RealTime PCR analysis of 293T cells transfected with LMP1 or ΔLMP1-MAVS.

<p>293T cells were transfected with pcDNA3.1, LMP1 plasmid, or ΔLMP1-MAVS plasmid and total RNA isolated following 36-hour culture. Normalized expression was determined relative to the pcDNA3.1 control.</p

ΔLMP1-MAVS enhances anti-Gag immune responses as an Ad5 viral vector vaccine adjuvant.

<p>BALB/c mice were left untreated or vaccinated with a combination of Ad5-Gag and either Ad5-GFP (control), Ad5-ΔLMP1-MAVS, or Ad5-LMP1. Two weeks following vaccination, mice were challenged with vaccinia-gag virus. Vaccinia titers were measured from ovaries after 5 days. NT: no treatment.</p

Gene array analysis of transfected 293T cells and primary CD4+ T cells cultured with 293T supernatant.

<p>Three independent wells of 293T cells were transfected with pcDNA3.1 empty vector or ΔLMP1-MAVS plasmid and total RNA isolated 36 hours later. Primary CD4+ T cells from 3 independent donors were cultured with 293T supernatant (collected 24 hours following pcDNA3.1 or ΔLMP1-MAVS transfection). Total CD4+ T cell RNA was isolated 36 hours later. (A) Venn Diagrams of the number of probe sets upregulated and down-regulated (>2-fold change) by ΔLMP1-MAVS. (B) Differential gene expression of 293T cells and CD4+ T cells. (C) List of genes upregulated by ΔLMP1-MAVS in both 293T cells and CD4+ T cells. Fold-change and P-values for each probe set are shown for CD4+ T cells following ΔLMP1-MAVS treatment. (D) List of genes upregulated by CD4+ T cells but not 293T cells. Fold-change between pcDNA3.1 and pΔLMP1-MAVS and P-values for each probe set are shown. (E) Gen Go networks analysis of pathways significantly upregulated by ΔLMP1-MAVS in transfected 293T cells, or CD4+ T cells cultured with ΔLMP1-MAVS transfected 293T supernatant.</p

Beta-interferon mediates inhibition of HIV-1 BaL replication.

<p>(A) The relative level of HIV-1 BaL strain viral replication in TZM-bl cells was measured following incubation with supernatant from 293T cells transfected with either pcDNA3.1 or pΔLMP1-MAVS plasmid, combined with 60 μg of isotype control antibody, anti-IFN-β antibody, or anti-IFN-α antibody. (B) Human CD4+ T cells were infected with HIV-1 BaL in the presence of increasing concentrations of interferon-α and compared to infection in the presence of 293T supernatant following transfection with either pcDNA3.1 or pΔLMP1-MAVS plasmid. (C) Supernatant from 293T cells transfected with pcDNA3.1 GFP, LMP1, or ΔLMP1-MAVS plasmid was assayed for IFN-α and IFN-β secretion by ELISA. NT: no treatment.</p

Inhibition of HIV and SIV viral infection of TZM-bl and primary human CD4+ T cells.

<p>The relative level of viral replication in TZM-bl cells was measured following incubation with supernatant from 293T cells transfected with pcDNA3.1 vector expressing GFP (control), LMP1, or ΔLMP1-MAVS plasmid. (A) Infection with HIV-1 BaL strain. (B) Infection with VSV-G pseudotyped single cycle SIV. (C) CD4+ T cells were isolated from a healthy donor by negative selection, activated, and cultured with 293T supernatant for 24 hours. Cells were then washed and infected with HIV-1 BaL at an MOI or 0.1 or 1. The concentration of p24 was measured 6 days later by ELISA assay. *p<0.05, **p<0.01, ***p<0.001.</p

Exosome-depleted 293T supernatant inhibits HIV and SIV replication.

<p>293T cells were transfected with pcDNA3.1 plasmids encoding GFP, LMP1, or ΔLMP1-MAVS and supernatant was isolated. Supernatant was depleted of exosomes by ultracentrifugation. (A) TZM-bl cells were cultured with isolated exosomes and infected with increasing concentrations of HIV-1 BaL. (B) TZM-bl cells were cultured with exosome-depleted supernatant, followed by infection with HIV-1 BaL. (C) TZM-bl cells were cultured with exosome-depleted supernatant, followed by infection with VSV-G pseudotyped single cycle SIV. *p<0.05, **p<0.01, ***p<0.001.</p

Ad5-LMP1 DC reduced viral titers of vaccinia-Gag in a prophylactic DC vaccination model.

<p>BMDC were transduced with Ad5-Gag and matured with either Ad5-LMP1 or Mimic in the presence of doxycycline. Mice were vaccinated with 1x10⁵ DC injected intradermally into the flank. Mice were given doxycycline in drinking water from the time of injection. 4 weeks post-vaccination, mice were challenged with 1x10⁷ viral particles vaccinia-Gag. Five days following challenge, ovaries were assayed for viral titer. Data combine the results from two independent experiments (n = 9 mice per group).</p

Biological activity of Ad5-LMP1.

<p><b>(A, B)</b> 293T cells were transfected with either NF-κB <b>(A)</b> or IFN-β <b>(B)</b> firefly luciferase reporter plasmid together with pRL-TK and either LMP1 or control expression plasmids. As positive controls, pcDNA3.1-FLAG-TRAF6 and pcDNA3.1-ΔRIG-I were used. 36-48h later, luciferase activity was measured in total cell lysate. <b>(C)</b> Viral stocks of Ad5-LMP1 were serially diluted and added to 293-SEAP reporter cells in triplicate, in the presence or absence of doxycycline. After 36h, secreted alkaline phosphatase was measured. Data represent typical results from three independent experiments.</p

Ad5-LMP1 enhanced bone marrow-derived dendritic cell maturation and cytokine secretion.

<p>BMDC were transduced with Ad5-LMP1 or control virus in the presence of doxycycline. Mimic was added as a positive control. <b>(A)</b> Gating strategy used to define CD11c+ DC from BMDC cultures. <b>(B)</b> On day 5, cells were stained for maturation markers, and analyzed by flow cytometry. <b>(C)</b> On day 5, supernatant was collected and analyzed using the BD mouse inflammation CBA kit. Note that Mimic cytokines were not removed from the Mimic control culture before analysis, and IL-6 and TNF-α levels may represent exogenous protein. Data represent results from three independent experiments.</p

Ad5-LMP1 enhanced maturation, cytokine production of human monocyte-derived dendritic cells.

<p>Human monocytes were isolated from buffy coat and cultured in the presence of GM-CSF and IL-4 for 5 days to generate DC. On day 5, cells were infected with Ad5-LMP1 or Ad5-GFP at an MOI of 100 in the presence of doxycycline. Mimic cytokine maturation cocktail was added on day 5 as a positive control. <b>(A)</b> Cells were analyzed by flow cytometry and representative forward vs. side scatter plots are displayed. <b>(B)</b> Images of DC were taken on day 7 before harvesting for maturation staining. Scale bar equals 100μm. <b>(C)</b> Representative gating strategy used to determine maturation of MDDC. <b>(D)</b> DC were stained for maturation markers, and analyzed by flow cytometry. <b>(E)</b> Supernatant was collected every 12 hours for 2 days and cytokine measured by CBA. TNF-α, IL-6 and IL-1β levels for Mimic sample may represent residual protein from the Mimic cocktail. Data represent results from three independent experiments.</p