Preclinical safety and tolerability of a repeatedly administered human leishmaniasis DNA vaccine

Riede, O; Seifert, K; Oswald, D; Endmann, A; Hock, C; Winkler, A; Salguero, F J; Schroff, M; Croft, S L; Juhls, C

Measurements were carried out with 200 ng of extracted total DNA in 384 well plates with a reaction volume of 20 μl on an AB7900HT instrument (Life Technologies, Carlsbad, CA, USA). Primers (Eurofins Genomics, Ebersberg, Germany) and hydrolysis MGB probe (Life Technologies) were designed to detect a consensus sequence present on each MIDGE-Th1 vector. The forward primer (5′-GTCGTTTAGTGAACCGTCAGATCA-3′) anneals to the CMV promoter region, the hydrolysis MGB probe (5′-FAM-TTTATTGCGGTAGTTTATCA-NFQ-3′) and the reverse primer (5′-GCACG ACTGCGTTAGCAATTTAA-3′) anneal to the intron. Limit of detection and lower limit of quantification of the assay were determined at 525 copies of MIDGE-Th1 vector per reaction (that is, 2625 copies per μg total DNA). All samples of extracted DNA were measured in triplicates. MIDGE-Th1 vector copy numbers were calculated according to the LEISHDNAVAX standard curve running in parallel on each plate and expressed as copy number per μg total DNA. Acceptability criteria of measurements included standard curve coefficient of correlation ⩾ 0.95 and detected copy numbers of No-template-controls below the limit of detection. In addition, the s.d. of the quantification cycle values of at least two replicate measurements had to be below 0.5, otherwise the measurement was repeated.