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Vaccine 31 (2013) 2130– 2136

Contents lists available at SciVerse ScienceDirect

Vaccine

j ourna l ho me pag e: www.elsev ier .com/ locate /vacc ine

dentification of conserved neutralizing linear epitopes within the VP1 protein ofoxsackievirus A16

inping Shi, Xulin Huang, Qingwei Liu, Zhong Huang ∗

ey Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, 411 Hefei Road, Shanghai 200025, China

r t i c l e i n f o

rticle history:eceived 28 November 2012eceived in revised form 4 February 2013ccepted 25 February 2013vailable online 13 March 2013

eywords:oxsackievirus A16ynthetic peptide

a b s t r a c t

Coxsackievirus A16 (CA16) is a major causative agent of hand, foot, and mouth disease. Immunizationwith inactivated whole-virus or recombinant virus-like particles (VLP) of CA16 elicits neutralizing anti-bodies that protect mice against lethal challenge. However, the epitope/s responsible for this inductionhas not been determined. In this investigation, we identified six neutralizing linear epitopes of CA16. Apanel of 95 synthetic peptides spanning the entire VP1 protein of CA16 were screened by ELISA for reac-tivity with neutralizing antisera against CA16 VLPs, which were generated in a previous study (Vaccine30:6642–6648). Fifteen high-binding peptides were selected and further examined for their inhibitoryeffect on neutralization by anti-VLP sera. Among them, six peptides with no overlap significantly inhibited

eutralizing antibodyinear epitope

neutralization. Mice immunized with these six peptides generated peptide-specific serum antibodies. Theanti-peptide antisera positively detected CA16 via immunofluorescent staining and Western blot assays.More importantly, they neutralized both homologous and heterologous CA16 strains, indicating thatthese six peptides represented neutralizing epitopes. Sequence alignment also showed that these epi-topes are extremely conserved among CA16 strains of different genotypes. These findings have important

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implications for the deve

. Introduction

Coxsackievirus A16 (CA16) is a member of the Humannterovirus A species of Enterovirus genus of Picornaviridae [1,2]. Itossesses a single-stranded positive-sense RNA genome of ∼7410ases [3]. The genome contains a single reading frame encoding

large polyprotein precursor, which is processed into structuralrotein P1 and nonstructural proteins P2 and P3. P1 can be furtherrocessed by a viral protease to yield capsid subunit proteins VP0,P1 and VP3; a proportion of VP0 may undergo autocleavage to pro-uce VP2 and VP4 [3]. Based on the VP1 sequence, CA16 strains cane clustered into two genogroups, A and B, with the latter consistsf two subgenotypes B1 (B1a, B1b and possibly B1c) and B2 [4–6].he prototype G-10 strain is the sole member of genogroup A. Theajority of the CA16 strains circulating internationally between

997 and 2007 belongs to subgenotype B1, whereas some strainssolated from Japan and Malaysia between 1981 and 2000 formubgenogroup B2 [5].

CA16 is one of the main causative agents of hand, foot and mouth

isease (HFMD) [5,7–10], which is currently prevalent in the Asia-acific region. In 2012, China reported a total of 2,198,442 HFMDases, to which CA16 may have attributed significantly. Specifically,

∗ Corresponding author. Tel.: +86 21 54653077; fax: +86 21 63843571.E-mail address: huangzhong@ips.ac.cn (Z. Huang).

264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.vaccine.2013.02.051

ent of peptide-based broadly protective CA16 vaccines.© 2013 Elsevier Ltd. All rights reserved.

a recent clinical survey indicated that 38.3% of the 266 laboratoryconfirmed HFMD cases was caused by CA16 infection during the2009 HFMD outbreak in China [11]. Individuals infected with CA16usually present with mild symptoms, including fever, oral ulcers,and rashes on the surface of hands, feet and buttocks [12–14]. How-ever, it has been reported that patients infected with CA16 couldalso develop severe complications, such as encephalitis [15–17],myocarditis [17–19], and pneumonitis [20], which may ultimatelylead to death [15,17–20]. Recently, a clinical study by Xu et al. showsthat about 21% of the severe HFMD cases with neurological compli-cations were caused by CA16 infection [16], indicating that CA16is posing a serious threat to the health of children living in theAsia-Pacific region, especially in China.

