Regulatory T Cells Suppress Natural Killer Cells during Plasmid DNA Vaccination in Mice, Blunting the CD8+ T Cell Immune Response by the Cytokine TGFβ

regulatory T cells (Tregs) suppress adaptive T cell-mediated immune responses to self- and foreign-antigens. Tregs may also suppress early innate immune responses to vaccine antigens and might decrease vaccine efficacy. NK and NKT cells are the first responders after plasmid DNA vaccination and are found at the site of inoculation. Earlier reports demonstrated that NKT cells could improve plasmid DNA efficacy, a phenomenon not found for NK cells. In fact, it has been shown that under certain disease conditions, NK cells are suppressed by Tregs via their release of IL-10 and/or TGFβ. Therefore, we tested the hypothesis that NK cell function is suppressed by Tregs in the setting of plasmid DNA vaccination. T cells. We found that this phenomenon is dependent on the secretion of cytokine TGFβ by Tregs, and independent of IL-10.Our data indicate a crucial function for Tregs in blocking plasmid DNA vaccine-elicited immune responses, revealing potentially novel strategies for improving the efficiency of plasmid DNA vaccines including chemical- or antibody-induced localized blockage of Treg-mediated suppression of NK cells at the site of plasmid DNA vaccine inoculation

Inhibition of HCV by the serpin antithrombin III

Background: Although there have been dramatic strides made recently in the treatment of chronic hepatitis C virus infection, interferon-α based therapy remains challenging for certain populations, including those with unfavorable IL28B genotypes, psychiatric co-morbidity, HIV co-infection, and decompensated liver disease. We have recently shown that ATIII, a serine protease inhibitor (serpin), has broad antiviral properties. Results: We now show that ATIII is capable of inhibiting HCV in the OR6 replicon model at micromolar concentrations. At a mechanistic level using gene-expression arrays, we found that ATIII treatment down-regulated multiple host cell signal transduction factors involved in the pathogenesis of cirrhosis and hepatocellular carcinoma, including Jun, Myc and BMP2. Using a protein interactive network analysis we found that changes in gene-expression caused by ATIII were dependent on three nodes previously implicated in HCV disease progression or HCV replication: NFκB, P38 MAPK, and ERK1/2. Conclusions: Our findings suggest that ATIII stimulates a novel innate antiviral host cell defense different from current treatment options

In Vivo Anti-HIV Activity of the Heparin-Activated Serine Protease Inhibitor Antithrombin III Encapsulated in Lymph-Targeting Immunoliposomes

Endogenous serine protease inhibitors (serpins) are anti-inflammatory mediators with multiple biologic functions. Several serpins have been reported to modulate HIV pathogenesis, or exhibit potent anti-HIV activity in vitro, but the efficacy of serpins as therapeutic agents for HIV in vivo has not yet been demonstrated. In the present study, we show that heparin-activated antithrombin III (hep-ATIII), a member of the serpin family, significantly inhibits lentiviral replication in a non-human primate model. We further demonstrate greater than one log10 reduction in plasma viremia in the nonhuman primate system by loading of hep-ATIII into anti-HLA-DR immunoliposomes, which target tissue reservoirs of viral replication. We also demonstrate the utility of hep-ATIIII as a potential salvage agent for HIV strains resistant to standard anti-retroviral treatment. Finally, we applied gene-expression arrays to analyze hep-ATIII-induced host cell interactomes and found that downstream of hep-ATIII, two independent gene networks were modulated by host factors prostaglandin synthetase-2, ERK1/2 and NFκB. Ultimately, understanding how serpins, such as hep-ATIII, regulate host responses during HIV infection may reveal new avenues for therapeutic intervention

Serpin Induced Antiviral Activity of Prostaglandin Synthetase-2 against HIV-1 Replication

The serine protease inhibitors (serpins) are anti-inflammatory proteins that have various functions. By screening a diverse panel of viruses, we demonstrate that the serpin antithrombin III (ATIII) has a broad-spectrum anti-viral activity for HIV-1, HCV and HSV. To investigate the mechanism of action in more detail we investigated the HIV-1 inhibition. Using gene-expression arrays we found that multiple host cell signal transduction pathways were activated by ATIII in HIV-1 infected cells but not in uninfected controls. Moreover, the signal pathways initiated by ATIII treatment, were more than 200-fold increased by the use of heparin-activated ATIII. The most up-regulated transcript in HIV-1 infected cells was prostaglandin synthetase-2 (PTGS2). Furthermore, we found that over-expression of PTGS2 reduced levels of HIV-1 replication in human PBMC. These findings suggest a central role for serpins in the host innate anti-viral response. Host factors such as PTGS2 elicited by ATIII treatment could be exploited in the development of novel anti-viral interventions

Effect of heparin activated ATIII (hep-ATIII) on gene expression in acutely HIV infected PBMC.

