Impact of SARS-CoV-2 RBD Mutations on the Production of a Recombinant RBD Fusion Protein in Mammalian Cells.

SARS-CoV-2 receptor-binding domain (RBD) is a major target for the development of diagnostics, vaccines and therapeutics directed against COVID-19. Important efforts have been dedicated to the rapid and efficient production of recombinant RBD proteins for clinical and diagnostic applications. One of the main challenges is the ongoing emergence of SARS-CoV-2 variants that carry mutations within the RBD, resulting in the constant need to design and optimise the production of new recombinant protein variants. We describe here the impact of naturally occurring RBD mutations on the secretion of a recombinant Fc-tagged RBD protein expressed in HEK 293 cells. We show that mutation E484K of the B.1.351 variant interferes with the proper disulphide bond formation and folding of the recombinant protein, resulting in its retention into the endoplasmic reticulum (ER) and reduced protein secretion. Accumulation of the recombinant B.1.351 RBD-Fc fusion protein in the ER correlated with the upregulation of endogenous ER chaperones, suggestive of the unfolded protein response (UPR). Overexpression of the chaperone and protein disulphide isomerase PDIA2 further impaired protein secretion by altering disulphide bond formation and increasing ER retention. This work contributes to a better understanding of the challenges faced in producing mutant RBD proteins and can assist in the design of optimisation protocols.

Protein disulfide isomerases (PDIs) negatively regulate ebolavirus structural glycoprotein expression in the endoplasmic reticulum (ER) via the autophagy-lysosomal pathway.

Zaire ebolavirus (EBOV) causes a severe hemorrhagic fever in humans and non-human primates with high morbidity and mortality. EBOV infection is dependent on its structural glycoprotein (GP), but high levels of GP expression also trigger cell rounding, detachment, and downregulation of many surface molecules that is thought to contribute to its high pathogenicity. Thus, EBOV has evolved an RNA editing mechanism to reduce its GP expression and increase its fitness. We now report that the GP expression is also suppressed at the protein level in cells by protein disulfide isomerases (PDIs). Although PDIs promote oxidative protein folding by catalyzing correct disulfide formation in the endoplasmic reticulum (ER), PDIA3/ERp57 adversely triggered the GP misfolding by targeting GP cysteine residues and activated the unfolded protein response (UPR). Abnormally folded GP was targeted by ER-associated protein degradation (ERAD) machinery and, unexpectedly, was degraded via the macroautophagy/autophagy-lysosomal pathway, but not the proteasomal pathway. PDIA3 also decreased the GP expression from other ebolavirus species but increased the GP expression from Marburg virus (MARV), which is consistent with the observation that MARV-GP does not cause cell rounding and detachment, and MARV does not regulate its GP expression via RNA editing during infection. Furthermore, five other PDIs also had a similar inhibitory activity to EBOV-GP. Thus, PDIs negatively regulate ebolavirus glycoprotein expression, which balances the viral life cycle by maximizing their infection but minimizing their cellular effect. We suggest that ebolaviruses hijack the host protein folding and ERAD machinery to increase their fitness via reticulophagy during infection.Abbreviations: 3-MA: 3-methyladenine; 4-PBA: 4-phenylbutyrate; ACTB: ß-actin; ATF: activating transcription factor; ATG: autophagy-related; BafA1: bafilomycin A₁; BDBV: Bundibugyo ebolavirus; CALR: calreticulin; CANX: calnexin; CHX: cycloheximide; CMA: chaperone-mediated autophagy; ConA: concanamycin A; CRISPR: clusters of regularly interspaced short palindromic repeats; Cas9: CRISPR-associated protein 9; dsRNA: double-stranded RNA; EBOV: Zaire ebolavirus; EDEM: ER degradation enhancing alpha-mannosidase like protein; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; Env: envelope glycoprotein; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; GP: glycoprotein; HA: hemagglutinin; HDAC6: histone deacetylase 6; HMM: high-molecular-mass; HIV-1: human immunodeficiency virus type 1; HSPA5/BiP: heat shock protein family A (Hsp70) member 5; IAV: influenza A virus; IP: immunoprecipitation; KIF: kifenesine; Lac: lactacystin; LAMP: lysosomal associated membrane protein; MAN1B1/ERManI: mannosidase alpha class 1B member 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MARV: Marburg virus; MLD: mucin-like domain; NHK/SERPINA1: alpha1-antitrypsin variant null (Hong Kong); NTZ: nitazoxanide; PDI: protein disulfide isomerase; RAVV: Ravn virus; RESTV: Reston ebolavirus; SARS-CoV: severe acute respiratory syndrome coronavirus; SBOV: Sudan ebolavirus; sGP: soluble GP; SQSTM1/p62: sequestosome 1; ssGP: small soluble GP; TAFV: Taï Forest ebolavirus; TIZ: tizoxanide; TGN: thapsigargin; TLD: TXN (thioredoxin)-like domain; Ub: ubiquitin; UPR: unfolded protein response; VLP: virus-like particle; VSV: vesicular stomatitis virus; WB: Western blotting; WT: wild-type; XBP1: X-box binding protein 1.

Direct Measurement of B Lymphocyte Gene Expression Biomarkers in Peripheral Blood Transcriptomics Enables Early Prediction of Vaccine Seroconversion.

Peripheral blood transcriptome is a highly promising area for biomarker development. However, transcript abundances (TA) in these cell mixture samples are confounded by proportions of the component leukocyte subpopulations. This poses a challenge to clinical applications, as the cell of origin of any change in TA is not known without prior cell separation procedure. We developed a framework to develop a cell-type informative TA biomarkers which enable determination of TA of a single cell-type (B lymphocytes) directly in cell mixture samples of peripheral blood (e.g., peripheral blood mononuclear cells, PBMC) without the need for subpopulation separation. It is applicable to a panel of genes called B cell informative genes. Then a ratio of two B cell informative genes (a target gene and a stably expressed reference gene) obtained in PBMC was used as a new biomarker to represent the target gene expression in purified B lymphocytes. This approach, which eliminates the tedious procedure of cell separation and directly determines TA of a leukocyte subpopulation in peripheral blood samples, is called the Direct LS-TA method. This method is applied to gene expression datasets collected in influenza vaccination trials as early predictive biomarkers of seroconversion. By using TNFRSF17 or TXNDC5 as the target genes and TNFRSF13C or FCRLA as the reference genes, the Direct LS-TA B cell biomarkers were determined directly in the PBMC transcriptome data and were highly correlated with TA of the corresponding target genes in purified B lymphocytes. Vaccination responders had almost a 2-fold higher Direct LS-TA biomarker level of TNFRSF17 (log 2 SMD = 0.84, 95% CI = 0.47-1.21) on day 7 after vaccination. The sensitivity of these Direct LS-TA biomarkers in the prediction of seroconversion was greater than 0.7 and area-under curves (AUC) were over 0.8 in many datasets. In this paper, we report a straightforward approach to directly estimate B lymphocyte gene expression in PBMC, which could be used in a routine clinical setting. Moreover, the method enables the practice of precision medicine in the prediction of vaccination response. More importantly, seroconversion could now be predicted as early as day 7. As the acquired immunology pathway is common to vaccination against influenza and COVID-19, these biomarkers could also be useful to predict seroconversion for the new COVID-19 vaccines.