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.

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.