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 ModelVIRUS 96219 18Virus Research xxx (2014) xxxxxx
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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
Adenoviagainst cannoviral vacas transgenin part to thtype 5 (Ad5ously admin
Correspon CorresponAfliated Hosp510120, China
E-mail add(X. Tian), lixiaoxbsu92@yahoo
1 These auth
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
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.
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: firstname.lastname@example.org (C. Xue), email@example.com@gird.cn (X. Li), firstname.lastname@example.org (Z. Zhou),.com (X. Su), email@example.com (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
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
o Sub,, Rong Zhoua,
Hospital of Guangzhou Medical University,
uangzhou 510120, China
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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
2.1. 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
escuzed wuriend g, th93 cu etd seined012 V
est tas i
Flash nm acencwth irus2 h ing d at ral sua 10-
Fig. 1. Diagram siduesof Ad3 hexon. sites tamino acid res rectannumbers show
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 alterna