No vaccine or drug is yet available to prevent or treat CA16 infec-tion. Recently, several vaccine companies and academic institutionsin China launched projects to develop CA16 vaccines [21,22]. Sev-eral CA16 vaccine candidates have been shown to induce in micethe production of antibodies capable of neutralizing CA16 infec-tion in vitro [21,22]. However, thus far, epitopes responsible for theelicitation of CA16 neutralizing antibodies remain unknown.

Our group has recently demonstrated that recombinant virus-like particles (VLPs) of CA16 were able to potently elicit neutralizing

antibodies in mice [22]. In this study, the anti-VLP neutralizingantisera were used to successfully identify six neutralizing linearepitopes within the VP1 protein of CA16. Sequence alignment indi-cates that these epitopes are extremely conserved among CA16

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enotypes. These findings have important implications for theevelopment of broadly protective CA16 vaccines.

. Materials and methods

.1. Cells, viruses and sera

RD and Vero cells were grown as described previously [23]. TwoA16 clinical strains, CA16/SZ05 and CA16/G08, were used in thistudy. Both strains were originally isolated in mainland China andelong to B1b subgenogroup. The CA16/SZ05 was described previ-usly [3]. Its genome had three nucleotide mutations at positions733 (C to T), 2760 (T to C), and 3161 (G to A) when comparedith the originally reported sequence (GenBank ID: EU262658)

24], resulting in two amino acid changes (T98 M and V107A) inhe VP1 protein. Both CA16/SZ05 and CA16/G08 were propagatedn RD cells. Virus titers were determined as described previously23]. Mouse sera against recombinant VLPs of CA16/SZ05 generatedreviously [22] were used for peptide screening and neutraliza-ion inhibition assays in this study. CA16/SZ05 is thus consideredomologous strain, and CA16/G08 considered heterologous one.

.2. Synthetic peptides

A series of 95 peptides covering the entire amino acid sequencef VP1 protein of the CA16 strain SZ05 [3] were synthesizedy GL Biochem (Shanghai, China). Each peptide consists of 15esidues with 12 overlapping with the adjacent peptide. Anrrelevant peptide (QLINTNGSWHINSTA) from hepatitis C virus2 protein [25], designated HCV1 in this study, was also synthe-ized and used as a negative control. In addition, six selected VP1eptides and the HCV1 peptide were linked to keyhole limpetemocyanin (KLH) using 4-(N-Maleimidomethyl)cyclohexane-1-arboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium saltSulfo-SMCC) by GL Biochem (Shanghai, China) for mouse immu-ization.

.3. Peptide-ELISA

Reactivity of synthetic peptides with the anti-CA16 VLP mouseera was measured by ELISA. Briefly, 96-well microtiter platesere coated with 50 �l/well of individual peptide (10 �g/ml in PBS

uffer) at 37◦C for 2 h. The unrelated HCV1 peptide was also useds a negative control. The wells were then incubated sequentiallyith 200 �l/well of PBST plus 5% milk at 37◦C for 1 h, 50 �l/well of

he mouse sera diluted 1:1000 in PBST plus 1% milk at 37◦C for 2 h,nd 50 �l/well of horseradish peroxidase (HRP) conjugated goatnti-mouse IgG diluted (1:5000) in PBST plus 1% milk at 37 ◦C for

h. Three washes with PBST were carried out between incubationteps. For color development, 50 �l/well of TMB mixture was addednd incubated for 5–10 min, followed by addition of 50 �l/wellf 1 N H3PO4 to stop the reaction. Absorbance was measured at50 nm in a 96-well plate reader.

