A pseudovirus system to fight viral antibody escape

From the Bloom Lab, Basic Sciences and Public Health Sciences Divisions

SARS-CoV-2 has evolved to acquire mutations in the spike protein - the part of the virus that protrudes from its surface and latches onto cells to infect them - that enhance the virus' ability to infect human cells or evade antibodies. A recent study led by Dr. Bernadeta Dadonaite, a staff scientist in the lab of Dr. Jesse Bloom, a professor in Fred Hutch’s Basic Science Division, created a novel deep mutational scanning (DMS) platform to map the mutations within the entire spike protein that affect viral entry and antibody neutralization. “This work provides a powerful and safe way to understand the effects of mutations to viral proteins,” Dr. Bloom said. “It will prove very useful for studying a range of important viruses that pose a hazard to human health,” he added. Their results from this study were recently published in Cell.

The DMS platform allows permutation of every amino acid in the spike protein and subsequent testing of viral entry and antibody neutralization. This was achieved by creating libraries of spike-pseudotyped lentiviruses. Lentiviral pseudotyping is a method that allows to produce non-replicative viruses that display foreign viral entry proteins, such as spike, on their surface. Using pseudotyping the authors produced DMS libraries for Delta and Omicron (BA.1) variants where each library contained thousands of different mutated spike sequences

“We developed a novel deep mutational scanning platform that can be used to understand the phenotypic effects of mutations in viral entry proteins,” Dr. Dadonaite said. Using this platform, “we showed how we can investigate the functional and antigenic effect of mutations throughout the spike protein,” she added. This platform will allow them to “better understand the effects of mutations in currently circulating and future SARS-CoV-2 variants.”

Deep mutational scanning workflow. Pseudovirus libraries that express a specific spike protein variant are made. These libraries are used to test antibody neutralization and spike-mediated infection.
Deep mutational scanning workflow. Pseudovirus libraries that express a specific spike protein variant are made. These libraries are used to test antibody neutralization and spike-mediated infection. Image provided by Bernadeta Dadonaite

Once these libraries were successfully established, the authors measured the effects of mutations in spike on virus infection and antibody neutralization. As a proof of principle, the authors demonstrated the ability of this platform to map escape from antibodies targeting different parts of spike. The antibodies mapped in the study included those targeting spike’s receptor-binding domain, N-terminal domain and S2 stem-helix. Antibody mapping experiments demonstrated how DMS can be used to preemptively predict the effects of mutations in spike on antibodies in clinical development as well as to explain the features that allow antibodies to broadly neutralize distinct coronavirus families. Notably, the DMS platform could also measure change in IC50 values (a measure of the potency of antibody neutralization) caused by escape mutations, which correlated well with the IC50 measured by traditional pseudovirus neutralization assay.

Lastly, the authors measured how well each mutation in spike mediates viral infection. Although the authors found most mutations were deleterious or neutral for spike-mediated pseudovirus infection, some mutations were predicted to increase spike-mediated pseudovirus infection by DMS. Combining data on the functional effects of mutation in spike with effects on antibody escape can be used to assess which mutations are tolerable and could potentially arise naturally.

Now, "we will aim to use our deep mutational scanning libraries in a polyclonal context, such as performing experiments with sera from previously vaccinated or infected individuals," said Dr. Dadonaite. "Such experiments could help inform the design of variant-proof vaccines and therapeutic monoclonal antibodies," she concluded.

The spotlighted research was funded by the National Institutes of Health, the Bill and Melinda Gates Foundation, the Howard Hughes Medical Institute (HHMI), the European Molecular Biology Organization (EMBO), the Viral Pathogenesis and Evolution training grant, the Cell and Molecular Biology (CMB) Training grant, and the National Science Foundation graduate research fellowship. 

Dadonaite B, Crawford KHD, Radford CE, Farrell AG, Yu TC, Hannon WW, Zhou P, Andrabi R, Burton DR, Liu L, Ho DD, Neher RA, Bloom JD. A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike. Cell. 2022 Oct 13:2022.10.13.512056. doi: 10.1101/2022.10.13.512056.