Adipokines, and not vitamin D, associate with antibody immune responses following dual BNT162b2 vaccination within individuals younger than 60 years

Abstract

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) led to a global health outbreak known as the COVID-19 pandemic which has been lasting since March 2020. Vaccine became accessible to people only at the beginning of 2021 which greatly helped reducing the mortality rate and severity of COVID-19 infection afterwards. The efficacy of vaccines was not fully known and studies documenting the immune responses following vaccination are continuing to emerge. Recent evidence indicate that natural infection prior vaccination may improve the antibody and cellular immune responses, while little is known about the factors influencing those processes. Here we investigated the antibody responses following BNT162b2 vaccination in relation to previous-infection status and age, and searched for possible biomarkers associated with the observed changes in immune responses. We found that the previous-infection status caused at least 8-times increase in the antibody titres, effect that was weaker in people over 60 years old and unaltered by the vitamin D serum levels. Furthermore, we identified adiponectin to positively associate with antibody responses and negatively correlate with pro-inflammatory molecules (MCP-1, factor D, CRP, PAI-1), especially in previously-infected individuals.

Keywords: BNT162b2 vaccination; MCP-1; PAI-1; SARS-CoV-2; adiponectin; age; antibody immune responses.

Figure 1

Spike RBD-specific IgG titre after two doses of BNT162b2 (BioNTech-Pfizer) vaccination. (A) Spike RBD-specific antibody titre after two doses of COVID-19 vaccination in both groups of subjects stratified by gender: infection-naïve (109 females, 40 males) and previously infected (30 females, 13 males) cases. The black and grey lines indicate the mean ± SEM (****P< 0.0001, ns, not significant; two-tailed Mann Whitney and Kruskal-Wallis followed by Dunn’s Multiple Comparison tests). (B) Spike RBD-specific antibody titre after two doses of COVID-19 vaccination in both groups of subjects stratified by age: infection-naïve (n = 149) and previously infected (n=33) cases. The black and grey lines indicate the mean ± SEM (****P< 0.0001, **P< 0.01, ns, not significant; two-tailed Mann Whitney and Kruskal-Wallis followed by Dunn’s Multiple Comparison tests). (C, D) Spike RBD-specific antibody titre in (C) infection-naïve and (D) infection-primed individuals stratified by age. Bars indicate the mean ± SEM (****P< 0.0001, ***P < 0.001, **P< 0.01, *P< 0.05, ns, not significant; two-tailed Mann Whitney and Kruskal-Wallis followed by Dunn’s Multiple Comparison tests).

Figure 2

Vitamin D levels moderately correlate with RBD specific IgG antibody titres in individuals under 60 years. (A) Vitamin D levels in the serum collected from both groups of subjects: infection-naïve (n = 103) and previously infected (n = 33) cases. The black and red lines indicate the mean ± SEM (ns, not significant; two-tailed Mann test). (B) Vitamin D levels according to gender in infection-naïve (77 females, 26 males) and previously infected (25 females, 8 males) cases. The black and grey lines indicate the mean ± SEM (ns, not significant; two-tailed Mann test). (C) The effect of age on vitamin D levels in infection-naïve subjects (R, correlation coefficient; *P< 0.05; F test). (D) The effect of age on vitamin D levels in infection-primed subjects (R, correlation coefficient; ns, not significant; F test). (E) The effect of vitamin D levels on the RBD-specific antibody response in infection-naïve subjects (R, correlation coefficient; *P< 0.05, ns, not significant; F test). The grey lines correspond to the group of individuals younger than 60 years, while the orange lines indicate the entire infection-naïve group. (F) The effect of vitamin D levels on the RBD-specific antibody response in infection-primed subjects (R, correlation coefficient; ns, not significant; F test). The grey lines correspond to the group of individuals younger than 60 years, while the orange lines indicate the entire infection-primed group.

Figure 3

Plasma profile of pro-inflammatory molecules following BNT162b2 vaccination in previously infected or naïve individuals. (A) MCP-1 levels, (B) CRP levels, (C) Factor D levels, and (D) PAI-1/SERPINE1 levels in the serum samples collected from infection-naïve (n = 92, 67 females, 25 males) and previously infected (n = 30, 23 females, 7 males) cases. The black and grey lines indicate the mean ± SEM (****P< 0.0001, ***P< 0.001, **P< 0.01, *P< 0.05, ns, not significant; two-tailed Mann Whitney and Kruskal-Wallis followed by Dunn’s Multiple Comparison tests). F, females; M, males.

Figure 4

Plasma profile of adipokines following BNT162b2 vaccination in previously infected or naïve individuals. (A) IL-6 levels, (B) IL-10 levels, (C) Adiponectin levels, and (D) Leptin levels in the serum samples collected from infection-naïve (n = 92, 67 females, 25 males) and previously infected (n = 30, 23 females, 7 males) cases. The black and grey lines indicate the mean ± SEM (****P< 0.0001, ns, not significant; two-tailed Mann Whitney and Kruskal-Wallis followed by Dunn’s Multiple Comparison tests). F, females; M, males.

Figure 5

Regression statistics describing the relationship between age, antibody response and various adipokines within the two main study groups: infection-naïve and infection-primed. Correlation coefficients (R) and statistical significances were computed for each pair of variables. (A) Heat map of R coefficients: left –upper corner corresponds to infection naïve cases, while the bottom-right corner corresponds to infection-primed cases. Linear regression analysis for (B) adiponectin levels and age (top) or adiponectin and RBD-specific antibody titre (bottom); (C) MCP-1 and adiponectin levels (top) or MCP-1 levels and antibody titre (bottom); (D) CRP and adiponectin levels (top) or CRP levels and antibody titre (bottom); (E) factor D and adiponectin levels (top) or factor D levels and antibody titre (bottom); (F) PAI-1/SERPINE1 and adiponectin levels (top) or PAI-1/SERPINE1 levels and antibody titre (bottom) in previously infected individuals. Data are presented as scatter plots with best-fit lines and 95% confidence bands (****P< 0.0001, ***P< 0.001, *P<0.05, ns, not significant; Spearman test).

Figure 6

mRNA expression of adipokines collected from GTEx database. (A) mRNA expression of SERPINE1 in fibroblasts, (B) mRNA expression of SERPINE1 and ADIPOQ in subcutaneous adipose tissue, (C) mRNA expression of CFD, ADIPOQ, and IL6 in visceral adipose tissue, and (D) mRNA expression of SERPINE1 and CFD in the lung. The bars indicate the mean ± SEM (****P< 0.0001, *P< 0.05; Kruskal-Wallis test).

Figure 7

Linear regression models generated to predict the antibody response within individuals younger than 60 years. Antibody responses in relation to regression-adjusted predicted values generated by associating (A) negative factors such as MCP-1 and factor D, (B) positive factors such as adiponectin and IL-6, or (C) combined factors such as MCP-1, factor D, adiponectin, IL-6, or (D) MCP-1 and adiponectin. Infection-primed cases are depicted as black dots, while the infection-naïve cases are shown in green.

Figure 8

ROC curves generated for negative and positive determinants of previous natural infection. ROC curves for (A) negative and (B) positive determinants. (C) ROC curves related to various associations of biomarkers. Model 2-1 comprises the values of MCP-1 and Factor D, model 2-2 comprises the values of adiponectin and IL-6, and model 2-3 comprises the biomarkers included in model 2-1 and 2-2. The AUC values between 0.8-0.9 define an excellent discrimination, while the AUC values > 0.9 denote an outstanding capacity of prediction.