| 1. |
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| 2. |
- Custodio, TF, et al.
(författare)
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Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2
- 2020
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Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 11:1, s. 5588-
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Tidskriftsartikel (refereegranskat)abstract
- The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed an unusual conformation of the spike where two RBDs are in the ‘up’ ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.
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| 3. |
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| 4. |
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| 5. |
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| 6. |
- Forsell, MNE, et al.
(författare)
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Increased human immunodeficiency virus type 1 Env expression and antibody induction using an enhanced alphavirus vector
- 2007
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Ingår i: The Journal of general virology. - : Microbiology Society. - 0022-1317 .- 1465-2099. ; 88:Pt 10, s. 2774-2779
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Tidskriftsartikel (refereegranskat)abstract
- Viral vectors encoding heterologous vaccine antigens are potent inducers of cellular immune responses, but they are generally less efficient at stimulating humoral immunity. To improve the induction of antibody responses by Semliki Forest virus-based vaccines, a vector encoding a translation-enhancer element and a novel internal signal sequence for increased expression and secretion of soluble antigens was designed. Approximately tenfold more human immunodeficiency virus type 1 gp120 was secreted into culture supernatants of infected cells using the enhanced vector compared with the parental vector. This translated into a significant increase in gp120-specific antibodies in immunized mice, suggesting that antigen-expression levels from the parental vector are limiting for induction of antibody responses. These data encourage the use of the enhanced vector for elicitation of immune responses against heterologous antigens during vaccination.
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| 7. |
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| 9. |
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| 10. |
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| 11. |
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| 12. |
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| 13. |
- Hanke, L, et al.
(författare)
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A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2 in vivo
- 2022
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Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13:1, s. 155-
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies binding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike have therapeutic promise, but emerging variants show the potential for virus escape. This emphasizes the need for therapeutic molecules with distinct and novel neutralization mechanisms. Here we describe the isolation of a nanobody that interacts simultaneously with two RBDs from different spike trimers of SARS-CoV-2, rapidly inducing the formation of spike trimer–dimers leading to the loss of their ability to attach to the host cell receptor, ACE2. We show that this nanobody potently neutralizes SARS-CoV-2, including the beta and delta variants, and cross-neutralizes SARS-CoV. Furthermore, we demonstrate the therapeutic potential of the nanobody against SARS-CoV-2 and the beta variant in a human ACE2 transgenic mouse model. This naturally elicited bispecific monomeric nanobody establishes an uncommon strategy for potent inactivation of viral antigens and represents a promising antiviral against emerging SARS-CoV-2 variants.
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| 14. |
- Hanke, L, et al.
(författare)
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An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction
- 2020
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Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 11:1, s. 4420-
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Tidskriftsartikel (refereegranskat)abstract
- SARS-CoV-2 enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ACE2) receptor. Directly preventing this interaction presents an attractive possibility for suppressing SARS-CoV-2 replication. Here, we report the isolation and characterization of an alpaca-derived single domain antibody fragment, Ty1, that specifically targets the receptor binding domain (RBD) of the SARS-CoV-2 spike, directly preventing ACE2 engagement. Ty1 binds the RBD with high affinity, occluding ACE2. A cryo-electron microscopy structure of the bound complex at 2.9 Å resolution reveals that Ty1 binds to an epitope on the RBD accessible in both the ‘up’ and ‘down’ conformations, sterically hindering RBD-ACE2 binding. While fusion to an Fc domain renders Ty1 extremely potent, Ty1 neutralizes SARS-CoV-2 spike pseudovirus as a 12.8 kDa nanobody, which can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. Ty1 is therefore an excellent candidate as an intervention against COVID-19.
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| 15. |
- Hanke, L, et al.
