Investigation of Kluyveromyces marxianus as a novel host for large\u2010scale production of porcine parvovirus virus\u2010like particles

Deqiang Yang,Jinkun Duan,Jungang Zhou,Lei Chen,Hong Lu,Yao Yu

doi:10.1186/s12934-021-01514-5

Deqiang Yang, Jinkun Duan + Show 4 more

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https://doi.org/10.1186/s12934-021-01514-5

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Abstract

BackgroundPorcine 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.ResultsAfter 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.ConclusionsThis 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.

Highlights

Porcine Parvovirus (PPV) is a Parvovirinae virus that can cause embryonic and fetal loss and death and mummification in affected fetal pigs
Expression of PPV VP2 in K. marxianus The VP2 gene of the Kresse strain was used to express in K. marxianus, since this PPV strain displayed an increased virulence compared with other virulent strains that could kill immunocompetent fetuses [40]
From SDS-PAGE, we found PPV virus-like particles (VLPs) were almost captured by the anion resins, and the purity of elution was lower than 10% as analyzed by HPLC (Fig. 7b)

Summary

Introduction

Porcine Parvovirus (PPV) is a Parvovirinae virus that can cause embryonic and fetal loss and death and mummification in affected fetal pigs. Yang et al Microb Cell Fact (2021) 20:24 of molecular biology techniques, six new serotypes of parvovirus have been clinically discovered in pigs, and designated as PPV2 to PPV7 [1]. The PPV virus can replicate and be shed from infected sows with no clinical symptoms, but transplacental infections usually result in death and mummification of the fetus before 70 days of gestation, in which fetuses have not developed antibodies to eliminate viruses and survived the infection. Vaccination against porcine parvovirus can’t prevent the virus infection and shedding, but it protects swine from SMEDI diseases [6]

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