Université Paris Descartes, Sorbonne Paris Cité, 75006 Paris, France.
Vaccine. 2012 Feb 8;30(7):1343-8. doi: 10.1016/j.vaccine.2011.12.065. Epub 2012 Jan 5.
VAR2CSA is considered as the main target of protective immunity against pregnancy-associated malaria. VAR2CSA high molecular weight complicates scaling up production of VAR2CSA recombinant protein for large-scale vaccination programmes. We previously demonstrated that antibodies induced by NTS-DBL1X-Id1-DBL2X efficiently block parasite binding to CSA in a similar manner to antibodies induced by the full-length extracellular part of VAR2CSA. In order to identifying the shortest fragment of VAR2CSA carrying major protective epitopes able to elicit inhibitory antibodies, we performed a refined antigenic mapping of NTS-DBL1X-Id1-DBL2X through a DNA vaccination technique.
Five single or double domains constructs encoding NTS-DBL1X, NTS-DBL1X-Id1, Id1, Id1-DBL2X and DBL2X were made and used to immunize mice. The NTS-DBL1X, NTS-DBL1X-Id1, and Id1-DBL2X fragments all raised high titer immune response, as measured by ELISA. The DBL2X fragment raised a weaker antibody titer, and the Id1 construct failed to elicit antibody. Sera from mice immunized with NTS-DBL1X or DBL2X constructs failed to block infected erythrocytes binding to CSA, whereas sera from mice immunized with NTS-DBL1X-Id1 showed partial inhibitory activity, and the Id1-DBL2X fragment elicited antisera that totally abrogated infected erythrocytes adhesion to CSA. IgG purified from Id1-DBL2X antisera showed a similar inhibitory profile than Id1-DBL2X antisera. Anti-FCR3 anti-Id1-DBL2X antibodies also efficiently block the adhesion of erythrocytes infected by the HB3 parasite line to CSA. Id1-DBL2X antisera recognized the surface of field isolates from pregnant women, and inhibited CSA-binding of all 8 isolates tested, although to a variable level.
We raised high-titer antibodies against several parts of the protein, and identified Id1-DBL2X as the minimal VAR2CSA fragment inducing antibodies with CSA-binding inhibitory efficiency in the same range as the full-length extracellular part of VAR2CSA.
VAR2CSA 被认为是针对妊娠相关疟疾的保护性免疫的主要靶标。VAR2CSA 高分子量使 VAR2CSA 重组蛋白的大规模生产复杂化，以用于大规模疫苗接种计划。我们之前证明，NTS-DBL1X-Id1-DBL2X 诱导的抗体以类似于 VAR2CSA 全长细胞外部分诱导的抗体的方式有效地阻止寄生虫与 CSA 的结合。为了鉴定携带主要保护性表位的 VAR2CSA 的最短片段，能够引发抑制性抗体，我们通过 DNA 疫苗接种技术对 NTS-DBL1X-Id1-DBL2X 进行了精细的抗原作图。
构建了五个编码 NTS-DBL1X、NTS-DBL1X-Id1、Id1、Id1-DBL2X 和 DBL2X 的单域或双域构建体，并用于免疫小鼠。ELISA 测量表明，NTS-DBL1X、NTS-DBL1X-Id1 和 Id1-DBL2X 片段均引起高滴度的免疫反应。DBL2X 片段引起的抗体滴度较弱，而 Id1 构建体未能引起抗体。用 NTS-DBL1X 或 DBL2X 构建体免疫的小鼠的血清未能阻止感染的红细胞与 CSA 结合，而用 NTS-DBL1X-Id1 免疫的小鼠的血清显示部分抑制活性，Id1-DBL2X 片段诱导的抗血清完全阻断感染的红细胞与 CSA 的粘附。从 Id1-DBL2X 抗血清中纯化的 IgG 显示出与 Id1-DBL2X 抗血清相似的抑制谱。抗 FCR3 抗 Id1-DBL2X 抗体也有效地阻止 HB3 寄生虫系感染的红细胞与 CSA 的粘附。Id1-DBL2X 抗血清识别来自孕妇的现场分离株的表面，并抑制所有 8 个测试分离株与 CSA 的结合，尽管水平不同。
我们针对该蛋白的几个部分产生了高滴度的抗体，并鉴定出 Id1-DBL2X 是诱导与 VAR2CSA 全长细胞外部分具有相似 CSA 结合抑制效率的抗体的最小 VAR2CSA 片段。
