Plasma Metabolomic Alterations Induced by COVID-19 Vaccination Reveal Putative Biomarkers Reflecting the Immune Response

Ioanna Dagla, Aikaterini Iliou, Dimitra Benaki, Evagelos Gikas, Emmanuel Mikros, Tina Bagratuni, Efstathios Kastritis, Meletios A Dimopoulos, Evangelos Terpos, Anthony Tsarbopoulos, Ioanna Dagla, Aikaterini Iliou, Dimitra Benaki, Evagelos Gikas, Emmanuel Mikros, Tina Bagratuni, Efstathios Kastritis, Meletios A Dimopoulos, Evangelos Terpos, Anthony Tsarbopoulos

Abstract

Vaccination is currently the most effective strategy for the mitigation of the COVID-19 pandemic. mRNA vaccines trigger the immune system to produce neutralizing antibodies (NAbs) against SARS-CoV-2 spike proteins. However, the underlying molecular processes affecting immune response after vaccination remain poorly understood, while there is significant heterogeneity in the immune response among individuals. Metabolomics have often been used to provide a deeper understanding of immune cell responses, but in the context of COVID-19 vaccination such data are scarce. Mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR)-based metabolomics were used to provide insights based on the baseline metabolic profile and metabolic alterations induced after mRNA vaccination in paired blood plasma samples collected and analysed before the first and second vaccination and at 3 months post first dose. Based on the level of NAbs just before the second dose, two groups, "low" and "high" responders, were defined. Distinct plasma metabolic profiles were observed in relation to the level of immune response, highlighting the role of amino acid metabolism and the lipid profile as predictive markers of response to vaccination. Furthermore, levels of plasma ceramides along with certain amino acids could emerge as predictive biomarkers of response and severity of inflammation.

Trial registration: ClinicalTrials.gov NCT04743388.

Keywords: COVID-19; LC-MS; NMR; ceramides; metabolomics; neutralizing antibodies (NAbs).

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Correlation of NMR metabolic features with immune response. (A) Manhattan-type plot showing the analysis of the 6555 CPMG NMR features with response at day 22. The signed −log10(p-value) is derived from Spearman correlation analysis with adjustment for age and sex. The dotted lines represent the threshold after multiple Bonferroni testing correction (blue line, p-value < 1.8 × 10−5) or FDR (black, p-value < 2.3 × 10−4), and the red dots represent the data points that remain significant after FDR. The horizontal axis is the NMR chemical shift (in ppm). In the upper panel, the 58 spectra (cyan) and their means (red) are depicted. Associations with (i) l-histidine (His), l-phenylalanine (Phe) and 3-methylhistidine (3-MH) and (ii) l-Glutamine (Gln) are shown. (B) Boxplots of the significant metabolites at all the 3 time points. In black, comparison of low vs. high responders within each time point. In orange, comparison between the three time points in the low responders group. In blue, comparison between the three time points in the high responders group. (C) Longitudinal change of the significant metabolites among the three time points. Asterisks indicate statistical significance using ANOVA: **** p < 0.0001, *** 0.0001 < p < 0.001, ** 0.001 < p < 0.01, * 0.01 < p < 0.05, ns: no significance.
Figure 2
Figure 2
Fold-change of high vs. low responders of metabolic ratios in Day 1. Columns show the numerator and rows the denominator of each ratio. For example, the l-Glutamine/d-Glucose ratio exhibits a 3.49-fold increase in the high responders compared to the low responders. The changes in the metabolic ratios found statistically significant using t-tests (p-value < 0.05) are in boxes. Asterisks (*) indicate statistical significance after FDR correction (p-value < 4.62 × 10−4). Color coding is based on conditional formatting of fold-change values.
Figure 3
Figure 3
NMR Multivariate Analysis. (A) Scores plots obtained from the OPLS-DA analysis of NMR CPMG from plasma samples of high vs. low responders at Day 1, Day 22 and 3 Months. The two groups are discriminated in all time points, with the discrimination being clearer at Day 1 and Day 22. (B) S-plot obtained from the OPLS-DA of NMR CPMG from plasma samples of high vs. low responders after first dose at Day 1. Spectral variables on the top-right corner (blue) are considered significantly increased in the high responders, while those on the lower-left corner (red) are increased in the low responders. Metabolites with p and p(corr)~0 (colored in grey) do not significantly impact the separation. Significant spectral variables and their assigned metabolites are shown.
Figure 4
Figure 4
Analysis of longitudinal encode-decode (LED) spectra. (A) LED NMR spectra (n = 58) with assignment. (B) Scores plots obtained from the OPLS-DA of high vs. low responders at Day 1 after first dose. The two groups are discriminated into (●) Low responders at Day 1 and (●) High responders at Day 1. (C) Boxplots of the statistically significant lipids. The p-value of t-test is indicated on the top. Lipids numbering is according to Table S5. Asterisks indicate statistical significance: *** 0.0001 < p < 0.001, ** 0.001 < p < 0.01, * 0.01 < p < 0.05.
Figure 5
Figure 5
LC–MS Multivariate Analysis. (A) Scores plots obtained from the partial least squares-discriminant analysis of ESI (−) MS analysis from plasma samples of high vs. low responders after first dose at Day 1 and (B) Scores plots obtained from the orthogonal partial least squares-discriminant analysis of ESI (−) MS analysis from plasma samples of high vs. low responders after 1st dose at Day 1. Clear clustering is observed between low and high responders: (■) Low responders at Day 1; (▲) High responders at Day 1.
Figure 6
Figure 6
Box and whiskers plots of the detected ceramides for the low vs. high responders. (A) Day 1: The differences were statistically significant; p-values < 0.05. (B) three months after the first dose (3M): The differences were not statistically significant; p-values > 0.05. Asterisks indicate statistical significance: *** 0.0001 < p < 0.001, ** 0.001 < p < 0.01, * 0.01< p < 0.05; ns: not significant.
Figure 7
Figure 7
Comparison of the Cer(d18:0/22:0) levels, employing ANOVA test, among the three time points; Day 1 (D1), Day 22 (D22) and 3 Months (3M) for low and high responders. Asterisks indicate statistical significance: *** 0.0001 p < 0.001, ** 0.001 < p < 0.01; ns: not significant.

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