Identification of two Th1 cell epitopes on the Babesia bovis-encoded 77-kilodalton merozoite protein (Bb-1) by use of truncated recombinant fusion proteins

Abstract

Previous studies have demonstrated the serologic and T-cell immunogenicity for cattle of a recombinant form of the apical complex-associated 77-kDa merozite protein of Babesia bovis, designated Bb-1. The present study characterizes the immunogenic epitopes of the Bb-1 protein. A series of recombinant truncated fusion proteins spanning the majority of the Bb-1 protein were expressed in Escherichia coli, and their reactivities with bovine peripheral blood mononuclear cells and T-cell clones derived from B. bovis-immune cattle and with rabbit antibodies were determined. Lymphocytes from two immune cattle were preferentially stimulated by the N-terminal half of the Bb-1 protein (amino acids 23 to 266, termed Bb-lA), localizing the T-cell epitopes to the Bb-1A portion of the molecule. CD4+ T-cell clones derived by stimulation with the intact Bb-1 fusion protein were used to identify two T-cell epitopes in the Bb-1A protein, consisting of amino acids SVVLLSAFSGN VWANEAEVSQVVK and FSDVDKTKSTEKT (residues 23 to 46 and 82 to 94). In contrast, rabbit antiserum raised against the intact fusion protein reacted only with the C-terminal half of the protein (amino acids 267 to 499, termed Bb-1B), which contained 28 tandem repeats of the tetrapeptide PAEK or PAET. Biological assays and Northern (RNA) blot analyses for cytokines revealed that following activation with concanavalin A, T-cell clones reactive against the two Bb-IA epitopes produced interleukin-2, gamma interferon, and tumor necrosis factors beta and alpha, but not interleukin-4, suggesting that the Bb-1 antigen preferentially stimulates the Thl subset of CD4+ T cells in cattle. The studies described here report for the first time the characterization, by cytokine production, of the Thl subset of bovine T cells and show that, as in mice, protozoal antigens can induce Thl cells in ruminants. This first demonstration of B. bovis-encoded Thl cell epitopes provides a rationale for incorporation of all or part of the Bb-1 protein into a recombinant vaccine.

Interleukin-10 is expressed by bovine type 1 helper, type 2 helper, and unrestricted parasite-specific T-cell clones and inhibits proliferation of all three subsets in an accessory-cell-dependent manner

“…Antigen-specific CD4+ Th cell clones were obtained from PBMC of cattle immune to either B. bovis or F. hepatica. The cloning procedures and characterization of the Th clones were described in detail in previous publications (2,5,7). The clones were cryopreserved and, upon thawing, were stimulated on a weekly basis with irradiated autologous PBMC as a source of AC, 10% bovine TCGF, and antigen.…”

“…The clones were cryopreserved and, upon thawing, were stimulated on a weekly basis with irradiated autologous PBMC as a source of AC, 10% bovine TCGF, and antigen. The majority of B. bovis-specific Th clones were stimulated with a crude membrane preparation of B. bovis merozoites (4), whereas one clone (C97.1C8) was stimulated with a recombinant form of the 77-kDa apical complex-associated protein expressed as a fusion protein with glutathione-S-transferase, designated Bb-1-GST (7). All F. hepatica-specific clones were stimulated with adult worm extract (2).…”

“…Northern (RNA) blot analysis. Northern blotting was performed to identify the expression of IL-2, IL-4, IFN-y, and IL-10 mRNAs as detailed previously (2,5,7,19). Briefly, equal amounts of RNA (20 ,ug were size-fractionated in formaldehyde-morpholinepropanesulfonic acid (MOPS)-1.6% agarose gels.…”

“…The present study was designed to further characterize IL-10 expression by and regulation of different bovine T-cell subsets and employed clones of bovine Th cells specific for either the hemoprotozoan parasite Babesia bovis (5,7) or the trematode parasite Fasciola hepatica (2). The CD4+ clones were recently characterized as either ThO, Thl, or Th2 cells by the expression patterns of IL-2, IL-4, and IFN--y mRNA.…”

The CD4+ T cell immunodominant Anaplasma marginale major surface protein 2 stimulates γδ T cell clones that express unique T cell receptors

“…␥␦ T cell clones 71.1G7 and 71.2B1 were obtained from animal 01B71 after stimulating PBMC for 2 weeks with 5 g per ml A. marginale (Florida strain) sonicate and cloning with A. marginale. As controls for assays measuring MSP2 peptide stimulation of MSP2-responsive ␥␦ T cell clones, WC1 ϩ ␥␦ T cell clone G1.2A9 obtained from cow G1, which was infected with Fasciola hepatica [28], and clones C15.1A7 and C15.2F12 obtained from cow C15, which was infected with Babesia bovis [29], were also used. Clone G1.2A9 was obtained by limiting dilution cloning of a cell line cultured with 25 g per ml F. hepatica adult worm antigen as described [28], and clones C15.1A7 and C15.2F12 were obtained by cloning a cell line cultured with 20 g per ml recombinant B. bovis spherical body protein-1-glutathione-S-transferase fusion protein as described [29].…”