Thermophilic Filamentous Fungus C1-Cell-Cloned SARS-CoV-2-Spike-RBD-Subunit-Vaccine Adjuvanted with Aldydrogel®85 Protects K18-hACE2 Mice against Lethal Virus Challenge

Nechooshtan, Ram; Ehrlich, Sharon; Vitikainen, Marika; Makovitzki, Arik; Dor, Eyal; Marcus, Hadar; Hefetz, Idan; Pitel, Shani; Wiebe, Marilyn G.; Huuskonen, Anne; Cherry, Lilach; Lupu, Edith; Sapir, Yehuda; Holtzman, Tzvi; Aftalion, Moshe; Gur, David; Tamir, Hadas; Yahalom-Ronen, Yfat; Ramot, Yuval; Kronfeld, Noam; Zarling, David; Vallerga, Anne K.; Tchelet, Ronen; Nyska, Abraham; Saloheimo, Markku; Emalfarb, Mark; Ophir, Yakir

doi:10.3390/vaccines10122119

Cited by 5 publications

(2 citation statements)

References 57 publications

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“…SARS-CoV-2 RBD has been expressed in various eukaryotic cell hosts, including yeast, mammalian cells such as HEK-293 and CHO cells, genetically engineered insect cells, and thermophilic filamentous fungi ( Argentinian AntiCovid, 2020 ; Chen et al, 2020 ; Dalvie et al, 2021 ; Klausberger et al, 2022 ; Sinegubova et al, 2021 ; Sun et al, 2021 ; Pan et al, 2021 ; Pollet et al, 2021 ; Yang et al, 2021 ; Nechooshtan et al, 2022 ). However, the presence of a free cysteine residue at position 538 in the RBD primary sequence poses a challenge for its expression due to disulfide bond scrambling ( Klausberger et al, 2022 ).…”

Section: Resultsmentioning

confidence: 99%

Development of a scalable single process for producing SARS-CoV-2 RBD monomer and dimer vaccine antigens

Boggiano-Ayo,

Palacios-Oliva,

Lozada-Chang

et al. 2023

Front. Bioeng. Biotechnol.

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We have developed a single process for producing two key COVID-19 vaccine antigens: SARS-CoV-2 receptor binding domain (RBD) monomer and dimer. These antigens are featured in various COVID-19 vaccine formats, including SOBERANA 01 and the licensed SOBERANA 02, and SOBERANA Plus. Our approach involves expressing RBD (319-541)-His6 in Chinese hamster ovary (CHO)-K1 cells, generating and characterizing oligoclones, and selecting the best RBD-producing clones. Critical parameters such as copper supplementation in the culture medium and cell viability influenced the yield of RBD dimer. The purification of RBD involved standard immobilized metal ion affinity chromatography (IMAC), ion exchange chromatography, and size exclusion chromatography. Our findings suggest that copper can improve IMAC performance. Efficient RBD production was achieved using small-scale bioreactor cell culture (2 L). The two RBD forms - monomeric and dimeric RBD - were also produced on a large scale (500 L). This study represents the first large-scale application of perfusion culture for the production of RBD antigens. We conducted a thorough analysis of the purified RBD antigens, which encompassed primary structure, protein integrity, N-glycosylation, size, purity, secondary and tertiary structures, isoform composition, hydrophobicity, and long-term stability. Additionally, we investigated RBD-ACE2 interactions, in vitro ACE2 recognition of RBD, and the immunogenicity of RBD antigens in mice. We have determined that both the monomeric and dimeric RBD antigens possess the necessary quality attributes for vaccine production. By enabling the customizable production of both RBD forms, this unified manufacturing process provides the required flexibility to adapt rapidly to the ever-changing demands of emerging SARS-CoV-2 variants and different COVID-19 vaccine platforms.

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Section: Resultsmentioning

confidence: 99%

Development of a scalable single process for producing SARS-CoV-2 RBD monomer and dimer vaccine antigens

Boggiano-Ayo,

Palacios-Oliva,

Lozada-Chang

et al. 2023

Front. Bioeng. Biotechnol.

