Sera of mice immunized with DNA vaccines encoding scaffolded RBD were diluted 50-fold, and the spike-pseudotyped virus infection of ACE2 and TMPRSS2-transfected HEK293 cells was measured by luminescence. The mean and SEM of six (RBD-AaLs) or five (all others) biological replicates are shown ( a). Afterwards, Spike bound to ACE2 was detected using streptactin-HRP. Sera of mice immunized with DNA vaccines comprising different scaffolded RBDs were diluted and pre-incubated with a spike protein. ( a– b) Neutralization of the binding of the spike protein to the hACE2 receptor determined by an ELISA-type assay and inhibition of pseudoviral infection of cells by mouse antisera. Neutralization of ACE2 binding and viral infection of RBD-scaffolded DNA vaccines. RBD-bann SARS-CoV-2 T-cell response nano-scaffolding domains vaccine. Results of the study support the use of a nanoscaffolding platform using the β-annulus peptide for vaccine design. The β-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Antibody titers and virus neutralization were most potently enhanced by fusion to the small β-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and β-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. 9 German Center for Infection Research (DZIF), Munich Partner Site, 38124 Braunschweig, Germany.8 Immunopathology of Virus Infections Laboratory, Institute of Virology, Technical University of Munich, 81675 Munich, Germany.7 Institute of Clinical Microbiology and Hygiene, University Hospital Regensburg, 93053 Regensburg, Germany.6 Molecular Microbiology (Virology), Institute of Medical Microbiology and Hygiene, University of Regensburg, 93053 Regensburg, Germany.5 Protein Production and Structure Core Facility PTPSP- EPFL SV PTECH PTPSP, 1015 Lausanne, Switzerland.4 Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, 1000 Ljubljana, Slovenia.3 Graduate School of Biomedicine, University of Ljubljana, 1000 Ljubljana, Slovenia.2 Department of Synthetic Biology and Immunology, National Institute of Chemistry, 1000 Ljubljana, Slovenia.1 EN-FIST, Centre of Excellence, 1000 Ljubljana, Slovenia.
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