After two days incubation at 37��C/5% CO2, 1 ��Ci of 3HTdR (5′-3H Thymidine spec.act http://www.selleckchem.com/products/Gefitinib.html 14.4 Ci/mmol, Amersham Biosciences) was added. After an additional 16�C18 hr of metabolic labelling, the cells were harvested on GF/C filters (Inotech AG, Basle, Switzerland) and analysed for incorporated radioactivity using a liquid scintillation counter (TriCarb 2500 TR, Packard Instruments, Meriden, CT). Spleen cells obtained from four mice immunised with the plasmid vector without insert constituted the negative control. The stimulation index (SI) was calculated as the ratio of radioactivity incorporated into cells from vaccinated mice and the count rate in cells from control mice.
Plaque reduction neutralisation test (PRNT) Heat-inactivated mouse sera including positive and negative controls, were serially diluted three-fold in PBS and incubated with a virus suspension containing about 30 plaque forming units (PFU) of RVFV. The mixtures were incubated for 90 min at 37��C and thereafter used to infect monolayers of BHK-21 cells in 6-well tissue culture plates (NUNC tissue culture, Nalgene Nunc International). After an adsorption period of 30 min at 37��C, the cells were rinsed with PBS and incubated with cell culture media containing 1% Carboxy-Methyl Cellulose (Aquacide II, Calbiochem?, Merck, CA) for six days at 37��C/5%CO2. The cells were subsequently fixed with 10% formaldehyde, washed with water and counter-stained with 1% crystal violet in water containing 20% ethanol and 0.7% NaCl. The PRNT50 titer was calculated as the reciprocal of the highest serum dilution that reduced the number of plaques by 50%, as compared to the virus control.
Statistical methods The outcome of the challenge was evaluated using the Fisher exact test (Epi Info?, Version 3.5). Quantitative variables were based on measurements of at least two independent experiments containing duplicate samples. Variables are expressed as means and the error bars represent the standard deviation. Results Antibody response after immunisation with cDNA encoding the N protein Genetic vaccination with cDNA encoding the N protein resulted in a strong humoral immune response in all mice. Anti-N specific antibodies (total Ig) were detected by ELISA already after the first immunisation and were followed by a large increase in titers after additional vaccination rounds (Fig (Fig1).1).
However, despite the strong antibody response observed after genetic vaccination with cDNA encoding the N protein, RVFV neutralising GSK-3 antibodies were not detected by PRNT (data not shown). Figure 1 Anti-N specific antibody responses (total Ig) after gene-gun vaccination with cDNA encoding the RVFV N protein. The curves correspond to the mean titers in individual mouse sera measured by ELISA. The error bars represent the standard deviation between …