Hexon-modified recombinant E1-deleted adenoviral vectors as bivalent vaccine carriers for Coxsackievirus A16 and Enterovirus 71

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Hexon-modified recombinant E1-deleted adenoviral vectors as bivalent vaccine carriers for Coxsackievirus A16 and Enterovirus 71 Chao Zhang, Yong Yang, Yudan Chi, Jieyun Yin, Lijun Yan, Zhiqiang Ku, Qingwei Liu, Zhong Huang ∗ , Dongming Zhou ∗ Vaccine Research Center, Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China

a r t i c l e

i n f o

Article history: Received 28 April 2015 Received in revised form 31 July 2015 Accepted 6 August 2015 Available online xxx Keywords: Adenovirus Hexon Coxsackievirus A16 Enterovirus 71 Bivalent vaccine

a b s t r a c t Hand, foot and mouth disease (HFMD) is a major public health concern in Asia; more efficient vaccines against HFMD are urgently required. Adenoviral (Ad) capsids have been used widely for the presentation of foreign antigens to induce specific immune responses in the host. Here, we describe a novel bivalent vaccine for HFMD based on the hexon-modified, E1-deleted chimpanzee adenovirus serotype 68 (AdC68). The novel vaccine candidate was generated by incorporating the neutralising epitope of Coxsackievirus A16 (CA16), PEP71, into hypervariable region 1 (HVR1), and a shortened neutralising epitope of Enterovirus 71 (EV71), sSP70, into HVR2 of the AdC68 hexon. In order to enhance the immunogenicity of EV71, VP1 of EV71 was cloned into the E1-region of the AdC68 vectors. The results demonstrated that these two epitopes were well presented on the virion surface and had high affinity towards specific antibodies, and VP1 of EV71 was also significantly expressed. In pre-clinical mouse models, the hexonmodified AdC68 elicited neutralising antibodies against both CA16 and EV71, which conferred protection to suckling mice against a lethal challenge of CA16 and EV71. In summary, this study demonstrates that the hexon-modified AdC68 may represent a promising bivalent vaccine carrier against EV71 and CA16 and an epitope-display platform for other pathogens. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) are the major pathogens responsible for hand, foot and mouth disease (HFMD) [1–5], which caused outbreaks in Linyi City in 2007 [6] and in Fuyang City in 2008 [7] in mainland China and leads to annual epidemics since then. Therefore, HFMD became a new threat to public health and has drawn considerable attention from the government. Unfortunately, a commercially available vaccine for HFMD has not been formulated to date. One type of EV71 vaccine, which is based on inactivated whole viruses, has completed phase 3 trials [8], and is now awaiting approval from the State Food and Drug Administration Department of China. However, individuals immunised with the approved single EV71

Abbreviations: Ad, adenovirus; CA16, Coxsackievirus A16; EV71, Enterovirus 71; HFMD, hand, foot and mouth disease; HVR, hypervariable region. ∗ Corresponding authors. E-mail addresses: [email protected] (Z. Huang), [email protected] (D. Zhou).

vaccine are still susceptible to CA16 infection [9]. Several other HFMD vaccines have been developed by using various approaches, such as bivalent vaccines for EV71 and CA16 based on inactivated viruses or virus-like particle, and EV71 vaccine based on the replication-competent human adenovirus type 3, none of these vaccines are close to clinical application [10–13]. EV71 and CA16 are small, non-enveloped, positive singlestranded RNA viruses. Neutralising antibodies against EV71 elicited by the SP70 epitope of EV71-VP1 could protect neonatal mice against lethal EV71 infections [14]. Similarly, neutralising antibodies against CA16 elicited by the PEP71 epitope of CA16-VP1 provided protections against different CA16 viruses [15]. Therefore, an epitope-based vaccine might represent a promising candidate for the prevention of HFMD. Compared with traditional methods of vaccination, which includes the uses of attenuated or inactivated pathogens, the epitope-based method of vaccine development might represent a safer and more effective approach [16,17]. Numerous methods have been developed for the delivery of peptides into host cells to induce immune responses [18]; one such method involves the use of adenovirus hexon as an epitopedisplaying system. Hexon is the most abundant capsid protein in

http://dx.doi.org/10.1016/j.vaccine.2015.08.016 0264-410X/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Zhang C, et al. Hexon-modified recombinant E1-deleted adenoviral vectors as bivalent vaccine carriers for Coxsackievirus A16 and Enterovirus 71. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.08.016

