Download - Construction and characterization of a recombinant human adenovirus type 3 vector containing two foreign neutralizing epitopes in hexon

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ARTICLE IN PRESSG ModelIRUS 96219 1–8

Virus Research xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Virus Research

j ourna l h o mepa ge: www.elsev ier .com/ locate /v i rusres

onstruction and characterization of a recombinant humandenovirus type 3 vector containing two foreign neutralizingpitopes in hexon

hunyan Xuea,1, Xingui Tiana,1, Xiao Lia, Zhichao Zhoua, Xiaobo Sub,∗, Rong Zhoua,∗∗

State Key Laboratory of Respiratory Disease, Guangzhou Institute of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University,uangzhou Medical University, Guangzhou 510120 , ChinaDepartment of Medical Genetics and Cell Biology, School of Basic Science, Guangzhou Medical University, Guangzhou 510120, China

r t i c l e i n f o

rticle history:eceived 13 December 2013eceived in revised form 25 January 2014ccepted 31 January 2014vailable online xxx

eywords:d vectorypervariable regionpitope displaymmune response

a b s t r a c t

The “antigen capsid-incorporation” strategy has been developed for adenovirus-based vaccines in thecontext of several diseases. Exogenous antigenic peptides incorporated into the adenovirus capsid struc-ture can induce a robust and boosted antigen-specific immune response. Recently, we sought to generatea multivalent adenovirus type 3 (Ad3) vaccine vector by incorporating multiple epitopes into the majoradenovirus capsid protein, hexon. In the present study, a multivalent recombinant Ad3 vaccine (R1R2A3)was constructed by homologous recombination, displaying two neutralizing epitopes from enterovirustype 71 (EV71) in hexon. The recombinant virus was confirmed by PCR, immunoblotting, and enzyme-linked immunosorbent assay, and injected into mice to analyze the epitope-specific humoral response.No differences were found between the viruses with two epitopes incorporated into the hypervariableregions (HVR1 and HVR2) of hexon and Ad3EGFP, based on thermostability and growth kinetic tests.Both the epitopes are thought to be exposed on the hexon-modified intact virion surface. The repeatedadministration of the modified adenovirus R1R2A3 to BALB/c mice boosted the humoral immune response
against both epitopes. Immunization with recombinant virus R1R2A3 elicited higher IgG titers and higherneutralization titers against EV71 in vitro than immunization with the modified adenovirus with only oneepitope incorporated into HVR1. In this study, the recombinant R1R2A3 virus expressing two exogenousneutralizing epitopes in hexon HVR1 and HVR2 induced specific immune responses to both foreign epi-topes. Our study contributes to a better understanding of hexon-modified Ad vector as a multiple-epitopedelivery vehicle.
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. Introduction

Adenoviral vectors have been widely used for vaccinationgainst cancer and infectious diseases. However, traditional ade-oviral vaccines, designed to express antigens that are encodeds transgenes, have yielded suboptimal clinical results, attributed

Please cite this article in press as: Xue, C., et al., Construction and chacontaining two foreign neutralizing epitopes in hexon. Virus Res. (201

n part to the preexisting immunity of the recipient to adenovirusype 5 (Ad5), arising from natural adenoviral infection or previ-usly administered Ad5 vectors (Nabs; Schagen et al., 2004; Zaiss

∗ Corresponding author. Tel.: +862034281614; fax: +8620 34281614.∗ ∗ Corresponding author at: State Key Laboratory of Respiratory Disease, The Firstffiliated Hospital of Guangzhou Medical University, 151 Yan Jiang Road, Guangzhou10120, China. Tel.: +862034281614; fax: +8620 34281614.

E-mail addresses: [email protected] (C. Xue), [email protected]. Tian), [email protected] (X. Li), [email protected] (Z. Zhou),[email protected] (X. Su), [email protected] (R. Zhou).1 These authors contributed equally to this work.

168-1702/$ – see front matter © 2014 Published by Elsevier B.V.ttp://dx.doi.org/10.1016/j.virusres.2014.01.027

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© 2014 Published by Elsevier B.V.

et al., 2009; Pandey et al., 2012). For this reason, the “antigencapsid-incorporation” strategy has been developed for adenovirus-based vaccines in the context of many diseases, and involves theincorporation of antigenic peptides within the capsid structureof adenovirus. Incorporating exogenous immunogenic peptidesinto the adenovirus capsid offers potential advantages, includinga potent humoral response similar to the response generated bynative adenoviral capsid proteins, and immune responses that canbe boosted against antigenic epitopes with repeated administration(Matthews, 2010; Shiratsuchi et al., 2010; Roberts et al., 2006).

