A modular platform to display multiple hemagglutinin subtypes on a single immunogen

Reviewer #1 (Public Review):

Summary:

In this manuscript by Thronlow Lamson et al., the authors develop a "beads-on-a-string" or BOAS strategy to link diverse hemagglutinin head domains, to elicit broadly protective antibody responses. The authors are able to generate varying formulations and lengths of the BOAS and immunization of mice shows induction of antibodies against a broad range of influenza subtypes. However, several major concerns are raised, including the stability of the BOAS, that only 3 mice were used for most immunization experiments, and that important controls and analyses related to how the BOAS alone, and not the inclusion of diverse heads, impacts humoral immunity.

Strengths:

Vaccine strategy is new and exciting.

Analyses were performed to support conclusions and improve paper quality.

Weaknesses:

Controls for how different hemagglutinin heads impact immunity versus the multivalency of the BOAS.

Only 3 mice were used for most experiments.

There were limited details on size exclusion data.

Reviewer #2 (Public Review):

Summary:

The authors describe a "beads-on-a-string" (BOAS) immunogen, where they link, using a non-flexible glycine linker, up to eight distinct hemagglutinin (HA) head domains from circulating and non-circulating influenzas and assess their immunogenicity. They also display some of their immunogens on ferritin NP and compare the immunogenicity. They conclude that this new platform can be useful to elicit robust immune responses to multiple influenza subtypes using one immunogen and that it can also be used for other viral proteins.

Strengths:

The paper is clearly written. While the use of flexible linkers has been used many times, this particular approach (linking different HA subtypes in the same construct resembling adding beads on a string, as the authors describe their display platform) is novel and could be of interest.

Weaknesses:

The authors did not compare to individuals HA ionized as cocktails and did not compare to other mosaic NP published earlier. It is thus difficult to assess how their BOAS compare.

Other weaknesses include the rationale as to why these subtypes were chosen and also an explanation of why there are different sizes of the HA1 construct (apart from expression). Have the authors tried other lengths? Have they expressed all of them as FL HA1?

Reviewer #3 (Public Review):

This work describes the tandem linkage of influenza hemagglutinin (HA) receptor binding domains of diverse subtypes to create 'beads on a string' (BOAS) immunogens. They show that these immunogens elicit ELISA binding titers against full-length HA trimers in mice, as well as varying degrees of vaccine mismatched responses and neutralization titers. They also compare these to BOAS conjugated on ferritin nanoparticles and find that this did not largely improve immune responses. This work offers a new type of vaccine platform for influenza vaccines, and this could be useful for further studies on the effects of conformation and immunodominance on the resulting immune response.

Overall, the central claims of immunogenicity in a murine model of the BOAS immunogens described here are supported by the data.

Strengths included the adaptability of the approach to include several, diverse subtypes of HAs. The determination of the optimal composition of strains in the 5-BOAS that overall yielded the best immune responses was an interesting finding and one that could also be adapted to other vaccine platforms. Lastly, as the authors discuss, the ease of translation to an mRNA vaccine is indeed a strength of this platform.

One interesting and counter-intuitive result is the high levels of neutralization titers seen in vaccine-mismatched, group 2 H7 in the 5-BOAS group that differs from the 4-BOAS with the addition of a group 1 H5 RBD. At the same time, no H5 neutralization titers were observed for any of the BOAS immunogens, yet they were seen for the BOAS-NP. Uncovering where these immune responses are being directed and why these discrepancies are being observed would constitute informative future work.

There are a few caveats in the data that should be noted:

(1) 20 ug is a pretty high dose for a mouse and the majority of the serology presented is after 3 doses at 20 ug. By comparison, 0.5-5 ug is a more typical range (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380945/, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9980174/). Also, the authors state that 20 ug per immunogen was used, including for the BOAS-NP group, which would mean that the BOAS-NP group was given a lower gram dose of HA RBD relative to the BOAS groups.

(2) Serum was pooled from all animals per group for neutralization assays, instead of testing individual animals. This could mean that a single animal with higher immune responses than the rest in the group could dominate the signal and potentially skew the interpretation of this data.

(3) In Figure S2, it looks like an apparent increase in MW by changing the order of strains here, which may be due to differences in glycosylation. Further analysis would be needed to determine if there are discrepancies in glycosylation amongst the BOAS immunogens and how those differ from native HAs.