Formulation and immunogenicity studies of type III secretion system needle antigens as vaccine candidates

Abstract

Bacterial infections caused by Shigella flexneri, Salmonella typhimurium, and Burkholderia pseudomallei are currently difficult to prevent due to the lack of a licensed vaccine. Here we present formulation and immunogenicity studies for the three type III secretion system (TTSS) needle proteins MxiH(Δ5), PrgI(Δ5), and BsaL(Δ5) (each truncated by five residues at its C terminus) as potential candidates for vaccine development. These antigens are found to be thermally stabilized by the presence of carbohydrates and polyols. Additionally, all adsorb readily to aluminum hydroxide apparently through a combination of hydrogen bonds and/or Van der Waals forces. The interaction of these proteins with the aluminum-based adjuvant changes with time resulting in varying degrees of irreversible binding. Peptide maps of desorbed protein, however, suggest that chemical changes are not responsible for this irreversible association. The ability of MxiH(Δ5) and PrgI(Δ5) to elicit strong humoral immune responses was tested in a murine model. When administered intramuscularly as monomers, the needle components exhibited dose dependent immunogenic behavior. The polymerized version of MxiH was exceptionally immunogenic even at low doses. The responses of both monomeric and polymerized forms were boosted by adsorption to an aluminum salt adjuvant.

Figure 1

Circular dichroism thermal melting curves for each MxiH ^Δ5 (), PrgI ^Δ5 () and BsaL ^Δ5 () at 0.2 mg/mL in isotonic 10 mM Histidine buffer at pH 6.0.

Figure 2

Changes in T_m (°C) as measured by circular dichroism spectroscopy for MxiH ^Δ5 () and PrgI ^Δ5 ().

Figure 3

Circular dichroism thermal melting curves for (A) MxiH ^Δ5 and (B) PrgI ^Δ5 at 222 nm in the presence () and absence (-) of 10% Sucrose and 5% Dextrose.

Figure 4

Adsorption isotherms for MxiH ^Δ5 (A), PrgI ^Δ5 (B) and BsaL ^Δ5 (C) with 0.5 mg/mL aluminum in isotonic 10 mM histidine buffer at pH 6. Solid lines represent 100% adsorption.

Figure 5

Absorbance values observed at 450 nm when each sample was plated and analyzed by a standard ELISA protocol.

Figure 6

Anti MxiH (A and B) and PrgI (C) IgG serum responses following intramuscular injections on study days 1, 14, 28 of Buffer Control (), Alhydrogel Control (), M1/M-His/P1 (), M10/M-Al/P10 (), M50/N-His/P50 (). N-Al/P-Soln ().