Antibody-independent, CD4+ T-cell-dependent protection against pneumococcal colonization elicited by intranasal immunization with purified pneumococcal proteins

Abstract

Immunity to pneumococcal colonization in mice by exposure to live or killed pneumococci has been shown to be antibody independent but dependent on CD4+ T cells. Here we show that intranasal immunization with pneumococcal proteins (pneumococcal surface protein C, adhesin A, and a pneumolysoid) can elicit a similar mechanism of protection. Colonization could be significantly reduced in mice congenitally deficient in immunoglobulins after intranasal immunization with this mixture of proteins; conversely, the depletion of CD4+ T cells in immunized wild-type mice at the time of challenge eliminated the protection afforded by immunization. Overall, our results show that intranasal immunization with a mixture of pneumococcal proteins protects against colonization in an antibody-independent, CD4+ T-cell-dependent manner.

FIG. 1.

Immunogenicity and protection against NP challenge by intranasal immunization with 3P-CT in wild-type mice. (A) Serum antibody concentrations in response to proteins PsaA, PspC, and PdT in immunized mice. Serum antibody concentrations in response to these proteins were measured in C57BL/6 mice that were immunized intranasally with CT alone, 3P-CT, or WCV-CT. Serum samples were obtained 3 weeks after the last immunizations. Antibody concentrations were determined by ELISA; median concentration and interquartile range are shown. (B) Results of nasopharyngeal challenge of immunized mice. Mice immunized as described above were challenged with strain 0603 4 weeks after the last immunization. Tracheal aspirates obtained 1 week later were serially diluted and plated for quantification of colonization. Density of colonization of each individual mouse is shown; lines represent the median densities of colonization.

FIG. 2.

Antibody independence and CD4⁺ T-cell dependence of protection against NP challenge by 3P-CT. (A) Nasopharyngeal challenge of immunized μMT (Ig deficient) mice with strain 0603. μMT mice were immunized with CT, 3P-CT, or WCV-CT and subsequently challenged as described in the text. Density of colonization of each mouse is shown; lines represent median densities of colonization. Mice immunized with 3P-CT or WCV-CT had significantly lower densities of colonization than mice that received CT alone. (B) Effect on protection against NP challenge of CD4⁺ or CD8⁺ T-cell depletion of immunized wild-type mice. C57BL/6 mice immunized with 3P-CT received anti-CD4⁺ or anti-CD8⁺ T-cell antibodies at the time of challenge. Whereas mice that received anti-CD8⁺ antibodies were significantly protected against colonization, the administration of anti-CD4⁺ antibodies abolished protection by the 3P-CT. Density of colonization of each mouse is shown; lines represent median densities of colonization. (C) Measurement of IL-17A secretion by splenocytes. Splenocytes from immunized and control mice were stimulated with individual proteins for 3 days as described in the text; supernatants were collected and assayed for IL-17A concentration by ELISA.