Fig. 5.
Immunogenicity of HA22-LR-8M in mice. (A) Groups of BALB/c mice (n = 9) received HA22 (250 μg/kg, ●), or HA22-LR-8M (500 μg/kg, ▲) intravenously every 14 d (arrows) and were bled 10 d after each injection. Antibody levels were measured as described in Materials and Methods. IgG antibody levels are expressed relative to IP30, a mAb against PE38. (B) IgM response. BALB/c mice (10 per group) were injected with HA22 (250 μg/kg, ●) or HA22-LR-8M (500 μg/kg, ▲). Mice were bled 10 d after injection and the 50-fold diluted sera were assayed for IgM by ELISA. (C) Secondary immune response to HA22 and HA22-LR-8M. Four weeks after primary immunization with HA22 (two injections of 5 μg with a 2-wk interval between injections; black arrows), mice were reimmunized with HA22 (C-a; black arrows) or HA22-LR-8M (C-b; gray arrows), and anti-PE IgG levels were measured. The SD of the HA22 group and HA22-LR-8M group on day 66 is 42.5 and 0.47, respectively. (D) Immune responses of mice with preexisting antibodies to HA22. Fifteen weeks after initial immunizations with HA22 (5 μg per mouse), eight mice with low titers (∼103) of anti-HA22-specific IgG were reimmunized with either HA22 (5 μg per mouse) or HA22-LR-8M (10 μg per mouse). Blood was drawn at week 17 and antibody levels were measured. HA22-specific IgG titer is shown. Data are expressed as the mean ± SD; ns, not significant; *P < 0.05.