Protective efficacy of formaldehyde-inactivated whole-virus vaccine and antivirals in a murine

32 Pages·2017·3.69 MB·English

JVI Accepted Manuscript Posted Online 19 April 2017
J. Virol. doi:10.1128/JVI.00333-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
1 Protective efficacy of formaldehyde-inactivated whole-virus vaccine and
2 antivirals in a murine model of coxsackievirus A10 infection
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4 Zhenjie Zhanga,1, Zhaopeng Donga,f,1, Juan Lia, Michael J. Carrb,c, Dongming Zhuanga,
5 Jianxing Wangd, Yawei Zhange, Shujun Dingd, Yigang Tonge, Dong Lif,*, Weifeng Shia,* D
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7 a Shandong Universities Key Laboratory of Etiology and Epidemiology of Emerging Infectious Diseases, Taishan a
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8 Medical University, Taian, China e
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9 b Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education (GI-CoRE), fr
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10 Hokkaido University, Sapporo, Japan

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11 c National Virus Reference Laboratory, University College Dublin, Dublin 4, Ireland tt
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12 d Department of Viral Infectious Diseases Control and Prevention, Shandong Center for Disease Control and //
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13 Prevention, Jinan, China i.
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14 e Beijing Institute of Microbiology and Epidemiology, Beijing, China m
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15 f School of Public Health, Taishan Medical University, Taian, China o
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16 Keywords: HFMD, Coxsackievirus A10, mouse model, antiviral, vaccine. on

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17 * Corresponding authors. E-mail addresses: [email protected] (W. Shi), [email protected] (D. Li). o
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18 1 These authors contributed equally to this work. e
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19 Running title: A neonatal model of coxsackievirus A10 infection e
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20 ,
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21 ABSTRACT Coxsackievirus A10 (CVA10) is one of the major pathogens associated with hand, 1
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22 foot and mouth disease (HFMD). CVA10 infection can cause herpangina and viral pneumonia, b
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23 which can be complicated by severe neurological sequelae. Morbidity and mortality of u
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24 CVA10-associated HFMD has been increasing in recent years, particularly in the pan-Pacific t
25 region. There are limited studies however on the pathogenesis and immunology of
26 CVA10-associated HFMD infections, and few antiviral drugs or vaccines have been reported. In
27 the present study, a cell-adapted CVA10 strain was employed to inoculate intramuscularly
28 five-day-old ICR mice, which developed significant clinical signs including: reduced mobility,
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29 lower weight gain and quadriplegia, with significant pathology in the brain, hind limb skeletal
30 muscles and lungs of infected mice in the moribund state. The severity of illness was associated
31 with abnormally high expression of the proinflammatory cytokine IL-6. Antiviral assays
32 demonstrated that ribavirin and gamma interferon administration could significantly inhibit
33 CVA10 replication both in vitro and in vivo. In addition, formaldehyde-inactivated CVA10
34 whole-virus vaccines induced immune responses in adult mice and maternal neutralizing D
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35 antibodies could be transmitted to neonatal mice providing protection against CVA10 clinical n
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36 strains. Furthermore, high titer antisera were effective against CVA10 and could relieve early a
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37 clinical symptoms and improve the survival rates of CVA10-challenged neonatal mice. In d

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38 summary, we present a novel murine model to study CVA10 pathology which will be extremely o
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39 useful to develop effective antivirals and vaccines to diminish the burden of HFMD-associated h
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40 disease. :
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41 IMPORTANCE Hand, foot and mouth disease cases in infancy, arising from coxsackievirus A10 i.
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42 (CVA10) infections, are typically benign, resolving without any significant adverse events. Severe m
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43 disease and fatalities can however occur in some children, necessitating the development of r
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44 vaccines and antiviral therapies. The present study has established a newborn mouse model of o
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45 CVA10 which, importantly, recapitulates many aspects of human disease, with respect to the
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46 neuropathology and skeletal muscle pathology. We found that high levels of the proinflammatory e
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47 cytokine interleukin 6 correlated with disease severity and that ribavirin and gamma interferon
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48 could decrease viral titers in vitro and in vivo. Whole-virus vaccines produced immune responses 1
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49 in adult mice and immunized mothers conferred protection to neonates against challenge from 2
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50 CVA10 clinical strains. Passive immunization with high titer antisera could also improve survival 8

