Immunodominant epitopes in Babesia bovis rhoptry-associated protein 1 that elicit memory CD4(+)-T-lymphocyte responses in B. bovis-immune individuals are located in the amino-terminal domain

Abstract

Babesia bovis rhoptry-associated protein 1 (RAP-1), which confers partial protection against B. bovis challenge, is recognized by antibodies and T lymphocytes from cattle that have recovered from infection and are immune to subsequent challenge. RAP-1 is a 60-kDa protein with an N-terminal (NT) region that contains four cysteine residues conserved among all Babesia RAP-1 family members and a C-terminal (CT) region that contains multiple, degenerate, tandem 23-amino-acid (aa) repeats. To define the location of CD4(+)-T-cell epitopes for vaccine development using a recombinant protein or minigene construct, a series of truncated recombinant RAP-1 proteins and peptides were tested for stimulation of T-cell lines derived from B. bovis-immune cattle. CD4(+)-T-cell lines from three B. bovis-immune cattle with different DRB3 haplotypes responded to the NT region of RAP-1, whereas T cells from only one animal responded weakly to the CT region. T-cell lines from the three individuals recognized two to six NT-region peptides spanning aa 134 to 316 and representing at least four dominant epitopes. Using RAP-1-specific CD4(+)-T-cell clones, two NT-region epitopes, EYLVNKVLYMATMNYKT (aa 187 to 203) and EAPWYKRWIKKFR (aa 295 to 307), and one CT-region repeat epitope, FREAPQATKHFL, which is present twice at aa positions 391 to 402 and 414 to 425, were identified. Several peptides representing degenerate repeats of the agonist CT-region peptide FREAPQATKHFL neither stimulated responses of T-cell clones specific for this peptide nor inhibited responses to the agonist peptide. Upon stimulation with specific antigen, T-cell clones specific for NT or CT epitopes produced gamma interferon. The presence of T-helper-cell epitopes in the NT domain of RAP-1, which is highly conserved among otherwise antigenically different strains of B. bovis, supports the inclusion of this region in vaccine constructs to be tested in cattle.

FIG. 1.

Schematic structure of B. bovis RAP-1. (A) RAP-1 consists of an NT region (aa 1 to 316) and a CT region (aa 317 to 565) containing tandem repeats of degenerate amino acid sequences. Arrows indicate locations of Th-cell epitopes identified in this study. (B) Truncated recombinant B. bovis RAP-1 proteins were used to identify CD4⁺-T-cell epitopes.

FIG. 2.

Proliferative responses of T-cell lines (CL) from the B. bovis-immune cattle C97 and C15 against recombinant B. bovis RAP-1 NT and RAP-1 CT antigens. Short-term T-cell lines from cows C97 and C15 were stimulated with 1, 5, and 25 μg of full-length RAP-1, RAP-1 NT, and RAP-1 CT/ml and proteins RAP-1 N2, RAP-1 NT2-1, and RAP-1 NT2-2 spanning the RAP-1 NT region (Fig. 1). Results are the mean counts per minute of triplicate cultures of T cells stimulated with antigen for 3 days ± 1 standard deviation and are representative of at least three experiments. Responses significantly higher than those for A. marginale MSP-5 control antigen are indicated with a † (P < 0.005) or ∗ (P < 0.05).

FIG. 3.

Proliferative responses of T-cell lines (CL) from the B. bovis-immune cattle C97, C15, and G3 against synthetic peptides (Table 2) spanning the RAP-1 NT region. Short-term T-cell lines from cows C97, C15, and G3 were stimulated with 1 and 10 μg of peptides P1 to P9/ml. As a negative control, a 30-mer peptide derived from A. marginale MSP-2 was used. Results are the mean counts per minute of triplicate cultures of T cells stimulated with antigen for 3 days ± 1 standard deviation. Responses significantly higher than those for A. marginale MSP-2 peptide are indicated with a † (P < 0.005) or ∗ (P < 0.05).

FIG. 3.

Proliferative responses of T-cell lines (CL) from the B. bovis-immune cattle C97, C15, and G3 against synthetic peptides (Table 2) spanning the RAP-1 NT region. Short-term T-cell lines from cows C97, C15, and G3 were stimulated with 1 and 10 μg of peptides P1 to P9/ml. As a negative control, a 30-mer peptide derived from A. marginale MSP-2 was used. Results are the mean counts per minute of triplicate cultures of T cells stimulated with antigen for 3 days ± 1 standard deviation. Responses significantly higher than those for A. marginale MSP-2 peptide are indicated with a † (P < 0.005) or ∗ (P < 0.05).

FIG. 4.

T-cell-epitope mapping for RAP-1 NT2-1- and RAP-1 NT2-2-specific CD4⁺-T-cell clones. (A) Response of RAP-1 NT2-1-specific T-cell clone 3B3 to peptides P1 to P5; (B) summary of the response of clone 3B3 to smaller peptides spanning peptides P3 and P4; (C) response of RAP-1 NT2-2-specific T-cell clone 1E7 to peptides P4 to P9; (D) summary of the response of clone 1E7 to smaller peptides spanning peptides P8 and P9. For all assays, T cells were cultured in duplicate with medium or peptide and APC for 3 days. Responses significantly higher than those for T cells in the medium are indicated with a † (P < 0.005) or ∗ (P < 0.05).

FIG. 5.

Determination of MHC class II restriction patterns of C97 RAP-1-specific T-cell clones. (A) RAP-1 NT2-1-specific CD4⁺-T-cell clone 3E1; (B) RAP-1 NT2-2-specific CD4⁺-T-cell clone 1E7; (C) RAP-1 CT-specific CD4⁺-T-cell clone 2H1. Autologous APC (2 × 10⁵) were incubated with a 4-μg/ml final concentration of MAb TH14B (anti-DR), MAb TH22A5 (anti-DQ), or isotype-matched control MAb Colis205D (isotype) in 96-well plates for 1 h before the addition of 3 × 10⁴ T cells and 12.5 μg of antigen per ml. Results are the mean counts per minute of duplicate cultures ± 1 standard deviation. Antigen-specific responses that are significantly lower than those of T cells cultured without MAb are indicated with a † (P < 0.005) or ∗ (P < 0.05).