High-resolution mapping of the neutralizing and binding specificities of polyclonal rabbit serum elicited by HIV Env trimer immunization
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Abstract
Mapping the epitope specificities of polyclonal serum is critical to rational vaccine design. However, most high-resolution mapping approaches involve isolating and characterizing individual monoclonal antibodies, which incompletely defines the full polyclonal response. Here we use two complementary approaches to directly map the specificities of the neutralizing and binding antibodies of polyclonal anti-HIV-1 sera from rabbits immunized with BG505 Env SOSIP trimers. To map the neutralizing specificity, we used mutational antigenic profiling to determine how all amino-acid mutations in Env affected viral neutralization. To map the binding specificity, we used electron microscopy polyclonal epitope mapping (EMPEM) to directly visualize the Fabs in serum bound to Env trimers. Mutational antigenic profiling showed that the dominant neutralizing specificities were the C3/V5 and/or 241/289 glycan hole epitopes, which were generally only a subset of the more diverse binding specificities mapped with EMPEM. Additional differences between binding and neutralization reflected antigenicity differences between virus and soluble Env trimer. Further, mutational antigenic profiling was able to refine epitope specificity in residue-level detail directly from sera, revealing subtle differences across rabbits. Together, mutational antigenic profiling and EMPEM allow for a holistic view of the binding and neutralizing specificity of polyclonal sera and could be used to finely evaluate and guide vaccine design.
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Reviewer #3:
This study combines two cutting-edge approaches for the study of polyclonal antibody responses to understand the molecular profiles of antibodies elicited by HIV envelope trimer immunization in a rabbit model. In one arm of the study, the authors performed mutational profiling of serum antibody neutralization escape variants, and in the second arm they used electron microscopy polyclonal epitope mapping (EMPEM) to track antibody binding sites. These authors performed large-scale data collection and present high-quality validation data and explorations of the resulting datasets that compare antibody binding and virus neutralization profiles. These approaches provide a comprehensive window into the molecular specificity and performance of HIV immunization and are expected to inform advanced HIV-1 vaccine designs.
Summary of …
Reviewer #3:
This study combines two cutting-edge approaches for the study of polyclonal antibody responses to understand the molecular profiles of antibodies elicited by HIV envelope trimer immunization in a rabbit model. In one arm of the study, the authors performed mutational profiling of serum antibody neutralization escape variants, and in the second arm they used electron microscopy polyclonal epitope mapping (EMPEM) to track antibody binding sites. These authors performed large-scale data collection and present high-quality validation data and explorations of the resulting datasets that compare antibody binding and virus neutralization profiles. These approaches provide a comprehensive window into the molecular specificity and performance of HIV immunization and are expected to inform advanced HIV-1 vaccine designs.
Summary of any substantive concerns:
The authors have done a nice job validating the integrity of the NGS data, and the strong data in Figs 4/5/2B show the power of the NGS-based neutralization mapping assays. This adds a solid confirmation of the study findings and demonstrates the quality of the techniques. Overall this is a solid study and the findings are informative. I see just a few methods updates and analyses that would help finalize the presentation of methods and data.
Additional information on the bioinformatic methods for data analysis is needed. How did the authors handle discrepancies in data across replicates or libraries, for example if a mutation that was enriched in one library or replicate, but deleted in another? Were there any quality filters or metrics used to estimate true signal vs. noise?
Differential selection statistics are mentioned briefly, along with citations to prior publications. Prior citations are definitely helpful. I think it is still important to state the key steps used in processing NGS data and the statistical techniques and quality metrics that were used. The authors should also state any criteria for acceptance or rejection or binning of individual data points, or acceptance/rejection of datasets or replicates, if quantitative criteria or metrics were used.
Several replicates showed a low percentage infectivity (Fig S1, e.g. animals 5724 and 2124), but the text indicates averages between 0.3% and 2.7% infectivity. Were some groups omitted from analysis, or were all groups included?
How well did the mutational profiles correlate between different libraries or replicates of the same samples?
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Reviewer #2:
This manuscript by Dingens et al. develops a novel application of mutational antigenic scanning to identify dominant neutralizing antibody epitopes in polyclonal sera from vaccinated animals, and compares the findings of such techniques with those from cryo-EM based unbiased mapping of binding antibodies and from conventional mutational mapping of neutralizing epitopes. Overall, I find the experiments and analyses to be of high quality, thorough and of sound reasoning, and the manuscript to be well written. I also commend the authors for the development of a facile and easy-to-use interactive viewer for exploring the mutational scanning data. I think the dual approach of mutational scanning and cryo-EM based mapping has the potential to be a powerful approach for dissecting antibody content of polyclonal sera …
Reviewer #2:
This manuscript by Dingens et al. develops a novel application of mutational antigenic scanning to identify dominant neutralizing antibody epitopes in polyclonal sera from vaccinated animals, and compares the findings of such techniques with those from cryo-EM based unbiased mapping of binding antibodies and from conventional mutational mapping of neutralizing epitopes. Overall, I find the experiments and analyses to be of high quality, thorough and of sound reasoning, and the manuscript to be well written. I also commend the authors for the development of a facile and easy-to-use interactive viewer for exploring the mutational scanning data. I think the dual approach of mutational scanning and cryo-EM based mapping has the potential to be a powerful approach for dissecting antibody content of polyclonal sera post-vaccination or in infected hosts.
The only major concern I could identify is the following. One of the main advantages of the mutational scanning approach is that it can identify novel epitopes targeted by antibody responses in a high-throughput manner. It is a little disappointing that this advantage was not leveraged in the current manuscript, perhaps due to the choice of the vaccine (BG505 SOSIP trimers where the epitopes have been thoroughly mapped in the literature) and the selection of vaccinated animals. Looking at Fig. 2, animal 5727 was the only animal whose serum showed some selection signatures outside of the regions considered in depth (at sites 507 and 509) - have the authors analyzed these escape mutations? If not, and only if possible within reasonable workload, I urge the authors to pursue this example or any other example where a potential novel epitope discovery could be possible.
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Reviewer #1:
Dingens et al. report a timely complementary study to map neutralizing and binding responses in polyclonal rabbit sera induced by immunization with the BG505 SOSIP Env trimer. Neutralizing responses are mapped using libraries of replication-competent HIV expressing all mutants of the BG505 Env, an approach developed in the Bloom laboratory. Binding responses were mapped using an EM-based method, EMPEM, developed in the Ward laboratory. The Env mutations that affect neutralization of the autologous BG505 strain in the BG505-SOSIP-immunized animals were largely known from other studies, as were the binding (not necessarily neutralizing) responses - the strength of this study is the combination of the two approaches. It is especially useful that the complex datasets have been deposited on-line where they can be interactively …
Reviewer #1:
Dingens et al. report a timely complementary study to map neutralizing and binding responses in polyclonal rabbit sera induced by immunization with the BG505 SOSIP Env trimer. Neutralizing responses are mapped using libraries of replication-competent HIV expressing all mutants of the BG505 Env, an approach developed in the Bloom laboratory. Binding responses were mapped using an EM-based method, EMPEM, developed in the Ward laboratory. The Env mutations that affect neutralization of the autologous BG505 strain in the BG505-SOSIP-immunized animals were largely known from other studies, as were the binding (not necessarily neutralizing) responses - the strength of this study is the combination of the two approaches. It is especially useful that the complex datasets have been deposited on-line where they can be interactively explored, including mapping onto Env trimer and monomer structures. Although results were anticipated, it is very nice to directly compare the neutralization epitopes to the binding epitopes determined by EMPEM. This is a well-written and beautifully illustrated paper.
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