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51 rates in newborn animals. y

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52 e
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53 Introduction
54 Human enteroviruses (EVs) are positive-stranded RNA virus members of the genus
55 Enterovirus within the family Picornaviridae, order Picornavirales, and comprise four species:
56 EV-A, EV-B, EV-C and EV-D (1). Coxsackievirus A10 (CVA10) belongs to species EV-A, which
57 currently consists of 16 serotypes, including 11 serotypes of coxsackievirus group A and 5
2
58 serotypes of EV (2, 3). In general, fatal cases of CVA10 infection have been rarely reported, as
59 EVA71 and CVA16 were the major epidemic strains prior to 2010 (4). However, numerous
60 outbreaks of CVA10 have occurred during recent years in different geographic regions, such as, in
61 China (5-8), Finland (9) and France (10). Clinical data demonstrated that CVA10 infections are
62 commonly mild and self-limiting; however, a small proportion of children experience severe
63 complications, such as, meningitis, encephalitis, acute flaccid paralysis and neurorespiratory D
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64 syndrome (11, 12). At present, due to the lack of effective antiviral drugs, CVA10 infection with n
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65 severe complications is mainly treated with symptomatic and supportive therapy. Due to the recent a
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66 increase in incidence and severity of CVA10 infection, it is of vital importance to understand the d

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67 pathogenesis associated with this agent and to develop therapeutic drugs and vaccines (13). o
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68 In the present study, we have established an animal model of CVA10 infection using h
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69 five-day-old neonatal mice, in which the rapid proliferation of CVA10 was associated with clear :
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70 pathology. Employing this model, we have investigated the correlation between the expression of i.
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71 virus-induced inflammatory cytokines and disease severity, the antiviral effects of different drugs m
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72 and therapeutic antibodies. Finally, we have evaluated the immunoprotective effect of candidate r
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73 CVA10 formaldehyde-inactivated whole-virus vaccines inoculated through different infection o
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74 routes.
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75 e
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76 Materials and methods
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77 Ethics statement 1
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78 The experimental animals were outbred, specific-pathogen-free Institute of Cancer Research 2
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79 (ICR) mice [Certificate No: SCXK (Beijing) 2012-0001]. The feeding and processing of the 8

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80 animals were subject to the guidelines by the Technical Committee on Laboratory Animal Science y

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81 of Standardization Administration of China, and the study was carried out in accordance with the e
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82 approved guidelines by the Ethics Committee of Taishan Medical College (Permission number:
83 2016061).
84 Viruses and cells
85 Rhabdomyosarcoma (RD) cells (ATCC, CCL-136) were cultured in minimum essential
86 medium (MEM; HyClone), containing 10% fetal bovine serum (Gibco), at 37°C under 5% CO.
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87 CVA10 clinical strains (TA151R, WH21R, QD102R and HZ302R) were isolated from fecal
88 samples of 2-4 year-old patients who presented with HFMD in different cities of Shandong
89 Province during 2015. The CVA10 viruses were serially passaged three times in RD cells and the
90 titers were determined by a 50% tissue culture infective dose (TCID ) assay. Total RNA was
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91 extracted and the complete viral genomes were sequenced by standard methods. The sequences
92 have been submitted to GenBank (accession numbers: KY272007-KY272010). D
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93 Mouse infection experiments n
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94 To evaluate the pathogenicity of the TA151R strain in neonatal mice under different a
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95 experimental conditions, an animal model of infection was established based on a two-factor d

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96 analysis of variance with the infective dose and the inoculation method as the two independent o
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97 factors (three levels for each factor). For each inoculation dose and inoculation route experiment, h
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98 five-day-old neonatal mice were inoculated with different doses (102, 1.5 x 103 and 2.25 x 104 :
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99 TCID50/mouse) and also by different inoculation routes: intracerebral (i.c.), intramuscular (i.m.) or i.a
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100 intraperitoneal (i.p.), respectively. For age-dependent susceptibility assays, the susceptibility to m
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101 CVA10 was compared for mice of different ages (3, 7, 9, 14 and 21 days), each inoculated by the r
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102 i.m. route with a dose of 1.5 x 103 TCID . The body weight, clinical signs and survival rates of o
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103 the mice were monitored for 14 days post-infection (dpi). The grade of clinical disease was scored
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104 as reported previously (14), and scoring was carried out as follows: 0, healthy; 1, lethargy and e
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105 inactivity; 2, hind limb weakness; 3, single limb paralysis; 4, double hind limb paralysis; and 5,
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106 death. The control mice were healthy throughout the experiments. The LD was calculated using 1
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107 the Reed and Muench formula (15). 2
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108 Histopathologic and immunohistochemical staining 8