<p>(<b>A</b>) Gene up-regulation of acutely infected PBMC after treatment with different doses of hep-ATIII, each dose compared to infected hep-ATIII untreated vehicle control. (<b>B</b>) Gene down-regulation of acutely infected PBMC after treatment with different doses of hep-ATIII, each dose compared to infected hep-ATIII untreated vehicle control. For signal transduction gene analysis, 10⁵ PBMC were infected with a 0.01 MOI of primary isolate HIV-1 (HIV 89.6) for 2 hrs at 37°C. Cells were washed and treated with 0.09, 0.17 and 0.4 µM hep-ATIII for 48 h. Total RNA was purified and a RT-PCR expression array was performed. The expression levels of genes of 20 different signal transduction pathways (84 genes) were analyzed. Genes with significant changes in gene expression (<i>p</i><0.05, n = 3) compared to controls are show. Significance was calculated using the ΔΔC_t method for three independent experiments.</p

Effect of ATIII on gene expression in uninfected PBMC.

<p>Gene-regulation of uninfected PBMC treated with different doses of ATIII, each dose compared to an uninfected, ATIII untreated vehicle control. For signal transduction gene analysis, 10⁵ PBMC were infected with a 0.01 MOI of primary isolate HIV-1 (HIV 89.6) for 2 h at 37°C. Cells were washed and treated with 6.8, 34 and 68 µM ATIII for 48 h. Total RNA was purified and a RT-PCR expression array was performed. The expression of 84 genes from 20 different signal transduction pathways was analyzed. Genes with significant changes in gene expression (<i>p</i><0.05, n = 3) compared to controls are shown. Significance was calculated using the ΔΔC_t method for three independent experiments.</p

Effect of ATIII on gene expression in acutely HIV infected PBMC.

<p>(<b>A</b>) Gene up-regulation of acutely infected PBMC after treatment with different doses of ATIII, each dose compared to infected untreated control. (<b>B</b>) Gene down-regulation of acutely infected PBMC after treatment with different doses of ATIII, each dose compared to an infected untreated control. For signal transduction gene analysis, 10⁵ PBMC were infected with a 0.01 MOI of primary isolate HIV-1 (HIV 89.6) for 2 h at 37°C. Cells were washed and treated with 6.8, 34 and 68 µM ATIII for 48 h. Total RNA was purified and a RT-PCR expression array was performed. The gene expression levels from 20 different signal transduction pathways were analyzed. Genes with significant changes in gene expression (<i>p</i><0.05, n = 3) compared to controls are shown. Significance was calculated using the ΔΔC_t method for three independent experiments.</p

Effect of acute HIV infection on PBMC gene expression.

<p>Gene expression profiling of PBMC acutely infected with HIV-1. For signal transduction gene analysis, 10⁵ PBMC were infected with a 0.01 MOI of the primary isolate HIV-1 (HIV 89.6), for 2 h at 37°C. Total RNA was purified two days after infection. A RT-PCR expression array was performed and gene expression of 84 genes from 20 different signal transduction pathways was analyzed. Genes with significant changes in gene expression (<i>p</i><0.05, n = 3) compared to uninfected vehicle treated controls are shown. Significance was calculated using the ΔΔC_t method for three independent experiments.</p

Damping of the antigen-specific CD8⁺ T cell immune response is mediated by a CD25⁺ T cell.

<p>(A) Representative gating of AL11 tetramer stains of CD25⁺ depleted or undepleted mice (isotype control) after plasmid DNA vaccination. (B) SIV-Gag AL11 epitope-specific CD8⁺ T cell responses in vaccinated anti-CD25- and isotype-matched antibody-treated mice. (C) Percentage of CD4⁺CD25^high FoxP3^high cells at day 7 with inset showing representative gating of splenocytes of mice depleted or undepeted of CD25⁺ cells. Epitope-specific CD8⁺ T cell responses were measured by D^b/AL11 tetramer staining of CD8⁺ T cells in 5 C57BL/b6 mice per group at the indicated times following plasmid DNA vaccine construct inoculation. CD25⁺ cells were inactivated by administration of anti-CD25 antibody 3 days prior to and on the day of vaccination. Line graphs represent mean values, and error bars represent SEM. Statistically significant differences at specific times were determined by Mann-Whitney test. Significant differences are indicated by asterisks (p<0.05).</p

Inhibition of HIV replication by PTGS2.

<p>(<b>A</b>) Viral replication measured in RNA copy number per ml of mock-transfected (empty vector) and pCMV-PTGS2-transfected (PTGS2) PBMC. (<b>B</b>) Protein expression of Western-blot of 20 µg lysate protein of mock-transfected (lane 1) or PTGS2-transfected PBMC (lane 2). PBMC were transiently transfected with plasmid DNA. After 2 days cells were infected with a 0.01 MOI of the HIV-1 primary isolate (HIV 89.6) for 2 hrs at 37°C. Cells were washed and supernatant was collected after 48 hr. HIV-1 RNA levels were measured using the COBAS Ampliprep/COBAS Taqman 48 system for three independent experiments. For the Western analysis, transfected Jurkat cellular homogenates (20 µg/lane) were separated by SDS-PAGE. Target proteins were identified by Western analysis using polyclonal antibody generated against the PTGS2 protein. Representative data of three independent experiments are shown. (<b>C</b>) PBMC were infected with HIV-1 primary isolate (HIV 89.6) at a MOI of 0.01. Hep-ATIII was added at concentrations of 0.09, 0.17 and 0.4 µM. After 48 hours, PTGS2 was quantified using the PTGS2-specific ELISA.</p