(författare)
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Multivariate mining of an alpaca immune repertoire identifies potent cross-neutralizing SARS-CoV-2 nanobodies
- 2022
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Ingår i: Science advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 8:12, s. eabm0220-
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Tidskriftsartikel (refereegranskat)abstract
- Conventional approaches to isolate and characterize nanobodies are laborious. We combine phage display, multivariate enrichment, next-generation sequencing, and a streamlined screening strategy to identify numerous anti–severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nanobodies. We characterize their potency and specificity using neutralization assays and hydrogen/deuterium exchange mass spectrometry (HDX-MS). The most potent nanobodies bind to the receptor binding motif of the receptor binding domain (RBD), and we identify two exceptionally potent members of this category (with monomeric half-maximal inhibitory concentrations around 13 and 16 ng/ml). Other nanobodies bind to a more conserved epitope on the side of the RBD and are able to potently neutralize the SARS-CoV-2 founder virus (42 ng/ml), the Beta variant (B.1.351/501Y.V2) (35 ng/ml), and also cross-neutralize the more distantly related SARS-CoV-1 (0.46 μg/ml). The approach presented here is well suited for the screening of phage libraries to identify functional nanobodies for various biomedical and biochemical applications.
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| 16. |
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| 17. |
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| 18. |
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| 19. |
- Kedersha, N, et al.
(författare)
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G3BP-Caprin1-USP10 complexes mediate stress granule condensation and associate with 40S subunits
- 2016
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Ingår i: The Journal of cell biology. - : Rockefeller University Press. - 1540-8140 .- 0021-9525. ; 212:7, s. 845-860
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Tidskriftsartikel (refereegranskat)abstract
- Mammalian stress granules (SGs) contain stalled translation preinitiation complexes that are assembled into discrete granules by specific RNA-binding proteins such as G3BP. We now show that cells lacking both G3BP1 and G3BP2 cannot form SGs in response to eukaryotic initiation factor 2α phosphorylation or eIF4A inhibition, but are still SG-competent when challenged with severe heat or osmotic stress. Rescue experiments using G3BP1 mutants show that G3BP1-F33W, a mutant unable to bind G3BP partner proteins Caprin1 or USP10, rescues SG formation. Caprin1/USP10 binding to G3BP is mutually exclusive: Caprin binding promotes, but USP10 binding inhibits, SG formation. G3BP interacts with 40S ribosomal subunits through its RGG motif, which is also required for G3BP-mediated SG formation. We propose that G3BP mediates the condensation of SGs by shifting between two different states that are controlled by binding to Caprin1 or USP10.
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| 20. |
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| 21. |
- Liu, LF, et al.
(författare)
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RNA processing bodies are disassembled during Old World alphavirus infection
- 2019
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Ingår i: The Journal of general virology. - : Microbiology Society. - 1465-2099 .- 0022-1317. ; 100:10, s. 1375-1389
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Tidskriftsartikel (refereegranskat)abstract
- RNA processing bodies (P-bodies) are non-membranous cytoplasmic aggregates of mRNA and proteins involved in mRNA decay and translation repression. P-bodies actively respond to environmental stresses, associated with another type of RNA granules, known as stress granules (SGs). Alphaviruses were previously shown to block SG induction at late stages of infection, which is important for efficient viral growth. In this study, we found that P-bodies were disassembled or reduced in number very early in infection with Semliki Forest virus (SFV) or chikungunya virus (CHIKV) in a panel of cell lines. Similar to SGs, reinduction of P-bodies by a second stress (sodium arsenite) was also blocked in infected cells. The disassembly of P-bodies still occurred in non-phosphorylatable eIF2α mouse embryonal fibroblasts (MEFs) that are impaired in SG assembly. Studies of translation status by ribopuromycylation showed that P-body disassembly is independent of host translation shutoff, which requires the phosphorylation of eIF2α in the SFV- or CHIKV-infected cells. Labelling of newly synthesized RNA with bromo-UTP showed that host transcription shutoff correlated with P-body disassembly at the same early stage (3–4 h) after infection. However, inhibition of global transcription with actinomycin D (ActD) failed to disassemble P-bodies as effectively as the viruses did. Interestingly, blocking nuclear import with importazole led to an efficient P-bodies loss. Our data reveal that P-bodies are disassembled independently from SG formation at early stages of Old World alphavirus infection and that nuclear import is involved in the dynamic of P-bodies.
