title: Effects of the fusion design and immunization route on the immunogenicity of Ag85A-Mtb32 in adenoviral vectored tuberculosis vaccine.
abstract: Vaccines containing multiple antigens may induce broader immune responses and provide better protection against Mycobacterium tuberculosis (Mtb) infection as compared to a single antigen. However, strategies for incorporating multiple antigens into a single vector and the immunization routes may affect their immunogenicity. In this study, we utilized recombinant adenovirus type 5 (rAd5) as a model vaccine vector, and Ag85A (Rv3804c) and Mtb32 (Rv0125) as model antigens, to comparatively evaluate the influence of codon usage optimization, signal sequence, fusion linkers, and immunization routes on the immunogenicity of tuberculosis (TB) vaccine containing multiple antigens in C57BL/6 mice. We showed that codon-optimized Ag85A and Mtb32 fused with a GSG linker induced the strongest systemic and pulmonary cell-mediated immune (CMI) responses. Strong CMI responses were characterized by the generation of a robust IFN-γ ELISPOT response as well as antigen-specific CD4(+) T and CD8(+) T cells, which secreted mono-, dual-, or multiple cytokines. We also found that subcutaneous (SC) and intranasal (IN)/oral immunization with this candidate vaccine exhibited the strongest boosting effects for Mycobacterium bovis bacille Calmette-Guérin (BCG)-primed systemic and pulmonary CMI responses, respectively. Our results supported that codon optimized Ag85A and Mtb32 fused with a proper linker and immunized through SC and IN/oral routes can generate the strongest systemic and pulmonary CMI responses in BCG-primed mice, which may be particularly important for the design of TB vaccines containing multiple antigens.
authors.affiliation: a State Key Laboratory of Respiratory Diseases; The First Affiliated Hospital of Guangzhou Medical University ; Guangzhou , China.
authors.forename: Yiling
authors.initials: Y
authors.lastname: Zhang
authors.forename: Liqiang
authors.initials: L
authors.lastname: Feng
authors.forename: Liang
authors.initials: L
authors.lastname: Li
authors.forename: Dimin
authors.initials: D
authors.lastname: Wang
authors.forename: Chufang
authors.initials: C
authors.lastname: Li
authors.forename: Caijun
authors.initials: C
authors.lastname: Sun
authors.forename: Pingchao
authors.initials: P
authors.lastname: Li
authors.forename: Xuehua
authors.initials: X
authors.lastname: Zheng
authors.forename: Yichu
authors.initials: Y
authors.lastname: Liu
authors.forename: Wei
authors.initials: W
authors.lastname: Yang
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Niu
authors.forename: Nanshan
authors.initials: N
authors.lastname: Zhong
authors.forename: Ling
authors.initials: L
authors.lastname: Chen
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000284:Administration, Oral; D000818:Animals; D001500:BCG Vaccine / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D016207:Cytokines / Q000378:metabolism; D005260:Female; D005822:Genetic Vectors; D007111:Immunity, Cellular; D008168:Lung / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D013154:Spleen / Q000276:immunology; D032581:Tuberculosis Vaccines / Q000008:administration & dosage / Q000235:genetics* / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000235:genetics* / Q000276:immunology*
chemical_list: D001500:BCG Vaccine; D016207:Cytokines; D032581:Tuberculosis Vaccines; D014614:Vaccines, Synthetic
keywords: APC, Allophycocyanin; BCG, Mycobacterium bovis bacille Calmette-Guérin; BSA, bovine serum album; CMI, cell-mediated immune responses; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, Dimethyl sulfoxide; ELISPOT, Enzyme-linked immune-sorbent spot; FACS, Fluorescence Activated Cell Sorter; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HA tag, hemagglutinin tag; HEK, human embryo kidney; ICS, Intracellular cytokine staining; IFN-γ, interferon gamma; IL-2, Interleukin 2; IM, intramuscular; IN, intranasal; Mtb, Mycobacterium tuberculosis; NBT/BCIP, Nitro blue tetrazolium/ 5-Bromo-4-chloro-3-indolyl phosphate; PBS, Phosphate Buffered Saline; PCR, polymerase chain reaction; PE, Phycoerythrin; PerCP, Peridinin-ChlorophylL-Protein Complex; RPMI, Roswell Park Memorial Institute; SC, subcutaneous; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SFC, spot-forming cells; TB, tuberculosis; TNF-α, tumor necrosis factor α; fusion strategies; immunization routes; immunogenicity; multiple antigens; mycobacterium tuberculosis; rAd5, recombinant adenovirus type 5; tPA, tissue plasminogen activator; vp, viral particles