Hierarchical sequence-affinity landscapes shape the evolution of breadth in an anti-influenza receptor binding site antibody
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eLife assessment
In this valuable study, authors convincingly show that epistasis between mutations plays an important role in the evolution of broadly neutralizing influenza antibodies. Although the data are convincing, several parts of the manuscript require more accurate description.
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Abstract
Broadly neutralizing antibodies (bnAbs) that neutralize diverse variants of a particular virus are of considerable therapeutic interest. Recent advances have enabled us to isolate and engineer these antibodies as therapeutics, but eliciting them through vaccination remains challenging, in part due to our limited understanding of how antibodies evolve breadth. Here, we analyze the landscape by which an anti-influenza receptor binding site (RBS) bnAb, CH65, evolved broad affinity to diverse H1 influenza strains. We do this by generating an antibody library of all possible evolutionary intermediates between the unmutated common ancestor (UCA) and the affinity-matured CH65 antibody and measure the affinity of each intermediate to three distinct H1 antigens. We find that affinity to each antigen requires a specific set of mutations – distributed across the variable light and heavy chains – that interact non-additively (i.e., epistatically). These sets of mutations form a hierarchical pattern across the antigens, with increasingly divergent antigens requiring additional epistatic mutations beyond those required to bind less divergent antigens. We investigate the underlying biochemical and structural basis for these hierarchical sets of epistatic mutations and find that epistasis between heavy chain mutations and a mutation in the light chain at the V H -V L interface is essential for binding a divergent H1. Collectively, this is the first work to comprehensively characterize epistasis between heavy and light chain mutations and shows that such interactions are both strong and widespread. Together with our previous study analyzing a different class of anti-influenza antibodies, our results implicate epistasis as a general feature of antibody sequence-affinity landscapes that can potentiate and constrain the evolution of breadth.
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Author Response
Reviewer #1 (Public Review):
Understanding the evolution of broadly neutralizing influenza antibodies is key to developing a more universal vaccine. In this study, Phillips et al. performed a comprehensive analysis of the evolutionary pathway of CH65, which is an H1-specific broadly neutralizing antibody. The authors generated a combinatorial mutant library with 2^16 members that contained all possible evolutionary intermediates between the unmutated common ancestor (UCA) and CH65, less two mutations that did not affect binding. The binding affinity of each member in the library was measured against HAs from MA90 and SI06, which were isolated 16 years apart, as well as MA90 with a UCA escape mutation G189E. The binding affinity was measured using a high-throughput approach that combined yeast display and Tite-Seq, …
Author Response
Reviewer #1 (Public Review):
Understanding the evolution of broadly neutralizing influenza antibodies is key to developing a more universal vaccine. In this study, Phillips et al. performed a comprehensive analysis of the evolutionary pathway of CH65, which is an H1-specific broadly neutralizing antibody. The authors generated a combinatorial mutant library with 2^16 members that contained all possible evolutionary intermediates between the unmutated common ancestor (UCA) and CH65, less two mutations that did not affect binding. The binding affinity of each member in the library was measured against HAs from MA90 and SI06, which were isolated 16 years apart, as well as MA90 with a UCA escape mutation G189E. The binding affinity was measured using a high-throughput approach that combined yeast display and Tite-Seq, with careful experimental validation. The results showed that epistasis between mutations within the heavy chain and also across heavy and light chains plays an important role in CH65 to evolve breadth. Although this study highly resembles a previous study by the authors that focused on another broadly neutralizing influenza antibody called CR9114 (Phillips et al., eLife 2021), there are several key differences. Firstly, CR9114 is a HA stem-directed antibody, whereas CH65 binds to the receptor-binding site of HA. Secondly, their previous study only studied the mutations in the heavy chain, whereas the present study looked at mutations in both heavy and light chains. Lastly, the present study provided a structural mechanism of epistasis by solving crystal structures. Such investigation of structural mechanisms was absent in their previous study. Overall, the data quality in this study is very high. In addition, the results have important implications for vaccine development.
We thank Reviewer #1 for their review of our work and have implemented each of their suggestions to improve the clarity of our manuscript.
Reviewer #2 (Public Review):
Although many broadly-neutralizing antibodies were discovered against virus accumulating mutations such as HIV, Influenza, and Sars-CoV-2, the methodology to induce such antibodies or design to generate them is highly demanded. The authors take the broadly-neutralizing antibody, CH65 as a model antibody and try to recapitulate the generation of the broadly-neutralizing antibody from an unmutated common ancestor over time. By performing Tite-Seq assays, Epistasis analysis, Pathway analysis, and Affinity measurement, and structural study, the authors proposed a scenario of the evolution of CH65.
Strengths
Combining the models and affinity/structure data, the authors enable us to show the possible track of gaining the breadth of the CH65 antibody from the unmutated repertoire. Using the Tite-Seq assay, the authors took a forward genetics approach which is high-throughput and non-bias and mimics the situation of the evolution of a B cell repertoire in an individual over time. The data is robust, and its outcome will provide an opportunity to build a prediction model to design the antibody in silico. Especially their identification of amino acid positions important for epistasis mode in antibody evolution is valuable. Antigen selection scenarios are decisive in this study.
