Activation of an Effective Immune Response after Yellow Fever Vaccination Is Associated with the Genetic Background and Early Response of IFN-γ and CLEC5A

Abstract

The yellow fever vaccine (YF17DD) is highly effective with a single injection conferring protection for at least 10 years. The YF17DD induces polyvalent responses, with a TH1/TH2 CD4⁺ profile, robust T CD8⁺ responses, and synthesis of interferon-gamma (IFN-γ), culminating in high titers of neutralizing antibodies. Furthermore, C-type lectin domain containing 5A (CLEC5A) has been implicated in innate outcomes in other flaviviral infections. Here, we conducted a follow-up study in volunteers immunized with YF17DD, investigating the humoral response, cellular phenotypes, gene expression, and single nucleotide polymorphisms (SNPs) of IFNG and CLEC5A, to clarify the role of these factors in early response after vaccination. Activation of CLEC5A⁺ monocytes occurred five days after vaccination (DAV). Following, seven DAV data showed activation of CD4⁺ and CD8⁺T cells together with early positive correlations between type II IFN and genes of innate antiviral response (STAT1, STAT2, IRF7, IRF9, OAS1, and RNASEL) as well as antibody levels. Furthermore, individuals with genotypes rs2430561 AT/AA, rs2069718 AG/AA (IFNG), and rs13237944 AC/AA (CLEC5A), exhibited higher expression of IFNG and CLEC5A, respectively. Together, we demonstrated that early IFN-γ and CLEC5A responses, associated with rs2430561, rs2069718, and rs13237944 genotypes, may be key mechanisms in the long-lasting immunity elicited by YF17DD.

Keywords: CLEC5A; interferon gamma; yellow fever vaccine.

Figure 1

Phenotypes of monocytes and T lymphocytes during vaccination follow-up. Peripheral blood mononuclear cells (PBMCs) from vaccinated volunteers (n = 38, 15 first time vaccinated and 23 s time vaccinated) were stimulated for 24 h with YF17DD in vitro. Bar-graphs show the cell population frequencies of (A) activated monocytes, represented as CD3⁻CD95⁻CD14⁺HLA-DR⁺, and (B) activated monocytes expressing CLEC5A, represented as CD3⁻CD95⁻CD14⁺HLA-DR⁺CLEC5A⁺. Cell population frequencies were calculated by frequency in YF17DD stimulated experimental condition minus frequency in unstimulated (mock) experimental condition. (C) Cells from one subject are represented as a density plot (FlowJo Tree Star^®) showing the kinetics of CLEC5A expression on activated monocytes. (D) Activated CD4⁺ T cells are represented as CD3⁺CD95⁻CD4⁺HLA-DR⁺. (E) Activated CD8⁺ T cells are represented as CD3⁺CD95⁻CD4⁻HLA-DR⁺. Cell population frequencies were compared between time points using Kruskal–Wallis with Dunns post-test, with significant p value represented as * p < 0.05, ** p < 0.01, *** p < 0.001. Each point corresponds to one individual analyzed, with median and standard error of groups. (F) Graphic representation of cellular phenotypes during vaccination follow-up. Lines represent these percentage difference distributions over time for CD14⁺HLA-DR⁺CLEC5A⁺ (green), CD14⁺HLA-DR⁺ (red), CD8⁺HLA-DR⁺ (purple), and CD4⁺HLA-DR⁺ (turquoise).

Figure 2

Gene expression profile of the early responses in first time YF17DD-vaccinated volunteers (n = 15). (A) Radar chart representing the average log10 fold change (2ΔΔCt) of each gene and day after vaccination (DAV). Mean 2ΔΔCt values of CLEC5A, DAP12, STAT1, STAT2, IRF7, IRF9, OAS1, RNASEL, IL6, IL12, CXCL10, NOS1, AIM2, IFI16, IFNGR1, and IFNG clustered by 4 DAV (red), 7 DAV (yellow), and 10 DAV (blue). Fold change (2ΔΔCt) was compared between DAV by Kruskall–Wallis test with Dunn’s post-test using the GraphPad Prism 5 software. The heatmaps represent the correlation R values according to the Z score for 2ΔΔCt values of genes analyzed and anti-YF antibody production at (B) 7 DAV, and (C) 10 DAV. Spearman correlation and heatmaps by library “corrplor” from R-project. p value is represented as p < 0.1, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

CLEC5A and IFNG SNPs associated with YF17DD immunologic responses. (A–C) Relative expression of CLEC5A and IFNG at 7 DAV according to SNP genotyping. The expression of CLEC5A and IFNG was stratified according to (A) rs13237944, (B) rs2430561, and (C) rs2069718 genotypes (n = 38). (D–F) Anti-YF antibody production according to SNP genotyping. Anti-YF antibody production at 60 DAV was stratified according to (A) rs13237944, (B) rs2430561, and (C) rs2069718 genotypes (n = 19). Each dot corresponds to one individual analyzed, with median and standard error of groups. Statistical analysis was determined using Mann–Whitney test. p value is represented as * p < 0.05, ** p < 0.01. DAV: days after vaccination.