title: In vivo protein expression and immune responses generated by DNA vaccines expressing mycobacterial antigens fused with a reporter protein.
abstract: We cloned six mycobacterial antigens into a mammalian expression vector as fusion proteins with the enhanced green fluorescent protein (EGFP). Plasmid DNA was injected intramuscularly, and the injection sites were examined 1 week later. Expression of each antigen-EGFP fusion protein was visualized as green fluorescence in muscle tissue sections. A plasmid expressing EGFP alone and a plasmid with a frameshift mutation served as positive and negative controls. Visualization of fluorescent protein in vivo was 100% specific when compared to in vitro results. In vivo sensitivity was only 37% based on individual injection sites, but increased to 100% when results from multiple injection sites were combined for each plasmid. EGFP alone was expressed in a higher proportion of myocytes than the antigen-EGFP fusion proteins (P < 0.001). There was a trend toward an inverse correlation between protein size and the proportion of myocytes with visible fluorescence (r = -0.68; P = 0.09). We compared antibody subtypes generated to Mycobacterium bovis antigen 85A, when it was expressed alone or as a fusion protein. Inclusion of EGFP modified the immune response toward a Th1 response, as indicated by the ratio of antigen 85A-specific IgG2a to IgG1 generated by each plasmid (antigen 85A alone 0.73 +/- 0.18 versus antigen 85A-EGFP 1.82 +/- 0.57, mean +/- S.D.; P < 0.01), though the magnitude of the antibody isotype shift was modest. Direct visualization of antigen-EGFP fusion proteins provided a simple and rapid method to confirm in vivo antigen expression.
authors.affiliation: Infectious Disease Section, Veterans Affairs Medical Center, Building 4, 508 Fulton Street, Durham, NC 27705, USA.
authors.forename: Anita
authors.initials: A
authors.lastname: Quinn
authors.forename: Weiwen
authors.initials: W
authors.lastname: Jiang
authors.forename: Maria
authors.initials: M
authors.lastname: Velaz-Faircloth
authors.forename: Alison J
authors.initials: AJ
authors.lastname: Cobb
authors.forename: Stanley C
authors.initials: SC
authors.lastname: Henry
authors.forename: Richard
authors.initials: R
authors.lastname: Frothingham
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis / Q000276:immunology; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D005260:Female; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000276:immunology; D007273:Injections, Intramuscular; D008164:Luminescent Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D009162:Mycobacterium avium / Q000235:genetics / Q000276:immunology*; D009163:Mycobacterium bovis / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity; D013045:Species Specificity; D018417:Th1 Cells / Q000276:immunology; D019444:Vaccines, DNA / Q000008:administration & dosage / Q000235:genetics* / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D007074:Immunoglobulin G; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA; D049452:Green Fluorescent Proteins