Understanding the Spike Protein in COVID-19 Vaccine in Recombinant Vesicular Stomatitis Virus (rVSV) Using Automated Capillary Western Blots

Abstract

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.

Enabling functionality and translation fidelity characterization of mRNA-based vaccines with a platform-based, antibody-free mass spectrometry detection approach

“…24 Among these methods, we selected automated Western blotting by Simple Western™ (SW) due to its relatively high throughput, automation, reproducibility, and availability of antibodies for antigen detection. [25][26][27] However, the CFT-SW approach still relies on suitable antibodies for antigen detection, which can pose challenges including increased assay development time and cost for antibody screening, high detection limits and variability when using poor quality antibodies, the need for alternative detection assays if suitable antibodies cannot be found, and limited ability to multiplex detection for analysis of mRNA mixtures due to antibody crossreactivity. Mass spectrometry, on the other hand, is a highly sensitive technique that lends itself well to a platform-based approach for protein detection and relative quantification, alleviating the need to perform antibody screening and re-development for each mRNA construct or antigen target.…”

Adaptation of an rVSV Ebola vaccine purification process for rapid development of a viral vaccine candidate for SARS‐CoV‐2

“…As previously described in Konstantinidis et al., [ 22 ] Rustandi et al., [ 23 ] and Gillespie et al. [ 24 ] Wes, Jess, Sally Sue, and/or Peggy Sue systems (Protein Simple, CA) and the appropriate size‐based assay test kits were used.…”

“…Process intermediate samples were tested for both viral protein and product impurities via Simple Western (SW), quantitative western blot analysis. As previously described in Konstantinidis et al, [22] Rustandi et al, [23] and Gillespie et al [24] Wes, Jess, Sally Sue, and/or Peggy Sue systems (Protein Simple, CA) and the appropriate size-based assay test kits were used.…”

Challenges and Opportunities in the Process Development of Chimeric Vaccines

“…In addition, a novel, simple western technology that represents a fast and automated capillary-based western assay has been developed. The technique has been successfully utilized for at-line qualitative and quantitative assessment of SARS-CoV-2 spike (S) protein in the V590 vaccine candidate production against COVID-19 [ 186 ]. In another study, in-station real-time cell analysis assisted by dimensionless cell index has proven to be a robust alternative method to count real-time chimeric yellow fever dengue virus titers [ 187 ].…”