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109 Five-day-old ICR mice were i.m. inoculated with the CVA10 clinical strain TA151R (240 y

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110 LD50) or uninfected culture medium. Tissue samples of brain, heart, lung, intestines and e
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111 contralateral hind limb skeletal muscles from control and CVA10-infected mice (grades 4 to 5,
112 n=3) were dehydrated and embedded in paraffin according to procedures as described previously
113 (16). A microtome was used to generate 4 μm sections for histopathological examination, which
114 were stained with hematoxylin and eosin.
115 Immunohistochemistry was performed using an avidin-biotin immunoperoxidase technique as
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116 described previously (17). Briefly, tissue sections were dewaxed, dehydrated and microwaved for
117 20 min at 99°C in a citrate buffer. Polyclonal mouse anti-CVA10 antibody (1:200 dilution; Abcam)
118 was applied for 2 hours at 37°C. A secondary biotinylated goat anti-mouse immunoglobulin G
119 (1:1000 dilution; Abcam) was added, followed by avidin-biotin-peroxidase complex and
120 3,3'-diaminobenzidine tetrahydrochloride (Beyotime) chromogen. Tissues were counterstained
121 with hematoxylin. The primary antibody was replaced by Tris-buffered saline or normal mouse D
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122 serum in duplicate IHC assays and used as negative controls. n
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123 Virus loads in post-challenge mouse tissues a
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124 After inoculation of five-day old mice with lethal doses (240 LD ) of TA151R via the i.m. d
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125 route, tissues and blood samples (blood, brain, heart, lung, intestines and contralateral hind limb o
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126 skeletal muscles) from experimental mice (n=3 per time point) were collected at 1, 3, 5 and 7 dpi, h
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127 and from control mice (n=3) at the same time points. Total RNA was extracted from individual :
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128 tissue/blood samples from infected mice and controls using Trizol reagent (Takara), and cDNA i.
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129 was generated by reverse transcription using random hexamers primers and M-MLV reverse m
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130 transcriptase (Takara), according to the manufacturer’s instructions. The cDNA was then used for r
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131 real-time PCR with a GoldStar Taqman Mixture kit (CWBIO) and primers F-CA o
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132 (5’-CCTGAATGCGGCTAATCC-3’), R-CA (5’-TTGTCACCATWAGCAGYCA-3’) and the
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133 hydrolysis probe (5’-FAM-CCGACTACTTTGGGWGTCCGTGT-BHQ1-3’) (18) in the ABI 7500 e
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134 Fast System. Real-time PCR thermocycling reactions were as follows: 3 min at 95°C, followed by
e
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135 40 cycles of 95°C for 15 s and 55°C for 35 s. The target amplicon amplified by the primers F-CV 1
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136 and R-CV was inserted into the pMD18-T plasmid, producing the plasmid pMD18-T-CV, which 2
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137 was used as a standard for absolute quantification of CVA10 copy numbers with virus loads 8

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138 expressed as log copies/mg of tissue or log copies/ml of blood. The standard curve was y
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139 generated from serially diluted pMD18-T-CV (ranging from 102 to 1010 copies/μl) by linear e
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140 regression. The 95% confidence interval of the negative control values determined in various
141 organs and tissues were regarded as the reference value for methodological sensitivity (0
142 copies/mg or ml).
143 Cytokine assays
144 After inoculation of five-day old mice with lethal doses (240 LD ) of TA151R via the i.m.
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145 route, peripheral blood was collected at 1, 2, 3, 4, 5, 6 and 7 dpi from the experimental and control
146 groups, and centrifuged for 10 min at 800 x g at 4°C. Sera were frozen at -80°C until further
147 examination. The levels of cytokines IL-1β, IL-6, IL-18, IFN-γ and tumor necrosis factor α
148 (TNF-α); that of the anti-inflammatory cytokine IL-10; and those of the chemokines IL-4 and
149 IL-13 were measured using mouse cytokine enzyme-linked immunosorbent assay (ELISA) kits
150 (Multisciences Biotechnology) according to the manufacturer’s instructions. Mean values of the D
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151 replicates were used for statistical analysis. n
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152 In vitro and in vivo studies of antiviral inhibitory activities a
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153 After the RD cells were inoculated with CVA10, the inhibitory effect of different drugs on d

