The MultiBac BEVS: Basics, applications, performance and recent developments

Gorda, Barbara; Toelzer, Christine; Aulicino, Francesco; Berger, Imre

doi:10.1016/bs.mie.2021.06.018

Cited by 9 publications

(4 citation statements)

References 50 publications

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“…Saccharomyces cerevisiae Nej1 was expressed in Sf 21 insect cells (sourced from the European Molecular Biology Laboratory, Grenoble, France) with a 10xhis tag on its N-terminus using the Nej1_pFL vector 81 . The cell pellet was suspended in lysis buffer containing 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 150 mM KCl, 10 mM β-mercaptoethanol, 50 mM imidazole, protease inhibitor cocktail (cOmplete EDTA free, Roche, ½ tablet for 250 ml of lysis buffer) and sonicated by three cycles of 1 min at 60% amplitude.…”

Section: Methodsmentioning

confidence: 99%

HLTF disrupts Cas9-DNA post-cleavage complexes to allow DNA break processing

Reginato,

Dello Stritto,

Wang

et al. 2024

Nat Commun

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The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3′-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing.

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Section: Methodsmentioning

confidence: 99%

HLTF disrupts Cas9-DNA post-cleavage complexes to allow DNA break processing

Reginato,

Dello Stritto,

Wang

et al. 2024

Nat Commun

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“…ADDoCoV was designed during the early pandemic before SARS-CoV-2 S structures became available, based on sequence comparison of the RBDs of SARS-CoV S, MERS-CoV S and SARS-CoV-2 S, and the structure of SARS-CoV bound to ACE2 receptor or neutralising antibodies, respectively [49][50][51][52] . Variations of the oligopeptide sequence corresponding to the ACE2 receptor binding motif (RBM) were then inserted into the ADDomer scaffold as described previously 28 and expressions carried out using the MultiBac baculovirus expression system following established protocols [53][54][55] . ADDoCoV, the candidate here described comprises a 33 amino acid sequence (AH epitope) in the VL insertion site (Fig.…”

Section: Protein Productionmentioning

confidence: 99%

Antibodies generatedin vitroandin vivoelucidate design of a thermostable ADDomer COVID-19 nasal nanoparticle vaccine

Buzas

Bunzel

Staufer

et al. 2023

Preprint

Self Cite

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COVID-19 continues to damage populations, communities and economies worldwide. Vaccines have reduced COVID-19-related hospitalisations and deaths, primarily in developed countries. Persisting infection rates, and highly transmissible SARS-CoV-2 Variants of Concern (VOCs) causing repeat and breakthrough infections, underscore the ongoing need for new treatments to achieve a global solution. Based on ADDomer, a self-assembling protein nanoparticle scaffold, we created ADDoCoV, a thermostable COVID-19 candidate vaccine displaying multiple copies of a SARS-CoV-2 receptor binding motif (RBM)-derived epitope. In vitro generated neutralising nanobodies combined with molecular dynamics (MD) simulations and electron cryo-microscopy (cryo-EM) established authenticity and accessibility of the epitopes displayed. A Gigabody comprising multimerized nanobodies prevented SARS-CoV-2 virion attachment with picomolar EC50. Antibodies generated by immunising mice cross-reacted with VOCs including Delta and Omicron. Our study elucidates nasal administration of ADDomer-based nanoparticles for active and passive immunisation against SARS-CoV-2 and provides a blueprint for designing nanoparticle reagents to combat respiratory viral infections.

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“…The ability of insect cell lines to grow in suspension in serum-free medium and to reach high density conditions positions this system as one with interesting advantages for the production of immunization agents [ 138 ]. In addition, this system allows the introduction of several genes in the baculovirus genome backbone under control of the strong viral promoters [ 139 ] and the use of recombinant baculovirus as transduction vectors for mammalian cells in the BacMam expression system [ 140 ]. However, even though insect cells BVES reproduces most of the post translational modifications of mammalian cells, the distinctive glycosylation pattern results in the synthesis of asialylated glycoproteins [ 141 ].…”

Section: Conventional Cell Factories For Recombinant Vaccinesmentioning

confidence: 99%

Recombinant vaccines in 2022: a perspective from the cell factory

et al. 2022

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The last big outbreaks of Ebola fever in Africa, the thousands of avian influenza outbreaks across Europe, Asia, North America and Africa, the emergence of monkeypox virus in Europe and specially the COVID-19 pandemics have globally stressed the need for efficient, cost-effective vaccines against infectious diseases. Ideally, they should be based on transversal technologies of wide applicability. In this context, and pushed by the above-mentioned epidemiological needs, new and highly sophisticated DNA-or RNA-based vaccination strategies have been recently developed and applied at large-scale. Being very promising and effective, they still need to be assessed regarding the level of conferred long-term protection. Despite these fast-developing approaches, subunit vaccines, based on recombinant proteins obtained by conventional genetic engineering, still show a wide spectrum of interesting potentialities and an important margin for further development. In the 80’s, the first vaccination attempts with recombinant vaccines consisted in single structural proteins from viral pathogens, administered as soluble plain versions. In contrast, more complex formulations of recombinant antigens with particular geometries are progressively generated and explored in an attempt to mimic the multifaceted set of stimuli offered to the immune system by replicating pathogens. The diversity of recombinant antimicrobial vaccines and vaccine prototypes is revised here considering the cell factory types, through relevant examples of prototypes under development as well as already approved products.

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The MultiBac BEVS: Basics, applications, performance and recent developments

Cited by 9 publications

References 50 publications

HLTF disrupts Cas9-DNA post-cleavage complexes to allow DNA break processing

HLTF disrupts Cas9-DNA post-cleavage complexes to allow DNA break processing

Antibodies generatedin vitroandin vivoelucidate design of a thermostable ADDomer COVID-19 nasal nanoparticle vaccine

Recombinant vaccines in 2022: a perspective from the cell factory

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