Anthrax sub-unit vaccine: the structural consequences of binding rPA83 to Alhydrogel®

Abstract

An anthrax sub-unit vaccine, comprising recombinant Protective Antigen (rPA83) and aluminium hydroxide adjuvant (Alhydrogel®) is currently being developed. Here, a series of biophysical techniques have been applied to free and adjuvant bound antigen. Limited proteolysis and fluorescence identified no changes in rPA83 tertiary structure following binding to Alhydrogel and the bound rPA83 retained two structurally important calcium ions. For adsorbed rPA83, differential scanning calorimetry revealed a small reduction in unfolding temperature but a large decrease in unfolding enthalpy whilst urea unfolding demonstrated unchanged stability but a loss of co-operativity. Overall, these results demonstrate that interactions between rPA83 and Alhydrogel have a minimal effect on the folded protein structure and suggest that antigen destabilisation is not a primary mechanism of Alhydrogel adjuvancy. This study also shows that informative structural characterisation is possible for adjuvant bound sub-unit vaccines.

Figure 1

Binding of rPA83 to Alhydrogel^®. A) Adsorption time course, showing the percentage of rPA83 adsorbed to Alhydrogel over various incubation times. On average 96 ± 0.44 % of protein was adsorbed at any given time B) Binding isotherm and the Langmuir linear regression plot (insert). In 30 mM MOPS, 100 mM NaCl, pH 7.0 buffer, the rPA83 protein had a binding capacity of 0.94 mg rPA83/mg Alhydrogel and an adsorption coefficient of 83.47 ml/mg. The isotherm was measured twice, is highly reproducible and one representative example is shown.

Figure 2

Tryptophan and Nile-red fluorescence of rPA83. A) Tryptophan emission spectrum 50 μg/ml rPA83 ± 1 mg/ml Alhydrogel^®, 30 mM MOPS, 100 mM NaCl, pH 7.0 Excitation wavelength = 280 nm and B) Nile-red emission spectrum (samples as above ± 2.6μM Nile Red) Excitation wavelength = 540 nm

Figure 3

Stern-Volmer plot of soluble rPA83, adsorbed rPA83 and solvent exposed Apo-rPA83 at increasing sodium iodide concentrations. Points represent mean (±SEM) values of three independent measurements.

Figure 4

Thermal unfolding of rPA83. A) Tryptophan fluorescence and barycentric mean analysis was used to calculate the fraction of folded rPA83 at the indicated temperature after thermal equilibrium has been reached. Points represent mean (±SEM) values of three independent measurements. B) Unfolding of rPA83 was monitored by tryptophan fluorescence at 340 nm while increasing temperature at the rate of 1 °C min⁻¹. The protein unfolding was investigated with and without 5 mM calcium.

Figure 5

A) The unfolding of soluble and absorbed rPA83 in urea determined by tryptophan fluorescence; B) DSC endotherms of rPA83, showing the difference between adsorbed rPA83 and soluble rPA83. Note that adsorbed rPA83 does not undergo the precipitation/aggregation step shown by the rapid fall in Cp in the soluble rPA83 sample.

Figure 6

Limited proteolysis of rPA83, Upper –SDS PAGE showing cleavage patterns A) Chymotrypsin, B) Pronase E and C) Trypsin; indicated by the arrow is an additional band produced by trypsin in adsorbed rPA83. Lane 1 = protein standards, lane 2 = rPA83, lane 3 = adsorbed rPA83, lane 4 = soluble rPA83 + protease, and lane 5 = adsorbed rPA83 + protease. Lower- structure of PA82 (PDB;1ACC). Additional cleavage site marked at T296 with unresolved loop indicated by dashed line as in [20]. Also shown are the calcium ions as black spheres, tryptophan residues as sticks and domain structure in roman numerals.