.4. Neutralization and neutralization-inhibition assays

For the neutralization assay, serum samples were seriallyiluted 2-fold with DMEM containing 2% FBS, and the CA16 stockCA16-SZ05 or CA16-G08) was diluted to a working concentrationf 1 TCID50/�l. The neutralization assay was conducted using 96-ell plates. In each well, 50 �l of diluted serum was mixed with

0 �l of CA16 virus containing 50 TCID50 and incubated for 1 h at

7 ◦C. Next, 100 �l of cell suspension containing 15,000 RD cells wasdded to wells containing the virus/serum mixtures and incubatedt 37 ◦C with 5% CO2. After three days, the cells were observed tovaluate the appearance of cytopathic effects (CPE), including cell

2013) 2130– 2136 2131

rounding, aggregation, and floatation. Neutralization titers wereread as the highest serum dilutions that could protect >95% cellsfrom CPE.

Neutralization-inhibition assay was carried out to determinethe effect of peptide treatment on the neutralization capacity ofthe anti-VLP sera. Briefly, synthetic peptides of different concen-trations were mixed with the anti-VLP antisera, which had beenserially diluted using DMEM containing 2% FBS. The mixtureswere incubated for 1 h at 37 ◦C and then subjected to neutral-ization assay as described above. After three days, the cells wereobserved to evaluate the appearance and degree of CPE. In addi-tion, the cells were also evaluated for viability using a MTT-basedmethod. Briefly, 20 �l of 0.1%(w/v) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) diluted in PBS was addedto wells and incubated for 4 h at 37 ◦C. The medium in the 96-wellplates was decanted gently and then 150 �l of DMSO was addedto each well to solubilize formazan crystals. The absorbance wasdetermined at 490 nm using a microtiter plate reader.

2.5. Mouse immunization

Groups of Balb/c female mice (5 animals/group) were immu-nized intraperitoneally (i.p.) with 50 �g of KLH-conjugatedpeptides plus complete Freund’s adjuvant (Sigma, St. Louis, MO,USA) on day 1. The animals were given three booster injectionswith the same antigens plus incomplete Freund’s adjuvant (Sigma)in two-week intervals. At two weeks after the last booster, the ani-mals were sacrificed and sera were collected. The animal protocolwas approved by the IACUC at the Institut Pasteur of Shanghai.

2.6. Antibody measurement

Peptide-specific IgG titers were determined by endpoint titerELISA as described previously [23,26] with the following modifi-cations: ELISA plates were coated with 500 ng/well of individualsynthetic peptide; a HRP-conjugated anti-mouse IgG antibody(Sigma) was used as the secondary antibody. Endpoint titer wasreported as the reciprocal of the highest serum dilution that hadan absorbance ≥0.1 OD unit above the blank (absorbance of thepre-immune samples).

2.7. SDS-PAGE and Western blot analyses

SDS-PAGE and Western blotting were performed as previouslydescribed [23]. Briefly, inactivated CA16 were separated on 12%polyacrylamide gels and transferred onto PVDF membranes. Mem-branes were then detected with one of the anti-peptide mouse seraand a HRP-conjugated anti-mouse IgG antibody (Sigma).

2.8. Immunofluorescent staining

Immunofluorescent staining was performed as described pre-viously [23] with the following modifications: anti-peptide mousesera diluted 1:100 in PBS, and Alexa Fluor 488-conjugated anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) diluted 1:1000 wereused for detection. The cells were also stained with 5 �g/ml of 4,6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature.The stained cells were examined using a conventional epifluores-cent microscope (Leica, Wetzlar, Germany).