The adenoviral capsid is composed of three major proteins:hexon, fiber, and penton base. Hexon is the largest and mostabundant capsid protein, with 720 copies per virion. Analysis ofthe protein sequences of different hexon proteins has revealed

racterization of a recombinant human adenovirus type 3 vector4), http://dx.doi.org/10.1016/j.virusres.2014.01.027

that there are seven discrete hypervariable regions (HVRs), whichform the most exposed surface of the virion and are found tobe the major targets of serotype-specific neutralizing antibodies(Crawford-Miksza and Schnurr, 1996; Gall et al., 1998; Rux et al.,

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dx.doi.org/10.1016/j.virusres.2014.01.027


http://www.sciencedirect.com/science/journal/01681702

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ARTICLEIRUS 96219 1–8

C. Xue et al. / Virus Re

003). The major capsid protein, hexon, has been used for anti-en capsid-incorporation strategies because hexon plays a naturalole in the generation of the anti-adenovirus immune response,nd it is numerously represented within the adenoviral virionShiratsuchi et al., 2010; Krause and McConnell, 2006). Previoustudies have verified that short heterologous peptides derived fromoliovirus, Pseudomonas aeruginosa, Bacillus anthracis, and HIV, asell as model epitopes, can be incorporated into the adenoviralexon HVRs without compromising viral viability (Worgall et al.,007; Matthews et al., 2010; Crompton et al., 1994). For exam-le, the replacement of HVR1 with a malarial B-cell epitope haseen shown to induce a substantially increased level of protectiveumoral immunity against malaria and circumvents any preexis-ing immunity to adenovirus (Shiratsuchi et al., 2010).

The immune response against an epitope inserted into hexon isependent on the incorporation site and the size of the incorpo-ated epitope (McConnell et al., 2006; Wu et al., 2005). Publishedtudies have focused on the incorporation of single epitopes orntigens into single HVRs. Recently, we sought to generate aultivalent vaccine Ad3 vector by incorporating epitopes in Ad3

exon. However, our previous study showed that the antiserumas induced against the new epitope but not against the multiple

pitopes that were simultaneously incorporated into single HVRsZhong et al., 2012). Therefore, the replacement of several HVRsith antigens might be a promising alternative way to generateultivalent adenoviral vectors. Our previous study confirmed thatVR1 and HVR2 of Ad3 are potential incorporation sites for vac-ine development (Tian et al., 2012). The present study focuses onhe creation of multivalent vaccine vectors displaying two differentpitopes in several HVRs of Ad3.

. Materials and methods

.1. Cells, virus strains, and plasmids

Sublines of HEp-2 cells, AD293 cells, and Vero cells were keptn our laboratory and cultured in Dulbecco’s modified Eagle’s

edium (DMEM; Gibco, Grand Island, USA) supplemented withenicillin (100 IU/ml), streptomycin (100 �g/ml), and 10% fetalalf serum (Tian et al., 2012). The following plasmids and virusessed in this study were obtained as previously described or areaintained in our laboratory (Tian et al., 2012; Zhang et al.,

009): the E3-defective adenovirus type 3 replication-competentlasmid pBRAdV3dE3egfp (pAd3egf), which expresses the reporterolecule enhanced green fluorescent protein (EGFP) and the corre-

ponding virus Ad3EGFP; hexon shuttle vector pBRHexonL/R; thelasmid pAd3egf-SP70 with the SP70 epitope in hexon HVR1 andhe corresponding virus R1SP70A3 (R1A3); and the EV71-08-02


train of the EV71 C4 genotype (GenBank accession no. FJ360545).he EV71 viral titer was determined as the TCID50 in Vero cells,ased on the typical cytopathic effect (CPE) produced by viral

nfection.

ig. 1. Diagram of the SP55 and SP70 epitope incorporation sites in Ad3 hexon. (A) Aminof Ad3 hexon. (B) R1R2A3, R1R4A3, or R1R5A3 corresponding to the hypervariable regionmino acid residues marked in HVR1 were replaced with the SP70 epitope, and the onesumbers show the positions of the amino acid residues in Ad3 hexon.

PRESS xxx (2014) xxx–xxx

2.2. Recombinant hexon-modified plasmid construction

To generate constructs containing SP70 epitope in HVR1 andSP55 epitope in HVR2, HVR4, or HVR5, the SP55 and SP70 epi-topes were genetically incorporated into the HVRs at the positionsmarked in Fig. 1. To achieve these genetic modifications, we firstgenerated the hexon fragments containing the sequences encodingthe SP70 and SP55 genes in different HVRs using an overlapping PCRmethod, as described previously by Tian et al. in 2012 (Tian et al.,2012). The corresponding primers are shown in Table 1.

The hexon fragments containing the SP70 and SP55 genes werepurified and cloned into the Ad3 hexon shuttle vector pBRHexonL/Rwith ClaI and BamHI restriction enzymes. To create recombinantAd3 vectors containing the SP70 and SP55 sequences in the HVRsof hexon, the three shuttle vectors were digested with EcoRI andSalI, and then used to cotransform Escherichia coli BJ5183 cells withthe AvrII- and PacI-linearized human adenovirus type 3 (HAdV3)plasmid pBRAdV3dE3egfp (pAd3egf). The resultant clones, whichcontained both SP70 and SP55, were obtained by homologousrecombination, and the constructs were then selected with PCRusing primers HexonF/sp55r or HexonF/sp70r (shown in Table 1)and confirmed by restriction digestion and sequence analysis.

