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The chemokine receptor CCR5 is the major fusion coreceptor for macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1). To define the structures of CCR5 that can support envelope (Env)mediated membrane fusion, we analyzed the activity of homologs, chimeras, and mutants of human CCR5 in a sensitive gene reporter cell-cell fusion assay. Simian, but not murine, homologs of CCR5 were fully active as HIV-1 fusion coreceptors. Chimeras between CCR5 and divergent chemokine receptors demonstrated the existence of two distinct regions of CCR5 that could be utilized for Env-mediated fusion, the amino-terminal domain and the extracellular loops. Dual-tropic Env proteins were particularly sensitive to alterations in the CCR5 amino-terminal domain, suggesting that this domain may play a pivotal role in the evolution of coreceptor usage in vivo. We identified individual residues in both functional regions, Asp-11, Lys-197, and Asp-276, that contribute to coreceptor function. Deletion of a highly conserved cytoplasmic motif rendered CCR5 incapable of signaling but did not abrogate its ability to function as a coreceptor, implying the independence of fusion and G-protein-mediated chemokine receptor signaling. Finally, we developed a novel monoclonal antibody to CCR5 to assist in future studies of CCR5 expression.

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Respiratory syncytial virus (RSV) is a leading cause of mortality in infants and young children. Despite the RSV disease burden, no vaccine is available, and treatment remains nonspecific. New drug candidates are needed to combat RSV. Toward this goal, we screened over 2,000 compounds to identify approved drugs with novel anti-RSV activity. Cardiac glycosides, inhibitors of the membrane-bound Na/K-ATPase, were identified to have anti-RSV activity. Cardiac glycosides diminished RSV infection in human epithelial type 2 cells and in primary human airway epithelial cells grown at an air-liquid interface. Digoxin, a U.S. Food and Drug Administration-approved cardiac glycoside, was also able to inhibit infection of primary nasal epithelial cells with community isolates of RSV. Our results suggest that the antiviral effects of cardiac glycosides may be dependent on changes in the intracellular Na and K composition. Consistent with this mechanism, we demonstrated that the ionophoric antibiotics salinomycin, valinomycin, and monensin inhibited RSV in human epithelial type 2 cells and primary nasal epithelial cells. Our data indicate that the K/Na-sensitive steps in the RSV life cycle occur within the initial 4 hours of viral infection but do not include virus binding/entry. Rather, our findings demonstrated a negative effect on the RSV transcription and/or replication process. Overall, this work suggests that targeting intracellular ion concentrations offers a novel antiviral strategy.

In Fig. 2d of this Article, owing to an error during the production process, the final rightmost x-axis label should be 'Ciliated bronchial epithelial cells' and not 'Lung fibroblasts'. This error has been corrected online.

Respiratory Syncytial Virus (RSV) is the leading cause of acute lower respiratory infections in young children and infection has been linked to the development of persistent lung disease in the form of wheezing and asthma. Despite substantial research efforts, there are no RSV vaccines currently available and an effective monoclonal antibody targeting the RSV fusion protein (palivizumab) is of limited general use given the associated expense. Therefore, the development of novel approaches to prevent RSV infection is highly desirable to improve pediatric health globally. We have developed a method to generate alveolar-like macrophages (ALMs) from pluripotent stem cells. These ALMs have shown potential to promote airway innate immunity and tissue repair and so we hypothesized that ALMs could be used as a strategy to prevent RSV infection. Here, we demonstrate that ALMs are not productively infected by RSV and prevent the infection of epithelial cells. Prevention of epithelial infection was mediated by two different mechanisms: phagocytosis of RSV particles and release of an antiviral soluble factor different from type I interferon. Furthermore, intratracheal administration of ALMs protected mice from subsequent virus-induced weight loss and decreased lung viral titres and inflammation, indicating that ALMs can impair the pathogenesis of RSV infection. Our results support a prophylactic role for ALMs in the setting of RSV infection and warrant further studies on stem cell-derived ALMs as a novel cell-based therapy for pulmonary viral infections.

Introduction Staphylococcus aureus seriously threatens human and animal health. IsdB137‐361 of the iron surface determinant B protein (IsdB) from S. aureus exhibits the strong immunogenicity, but its immunoprotective effect is still to be further promoted. Because PEI‐PLGA nanoparticles are generated by PEI conjugate with PLGA to develop great potential as a novel immune adjuvant, the immunogenicity of IsdB137‐361 is likely be strengthened by PEI‐PLGA. Methods Here, PEI‐PLGA nanoparticles containing IsdB137‐361 proteins were prepared by optimizing the entrapment efficiency. Mice were immunized with IsdB137‐361‐PEI‐PLGA nanoparticles to assess their anti‐S. aureus effects. The level of IFN‐γ, IL‐4, IL‐17, and IL‐10 cytokines from spleen lymphocytes in mice and generation of the antibodies against IsdB137‐361 in serum was assessed by ELISA, the protective immune response was appraised by S. aureus challenge. Results IsdB137‐361 proteins loaded by PEI‐PLGA were able to stimulate effectively the proliferation of spleen lymphocytes and increase the secretion of IFN‐γ, IL‐4, IL‐17, and IL‐10 cytokine from spleen lymphocytes, and significantly enhance generation of the antibodies against IsdB137‐361 in serum, reduce the level of bacterial load in liver, spleen and kidney, and greatly improve the survival rate of mice after challenge. Conclusion These data showed that PEI‐PLGA nanoparticles can significantly enhance the immunogenicity of IsdB137‐361 proteins, and provide an important reference for the development of novel immune adjuvant.

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