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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.

The blockade of programmed cell death-1 (PD1) and its ligand PDL1 has been proven to be a successful immunotherapy against several cancers. Similar to cancer, PD1 contributes to the establishment of several chronic infectious diseases, including malaria. While monoclonal antibodies (mAbs) targeting checkpoint receptors are revolutionary in cancer treatment, the immune-related adverse events (irAEs) may prevent their utilization in prophylactic and therapeutic treatments of infectious diseases. The irAEs are, in part, due to the prolonged half-life of mAbs resulting in prolonged activation of the immune system. As an alternative modality to mAbs, peptides represent a viable option because they possess a shorter pharmacokinetic half-life and offer more formulation and delivery options. Here, we report on a 22-amino acid immunomodulatory peptide, LD01, derived from a Bacillus bacteria. When combined prophylactically with an adenovirus-based or irradiated sporozoite-based malaria vaccine, LD01 significantly enhanced antigen-specific CD8+ T cell expansion. Therapeutically, LD01 treatment of mice infected with a lethal malaria strain resulted in survival that was associated with lower numbers of FOXP3+Tbet+CD4+ regulatory T cells. Taken together, our results demonstrate that LD01 is a potent immunomodulator that acts upon the adaptive immune system to stimulate T cell responses both prophylactically and therapeutically.

Background Maintenance of a symbiotic relationship with microbes has led to the evolution of a complex host immune recognition system. How microbes achieve homeostasis with their hosts is an unexploited source of novel chemistry and targets for cancer immunotherapy. Peptides are a proven and powerful medium through which microbes communicate amongst themselves and with the host immune system [Hilchie et al. Nat Chem Biol. 2013 and Hancock Nat Rev Microbiol 2012]. Accordingly, we have developed a platform to mine bioactive peptides from microbial databases/libraries. As proof of concept of our screening technology we targeted PD1 to (A) determine if the PD1 “rheostat” is a target of the microbial immune manipulation, and (B) characterize the activity of the peptide(s) towards developing a novel checkpoint inhibitor therapeutic modality. Methods A microbiome database was queried against previously identified PD1 binding peptides [Kotraiah V Front. Immunol. 2020] and identified peptides were computationally modeled to determine binding site. Reporter assays and ex vivo models were used to measure binding and quantify receptor antagonism. Syngeneic tumor models responsive and unresponsive to anti-PD1 monoclonal antibodies were used to test the efficacy of the peptides. Further, an infectious disease therapeutic model was used to measure and characterize the T-cell response of the peptides in vivo. Results A 22-amino acid peptide, BtLD01, from Bacillus thuringiensis was computationally shown to target a novel allosteric binding site on PD1 which is shared by the related CD28 family receptor, CTLA4. Indeed, BtLD01 antagonizes PD1 and CTLA4 simultaneously as measured in a cell-based reporter assay. Using ex vivo assays we show that BtLD reverses exhaustion-like phenotype in human PBMC recall and that they can stimulate T-Cell proliferation in the presence of PD-L1 blockade. Intriguingly, BtLD checkpoint inhibition is not dependent on PD-L1 blockade but by potentially recruiting or stabilizing the PD1:PD-L1 complex. In vivo studies with sygeneic, mouse tumor models displayed activity in both anti-PD1 mAb responsive and unresponsive tumors. Further, in a lethal malaria model of T-cell activation, we saw that BtLD01 not only protects mice and forms a memory recall response, but also suppresses Tregs. Conclusions Novel Microbially-Derived BtLD peptides target PD1/CTLA4 receptors. The unique allosteric binding site allows peptides to function non-competitively with PD-L1. This novel combined checkpoint inhibition mechanism of activating Tcells and suppressing Tregs along with the rapid and transient modulating nature of peptide may provide a safe and potent means to treat tumors refractory to anti-PD1 mAb therapy alone in clinic. Citation Format: Gabriel M. Gutierrez, Timothy W. Phares, Vinayaka Kotraiah, Peter Buontempo, James Pannucci, Llew Keltner. A dual CTLA4 and PD1 microbially derived inhibitor shows efficacy in anti-PD1 mAb unresponsive tumor models through a unique allosteric mechanism of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 292.