2.3. Generation of recombinant virus

To rescue the recombinant virus, these modified plasmids werelinearized with AsiSI. AD293 cells were transfected with 4 �g ofeach purified DNA using the NanoJuice Transfection Kit (Novagen,USA) and grown in dishes of 30 mm diameter. After the plaquesformed, the viruses were processed for large-scale propagationin AD293 cells and then purified with CsCl gradient centrifuga-tion (Wu et al., 2002). The purified viral DNA was confirmed withPCR and sequence analysis. The viral particle (VP) titers weredetermined with spectrophotometry using a conversion factor of1.1 × 1012 VPs per absorbance unit at 260 nm.

2.4. Thermostability assay and growth characteristics

To test the heat stability of the hexon-modified adenovirus, thevirus was incubated in DMEM containing 2% FBS at 45 ◦C for 0,5, 10, 20, 40, or 60 min before it was used to infect HEp-2 cells.Fluorescence was then measured 48 h after infection with a Var-ioskan Flash Multimode Reader (Thermo Scientific), with excitationat 488 nm and recording the light emitted at 570 nm. Backgroundfluorescence was normalized to wells containing cells only.

Growth curves were generated by infecting HEp-2 cells withadenovirus at five VPs/cell and the infected cells were collectedevery 12 h for 72 h. The harvested cells were suspended in DMEMcontaining 2% FBS, subjected to three freeze–thaw cycles, and cen-


trifuged at 10,000 × g for 30 min at 4 ◦C to remove the cell debris.The viral suspension was then diluted with DMEM containing 2%FBS in a 10-fold dilution series and each dilution was used to infectHEp-2 cells cultured in 24-well plates. The number of infectious

acid residues of the SP70 and SP55 epitopes that were incorporated into the HVRs (HVR) sites that were modified with the SP55 and SP70 epitopes. The underlined

in the rectangles in regions 2, 4, and 5 were replaced with the SP55 epitope. The

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Table 1Primers used to incorporate the SP55 epitope into the HVRs of the Ad3 hexon and for PCR identification.

Primer Sequencea

HexonF 5′-AGGCTGAGTTGCTTTCAAGATGGCCACC-3′

HexonR 5′-CATGGGATCCACCTCAAAGTCATGTCCAGC-3′

A3-h2-sp55U 5′-GACATTACCCCAGATTCCAGGGAATCCCTTGCATGGCAAACTGCCACCAACCCCAAGCCCATTTATGCCGATAAAAC-3′

A3-h2-sp55R 5′-AATGGGCTTGGGGTTGGTGGCAGTTTGCCATGCAAGGGATTCCCTGGAATCTGGGGTAATGTCTTTCCCAATTTGCA-3′

A3-h4-sp55U 5′-AACCAACACCAGATTCCAGGGAATCCCTTGCATGGCAAACTGCCACCAACCCCGAAGGAGGGGTTGAAACTGAGG-3′

A3-h4-sp55R 5′-CCTCCTTCGGGGTTGGTGGCAGTTTGCCATGCAAGGGATTCCCTGGAATCTGGTGTTGGTTTTACTTTTCTGTTTT-3′

A3-h5-sp55U 5′-GGGATGCTCCAGATTCCAGGGAATCCCTTGCATGGCAAACTGCCACCAACCCCGCAGGAGCTTTAGCGCCTGAAAT-3′

A3-h5-sp55R 5′-CTCCTGCGGGGTTGGTGGCAGTTTGCCATGCAAGGGATTCCCTGGAATCTGGAGCATCCCTACCATCGAAAAATTC-3′

sp55r 5′-GGTGGCAGTTTGCCATGCAAGG-3′

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sp70r 5′-GATCTTTCTCCTGTTTGTGTTCTC

a The underlined letters represent the sequences encoding the SP55 epitope.

articles at each time point was determined by the measurementf fluorescence counting 48 h after infection, as described by Zhongt al. in 2012 (Zhong et al., 2012).

.5. Production of recombinant SP55 and SP70 fusion peptides

A pGEX-4T-3 vector was used to produce the SP55 peptide andP70 peptide with an N-terminal glutathione S-transferase (GST)ag (SP70–GST and SP55–GST, respectively). The GST fusion pep-ides were purified by affinity chromatography using GST-Bindesin (Novagen) under native conditions and stored as aliquots at80 ◦C to avoid repetitive freeze–thaw cycles. The recombinant VP1rotein was prepared previously (Wang et al., 2013).

.6. Mouse immunization

Groups of 4–6-week-old female BALB/c mice were used formmunization. Groups of five mice were analyzed in each exper-ment or at each time point. The mice were vaccinated byntraperitoneal injection with 1 × 1010 VPs of recombinant viruses1A3, R1R2A3, Ad3EGFP, heat-inactivated EV71 strain (10 �g ofotal protein), 50 �g of SP70–GST, 50 �g of SP55–GST, or 50 �g ofST protein with complete adjuvant. Three boosters were given at, 4, and 6 weeks, with the same dose of the same vectors or pep-ide, by intraperitoneal injection. Blood was collected from the tailein before each immunization. The sera were separated from thelood cells and kept for serum analysis. The blood was collected via

retroorbital eye bleed 10 days after the final injection. The seraere separated from the blood cells and stored at −80 ◦C for analy-

is of the anti-VP1 antibody response and the in vitro neutralizationf EV71. All animal procedures were approved by the First Affiliatedospital of Guangzhou Medical University Ethics Committee.

