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

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  • Please citcontainin

    ARTICLE IN PRESSG ModelVIRUS 96219 18Virus Research xxx (2014) xxxxxx

    Contents lists available at ScienceDirect

    Virus Research

    j ourna l h o mepa ge: www.elsev ier .com

    Construction and characterization of a recombinaadenovirus type 3 vector containing two foreign epitope

    Chunyan iaobQ1a State Key Lab liatedGuangzhou Meb Department o rsity, G

    a r t i c l

    Article history:Received 13 December 2013Received in revised form 25 January 2014Accepted 31 January 2014Available online xxx

    Keywords:Ad vectorHypervariableEpitope displaImmune respo

    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-specic 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 enterovirus

    1. Introdu

    Adenoviagainst cannoviral vacas transgenin part to thtype 5 (Ad5ously admin

    Correspon CorresponAfliated Hosp510120, China

    E-mail add(X. Tian), lixiaoxbsu92@yahoo

    1 These auth

    0168-1702/$ http://dx.doi.o

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    30e this article in press as: Xue, C., et al., Construction and characterization of a recombinant human adenovirus type 3 vectorg two foreign neutralizing epitopes in hexon. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.01.027

    regionynse

    type 71 (EV71) in hexon. The recombinant virus was conrmed by PCR, immunoblotting, and enzyme-linked immunosorbent assay, and injected into mice to analyze the epitope-specic 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-modied intact virion surface. The repeatedadministration of the modied adenovirus R1R2A3 to BALB/c mice boosted the humoral immune responseagainst both epitopes. Immunization with recombinant virus R1R2A3 elicited higher IgG titers and higherneutralization titers against EV71 in vitro than immunization with the modied adenovirus with only oneepitope incorporated into HVR1. In this study, the recombinant R1R2A3 virus expressing two exogenousneutralizing epitopes in hexon HVR1 and HVR2 induced specic immune responses to both foreign epi-topes. Our study contributes to a better understanding of hexon-modied Ad vector as a multiple-epitopedelivery vehicle.

    2014 Published by Elsevier B.V.

    ction

    ral vectors have been widely used for vaccinationcer and infectious diseases. However, traditional ade-cines, designed to express antigens that are encodedes, have yielded suboptimal clinical results, attributede preexisting immunity of the recipient to adenovirus), arising from natural adenoviral infection or previ-istered Ad5 vectors (Nabs; Schagen et al., 2004; Zaiss

    ding author. Tel.: +862034281614; fax: +8620 34281614.ding author at: State Key Laboratory of Respiratory Disease, The Firstital of Guangzhou Medical University, 151 Yan Jiang Road, Guangzhou. Tel.: +862034281614; fax: +8620 34281614.resses: 372546283@qq.com (C. Xue), tianxingui7902@aliyun.com@gird.cn (X. Li), zzcmhxy003@163.com (Z. Zhou),.com (X. Su), zhourong@gird.cn (R. Zhou).ors contributed equally to this work.

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

    see front matter 2014 Published by Elsevier B.V.rg/10.1016/j.virusres.2014.01.027

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    48s in hexon

    Xuea,1, Xingui Tiana,1, Xiao Lia, Zhichao Zhoua, Xoratory of Respiratory Disease, Guangzhou Institute of Respiratory Disease, The First Afdical University, Guangzhou 510120 , Chinaf Medical Genetics and Cell Biology, School of Basic Science, Guangzhou Medical Unive

    e i n f o a b s t r a c t/ locate /v i rusres

    nt humanneutralizing

    o Sub,, Rong Zhoua,

    Hospital of Guangzhou Medical University,

    uangzhou 510120, China

  • Please cit izaticontainin p://d

    ARTICLE IN PRESSG ModelVIRUS 96219 182 C. Xue et al. / Virus Research xxx (2014) xxxxxx

    2003). The major capsid protein, hexon, has been used for anti-gen capsid-incorporation strategies because hexon plays a naturalrole in the generation of the anti-adenovirus immune response,and it is numerously represented within the adenoviral virionQ2(Shiratsuchi et al., 2010; Krause and McConnell, 2006). Previousstudies have veried that short heterologous peptides derived frompoliovirus, Pseudomonas aeruginosa, Bacillus anthracis, and HIV, aswell as model epitopes, can be incorporated into the adenoviralhexon HVRs without compromising viral viability (Worgall et al.,2007; Matthews et al., 2010; Crompton et al., 1994). For exam-ple, the replacement of HVR1 with a malarial B-cell epitope hasbeen shown to induce a substantially increased level of protectivehumoral immunity against malaria and circumvents any preexis-ting immunity to adenovirus (Shiratsuchi et al., 2010).

