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

Biologic activity of the vaccine lot and assessment of antibodies against dsDNA. In all, 100 BALB/c mice (50 male, 50 female) were randomized and allocated to satellite study groups of the toxicity study resembling the repeated-dose treatment schedule (Table 1) . At first treatment, animals were 44-49 days (males) or 60-65 days (females) of age with a body weight of 17-19.3 g (males) or 17-18.4 (females) . At study termination, blood samples were taken and serum obtained for enzyme-linked immunosorbent assay to test for antibodies against vaccine antigens and for radioimmunoassay to test for antibodies against dsDNA. These analyses were performed under non-GLP Regulations conditions. LEISHDNAVAX-specific antibodies were detected performing an enzyme-linked immunosorbent assay as previously described 22 with minor modifications. Plates (Nunc MaxiSorp, Thermo Scientific, Roskilde, Denmark) were coated per well with 100 μl of 5 μg ml − 1 antigen-mix (KMP11, CPA, CPB, P74, TSA 1:1:1:1:1) in PBS (Fisher Scientific, Paisley, UK). Recombinant proteins were obtained from Proteogenix, Oberhausbergen, France (CPA, CPB, P74, TSA) or were kindly provided by Professor C Jaffe (KMP11). Antigen mix was assembled at LPT. Plates were incubated overnight at 4°C, then washed three times with wash buffer (0.05% (v/v) TWEEN in PBS) and subsequently blocked with assay diluent (5% (w/v) bovine serum albumin (Sigma Aldrich, St Louis, USA) in PBS) for 1 h at room temperature. Plates were washed three times with wash buffer and 100 μl per well of 1:50 diluted serum samples were added. The plates were incubated for 2 h at room temperature and subsequently washed five times with wash buffer. Hundred micolitre per well of anti-mouse IgG-HRP (Conc.: 11.3 mg ml − 1 ), (Sigma Aldrich) were added at a 1:5000 dilution and plates were incubated for 2 h at room temperature. After washing five times developer solution containing 3,3',5,5'-tetramethylbenzidine (Sigma Aldrich) was added and the reaction stopped by adding acid solution when the color developed. Absorbance was measured at 450 nm.