Mucosal Immunization Has Benefits over Traditional Subcutaneous Immunization with Group A Streptococcus Antigens in a Pilot Study in a Mouse Model

Abstract

Group A Streptococcus (GAS) is a major human pathogen for which there is no licensed vaccine. To protect against infection, a strong systemic and mucosal immune response is likely to be necessary to prevent initial colonization and any events that might lead to invasive disease. A broad immune response will be necessary to target the varied GAS serotypes and disease presentations. To this end, we designed a representative panel of recombinant proteins to cover the stages of GAS infection and investigated whether mucosal and systemic immunity could be stimulated by these protein antigens. We immunized mice sublingually, intranasally and subcutaneously, then measured IgG and IgA antibody levels and functional activity through in vitro assays. Our results show that both sublingual and intranasal immunization in the presence of adjuvant induced both systemic IgG and mucosal IgA. Meanwhile, subcutaneous immunization generated only a serum IgG response. The antibodies mediated binding and killing of GAS cells and blocked binding of GAS to HaCaT cells, particularly following intranasal and subcutaneous immunizations. Further, antigen-specific assays revealed that immune sera inhibited cleavage of IL-8 by SpyCEP and IgG by Mac/IdeS. These results demonstrate that mucosal immunization can induce effective systemic and mucosal antibody responses. This finding warrants further investigation and optimization of humoral and cellular responses as a viable alternative to subcutaneous immunization for urgently needed GAS vaccines.

Keywords: Streptococcus pyogenes; group A Streptococcus; intranasal; mucosal; multicomponent; strep A; sublingual; vaccines.

Figure 1

Sera IgG response to vaccine antigens. Test bleeds from Day 21 (light grey) and terminal bleeds from Day 36 (dark grey) were collected and tested by ELISA for an IgG response to each vaccine antigen in individual mice for sublingual (SL), intranasal (IN) or subcutaneous (SC) administration or for the PBS control with (+) or without (-) adjuvant. Kruskal-Wallis ANOVA was performed to compare groups with the PBS control. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. One-way ANOVA was performed to test for differences between the D21 and D36 response. #, p < 0.05; ##, p < 0.01.

Figure 2

Mucosal secreted IgA response to vaccine antigens. Distal secreted IgA was detected in terminal intestinal washes from individual mice for sublingual (SL), intranasal (IN), subcutaneous (SC) and PBS control with (+) or without (-) adjuvant. Kruskal-Wallis ANOVA was performed to compare groups with the PBS control. * p < 0.05; ** p < 0.01, *** p < 0.001.

Figure 3

Antibodies can bind to GAS cells and promote killing by OPA. GAS cultures were incubated with sera from sample groups and investigated for IgG binding by flow cytometry (A) and killing activity (B). POS, positive control; SL, sublingual; IN, intranasal; SC, subcutaneous; PBS, negative control group. (A) Immunostaining was conducted with sera samples and analysed by flow cytometry. Specific binding was demonstrated with a shift in FITC fluorescence. Mean fluorescent intensity (MFI) is shown numerically for each panel with (+) and without (-) adjuvant indicated. Dashed line, PBS control; light grey, without adjuvant; dark grey, with adjuvant. (B) Opsonophagocytosis assays (OPA) were conducted to demonstrate killing activity of sera in the presence of DMF-differentiated HL60 cells and baby rabbit complement. Percentage killing was calculated by CFU relative to a non-immune standard normal mouse sera control. Adjuvanted groups are indicated with black bars and non-adjuvanted groups are indicated with grey bars. Data are presented as the mean % killing +/- standard deviation. One-way ANOVA comparison with non-immune sera is indicated. *, p < 0.05.

Figure 4

Antibodies can bind to GAS cells with functional activity. GAS cultures were incubated with immune sera samples diluted 1/100 (A) or mucosal secretions (intestinal) normalised to 50 mg/mL (B) and investigated for blocking of binding to HaCaT monolayers. POS, positive control; SL, sublingual; IN, intranasal; SC, subcutaneous; PBS, negative control group. Adjuvanted groups are indicated with black bars and non-adjuvanted groups are indicated with grey bars. One-way ANOVA comparison with PBS control is indicated: *, p < 0.05; ***, p < 0.001; ****, p < 0.0001. One-way ANOVA comparison between adjuvanted and non-adjuvanted controls is indicated: +++, p < 0.001; ++++, p < 0.0001.

Figure 5

Antibodies have functional activity against specific vaccine antigens. Functional assays were conducted for SpyCEP (A) and Mac/IdeS (B) to test activity of immune sera. POS = positive control. Black bars, adjuvanted groups; grey bars, non-adjuvanted groups. One-way ANOVA significance is indicated above individual bars for comparison with the PBS control (****, p < 0.0001), and significance between groups is indicated with bars (+, p < 0.05; ++++, p < 0.0001). (A) An IL-8 cleavage assay was conducted with SpyCEP positive culture supernatants in the presence of sera samples with IL-8 cleavage compared to a no-sera control. Low percentage cleavage indicates high neutralisation of SpyCEP activity by the sample. (B) An IgG cleavage assay was conducted with recombinant Mac in the presence of sera samples. Cleavage was compared to a no-sera control as indicated by ELISA. Low percentage cleavage indicates high neutralisation of Mac activity by the sample.