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adenovirus, and has room for presenting foreign antigens. The foreign peptides incorporated on the surface of hexon could elicit strong immune response in hosts [19,20]. In this study, we developed a hexon-modified replicationdeficient (E1-region-deleted) adenovirus AdC68 that may be used for epitope display in the synthesis of a bivalent vaccine against HFMD. AdC68 is type of chimpanzee-origin adenovirus, which exhibits low seroprevalence in the human population [21–23] and has been applied widely for vaccination. PEP71 of CA16 and a shortened SP70 segment (sSP70) of EV71 were incorporated into hypervariable region 1 (HVR1) and HVR2, respectively, of the AdC68 hexon. Because VP1 can induce immune protection against EV71 challenge in mice [24–26], an optimised EV71 VP1 sequence (optimised for mammals) was cloned into the E1 region of AdC68 in order to enhance the immune response to EV71. 2. Materials and methods 2.1. Cells and viruses RD and Vero cells were grown as described previously [27]. The EV71 strains used in this study included EV71/G082 [28] and a mouse-adapted EV71 strain termed EV71/MAV-W [29]. Two CA16 strains, CA16/SZ05 and CA16/G08, were cultured as described previously [30]. All viruses were titrated for 50% tissue culture infectious dose (TCID50 ) using RD cells, as described previously [31]. 2.2. Adenovirus (Ad) and anti-Ad neutralising antibody titration The AdC68 expressing PEP71 and sSP70 within the hexon were constructed as shown in Fig. 1a. Generally, the construction, rescue, amplification and purification of the Ads were performed as previously described [20]. Namely, a shuttle vector containing Ad hexon plus a bit of penton was constructed and termed as pcDNA3.1MM. Based on the shuttle vector, residues 142–144 (ETA) in HVR1 of hexon were deleted and replaced with the PEP71 sequence (FGEHLQANDLDYGQC), and residues 172–174 (TDD) in HVR2 of hexon were deleted and replaced with sSP70 sequence (HKQEKD). Finally, the modified hexon was cloned back to the AdC68 vector. The Ads and nomenclatures used throughout this manuscript are listed in Table 1, and the anti-Ad serum was titrated as described previously [32]. 2.3. Detection of the epitopes and EV71 VP1 expression The four Ads were serially diluted (twofold) in phosphatebuffered saline (PBS) from 5.0 × 1010 vp to 0.6 × 1010 vp in a reaction volume of 50 ␮L. The ELISA was then performed as previously described [14]. To detect the CA16 PEP71 epitope, anti-PEP71 serum was harvested as described previously [15], diluted (1:1000), and added to the plate (50 ␮L/well). To detect the EV71 sSP70 epitope, monoclonal antibody for sSP70 was diluted (1:1000) and added to the plate (50 ␮L/well). Each sample was represented in Table 1 A list of the new vectors and the nomenclature used throughout manuscript. Vector name

Backbone

Insert

AdC68-Hx-PEP71(R1)-sSP70 (R2) AdC68VP1-Hx-PEP71 (R1)-sSP70 (R2) AdC68VP1

AdC68 with PEP71 in HVR1 and sSP70 in HVR2 AdC68 with PEP71 in HVR1 and sSP70 in HVR2 AdC68 with wt hexon

