228 results on '"Durocher Y"'
Search Results
2. Antiperlecan Antibodies Are Novel Accelerators of Immune-Mediated Vascular Injury
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Cardinal, H., Dieudé, M., Brassard, N., Qi, S., Patey, N., Soulez, M., Beillevaire, D., Echeverry, F., Daniel, C., Durocher, Y., Madore, F., and Hébert, M.J.
- Published
- 2013
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3. Transcriptome profiling of a TGF-β-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies
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Lenferink, A E G, Cantin, C, Nantel, A, Wang, E, Durocher, Y, Banville, M, Paul-Roc, B, Marcil, A, Wilson, M R, and O'Connor-McCourt, M D
- Published
- 2010
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4. Glioblastoma-secreted factors induce IGFBP7 and angiogenesis by modulating Smad-2-dependent TGF-β signaling
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Pen, A, Moreno, M J, Durocher, Y, Deb-Rinker, P, and Stanimirovic, D B
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- 2008
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5. Nanovesicles Released by Apoptotic Endothelial Cells Favor the Production of Non-HLA Antibodies and Neointima Formation in a Murine Model of Vascular Rejection.: Abstract# 1253 Poster Board #-Session: P120-III
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Dieudé, M., Pallet, N., Qi, S., Brassard, N., Kokta, V., Durocher, Y., Thibault, P., and Hébert, M.-J.
- Published
- 2012
6. Oriented immobilization of epidermal growth factor for tissue engineering applications: OP019
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Boucher, C, Liberelle, B, St-Laurent, G, Loignon, M, Jolicoeur, M, Durocher, Y, and De Crescenzo, G
- Published
- 2009
7. Possible common central pathway for resistin and insulin in regulating food intake
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Cifani, C., Durocher, Y., Pathak, A., Penicaud, L., Smih, F., Massi, M., Rouet, P., and Polidori, C.
- Published
- 2009
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8. Stable expression of chimeric heavy chain antibodies in CHO cells
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Agrawal, V., Slivac, I., Perret, S., Bisson, L., St-Laurent, G., Murad, Y., Zhang, J., and Durocher, Y.
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Recombinant Fusion Proteins ,Genetic Vectors ,puromycin ,cloning ,gene order ,plasmid vector ,Gene Expression ,animal cell ,CHO Cells ,Transfection ,hygromycin B ,drug activity ,Cricetinae ,gene vector ,chimeric heavy chain antibody ,blasticidin S ,genetics ,suspension cell culture ,heavy chain ,protein expression ,hybrid protein ,antibody production ,CHO cell ,isolation and purification ,phleomycin ,toxicity ,Antibodies, Monoclonal ,immunoglobulin heavy chain ,chimeric antibody ,unclassified drug ,culture technique ,hamster ,antibiotic g 418 ,monoclonal antibody ,genetic transfection ,polyethyleneimine ,Immunoglobulin Heavy Chains ,metabolism - Abstract
Camelid single domain antibodies fused to noncamelid Fc regions, also called chimeric heavy chain antibodies (cHCAb), offer great potential as therapeutic and diagnostic candidates due to their relatively small size (80 kDa) and intact Fc. In this chapter, we describe two approaches, limiting dilution and minipools, for generating nonamplified Chinese hamster ovary cell lines stably expressing cHCAb in suspension and serum-free cultures using a stringent antibiotic selection. Neither of the protocols necessitates the acquisition or implementation of expensive automated infrastructures and thus could be applied in any lab with minimal cell culture setup. The given protocol allows the isolation of stable clones capable of generating up to 100 mg/L of antibody in batch mode performed in shaker flasks. © 2012 Springer Science+Business Media, LLC.
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- 2012
9. recombinant protein production by transient transfection of a suspension-growing cells
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Durocher, Y., Perret, S., and Kamen, A.
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biotechnology - Abstract
Canadian Society for Biochemistry and Molecular and Cellular Biology, Toronto, June, 2001
- Published
- 2009
10. Development of a reporter gene assay for both Gs and Gqcoupled prostanoid receptors stably expressed in HEK293 EBNA
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Durocher, Y., Perret, S., Thibaudeau, E., Gaumond, M. H., Kamen, A., Stocco, R., and Abramovitz, M.
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health care facilities, manpower, and services ,education ,lipids (amino acids, peptides, and proteins) ,health care economics and organizations ,biotechnology - Abstract
The 11 International Conference on advances in prostaglandin and leukotriene research, Florence, Italy, June 2000
- Published
- 2009
11. A reporter gene assay using the human placental secreted alkaline phosphatase (SEAP) or the green fluorescent protein for G proteiencoupled receptors
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Durocher, Y., Abramovitz, M., Perret, S., and Kamen, A.
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biotechnology - Abstract
IBC's Second Annual International Conference on G ProteineCoupled Receptors, Philadelphia, P.A., October 1996
- Published
- 2009
12. 318 - Development of AVID200, a novel TGF-β targeting immunotherapy: Characterization of immunomodulatory effects
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O'Connor-McCourt, M., Lenferink, A., Zwaagstra, J., Sulea, T., Weeratna, R., Maleki, S., Baardsnes, J., Collins, C., Cantin, C., Durocher, Y., Singh, R., Figueredo, R., Krishnan, L., Koropatnick, J., and Tikhomirov, I.
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- 2016
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13. Potency and selectivity of human EPI prostanoid receptor antagonists
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Abramovitz, M., Ungrin, M. D., Stocco, R., Cuncic, R., Durocher, Y., Sawyer, N., and Metters, K. M.
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biotechnology - Published
- 2001
14. Hyperphosphorylation of beta-catenin on serine-threonine residues and loss of cell-cell contacts induced by calyculin A and okadaic acid in human epidermal cells
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Serres, M., Grangeasse, C., Haftek, M., Durocher, Y., Duclos, B., Schmitt, D., and Deleage, Gilbert
- Subjects
[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology - Abstract
Phosphorylation and dephosphorylation events may critically control junction assembly and stability, as well as regulate the formation of the cadherin-cytoskeleton complex, thus influencing the adhesive function of cells. In the present study, we have used specific activators and inhibitors of protein kinases and phosphatases to analyze the role of protein phosphorylation in the maintenance of epithelial architecture. Okadaic acid and calyculin A cell treatments induced two major effects: a dramatic alteration of the keratin network of epidermal cells and a complete disruption of cell-cell contacts. This loss in cell-cell contacts was not tissue and species restricted and the interactions of keratinocytes with the matrix were not involved. The observed changes were highly specific for these drugs and were obtained in the range of concentrations corresponding to the inhibition of protein phosphatase 1 (PP1). They were time- and dose-dependent, and reversible, excluding a cytotoxic effect of the drugs. A decrease in electrophoretic mobility of beta-catenin, a major protein involved in the regulation of intercellular adherens junctions, was observed in keratinocytes and fibroblasts treated with okadaic acid and calyculin A, suggesting a change in the protein phosphorylation level and/or protein conformation. Data from beta-catenin immunocomplex autoradiography performed after 32P in vivo incorporation in untreated and okadaic acid or calyculin A-treated HaCaT cells, demonstrated a higher level of phosphorylation of beta-catenin in treated cells compared to untreated ones. Analysis of 32P-labeled phosphoaminoacids demonstrated that beta-catenin was exclusively phosphorylated on serine-threonine residues but not on tyrosine residues. Immunoprecipitations and Western blotting using anti-phosphoserine and anti-phosphotyrosine antibodies confirmed these data. The change in beta-catenin phosphorylation on serine-threonine residues may play a role in the control of the cohesion between epithelial cells and may be involved in the regulation of the transduction signal.Phosphorylation and dephosphorylation events may critically control junction assembly and stability, as well as regulate the formation of the cadherin-cytoskeleton complex, thus influencing the adhesive function of cells. In the present study, we have used specific activators and inhibitors of protein kinases and phosphatases to analyze the role of protein phosphorylation in the maintenance of epithelial architecture. Okadaic acid and calyculin A cell treatments induced two major effects: a dramatic alteration of the keratin network of epidermal cells and a complete disruption of cell-cell contacts. This loss in cell-cell contacts was not tissue and species restricted and the interactions of keratinocytes with the matrix were not involved. The observed changes were highly specific for these drugs and were obtained in the range of concentrations corresponding to the inhibition of protein phosphatase 1 (PP1). They were time- and dose-dependent, and reversible, excluding a cytotoxic effect of the drugs. A decrease in electrophoretic mobility of beta-catenin, a major protein involved in the regulation of intercellular adherens junctions, was observed in keratinocytes and fibroblasts treated with okadaic acid and calyculin A, suggesting a change in the protein phosphorylation level and/or protein conformation. Data from beta-catenin immunocomplex autoradiography performed after 32P in vivo incorporation in untreated and okadaic acid or calyculin A-treated HaCaT cells, demonstrated a higher level of phosphorylation of beta-catenin in treated cells compared to untreated ones. Analysis of 32P-labeled phosphoaminoacids demonstrated that beta-catenin was exclusively phosphorylated on serine-threonine residues but not on tyrosine residues. Immunoprecipitations and Western blotting using anti-phosphoserine and anti-phosphotyrosine antibodies confirmed these data. The change in beta-catenin phosphorylation on serine-threonine residues may play a role in the control of the cohesion between epithelial cells and may be involved in the regulation of the transduction signal.