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show abstract

“…Despite the unanswered mechanistic questions, aluminum adjuvants have been widely and successfully used for decades in many licensed vaccines, and continue to be included in novel, investigational vaccine formulations. For example, aluminum adjuvants were recently used in novel vaccine candidates containing attenuated SARS-CoV-2 virus [10], and recombinant subunits of the SARS-CoV-2 spike receptor binding domain antigen [11].…”

Section: Introductionmentioning

confidence: 99%

Analysis of the Adsorbed Vaccine Formulations Using Water Proton Nuclear Magnetic Resonance—Comparison with Optical Analytics

et al. 2023

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Purpose To evaluate w NMR, an emerging noninvasive analytical technology, for characterizing aluminum-adjuvanted vaccine formulations. Methods w NMR stands for water proton nuclear magnetic resonance. In this work, w NMR and optical techniques (laser diffraction and laser scattering) were used to characterize vaccine formulations containing different antigen loads adsorbed onto AlPO 4 adjuvant microparticles, including the fully dispersed state and the sedimentation process. All w NMR measurements were done noninvasively on sealed vials containing the adsorbed vaccine suspensions, while the optical techniques require transferring the adsorbed vaccine suspensions out of the original vial into specialized cuvette/tube for analysis. For analyzing fully dispersed suspensions, optical techniques also require sample dilution. Results w NMR outperformed laser diffraction in differentiating high- and low-dose formulations of the same vaccine, while w NMR and laser scattering achieved comparable results on vaccine sedimentation kinetics and the compactness of fully settled vaccines. Conclusion w NMR could be used to analyze aluminum-adjuvanted formulations and to differentiate between formulations containing different antigen loads adsorbed onto aluminum adjuvant microparticles. The results demonstrate the capability of w NMR to characterize antigen-adjuvant complexes and to noninvasively inspect finished vaccine products. Supplementary Information The online version contains supplementary material available at 10.1007/s11095-023-03528-7.

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Filamentous fungus-produced human monoclonal antibody provides protection against SARS-CoV-2 in hamster and non-human primate models

Kaiser,

Hernandez,

Krüger

et al. 2024

Nat Commun

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Monoclonal antibodies are an increasingly important tool for prophylaxis and treatment of acute virus infections like SARS-CoV-2 infection. However, their use is often restricted due to the time required for development, variable yields and high production costs, as well as the need for adaptation to newly emerging virus variants. Here we use the genetically modified filamentous fungus expression system Thermothelomyces heterothallica (C1), which has a naturally high biosynthesis capacity for secretory enzymes and other proteins, to produce a human monoclonal IgG1 antibody (HuMab 87G7) that neutralises the SARS-CoV-2 variants of concern (VOCs) Alpha, Beta, Gamma, Delta, and Omicron. Both the mammalian cell and C1 produced HuMab 87G7 broadly neutralise SARS-CoV-2 VOCs in vitro and also provide protection against VOC Omicron in hamsters. The C1 produced HuMab 87G7 is also able to protect against the Delta VOC in non-human primates. In summary, these findings show that the C1 expression system is a promising technology platform for the development of HuMabs in preventive and therapeutic medicine.

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Thermophilic Filamentous Fungus C1-Cell-Cloned SARS-CoV-2-Spike-RBD-Subunit-Vaccine Adjuvanted with Aldydrogel®85 Protects K18-hACE2 Mice against Lethal Virus Challenge

Cited by 5 publications

References 57 publications

Development of a scalable single process for producing SARS-CoV-2 RBD monomer and dimer vaccine antigens

Development of a scalable single process for producing SARS-CoV-2 RBD monomer and dimer vaccine antigens

Analysis of the Adsorbed Vaccine Formulations Using Water Proton Nuclear Magnetic Resonance—Comparison with Optical Analytics

Filamentous fungus-produced human monoclonal antibody provides protection against SARS-CoV-2 in hamster and non-human primate models

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