Self-Adjuvanting Calcium-Phosphate-Coated Microcrystal-Based Vaccines Induce Pyroptosis in Human and Livestock Immune Cells.

Successful vaccines require adjuvants able to activate the innate immune system, eliciting antigen-specific immune responses and B-cell-mediated antibody production. However, unwanted secondary effects and the lack of effectiveness of traditional adjuvants has prompted investigation into novel adjuvants in recent years. Protein-coated microcrystals modified with calcium phosphate (CaP-PCMCs) in which vaccine antigens are co-immobilised within amino acid crystals represent one of these promising self-adjuvanting vaccine delivery systems. CaP-PCMCs has been shown to enhance antigen-specific IgG responses in mouse models; however, the exact mechanism of action of these microcrystals is currently unclear. Here, we set out to investigate this mechanism by studying the interaction between CaP-PCMCs and mammalian immune cells in an in vitro system. Incubation of cells with CaP-PCMCs induced rapid pyroptosis of peripheral blood mononuclear cells and monocyte-derived dendritic cells from cattle, sheep and humans, which was accompanied by the release of interleukin-1ß and the activation of Caspase-1. We show that this pyroptotic event was cell-CaP-PCMCs contact dependent, and neither soluble calcium nor microcrystals without CaP (soluble PCMCs) induced pyroptosis. Our results corroborate CaP-PCMCs as a promising delivery system for vaccine antigens, showing great potential for subunit vaccines where the enhancement or find tuning of adaptive immunity is required.

Predictors of Survival after Vaccination in a Pneumonic Plague Model.

Background: The need for an updated plague vaccine is highlighted by outbreaks in endemic regions together with the pandemic potential of this disease. There is no easily available, approved vaccine. Methods: Here we have used a murine model of pneumonic plague to examine the factors that maximise immunogenicity and contribute to survival following vaccination. We varied vaccine type, as either a genetic fusion of the F1 and V protein antigens or a mixture of these two recombinant antigens, as well as antigen dose-level and formulation in order to correlate immune response to survival. Results: Whilst there was interaction between each of the variables of vaccine type, dose level and formulation and these all contributed to survival, vaccine formulation in protein-coated microcrystals (PCMCs) was the key contributor in inducing antibody titres. From these data, we propose a cut-off in total serum antibody titre to the F1 and V proteins of 100 µg/mL and 200 µg/mL, respectively. At these thresholds, survival is predicted in this murine pneumonic model to be >90%. Within the total titre of antibody to the V antigen, the neutralising antibody component correlated with dose level and was enhanced when the V antigen in free form was formulated in PCMCs. Antibody titre to F1 was limited by fusion to V, but this was compensated for by PCMC formulation. Conclusions: These data will enable clinical assessment of this and other candidate plague vaccines that utilise the same vaccine antigens by identifying a target antibody titre from murine models, which will guide the evaluation of clinical titres as serological surrogate markers of efficacy.

Effect of mixing, concentration and temperature on the formation of mesostructured solutions and their role in the nucleation of DL-valine crystals.

We report investigations on the formation of mesostructured solutions in DL-valine-water-2-propanol mixtures, and the crystallization of DL-valine from these solutions. Mesostructured liquid phases, similar to those previously observed in aqueous solutions of glycine and DL-alanine, were observed using Dynamic Light Scattering and Brownian microscopy, in both undersaturated and supersaturated solutions below a certain transition temperature. Careful experimentation was used to demonstrate that the optically clear mesostructured liquid phase, comprising colloidal mesoscale clusters dispersed within bulk solution, is thermodynamically stable and present in equilibrium with the solid phase at saturation conditions. Solutions prepared by slow cooling contained mesoscale clusters with a narrow size distribution and a mean hydrodynamic diameter of around 200 nm. Solutions of identical composition prepared by rapid isothermal mixing of valine aqueous solutions with 2-propanol contained mesoscale clusters which were significantly larger than those observed in slowly cooled solutions. The presence of larger mesoscale clusters was found to correspond to faster nucleation. Observed induction times were strongly dependent on the rapid initial mixing step, although solutions were left undisturbed afterwards and the induction times observed were up to two orders of magnitude longer than the initial mixing period. We propose that mesoscale clusters above a certain critical size are likely to be the location of productive nucleation events.

