Improvement influenza vaccine immune responses with traditional Chinese medicine and its active ingredients

Abstract

The current influenza vaccines are unable to provide effective protection in many cases, like influenza viruses strain antigenic drift or shift, and the influenza continues to cause significant annual morbidity and mortality. Improving the immune response to influenza vaccination is an unmet need. Traditional Chinese medicine (TCM) and its active ingredients are commonly known to have immunomodulatory properties. We therefore compared influenza vaccination alone or formulated with Astragali Radix (Huangqi in Chinese), and several representative ingredients of TCM, including lentinan (polysaccharide), panax notoginseng saponins (saponin), breviscapine (flavone), andrographolide (terpenoid), and a Chinese herbal compound (kangai) for their potential to enhance immune responses to influenza vaccine in mice. We found that all these TCM-adjuvants were able to increase hemagglutination inhibition (HAI) antibody titers, splenocyte proliferation, splenic T cell differentiation, bone marrow dendritic cell maturity, and both Th1 and Th2 cytokine secretion of influenza vaccine to varying degrees, and that had the characteristics of no excessive inflammatory responses and bidirectional regulation simultaneously. Taken together, our findings show that Astragali Radix exerts a more comprehensive effect on vaccine immunity, on both innate and adaptive immunity. The effects of lentinan and andrographolide on adaptive immunity were more significant, while the effects of breviscapine on innate immunity were stronger, and the other two TCM adjuvants were weaker. As the first report of a comprehensive evaluation of TCM adjuvants in influenza vaccines, the results suggest that TCM and their active ingredients are good candidates for enhancing the immune response of influenza vaccines, and that suitable TCMs can be selected based on the adjuvant requirements of different vaccines.

Keywords: active ingredients; adjuvant; immune responses; influenza vaccine; traditional Chinese medicine.

Figure 1

Schematic diagram of vaccination and sample collection.

Figure 2

Dynamic changes in the percentage of original weight among groups. A, PBS group; B, influenza vaccination alone group; C, influenza vaccine and aluminum hydroxide co-immunized group; D, influenza vaccine and lentinan co-immunized group; E, influenza vaccine and panax notoginseng saponins co-immunized group; F, influenza vaccine and breviscapine co-immunized group; G, influenza vaccine and andrographolide co-immunized group; H, influenza vaccine and Astragali Radix co-immunized group; I, influenza vaccine and kangai co-immunized group. The letters in the following figures have the same meaning.

Figure 3

The response of HAI antibody titers to H1N1 virus measured by the hemagglutination inhibition assay on days 14 and 30 after booster vaccination. Each group was compared with the group given influenza vaccination alone (^*p < 0.05, ^**p < 0.01, ^***p < 0.01), and influenza vaccine and Astragali Radix co-immunized group (^△p < 0.05).

Figure 4

Changes of spleen index (A) and thymus index (B) among groups. Significant differences were observed between the PBS group and the vaccination groups (^#p < 0.05, ^##p < 0.01, ^###p < 0.001), and between the influenza vaccination alone group and the other vaccination groups (^*p < 0.05, ^**p < 0.01, ^***p < 0.001).

Figure 5

Lymphocyte proliferation of the spleen stimulated with Con A (A) and LPS (B) among groups. One-way ANOVA followed by Tukey’s multiple comparison test was conducted to compare two groups. Significant differences were defined between the PBS group and the vaccination groups (^#p < 0.05, ^##p < 0.01, ^###p < 0.001), and between the influenza vaccination alone group and other vaccination groups (^*p < 0.05, ^**p < 0.01), and between the influenza vaccine and Astragali Radix co-immunized group and other TCM-adjuvant groups (^△△p < 0.01).

Figure 6

Changes in the expression of the Spleen T lymphocyte immunophenotype between groups detected by flow cytometry assay. (A) CD45⁺CD3⁺; (B) CD3⁺CD4⁺; (C) CD3⁺CD8⁺; (D) CD3⁺CD4⁺/CD3⁺CD8⁺; (E) CD3⁺CD4⁺CD25⁺; (F) representative flow cytometry plots of each detected T lymphocyte. A significant differences were defined between the influenza vaccination alone group and other vaccination groups (^*p < 0.05, ^**p < 0.01, ^***p < 0.001), and between the influenza vaccine and Astragali Radix co-immunized group and other TCM-adjuvant groups (^△△p < 0.01, ^△△△p < 0.001).

Figure 7

Changes in the expression of bone marrow co-stimulatory signaling molecules across the indicated groups as detected by flow cytometry. (A) CD45⁺; (B) CD11c⁺MHC II⁺; (C) CD11c⁺CD80⁺; (D) CD11c⁺CD86⁺; (E) representative flow cytometry plots of each detected co-stimulatory signaling molecules. Significant differences were defined between the influenza vaccination alone group and other vaccination groups (^*p < 0.05, ^**p < 0.01, ^***p < 0.001), and between the influenza vaccine and Astragali Radix co-immunized group and other TCM-adjuvant groups (^△p < 0.05, ^△△p < 0.01, ^△△△p < 0.001).

Figure 8

Changes in serum cytokine content (A, IL-4; B, IFN-γ; C, TNF-α; D, IL-12; E, IL-1β; F, MCP-1) among groups. The significant differences were defined between the PBS group and the vaccination groups (^#p < 0.05, ^##p < 0.01, ^###p < 0.001), and between the influenza vaccination alone group and the other vaccination groups (^*p < 0.05, ^**p < 0.01, ^***p < 0.001), and between the influenza vaccine and Astragali Radix co-immunized group and other TCM-adjuvant groups (^△p < 0.05, ^△△p < 0.01, ^△△△p < 0.001).

Figure 9

Schematic diagram of the mechanisms induced by TCM and its active ingredients to improve influenza vaccine immune responses. Figure drawn using Figdraw.