Mucosal immunization with the live attenuated vaccine SPY1 induces humoral and Th2-Th17-regulatory T cell cellular immunity and protects against pneumococcal infection

Abstract

Mucosal immunization with attenuated vaccine can protect against pneumococcal invasion infection, but the mechanism was unknown. Our study found that mucosal delivery with the live attenuated SPY1 vaccine strain can confer T cell- and B cell-dependent protection against pneumococcal colonization and invasive infection; yet it is still unclear which cell subsets contribute to the protection, and their roles in pneumococcal colonization and invasion remain elusive. Adoptive transfer of anti-SPY1 antibody conferred protection to naive μMT mice, and immune T cells were indispensable to protection examined in nude mice. A critical role of interleukin 17A (IL-17A) in colonization was demonstrated in mice lacking IL-17A, and a vaccine-specific Th2 immune subset was necessary for systemic protection. Of note, we found that SPY1 could stimulate an immunoregulatory response and that SPY1-elicited regulatory T cells participated in protection against colonization and lethal infection. The data presented here aid our understanding of how live attenuated strains are able to function as effective vaccines and may contribute to a more comprehensive evaluation of live vaccines and other mucosal vaccines.

FIG 1

Intranasal vaccination with SPY1 is strongly protective against infection with heterogenic S. pneumoniae strains. (A) C57BL/6 mice were i.n. immunized with SPY1 plus CT or with CT alone four times, and at 2 weeks after the final vaccination, mice (n = 8) were i.n. challenged with 1 × 10⁸ CFU of CMCC 31693 (serotype 19F) and TIGR4. At 72 h postchallenge mice were sacrificed, and bacterial loads in nasal washes and lungs were determined. (B) The immunized or control mice (n = 15) were i.n. infected with either pneumococcal strain NCTC 7466 (D39, serotype 2; 5 × 10⁷ CFU), strain CMCC 31436 (serotype 3; 1.5 × 10⁸ CFU), strain CMCC 31203 (serotype 3; 4 × 10⁸ CFU), strain CMCC 31207 (serotype 6B; 5 × 10⁸ CFU), or strain CMCC 31614 (serotype 14; 4 × 10⁸ CFU). The survival rates were observed for 21 days postchallenge. *, P < 0.05; ** P < 0.01; ***, P < 0.001.

FIG 2

Immunization with SPY1 stimulated both humoral and cellular immunity. C57BL/6 mice were i.n. immunized with SPY1 plus CT or with CT alone four times, and on day 7 after the final vaccination, SPY1-specific IgG titers in serum and SPY1-specific IgA titers in saliva (sIgA) (A) and concentrations of IFN-γ, IL-4, IL-17A, and IL-10 in supernatants of stimulated splenocytes (B) were detected by ELISA. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 3

Both humoral and cellular immunity participated in SPY1-mediated protection in vivo and in vitro. μMT and nude mice were immunized with SPY1 plus CT or with CT alone and challenged with 1 × 10⁸ CFU of strain CMCC 31693 (serotype 19F) and 5 × 10⁷ CFU of NCTC 7466 (D39, serotype 2). The bacterial densities in nasal washes and lungs were detected (A), and the survival rates were observed for 21 days postinfection (B). Serum (200 μl) or approximately 1 × 10⁸ whole spleen cells (WSC) from immunized or control mice were i.p. transferred to μMT mice or nude mice. Twenty-four hours later, the mice were i.n. infected with 5 × 10⁷ CFU of NCTC 7466 (D39, serotype 2) or 1 × 10⁸ CFU of CMCC 31693 (serotype 19F). CFU counts in the upper respiratory tract (C) and survival rates (D) were observed. The data points represent the means (± standard deviations) of values from 4 to 6 mice (A and C) or from 10 to 15 mice (B and D). (E) Bacteria were added to HL-60 cells at a ratio of 10:1 (bacteria to cells; 4 × 10⁵ cells). Killing was defined as the percent reduction in CFU counts in wells containing cocultures of phagocyte cells and bacteria compared to wells containing only bacteria. Data represent the means ± standard errors of the means. Results are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