Hypoxia drives sickle cell disease (SCD) by inducing sickle cell haemoglobin to polymerize and deform red blood cells (RBC) into the sickle shape. A novel carboxyhaemoglobin-based oxygen carrier (PEG-COHb; PP-007) promotes unsickling in vitro by relieving RBC hypoxia. An in vivo rat model of vaso-occlusive crisis (VOC) capable of accommodating a suite of physiological and microcirculatory measurements was used to compare treatment with PEG-COHb to a non-oxygen carrying control solution (lactated ringer's [LRS]). Male Sprague-Dawley rats were anesthetized and surgically prepared to monitor microvascular interstitial oxygenation (P

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.

Background: Pain during Vaso-Occlusive Crisis (VOC) is a hallmark of sickle cell disease (SCD) as deoxygenated (sickled) red blood cells (RBC) aggregate in the microvasculature due to Hemoglobin S (HbS) polymerization. PEGylated carboxyhemoglobin bovine (PEG-COHb; SANGUINATE®) was designed as a gas transfer agent to depolymerize HbS resulting from hypoxia. Prior in vitro studies have shown PEG-COHb capability to transfer carbon monoxide (CO) and oxygen (O2) to SCD hypoxic RBCs, reversing sickling. A prospective, randomized, single-dose Phase II clinical study is in progress (NCT02411708) evaluating the safety and efficacy of PEG-COHb in the treatment of severe VOC including pain reduction in the ambulatory setting. This study is also evaluating the levels of RBC sickling following treatment and association with pain scoring. Methods: Participants were randomized to a PEG-COHb (320 mg/kg) or saline (placebo) arm, in addition to standard treatment and IV opioid per institutional practice. Pain was assessed using a 10-point pain scale at screening, pre-infusion, hourly until discharge, 24-hours, 72-hours, and Day 7 post-infusion. Blood was collected at pre-infusion, time for readiness to discharge and 72 hours post-infusion to assess RBC shape morphology changes using image-based flow cytometry. Results: Interim results showed PEG-COHb treated subjects experienced significant reduction at discharge in pain score of 4.3 (±0.3) compared to 2.4 (±0.6) reduction in the placebo arm (p Conclusions: PEG-COHb appeared to be safe, well tolerated, and associated with significantly reduced pain scores. The flow cytometry analysis of blood samples revealed PEG-COHb treatment reverted RBC to a more normal round morphology when compared to the placebo group. Individual patient data showed a trend between RBC unsickling (roundness) and pain score reductions. These results are consistent with depolymerization of HbS resulting from transfer of CO and/or O2 to sickled RBCs and O2 delivery to hypoxic tissue. This study supports advanced clinical trials of PEG-COHb as a potential therapy to reduce the pain associated with VOC. Disclosures Buontempo: Prolong Pharmaceuticals: Employment. Jubin: Prolong Pharmaceuticals: Employment. Buontempo: Prolong Pharmaceuticals: Employment. Real: Prolong Pharmaceuticals: Employment. Kazo: Prolong Pharmaceuticals: Employment. O'Brien: Prolong Pharmaceuticals: Employment. Adeel: Prolong Pharmaceuticals: Employment. Abuchowski: Prolong Pharmaceuticals: Employment, Membership on an entity's Board of Directors or advisory committees.

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.

e14085Background: Anti-Neutropenia Factor - RHO (ANF-RHO) is a novel pegylated version of native human recombinant G-CSF protein. ANF-RHO has distinct biophysical and biological properties that pro...

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.

