Hudspeth Elissa M, Wang Qian, Seid Christopher A, Hammond Molly, Wei Junfei, Liu Zhuyun, Zhan Bin, Pollet Jeroen, Heffernan Michael J, McAtee C Patrick, Engler David A, Matsunami Risë K, Strych Ulrich, Asojo Oluwatoyin A, Hotez Peter J, Bottazzi Maria Elena
a Department of Pediatrics (Section of Tropical Medicine) , National School of Tropical Medicine, Baylor College of Medicine , Houston , TX , USA.
b Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development , Houston , TX , USA.
Hum Vaccin Immunother. 2016 Jul 2;12(7):1707-20. doi: 10.1080/21645515.2016.1139254. Epub 2016 Feb 2.
Leishmania donovani is the major cause of visceral leishmaniasis (kala-azar), now recognized as the parasitic disease with the highest level of mortality second only to malaria. No human vaccine is currently available. A 36 kDa L. donovani nucleoside hydrolase (LdNH36) surface protein has been previously identified as a potential vaccine candidate antigen. Here we present data on the expression of LdNH36 in Pichia pastoris and its purification at the 20 L scale to establish suitability for future pilot scale manufacturing. To improve efficiency of process development and ensure reproducibility, 4 N-linked glycosylation sites shown to contribute to heterogeneous high-mannose glycosylation were mutated to glutamine residues. The mutant LdNH36 (LdNH36-dg2) was expressed and purified to homogeneity. Size exclusion chromatography and light scattering demonstrated that LdNH36-dg2 existed as a tetramer in solution, similar to the wild-type recombinant L. major nucleoside hydrolase. The amino acid mutations do not affect the tetrameric interface as confirmed by theoretical modeling, and the mutated amino acids are located outside the major immunogenic domain. Immunogenic properties of the LdNH36-dg2 recombinant protein were evaluated in BALB/c mice using formulations that included a synthetic CpG oligodeoxynucleotide, together with a microparticle delivery platform (poly(lactic-co-glycolic acid)). Mice exhibited high levels of IgG1, IgG2a, and IgG2b antibodies that were reactive to both LdNH36-dg2 and LdNH36 wild-type. While the point mutations did affect the hydrolase activity of the enzyme, the IgG antibodies elicited by LdNH36-dg2 were shown to inhibit the hydrolase activity of the wild-type LdNH36. The results indicate that LdNH36-dg2 as expressed in and purified from P. pastoris is suitable for further scale-up, manufacturing, and testing in support of future first-in-humans phase 1 clinical trials.
杜氏利什曼原虫是内脏利什曼病（黑热病）的主要病因，目前被认为是死亡率仅次于疟疾的寄生虫病。目前尚无人类疫苗。先前已鉴定出一种36 kDa的杜氏利什曼原虫核苷水解酶（LdNH36）表面蛋白作为潜在的疫苗候选抗原。在此，我们展示了LdNH36在毕赤酵母中的表达及其20 L规模的纯化数据，以确定其是否适合未来的中试规模生产。为提高工艺开发效率并确保可重复性，将显示有助于异质性高甘露糖糖基化的4个N-糖基化位点突变为谷氨酰胺残基。突变型LdNH36（LdNH36-dg2）表达并纯化至均一性。尺寸排阻色谱和光散射表明，LdNH36-dg2在溶液中以四聚体形式存在，类似于野生型重组硕大利什曼原虫核苷水解酶。理论建模证实，氨基酸突变不影响四聚体界面，且突变的氨基酸位于主要免疫原性结构域之外。使用包含合成CpG寡脱氧核苷酸以及微粒递送平台（聚乳酸-乙醇酸共聚物）的制剂，在BALB/c小鼠中评估了LdNH36-dg2重组蛋白的免疫原性。小鼠表现出高水平的IgG1、IgG2a和IgG2b抗体，这些抗体对LdNH36-dg2和LdNH36野生型均有反应。虽然点突变确实影响了该酶的水解活性，但LdNH36-dg2引发的IgG抗体显示可抑制野生型LdNH36的水解活性。结果表明，在毕赤酵母中表达并纯化的LdNH36-dg2适合进一步扩大规模、生产和测试，以支持未来的首次人体1期临床试验。
