A virus-like particle vaccine protects mice against coxsackievirus A10 lethal infection

Accepted Manuscript A virus-like particle vaccine protects mice against coxsackievirus A10 lethal infection Yu Zhou, Chao Zhang, Qingwei Liu, Sitang Gong, Lanlan Geng, Zhong Huang PII:

S0166-3542(17)30713-1

DOI:

10.1016/j.antiviral.2018.02.016

Reference:

AVR 4255

To appear in:

Antiviral Research

Received Date: 1 November 2017 Revised Date:

3 February 2018

Accepted Date: 17 February 2018

Please cite this article as: Zhou, Y., Zhang, C., Liu, Q., Gong, S., Geng, L., Huang, Z., A virus-like particle vaccine protects mice against coxsackievirus A10 lethal infection, Antiviral Research (2018), doi: 10.1016/j.antiviral.2018.02.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT A virus-like particle vaccine protects mice against coxsackievirus A10 lethal infection

Yu Zhou

a, #

, Chao Zhang

a, b, #

, Qingwei Liu a, Sitang Gong b, Lanlan Geng b, *, Zhong

a

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Huang a, b, *

Unit of Vaccinology & Antiviral Strategies, CAS Key Laboratory of Molecular Virology

& Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences,

b

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University of Chinese Academy of Sciences, Shanghai 200031, China

Joint Center for Infection and Immunity, Guangzhou Institute of Pediatrics,

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Department of Gastroenterology, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou 510623, China

These authors contributed equally to this work.

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#

* Correspondence: Zhong Huang, Institut Pasteur of Shanghai, 320 Yueyang Road, Shanghai 200031, China; Tel.:+86 21 54923067; E-mail: [email protected] (Z. Huang)

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* Correspondence: Lanlan Geng, Guangzhou Women and Children's Medical Center, 9 Jinhui Road, Guangzhou 510623, China; Tel.:+86 20-81330558; E-mail:

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[email protected] (L. Geng)

ACCEPTED MANUSCRIPT ABSTRACT Coxsackievirus A10 (CVA10) has emerged worldwide as one of the main pathogens of hand, foot, and mouth disease (HFMD) in recent years. However, there is currently no commercial vaccine available to prevent CVA10 infection. Here we report the

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development of a recombinant virus-like particle (VLP) based candidate vaccine for CVA10. Co-expression of the capsid protein precursor P1 and the protease 3CD of CVA10 in Pichia pastoris resulted in cleavage of P1 into three capsid subunit proteins VP0, VP1, and VP3. These three subunit proteins co-assembled into CVA10 VLPs,

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which were visualized as spherical particles with a diameter of ~30 nm under electron microscope. Immunization studies showed that CVA10 VLP could efficiently induce

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antigen-specific serum antibodies in mice. The anti-VLP sera were able to potently neutralize homologous and heterologous CVA10 strains. Importantly, passively transferred anti-VLP sera fully protected recipient neonatal mice from lethal CVA10 infection. In addition, neonatal mice born to the VLP-immunized dams were also

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completely protected from CVA10 lethal challenge. Collectively, these data show that CVA10 VLP represents a promising CVA10 vaccine candidate.

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Vaccine

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Keywords: Hand, foot, and mouth disease; Coxsackievirus A10; Virus-like particle;

ACCEPTED MANUSCRIPT 1. Introduction Hand, foot, and mouth disease (HFMD) is a common contagious disease that primarily affects infants and children under five years old (Repass et al., 2014). HFMD is usually caused by a few enteroviruses, including enterovirus 71 (EV71),

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coxsackievirus A16 (CVA16), coxsackievirus A6 (CVA6), and coxsackievirus A10 (CVA10) (Bian et al., 2015; Xing et al., 2014; Zhuang et al., 2015). Previous epidemiological studies revealed that EV71 and CVA16 had been the major pathogens of HFMD worldwide for decades (Mao et al., 2014; Wong et al., 2010).