2.9. Sequence alignment

The genome sequences of eleven CA16 strains were down-loaded from the NCBI PubMed website. The sequences of the VP1proteins of these strains were aligned using DNAMAN program.Neutralizing epitopes identified in this study were examined for

2132 J. Shi et al. / Vaccine 31 (2013) 2130– 2136

Table 1Effect of the selected peptides on neutralization by the anti-VLP serum.

Peptide Inhibition of neutralization capacity ofanti-VLP serum diluted

1:8000 1:4000

#21 (ctr) − −#29 − −#32 + +#37 + −#45 − −#49 − −#50 − −#55 + −#63 + −#71 + −#79 − −#84 − −#85 − −#87 − −#90 + −#91 + +

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Table 2Neutralization-inhibiting effect of the selected peptides at different concentrations.

Peptide Residues Sequence Inhibitory effect ofpeptides at differentconcentrations (�g/ml)

25 50 100 200

#21(ctr) aa. 61–75 KNLIETRCVLNHHST − − − −#32 aa. 94–108 TMPTMGTQNTDGYAN − + + +#37 aa. 109–123 WDIDLMGYAQLRRKC − − + +#55 aa. 163–177 PTSRDSFAWQTATNP − − + +#63 aa. 187–201 PAQVSVPFMSPASAY − − + +#71 aa. 211–225 FGEHLQANDLDYGQC − − + +#90 aa. 268–282 NQPYLFKTNPNYKGN − + + +#91 aa. 271–285 YLFKTNPNYKGNDIK + + + +

The neutralizing serum was diluted 1:5000.

antibody described previously [23]. Western blot analysis furthershowed that all the anti-peptide sera specifically recognized a∼34 kDa band (Fig. 2B) representing the VP1 protein of CA16, as

Table 3Functional activity of anti-peptide antibodies.

Sera Peptide-binding antibody titer Neutralization titers against

Range GMT CA16-SZ05 CA16-G08

Preimmune <100 <100 <8 <8Anti-PEP32 2000–8000 2639 16 16Anti-PEP37 64,000–128,000 97,006 32 16Anti-PEP55 5000–40,000 11,542 16 16

reatments which resulted in >50% cells showing CPE were considered to havenhibitory effect on neutralization by the anti-VLP serum and therefore marked as+”; otherwise “−”.

equence similarity to human proteins using the NCBI BLASTP pro-ram.

.10. Structure homology modeling

A three-dimensional structural model of CA16 was generatedy homology modeling with the crystal structure of EV71 (PDB ID:VBS) as the template.

.11. Statistics

Statistical significance was determined by the Student’s two-ailed t-test using GraphPad Prism version 4.

. Results

.1. Mapping of B-cell linear epitopes within the VP1 protein byeptide ELISA

It has been previously shown that the immunization of miceith CA16 VLPs potently elicit the production of neutralizing anti-

odies [22]. To identify the responsible neutralizing epitopes, weested anti-CA16 VLP mouse serum (neutralizing titer of 32,000gainst the CA16-SZ05 strain) for reactivity against a panel of 95verlapping synthetic peptides derived from the CA16 VP1 pro-ein. Fig. 1A shows the profile of reactivity of individual peptidesith the antisera. The HCV1 control peptide yielded a background

evel of readings. Some CA16 peptides exhibited high bindingctivities, suggesting they contain B-cell epitopes. Based on meanalues of ELISA readings from three independent experiments, theop 15 high-binding peptides (Table 1) were selected for furthernalysis.

.2. Peptide treatment affects neutralization capacity of anti-VLPera

We next tested whether binding of the selected peptides to theeutralizing antisera affected its neutralization capacity. Peptide

21 (designated PEP21), which did not show significant reactiv-

ty with the antisera (Fig. 1A), was used as a negative control. Ashown in Table 1, 8 out of the 15 high-binding peptides had nonhibitory effect on the neutralization capacity of the anti-VLP sera

Treatments which resulted in >50% cells showing CPE were considered to haveinhibitory effect on neutralization by the anti-VLP serum and therefore marked as“+”; otherwise “−”.

