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Bloom lab @jbloomlab.bsky.social · 17/09/2026

For H3N2, some new subclade K strains, especially those with mutations in antigenic region D (eg, V223I) have mildly reduced neutralization by human sera. See this paper from @scottehensley.bsky.social for possible explanation: www.medrxiv.org/content/10.6...

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Bloom lab @jbloomlab.bsky.social · 17/09/2026

But effect of G155E mutation in H1N1 is highly variable across people. For most sera, strains with G155E have only mildly reduced neutralization---but some sera neutralize G155E strains much more poorly. Individuals who are strongly affected by G155E tend to be younger.

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Bloom lab @jbloomlab.bsky.social · 17/09/2026

For H1N1, many new natural strains have modestly reduced neutralization by human sera relative to strain in 2026-2027 Northern Hemisphere vaccine. Strains w lowest neutralization often have G155E, which has emerged repeatedly in natural strains, especially in D.3.1.1 subclade.

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Bloom lab @jbloomlab.bsky.social · 17/09/2026

We assembled set of 78 H3N2 and 62 H1N1 HAs from naturally occurring human seasonal strains that largely cover recent diversity of influenza circulating in human population. We measured neutralization of these viruses by 325 recent human sera.

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Bloom lab @jbloomlab.bsky.social · 17/09/2026

We recently developed sequencing-based neutralization assays that measure how serum antibodies neutralize many viral strains. This enables us to measure in near real-time how new naturally emerging strains are neutralized by current human sera.

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Bloom lab @jbloomlab.bsky.social · 17/09/2026

As background, human seasonal influenza evolves rapidly. HA protein of H3N2 influenza has accumulated >45 amino-acid mutations over last 18 years. This evolution erodes immunity, so vaccine updates considered twice per year (Sept & Feb for Southern & Northern hemisphere vaccines).

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Bloom lab @jbloomlab.bsky.social · 23/07/2026

Note our study used pseudoviruses and conditionally replicative virions to ensure biosafety, and reports deep mutational scanning only for HA usage of tufted duck MHC-II to limit any information hazard concerns.

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Bloom lab @jbloomlab.bsky.social · 23/07/2026

We also showed H7 HA binds tufted duck MHC-II similarly to H5 HA, & some H1, H2, H3, & H9 HAs also can use avian or human MHC-II. But patterns vary among strains. For instance, an avian influenza HA and the 1918 HA can use tufted duck MHC-II, but later human strains cannot.

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Bloom lab @jbloomlab.bsky.social · 23/07/2026

Structure & deep mutational scanning suggest identity of peptide bound to MHC-II could influence interaction of HA & MHC-II. Also, HA binding would likely block ability of MHC-II to interact with T-cell (perhaps analogous to how EBV gp42 can bind to MHC-II to block T cell activation).

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Bloom lab @jbloomlab.bsky.social · 23/07/2026

So although structure only 4.8 A, it is corroborated by deep mutational scanning of both HA and MHC-II showing that sites in both proteins that affect binding are at structural interface. (Sites where mutations decrease binding are red in structure below)

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Bloom lab @jbloomlab.bsky.social · 23/07/2026

To validate structure, we used inverted pseudotyping deep mutational scanning to measure how mutations to tufted duck MHC-II affect binding to H5 HA. Most mutations with big impact in alpha chain, but beta-chain mutations near peptide-binding groove also have effect.

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Bloom lab @jbloomlab.bsky.social · 23/07/2026

To determine structure, we produced H5 HA protein w mutations that increased binding to tufted duck MHC-II. This HA increased fraction of particles bound to MHC-II in ns-EM, and we were able to use it solve cryo-EM structure of H5 HA bound to tufted duck MHC-II.

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Bloom lab @jbloomlab.bsky.social · 23/07/2026

The mutations that reduced MHC-II entry clustered in a region on HA head defining the MHC-II binding surface. We also directly measured how HA mutations affect binding to tufted duck MHC-II, and identified same binding surface.

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Bloom lab @jbloomlab.bsky.social · 23/07/2026

To understand how HA interacts w MHC-II, we measured how all H5 HA mutations affect pseudovirus entry via sialic acid or tufted duck MHC-II. Identified loss-of-function mutants that could only use MHC-II or sialic acid.

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Bloom lab @jbloomlab.bsky.social · 23/07/2026

We tested 80 H5 HAs: most but not all could enter cells via tufted duck & to lesser extent human MHC-II. Note MHC-II highly variable within and between species. See dms-vep.org/Flu-H5N1-Ame... for interactive version of below plot.

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Bloom lab @jbloomlab.bsky.social · 23/07/2026

We first measured ability of two H5 HAs to enter cells via sialic acid or MHC-II. As shown below, both HAs could use tufted duck & to lesser extent human MHC-II. [Note: experiments used pseudoviruses, which can only undergo single round of cell entry, providing safe way to study HA]

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Bloom lab @jbloomlab.bsky.social · 23/07/2026

In new study led by @bdadonaite.bsky.social, we show many influenza HAs (H5, H7, H9, H1, H2, H3) can use avian or human MHC-II to enter cells. We then use novel combo of deep mutational scanning & cryoEM to define how H5 HA binds to avian MHC-II. Preprint: doi.org/10.64898/202...

