Biology subjects

Tchelet, R.

Publications and source records attributed to Tchelet, R..

3 recordsLinked to original sources

bioRxiv2025

Expression and Characterization of SARS-CoV-2 Spike Protein in Thermothelomyces heterothallica C1

The COVID-19 pandemic demonstrated a pressing need for rapid, adaptive, and scalable manufacturing of vaccines and reagents. With the transition into an endemic disease and rising threats of other emerging pandemics, production of these biologicals requires a stable and sustainable supply chain and accessible distribution methods. In this study, we demonstrate the strength of an engineered filamentous fungal platform, Thermothelomyces heterothallica C1, for high volumetric productivity of the full-length spike glycoprotein. Spike protein produced in this system is highly thermostable and immunization of mice with spike made in C1 or mammalian platforms resulted in a similar humoral response. Additionally, it was shown that the native N-glycan profile can be redecorated with complex sialylated structures, if necessary, resulting in a more human-like glycan profile, without impacting binding characteristics as shown experimentally and in simulations. Through extensive physicochemical analysis, the C1 produced spike performs similarly to spike proteins produced in other commercially available systems. The data presented is evidence that C1 can be a strong platform for production of complex glycosylated recombinant proteins such as subunit antigen vaccines.

bioengineering↗

bioRxiv2021

Protein-based RBD-C-tag COVID-19 Vaccination Candidate Elicits Protection Activity against SARS-COV-2 Variant Infection

The identification of a vaccination candidate against COVID-19 providing protecting activity against emerging SARS-COV-2 variants remains challenging. Here, we report protection activity against a spectrum of SARS-COV-2 and variants by immunization with protein-based recombinant RBD-C-tag administered with aluminum-phosphate adjuvant intramuscularly. Immunization of C57BL/6 mice with RBD-C-tag resulted in the in vivo production of IgG antibodies recognizing the immune-critical spike protein of the SARS-COV-2 virus as well as the SARS-COV-2 variants alpha ("United Kingdom"), beta ("South Africa"), gamma ("Brazil/Japan"), and delta ("India") as well as wt-spike protein. RBD-C-tag immunization led to a desired Th1 polarization of CD4 T cells producing IFN{gamma}. Importantly, RBD-C-tag immunization educated IgG production delivers antibodies that exert neutralizing activity against the highly transmissible SARS-COV-2 virus strains "Washington", "South Africa" (beta), and "India" (delta) as determined by conservative infection protection experiments in vitro. Hence, the protein-based recombinant RBD-C-tag is considered a promising vaccination candidate against COVID-19 and a broad range of emerging SARS-COV-2 virus variants.

microbiology↗

bioRxiv2021

In-solution buffer-free digestion for the analysis of SARS-CoV-2 RBD proteins allows a full sequence coverage and detection of post-translational modifications in a single ESI-MS spectrum

Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, are among the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI-MS spectrum, BFD allowed very high sequence coverage ([≥] 99 %) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2-8 amino acids), the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, glutathionylation, cyanilation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80-90 %) and did not detect peptides carrying some of the above-mentioned post-translational modifications. The two C-terminal peptides of a dimer [RBD(319-541)-(His)6]2 linked by an intermolecular disulfide bond (Cys538-Cys538) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD and the low-abundance scrambling variants, free cysteine residues, O-glycoforms and incomplete processing of the N-terminal end, if present. Artifacts that might be generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented and we foresee that it can be also helpful to the characterization of mutated RBD.

biochemistry↗