Genetic immunization converts the trypanosoma cruzi B-Cell mitogen proline racemase to an effective immunogen

Abstract

Trypanosoma cruzi is the etiologic agent of Chagas' disease. Acute T. cruzi infection results in polyclonal B-cell activation and delayed specific humoral immunity. T. cruzi proline racemase (TcPRAC), a T. cruzi B-cell mitogen, may contribute to this dysfunctional humoral response. Stimulation of murine splenocytes with recombinant protein (rTcPRAC) induced B-cell proliferation, antibody secretion, interleukin-10 (IL-10) production, and upregulation of CD69 and CD86 on B cells. Marginal zone (MZ) B cells are more responsive to T-cell-independent (TI) rTcPRAC stimulation than are follicular mature (FM) B cells in terms of proliferation, antibody secretion, and IL-10 production. During experimental T. cruzi infection, TcPRAC-specific IgG remained undetectable when responses to other T. cruzi antigens developed. Conversely, intradermal genetic immunization via gene gun (GG) delivered TcPRAC as an immunogen, generating high-titer TcPRAC-specific IgG without B-cell dysfunction. TcPRAC GG immunization led to antigen-specific splenic memory B-cell and bone marrow plasma cell formation. TcPRAC-specific IgG bound mitogenic rTcPRAC, decreasing subsequent B-cell activation. GG immunization with rTcPRAC DNA was nonmitogenic and did not affect the generation of specific IgG to another T. cruzi antigen, complement regulatory protein (CRP). These data demonstrate the utility of genetic immunization for the conversion of a protein mitogen to an effective antigen. Furthermore, coimmunization of TcPRAC with another T. cruzi antigen indicates the usefulness of this approach for multivalent vaccine development.

FIG. 1.

Y strain-derived rTcPRAC-induced B-cell proliferation. (A) Empty vector IMAC-purified protein (lane 1) and IMAC-purified rTcPRAC (lane 2). (B) rTcPRAC-induced B-cell proliferation compared with that for negative control in the absence of PMB-treated medium or that for 10 μg/ml LPS as a positive control. (C) Time course analysis of rTcPRAC-induced B-cell proliferation. Statistics compare each time point with the previous one. Data represent the mean percentages for triplicate repeats ± SD. ****, P < 0.00001; ***, P < 0.0001; **, P < 0.001 (Student's t test).

FIG. 2.

rTcPRAC-induced B-cell proliferation, surface activation phenotype, IgG secretion, and IL-10 secretion. BALB/c splenocytes were stimulated for 72 h with rTcPRAC. (A) Increased B-cell proliferation compared to that of controls (P < 0.0001) was dose dependent (P = 0.02; R² = 0.86; Pearson test). (B) Increased expression of CD69 and CD86 on B cells compared to that on negative controls (P < 0.0001) was dose dependent (P = 0.008 and R² = 0.93 for CD69 and P = 0.009 and R² = 0.92 for CD86). (C) CD69 and CD86 expression on B cells correlated with TcPRAC-induced B-cell proliferation (P < 0.001 and R² = 0.98 for CD69 and P = 0.01 and R² = 0.90 for CD86). (D) Increased IgG secretion compared to that in controls (P < 0.0001) was dose dependent (P = 0.03; R² = 0.94; Pearson test). (E) Increased IL-10 production compared to that in controls (P < 0.0001) was dose dependent (P = 0.02; R² = 0.88). (F) IL-10 production correlated with both B-cell proliferation and IgG production (P = 0.0001 and R² = 0.996 for B-cell proliferation and P = 0.0032 and R² = 0.962 for IgG production). Data are presented as means for duplicate or triplicate repeats ± SEM. Two-way ANOVA was used to compare treatment groups, and the Pearson test was used for correlation.

FIG. 3.

Differential rTcPRAC-induced TI MZ and FM B-cell stimulation. Splenocytes (spln), sorting-purified MZ B cells, or sorting-purified FM B cells were stimulated with 2.5 to 10 μg/ml rTcPRAC, negative control (cntrl), or 1 μg/ml LPS or were left unstimulated (medium control). For flow cytometry data, the CD19⁺ live cell gates were analyzed. (A) B-cell proliferation was measured by CFSE diminution after stimulation. (B) B-cell activation by surface phenotype was assessed after stimulation with 5 μg/ml rTcPRAC. (C) rTcPRAC-induced antibody and IL-10 secretion was assessed in culture supernatants from splenocyte, MZ B-cell, and FM B-cell stimulations. Data are presented as means for duplicate or triplicate repeats ± SEM. ***, P < 0.0001; **, P < 0.001; *, P < 0.05 (two-way ANOVA and Bonferroni posttest analysis).