None

AdC68-empty

AdC68 with wt hexon

Optimised VP1 of EV71 Optimised VP1 of EV71 None

triplicate on the plate. Affinity binding assay was performed as follows: briefly, each well of the ELISA plate was coated with 2.0 × 1010 vp of purified Ads, and different dilutions of anti-PEP71 or anti-sSP70 antibodies were used as the primary antibodies; the other conditions and protocols were the same as those used for the detection of the epitopes. To detect EV71 VP1 expression, different titres of Ads in Table 1 were used to infect HEK 293 cells prepared in a 6-well plate. After 24 h, the cells were harvested for western blot analysis as previously described [13]. 2.4. Mouse immunisation Female BALB/c mice (6–8 weeks old, eight mice per group) were used to evaluate vaccine induced immune responses. Four groups of mice were vaccinated intramuscularly with a total of 5 × 1010 vp from different Ads. The animals were given two booster injections with the same dose of Ads at two-week intervals. Blood was harvested every two weeks after vaccination; two weeks after the last booster, the animals were sacrificed, and sera were collected. The animal studies were approved by the Institutional Animal Care and Use Committee of the Institut Pasteur of Shanghai. 2.5. Antibody measurement and isotyping of total immunoglobulin (Ig) G To measure specific antibody responses and the isotyping of total IgG in the serum samples, 96-well enzyme-linked immunosorbent assay (ELISA) plates were coated with 50 ␮L of inactivated EV71 or CA16 (10 ng/well) and incubated for 3 h at 37 ◦ C. The antisera was diluted 1:100, and the ELISA was then performed as previously described[14], but for the isotyping, the secondary antibodies were the HRP-conjugated anti-mouse IgG1, IgG2a, and IgG2b antibodies (Southern biotechnology, Birmingham, AL, USA) were diluted (1:5000). 2.6. Anti-EV71 and anti-CA16 neutralisation assay Serum samples were diluted serially twofold using Dulbecco’s modified Eagle’s medium (DMEM) containing 2% FBS. EV71/G082 and CA16/G08 stocks were diluted to working concentrations of 2 TCID50 /␮L and 0.2 TCID50 /␮L, respectively. The neutralisation assay was performed using 96-well plates. In each well, 50 ␮L of diluted antiserum was mixed with 50 ␮L of EV71 (100 TCID50 ) or 50 ␮L CA16 (10 TCID50 ) and incubated for 2 h at 37 ◦ C. Next, 100 ␮L of cell suspension containing 15,000 Vero cells was added to wells containing the virus/antiserum mixtures, and the plates were incubated at 37 ◦ C in an atmosphere of 5% CO2 . After 3 days, the cells were observed to evaluate the appearance of cytopathic effects (CPEs). Each sample was represented in duplicate. Neutralisation titre was defined as the highest serum dilution that could protect 50% of the cells from CPE. 2.7. Passive immunisation and virus challenge The protective efficacy of the experimental vaccines was evaluated by two in vivo assays. In the first assay, five groups of 2-day-old neonatal ICR mice were inoculated intraperitoneally (i.p.) with 100 ␮L of serum from the four different vaccination groups or 100 ␮L of PBS (control), followed by inoculation (i.p.) with 2.0 × 105 TCID50 of CA16/G08 after 24 h. In the second assay, five groups of neonatal ICR mice at 7 days of age were first inoculated (i.p.) with 100 ␮L of serum from the 4 different vaccination groups or 100 ␮L of PBS (control), followed by inoculation (i.p.) with 1.3 × 104 TCID50 of EV71/MAV-W. The challenged mice were monitored daily for

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Fig. 1. Construction of hexon-modified vectors. (a) The flowchart depicts the cloning procedures used for the HVR1 and HVR2 hexon-modified E1-deleted AdC68 vectors. The upper panel shows the entire sequence of the E1-deleted molecular clone of AdC68 including the Mlu I sites that were used to excise the gene encoding the hexon. The lower part shows the pcDNA3.1 clone containing the viral hexon, including the Mlu I sites used to insert the PEP71 sequence into HVR1 and the sSP70 into HVR2. (b) Neutralising epitopes displayed on the hypervariable regions (HVRs) of the hexon. The hexon forms a homo-trimer presenting PEP71 (red) on HVR1 and sSP70 (magenta) on HVR2 (the top and side views). This protein was modelled using the Swiss-Model server.

survival and clinical score for 18 days. Clinical scores were graded as follows: 0, healthy; 1, reduced mobility; 2, limb weakness; 3, paralysis; and 4, death.

