26 research outputs found
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Steric trapping reveals a cooperativity network in the intramembrane protease GlpG.
Membrane proteins are assembled through balanced interactions among proteins, lipids and water. Studying their folding while maintaining the native lipid environment is necessary but challenging. Here we present methods for analyzing key elements of membrane protein folding including thermodynamic stability, compactness of the unfolded state and folding cooperativity under native conditions. The methods are based on steric trapping, which couples the unfolding of a doubly biotinylated protein to the binding of monovalent streptavidin (mSA). We further advanced this technology for general application by developing versatile biotin probes possessing spectroscopic reporters that are sensitized by mSA binding or protein unfolding. By applying these methods to the Escherichia coli intramembrane protease GlpG, we elucidated a widely unraveled unfolded state, subglobal unfolding of the region encompassing the active site, and a network of cooperative and localized interactions to maintain stability. These findings provide crucial insights into the folding energy landscape of membrane proteins
Application of built-in adjuvants for epitope-based vaccines
Several studies have shown that epitope vaccines exhibit substantial advantages over conventional vaccines. However, epitope vaccines are associated with limited immunity, which can be overcome by conjugating antigenic epitopes with built-in adjuvants (e.g., some carrier proteins or new biomaterials) with special properties, including immunologic specificity, good biosecurity and biocompatibility, and the ability to vastly improve the immune response of epitope vaccines. When designing epitope vaccines, the following types of built-in adjuvants are typically considered: (1) pattern recognition receptor ligands (i.e., toll-like receptors); (2) virus-like particle carrier platforms; (3) bacterial toxin proteins; and (4) novel potential delivery systems (e.g., self-assembled peptide nanoparticles, lipid core peptides, and polymeric or inorganic nanoparticles). This review primarily discusses the current and prospective applications of these built-in adjuvants (i.e., biological carriers) to provide some references for the future design of epitope-based vaccines
Growth enhancement of porcine epidemic diarrhea virus (PEDV) in Vero E6 cells expressing PEDV nucleocapsid protein.
More recently emerging strains of porcine epidemic diarrhea virus (PEDV) cause severe diarrhea and especially high mortality rates in infected piglets, leading to substantial economic loss to worldwide swine industry. These outbreaks urgently call for updated and effective PEDV vaccines. Better understanding in PEDV biology and improvement in technological platforms for virus production can immensely assist and accelerate PEDV vaccine development. In this study, we explored the ability of PEDV nucleocapsid (N) protein in improving viral yields in cell culture systems. We demonstrated that PEDV N expression positively affected both recovery of PEDV from infectious clones and PEDV propagation in cell culture. Compared to Vero E6 cells, Vero E6 cells expressing PEDV N could accelerate growth of a slow-growing PEDV strain to higher peak titers by 12 hours or enhance the yield of a vaccine candidate strain by two orders of magnitude. Interestingly, PEDV N also slightly enhances replication of porcine reproductive and respiratory virus, a PEDV relative in the Nidovirales order. These results solidify the importance of N in PEDV recovery and propagation and suggest a potentially useful consideration in designing vaccine production platforms for PEDV or closely related pathogens
Lipopeptide-Coated Iron Oxide Nanoparticles as Potential Glycoconjugate-Based Synthetic Anticancer Vaccines
Although
iron oxide magnetic nanoparticles (NPs) have been widely
utilized in molecular imaging and drug delivery studies, they have
not been evaluated as carriers for glycoconjugate-based anticancer
vaccines. Tumor-associated carbohydrate antigens (TACAs) are attractive
targets for the development of anticancer vaccines. Due to the weak
immunogenicity of these antigens, it is highly challenging to elicit
strong anti-TACA immune responses. With their high biocompatibilities
and large surface areas, magnetic NPs were synthesized for TACA delivery.
The magnetic NPs were coated with phospholipid-functionalized TACA
glycopeptides through hydrophobic–hydrophobic interactions
without the need for any covalent linkages. Multiple copies of glycopeptides
were presented on NPs, potentially leading to enhanced interactions
with antibody-secreting B cells through multivalent binding. Mice
immunized with the NPs generated strong antibody responses, and the
glycopeptide structures important for high antibody titers were identified.
The antibodies produced were capable of recognizing both mouse and
human tumor cells expressing the glycopeptide, resulting in tumor
cell death through complement-mediated cytotoxicities. These results
demonstrate that magnetic NPs can be a new and simple platform for
multivalently displaying TACA and boosting anti-TACA immune responses
without the need for a typical protein carrier
Lipopeptide-Coated Iron Oxide Nanoparticles as Potential Glycoconjugate-Based Synthetic Anticancer Vaccines
Lipopeptide-Coated Iron Oxide Nanoparticles as Potential Glycoconjugate-Based Synthetic Anticancer Vaccines
Although
iron oxide magnetic nanoparticles (NPs) have been widely
utilized in molecular imaging and drug delivery studies, they have
not been evaluated as carriers for glycoconjugate-based anticancer
vaccines. Tumor-associated carbohydrate antigens (TACAs) are attractive
targets for the development of anticancer vaccines. Due to the weak
immunogenicity of these antigens, it is highly challenging to elicit
strong anti-TACA immune responses. With their high biocompatibilities
and large surface areas, magnetic NPs were synthesized for TACA delivery.
The magnetic NPs were coated with phospholipid-functionalized TACA
glycopeptides through hydrophobic–hydrophobic interactions
without the need for any covalent linkages. Multiple copies of glycopeptides
were presented on NPs, potentially leading to enhanced interactions
with antibody-secreting B cells through multivalent binding. Mice
immunized with the NPs generated strong antibody responses, and the
glycopeptide structures important for high antibody titers were identified.
The antibodies produced were capable of recognizing both mouse and
human tumor cells expressing the glycopeptide, resulting in tumor
cell death through complement-mediated cytotoxicities. These results
demonstrate that magnetic NPs can be a new and simple platform for
multivalently displaying TACA and boosting anti-TACA immune responses
without the need for a typical protein carrier
SARS-CoV-2 Delta (B.1.617.2) variant replicates and induces syncytia formation in human induced pluripotent stem cell-derived macrophages
Alveolar macrophages are tissue-resident immune cells that protect epithelial cells in the alveoli from invasion by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, the interaction between macrophages and SARS-CoV-2 is inevitable. However, little is known about the role of macrophages in SARS-CoV-2 infection. Here, we generated macrophages from human induced pluripotent stem cells (hiPSCs) to investigate the susceptibility of hiPSC-derived macrophages (iMΦ) to the authentic SARS-CoV-2 Delta (B.1.617.2) and Omicron (B.1.1.529) variants as well as their gene expression profiles of proinflammatory cytokines during infection. With undetectable angiotensin-converting enzyme 2 (ACE2) mRNA and protein expression, iMΦ were susceptible to productive infection with the Delta variant, whereas infection of iMΦ with the Omicron variant was abortive. Interestingly, Delta induced cell-cell fusion or syncytia formation in iMΦ, which was not observed in Omicron-infected cells. However, iMΦ expressed moderate levels of proinflammatory cytokine genes in response to SARS-CoV-2 infection, in contrast to strong upregulation of these cytokine genes in response to polarization by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Overall, our findings indicate that the SARS-CoV-2 Delta variant can replicate and cause syncytia formation in macrophages, suggesting that the Delta variant can enter cells with undetectable ACE2 levels and exhibit greater fusogenicity