Correlative imaging using super‐resolution fluorescence microscopy and soft X‐ray tomography at cryogenic temperatures provides a new way to assess virosome solutions for vaccine development

Abstract

Active virosomes (AVs) are derivatives of viruses, broadly similar to ‘parent’ pathogens, with an outer envelope that contains a bespoke genome coding for four to five viral proteins capable of eliciting an antigenic response. AVs are essentially novel vaccine formulations that present on their surface selected viral proteins as antigens. Once administered, they elicit an initial ‘anti‐viral’ immune response. AVs are also internalised by host cells where their cargo viral genes are used to express viral antigen(s) intracellularly. These can then be transported to the host cell surface resulting in a second wave of antigen exposure and a more potent immuno‐stimulation. A new 3D correlative microscopy approach is used here to provide a robust analytical method for characterisation of Zika‐ and Chikungunya‐derivatised AV populations including vesicle size distribution and variations in antigen loading. Manufactured batches were compared to assess the extent and nature of batch‐to‐batch variations. We also show preliminary results that verify antigen expression on the surface of host cells. We present here a reliable and efficient high‐resolution 3D imaging regime that allows the evaluation of the microstructure and biochemistry of novel vaccine formulations such as AVs.

A guide into the world of high-resolution 3D imaging: the case of soft X-ray tomography for the life sciences

“…CryoSXT is a tool which is bridging the divide from research to product development and beyond. It's potentials span across cross-disciplinary areas from medicine to environmental monitoring for ecosystem sustainability [ 54 ].…”

“…Users can receive training on the use of instruments and software and are expected to collect their own data (remotely or in-person on site) and are highly encouraged to deposit it once published to public archives to allow unfettered access and opportunity for scrutiny to colleagues in the same or related fields [46]. The user base at beamline B24 are predominantly academics investigating areas of interest within the biomedical sciences and who target systems such as: pathogen-host interactions, immunity, cell death, cancer, vaccine and drug development as well as basic cellular biology mechanisms and response to environmental stressors [22,[47][48][49][50][51][52][53][54][55].…”

A combination of soft X-ray and laser light sources offer 3D high content information on the native state of the cellular environment

“…Concurrently, momentum will be maintained in our outreach and education efforts. We have published a case study showcasing the application and potential of our technology in the characterisation and assessment of new vaccine formulations [18], and we will work to expand our user base in this area as well as for other projects in basic and translational biomedical research. The beamline also has set plans for developing further functionality within the lifetime of Diamond I to include (a) Xray absorption near edge spectroscopy (XANES), which will enable the chemical identification of biologically relevant elements in the samples such as K, P and Ca; and (b) phase-contrast imaging at 2.5 keV.…”

On-Line Fluorescence Microscopy for Identification and Imaging of Apoptotic Cell with Synchrotron-Based Soft X-ray Tomography

“…Off-line fluorescence microscopies (FMs) are usually combined to identify types of organelles and cells [ 6 , 7 , 8 ]. With correlative off-line FM and cryo-SXT, researchers can easily obtain the state/function information and ultrastructure of the imaged cells [ 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ], but deformation and displacement usually occur due to its being ex-situ.…”