as per the PEP21 control; whereas the other 7 peptides negativelyaffected its neutralization by 1:8000 diluted antisera. PEP32 andPEP91 were able to further abrogate the neutralization capacity of1:4000 diluted antisera. Examples of neutralization inhibition byPEP32 and PEP91 were shown in Fig. 1B. The capacity of the 15 high-binding peptides to inhibit neutralization was also determined byquantification of cell viability using a MTT method, and the results(Fig. 1C) were in agreement with those based on CPE observation.These 7 inhibiting peptides were further evaluated for their doseeffect on neutralization inhibition. Table 2 shows that all 7 inhib-iting peptides exhibited inhibitory effects at the concentrations of100 and 200 �g/ml, whereas PEP91 could inhibit neutralization aslow as 25 �g/ml. These results strongly suggest that the 7 inhibitingpeptides contain neutralizing epitopes.

3.3. Immunogenicity of the inhibiting peptides in mice

To determine the immunogenicity of the inhibiting peptides,all but PEP90, which overlaps with PEP91 but exhibited lowerinhibitory effects, were chemically linked to KLH and then used toimmunize mice. Antibody titers in the peptide-immunized mousesera were determined by ELISA using the corresponding peptidesas the capture antigen. Peptide-specific antibodies were detectedfor all immunized mice, although the titers for different groupsvaried (Table 3). Control antisera from KLH-linked HCV1 peptide-immunized mice did not show significant cross-reactivity with anyCA16 peptides (data not shown). Peptide-specific antisera werepooled for each group and then evaluated further for their abilityto detect CA16. As demonstrated in Fig. 2A, all of the six anti-peptide antisera, but not the preimmune sera, detected positivelyCA16-infected cells in IFA, as did a guinea pig anti-VP0 polyclonal

Anti-PEP63 1000–8000 2639 16 32Anti-PEP71 80,000–160,000 139,288 32 32Anti-PEP91 4000–16,000 6063 32 16

GMT, geometric mean.

J. Shi et al. / Vaccine 31 (2013) 2130– 2136 2133

Fig. 1. Peptide screening. (A) Reactivity of the anti-VLP mouse sera with synthetic peptides measured by peptide-ELISA. A panel of 95 overlapping peptides spanning theentire sequence of VP1 of CA16 was used as coating antigen in ELISA. Anti-VLP sera were diluted 1:1000 and used for detection. An unrelated HCV peptide, designatedHCV1, was used as a negative control in the assay. The data are results (mean ± SEM) from three independent experiments. (B) Inhibitory effect of PEP32 and PEP91 on CA16neutralization by the anti-VLP sera. RD cells were treated with combinations of CA16 virus, anti-VLP antisera, and peptide, as indicated. The anti-VLP dilution used was1:4000. The peptide concentration used was 50 �g/ml. After three days, the cells were observed for CPE, such as cell rounding, aggregation, and floatation. Treatments whichr tralizb nti-Vp ed uso readi

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esulted in >50% cells showing CPE were considered to have inhibitory effect on neuy a MTT-based method. RD cells were treated with combinations of CA16 virus, aeptide concentration used was 50 �g/ml. After three days, cell viability was quantifif triplicate wells. The dash line indicates the cutoff, which is 50% of the OD 490 nm

id an anti-VP1 serum reported previously [23]; whereas a majorand of ∼28 kDa and a minor band of ∼38 kDa, representing the VP2nd VP0 proteins, respectively, were detected only by anti-VP0 thatlso recognizes VP2 [23].

