FIG 5.

Comparison of heat-shocked MAV versus non-heat-shocked MAV. (A) MAV preparations were made with (MAV^IPS004) and without (MAV^IPS005) the Hsp induction step. Protein bands were compared using a Coomassie gel (top). Three micrograms (lanes 7 and 9) and 5 μg (lanes 8 and 10) of each MAV was loaded; immunoblots of the MAV preparations were also probed for the presence of the key S. pneumoniae protein antigens (PlyD6, PspA) and Hsps (Hsp70, Hsp60). (B) Capillary gel electrophoresis (CGE) analysis was conducted to determine the protein constituents of each preparation. Each peak is denoted by a number, and interpeak regions are marked by a letter. Quantification of peaks is shown in the bar chart on top. CGE traces are shown below. (C) Both MAV^IPS004 and MAV^IPS005 were used to generate antisera using vaccination experiments in mice. Sera recovered from mice vaccinated with either preparation were analyzed using flow cytometry assays of IgG binding to live S. pneumoniae bacteria (serotypes 1, 2 [D39], 4 [TIGR4], 6B, 8, 19A, 22F, and 23F), and results are represented as the mean fluorescence intensity (MFI) in the appropriate gate. Q1, quadrant 1. (D and E) ELISAs detecting anti-Ply (D) and anti-PspA (E) responses were conducted in duplicate. Sera from the experiments described above were diluted, as shown on the x axis, and the OD₄₅₀ was measured for each MAV and a buffer control. Abbreviations: MAV, multiantigen vaccine; HS, heat shocked; NHS, non-heat shocked; MK, molecular weight marker; AUC, area under the curve.