Markku Saloheimo

SARS-CoV-2 is evolving with increased transmission, host range, pathogenicity, and virulence. The original and mutant viruses escape host innate (Interferon) immunity and adaptive (Antibody) immunity, emphasizing unmet needs for high-yield, commercial-scale manufacturing to produce inexpensive vaccines/boosters for global/equitable distribution. We developed DYAI-100A85, a SARS-CoV-2 spike receptor binding domain (RBD) subunit antigen vaccine expressed in genetically modified thermophilic filamentous fungus, Thermothelomyces heterothallica C1, and secreted at high levels into fermentation medium. The RBD-C-tag antigen strongly binds ACE2 receptors in vitro. Alhydrogel^®‘85’-adjuvanted RDB-C-tag-based vaccine candidate (DYAI-100A85) demonstrates strong immunogenicity, and antiviral efficacy, including in vivo protection against lethal intranasal SARS-CoV-2 (D614G) challenge in human ACE2-transgenic mice. No loss of body weight or adverse events occurred. DYAI-100A85 also demonstrates excellent safety profile in repeat-dose GLP toxicity study. In summary, subcutaneous prime/boost DYAI-100A85 inoculation induces high titers of RBD-specific neutralizing antibodies and protection of hACE2-transgenic mice against lethal challenge with SARS-CoV-2. Given its demonstrated safety, efficacy, and low production cost, vaccine candidate DYAI-100 received regulatory approval to initiate a Phase 1 clinical trial to demonstrate its safety and efficacy in humans.

(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.

Emerging infectious diseases represent an increasing threat to human and animal health. Therefore, safe and effective vaccines that could be available within a short time frame after an outbreak are required for adequate prevention and control. Here, we developed a robust and versatile self-assembling multimeric protein scaffold particle (MPSP) vaccine platform using lumazine synthase (LS) from Aquifex aeolicus. This scaffold allowed the presentation of peptide epitopes by genetic fusion as well as the presentation of large antigens by bacterial superglue-based conjugation to the pre-assembled particle. Using the orthobunyavirus model Schmallenberg virus (SBV) we designed MPSPs presenting major immunogens of SBV and assessed their efficacy in a mouse model as well as in cattle, a target species of SBV. All prototype vaccines conferred protection from viral challenge infection and the multivalent presentation of the selected antigens on the MPSP markedly improved their immunogenicity compared to the monomeric subunits. Even a single shot vaccination protected about 80% of mice from an otherwise lethal dose of SBV. Most importantly, the MPSPs induced a virtually sterile immunity in cattle. Altogether, LS represents a promising platform for modular and rapid vaccine design.

During the past few years, the first industrial-scale cellulosic ethanol plants have been inaugurated. Although the performance of the commercial cellulase enzymes used in this process has greatly improved over the past decade, cellulases still represent a very significant operational cost. Depending on the region, transport of cellulases from a central production facility to a biorefinery may significantly add to enzyme cost. The aim of the present study was to develop a simple, cost-efficient cellulase production process that could be employed locally at a Brazilian sugarcane biorefinery. Our work focused on two main topics: growth medium formulation and strain improvement. We evaluated several Brazilian low-cost industrial residues for their potential in cellulase production. Among the solid residues evaluated, soybean hulls were found to display clearly the most desirable characteristics. We engineered a Trichoderma reesei strain to secrete cellulase in the presence of repressing sugars, enabling the use of sugarcane molasses as an additional carbon source. In addition, we added a heterologous β-glucosidase to improve the performance of the produced enzymes in hydrolysis. Finally, the addition of an invertase gene from Aspegillus niger into our strain allowed it to consume sucrose from sugarcane molasses directly. Preliminary cost analysis showed that the overall process can provide for very low-cost enzyme with good hydrolysis performance on industrially pre-treated sugarcane straw. In this study, we showed that with relatively few genetic modifications and the right growth medium it is possible to produce considerable amounts of well-performing cellulase at very low cost in Brazil using T. reesei. With further enhancements and optimization, such a system could provide a viable alternative to delivered commercial cellulases.

The filamentous fungus Trichoderma reesei has tremendous capability to secrete over 100 g/L of proteins and therefore it would make an excellent host system for production of high levels of therapeutic proteins at low cost. We have developed T. reesei strains suitable for production of therapeutic proteins by reducing the secreted protease activity. Protease activity has been the major hindrance to achieving high production levels. We have constructed a series of interferon alpha-2b (IFNα-2b) production strains with 9 protease deletions to gain knowledge for further strain development. We have identified two protease deletions that dramatically improved the production levels. Deletion of the subtilisin protease slp7 and the metalloprotease amp2 has enabled production levels of IFNα-2b up to 2.1 and 2.4 g/L, respectively. With addition of soybean trypsin protease inhibitor the level of production improved to 4.5 g/L, with an additional 1.8 g/L still bound to the secretion carrier protein. High levels of IFNα-2b were produced using T. reesei strains with reduced protease secretion. Further strain development can be done to improve the production system by reducing protease activity and improving carrier protein cleavage.

