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

A Doekhie,Y-C Chen,J M H Van Den Elsen,K J Edler,S A Wells,F Koumanov,A Smith,A Sartbaeva,A P Silve,R Dattani,K J Marchbank,Y Yang

doi:10.1038/s41598-020-65876-3

Abstract

Our recently developed ensilication approach can physically stabilize proteins in silica without use of a pre-formed particle matrix. Stabilisation is done by tailor fitting individual proteins with a silica coat using a modified sol-gel process. Biopharmaceuticals, e.g. liquid-formulated vaccines with adjuvants, frequently have poor thermal stability; heating and/or freezing impairs their potency. As a result, there is an increase in the prevalence of vaccine-preventable diseases in low-income countries even when there are means to combat them. One of the root causes lies in the problematic vaccine ‘cold chain’ distribution. We believe that ensilication can improve vaccine availability by enabling transportation without refrigeration. Here, we show that ensilication stabilizes tetanus toxin C fragment (TTCF), a component of the tetanus toxoid present in the diphtheria, tetanus and pertussis (DTP) vaccine. Experimental in vivo immunization data show that the ensilicated material can be stored, transported at ambient temperatures, and even heat-treated without compromising the immunogenic properties of TTCF. To further our understanding of the ensilication process and its protective effect on proteins, we have also studied the formation of TTCF-silica nanoparticles via time-resolved Small Angle X-ray Scattering (SAXS). Our results reveal ensilication to be a staged diffusion-limited cluster aggregation (DLCA) type reaction. An early stage (tens of seconds) in which individual proteins are coated with silica is followed by a subsequent stage (several minutes) in which the protein-containing silica nanoparticles aggregate into larger clusters. Our results suggest that we could utilize this technology for vaccines, therapeutics or other biopharmaceuticals that are not compatible with lyophilization.

Highlights

Biopharmaceuticals are biologically derived active compounds intended for therapeutic or diagnostic usage
Using tetanus toxin C fragment (TTCF) as a model, we demonstrate that ensilication could be a solution to overcome the challenges of biopharmaceutical ‘cold chain’ transportation
In our previous paper we observed the retention of protein primary and tertiary structure via ensilication, including heat treated material, using biochemical methods[14]

Summary

Introduction

Biopharmaceuticals are biologically derived active compounds intended for therapeutic or diagnostic usage. 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. 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.

Results

Conclusion