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Bloom lab @jbloomlab.bsky.social · 25/02/2026

We also defined how F mutations affect neutralization by a panel of monoclonal antibodies. This allowed us to quantify the resilience of different antibodies to escape, and predict which antibodies also neutralize the related Hendra virus.

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Bloom lab @jbloomlab.bsky.social · 25/02/2026

A strategy for vaccines is to stabilize F in pre-fusion conformation. We identified sites where mutations to proline (which blocks helix formation) are disfavored. This identifies new candidate mutations for stabilizing F vaccine immunogens.

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Bloom lab @jbloomlab.bsky.social · 25/02/2026

For this study, we used pseudoviruses that can only undergo a single round of cell entry (& so are not human pathogens) to measure how mutations to F affect its fusion function. We found F is more functionally constrained than the other Nipah surface protein, RBP.

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Bloom lab @jbloomlab.bsky.social · 21/02/2026

For instance, in interactive plot below I've moused over to highlight serum from an individual who mostly has high titers, but has dramatically reduced titers just to strains with mutations at site 135. There is no way to represent that sort of thing w just medians and ranges across sera.

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Bloom lab @jbloomlab.bsky.social · 20/02/2026

For H1N1 influenza, a new subclade (D.3.1.1) has also recently spread to become dominant, and our data show that this new subclade has reduced neutralization by human sera See jbloomlab.github.io/flu-seqneut-... for interactive version of below plot

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Bloom lab @jbloomlab.bsky.social · 20/02/2026

The mutations that further reduce neutralization of subclade K are in antigenic regions D & E, which were less mutated in parent subclade K See below from recent @scottehensley.bsky.social preprint (doi.org/10.64898/202...) & stay tuned for study from their group that explains this observation

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Bloom lab @jbloomlab.bsky.social · 20/02/2026

For vaccine update decisions, we care about what is NEXT. Here our data help by showing that within subclade K strains there are already new subvariants w further reduced neutralization. These subvariants have additional mutations as shown below & interactively at nextstrain.org/community/jb...

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Bloom lab @jbloomlab.bsky.social · 20/02/2026

Probably because of these lower titers, subclade K has rapidly become dominant among H3N2, rising from <1% to 95% frequency in ~9 months. See below image from this Nextstrain link (nextstrain.org/seasonal-flu...).

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Bloom lab @jbloomlab.bsky.social · 20/02/2026

Resulting datasets are very rich. Below are H3N2 data (also under first post in this thread). There is extreme variability among human sera; the median serum has ~1.5-2-fold lower neutralization of subclade K. See jbloomlab.github.io/flu-seqneut-... to explore interactive plot

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Bloom lab @jbloomlab.bsky.social · 20/02/2026

Specifically, we first assembled a set of 57 H3N2 and 34 H1N1 strains that largely cover the current diversity of human seasonal influenza (see image below). We then measured neutralization of all 91 strains against 302 sera from humans of a range of ages (0 to 103 years) and geographic locations.

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Bloom lab @jbloomlab.bsky.social · 20/02/2026

We have posted data providing real-time measurement of human neutralizing antibody landscape to seasonal influenza. Data explain spread of subclades K (H3N2) & D.3.1.1 (H1N1), identify subclade K subvariants w reduced neutralization, & can inform choice of strains for next vaccine.

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Bloom lab @jbloomlab.bsky.social · 08/09/2025

Above visualizations just scratch surface of data: there is tremendous heterogeneity across sera from different individuals not easily summarized by median/mean. Indeed, we previously found this heterogeneity may be important for influenza evolution: elifesciences.org/reviewed-pre...

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Bloom lab @jbloomlab.bsky.social · 08/09/2025

We then measured how 188 human sera recently collected at four different sites neutralized all 140 influenza strains in library. Titers are summarized below; can be examined interactively at jbloomlab.github.io/flu-seqneut-... & jbloomlab.github.io/flu-seqneut-...

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Bloom lab @jbloomlab.bsky.social · 08/09/2025

In spring of 2025, we designed library of naturally occurring human seasonal influenza strains that represented diversity of available sequences at that time; this library continues to cover most sequenced diversity of H3N2 and H1N1 hemagglutinin today.

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Bloom lab @jbloomlab.bsky.social · 08/09/2025

To do this, we used sequencing-based neutralization assays that measure many neutralization curves simultaneously (journals.asm.org/doi/10.1128/... & elifesciences.org/reviewed-pre...) Approach enabled one grad student (@ckikawa.bsky.social) to measure ~26,000 neutralization curves in ~5 months.

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Bloom lab @jbloomlab.bsky.social · 08/09/2025

But because it takes time to perform experiments, measurement of how current strains are neutralized by human serum antibodies can lag timeline for vaccine strain selection. Our goal was to use new approach to characterize human antibody landscape at scale in near real-time.