Population and size distribution of solute-rich mesospecies within mesostructured aqueous amino acid solutions.

Aqueous solutions of highly soluble substances such as small amino acids are usually assumed to be essentially homogenous systems with some degree of short range local structuring due to specific interactions on the sub-nanometre scale (e.g. molecular clusters, hydration shells), usually not exceeding several solute molecules. However, recent theoretical and experimental studies have indicated the presence of much larger supramolecular assemblies or mesospecies in solutions of small organic and inorganic molecules as well as proteins. We investigated both supersaturated and undersaturated aqueous solutions of two simple amino acids (glycine and DL-alanine) using Dynamic Light Scattering (DLS), Brownian Microscopy/Nanoparticles Tracking Analysis (NTA) and Cryogenic Transmission Electron Microscopy (Cryo-TEM). Colloidal scale mesospecies (nanodroplets) were previously reported in supersaturated solutions of these amino acids and were implicated as intermediate species on non-classical crystallization pathways. Surprisingly, we have found that the mesospecies are also present in significant numbers in undersaturated solutions even when the solute concentration is well below the solid-liquid equilibrium concentration (saturation limit). Thus, mesopecies can be observed with mean diameters ranging from 100 to 300 nm and a size distribution that broadens towards larger size with increasing solute concentration. We note that the mesospecies are not a separate phase and the system is better described as a thermodynamically stable mesostructured liquid containing solute-rich domains dispersed within bulk solute solution. At a given temperature, solute molecules in such a mesostructured liquid phase are subject to equilibrium distribution between solute-rich mesospecies and the surrounding bulk solution.

Biophysical characterisation of thermal-induced precipitates of recombinant anthrax protective antigen: evidence for kinetically trapped unfolding domains in solid-state.

Insoluble aggregation or precipitation is one of the most common degradation pathways observed for biotherapeutics; despite this, the structural mechanisms by which this occurs remain poorly understood due to difficulties associated with biophysical characterisation of protein particulates. To address this knowledge gap, we developed a solid-state circular dichroism (CD) technique, which allows in situ measurements of the secondary and tertiary structural changes associated with the formation of visible therapeutic protein aggregates. We demonstrate how solid-state CD, in conjunction with other biophysical and computational methods can aid in gaining valuable insights into the mechanisms and pathways of thermal-induced precipitation of Bacillus anthracis recombinant protective antigen (rPA), the primary immunogen of anthrax subunit vaccine. Using these methods, we show the domains d3 and d4 are the most labile of the four structurally distinct domains of rPA and play the critical role in nucleating the cascade of unfolding and aggregation. During the assembly process, the domains d1 and d2 become kinetically trapped within the insoluble aggregate and reveal previously intractable distinct tertiary structural elements of the rPA native structure. These findings reveal a uniquely detailed insight into the role of rPA domains on protein stability and provide a mechanistic framework for thermal-induced unfolding and precipitation. It also shows that solid-state CD provides a novel approach in characterising protein precipitation that may facilitate rational improvements to the stability of biopharmaceuticals.

Bioprocessing of bacteriophages via rapid drying onto microcrystals.

