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.

中文翻译：

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卵形疟原虫裂殖子表面蛋白4保守片段的免疫原性分析

迫切需要一种有效的疫苗来控制和根除疟疾，这是全球最严重的传染病之一。疟原虫裂殖子表面蛋白4（MSP4）已被列为疟疾的血液亚基候选疫苗。椭圆形疟原虫物种的感染，包括椭圆形疟原虫wallikeri和椭圆形疟原虫，也是热带地区疟疾负担的来源，在热带地区有时与其他疟原虫物种混合。但是，对椭圆形假单胞菌MSP4知之甚少。msp4基因通过聚合酶链反应扩增，使用的基因组DNA是从46个感染卵形疟原虫的患者的血液样本中提取的。并对扩增产物进行测序。开放阅读框被预测为由119个和97个氨基酸组成的椭圆形麻风MSP4（PocMSP4）和椭圆形沃利壁霉MSP4（PowMSP4）的免疫原性肽，分别选择蛋白质表达。重组蛋白（rPoMSP4）在大肠杆菌中表达，在BALB / c小鼠中进行纯化，分析和免疫。通过蛋白质印迹和酶联免疫吸附试验评估抗MSP4-免疫球蛋白（Ig）G抗体的特异性，并通过淋巴细胞增殖试验分析细胞免疫应答。PocMSP4和PowMSP4的完整肽序列在所有临床分离株中都完全保守，除了在羧基末端的表皮生长因子样结构域中，在一个P. o。中仅观察到一个突变。Wallikeri隔离。此外，截短的PoMSP4片段已成功表达并纯化为〜32 kDa蛋白。重要的是，高抗体反应的终点滴度范围为1：10,000至1：2,560，在所有免疫小鼠组中均观察到000，对PocMSP4（80.5％）和PowMSP4（92.3％）具有较高的IgG亲和力。此外，rPocMSP4和rPowMSP4与抗PowMSP4特异性抗体或抗PocMSP4特异性抗体发生交叉反应。此外，抗PoMSP4 IgG抗体在与rPoMSP1和rPoAMA1反应中显示出广泛的免疫特异性。最后，在脾细胞增殖试验中，经PocMSP4和PowMSP4免疫的小鼠诱导了PocMSP4（36％）和PowMSP4细胞（15.8％）的细胞免疫应答。这项研究的发现表明，PoMSP4蛋白序列具有保守性，并且在rPoMSP4中观察到高免疫原性。此外，在PocMSP4和PowMSP4免疫的小鼠中诱导免疫反应表明，体液和细胞免疫反应在PoMSP4的保护中均起着关键作用。5％）和PowMSP4（92.3％）。此外，rPocMSP4和rPowMSP4与抗PowMSP4特异性抗体或抗PocMSP4特异性抗体发生交叉反应。此外，抗PoMSP4 IgG抗体在与rPoMSP1和rPoAMA1反应中显示出广泛的免疫特异性。最后，在脾细胞增殖试验中，经PocMSP4和PowMSP4免疫的小鼠诱导了PocMSP4（36％）和PowMSP4细胞（15.8％）的细胞免疫应答。这项研究的发现表明，PoMSP4蛋白序列具有保守性，并且在rPoMSP4中观察到高免疫原性。此外，在PocMSP4和PowMSP4免疫的小鼠中诱导免疫反应表明，体液和细胞免疫反应在PoMSP4的保护中均起着关键作用。5％）和PowMSP4（92.3％）。此外，rPocMSP4和rPowMSP4与抗PowMSP4特异性抗体或抗PocMSP4特异性抗体发生交叉反应。此外，抗PoMSP4 IgG抗体在与rPoMSP1和rPoAMA1反应中显示出广泛的免疫特异性。最后，在脾细胞增殖试验中，经PocMSP4和PowMSP4免疫的小鼠诱导了PocMSP4（36％）和PowMSP4细胞（15.8％）的细胞免疫应答。这项研究的发现表明，PoMSP4蛋白序列具有保守性，并且在rPoMSP4中观察到高免疫原性。此外，在PocMSP4和PowMSP4免疫的小鼠中诱导免疫反应表明，体液免疫反应和细胞免疫反应均对PoMSP4在保护中起关键作用。抗PoMSP4 IgG抗体在与rPoMSP1和rPoAMA1的反应中显示出广泛的免疫特异性。最后，在脾细胞增殖试验中，经PocMSP4和PowMSP4免疫的小鼠诱导了PocMSP4（36％）和PowMSP4细胞（15.8％）的细胞免疫应答。这项研究的发现表明，PoMSP4蛋白序列具有保守性，并且在rPoMSP4中观察到高免疫原性。此外，在PocMSP4和PowMSP4免疫的小鼠中诱导免疫反应表明，体液和细胞免疫反应在PoMSP4的保护中均起着关键作用。抗PoMSP4 IgG抗体在与rPoMSP1和rPoAMA1反应中显示出广泛的免疫特异性。最后，在脾细胞增殖试验中，经PocMSP4和PowMSP4免疫的小鼠诱导了PocMSP4（36％）和PowMSP4细胞（15.8％）的细胞免疫应答。这项研究的发现表明，PoMSP4蛋白序列具有保守性，并且在rPoMSP4中观察到高免疫原性。此外，在PocMSP4和PowMSP4免疫的小鼠中诱导免疫反应表明，体液和细胞免疫反应在PoMSP4的保护中均起着关键作用。这项研究的发现表明，PoMSP4蛋白序列具有保守性，并且在rPoMSP4中观察到高免疫原性。此外，在PocMSP4和PowMSP4免疫的小鼠中诱导免疫反应表明，体液免疫反应和细胞免疫反应均对PoMSP4在保护中起关键作用。这项研究的发现表明，PoMSP4蛋白序列具有保守性，在rPoMSP4中观察到高免疫原性。此外，在PocMSP4和PowMSP4免疫的小鼠中诱导免疫反应表明，体液和细胞免疫反应在PoMSP4的保护中均起着关键作用。