in our laboratories to address this potentially significant issue. In summary, we used the clinically isolated BJCA08 viral strain to develop the first CA16 neonatal mouse model for the evaluation of the vaccine s the protective efficacy. This animal model employed survival rate as an evaluation indicator with a good reproducibility. Pathological observation, IHC staining, and quantitative real time PCR revealed that BJCA08 had a strong tropism to skeletal and cardiac muscles, and caused necrosis. Using a maternal antibody protection study and an anti-serum protection study, we demonstrated that the specific CA16 neutralizing antibody might block invasion of the virus, and were able to evaluate the protective efficacy of the CA16 vaccine. In order to further improve this model, particularly on better understanding of virus transmission in vivo, and natural infection process in human beings, we are collecting CA16 virus strains with more virulent nature to address the aforementioned issues. Acknowledgements We wish to acknowledge the assistance of the Institute of Medical Biology, Chinese Academy of Medical Science for the CA16/G10 and goat anti-g10 serum. We wish to acknowledge the assistance of Beijing Wantai Biological Pharmacy Enterprise Co., Ltd. for the anti-ca16 MAb T26. This work was supported by National Science Project(No. 2008BAI69B01)and National 11th Five 451 Major Special Projects(No. 2009ZX )Funding Program. 18

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21 Severe hand, foot, and mouth disease caused by mixed infection of enterovirus 71 and coxsackie A16: report of 6 cases. Chinese General Practice. 14(10): Zhang YJ, Bai J, Huang XL Clinical analysis of 54 cases of hand, foot and mouth disease with central nervous system damage. Chin J Crit Care Med, 30 (12): Zhu Z, Zhu SL, Guo XB, Wang JT, Wang DY, Yan DM, Tan XJ, Tang LY, Zhu H, Yang ZH, Jiang XH, Ji YX, Zhang Y, Xu WB Retrospective seroepidemiology indicated that human enterovirus 71 and coxsackievirus A16 circulated wildly in central and southern China before large-scale outbreaks from Virol J., 7: Zhang W, Wang YG, Yang ZH, Pang BD, Wu H, Yang QZ, Jin M, Yang JL, Li XW, Liu QQ, Zhang YL Severe Hand-Foot-and-Mouth Disease Caused by Mixed Infection of Enterovirus 71 and Coxsackie A16:Report of 6 Cases. Chinese General Practice.29: Chiu CH, Chu C, He CC, Lin TY Protection of neonatal mice from lethal enterovirus 71 infection by maternal immunization with attenuated Salmonella enterica serovar Typhimurium expressing VP1 of enterovirus 71. Microbes Infect. 8: Chan YF, AbuBakar S Human enterovirus 71 subgenotype B3 lacks coxsackievirus A16-like neurovirulence in mice infection. Virol J. 2: Dong C, Liu L, Zhao H, et al Immunoprotection elicited by an enterovirus type 71 experimental inactivated vaccine in mice and rhesus monkeys. Vaccine. 29 (37): Paoletti LC, Pinel J, Kennedy RC, Kasper DL Maternal antibody transfer in baboons and mice vaccinated with a Group B streptococcal polysaccharide conjugate. Journal of Infectious Diseases. 181 (2): Yu, CK., Chen CC, Chen CL, Wang JR, Liu CC, Yan JJ, and Su IJ Neutralizing antibody provided protection against enterovirus type 71 lethal challenge in neonatal mice. J. Biomed. Sci. 7: Li YP, Liang ZL, Gao Q, Huang LR, Mao QY, Wen SQ, Liu Y, Yin WD, Li RC, Wang JZ Safety and immunogenicity of a novel human Enterovirus 71 (EV71) vaccine: A randomized, placebo-controlled, double-blind, Phase I clinical trial. Vaccine. 30(22): Chung YC;Ho MS;Wu JC;Chen WJ;Huang JH;Chou ST;Hu YC Immunization with virus-like particles of enterovirus 71 elicits potent immune responses and protects mice against lethal challenge. Vaccine. 26(15): Chen CW, Lee YP, Wang YF, Yu CK Formaldehyde-inactivated human enterovirus 71 vaccine is compatible for co-immunization with a commercial pentavalent vaccine. 29:

22 Table 1 Grading score for clinical symptoms of BJCA08/CA16 infected mice Grade Clinical signs 0 Healthy 1 Lethargy and inactivity 2 Wasting 3 Limb-shake weakness 4 Hind limb paralysis 5 Moribund and death Table 2 Nucleotides identity of BJCA08 with other CA16 virus strains in VP1 region Genotype (or Subtype) A B C1 C1 C2 Isolate G10 SHZH00 SHZH05 GZ08 TW01 GeneBank accession No. (Gene/s) FIGURE LEGENDS: U05876 AY EU FJ AF Location South China, China, China, Africa Shenzhen Shenzhen Guangdong Year of isolation Nucleotides identity with BJCA08 in VP1 region (%) Fig.1. Intracerebral inoculation of BJCA08/CA16 results in age-related disease and mortality. ICR mice (n=8-10 per age) were intracerebrally inoculated with BJCA08 ( PFU/mouse) at 1, 3, 5, 7, or 14 days of age. BJCA08-induced mean score of clinical disease and BJCA08-induced mortality were monitored and recorded daily after infection. Representative results of two similar experiments were shown. The Mantel-Cox log-rank test was used to compare the survival rates of pups between each age group and the one-day-old group at 21 days of post-infection. *** p < China Taiwan Fig.2. Intracerebral inoculation of BJCA08/CA16 results in dose-related disease and mortality. One-day-old ICR mice (n=8-10 per group) were intracerebrally inoculated with increasing dosages of BJCA08 (from 0.3, 2.4, , to PFU/mouse). Control animals were given a medium instead of a virus. The mortality and clinical disease were monitored and recorded daily after infection. 22

