Characterization of alternative protein–liposome attachment strategies. (a) Fraction of protein bound to liposomes as measured by an EMSA assay using Ni-NTA, Co-NTA, BSB, and BS attachment. Incubations were conducted for 10, 20, 30, or 60 min. Error bars represent the standard deviation of three technical replicates. (b) Composition of CPS antibody pool segmented by IgG (blue), IgM (red), IgA (green), and IgE (purple) post-vaccination. (c) Relative antibody titer for alternative protein–liposome attachment formulations relative to PCV13 and PPSV23 following first (black) and second (blue) administrations. Error bars represent the standard deviation of four biological replicates. (d) OPA assay using alternative protein–liposome attachment formulations; dashed line represents 50% killing of bacteria. Error bars represent the 95% confidence interval of four replicates.