In vivo protein expression and immune responses generated by DNA vaccines expressing mycobacterial antigens fused with a reporter protein.

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Quinn, A; Jiang, W; Velaz-Faircloth, M; Cobb, AJ; Henry, SC; Frothingham, R

Published Date

  • August 19, 2002

Published In

Volume / Issue

  • 20 / 25-26

Start / End Page

  • 3187 - 3192

PubMed ID

  • 12163270

International Standard Serial Number (ISSN)

  • 0264-410X

Digital Object Identifier (DOI)

  • 10.1016/s0264-410x(02)00253-0


  • eng

Conference Location

  • Netherlands