- Published
- 1997
15. An efficient and scalable process for helper-dependent adenoviral vector production using polyethylenimine-adenofection.
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Dormond, E., Meneses-Acosta, A., Jacob, D., Durocher, Y., Gilbert, R., Perrier, M., and Kamen, A.
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- 2009
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16. At-line monitoring of bioreactor protein production by surface plasmon resonance.
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Jacquemart, R., Chavane, N., Durocher, Y., Hoemann, C., De Crescenzo, G., and Jolicoeur, M.
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- 2008
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17. Transient gene expression in HEK293 cells: Peptone addition posttransfection improves recombinant protein synthesis.
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Pham, P.L., Perret, S., Cass, B., Carpentier, E., St-Laurent, G., Bisson, L., Kamen, A., and Durocher, Y.
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- 2005
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18. Prostatic epithelial cells in culture: Phosphorylation of protein tyrosyl residues and tyrosine protein kinase activity.
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Bourassa, C., Nguyen, L. T., Durocher, Y., Roberts, K. D., and Chevalier, S.
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- 1991
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19. Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII
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Swiech Kamilla, Kamen Amine, Ansorge Sven, Durocher Yves, Picanço-Castro Virgínia, Russo-Carbolante Elisa MS, Neto Mário SA, and Covas Dimas T
- Subjects
Biotechnology ,TP248.13-248.65 - Abstract
Abstract Background Hemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 μg/106 cells) and repeated transfections done at 34° and 37°C. Results We observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated transfections at 34°C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-transfection. Conclusion Serum-free suspension transient transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability.
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- 2011
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20. Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells
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Afkhamizarreh Fatemeh, Bisson Louis, Cass Brian, Boulais Denise, Kelly John, Perret Sylvie, Loignon Martin, and Durocher Yves
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Biotechnology ,TP248.13-248.65 - Abstract
Abstract Background Mammalian cells are becoming the prevailing expression system for the production of recombinant proteins because of their capacity for proper protein folding, assembly, and post-translational modifications. These systems currently allow high volumetric production of monoclonal recombinant antibodies in the range of grams per litre. However their use for large-scale expression of cytokines typically results in much lower volumetric productivity. Results We have engineered a HEK293 cell clone for high level production of human recombinant glycosylated IFNα2b and developed a rapid and efficient method for its purification. This clone steadily produces more than 200 mg (up to 333 mg) of human recombinant IFNα2b per liter of serum-free culture, which can be purified by a single-step cation-exchange chromatography following media acidification and clarification. This rapid procedure yields 98% pure IFNα2b with a recovery greater than 70%. Purified IFNα2b migrates on SDS-PAGE as two species, a major 21 kDa band and a minor 19 kDa band. N-terminal sequences of both forms are identical and correspond to the expected mature protein. Purified IFNα2b elutes at neutral pH as a single peak with an apparent molecular weight of 44,000 Da as determined by size-exclusion chromatography. The presence of intramolecular and absence of intermolecular disulfide bridges is evidenced by the fact that non-reduced IFNα2b has a greater electrophoretic mobility than the reduced form. Treatment of purified IFNα2b with neuraminidase followed by O-glycosidase both increases electrophoretic mobility, indicating the presence of sialylated O-linked glycan. A detailed analysis of glycosylation by mass spectroscopy identifies disialylated and monosialylated forms as the major constituents of purified IFNα2b. Electron transfer dissociation (ETD) shows that the glycans are linked to the expected threonine at position 106. Other minor glycosylated forms and non-sialylated species are also detected, similar to IFNα2b produced naturally by lymphocytes. Further, the HEK293-produced IFNα2b is biologically active as shown with reporter gene and antiviral assays. Conclusion These results show that the HEK293 cell line is an efficient and valuable host for the production of biologically active and glycosylated human IFNα2b.
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- 2008
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21. THE LG3 PERLECAN FRAGMENT RELEASED BY APOPTOTIC ENDOTHELIAL CELLS ACCELERATES VASCULAR REJECTION.
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Dieudé, M., Soulez, M., Brassard, N., Hamelin, K., Qi, S., Durocher, Y., and Hébert, M. -.
- Published
- 2010
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22. ROLE OF LG3 IN THE SURVIVAL OF VASCULAR SMOOTH MUSCLE CELLS: POTENTIAL IMPACT IN CHRONIC TRANSPLANT VASCULOPATHY.
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Soulez, M., Wu, S., Brassard, N., Raymond, M., Durocher, Y., and Hébert, M. -.
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- 2010
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23. Detection of phosphotyrosine in glutaraldehyde-crosslinked and alkali-treated phosphoproteins following their partial acid hydrolysis in gels
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Durocher, Y. and Chevalier, S.
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- 1994
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24. FMD empty capsids combined with the Immunostant Particle Adjuvant -ISPA or ISA206 induce protective immunity against foot and mouth disease virus.
- Author
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Bidart, J., Mignaqui, A., Kornuta, C., Lupi, G., Gammella, M., Soria, I., Galarza, R., Ferella, A., Cardillo, S., Langellotti, C., Quattrocchi, V., Durocher, Y., Wigdorovitz, A., Marcipar, I., and Zamorano, P.
- Subjects
- *
FOOT & mouth disease virus , *VIRUS-like particles , *ANTIBODY titer , *VACCINE trials - Abstract
• We used FMDV strain A/Argentina/2001 Virus-like Particles (VLPs) obtained in mammalian cell cultures to formulate vaccines. • VLPs-ISPA and VLPs-ISA206 induced FMDV-specific antibody titers, VNT and virus-specific T response in a murine model. • VLPs-ISPA and VLPs-ISA206 protected 100 % of vaccinated mice. • In cattle, VLPs-ISPA and VLPs-ISA206 induced FMDV-specific antibody titers, VNT and IgG2 antibodies. • VLPs-ISPA and VLPs-ISA206 formulations elicited total and neutralizing anti-FMDV Ab titers corresponding to an Expected Percentage of Protection (EPP) above 90 % in cattle. Foot and Mouth Disease Virus (FMDV) causes economy losses and is controlled by vaccination in many countries. Vaccine formulations based on empty capsids or Virus-Like Particles (VLPs) have the advantage of avoiding the biological hazard of using infectious FMDV, albeit are poorly immunogenic. Recently, we have described that ISPA a new Immune Stimulating Complex adjuvant, is useful to improve the response against FMD of vaccines that use inactivated virus. Now, the adjuvant effects of ISPA and ISA 206 (water/oil/water) on a VLPs-based FMD vaccine were evaluated. VLPs (strain A/Argentina/2001) were obtained in mammalian cell cultures and their elicitation of an immune response against FMDV with and without ISPA or ISA 206 was evaluated in mice as a first approach. Notably, VLPs-ISPA and VLPs-ISA 206 vaccines induced protection against viral challenge in 100 % of mice, while protection induced by VLPs alone was of 40 %. Total and neutralizing FMDV antibodies were higher in the VLPs-ISPA and VLPs-ISA 206 groups compared to the VLPs group. VLPs-ISPA induced significantly higher (p < 0.001) IgG1, IgG2a, IgG2b and IgG3 titers than the VLPs vaccine. Moreover, in comparison with non-adjuvanted VLPs, VLPs-ISPA and VLPs-ISA 206 elicited an increased virus-specific T response, including higher IFNγ+/CD8 + lymphocyte production in mice. When these vaccines were tested in calves, antibody titers reached an Expected Percentage of Protection (EPP) above 90 % in the case of the VLPs-ISPA and VLPs-ISA 206 vaccines, while, in the VLPs group, EPP reached 25 %. IFNγ levels secreted by mononuclear cells of VLP-ISPA-vaccinated cattle were significantly higher than in the VLPs group. Overall, the results demonstrate that VLPs-ISPA or VLPs-ISA 206 are promising formulations for the development of a novel FMD vaccine. [ABSTRACT FROM AUTHOR]
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- 2021
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25. An unconventional strategy for purifying recombinant SARS-CoV-2 spike protein.
