Ralf Geiben Lynn

Natural serine protease inhibitors (serpins) elicit sensing of a microbial cell intruder and activate an intrinsic cellular immune response in HIV and HCV infected cells. Here, we demonstrate in vitro inhibition of HSV with serpin antithrombin III (ATIII) early during infection pointing towards inhibition of an entry event. We also found reduction of mortality from 90% to 40% in an abrasion mice model demonstrating a strong reduction of infection in vivo. Our data also indicated that this… Show more

Natural serine protease inhibitors (serpins) elicit sensing of a microbial cell intruder and activate an intrinsic cellular immune response in HIV and HCV infected cells. Here, we demonstrate in vitro inhibition of HSV with serpin antithrombin III (ATIII) early during infection pointing towards inhibition of an entry event. We also found reduction of mortality from 90% to 40% in an abrasion mice model demonstrating a strong reduction of infection in vivo. Our data also indicated that this treatment might be suitable for drug-resistant viruses since high inhibition of an acyclovir-resistant HSV-1 strain was found. Thus, an ATIII tropical treatment might be used for immunocompromised patients where prolonged treatment leads to drug resistant HSV-1 strains. Understanding how ATIII regulates HSV-1 infections may reveal new avenues for therapeutic interventions. Show less

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… Show more

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. Show less

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 ex vivo in a dose-dependent manner, suggesting augmentation of… Show more

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 ex vivo 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-β 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. Show less

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 nonhuman… Show more

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 nonhuman 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 NFkB. Ultimately, understanding how serpins, such as hep-ATIII, regulate host responses during HIV infection may reveal new avenues for therapeutic intervention. Show less

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… Show more

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. Show less

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… Show more

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. Show less

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… Show more

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 TGFb. 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 TGFb 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. Show less

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… Show more

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. Show less