The use of in silico analysis to engineer the best immunogenic epitope and produce the corresponding prophylactic antigen-based vaccines with C1 production platform in order to rapidly respond to viral pandemics

Please click Additional Files below to see the full abstrac

The use of in silico analysis to engineer the best immunogenic epitope and produce the corresponding prophylactic antigen-based vaccines with C1 production platform in order to rapidly respond to viral pandemics

Please click Additional Files below to see the full abstract

The C1 gene expression system, disrupting the way biologic vaccines, and drugs are developed & manufactured

Dyadic International, Inc. is a global biotechnology company which is developing what it believes will be a potentially significant biopharmaceutical protein production system based on the fungus Myceliophthora thermophila, nicknamed C1. The C1 microorganism, which enables the development and large scale manufacture of low cost proteins, has the potential to be further developed into a safe and efficient expression system that may help speed up the development, production and performance of biologic vaccines and drugs at flexible commercial scales. Dyadic is using the C1 technology and other technologies to conduct research, development and commercial activities for the development and manufacturing of human and animal vaccines, monoclonal antibodies, biosimilars and/or biobetters, and other therapeutic proteins. Dyadic pursues research & development collaborations, licensing arrangements and other commercial opportunities with its partners and collaborators to leverage the value and benefits of these technologies in developing and manufacturing biopharmaceuticals which these technologies help produce. In particular, as the aging population grows in developed and undeveloped countries, Dyadic believes the C1 technology may help bring biologic drugs to market faster, in greater volumes, at lower cost, and with new properties to drug developers and manufacturers and, hopefully, improve access and cost to patients and the healthcare system, but most importantly save lives

C1: How the C1 platform will change the production approach for therapeutic proteins

For over 30 years, Dyadic has proven itself, both commercially and scientifically, to be a high quality and highly productive producer of enzymes and proteins for specialty chemical applications using a proprietary and patented expression system based on the Myceliopthora thermophila fungus, nicknamed C1. The C1 platform technology, a hyper-productive fungal expression system, was used to develop & manufacture large quantities of desired proteins at industrial scale at significantly lower capital and operating expenditures. In this presentation we shall demonstrate how the benefits of C1 as a successful production host is now being harnessed by Dyadic to produce biological medicines and vaccines. Using new and improved C1 base strains along with better molecular genetics tools that have been developed over the past several years, we demonstrate the ability of C1 to express mAbs that are secreted, folded correctly and reach high yields. MAbs produced in C1 have almost identical binding kinetics to mAbs produced using CHO cells. In addition, our research program includes comprehensive approach to identify and knock-out proteases for further enhancing protein stability and improving yields. We have also achieved encouraging results, knowledge and experience in the rVaccine space from our prior research collaboration with Sanofi Pasteur to express rVaccines against Influenza virus. Results of a mice test that was conducted by Sanofi Pasteur, clearly demonstrated that HA produced by C1, generated high immunogenicity response against the virus without any adverse affects. Like other filamentous fungal strains, C1 has high mannose glycoform structures. However, unlike most fungi and yeasts, C1 does not have ‘high’ mannose (branched 30-50 mannose species), but rather has ‘oligo’ mannose structure (branched 5-9 mannose species). In addition, no O-glycosylation has been observed on C1 secreted proteins, in contrast to Pichia that O-glycosylates all secreted antibodies, necessitating deletion of the O-glycosylation machinery. Using the benefits of those advantages, we have started Glycoengineering program aiming to develop C1 strain that produces proteins with defined human-like glycan patterns. The progress we have already made in C1 glycoengineering will also be presented. Thus, Dyadic firmly believe that the C1 strains that we are developing with offer certain competitive advantages over other leading pharmaceutical expression systems, such as CHO cells, has the potential to become the production system of choice for therapeutic protein and vaccines manufacturing

Dopamine D2 receptor gene variants and response to rasagiline in early Parkinson's disease:a pharmacogenetic study

The treatment of early Parkinson's disease with dopaminergic agents remains the mainstay of symptomatic therapy for this incurable neurodegenerative disorder. However, clinical responses to dopaminergic drugs vary substantially from person to person due to individual-, drug- and disease-related factors that may in part be genetically determined. Using clinical data and DNA samples ascertained through the largest placebo-controlled clinical trial of the monoamine oxidase B inhibitor, rasagiline (ClinicalTrials.gov number, NCT00256204), we examined how polymorphisms in candidate genes associate with the clinical response to rasagiline in early Parkinson's disease. Variants in genes that express proteins involved in the pharmacokinetics and pharmacodynamics of rasagiline, and genes previously associated with the risk to develop Parkinson's disease were genotyped. The LifeTechnologies OpenArray NT genotyping platform and polymerase chain reaction-based methods were used to analyse 204 single nucleotide polymorphisms and five variable number tandem repeats from 30 candidate genes in 692 available DNA samples from this clinical trial. The peak symptomatic response to rasagiline, the rate of symptom progression, and their relation to genetic variation were examined controlling for placebo effects using general linear and mixed effects models, respectively. Single nucleotide polymorphisms, rs2283265 and rs1076560, in the dopamine D2 receptor gene (DRD2) were found to be significantly associated with a favourable peak response to rasagiline at 12 weeks in early Parkinson's disease after controlling for multiple testing. From a linear regression, the betas were 2.5 and 2.38, respectively, with false discovery rate-corrected P-values of 0.032. These polymorphisms were in high linkage disequilibrium with each other (r(2) = 0.96) meaning that the same clinical response signal was identified by each of them. No polymorphisms were associated with slowing the rate of worsening in Parkinson symptoms from Weeks 12 to 36 after correction for multiple testing. This is the largest and most comprehensive pharmacogenetics study to date examining clinical response to an anti-parkinsonian drug and the first to be conducted in patients with early stage Parkinson's disease receiving monotherapy. The results indicate a clinically meaningful benefit to rasagiline in terms of the magnitude of improvement in parkinsonian symptoms for those with the favourable response genotypes. Future work is needed to elucidate the specific mechanisms through which these DRD2 variants operate in modulating the function of the nigrostriatal dopaminergic system

