In Vitro Induction of T Helper 17 Cells by Synergistic Activation of Human Monocyte-Derived Langerhans Cell-Like Cells with Bacterial Agonists.

In the case of epidermal barrier disruption, pathogens encounter skin-resident Langerhans cells (LCs) and are recognized by pathogen recognition receptors such as Toll-like receptors (TLRs). As the majority of microorganisms exhibit more than one TLR ligand, the mechanisms of subsequent T cell differentiation are complex and far from clear. In this study, we investigated combinatory effects on Th cell polarization by bacterial cell wall compounds peptidoglycan (PGN) and lipopolysaccharide (LPS) and by bacterial nucleic acid (DNA). Expression of maturation markers CD40, CD80, HLA-DR and CCR7 and the release of IL-1ß, IL-6 and IL-23 was strongly enhanced by simultaneous exposure to PGN, LPS and DNA in LCs. As all these factors were potential Th17 driving cytokines, we investigated the potency of combinatory TLR stimuli to induce Th17 cells via LC activation. High amounts of IL-17A and IL-22, key cytokines of Th17 cells, were detected. By intracellular costaining of IL-17⁺T cells, IL-22- (Th17) and IL-22⁺ (immature Th17) cells were identified. Interestingly, one population of LPS stimulated cells skewed into IL-9⁺Th cells, and LPS synergized with PGN while inducing high IL-22. In conclusion, our data indicates that when mediated by a fine-tuned signal integration via LCs, bacterial TLR agonists synergize and induce Th17 differentiation.

Efficacy of rintatolimod in the treatment of chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME).

Chronic fatigue syndrome/ Myalgic encephalomyelitis (CFS/ME) is a poorly understood seriously debilitating disorder in which disabling fatigue is an universal symptom in combination with a variety of variable symptoms. The only drug in advanced clinical development is rintatolimod, a mismatched double stranded polymer of RNA (dsRNA). Rintatolimod is a restricted Toll-Like Receptor 3 (TLR3) agonist lacking activation of other primary cellular inducers of innate immunity (e.g.- cytosolic helicases). Rintatolimod also activates interferon induced proteins that require dsRNA for activity (e.g.- 2'-5' adenylate synthetase, protein kinase R). Rintatolimod has achieved statistically significant improvements in primary endpoints in Phase II and Phase III double-blind, randomized, placebo-controlled clinical trials with a generally well tolerated safety profile and supported by open-label trials in the United States and Europe. The chemistry, mechanism of action, clinical trial data, and current regulatory status of rintatolimod for CFS/ME including current evidence for etiology of the syndrome are reviewed.

The effect of mucoadhesive excipient on the nasal retention time of and the antibody responses induced by an intranasal influenza vaccine.

INTRODUCTION: Recently, we reported that intranasal vaccination of humans with whole inactivated influenza vaccine in the absence of mucosal adjuvant induced neutralizing antibody responses in the serum and nasal mucus. The mucoadhesive excipient carboxy-vinyl polymer (CVP) increases the viscosity and therefore mucoadhesiveness of intranasal medicaments and is an authorized excipient in Japan. In the present study, we analyzed the effect of adding CVP on intranasal whole inactivated influenza vaccine antigen dynamics and antibody responses. METHODS: Mice and nonhuman primates (NHPs) were intranasally administered the [(18)F]-radiolabeled vaccine and subjected to positron emission tomography analysis for 6h. Dendritic cells were stimulated in vitro with the vaccine mixed with or without a mucosal adjuvant (Ampligen) and/or CVP, after which the tumor necrosis factor (TNF)-α and interferon (IFN)-ß levels in the supernatants were measured. Cynomolgus monkeys were immunized intranasally with the vaccine mixed with Ampligen and/or CVP and their vaccine-specific serum IgG and IgA titers were measured on days 0 and 33. RESULTS: The vaccine was retained significantly longer in the nasal cavity of both mice and NHPs when it was delivered with CVP rather than PBS. Accumulation of the radiolabeled vaccine in the central nervous system was not detected in either model regardless of whether CVP was used. CVP only very weakly increased the TNF-α production of vaccine-stimulated dendritic cells. IFN-ß production was not observed regardless of the presence or absence of CVP. CVP increased the vaccine-specific IgA antibody responses of the intranasally vaccinated cynomolgus macaques. CONCLUSION: CVP increased intranasal retention of whole inactivated influenza vaccine, did not promote antigen redirection to the central nervous system, and improved mucosal antibody responses. The mechanism probably relates to its mucoadhesive properties rather than its ability to directly stimulate the immune system. Intranasal vaccines with CVP may be a promising candidate vaccine formulation for humans.

