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. 2018 Dec 28;7:e37015. doi: 10.7554/eLife.37015

Figure 2. Variation in the immune profiles of trial participants.

The IgG-binding data for each sample were projected across two dimensions using t-distributed stochastic neighbour embedding. Points are coloured according to the cohort of the individual contributing the sample. The shape represents the day of the trial on which the sample was collected. Ellipses surround sets of samples taken from the same individual. The separation of each ellipse shows the distinctive immune ‘fingerprint’ of each individual, which is maintained over the course of the trial.

Figure 2—source data 1. Panproteome array IgG binding data.
elife-37015-fig2-data1.tds (10.2MB, tds)
DOI: 10.7554/eLife.37015.010

Figure 2.

Figure 2—figure supplement 1. Reproducibility of probe signals.

Figure 2—figure supplement 1.

In a pilot experiment, the serum samples were applied to an array consisting of probes representing the proteome of Streptococcus pneumoniae TIGR4. These were also present on the panproteome array, but they were not included in subsequent analyses. These two independent experiments provided an estimate of the variation between technical replicates, although the differences in the array designs means there is substantial systematic variation between them. The Pearson correlation (R2) was calculated between all pairwise comparisons from the two sets of replicates, and the overall distribution for different types of comparison shown as violin plots for (A) all probes and (B) the 1165 immunoreactive probes (a maximum IgG binding of at least one across the S. pneumoniae TIGR4 probes from the panproteome dataset). There was no significant difference between the correlations observed across all probes for technical replicates and other comparisons between samples from the same individual (Wilcoxon rank sum tests; N = 312, W = 13239, p = 0.073 for all probes). However, restricting the analysis to the immunoreactive probes found technical replicates to exhibit a significantly stronger correlation with one another, relative to samples from the same individual at other timepoints (N = 312, W = 13480, p = 0.036 for immunoreactive probes), consistent with the WCV-induced changes associated with antibody-binding target being reproducibly detectable. This high similarity between technical replicates and samples from the same individual is necessary for the persistence of an immune fingerprint in longitudinal samples (Figure 2). Comparisons between these two categories were significantly more correlated than comparisons with the next most similar category, different individuals in the same cohort at the same timepoint (Wilcoxon rank sum tests; N = 601, W = 48170, p < 10−16 for all probes; N = 601, W = 48738, p < 10−16 for immunoreactive probes). As there was relatively little variation between technical replicates, but reproducibly high variation between trial participants, biological replicates were prioritised over technical replicates in designing the study.
Figure 2—figure supplement 2. Evidence of distinct antibody fingerprints of trial participants from non-DCL probes.

Figure 2—figure supplement 2.

The t-SNE analysis shown in Figure 2 was repeated, excluding the probes corresponding to the DCL. Longitudinal samples from the same individual again cluster together, demonstrating the distinctiveness of individuals’ antibody repertoires was apparent even when the allelic variation of PspA, PspC, ZmpA and ZmpB were excluded. The longer-range structure of the projection is stochastic, and these differences with Figure 2 are not biologically relevant.
Figure 2—figure supplement 3. Evidence of distinct antibody fingerprints of trial participants from proteins conserved across S. pneumoniae, S. pseudopneumoniae and S. mitis.

Figure 2—figure supplement 3.

The t-SNE analysis shown in Figure 2 was repeated, but only using those probes corresponding to proteins on the array conserved with ≥90% sequence identity in S. mitis and S. pseudopneumoniae. Longitudinal samples from the same individual again cluster together, demonstrating the distinctiveness of individuals’ antibody repertoires was apparent even to proteins exhibiting little variation across S. pneumoniae isolates. The longer-range structure of the projection is stochastic, and these differences with Figure 2 are not biologically relevant.