In new study led by @bdadonaite, we measure how all mutations to H5 influenza HA affect four molecular phenotypes relevant to pandemic risk:. Results can inform surveillance of ongoing evolution of H5N1.

All the data are available for visualization and download at Thanks to @timcyuu for leading study, and also @CKikawa, Bernadeta Dadonaite, Andrea Loes, & Jane Englund.

Overall, these results show how pleiotropic effects of mutations constrain HA evolution. Epistasis can alleviate these constraints w respect to cell entry in receptor binding pocket, but we find no evidence that mutations that strongly destabilize HA ever fix in H3N2 evolution.

To examine implications for antigenic evolution, we measured how all HA mutations affected serum antibody neutralization. Mutations at key trimer interface sites that cause substantial antibody escape but destabilize HA have never fixed.

We found extensive epistatic entrenchment w respect to cell entry in receptor binding pocket (see below), consistent w prior work by @wchnicholas @RobertPdeVries1 . But minimal epistatic entrenchment w respect to HA stability.

We then looked for evidence of epistasis by examining whether mutations had become entrenched. A mutation is entrenched if reverting it to the ancestral amino acid is deleterious in recent HAs, as schematized below.

As can be seen below, constraint due to mutational impacts on cell entry are widely distributed across HA including receptor-binding pocket and fusion peptide. But mutational constraint due to HA stability is concentrated at trimer and HA1-HA2 interface.

We used pseudovirus deep mutational scanning to characterize all mutations to a recent H3N2 HA. This approach uses virions that can only undergo one round of cell entry & so are not pathogens capable of causing disease. All measurements available here:

In new study led by @timcyuu, we measure how mutations to H3 flu HA affect cell entry, stability & antibody escape. We find pleiotropic effects of mutations on these phenotypes shape evolution: epistasis alleviates cell-entry but not stability constraints.

Final version of our pseudovirus deep mutational scanning of rabies glycoprotein now published in @cellhostmicrobe: Main addition from preprint summarized above are additional validations of prefusion stabilization candidate mutations in Fig S4 and S5.

The final published version of our study (described in thread above) on the differences in SARS2 antibody specificities in imprinted adults versus recently infected infants is now out in @JVirology:

@bblarsen1 @CaelanRadford @VUMC_Vaccines @veeslerlab The final version of our pseudovirus deep mutational scanning of the Nipah virus receptor-binding protein is now published.

All data described above are at Thanks to @CKikawa & Andrea Loes for leading study, w important contributions from @huddlej, @marlinfiggins, @Miriampippaart, Tachianna Griffiths, @ETroisi10, Heidi Peck, Ian Barr, Jan Englund, @SCOTTeHENSLEY, @trvrb.

We hope that high-throughput measurements of neutralization of many recent influenza strains by many human sera, which are feasible to make w these new methods, can help w efforts to forecast influenza evolution for vaccine strain selection.

The actual growth rates of viral strains were highly correlated w fraction of sera w low titers to strains, as shown below. But there was no correlation if we just pooled all the sera and measured titers: per-serum measurements are needed to capture population heterogeneity.

Can these measurements of antibody immunity across human population help us understand influenza evolution?. To address that question, we worked w @trvrb @huddlej @marlinfiggins @Miriampippaart to estimate growth rates of H3N2 strains in 2023 using multinomial logistic regression

Below are titers of a large set of sera against all the strains. The plot shows the extensive heterogeneity of population antibody immunity: titers against different strains vary widely across individuals.

Different sera were better or worse at neutralizing different viral strains. Below plot shows a 14-year old child neutralized most strains but had lower titers to strains mutated at site 145; a 24-year old neutralized those strains well but had lower titers to other strains.

We used this assay to measure neutralizing titers of a large set of >100 children and adult sera against a panel of viruses representing the H3N2 influenza diversity circulating in humans in 2023.

We recently developed high-throughput sequencing-based assay to measure how serum antibodies neutralize different influenza strains. With assay, we can measure 1,872 neutralization curves per 96-well plate, compared to traditional assays that yield 8 or 12 curves per plate.

As background, human seasonal influenza evolves to erode immunity. Vaccine updated regularly to keep pace w evolution, but forecasting which viral strains will dominate next season is hard. One limitation is we don’t fully understand human immune landscape that drives evolution.