Laboratory of Pathogen Infection and Immunity, Department of Public Health and Preventive Medicine, Wuxi School of Medicine, Jiangnan University, Wuxi, 214122, Jiangsu, People's Republic of China.
Key Laboratory of National Health and Family Planning Commision on Parasitic Disease Control and Prevention, Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology, Jiangsu Institute of Parasite Diseases, Wuxi, 214064, Jiangsu, People's Republic of China.
Malar J. 2020 Mar 30;19(1):126. doi: 10.1186/s12936-020-03207-7.
There is an urgent need for an effective vaccine to control and eradicate malaria, one of the most serious global infectious diseases. Plasmodium merozoite surface protein 4 (MSP4) has been listed as a blood-stage subunit vaccine candidate for malaria. Infection with Plasmodium ovale species including P. ovale wallikeri and P. ovale curtisi, is also a source of malaria burden in tropical regions where it is sometimes mixed with other Plasmodium species. However, little is known about P. ovale MSP4.
The msp4 gene was amplified through polymerase chain reaction using genomic DNA extracted from blood samples of 46 patients infected with P. ovale spp. and amplified products were sequenced. Open reading frames predicted as immunogenic peptides consisting of 119 and 97 amino acids of P. ovale curtisi MSP4 (PocMSP4) and P. ovale wallikeri MSP4 (PowMSP4), respectively, were selected for protein expression. Recombinant proteins (rPoMSP4) were expressed in Escherichia coli, purified, analysed, and immunized in BALB/c mice. The specificity of anti-MSP4-immunoglobulin (Ig) G antibodies was evaluated by Western blot and enzyme-linked immunosorbent assays, and cellular immune responses were analysed via lymphocyte proliferation assays.
Full peptide sequences of PocMSP4 and PowMSP4 were completely conserved in all clinical isolates, except in the epidermal growth factor-like domain at the carboxyl terminus where only one mutation was observed in one P. o. wallikeri isolate. Further, truncated PoMSP4 segments were successfully expressed and purified as ~ 32 kDa proteins. Importantly, high antibody responses with end-point titres ranging from 1:10,000 to 1:2,560,000 in all immunized mouse groups were observed, with high IgG avidity to PocMSP4 (80.5%) and PowMSP4 (92.3%). Furthermore, rPocMSP4 and rPowMSP4 cross-reacted with anti-PowMSP4-specific or anti-PocMSP4-specific antibodies. Additionally, anti-PoMSP4 IgG antibodies showed broad immuno-specificity in reacting against rPoMSP1 and rPoAMA1. Lastly, PocMSP4- and PowMSP4-immunized mice induced cellular immune responses with PocMSP4 (36%) and PowMSP4 cells (15.8%) during splenocyte proliferation assays.
Findings from this study suggest conservation in PoMSP4 protein sequences and high immunogenicity was observed in rPoMSP4. Furthermore, induction of immune responses in PocMSP4- and PowMSP4-immunized mice informed that both humoral and cellular immune responses play crucial roles for PoMSP4 in protection.
迫切需要一种有效的疫苗来控制和消灭疟疾，这是最严重的全球传染病之一。疟原虫裂殖子表面蛋白 4（MSP4）已被列为疟疾的血液阶段亚单位疫苗候选物。感染卵形疟原虫物种，包括卵形疟原虫沃利克里和卵形疟原虫库蒂斯，也是热带地区疟疾负担的一个来源，在那里它有时与其他疟原虫物种混合。然而，关于卵形疟原虫 MSP4 知之甚少。
使用从感染卵形疟原虫 spp. 的 46 名患者的血液样本中提取的基因组 DNA 通过聚合酶链反应扩增 msp4 基因，并对扩增产物进行测序。选择分别由 119 和 97 个氨基酸组成的卵形疟原虫库蒂斯 MSP4（PocMSP4）和卵形疟原虫沃利克里 MSP4（PowMSP4）的免疫原性肽的开放阅读框进行蛋白表达。重组蛋白（rPoMSP4）在大肠杆菌中表达、纯化、分析，并在 BALB/c 小鼠中免疫。通过 Western blot 和酶联免疫吸附试验评估抗-MSP4-免疫球蛋白（IgG）抗体的特异性，并通过淋巴细胞增殖试验分析细胞免疫反应。
除了在羧基末端的表皮生长因子样结构域中仅观察到一个突变外，所有临床分离株中 PocMSP4 和 PowMSP4 的全长肽序列完全保守。此外，成功表达和纯化了截断的 PoMSP4 片段，作为~32 kDa 的蛋白质。重要的是，所有免疫小鼠组均观察到高抗体反应，终点滴度范围为 1:10,000 至 1:2,560,000，对 PocMSP4（80.5%）和 PowMSP4（92.3%）的 IgG 亲和力高。此外，rPocMSP4 和 rPowMSP4 与抗 PowMSP4 特异性或抗 PocMSP4 特异性抗体发生交叉反应。此外，抗 PoMSP4 IgG 抗体在反应 rPoMSP1 和 rPoAMA1 时表现出广泛的免疫特异性。最后，PocMSP4 和 PowMSP4 免疫的小鼠在脾细胞增殖试验中诱导 PocMSP4（36%）和 PowMSP4 细胞（15.8%）的细胞免疫反应。
本研究结果表明 PoMSP4 蛋白序列保守，rPoMSP4 具有高免疫原性。此外，PocMSP4 和 PowMSP4 免疫的小鼠诱导免疫反应表明，PoMSP4 在保护中发挥作用的体液和细胞免疫反应都很重要。
