The role of adjuvant in mediating antigen structure and stability

Abstract

The purpose of this study was to probe the fate of a model antigen, a cysteine-free mutant of bacteriophage T4 lysozyme, to the level of fine structural detail, as a consequence of its interaction with an aluminum (Al)-containing adjuvant. Fluorescence spectroscopy and differential scanning calorimetry were used to compare the thermal stability of the protein in solution versus adsorbed onto an Al-containing adjuvant. Differences in accessible hydrophobic surface areas were investigated using an extrinsic fluorescence probe, 8-Anilino-1-naphthalenesulfonic acid (ANS). As has been observed with other model antigens, the apparent thermal stability of the protein decreased following adsorption onto the adjuvant. ANS spectra suggested that adsorption onto the adjuvant caused an increase in exposure of hydrophobic regions of the protein. Electrostatic interactions drove the adsorption, and disruption of these interactions with high ionic strength buffers facilitated the collection of two-dimensional (15) N heteronuclear single quantum coherence nuclear magnetic resonance data of protein released from the adjuvant. Although the altered stability of the adsorbed protein suggested changes to the protein's structure, the fine structure of the desorbed protein was nearly identical to the protein's structure in the adjuvant-free formulation. Thus, the adjuvant-induced changes to the protein that were responsible for the reduced thermal stability were not observed upon desorption.

Figure 1.

Thermal unfolding based on intrinsic fluorescence data. Squares—T4 lysozyme in solution. Triangles—T4 lysozyme adsorbed onto adjuvant. The apparent T_ms are approximately 64 ± 0.5°C and 43°C for the solution state and adsorbed protein, respectively. For comparison, the apparent T_ms of these formulations according to DSC data (thermograms not shown) were 65 ± 0.5°C (solution state) and 45 ± 0.10°C (adsorbed).

Figure 2.

Averaged emission spectra from ANS-binding experiments. Solid line—ANS in WT_* T4 solution. Dashed line—ANS with adsorbed WT_* T4 lysozyme. The spectra have been corrected by subtracting the appropriate blank spectrum: ANS in buffer, with or without adjuvant.

Figure 3.

¹⁵N HSQC spectra of WT_* T4 lysozyme in solution. (a) The spectrum of the protein in MOPS buffer containing 50mM NaCl in the absence of adjuvant. (b) The spectrum obtained in the same MOPS buffer containing 50mM NaCl in the presence of adjuvant. The reduction in signal intensity is due to the binding of protein to adjuvant. (c) The spectrum obtained in the presence of the same MOPS buffer in 200mM NaCl and adjuvant. Recovery of the signal is due to the desorption of protein from adjuvant. The spectrum suggests minimal perturbation to the protein as a result of binding.

Figure 4.

Effect of increasing amounts of salt on adsorbed WT_* T4 lysozyme. The graph shows the percentage of the previously adsorbed protein that is desorbed as when sodium chloride is added to the sample—determined by to UV–visible spectroscopy analysis of the supernatant.

Figure 5.

(a) The effect of salt on the chemical shift values for residues in WT_* T4 lysozyme. The graph shows the magnitude of the change in chemical shift due to salt (0 vs. 200mM) as a function of position in the protein in the absence of adjuvant. The inset shows a small region of the spectra containing the peaks for three residues (84, 105, and 144) at three salt concentrations 0mM NaCl (black), 100mM NaCl (cyan), and 200mM NaCl (red) in MOPS buffer. (b) Chemical shift changes of desorbed WT_* T4 lysozyme in the presence of adjuvant relative to the adjuvant-free solution state sample in the presence of identical concentrations of NaCl. Red—100mM NaCl. Orange—150mM NaCl. Yellow—200mM NaCl. The inset shows a small region of the ¹⁵N HSQC spectrum of the protein as in panel (a) in the presence of 200mM NaCl with adjuvant (black) and without adjuvant (red).

Figure 6.

The adjuvant-dependent differences in chemical shifts at 100mM NaCl mapped onto the structure of the protein. Residues with the largest chemical shift changes (0.04–0.06 ppm) are shown in red and residues with moderate changes (0.025–0.04 ppm) are shown in yellow. The figure was generated using Pymol (The PyMOL Molecular Graphics System, version 1.2r3pre; Schrödinger, LLC (Portland, Oregon) using the PDB file 1L63.