Norimine Junzo, Suarez Carlos E, McElwain Terry F, Florin-Christensen Monica, Brown Wendy C
Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology Animal Disease Research Unit, Washington State University, Pullman, Washington 99164, USA.
Infect Immun. 2002 Apr;70(4):2039-48. doi: 10.1128/IAI.70.4.2039-2048.2002.
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.
牛巴贝斯虫棒状体相关蛋白1（RAP-1）能提供部分抗牛巴贝斯虫攻击的保护作用，可被已从感染中恢复并对后续攻击具有免疫力的牛的抗体和T淋巴细胞识别。RAP-1是一种60 kDa的蛋白质，其N端区域含有在所有巴贝斯虫RAP-1家族成员中保守的四个半胱氨酸残基，C端区域含有多个简并的串联23个氨基酸（aa）重复序列。为了使用重组蛋白或小基因构建体确定用于疫苗开发的CD4(+) - T细胞表位的位置，测试了一系列截短的重组RAP-1蛋白和肽对源自牛巴贝斯虫免疫牛的T细胞系的刺激作用。来自三只具有不同DRB3单倍型的牛巴贝斯虫免疫牛的CD4(+) - T细胞系对RAP-1的N端区域有反应，而只有一只动物的T细胞对C端区域反应较弱。来自这三个个体的T细胞系识别了两个至六个跨越aa 134至316且代表至少四个显性表位的N端区域肽。使用RAP-1特异性CD4(+) - T细胞克隆，鉴定出两个N端区域表位，EYLVNKVLYMATMNYKT（aa 187至203）和EAPWYKRWIKKFR（aa 295至307），以及一个C端区域重复表位，FREAPQATKHFL，其在aa位置391至402和414至425处出现两次。几个代表激动剂C端区域肽FREAPQATKHFL简并重复的肽既不刺激针对该肽的T细胞克隆的反应，也不抑制对激动剂肽的反应。在用特异性抗原刺激后，针对N端或C端表位的T细胞克隆产生γ干扰素。RAP-1的N端结构域中存在T辅助细胞表位，该结构域在抗原性不同的牛巴贝斯虫菌株中高度保守，这支持将该区域纳入待在牛中测试的疫苗构建体中。
