Effect of immunization with cryptococcal antigenic fractions on survival of C57BL/6J mice challenged with C. neoformans. C57BL/6J mice received two i.p. injections 3 weeks apart of PBS, Ribi adjuvant system without antigen (ADJ), or Ribi adjuvant system admixed with 100 μg of crude MP or FT antigen. One week following the last injection, mice received an i.v. challenge of 10⁶ live C. neoformans cells. Mice were monitored for signs of disease and sacrificed when signs of disease were severe. P < 0.001 for comparison of survival of the MP, CR, and FT groups versus the ADJ control. P = 0.005 for comparison of the ADJ and PBS groups. The figure is representative of three to four independent experiments (summarized in Table 1), each of which had similar results.

Effect of immunization with cryptococcal antigenic fractions on organ fungal burden. C57BL/6J mice received two i.p. injections 3 weeks apart of PBS, Ribi adjuvant system without antigen (ADJ), or the Ribi adjuvant system admixed with 100 μg of crude or MP antigen. One week following the last injection, mice received an i.v. challenge of 10⁶ live C. neoformans cells. Ten days postinfection, mice were sacrificed, and the number of CFU in the brain and kidneys was determined. Data are expressed as CFU per gram of tissue. P < 0.05 for comparison of adjuvant with CR or MP for brain and kidneys.

Effect of deglycosylation of MP on survival and organ fungal burden following cryptococcal challenge. C57BL/6J mice received two i.p. injections 3 weeks apart of PBS, Ribi adjuvant system without antigen (ADJ), or Ribi adjuvant system admixed with 50 μg of MP or β-eliminated MP (MP-BE). One week following the last injection, mice received an i.v. challenge of 10⁶ live C. neoformans cells. (A) Mice (10 per group) were monitored for signs of disease and sacrificed when signs of disease were severe. P < 0.001 for comparison of survival of the ADJ group with that of the PBS, MP, and BE-MP groups. (B) Ten days postinfection, mice (three to four per group) were sacrificed, and the numbers of CFU per gram of tissue in the brain and kidneys were determined.

Effect of MP immunization on tissue cytokine levels following infection. C57BL/6J mice received two i.p. injections 3 weeks apart of Ribi adjuvant system without antigen (Adjuvant-infected) orRibi adjuvant system admixed with 100 μg of MP antigen (MP-infected). One week following the last injection, mice received an i.v. challenge of 10⁶ live C. neoformans cells. Ten days postinfection, mice were sacrificed, and cytokine levels in homogenized organs were assayed. Organ cytokine levels in uninfected, unimmunized mice (Non-infected) were also determined to establish baseline levels. Data are expressed as picograms of cytokine per gram of tissue. n = 3 mice per group. ND, not detectable. Asterisks denote P < 0.05 for comparison of adjuvant-infected with MP-infected groups.

Influence of dose and boiling on MP-dependent protection. Groups of 10 C57BL/6J mice received two i.p. injections 3 weeks apart of PBS, Ribi adjuvant system without antigen (ADJ), or Ribi adjuvant system admixed with 50 μg of native MP (MP 50), 5 μg of native MP (MP 5), or 50 μg of boiled MP (MP-B). One week following the last injection, mice received an i.v. challenge of 10⁶ live C. neoformans cells. Mice were monitored for signs of disease and sacrificed when signs of disease were severe. P < 0.05 for comparison of the ADJ with the MP 50, MP 5, and MP-B groups.

Histopathological examination of tissues from immunized mice. C57BL/6J mice received two i.p. injections 3 weeks apart of Ribi adjuvant system without antigen or Ribi adjuvant system admixed with 100 μg of MP antigen. One week following the last injection, mice received an i.v. challenge of 10⁶ live C. neoformans cells. Ten days postinfection, mice were sacrificed, and their brains, livers, and kidneys were analyzed. (A) Brain specimens from mice immunized with MP plus adjuvant contained individual fungal organisms (arrow) or colonies, some of which were infiltrated with mononuclear cells (arrowhead). (B) Brain specimens from control mice immunized with adjuvant also contained fungal colonies, some of which were rather large, but no inflammation. (C) Liver specimens from mice immunized with MP plus adjuvant contained multifocal pyogranulomas (arrowheads), some of which contained prominent aggregates of eosinophils. The few organisms observed in these sections were associated with inflammatory foci. (D) Control mouse liver specimens contained numerous fungal organisms, which were randomly distributed throughout the liver, present individually or in colonies (arrows). Compared to mice immunized with MP plus adjuvant, control mouse liver specimens also contained an increased number of pyogranulomas, only some of which were associated with the fungal colonies. (E) Examination of kidney specimens from mice immunized with MP plus adjuvant revealed mild, multifocal interstitial nephritis, dominated by mononuclear cells and fewer neutrophils (arrowhead). Occasionally, these foci contained small numbers of fungal organisms (arrow). (F) In contrast, control mouse kidney specimens contained numerous fungal organisms, which were present individually or in small colonies (arrows). These kidney specimens contained fewer foci of interstitial nephritis, and only occasionally were the inflammatory foci associated with organisms. The results are representative of two to three mice per group.

Contribution of T and B cells to cryptococcal fraction-dependent protection. C57BL/6J wild-type, B-cell-deficient (knockout [KO]) (MuMT B6.129S2-Igh-6^tm1Cgn), or T cell-deficient (B6.129P2-Tcrb^tm1Mom Tcrd^tm1Mom) mice received two i.p. injections 3 weeks apart of Ribi adjuvant system without antigen (ADJ) or Ribi adjuvant system admixed with 100 μg of MP or FT antigen. One week following the last injection, mice received an i.v. challenge of 10⁶ live C. neoformans cells. Mice were monitored for signs of disease and sacrificed when signs of disease were severe. P < 0.001 for comparison of survival of the FT group versus ADJ for the wild-type and B-cell-deficient mice. P = 0.015 and P < 0.001 for comparison of survival of the MP group versus ADJ for the wild-type and B-cell-deficient mice, respectively.

Effect of immunization with cryptococcal antigenic fractions on survival of CBA/J mice challenged with C. neoformans. The conditions are identical to those described in the legend to Fig. 1, except CBA/J mice were utilized. P < 0.001 for comparison of survival of the PBS and CR groups versus the ADJ control. P = 0.03 for comparison of the ADJ and MP groups.