3. Results

2.8. Statistical analysis

The modified hexon was modelled online using the Swiss-Model server, as shown in Fig. 1b. This modelling study revealed that both PEP71 and sSP70 were displayed on the surface of the Ad virion. And all the Ads used in this study were listed in Table 1. Western blot was performed to verify the expression of EV71 VP1. The results revealed that both AdC68VP1-Hx-PEP71 (R1)-sSP70 (R2)

Statistical significance was determined by one-way analysis of variance (ANOVA) and chi-squared test using GraphPad Prism version 6 (GraphPad Software, La Jolla, CA, USA). A P-value of less than 0.05 was considered statistically significant.

3.1. Presentation of different epitopes on the virion surface and expression of EV71 VP1

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Fig. 2. Epitopes displayed on the adenoviral (Ad) virion and expression of EV71 VP1 on the E1 cassette. (a) HEK293 cells were infected with different concentrations of AdC68VP1-Hx-PEP71 (R1)-sSP70 (R2) or AdC68VP1 expressing the EV71 VP1 and were analysed by western blotting with the anti-VP1 polyclonal antibody. ␤-Actin antibody was used as the loading control. NC, negative control (AdC68-empty); vp, virus particles. (b) Plates were coated with different titres of purified AdC68. The PEP71 epitope was detected with anti-PEP71 polyclonal antibody, followed by incubation with the secondary antibody and trimethylboron (TMB) substrate. The graph shows the mean absorbance (OD ± standard deviation [SD]) of the substrate in the wells. (c) Plates were coated with 2.0 × 1010 vp of purified Ads, and different dilutions of anti-PEP71 antibodies were used as the primary antibodies. (d) Plates were coated with different titres of purified AdC68 and the sSP70 epitope was detected with anti-sSP70 monoclonal antibody. (e) Plates were coated with 2.0 × 1010 vp of purified Ads, and different dilutions of anti-sSP70 antibodies were used as the primary antibodies. One-way ANOVA with Tukey adjustment was applied to compare the OD values between groups for b–e. No significant difference was found between two hexon-modified Ads groups. All OD values in groups of hexon-modified Ads were significant higher than those in groups of wild-type-hexon Ads (P < 0.01 for each).

and AdC68VP1 sufficiently expressed the VP1 protein of EV71, and VP1 was expressed in a dose-dependent pattern; the expression level was higher at 1011 vp/well than at 109 vp/well (Fig. 2a). To assess whether the PEP71 and sSP70 epitopes are displayed on the surface of the modified hexon, different concentrations of purified Ads were coated on the wells of ELISA plates and incubated with anti-sSP70 or anti-PEP71 antibodies. The results revealed significant binding of the anti-sSP70 and anti-PEP71 antibodies to AdC68VP1-Hx-PEP71 (R1)-sSP70 (R2) and AdC68-Hx-PEP71 (R1)sSP70 (R2), whereas no binding was seen in response to AdC68VP1 and AdC68-empty (Fig. 2b and d), indicating that the sSP70 and PEP71 epitopes were optimally displayed on the hexon. In order to determine the dose-response relationship of the anti-PEP71 or anti-sSP70 antibody response to the hexon-modified Ad virions, a dose-response ELISA assay was performed. As expected, the antiPEP71 or anti-sSP70 antibodies bound to the hexon-modified Ad virions in a dose-dependent manner, whereas wild type hexon Ads, AdC68-empty, and AdC68VP1 showed no obvious binding (Fig. 2c and e). Statistical analysis revealed that all OD values in groups of hexon-modified Ads were significant higher than those in groups of wild-type-hexon Ads (P < 0.01 for each, Fig. 2b–e).

3.2. Anti-CA16 and anti-EV71 antibodies were produced in the mice after vaccination Mice immunised with AdC68-Hx-PEP71 (R1)-sSP70 (R2) and AdC68VP1-Hx-PEP71 (R1)-sSP70 (R2) exhibited similar increasing patterns of total IgG levels for both EV71 and CA16, because the Ad had both PEP71 (on HVR1) and sSP70 (on HVR2) epitopes. On the 6th week, total IgG of anti-EV71 and anti-CA16 in vaccinated group of AdC68VP1-Hx-PEP71 (R1)-sSP70 (R2) were higher than those in AdC68-empty group (P < 0.0001; P < 0.01). This result confirmed that the hexon-modified Ads could function as bivalent vaccine carriers. As expected, mice immunised with AdC68VP1 had very low CA16 antibody titres, but high EV71 antibody titres (Fig. 3a and b). The anti-CA16 or anti-EV71 IgG subtypes, IgG1, IgG2a, and IgG2b, were also assessed in sera obtained 6 weeks after immunisation to determine the nature of immune response being stimulated. Fig. 3c and d depicts the results of the isotyping experiment for anti-CA16 and anti-EV71 sera, respectively. In the anti-CA16 sera isotyping experiment, mice immunised with AdC68-Hx-PEP71 (R1)-sSP70 (R2) or AdC68VP1-Hx-PEP71 (R1)-sSP70 (R2) had the