.7. In vitro neutralization

To determine whether anti-SP70–GST and anti-SP55–GST anti-odies neutralized the hexon-modified recombinant virus R1R2A3nd whether the hexon-modified recombinants could escapeeutralization by anti-Ad3EGFP, the serum samples were heat-

nactivated for 45 min at 56 ◦C and then diluted 2-fold in DMEMn 96-well plates. A viral suspension with a titer of 100 TCID50 in


0 �l was added to each serum sample and incubated at 37 ◦C for h. AD293 cells were then added to each well and incubated for–5 days. Serum, virus, and cell controls were included in this test.he plates were monitored for CPEs with light microscopy.

2.8. Immunoblotting analysis

An immunoblotting analysis was performed to detect therecombinant adenovirus R1R2A3. Purified EV71 virions, Ad3EGFP,and recombinant adenovirus R1R2A3 cultures were fractionated ona 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) gel, and blotted onto polyvinylidene difluoride (PVDF)membrane. The membrane was exposed to blocking solution (5%fat-free milk) in phosphate-buffered saline containing 0.05% Tween20 (PBST) at room temperature for 1 h. It was then incubated witha 1:250 dilution of anti-SP55–GST or anti-SP70–GST antibody for1 h, followed by a horseradish peroxidase (HRP)-labeled goat anti-mouse IgG secondary antibody diluted 1:10,000 (Bio-Rad). Thesignals were detected with a chemiluminescent peroxidase sub-strate (ECL Plus, Millipore).

To analyze whether the mice that received R1R2A3 producedantibodies directed against SP70 or SP55, 8-week sera fromR1R2A3-immunized mice were used for an immunoblotting anal-ysis. EV71-infected cell lysate, SP70–GST, and SP55–GST wereseparated on a 10% SDS-PAGE gel and transferred to PVDF mem-brane. Anti-R1R2A3 serum was diluted 1:500 and used as theprimary antibody, and serum from Ad3EGFP-immunized mice wasused as the control. The signal was amplified with a 1:9000 dilutionof secondary antibody. The signal was detected as described aboveusing ECL Plus.

2.9. Indirect ELISA analysis

An enzyme-linked immunosorbent assay (ELISA) was used todetermine whether the SP70 or SP55 epitope was presented onthe surface of the recombinant virion. Carbonate buffer (pH 9.6)containing 109 VP/100 �l of recombinant adenovirus R1R2A3 wasimmobilized in a 96-well plate overnight at 4 ◦C. The plate wasthen blocked with 2% bovine serum albumin (BSA) in PBST at 37 ◦Cfor 2 h. Serially diluted anti-SP55GST antibody, anti-SP70GST anti-body, or anti-GST antibody was then added to each well and theplates incubated for 1.5 h at 37 ◦C. The wells were washed threetimes and incubated with a 1:8000 dilution of HRP-conjugated goatanti-mouse IgG secondary antibody for 1 h. After the plates waswashed four times, the reactions were developed with tetramethyl-benzidine (TMB) substrate, stopped with 2 M sulfuric acid, and theabsorbance (optical density) at 450 nm (OD450) measured with amicroplate reader (Thermo Scientific Multiskan MK3).


To test the anti-SP70 and anti-SP55 antibody responses inimmunized mice, ELISA plates were coated with 50 mM carbonatebuffer (pH 9.6) containing 1 �g/ml SP70–GST or SP55–GST pro-tein overnight at 4 ◦C. The wells were washed twice with PBST and

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Fig. 2. PCR amplification of the SP55 and SP70 epitope insertions from recombinantadenovirus plasmids. PCR products were analyzed by 0.8% agarose gel electrophore-sis. M: marker DL5,000 (Takara); lanes 1, 3, and 5 are pR1R2A3, pR1R4A3, andpR1R5A3, respectively, amplified with primers HexonF/sp55r. Lanes 2, 4, and 6are pR1R2A3, pR1R4A3, and pR1R5A3, respectively, amplified with primers Hex-

Because hexon is the most abundant protein in the adenovi-rus capsid, changing its structural components may lead to virioninstability compared with that of its wild-type counterpart. Totest whether the incorporation of exogenous peptides SP55 and

Fig. 3. SP70 and SP55 epitopes incorporated into hexon HVR1 and HVR2. (A) Res-cued virus was amplified and viral DNA was used to confirm that SP70 and SP55were correctly incorporated into hexon hypervariable region (HVR) 1 and HVR2,respectively. M: DNA marker DL15,000 (Takara, China); lanes 1, 2, and 3 are rescuedvirus R1R2A3, amplified with primers HexonF/HexonR, HexonF/sp55r, and Hex-onF/sp70r, respectively; lanes 4, 5, and 6 are the negative control Ad3EGFP amplified

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locked with 2% BSA/PBST at 37 ◦C for 2 h. After three washes withBST, serially diluted R1R2A3 sera that had been collected at dif-erent time points were added to each well and incubated at 37 ◦Cor 1.5 h. The plates were washed four times with PBST and incu-ated for 1 h at 37 ◦C with a 1:9000 dilution of HRP-conjugated goatnti-mouse IgG secondary antibody. The signals were developedith TMB substrate. The OD450 was read on a microplate reader.

he endpoint titer was defined as the highest dilution at whichhe OD450 was at least 0.1 higher than that of wells that receivedo serum. Serum derived from uninfected mice was used as theegative control.