Weakness
The proposed scenarios cannot be tested using human CH65. The readers would have great interest in how these hypothetical scenarios are fitting to the evolution occurring in vivo situation, especially in a quantitative way. The broadly neutralizing antibodies often react with self-antigens as the authors cite previous work(ref 19). How do these environmental factors affect the evolution of the antibody? These already-known facts could be mentioned and discussed in detail.
We thank Reviewer #2 for these comments and agree that applying these insights to understand in vivo antibody affinity maturation would be fascinating. As the Reviewer points out, our study is limited to examining antigen affinity and neglects other properties that are known to impact antibody affinity maturation (e.g., autoreactivity). As we mention in the Discussion, our work shows how the acquisition of breadth is shaped by mutations that interact epistatically to determine binding affinity, and future work is required to understand how these mutations and interactions may also impact the myriad other properties relevant to antibody maturation.
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eLife assessment
In this valuable study, authors convincingly show that epistasis between mutations plays an important role in the evolution of broadly neutralizing influenza antibodies. Although the data are convincing, several parts of the manuscript require more accurate description.
-
Reviewer #1 (Public Review):
Understanding the evolution of broadly neutralizing influenza antibodies is key to developing a more universal vaccine. In this study, Phillips et al. performed a comprehensive analysis of the evolutionary pathway of CH65, which is an H1-specific broadly neutralizing antibody. The authors generated a combinatorial mutant library with 2^16 members that contained all possible evolutionary intermediates between the unmutated common ancestor (UCA) and CH65, less two mutations that did not affect binding. The binding affinity of each member in the library was measured against HAs from MA90 and SI06, which were isolated 16 years apart, as well as MA90 with a UCA escape mutation G189E. The binding affinity was measured using a high-throughput approach that combined yeast display and Tite-Seq, with careful …
Reviewer #1 (Public Review):
Understanding the evolution of broadly neutralizing influenza antibodies is key to developing a more universal vaccine. In this study, Phillips et al. performed a comprehensive analysis of the evolutionary pathway of CH65, which is an H1-specific broadly neutralizing antibody. The authors generated a combinatorial mutant library with 2^16 members that contained all possible evolutionary intermediates between the unmutated common ancestor (UCA) and CH65, less two mutations that did not affect binding. The binding affinity of each member in the library was measured against HAs from MA90 and SI06, which were isolated 16 years apart, as well as MA90 with a UCA escape mutation G189E. The binding affinity was measured using a high-throughput approach that combined yeast display and Tite-Seq, with careful experimental validation. The results showed that epistasis between mutations within the heavy chain and also across heavy and light chains plays an important role in CH65 to evolve breadth. Although this study highly resembles a previous study by the authors that focused on another broadly neutralizing influenza antibody called CR9114 (Phillips et al., eLife 2021), there are several key differences. Firstly, CR9114 is a HA stem-directed antibody, whereas CH65 binds to the receptor-binding site of HA. Secondly, their previous study only studied the mutations in the heavy chain, whereas the present study looked at mutations in both heavy and light chains. Lastly, the present study provided a structural mechanism of epistasis by solving crystal structures. Such investigation of structural mechanisms was absent in their previous study. Overall, the data quality in this study is very high. In addition, the results have important implications for vaccine development.
-
Reviewer #2 (Public Review):
Although many broadly-neutralizing antibodies were discovered against virus accumulating mutations such as HIV, Influenza, and Sars-CoV-2, the methodology to induce such antibodies or design to generate them is highly demanded. The authors take the broadly-neutralizing antibody, CH65 as a model antibody and try to recapitulate the generation of the broadly-neutralizing antibody from an unmutated common ancestor over time. By performing Tite-Seq assays, Epistasis analysis, Pathway analysis, and Affinity measurement, and structural study, the authors proposed a scenario of the evolution of CH65.
Strengths
Combining the models and affinity/structure data, the authors enable us to show the possible track of gaining the breadth of the CH65 antibody from the unmutated repertoire. Using the Tite-Seq assay, the …Reviewer #2 (Public Review):
Although many broadly-neutralizing antibodies were discovered against virus accumulating mutations such as HIV, Influenza, and Sars-CoV-2, the methodology to induce such antibodies or design to generate them is highly demanded. The authors take the broadly-neutralizing antibody, CH65 as a model antibody and try to recapitulate the generation of the broadly-neutralizing antibody from an unmutated common ancestor over time. By performing Tite-Seq assays, Epistasis analysis, Pathway analysis, and Affinity measurement, and structural study, the authors proposed a scenario of the evolution of CH65.
Strengths
Combining the models and affinity/structure data, the authors enable us to show the possible track of gaining the breadth of the CH65 antibody from the unmutated repertoire. Using the Tite-Seq assay, the authors took a forward genetics approach which is high-throughput and non-bias and mimics the situation of the evolution of a B cell repertoire in an individual over time. The data is robust, and its outcome will provide an opportunity to build a prediction model to design the antibody in silico. Especially their identification of amino acid positions important for epistasis mode in antibody evolution is valuable. Antigen selection scenarios are decisive in this study.Weakness
The proposed scenarios cannot be tested using human CH65. The readers would have great interest in how these hypothetical scenarios are fitting to the evolution occurring in vivo situation, especially in a quantitative way. The broadly neutralizing antibodies often react with self-antigens as the authors cite previous work(ref 19). How do these environmental factors affect the evolution of the antibody? These already-known facts could be mentioned and discussed in detail. -
