RESUMO
Site-specific protein modifications are vital for biopharmaceutical drug development. Gluconoylation is a non-enzymatic, post-translational modification of N-terminal HisTags. We report high-yield, site-selective inâ vitro α-aminoacylation of peptides, glycoproteins, antibodies, and virus-like particles (VLPs) with azidogluconolactone at pHâ 7.5 in 1â h. Conjugates slowly hydrolyse, but diol-masking with borate esters inhibits reversibility. In an example, we multimerise azidogluconoylated SARS-CoV-2 receptor-binding domain (RBD) onto VLPs via click-chemistry, to give a COVID-19 vaccine. Compared to yeast antigen, HEK-derived RBD was immunologically superior, likely due to observed differences in glycosylation. We show the benefits of ordered over randomly oriented multimeric antigen display, by demonstrating single-shot seroconversion and best virus-neutralizing antibodies. Azidogluconoylation is simple, fast and robust chemistry, and should accelerate research and development.
Assuntos
Azidas/química , Vacinas contra COVID-19/química , Gluconatos/química , Glicina/química , Histidina/química , Lactonas/química , Vacinas de Partículas Semelhantes a Vírus/química , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Azidas/imunologia , Vacinas contra COVID-19/imunologia , Gluconatos/imunologia , Glicina/imunologia , Histidina/imunologia , Humanos , Lactonas/imunologia , Modelos Moleculares , Estrutura Molecular , Vacinas de Partículas Semelhantes a Vírus/imunologiaRESUMO
OBJECTIVE: We previously demonstrated that altered zinc homeostasis is an important feature of pediatric sepsis, thus raising the possibility of zinc supplementation as a therapeutic strategy in sepsis. Herein, we tested the hypothesis that prophylactic zinc supplementation would be beneficial in a murine model of peritoneal sepsis. DESIGN: Murine model of sepsis (intraperitoneal fecal-slurry injection). SETTING: Basic science research laboratory. SUBJECTS: C57BL/6 male mice. INTERVENTIONS: Intraperitoneal fecal-slurry injection, with or without zinc supplementation (10 mg/kg of intraperitoneal zinc gluconate for 3 days prior to intraperitoneal fecal-slurry injection). MEASUREMENTS AND MAIN RESULTS: Survival over 3 days following intraperitoneal fecal-slurry injection, markers of inflammation, bacterial load studies, and immunophenotyping studies. Zinc-supplemented mice demonstrated a significant survival advantage compared to control (nonsupplemented) mice. Zinc-supplemented mice also demonstrated moderate reductions of inflammation and immune activation. The survival advantage primarily correlated with reduced in vivo bacterial load in zinc-supplemented mice, compared to controls. In addition, peritoneal macrophages harvested from zinc-supplemented mice demonstrated a significantly enhanced phagocytosis capacity for Escherichia coli and Staphylococcus aureus, compared to peritoneal macrophages harvested from control mice. CONCLUSION: Prophylactic zinc supplementation reduces bacterial load and is beneficial in a murine model of peritoneal sepsis.