Department of Microbiology, Monash University, Clayton, Victoria, Australia.
Malar J. 2011 Sep 16;10:266. doi: 10.1186/1475-2875-10-266.
Malaria remains a global public health challenge. It is widely believed that an effective vaccine against malaria will need to incorporate multiple antigens from the various stages of the parasite's complex life cycle. Plasmodium falciparum Merozoite Surface Protein 4 (MSP4) is a vaccine candidate that has been selected for development for inclusion in an asexual stage subunit vaccine against malaria.
Nine monoclonal antibodies (Mabs) were produced against Escherichia coli-expressed recombinant MSP4 protein and characterized. These Mabs were used to develop an MSP4-specific competition ELISA to test the binding specificity of antibodies present in sera from naturally P. falciparum-infected individuals from a malaria endemic region of Vietnam. The Mabs were also tested for their capacity to induce P. falciparum growth inhibition in vitro and compared against polyclonal rabbit serum raised against recombinant MSP4.
All Mabs reacted with native parasite protein and collectively recognized at least six epitopes. Four of these Mabs recognize reduction-sensitive epitopes within the epidermal growth factor-like domain found near the C-terminus of MSP4. These sera were shown to contain antibodies capable of inhibiting the binding of the six Mabs indicating infection-acquired responses to the six different epitopes of MSP4. All of the six epitopes were readily recognized by human immune sera. Competition ELISA titres varied from 20 to 640, reflecting heterogeneity in the intensity of the humoral response against the protein among different individuals. The IgG responses during acute and convalescent phases of infection were higher to epitopes in the central region than to other parts of MSP4. Immunization with full length MSP4 in Freund's adjuvant induced rabbit polyclonal antisera able to inhibit parasite growth in vitro in a manner proportionate to the antibody titre. By contrast, polyclonal antisera raised to individual recombinant fragments rMSP4A, rMSP4B, rMSP4C and rMSP4D gave negligible inhibition. Similarly, murine Mabs alone or in combination did not inhibit parasite growth.
The panel of MSP4-specific Mabs produced were found to recognize six distinct epitopes that are also targeted by human antibodies during natural malaria infection. Antibodies directed to more than three epitope regions spread across MSP4 are likely to be required for P. falciparum growth inhibition in vitro.
疟疾仍然是全球公共卫生面临的挑战。人们普遍认为，一种有效的疟疾疫苗将需要包含寄生虫复杂生命周期各个阶段的多种抗原。恶性疟原虫裂殖子表面蛋白 4（MSP4）是一种已被选中用于开发的疫苗候选物，用于包含在针对疟疾的无性阶段亚单位疫苗中。
针对大肠杆菌表达的重组 MSP4 蛋白产生了 9 种单克隆抗体（Mabs），并对其进行了表征。这些 Mabs 用于开发 MSP4 特异性竞争 ELISA，以测试来自越南疟疾流行地区自然感染恶性疟原虫的个体血清中存在的抗体的结合特异性。还测试了这些 Mabs 在体外抑制恶性疟原虫生长的能力，并与针对重组 MSP4 产生的多克隆兔血清进行了比较。
所有 Mabs 均与天然寄生虫蛋白反应，并共同识别至少 6 个表位。其中 4 种 Mabs 识别 MSP4 近 C 端表皮生长因子样结构域内的还原敏感表位。这些血清表明含有能够抑制 6 种 Mabs 结合的抗体，表明感染后对 MSP4 的 6 个不同表位产生了获得性反应。所有 6 个表位均容易被人类免疫血清识别。竞争 ELISA 滴度范围为 20 至 640，反映了不同个体对蛋白质的体液反应强度存在异质性。在感染的急性和恢复期，针对中央区域的表位的 IgG 反应高于 MSP4 的其他部分。用弗氏佐剂全长 MSP4 免疫诱导的兔多克隆抗血清能够以与抗体滴度成比例的方式在体外抑制寄生虫生长。相比之下，针对单个重组片段 rMSP4A、rMSP4B、rMSP4C 和 rMSP4D 的多克隆抗血清几乎没有抑制作用。同样，单独的鼠 Mabs 或组合也不能抑制寄生虫生长。
所产生的 MSP4 特异性 Mabs 被发现识别 6 个独特的表位，这些表位在自然疟疾感染期间也是人类抗体的靶标。针对 MSP4 上 3 个以上表位区域的抗体可能是体外抑制恶性疟原虫生长所必需的。