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However, during recent years, the incidence of CVA10 infections has increased markedly in many countries, such as China (Chen et al., 2017; He et al., 2013; Yang

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et al., 2015), Finland (Blomqvist et al., 2010), Singapore (Wu et al., 2010), and France (Mirand et al., 2012). For instance, CVA10 infections accounted for 39% of severe HFMD cases in Xiamen, China in 2015 (Chen et al., 2017). These studies indicate that CVA10 is becoming one of the predominant causative agents of HFMD. Moreover,

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CVA10 is frequently found to co-circulate with CVA6, CVA16, and/or EV71 during HFMD outbreaks (Blomqvist et al., 2010; Mirand et al., 2012; Wu et al., 2010), increasing the possibility of co-infections and viral genetic recombination. Infection with CVA10 usually results in a mild and self-limiting disease; however, severe

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life-threatening complications and even death have been associated with CVA10 infection (Fuschino et al., 2012; Lu et al., 2012). Currently, there is no commercial

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vaccine available for preventing CVA10 infection. In the past, most efforts to develop vaccines against HFMD have mainly targeted

EV71 and CVA16 (Cai et al., 2014; Liu et al., 2012; Yi et al., 2017). Thus far, formalin-inactivated EV71 vaccines have been approved for marketing in China (Yi et al., 2017) whereas CVA16 vaccine development is still in preclinical stage (Cai et al., 2013; Chong et al., 2012; Liu et al., 2012). However, all of these EV71 and CVA16 vaccines failed to provide cross-protection against CVA10 infection (Liu et al., 2014). Recently, we and other groups have attempted to develop CVA10 vaccines by

ACCEPTED MANUSCRIPT employing the traditional inactivated whole virus vaccine approach (Li et al., 2017; Liu et al., 2016; Shen et al., 2016; Zhang et al., 2017). Although proof-of-concept for inactivated CVA10 vaccines has been demonstrated, future production of such vaccines would require growth of large quantities of live viruses under

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bio-containment and subsequent complete inactivation of viral infectivity, thus raising significant safety concerns. Compared to the inactivated whole-virus vaccine approach, virus-like particles (VLPs), which morphologically resemble authentic viruses, can be generated in recombinant expression systems and are therefore

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devoid of infectious viral genomes. VLP-based vaccines are in general safe, highly immunogenic and protective, as exemplified by licensed human papillomavirus (HPV)

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vaccines (Kushnir et al., 2012; Roldao et al., 2010). Our group has recently produced recombinant VLPs of EV71, CVA16, and CVA6, respectively, in yeast, and demonstrated their vaccine potency in preclinical studies (Zhang et al., 2015; Zhang et al., 2016; Zhou et al., 2016). In this study, we investigated the possibility of

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developing a VLP-based vaccine for CVA10. Our results showed that CVA10 VLP could be readily produced in Pichia pastoris yeast and this VLP could elicit protective immunity against CVA10 infection in mice.

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2. Materials and methods 2.1. Cells and Viruses

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Human rhabdomyosarcoma cells (RD cells; ATCC, CCL-136) were grown as

described previously (Ku et al., 2012). Pichia pastoris (P. pastoris) strain PichiaPink (Invitrogen, USA) was grown according to manufacturer's instructions. CVA10 prototype strain CVA10/Kowalik (GenBank ID: AY421767), and two CVA10 clinical isolates CVA10/S0148b (GenBank ID: KX094564) and CVA10/S0273b (GenBank ID: KX094565) were described previously (Shen et al., 2016). EV71 strain EV71/G082 and CVA16 strain CVA16/SZ05 were described previously (Cai et al., 2013; Ku et al., 2012). All viruses were titrated for the 50% tissue culture infectious dose (TCID50)

ACCEPTED MANUSCRIPT using RD cells, according to the Reed–Muench method (Reed and Muench, 1938).

2.2. Antibodies Polyclonal antibodies against VP0, VP1 or VP3 proteins of CVA10/Kowalik were

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described previously (Shen et al., 2016). Polyclonal antibody against inactivated CVA10/S0148b was generated in a previous study (Shen et al., 2016).

