Csalane Besenyei, Greta; (2024) Engineering Phaeodactylum for vaccine production. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The evolution of molecular biology tools enabled the development of a variety of recombinant vaccine production platforms with a prospect to alleviate the bottlenecks of traditional vaccine manufacturing strategies. Phaeodactylum tricornutum, the photoautotroph model diatom, has been established as a eukaryotic microalgal expression platform producing a recombinant Hepatitis B surface antigen and its respective human antibody, offering the unique opportunity for optional glycosylation and targeting proteins into the culture medium. The selection of recombinant cell lines intended for whole-cell oral vaccine applications requires routinely used antibiotic selection markers to be replaced with alternative selection methods due to the risk of horizontal gene transfer. ptUMPS is one of the two endogenous, auxotrophic selection markers discovered so far in P. tricornutum, however, its use as a complementation marker for the positive selection of rescued, ex-auxotrophic mutants without the use of 5-FOA has not yet been explored. In this study, the production of a recombinant salmonid alphavirus (SAV) vaccine antigen (~120 kDa) is demonstrated in P. tricornutum using a robust selection protocol relying exclusively on the ptUMPS uracil auxotrophic selectable marker and standard f/2 selective medium. Furthermore, a faster selection protocol in liquid medium was established by shortening the uracil starvation phase to 6 weeks, requiring 3 transfers at inoculum rates not higher than 106 cells∙mL-1 . The isolated SAV clones have shown stable expression for >18 months in standard f/2 medium in the absence of selective pressure. In addition, the SAV antigen was expressed under the control of the highly sensitive, inducible alkaline phosphatase promoter with a significantly higher yield (24.1 μg∙L-1 culture) under phosphate replete conditions (<3.6 μM) compared to constitutive SAV expression driven by the fcpA promoter. Finally, process scale-up to 20L batch volume - cultured photosynthetically in single-use hanging bags and induced using in situ flocculation without the use of centrifugation - demonstrated the adaptability of the above process for implementation in outdoor aquaculture sites and highlighted the potential of this emerging platform for further development towards oral vaccine applications.

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