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| 22. |
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| 23. |
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| 24. |
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| 25. |
- McInerney, GM, et al.
(författare)
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Direct cleavage, proteasomal degradation and sequestration: three mechanisms of viral subversion of type I interferon responses
- 2009
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Ingår i: Journal of innate immunity. - : S. Karger AG. - 1662-8128 .- 1662-811X. ; 1:6, s. 599-606
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Tidskriftsartikel (refereegranskat)abstract
- The host type I interferon (IFN) system is central in antiviral defence and represents one of the greatest obstacles for a virus to overcome in order to establish a productive infection. Viruses have evolved many different mechanisms to repress the effects of the type I IFN system. For example, a number of viruses encode viral proteases, which can directly cleave and inactivate key components of the type I IFN induction and signalling pathway. Others recruit the ubiquitin proteasome system to destabilise proteins that are important for IFN responses. There are also many known viral proteins, which bind to and sequester proteins of the type I IFN system in an inactive state. Here, we review each of these different mechanisms of viral escape from the type I IFN response with examples from a range of viruses.
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| 26. |
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| 27. |
- McInerney, GM, et al.
(författare)
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Importance of eIF2alpha phosphorylation and stress granule assembly in alphavirus translation regulation
- 2005
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Ingår i: Molecular biology of the cell. - : American Society for Cell Biology (ASCB). - 1059-1524 .- 1939-4586. ; 16:8, s. 3753-3763
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Tidskriftsartikel (refereegranskat)abstract
- Alphavirus infection results in the shutoff of host protein synthesis in favor of viral translation. Here, we show that during Semliki Forest virus (SFV) infection, the translation inhibition is largely due to the activation of the cellular stress response via phosphorylation of eukaryotic translation initiation factor 2α subunit (eIF2α). Infection of mouse embryo fibroblasts (MEFs) expressing a nonphosphorylatable mutant of eIF2α does not result in efficient shutoff, despite efficient viral protein production. Furthermore, we show that the SFV translation enhancer element counteracts the translation inhibition imposed by eIF2α phosphorylation. In wild-type MEFs, viral infection induces the transient formation of stress granules (SGs) containing the cellular TIA-1/R proteins. These SGs are disassembled in the vicinity of viral RNA replication, synchronously with the switch from cellular to viral gene expression. We propose that phosphorylation of eIF2α and the consequent SG assembly is important for shutoff to occur and that the localized SG disassembly and the presence of the enhancer aid the SFV mRNAs to elude general translational arrest.
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| 28. |
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| 29. |
- Merkl, P, et al.
(författare)
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Antiviral Activity of Silver, Copper Oxide and Zinc Oxide Nanoparticle Coatings against SARS-CoV-2
- 2021
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Ingår i: Nanomaterials (Basel, Switzerland). - : MDPI AG. - 2079-4991. ; 11:5
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Tidskriftsartikel (refereegranskat)abstract
- SARS-CoV-2 is responsible for several million deaths to date globally, and both fomite transmission from surfaces as well as airborne transmission from aerosols may be largely responsible for the spread of the virus. Here, nanoparticle coatings of three antimicrobial materials (Ag, CuO and ZnO) are deposited on both solid flat surfaces as well as porous filter media, and their activity against SARS-CoV-2 viability is compared with a viral plaque assay. These nanocoatings are manufactured by aerosol nanoparticle self-assembly during their flame synthesis. Nanosilver particles as a coating exhibit the strongest antiviral activity of the three studied nanomaterials, while copper oxide exhibits moderate activity, and zinc oxide does not appear to significantly reduce the virus infectivity. Thus, nanosilver and copper oxide show potential as antiviral coatings on solid surfaces and on filter media to minimize transmission and super-spreading events while also providing critical information for the current and any future pandemic mitigation efforts.
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| 30. |
- Moliner-Morro, A, et al.