2.3. Vector construction

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RNA was isolated from CVA10/Kowalik-infected Vero cells using TRIzol reagent (Invitrogen, USA), and reverse transcription was performed with M-MLV reverse

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transcriptase (Promega, USA) and oligo (dT) primers to synthesize cDNA. Then 3CD gene fragment was amplified by PCR from the cDNA and subsequently cloned into the expression vector pPink-HC (Invitrogen, USA), to generate a plasmid called pCVA10-001. The P1 gene of CVA10/S0273b was codon-optimized, synthesized by

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GenScript (Nanjing, China), and subsequently inserted into pPink-HC, yielding a plasmid termed pCVA10-002. For co-expression of P1 and 3CD, the 3CD expression cassette was amplified by PCR from pCVA10-001 and cloned into pCVA10-002 from the Bgl

site by using ClonExpress II One Step Cloning Kit (Vazyme, China), yielding

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plasmid pCVA10-003.

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2.4. Yeast transformation and screening Prior to transformation, the plasmid pCVA10-003 was linearized by digestion with

EcoN . PichiaPink™ Strain 1 (Invitrogen, USA) cells were transformed with the linearized DNA by electroporation and subsequently plated onto PAD plates lacking adenine for selection of transformants according to the manufacturer's instructions. To screen high-expression recombinant strains, 24 colonies were randomly chosen from the plates, and small-scale expression experiments were performed as described previously (Zhang et al., 2015). Induced cells from each culture were harvested and

ACCEPTED MANUSCRIPT subjected to glass bead lysis followed by a centrifugal clarification step according to the manufacturer's instructions. The resulting lysates from each clone were analyzed for expression of CVA10 proteins by ELISA and Western blotting assays as described

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below.

2.5. ELISA and Western blotting

For ELISA, 96-well microplates were coated overnight at 4°C with 5 µl/well of yeast lysates plus 45 µl/well of PBS buffer, followed by blocking with 200 µl/well of 5%

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non-fat milk in PBST at 37°C for 1 h; 50 µl/well of mouse polyclonal antibody against inactivated CVA10/S0148b (diluted 1:500 in 1% milk/PBST) was added and incubated

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at 37°C for 2 h; then 50 µl of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Sigma, USA) was added to each well and incubated at 37°C for 1 h. The microplates were washed with washing buffer (PBST) for three times between each step. Finally, color was developed with TMB substrate (New Cell & Molecular

microplate reader.

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Biotech, China), and the absorbance at 450 nm was measured by a 96-well

Western Blotting was performed as described previously (Ku et al., 2013), except that polyclonal antibodies against VP0, VP1 or VP3 proteins of CVA10 were used as

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the detection antibodies.

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2.6. Preparation of CVA10 VLPs and control antigen To generate CVA10 VLPs, the yeast clone exhibiting the highest expression level

was selected for large-scale protein expression, followed by protein purification according to protocols described in a previous study (Zhang et al., 2015). For comparison, the negative control antigen was prepared from an empty vector pPink-HC transformed yeast strain following the same procedures. The purified VLPs and negative control antigen were quantified using a Bradford protein assay and used in subsequent experiments.

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2.7. Electron microscopy Purified CVA10 VLP sample was diluted to a concentration of 100 ng/µl, negatively stained with 0.5% aqueous uranyl acetate, and subsequently observed under Tecnai

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G2 Spirit transmission electron microscope (FEI, USA) operated at 120 kV.

2.8. Mouse immunization

The animal studies were approved by the Institutional Animal Care and Use

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Committee at the Institut Pasteur of Shanghai. All mice used in this study were purchased from Shanghai Laboratory Animal Center (SLAC, China).

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To prepare the experimental vaccines, purified CVA10 VLPs and negative control antigen (1 µg/dose) were adsorbed separately onto the aluminum hydroxide adjuvant (Invivogen, USA; 500 µg/dose) according to the manufacturer's instructions. Two groups of 6 female ICR mice, 6-8 weeks of age, were immunized with the

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experimental vaccines via the intraperitoneal route at weeks 0, 2, and 4. Blood samples were collected from the orbital venous plexus of each mouse at week 6 and the resulting sera were subjected to heat inactivation at 56°C for 30 min to destroy

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complement activity prior to use.