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highest level of IgG2a and low levels of IgG2b or IgG1. However, mice immunised with AdC68VP1 or AdC68-empty had almost no detectable IgG. In the anti-EV71 sera isotyping experiment, mice immunised with AdC68VP1-Hx-PEP71 (R1)-sSP70 (R2) had much higher levels of IgG1, IgG2a, and IgG2b than the other three groups. Of the groups which had detectable IgG levels, IgG1, IgG2a and IgG2b were all well induced, and the IgG2a level was higher than the IgG1 and IgG2b levels. However, compared to anti-CA16 sera, the Ads induced balanced levels of IgG1, IgG2a and IgG2a in anti-EV71 sera.

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Neutralising antibodies against EV71 or CA16 were tested by in vitro neutralisation assays. In the case of the anti-CA16 neutralising antibodies, mice immunised with the PEP71-chimeric-hexon Ads had higher neutralising antibody titres than those immunised with wild type-hexon Ads, indicating that PEP71 displayed on the HVR1 of AdC68 could elicit neutralising antibody response against CA16, and there’s no significant difference in neutralising titres generated between two hexon-modified Ads (Fig. 3e). As expected, the anti-EV71 neutralising antibody titres generated in mice immunised with AdC68VP1-Hx-PEP71 (R1)-sSP70 (R2) was the highest

Fig. 3. Total immunoglobulin (Ig) G production, isotyping of total IgG in response to EV71 and CA16 vaccination, and neutralising antibody test. Inactivated EV71 or CA16 (10 ng/well) was coated on the enzyme-linked immunosorbent assay (ELISA) plate. Anti-sera were added, followed by the secondary antibody and TMB substrate. (a) Total anti-CA16 IgG; (b) total anti-EV71 IgG; (c) and (d) isotyping of anti-sera isolated from mice six weeks after vaccination; (e) and (f) neutralising antibody test in vitro against CA16 and EV71. One-way ANOVA was applied to compare the neutralising antibody titres between groups. P-values reaching statistical significance were indicated in the figure. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

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Fig. 4. Clinical score and survival rates of suckling mice challenged with lethal doses of EV71 or CA16. The number of suckling mice is indicated in the bracket following the name of each group, and the clinical scores and survival rates were recorded every day. (a) Change of clinical score of suckling mice challenged with CA16. (b) Survival rate of CA16 challenged mice. P-values for the AdC68-Hx-PEP71 (R1)-sSP70, AdC68VP1-Hx-PEP71 (R1)-sSP70 and AdC68VP1 were 0.003, 0.032, 0.105, respectively, vs. the AdC68-empty group. No significant differences were detected between the AdC68-empty and phosphate-buffered saline (PBS) groups. (c) Change of clinical score of suckling mice challenged with EV71. (d) Survival rate of EV71 challenged mice. P-values for AdC68-Hx-PEP71 (R1)-sSP70, AdC68VP1-Hx-PEP71 (R1)-sSP70 and AdC68VP1 were 0.016, 0.001, 0.036, respectively, vs. the AdC68-empty group. No significant differences were detected between the AdC68-empty and PBS groups. A chi-squared test was performed to compare the survival rates.

