Evaluation of DNA Vaccine Candidates against Foot-and-Mouth Disease Virus in Cattle

Figures

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  Figure 1. (A) All DNA vaccine constructs consisted of circular double-stranded DNA. Traditional plasmids, pTarget O1P1-3C and mpTarget O1P1-3CLT, are of larger size than minicircles O1P1-3C or O1P1-HIV-3CT. Minicircle and plasmid features; 1: SV40 polyA, 2: attP/attB, 3: CMV promoter, 4: O1P1, 5: 3C protease, 6: HIV frameshift, 7: ORI, 8: CMV enhancer, 9: Intron, 10: SGLuc biomarker, 11: Neomycin selection marker, and 12: AmpR. (B) Western blots of transfected cell lysates demonstrate fully processed VPs with O1P1-3C minicircles and mpTarget O1P1-3CLT plasmid, whereas lysates of the O1P1-HIV-3CT minicircle transfected cells demonstrate both fully processed VPs and partially processed intermediates.

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  Figure 2. TEM of cells transfected with either O1P1-3C minicircles, pTarget O1P1-3C, or mpTarget O1P1-3CLT plasmid, showing FMDV VLP crystalline arrays; the black bar represents 500 nm. Cells transfected with pTarget O1P1-3C were evaluated by immuno-EM utilizing gold labeled F1412SA antibody.

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  Figure 3. Antigen extracted from cells transfected with mpTarget O1P1-3CLT plasmid was found to (A) sediment at the appropriate density using a cesium chloride density gradient and (B) produce VNTs in two guinea pigs, ear tags D17-57 and D17-58, inoculated in a non-challenge study at 7 (blue) and 14 (orange) days post-vaccination. VNTs were further enhanced at 28 days post-vaccination (gray) following a boost at day 21.

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  Figure 4. (A) Relative luciferase units per half second of IBRS2 cells transfected with either traditional plasmid, pTarget (blue), or minicircles (red), expressing the SGLuc reporter and monitored over three days of expression. (B) Minicircles are less than half the size of pTarget, they do not contain additional features to enhance transgene expression, such as chimeric intron and CMV enhancer sequences.