2.9. Serum antibody measurement and neutralization assay

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Specific anti-CVA10 IgG antibodies in mouse sera were detected by indirect

ELISA. Briefly, 96-well microplates were coated overnight at 4°C with 10 ng/well of CVA10 VLPs, and then blocked with 200 µl/well of 5% non-fat milk in PBST at 37°C for 1 h; 50 µl of serially diluted serum samples were added and incubated at 37°C for 2 h; subsequently, 50 µl of HRP-conjugated goat anti-mouse IgG (Sigma, USA) was added to each well and the plates were incubated at 37°C for 1 h. The microplates were washed with PBST for three times between each step. After color development, the absorbance at 450 nm was measured by a 96-well microplate reader. Anti-CVA10

ACCEPTED MANUSCRIPT IgG endpoint titers were defined as the highest serum dilutions at which the absorbances were 0.1 OD unit higher than those of pre-immune serum samples. The neutralizing antibody titers of vaccinated sera against CVA10 were determined by cytopathic effect (CPE)-based neutralization assay which has been

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described previously (Shen et al., 2016).

2.10. In vivo protection assays

In vivo protective efficacy of CVA10 VLPs was evaluated using two different

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assays. In one assay, groups of naive ICR mice (6 days old) were injected intraperitoneally (i.p.) with 75 µl of pooled antisera from the CVA10 VLP group or the

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control antigen group. One day later, all neonatal mice were challenged i.p. with 4.65 × 105 TCID50 of CVA10/Kowalik or 8.9 × 105 TCID50 of CVA10/S0148b, followed by daily observation of survival and clinical score for 15 days. Clinical scores were graded as follows: 0, healthy; 1, reduced mobility; 2, limb weakness; 3, paralysis; 4,

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death.

In another assay, groups of adult female ICR mice that had been immunized with CVA10 VLPs or negative control antigen (described above) were allowed to mate with naive ICR male mice five weeks after the third immunization. 7-day-old neonatal mice

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born to the immunized mice were i.p. inoculated with 4.65 × 105 TCID50 of CVA10/Kowalik or 8.9 × 105 TCID50 of CVA10/S0148b, followed by daily monitoring of

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survival and clinical score for 15 days using the same criteria as described above.

2.11. Statistics

Data processing and statistical analysis were performed using GraphPad Prism

version 7.

3. Results 3.1. Expression and characterization of CVA10 VLPs in P. pastoris

ACCEPTED MANUSCRIPT Previous studies have demonstrated that co-expression of the P1 (structural protein precursor) and 3CD (protease) proteins of EV71, CVA16 or CVA6 in P. pastoris leads to the cleavage of P1 precursor into the individual capsid proteins VP0, VP1 and VP3, which assemble spontaneously into VLPs (Feng et al., 2016; Zhang et

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al., 2015; Zhang et al., 2016; Zhou et al., 2016). In the present study, we took the same approach to generate CVA10 VLPs in P. pastoris. To co-express CVA10 P1 and 3CD proteins, the construct pCVA10-003 that carried both P1 and 3CD expression cassettes was made (Fig. 1A), and then transformed into P. pastoris strain PichiaPink.

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24 randomly selected clones were subjected to small-scale expression and the resultant yeast lysates were analyzed for CVA10 protein expression by ELISA with

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mouse polyclonal antibody against inactivated CVA10 as the detection antibody. A yeast clone transformed with empty vector served as the negative control. As shown in Fig. 1B, CVA10 proteins were detected for most of the pCVA10-003 transformants; two clones #7 and #15 (designated CVA10-003-7 and CVA10-003-15, respectively)

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showing strong signals were further analyzed by Western blotting with VP0-, VP1-, and VP3-specific antibodies. As shown in Fig. 1C, no specific bands were observed for the control sample (lysate from the empty vector-transformed yeast); whereas the clones CVA10-003-7 and CVA10-003-15 produced specific protein bands between 25

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KD and 40 KD, representing VP0, VP1 and VP3 subunit proteins, respectively. These results suggested that the P1 protein was expressed in P. pastoris and successfully

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processed by protein 3CD to produce capsid subunit proteins. To assess VLP assembly, cell lysate from the pCVA10-003-transformed yeast was

subjected to sucrose gradient ultracentrifugation, and the resulting gradient fractions were detected for the presence and distribution of the processed CVA10 subunit proteins. ELISA analysis with CVA10 VP0-specific antibody showed that no obvious signal was observed for the control sample (fractions of lysate from the empty vector-transformed yeast), whereas the factions #7 and #8 of the CVA10-003-7 yeast clone displayed strong reactivity (Fig. 2A). Moreover, Western blotting analysis of