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Ingawale M, Riaz M, Durocher Y, and Ghosh R
- Abstract
The soluble domain of the trimeric SARS-CoV-2 spike protein is a promising candidate for a COVID-19 vaccine. Purification of this protein from mammalian cell culture supernatant using conventional resin-based chromatography is challenging as its large size (∼550 kDa) restricts its access and mobility within the pores of the resin particles. This reduces binding capacity and process robustness very significantly as extremely low flow rates need to be used during purification. Convection-based ion-exchange membrane chromatography has been found to be suitable in this respect. However, the high ionic strength of mammalian cell culture supernatant makes it difficult to bind this protein on charged membranes without dilution with a suitable buffer. An unconventional strategy involving size-exclusion chromatography as the first step, followed by cation exchange membrane chromatography as the second step is proposed in this paper. In the size exclusion chromatography step, the spike protein is excluded from the pores and can therefore be isolated in the void volume fraction. This step removes small molecule impurities and also serves as a desalting and buffer exchange step, making the partially purified material suitable for the cation exchange membrane chromatography step. The proposed process is variant-independent, fast and scalable and addresses some of the challenges associated with the currently used purification methods., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2024
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26. CHO stable pool fed-batch process development of SARS-CoV-2 spike protein production: Impact of aeration conditions and feeding strategies.
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Reyes SJ, Pham PL, Durocher Y, and Henry O
- Abstract
Technology scale-up and transfer are a fundamental and critical part of process development in biomanufacturing. Important bioreactor hydrodynamic characteristics such as working volume, overhead gas flow rate, volumetric power input (P/V), impeller type, agitation regimen, sparging aeration strategy, sparger type, and k
L a must be selected based on key performance indicators (KPI) to ensure a smooth and seamless process scale-up and transfer. Finding suitable operational setpoints and developing an efficient feeding regimen to ensure process efficacy and consistency are instrumental. In this investigation, process development of a cumate inducible Chinese hamster ovary (CHO) stable pool expressing trimeric SARS-CoV-2 spike protein in 1.8 L benchtop stirred-tank bioreactors is detailed. Various dissolved oxygen levels and aeration air caps were studied to determine their impact on cell growth and metabolism, culture longevity, and endpoint product titers. Once hydrodynamic conditions were tuned to an optimal zone, various feeding strategies were explored to increase culture performance. Dynamic feedings such as feeding based on current culture volume, viable cell density (VCD), oxygen uptake rate (OUR), and bio-capacitance signals were tested and compared to standard bolus addition. Increases in integral of viable cell concentration (IVCC) (1.25-fold) and protein yield (2.52-fold), as well as greater culture longevity (extension of 5 days) were observed in dynamic feeding strategies when compared to periodic bolus feeding. Our study emphasizes the benefits of designing feeding strategies around metabolically relevant signals such as OUR and bio-capacitance signals., (© 2024 His Majesty the King in Right of Canada and The Author(s). Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers. Reproduced with the permission of the Minister of Innovation, Science, and Economic Development.)- Published
- 2024
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27. Characterization of biotinylated human ACE2 and SARS-CoV-2 Omicron BA.4/5 spike protein reference materials.
- Author
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Stocks BB, Thibeault MP, L'Abbé D, Umer M, Liu Y, Stuible M, Durocher Y, and Melanson JE
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- Humans, Biotinylation, COVID-19 virology, COVID-19 Serological Testing methods, Spike Glycoprotein, Coronavirus chemistry, Spike Glycoprotein, Coronavirus metabolism, Angiotensin-Converting Enzyme 2 metabolism, Angiotensin-Converting Enzyme 2 chemistry, SARS-CoV-2, Reference Standards
- Abstract
Accurate diagnostic and serology assays are required for the continued management of the COVID-19 pandemic yet spike protein mutations and intellectual property concerns with antigens and antibodies used in various test kits render comparability assessments difficult. As the use of common, well-characterized reagents can help address this lack of standardization, the National Research Council Canada has produced two protein reference materials (RMs) for use in SARS-CoV-2 serology assays: biotinylated human angiotensin-converting enzyme 2 RM, ACE2-1, and SARS-CoV-2 Omicron BA.4/5 spike protein RM, OMIC-1. Reference values were assigned through a combination of amino acid analysis via isotope dilution liquid chromatography tandem mass spectrometry following acid hydrolysis, and ultraviolet-visible (UV-Vis) spectrophotometry at 280 nm. Vial-to-vial homogeneity was established using UV-Vis measurements, and protein oligomeric status, monitored by size exclusion liquid chromatography (LC-SEC), was used to evaluate transportation, storage, and freeze-thaw stabilities. The molar protein concentration in ACE2-1 was 25.3 ± 1.7 µmol L
-1 (k = 2, 95% CI) and consisted almost exclusively (98%) of monomeric ACE2, while OMIC-1 contained 5.4 ± 0.5 µmol L-1 (k = 2) spike protein in a mostly (82%) trimeric form. Glycoprotein molar mass determination by LC-SEC with multi-angle light scattering detection facilitated calculation of corresponding mass concentrations. To confirm protein functionality, the binding of OMIC-1 to immobilized ACE2-1 was investigated with surface plasmon resonance and the resulting dissociation constant, KD ~ 4.4 nM, was consistent with literature values., (© 2024. Crown.)- Published
- 2024
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28. A Novel Antigen Design Strategy to Isolate Single-Domain Antibodies that Target Human Nav1.7 and Reduce Pain in Animal Models.
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Martina M, Banderali U, Yogi A, Arbabi Ghahroudi M, Liu H, Sulea T, Durocher Y, Hussack G, van Faassen H, Chakravarty B, Liu QY, Iqbal U, Ling B, Lessard E, Sheff J, Robotham A, Callaghan D, Moreno M, Comas T, Ly D, and Stanimirovic D
- Abstract
Genetic studies have identified the voltage-gated sodium channel 1.7 (Na
v 1.7) as pain target. Due to the ineffectiveness of small molecules and monoclonal antibodies as therapeutics for pain, single-domain antibodies (VH Hs) are developed against the human Nav 1.7 (hNav 1.7) using a novel antigen presentation strategy. A 70 amino-acid peptide from the hNav 1.7 protein is identified as a target antigen. A recombinant version of this peptide is grafted into the complementarity determining region 3 (CDR3) loop of an inert VH H in order to maintain the native 3D conformation of the peptide. This antigen is used to isolate one VH H able to i) bind hNav 1.7, ii) slow the deactivation of hNav 1.7, iii) reduce the ability of eliciting action potentials in nociceptors, and iv) reverse hyperalgesia in in vivo rat and mouse models. This VH H exhibits the potential to be developed as a therapeutic capable of suppressing pain. This novel antigen presentation strategy can be applied to develop biologics against other difficult targets such as ion channels, transporters and GPCRs., (© 2024 His Majesty the King in Right of Canada. Advanced Science published by Wiley‐VCH GmbH. Reproduced with the permission of the Minister of Innovation, Science, and Economic Development.)- Published
- 2024
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29. Lipid nanoparticle encapsulation of a Delta spike-CD40L DNA vaccine improves effectiveness against Omicron challenge in Syrian hamsters.
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Tamming L, Duque D, Tran A, Lansdell C, Frahm G, Wu J, Fekete EEF, Creskey M, Thulasi Raman SN, Laryea E, Zhang W, Pfeifle A, Gravel C, Stalker A, Hashem AM, Chen W, Stuible M, Durocher Y, Safronetz D, Cao J, Wang L, Sauve S, Rosu-Myles M, Zhang X, Johnston MJW, and Li X
- Abstract
The effectiveness of mRNA vaccines largely depends on their lipid nanoparticle (LNP) component. Herein, we investigate the effectiveness of DLin-KC2-DMA (KC2) and SM-102-based LNPs for the intramuscular delivery of a plasmid encoding B.1.617.2 (Delta) spike fused with CD40 ligand. LNP encapsulation of this CD40L-adjuvanted DNA vaccine with either LNP formulation drastically enhanced antibody responses, enabling neutralization of heterologous Omicron variants. The DNA-LNP formulations provided excellent protection from homologous challenge, reducing viral replication, and preventing histopathological changes in the pulmonary tissues. Moreover, the DNA-LNP vaccines maintained a high level of protection against heterologous Omicron BA.5 challenge despite a reduced neutralizing response. In addition, we observed that DNA-LNP vaccination led to the pulmonary downregulation of interferon signaling, interleukin-12 signaling, and macrophage response pathways following SARS-CoV-2 challenge, shedding some light on the mechanisms underlying the prevention of pulmonary injury. These results highlight the potential combination of molecular adjuvants with LNP-based vaccine delivery to induce greater and broader immune responses capable of preventing inflammatory damage and protecting against emerging variants. These findings could be informative for the future design of both DNA and mRNA vaccines., Competing Interests: The authors declare no competing interests., (Crown Copyright © 2024 Published by Elsevier Inc. on behalf of The American Society of Gene and Cell Therapy.)
