108 109 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 Soft CondensedMatter Group Beamline I22 A potential newmethod for vaccines without refrigeration Related publication: Doekhie A., Dattani R., ChenY. C.,YangY., Smith A., Silve A. P., Koumanov F.,Wells S. A., Edler K. J., Marchbank K. J., Elsen J. M. H. va. den & Sartbaeva A. Ensilicated tetanus antigen retains immunogenicity: in vivo study and time-resolved SAXS characterization. Sci. Rep. 10 , 9243 (2020). DOI: 10.1038/s41598-020-65876-3 Publication keywords: Vaccine;Thermal stability; SAXS; in vivo; in situ ;Tetanus toxoid; Silica; Ensilication; Protein; Protein unfolding; Aggregation; Denaturation A lmost all vaccines require refrigeration or freezing conditions for storage and transport. A vaccine cold chain has been developed to distribute vaccines worldwide in our fight against infectious diseases. However, if vaccines were not dependent on refrigeration or freezing, they would be available tomore people, leading to higher vaccination rates and eradicatingmore diseases. An international team of researchers, led by Dr Asel Sartbaeva, has developed a novel method of making existing vaccines thermally stable so that they will not depend on cold chain distribution. The process uses silica (SiO 2 ) to create layers of inorganic materials around individual vaccine components. This ensilication results innanoparticles of silicawith protein in themiddle. The teamperformed insitu Small Angle X-ray Scattering (SAXS) measurements during ensilication , on Diamond Light Source’s Time-Resolved SAXS & Diffraction beamline (I22). They chose to study tetanus toxin C fragment (TTCF), an inactive component of the tetanus toxin present in the diphtheria, tetanus and pertussis (DTP) vaccine. Their results demonstrated that ensilicationmaintained the vaccine effect. All biopharmaceuticals have unique functions that require different environments for their operation. For some, lyophilisation (freeze-drying) may be the optimum choice for stabilising and transporting the bioactive compounds. However, this new methodology provides another solution for biopharmaceutical stabilisation and could help to increase vaccine transport and administration all around theworld. Vaccination is one of the greatest interventions of modern medicine. It is thought to have prevented at least 10 million deaths between 2010 and 2015 1 andhashelpedtosuccessfullyeradicatediseasessuchassmallpoxandrinderpest. In the 1970s, the vaccine cold chain was devised for storage and transport of vaccines in refrigerated conditions, between 2 and 8 °C, the temperature regime that suits most vaccines for long term storage and transport (Fig. 1). While in mostdevelopedcountriesthiswasaneasierundertakingduetotheavailabilityof refrigerators,electricity, infrastructureandstafftraining,developingcountriesare still lagging behind on even the most basic vaccines, leading to a large difference in infant vaccination andmortality from infectious, vaccine-preventable diseases. The cold chain dependency prevents effective distribution of many vaccines inpoorercountries,necessitatingthedevelopmentofthermallystable,coldchain independent vaccines. One such method of vaccine thermal stabilisation is being developed at the University of Bath and is called ensilication. Ensilication is a method of applying a silica, SiO 2 , network in layers on top of vaccine components to prevent them from denaturation. Most vaccine components include proteins, protein complexes, viruses, viral particles, protein constructs, enzymes and toxoids, thus their primary structure consists mainly of amino acid chains. Protein unfolding and aggregation are the two most damaging processes for amino acid chains that lead to denaturation due to temperature increase. The silica network, when applied, prevents this amino chain unfolding and aggregation, thus creating protection against temperature degradation. In this study on beamline I22, our aim was to gain understanding of the ensilication mechanism by performing in situ ensilication and collecting high resolution data on the onset of ensilication process. Previously, our hypothesis was that silica could be grown to fully surround the target biological and follow the targets’surface structure, mimicking the surface shape and size. At I22, we devised an experimental set up for in situ ensilication (Fig. 2) consisting of a line through which we continuously pumped the solution with a target biological and a syringe pump that would inject prehydrolysed silica at a specific time. Data were collected just before the injection and for up to 60 minutes after the injection.This allowed us to observe the progress of ensilication at the