An antibody-free evaluation of an mRNA COVID-19 vaccine.

This manuscript describes the use of an analytical assay that combines transfection of mammalian cells and isotope dilution mass spectrometry (IDMS) for accurate quantification of antigen expression. Expired mRNA COVID-19 vaccine material was stored at 4 °C, room temperature (∼25 °C), and 56 °C over a period of 5 weeks. The same vaccine was also exposed to 5 freeze-thaw cycles. Every week, the spike protein antigenic expression in mammalian (BHK-21) cells was evaluated. Housekeeping proteins, ß-actin and GAPDH, were simultaneously quantified to account for the variation in cell counts that occurs during maintenance and growth of cell cultures. Data show that vaccine stored at elevated temperatures results in reduced spike protein expression. Also, maintaining the vaccine in ultracold conditions or exposing the vaccine to freeze-thaw cycles had less effect on the vaccine's ability to produce the antigen in mammalian cells. We describe the use of IDMS as an antibody-free means to accurately quantify expressed protein from mammalian cells transfected with mRNA vaccine.

Quantification of SARS-CoV-2 spike protein expression from mRNA vaccines using isotope dilution mass spectrometry.

The advent of mRNA vaccine technology has been vital in rapidly creating and manufacturing COVID-19 vaccines at an industrial scale. To continue to accelerate this leading vaccine technology, an accurate method is needed to quantify antigens produced by the transfection of cells with a mRNA vaccine product. This will allow monitoring of protein expression during mRNA vaccine development and provide information on how changes to vaccine components affects the expression of the desired antigen. Developing novel approaches that allow for high-throughput screening of vaccines to detect changes in antigen production in cell culture prior to in vivo studies could aid vaccine development. We have developed and optimized an isotope dilution mass spectrometry method to detect and quantify the spike protein expressed after transfection of baby hamster kidney cells with expired COVID-19 mRNA vaccines. Five peptides of the spike protein are simultaneously quantified and provide assurance that protein digestion in the region of the target peptides is complete since results between the five peptides had a relative standard deviation of less than 15 %. In addition, two housekeeping proteins, actin and GAPDH, are quantified in the same analytical run to account for any variation in cell growth within the experiment. IDMS allows a precise and accurate means to quantify protein expression by mammalian cells transfected with an mRNA vaccine.

Rapid Deorphanization of Human Olfactory Receptors in Yeast.

Olfactory receptors are ectopically expressed (exORs) in more than 16 different tissues. Studying the role of exORs is hindered by the lack of known ligands that activate these receptors. Of particular interest are exORs in the colon, the section of the gastrointestinal tract with the greatest diversity of microbiota where ORs may be participating in host-microbiome communication. Here, we leverage a G-protein-coupled receptor (GPCR)-based yeast sensor strain to generate sensors for seven ORs highly expressed in the colon. We screen the seven colon ORs against 57 chemicals likely to bind ORs in olfactory tissue. We successfully deorphanize two colon exORs for the first time, OR2T4 and OR10S1, and find alternative ligands for OR2A7. The same OR deorphanization workflow can be applied to the deorphanization of other ORs and GPCRs in general. Identification of ligands for OR2T4, OR10S1, and OR2A7 will enable the study of these ORs in the colon. Additionally, the colon OR-based sensors will enable the elucidation of endogenous colon metabolites that activate these receptors. Finally, deorphanization of OR2T4 and OR10S1 supports studies of the neuroscience of olfaction.