FIG. 4.

rTcPRAC-specific IgG is undetectable during experimental T. cruzi infection. Susceptible BALB/c or resistant C57BL/6 mice were infected with T. cruzi. (A) Serial serum samples were analyzed for responses to whole parasites, rTcPRAC, or negative control to establish background binding. Data represent triplicate repeats with pooled sera from five mice in each experiment. (B) Western blot analysis of IgG reactivity to rCRP (500 ng) (lane 1), rTcPRAC (500 ng) (lane 2), and parasite membrane proteins (10 μg) (lane 3) in BALB/c sera collected at 125 days postinfection (diluted 1:500).

FIG. 5.

GG immunization with rTcPRAC DNA elicits an immunogenic response. BALB/c mice were primed and boosted at 1-month intervals. Ten days after each boost, serum was collected. (A) TcPRAC-specific IgG (diluted 1:2,000) binds to rTcPRAC (7 μg of protein loaded) (lane 2) without binding the negative control (lane 1). (B) Analysis of anti-rTcPRAC IgG1 and IgG2 responses by ELISA. Horizontal lines represent the mean RET (±SEM) for five mice. (C) (Left) Representative histograms showing IgG bound to the surfaces of live parasites, representing mice after 125 days of infection (chronic), TcPRAC-immune mice, naïve mice, or mice with fetal bovine serum as a negative control (no mouse antibody). (Right) Mean florescence intensity (MFI) after treatment of parasites with naïve or TcPRAC immune sera from five separate mice. (D) Bone marrow (BM) cells were isolated after boost 3 and analyzed by rTcPRAC-specific ELISPOT assay for antibody-secreting plasma cells. Data represent the mean number of ASC (±SEM) in 8 mice. Splenocytes were isolated after boost 3 and analyzed for rTcPRAC-specific memory B cells. Data represent the mean number of ASC (±SEM) in 4 mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student's t test).

FIG. 6.

GG immunization with rTcPRAC DNA was nonmitogenic and led to successful coimmunization. (A) BALB/c mice were primed by GG immunization with rTcPRAC or CRP DNA. Serum samples were collected prior to priming and every 3 days for 12 days postpriming. Staining of whole blood for B cells (CD19⁺) and T cells (CD3⁺) indicated that there was no premature expansion of peripheral B cells after rTcPRAC priming compared to CRP priming. Data represent repeat measures on three mice (right). Analysis of the IgM concentrations in the sera of rTcPRAC-immunized mice showed comparable levels and timing of the postprime increase compared to those for CRP-immunized mice. Data represent serum samples from three mice, with the mean indicated by a line. (B) BALB/c mice were immunized with CRP, rTcPRAC, or both immunogens in combination and then analyzed for specific responses via ELISA. Data represent the mean RET for five mice for each experimental group. *, P < 0.05 (Student's t test or Bonferroni posttest from two-way ANOVA). (C) Western blot analysis demonstrates a lack of cross-reactivity between TcPRAC and CRP in immunized mice. Lane 1 contains rCRP (500 ng), and lane 2 contains rTcPRAC (500 ng). Blots were probed with three pooled booster sera, diluted 1:500, from rTcPRAC-immune (left) or CRP-immune (right) mice.

FIG. 7.

GG rTcPRAC immunization generates specific IgG that binds mitogenic rTcPRAC. rTcPRAC-immune (TcP-imm) IgG or control CRP-immune (CRP-imm) IgG was bound to protein A/G resin and then incubated with rTcPRAC. (A) Equal volumes of nonbound protein after incubation of 30 μg rTcPRAC with TcP-imm IgG (rTcPRAC depleted) or CRP-imm IgG (TcPRACA control) were added to splenocyte cultures. After stimulation, live cells were assessed for B-cell activation in terms of B-cell proliferation and surface phenotype (CD69 and CD86) by flow cytometry. IL-10 and antibody (IgM and IgG) secretion was assessed by ELISA. Data represent the means for triplicate repeats (±SEM) for each measure. (B) rTcPRAC eluted from TcP-imm IgG was compared to nonspecifically bound protein eluted from control CRP-imm IgG for the ability to stimulate B cells. (Left) Cells were stimulated with eluted protein. After stimulation, cells were analyzed for B-cell proliferation and activation (CD69 and CD86) by flow cytometry. Data represent the means for triplicate repeats (±SD) for each measure. (Right) Cells were stimulated with eluted protein. After stimulation, the culture supernatant was analyzed for IL-10 secretion by ELISA. Eluted rTcPRAC induced dose-dependent IL-10 secretion compared to control eluted protein. Data represent the means for triplicate repeats (±SEM). *, P < 0.05; **, P < 0.001; ***, P < 0.0001 (Bonferroni posttests after two-way ANOVA or Student's t test). (C) Application of rTcPRAC to TcPRAC-specific and control IgG resulted in rTcPRAC in the flowthrough fraction from the control IgG column and in the elution fraction from the TcPRAC-specific IgG column. TcPRAC was visualized by use of anti-TcPRAC IgG from polyclonal sera from protein-immunized rabbits.