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- 2024
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30. Role of N343 glycosylation on the SARS-CoV-2 S RBD structure and co-receptor binding across variants of concern.
- Author
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Ives CM, Nguyen L, Fogarty CA, Harbison AM, Durocher Y, Klassen J, and Fadda E
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- Glycosylation, Humans, COVID-19 virology, COVID-19 metabolism, Polysaccharides metabolism, Polysaccharides chemistry, Protein Domains, Binding Sites, Protein Conformation, Mutation, Spike Glycoprotein, Coronavirus metabolism, Spike Glycoprotein, Coronavirus chemistry, Spike Glycoprotein, Coronavirus genetics, SARS-CoV-2 metabolism, SARS-CoV-2 chemistry, SARS-CoV-2 genetics, Molecular Dynamics Simulation, Protein Binding
- Abstract
Glycosylation of the SARS-CoV-2 spike (S) protein represents a key target for viral evolution because it affects both viral evasion and fitness. Successful variations in the glycan shield are difficult to achieve though, as protein glycosylation is also critical to folding and structural stability. Within this framework, the identification of glycosylation sites that are structurally dispensable can provide insight into the evolutionary mechanisms of the shield and inform immune surveillance. In this work, we show through over 45 μs of cumulative sampling from conventional and enhanced molecular dynamics (MD) simulations, how the structure of the immunodominant S receptor binding domain (RBD) is regulated by N -glycosylation at N343 and how this glycan's structural role changes from WHu-1, alpha (B.1.1.7), and beta (B.1.351), to the delta (B.1.617.2), and omicron (BA.1 and BA.2.86) variants. More specifically, we find that the amphipathic nature of the N -glycan is instrumental to preserve the structural integrity of the RBD hydrophobic core and that loss of glycosylation at N343 triggers a specific and consistent conformational change. We show how this change allosterically regulates the conformation of the receptor binding motif (RBM) in the WHu-1, alpha, and beta RBDs, but not in the delta and omicron variants, due to mutations that reinforce the RBD architecture. In support of these findings, we show that the binding of the RBD to monosialylated ganglioside co-receptors is highly dependent on N343 glycosylation in the WHu-1, but not in the delta RBD, and that affinity changes significantly across VoCs. Ultimately, the molecular and functional insight we provide in this work reinforces our understanding of the role of glycosylation in protein structure and function and it also allows us to identify the structural constraints within which the glycosylation site at N343 can become a hotspot for mutations in the SARS-CoV-2 S glycan shield., Competing Interests: CI, LN, CF, AH, YD, JK, EF No competing interests declared, (© 2024, Ives, Nguyen et al.)
- Published
- 2024
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31. Intranasal administration of unadjuvanted SARS-CoV-2 spike antigen boosts antigen-specific immune responses induced by parenteral protein subunit vaccine prime in mice and hamsters.
- Author
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Agbayani G, Akache B, Renner TM, Tran A, Stuible M, Dudani R, Harrison BA, Duque D, Bavananthasivam J, Deschatelets L, Hemraz UD, Régnier S, Durocher Y, and McCluskie MJ
- Subjects
- Animals, Mice, Cricetinae, Female, Antibodies, Neutralizing immunology, Antibodies, Neutralizing blood, Immunization, Secondary, Adjuvants, Immunologic administration & dosage, Mice, Inbred BALB C, Immunity, Mucosal immunology, Humans, Vaccination methods, Administration, Intranasal, Spike Glycoprotein, Coronavirus immunology, Spike Glycoprotein, Coronavirus administration & dosage, SARS-CoV-2 immunology, COVID-19 Vaccines immunology, COVID-19 Vaccines administration & dosage, Vaccines, Subunit immunology, Vaccines, Subunit administration & dosage, COVID-19 prevention & control, COVID-19 immunology, Antibodies, Viral blood, Antibodies, Viral immunology
- Abstract
With the continued transmission of SARS-CoV-2 across widely vaccinated populations, it remains important to develop new vaccines and vaccination strategies capable of providing protective immunity and limiting the spread of disease. Heterologous prime-boost vaccination based on the selection of different vaccine formulations and administration routes for priming and booster doses presents a promising strategy for inducing broader immune responses in key systemic and respiratory mucosal compartments. Intranasal vaccination can induce mucosal immune responses at the site of SARS-CoV-2 infection; however, the lack of clinically approved mucosal adjuvants makes it difficult to induce robust immune responses with protein subunit vaccines. Herein, we evaluated the immunogenicity of heterologous prime-boost regimens in mice and hamsters based on a parenteral vaccination of the antigen in combination with sulfated lactosylarchaeol (SLA) archaeosomes, a liposome adjuvant comprised of a single semisynthetic archaeal lipid, followed by an intranasally administered unadjuvanted SARS-CoV-2 spike antigen. Intranasal administration of unadjuvanted spike to mice and hamsters increased serum spike-specific IgG titers and spike-neutralizing activity compared with nonboosted animals. Spike-specific IgA responses were also detected in the bronchoalveolar lavage fluid in the lungs of mice that received an intranasal boost. In hamsters, the intranasal boost showed high efficacy against SARS-CoV-2 infection by protecting from body weight loss and reducing viral titers in the lungs and nasal turbinate. Overall, our heterologous intramuscular prime-intranasal boost with SLA-adjuvanted and unadjuvanted spike, respectively, demonstrated the potential of protein subunit formulations to promote antigen-specific systemic and mucosal immune responses., (© 2024 National Research Council Canada. European Journal of Immunology published by Wiley‐VCH GmbH. Reproduced with the permission of the Minister of Innovation, Science, and Economic Development.)
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- 2024
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32. A low-temperature SPR-based assay for monoclonal antibody galactosylation and fucosylation assessment using FcγRIIA/B.
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Gaudreault J, Forest-Nault C, Gilbert M, Durocher Y, Henry O, and De Crescenzo G
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- Surface Plasmon Resonance, Glycosylation, Temperature, Trastuzumab, Antibodies, Monoclonal chemistry, Receptors, IgG metabolism
- Abstract
Monoclonal antibodies (MAbs) are powerful therapeutic tools in modern medicine and represent a rapidly expanding multibillion USD market. While bioprocesses are generally well understood and optimized for MAbs, online quality control remains challenging. Notably, N-glycosylation is a critical quality attribute of MAbs as it affects binding to Fcγ receptors (FcγRs), impacting the efficacy and safety of MAbs. Traditional N-glycosylation characterization methods are ill-suited for online monitoring of a bioreactor; in contrast, surface plasmon resonance (SPR) represents a promising avenue, as SPR biosensors can record MAb-FcγR interactions in real-time and without labeling. In this study, we produced five lots of differentially glycosylated Trastuzumab (TZM) and finely characterized their glycosylation profile by HILIC-UPLC chromatography. We then compared the interaction kinetics of these MAb lots with four FcγRs including FcγRIIA and FcγRIIB at 5°C and 25°C. When interacting with FcγRIIA/B at low temperature, the differentially glycosylated MAb lots exhibited distinct kinetic behaviors, contrary to room-temperature experiments. Galactosylated TZM (1) and core fucosylated TZM (2) could be discriminated and even quantified using an analytical technique based on the area under the curve of the signal recorded during the dissociation phase of a SPR sensorgram describing the interaction with FcγRIIA (1) or FcγRII2B (2). Because of the rapidity of the proposed method (<5 min per measurement) and the small sample concentration it requires (as low as 30 nM, exact concentration not required), it could be a valuable process analytical technology for MAb glycosylation monitoring., (© 2024 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)
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- 2024
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33. Multivariate data analysis of process parameters affecting the growth and productivity of stable Chinese hamster ovary cell pools expressing SARS-CoV-2 spike protein as vaccine antigen in early process development.
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Reyes SJ, Lemire L, Molina RS, Roy M, L'Ecuyer-Coelho H, Martynova Y, Cass B, Voyer R, Durocher Y, Henry O, and Pham PL
- Abstract
The recent COVID-19 pandemic revealed an urgent need to develop robust cell culture platforms which can react rapidly to respond to this kind of global health issue. Chinese hamster ovary (CHO) stable pools can be a vital alternative to quickly provide gram amounts of recombinant proteins required for early-phase clinical assays. In this study, we analyze early process development data of recombinant trimeric spike protein Cumate-inducible manufacturing platform utilizing CHO stable pool as a preferred production host across three different stirred-tank bioreactor scales (0.75, 1, and 10 L). The impact of cell passage number as an indicator of cell age, methionine sulfoximine (MSX) concentration as a selection pressure, and cell seeding density was investigated using stable pools expressing three variants of concern. Multivariate data analysis with principal component analysis and batch-wise unfolding technique was applied to evaluate the effect of critical process parameters on production variability and a random forest (RF) model was developed to forecast protein production. In order to further improve process understanding, the RF model was analyzed with Shapley value dependency plots so as to determine what ranges of variables were most associated with increased protein production. Increasing longevity, controlling lactate build-up, and altering pH deadband are considered promising approaches to improve overall culture outcomes. The results also demonstrated that these pools are in general stable expressing similar level of spike proteins up to cell passage 11 (~31 cell generations). This enables to expand enough cells required to seed large volume of 200-2000 L bioreactor., (© 2024 National Research Council Canada. Biotechnology Progress published by Wiley‐VCH GmbH on behalf of American Institute of Chemical Engineers. Reproduced with the permission of the Minister of Innovation, Science, and Economic Development.)
