Viruses are remarkable in their ability to disguise themselves and evade the immune system. When the body’s immune defenses recognize and attack them, viruses can adapt quickly by mutating, leading to new variants that the immune system no longer recognizes. This constant evolutionary battle between viruses and our immune system presents a significant challenge for vaccine development. In response to this, researchers have developed innovative methods to identify the mutations that allow viruses to escape immune detection, which is essential for keeping vaccines effective in the face of new variants.
A promising new method, reverse mutational scanning, has been developed by researchers at the Helmholtz Centre for Infection Research (HZI), in collaboration with the Hannover Medical School (MHH). This method allows scientists to quickly and accurately identify the specific mutations in a virus that enable it to evade immune detection. The research was recently published in the journal Nature Communications.
Viruses and Their Immune Evasion Tactics
Viruses are masters at adapting to their environment, and they do so through genetic mutations. These mutations can alter the virus’s structure, allowing it to evade detection by the immune system. This process can occur rapidly, particularly when the virus faces pressure from immune responses, such as those triggered by vaccination or previous infection.
A virus with mutations that help it evade immune recognition has a survival advantage. It is more likely to thrive and spread, eventually becoming the dominant strain. This dynamic has been particularly evident with the SARS-CoV-2 virus, which has mutated several times, leading to the emergence of new variants that challenge the effectiveness of vaccines and treatments.
Prof. Luka Cicin-Sain, head of the Viral Immunology department at HZI, explains the impact of immune escape: “If the new virus variant can successfully escape the immune response, it is no longer sufficient to have recovered from one of the previous variants or to have been vaccinated with a previously effective vaccine.” This highlights the need for vaccines to be adaptable and responsive to the ongoing evolution of the virus.
To meet this challenge, scientists must identify the mutations responsible for immune escape so that vaccines can be updated to target the new variants effectively. This is where the new method of reverse mutational scanning comes into play.
The Need for Rapid Identification of Immune Escape Mutations
When a virus mutates, it does so by altering specific parts of its structure. These changes can affect the way the virus interacts with immune cells and antibodies. If these mutations occur in parts of the virus that are recognized by the immune system, they may prevent the antibodies from binding effectively and neutralizing the virus. However, not all mutations necessarily contribute to immune escape. Therefore, it’s crucial to distinguish which specific mutations enable the virus to evade immune recognition.
Traditionally, researchers have used a method called mutational scanning to study how different mutations affect the virus’s ability to escape immune detection. In this process, each mutation found in a new variant is introduced into the original virus, creating a library of mutants. By comparing these mutants to the original virus and to the new variant, scientists can identify which mutations have the most significant impact on immune escape.
However, the approach used in the recent study by the HZI researchers is slightly different. Instead of starting with the original virus and introducing mutations one by one, the team reversed the process. They started with the new immune-evading variant and systematically reintroduced mutations that made it resemble the original strain. This allowed them to pinpoint which specific mutations in the new variant were responsible for immune escape.
Reverse Mutational Scanning: A Breakthrough Method
The process behind reverse mutational scanning is both novel and highly effective. The researchers chose the SARS-CoV-2 BA.2.86 variant as a case study. This variant had mutations that allowed it to evade immunity, but the specific mutations responsible for immune escape were not yet fully understood. The team’s goal was to determine which of the 33 mutations in the spike protein of BA.2.86 were key to immune escape.
To accomplish this, the researchers used pseudoviruses—modified viruses that carry the spike protein of SARS-CoV-2 but cannot replicate. These pseudoviruses are harmless and provide a safe way to study the virus’s interactions with the immune system. By creating pseudoviruses that contained the 33 mutations of BA.2.86, and then systematically reversing each mutation, the researchers could assess how the immune system responded to each variant.
“We created various pseudoviruses in which one of the 33 different mutations was reversed, in the direction of the original virus BA.2,” explains Dr. Najat Bdeir, first author of the study. The researchers then tested these pseudoviruses using immune cells from 40 healthcare workers who had been vaccinated multiple times, including with the vaccine effective against the Omicron XBB.1.5 variant.
Why “Reverse” Mutational Scanning Works
The key advantage of reverse mutational scanning lies in the order of operations. As Prof. Cicin-Sain explains, starting with the new variant and reversing the mutations is critical because the immune system is highly complex and diverse. The immune system produces antibodies that target different regions of the virus, meaning that if a mutation occurs in one region, other parts of the virus may still be recognized and neutralized by antibodies. By starting with the immune-evading variant and reversing mutations, researchers can more accurately determine which mutations are most responsible for immune escape.
“If we start from the original variant and insert a mutation in an area that is actually recognized by antibodies, there is a high probability that antibodies binding to another part of the virus will still recognize and neutralize the virus. The actual contribution of the mutation to immune escape cannot be adequately detected in this way,” says Cicin-Sain. “So we have to start from the new variant and work backwards from there—backwards takes us forwards here.”
Identifying Key Mutations in SARS-CoV-2 Variants
Using reverse mutational scanning, the researchers were able to identify three key mutations in the spike protein of BA.2.86 that were responsible for immune escape. These mutations allowed the virus to evade the immune response generated by previous infections or vaccinations. The findings underscore the importance of understanding how new variants evolve and how their mutations impact immune recognition.
This method of identifying immune-evading mutations is not only applicable to SARS-CoV-2 but could be used to study other viruses and their variants as well. The researchers believe that reverse mutational scanning could play a vital role in vaccine development, helping to accelerate the adaptation of vaccines to new viral variants as they emerge.
Implications for Future Vaccine Development
The implications of this study are far-reaching. As Cicin-Sain notes, the goal is to outsmart the viruses and reduce their advantages, not just for current variants but also for future pandemics. The ability to quickly identify mutations responsible for immune escape means that vaccines can be adapted much more rapidly, potentially staying ahead of the virus’s evolution.
One promising future direction is the use of machine learning and artificial intelligence (AI) to predict which mutations could lead to immune escape. By training AI models on data generated through reverse mutational scanning, researchers could gain insights into potential future variants and prepare vaccines in advance. “If we could produce vaccines based on pre-adapted vaccines, we would be faster than the virus,” says Cicin-Sain.
Collaborative Effort and Future Directions
This study was a collaborative effort involving researchers from the Viral Immunology Department at HZI, the Structure and Function of Proteins Department at HZI, and several other institutes, including the German Primate Center and the German Center for Infection Research (DZIF). The success of this method highlights the importance of interdisciplinary collaboration in advancing scientific knowledge and tackling complex challenges like viral immune escape.
Looking ahead, reverse mutational scanning has the potential to be a game-changer in the field of infectious disease research. It can help scientists identify key mutations in real-time, enabling faster vaccine development and more effective interventions. Moreover, this approach could be applied to a wide range of viruses, from influenza to HIV and beyond, offering a powerful tool in the fight against infectious diseases.
Conclusion
Viruses are incredibly adept at evolving to evade the immune system, and new variants that escape immune detection pose significant challenges for public health. However, the development of reverse mutational scanning provides a promising solution, enabling researchers to identify the mutations responsible for immune escape in a timely and reliable manner. This new method could accelerate vaccine development and help prepare for future pandemics, ensuring that vaccines remain effective against emerging viral threats.
By understanding and outsmarting viral evolution, we take an important step toward protecting public health and staying ahead of the curve in the ongoing battle against infectious diseases.
Reference: Najat Bdeir et al, Reverse mutational scanning of SARS-CoV-2 spike BA.2.86 identifies epitopes contributing to immune escape from polyclonal sera, Nature Communications (2025). DOI: 10.1038/s41467-025-55871-5