State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, 2005 Songhu Road, Shanghai, 200438, People's Republic of China.
Shanghai Engineering Research Center of Industrial Microorganisms, 2005 Songhu Road, Shanghai, 200438, People's Republic of China.
Microb Cell Fact. 2021 Jan 25;20(1):24. doi: 10.1186/s12934-021-01514-5.
Porcine Parvovirus (PPV) is a Parvovirinae virus that can cause embryonic and fetal loss and death and mummification in affected fetal pigs. Unlike conventional vaccines, virus-like particles (VLPs) inherit the natural structure of their authentic virions and highly immunostimulatory that can induce strong humoral immune and T cell responses with no risk of pathogenicity. The production of PPV VLPs is still a challenge based on traditional expression platforms due to their low yields and high culture costs. Kluyveromyces marxianus is a safe and fast-growing eukaryote that can get high biomass with low-cost cultures. In this study, we investigated the expression and downstream processes of PPV VLPs in K. marxianus, and the potential for effective stand-alone vaccines.
After optimization according to the codon bias of K. marxianus, the VP2 protein from Kresse strain was highly expressed. In a 5 L fermentator, the yield of PPV VLPs reached 2.5 g/L, quantified by HPLC, using a defined mineral medium after 48 h fermentation. Two strategies were established to purify intracellular PPV VLPs: (i) Using the cation exchange chromatography coupled with Sephacryl® S-500 HR chromatography to purify VLPs from the supernatants of pH adjusted cell lysates. (ii) Using anion exchange chromatography followed by cross-flow diafiltration to recover the VLPs precipitated in pH adjusted cell lysates. The purity of PPV VLPs reached about 95%, and total recovery was more than 60%. Vaccination of mice with the purified PPV VLPs induced high titers of specific IgG antibodies in sera, and showed hemagglutination inhibitions on both swine and guinea pig erythrocytes. Spleen lymphocyte proliferation and cytokines detection suggested the PPV VLPs produced by K. marxianus provoked the cellular immune and humoral immunity responses in mice.
This is the highest production of recombinant PPV VLPs achieved to date. The superiorities, Generally Recognized As Safe (GRAS), high production, short lead time, and low cost, make K. marxianus a greatly competitive platform for bioproduction of PPV VLPs vaccine.
猪细小病毒（PPV）是细小病毒科的一种病毒，可导致受感染胎儿猪的胚胎和胎儿死亡以及木乃伊化。与传统疫苗不同，病毒样颗粒（VLPs）继承了其天然病毒粒子的自然结构，具有高度免疫刺激性，可诱导强烈的体液免疫和 T 细胞反应，且无致病性风险。由于产量低和培养成本高，基于传统表达平台生产 PPV VLPs 仍然是一个挑战。马克斯克鲁维酵母是一种安全且生长迅速的真核生物，可利用低成本培养获得高生物量。在本研究中，我们研究了 PPV VLPs 在马克斯克鲁维酵母中的表达和下游工艺，以及作为有效独立疫苗的潜力。
根据马克斯克鲁维酵母的密码子偏好性进行优化后，Kresse 株的 VP2 蛋白得到了高度表达。在 5 L 发酵罐中，经过 48 h 发酵，使用定义的矿物培养基，通过 HPLC 定量，VP2 蛋白的产量达到了 2.5 g/L。建立了两种从细胞内纯化 PPV VLPs 的策略：（i）使用阳离子交换色谱结合 Sephacryl® S-500 HR 色谱从 pH 调整后的细胞裂解物上清液中纯化 VLPs。（ii）使用阴离子交换色谱，然后进行切向流超滤，回收在 pH 调整后的细胞裂解物中沉淀的 VLPs。PPV VLPs 的纯度达到约 95%，总回收率超过 60%。用纯化的 PPV VLPs 免疫小鼠可诱导血清中特异性 IgG 抗体的高滴度，并对猪和豚鼠红细胞均具有血凝抑制作用。脾淋巴细胞增殖和细胞因子检测表明，马克斯克鲁维酵母生产的 PPV VLPs 可引起小鼠的细胞免疫和体液免疫反应。
这是迄今为止重组 PPV VLPs 的最高产量。马克斯克鲁维酵母具有优势，被普遍认为安全（GRAS）、高产量、短生产时间和低成本，使其成为生产 PPV VLPs 疫苗的极具竞争力的生物生产平台。