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- 2024
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34. CHO cells for virus-like particle and subunit vaccine manufacturing.
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Sanchez-Martinez ZV, Alpuche-Lazcano SP, Stuible M, and Durocher Y
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- Cricetinae, Animals, Humans, CHO Cells, Cricetulus, Antibodies, Neutralizing, Antibodies, Viral, Herpesvirus 3, Human, Vaccines, Subunit, Respiratory Syncytial Virus Infections prevention & control, Respiratory Syncytial Virus, Human, Vaccines, Virus-Like Particle, Respiratory Syncytial Virus Vaccines
- Abstract
Chinese Hamster Ovary (CHO) cells, employed primarily for manufacturing monoclonal antibodies and other recombinant protein (r-protein) therapeutics, are emerging as a promising host for vaccine antigen production. This is exemplified by the recently approved CHO cell-derived subunit vaccines (SUV) against respiratory syncytial virus (RSV) and varicella-zoster virus (VZV), as well as the enveloped virus-like particle (eVLP) vaccine against hepatitis B virus (HBV). Here, we summarize the design, production, and immunogenicity features of these vaccine and review the most recent progress of other CHO-derived vaccines in pre-clinical and clinical development. We also discuss the challenges associated with vaccine production in CHO cells, with a focus on ensuring viral clearance for eVLP products., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)
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- 2024
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35. Outer membrane vesicles derived from Bordetella pertussis are potent adjuvant that drive Th1-biased response.
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Pschunder B, Locati L, López O, Martin Aispuro P, Zurita E, Stuible M, Durocher Y, and Hozbor D
- Subjects
- Animals, Mice, Female, Pertussis Vaccine immunology, Pertussis Vaccine administration & dosage, Antibodies, Bacterial immunology, Antibodies, Bacterial blood, Spike Glycoprotein, Coronavirus immunology, Mice, Inbred BALB C, SARS-CoV-2 immunology, Bacterial Outer Membrane Proteins immunology, Humans, COVID-19 immunology, COVID-19 prevention & control, Tetanus Toxoid immunology, Bordetella pertussis immunology, Adjuvants, Immunologic administration & dosage, Th1 Cells immunology, Whooping Cough immunology, Whooping Cough prevention & control, Immunoglobulin G blood, Immunoglobulin G immunology
- Abstract
For several years, we have been committed to exploring the potential of Bordetella pertussis -derived outer membrane vesicles (OMV
Bp ) as a promising third-generation vaccine against the reemerging pertussis disease. The results of our preclinical trials not only confirm its protective capacity against B. pertussis infection but also set the stage for forthcoming human clinical trials. This study delves into the examination of OMVBp as an adjuvant. To accomplish this objective, we implemented a two-dose murine schedule to evaluate the specific immune response induced by formulations containing OMVBp combined with 3 heterologous immunogens: Tetanus toxoid (T), Diphtheria toxoid (D), and the SARS-CoV-2 Spike protein (S). The specific levels of IgG, IgG1, and IgG2a triggered by the different tested formulations were evaluated using ELISA in dose-response assays for OMVBp and the immunogens at varying levels. These assays demonstrated that OMVBp exhibits adjuvant properties even at the low concentration employed (1.5 μg of protein per dose). As this effect was notably enhanced at medium (3 μg) and high concentrations (6 μg), we chose the medium concentration to determine the minimum immunogen dose at which the OMV adjuvant properties are significantly evident. These assays demonstrated that OMVBp exhibits adjuvant properties even at the lowest concentration tested for each immunogen. In the presence of OMVBp , specific IgG levels detected for the lowest amount of antigen tested increased by 2.5 to 10 fold compared to those found in animals immunized with formulations containing adjuvant-free antigens (p<0.0001). When assessing the adjuvant properties of OMVBp compared to the widely recognized adjuvant alum, we detected similar levels of specific IgG against D, T and S for both adjuvants. Experiments with OMVs derived from E. coli (OMVE.coli ) reaffirmed that the adjuvant properties of OMVs extend across different bacterial species. Nonetheless, it's crucial to highlight that OMVBp notably skewed the immune response towards a Th1 profile (p<0.05). These collective findings emphasize the dual role of OMVBp as both an adjuvant and modulator of the immune response, positioning it favorably for incorporation into combined vaccine formulations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Pschunder, Locati, López, Martin Aispuro, Zurita, Stuible, Durocher and Hozbor.)- Published
- 2024
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36. Recombinant Protein Production from Stable CHO Cell Pools.
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Delafosse L, Lord-Dufour S, Pelletier A, Perret S, Burlacu A, Ouimet M, Cass B, Joubert S, Stuible M, and Durocher Y
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- CHO Cells, Animals, SARS-CoV-2 genetics, SARS-CoV-2 metabolism, Cricetinae, Culture Media, Serum-Free, Cricetulus, Recombinant Proteins genetics, Recombinant Proteins biosynthesis, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Transfection methods, Polyethyleneimine chemistry, Spike Glycoprotein, Coronavirus genetics, Spike Glycoprotein, Coronavirus metabolism, Spike Glycoprotein, Coronavirus biosynthesis, Spike Glycoprotein, Coronavirus isolation & purification
- Abstract
The continuous improvement of expression platforms is necessary to respond to the increasing demand for recombinant proteins that are required to carry out structural or functional studies as well as for their characterization as biotherapeutics. While transient gene expression (TGE) in mammalian cells constitutes a rapid and well-established approach, non-clonal stably transfected cells, or "pools," represent another option, which is especially attractive when recurring productions of the same protein are required. From a culture volume of just a few liters, stable pools can provide hundreds of milligrams to gram quantities of high-quality secreted recombinant proteins.In this chapter, we describe a highly efficient and cost-effective procedure for the generation of Chinese Hamster Ovary cell stable pools expressing secreted recombinant proteins using commercially available serum-free media and polyethylenimine (PEI) as the transfection reagent. As a specific example of how this protocol can be applied, the production and downstream purification of recombinant His-tagged trimeric SARS-CoV-2 spike protein ectodomain (SmT1) are described., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2024
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37. A Biosensor Assay Based on Coiled-Coil-Mediated Human ACE2 Receptor Capture for the Analysis of Its Interactions with the SARS-CoV-2 Receptor Binding Domain.
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Forest-Nault C, Koyuturk I, Gaudreault J, Pelletier A, L'Abbé D, Cass B, Bisson L, Burlacu A, Delafosse L, Stuible M, Henry O, De Crescenzo G, and Durocher Y
- Subjects
- Humans, SARS-CoV-2 metabolism, Angiotensin-Converting Enzyme 2 metabolism, Spike Glycoprotein, Coronavirus metabolism, Protein Binding, COVID-19, Biosensing Techniques methods
- Abstract
Surface plasmon resonance (SPR)-based biosensing enables the characterization of protein-protein interactions. Several SPR-based approaches have been designed to evaluate the binding mechanism between the angiotensin-converting enzyme 2 (ACE2) receptor and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein leading to a large range of kinetic and thermodynamic constants. This chapter describes a robust SPR assay based on the K5/E5 coiled-coil capture strategy that reduces artifacts. In this method, ACE2 receptors were produced with an E5-tag and immobilized as ligands in the SPR assay. This chapter details methods for high-yield production and purification of the studied proteins, functionalization of the sensor chip, conduction of the SPR assay, and data analysis., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2024
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38. Heterologous booster with a novel formulation containing glycosylated trimeric S protein is effective against Omicron.
