Figure 1: Experimental overview

Figure 2: Experimental overview

In year 0, we performed experiments on the UGA 9 cohort. This cohort consisted of 40 participants between the ages of 19 and 89 (Table 1). 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 (Table 2 shows flu strains present in the vaccine). Samples were collected according to Figure 1.   

Table 1 : Cohort demographics

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. 

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 2. 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. 

Bulk RNA-seq (PBMC) 

RNA was extracted 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 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.