Finding a new vaccine in the ricin protein fold

Abstract

Previous attempts to produce a vaccine for ricin toxin have been hampered by safety concerns arising from residual toxicity and the undesirable aggregation or precipitation caused by exposure of hydrophobic surfaces on the ricin A-chain (RTA) in the absence of its natural B-chain partner. We undertook a structure-based solution to this problem by reversing evolutionary selection on the 'ribosome inactivat-ing protein' fold of RTA to arrive at a non-functional, compacted single-domain scaffold (sequence RTA1-198) for presentation of a specific protective epitope (RTA loop 95-110). An optimized protein based upon our modeling design (RTA1-33/44-198) showed greater resistance to thermal denaturation, less precipitation under physiological conditions and a reduction in toxic activity of at least three orders of magnitude compared with RTA. Most importantly , RTA1-198 or RTA1-33/44-198 protected 100% of vaccinated animals against supra-lethal challenge with aero-solized ricin. We conclude that comparative protein analysis and engineering yielded a superior vaccine by exploiting a component of the toxin that is inherently more stable than is the parent RTA molecule.

RiVax, a recombinant ricin subunit vaccine, protects mice against ricin delivered by gavage or aerosol

“…The authors reported no signs of ricin intoxication though they did not present any data concerning lung function or tissue damage. The other recombinant RTA vaccine contains two large deletions, to eliminate the enzymatic activity as well as to remove a hydrophobic region (normally associated with RTB in the holotoxin) to make the vaccine more stable and soluble [ 32 ]. This vaccine administered i.m., with or without adjuvant, protected mice against lethal ricin challenges given either i.p.…”

Coarse‐grained lattice model simulations of sequence‐structure fitness of a ribosome‐inactivating protein

“…As described further below, the broad nature of C v over the wide range of temperatures indicates that the model for unfolding-folding is nearly continuous and lacks significant cooperativity, leading to what can best be described as a polyamorphic transition between a highly-frustrated ''glassy'' state and a configurationally labile protein. 27,28 While the SICHO model may not have realistically reproduced the high-resolution calorimetric features of the RTA molecules, the difference given by DT f between the two structures is approximately 8 K and is in remarkable concurrence with the experimental determination of roughly 7 K. 16,17 This correct prediction of a small change in protein stability needs, however, to be cautiously weighted by the ambiguity in determining T f from a broad C v function. Although each heat capacity has converged from Monte Carlo sampling over 5 3 10 10 lattice configurations and the error in the WHAM numerical 16 The bumps in the all-atom C v are an artifact of the high T conversion.…”

“…We designate this modeled structure as RTA1-33/44-198. 16 The remaining two modeled structures are RTA with residues located in the C-terminal region 199-267 replaced with the hydrophobic residue Ile (the protein chain is denoted as RTA-Ile), and the second are the same residues replaced with the sequence corresponding to pokeweed antivirus protein (PAP), using a structure-structure alignment with the PDB structure 1pag. We denote our hybrid as RTA/PAP.…”

“…An immunogenic polypeptide was recently developed for RTA as a modified RIP fold by dedifferentiation of the molecule into a nontoxic, compact single-domain scaffold for presentation of protective B-cell epitopes. 16 Experimental melting temperatures were reported and indicated that the altered protein fold showed greater resistance to thermal denaturation than the native RTA structure. 16,17 Here, we estimate the unfolding-folding transition temperature (T f ) from the heat capacity and compare our results with experiments.…”

“…16 Experimental melting temperatures were reported and indicated that the altered protein fold showed greater resistance to thermal denaturation than the native RTA structure. 16,17 Here, we estimate the unfolding-folding transition temperature (T f ) from the heat capacity and compare our results with experiments. We show that, regardless of the low resolution of conformational states generated from the lattice model, the potential energy function of the reduced protein representation and the scoring of reconstructed all-atom structures from lattice chains using the CHARMM19 forcefield with a generalized Born (GB) implicit solvent model 18 are sufficiently accurate to yield the correct relative change in T f .…”