Extracellular pH is one of the several environmental factors affecting protein production by filamentous fungi. Regulatory mechanisms ensure that extracellular enzymes are produced under pH-conditions in which the enzymes are active. In filamentous fungi, the transcriptional regulation in different ambient pH has been studied especially in Aspergilli, whereas the effects of pH in the industrial producer of hydrolytic enzymes, Trichoderma reesei, have mainly been studied at the protein level. In this study, the pH-dependent expression of T. reesei genes was investigated by genome-wide transcriptional profiling and by analysing the effects of deletion of the gene encoding the transcriptional regulator pac1, the orthologue of Aspergillus nidulans pacC gene. Transcriptional analysis revealed the pH-responsive genes of T. reesei, and functional classification of the genes identified the activities most affected by changing pH. A large number of genes encoding especially transporters, signalling-related proteins, extracellular enzymes and proteins involved in different metabolism-related functions were found to be pH-responsive. Several cellulase- and hemicellulase-encoding genes were found among the pH-responsive genes. Especially, genes encoding hemicellulases with the similar type of activity were shown to include both genes up-regulated at low pH and genes up-regulated at high pH. However, relatively few of the cellulase- and hemicellulase-encoding genes showed direct PACI-mediated regulation, indicating the importance of other regulatory mechanisms affecting expression in different pH conditions. New information was gained on the effects of pH on the genes involved in ambient pH-signalling and on the known and candidate regulatory genes involved in regulation of cellulase and hemicellulase encoding genes. In addition, co-regulated genomic clusters responding to change of ambient pH were identified. Ambient pH was shown to be an important determinant of T. reesei gene expression. The pH-responsive genes, including those affected by the regulator of ambient pH sensing, were identified, and novel information on the activity of genes encoding carbohydrate active enzymes at different pH was gained.

Trichoderma reesei is known as a good producer of industrial proteins but has hitherto been less successful in the production of therapeutic proteins. In order to elucidate the bottlenecks of heterologous protein production, human α-galactosidase A (GLA) was chosen as a model therapeutic protein. Fusion partners were designed to compare the effects of secretion using a cellobiohydrolase I (CBHI) carrier and intracellular production using a gamma zein peptide from maize (ZERA) which accumulates inside the endoplasmic reticulum (ER). The two strategies were compared on the basis of expression levels, purification performance, enzymatic activity, bioreactor cultivations, and transcriptional profiling. Constructs were cloned into the cbh1 locus of the T. reesei strain Rut-C30. The secretion and intracellular strains produced 20 mg/l and 636 mg/l of GLA respectively. Purifications of secreted product were accomplished using Step-Tactin affinity columns and for intracellular product, a method was developed for gravity-based density separation and protein body solubilisation. The secreted protein had similar specific activity to that of the commercially available mammalian form. The intracellular version had 5-10-fold lower activity due to the enzymes incompatibility with alkaline pH. The secretion strain achieved 10% lower total biomass than either the parental or the intracellular strain. The patterns of gene induction for intracellular and parental strains were similar, whereas the secretion strain had a broader spectrum of gene expression level changes. Identification of the genes involved indicated strong secretion stress in the secretion strain and to a lesser extent also in intracellular production. Genes involved in the unfolded protein response (UPR) and ER-associated degradation were induced by GLA production, including; hac1, pdi1, prp1, cnx1, der1, and bap31. Active human α-galactosidase could most effectively be produced intracellularly in Trichoderma reesei at >0.5 g/l by avoidance of the extracellular environment, although purification was challenging due to specific activity losses. Strain analysis revealed that in addition to the issues with secreted proteases, the processes of secretion stress including UPR and ER degradation remain as bottlenecks for heterologous protein production. Genetic engineering to eliminate these bottlenecks is the logical path towards establishing a strain capable of producing sensitive heterologous proteins.

The soft rot ascomycetal fungus Trichoderma reesei is utilized for industrial production of secreted enzymes, especially lignocellulose degrading enzymes. T. reesei uses several different enzymes for the degradation of plant cell wall-derived material, including 9 characterized cellulases, 15 characterized hemicellulases and at least 42 genes predicted to encode cellulolytic or hemicellulolytic activities. Production of cellulases and hemicellulases is modulated by environmental and physiological conditions. Several regulators affecting the expression of cellulase and hemicellulase genes have been identified but more factors still unknown are believed to be present in the genome of T. reesei.

Trichoderma reesei is a soft rot Ascomycota fungus utilised for industrial production of secreted enzymes, especially lignocellulose degrading enzymes. About 30 carbohydrate active enzymes (CAZymes) of T. reesei have been biochemically characterised. Genome sequencing has revealed a large number of novel candidates for CAZymes, thus increasing the potential for identification of enzymes with novel activities and properties. Plenty of data exists on the carbon source dependent regulation of the characterised hydrolytic genes. However, information on the expression of the novel CAZyme genes, especially on complex biomass material, is very limited.