among all the groups (Fig. 3f). However, mice immunised with AdC68VP1 had lower titres of anti-EV71 neutralising antibodies than mice immunised with AdC68-Hx-PEP71 (R1)-sSP70 (R2) or AdC68VP1-Hx-PEP71 (R1)-sSP70 (R2). 3.3. Protection against lethal EV71 and CA16 challenge in suckling mice The in vivo protective efficacy against lethal EV71 or CA16 challenge was evaluated in suckling mice. As shown in Fig. 4a, when the CA16-infected mice were administered anti-sera or PBS, the clinical score of all sucking mice rose until the sixth day. On the seventh day, the clinical score of suckling mice administered with antiPEP71-chimeric-hexon-Ads sera began to drop. Of the five groups

of suckling mice, the groups injected with anti-PEP71-chimerichexon-Ads sera had lower clinical scores than the other three groups. These results were consistent with the survival curve of the suckling mice (Fig. 4b); suckling mice injected with anti-PEP71chimeric-hexon-Ads sera had higher survival rates than the other three groups. Similarly, when the EV71-infected mice were administered antisera or PBS, the clinical symptoms of the suckling mice rose until the seventh day (Fig. 4c). On the eighth day, the clinical score of the suckling mice administered anti-sSP70-chimeric-hexon-Ads sera or anti-AdC68VP1 serum began to drop down. Of the five groups of suckling mice, the groups injected with anti-AdC68VP1-Hx-PEP71 (R1)-sSP70 (R2) sera had the lowest clinical scores. These results are consistent with the survival curve of the suckling mice (Fig. 4d);

Fig. 5. Neutralising antibodies against wild-type-hexon AdC68. Sera collected 2 weeks after the final boost were tested by neutralising assay. One-way ANOVA was applied to compare the neutralising antibody titres between groups. P-values reaching statistical significance were indicated in the figure. *P < 0.05; **P < 0.01; ***P < 0.001.

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suckling mice injected with anti-AdC68VP1-Hx-PEP71 (R1)-sSP70 (R2) sera exhibited a survival rate of 100% whereas mice injected with anti-AdC68-empty sera exhibited a survival rate of no more than 50%. These results indicate that the antisera from mice immunised with the sSP70-chimeric-hexon-Ads could conferred passive protection against lethal challenge of EV71 in suckling mice, and that the additional expression of EV71 VP1 in the Ad could enhances the protection efficacy of the anti-sera against EV71 challenge. 3.4. In vitro neutralisation of hexon-modified Ads Anti-vector immunity has restricted the use of current AdHu5, because the boosting strategy is ineffective if immunity to the Ads is present [33]. We measured the neutralising antibodies against wild-type-hexon AdC68 in all groups. Interestingly, the anti-hexonmodified-Ad sera did not neutralise wild type-hexon Ads in this study (Fig. 5). In serum samples collected two weeks after the final boost, the average neutralising antibody titre against hexonmodified Ads was <100 while the antisera from the AdC68VP1 and AdC68-empty vaccinated groups exhibited titres of >800. This result indicates that the hexon-modified vectors may be used in the prime-boost strategy to overcome pre-existing neutralising antibodies against the vector. 4. Discussion Intense focus has been directed towards developing a vaccine for HFMD in recent times. The causative agents for HFMD include EV71 and CA16, and recently, other enteroviruses such as CA6 or CA10 have also reached epidemic levels [34]. Therefore, a vaccine that can confer protection against multiple epidemic pathogens is highly desirable. Recently, different types of EV71 vaccines have been developed, such as recombinant VP1 protein, DNA vaccine, and a peptide-based vaccine targeting neutralising epitopes [35–37]. Of the various EV71 neutralising epitopes, SP70 has been identified as one of the best neutralising epitopes on VP1 [38]. Ye et al. demonstrated that the fusion product of HBc and SP70 could selfassemble into virus-like particles (VLPs) with the epitope displayed on the surface, and immunisation of mice with HBcSP70 VLPs could induce neutralising antibodies at titres ranging from 64 to 128 [14]. In a previous study, Foo et al. demonstrated that 50 ␮g of synthetic SP70 peptide conjugated to the carrier protein, diphtheria toxoid, could elicit low titres (16–32) of neutralising antibodies [38]. These results suggested that the SP70 linear epitope might elicit neutralising antibodies against EV71 and might have applications as a vaccine candidate. Besides EV71, CA16 is one of the most prevalent pathogen in HFMD. PEP71, an epitope on VP1 of CA16, has been identified as the neutralising epitope of CA16, and immunising mice with keyhole limpet haemocyanin-conjugated PEP71 could elicit neutralising antibodies against CA16 [15]. However, few studies have attempted to develop a vaccine against CA16 based on the neutralising peptide PEP71. Ads have been used widely in vaccination; they harbour several structural locations suitable for incorporating foreign antigens, such as the penton, protein IX, and fibres [39,40]. For each adenoviral virion, 720 copies of epitopes are displayed if the antigen is incorporated into a single HVR. Therefore, epitope incorporation on the hexon of adenovirus is a novel and interesting strategy for vaccine development. Incorporating the neutralising epitopes of different antigens into the HVRs of the hexons may generate a multivalent vaccine, which can offer protection against various infectious diseases and pathogenic microbes [41]. A previous study revealed that the antigens could be incorporated into different HVRs of AdC68 to produce a multivalent vaccine [42]. Here, we report the design of an adenoviral vector as a bivalent vaccine

