As we approach the five-year anniversary of the COVID-19 pandemic, it is worth reflecting not only on the paradigm-shifting impact of the initial outbreak, but also on how SARS-CoV-2 continues to evolve. Much of this evolution centers on the virus’s spike protein—the molecular key that allows the virus to enter human cells and the primary target of the immune response generated by infection and vaccination. The spike protein binds to the ACE2 receptor on the cell surface, a process that depends on subtle structural movements that expose or conceal the receptor-binding domain. Because spike sits at the intersection of viral infectivity and immune recognition, even small changes can have outsized effects on how efficiently the virus spreads and how well it evades existing immunity.
Understanding how viruses evolve under immune pressure requires tools that can keep pace with viral evolution. Dr. Jesse Bloom’s research group in the Basic Sciences Division specializes in developing high-throughput experimental approaches to map how mutations alter viral proteins, particularly in the context of antibody recognition and viral fitness. Over the past several years, the Bloom lab has applied these methods to SARS-CoV-2 to systematically chart how spike mutations affect infectivity and immune escape, generating datasets that have helped interpret emerging variants and anticipate evolutionary trends.
To systematically test how spike mutations influence viral behavior, the Bloom lab’s newest study in the Journal of Virology used a technique called pseudovirus-based deep mutational scanning. In this approach, they generate large libraries of harmless, single-round–infecting viral particles that each carry a slightly different version of the SARS-CoV-2 spike protein, tagged with a unique genetic barcode. These pseudoviruses can enter cells but cannot replicate or spread, allowing thousands of spike variants to be tested safely. By tracking how each barcoded variant performs across different assays—such as cell entry, ACE2 binding, or antibody neutralization—the researchers can directly link specific mutations to functional outcomes.
The study centered on a spike sequence—KP.3.1.1—which represents recently circulating SARS-CoV-2 variant, and was specifically chosen because it reflects many of the antigenic changes now common across contemporary lineages and closely resembles spike proteins used in current vaccine formulations. The mutation libraries were designed to emphasize changes most relevant to ongoing viral evolution, capturing both mutations already observed in circulating viruses and changes at sites known to strongly influence antibody recognition and spike conformation.
The analysis showed that different classes of mutations have distinct effects on spike function: mutating stop codons strongly impaired entry, the effects of amino acid substitutions ranged from highly deleterious to well tolerated depending on the site, and mutations resulting in short deletions—particularly in the N-terminal domain—were often compatible with efficient cell entry, mirroring patterns seen in circulating variants. Their results also highlight that ACE2 binding and cell entry are related but distinct properties of spike, with many mutations—especially those near the base of the receptor-binding domain (RBD), the part of spike that directly engages the ACE2 receptor on human cells—altering receptor engagement by changing how often the RBD adopts an accessible conformation.
A key insight emerged around the receptor-binding domain’s dynamic positioning. Mutations that push the RBD into an “up” conformation increase ACE2 binding but also expose vulnerable sites targeted by neutralizing antibodies. Conversely, mutations favoring the “down” conformation help the virus evade antibodies but reduce receptor engagement. This evolutionary balancing act was reflected in strong correlations between mutation effects on ACE2 binding and serum neutralization escape —the ability of spike variants to resist neutralization by antibodies generated through prior infection or vaccination—underscoring conformational changes as a central mechanism shaping spike evolution.
As associate researcher Bernadeta Dadonaite explains, “We show that after infection or vaccination with recent SARS-CoV-2 variants there is a shift in neutralizing epitope immunodominance. Notably, new circulating SARS-CoV-2 variants already carry mutations at some of these dominant epitopes illustrating how population immunity is driving SARS-CoV-2 evolution.”
The team further tested how spike mutations influence neutralization by three monoclonal antibodies currently used clinically for COVID-19 prevention and treatment: BD55-1205, SA55, and VYD222. They found that these antibodies are sensitive not only to mutations within their direct binding sites but also to mutations at distant positions that alter RBD conformation, thereby modulating epitope accessibility. This finding highlights the ongoing challenge posed by SARS-CoV-2’s mutational flexibility and the importance of continuous surveillance to maintain antibody efficacy.
Dadonaite highlights the broader implications: “This paper illustrates how the motion of the receptor binding domain (RBD) in SARS-CoV-2 spike is an important mechanism of evasion from neutralizing antibodies targeting spike. We think SARS-CoV-2 spike might be evolving to have its RBD in a more closed conformation to evade neutralizing antibodies and we would like to explore this aspect of immune evasion further. Previous research has also shown that changes in the RBD motion can facilitate cross-species transfer of coronaviruses in non-human hosts, further highlighting the importance of understanding this spike feature for pandemic preparedness.”
Together, these comprehensive maps of spike mutation effects illuminate the evolutionary pressures and functional constraints shaping SARS-CoV-2’s ongoing adaptation. By revealing how conformational changes influence viral entry, receptor binding, and immune escape, this work provides critical insights for anticipating future variant trajectories and improving vaccines and antibody therapies.
The spotlighted research was funded by National Institutes of Health, the Washington Research Foundation, and the National Science Foundation.
Dadonaite B, Harari S, Larsen BB, Kampman L, Harteloo A, Elias-Warren A, Chu HY, Bloom JD. 2025. Spike mutations that affect the function and antigenicity of recent KP.3.1.1-like SARS-CoV-2 variants. Journal of Virology. doi: 10.1128/jvi.01423-25.
Science Spotlight writer Jenny Waters is a postdoctoral research fellow in the Hsieh lab at Fred Hutch. She studies how mRNA translation coordinates bladder cancer transformation and metastasis by post-transcriptionally regulating expression of oncogenic proteins. Outside of the lab, Jenny enjoys spending time with her dogs, convincing her husband to join her on trail runs, and pretending every steep hill is just a "gentle incline."
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