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Sepsis-induced immunosuppression involves both innate and adaptive immunity and is associated with the increased expression of checkpoint inhibitors, such as programmed cell-death protein 1 (PD-1). The expression of PD-1 is associated with poor outcomes in septic patients, and in models of sepsis, blocking PD-1 or its ligands with antibodies increased survival and alleviated immune suppression. While inhibitory antibodies are effective, they can lead to immune-related adverse events (irAEs), in part due to continual blockade of the PD-1 pathway, resulting in hyperactivation of the immune response. Peptide-based therapeutics are an alternative drug modality that provide a rapid pharmacokinetic profile, reducing the incidence of precipitating irAEs. We recently reported that the potent, peptide-based PD-1 checkpoint antagonist, LD01, improves T-cell responses. The goal of the current study was to determine whether LD01 treatment improved survival, bacterial clearance, and host immunity in the cecal-ligation and puncture (CLP)-induced murine polymicrobial sepsis model. LD01 treatment of CLP-induced sepsis significantly enhanced survival and decreased bacterial burden. Altered survival was associated with improved macrophage phagocytic activity and T-cell production of interferon-γ. Further, myeloperoxidase levels and esterase-positive cells were significantly reduced in LD01-treated mice. Taken together, these data establish that LD01 modulates host immunity and is a viable therapeutic candidate for alleviating immunosuppression that characterizes sepsis and other infectious diseases.

Infection of certain cell types by HIV results in formation of syncytia. This process can be blocked by antibodies or compounds that prevent interaction of viral envelope protein with host cell receptors. Here the authors describe an automated imaging-based assay for inhibitors of cell-cell fusion mediated by interaction of HIV gp120 with CXCR4 coreceptor. The assay quantifies syncytia formation between U87MG astrocytoma cells constitutively expressing CD4/CXCR4 and morphologically distinct Jurkat T lymphoma cells inducibly expressing HIV env. Each cell type was differentially labeled with vital dyes. Fusion was quantified by measuring size, shape, and color of Jurkat cells and Jurkat-harboring cell syncytia. Dose-response experiments with reference inhibitors AMD 3100 and KRH-1636 yielded potencies consistent with those obtained using standard antiviral assays. This assay complements virus-based infectivity assays for identification of inhibitors of membrane fusion events triggered by interaction of HIV gp120 with host CXCR4.

SCH 43478 and analogs are a class of non-nucleoside antiviral agents that have potent and selective activity against herpes simplex virus type 2 (HSV-2). The IC50 for these compounds in plaque reduction analysis using Vero cells ranges from 0.8 to 2.0 microg/ml. All compounds have a LC50 > 100 microg/ml in cytotoxicity analysis. Mechanism of action studies suggest that these molecules have an effect on the transactivation of viral immediate early (alpha) gene expression. Time of addition studies indicate that antiviral activity of these analogs is limited to the initial 2-3 h after infection and is not due to inhibition of viral adsorption or penetration. Analysis of HSV protein expression demonstrates that SCH 49286 inhibits the accumulation of viral immediate early (alpha) gene products. SCH 43478 demonstrates statistically significant efficacy (P < 0.05) in the guinea pig genital model of HSV infection. Following subcutaneous administration in a therapeutic treatment regimen, SCH 43478 (90 mg/kg/day) is efficacious in reducing the number and severity of lesions and the neurological complications of acute HSV infection. Thus, SCH 43478 and analogs are anti-herpesvirus agents with a unique mechanism of action.