    The immune response against an epitope inserted into hexon isdependent on the incorporation site and the size of the incorpo-rated epitostudies havantigens inmultivalenthexon. Howwas induceepitopes th(Zhong et awith antigemultivalentHVR1 and Hcine develothe creationepitopes in

    2. Materia

    2.1. Cells, v

    Sublinesin our labomedium (Dpenicillin (calf serum used in thimaintained2009): the plasmid pBmolecule ensponding vplasmid pAthe correspstrain of theThe EV71 vbased on tinfection.

    2.2. Recombinant hexon-modied 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 modications, we rstgenerated 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 werepuried 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 with

    rII- ad pBed

    binatrimnrm

    nera

    escuzed wuriend g, th93 cu etd seined012 V

    ermo

    est tas i

    20, 4scenc

    Flash nm acencwth irus2 h ing d at ral sua 10-

    cells

    Fig. 1. Diagram siduesof Ad3 hexon. sites tamino acid res rectannumbers show

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    112e this article in press as: Xue, C., et al., Construction and characterg two foreign neutralizing epitopes in hexon. Virus Res. (2014), htt

    pe (McConnell et al., 2006; Wu et al., 2005). Publishede focused on the incorporation of single epitopes orto single HVRs. Recently, we sought to generate a

    vaccine Ad3 vector by incorporating epitopes in Ad3ever, our previous study showed that the antiserumd against the new epitope but not against the multipleat were simultaneously incorporated into single HVRsl., 2012). Therefore, the replacement of several HVRsns might be a promising alternative way to generate

    adenoviral vectors. Our previous study conrmed thatVR2 of Ad3 are potential incorporation sites for vac-

    pment (Tian et al., 2012). The present study focuses on of multivalent vaccine vectors displaying two different

    several HVRs of Ad3.

    ls and methods

    irus strains, and plasmids

    of HEp-2 cells, AD293 cells, and Vero cells were keptratory and cultured in Dulbeccos modied EaglesMEM; Gibco, Grand Island, USA) supplemented with100 IU/ml), streptomycin (100 g/ml), and 10% fetal(Tian et al., 2012). The following plasmids and virusess study were obtained as previously described or are

    in our laboratory (Tian et al., 2012; Zhang et al.,E3-defective adenovirus type 3 replication-competentRAdV3dE3egfp (pAd3egf), which expresses the reporterhanced green uorescent protein (EGFP) and the corre-irus Ad3EGFP; hexon shuttle vector pBRHexonL/R; thed3egf-SP70 with the SP70 epitope in hexon HVR1 andonding virus R1SP70A3 (R1A3); and the EV71-08-02

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

    the Avplasmicontainrecomusing pand co

    2.3. Ge

    To rlinearieach pUSA) aformedin AD2tion (WPCR andeterm1.1 1

    2.4. Th

    To tvirus w5, 10, Fluoreioskanat 488uores

    Groadenovevery 1containtrifugeThe viFBS in HEp-2

    of the SP55 and SP70 epitope incorporation sites in Ad3 hexon. (A) Amino acid re (B) R1R2A3, R1R4A3, or R1R5A3 corresponding to the hypervariable region (HVR) idues marked in HVR1 were replaced with the SP70 epitope, and the ones in the

    the positions of the amino acid residues in Ad3 hexon.on of a recombinant human adenovirus type 3 vectorx.doi.org/10.1016/j.virusres.2014.01.027

    nd PacI-linearized human adenovirus type 3 (HAdV3)RAdV3dE3egfp (pAd3egf). The resultant clones, whichboth SP70 and SP55, were obtained by homologousion, and the constructs were then selected with PCRers HexonF/sp55r or HexonF/sp70r (shown in Table 1)ed by restriction digestion and sequence analysis.

    tion of recombinant virus

    e the recombinant virus, these modied plasmids wereith AsiSI. AD293 cells were transfected with 4 g ofd DNA using the NanoJuice Transfection Kit (Novagen,rown in dishes of 30 mm diameter. After the plaquese viruses were processed for large-scale propagationells and then puried with CsCl gradient centrifuga-

    al., 2002). The puried viral DNA was conrmed withquence analysis. The viral particle (VP) titers were

    with spectrophotometry using a conversion factor ofPs per absorbance unit at 260 nm.

