Intranasal immunization confers protection against murine Pneumocystis carinii lung infection

Abstract

To evaluate the feasibility of mucosal immunization against Pneumocystis carinii (Pc) experimental infection, female BALB/c mice were intranasally immunized three times with soluble Pc antigens plus cholera toxin fraction B (Pc-CTB); control groups received either Pc antigen, CTB, or phosphate-buffered saline (PBS) alone. Two weeks after the last immunization, five animals from each group were sacrificed, and cellular and humoral immune responses were evaluated. The remaining five mice were CD4 depleted using a monoclonal antibody against mouse CD4 and inoculated with viable Pc. Significantly higher specific lymphoproliferative responses from tracheobronchial lymph node cells, immunoglobulin M (IgM) and IgG antibody levels in serum, and bronchoalveolar lavage (BAL)-derived IgA antibody concentrations were observed in the Pc-CTB group of mice relative to control groups (P < 0.01). Five weeks after challenge, no Pc organisms were observed in the lung smears of the Pc-CTB group, while the animals receiving antigen, adjuvant, or PBS had progressively higher numbers of Pc microorganisms. By Western blot analysis, a strongly reactive 55- to 60-kDa antigen was recognized by BAL IgA and by serum IgG. In summary, mucosal immunization elicited specific cellular and humoral immune responses and protected against Pc lung infection after immunosuppression.

FIG. 1

Humoral immune responses of IgG, IgA, IgG1, and IgG2a from BALs to mouse Pc antigen at the end of the experiment. After immunization, animals were CD4 cell depleted with rat anti-mouse CD4 MAb. After 10 days of antibody treatment, mice were inoculated with 1.5 × 10⁶ viable Pc. After five more weeks of biweekly anti-CD4 MAb injections, the remaining animals were sacrificed for analysis of specific IgG, IgA, IgG1, and IgG2a in BALs by ELISA, as described in Materials and Methods. A 405 nm, absorbance at 405 nm.

FIG. 2

SDS-PAGE and Western blot analysis of IgG and IgA antibodies from Pc-CTB-immunized mice to different Pc antigens. (A) Results of SDS-PAGE showing molecular weight markers (MWM) and sPc antigen. (B) Results of IgA Western blot analysis using 1:2 diluted BAL from Pc-CTB-immunized animals and sPc antigen. (C) Results of IgG Western blot analysis using 1:100 diluted serum from Pc-CTB-immunized animals and sPc antigen. Lanes: 1, sPc (no treatment); 2, proteinase K treatment. The arrow indicates a 55- to 60-kDa antigen.