SCH 47802 and its derivatives are potent inhibitors of enteroviruses in vitro. The IC50 for SCH 47802 ranges from 0.03 to 10 micrograms/ml when tested against a spectrum of enteroviruses in plaque reduction assays. The compounds have in vitro therapeutic indices of at least 81 based on viral cytopathic effect (CPE) assays. The in vitro activity of SCH 47802 translates into in vivo activity in the murine model of poliovirus encephalitis. In an oral dosing regimen, SCH 47802 protects mice from mortality at 60 mg/kg per day. Consistent with the in vivo efficacy, pharmacokinetic analyses after oral dosing with SCH 47802 demonstrate serum levels of the compound above the in vitro IC50 for poliovirus for at least 4 h. SCH 47802 and its active analogs stabilize poliovirus to thermal inactivation indicating that the compounds bind to the virus capsid. Mechanistic studies with poliovirus indicate that SCH 47802 acts early in viral infection. This series of molecules represents potential candidates for the treatment of human enterovirus infections.

The activity of a new water-soluble molecule, SCH 38057, against picornaviruses is described. SCH 38057 inhibited plaque formation of selected entero- and rhinoviruses in a range of 10.2 to 29.1 microM (50% endpoint) and had a therapeutic index of 10 against poliovirus type 2 (polio 2) in HeLa cells. When administered orally or subcutaneously, SCH 38057 protected mice infected with either coxsackievirus B3 (CVB3) or echovirus-9 from mortality. The molecule provided a low level of protection against thermal inactivation of virus, indicating that SCH 38057 interacts with the picornavirus capsid. Binding studies with [3H]SCH 38057 revealed that the molecule binds to CVB3 and human rhinovirus 14 (HRV14) in a ratio of 29 and 19 molecules per viral particle, respectively. The affinity constant for SCH 38057 binding to CVB3 was 7.0 x 10(-4) M. When added to cultures of infected cells at 3 h after infection, SCH 38057 markedly inhibited viral RNA synthesis. This finding with lack of inhibition of attachment and loss of infectious virus after attachment were interpreted to indicate that, although SCH 38057 binds to the viral capsid, the molecule exerts its antiviral effect after the initial stage of picornavirus uncoating, i.e., after conversion of the 156S infectious viral particle to smaller subviral species.

SCH 38057 (1-[6-(2-chloro-4-methoxyphenoxy)-hexyl]imidazole hydrochloride) is a new, water-soluble antiviral compound that has inhibitory activities against a number of picornavirus infections. The structure of the human rhinovirus 14 (HRV14) complex with SCH 38057 was determined at 3.0 A resolution by single-crystal diffraction techniques using synchrotron X-radiation. SCH 38057 was found to bind at the innermost end of the hydrophobic pocket within the capsid protein VP1, a locus of binding of other antipicornaviral agents; however, the complex differs from previously reported complexes in two important aspects. It leaves a considerable volume near the entrance to the binding pocket unoccupied. In addition, the alterations in the conformation of the VP1 polypeptide are similar to, but more extensive than those observed in HRV14 complexes with other antiviral agents. Although only 9 amino acids of VP1 have close contacts with the SCH 38057 molecule (within 3.6 A), at least 36 amino acids from both VP1 and VP3 have significantly altered conformations (C alpha movement0.5 A versus native). The structures of complexes of HRV14 with SCH 38057 and WIN 51711 are compared. Aromatic ring interactions between picornavirus capsid residues and antiviral inhibitors are proposed to be among the major determinants for positioning of these compounds.

Vicriviroc (VCV) is a small-molecule CCR5 coreceptor antagonist currently in clinical trials for treatment of R5-tropic human immunodeficiency virus type 1 (HIV-1) infection. With this drug in development, identification of resistance mechanisms to VCV is needed to allow optimal outcomes in clinical practice. In this study we further characterized VCV resistance in a lab-adapted, VCV-resistant RU570 virus (RU570-VCV res ). We show that K305R, R315Q, and K319T amino acid changes in the V3 loop, along with P437S in C4, completely reproduced the resistance phenotype in a chimeric ADA envelope containing the C2-V5 region from RU570 passage control gp120. The K305R amino acid change primarily impacted the degree of resistance, whereas K319T contributed to both resistance and virus infectivity. The P437S mutation in C4 had more influence on the relative degree of virus infectivity, while the R315Q mutation contributed to the virus concentration-dependent phenotypic resistance pattern observed for RU570-VCV res . RU570-VCV res pseudovirus entry with VCV-bound CCR5 was dramatically reduced by Y10A, D11A, Y14A, and Y15A mutations in the N terminus of CCR5, whereas these mutations had less impact on entry in the absence of VCV. Notably, an additional Q315E/I317F substitution in the crown region of the V3 loop enhanced resistance to VCV, resulting in a stronger dependence on the N terminus for viral entry. By fitting the envelope mutations to a molecular model of a recently described docked N-terminal CCR5 peptide consisting of residues 2 to 15 in complex with HIV-1 gp120 CD4, potential new interactions in gp120 with the N terminus of CCR5 were uncovered. The cumulative results of this study suggest that as the RU570 VCV-resistant virus adapted to use the drug-bound receptor, it also developed an increased reliance on the N terminus of CCR5.