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Bottero D, Rudi E, Martin Aispuro P, Zurita E, Gaillard E, Gonzalez Lopez Ledesma MM, Malito J, Stuible M, Ambrosis N, Durocher Y, Gamarnik AV, Wigdorovitz A, and Hozbor D
- Subjects
- Humans, Animals, Adjuvants, Immunologic, Disease Models, Animal, RNA, Messenger, ChAdOx1 nCoV-19, 2019-nCoV Vaccine mRNA-1273
- Abstract
In this study, we evaluated the efficacy of a heterologous three-dose vaccination schedule against the Omicron BA.1 SARS-CoV-2 variant infection using a mouse intranasal challenge model. The vaccination schedules tested in this study consisted of a primary series of 2 doses covered by two commercial vaccines: an mRNA-based vaccine (mRNA1273) or a non-replicative vector-based vaccine (AZD1222/ChAdOx1, hereafter referred to as AZD1222). These were followed by a heterologous booster dose using one of the two vaccine candidates previously designed by us: one containing the glycosylated and trimeric spike protein (S) from the ancestral virus (SW-Vac 2µg), and the other from the Delta variant of SARS-CoV-2 (SD-Vac 2µg), both formulated with Alhydrogel as an adjuvant. For comparison purposes, homologous three-dose schedules of the commercial vaccines were used. The mRNA-based vaccine, whether used in heterologous or homologous schedules, demonstrated the best performance, significantly increasing both humoral and cellular immune responses. In contrast, for the schedules that included the AZD1222 vaccine as the primary series, the heterologous schemes showed superior immunological outcomes compared to the homologous 3-dose AZD1222 regimen. For these schemes no differences were observed in the immune response obtained when SW-Vac 2µg or SD-Vac 2µg were used as a booster dose. Neutralizing antibody levels against Omicron BA.1 were low, especially for the schedules using AZD1222. However, a robust Th1 profile, known to be crucial for protection, was observed, particularly for the heterologous schemes that included AZD1222. All the tested schedules were capable of inducing populations of CD4 T effector, memory, and follicular helper T lymphocytes. It is important to highlight that all the evaluated schedules demonstrated a satisfactory safety profile and induced multiple immunological markers of protection. Although the levels of these markers were different among the tested schedules, they appear to complement each other in conferring protection against intranasal challenge with Omicron BA.1 in K18-hACE2 mice. In summary, the results highlight the potential of using the S protein (either ancestral Wuhan or Delta variant)-based vaccine formulation as heterologous boosters in the management of COVID-19, particularly for certain commercial vaccines currently in use., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Bottero, Rudi, Martin Aispuro, Zurita, Gaillard, Gonzalez Lopez Ledesma, Malito, Stuible, Ambrosis, Durocher, Gamarnik, Wigdorovitz and Hozbor.)
- Published
- 2023
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39. Influence of variant-specific mutations, temperature and pH on conformations of a large set of SARS-CoV-2 spike trimer vaccine antigen candidates.
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Stuible M, Schrag JD, Sheff J, Zoubchenok D, Lord-Dufour S, Cass B, L'Abbé D, Pelletier A, Rossotti MA, Tanha J, Gervais C, Maurice R, El Bakkouri M, Acchione M, and Durocher Y
- Subjects
- Animals, Cricetinae, Humans, COVID-19 Vaccines genetics, Temperature, Cricetulus, Antigens, Mutation, Hydrogen-Ion Concentration, Antibodies, Neutralizing, SARS-CoV-2, COVID-19
- Abstract
SARS-CoV-2 subunit vaccines continue to be the focus of intense clinical development worldwide. Protein antigens in these vaccines most commonly consist of the spike ectodomain fused to a heterologous trimerization sequence, designed to mimic the compact, prefusion conformation of the spike on the virus surface. Since 2020, we have produced dozens of such constructs in CHO cells, consisting of spike variants with different mutations fused to different trimerization sequences. This set of constructs displayed notable conformational heterogeneity, with two distinct trimer species consistently detected by analytical size exclusion chromatography. A recent report showed that spike ectodomain fusion constructs can adopt an alternative trimer conformation consisting of loosely associated ectodomain protomers. Here, we applied multiple biophysical and immunological techniques to demonstrate that this alternative conformation is formed to a significant extent by several SARS-CoV-2 variant spike proteins. We have also examined the influence of temperature and pH, which can induce inter-conversion of the two forms. The substantial structural differences between these trimer types may impact their performance as vaccine antigens., (© 2023. Springer Nature Limited.)
- Published
- 2023
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40. Repressing expression of difficult-to-express recombinant proteins during the selection process increases productivity of CHO stable pools.
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Maltais JS, Lord-Dufour S, Morasse A, Stuible M, Loignon M, and Durocher Y
- Subjects
- Cricetinae, Animals, Cricetulus, CHO Cells, Recombinant Proteins metabolism, Antibodies, Monoclonal, Cytokines
- Abstract
More than half of licensed therapeutic recombinant proteins (r-proteins) are manufactured using constitutively-expressing, stably-transfected Chinese hamster ovary (CHO) clones. While constitutive CHO expression systems have proven their efficacy for the manufacturing of monoclonal antibodies, many next-generation therapeutics such as cytokines and bispecific antibodies as well as biological targets such as ectodomains of transmembrane receptors remain intrinsically challenging to produce. Herein, we exploited a cumate-inducible CHO platform allowing reduced expression of various classes of r-proteins during selection of stable pools. Following stable pool generation, fed-batch productions showed that pools generated without cumate (OFF-pools) were significantly more productive than pools selected in the presence of cumate (ON-pools) for 8 out of the 10 r-proteins tested, including cytokines, G-protein coupled receptors (GPCRs), the HVEM membrane receptor ectodomain, the multifunctional protein High Mobility Group protein B1 (HMGB1), as well as monoclonal and bispecific T-cell engager antibodies. We showed that OFF-pools contain a significantly larger proportion of cells producing high levels of r-proteins and that these cells tend to proliferate faster when expression is turned off, suggesting that r-protein overexpression imposes a metabolic burden on the cells. Cell viability was lower and pool recovery was delayed during selection of ON-pools (mimicking constitutive expression), suggesting that high producers were likely lost or overgrown by faster-growing, low-producing cells. We also observed a correlation between the expression levels of the GPCRs with Binding immunoglobulin Protein, an endoplasmic reticulum (ER) stress marker. Taken together, these data suggest that using an inducible system to minimize r-protein expression during stable CHO pool selection reduces cellular stresses, including ER stress and metabolic burden, leading to pools with greater frequency of high-expressing cells, resulting in improved volumetric productivity., (© 2023 National Research Council Canada and The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC. Reproduced with the permission of the Minister of Innovation, Science, and Economic Development.)
- Published
- 2023
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41. SARS-CoV-2 spike antigen-specific B cell and antibody responses in pre-vaccination period COVID-19 convalescent males and females with or without post-covid condition.
- Author
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Limoges MA, Quenum AJI, Chowdhury MMH, Rexhepi F, Namvarpour M, Akbari SA, Rioux-Perreault C, Nandi M, Lucier JF, Lemaire-Paquette S, Premkumar L, Durocher Y, Cantin A, Lévesque S, Dionne IJ, Menendez A, Ilangumaran S, Allard-Chamard H, Piché A, and Ramanathan S
- Subjects
- Humans, Female, Male, SARS-CoV-2, Post-Acute COVID-19 Syndrome, Antibody Formation, Pandemics, Immunoglobulin G, COVID-19
- Abstract
Background: Following SARS-CoV-2 infection a significant proportion of convalescent individuals develop the post-COVID condition (PCC) that is characterized by wide spectrum of symptoms encompassing various organs. Even though the underlying pathophysiology of PCC is not known, detection of viral transcripts and antigens in tissues other than lungs raise the possibility that PCC may be a consequence of aberrant immune response to the viral antigens. To test this hypothesis, we evaluated B cell and antibody responses to the SARS-CoV-2 antigens in PCC patients who experienced mild COVID-19 disease during the pre-vaccination period of COVID-19 pandemic., Methods: The study subjects included unvaccinated male and female subjects who developed PCC or not (No-PCC) after clearing RT-PCR confirmed mild COVID-19 infection. SARS-CoV-2 D614G and omicron RBD specific B cell subsets in peripheral circulation were assessed by flow cytometry. IgG, IgG3 and IgA antibody titers toward RBD, spike and nucleocapsid antigens in the plasma were evaluated by ELISA., Results: The frequency of the B cells specific to D614G-RBD were comparable in convalescent groups with and without PCC in both males and females. Notably, in females with PCC, the anti-D614G RBD specific double negative (IgD
- CD27- ) B cells showed significant correlation with the number of symptoms at acute of infection. Anti-spike antibody responses were also higher at 3 months post-infection in females who developed PCC, but not in the male PCC group. On the other hand, the male PCC group also showed consistently high anti-RBD IgG responses compared to all other groups., Conclusions: The antibody responses to the spike protein, but not the anti-RBD B cell responses diverge between convalescent males and females who develop PCC. Our findings also suggest that sex-related factors may also be involved in the development of PCC via modulating antibody responses to the SARS-CoV-2 antigens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Limoges, Quenum, Chowdhury, Rexhepi, Namvarpour, Akbari, Rioux-Perreault, Nandi, Lucier, Lemaire-Paquette, Premkumar, Durocher, Cantin, Lévesque, Dionne, Menendez, Ilangumaran, Allard-Chamard, Piché and Ramanathan.)- Published
- 2023
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42. Simplifying glycan monitoring of complex antigens such as the SARS-CoV-2 spike to accelerate vaccine development.
