Vaccine protein stabilization in silica

Successful eradication or control of prevailing infectious diseases is linked to vaccine efficacy, stability and distribution. The majority of protein based vaccines are transported at fridge temperatures (‘cold-chain’) to maintain their potency. However, this has been shown to be problematic. Proteins are inherently susceptible to thermal fluctuations, occurring during transportation, causing them to denature. This leads to ineffective vaccines and an increase in vaccine preventable diseases, especially in low-income countries. Our research utilizes silica to preserve and eventually distribute vaccines at room temperature, thereby decreasing the load on ‘cold-chain’ logistics. The methodology is based upon sol-gel chemistry where soluble silica is employed to encapsulate, ensilicate, vaccine proteins1. This yields protein-loaded silica nanoparticles in the form of a dry powder (figure 1). The material is stored at room temperature and stress tested (heating, 80°C, 2 hours). Subsequently, ensilicated protein is released using a fast chemical process. Silica, silicon dioxide, is an inert biocompatible material with certain ceramic properties that is beneficial in this scenario. The proof-of-concept work was done with a common vaccine antigen: tetanus toxin C fragment2. This protein is the immunogenic part of the full tetanus neurotoxin. Analysis of TTCF protein before and after stabilization in silica revealed full retention of protein structure at various levels. Additionally, specific antibody binding indicated retention of immunogenic epitopes (figure 2). These finding suggest that this methodology could reduce or perhaps eliminate vaccine waste. More work will be undertaken to verify protein stabilization and functional retention in vivo. Please click Additional Files below to see the full abstract

Ensilication Improves the Thermal Stability of the Tuberculosis Antigen Ag85b and an Sbi-Ag85b Vaccine Conjugate

There is an urgent need for the development of vaccine thermostabilisation methodologies as the maintenance of a continuous and reliable cold chain remains a major hurdle to the global distribution of safe and effective vaccines. Ensilication, a method that encases proteins in a resistant silica cage has been shown to physically prevent the thermal denaturation of a number of model proteins. In this study we investigate the utility of this promising approach in improving the thermal stability of antigens and vaccine conjugates highly relevant to the development of candidate tuberculosis vaccines, including antigen 85b conjugated with the Staphylococcus aureus-protein based adjuvant Sbi. Here we analyse the sensitivity of these constructs to thermal denaturation and demonstrate for the first time the benefits of ensilication in conferring these vaccine-relevant proteins with protection against temperature-induced loss of structure and function without the need for refrigeration. Our results reveal the potential of ensilication in facilitating the storage and transport of vaccines at ambient temperatures in the future and therefore in delivering life-saving vaccines globally, and in particular to remote areas of developing countries where disease rates are often highest

Dataset for "Physiochemical Changes to TTCF Ensilication Investigated Using Time-Resolved SAXS"

This dataset contains the SAXS data obtained at the European Synchrotron Radiation Facility beamline ID02. All normalised SAXS data files and processed fitting including visualisation data via MatLab is included. All relevant data to the presented figures/table in the article is contained within this archive.Pre-hydrolysed TEOS was prepared by mixing TEOS and ultrapure H2O 1:1 and the addition HCl to catalyse the reaction. This was added to 10 ml of 1 mg/ml TTCF solution, once both phases turned homogenous, at 1:50 ratio at pH 7 in a glass beaker. Using a sterile syringe, 1 ml of sample was taken and injected into a quartz capillary. SAXS measurements were performed on the Time-Resolved Ultra Small-Angle Scattering beam-line ID02 at the European Synchrotron Research Facility (ESRF), Grenoble, France [23]. The incident X-ray energy was 12.46 keV and sample-detector distance was employed at 1.5 m. SAXS data were acquired using the Rayonix MX-170HS detector with exposure times between 0.01 and 0.03 sec. The measured 2D patterns after normalization by incident flux, sample transmission, and the solid angle were azimuthally averaged to obtain the 1D static scattering profiles as a function of the magnitude of scattering vector, q. Where q is given by, q=(4π sin⁡θ)/λ, with λ the incident X-ray wavelength (=0.0996 nm) and θ the scattering angle. This gave a q-range of 0.006<q<0.5 Å^(-1). The scattering background in each case was measured using Tris buffer and the normalized background subtracted data is represented by I(q).SASview 4.0 MatLab 2015

Ensilication Improves the Thermal Stability of the Tuberculosis Antigen Ag85b and an Sbi-Ag85b Vaccine Conjugate