To determine the anti-VP1 humoral responses, ELISA platesere coated with 0.1 �g of recombinant VP1 protein in 100 �l of

0 mM carbonate buffer (pH 9.6) per well. After the wells werelocked with 2% BSA/PBST, they were washed three times and

ncubated with 8-week immunized sera from R1A3-, R1R2A3- SP55–GST-, SP70–GST-, Ad3EGFP-, or GST-injected mice at7 ◦C for 1.5 h, followed by incubation with HRP-conjugatedoat anti-mouse IgG antibody. ELISAs were then performed asescribed above. Each assay was performed independently at leasthree times and with at least two parallel reactions for eachell.

.10. Neutralization of EV71 in vitro

The titers of NAbs against EV71 were measured in a microneu-ralization test in vitro. The antisera from different antigen-injected

ice were collected 10 days after the fourth injection, accordingo the immunization schedule. The final immunized serum sam-les were inactivated at 56 ± 0.5 ◦C for 30 min, and 50 �l of eacherially diluted sample was mixed with an equal volume of 100CID50 of the EV71-08-02 strain and incubated at 37 ◦C for 1 h. Theixtures were then adsorbed onto a 96-well microtiter plate con-

aining Vero cells (1 × 105 cell/ml) seeded the previous day. Eachssay set included a cell control, a virus control, and antiserum fromeat-inactivated EV71 virus, which was used as a positive control.he plates were placed in a CO2 incubator at 37 ◦C for 5 days, afterhich the CPEs were observed with microscopy. The NAb titer wasefined as the highest dilution of serum capable of inhibiting viralrowth.

.11. Statistical analysis

The data are presented as means ±standard errors. Compar-sons among groups were made with analysis of variance andonferroni’s test to account for multiple comparisons. Statisticalignificance was defined as p < 0.05.

. Results

.1. Generation of hexon-modified Ad3 containing both SP55 andP70

The recombinant plasmids pR1R2A3, pR1R4A3, and pR1R5A3ncoding the 15 amino acids each of SP55 and SP70 werebtained by homologous recombination in E. coli and identifiedy PCR (Fig. 2), and confirmed by restriction enzyme digestionnd full-length hexon sequencing analysis (data not shown). Inells transfected with pR1R4A3 or pR1R5A3, no evidence of viralrowth was seen, even 2 weeks after infection, and repeatedttempts to package the virus failed. However, in cells transfected


ith pR1R2A3, the fluorescent signal from individual cells wasetectable 7 days after transfection, indicating that the R1R2A3irus had been rescued. After amplification on AD293 cells, the res-ued R1R2A3 virus was confirmed by PCR (Fig. 3A) and sequencing.

onF/sp70r. Lanes 7 and 8 are the negative control pAd3egf, amplified with primersHexonF/sp55r and HexonF/sp70r, respectively.

To determine whether R1R2A3 expressed the SP55 and SP70epitopes in the hexon region, purified EV71 and recombinant ade-novirus R1R2A3 or Ad3EGFP were subjected to immunoblottinganalysis with anti-SP55–GST and anti-SP70–GST antibodies. Thedetection of a hexon fusion protein band of the expected size (about110 kDa) suggested that the SP70 and SP55 epitopes were success-fully incorporated into the hexon capsid protein (Fig. 3B, lane 2,lane 5). A band was visible for EV71 consistent with the expectedsize (33 kDa) of the EV71 VP1 peptide (Fig. 3B, lane 1, lane 6), andno band was found in the Ad3EGFP control (Fig. 3B, lane 3 andlane 4).


with the primers HexonF/HexonR, HexonF/sp55r, and HexonF/sp70r, respectively.(B) Immunoblotting analysis of the SP70 and SP55 epitopes in the hexon protein.Purified EV71 virions (lanes 1, 6), the 33-kDa band represents the VP1 protein;recombinant virus R1R2A3 (lanes 2, 5), the 110-kDa band represents the hexonfusion protein; unmodified control Ad3EGFP (lanes 3, 4).



