Decoration of norovirus-like particles with coronavirus (SARS-CoV-2) antigens to create potential vaccine candidate
Music, Amna (2021)
Music, Amna
2021
Master's Programme in Biomedical Technology
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2021-05-10
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202104263592
https://urn.fi/URN:NBN:fi:tuni-202104263592
Tiivistelmä
Background and aims: Pathogens that can cause severe epidemic/pandemic outbreaks, like the current coronavirus (SARS-CoV-2), have the potential to inflict deleterious effects on a global level. In this project, we aimed to create a modular and fast-to-produce vaccine platform based on norovirus-like particles. We wanted to address the potential challenge of a previously developed SpyCatcher/SpyTag noroVLP platform by utilizing a similar three-part SnoopLigase system to decorate noroVLPs with SARS-CoV-2 antigens. Both methods are based on the formation of isopeptide bonds between peptide tags that can be fused with antigens. In this project receptor-binding domain (RBD) and receptor-binding motif (RBM) of SARS-CoV-2 were fused to SnoopTagJr tag and DogTag was expressed on the surface of noroVLP. SnoopLigase was used to form a bond between respective tags and generate potential vaccine candidates against SARS-CoV-2.
Materials and methods: SnoopLigase was expressed in E. coli BL21(DE3) Star cell line. The DogTag-noroVLPs and HisTag-SnoopTagJr-RBD were produced using the baculovirus-insect cells expression system. SnoopTagJr-RBM was chemically synthesized by a commercial provider. SnoopLigase and SnoopTagJr-RBD were purified with Affinity Chromatography. Purified SnoopLigase was biotinylated both chemically and enzymatically. The DogTag-noroVLPs were first purified with sucrose-gradient centrifugation and then polished with Ion Exchange Chromatography. SDS-PAGE and Western Blotting were utilized for purity assessment and characterization. Conjugation between DogTag-noroVLPs and SnoopTagJr-SARS-CoV-2 antigens was catalysed with SnoopLigase in the different onset of conditions. Dynamic light scattering, Differential scanning calorimetry, and Transmission electron microscopy were used to assess the homogeneity, stability, and morphology of particles.
Results: The SnoopLigase production was successful, with a high purity sample and great yield of 20 mg/L. Both enzymatic and chemical biotinylation of SnoopLigase was accomplished. The DogTag-noroVLPs production in Hi5 insect cells and two-step purification resulted in 90% pure particles with a yield of 16 mg/L. The size of DogTag-noroVLPs was estimated to be ~58 nm and the Tm of 64 °C. The particles were stable for one month at +4 °C. SnoopTagJr-RBD was produced similarly, however, the production yield was 6 mg/L and purity ~80%. SnoopLigase was efficient in forming the conjugation complex between DogTag-noroVLPs and SnoopTagJr-antigens. Differently biotinylated SnoopLigase has shown the same results. Control reactions were able to confirm the efficiency of SnoopLigase to form conjugates. Removal of the SnoopLigase from the end-product was not successful and requires additional optimization and trials.
Conclusion: Decoration of the norovirus-like particles with SARS-CoV-2 antigens by SnoopLigase system was successful. The SnoopTagJr-RBD production needs optimization to obtain a higher yield. The long-term storage of DogTag-noroVLPs should be explored in further research. Removal of the SnoopLigase requires certain adjustments and the stability of conjugated particles needs to be measured before pre-clinical trials. However, with a couple of modifications, this vaccine platform has plenty of potential for future application in vaccine development.
Keywords: norovirus, virus-like particle (VLP), SARS-CoV-2, antigens, vaccine, SnoopLigase, biotinylation, decoration
The originality of this thesis has been checked using the Turnitin OriginalityCheck service.
Materials and methods: SnoopLigase was expressed in E. coli BL21(DE3) Star cell line. The DogTag-noroVLPs and HisTag-SnoopTagJr-RBD were produced using the baculovirus-insect cells expression system. SnoopTagJr-RBM was chemically synthesized by a commercial provider. SnoopLigase and SnoopTagJr-RBD were purified with Affinity Chromatography. Purified SnoopLigase was biotinylated both chemically and enzymatically. The DogTag-noroVLPs were first purified with sucrose-gradient centrifugation and then polished with Ion Exchange Chromatography. SDS-PAGE and Western Blotting were utilized for purity assessment and characterization. Conjugation between DogTag-noroVLPs and SnoopTagJr-SARS-CoV-2 antigens was catalysed with SnoopLigase in the different onset of conditions. Dynamic light scattering, Differential scanning calorimetry, and Transmission electron microscopy were used to assess the homogeneity, stability, and morphology of particles.
Results: The SnoopLigase production was successful, with a high purity sample and great yield of 20 mg/L. Both enzymatic and chemical biotinylation of SnoopLigase was accomplished. The DogTag-noroVLPs production in Hi5 insect cells and two-step purification resulted in 90% pure particles with a yield of 16 mg/L. The size of DogTag-noroVLPs was estimated to be ~58 nm and the Tm of 64 °C. The particles were stable for one month at +4 °C. SnoopTagJr-RBD was produced similarly, however, the production yield was 6 mg/L and purity ~80%. SnoopLigase was efficient in forming the conjugation complex between DogTag-noroVLPs and SnoopTagJr-antigens. Differently biotinylated SnoopLigase has shown the same results. Control reactions were able to confirm the efficiency of SnoopLigase to form conjugates. Removal of the SnoopLigase from the end-product was not successful and requires additional optimization and trials.
Conclusion: Decoration of the norovirus-like particles with SARS-CoV-2 antigens by SnoopLigase system was successful. The SnoopTagJr-RBD production needs optimization to obtain a higher yield. The long-term storage of DogTag-noroVLPs should be explored in further research. Removal of the SnoopLigase requires certain adjustments and the stability of conjugated particles needs to be measured before pre-clinical trials. However, with a couple of modifications, this vaccine platform has plenty of potential for future application in vaccine development.
Keywords: norovirus, virus-like particle (VLP), SARS-CoV-2, antigens, vaccine, SnoopLigase, biotinylation, decoration
The originality of this thesis has been checked using the Turnitin OriginalityCheck service.