Constuction And Characterization Of A Recombinant Adenovirus Type3Vector Containing Two Foreign Neutralizing Epitopes In Hexon

ObjectiveTo construct a recombinant adenovirus type3(Ad3) vector by incorporatingmultiple epitopes into the major adenovirus capsid protein, hexon.MethodsThe hexon mutated gene fragment hexon-sp55sp70was obtained byoverlapping PCR with the plasmid pBRAd△E3GFP-sp70of adenovirus type3, andwas cloned into adenovirus shuttle vector pBRHexonL/R. The linearized shuttlevector and the backbone plasmid, a recombinant Ad3expressing enhanced greenfluorescence protein in E3region were co-transformed into E. coli BJ5183cells.The recombinant plasmid pBRsp70sp55Ad3egf were obtained by homologousrecombination. The resultant recombinants were identified by PCR, digestion ofrestriction enzyme and sequencing, then were linearized and transfected into AD293cells to rescue recombinant adenoviruses. After amplification and purification, thethermostability and growth characteristics of the rescued adenovirus were analyzed.The fused expression in hexon protein and the exposition on virion surface of SP55and SP70epitopes were identified by enzyme-linked immune-sorbent assay (ELISA)and Western-blot. After multiple immunizations of BALB/c mice, theepitope-specific humoral immune responses were detected by ELISA, western blotand micro-neutralization test in vitro.Results The recombinant adenovirus R1R2A3incorporated with SP55in HVR2andSP70in HVR1was successfully rescued on AD293cells while R1R4A3andR1R5A3were failed. The hexon-modified R1R2A3showed the similar stability andgrowth characteristic to that of unmodified Ad3EGFP. The SP55and SP70epitopeswere expressed in the hexon protein and exposed on the surface of the modifiedadenovirus particles. The SP55and SP70epitope-specific antibodies were inducedafter the first injection of BALB/c mice with R1R2A3, and after the repeatedadministration, the immune responses were boosted. The recombinant adenovirusinduced higher IgG antibody titers compared with SP55-GST and SP70-GST andthe antiserum from mice immunized with R1R2A3could neutralize EV71vitro.ConclusionThe recombinant R1R2A3virus expressing two exogenous neutralizingepitopes in hexon HVR1and HVR2was successfully constructed, and inducedspecific immune responses to both foreign epitopes after immunization. Our studycontributes to a better understanding of hexon-modified Ad vector as amultiple-epitope delivery vehicle.