(författare)
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Nanobodies in the limelight: Multifunctional tools in the fight against viruses
- 2022
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Ingår i: The Journal of general virology. - : Microbiology Society. - 1465-2099 .- 0022-1317. ; 103:5
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies are natural antivirals generated by the vertebrate immune system in response to viral infection or vaccination. Unsurprisingly, they are also key molecules in the virologist’s molecular toolbox. With new developments in methods for protein engineering, protein functionalization and application, smaller antibody-derived fragments are moving in focus. Among these, camelid-derived nanobodies play a prominent role. Nanobodies can replace full-sized antibodies in most applications and enable new possible applications for which conventional antibodies are challenging to use. Here we review the versatile nature of nanobodies, discuss their promise as antiviral therapeutics, for diagnostics, and their suitability as research tools to uncover novel aspects of viral infection and disease.
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| 31. |
- Moliner-Morro, A, et al.
(författare)
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Picomolar SARS-CoV-2 Neutralization Using Multi-Arm PEG Nanobody Constructs
- 2020
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Ingår i: Biomolecules. - : MDPI AG. - 2218-273X. ; 10:12
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Tidskriftsartikel (refereegranskat)abstract
- Multivalent antibody constructs have a broad range of clinical and biotechnological applications. Nanobodies are especially useful as components for multivalent constructs as they allow increased valency while maintaining a small molecule size. We here describe a novel, rapid method for the generation of bi- and multivalent nanobody constructs with oriented assembly by Cu-free strain promoted azide-alkyne click chemistry (SPAAC). We used sortase A for ligation of click chemistry functional groups site-specifically to the C-terminus of nanobodies before creating C-to-C-terminal nanobody fusions and 4-arm polyethylene glycol (PEG) tetrameric nanobody constructs. We demonstrated the viability of this approach by generating constructs with the SARS-CoV-2 neutralizing nanobody Ty1. We compared the ability of the different constructs to neutralize SARS-CoV-2 pseudotyped virus and infectious virus in neutralization assays. The generated dimers neutralized the virus similarly to a nanobody-Fc fusion variant, while a 4-arm PEG based tetrameric Ty1 construct dramatically enhanced neutralization of SARS-CoV-2, with an IC50 in the low picomolar range.
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| 32. |
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| 33. |
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| 34. |
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| 35. |
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| 36. |
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| 37. |
- Reuper, H, et al.
(författare)
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Arabidopsis thaliana G3BP Ortholog Rescues Mammalian Stress Granule Phenotype across Kingdoms
- 2021
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Ingår i: International journal of molecular sciences. - : MDPI AG. - 1422-0067. ; 22:12
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Tidskriftsartikel (refereegranskat)abstract
- Stress granules (SGs) are dynamic RNA–protein complexes localized in the cytoplasm that rapidly form under stress conditions and disperse when normal conditions are restored. The formation of SGs depends on the Ras-GAP SH3 domain-binding protein (G3BP). Formations, interactions and functions of plant and human SGs are strikingly similar, suggesting a conserved mechanism. However, functional analyses of plant G3BPs are missing. Thus, members of the Arabidopsis thaliana G3BP (AtG3BP) protein family were investigated in a complementation assay in a human G3BP knock-out cell line. It was shown that two out of seven AtG3BPs were able to complement the function of their human homolog. GFP-AtG3BP fusion proteins co-localized with human SG marker proteins Caprin-1 and eIF4G1 and restored SG formation in G3BP double KO cells. Interaction between AtG3BP-1 and -7 and known human G3BP interaction partners such as Caprin-1 and USP10 was also demonstrated by co-immunoprecipitation. In addition, an RG/RGG domain exchange from Arabidopsis G3BP into the human G3BP background showed the ability for complementation. In summary, our results support a conserved mechanism of SG function over the kingdoms, which will help to further elucidate the biological function of the Arabidopsis G3BP protein family.
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| 38. |
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| 39. |
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| 40. |
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| 41. |
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| 42. |
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| 43. |
- Szurgot, I, et al.