onset of the reaction and through ensilication and further growth of the silica nanoparticles until they precipitated out of the solution. About 40 such ensilication runs during our two-day experiment were performed, on three different targets, an enzyme, a toxoid and a protein complex. Each set up required at least an hour of preparation, as silica prehydrolysis takes about 50-60 minutes andthelinehadtobethoroughlycleanedbeforeeachruntoavoidcontamination. The analysed data show that the ensilication is a diffusion driven process when silica network is being formed. This was clearly seen from the early onset datawheretheshapeofthe initialsilicananoparticlesresembledtheshapeofthe target molecule. As the particles grew, they eventually became more spherical, as we have previously seen from Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) studies. We knew before that the more positively charged the surface of the target, the faster the ensilication, indicating that these positively charged areas attract silica first. Silica then rapidly grows in a network as it diffuses around the target and rapidly (within seconds) creates the network surrounding the target.What was interesting to see from these datawas that the shape of the target dictated the shape of the silica nanoparticle at least in the first stages of ensilication, confirming our hypothesis that the silica 'shell' mimics the shape of the target. A comprehensive understanding for the ensilication mechanism was built, where we have defined three stages for the ensilication (Fig. 3). Stage I – nucleation, where silica monomers get attracted to the positively charged areas on the surface of the target and the first silica layer start growing from those nucleation areas. Stage II – a rapid growth of further silica layers. Here the growing silica nanoparticle still mimics the shape of the original target. For example, tetanus toxoid C fragment, TTCF, has an oval shape, which is clearly seen during the Stages I and II. Stage III is associated with growth through silica aggregation. Particles that have resembled the target shape during Stages I and II rapidly change their shape during Stage III. We know that silica particles aggregate when they reach a specific size, exactly as we are seeing here. During suchaggregation,which isalsocalledamassfractalgrowth,weseeaformationof largely spherical silica particles that we frequently observewith the aid of an SEM. Inconclusion,thisstudyhelpedus inexplainingthemechanismofensilication of biologicals. Understanding the mechanism helps in controlling ensilication, controlling the size of particle formation and in eventually explaining why some biologicals are easier to ensilicate compared to others. Ensilication may help us in thermal stabilisation of vaccines, which could be a game- changing discovery that may help in expanding vaccination and potentially savingmillions of lives. Reference: 1. https://www.who.int/ publications/10-year-review/ vaccines/en/ Funding acknowledgement: Diamond Light Source beamtime at I22 was granted on project SM-14148. Dr Asel Sartbaeva thanks AnnettTrust and the University of Bath Alumni Fund,The Royal Society and the British Council Newton Fund for funding. Corresponding author: Dr Asel Sartbaeva, University of Bath,
[email protected] Figure 1: Vaccine cold chain. Figure modified from Sartbaeva A., “Vaccines: the end of the cold war? How the award-winning ensilication technology could remove the need to refrigerate life-saving vaccines”, The Chemical Engineer, 921, 24 -29, 2018. Figure 2: Flow cell setup for in situ SAXS experiment at beamline I22. Remote controlled syringe pump injected a specific amount of hydrolysed tetraethyl orthosilicate (TEOS) into the protein solution agitated using a magnetic stirrer. The peristaltic pump flowed the solution through the flow-cell where images were taken on the evolution of silica condensation with protein-silica particle growth. Figure modified from Fig. S3 in 10.1038/s41598-020-65876-3 and originally drawn by Dr Aswin Doekhie. Polymeric associates Poly-condensation Particles aggregate Non-porous and dry Milliseconds Seconds Minutes Dried Stage I Nucleation Stage II Particle formation Stage III Mass fractal growth Final material Figure 3: Schematic representation of ensilication stages. Nucleation, induced via electrostatics, initiates ensilication at positive external residues present on the protein. Poly-condensation of TEOS results in ensilication of individual proteins which triggers aggregation. Vacuum filtration of the then turbid solution results in dried powder material containing protein loaded silica nanoparticles. Modified from Fig. 4 in 10.1038/s41598-020- 65876-3