Background: Patients receiving pegfilgrastim (Neulasta) for the treatment of neutropenia can experience bone pain following the frequent injections required to achieve effective neutrophil levels. Bone pain is thought to be caused by the rapid expansion of bone marrow hematopoietic cells and accompanying secretion of certain cytokines. ANF-Rho (ANF) is a novel modified granulocyte colony stimulating factor that was found in preclinical animal studies to be approximately 2-4 times more active than pegfilgrastim. A Phase I study was conducted to determine its safety profile and the potential to reduce the occurrence of side effects such as bone pain as well as its dose-response relationship in healthy adults. Methods: This was a first-in-human, double-blind, randomized, placebo-controlled, single ascending dose Phase 1 study in 76 healthy subjects. Subjects received a single subcutaneous (sc) dose of ANF over a range of 6 doses (5-50 ug/kg), placebo (saline) or the recommended clinical dose of pegfilgrastim (100μg/kg). The primary outcome measure was safety and tolerability. Secondary outcomes included pharmacokinetics (PK) and pharmacodynamic (PD) effect on absolute neutrophil count (ANC) and number of CD34+ progenitor cells. The Bond and Lader Visual Analogue Scale (VAS) was used to assess the severity of bone pain. Results: ANF was well to moderately-well tolerated up to a dose level of 50 µg/kg and appeared to be better tolerated than pegfilgrastim. There were no deaths or withdrawals due to adverse events (AEs) during this study. A total of 354 AEs were reported by 71 (93%) of the subjects. The majority of the AEs were of mild intensity (87%), and 13% of the AEs were of moderate intensity. The most frequently reported were musculoskeletal and connective tissue disorders. Mean bone pain scores were lower in the 5 to 30 µg/kg ANF groups compared to the pegfilgrastim group and were similar for the 50 µg/kg ANF and pegfilgrastim group. There were no clinically significant findings for ANF with respect to clinical laboratory, vital signs, ECG, Holter monitoring, physical examination, and local tolerability. One patient was positive for a low level of anti-drug antibody to ANF. The t1/2 of ANF ranged between 38.5 and 51 hours (hr) and is longer compared to pegfilgrastim (28hr). The tmax of ANF is 36 hr and is dose-independent and occurs later than for the pegfilgrastim (16 hr). ANF-Rho has a Cmax and AUC0-t and AUC0-inf values that increase with dose without a dose-linear relationship, a Cmax approximately 1.8-fold lower for the 50 µg/kg dose and a mean AUC0-t and AUC0-inf that are approximately 1.5-fold higher compared to treatment with 100μg/kg pegfilgrastim. There was a more gradual and prolonged increase of ANC and CD34+ cell by ANF as compared to pegfilgrastim. A maximum mean ANC (8.6 x109/L (5 µg/kg) and 45x109/L (50 µg/kg) was reached between Day 6 and Day 10 as compared to Day 4 for pegfilgrastim (21.5x109/L) (Fig A, B). A maximum number of CD34+ cells (10.74 (5 µg/kg) and 71.4 (50 µg/kg) cells/µL) was reached on Day 7 as compared to Day 5 for pegfilgrastim (66.98 cells/µL). The 20 µg/kg ANF dose showed a PD effect (ANC and CD34+) that was comparable to treatment with 100μg/kg of pegfilgrastim, whereas Cmax and AUC values were 4.3-fold and 1.7-fold, respectively, lower for the 20 µg/kg ANF dose compared to the pegfilgrastim. This PD effect appeared to be more sustained in the ANF-Rho dose groups compared to pegfilgrastim. Summary: In healthy volunteers, ANF was administered at a dose of 50µg/kg without significant adverse effects. ANF appeared to be better (5 to 30 µg/kg) or equally well (50 µg/kg) tolerated and had lower mean bone pain scores as compared to pegfilgrastim. ANF achieved CD34+and ANC numbers at significantly lower doses and had a significantly longer circulating half-life than pegfilgrastim. These results suggest that ANF can be provided less frequently at a lower dose and with fewer side effects. Phase 2 trials of ANF are planned to begin shortly in patients. Figure 1. Figure 1. Disclosures Parmar: Prolong Pharmaceuticals: Employment. Byczkowski:Prolong Pharmaceuticals: Employment. Jubin:Prolong Pharmaceuticals: Employment. Abuchowski:Prolong Pharmaceuticals: Employment. Buontempo:Prolong Pharmaceuticals: Employment. Gitlin:Prolong Pharmaceuticals: Employment. Boro:Prolong Pharmaceuticals: Employment. Misra:Prolong Pharmaceuticals: Employment.

