Influenza Virus H1N1 inhibition by serine protease inhibitor (serpin) antithrombin III.

Endogenous serine protease inhibitors (serpins) are anti-inflammatory mediators with multiple biologic functions. Serpins are also part of the early innate immune response to viral infection that includes mannose binding lectins, soluble CD14, defensins and antimicrobial peptides. Recently, serpin antithrombin III (ATIII) was shown to have broad-spectrum antiviral activity against HIV, HSV and HCV. We tested ATIII's antiviral activity against a variety of influenza virus strains. In our studies we found strong in vitro inhibition of influenza virus A H1N1 isolates. Our data also demonstrate that ATIII potency was more than 100-fold that of ribavirin. We also found that inhibition was dependent on viral hemagglutinin with decreasing efficacy in the order of H1N1 > H3N2 > H5N1 >> Flu B. In vivo efficacy is currently still lacking demonstrating need for more advanced delivery methods for this biomolecule. Understanding how ATIII regulates influenza virus inhibition may reveal new avenues for therapeutic interventions.

Natural Killer cell-dependent and non-dependent anti-viral activity of 2-Cys Peroxiredoxin against HIV.

2-cys peroxiredoxins (Prx), a group of anti-oxidative enzyme proteins, act directly on virally-infected cells to inhibit HIV-1 replication, and indirectly through destruction of HIV infected cells by stimulation of Natural Killer (NK) cell-mediated immune responses. We assayed for antibody-dependent NK cell mediated viral inhibition (ADCVI) using plasma from SIV-infected rhesus macaques. We found that Prx-1 strongly increased ADCVI in a dose-dependent manner, suggesting augmentation of NK cell killing. We also investigated the effect of Prx-1 on NK cell-independent HIV-1 and HIV-2 inhibition. We found that primary HIV isolates were potently inhibited at nM concentrations, regardless of viral clade, receptor usage or anti-retroviral drug resistance. During NK cell independent inhibition, we found that Prx-1 reversed the HIV-1 induced gene expression of Heat shock protein 90 kDa alpha (cystolic), class A member 2, (HSP90), a protein of the stress pathway. Prx-1 highly activated Cyclin-dependent kinase inhibitor 2B (CDKN2B), a gene of the TGF-ß pathway, and Baculoviral IAP repeat-containing 2 (Birc-2), an anti-apoptotic gene of the NF-κB pathway. We identified gene-expression networks highly dependent on the NFκB and ERK1/2 pathways. Our findings demonstrate that Prx-1 inhibits HIV replication through NK cell-dependent and NK cell-independent mechanisms.

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 log(10) 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.

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.

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.

Modulation of plasmid DNA vaccine antigen clearance by caspase 12 RNA interference potentiates vaccination.

The magnitude of the immune responses elicited by plasmid DNA vaccines might be limited, in part, by the duration of vaccine antigen expression in vivo. To explore strategies for improving plasmid DNA vaccine efficacy, we studied the apoptotic process in myocytes of mice vaccinated intramuscularly. We found that after vaccination, the proapoptotic protein caspase 12 (Casp12) was upregulated in myocytes coincident with the loss of vaccine antigen expression. To harness this observation to improve plasmid DNA vaccine efficacy, we used RNA interference technology, coadministering plasmid DNA expressing a short hairpin RNA (shRNA) of Casp12 with plasmid DNA vaccine constructs. This treatment with shRNA Casp12, administered twice within the first 10 days following vaccine administration, increased antigen expression 7-fold, the antigen-specific CD8(+) T cell immune response 6-fold, and antigen-specific antibody production 5-fold. This study demonstrates the critical role for Casp12 in plasmid DNA vaccine-induced immune responses and shows that increased antigen expression mediated by down-modulation of Casp12 can be used to potentiate vaccine efficacy.

Regulatory T cells suppress natural killer cells during plasmid DNA vaccination in mice, blunting the CD8+ T cell immune response by the cytokine TGFbeta.

BACKGROUND: CD4(+)CD25(+) 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 TGFbeta. Therefore, we tested the hypothesis that NK cell function is suppressed by Tregs in the setting of plasmid DNA vaccination. METHODOLOGY/PRINCIPAL FINDINGS: In this study we show that Tregs directly inhibit NK cell function during plasmid DNA vaccination by suppressing the potentially 10-fold, NK cell-mediated, augmentation of plasmid DNA antigen-specific CD8(+) T cells. We found that this phenomenon is dependent on the secretion of cytokine TGFbeta by Tregs, and independent of IL-10. CONCLUSIONS: 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.