(författare)
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DNA-launched RNA replicon vaccines induce potent anti-SARS-CoV-2 immune responses in mice
- 2021
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Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11:1, s. 3125-
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Tidskriftsartikel (refereegranskat)abstract
- The outbreak of the SARS-CoV-2 virus and its rapid spread into a global pandemic made the urgent development of scalable vaccines to prevent coronavirus disease (COVID-19) a global health and economic imperative. Here, we characterized and compared the immunogenicity of two alphavirus-based DNA-launched self-replicating (DREP) vaccine candidates encoding either SARS-CoV-2 spike glycoprotein (DREP-S) or a spike ectodomain trimer stabilized in prefusion conformation (DREP-Secto). We observed that the two DREP constructs were immunogenic in mice inducing both binding and neutralizing antibodies as well as T cell responses. Interestingly, the DREP coding for the unmodified spike turned out to be more potent vaccine candidate, eliciting high titers of SARS-CoV-2 specific IgG antibodies that were able to efficiently neutralize pseudotyped virus after a single immunization. In addition, both DREP constructs were able to efficiently prime responses that could be boosted with a heterologous spike protein immunization. These data provide important novel insights into SARS-CoV-2 vaccine design using a rapid response DNA vaccine platform. Moreover, they encourage the use of mixed vaccine modalities as a strategy to combat SARS-CoV-2.
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| 44. |
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| 45. |
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| 46. |
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| 47. |
- Van Huizen, E, et al.
(författare)
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Activation of the PI3K-AKT Pathway by Old World Alphaviruses
- 2020
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Ingår i: Cells. - : MDPI AG. - 2073-4409. ; 9:4
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Tidskriftsartikel (refereegranskat)abstract
- Alphaviruses can infect a broad range of vertebrate hosts, including birds, horses, primates, and humans, in which infection can lead to rash, fever, encephalitis, and arthralgia or arthritis. They are most often transmitted by mosquitoes in which they establish persistent, asymptomatic infections. Currently, there are no vaccines or antiviral therapies for any alphavirus. Several Old World alphaviruses, including Semliki Forest virus, Ross River virus and chikungunya virus, activate or hyperactivate the phosphatidylinositol-3-kinase (PI3K)-AKT pathway in vertebrate cells, potentially influencing many cellular processes, including survival, proliferation, metabolism and autophagy. Inhibition of PI3K or AKT inhibits replication of several alphaviruses either in vitro or in vivo, indicating the importance for viral replication. In this review, we discuss what is known about the mechanism(s) of activation of the pathway during infection and describe those effects of PI3K-AKT activation which could be of advantage to the alphaviruses. Such knowledge may be useful for the identification and development of therapies.
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| 48. |
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| 49. |
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| 50. |
- Yang, EM, et al.
(författare)
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Elucidation of TRIM25 ubiquitination targets involved in diverse cellular and antiviral processes
- 2022
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Ingår i: PLoS pathogens. - : Public Library of Science (PLoS). - 1553-7374. ; 18:9, s. e1010743-
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Tidskriftsartikel (refereegranskat)abstract
- The tripartite motif (TRIM) family of E3 ubiquitin ligases is well known for its roles in antiviral restriction and innate immunity regulation, in addition to many other cellular pathways. In particular, TRIM25-mediated ubiquitination affects both carcinogenesis and antiviral response. While individual substrates have been identified for TRIM25, it remains unclear how it regulates diverse processes. Here we characterized a mutation, R54P, critical for TRIM25 catalytic activity, which we successfully utilized to “trap” substrates. We demonstrated that TRIM25 targets proteins implicated in stress granule formation (G3BP1/2), nonsense-mediated mRNA decay (UPF1), nucleoside synthesis (NME1), and mRNA translation and stability (PABPC4). The R54P mutation abolishes TRIM25 inhibition of alphaviruses independently of the host interferon response, suggesting that this antiviral effect is a direct consequence of ubiquitination. Consistent with that, we observed diminished antiviral activity upon knockdown of several TRIM25-R54P specific interactors including NME1 and PABPC4. Our findings highlight that multiple substrates mediate the cellular and antiviral activities of TRIM25, illustrating the multi-faceted role of this ubiquitination network in modulating diverse biological processes.
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