Background: In Sickle Cell disease (SCD), a single amino acid substitution in the beta globin chain converts HbA to sickle genotype HbS. This genetic change promotes HbS polymerization upon deoxygenation that can promote occlusion of small blood vessels that is often associated with increased blood viscosity, and circulatory inflammation. PEGylated-carboxyhemoglobin (PEG-COHb; SANGUINATE) was designed as a novel therapeutic agent to initially release carbon monoxide (CO) and then transfer oxygen (O2) to hypoxic tissue and cells. Delivery of either CO and/or O2 to hypoxic, sickled red blood cells (RBCs) should return cells to a more normal cell morphology and help re-establish normal blood flow and rheology. PEG-COHb was shown to mediate transfer of either a CO or O2/CO mixture and restore normal morphology to hypoxic, sickled RBCs in vitro. Studies are now focused on the potential therapeutic implications of delaying or slowing sickling, which should maintain normal blood flow through hypoxic microvasculature. Unsickling is expected to be expedited by O2 transfer by PEG-COHb. To examine these potential therapeutic effects, current in vitro studies examined the effects of time and dose of PEG-COHb to not only reverse, but also prevent or delay sickling by transferring CO as well as expedite atmospheric O2 transfer to the sickled RBCs. Methods: Reversal of sickling studies were conducted by deoxygenating RBCs from healthy (control) and SCD volunteers in followed by treatment with either PEG-COHb, fully oxygenated PEG-Hb (PEG-O2Hb) or PEG-BSA for 2 hours. For prevention of sickling studies, fully oxygenated RBC suspensions were treated with increasing amounts of PEG-COHb and then subjected to hypoxia for 3 hours. Time-dose effects were quantified by area under the curve (AUC) analysis. O2 transfer studies were conducted by treating hypoxic, sickled RBCs to increasing concentrations of PEG-COHb and raising the pO2 from 3.8mm to 40mm. In all studies, the fractions of CO-Hb, O2-Hb and reduced Hb were determined by co-oximetry and sickled RBCs were quantified by imaging flow cytometry of fixed RBC specimens. Results: PEG-COHb-mediated delivery of either CO or O2 can unsickle hypoxic SCD RBCs. Controls exhibited gas exchange similar to SCD RBCs. Interestingly, sickle reversion time-course studies showed differential kinetics between the CO and O2 capacity to cause unsickling. AUC analysis at 20 minutes demonstrated that both CO and O2 reversed sickling by 41% and 42%, respectively. PEG-O2Hb was able to exert substantial unsickling by 5 minutes, where PEG-COHb showed a delayed, more pronounced effect, peaking approximately 20 to 40 minutes post-treatment. When fully oxygenated SCD RBCs were pretreated with PEG-COHb prior to oxygenation, sickling was inhibited with an IC50 of 2.5±0.6 mg per mL in deoxygenated saline (PBS). In addition, treatment concentrations below IC50 values had increased time-dose AUC values indicating that although, not completly inhibited, sickling was delayed. Oxygen transfer facilitation studies indicated that PEG-COHb increased the rate of unsickling as measured by AUC by 50% and 15% at 4 and 2 mg per mL, respectively. These levels are within the expected therapeutic dosage of SANGUINATE. Summary: RBCs from patients with SCD undergo a conformational shift upon deoxygenation resulting in HbS polymerization and morphological changes of the RBCs. The occlusive and fragile properties of sickled RBCs are responsible for the development of the numerous comorbidities associated with SCD. It is only when the fraction of oxygenated or carboxylated HbS reaches a sufficient level that reversion to normal cell morphology occurs which promotes vascular perfusion. These experiments showed a concentration and time-dependent effect of PEG-COHb ability to deliver both O2 and CO to sickled RBC. These data suggested that PEG-COHb is a promising gas transfer agent that has the potential to improve sickle cell morphology by reversing sickling; the underlying pathology of sickle cell disease co-morbidities. Disclosures Buontempo: Prolong Pharmaceuticals: Employment. Yglesias:Prolong Pharmaceuticals: Employment. Chen:Prolong Pharmaceuticals: Employment. Buontempo:Prolong Pharmaceuticals: Employment. Jubin:Prolong Pharmaceuticals: Employment. Abuchowski:Prolong Pharmaceuticals: Employment.