We present an alternative bioprocess for bacteriophages involving room temperature coprecipitation of an aqueous mixture of phage (Siphoviridae) and a crystallizable carrier (glutamine or glycine) in excess of water miscible organic solvent (isopropanol or isobutanol). The resultant suspension of phage-coated microcrystals can be harvested by filtration and the residual solvent removed rapidly by air-drying at a relative humidity of 75%. Albumin or trehalose added at 5% w/w of the crystalline carrier provide for better stabilization of the phage during co-precipitation. Free-flowing dry powders generated from an aqueous solution of phage (∼13 log(10) pfu/mL) can be reconstituted in the same aqueous volume to a phage titer of almost 10 log(10) pfu/mL; high enough to permit subsequent formulation steps following bioprocessing. The phage-coated microcrystals remain partially stable at room temperature for at least one month, which compares favorably with phage immobilized into polyester microcarriers or lyophilized with excipient (1-5% polyethylene glycol 6000 or 0.1-0.5 M sucrose). We anticipate that this bioprocessing technique will have application to other phage families as required for the development of phage therapies.

Isolation of recombinant proteins from culture broth by co-precipitation with an amino acid carrier to form stable dry powders.

Protein-coated microcrystals can be generated by co-precipitation of protein and a water-soluble crystalline carrier by addition to excess water miscible organic solvent. We have investigated this novel process for its utility in the concentration and partial purification of a recombinant protein exported into the culture broth during expression by Pichia pastoris. Co-precipitation with a L-glutamine carrier selectively isolated the protein content of the culture broth, with a minimal number of steps, and simultaneously removed contaminants including a novel yeast metabolite. This pigment co-elutes during aqueous chromatography but its elucidation as a benzoylated glycosamine suggested a simple route of removal by partition during the co-precipitation process. Scale-up of the process was readily achieved through in-line mixing and subsequent reconstitution of the dried protein-coated microcrystals yielded natively folded, bioactive protein. Additional washing of the crystals with saturated L-glutamine facilitated further purification of the recombinant protein immobilized on the L-glutamine carrier. Thus, we present a novel method for the harvesting of recombinant protein from culture broth as a dry powder, which may be of general applicability to bioprocessing.

Optical spectroscopic methods for probing the conformational stability of immobilised enzymes.

We report the development of biophysical techniques based on circular dichroism (CD), diffuse reflectance infrared Fourier transform (DRIFT) and tryptophan (Trp) fluorescence to investigate in situ the structure of enzymes immobilised on solid particles. Their applicability is demonstrated using subtilisin Carlsberg (SC) immobilised on silica gel and Candida antartica lipase B immobilised on Lewatit VP.OC 1600 (Novozyme 435). SC shows nearly identical secondary structure in solution and in the immobilised state as evident from far UV CD spectra and amide I vibration bands. Increased near UV CD intensity and reduced Trp fluorescence suggest a more rigid tertiary structure on the silica surface. After immobilised SC is inactivated, these techniques reveal: a) almost complete loss of near UV CD signal, suggesting loss of tertiary structure; b) a shift in the amide I vibrational band from 1658 cm(-1) to 1632 cm(-1), indicating a shift from alpha-helical structure to beta-sheet; c) a substantial blue shift and reduced dichroism in the far UV CD, supporting a shift to beta-sheet structure; d) strong increase in Trp fluorescence intensity, which reflects reduced intramolecular quenching with loss of tertiary structure; and e) major change in fluorescence lifetime distribution, confirming a substantial change in Trp environment. DRIFT measurements suggest that pressing KBr discs may perturb protein structure. With the enzyme on organic polymer it was possible to obtain near UV CD spectra free of interference by the carrier material. However, far UV CD, DRIFT and fluorescence measurements showed strong signals from the organic support. In conclusion, the spectroscopic methods described here provide structural information hitherto inaccessible, with their applicability limited by interference from, rather than the particulate nature of, the support material.

Simultaneous detection of alkaline phosphatase and beta-galactosidase activity using SERRS.

Surface enhanced resonance Raman scattering (SERRS) is an alternative to fluorescence for use in bioanalysis however due to the different optical mechanism it requires specifically designed reporters. Recently we have reported the use of 8-hydroxyquinolinyl azo dyes and their ester derivatives as reporters of lipase activity using SERRS. Acylation of the 8-hydroxy moiety significantly reduces surface enhancement of the Raman response and subsequent lipase catalysed ester hydrolysis enables the analyte to bind to silver nanoparticles, thus providing surface enhancement and the SERRS signal is 'switched on'. By following this principle, phosphorylated and galactosylated analogues of 8-hydroxyquinolinylazo dyes were prepared and shown to act as reporters of enzymatic activity for alkaline phosphatase and beta-galactosidase respectively when using SERRS.