- Author
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Sauvageau J, Koyuturk I, St Michael F, Brochu D, Goneau MF, Schoenhofen I, Perret S, Star A, Robotham A, Haqqani A, Kelly J, Gilbert M, and Durocher Y
- Abstract
Glycosylation is a key quality attribute that must be closely monitored for protein therapeutics. Established assays such as HILIC-Fld of released glycans and LC-MS of glycopeptides work well for glycoproteins with a few glycosylation sites but are less amenable for those with multiple glycosylation sites, resulting in complex datasets that are time consuming to generate and difficult to analyze. As part of efforts to improve preparedness for future pandemics, researchers are currently assessing where time can be saved in the vaccine development and production process. In this context, we evaluated if neutral and acidic monosaccharides analysis via HPAEC-PAD could be used as a rapid and robust alternative to LC-MS and HILIC-Fld for monitoring glycosylation between protein production batches. Using glycoengineered spike proteins we show that the HPAEC-PAD monosaccharide assays could quickly and reproducibly detect both major and minor glycosylation differences between batches. Moreover, the monosaccharide results aligned well with those obtained by HILIC-Fld and LC-MS., (© 2023. Springer Nature Limited.)
- Published
- 2023
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43. Coiled-Coil-Based Biofunctionalization of 100 nm Gold Nanoparticles with the Trastuzumab Antibody for the Detection of HER2-Positive Cancer Cells.
- Author
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Dégardin M, Liberelle B, Oliverio R, Baniahmad SF, Darviot C, Largillière I, Henry O, Durocher Y, Banquy X, Meunier M, and De Crescenzo G
- Subjects
- Humans, Female, Trastuzumab pharmacology, Trastuzumab chemistry, Trastuzumab metabolism, Gold chemistry, Peptides chemistry, Metal Nanoparticles chemistry, Breast Neoplasms drug therapy, Breast Neoplasms metabolism
- Abstract
We compared different biofunctionalization strategies for immobilizing trastuzumab, an IgG targeting the HER2 biomarker, onto 100 nm spherical gold nanoparticles because of the E/K coiled-coil peptide heterodimer. First, Kcoil peptides were grafted onto the gold surface while their Ecoil partners were genetically encoded at the C-terminus of trastuzumab's Fc region, allowing for a strong and specific interaction between the antibodies and the nanoparticles. Gold nanoparticles with no Kcoil peptides on their surface were also produced to immobilize Ecoil-tagged trastuzumab antibodies via the specific adsorption of their negatively charged Ecoil tags on the positively charged gold surface. Finally, the nonspecific adsorption of wild-type trastuzumab on the gold surface was also assessed, with and without Kcoil peptides grafted on it beforehand. We developed a thorough workflow to systematically compare the immobilization strategies regarding the stability of nanoparticles, antibody coverage, and ability to specifically bind to HER2-positive breast cancer cells. All nanoparticles were highly monodisperse and retained their localized surface plasmon resonance properties after biofunctionalization. A significant increase in the amount of immobilized antibodies was observed with the two oriented coil-based strategies compared to nonspecific adsorption. Finally, all biofunctionalization strategies allowed for the detection of HER2-positive breast cancer cells, but among the investigated approaches, we recommend using the E/K coiled-coil-based strategy for gold nanoparticle biofunctionalization because it allows for the qualitative and quantitative detection of HER2-positive cells with a higher contrast compared to HER2-negative cells.
- Published
- 2023
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44. Preclinical evaluation of manufacturable SARS-CoV-2 spike virus-like particles produced in Chinese Hamster Ovary cells.
- Author
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Alpuche-Lazcano SP, Stuible M, Akache B, Tran A, Kelly J, Hrapovic S, Robotham A, Haqqani A, Star A, Renner TM, Blouin J, Maltais JS, Cass B, Cui K, Cho JY, Wang X, Zoubchenok D, Dudani R, Duque D, McCluskie MJ, and Durocher Y
- Abstract
Background: As the COVID-19 pandemic continues to evolve, novel vaccines need to be developed that are readily manufacturable and provide clinical efficacy against emerging SARS-CoV-2 variants. Virus-like particles (VLPs) presenting the spike antigen at their surface offer remarkable benefits over other vaccine antigen formats; however, current SARS-CoV-2 VLP vaccines candidates in clinical development suffer from challenges including low volumetric productivity, poor spike antigen density, expression platform-driven divergent protein glycosylation and complex upstream/downstream processing requirements. Despite their extensive use for therapeutic protein manufacturing and proven ability to produce enveloped VLPs, Chinese Hamster Ovary (CHO) cells are rarely used for the commercial production of VLP-based vaccines., Methods: Using CHO cells, we aimed to produce VLPs displaying the full-length SARS-CoV-2 spike. Affinity chromatography was used to capture VLPs released in the culture medium from engineered CHO cells expressing spike. The structure, protein content, and glycosylation of spikes in VLPs were characterized by several biochemical and biophysical methods. In vivo, the generation of neutralizing antibodies and protection against SARS-CoV-2 infection was tested in mouse and hamster models., Results: We demonstrate that spike overexpression in CHO cells is sufficient by itself to generate high VLP titers. These VLPs are evocative of the native virus but with at least three-fold higher spike density. In vivo, purified VLPs elicit strong humoral and cellular immunity at nanogram dose levels which grant protection against SARS-CoV-2 infection., Conclusions: Our results show that CHO cells are amenable to efficient manufacturing of high titers of a potently immunogenic spike protein-based VLP vaccine antigen., (© 2023. Springer Nature Limited.)
- Published
- 2023
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45. BCG administration promotes the long-term protection afforded by a single-dose intranasal adenovirus-based SARS-CoV-2 vaccine.
- Author
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Perera DJ, Domenech P, Babuadze GG, Naghibosadat M, Alvarez F, Koger-Pease C, Labrie L, Stuible M, Durocher Y, Piccirillo CA, Lametti A, Fiset PO, Elahi SM, Kobinger GP, Gilbert R, Olivier M, Kozak R, Reed MB, and Ndao M
- Abstract
Recent publications have explored intranasal (i.n.) adenovirus-based (Ad) vaccines as an effective strategy for SARS-CoV-2 in pre-clinical models. However, the effects of prior immunizations and infections have yet to be considered. Here, we investigate the immunomodulatory effects of Mycobacterium bovis BCG pre-immunization followed by vaccination with an S-protein-expressing i.n. Ad, termed Ad(Spike). While i.n. Ad(Spike) retains some protective effect after 6 months, a single administration of BCG-Danish prior to Ad(Spike) potentiates its ability to control viral replication of the B.1.351 SARS-CoV-2 variant within the respiratory tract. Though BCG-Danish did not affect Ad(Spike)-generated humoral immunity, it promoted the generation of cytotoxic/Th1 responses over suppressive FoxP3
+ TREG cells in the lungs of infected mice. Thus, this vaccination strategy may prove useful in limiting future pandemics by potentiating the long-term efficacy of mucosal vaccines within the context of the widely distributed BCG vaccine., Competing Interests: The authors declare that there are no competing interests involved in this work., (© 2023.)- Published
- 2023
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46. A CHO stable pool production platform for rapid clinical development of trimeric SARS-CoV-2 spike subunit vaccine antigens.
- Author
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Joubert S, Stuible M, Lord-Dufour S, Lamoureux L, Vaillancourt F, Perret S, Ouimet M, Pelletier A, Bisson L, Mahimkar R, Pham PL, L Ecuyer-Coelho H, Roy M, Voyer R, Baardsnes J, Sauvageau J, St-Michael F, Robotham A, Kelly J, Acel A, Schrag JD, El Bakkouri M, and Durocher Y
- Subjects
- Cricetinae, Animals, Humans, Cricetulus, CHO Cells, Antibodies, Monoclonal, COVID-19 Vaccines genetics, Recombinant Proteins metabolism, Vaccines, Subunit genetics, SARS-CoV-2 metabolism, COVID-19 prevention & control
- Abstract
Protein expression from stably transfected Chinese hamster ovary (CHO) clones is an established but time-consuming method for manufacturing therapeutic recombinant proteins. The use of faster, alternative approaches, such as non-clonal stable pools, has been restricted due to lower productivity and longstanding regulatory guidelines. Recently, the performance of stable pools has improved dramatically, making them a viable option for quickly producing drug substance for GLP-toxicology and early-phase clinical trials in scenarios such as pandemics that demand rapid production timelines. Compared to stable CHO clones which can take several months to generate and characterize, stable pool development can be completed in only a few weeks. Here, we compared the productivity and product quality of trimeric SARS-CoV-2 spike protein ectodomains produced from stable CHO pools or clones. Using a set of biophysical and biochemical assays we show that product quality is very similar and that CHO pools demonstrate sufficient productivity to generate vaccine candidates for early clinical trials. Based on these data, we propose that regulatory guidelines should be updated to permit production of early clinical trial material from CHO pools to enable more rapid and cost-effective clinical evaluation of potentially life-saving vaccines., (© 2023 National Research Council Canada. Biotechnology and Bioengineering published by Wiley Periodicals LLC. Reproduced with the permission of the Minister of Innovation, Science, and Economic Development.)