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: Red blood cell (RBC) sickling is the hallmark phenotype of Sickle Cell Disease (SCD) resulting from an inherited hemoglobin (HbS) point mutation genotype. Sickling is a direct consequence of HbS polymerization that occurs as RBC encounter low oxygen environments. Sickled RBCs have increased rigidity restricting their passage through the microvasculature that can lead to a vaso-occlusive crisis (VOC). VOCs are painful, debilitating and can be associated with more severe co morbidities including acute chest syndrome, leg ulcers and stroke. Treatments for VOC are limited and include IV hydration and opioid for pain control. Extensive VOC events often require hospitalization, supplemental oxygen and transfusions. Improved therapies that can be administered early during VOC that directly target the causative RBC sickling events are warranted. PEGylated carboxyhemoglobin bovine (Sanguinate™ SG) is currently in clinical development being evaluated for the ability to treat the comorbidities of SCD including VOC. SG carries oxygen (O2) and/or carbon monoxide (CO) in the mammalian vasculature for delivery to tissues to synergistically treat inflammation and vasoconstriction and other comorbidities characterized by tissue hypoxia. Early intervention of VOC with SG treatment could limit the crisis event and reduce pain severity while providing a timely crisis resolution thereby reducing the likelihood of more severe VOC clinically related complications. Methods: SG treatment effects were evaluated under controlled environmental conditions to determine its capacity for gas exchange with RBCs obtained with IRB consent from healthy and SCD volunteers. Carboxyhemoglobin and oxyhemoglobin levels were monitored to determine dose and time effects as well as the repetitive capacity of SG to facilitate gas transfer processes. RBC treated samples were also analyzed by light microscopy and image capture flow cytometry to visualize and quantify the effects of SG treatment on reversing sickled SCD RBC. A PEGylated bovine serum albumin (PBSA) product was also produced and analyzed in parallel as a control agent. Results: SG addition to normal oxygenated RBC resulted in CO and O2 exchange between RBC and SG that followed mass balance and reached equilibrium in closed system. Kinetic analysis revealed SG rapidly transferred its CO component to oxygenated RBC with concomitant O2 loading of SG. Using experimentally loaded RBC with CO and SG with O2 produced similar reciprocal gas exchange results. Additionally the primary RBC/ SG reaction products were isolated and cycled demonstrating the ability of SG to continually facilitate gas transfer through multiple exposure events. Similar studies using SG, oxygenated SG (produced by an RBC exchange reaction) or PBSA control were conducted with SCD RBC. Sickling was induced by incubation of RBC in a hypoxic chamber for 3 hours prior to SG or control treatments. After 2 hours of treatment, cells were fixed by addition of glutaraldehyde. Photomicroscopy showed a marked reduction in the sickled RBC population with both SG or oxygenated SG treatments but not with the PBSA control (Fig. 1). Results from imaging flow cytometry further supported the microscopy findings and revealed a significant quantitative reduction in the percentage of sickled RBC levels. Summary: RBC rheology and lifespan are negatively impacted by repetitive HbS polymerization/de-polymerization cycling and treatments that reverse RBC sickling during a VOC event could be expected to provide broad clinical benefits through reduction of SCD comorbidities. SG was designed to promote CO and oxygen transfer in concentration dependent manners providing physiological supplementation of O2 transport/delivery in conditions of hemolytic or ischemic anemia. These ex vivo data demonstrated for the first time that under controlled conditions, a therapeutic agent could be observed serving as an active gas transport agent providing either CO or O2 to sickled RBCs, prompting rapid unsickling. Furthermore, the use of image capture flow cytometry provided a quantitative measurement of sickled RBC fraction decrease. This mechanism may provide a useful biomarker test in future clinical studies to monitor SG treatment effects on SCD patients. Fig. 1 SG Treatment of Sickled RBC. Hypoxic SCD RBC treated with SG, oxygenated SG or PBSA for 2 hrs prior to fixation and image capture (40X mag). Fig. 1. SG Treatment of Sickled RBC. Hypoxic SCD RBC treated with SG, oxygenated SG or PBSA for 2 hrs prior to fixation and image capture (40X mag). Disclosures Jubin: Prolong Pharmaceuticals: Employment. Buontempo:Prolong Pharmaceuticals: Employment. Yglesias:Prolong Pharmaceuticals: Employment. Abuchowski:Prolong Pharmaceuticals: Employment. Chen:Prolong Pharmaceuticals: Employment. Kazo:Prolong Pharmaceuticals: Employment. Kazo:Prolong Pharmaceuticals: Employment.

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.