Isatis indigotica root polysaccharides as adjuvants for an inactivated rabies virus vaccine

Abstract

Adjuvants can enhance vaccine immunogenicity and induce long-term enhancement of immune responses. Thus, adjuvants are important for vaccine research. Polysaccharides isolated from select Chinese herbs have been demonstrated to possess various beneficial functions and excellent adjuvant abilities. In the present study, the polysaccharides IIP-A-1 and IIP-2 were isolated from Isatis indigotica root and compared with the common vaccine adjuvant aluminum hydroxide via intramuscular co-administration of inactivated rabies virus rCVS-11-G into mice. Blood was collected to determine virus neutralizing antibody (VNA) titers and B and T lymphocyte activation status. Inguinal lymph node samples were collected and used to measure B lymphocyte proliferation. Splenocytes were isolated, from which antigen-specific cellular immune responses were detected via ELISpot, ELISA and intracellular cytokine staining. The results revealed that both types of polysaccharides induce more rapid changes and higher VNA titers than aluminum hydroxide. Flow cytometry assays revealed that the polysaccharides activated more B lymphocytes in the lymph nodes and more B and T lymphocytes in the blood than aluminum hydroxide. Antigen-specific cellular immune responses showed that IIP-2 strongly induced T lymphocyte proliferation in the spleen and high levels of cytokine secretion from splenocytes, whereas aluminum hydroxide induced proliferation in only a small number of lymphocytes and the secretion of only small quantities of cytokines. Collectively, these data suggest that the polysaccharide IIP-2 exhibits excellent adjuvant activity and can enhance both cellular and humoral immunity.

Keywords: Adjuvant; Antibody titer; Cellular immunity; Polysaccharide; Rabies vaccine.

Fig. 1

Production of rabies-specific antibodies in mice following the administration of rCVS-11-G mixed with different adjuvants or no adjuvant (control). Mice were immunized twice by intramuscular injection in the hind leg at 2-week intervals. Blood was collected from 6 randomly selected mice of each group at 3, 7, 14, 21, 28 and 42 days after the first immunization. Rabies-specific antibody titers were measured using an FAVN test. Representative data are shown as the means ± SDs of 6 mice from each group and were analyzed using one-way ANOVA (*p < 0.05, **p < 0.01).

Fig. 2

ELISpot analysis of IFN-γ and IL-4 secretion and ICS assays for antigen-specific CD4⁺⁺ and CD8⁺⁺ T cell secretion of IFN-γ and IL-4 in mouse splenocytes. Spleens were collected from 3 mice per group 14 days after the second vaccination, and splenocytes were assayed by ELISpot and ICS assays. SFCs secreting IL-4 (A) and IFN-γ (B) were measured using a commercial ELISpot kit. RABV-specific CD4⁺ and CD8⁺ T cells were measured via ICS assays. Spleens were collected from 3 mice per group 14 days after the second vaccination and were stained with mouse anti-CD4, anti-CD8, anti-IFN-γ and anti-IL-4 monoclonal antibodies. CD4⁺ cells secreting IL-4 (C) or IFN-γ (D) and CD8⁺ cells secreting IL-4 (E) or IFN-γ (F) are shown in Fig. 3. The data represent the subtraction value means and SDs of 3 mice and were analyzed using one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 3

Flow cytometry analysis of B cells isolated from lymph nodes. Lymph nodes were collected from 3 mice per group and cultured as described in Section 2. Different adjuvants activated CD19⁺CD40⁺ B cells at 3 (A) and 6 (B) days after the first immunization. Representative data are shown as the means ± SDs of 3 mice per group and were analyzed using one-way ANOVA (*p < 0.05, **p < 0.01).

Fig. 4

Blood was collected from mice on day 6 after primary immunization. The proliferation of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells and CD19⁺CD40⁺ B cells following treatments with different adjuvants was measured by flow cytometry. (A) and (B) show CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells, and (C) shows B cells following activation with different adjuvants. The cells in (A), (B) and (C) were collected from the blood of 3 mice per group. The data represent the subtraction value means of 3 mice with SDs and were analyzed using one-way ANOVA (*p < 0.05, **p < 0.01).

Fig. 5

ELISA results showing the quantities of IL-2, IL-4, IL-10 and IFN-γ secreted by splenocytes. Spleens were isolated from 6 mice per group 14 days after the final vaccination, and isolated splenocytes were cultured and stained as described in Section 2. IL-2 (A), IL-4 (B), IL-10 (C) and IFN-γ (D) levels were measured using a commercial ELISA kit. Representative data are shown as the means ± SDs of 6 mice per group and were analyzed using one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 6

Challenge test in mice. All mice in each group (n = 10) were challenged with 100× IMLD₅₀ of the RABV street stain HuNPB3 by intramuscular injection 14 days after the second vaccination and were observed for 21 days. The results are shown in (A). The survival numbers from each group at different times post-challenge are shown. Post-exposure immune test in mice. Mice were challenged with 10× IMLD₅₀ HuNPB3 24 h before immunization and were observed for 21 days. The results are shown in (B). The survival numbers from each group at different times post-challenge are shown. Significant differences of survival rates between groups were assessed by Kaplan–Meier (**p < 0.01, ***p < 0.001).