There is an urgent need for the development of vaccine thermostabilisation methodologies as the maintenance of a continuous and reliable cold chain remains a major hurdle to the global distribution of safe and effective vaccines. Ensilication, a method that encases proteins in a resistant silica cage has been shown to physically prevent the thermal denaturation of a number of model proteins. In this study we investigate the utility of this promising approach in improving the thermal stability of antigens and vaccine conjugates highly relevant to the development of candidate tuberculosis vaccines, including antigen 85b conjugated with the Staphylococcus aureus-protein based adjuvant Sbi. Here we analyse the sensitivity of these constructs to thermal denaturation and demonstrate for the first time the benefits of ensilication in conferring these vaccine-relevant proteins with protection against temperature-induced loss of structure and function without the need for refrigeration. Our results reveal the potential of ensilication in facilitating the storage and transport of vaccines at ambient temperatures in the future and therefore in delivering life-saving vaccines globally, and in particular to remote areas of developing countries where disease rates are often highest

Dataset for "Ensilicated tetanus antigen retains immunogenicity: in vivo study and time-resolved SAXS characterization"

This dataset contains the SAXS data obtained at i22 (Diamond Light Source) and ID02 beamline (ESRF). Other data such as ELISA and Circular Dichroism are also included. All data is organised by figures presented in the research paper. Majority of data is stored in excel or csv format with processing done in MatLab for graphical presentation.Time-resolved (ultra) SAXS (ID02, ESRF): Small Angle X-Ray Scattering (SAXS)21 measurements were performed on the Time-Resolved Ultra Small-Angle Scattering beamline ID02 at the ESRF, Grenoble, France. The incident X-ray energy was 12.46 keV and two sample-detector distances were employed: 1.5 m (SAXS) and 10 m (USAXS). SAXS data were acquired using the Rayonix MX-170HS detector with exposure times between 0.01 and 0.03 seconds at room temperature (20 °C). Pre-hydrolysed TEOS was added to 10 ml of 1 mg/ml TTCF solution at 1:50 (v/v) ratio at pH 7 ex situ, initiating the ensilication reaction. Using a sterile syringe, 1 ml of this ensilication mixture was injected into a quartz capillary (figure S2) after which the beamline hutch was checked for safety and closed for the start of measurement. We measured the delay on hutch closure to be approximately 1 minute. As a result, the total time delay between the start of the reaction and the first measurements was between 1 and 2 minutes. The measured 2D patterns, after normalisation by incident flux, sample transmission, and solid angle, were azimuthally averaged to obtain the 1D static scattering profiles as a function of the magnitude of the scattering vector q=4π/λ sin(θ/2), with λ the incident X-ray wavelength (=0.996 Å-1) and θ the scattering angle. This gave two overlapping q-ranges of 0.0008≤q≤0.008 and 0.006≤q≤0.5 Å-1. The scattering background in each case was measured using Tris buffer and the normalised background subtracted data are represented by I(q). Fitting of SAXS data: Data fitting was done using several models within SASview to probe the various changes observed in the scattering signal. Good residual fits were found using a combination of power law, ellipsoid, broad peak and mass fractal models at different stages of the ensilication process. The ellipsoid model provided shape information (the polar and equatorial radii, Rpolar and Requatorial respectively) on the protein and the initial growth of its silica coating. The broad peak model gave a characteristic length scale for scattering consistent with the particle sizes from the ellipsoid fits, and was utilised as a transition model towards the mass fractal growth. The latter provided the fractal radius of silica particulates and fractal dimension, Rfrac and Df respectively, which are indicative of reaction type. All models were assessed on χ2 as a goodness-of-fit indicator. Detailed information about the fitting parameters are presented in the Supplementary Information. in situ time-resolved SAXS (i22, Diamond Light Source): Small Angle X-Ray Scattering (SAXS) measurements during ensilication in situ were performed on the Time-Resolved Small-Angle Scattering beamline i22 at Diamond Light Source, Didcot, UK. TTCF at 1 mg/ml in (50 mM Tris pH 7.0) buffer, 25 ml volume, was circulated using a peristaltic pump at 2 ml / min through Teflon tubing with an internal diameter of 1.6 mm. The sample solution passed through a 1.5 mm capillary flow cell in a loop before addition of hydrolysed TEOS via a syringe injector. Both pump and injector were remotely controlled (figure S3). SAXS frames were taken at 1 frame/sec for 120 seconds. Pre-hydrolysed TEOS was added to the sample at 1:50 (v/v) ratio after 3 seconds from start of measurement, so that data on the native protein could be acquired before ensilication began. The incident X-ray energy was 12.4 keV. SAXS data were acquired using the Pilatus P3-2M detector at 2.2 m sample distance with 0.8 seconds exposure time. The collected 2D data were processed using a pipeline setup in the Data Analysis WorkbeNch (DAWN) software27. The pipeline was set up with detector calibration, SAXS mask, Poisson error, time with flux and transmission correction. Azimuthal integration produced 1D data in I vs q, where q=4π/λ sin(θ/2), with λ the incident X-ray wavelength (0.9998 Å) and θ the scattering angle. The q range was between 0.008≤q≤0.75 Å-1. The experiment was performed at room temperature (20 °C). Background subtraction scattering was done using a double subtraction method (figure S4 & table S1). Empty capillary scattering was subtracted from Tris buffer scattering. The sample SAXS was then processed by subtracting Tris buffer (minus capillary scattering) and capillary scattering only (table S1).Diamond Light Source data was processed via DAWN. ESRF data was processed using SAXSutilities. SASview was used to analyse the datasets.Data are stored per figure presented in the original work and supplementary information. MatLab code is supplemented for generating figures