Non-classical natural killer T cells modulate plasmid DNA vaccine antigen expression and vaccine-elicited immune responses by MCP-1 secretion after interaction with a beta2-microglobulin-independent CD1d.

The magnitude and durability of a plasmid DNA vaccine-induced immune response is shaped by immune effector molecules at the site of vaccination. In the present study, we show that antigen expression is modified by type II NKT cells, after interaction with a beta2-microglobulin-independent CD1d receptor. After activation, during the first days following plasmid DNA vaccination, NKT cells release IL-5 and MCP-1, leading to a T helper 0 (T(H)0) cytokine/chemokine profile and a stronger CD8(+)/CD4(+) T cell immune response. Our data indicate that this phenomenon was induced through the strong T(H)1 chemokine MCP-1 during the early phases of plasmid DNA vaccination because injecting the type II NKT cell-associated MCP-1 during the first 5 days led to 2-3-fold increases in vaccine-elicited T cell responses. This study demonstrates a critical role for NKT cells in plasmid DNA vaccine-induced immune responses. Manipulation of NKT cell function or co-administration of MCP-1 may represent novel methods for enhancing immune responses to plasmid DNA vaccines.

CD4+ T lymphocytes mediate in vivo clearance of plasmid DNA vaccine antigen expression and potentiate CD8+ T-cell immune responses.

There is evidence that the limited immunogenicity of plasmid DNA vaccines is the result, at least in part, of the rapid clearance of vaccine antigen expression by antigen-specific immune responses. However, the cell types responsible for the clearance of plasmid DNA vaccine antigens are not known. Here we demonstrate that macrophages, NK cells, and CD8(+) T cells did not significantly contribute to the DNA antigen clearance but CD4(+) T cells played the crucial role in attenuating plasmid DNA vaccine antigen expression. Adoptive transfer experiments demonstrate that CD4(+) T cells facilitated DNA vaccine antigen clearance in a Fas/FasL-dependent manner. Furthermore, we show that depletion of CD4(+) T cells prevented the clearance of vaccine antigen and the appearance of a CD8(+) T-cell immune response. Inoculation of major histocompatibility complex class II KO mice with the plasmid DNA led to persistent antigen expression and abolition of a CD8(+) T-cell immune response. Importantly, the prolongation of antigen expression by disrupting the CD4(+) T-cell Fas/FasL myocytes signaling led to a 3- to 5-fold increase of antigen-specific CD8(+) T-cell responses. These data demonstrate a dominant role of CD4(+) T cell-mediated cytotoxicity in plasmid DNA vaccine antigen clearance.

Kinetics of recombinant adenovirus type 5, vaccinia virus, modified vaccinia ankara virus, and DNA antigen expression in vivo and the induction of memory T-lymphocyte responses.

While a new generation of vaccine vectors has been developed for eliciting cellular immune responses, little is known about the optimal routes for their administration or about the ramifications of the kinetics of in vivo vaccine antigen expression for immunogenicity. We evaluated the kinetics of vaccine antigen expression by real-time in vivo photon imaging and showed dramatic differences in these kinetics using different vectors and different routes of administration. Further, using a gamma interferon enzyme-linked immunospot assay to measure T-lymphocyte immune responses, we observed an association between the kinetics of vaccine antigen expression in vivo and the magnitude of vaccine-elicited memory T-lymphocyte responses. These results highlight the utility of the real-time in vivo photon-imaging technology in evaluating novel immunization strategies and suggest an association between the kinetics of vaccine antigen clearance and the magnitude of vaccine-elicited T-lymphocyte memory immune responses.

Anti-viral activity of human antithrombin III.

