Figure 5. Optimizing antibody titers to co-assembled fibers using T cell epitope titration.

(a) Fibers were co-assembled with E214Q11 and 5 different concentrations of PADREQ11, and mice were immunized and boosted (half-dose) with these formulations according the schedule shown. (b) The antibody response against E214 was measured over time by ELISA and (d) followed a bell-shaped dose response curve, shown 7 days after the first and second boosts. (c) Antibody isotypes were measured by ELISA and normalized to the total IgG response. Isotypes in responding groups consisted equally of IgG1 and IgG2b at all doses of PADREQ11, with only the lowest dose (0.005 mM) showing a significant polarization to IgG1 (serum from week 9; analyzed for each dose using a t-test; *, p<0.01). Contributions from IgG2c or IgG3 were negligible at all doses. (d) Competitive ELISA was used to compare antibody affinity for E214 in serum samples collected after the second boost. ELISA plates were coated with E214 at high (1 ug/mL) or low (0.1 ug/mL) density to assess lower and higher affinity antibodies, respectively. No significant differences were observed between doses of PADREQ11 or with the CRM+alum group, by 2-way ANOVA. N = 5 mice per group. Mean + standard deviation is shown.