Immunofluorescence analysis

(Fig. 2A) and intracellular FACS staining (Fig. 2B, upper graphs) revealed that 30–40% of cells in A549 cell cultures infected with HTNV at a MOI of 1.5 expressed hardly any detectable HTNV nucleocapsid (N) protein. Nevertheless, these HTNV N protein-negative cells from HTNV-infected A549 cell cultures showed an increase in HLA-I surface expression comparable to HTNV N protein-positive cells (Fig. 2B, lower graphs). Moreover, uninfected A549 cells upregulated HLA-I in response to UV-inactivated supernatant Crenolanib derived from HTNV-infected A549 cell cultures (data not shown). This indicates that HTNV mediates HLA-I upregulation on both actively infected and bystander Selleckchem Gefitinib cells. To further dissect HTNV-induced upregulation of HLA-I expression,

we tested whether HTNV transactivates the regulatory elements of single HLA-I genes in A549 cells. The promoter activities of all classical HLA-I genes were enhanced upon HTNV infection (Fig. 3). In contrast, HTNV did not significantly increase the promoter activity of nonclassical HLA-I genes (HLA-E, -F, -G) (Fig. 3). In summary, these findings show that HTNV-induced HLA-I surface expression is replication dependent, affects actively infected and bystander cells, and is based on activation of transcription factors that drive HLA-I gene expression. Next, we examined whether generation of peptides by the proteasome plays a role in HTNV-induced HLA-I upregulation. For this purpose, A549 cells were treated with epoximicin, a specific

and irreversible proteasome inhibitor or DMSO as a control. In the presence of epoxomicin, HTNV-infected A549 cells failed to significantly increase cell surface HLA-I expression (Fig. 4A). This finding prompted us to investigate the effect of HTNV on expression of TAP molecules because they transport proteasome-derived peptides into the lumen of the ER and represent a bottleneck in the HLA-I pathway. Dual luciferase reporter assays revealed enhanced activity of Sinomenine the promoter elements regulating TAP1 expression after HTNV infection (Fig. 4B). Moreover, we found increased expression of TAP1 protein in HTNV-infected as compared to uninfected A549 cells by performing intracellular FACS analysis (Fig. 4C). In conclusion, enhanced HLA-I expression after hantavirus infection requires a functional proteasome and increased TAP1 expression. We now analyzed IFN production in HTNV-infected A549 cells because the promoter regions of HLA-I and TAP genes encompass IFN-stimulated response elements. By using quantitative RT-PCR (qRT-PCR), no increase in the number of transcripts encoding IFN-α was detected at 4 days post infection (p.i.) compared to untreated A549 cells whereas IFN-β mRNA expression was enhanced (Fig. 5A). The positive control, A549 cells treated with IFN-α, upregulated IFN-α but not IFN-β encoding transcripts.

, 2004; Wang et al., 2007; Shen et al., 2009). However, the magnitude of the antigen-specific titers was not enhanced by PA co-delivered with the LFn fusions. This may reflect a low extracellular concentration/dose following expression that may limit the potential of the LFn fusions to come in contact with and bind to PA. Previous reports demonstrating an

additive immune response with PA and LFn used recombinant protein (Ballard et al., 1996; Lu et al., 2000) or targeted endogenously expressed PA and LFn from DNA vaccines to intracellular compartments (Price et al., 2001). In general, the antibody responses to the quadra-valent cocktail were consistent with the single antigen or this website fusion formula; however, the anti-F1 response was significantly reduced (P = 0.05). selleckchem This may reflect competition between the endogenously produced fusion proteins for the same binding site on PA following expression and cellular binding. Twenty-one days after the final immunization, the mice were aerosol challenged with either 2.75 × 104 B. anthracis STI (10 LD50) spores per mouse or 1 × 105 CFU of Y. pestis

GB (10 LD50) per mouse using a Collison spray conditioned in a modified Henderson aerosol apparatus (Williamson et al., 2000). Significance between groups was determined by log rank tests in conjunction with the Bonferroni multiple comparison method where P < 0.02 was defined as significant. The inhaled anthrax dose defeated 80% of the sham-vaccinated (pDNAVACCultra2 Sirolimus empty) mice, with a mean time to death (MTD) of 5 days. Groups receiving the PA and/or LFn expressing constructs were completely protected (100%, P < 0.02; Fig. 2a),

which is consistent with previous reports (Price et al., 2001; Hermanson et al., 2004; Livingston et al., 2010) and lends credence to the inclusion of nontoxic regions of LF in future anthrax vaccines (Baillie et al., 2010). The plague challenge was also lethal in the sham and phPA-vaccinated mice, resulting in a MTD of 3 days (Fig. 2b). Immunizations with phV-LFn or phLFn-F1 prolonged the MTD by 1 day relative to the sham (P < 0.02) but were still weakly protective against Y. pestis despite the relatively high antibody titers elicited by these fusions (Fig. 1c and d). In contrast, the protective efficacy of the phV-LFn construct was enhanced following co-immunization with phPA (83% survival). Immunization with all three constructs was also modestly protective against plague (66%). The mechanism behind this enhancement remains unclear; as previously noted, the antibody titers to the fusions were not synergistically increased in the presence of phPA. It is feasible that the CpG motifs within the plasmid backbone provided additional, nonspecific immune-stimulation (Williamson et al.