An Alternative Platform for Rapid Production of Effective Subunit Vaccines - Tetrahymena thermophila offers numerous advantages as an expression system, including rapid cell growth and high cell densi

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An Alternative Platform for Rapid Production of Effective Subunit Vaccines
Tetrahymena thermophila offers numerous advantages as an expression system, including rapid cell growth and high cell densities, eukaryotic protein folding, and active synthesis of membrane and secreted proteins.


BioPharm International Supplements


ABSTRACT

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.


(TETRAGENETICS, INC.)
Although cell-mediated immunity can play a significant role in clearing microbial pathogens, antibodies targeting secreted proteins or exposed antigens on microbial surfaces are often sufficient to generate protection against infectious agents. In such cases, vaccines that elicit strong, long-lasting antibody responses are highly effective in preventing disease. When compared with live and killed whole pathogens, subunit vaccines have distinct advantages particularly when dealing with newly emerging infectious agents such as pandemic influenza virus that have the potential to cause widespread disease. Subunit antigens can be produced rapidly on a large-scale as recombinant proteins without relying on attenuation or killing as steps in the production process. Subunit proteins are typically less reactogenic than live or killed pathogens and can be delivered at high antigenic mass. Furthermore, their production obviates the need to work directly with pathogens, which can be difficult to grow and dangerous. Finally, because recombinant antigens can be manufactured in microbial systems, the potential for contamination by adventitious agents that infect mammalian cells is minimized.

Despite the advantages of recombinant subunit proteins, their protective efficacy may be lower than that of native antigens because of a number of factors. Primary among these is absence of pathogen-associated molecules and other "danger signals" required to stimulate robust immune responses to purified antigens.1,2 Although these can be provided by adding immuonstimulatory substances, or adjuvants, to the vaccine formulation, there are few such substances currently approved by the FDA.3 Equally important is the problem of protein folding and post-translation processing. In the case of viruses and parasites, the preponderance of vaccine candidates are membrane or secreted proteins that must fold properly to elicit protective antibodies in the host. This requires accurate disulfide bond formation, which is often difficult to achieve in bacterial expression hosts.4 Some microbial systems (including yeast) also contain rigid cell walls that impede downstream protein purification, and contain endogenous pyrogens that must be removed in the production process.

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.


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