FIG 4

Acquired immunity to pneumococcal colonization and invasive infection are dominated by SPY1-specific Th17 and Th2 immune responses, respectively. IFN-γ, IL-4, or IL-17A knockout (KO) C57BL/6 mice were i.n. immunized with SPY1 plus CT or with CT alone and challenged with 1 × 10⁸ CFU of S. pneumoniae strain CMCC 31693 (serotype 19F) and 5 × 10⁷ CFU of S. pneumoniae NCTC 7466 (D39, serotype 2). At 72 h postinfection, mice infected with S. pneumoniae strain CMCC 31693 (serotype 19F) were sacrificed, and bacterial loads in nasal washes (A) and lungs (B) were determined. (C) The percentages of neutrophils and macrophages in lungs from the above groups were detected by flow cytometry (n = 4 to 6 mice for each group). (D to F) Survival rates of mice infected with S. pneumoniae NCTC 7466 (D39, serotype 2) were observed for 21 days postchallenge (n = 10 to 15 mice for each group). *, P < 0.05; **, P < 0.01; ***, P < 0.001. wt, wild type; PMNs, polymorphonuclear cells.

FIG 5

IL-17A-mediated inflammatory cell infiltrations and increased expression of IL-10 in C57BL/6 mice immunized with SPY1. (A) Wild-type C57BL/6 mice and IL-17A knockout (KO) C57BL/6 mice were i.n. immunized with SPY1 plus CT or with CT alone and challenged with 1 × 10⁸ CFU of S. pneumoniae strain CMCC 31693 (serotype 19F). At 72 h postinfection, mice were sacrificed, and heads were removed, fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin. The presence of inflammatory cell infiltrations in the submucosa at the junction of the olfactory and respiratory epithelium was observed. Images are from the following: frame a, C57BL/6 mice immunized with SPY1 plus CT; frame b, C57BL/6 mice immunized with CT alone; frame c, naive C57BL/6 mice; frame d, IL-17A knockout C57BL/6 mice immunized with SPY1 plus CT; frame e, IL-17A knockout C57BL/6 mice immunized with CT alone; frame f, naive IL-17A knockout C57BL/6 mice. Original magnification for sections stained with hematoxylin and eosin, ×100. (B) Immunohistochemistry staining demonstrated the expression of the immunoregulatory cytokine IL-10 in lung tissues. C57BL/6 mice were i.n. immunized with 1 × 10⁸ CFU of SPY1 plus 1 μg of CT or with 1 μg of CT four times at 1-week intervals. On day 7 after the last vaccination, lungs were aseptically removed, fixed in 4% paraformaldehyde, embedded in paraffin, and stained with anti-IL-10 antibodies and streptavidin horseradish peroxidase chemistry according to standard protocols. Images are from the following: frame a, C57BL/6 mice immunized with CT alone; frame b, C57BL/6 mice immunized with SPY1 plus CT; frame c, naive C57BL/6 mice. Original magnification for immunofluorescence staining, ×100. Images are representative of staining observed in the lungs of mice within the group (n = 4 to 6 mice).

FIG 6

SPY1-specific Treg cells are protective against S. pneumoniae infection. (A) C57BL/6 mice were i.n. immunized with 1 × 10⁸ CFU of SPY1 plus 1 μg of CT or with 1 μg of CT four times at 1-week intervals. On day 7 after the last vaccination, lungs from immunized and control mice were aseptically removed and homogenized, and single lung cells were stained with anti-mouse CD4-FITC and anti-mouse CD25-APC, followed by anti-mouse Foxp3-APC according to the manufacturer's instructions; cells were analyzed using a Becton Dickinson FACSCalibur flow cytometer. The percentages of CD4⁺ T cells which were CD25⁺ Foxp3⁺ Treg were calculated. During the vaccination period, another group of immunized mice were i.p. injected with peptide P17 to downregulate Treg cell activity. On day 7 after the last vaccination, percentages of CD4⁺ CD25⁺ Foxp3⁺ T cells (B), the concentration of IL-10 (C), and the percentages of neutrophils and macrophages in lungs were determined (E). At 2 weeks after the final immunization, P17- or PBS-treated mice were i.n. challenged with 1 × 10⁸ CFU of CMCC 31693 (serotype 19F) and 5 × 10⁷ CFU of NCTC 7466 (D39, serotype 2). The bacterial densities in nasal washes and lungs were detected (D) and the survival rates were observed until day 21 postinfection (F). The data points represent the median (± standard deviations) of values from 4 to 6 mice (A, B, C, D, and E) or from 10 to 15 mice (F).