SERRS-based enzymatic probes for the detection of protease activity.

Measurement of protease activity, for the first time using SERRS as a detection method, is reported herein. Synthetic introduction of phenylalanine to a benzotriazole azo dye allows the SERRS response to be "switched off" and subsequent exposure to protease restores the SERRS response. The substrates exhibit varying reactivity for a range of proteases and allow for in situ, real-time analysis of protease reactivity. A limit of detection for one protease, Subtilisin carlsberg, was investigated and was established to be 50 ng ml-1.

A rapid and direct method for the determination of active site accessibility in proteins based on ESI-MS and active site titrations.

We have developed an electrospray ionisation mass spectrometry (ESI-MS) technique that can be applied to rapidly determine the number of intact active sites in proteins. The methodology relies on inhibiting the protein with an active-site irreversible inhibitor and then using ESI-MS to determine the extent of inhibition. We have applied this methodology to a test system: a serine protease, subtilisin Carlsberg, and monitored the extent of inhibition by phenylmethylsulfonyl fluoride (PMSF), an irreversible serine hydrolase inhibitor as a function of the changes in immobilisation and hydration conditions. Two types of enzyme preparation were investigated, lyophilised enzymes and protein-coated microcrystals (PCMC).

Investigation of enzyme activity by SERRS using poly-functionalised benzotriazole derivatives as enzyme substrates.

New methods of measuring biologically relevant concentrations of enzymes are necessary to allow greater understanding of biological systems. We have previously shown that aryl azo benzotriazolyl alkyl esters can act as enzyme substrates, with the progress of the reaction being monitored using SERRS (see Nat. Biotechnol., 2004, 22, 1133, ref. ). This is a wholly novel analytical application of SERRS, and the low detection levels of the technique allow for an ultra-sensitive enzyme assay. Masked enzyme substrates are used that are invisible to SERRS until enzymatic hydrolysis. Turnover of the substrate by the enzyme leads to the release of the surface-seeking dye necessary for SERRS, and intense signals are produced. Here we report an improved synthesis of 2H-benzotriazolyl alkyl esters via nucleophilic substitution of a chloromethyl ester by benzotriazolyl azo dyes, giving up to a ten-fold increase on previously reported yields. Introduction of electron-withdrawing groups to the benzotriazole ring allows control over the SERRS properties of the compounds. This is of great significance in expanding the synthetic flexibility and subsequently the fundamental use of these compounds as ultra-sensitive and selective reporters of enzyme activity.

Proteomic methods applied to the analysis of immobilized biocatalysts.

Methods adapted from proteomics can directly characterize proteins present in immobilized biocatalysts. Complete hydrolysis followed by HPLC analysis of Tyr and Phe estimates total protein bound, and is preferable to conventional difference methods, as tested with subtilisin Carlsberg on silica. This new method shows that various treatments give quantitative desorption of proteins immobilized by adsorption. Intact desorbed proteins may be analyzed by electrospray mass spectrometry. The Candida antarctica lipase B from Novozyme 435 was shown to be heavily glycosylated, while the lipase from Lipozyme RM IM was a mixture of four N-terminally truncated forms. Peptides from selective cleavage were analyzed by tandem mass spectrometry, leading to automatic identification of proteins present. A second major protein present in Lipozyme RM IM was thus found to be alpha-amylase from Aspergillus oryzae. These methods should be valuable complements to activity measurements in understanding immobilized enzyme activity and stability.

Circular dichroism studies of subtilisin Carlsberg immobilised on micron sized silica particles.