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Bloom lab @jbloomlab.bsky.social · 05/09/2025

Data in interactive form at dms-vep.org/CHIKV-181-25... Thanks to Xiaohui Ju for leading study Special thanks to @msdiamondlab.bsky.social for help Also Will Hannon, Caelan Radford, Brendan Larsen, Daved Fremont, Ofer Zimmerman, Tomasz Kaszuba, Chris Nelson, Israel Baltazar-Perez, Samantha Nelson

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Bloom lab @jbloomlab.bsky.social · 05/09/2025

After using pseudoviruses & reporter particles to show mutations *loss* of function, we engineered into Chikungunya virus: mutants lost ability to infect human or mosquito cells. So we reduced natural tropism for both human & mosquito cells to just one type of cell.

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Bloom lab @jbloomlab.bsky.social · 05/09/2025

We next used non-replicative single-cycle alphavirus reporter particles (which provide another safe way to study mutations) to validate that mutations identified in deep mutational scanning indeed specifically impaired entry in human or mosquito cells only.

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Bloom lab @jbloomlab.bsky.social · 05/09/2025

Sites where mutations specifically impair entry in 293T-MXRA8 cells mostly at MXRA8 binding interface. We also find sites where mutations specifically impair entry in C6/36 cells. Although mosquito receptor unknown, we hypothesize these sites at its binding interface.

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Bloom lab @jbloomlab.bsky.social · 05/09/2025

Most mutations similarly affect entry in all three cells, but some have cell-specific effects. For instance, mutations at E2 site 119 are generally tolerated in C6/36 and 293T-TIM1 cells, but deleterious in 293T-MXRA8 cells. (See dms-vep.org/CHIKV-181-25... for interactive plot.)

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Bloom lab @jbloomlab.bsky.social · 05/09/2025

We first measured how mutations affect entry in 293T cells expressing human receptor MXRA8. Below is constraint mapped on structure; see dms-vep.org/CHIKV-181-25... for interactive heatmap of these data.

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Bloom lab @jbloomlab.bsky.social · 05/09/2025

We used pseudovirus deep mutational scanning to measure effects of all mutations to envelope proteins in context of single-cycle pseudotyped particles that provide a safe way to study viral protein mutations outside context of fully infectious virus.

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Bloom lab @jbloomlab.bsky.social · 05/09/2025

Chikungunya virus enters cell using its envelope proteins, which are also target of neutralizing antibodies and vaccine design. A receptor for these viral proteins in mammalian cells is the protein MXRA8, but receptor in mosquito cells is unknown.

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Bloom lab @jbloomlab.bsky.social · 05/09/2025

As background, Chikungunya virus has transmission cycle that involves infecting both mosquitoes & humans or other primates. Infection can cause fever and severe joint pain in humans. Outbreaks are growing due to expanding mosquito range: www.nytimes.com/2025/08/19/h...

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Bloom lab @jbloomlab.bsky.social · 20/08/2025

Finally, we measured how mutations affect neutralization by three relevant monoclonal antibodies. As shown below, all antibodies adversely affected by mutating site 505 which fortunately remains highly constrained for ACE2 binding. We discuss this interesting site more in preprint.

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Bloom lab @jbloomlab.bsky.social · 20/08/2025

We used this fact to estimate how much mutations at each site affect RBD up-down motion, as shown below. Many of these sites have mutated during SARS-CoV-2 evolution in humans, demonstrating importance of RBD motion and its effects on ACE2 binding & antibody neutralization.

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Bloom lab @jbloomlab.bsky.social · 20/08/2025

This tradeoff between serum antibody escape and ACE2 binding is because mutations that put the RBD more up enable ACE2 binding but also promote antibody binding. Mutations that put the RBD more down do the opposite.

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Bloom lab @jbloomlab.bsky.social · 20/08/2025

Some mutations outside the RBD have a strong effect on serum antibody neutralization. But for ACE2-distal or non-RBD mutations, there is a strong tradeoff between serum antibody escape and ACE2 binding as shown below.

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Bloom lab @jbloomlab.bsky.social · 20/08/2025

Despite imprinting, in some individuals recent infection or vaccination appreciably shift immunodominant neutralizing epitopes. So new exposures are altering neutralizing serum antibody repertoire, although our data do not define mechanism (see preprint for hypotheses).

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Bloom lab @jbloomlab.bsky.social · 20/08/2025

We measured how mutations affected neutralization by sera collected from humans before or after vaccination or infection w recent JN.1-descendant variant. Key sites of escape are shown below; some of the sites (eg, 475 and 478) are mutated in very recent variants.

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Bloom lab @jbloomlab.bsky.social · 20/08/2025

We next measured how mutations affect full-spike ACE2 binding. Most mutations had similar impacts in KP.3.1.1 and the older XBB.1.5 spike, but some recent mutations (eg, A435S and Q493E) enhance ACE2 binding KP.3.1.1 after impairing it in XBB.1.5.

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Bloom lab @jbloomlab.bsky.social · 20/08/2025

We first measured how all mutations affect cell entry; see this interactive page for those data: dms-vep.org/SARS-CoV-2_K... A number of mutations that have spread recently are more tolerated in KP.3.1.1 than older XBB.1.5 variant, suggesting epistatic shifts favored their emergence.

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