Passive antibody or serum transfer

Rhesus Macaques were immunized with 50 or 250 μg MCSP and 250 μl AddaVax at the indicated frequencies and intervals (see “Results” section). Four weeks after each immunization, macaque blood was collected and serum was isolated. All samples were pooled and some of the sera were used to obtain purified IgG, using protein A affinity chromatography (GE Healthcare, Marlborough, MA). IgG from pooled samples that were collected from the macaques before immunization was purified as the control for passive antibody transfer. To determine ELISA titers achieved in mice by transfer of macaque sera, 200 μl of macaque sera with a reciprocal ELISA titer of 512,000 was inoculated by tail vein injection into three C57Bl/6 mice and mice were bled by tail vein 15 and 60 min post tail vein inoculation. The mouse sera at both time points in all three mice had a reciprocal titer of 64,000, an eightfold dilution of the injected macaque sera. Subsequent challenge studies in mice assumed this eightfold dilution for evaluating protective capabilities in mice of titers achieved in the macaques. For challenge studies C57BL/6 mice were injected by tail vein with 200 μl purified IgG from immunized macaques or immune sera or control IgG or sera. Purified IgG was used in those settings in which higher concentrations of antibody in volumes appropriate for IV injection were required to attain the desired high titer in the mouse. Thirty minutes following injection of antibody or sera, mice were inoculated via tail vein with 5 × 10³ transgenic P. berghei sporozoites. Liver stage sporozoite copy number was measured 48 h following infection.