Affinity purification of SARS-COV-2 spike protein receptor binding domain produced in a C1 fungal expression system

The Receptor Binding Domain (RBD)of the spike protein of SARS-CoV-2 has shown promise for diagnosis, treatment, and development of vaccines for COVID-19. However, two problems persist with large scale production of RBD: 1) lack of high productivity upstream cell culture, 2) absence of a commercial, highly selective affinity resin. In an effort to overcome these limitations, we evaluated two novel technologies for the production and purification of RBD. Briefly, RBD was expressed using C1, an engineered fungal strain of Thermothelomyces heterothallica (DyadicInternational1). The C1 platform expresses glycosylated antigens with high productivity, stability, and purity. RBD was purified using a novel affinity resin2 known to produce yields of 90% to 95% purity in one chromatography step. Affinity purification did not affect protein quality, as demonstrated by ACE-2 binding of RBD. The novel affinity resin showed excellent base stability, consistent product quality, and similar ACE-2 binding activity over 40 cycles. Please click Download on the upper right corner to see the full abstract

Development of the filamentous fungus Thermothelomyces heterothallica C1 into a next-generation production platform for human and animal vaccines

engineering. The thermophilic fungus Thermothelomyces heterothallica is a robust and versatile fungal expression system for the rapid production of proteins at very high levels. In the last 6 years, the C1 protein production platform has been further improved to become a safe and efficient expression system with the prime objective of speeding up the development and production of commercial scale human and animal vaccines, monoclonal antibodies, biosimilars, as well as other therapeutic proteins at larger quantities and lower cost. C1 is a very efficient platform to produce antigens, even to generate multicomponent vaccines. The production levels of engineered C1 strains are similar in terms of yield and purity, reaching in some cases more than 2.5 g/L (in 4-5 days). In contrast to other vaccine platforms, C1 has a higher safety profile, and production can be scaled up in a more cost-effective manner using standard microbial E. coli fermenters. Stable cell lines have been developed to produce different antigens as influenza, neuraminidase, west Nile, rabies, rift valley fever..etc. Please click Download on the upper right corner to see the full abstract

Genome-wide analysis of Sphingomonas wittichii RW1 behaviour during inoculation and growth in contaminated sand.

The efficacy of inoculation of single pure bacterial cultures into complex microbiomes, for example, in order to achieve increased pollutant degradation rates in contaminated material (that is, bioaugmentation), has been frustrated by insufficient knowledge on the behaviour of the inoculated bacteria under the specific abiotic and biotic boundary conditions. Here we present a comprehensive analysis of genome-wide gene expression of the bacterium Sphingomonas wittichii RW1 in contaminated non-sterile sand, compared with regular suspended batch growth in liquid culture. RW1 is a well-known bacterium capable of mineralizing dibenzodioxins and dibenzofurans. We tested the reactions of the cells both during the immediate transition phase from liquid culture to sand with or without dibenzofuran, as well as during growth and stationary phase in sand. Cells during transition show stationary phase characteristics, evidence for stress and for nutrient scavenging, and adjust their primary metabolism if they were not precultured on the same contaminant as found in the soil. Cells growing and surviving in sand degrade dibenzofuran but display a very different transcriptome signature as in liquid or in liquid culture exposed to chemicals inducing drought stress, and we obtain evidence for numerous 'soil-specific' expressed genes. Studies focusing on inoculation efficacy should test behaviour under conditions as closely as possible mimicking the intended microbiome conditions

Preclinical immunogenicity and protective efficacy of a SARS-CoV-2 RBD-based vaccine produced with the thermophilic filamentous fungal expression system Thermothelomyces heterothallica C1

INTRODUCTION: The emergency use of vaccines has been the most efficient way to control the coronavirus disease 19 (COVID-19) pandemic. However, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern has reduced the efficacy of currently used vaccines. The receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein is the main target for virus neutralizing (VN) antibodies. METHODS: A SARS-CoV-2 RBD vaccine candidate was produced in the Thermothelomyces heterothallica (formerly, Myceliophthora thermophila) C1 protein expression system and coupled to a nanoparticle. Immunogenicity and efficacy of this vaccine candidate was tested using the Syrian golden hamster (Mesocricetus auratus) infection model. RESULTS: One dose of 10-μg RBD vaccine based on SARS-CoV-2 Wuhan strain, coupled to a nanoparticle in combination with aluminum hydroxide as adjuvant, efficiently induced VN antibodies and reduced viral load and lung damage upon SARS-CoV-2 challenge infection. The VN antibodies neutralized SARS-CoV-2 variants of concern: D614G, Alpha, Beta, Gamma, and Delta. DISCUSSION: Our results support the use of the Thermothelomyces heterothallica C1 protein expression system to produce recombinant vaccines against SARS-CoV-2 and other virus infections to help overcome limitations associated with the use of mammalian expression system