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Jennifer Juno @jenjuno.viralvaxlab.com · 03/03/2026

We used tetramers to confirm that both HA91 and SMARTA peptides were processed and primed similar numbers of CD4 T cells. What differed was their TFH differentiation - HA91-specific T cells failed to develop into a CD90low GC TFH population. As a result, no stem-specific GC formed.

Number of epitope-specific CD4 T cells determined by tetramer staining

Number and phenotype of tetramer-specific TFH cells

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Jennifer Juno @jenjuno.viralvaxlab.com · 03/03/2026

We next engineered variants of our stem antigen ‘tagged’ with one of these peptides or a well known control epitope – OTII or SMARTA. Some of the peptides were able to rescue the immunogenicity of the stem protein (like HA523 and GP61). Surprisingly, though, the dominant HA91 peptide had no impact.

Design and primary immunogenicity of stem-peptide antigens

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Jennifer Juno @jenjuno.viralvaxlab.com · 03/03/2026

But what about recombinant protein immunogens? To what extent can individual CD4 epitopes rescue or tune immunogenicity? We started by mapping the CD4 epitopes in PR8 HA, and found a highly dominant peptide (HA91) as well as three subdominant peptides.

Map of immunogenic peptides in influenza HA

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Jennifer Juno @jenjuno.viralvaxlab.com · 03/03/2026

It turns out that after repeated boosting, we could find extremely subdominant ferritin-specific CD4 cells (but nothing for stem!) - meaning that these rare responses can support a GC reaction when the antigen is arrayed on a nanoparticle

Proliferation of ferritin-specific CD4 T cells following 3 doses of stem-ferritin nanoparticle vaccination

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Jennifer Juno @jenjuno.viralvaxlab.com · 03/03/2026

So how can we make stem more immunogenic? Up first: stem-ferritin nanoparticles, which elicit a strong IgG response after one immunisation in the mice. This was a bit of a mystery to us, since neither the stem protein nor ferritin nanoparticle contain detectible CD4 epitopes in BL/6 mice.

Immunogenicity of stem versus stem-ferritin nanoparticles in BL/6 mice

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Jennifer Juno @jenjuno.viralvaxlab.com · 20/12/2024

8/9 In short, we suggest in vitro stimulation activates ag-specific T cells, which secrete cytokines and activate Treg and Th17/22 cells. Without careful phenotyping, all of these cells can get picked up as AIM+, accounting for the Th17-like memory cells found in many virus-specific AIM datasets.

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Jennifer Juno @jenjuno.viralvaxlab.com · 20/12/2024

7/9 Mitch sorted tetramer-specific cells, labelled them, and added them back into PBMC before stimulating with the relevant peptide. He was able to show that ag-specific CD4 cells downregulate CXCR4 while upregulating 4-1BB/CD137, giving us a more accurate way to define antigen specificity.

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Jennifer Juno @jenjuno.viralvaxlab.com · 20/12/2024

5/9 Using RNAseq and confirmatory FACS, Mitch showed that CD39+ Tregs and Th17/Th22 cells expressing CCR6 and high levels of CD26 were being activated through the transwell via a mechanism involving IL-2. These same subsets are also found during standard peptide-based AIM assays.

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Jennifer Juno @jenjuno.viralvaxlab.com · 20/12/2024

4/9 Possibly! In a transwell assay, anti-CD3/CD28 stimulation can drive T cells in an adjacent chamber to express common activation markers (CD25, OX-40, 4-1BB, CD40L). All of these responding cells had a memory phenotype, and were strongly enriched for CCR6 expression.

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Jennifer Juno @jenjuno.viralvaxlab.com · 20/12/2024

2/9 Recently, though, we’ve been bothered by something we couldn’t explain: we find a lot of Th17-like, CCR6+ SARS-CoV-2 spike-specific CD4 T cells in our AIM assays, but we never see these cells when we use spike-specific class II tetramers. Why?

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