Messenger RNA (mRNA) vaccines have emerged as a flexible platform for vaccine development. The evolution of lipid nanoparticles as effective delivery vehicles for modified mRNA encoding vaccine antigens was demonstrated by the response to the COVID-19 pandemic. The ability to rapidly develop effective SARS-CoV-2 vaccines from the spike protein genome, and to then manufacture multibillions of doses per year was an extraordinary achievement and a vaccine milestone. Further development and application of this platform for additional pathogens is clearly of interest. This comes with the associated need for new analytical tools that can accurately predict the performance of these mRNA vaccine candidates and tie them to an immune response expected in humans. Described here is the development and characterization of an imaging based in vitro assay able to quantitate transgene protein expression efficiency, with utility to measure lipid nanoparticles (LNP)-encapsulated mRNA vaccine potency, efficacy, and stability. Multiple biologically relevant adherent cell lines were screened to identify a suitable cell substrate capable of providing a wide dose–response curve and dynamic range. Biologically relevant assay attributes were examined and optimized, including cell monolayer morphology, antigen expression kinetics, and assay sensitivity to LNP properties, such as polyethylene glycol-lipid (or PEG–lipid) composition, mRNA mass, and LNP size. Collectively, this study presents a strategy to quickly optimize and develop a robust cell-based potency assay for the development of future mRNA-based vaccines.
Severe
acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
is
the viral agent that is responsible for the coronavirus disease-2019
(COVID-19) pandemic. One of the live virus vaccine candidates Merck
and Co., Inc. was developing to help combat the pandemic was V590.
V590 was a live-attenuated, replication-competent, recombinant vesicular
stomatitis virus (rVSV) in which the envelope VSV glycoprotein (G
protein) gene was replaced with the gene for the SARS-CoV-2 spike
protein (S protein), the protein responsible for viral binding and
fusion to the cell membrane. To assist with product and process development,
a quantitative Simple Western (SW) assay was successfully developed
and phase-appropriately qualified to quantitate the concentration
of S protein expressed in V590 samples. A strong correlation was established
between potency and S-protein concentration, which suggested that
the S-protein SW assay could be used as a proxy for virus productivity
optimization with faster data turnaround time (3 h vs 3 days). In
addition, unlike potency, the SW assay was able to provide a qualitative
profile assessment of the forms of S protein (S protein, S1 subunit,
and S multimer) to ensure appropriate levels of S protein were maintained
throughout process and product development. Finally, V590 stressed
stability studies suggested that time and temperature contributed
to the instability of S protein demonstrated by cleavage into its
subunits, S1 and S2, and aggregation into S multimer. Both of which
could potentially have a deleterious effect on the vaccine immunogenicity.
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