Several small molecule drugs that bind to the host CCR5 co-receptor and prevent viral entry have been developed for the treatment of HIV-1 infection. The innate variability found in HIV-1 envelope and the complex viral/cellular interactions during entry makes defining resistance to these inhibitors challenging. Here we found that mapping determinants in the gp160 gene from a primary isolate RU570-VCV res , selected in culture for resistance to the CCR5 entry inhibitor vicriviroc, was complicated by inactivity of the cloned envelope gene in pseudovirus assays. We therefore recombined the envelope from RU570-VCV res into a highly active and susceptible ADA gp160 backbone. The chimeric envelopes generated robust signals in the pseudovirus assay and a 200 amino acid fragment, encompassing a C2-V5 region of the RU570-VCV res envelope, was required to confer resistance in both the single-cycle assay and in replicating virus. In contrast, a chimeric envelope that contained only the V3-loop region from this resistant virus was completely susceptible suggesting that the V3-loop changes acquired are context dependent.

Most type I interferons (IFNs) are expressed by the majority of cell types in response to viral infection. In contrast, IFN-kappa has been reported to have a cellular distribution limited to keratinocytes and certain lymphoid cell populations. Recombinant expressed IFN-kappa has been shown previously to possess weak antiviral activity when directly compared with IFN-beta. In order to expand on the antiviral potential of IFN-kappa, we transiently transfected human cell lines to circumvent the need to purify recombinant proteins and to avoid the possible loss of biologic activity by the purification process. We evaluated the transcriptional signaling and antiviral activity of IFN-kappa in parallel with IFN-alpha2b with mammalian expression vectors to express each protein transiently. Both IFN-kappa and IFN-alpha2b exhibited comparable transcriptional and antiviral activities. However, in contrast to IFN-alpha2b transcriptional signaling and antiviral activity, IFN-kappa activity was not detectable in conditioned cell culture medium. Subsequent experiments revealed there was a direct relationship between IFN-kappa-expressing cells and antiviral activity. These results were confirmed in immunocytochemical studies. Furthermore, IFN-kappa exhibited cell-associated antiviral activity against a hepatitis C virus (HCV) replicon cell line. This novel IFN signaling strategy may represent an important distinct and divergent mechanism for limiting viral infections.

Research since 1983 has demonstrated that human hepatocytes can be isolated, cultured, and used for biological investigations, including studies of gene transcription and drug metabolism (1,2). In addition, the ability to cyropreserve hepatocytes has facilitated clinical research of hepatitic cell transplantation (3). We have used primary human heptocytes as host tissue for viral infection with hepatitis C. The availability of HCV-infected livers has also allowed for the culturing and analysis of HCV-positive cells. Our laboratory (4) and others (5) have confirmed the ability of these cells to display molecular markers of HCV replication. This chapter will review the basic steps of hepatocyte isolation and culturing and analysis for HCV by RT-PCR. We have also attempted to indicate alternative techniques that may be better suited to an individual investigator's needs.

