Characterization of (18)F-dipicolylamine (DPA) derivatives in cells infected with influenza virus.

OBJECTIVE: Bis(Zn-dipicolylamine (Zn-DPA)) coordination complexes represent a new class of synthetic small molecules that can target anionic phosphatidylserine (PS) in the apoptotic cells with high affinity and specificity. In this study, we labeled Zn-DPA and Cy7-Zn-DPA with different (18)F-prosthetic groups and characterized their uptake in A549 cells infected with influenza A virus from the 2009 pandemic (H1N1pdm). METHODS: DPA was labeled with N-succinimidyl 4-(18)F-fluorobenzoate ((18)F-SFB), 4-nitrophenyl 2-(18)F-fluoropropionate ((18)F-NFP), 2-(18)F-Fluoroethyl toslyate ((18)F-FET), and (18)F-aluminum (Al(18)F), respectively. Cy7-DPA was labeled with (18)F-SFB and (18)F-NFP only. The tracers were reconstituted with zinc nitrate before use. Apoptosis in A549 cells was induced by infection with the H1N1pdm virus for 48 h. Three µCi of each tracer was added to each well and incubated at 37 °C. The effect of different prosthetic groups, different MOI, and incubation time on percent cellular uptake was studied. Cell internalization and efflux was evaluated within 2h of incubation. The competitive binding assay was performed with increasing concentration (10(-12)-10(-5)M) of Zn-DPA or Cy7-Zn-DPA prior to the addition of either (18)F-FB-Zn-DPA or (18)F-FB-Cy7-Zn-DPA into each well. IC50 values for the two Zn-DPA analogues were estimated by GraphPad Prism 6.0. RESULTS: Among all the four prosthetic groups, the (18)F-SFB method provided the highest conjugation yield for DPA and the highest uptake ratio between the infection cells and the control when both Zn-DPA and Cy7-Zn-DPA were present in the complex. The uptake ratio was similar for (18)F-FB-Zn-DPA and (18)F-FB-Cy7-Zn-DPA. Uptake of (18)F-FB-Zn-DPA and (18)F-FB-Cy7-Zn-DPA was proportional to the degree of apoptosis with a plateau at MOI 3. Uptake of (18)F-FB-Cy7-Zn-DPA also increased over incubation time and reached a plateau at 1h, whereas uptake of (18)F-FB-Zn-DPA did not show any significant change over time. Cell internalization studies showed that more than 70% of (18)F-FB-Zn-DPA remained on the cell surface over a time course of 2 hr in the cell media, but over 90% of (18)F-FB-Cy7-Zn-DPA was internalized within 15 min of incubation. IC50 values were estimated to be 1.5±0.3 nM and 26.2±5.1 nM for Zn-DPA and Cy7-Zn-DPA, respectively. CONCLUSIONS: (18)F-SFB was the optimal labeling method for Zn-DPA and Cy7-Zn-DPA with respect to radiochemistry and provided complexes with high target-to-background ratios. (18)F-FB-Zn-DPA and (18)F-FB-Cy7-Zn-DPA appeared to have a completely different internalization mechanism, while Zn-DPA showed higher binding affinity than Cy7-Zn-DPA. Based on these favorable characteristics, (18)F-FB-Zn-DPA and (18)F-FB-Cy7-Zn-DPA should be further evaluated as potential imaging agents for viral infection.

Early host responses of seasonal and pandemic influenza A viruses in primary well-differentiated human lung epithelial cells.

Replication, cell tropism and the magnitude of the host's antiviral immune response each contribute to the resulting pathogenicity of influenza A viruses (IAV) in humans. In contrast to seasonal IAV in human cases, the 2009 H1N1 pandemic IAV (H1N1pdm) shows a greater tropism for infection of the lung similar to H5N1. We hypothesized that host responses during infection of well-differentiated, primary human bronchial epithelial cells (wd-NHBE) may differ between seasonal (H1N1 A/BN/59/07) and H1N1pdm isolates from a fatal (A/KY/180/10) and nonfatal (A/KY/136/09) case. For each virus, the level of infectious virus and host response to infection (gene expression and apical/basal cytokine/chemokine profiles) were measured in wd-NHBE at 8, 24, 36, 48 and 72 hours post-infection (hpi). At 24 and 36 hpi, KY/180 showed a significant, ten-fold higher titer as compared to the other two isolates. Apical cytokine/chemokine levels of IL-6, IL-8 and GRO were similar in wd-NHBE cells infected by each of these viruses. At 24 and 36 hpi, NHBE cells had greater levels of pro-inflammatory cytokines including IFN-α, CCL2, TNF-α, and CCL5, when infected by pandemic viruses as compared with seasonal. Polarization of IL-6 in wd-NHBE cells was greatest at 36 hpi for all isolates. Differential polarized secretion was suggested for CCL5 across isolates. Despite differences in viral titer across isolates, no significant differences were observed in KY/180 and KY/136 gene expression intensity profiles. Microarray profiles of wd-NHBE cells diverged at 36 hpi with 1647 genes commonly shared by wd-NHBE cells infected by pandemic, but not seasonal isolates. Significant differences were observed in cytokine signaling, apoptosis, and cytoskeletal arrangement pathways. Our studies revealed differences in temporal dynamics and basal levels of cytokine/chemokine responses of wd-NHBE cells infected with each isolate; however, wd-NHBE cell gene intensity profiles were not significantly different between the two pandemic isolates suggesting post-transcriptional or later differences in viral-host interactions.

Phenotypic differences in virulence and immune response in closely related clinical isolates of influenza A 2009 H1N1 pandemic viruses in mice.

To capture the possible genotypic and phenotypic differences of the 2009 influenza A virus H1N1 pandemic (H1N1pdm) strains circulating in adult hospitalized patients, we isolated and sequenced nine H1N1pdm viruses from patients hospitalized during 2009-2010 with severe influenza pneumonia in Kentucky. Each viral isolate was characterized in mice along with two additional H1N1 pandemic strains and one seasonal strain to assess replication and virulence. All isolates showed similar levels of replication in nasal turbinates and lung, but varied in their ability to cause morbidity. Further differences were identified in cytokine and chemokine responses. IL-6 and KC were expressed early in mice infected with strains associated with higher virulence. Strains that showed lower pathogenicity in mice had greater IFNγ, MIG, and IL-10 responses. A principal component analysis (PCA) of the cytokine and chemokine profiles revealed 4 immune response phenotypes that correlated with the severity of disease. A/KY/180/10, which showed the greatest virulence with a rapid onset of disease progression, was compared in additional studies with A/KY/136/09, which showed low virulence in mice. Analyses comparing a low (KY/136) versus a high (KY/180) virulent isolate showed a significant difference in the kinetics of infection within the lower respiratory tract and immune responses. Notably by 4 DPI, virus titers within the lung, bronchoalveolar lavage fluid (BALf), and cells within the BAL (BALc) revealed that the KY/136 replicated in BALc, while KY/180 replication persisted in lungs and BALc. In summary, our studies suggest four phenotypic groups based on immune responses that result in different virulence outcomes in H1N1pdm isolates with a high degree of genetic similarity. In vitro studies with two of these isolates suggested that the more virulent isolate, KY/180, replicates productively in macrophages and this may be a key determinant in tipping the response toward a more severe disease progression.