.4. Neutralization capacity of the anti-peptide mouse sera

The ability of the mouse sera to neutralize CA16 infection wasetermined by a micro-neutralization assay. As shown in Fig. 2C, allhe anti-peptide antisera neutralized the homologous strain CA16-Z05, with titers ranging from 8–64; whereas the preimmune serar a control antisera (raised against the KLH-conjugated HCV1 pep-ide) failed to neutralize CA16 infections even at 1:8, the lowestilution tested, and were therefore assigned a titer of 4 for geomet-ic mean titer (GMT) computation; in contrast, all the anti-peptidentisera neutralized the homologous strain CA16-SZ05, with titersanging from 8–64 (Fig. 2C). Statistical analyses indicate no signif-

cant differences in neutralizing capacity among antisera obtainedrom the six peptide-immunized groups (P > 0.05). The neutraliza-ion experiments were repeated three times with nearly identicalesults. Furthermore, the anti-peptide antisera pooled for each

ation by the anti-VLP serum. (C) Inhibitory effect of the selected peptides measuredLP antisera, and peptide, as indicated. The anti-VLP dilution used was 1:8000. Theing a MTT method as described in Materials and Methods. The data are mean ± SEMng of the cells treated only with anti-VLP sera.

group were found to neutralize both CA16-SZ05 and a heterologousstrain, CA16-G08 (Table 3).

3.5. Sequence alignment

Eleven representative CA16 strains from the A, B1 (B1a and B1b),and B2 genotypes were compared for homology in the VP1 aminoacid sequence. The Alignment shows that the amino acid sequencesof the PEP55, PEP63, and PEP91, were identical among thesestrains (Fig. 3); whereas the sequences corresponding to PEP32and PEP37 were highly conserved with 93.3% homology. Interest-ingly, although the homology for PEP71 was not high among theeleven strains, variation was solely caused by the G10 stain whichis the only member of genogroup A; the sequences correspondingto PEP71 were identical among non-A genotypes (Fig. 3).

4. Discussion

Enterovirus 71 (EV71) and CA16 are the two main causativeagents of HFMD [7,8,12,27]. No specific vaccine for these twoviruses is currently available. Passive transfer of neutralizing

2134 J. Shi et al. / Vaccine 31 (2013) 2130– 2136

Fig. 2. Functional characterization of the mouse antisera against the selected peptides. (A) Anti-peptide antisera detected positively CA16-infected cells in IFA. Vero cellsinfected with CA16 were incubated with 1:100 diluted preimmune sera or anti-peptide sera for 1 h at 37 ◦C, followed by incubation with Alexa Fluor 488-conjugated anti-mouse IgG antibody for 30 min at 37 ◦C. Guinea pig antiserum against recombinant VP0 protein of CA16 was used as a positive control. Cells were also stained briefly withDAPI. Images were taken with Alexa Fluor 488 and a DAPI filters, and subsequently merged. (B) Western blot analysis. Inactivated CA16 was separated on SDS-PAGE andblotted onto PVDF membranes. The membranes were probed with one of the antibodies indicated, followed by incubation with the corresponding HRP-conjugated secondaryantibodies. (C) Anti-peptide antisera neutralized CA16 in vitro. Neutralizing titers of the antisera were determined based on CPE observation as described in Materials andMethods. Preimmune and control (anti-HCV1) antisera did not show any neutralization at 1:8 (the lowest dilution tested) and were therefore assigned a titer of 4 for geometricm ates th

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ean titer (GMT) computation. Each symbol represents a mouse, and the line indic

ntisera or monoclonal antibodies provided protection againstethal EV71 or CA16 challenge [21,22,28,29], indicating that neu-ralizing antibodies play an important role in protection in vivo.dentification of neutralizing epitopes from these two viruses

ill allow better understanding of mechanisms of protection androvide valuable information in designing safe and effective vac-ines. Several neutralizing linear epitopes from EV71 have beendentified, including SP55 [30], SP70 [30], VP1-43 [31] and K1 [32]

ithin VP1 protein, VP2-28 within VP2 [31], and K2 and K3 withinP3 [32]. Interestingly, IgG antibodies (10 �g/ml) purified fromP70-, K1-, K2- or K3-immunized mouse sera were found to beble to cross-neutralize CA16 with varied efficiencies [32]. In theresent study, we report the first identification of six neutralizing

inear epitopes from VP1 protein of CA16. Among these six epitopes,

EP71 (residues 211–225) locates at a region overlapping with SP70residues 208–222) and VP1-43 (residues 211–220) of EV71; thether five CA16 epitopes do not overlap with known neutralizingpitopes of EV71.

e GMT of the group.