Immobilised enzymes are widely used in industry, but the reasons for loss of activity of such biocatalysts are usually not known. We have used circular dichroism (CD) to investigate the structure of one such system, i.e., subtilisin Carlsberg (SC) immobilised on silica gel particles (60 microm). A number of technical problems have to be overcome in order to obtain appropriate data from which conclusions can be drawn. A rotating cell holder has been developed to avoid sedimentation of the silica particles during the collection of spectra. By moving the cell holder as close as possible to the detector window, the effects of differential scattering can be minimised. However, the effects of absorption flattening limit the extent to which reliable quantitative information on secondary structure content can be obtained from far UV CD studies. We have used an empirical approach based on absorbance units derived from the high-tension voltage to correct for absorption flattening effects. After applying the correction there was satisfactory agreement with the solution spectra. Comparison of the fresh and used (inactive) SC-silica gel spectra in organic media reveals substantial change in the secondary structure. Additional evidence for loss of native conformation is provided by the significant decrease in the near UV CD spectrum. These results for the first time clearly demonstrate the origin of enzyme instability in the immobilised state.

DNA-coated microcrystals.

Coprecipitation leads to self-assembly of bioactive DNA on the surface of salt, sugar or amino-acid crystals and provides a rapid inexpensive immobilization method suitable for preparing dry-powder formulations of nucleic acids, useful for storage, imaging and drug delivery.

Nanoparticle-coated microcrystals.

Coprecipitation provides a rapid high-yield method for self-assembly of nanoparticles on the surface of flat water-soluble crystalline surfaces and a simple immobilisation technique prior to storage or thermal and chemical modification.

Rapid and ultra-sensitive determination of enzyme activities using surface-enhanced resonance Raman scattering.

Measurement of enzyme activity and selectivity at in vivo concentrations is highly desirable in a range of fields including diagnostics, functional proteomics and directed evolution. Here we demonstrate how surface-enhanced resonance Raman scattering (SERRS), measured using silver nanoparticles, can be used to detect the activity of hydrolases at ultra-low levels. This approach was made possible by designing 'masked' enzyme substrates that are initially completely undetected by SERRS. Turnover of the substrate by the enzyme leads to the release of a surface targeting dye, and intense SERRS signals proportional to enzyme activity are generated. The method was used to rapidly screen the relative activities and enantioselectivities of fourteen enzymes including examples of lipases, esterases and proteases. In the current format the sensitivity of the technique is sufficient to detect 500 enzyme molecules, which offers the potential to detect multiple enzyme activities simultaneously and at levels found within single cells.

Reversible acetonitrile-induced inactivation/activation of thermolysin.

Thermolysin is catalytically inactive in mixtures of 10-15 % acetonitrile in aqueous buffer. Unexpectedly, dilution of the inactive enzyme with acetonitrile leads to complete recovery of the catalytic activity in a similar way to dilution with aqueous buffer. Circular dichroism and fluorescence studies of thermolysin in the same solvent mixtures reveal discontinuous changes in the overall secondary and tertiary protein structure that correlate well with the reversible differences in catalytic activity. The spectra on either side of the minimum activity point are different from each other, a fact indicating that the enzyme may be able to access two active conformations which are thermodynamically stable in different solvent environments.

Solvent selection for solid-to-solid synthesis.

Thermolysin catalyzed solid-to-solid synthesis of the model peptide Z-L-Phe-L-Leu-NH(2) is practically feasible in water and a range of organic solvents with different physicochemical properties. Excellent overall conversions were obtained in acetonitrile, ethyl acetate, n-hexane, methanol, 2-propanol, tert-amyl alcohol, tetrahydrofuran, toluene and water, while no product precipitation was observed in dichloromethane resulting in a much lower yield. In precipitation driven synthesis the product accumulates both in solution and in the solid phase. It was shown that the highest overall yields (yield in the liquid plus yield in the solid) can be expected in solvents where the substrate solubilities are minimized. The best yields of solid product can be expected in solvents where both product and substrate solubilities are lowest. This was in agreement with experimental observations and should be generally valid.