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154 CVA10 replication in vitro was measured using a CCK-8 kit (Multisciences Biotechnology), o
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155 following the manufacturer's instructions. The RD cells were placed in 96-well plates (5 x 103 h
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156 cells/well) and incubated overnight. When >80% confluent, the monolayers were infected with :
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157 TA151R at a multiplicity of infection (MOI) of 0.001. After adsorption of CVA10 for 1 h, the i.
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158 supernatant was discarded and the RD cells were treated with the following different m
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159 concentrations of drugs: ribavirin (20 μg), IFN-α2a (125 U), IFN-α2a (125 U), IFN-β (12.5 U), r
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160 IFN-γ (25 U), IFN-λ1 (125 ng), IL-1β (1250 U) and IL-6 (12.5 U). MEM medium was used as o
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161 negative control (NC). Four hours after treatment, the supernatant was discarded and 500 μl of
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162 MEM containing 2% fetal bovine serum was added to each well. The cells were incubated at 37°C e
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163 under 5% CO for 36 h. The supernatant was discarded, and the cells were washed with PBS three
2 e
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164 times. After adding 200 μl of 10% CCK-8 solution, the cells were incubated for 1.5 h and the 1
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165 absorbance of each cell was measured at 450 nm using a microplate reader (ELX-800, BIOTEK). 2
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166 The untreated RD cells were regarded as a reference with 100% growth activity. The 8

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167 MEM-treated cells were used as a negative control. The ratio of growth activities was calculated y

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168 by comparing the absorbance (OD) of the cell in the control group and the reference group, as e
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169 follows:
170 SP(%) = A −A1 ⅹ100%
A2−A1
171 Where, SP represents the survival rate of the drug-treated RD cells. A, A1 and A2 are the OD
172 values of cells in the experimental group, the reference group and the negative control group,
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173 respectively. After the RD cells were treated with TA151R for 48 h, the viral loads of the
174 supernatants in the experimental and the reference groups were determined by RT-qPCR every 8 h.
175 Mean values of the replicates were used for statistical analysis and virus loads were expressed as
176 log copies/ml.
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177 In order to further examine the antiviral activities of the drugs in the neonatal mice, an in vivo
178 antiviral assay was carried out. A lethal dose of TA151R (240 LD ) was injected into the D
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179 five-day-old mice via an i.m. route. After 1 h of inoculation, different doses of the drugs were n
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180 injected into the mice of the experimental group by an intravenous (i.v.) injection route, while a
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181 normal saline (NS) was injected into the mice of the control group. The clinical signs and d

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182 mortality were monitored and recorded daily until 14 dpi. o
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183 Preparation of formaldehyde-inactivated CVA10 strain TA151R whole-virus vaccines and h
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184 determination of anti-CVA10 antibody titers :
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185 An inactivated CVA10 suspension with a formaldehyde concentration of 1:4000 (v/v) was i.
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186 prepared by mixing 37% formaldehyde with CVA10 strain TA151R (1.1 x 107 TCID /ml). 10 ml m
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187 of the suspension was mixed with Freund's adjuvant or Freund's complete adjuvant (Sigma) in r
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188 equal volumes to produce an emulsion. The viral emulsion was then incubated at 37°C for 3 days o
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189 (19). Ultrasonic emulsification was employed to prevent the formation of virus aggregates as
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190 follows: 2 ml of antigen/adjuvant mixture (1:1) was fully emulsified using sonication for 5 e
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191 seconds at 5 seconds intervals, for a total of 8 times in an ice bath. No viable virus was detected
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192 after repeated RD cell culture and blind passage for up to 3 weeks. Eight-week-old adult ICR mice 1
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193 were inoculated with 50 μl of inactivated TA151R emulsion by the i.m. route. Blood of adult mice 2
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194 was collected 10 days after two immunizations with an interval of two weeks. The CVA10 8

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195 antiserum was separated by centrifugation at 4,000 x g for 10 min at 4°C, and stored at -80°C until y