The crystal structure of CA16 has not been determined. To pre-dict the location of the six CA16 neutralizing epitopes on virions, athree-dimensional structure model of CA16 (Supplemental Figure1) was generated using the available EV71 crystal structures [33,34]as a template. In this model, PEP32, PEP55 and PEP71 were locatedat BC, EF and GH loops, respectively, and they were well exposed onthe virion surface; PEP63 and PEP91 were less exposed and moreburied with a few residues displayed near the bottom of the pro-posed canyon; PEP37 appeared to lie at the bottom of the canyon(Supplemental Figure 1). The exact neutralization mechanisms foreach anti-peptide sera remain to be determined.

In the present study, immunization of mice with the six pep-tides elicited peptide-specific antibodies. The specific antibodytiters determined by peptide-binding ELISA appeared to vary sig-

nificantly between peptide-immunized groups (Table 3). Thesevariations suggest that there is difference in the quantity and affin-ity of the peptide-specific antibodies, possibly a reflection of theintrinsic nature of peptide-specific B-cell repertories. Interestingly,

J. Shi et al. / Vaccine 31 (2013) 2130– 2136 2135

Fig. 3. Sequence alignment. The VP1 protein sequences of eleven CA16 strains belonging to different genogroups were aligned using the DNAMAN program as described inMaterials and Methods.

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ased on the CPE observation, the overall neutralizing capacitiesmong these anti-peptide antisera were not statistically different.hether this is true remains to be precisely determined using aore quantitative neutralization assay with IgG antibodies purified

rom the anti-peptide sera.It has been reported enterovirus infection may be associated

ith autoimmune diseases [35–37], possibly due to the induc-ion of harmful autoantibodies and T cells by viral epitopes withigh sequence similarity to human proteins. Thus, vaccines whichontain inactivated whole-viruses or VLPs consisting of all struc-ural proteins also have the potential risk of causing autoimmuneiseases [38]. Carefully selected epitopes which elicit only neutral-

zing but not deleterious antibodies can eliminate this risk and arehus ideal candidates for development of peptide-based vaccines.reviously, Kirk et al. [32] showed that four neutralizing epitopesrom EV71 did not share significant sequence similarities with anyuman proteins for which the sequence is known. We have alsoxamined the six CA16 neutralizing epitopes identified in this studysing the NCBI BLASTP program and found that there was no sig-ificant sequence similarity between the CA16 epitope peptidesnd any known human proteins. These results indicate that the sixdentified CA16 neutralizing epitopes are excellent candidate fornclusion in a vaccine.

CA16 is a RNA virus which undergoes constant mutation andvolution, and it contains multiple genotypes. Sequence alignmenthows that the six neutralizing epitopes are extremely conservedmong representative CA16 strains from different genotypes. Inddition, our data show that immunization of mice with thesepitope peptides elicited serum antibodies capable of neutraliz-ng both homologous and heterologous CA16 strains. These resultsemonstrate that the six epitopes are promising vaccine candidates

ith broadly protective potentials.

In conclusion, we have successfully identified six CA16 neu-ralizing linear epitopes within the VP1 protein. Our findings havemportant implications for CA16 vaccine design.

Acknowledgements

We thank Drs Bing Sun, Qi Jin and Wei Liu for providing the CA16viruses. We also thank Drs. Xia Jin, Andy Tsun and Yunfang Zhangfor their excellent editorial contribution. This work was supportedby grants from the Chinese Academy of Sciences.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2013.02.051.

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