In a recent study posted to the bioRxiv* preprint server, researchers used various analytical characterization techniques to determine the biophysical attributes of the Imperial College London self-amplifying viral ribonucleic acid (RNA) vaccine (IMP-1) developed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Background

Since the onset of the coronavirus disease 2019 (COVID-19) pandemic in late 2019, vaccine technology, especially messenger RNA vaccine development, has advanced significantly in an attempt to mitigate the severity and transmission of SARS-CoV-2 infections.

Self-amplifying (sa) RNA vaccines are an improvement over mRNA vaccines, as the self-amplifying replicon allows the host cell machinery to make multiple copies of the target antigen RNA. While this provides the advantage of administering 10-to-100-fold lower vaccine doses, the self-amplifying code makes the vaccine bulkier than regular mRNA vaccines.

The advantages of RNA vaccines lie in the ease with which they can be designed, manufactured, or altered. The mRNA vaccine production process involves various steps, beginning with the in vitro transcription of the RNA molecule and progressing to purification and encapsulation in a lipid nanoparticle. Each step of the process involves various reagents and multiple sub-process, and the smallest error in any of those can result in an ineffective or incomplete vaccine.

Therefore, analytical characterization and quality control are essential to produce effective mRNA vaccines.

About the study

In the present study, the researchers used ultraviolet (UV) spectroscopy, biological small angle scattering (BioSAXS), dynamic light scattering (DLS), and circular dichroism (CD) to characterize the biophysical attributes of IMP-1. This saRNA vaccine comprises a genetic code for pre-fusion stabilized SARS-CoV-2 spike protein and a Venezuelan equine encephalitis virus (VEEV) replicase.

The IMP-1 mRNA was transcribed in vitro, purified, and concentrated using tangential flow filtration and chromatography. The purity and concentration of the IMP-1 mRNA were assessed using the A_260/280 UV spectrometry assay. The ratio between 260 nm and 280 nm absorbances is used to determine the purity of the RNA, and ratios lower than two indicate protein contamination.

The researchers then performed DLS, a solution-based light scattering technique to determine the polydispersity and size of the RNA molecules in sodium citrate or sodium phosphate buffer. IMP-1 RNA in sodium citrate was also used in the BioSAXS experiments to determine the size and shape of the RNA. BioSAXS is a high-throughput process requiring very low amounts of purified samples and can be performed using offline home X-ray or synchrotron facilities.

Lastly, CD was performed on RNA in a sodium phosphate buffer and water to understand the structure and chirality or conformation of the RNA molecule. The CD spectra were measured using a spectrophotometer using a 320 nm to 180 nm wavelength range.

Results

The results indicated that the IMP-1 RNA molecule comprises 11,551 base pairs and weighs 3.71 MDa. The UV spectroscopy A_260/280 ratio was 2.18, indicating that the RNA contained no protein contaminants.

The DLS and BioSAXS experiments determined the RNA molecule diameter to be 873.63 Å. The DLS method was performed on two different buffers, and the Z-average diameter and polydispersity index (PDI) for the RNA were slightly but significantly different for both buffers, indicating that buffer type, ionic strength, and pH influence the RNA size.

The CD spectra, which reveal the structural and conformational features of the RNA molecule, indicated a right-hand A-form RNA helix and stacking interactions in the IMP-1 RNA molecule. The spectra from the measurements using water revealed a smaller helical signal, indicating that, as with DLS, the CD results are dependent on the buffer. As an analytical technique, CD is highly useful for observing the effect of buffer, pH, temperature, denaturants, and salts on the structure and conformation of RNA.

The authors also discussed the challenges in delivering the negatively charged mRNA molecules and using lipid nanoparticle encapsulation to stabilize and effectively deliver the vaccine.

Conclusions

To summarize, the study explored various analytical characterization techniques to study the purity, size, polydispersity, structure, and conformation of the IMP-1 mRNA molecule transcribed from the plasmid carrying the genetic code for SARS-CoV-2 spike protein and a VEEV replicase.

The UV spectrometry, DLS, BioSAXS, and CD techniques explored in the study provided valuable information about the concentration, size, shape, and structure of the RNA molecule. The study also brought to light the effects of buffer, pH, and ionic strength on the structure of the RNA. The authors believe that additional techniques such as differential scanning calorimetry and small angle neutron scattering can better understand the thermodynamics, stability, and structure of the RNA molecule at relevant physiological temperatures.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Dr. Chinta Sidharthan

Chinta Sidharthan is a writer based in Bangalore, India. Her academic background is in evolutionary biology and genetics, and she has extensive experience in scientific research, teaching, science writing, and herpetology. Chinta holds a Ph.D. in evolutionary biology from the Indian Institute of Science and is passionate about science education, writing, animals, wildlife, and conservation. For her doctoral research, she explored the origins and diversification of blindsnakes in India, as a part of which she did extensive fieldwork in the jungles of southern India. She has received the Canadian Governor General’s bronze medal and Bangalore University gold medal for academic excellence and published her research in high-impact journals.