Immune System Modulation by the Adjuvants Poly (I:C) and Montanide ISA 720

Abstract

Adjuvants are essential for vaccine development, especially subunit-based vaccines such as those containing recombinant proteins. Increasing the knowledge of the immune response mechanisms generated by adjuvants should facilitate the formulation of vaccines in the future. The present work describes the immune phenotypes induced by Poly (I:C) and Montanide ISA 720 in the context of mice immunization with a recombinant protein based on the Plasmodium vivax circumsporozoite protein (PvCSP) sequence. Mice immunized with the recombinant protein plus Montanide ISA 720 showed an overall more robust humoral response, inducing antibodies with greater avidity to the antigen. A general trend for mixed Th1/Th2 inflammatory cytokine profile was increased in Montanide-adjuvanted mice, while a balanced profile was observed in Poly (I:C)-adjuvanted mice. Montanide ISA 720 induced a gene signature in B lymphocytes characteristic of heme biosynthesis, suggesting increased differentiation to Plasma Cells. On the other hand, Poly (I:C) provoked more perturbations in T cell transcriptome. These results extend the understanding of the modulation of specific immune responses induced by different classes of adjuvants, and could support the optimization of subunit-based vaccines.

Keywords: Plasmodium vivax; immune response; montanide; poly (I:C); vaccine.

Figure 1

Montanide ISA 720 and Poly (I:C) elicit different antibody response profiles. C57BL/6 mice (n = 6) were vaccinated with three doses of PvCSP + Montanide ISA 720 or PvCSP + Poly (I:C). (A) PvCSP-specific IgG titers (Log) elicited 14 days after each immunizing dose were determined by ELISA. (B) Anti-PvCSP IgG1, IgG2b, IgG2c, and IgG3 subclasses were determined by ELISA 14 days after the last immunization. (C) Antibody affinity to PvCSP was measured after 30min of incubation in urea gradient solutions. Statistical significance was calculated on 6M urea point. Asterisks denote statistical differences; *p < 0.05; ****p < 0.0001; ns, not significant differences.

Figure 2

Montanide ISA 720 favors serum inflammatory cytokines, while Poly (I:C) elicits a balanced profile. The serum levels of cytokines were measured in mice sera obtained 14 days after the last immunization, by Luminex using the Milliplex MAP Mouse Cytokine/Chemokine Magnetic Bead Panel - Premixed 32 Plex. Sera samples were measured in triplicates; only the cytokines with the highest expression are depicted. Asterisks denote statistical differences; *p < 0.05; **p < 0.01.

Figure 3

Transcriptome analysis of B cells show a strong “heme metabolism” gene signature in Montanide-adjuvanted mice. Differentially expressed genes (DEGs) were determined with the R-Bioconductor DESeq2 package, using the raw reads from the different samples as input. Genes that displayed a p-adjusted < 0.05 were considered for downstream analysis. (A, B), Venn diagrams depicting DEGs detected only in Poly (I:C)-adjuvanted, only in Montanide-adjuvanted or in both groups of mice. (C), volcano plot highlighting the Poly (I:C)-induced DEGs. (D), gene enrichment analysis of the 4-fold-induced genes by Poly (I:C). (E), HOMER motif analysis in the promoters of 3-fold induced genes by Poly (I:C), performed with the Enrichr webtool. (F), volcano plot highlighting the Montanide-induced DEGs. (G), gene enrichment analysis of the 4-fold-induced genes by Montanide ISA 720. (H), HOMER motif analysis in the promoters of 3-fold induced genes by Montanide ISA 720, performed with the Enrichr webtool.

Figure 4

Poly (I:C) and Montanide ISA 720 induced almost the same number of DEGs in T CD4⁺ lymphocytes, although specific gene signatures were more significantly detected in Poly (I:C)-adjuvanted mice. Differentially expressed genes (DEGs) were determined with the R-Bioconductor DESeq2 package, using the raw reads from the different samples as input. Genes that displayed a p-adjusted < 0.05 were considered for downstream analysis. (A, B), Venn diagrams depicting DEGs detected only in Poly (I:C)-adjuvanted, only in Montanide-adjuvanted or in both groups of mice. (C), volcano plot highlighting the Poly (I:C)-induced DEGs. (D), gene enrichment analysis of the 4-fold-induced genes by Poly (I:C), performed with the Enrichr webtool. (E), volcano plot highlighting the Montanide-induced DEGs. (F), gene enrichment analysis of the 4-fold-induced genes by Montanide ISA 720, performed with the Enrichr webtool.

Figure 5

Poly (I:C) induced stronger perturbation in T CD8⁺ lymphocyte transcriptome in comparison to Montanide ISA 720. Differentially expressed genes (DEGs) were determined with the R-Bioconductor DESeq2 package, using the raw reads from the different samples as input. Genes that displayed a p-adjusted < 0.05 were considered for downstream analysis. (A, B), Venn mulsionMontanide-adjuvanted or in both groups of mice. (C), volcano plot highlighting the Poly (I:C)-induced DEGs. (D), gene enrichment analysis of the 4-fold-induced genes by Poly (I:C), performed with the Enrichr webtool. (E), volcano plot highlighting the Montanide-induced DEGs. (F), gene enrichment analysis of the 4-fold-induced genes by Montanide ISA 720, performed with the Enrichr webtool. e.