Long-Term Stability of a Vaccine Formulated with the Amphipol-Trapped Major Outer Membrane Protein from Chlamydia trachomatis

Abstract

Chlamydia trachomatis is a major bacterial pathogen throughout the world. Although antibiotic therapy can be implemented in the case of early detection, a majority of the infections are asymptomatic, requiring the development of preventive measures. Efforts have focused on the production of a vaccine using the C. trachomatis major outer membrane protein (MOMP). MOMP is purified in its native (n) trimeric form using the zwitterionic detergent Z3–14, but its stability in detergent solutions is limited. Amphipols (APols) are synthetic polymers that can stabilize membrane proteins (MPs) in detergent-free aqueous solutions. Preservation of protein structure and optimization of exposure of the most effective antigenic regions can avoid vaccination with misfolded, poorly protective protein. Previously, we showed that APols maintain nMOMP secondary structure and that nMOMP/APol vaccine formulations elicit better protection than formulations using either recombinant or nMOMP solubilized in Z3–14. To achieve a greater understanding of the structural behavior and stability of nMOMP in APols, we have used several spectroscopic techniques to characterize its secondary structure (circular dichroism), tertiary and quaternary structures (immunochemistry and gel electrophoresis) and aggregation state (light scattering) as a function of temperature and time. We have also recorded NMR spectra of 15N-labeled nMOMP and find that the exposed loops are detectable in APols but not in detergent. Our analyses show that APols protect nMOMP much better than Z3–14 against denaturation due to continuous heating, repeated freeze/thaw cycles, or extended storage at room temperature. These results indicate that APols can help improve MP-based vaccine formulations.

Amphipols for Each Season

“…First, a detailed analysis of the mechanism of stabilization of OmpA by A8-35 against urea-induced denaturation shows that its origin is not thermodynamic, but kinetic: under the (rather extreme) conditions used, A8-35-trapped OmpA is thermodynamically less stable than in detergent solution, whereas the free energy barrier for moving from the folded to the unfolded state is strongly increased, resulting in a much slower denaturation rate (Pocanschi et al 2013). Whether a similar mechanism accounts for the resistance of APol-trapped MPs to heat-induced denaturation in the absence of urea (see e.g., Dahmane et al 2009, 2013; Feinstein et al 2014; Tifrea et al 2011) remains of course to be seen. The second line of support originates from a recent MD comparison of the dynamics of OmpX in complex with either A8-35, the detergent dihexanoylphosphatidylcholine (DHPC), or a lipid bilayer.…”

“…The NMR spectra of A8-35-trapped BR are of a sufficient quality to expect that, given proper labeling, it should be possible to collect high-resolution data on the structure and dynamics of APol-trapped GPCRs (Elter et al 2014; Etzkorn et al 2013). Preliminary data show that the extramembrane loops of the major outer membrane protein (MOMP) from Chlamydia trachomatis trapped in A8-35 are amenable to a solution NMR study (Feinstein et al 2014). Tryptophan aromatic rings, which typically, in a membrane, interact with lipid headgroups, appear to be buried in MOMP/DPC complexes and accessible in MOMP/A8-35 ones, presumably because of weaker interactions with carboxylate polar moieties than with phosphorylcholine ones (Feinstein et al 2014; Tifrea et al 2014).…”

Folding and stability of integral membrane proteins in amphipols

“…Refs. 137 [45,46, [52][53][54][55]). APols bind to TMPs by adsorbing specifically onto 138 their hydrophobic transmembrane surface, as demonstrated by 139 NMR spectroscopy [56][57][58][59][60], electron microscopy (EM) [61][62][63][64][65][66][67][68][69], 140 and molecular dynamics (MD) simulations [70].…”

“…TMPs can be overexpressed either homol- 52 ogously or heterologously, for example in Escherichia coli, yeast, etc. 53 Plasmid-based overexpression can be designed to target TMPs either 54 to a membrane, which often results in low yields, or to cytoplasmic 55 inclusion bodies, which yields larger amounts of protein, but in a 56 misfolded and aggregated form that has to be folded to a functional 57 state -a difficult achievement. Folding outside the cell environment 58 is also necessary when TMPs are expressed in vitro in a cell-free sys- 59 tem.…”

“…[60,91,158,159] [64], which may be related to stabilization. In vaccination, better 1072 protection against a bacterial disease is obtained following immu-1073 nization against an APol-trapped than a detergent-solubilized 1074 outer TMP used as immunogen, which may be related either to 1075 its stabilization and/or to a better presentation to the immune 1076 system [53,55,161]. Also worth noting is the developing use of 1077 tagged APols for immobilizing TMPs onto solid surfaces 1078 [141,142,[162][163][164].…”