C. Xue et al. / Virus Research xxx (2014) xxx–xxx 5

Fig. 4. Thermostability and growth kinetics of R1R2A3 expressing two epitopes inhexon. (A) Thermostability assay of R1R2A3. Hexon-modified R1R2A3 and unmo-dified Ad3EGFP were incubated at 45 ◦C for 0, 5, 10, 20, 40, or 60 min before theywere used to infect HEp-2 cells. Fluorescence was measured with a Varioskan FlashMultimode Reader (Thermo Scientific). Relative infectivity is expressed as a per-centage of the fluorescence of infectious particles remaining in each sample withthat in the sample without incubating at 45 ◦C. (B) Growth curve of recombinantR1R2A3. The cells were infected with R1R2A3 or Ad3EGFP at 5 VPs/cell and har-vw

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Fig. 5. ELISA analysis of SP55 and SP70 epitopes exposed on recombinant R1R2A3virion surfaces. Recombinant R1R2A3 or control Ad3EGFP, at final concentrations of109 VP/ml, were immobilized on ELISA plates and incubated with 4-fold serial dilu-tions of (A) anti-SP55–GST antiserum, (B) anti-SP70–GST antiserum, or (C) controlanti-GST antiserum. Binding was detected with an HRP-conjugated goat anti-mouse302

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ested every 12 h for 72 h. The number of infectious particles (i.p.) was determinedith the fluorescent counting method. i.p./ml = n × fold dilution.

P70 into the capsid hexon molecules affected viral thermostabi-ity, R1R2A3 and Ad3EGFP were incubated at 45 ◦C for different timentervals before they were used to infect HEp-2 cells. The growthharacteristics of R1R2A3 and Ad3EGFP were also compared tovaluate whether the insertion of the peptides had any effect onirus assembly. Hexon-modified R1R2A3 showed similar stabilityo that of unmodified Ad3EGFP (Fig. 4A), and there was no differ-nce in the viral yields of R1R2A3 and Ad3EGFP (Fig. 4B). These datauggest that the incorporation of heterologous peptides within theexon HVRs does not affect virion stability or growth kinetics.

.2. Presentation of both SP70 and SP55 epitopes on the virionurface

To analyze the presentation of these two epitopes on the


urface of the virion, purified virions or Ad3EGFP was immobi-ized on ELISA plates and incubated with anti-SP55–GST antibodyFig. 5A), anti-SP70–GST antibody (Fig. 5B), and control anti-GSTntibody (Fig. 5C). R1R2A3 was recognized by the corresponding

IgG secondary antibody. These results suggest that the SP55 and SP70 epitopes incor-porated into hexon were accessible to anti-SP55–GST and anti-SP70–GST antibodiesat the virion level, indicating that the epitopes were exposed on the virion surface.

anti-SP55–GST and anti-SP70–GST antibodies, whereas the con-trol Ad3EGFP containing the wild-type hexon was not recognizedby any of these antibodies (Fig. 5A and B). This indicates that theSP55 and SP70 epitopes were exposed on the surfaces of the viralparticles when incorporated within the hexon HVRs.

3.3. Incorporation of SP55 and SP70 within HVRs partlycircumvents the neutralization of Ad3EGFP antiserum

The SP55–GST and SP70–GST-peptide-immunized sera showed


the same neutralizing antibody titers against recombinant R1R2A3(Table 2). The neutralization titer of anti-Ad3EGFP serum againsthexon-modified R1R2A3 was significantly lower than that againstAd3EGFP, which indicates that the replacement of HVR1 and HVR2

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6 C. Xue et al. / Virus Research xxx (2014) xxx–xxx

Fig. 6. SP55 and SP70 epitopes incorporated within HVRs elicit IgG immune responses. (A) An immunization time line showing when the immunizations were performed;arrows show each injection time. Primary and postimmunization sera were collected before each immunization for the analysis of the antibody response in vaccinated mice.(B) EV71-infected cell lysate (lane 1), SP55–GST fusion protein (lane 2), SP70–GST fusion protein (lane 3), and GST (lane 4) were fractioned on an SDS-PAGE gel and thentransferred to PVDF membrane. The membrane was immunoblotted with antiserum from R1R2A3-injected mice. An HRP-linked antibody was used to detect the mouseantibodies and the signal was amplified with ECL reagent. (C) 1 �g/ml SP55–GST and SP70–GST were bound to ELISA plates and incubated with R1R2A3-immunized seracollected before the first injection (0 w) and at 2, 4, 6, and 8 weeks. Binding was detected with IgG-specific HRP-conjugated goat anti-mouse IgG secondary antibody. (D)A LISA. Tg p. ∗∗pS

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nti-VP1 IgG levels in sera collected at 8 weeks were quantified with an indirect Eroup of mice. Error bars represent the standard error of the means for each grouP55–GST, or SP70–GST.

ith exogenous epitopes made R1R2A3 partly resistant to anti-d3EGFP serum.

.4. Both SP55-specific and SP70-specific antibodies werenduced in mice after immunization

To determine whether immunized mice produced antibod-es against the SP55 and SP70 epitopes, mice were immunized

ith R1A3, R1R2A3, SP70–GST, SP55–GST, Ad3EGFP, or GST.he injections were performed at 0, 2, 4, and 6 weeks andhe sera were collected before each injection (Fig. 6A). EV71-nfected cell lysate, SP55–GST protein, and SP70–GST protein

ere subjected to immunoblotting analysis with antiserum from1R2A3-immunized mice, and anti-Ad3EGFP serum was used ashe control. Serum from mice vaccinated with R1R2A3 recognizedhe EV71 antigen in the lysate showing a protein band of approxi-

ately 33 kDa, consistent with the EV71 VP1 protein, and reactedith the SP55–GST and SP70-GST proteins (Fig. 6B). No protein


and was detected with antiserum from mice immunized withhe control Ad3EGFP (data not shown). These results indicate that

humoral immune response had been elicited against these twopitopes inserted into hexon.

able 2n vitro neutralization (mean ±SD, n = 5).