ACCEPTED MANUSCRIPT fractions of the CVA10-003-7 clone revealed that VP0, VP1 and VP3 proteins with the expected molecular weights co-migrated among fractions #7 to #12 (Fig. 2B), suggesting that the three proteins co-assembled into particles. The peak fractions were pooled and further analyzed for particle size and shape by electron microscopic

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analysis. As shown in Fig. 2C, a large number of spherical hollow particles with a diameter of ~30 nm were clearly seen. Taken together, these results demonstrated the successful formation of CVA10 VLPs comprising VP0, VP1 and VP3 in P. pastoris.

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3.2. Antibody responses induced by immunization with CVA10 VLPs

Prior to immunization studies, CVA10 VLPs were generated from induced yeast

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cells of the CVA10-003-7 clone, and the control antigen was prepared from yeast cells transformed with the empty vector following the same procedures. The purified antigens were then characterized by means of SDS-PAGE and western blotting. As shown in Fig. 3A, the CVA10 VLP sample yielded three major protein bands of 39 kDa,

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37 kDa and 29 kDa, corresponding to VP0, VP1, and VP3, respectively; whereas no specific band was detectable for the control antigen. To study the immunogenicity, purified CVA10 VLPs and control antigen (1 µg/dose) were separately formulated with aluminum adjuvant, and then used to immunize adult

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female ICR mice three times at two-week intervals. Two weeks after the last immunization, blood samples were taken from each mouse and the resultant sera

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were detected by ELISA for CVA10-specific IgG antibodies. All antisera of the VLP group reacted strongly with the coating antigen (CVA10 VLPs) (Fig. 3B), with a geometric mean titer (GMT) of 45254 (Fig. 3C); on the contrary, the antisera from the control antigen-immunized mice did not show any detectable binding activity towards CVA10 VLPs (Fig. 3B and C). These results demonstrated that mice could develop strong antibody responses against CVA10 following immunization with VLPs. The same antiserum samples were further detected for the ability to neutralize CVA10 infection of susceptible RD cells. As shown in Fig. 3D-F, the control antisera

ACCEPTED MANUSCRIPT did not show any neutralization effect even at the minimum dilution tested (1:16) and were therefore denoted as a titer of 8 for GMT calculation. In contrast, antisera from VLP-immunized mice effectively neutralized the homologous strain CVA10/S0273b with a GMT of 287, and importantly, the anti-VLP sera could also cross-neutralize the

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heterologous CVA10 strains CVA10/Kowalik and CVA10/S0148b with GMTs of 101 and 181, respectively (Fig. 3D-F). However, all VLP-immunized mouse sera did not show any cross-neutralizing activities against other HFMD-causing enteroviruses, including EV71 strain EV71/G082 and CVA16 strain CVA16/SZ05, at the lowest

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dilution tested (1:16) (Data not shown). These results suggest that antibodies elicited

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by VLPs could only neutralize CVA10, but not other viruses of the Enterovirus genus.

3.3. Passively transferred anti-VLP sera fully protected neonatal mice from lethal CVA10 infections

Our previous study has demonstrated that CVA10 strains CVA10/Kowalik and

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CVA10/S0148b were highly lethal to neonatal ICR mice (Shen et al., 2016), and therefore, the two strains were used as the challenge virus strains in this study. To assess the in vivo protective efficacy of anti-VLP sera, groups of naive ICR mice at 6 days of age were injected i.p. with pooled antisera from the CVA10 VLP

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group or the control antigen group described above, and one day later infected with lethal doses of CVA10/Kowalik or CVA10/S0148b, followed by daily observation of

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survival and clinical score for 15 days. After CVA10/Kowalik challenge, the mice which received control antisera gradually developed clinical symptoms, including retardation, limb weakness and paralysis, and all of them died between 5 and 9 days post-infection (dpi); in contrast, all of the mice treated with anti-VLP sera remained healthy throughout the experiment (Fig. 4A and B). Similarly, after CVA10/S0148b infection, neonatal mice injected with the control antisera began to get sick at 3 dpi, and all died within 7dpi; whereas all of the suckling mice given VLP immune sera survived without any obvious symptoms (Fig. 4C and D). Taken together, these

ACCEPTED MANUSCRIPT results indicate that antibodies in the anti-VLP sera could provide protection against lethal CVA10 infection in suckling mice.

