Epitope-specific regulation of immunoglobulin class switching in mice immunized with malarial merozoite surface proteins

Abstract

Antibodies that bind to Fc receptors and activate complement are implicated in the efficient control of pathogens, but the processes that regulate their induction are still not well understood. To investigate antigen-dependent factors that regulate class switching, we have developed an in vivo model of class switching to immunoglobulin G2b (IgG2b) using the malaria antigen Plasmodium falciparum merozoite surface protein 2 (MSP2). C57BL/6 mice were immunized with recombinant proteins representing discrete domains of MSP2, and a T-cell epitope (C8) was identified within the conserved C terminus of the protein that preferentially induces IgG2b antibodies. The ability of C8 to induce IgG2b is ablated in both homozygous gamma interferon-negative and interleukin 10-negative mice. The IgG2b-inducing properties of C8 override the IgG1-inducing properties of both the fusion protein partner, glutathione S-transferase, and the adjuvant. Furthermore, when attached to other proteins that normally induce IgG1 responses, C8 induces a switch to IgG2b secretion. This is the first description of a defined T-cell epitope that drives specific IgG2b subclass switching, and our data offer proof of the concept that chimeric vaccines incorporating specific T-cell "switch epitopes" might be used to enhance qualitative aspects of the antibody response.

FIG. 1.

Schematic showing the predicted protein structure of MSP2 and the derivation of the recombinant proteins. Filled blocks indicate sequences that are conserved among all P. falciparum isolates. Hatched blocks indicate dimorphic sequences which differ between A family and B family proteins but which are conserved within families. Checkered blocks indicate the highly polymorphic central region of the molecule, which contains tandemly repeated amino acid sequences. The recombinant proteins representing the dimorphic sequences (Di-A and Di-B) comprise the N-terminal dimorphic sequence fused to the C-terminal dimorphic sequence with exclusion of the intervening polymorphic sequence.

FIG. 2.

Total IgG and IgG subclass antibody responses to MSP1₁₉ and MSP2 (serogroup A) proteins in C57BL/6 mice. Median midpoint titers of IgG and IgG subclasses (measured by ELISA) are shown for sera (six mice per group) collected prior to immunization and 2 weeks after each immunization with GST, MSP2, or MSP1₁₉ proteins. Horizontal dotted lines indicate a titer of 10⁵. (a) Total IgG titers. Sera were tested against the same protein that was used for immunization. (b) IgG subclass titers to GST alone. Geometric mean titers are shown for all groups of mice combined (n = 36). (c to h) IgG subclass titers for mice immunized with different recombinant proteins. Sera were tested against the same protein that was used for immunization. (c) Full-length MSP2-His₆ (MSP2A); (d) Di-A-GST; (e) Poly-A-GST; (f) Con-N; (g) Con-C-GST; (h) unrelated antigen-GST (MSP1₁₉).

FIG. 3.

Conserved C terminus of MSP2 induces rapid secretion of IL-6 and IL-10 by spleen cells from immunized C57BL/6 mice. Four weeks after the third immunization, spleen cells from mice (six per group) immunized with either MSP2A, Con-C, or MSP1₁₉ were collected and restimulated in vitro for 3 or 5 days with either the original immunizing antigen (a, b) or the C8 peptide (c, d). Culture supernatants were assayed by a cytokine bead assay. Data are presented as geometric mean cytokine concentrations (pg/ml). Cytokine values for cells cultured with GST alone (for a and b) or for cells cultured with an irrelevant peptide (for c and d) have been subtracted. ND, no detectable cytokine.

FIG. 4.

B cells from IFN-γ-deficient and IL-10-deficient mice fail to class switch to IgG2b. Wild-type (WT), IFN-γ^−/− (a), and IL-10^−/− (b) mice (six per group) were immunized with MSP1₁₉, MSP2A, or Con-C, and median midpoint titers of IgG1 and IgG2b were measured by ELISA 2 weeks after the final immunization. The statistical significance of differences between wild-type and cytokine-deficient mice was determined using nonparametric (Wilcoxon signed rank) tests and is indicated. *, P < 0.05; **, P < 0.005.

FIG. 5.

The C8 peptide of MSP2 drives class switching of the anti-GST antibody response to IgG2b. Mice (four per group) immunized with (a) MSP1₁₉-GST, (b) MSP2A-GST, or (c) Con-C-GST were tested for antibodies to GST alone. Data represent median midpoint titers of each IgG subclass.

FIG. 6.

Peptide C8 drives class switching of the antibody response to MSP1₁₉, GST, and BSA to IgG2b. Ratios of midpoint titers of IgG2b to IgG1 antibodies to (a) MSP11₁₉ (b) GST, and (c) BSA in the sera of C57BL/6 mice (four per group) immunized with (a, b) MSP1₁₉-GST, MSP1₁₉-GST conjugated with peptide C8 (C8-MSP1₁₉-GST), MSP1₁₉-GST conjugated with an irrelevant peptide (L-MS1₁₉-GST), (c) BSA alone, BSA conjugated to peptide L (L-BSA), or BSA conjugated to C8 (C8-BSA). Each bar represents the IgG2b/IgG1 ratio for serum from one mouse. * indicates a serum where the ratio was 1.0.