Immunogenicity and efficacy of Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan peptide mimotope-protein conjugates in human immunoglobulin transgenic mice

Abstract

Peptide mimotopes of capsular polysaccharides have been proposed as antigens for vaccines against encapsulated pathogens. In this study, we determined the antibody response to and efficacy of P13, a peptide mimetic of the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM), in mice that produce human antibodies. P13 was conjugated to tetanus toxoid (TT) or diphtheria toxoid (DT) and administered subcutaneously in Alhydrogel with or without CpG to mice transgenic for human immunoglobulin loci (XenoMouse mice) and expressing either immunoglobulin G2 (IgG2) (G2 mice) or IgG4 (G4 mice). Mice were vaccinated and revaccinated two or three times. The serum antibody responses of the mice to GXM and P13 and antibody idiotype expression were analyzed by an enzyme-linked immunosorbent assay. The results showed that both P13-TT and P13-DT were antigenic, inducing a mimetic response to P13 in both G2 and G4 mice, and immunogenic, inducing a mimotope response including VH3 (idiotype)-positive antibodies to GXM in G2 but not G4 mice. CpG led to higher titers of IgG to P13 and GXM in P13-TT-vaccinated G2 mice. C. neoformans challenge of P13-protein conjugate-vaccinated and control G2 mice induced anamnestic IgG- and VH3-positive responses to GXM and was associated with a significantly decreased risk of death and a prolongation of survival in P13-DT-vaccinated mice compared to phosphate-buffered saline-treated or protein carrier-vaccinated mice. These findings reveal that P13 elicited a human antibody response with VH3 expression in human immunoglobulin transgenic mice that has been observed for human antibodies to GXM and support the concept that peptide mimotope-based vaccines may hold promise for the treatment of C. neoformans infections.

FIG. 1.

Titers of IgM (A, C, E, and G) and IgG (B, D, F, and H) to P13 in sera from P13-TT-vaccinated (A to D) and P13-DT-vaccinated (E to H) G2 and G4 mice. The y axis shows the inverse titer for the times after primary vaccination shown on the x axis. Hatched bars and black bars represent mice that did not and mice that did receive CpG, respectively. Overall, CpG increased the antigenicity of P13, the antigenicities of P13-TT and P13-DT were comparable, and G2 mice had a more robust response than G4 mice. Error bars represent the standard error of the mean of replicate samples. Statistically significant differences between CpG-treated and untreated mice, between P13-TT- and P13-DT-vaccinated mice, and between G2 and G4 mice are described in the text. There were five mice per group. Pre, preimmunization.

FIG. 2.

Titers of IgM (A, C, E, and G) and IgG (B, D, F, and H) to GXM (serotype D, 24067) in sera from P13-TT-vaccinated (A to D) and P13-DT-vaccinated (E to H) G2 and G4 mice. The y axis shows the inverse titer for the times after primary vaccination shown on the x axis. Hatched bars and black bars represent mice that did not and mice that did receive CpG, respectively. Overall, CpG increased the IgG response to GXM, the immunogenicities of P13-TT and P13-DT were comparable, and G4 mice did not manifest a GXM response. Error bars represent the standard error of the mean of replicate samples. Statistically significant differences between CpG-treated and untreated mice and between P13-TT- and P13-DT-vaccinated mice are described in the text. There were five mice per group. Pre, preimmunization.

FIG. 3.

Reactivity of sera from CpG- or P13-protein conjugate-vaccinated G2 mice with GXM from serotype A C. neoformans strain H99 or SB4. The y axis shows the inverse titer of antibodies for the immunogens on the x axis for sera obtained either 7 days or 30 days after primary vaccination in the C. neoformans challenge experiment. Each bar represents pooled sera from five vaccinated mice. Pre, preimmunization.

FIG. 4.

Titers of antibodies expressing the V_H3 determinants recognized by mouse MAbs D12 and 16.84 in P13-DT-vaccinated G2 and G4 mice. The y axis shows the inverse titer of antibodies for the times after primary vaccination shown on the x axis for G4 and G2 mice. Error bars represent the standard error of the mean. An asterisk indicates a P value of <0.05 for a comparison of titers in G2 and G4 mice, as determined by the Mann-Whitney U test. There were five mice per group. Pre, preimmunization.

FIG. 5.

Survival of P13-TT-, P13-DT-, and CpG-vaccinated and control G2 mice after C. neoformans infection. (A) The y axis shows the Kaplan-Meier survival plot for mice on the days after primary vaccination shown on the x axis. Mice were challenged 35 days after primary vaccination with C. neoformans strain 24067 and rechallenged 65 days later (on day 100 postvaccination), as shown by the arrow. An asterisk indicates a P value of <0.03 for a comparison of the survival of P13-DT-vaccinated mice to that of PBS-, DT-, and TT-treated mice, as determined by a Kaplan-Meier log rank test. (B) Cox hazard regression analysis of the survival data shown in panel A, depicting the increasing likelihood of death over time for each of the groups. A number sign indicates a P value of 0.031 for the reduction in the likelihood of death for P13-DT vaccination compared to PBS vaccination and a P value of 0.0571 for the overall reduction in risk of death for P13-DT vaccination compared to PBS vaccination. There were five mice per group.

FIG. 6.

Serum antibody profiles of P13-DT-, P13-TT-, and CpG-vaccinated G2 mice in the C. neoformans challenge experiment depicted in Fig. 5. Days 7 and 30 represent days after primary vaccination, and days 65 and 70 represent days after C. neoformans challenge. The y axis shows the inverse titer for the immunogens shown on the x axis at the designated times. GXM was from strain 24067. Error bars represent the standard error of the mean. An asterisk indicates a P value of ≤0.01 for a comparison of preimmunization (Pre) sera and sera obtained at days 7 and/or 30 after primary vaccination. A number sign indicates a P value of ≤0.01 for a comparison of day 30 after primary vaccination and days 65 and/or 70 after C. neoformans challenge. An ampersand indicates a P value of ≤0.01 for a comparison of day 65 after C. neoformans challenge and day 70 after the first C. neoformans challenge (day 5 after rechallenge). Comparisons were made by a one-way ANOVA with the Bonferroni posttest.