23 One representation of two independent experiments was shown. LD 50 was calculated as 1.2 PFU/mouse and PFU/mouse (260LD 50 ) was chosen as the challenge dose Fig.3. Histological examination for infected mice after intracerebral challenge with lethal doses of BJCA08/CA16. One-day-old ICR mice were intracerebrally inoculated with medium (mock control) or BJCA08 (260LD 50 ). (a) A representative picture of wasting caused by BJCA08 at day 8 of post-infection was shown (arrow). (b) A representative picture of right hind limb paralysis caused by BJCA08 at day 8 of post-infection was shown (arrow). Mice on the right side of figure a and b labeled in yellow were naive age-matched controls. Representative sections were shown. Infected mice (grades 4 to 5) exhibited severe necrotizing myositis in hind limb skeletal muscles (c, arrow). Severe necrotizing myositis in skeletal muscles near the spine (d, arrow) was also detected in infected mice (grades 4 to 5). A moribund mouse due to BJCA08 exhibited focal myocarditis (e, arrow); in contrast, no histological change was observed in the heart of the mock control mice (f). No histological change in the brain was observed in the brain of the infected mice (grades 3 to 5) (g) and the mock control mice (h). Samples in panels (c) to (h) were stained with hematoxylin and eosin stain; magnifications 400 (c to h). Fig.4. Immunohistochemical results for infected mice after intracerebral challenge with lethal doses of BJCA08/ CA16. One-day-old ICR mice were intracerebrally inoculated with medium (mock control) or BJCA08 (260LD 50 ). Representative sections are shown. Infected mice (grades 4 to 5) exhibited numerous viral antigenpositive fibers in hind-limb skeletal muscles (a, arrow); in contrast, no viral antigen was observed in hind-limb skeletal muscles of the mock control mice (b). Numerous viral antigen-positive fibers (c, arrow) were also detected in skeletal muscles near the spine, but no viral antigen was observed in intralmedullary nerve cells (d) of infected mice (grades 4 to 5). A moribund mouse due to BJCA08, exhibited numerous viral antigen-positive fibers in the heart (e, arrow); in contrast, no viral antigens 23

24 were observed in the heart of the mock control mice (f). Viral antigens were detected in ependymal cells and chroid plexus epithelium (g, arrow), but no viral antigen was found in other parts of the brain of the infected mice (grades 3 to 5) (h). Samples in panels (a)-(h) were subjected to IHC analysis with DAB (3,3 -diaminobenzidine) chromogen and hematoxylin counterstain; magnifications, 400 (a to h) Fig.5. Mean tissue viral loads in BJCA08/CA16-infected mice. One-day-old ICR mice were intracerebrally inoculated with BJCA08 (260LD 50 ). Virus loads were assessed by real-time quantitative reverse transcriptase PCR in samples of the intestine, lung, liver, muscle, brain, spleen, kidney, heart, and blood from the infected mice. Samples were collected at the times indicated. Results represent the mean virus load (log 10 copeies) per milligram tissue or per milliliter of blood ± SD (three mice per group). Fig.6. Passive immunization with anti-g10/ca16 serum protected pups against CA16 challenge in vivo. One-day-old ICR mice (n=8-10 per group) were intraperitoneally inoculated with 50ul of 10-fold serially diluted anti-g10 serum (NT titer 1280) or medium. Within one hour after inoculation, each mouse was intracerebrally challenged with 260LD 50 of BJCA08. The mortality and clinical disease were monitored and recorded daily after infection. The Mantel-Cox log-rank test was used to compare the survival of pups between each anti-serum group and the medium control group at 21 days of post-infection. *** p < Fig.7. BJCA08/CA16 in vitro pretreated with anti-g10/ca16 serum could significantly lose its lethal effect in neonatal mice. Serially diluted anti-g10 serum (NT titer 1280) or medium were, respectively, incubated with an equal volume of 300 PFU BJCA08 at 37 C for one hour. One-day-old ICR mice (n=8-10 per group) were intracerebrally inoculated with the mixture described above. The mortality and clinical disease were 24

25 monitored and recorded daily after infection. The Mantel-Cox log-rank test was used to compare the survival of pups between each anti-serum group and the medium control group at 21 days of post- infection. *** p < ** p < Fig.8. Maternal immunization with inactivated whole-virus CA16 antigen protected newborn mice against lethal challenge. Adult female ICR mice (n=2 per group) were, respectively, intraperitoneally injected with formaldehydeinactivated G10 antigen ( PFU/mouse), BJCA08 antigen ( PFU/mouse) or medium twice at a 2-week intervals, and allowed to mate at 1 hour after the first injection. After delivery, pups were challenged with BJCA08 (300PFU/mouse) on postnatal day 1. The mortality and clinical disease were monitored and recorded daily after infection. The Mantel-Cox log-rank test was used to compare the survival of pups between each maternal immunization group and the medium control group at 21 days of post-infection. Dunn's multiple comparison test was used to compare the weight of pups between each maternal immunization group and the medium control group at 21 days of post-infection. *** p <

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