The Highly Productive Thermothelomyces heterothallica C1 Expression System as a Host for Rapid Development of Influenza Vaccines

Abstract

(1) Influenza viruses constantly change and evade prior immune responses, forcing seasonal re-vaccinations with updated vaccines. Current FDA-approved vaccine manufacturing technologies are too slow and/or expensive to quickly adapt to mid-season changes in the virus or to the emergence of pandemic strains. Therefore, cost-effective vaccine technologies that can quickly adapt to newly emerged strains are desirable. (2) The filamentous fungal host Thermothelomyces heterothallica C1 (C1, formerly Myceliophthora thermophila) offers a highly efficient and cost-effective alternative to reliably produce immunogens of vaccine quality at large scale. (3) We showed the utility of the C1 system expressing hemagglutinin (HA) and a HA fusion protein from different H1N1 influenza A virus strains. Mice vaccinated with the C1-derived HA proteins elicited anti-HA immune responses similar, or stronger than mice vaccinated with HA products derived from prototypical expression systems. A challenge study demonstrated that vaccinated mice were protected against the aggressive homologous viral challenge. (4) The C1 expression system is proposed as part of a set of protein expression systems for plug-and-play vaccine manufacturing platforms. Upon the emergence of pathogens of concern these platforms could serve as a quick solution for producing enough vaccines for immunizing the world population in a much shorter time and more affordably than is possible with current platforms.

Filamentous Fungus-Produced Human Monoclonal Antibody Provides SARS-CoV-2 Protection in Hamster and Non-Human Primate Models

“…Several studies have demonstrated the bene ts of C1 produced proteins as promising vaccine candidates for various viral pathogens such as In uenza A virus, SARS-CoV-2, and orthobunyaviruses. These studies have used different subunits, such as the hemagglutinin for In uenza A virus, the RBD for SARS-CoV-2 and part of the Gc glycoprotein for Schmallenberg virus 18,19,23 . A previous study testing C1-derived SARS-CoV-2 RBD protein in rabbits for toxicology did not show any local or systemic side effects of fungal produced proteins, indicating its potential safety for use in humans 24 .…”

“…During the last decades, the C1 expression system has been genetically modi ed continuously resulting in a well-characterized genetic toolbox and technology platform 17 . Besides the industrial use, heterologous proteins expressed by the C1 expression system have been successfully used such as the successful production of SARS-CoV-2 Full Spike protein, receptor binding domain (RBD)-based vaccine candidates against COVID-19 and hemagglutinin and neuraminidase in uenza antigens in animal models, showing their immunogenicity and effectiveness [18][19][20] .…”

Toxicity and Local Tolerance of a Novel Spike Protein RBD Vaccine Against SARS-CoV-2, Produced Using the C1 Thermothelomyces Heterothallica Protein Expression Platform

“…The expression vector was transformed into C1 DNL-155 strain as described and cell clones producing RBD-C-tag protein were selected. 14 …”

“…C1-RBD was manufactured in a 10-L Bioflo 120 Fermenter (Eppendorf, Hamburg). Five liters of high concentration yeast extract medium with glucose 14 was inoculated with a seed culture of 500 ml the C1 DNL-155 production strain, Fermentation conditions were as decribed. 14 …”

“… 12 Since 2016 C1 is explicitly being used for the rapid development and production of therapeutic proteins and vaccines. 13 , 14 Characterization of the glycoprotein structure of the recombinant RBD-C-tag 333-527 produced in C1 has been previously compared with RBD produced in Yeast, E. coli , and Mammalian cells using buffer-free protein digestion with electrospray ionization-mass spectrometry analysis. 11 This confirmed its identity with the typical heterogeneity of N-glycans and the expected molecular masses, as well as the integrity of the N-terminal and C-terminal ends and formation of the four disulfide bonds.…”