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196 analysis. e
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197 The geometric mean titer (GMT) of the neutralizing antibody in the CVA10 antiserum was
198 determined by in vitro microneutralization assays as described previously (20) with slight
199 modifications. Briefly, the anti-CVA10 serum samples were 10-fold serially diluted (1:10 to
200 1:10,000) using MEM containing 2% FBS. 50 μl of diluted serum was mixed with 100 TCID of
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201 CVA10 strain TA151R in 96-plates, respectively, and incubated at 37°C for 1 h. Then, 5.0 x 103
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202 RD cells were added to each well of the 96-well plate and cultured at 37°C under 5% CO. Three
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203 days later, the cells were inspected for cytopathic effect (CPE). Neutralization titers were
204 determined as the highest serum dilution that could fully prevent cells from CPE.
205 Effects of passive immunization and maternal antibody on five-day-old neonatal mice
206 In order to study the protective effects of passive immunization, the CVA10 antiserum after
207 10-fold serial dilution (1:10-1:10,000) was injected into five-day-old neonatal mice by i.v. D
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208 injection (n=10), while the same volume of NS was injected into the mice of the control group. n
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209 After 24 h, a lethal dose of TA151R (240 LD ) was inoculated into the mice in both groups by the a
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210 i.m. route. The clinical signs and survival rates were monitored and recorded daily until 14 dpi. d

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211 To study the immunoprotective effect of the maternal antibodies, the neonatal mice were o
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212 subcutaneously inoculated with 200 μl of Freund's complete adjuvant-inactivated CVA10 h
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213 emulsion at 8 weeks of age and the immune response was then boosted by inoculation with 200 μl :
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214 Freund's adjuvant-inactivated CVA10 emulsion 2 weeks later. The female and male mice were i.
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215 mated after the first immunization, and the dams delivered 7-10 days after the second m
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216 immunization. Mice in the control group were immunized with the same volume of NS. A lethal r
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217 dose (240 LD ) of TA151R and the CVA10 clinical isolates WH21R (240 LD ), QD102R (240 o
50 50 n

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218 LD ) and HZ302R (240 LD ) (Fig. 1) were i.m. injected into the mice in the experimental group
50 50 o
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219 (n=10) and the control group (n=10). The clinical signs and survival rates were monitored and e
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220 recorded for 14 dpi, and the immunoprotective effect of the maternal antibody on the neonates was
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221 evaluated. 1
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222 Therapeutic effect of the CVA10 antiserum on the CVA10-challenged neonatal mice 2
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223 In order to study the therapeutic effect of the CVA10 antiserum on the infected neonatal mice, 8

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224 a lethal dose of TA151R (240 LD ) was first administered to the five-day-old neonatal mice by y
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225 the i.m. route sufficient to induce 100% mortality. The infected neonatal mice were quarantined. e
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226 The neonatal mice with a clinical score of <3 and ≥3 were selected as the early and late
227 treatment groups (n=10 in each group), respectively. The CVA10 antiserum with different dilution
228 ratios was injected into the neonatal mice by i.v. injection in the early and late treatment groups.
229 The dilution ratios of the CVA10 antiserum were 1:10-1:10,000, and 1:1-1:100 for the early
230 treatment and late treatment groups, respectively. The body weight, clinical signs and survival
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231 rates of the mice were monitored and recorded daily between 7 and 11 days posttreatment (dpt) to
232 evaluate the therapeutic effect of the antiserum on the neonatal mice.
233 Statistical analysis
234 All statistical analysis was performed with GraphPad Prism Version 5.0 (GraphPad 4 Software,
235 San Diego, CA, USA). The frequencies of survival and mortality in treated mice versus control
236 mice were assessed using Fisher’s exact two-tailed test. The results were expressed as means and D
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237 standard deviations (SD) from the means. Differences in the mean tissue viral titers, serum n
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238 cytokines concentrations and TCID were determined using a two-tailed Mann-Whitney U test or a
50 d
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239 Kruskal-Wallis test. LD was calculated as described by the Reed and Muench method (15). P d
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240 values <0.05 after two-tailed t-testing was regarded as significant. o
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241 Results tt
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242 Establishment of a mouse model of CVA10 infection //
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243 Five-day-old neonatal mice were inoculated with CVA10 strain TA151R at different doses (1.0 a
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244 x 102, 1.5 x 103 and 2.25 x 104 TCID ) via the i.m., i.p, and i.c. routes, respectively. Clinical signs,
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245 such as reduction in movement, appeared at 5-6 days after low dose inoculation (1.0 x 102 TCID ) g
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246 and the maximum clinical score was 3 (rounding to the nearest whole number, Figs. 2A to 2C). At n

