High-titer manufacturing of SARS-CoV-2 spike-pseudotyped VSV in stirred-tank bioreactors

Abstract

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic highlighted the importance of vaccine innovation in public health. Hundreds of vaccines built on numerous technology platforms were rapidly developed against SARS-CoV-2 since 2020. Like all vaccine platforms, an important bottleneck to viral-vectored vaccine development is manufacturing. Here, we describe a scalable manufacturing protocol for replication-competent SARS-CoV-2 Spike-pseudotyped Vesicular Stomatitis Virus (S-VSV)-vectored vaccines using Vero cells grown on microcarriers in a stirred-tank bioreactor. Using Cytodex 1 microcarriers over 6 days of fed-batch culture, Vero cells grew to a density of 3.95 + 0.42 ×10⁶ cells/mL in 1 L stirred-tank bioreactors. Ancestral strain S-VSV reached a peak titer of 2.05 + 0.58 ×10⁸ plaque-forming units (pfu)/mL at 3 days post-infection. When compared to growth in plate-based cultures this was a 29-fold increase in virus production, meaning a 1 L bioreactor produces the same amount of virus as 1284 15cm plates. Additionally, the omicron BA.1 S-VSV reached a peak titer of 5.58 + 0.35 ×10⁶ pfu/mL. Quality control testing showed plate- and bioreactor-produced S-VSV had similar particle-to-pfu ratios and elicited comparable levels of neutralizing antibodies in immunized hamsters. This method should enhance preclinical and clinical development of pseudotyped VSV-vectored vaccines in future pandemics.