Antiserum Virus neutralization titer

R1R2A3 Ad3EGFPAnti-SP55-GST 800 ± 240 <8Anti-SP70-GST 800 ± 240 <8Anti-GST <8 <8Anti-Ad3EGFP 2000 8000

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he OD450 reflects the levels of serum antibodies. Data are the means ±SD for each < 0.05, when antiserum from R1R2A3 was compared with antiserum from R1A3,

The serum levels of anti-SP70 and anti-SP55 total IgG werequantified by indirect ELISAs using the SP55–GST and SP70–GSTproteins as the capture antigens. As expected, no IgG against SP55 orSP70 was detected in the preimmune serum (Fig. 6C). However, allmice immunized with R1R2A3 had detectable levels of IgG againstSP55 and SP70 2 weeks after the first injection. In the sera collected2 weeks after the second injection (4 weeks), the IgG levels hadincreased about 50-fold. Anti-SP70 and anti-SP55 IgG levels hadincreased again after the third immunization (6 weeks) and hadincreased to approximately 106 at 8 weeks. In sera collected fromAd3EGFP-injected mice, no anti-epitope antibody was detectableat any time point (data not shown).

3.5. Higher antibody titers against EV71 elicited by theincorporation of two epitopes than one epitope in mice

To determine the intensity of the humoral immune responsestimulated, anti-VP1 IgG levels were also compared between miceinjected with the modified adenovirus and those injected withthe recombinant protein. Eight-week serum was used as theprimary antibody for the ELISA analysis of plates coated withrecombinant EV71 VP1. As expected, serum from the negative con-trol Ad3EGFP- or GST-immunized mice contained no detectableanti-VP1 antibodies (Fig. 6D). Mice injected with R1A3, R1R2A3,SP55–GST, or SP70–GST all showed detectable levels of anti-VP1IgG. Mice injected with R1R2A3, which had two epitopes incor-


porated into the capsid protein, showed the highest anti-VP1 IgGlevels (OD450 ≈ 1.78; n = 5) compared with the R1A3-, SP55–GST-, and SP70–GST-immunized mice (p < 0.05). These results suggestthat R1R2A3, with two epitopes in the hexon capsid protein, elicited

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Fig. 7. Neutralization of EV71 in vitro. Serial dilutions of antisera collected fromBALB/c mice (five mice per group) after 8 weeks were incubated with 100 TCID50 ofthe EV71-08-02 virus strain at 37 ◦C for 1 h before they were used to infect Vero cellsin 96-well plates. After incubation at 37 ◦C for 5–6 days, the neutralizing antibodytiters were read as the highest dilution of serum that protected cells from CPEs.∗

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p < 0.05, when R1R2A3 was compared with R1A3, SP70–GST, or SP55–GST. p < 0.05,hen SP70–GST or SP55–GST was compared with R1A3 or R1R2A3. Control antiseraere from mice immunized with EV71 or Ad3EGFP.

stronger anti-VP1 humoral immune response in immunized micehan R1A3 with only a single epitope.

In view of the anti-VP1 immune response produced by recom-inant R1R2A3, we next characterized its neutralization activitygainst EV71 in vitro. The 8-week antisera from SP55–GST-

SP70–GST-, A3EGFP-, R1A3-, R1R2A3-, and EV71-immunizedice were analyzed in a neutralization assay using Vero cells

nfected with 100 TCID50 of the EV71-08-02 strain. Whereas theontrol antiserum from mice immunized with heat-inactivatedV71 had a mean neutralizing titer of 1:150, the antiserum from1R2A3-injected mice showed the highest neutralizing titer whenompared with antiserum from R1A3, SP70–GST, or SP55–GST-mmunized mice (p < 0.05; Fig. 7). Mice that received SP70–GST orP55–GST showed titers of 1:24 or 1:12, respectively, lower thanhat of the R1A3- or R1R2A3-injected mice (p < 0.05; Fig. 7). Thiss similar to the trend seen with anti-VP1 IgG (Fig. 6D). No NAbsgainst EV71 were observed in the sera from Ad3EGFP-immunizedice. These results indicate that the hexon-modified virus R1R2A3,ith two heterologous epitopes in its hexon HVRs, elicited strongereutralizing antibodies against EV71 than did R1A3, with only aingle epitope.

