The freshwater ciliate, Tetrahymena thermophila, has recently emerged as a novel manufacturing platform for recombinant subunit vaccines. It combines ease of growth with facile genetics in a complex unicellular eukaryote that can be grown rapidly in inexpensive media on an industrial scale. T. thermophila devotes a large part of its metabolism to membrane protein production, and can release proteins to the extracellular space by constitutive and stimulus-dependent pathways of secretion. In this article, we show high-level expression of correctly folded parasite and viral proteins in a Tetrahymena system and provide direct evidence that regulated secretion can be harnessed as an effective pathway for producing influenza hemagglutinin (HA). HA can be targeted to dense core granules in vivo and be recovered following stimulus-dependent secretion in association with a proteinaceous gel termed PRISM. PRISM offers a convenient matrix for protein purification, but at the same time, has intrinsic properties with the potential to induce potent immune responses to co-administered antigens.
To address some of the challenges surrounding potency, investigators have turned to eukaryotic expression hosts for improved protein folding (especially insect cells and fungi),5–7 and using virus-like particles (VLPs) that can present antigens in a repetitive, high-density format that can generate strong B- and T-cell responses.8,9 Still, VLPs are not applicable to all vaccine antigens and can be difficult or slow to produce in high yield.
As an alternative to these approaches, we have focused on Tetrahymena thermophila, a eukaryotic microbe that is capable of rapid, scalable growth. Tetrahymena lacks a cell wall and adds mammalian-like post-translational modifications (PTMs) onto proteins.10–13 More importantly, T. thermophila devotes a large-part of its metabolism to membrane protein production owing to the hundreds of cilia that extend from its surface. Moreover, Tetrahymena not only constitutively secretes proteins, but also stores large amounts of protein in hundreds-to-thousands of dense core granules that can be induced to secrete at will.14,15 Indeed, the material released from these granules takes the form of a proteinaceous gel (termed PRISM), which can easily be harvested from cells by low-speed centrifugation, providing a natural matrix for streamlined protein purification. PRISM has an underlying crystalline structure, and like VLPs, offers the opportunity to present antigens in a repetitive format that is optimal for cross-linking of the immunoglobulin (Ig) receptor on B-cells. In this article, we demonstrate the expression of correctly folded viral and eukaryotic vaccine antigens in the Tetrahymena system and provide preliminary evidence that PRISM may be an ideal matrix for rapid production of highly potent, low-cost vaccines.