- Published
- 2023
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47. Tuning the immune response: sulfated archaeal glycolipid archaeosomes as an effective vaccine adjuvant for induction of humoral and cell-mediated immunity towards the SARS-CoV-2 Omicron variant of concern.
- Author
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Renner TM, Akache B, Stuible M, Rohani N, Cepero-Donates Y, Deschatelets L, Dudani R, Harrison BA, Baardsnes J, Koyuturk I, Hill JJ, Hemraz UD, Régnier S, Lenferink AEG, Durocher Y, and McCluskie MJ
- Subjects
- Cricetinae, Animals, Mice, SARS-CoV-2, Glycolipids, Sulfates, CHO Cells, Liposomes, Spike Glycoprotein, Coronavirus genetics, Cricetulus, Immunity, Cellular, Adjuvants, Immunologic, Adjuvants, Pharmaceutic, Archaea, COVID-19 Vaccines, Adjuvants, Vaccine, COVID-19 prevention & control
- Abstract
Liposomes composed of sulfated lactosyl archaeol (SLA) have been shown to be a safe and effective vaccine adjuvant with a multitude of antigens in preclinical studies. In particular, SLA-adjuvanted SARS-CoV-2 subunit vaccines based on trimeric spike protein antigens were shown to be immunogenic and efficacious in mice and hamsters. With the continued emergence of SARS-CoV-2 variants, we sought to evaluate next-generation vaccine formulations with an updated antigenic identity. This was of particular interest for the widespread Omicron variant, given the abundance of mutations and structural changes observed within its spike protein compared to other variants. An updated version of our resistin-trimerized SmT1 corresponding to the B.1.1.529 variant was successfully generated in our Chinese Hamster Ovary (CHO) cell-based antigen production platform and characterized, revealing some differences in protein profile and ACE2 binding affinity as compared to reference strain-based SmT1. We next evaluated this Omicron-based spike antigen for its immunogenicity and ability to generate robust antigen-specific immune responses when paired with SLA liposomes or AddaS03 (a mimetic of the AS03 oil-in-water emulsion adjuvant system found in commercialized SARS-CoV-2 protein vaccines). Immunization of mice with vaccine formulations containing this updated antigen with either adjuvant stimulated neutralizing antibody responses favouring Omicron over the reference strain. Cell-mediated responses, which play an important role in the neutralization of intracellular infections, were induced to a much higher degree with the SLA adjuvant relative to the AddaS03-adjuvanted formulations. As such, updated vaccines that are better capable of targeting towards SARS-CoV-2 variants can be generated through an optimized combination of antigen and adjuvant components., Competing Interests: The authors declare no competing non-financial interests but the following competing financial interests: BA, UH, SR and MM are inventors on an SLA archaeosome-related patent application. YD is an inventor of a patent application related to the SmT1 antigen. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Renner, Akache, Stuible, Rohani, Cepero-Donates, Deschatelets, Dudani, Harrison, Baardsnes, Koyuturk, Hill, Hemraz, Régnier, Lenferink, Durocher and McCluskie.)
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- 2023
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- View/download PDF
48. Assessment of the longitudinal humoral response in non-hospitalized SARS-CoV-2-positive individuals at decentralized sites: Outcomes and concordance.
- Author
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Djaïleb A, Lavallée É, Parker MF, Cayer MP, Desautels F, de Grandmont MJ, Stuible M, Gervais C, Durocher Y, Trottier S, Boudreau D, Masson JF, Brouard D, and Pelletier JN
- Subjects
- Adult, Humans, SARS-CoV-2, Pandemics, Antibodies, Viral, COVID-19
- Abstract
Introduction: Early in the COVID-19 pandemic, reagent availability was not uniform, and infrastructure had to be urgently adapted to undertake COVID-19 surveillance., Methods: Before the validation of centralized testing, two enzyme-linked immunosorbent assays (ELISA) were established independently at two decentralized sites using different reagents and instrumentation. We compared the results of these assays to assess the longitudinal humoral response of SARS-CoV-2-positive (i.e., PCR-confirmed), non-hospitalized individuals with mild to moderate symptoms, who had contracted SARSCoV-2 prior to the appearance of variants of concern in Québec, Canada., Results: The two assays exhibited a high degree of concordance to identify seropositive individuals, thus validating the robustness of the methods. The results also confirmed that serum immunoglobulins persist ≥ 6 months post-infection among non-hospitalized adults and that the antibodies elicited by infection cross-reacted with the antigens from P.1 (Gamma) and B.1.617.2 (Delta) variants of concern., Discussion: Together, these results demonstrate that immune surveillance assays can be rapidly and reliably established when centralized testing is not available or not yet validated, allowing for robust immune surveillance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Djaïleb, Lavallée, Parker, Cayer, Desautels, de Grandmont, Stuible, Gervais, Durocher, Trottier, Boudreau, Masson, Brouard and Pelletier.)
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- 2023
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49. A fast, efficient, and scalable method for purifying recombinant SARS-CoV-2 spike protein.
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Butani N, Xu Y, Pan S, Durocher Y, and Ghosh R
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- COVID-19 Vaccines, SARS-CoV-2 genetics, Spike Glycoprotein, Coronavirus biosynthesis, Spike Glycoprotein, Coronavirus isolation & purification, Recombinant Proteins biosynthesis, Recombinant Proteins isolation & purification
- Abstract
Recombinant SARS-CoV-2 trimeric spike protein produced by mammalian cell culture is a potential candidate for a COVID-19 vaccine. However, this protein is much larger than most typical biopharmaceutical proteins and its large-scale manufacture is therefore challenging. Particularly, its purification using resin-based chromatography is difficult as the diffusive transport of this protein to and from its binding site within the pores of the stationary phase particles is slow. Therefore, very low flow rates need to be used during binding and elution, and this slows down the purification process. Also, due to its large size, the binding capacity of this protein on resin-based media is low. Membrane chromatography is an efficient and scalable technique for purifying biopharmaceuticals. The predominant mode of solute transport in a membrane is convective and hence it is considered better than resin-based chromatography for purifying large proteins. In this paper, we propose a membrane chromatography-based purification method for fast and scalable manufacture of recombinant SARS-CoV-2 trimeric spike protein. A combination of cation exchange z
2 laterally-fed membrane chromatography and size exclusion chromatography was found to be suitable for obtaining a homogeneous spike protein sample from mammalian cell culture supernatant. The proposed method is both fast and scalable and could be explored as a method for manufacturing vaccine grade spike protein., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2022. Published by Elsevier B.V. All rights reserved.)- Published
- 2023
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50. Affinity-controlled capture and release of engineered monoclonal antibodies by macroporous dextran hydrogels using coiled-coil interactions.
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Baniahmad SF, Oliverio R, Obregon-Gomez I, Robert A, Lenferink AEG, Pazos E, Virgilio N, Banquy X, De Crescenzo G, and Durocher Y
- Subjects
- Animals, Cricetinae, Hydrogels chemistry, Cricetulus, Peptides chemistry, Trastuzumab chemistry, Antibodies, Monoclonal, Dextrans
- Abstract
Long-term delivery is a successful strategy used to reduce the adverse effects of monoclonal antibody (mAb)-based treatments. Macroporous hydrogels and affinity-based strategies have shown promising results in sustained and localized delivery of the mAbs. Among the potential tools for affinity-based delivery systems, the de novo designed Ecoil and Kcoil peptides are engineered to form a high-affinity, heterodimeric coiled-coil complex under physiological conditions. In this study, we created a set of trastuzumab molecules tagged with various Ecoil peptides and evaluated their manufacturability and characteristics. Our data show that addition of an Ecoil tag at the C-termini of the antibody chains (light chains, heavy chains, or both) does not hinder the production of chimeric trastuzumab in CHO cells or affect antibody binding to its antigen. We also evaluated the influence of the number, length, and position of the Ecoil tags on the capture and release of Ecoil-tagged trastuzumab from macroporous dextran hydrogels functionalized with Kcoil peptide (the Ecoil peptide-binding partner). Notably, our data show that antibodies are released from the macroporous hydrogels in a biphasic manner; the first phase corresponding to the rapid release of residual, unbound trastuzumab from the macropores, followed by the affinity-controlled, slow-rate release of antibodies from the Kcoil-functionalized macropore surface.
- Published
- 2023
- Full Text
- View/download PDF
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