    stability assay and growth characteristics

    he heat stability of the hexon-modied adenovirus, thencubated in DMEM containing 2% FBS at 45 C for 0,0, or 60 min before it was used to infect HEp-2 cells.e was then measured 48 h after infection with a Var-

    Multimode Reader (Thermo Scientic), with excitationnd recording the light emitted at 570 nm. Backgrounde was normalized to wells containing cells only.curves were generated by infecting HEp-2 cells with

    at ve VPs/cell and the infected cells were collectedfor 72 h. The harvested cells were suspended in DMEM2% FBS, subjected to three freezethaw cycles, and cen-10,000 g for 30 min at 4 C to remove the cell debris.spension was then diluted with DMEM containing 2%fold dilution series and each dilution was used to infect

    cultured in 24-well plates. The number of infectious

    of the SP70 and SP55 epitopes that were incorporated into the HVRshat were modied with the SP55 and SP70 epitopes. The underlinedgles in regions 2, 4, and 5 were replaced with the SP55 epitope. The

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  • Please citcontainin

    ARTICLE IN PRESSG ModelVIRUS 96219 18C. Xue et al. / Virus Research xxx (2014) xxxxxx 3

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

    Primer Sequencea

    HexonF 5-AGGCTGAGTTGCTTTCAAGATGGCCACC-3

    HexonR A3-h2-sp55U TGGC

    A3-h2-sp55R CAAG

    A3-h4-sp55U GGCA

    A3-h4-sp55R AGGG

    A3-h5-sp55U TGGCA

    A3-h5-sp55R AGGG

    sp55r sp70r

    a The under

    particles atof uoresceet al. in 201

    2.5. Produc

    A pGEX-SP70 peptidtag (SP70Gtides were Resin (Nova80 C to avprotein was

    2.6. Mouse

    Groups immunizatiiment or aintraperitonR1A3, R1R2total proteiGST protein2, 4, and 6 wtide, by intrvein beforeblood cells aa retroorbitwere separasis of the anof EV71. AllHospital of

    2.7. In vitro

    To deterbodies neutand whethneutralizatiinactivatedin 96-well p50 l was a1 h. AD293 35 days. SeThe plates w

    mun

    immbinanombsodiuAGErane.e milST) a0 dilulowe

    IgG wer(ECL analydies 3-imV71-ted o

    Antiy ans the ndarCL P

    direc

    enzyine rface

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    1845-CATGGGATCCACCTCAAAGTCATGTCCAGC-3

    5-GACATTACCCCAGATTCCAGGGAATCCCTTGCAAAGCCCATTTATGCCGATAAAAC-3

    5-AATGGGCTTGGGGTTGGTGGCAGTTTGCCATGGGTAATGTCTTTCCCAATTTGCA-3

    5-AACCAACACCAGATTCCAGGGAATCCCTTGCATGAAGGAGGGGTTGAAACTGAGG-3

    5-CCTCCTTCGGGGTTGGTGGCAGTTTGCCATGCATGTTGGTTTTACTTTTCTGTTTT-3

    5-GGGATGCTCCAGATTCCAGGGAATCCCTTGCAGCAGGAGCTTTAGCGCCTGAAAT-3

    5-CTCCTGCGGGGTTGGTGGCAGTTTGCCATGCAAGCATCCCTACCATCGAAAAATTC-3

    5-GGTGGCAGTTTGCCATGCAAGG-3

    5-GATCTTTCTCCTGTTTGTGTTCTCC-3

    lined letters represent the sequences encoding the SP55 epitope.

    each time point was determined by the measurementnce counting 48 h after infection, as described by Zhong2 (Zhong et al., 2012).

    tion of recombinant SP55 and SP70 fusion peptides

    4T-3 vector was used to produce the SP55 peptide ande with an N-terminal glutathione S-transferase (GST)ST and SP55GST, respectively). The GST fusion pep-puried by afnity chromatography using GST-Bindgen) under native conditions and stored as aliquots atoid repetitive freezethaw cycles. The recombinant VP1

    prepared previously (Wang et al., 2013).

    immunization

    of 46-week-old female BALB/c mice were used foron. G...

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