Biology subjects

Pollet, J.

Publications and source records attributed to Pollet, J..

4 recordsLinked to original sources

bioRxiv2020

Cloning, Expression and Biophysical Characterization of a Yeast-expressed Recombinant SARS-CoV-2 Receptor Binding Domain COVID-19 Vaccine Candidate

BackgroundCoronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has now spread worldwide to infect over 110 million people, with approximately 2.5 million reported deaths. A safe and effective vaccine remains urgently needed. MethodWe constructed three variants of the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (residues 331-549) in yeast as follows: (1) a "wild type" RBD (RBD219-WT), (2) a deglycosylated form (RBD219-N1) by deleting the first N-glycosylation site, and (3) a combined deglycosylated and cysteine-mutagenized form (C538A-mutated variant (RBD219-N1C1)). We compared the expression yields, biophysical characteristics, and functionality of the proteins produced from these constructs. Results and conclusionsThese three recombinant RBDs showed similar secondary and tertiary structure thermal stability and had the same affinity to their receptor, angiotensin-converting enzyme 2 (ACE-2), suggesting that the selected deletion or mutations did not cause any significant structural changes or alteration of function. However, RBD219-N1C1 had a higher fermentation yield, was easier to purify, was not hyperglycosylated, and had a lower tendency to form oligomers, and thus was selected for further vaccine development and evaluation. General significanceBy genetic modification, we were able to design a better-controlled and more stable vaccine candidate, which is an essential and important criterion for any process and manufacturing of biologics or drugs for human use.

immunology↗

bioRxiv2020

SARS-CoV-2 RBD219-N1C1: A Yeast-Expressed SARS-CoV-2 Recombinant Receptor-Binding Domain Candidate Vaccine Stimulates Virus Neutralizing Antibodies and T-cell Immunity in Mice

There is an urgent need for an accessible and low-cost COVID-19 vaccine suitable for low- and middle-income countries. Here we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) protein, expressed at high levels in yeast (Pichia pastoris), as a suitable vaccine candidate against COVID-19. After introducing two modifications into the wild-type RBD gene to reduce yeast-derived hyperglycosylation and improve stability during protein expression, we show that the recombinant protein, RBD219-N1C1, is equivalent to the wild-type RBD recombinant protein (RBD219-WT) in an in vitro ACE-2 binding assay. Immunogenicity studies of RBD219-N1C1 and RBD219-WT proteins formulated with Alhydrogel(R) were conducted in mice, and, after two doses, both the RBD219-WT and RBD219-N1C1 vaccines induced high levels of binding IgG antibodies. Using a SARS-CoV-2 pseudovirus, we further showed that sera obtained after a two-dose immunization schedule of the vaccines were sufficient to elicit strong neutralizing antibody titers in the 1:1,000 to 1:10,000 range, for both antigens tested. The vaccines induced IFN-{gamma}, IL-6, and IL-10 secretion, among other cytokines. Overall, these data suggest that the RBD219-N1C1 recombinant protein, produced in yeast, is suitable for further evaluation as a human COVID-19 vaccine, in particular, in an Alhydrogel(R) containing formulation and possibly in combination with other immunostimulants.

immunology↗

bioRxiv2020

Splice switching oligonucleotide mediated gene knockdown in B cells and plasma cells

The need to identify new therapeutic approaches to the treatment of cancers of the B lymphoid lineage is crucial. Unlike CRISPR/Cas technology, antisense strategies result in transient modifications of gene expression and lack mutagenic effects at the DNA level. Here, we provide evidence for efficient knockdown of c-REL and RELA expression after treatment with splice switching antisense oligonucleotides (SSO) inducing exon skipping and reading frameshifts. We also developed a tool to facilitate the choice of exons for on purpose inhibition of mouse and human gene expression. Interestingly, treatments with morpholino SSO targeting the c-REL exon 2 donor splice site or RELA exon 5 acceptor splice site elicited very efficient knockdown in diffuse large B cell lymphoma (DLBCL) cell lines and antibody-secreting cells derived from primary human B cells. Consistent with the clinical relevance of c-REL activation in DLBCLs, treatment with c-REL SSO induced major alterations in NF-{kappa}B and TNF signalling pathways and strongly decreased cell viability. Altogether, SSO-mediated knockdown is a powerful approach to transiently inhibit the expression of given genes in B-lineage cells that should pave the way for cancer treatments, provided optimized ligand-conjugations for in vivo delivery. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/302984v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@170de4forg.highwire.dtl.DTLVardef@113229corg.highwire.dtl.DTLVardef@145377forg.highwire.dtl.DTLVardef@13e8c1e_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2020

Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1) Formulated with Alum Induces Protective Immunity and Reduces Immune Enhancement

We developed a severe acute respiratory syndrome (SARS) subunit recombinant protein vaccine candidate based on a high-yielding, yeast-engineered, receptor-binding domain (RBD219-N1) of the SARS beta-coronavirus (SARS-CoV) spike (S) protein. When formulated with Alhydrogel®, RBD219-N1 induced high-level neutralizing antibodies against both pseudotyped virus and a clinical (mouse-adapted) isolate of SARS-CoV. Here, we report that mice immunized with RBD219-N1/Alhydrogel® were fully protected from lethal SARS-CoV challenge (0% mortality), compared to ∼ 30% mortality in mice when immunized with the SARS S protein formulated with Alhydrogel®, and 100% mortality in negative controls. An RBD219-N1 formulation Alhydrogel® was also superior to the S protein, unadjuvanted RBD, and AddaVax (MF59-like adjuvant)-formulated RBD in inducing specific antibodies and preventing cellular infiltrates in the lungs upon SARS-CoV challenge. Specifically, a formulation with a 1:25 ratio of RBD219-N1 to Alhydrogel® provided high neutralizing antibody titers, 100% protection with non-detectable viral loads with minimal or no eosinophilic pulmonary infiltrates. As a result, this vaccine formulation is under consideration for further development against SARS-CoV and potentially other emerging and re-emerging beta-CoVs such as SARS-CoV-2.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