Soluble inhibitory factors produced by CD8+ T-cells have been shown to inhibit HIV-1 replication and may play a critical role in vivo in anti-viral host defense. CD8+ T-cell-modified antithrombin III (ATIII) accounts for some of the described CD8+ T-cell anti-viral activity. We demonstrate that CD4+ T-cells, CD8+ T-cells, and natural killer cells react to an ATIII gradient by cell migration. Furthermore, exogenously added ATIII induced a G-protein-coupled signal transduction process in CD4+ T-cells and inhibited TNF-alpha-induced NF-kappaB activation. Heat and/or heparin treatment prior to the anti-viral inhibition test increased the anti-HIV activity up to 1000-fold. Our data indicate that anti-viral inactive ATIII can be activated having promising anti-viral properties as complementary candidate for the treatment of HIV infection.

HIV-1 antiviral activity of recombinant natural killer cell enhancing factors, NKEF-A and NKEF-B, members of the peroxiredoxin family.

CD8(+) T-cells are a major source for the production of non-cytolytic factors that inhibit HIV-1 replication. In order to characterize further these factors, we analyzed gene expression profiles of activated CD8(+) T-cells using a human cDNA expression array containing 588 human cDNAs. mRNA for the chemokine I-309 (CCL1), the cytokines granulocyte-macrophage colony-stimulating factor and interleukin-13, and natural killer cell enhancing factors (NKEF) -A and -B were up-regulated in bulk CD8(+) T-cells from HIV-1 seropositive individuals compared with seronegative individuals. Recombinant NKEF-A and NKEF-B inhibited HIV-1 replication when exogenously added to acutely infected T-cells at an ID(50) (dose inhibiting HIV-1 replication by 50%) of approximately 130 nm (3 microg/ml). Additionally, inhibition against dual-tropic simian immunodeficiency virus and dual-tropic simian-human immunodeficiency virus was found. T-cells transfected with NKEF-A or NKEF-B cDNA were able to inhibit 80-98% HIV-1 replication in vitro. Elevated plasma levels of both NKEF-A and NKEF-B proteins were detected in 23% of HIV-infected non-treated individuals but not in persons treated with highly active antiviral therapy or uninfected persons. These results indicate that the peroxiredoxin family members NKEF-A and NKEF-B are up-regulated in activated CD8(+) T-cells in HIV infection, and suggest that these antioxidant proteins contribute to the antiviral activity of CD8(+) T-cells.

Anti-human immunodeficiency virus noncytolytic CD8+ T-cell response: a review.

The CD8+ T-cell immune response for human immunodeficiency virus (HIV) is divided into a cytolytic and noncytolytic mechanism. The mechanism of cell-mediated cytotoxic immunity for the partial control of human immunodeficiency virus type 1 (HIV-1) replication in infected individuals is well-characterized, and the direct killing of virus-infected cells by antigen-specific cytotoxic T-lymphocytes (CTL) is widely correlated with disease outcome. However, the mechanism of the noncytolytic component is not well understood. In part, this is because the main inhibitory factor or factors called CD8+ T-cell antiviral factor (CAF), have not yet been purified. In addition, results between the investigators are difficult to compare because of technical differences between laboratories, including the use of different in vitro cell expansion and stimulation methods for the CD8+ T cells, the necessity of sequential biochemical purification steps with restricted amounts of material, the complex analysis and interpretation of gene expression arrays, the use of different HIV strains, and the use of different short- or long-term inhibition assays using primary or immortalized target cells. Nevertheless, the diminishing efficacy of highly active antiretroviral therapy (HAART) because of the development of resistant HIV and the persistence of latent HIV provides a strong rationale for an immune therapy approach using antiviral factor(s) of the CD8+ T-cell noncytolytic immune response.

Purification of a modified form of bovine antithrombin III as an HIV-1 CD8+ T-cell antiviral factor.

CD8(+) T-cells secrete soluble factor(s) capable of inhibiting both R5- and X4-tropic strains of human immunodeficiency virus type 1 (HIV-1). CCR5 chemokine ligands, released from activated CD8(+) T-cells, contribute to the antiviral activity of these cells. These CC-chemokines, however, do not account for all CD8(+) T-cell antiviral factor(s) (CAF) released from these cells, particularly because the elusive CAF can inhibit the replication of X4 HIV-1 strains that use CXCR4 and not CCR5 as a coreceptor. Here we demonstrate that activated CD8(+) T-cells of HIV-1-seropositive individuals modify serum bovine antithrombin III into an HIV-1 inhibitory factor capable of suppressing the replication of X4 HIV-1. These data indicate that antithrombin III may play a role in the progression of HIV-1 disease.