. Discussion

In recent years, the antigen capsid-incorporation strategy hasffered a novel and exciting approach for adenovirus-based vaccineesign because of the plasticity of the adenoviruses. Incorporatingultiple epitopes from various infectious agents or tumor anti-

ens into the adenovirus capsid to generate multivalent vaccineectors is a promising technique. This approach might induce mul-ivalent immunity against various infectious diseases or pathogensf multiple serotypes (Olugbile et al., 2011; Shao et al., 2010). Anmmunotherapeutic approach that induces polyvalent immunity toumor antigens can generate vaccines with reduced potential formmunological escape (Aguilar et al., 2011). The adenoviral vectorlatform potentially allows the incorporation of a foreign epitope

nto four structurally distinct locations: hexon, protein IX, pen-on base, and/or fiber (Krause and Li, 2006; Matthews et al., 2008;

athis et al., 2011). Adenovirus hexon could present an array of 720


pitope copies per virion if an antigenic epitope were inserted into single HVR. Previous studies have suggested that antigenic epi-opes can be incorporated into HVR1, HVR2, and HVR5 of Ad5 hexonShiratsuchi and Matthews, 2010; Wu et al., 2005; McConnell et al.,

PRESS xxx (2014) xxx–xxx 7

2006). Our previous studies also suggested that foreign epitopescan be incorporated into HVR1, HVR2, and HVR7 of the Ad3 vec-tor to elicit antibodies (Tian et al., 2012). It is possible to generatemultivalent vaccine vectors by incorporating multiple epitopes intosingle HVRs. However, our previous findings suggested that polyva-lent immunity is not generated in this way. When multiple epitopeswere simultaneously incorporated into single HVRs, antiserum wasraised against one new epitope but not against multiple epitopes(Zhong et al., 2012).

There has been no report of the successful incorporation ofmultiple epitopes into hexon to produce a multivalent vaccineuntil now. Therefore, we used a new strategy in the present study,which is the first of its kind to generate a multivalent adenoviralvector by displaying two different epitopes, which were geneti-cally incorporated into two separate HVRs of hexon. In this study,we successfully incorporated SP70 into Ad3 HVR1 and SP55 intoAd3 HVR2 to construct the multiepitope vector, R1R2A3, whichwe confirmed with PCR and immunoblotting analysis, and puri-fied whole-virus ELISAs (Fig. 5). Assays of thermostability andgrowth kinetics suggested that the incorporation of the two epi-topes into hypervariable regions (HVR1 and HVR2) of hexon did notadversely affect the stability of the virus (Fig. 4). Both epitopes werethought to be exposed on the intact hexon-modified virion sur-face (Fig. 5). More importantly, the repeated administration of thehexon-modified adenovirus to BALB/c mice boosted their humoralimmune responses against both exogenous epitopes (Fig. 6C). Thereappeared to be no significant difference between the antibody titersdirected against SP70 and SP55 generated after vaccination withR1R2A3. This is noteworthy, because it is important for a multi-valent vaccine to induce a balanced immune response against itsvarious exogenous epitopes. Immunization with R1R2A3 elicitedhigher IgG titers and higher neutralization titers in vitro againstEV71 than immunization with a modified adenovirus carrying onlyone epitope in HVR1 (Fig. 6D, Fig. 7).

In our previous study, the incorporation of the single SP70 pep-tide into HVR4 or HVR5 of Ad3 failed to rescue the virus. In thisstudy, although the positions of the HVR5 or HVR4 regions forSP55 epitope incorporation were altered, the rescue of recombi-nant Ad3 still failed. McConnell et al. demonstrated that HVR5 ofAd5 can accommodate a peptide of up to 36 amino acids with-out any adverse effect on viral infectivity, growth, or stability(McConnell et al., 2006). It may be the space structural differ-ence of the hexon between Ad5 and Ad3 that led to the differentresults.

Antivector immunity represents a key limitation of the currentlyavailable Ad5 vectors based on gene transfer, because boostingagainst transfer gene production is ineffective if there is an immuneresponse to the adenoviral vector. Shiratsuchi et al. found thatreplacing the Ad5 vector HVR1 with a malarial B-cell epitopeimproved its immunogenicity and circumvented any preexistingimmunity to adenovirus in mice (Shiratsuchi et al., 2010). Table 2compares the infectivity of R1R2A3 with that of the Ad3EGFP vectorin the presence or absence anti-Ad3 Nabs. R1R2A3 was neutral-ized to a lesser degree than Ad3EGFP in the presence of anti-Ad3Nabs. We plan to investigate whether R1R2A3 can circumvent anypreexisting immunity to Ad3 in mice in a future study.

5. Conclusion

In summary, in this study, we first generated a multivalent ade-noviral vaccine vector presenting two neutralizing epitopes from


EV71 in separate HVRs on the same virion. Our results indicatethat the replacement of several HVRs with exogenous epitopesis a promising approach to the generation of multivalent vaccinevectors for pathogens and cancers.

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ompeting interests

The authors declare that they have no competing interests.

ncited references

Krause et al. (2006) and Li et al. (2006).

cknowledgments

CYX performed most of the experiments, analyzed the data, andrafted the manuscript; XGT guided the experiments and draftedarts of the manuscript; XGT and RZ designed the study and crit-

cally revised the manuscript; XBS, XL, and ZCZ helped with thexperiments. All of the authors have read and approved the finalanuscript. This research was supported by the National Nature

cience Foundation of China (NSCF 31370194), the National Naturecience Foundation of China (NSCF 31200132), and the Guang-ong and Macau Joint Center of Innovative Drugs for Respiratoryathogens (2010B091000018).

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