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against EV71 and CA16. CA16 PEP71 and EV71 sSP70 were successfully incorporated into HVR1 and HVR2, respectively, of AdC68. Both the epitopes were sufficiently displayed on the virion surface and elicited immune responses against EV71 and CA16. Normally, IgG1 production indicates Th2-type response, whereas IgG2a and IgG2b are predominantly produced in Th1type response. In this study, the total IgG titres of both anti-CA16 and anti-EV71 antibodies increased after every booster doze and reached a peak value six weeks after priming; the hexon-modified Ads elicited both Th1- and Th2-type responses, but PEP71 induced stronger Th1-type immune response, as evidenced by the fact that the IgG2a level was much higher than the IgG1 level. On the other hand, sSP70 on HVR2 elicited balanced levels of IgG1, IgG2a, and IgG2a. These data further confirm that HVR1 and HVR2 are suitable sites for the display of foreign antigens. Furthermore, previous studies have revealed that replacing HVR1 of the AdHu5 with a malarial B cell epitope improves immunogenicity and circumvents pre-existing immunity to adenovirus in mice [43], and mutations within HVR of AdC68 hexon permitted virus to escape neutralisation against wild-type hexon AdC68 [44], which are consistent with our result, suggesting hexon-modified Ads have different immunogenicity from the wild type Ads. Neutralising antibodies play crucial roles in protecting cells from CPE or animals from lethal challenge. In this study, the hexonmodified Ads induced a robust neutralising antibody response against EV71, and provided 100% protection against lethal challenge to suckling mice. However, the neutralising antibody response against CA16 induced by the hexon-modified Ads was quite low, and could only confer partial protection to suckling mice against a lethal challenge of CA16. Previous studies have demonstrated that an inactivated CA16 vaccine can induce high-titre neutralising antibodies (up to 1:256) against CA16 in vitro [13]. These results suggest that the single linear epitope PEP71 is not the best neutralising epitope for CA16. Therefore, more effective antigenic epitopes for CA16 need to be screened in future studies. In conclusion, we have successfully constructed a hexonmodified adenovirus by incorporating PEP71 and sSP70 on HVR1 and HVR2, respectively. The modified virus particle might represent a bivalent vaccine candidate for HFMD. Immune responses induced by this vaccine candidate conferred passive protection to suckling mice challenged with CA16 or EV71. Importantly, our results demonstrated that hexon-modified AdC68 is an effective and safe vehicle for delivering or presenting antigens to the immune system. This strategy may be adopted to generate multivalent vaccines against several diseases or cancers in future studies.

Acknowledgements This work was supported by grants from the ‘Knowledge Innovation Program’ (NO Y014P31503) and the ‘100 Talent Program’ (NO Y316P11503) from the Chinese Academy of Sciences and the Shanghai Pasteur Foundation. Conflict of interest statement: There is no conflict of interest to declare.

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Please cite this article in press as: Zhang C, et al. Hexon-modified recombinant E1-deleted adenoviral vectors as bivalent vaccine carriers for Coxsackievirus A16 and Enterovirus 71. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.08.016

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Please cite this article in press as: Zhang C, et al. Hexon-modified recombinant E1-deleted adenoviral vectors as bivalent vaccine carriers for Coxsackievirus A16 and Enterovirus 71. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.08.016