High-dimensional CyTOF analysis of dengue virus-infected human DCs reveals distinct viral signatures.

Dengue virus (DENV) is the most prevalent mosquito-borne virus causing human disease. Of the 4 DENV serotypes, epidemiological data suggest that DENV-2 secondary infections are associated with more severe disease than DENV-4 infections. Mass cytometry by time-of-flight (CyTOF) was used to dissect immune changes induced by DENV-2 and DENV-4 in human DCs, the initial targets of primary infections that likely affect infection outcomes. Strikingly, DENV-4 replication peaked earlier and promoted stronger innate immune responses, with increased expression of DC activation and migration markers and increased cytokine production, compared with DENV-2. In addition, infected DCs produced higher levels of inflammatory cytokines compared with bystander DCs, which mainly produced IFN-induced cytokines. These high-dimensional analyses during DENV-2 and DENV-4 infections revealed distinct viral signatures marked by different replication strategies and antiviral innate immune induction in DCs, which may result in different viral fitness, transmission, and pathogenesis.

HIV-1 Vpu antagonism of tetherin inhibits antibody-dependent cellular cytotoxic responses by natural killer cells.

UNLABELLED: The type I interferon-inducible factor tetherin retains virus particles on the surfaces of cells infected with vpu-deficient human immunodeficiency virus type 1 (HIV-1). While this mechanism inhibits cell-free viral spread, the immunological implications of tethered virus have not been investigated. We found that surface tetherin expression increased the antibody opsonization of vpu-deficient HIV-infected cells. The absence of Vpu also stimulated NK cell-activating FcγRIIIa signaling and enhanced NK cell degranulation and NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). The deletion of vpu in HIV-1-infected primary CD4(+) T cells enhanced the levels of antibody binding and Fc receptor signaling mediated by HIV-positive-patient-derived antibodies. The magnitudes of antibody binding and Fc signaling were both highly correlated to the levels of tetherin on the surfaces of infected primary CD4 T cells. The affinity of antibody binding to FcγRIIIa was also found to be critical in mediating efficient Fc activation. These studies implicate Vpu antagonism of tetherin as an ADCC evasion mechanism that prevents antibody-mediated clearance of virally infected cells. IMPORTANCE: The ability of the HIV-1 accessory factor to antagonize tetherin has been considered to primarily function by limiting the spread of virus by preventing the release of cell-free virus. This study supports the hypothesis that a major function of Vpu is to decrease the recognition of infected cells by anti-HIV antibodies at the cell surface, thereby reducing recognition by antibody-dependent clearance by natural killer cells.

Human chorionic gonadotropin interactions with immobilized anti-hCG studied by quartz-crystal microbalance with dissipation monitoring.

Previous studies have found that commercial human pregnancy tests are often too insensitive to function to their advertised >99% accuracy. Improper orientation of proteins used for recognition of ligands in sensors can often prevent the binding site from being available to the ligand, thereby decreasing sensor sensitivity. We have developed a simple method for the immobilization of anti-human chorionic gonadotropin on a sensor surface that maximizes its sensitivity by ensuring ligand binding sites are exposed and densely packed. This surface also has an improved regenerative capacity over previously reported human chorionic gonadotropin sensors, retaining 99% of initial sensitivity after six regeneration cycles with 8M urea.

A QCM study of the immobilization of beta-galactosidase on polyelectrolyte surfaces: effect of the terminal polyion on enzymatic surface activity.

This work describes the immobilization of beta-galactosidase onto polyelectrolyte multilayer assemblies of the polyanion poly[1-[4-(3-carboxy-4-hydroxyphenylazo)benzenesulfonamido]-1,2-ethanediyl, sodium salt] (PAZO) and the polycation poly(ethylenimine) (PEI) constructed by electrostatic self-assembly (ESA). A single layer of beta-galactosidase was deposited over a precursor film comprising up to five bilayers of the PEI/PAZO polyelectrolyte pair. The enzyme was deposited on both the polycationic (PEI) and the polyanionic (PAZO) surfaces. Quartz crystal microbalance with dissipation monitoring (QCM-D), single-wavelength ellipsometry, and UV-visible absorption spectroscopy revealed differences in both the amount of beta-galactosidase incorporated in each of the multilayer assemblies and the resulting enzyme packing density in the films. The enzymatic films were immersed in a reaction solution containing o-nitrophenyl-beta-d-galactopyranoside (ONPG), and absorbance measurements were used to monitor the concentration of o-nitrophenyl (ONP), the product of the beta-galactosidase catalyzed by hydrolysis of ONPG. Although our data indicate that comparable amounts of beta-galactosidase are incorporated onto both surfaces, enzymatic activity is substantially inhibited when the beta-galactosidase is immobilized on the polyanionic surface compared to the enzyme on the polycationic surface. The difference in catalytic activities reflects the different abilities of the two polyelectrolytes to screen the protein's active site from the substrate environment. In both assemblies, the protein interpenetrated the PEI/PAZO multilayer, disrupting the J-aggregated state of the PAZO chromophores. This work demonstrates that the charge, conformation, and composition of underlying polyelectrolyte cushions have a significant effect on the structure and function of an immobilized protein within functional nanoassemblies.