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247 9-10 days following low dose inoculation (1.0 x 102 TCID ), recovery and weight gain were o
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248 observed (Figs. 2D to 2F), and the final survival rates were 70%, 80% and 100% (Figs. 2G to 2I), m
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249 for the mice inoculated via the i.m., i.p, and i.c. routes, respectively. These results suggest that the e
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250 low infection dose (1.0 x 102 TCID ) is unsuitable for five-day-old neonatal ICR mice. Mice 8
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251 infected with 1.5 x 103 TCID dose via the i.c. route did not show typical neurological symptoms 0
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252 (i.e. single or double hind limb paralysis) and only transient neonatal myasthenia gravis was
b
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253 observed. The average clinical score was 1.75 (Fig. 2C), and the survival rate was 60% (Fig. 2I). g
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254 At the same dose, the mice inoculated via the i.m. and i.p. routes began to show clinical symptoms s
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255 3 days after inoculation and the symptoms gradually worsened over time. The clinical score was
256 4-5 at 6-7 days after inoculation with typical neurological symptoms (Figs. 2A and 2B). Compared
257 with the control group, the body weight of the mice in these latter two groups decreased by 28.71%
258 and 19.19% (Figs. 2D and 2E), respectively. However, clinical scores of the mice inoculated via
259 the i.p. route had large variation and uneven distribution. The high dose (2.25 x 104 TCID ) of
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260 CVA10 resulted in a rapid disease onset in the neonatal mice and a short survival time (Figs.
261 2G-2I), especially in the group inoculated via the i.m. route, where all mice were dead 6-7 days
262 after inoculation (Fig. 2G). This result indicated that the high dose (2.25 x 104 TCID ) was also
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263 not suitable for the establishment of the CVA10 model. An intermediate dose (1.5 x 103 TCID )
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264 of CVA10 was administered to mice at different ages (3, 7 and 9 days) via the i.m. route and the
265 3-day-old mice had high clinical scores and short survival times (Figs. 2J and 2K); the 7 and 9 D
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266 day-old mice had a low survival rate of 10% and 20% (Fig. 2L), respectively. Finally, the neonatal n
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267 murine model of CVA10 infection was established by inoculating five-day-old mice with 1.5 x 103 a
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268 TCID of CVA10 via the i.m. route. The average clinical score was 4-5, and the mice died 8-9 d
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269 days after inoculation. In this model, the disease onset time and mortality rates were stable and o
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270 had good reproducibility. h
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271 :
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272 Pathology and immunohistochemistry i.
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273 HE and IHC staining of the major tissues were performed 5 days after CVA10 inoculation in m
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274 order to determine the pathological changes and antigenic distribution in tissues derived from the r
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275 CVA10-infected neonatal mice. The results showed that the virus had strong tropism to the o
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276 skeletal muscle and lung tissues (Fig. 3). Viral replication was associated with serious pathological
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277 damage, loose fiber and massive necrosis, accompanied by large numbers of lymphatic infiltrates, e
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278 muscle bundle fracture and fibrosis (Fig. 3A). In addition, the lungs showed interstitial fibrosis
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279 and inflammatory hyperemia, which is a typical clinical manifestation of severe interstitial 1
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280 pneumonia in neonates (Fig. 3B) with the virus diffusely distributed throughout the lung (Fig. 3J). 2
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281 In the brain tissue, the neurons and glial cells showed diffuse edema, and the nucleus was not 8

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282 completely lysed (Fig. 3C); the virus was mainly present in the cerebral cortex (Fig. 3K). y

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283 Compared with EVA71 and CVA16 infections, CVA10 showed myocardial fiber dissolution and e
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284 increased numbers of inflammatory cells, with focal fatty change (Fig. 3D) and detectable viral
285 antigen (Fig. 3L). The other organs of the infected mice, such as liver, intestine, kidney, spleen,
286 and lymph, were also examined, but no significant histological changes or viral antigens were
287 observed (data not shown).
288
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