Figure 1: Experimental overview

In year 1, participants were enrolled at La Jolla Institute for Immunology in the 2025-2026 influenza season (IRB #VD-319). This cohort consisted of 40 participants between the ages of 19 and 65 (Table 1). Each participant received a standard Fluzone dose (15 μg of antigen). Samples were collected according to Figure 1.  

Figure 2: Experimental overview

In year 0, we performed experiments on samples collected from the UGA 9 cohort. This cohort consisted of 40 participants between the ages of 19 and 89 (Table 2). The study protocol was approved by the Institutional Review Board of the University of Georgia (IRB #20224877). Each participant received either a standard Fluzone dose (15 μg of antigen for ages < 65) or a high Fluzone dose (60 μg of antigen for ages >= 65) in the 2024-2025 influenza season. Samples were collected according to Figure 2.   

Table 2 : Cohort demographics (UGA-9 / Year 0)

Hemagglutination Assay (Sera)

Influenza hemagglutination-inhibition (HAI) assays were performed to quantify HA-specific antibodies by assessing the ability to inhibit the binding of influenza virus to sialic acid receptors on erythrocytes. HAI titers were defined as the reciprocal of the highest serum dilution that completely inhibited hemagglutination. 

Plasma cytokines

Plasma samples were analyzed in duplicate using two 12-plex Legendplex cytokine bead panels on a Cytek Aurora analyzer. Following automated bead gating and manual quality control – which led to the exclusion of one outlier replicate plate – mean fluorescence intensities were converted to concentrations using a 5-PL standard curve, with values below a cytokine- and experiment-specific limit of detection (LODHigh) thresholded to that limit. Finally, individual baseline values were established by averaging the two pre-vaccination visits (D-14 and D0).

Spectral cytometry (PBMC)

We performed spectral cytometry on PBMCs, staining for surface antibodies listed in Table 3. Gating strategy for all immune cell populations is available in Figure 3. For a given cell population, frequencies represent the proportion of cells within the indicated parent population. Samples with either less than 50% live PBMC at thawing or less than 30,000 live singlet cells recorded were excluded from the dataset. 

AIM (antigen-reactive T cells)

PBMC were thawed and stimulated for 24h with (last 4h of stimulation with Golgi Stop/Plug):

• DMSO: negative control
• GLO: HA universal peptide pool: overlapping 15-mer covering the HA proteins from the virus strains contained in vaccines spanning 2017-2019 seasons + epitopes with experimental data available in IEDB.
• PR8 backbone pool : overlapping 15-mer peptides covering all non-HA and non-NA proteins of the H1N1 A/Puerto Rico/8/1934 (PR8) strain (8 proteins total: M1, M2, NP, NEP, NS1, PA, PB1, PB2).
• CON: Conserved HA/NA pool: overlapping 15-mer peptides covering conserved HA/NA epitopes from H1N1 and H3N2 vaccine strains from the past 10 flu seasons, starting in 2024/2025 season
• SEA: Seasonal HA/NA pool: overlapping 15-mer peptides covering novel (i.e., absent from the conserved HA/NA pool) HA/NA epitopes from H1N1 and H3N2 strains in the 2025/2026 flu season.
• POL: Polyclonal stimulation: antiCD3+anti-CD28, positive control.

Following stimulation, cells were stained with a 23-color surface and intracellular panel, and analyzed on a Cytek Aurora spectral analyzer. Antigen-specific CD4 T cells were defined as Live/CD14-CD19-/CD3+/CD4+CD8-/OX40+CD137+.

During data processing, DMSO (negative control) frequencies were subtracted from cell frequencies, and values <0.01 were assigned to 0.01 (the assay's limit of detection).

Bulk RNA-Seq (PBMC) 

RNA was extracted from PBMCs using the miRNeasy Mini Kit (Qiagen). Libraries were prepped using Watchmaker mRNA Library Prep Kit (Watchmaker Genomics). Libraries were sequenced on a NovaSeq6000 (Illumina) system, targeting 20 million reads per sample. Processing was performed using the default nf-core/rna-seq pipeline and the ensembl reference genome v113.

Bulk BCR-Seq (PBMC)

RNA was extracted from PBMCs using the miRNeasy Mini Kit (Qiagen) bulk BCR-seq library preparation, using the Takara SMART-Seq® Human BCR (with UMIs) (Takara Biosciences). Libraries were sequenced on an AVITI (Element Biosciences), targeting 1 million reads for heavy chain samples and 2 million reads for light chain samples. Processing was performed using the default nf-core/airrflow pipeline.

HLA typing

Genomic DNA from PBMC was typed at the American Society for Histocompatibility and Immunogenetics (ASHI)–accredited Medical Genomics Laboratories, Murdoch University (Western Australia). Locus-specific, sample-indexed (multiplex identifier, MID) primers targeted polymorphic exons for Class I HLA-A, -B, and -C (exons 2–3) and Class II HLA-DQA1/-DQB1 (exons 2–3), -DRB1/3/4/5 (exon 2), and -DPB1 (exon 2). Libraries were prepared using NEBNext Ultra II Library Prep Kit and sequenced on an Illumina MiSeq (v3 600-cycle, 2 × 300 bp). Reads were demultiplexed by MID barcodes, adapter/primer sequences trimmed, quality-filtered, and merged. Alleles were called using the IIID HLA Allele Caller and the IIID HLA Analysis Suite, with the IMGT/HLA Database as the reference.