T(H)17-based vaccine design for prevention of Streptococcus pneumoniae colonization

Abstract

Streptococcus pneumoniae is a leading cause of mortality in young children. While successful conjugate polysaccharide vaccines exist, a less expensive serotype-independent protein-based pneumococcal vaccine offers a major advancement for preventing life-threatening pneumococcal infections, particularly in developing nations. IL-17A-secreting CD4+ T cells (T(H)17) mediate resistance to mucosal colonization by multiple pathogens including S. pneumoniae. Screening an expression library containing >96% of predicted pneumococcal proteins, we identified antigens recognized by T(H)17 cells from mice immune to pneumococcal colonization. The identified antigens also elicited IL-17A secretion from colonized mouse splenocytes and human PBMCs suggesting that similar responses are primed during natural exposure. Immunization of two mouse strains with identified antigens provided protection from pneumococcal colonization that was significantly diminished in animals treated with blocking CD4 or IL-17A antibodies. This work demonstrates the potential of proteomic screening approaches to identify specific antigens for the design of subunit vaccines against mucosal pathogens via harnessing T(H)17-mediated immunity.

Figure 1. SP2108 and SP0148 are recognized by T_H17 cells of mice colonized with pneumococcus and adult human volunteers

(A) Antigen-specific IL-17A secretion by splenocytes isolated from mice after intranasal exposure to live pneumococcus three times at one week intervals. Bars represent median IL-17A values with interquartile range. *p<0.05, **p<0.01, ***p<0.001 by Mann-Whitney test when compared with secretion of IL-17A in response to medium (DMEM) alone. NS=not statistically different (B) Antigen-specific IL-17A secretion by splenocytes of C57BL/6J mice ten days following a single intranasal exposure to live pneumococcus. (C) The amount of IL-17A secreted by human PBMC stimulated with the indicated antigen. 9 of 11 donors had a response to WCA above the 50 pg/ml IL-17A cutoff (dashed grey line). Only WCA responders are shown here and were included in analysis. Each symbol represents the IL-17A value from a single donor with the GFP-induced IL-17A response subtracted. Median GFP value was 52 pg/mL. Bars represent median values. *p<0.05, **p<0.01 by paired Wilcoxon rank sum test when compared with IL-17A secretion following stimulation with GFP for the protein antigens or DMEM alone (median DMEM value at limit of detection; dotted black line) for WCA. NS=not statistically different.

Figure 2. Intranasal immunization with SP2108, SP0148 and SP0882 induces T_H17 responses and protection against pneumococcal colonization

(A) Enumeration of pneumococcus in nasal washes seven days after intranasal challenge by pneumococcus in C57BL/6 mice that were previously immunized with the indicated antigen and cholera toxin. Bars represent median CFU per immunization group. * p<0.05, **p<0.01, ***p<0.001 compared with ICP47 immunized group by Mann Whitney test (B–C) IL-17A secretion by whole blood cells isolated from the same immunized mice prior to challenge when stimulated with (B) the immunizing protein or (C) WCA. Bars represent median IL-17A values with interquartile range. *p<0.05, **p<0.01, ***p<0.001 by Mann-Whitney test when compared with ICP47 immunized group. NS=not statistically different.

Figure 3. SP2108 and SP0148 protect mice from pneumococcal colonization in a CD4⁺ T cell and IL-17A dependent fashion

(A) Enumeration of pneumococcus in nasal washes seven days after intranasal challenge of C57BL/6 mice that were previously immunized with a mixture of SP2108, SP0148 (Pair) and cholera toxin (CT) and then treated with anti-CD4, anti-IL-17A or an isotype control antibody. Bars represent median CFU per group. p value for bracketed groups is indicated. NS= not statistically different. (B) Flow cytometric analysis of CD4⁺ cells in spleens of immunized mice treated with anti-CD4, anti-IL-17A or an isotype control antibody.