3.4. Maternal immunization with VLPs offered complete protection against lethal

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CVA10 challenges in suckling mice The in vivo protective efficacy of CVA10 VLPs was further determined by maternal immunization/viral challenge assay. Groups of adult female ICR mice that had been immunized three times with CVA10 VLPs or the control antigen were allowed to

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produce litters. 7-day-old neonatal mice born to these immunized mothers were inoculated with lethal doses of CVA10/Kowalik or CVA10/S0148b, and then observed

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daily for clinical signs and survival for 15 days. As shown in Fig. 5A and B, following CVA10/Kowalik infection, pups born to the control antigen-immunized dams gradually showed symptoms as described above and eventually all died by 10 dpi; whereas all offspring from dams immunized with VLPs survived and had no signs of disease.

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Similarly, when infected with CVA10/S0148b, a 100% survival rate was observed in suckling mice born to VLP-immunized dams, while no pups born from the control dams survived (Fig. 5C and D). These results suggest that maternal antibodies

4. Discussion

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elicited by VLPs could effectively protect the offspring from lethal CVA10 infection.

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We report here, to our knowledge, the first development and evaluation of a

VLP-based vaccine candidate for CVA10. We showed that CVA10 VLP could be produced in P. pastoris yeast co-expressing the precursor P1 and the protease 3CD. The P1 precursor was processed presumably by 3CD to yield VP0, VP1, and VP3 subunits (Fig. 1C and Fig. 3A), which together assembled into VLPs (Fig. 2C). This result indicates that 3CD, albeit produced in a heterologous system, may have folded correctly and retained its protease activity. The same strategy has been previously used to successfully generate VLPs of EV71 (Zhang et al., 2015), CVA16 (Zhang et

ACCEPTED MANUSCRIPT al., 2016), and CVA6 (Zhou et al., 2016), respectively. Hence, this P1/3CD co-expression strategy appears to be generally applicable to enteroviruses. Our study showed that CVA10 VLP could stimulate the production of high-titer VLP-binding serum antibodies in mice (Fig. 3B and C), indicating that CVA10 VLP is

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highly immunogenic. It was found that the anti-VLP sera could potently neutralize the homologous and two heterologous CVA10 strains including the prototype strain Kowalik (Fig. 3D-F). In addition, amino acid sequence alignment (Data not shown) showed that the P1 region of the VLP vaccine strain CVA10/S0273b is ~98% identical

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to those of the other CVA10 clinical isolates, suggesting that the CVA10/S0273b strain is a representative of the current circulating CVA10 strains. These data demonstrate

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that the VLP based on the P1 sequence of CVA10/S0273b can induce broad-spectrum neutralizing antibodies against CVA10 virus.

In this study, the in vivo protective efficacy of CVA10 VLPs was determined by two passive immunization/virus challenge assays. Both passively transferred anti-VLP

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sera and maternal antibodies elicited by VLPs could confer full protection against lethal CVA10 challenges in suckling mice (Fig. 4 and 5). It is worth noting that the challenge viruses (CVA10/Kowalik and CVA10/S0148b) are heterologous strains relative to CVA10/S0273b (vaccine strain). CVA10/S0273b was not used in the

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challenge experiments, because the virus is much less virulent in vivo than CVA10/Kowalik and CVA10/S0148b (neonatal mice inoculated with CVA10/S0273b

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did not develop severe clinical disease such as paralysis or death (Data not shown). Nonetheless, the results from the challenge experiments demonstrate that the CVA10 VLP vaccine can efficiently confer intratypic cross-protection in mice. Collectively, our data demonstrate that yeast-produced CVA10 VLP is highly

immunogenic and confers broad protection against lethal CVA10 infections in mice, thus representing a promising candidate vaccine for CVA10. Ideally, CVA10 VLP can be combined with VLPs derived from other HFMD-causing enteroviruses, such as EV71, CVA16, and CVA6, to formulate a VLP-based polyvalent vaccine with broad

ACCEPTED MANUSCRIPT protective spectrum against HFMD.