Background: Polioviruses are human pathogens and the causative agents of poliomyelitis. Polioviruses are icosahedral single-standed RNA viruses, which belong to the picornavirus family, and occur as three distinct serotypes. All three serotypes of poliovirus can infect primates, but only type 2 can infect mice. The crystal structures of a type 1 and a type 3 poliovirus are already known. Structural studies of poliovirus type 2 Lansing (PV2L) were initiated to try to enhance our understanding of the differences in host range specificity, antigenicity and receptor binding among the three serotypes of poliovirus. Results: The crystal structure of the mouse neurovirulent PV2L complexed with a potent antiviral agent, SCH48973, was determined at 2.9Aresolution. Structural differences among the three poliovirus serotypes occur primarily in the loop regions of the viral coat proteins (VPs), most notably in the loops of VP1 that cluster near the fivefold axes of the capsid, where the BC loop of PV2L is disordered. Unlike other known structures of enteroviruses, the entire polypeptide chain of PV2L VP4 is visible in the electron density and RNA bases are observed stacking with conserved aromatic residues (Tyr4020 and Phe4046) of VP4. The broad-spectrum antiviral agent SCH48973 is observed binding in a pocket within the β -barrel of VP1, in approximately the same location that natural ‘pocket factors' bind to polioviruses. SCH48973 forms predominantly hydrophobic interactions with the pocket residues. Conclusions: Some of the conformational changes required for infectivity and involved in the control of capsid stability and neurovirulence in mice may occur in the vicinity of the fivefold axis of the poliovirus, where there are significant structural differences among the three poliovirus serotypes in the surface exposed loops of VP1 (BC, DE, and HI). A surface depression is located at the fivefold axis of PV2L that is not present in the other two poliovirus serotypes. The observed interaction of RNA with VP4 supports the observation that loss of VP4 ultimately leads to the loss of viral RNA. A model is proposed that suggests dual involvement of the virion fivefold and pseudo-threefold axes in receptor-mediated initiation of infection by picornaviruses.

SCH 48973 is a novel molecule with potent, selective, antienterovirus activity. In assays of the cytopathic effect against five picornaviruses, SCH 48973 had antiviral activity (50% inhibitory concentrations [IC50s]) of 0.02 to 0.11 microg/ml, with no detectable cytotoxicity at 50 microg/ml. SCH 48973 inhibited 80% of 154 recent human enterovirus isolates at an IC50 of 0.9 microg/ml. The antiviral activity of SCH 48973 is derived from its specific interaction with viral capsid, as confirmed by competition binding studies. The affinity constant (Ki) for SCH 48973 binding to poliovirus was 8.85 x 10(-8) M. In kinetic studies, a maximum of approximately 44 molecules of SCH 48973 were bound to poliovirus capsid. SCH 48973 demonstrated efficacy in a murine poliovirus model of enterovirus disease. SCH 48973 increased the survival of infected mice when it was administered orally at dosages of 3 to 20 mg/kg of body weight/day. Oral administration of SCH 48973 also reduced viral titers in the brains of infected mice. On the basis of its in vitro and in vivo profiles, SCH 48973 represents a potential candidate for therapeutic intervention against enterovirus infections.

Previous studies have demonstrated that HIV-1 develops resistance to CCR5 antagonists by gaining the ability to use drug-occupied co-receptor. However, the effects of CCR5 antagonists on the affinity of virus-co-receptor interactions have been difficult to quantify. We developed a pharmacological model for allosteric interaction at G-protein coupled receptors to analyze the effect of different CCR5 antagonists on infection by three laboratory adapted viruses with low, moderate and high susceptibility to the inhibitors. Infection data for these viruses fitted a model in which susceptibility to inhibition by CCR5 antagonists was directly related to fold reduction in virus affinity for CCR5. Dissociation constants for CCR5 antagonists calculated from the modeled data were consistent with values obtained by standard methods, suggesting that this approach can quantify pharmacologically relevant changes in co-receptor:ligand affinity in the context of infection of whole cells by authentic HIV-1 particles.