SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness.

A vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to control the coronavirus disease 2019 (COVID-19) global pandemic. Structural studies have led to the development of mutations that stabilize Betacoronavirus spike proteins in the prefusion state, improving their expression and increasing immunogenicity1. This principle has been applied to design mRNA-1273, an mRNA vaccine that encodes a SARS-CoV-2 spike protein that is stabilized in the prefusion conformation. Here we show that mRNA-1273 induces potent neutralizing antibody responses to both wild-type (D614) and D614G mutant2 SARS-CoV-2 as well as CD8+ T cell responses, and protects against SARS-CoV-2 infection in the lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a phase III trial to evaluate its efficacy.

SARS-CoV-2 mRNA Vaccine Development Enabled by Prototype Pathogen Preparedness.

A SARS-CoV-2 vaccine is needed to control the global COVID-19 public health crisis. Atomic-level structures directed the application of prefusion-stabilizing mutations that improved expression and immunogenicity of betacoronavirus spike proteins. Using this established immunogen design, the release of SARS-CoV-2 sequences triggered immediate rapid manufacturing of an mRNA vaccine expressing the prefusion-stabilized SARS-CoV-2 spike trimer (mRNA-1273). Here, we show that mRNA-1273 induces both potent neutralizing antibody and CD8 T cell responses and protects against SARS-CoV-2 infection in lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a Phase 2 clinical trial with a trajectory towards Phase 3 efficacy evaluation.

Establishment of higher passage PER.C6 cells for adenovirus manufacture.

PER.C6 cells, an industrially relevant cell line for adenovirus manufacture, were extensively passaged in serum-free suspension cell culture to better adapt them to process conditions. The changes in cell physiology that occurred during this passaging were characterized by investigating cell growth, cell size, metabolism, and cultivation of replication-deficient adenovirus. The changes in cell physiology occurred gradually as the population doubling level, the number of times the cell population had doubled, increased. Higher passage PER.C6 (HP PER.C6) proliferated at a specific growth rate of 0.043 h(-1), 2-fold faster than lower passage PER.C6, and were capable of proliferation from lower inoculation cell densities. HP PER.C6 cell volume was 16% greater, and cellular yields on glucose, lactate, oxygen, and amino acids were greater as well. In batch cultures, HP PER.C6 cells volumetrically produced 3-fold more adenovirus, confirmed with three different constructs. The increase in productivity was also seen on a cell-specific basis. Although HP PER.C6 were more sensitive to the "cell density effect", requiring lower infection cell densities for optimal specific productivity, they proliferated more after infection than lower passage PER.C6, increasing the number of cells available for virus production. The extensive passaging established HP PER.C6 cells with several desirable attributes for adenovirus manufacture.

Pilot-scale adenovirus seed production through concurrent virus release and concentration by hollow fiber filtration.

Recovery of recombinant adenoviruses from infected mammalian cell cultures often requires multiple unit operations such as cell lysis for virus release, microfiltration for clarification, and ultrafiltration for concentration. While development of these multiple unit operations is relatively straightforward, implementation under aseptic conditions in a closed system can be challenging for the production of virus seed at industrial scales. In this study, we have developed a simple, single-step, scaleable process to effectively recover adenoviruses from infected PER.C6 cell cultures for the production of concentrated adenovirus seeds under aseptic conditions. Specifically, hollow fiber tangential flow filtration technology was applied to maximize cell lysis of infected cultures for virus release while simultaneously concentrating the virus to an appropriate level of volume reduction. Hollow fiber filters with small lumen diameter of 0.5 mm were chosen to maximize the wall shear for a highly effective cell lysis and virus release. Cell lysis and virus release were shown to correlate with the exposure time in the hollow fiber cartridge: the shear zone. In most cases, a virus recovery yield of more than 80% and a 15- to 20-fold concentration (or up to 95% volume reduction) was achieved in less than 2 h of processing time. The virus seeds prepared using this process at lab scale and at 300-L scale without clarification have been successfully tested for sterility and potency and used for subsequent infection with consistent virus productivity. This process should enable rapid production of adenovirus seeds with minimal unit operations and high efficiency recovery for adenovirus production at 1000-L scale.