Recombinant proteins of Plasmodium malariae merozoite surface protein 1 (PmMSP1): Testing immunogenicity in the BALB/c model and potential use as diagnostic tool

BackgroundPlasmodium malariae is the third most prevalent human malaria-causing species and has a patchy, but ample distribution in the world. Humans can host the parasite for years without presenting significant symptoms, turning its diagnosis and control into a difficult task. Here, we investigated the immunogenicity of recombinant proteins of P. malariae MSP1.MethodsFive regions of PmMSP1 were expressed in Escherichia coli as GST-fusion proteins and immunized in BALB/c mice. The specificity, subtyping, and affinity of raised antibodies were evaluated by enzyme-linked immunosorbent assays. Cellular immune responses were analyzed by lymphoproliferation assays and cytokine levels produced by splenocytes were detected by cytometry.ResultsWe found that N-terminal, central regions, and PmMSP119 are strongly immunogenic in mice. After three doses, the induced immune responses remained high for 70 days. While antibodies induced after immunization with N-terminal and central regions showed similar affinities to the target antigens, affinities of IgG against PmMSP119 were higher. All proteins induced similar antibody subclass patterns (predominantly IgG1, IgG2a, and IgG2b), characterizing a mixed Th1/Th2 response. Further, autologous stimulation of splenocytes from immunized mice led to the secretion of IL2 and IL4, independently of the antigen used. Importantly, IgG from P. malariae-exposed individuals reacted against PmMSP1 recombinant proteins with a high specificity. On the other hand, sera from P. vivax or P. falciparum-infected individuals did not react at all against recombinant PmMSP1 proteins.ConclusionRecombinant PmMSP1 proteins are very useful diagnostic markers of P. malariae in epidemiological studies or in the differential diagnosis of malaria caused by this species. Immunization with recombinant PmMSP1 proteins resulted in a significant humoral immune response, which may turn them potential component candidates for a vaccine against P. malariae.

背景：三日疟原虫（Plasmodium malariae）是全球第三大流行的人类致病疟原虫虫种，其分布呈斑块状但范围广泛。人类可携带该寄生虫数年而无明显临床症状，这使其诊断与防控均颇具挑战。本研究探讨了三日疟原虫裂殖子表面蛋白1（PmMSP1）重组蛋白的免疫原性。
方法：我们将PmMSP1的5个区域在大肠杆菌（Escherichia coli）中表达为谷胱甘肽S-转移酶融合蛋白（GST-fusion proteins），并免疫BALB/c小鼠。通过酶联免疫吸附试验（enzyme-linked immunosorbent assays, ELISA）评估所诱导抗体的特异性、亚型与亲和力。采用淋巴细胞增殖试验分析细胞免疫应答，并通过流式细胞术检测脾细胞分泌的细胞因子水平。
结果：研究发现，PmMSP1的N端区域、中央区域以及PmMSP119在小鼠体内均具有较强免疫原性。经3次免疫后，诱导的免疫应答可维持70天且水平仍较高。针对N端与中央区域免疫所诱导的抗体，其与靶抗原的亲和力相近；而针对PmMSP119的IgG抗体亲和力更高。所有重组蛋白均诱导了相似的抗体亚型谱（以IgG1、IgG2a及IgG2b为主），提示机体产生了混合的辅助性T细胞1/辅助性T细胞2（Th1/Th2）型免疫应答。进一步实验显示，对免疫小鼠的脾细胞进行自体抗原刺激后，无论使用何种抗原，均可检测到白细胞介素2（IL-2）与白细胞介素4（IL-4）的分泌。值得注意的是，曾暴露于三日疟原虫的感染者血清中的IgG可与PmMSP1重组蛋白发生特异性结合；与之相反，间日疟原虫（Plasmodium vivax, P. vivax）或恶性疟原虫（Plasmodium falciparum, P. falciparum）感染者的血清则完全不与重组PmMSP1蛋白发生反应。
结论：重组PmMSP1蛋白可作为三日疟原虫感染的有效诊断标志物，适用于流行病学研究或该虫种所致疟疾的鉴别诊断。采用重组PmMSP1蛋白免疫可诱导显著的体液免疫应答，提示其有望成为抗三日疟原虫疫苗的潜在候选组分。