Production of self-assembled vaccine antigens in Komagataella phaffii
File(s)
Author(s)
Mastropietro Garcia, Giuliana
Type
Thesis
Abstract
Developing countries face significant challenges in vaccine delivery, primarily due to the cost of vaccines and distribution systems. To overcome these obstacles, new technologies are emerging to revolutionise vaccine manufacturing and distribution for global accessibility and affordability. One of these technologies involve a novel platform utilising yeast-expressed self-assembling antigens, including virus-like particles (VLPs).
In this project the methylotrophic yeast Komagataella phaffii was employed for its ability to achieve high cell densities using methanol as its carbon and energy source, producing high titres of secreted heterologous protein with minimal endogenous proteins. By genetically modifying this yeast, Hepatitis E (HEV), Chikungunya (CHKV) ECSA and CAR genotypes, and Human Papilloma Virus (HPV) serotypes -6, -11, -16 and -18 VLPs and the rabies virus glycoprotein were expressed.
Evaluation of the expressing strains involved various techniques such as polyacrylamide electrophoresis, western blot, and ELISA, complemented by characterisation through transmission electron microscopy and dynamic light scattering for VLPs. Secretion saturation in K. phaffii, due to increased trafficking through the secretory pathway, prompted a transcriptomic analysis to identify stress responses and adverse metabolic changes in protein secretion. Notably, a reduction in expression temperature increased the yield of HEV, HPV-6, and HPV-16 recombinant proteins by 14-, 9- and 20-fold, respectively.
The transcriptomic analysis of the HPV-11 VLP-expressing strain revealed a bottleneck in protein translocation, leading to the application of two strategies: the push-and-pull strategy involving chaperone overexpression and signal peptide substitution for improved translocation. The latter strategy demonstrated promising results in enhancing recombinant protein secretion.
This project underscores the ability of K. phaffii to efficiently secrete previously unexpressed HPV-6 and HPV-11 L1 recombinant proteins, along with the HEV capsid protein. Furthermore, it lays the groundwork for expressing CHKV ECSA, CAR, and modified rabies glycoprotein recombinantly, addressing critical vaccine production needs for diseases prevalent in low- and middle-income countries.
In this project the methylotrophic yeast Komagataella phaffii was employed for its ability to achieve high cell densities using methanol as its carbon and energy source, producing high titres of secreted heterologous protein with minimal endogenous proteins. By genetically modifying this yeast, Hepatitis E (HEV), Chikungunya (CHKV) ECSA and CAR genotypes, and Human Papilloma Virus (HPV) serotypes -6, -11, -16 and -18 VLPs and the rabies virus glycoprotein were expressed.
Evaluation of the expressing strains involved various techniques such as polyacrylamide electrophoresis, western blot, and ELISA, complemented by characterisation through transmission electron microscopy and dynamic light scattering for VLPs. Secretion saturation in K. phaffii, due to increased trafficking through the secretory pathway, prompted a transcriptomic analysis to identify stress responses and adverse metabolic changes in protein secretion. Notably, a reduction in expression temperature increased the yield of HEV, HPV-6, and HPV-16 recombinant proteins by 14-, 9- and 20-fold, respectively.
The transcriptomic analysis of the HPV-11 VLP-expressing strain revealed a bottleneck in protein translocation, leading to the application of two strategies: the push-and-pull strategy involving chaperone overexpression and signal peptide substitution for improved translocation. The latter strategy demonstrated promising results in enhancing recombinant protein secretion.
This project underscores the ability of K. phaffii to efficiently secrete previously unexpressed HPV-6 and HPV-11 L1 recombinant proteins, along with the HEV capsid protein. Furthermore, it lays the groundwork for expressing CHKV ECSA, CAR, and modified rabies glycoprotein recombinantly, addressing critical vaccine production needs for diseases prevalent in low- and middle-income countries.
Version
Open Access
Date Issued
2024-02-21
Date Awarded
2024-06-01
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Polizzi, Karen
Publisher Department
Chemical Engineering
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)