METHODS: A total of 93 P. ovale curtisi samples were collected from migrant workers who returned to China from 17 countries in Africa between 2012 and 2016. The genetic polymorphism, natural selection and copy number variation (CNV) were investigated by sequencing and real-time PCR. The antigenicity and immunogenicity of the recombinant PocDBP-RII (rPocDBP-RII) protein were further examined, and the humoral and cellular responses of immunized mice were assessed using protein microarrays and flow cytometry.
RESULTS: Efficiently expressed and purified rPocDBP-RII (39 kDa) was successfully used as an antigen for immunization in mice. The haplotype diversity (Hd) of PocDBP-RII gene was 0.105, and the nucleotide diversity index (π) was 0.00011. No increased copy number was found among the collected isolates of P. ovale curtisi. Furthermore, rPocDBP-RII induced persistent antigen-specific antibody production with a serum IgG antibody titer of 1: 16,000. IFN-γ-producing T cells, rather than IL-10-producing cells, were activated in response to the stimulation of rPocDBP-RII. Compared to PBS-immunized mice (negative control), there was a higher percentage of CD4+CD44highCD62L- T cells (effector memory T cells) and CD8+CD44highCD62L+ T cells (central memory T cells) in rPocDBP-RII‑immunized mice.
CONCLUSIONS: PocDBP-RII sequences were highly conserved in clinical isolates of P. ovale curtisi. rPocDBP-RII protein could mediate protective blood-stage immunity through IFN-γ-producing CD4+ and CD8+ T cells and memory T cells, in addition to inducing specific antibodies. Our results suggested that rPocDBP-RII protein has potential as a vaccine candidate to provide assessment and guidance for malaria control and elimination activities.
方法:从2012年至2016年期间从17个非洲国家返回中国的移民工人中收集了93份卵卵圆竹样本。遗传多态性,通过测序和实时PCR研究自然选择和拷贝数变异(CNV)。进一步检测了重组PocDBP-RII(rPocDBP-RII)蛋白的抗原性和免疫原性,使用蛋白质微阵列和流式细胞术评估免疫小鼠的体液和细胞反应。
结果:有效表达和纯化的rPocDBP-RII(39kDa)成功地用作小鼠免疫的抗原。PocDBP-RII基因的单倍型多样性(Hd)为0.105,核苷酸多样性指数(π)为0.00011。在收集到的卵圆藻分离株中没有发现拷贝数增加。此外,rPocDBP-RII诱导持续的抗原特异性抗体产生,血清IgG抗体滴度为1:16,000。产生IFN-γ的T细胞,而不是产生IL-10的细胞,响应于rPocDBP-RII的刺激而被激活。与PBS免疫的小鼠(阴性对照)相比,在rPocDBP-RII免疫的小鼠中,CD4CD44highCD62L-T细胞(效应记忆T细胞)和CD8CD44highCD62LT细胞(中枢记忆T细胞)的百分比更高。
结论:PocDBP-RII序列在卵圆竹临床分离株中高度保守。rPocDBP-RII蛋白可通过产生IFN-γ的CD4+和CD8+T细胞和记忆T细胞介导保护性血液阶段免疫,除了诱导特异性抗体。我们的结果表明,rPocDBP-RII蛋白具有作为候选疫苗的潜力,可以为疟疾的控制和消除活动提供评估和指导。