Dataset for "Thermal resilience of ensilicated lysozyme via calorimetric and in vivo analysis"

This dataset contains a variety of data formats used to characterise the thermal resilience of ensilicated lysozyme. All raw data formats have been converted to *.csv or similar Excel format, which is editable. MATLAB is used to generate all graphs and files have been included in each folder where applicable. Specifically, data were generated using analytical methods such a differential scanning calorimetry, circular dichroism spectroscopy, enzyme-linked immunosorbent assay (ELISA) and thermogravimetric analysis. Data is sorted based on the figure order described in the article, including supplementary figures. Each folder contains a brief explanation on the contents.Figure 1 - Field-Emission Scanning Electron Microscopy (FE-SEM) - samples were desiccated overnight before imaging and place on sticky carbon tape and imaged at various resolutions. Chromium was used to spin coat the samples. Figure 2 - Micro Differential Scanning Calorimetry (µDSC) and Thermogravimetric analysis (TGA) coupled with Differential Thermal Analysis (DTA) and Mass Spectrometry (MS) or TGA-DTA-MS - liquid samples were analysed using the µDSC apparatus. Ensilicated and lyophilised material was analysed using TGA-DTA-MS. Measurement output was in weight loss (mg, milligram), heat flow (µDSC, DTA) and ion count (A, ampere). Figure 3 - Enzcheck (trademark) Lysozyme assay kit (ThermoFisher) - samples catalytic activity compared to standard protein with validated activity in units(U)/ml. Substrate conversion measured using UV-vis. Data output in arbitrary format converted to U/ml. Figure 4 - Enzyme Linked Immunosorbent Assay (ELISA) - Serum IgG responses to coated antigen measured by substrate conversion using UV-vis and normalised using reference antibody.MATLAB r2015b used for graphical presentation. Excel used for processing data and Calisto software (Setsys) used for exporting DSC/DTA data

Physiochemical Changes to TTCF Ensilication Investigated Using Time-Resolved SAXS

Successful eradication or control of prevailing infectious diseases is linked to vaccine efficacy, stability, and distribution. The majority of protein-based vaccines are transported at fridge (2–8 °C) temperatures, cold chain, to retain potency. However, this has been shown to be problematic. Proteins are inherently susceptible to thermal fluctuations, occurring during transportation, causing them to denature. This leads to ineffective vaccines and an increase in vaccine-preventable diseases, especially in low-income countries. Our research utilises silica to preserve vaccines at room temperature, removing the need for cold chain logistics. The methodology is based upon sol–gel chemistry in which soluble silica is employed to encapsulate and ensilicate vaccine proteins. This yields a protein-loaded silica nanoparticle powder which is stored at room temperature and subsequently released using a fast chemical process. We have previously shown that tetanus toxin C fragment (TTCF) ensilication is a diffusion-limited cluster aggregation (DLCA)-based process using time-resolved small-angle x-ray scattering (SAXS). Here, we present our expanded investigation on the modularity of this system to further the understanding of ensilication via time-resolved SAXS. Our results show that variations in the ensilication process could prove useful in the transition from batch to in-flow manufacturing of ensilicated nanoparticles

Dataset for "Physiochemical Changes to TTCF Ensilication Investigated Using Time-Resolved SAXS"

This dataset contains the SAXS data obtained at the European Synchrotron Radiation Facility beamline ID02. All normalised SAXS data files and processed fitting including visualisation data via MatLab is included. All relevant data to the presented figures/table in the article is contained within this archive