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Infection and Immunity, April 2002, p. 2039-2048, Vol. 70, No. 4
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.4.2039-2048.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Junzo Norimine,1 Carlos E. Suarez,2 Terry F. McElwain,1 Monica Florin-Christensen,1,{dagger} and Wendy C. Brown1*

Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology ,1 Animal Disease Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Washington State University, Pullman, Washington 991642

Received 1 November 2001/ Returned for modification 21 December 2001/ Accepted 2 January 2002

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.

* Corresponding author. Mailing address: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040. Phone: (509) 335-6067. Fax: (509) 335-8529. E-mail: wbrown{at}vetmed.wsu.edu.

Editor: W. A. Petri, Jr.

{dagger} Present address: Institute of Neuroscience (CONICET), RA-1663 San Miguel, Argentina.

Infection and Immunity, April 2002, p. 2039-2048, Vol. 70, No. 4
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.4.2039-2048.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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