Dimorphic sequences of rap-1 genes encode B and CD4+ T helper lymphocyte epitopes in the Babesia bigemina rhoptry associated protein-1.
The rhoptry-associated protein-1 (RAP-1) of Babesia bigemina induces protective immune responses in cattle and contains neutralization-sensitive B cell epitopes. RAP-1 variants containing blocks of sequence dimorphism in the amino and carboxy terminal ends are encoded by four nonallelic genes in B. bigemina. Epitopes recognized by RAP-1 specific monoclonal antibodies (MAbs) and bovine CD4+ T cell clones were mapped to determine whether these epitopes are localized in the amino and carboxy terminal dimorphic regions. Four B cell epitopes, including a neutralization-sensitive epitope, required both the amino terminal variant type 1 (NT-1) and non-dimorphic sequences for conformation. Intrachain disulfide bonds were required for at least one of these epitopes, since reduction and alkylation of cysteine residues abolished MAb binding. A fifth B cell epitope was mapped to the carboxy terminal variant type 1 (CT-1). As expected, the neutralizing MAb and two other MAbs requiring NT-1 for epitope binding recognized only the two RAP-1 variants with the NT-1 sequence, while the MAb binding an epitope in CT-1 did not bind RAP-1 variants with CT-2. In contrast, the fourth MAb requiring NT-1 for binding recognized all rap-1 gene products, indicating that dimorphic residues are not part of the epitope recognized by this MAb. Bovine CD4+ T cell clones characterized previously as responding in a strain dependent fashion recognized at least one epitope in CT-1, and did not cross-react with CT-2. A second group of bovine CD4+ T cell clones that responded to multiple parasite strains recognized an epitope in a non-dimorphic region of RAP-1. These data indicate that dimorphic regions of RAP-1 encode unique B and T helper lymphocyte epitopes and may be required for enhanced protective immune responses in cattle.
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman 99164-7040, USA. ihe@vetmed.wsu.edu
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I
Hötzel
W C
WC
Brown
T F
TF
McElwain
S D
SD
Rodríguez
G H
GH
Palmer
D000818:Animals; D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan / Q000737:chemistry / Q000235:genetics*; D001402:B-Lymphocytes / Q000276:immunology; D001403:Babesia / Q000235:genetics* / Q000276:immunology*; D001483:Base Sequence; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D002417:Cattle; D017931:DNA Primers / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D017125:Genes, Protozoan; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; D017931:DNA Primers; D004220:Disulfides; D000939:Epitopes; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C097551:rhoptry-associated protein-1, Babesia