title: Incorporation of immunostimulatory motifs in the transcribed region of a plasmid DNA vaccine enhances Th1 immune responses and therapeutic effect against Mycobacterium tuberculosis in mice.
abstract: T-helper type 1 (Th1) immune response is involved in the development of protective immunity against Mycobacterium tuberculosis. Thus, an increase in Th1 and cellular immune responses should lead to enhanced anti-mycobacterial activity. In this study, we aimed to improve Th1 immune responses to a DNA vaccine by adding potentially immunostimulatory nucleotide sequences into the transcribed region downstream of the antigen. The Mycobacterium leprae gene for hsp65, codon-optimized for expression in mammalian cells, was inserted into pVAX1 with and without 3'-sequences containing CpG and dsRNA motifs. When the plasmid contained both motifs, transfected murine macrophage-like RAW264.7 cells showed markedly increased levels of mRNA for immune molecules of Th1 (IFN-α, IL-12) and Th17 (IL-17, IL-23 and IL-6) responses and for T cell co-stimulatory molecules (CD80 and CD86) but not for a Th2 response (IL-4 and IL-10). Immunized mice showed substantially increased serum anti-Hsp65 IgG2a antibody levels and IFN-γ production by spleen cells, confirming enhancement of the Th1 response in vivo. Furthermore, when non-vaccinated mice were infected with H37Rv by low-dose aerosol challenge, and then 4 weeks later were treated with plasmids by intramuscular injection, the mice that had been treated with plasmids containing immunostimulatory motifs showed an enhanced reduction in mycobacterial loads in lung and spleen. We conclude that DNA vaccines may be made more highly immunogenic and more effective for treatment by including transcribed stimulatory sequences.
authors.affiliation: Shanghai Public Health Clinical Center Affiliated to Fudan University, Shanghai 201508, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Wu
authors.forename: Hui
authors.initials: H
authors.lastname: Ma
authors.forename: Qing
authors.initials: Q
authors.lastname: Qu
authors.forename: Wen-Jiang
authors.initials: WJ
authors.lastname: Zhou
authors.forename: Yu-Ping
authors.initials: YP
authors.lastname: Luo
authors.forename: Harry
authors.initials: H
authors.lastname: Thangaraj
authors.forename: Dougtas B
authors.initials: DB
authors.lastname: Lowrie
authors.forename: Xiao-Yong
authors.initials: XY
authors.lastname: Fan
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics / Q000276:immunology; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D018122:B7-1 Antigen / Q000096:biosynthesis; D051940:B7-2 Antigen / Q000096:biosynthesis; D058491:Bacterial Load; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D018834:Chaperonin 60 / Q000235:genetics / Q000276:immunology; D016207:Cytokines / Q000096:biosynthesis; D015870:Gene Expression; D020869:Gene Expression Profiling; D007074:Immunoglobulin G / Q000097:blood; D007155:Immunologic Factors / Q000235:genetics / Q000276:immunology; D008168:Lung / Q000382:microbiology; D008264:Macrophages / Q000276:immunology; D051379:Mice; D009166:Mycobacterium leprae / Q000235:genetics / Q000276:immunology; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000276:immunology*; D010957:Plasmids; D013154:Spleen / Q000382:microbiology; D018417:Th1 Cells / Q000276:immunology*; D014376:Tuberculosis / Q000276:immunology / Q000517:prevention & control*; D032581:Tuberculosis Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D019444:Vaccines, DNA / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D018122:B7-1 Antigen; D051940:B7-2 Antigen; D001426:Bacterial Proteins; D018834:Chaperonin 60; D016207:Cytokines; D007074:Immunoglobulin G; D007155:Immunologic Factors; D032581:Tuberculosis Vaccines; D019444:Vaccines, DNA; D014614:Vaccines, Synthetic; C074119:heat-shock protein 65, Mycobacterium