Structure-based Design of Chimeric Influenza Hemagglutinins to Elicit Cross-group Immunity

Reviewer #1 (Public review):

Summary:

The study by Castro et al. presents an interesting blueprint for designing influenza immunogens that can induce cross-group influenza-specific antibodies. The authors used a structure-based design to transplant receptor binding site (RBS) residues from H5 and H3 into an H1 scaffold. In addition, they assembled the transplanted structures as heterotrimers. They characterized the constructs structurally and used them to immunize mice to define ELISA binding and neutralizing antibodies (Abs) to different influenza strains.

Strengths and Weaknesses:

The authors succeeded in generating the different, correctly folded immunogens. The heterotrimers would benefit from more characterization: it remains unclear whether they are even formed or whether the sample is a mix of homotrimers and whether some combinations are more likely than others. While some of these questions are complex to answer, authors should at least confirm the presence of heterotrimers.

While all constructs were able to elicit H1-specific Abs, different immunogens displayed differential ability to induce a response to the transplanted epitope. While H3-transplant resulted in H3-specific Abs, this was not the case for H5 or the heterotrimers. The importance of the finding is that authors are able to elicit polyclonal Abs neutralizing group 1 and group 2 influenza viruses with a single immunogen. A more in-depth discussion on why the H3-transplant but not the H5-transplant resulted in those specific Abs could be beneficial.

Overall, the work is a proof of concept that H1-H3 chimeric proteins can be produced and an important first step towards computational vaccines, inducing Abs to multiple groups.

Reviewer #2 (Public review):

Summary:

The manuscript from Castro et al describes the engineering of influenza hemagglutinin H1-based head domains that display receptor-binding-site residues from H5 and H3 HAs. The initial head-only chimeras were able to bind to FluA20, which recognizes the trimer interface, but did not bind well to H5 or H3-specific antibodies. Furthermore, these constructs were not particularly stable in solution as assessed by low melting temperatures. Crystal structures of each chimeric head in complex with FluA20 were obtained, demonstrating that the constructs could adopt the intended conformation upon stabilization with FluA20. The authors next placed the chimeric heads onto an H1 stalk to create homotrimeric HA ectodomains, as well as a heterotrimeric HA ectodomain. The homotrimeric chimeric HAs were better behaved in solution, and H3- and H5-specific antibodies bound to these trimers with affinities that were only about 10-fold weaker compared to their respective wildtype HAs. The heterotrimeric chimeric HA showed transient stability in solution and could bind more weakly to the H3- and H5-specific antibodies. Mice immunized with these trimers elicited cross-reactive binding antibodies, although the cross-neutralizing titers were less robust. The most positive result was that the H1H3 trimer was able to elicit sera that neutralized both H1 and H3 viruses.

Strengths:

The manuscript is very well-written with clear figures. The biophysical and structural characterizations of the antigen were performed to a high standard. The engineering approach is novel, and the results should provide a basis for further iteration and improvement of RBS transplantation.

Weaknesses:

The main limitation of the study is that there are no statistical tests performed for the immunogenicity results shown in Figures 4 and 5. It is therefore unknown whether the differences observed are statistically significant. Additionally, fits of the BLI data in Figure 3 to the binding model used to determine the binding constants should be shown.