Acknowledgements This work was supported by grants from the National Natural Science Foundation of

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China (31370930), the Science and Technology Commission of Shanghai Municipality (15XD1524900), the Youth Innovation Promotion Association of CAS (2016249), the K.C.Wong Education Foundation, and the TOTAL Foundation. We thank Chaoyun

Shanghai (NCPSS) for electron microscopy service.

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ACCEPTED MANUSCRIPT Figure legends Fig. 1. Co-expression of P1 and 3CD proteins of CVA10 in P. pastoris. (A) Diagram of the plasmid pCVA10-003. TRP2-L and TRP2-R, the up- and down-stream parts of the TRP region; PAOX1, AOX1 promoter; CYC1 TT, CYC1 transcription region;

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phosphoribosylaminoimidazole carboxylase, used as the selection marker. (B) ELISA analysis of the crude cell lysates from yeast clones transformed with pCVA10-003. Lysate from a yeast clone transformed with empty vector served as the negative

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control (ctr). A polyclonal antibody against inactivated CVA10 was used as the detection antibody. Data are mean ± SD of triplicate wells. (C) Western blotting

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analyses of yeast lysates with the indicated CVA10 capsid protein-specific antibodies. Lane M, marker; lane 1, empty vector-transformed yeast; lanes 2 and 3, pCVA10-003-transformed yeast clones #7 and #15, respectively.

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Fig. 2. Characterization of CVA10 VLP assembly. (A-B) Sucrose gradient sedimentation analysis. Lysate from pCVA10-003-transformed yeast was sedimented through 10-50% sucrose gradients. Twelve fractions were collected from the top and then subjected to (A) ELISA with VP0-specific antibody and (B) Western blotting with

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polyclonal antibodies against CVA10 VP0, VP1 and VP3 proteins, respectively. Fractions of lysate from the empty vector-transformed yeast served as the negative

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control (ctr). (C) Transmission electron microscopy analysis of CVA10 VLPs. Bar = 100 nm.

Fig. 3. Neutralizing antibody responses in mice after immunization with CVA10 VLPs. (A) SDS-PAGE and western blotting analysis of purified CVA10 VLPs and control antigen. Lane M, protein marker; ctr, the control antigen generated from a yeast clone transformed with empty vector; VLP, purified CVA10 VLPs. (B) Reactivity of antisera towards CVA10 VLPs measured by ELISA. Mouse antisera taken 2 weeks

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mean titer (GMT) calculation. (D-F) Neutralizing titers against CVA10 strains (D) CVA10/S0273b, (E) CVA10/Kowalik, and (F) CVA10/S0148b. Antisera taken 2 weeks following the last immunization were used in the assay. The lowest serum dilution tested is 1:16. The control antisera did not show any neutralization effect at the 1:16

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dilution and were assigned a titer of 8 for GMT computation. Each symbol represents

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a mouse, and the solid line indicates the GMT of the group.

Fig. 4. Protective efficacy of anti-VLP sera against CVA10 infections in mice. Groups of naive ICR mice (6 days old) were injected i.p. with pooled anti-CVA10 VLP or control sera. One day later, all neonatal mice were i.p. infected with (A and B)

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CVA10/Kowalik or (C and D) CVA10/S0148b, followed by daily observation of (A and C) survival and (B and D) clinical score for 15 days. Clinical scores were graded as follows: 0, healthy; 1, reduced mobility; 2, limb weakness; 3, paralysis; 4, death. The

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numbers of mice in each group were indicated in brackets.

Fig. 5. Maternal immunization with CVA10 VLP protected offspring mice against

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lethal challenge. 7-day-old ICR mice born to the control antigen or VLP-immunized dams were i.p. inoculated with (A and B) CVA10/Kowalik or (C and D) CVA10/S0148b, followed by daily monitoring of (A and C) survival and (B and D) clinical score for 15 days using the same criteria as described in the legend of Figure 4. The numbers of mice in each group were indicated in brackets.

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CVA10 VLPs could be produced in P. pastoris yeast co-expressing the precursor P1 and the protease 3CD.

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Mice immunized with the VLPs produced high level serum neutralizing antibodies

CVA10 VLP vaccine could confer complete protection against lethal viral

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challenges in suckling mice.

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against CVA10.