Aging Impairs the Ability of Conventional Dendritic Cells to Cross-Prime CD8+ T Cells upon Stimulation with a TLR7 Ligand.

The aging process is accompanied by altered immune system functioning and an increased risk of infection. Dendritic cells (DCs) are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established. Here we examined the function of conventional DCs (cDCs) in old mice after TLR7 stimulation, focusing on their ability to cross-prime CD8+ T cells. Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag) model, we found that cDCs from old mice have a poor ability to stimulate a CD8+ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation. Remarkably, CD8α+ cDCs from old mice have an impaired ability to activate naïve CD8+ T cells and, moreover, a lower capacity to mature and to process exogenous Ag. Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8+ T cells and the generation of effector cytotoxic T cells.

Intranasal seasonal influenza vaccine and a TLR-3 agonist, rintatolimod, induced cross-reactive IgA antibody formation against avian H5N1 and H7N9 influenza HA in humans.

The intranasal use of rintatolimod, a specific TLR-3 agonist, combined with trivalent seasonal influenza vaccine generated cross-protection against highly pathogenic H5N1 avian influenza in mice. The purpose of this clinical trial is to assess the safety and impact of rintatolimod on intranasal influenza vaccine in healthy adults. During Stage I of this Phase I/II clinical trial, 12 volunteers were immunized intranasally with 3 doses of FluMist seasonal influenza vaccine on Days 0, 28, and 56 followed by intranasal rintatolimod (50 µg, 200 µg, or 500 µg) 3 days later. Parotid saliva and nasal wash samples were collected at baseline and on Days 25, 53, 84, and 417. The samples were tested for IgA and IgG specific antibodies (Ab) directed against the homologous FluMist viral hemagglutinins (HAs). In addition, viral specific responses against influenza A HAs were tested for IgA Ab cross-reactivity against 3 H5 clades: HA (H5N1) A/Indonesia/5/2005, HA (H5N1) A/Hong Kong/483/97 and HA (H5N1) A/Vietnam/1194/2004, as well as, two H7 strains, HA (H7N9) A/Shanghai/2/2013 and HA (H7N3) A/chicken/Jalisco/CPA1. The combination of the intranasal FluMist along with the rintatolimod generated specific secretory IgA responses of at least 4-fold over baseline against at least one of the homologous vaccine strains included in the vaccine in 92% of the vaccinees. Additionally, this vaccination strategy induced cross-reactive secretory IgA against highly pathogenic avian influenza virus strains H5N1, H7N9, and H7N3 with pandemic potential for humans. The combination of rintatolimod and FluMist was well-tolerated.

Discordant biological and toxicological species responses to TLR3 activation.

Toll-like receptors (TLRs) are highly conserved type 1 membrane proteins that initiate a multiplicity of transient gene transcriptions, resulting in innate and adaptive immune responses. These essential immune responses are triggered by common TLR pattern recognition receptors of microbial products expressed through the cytoplasmic carboxy-terminal Toll/IL-1 domain. Toll/IL-1 adapter protein cascades are induced by an activated Toll/IL-1 to induce transient transcription responses. All TLRs, with the exception of TLR3, use an MyD88 adapter to Toll/IL-1 to initiate a proinflammatory cascade. TLR3 uses the toll receptor 3/4 induction factor adapter to initiate a different cytosolic adapter cascade with double-stranded RNA agonists. This non-MyD88 pathway induces both NF-κB and type 1 interferon responses. By using a TLR3-restricted double-stranded RNA agonist, rintatolimod, we demonstrate significant unexpected differences in toxic responses between rats and primates. The mechanism of this differential response is consistent with a relative down-regulation of the NF-κB inflammatory cytokine induction pathway in the cynomolgus monkey and humans, but not observed systemically in rat. Our findings suggest evaluation of TLR3 agonists in drug therapy.

TLR7 triggering with polyuridylic acid promotes cross-presentation in CD8α+ conventional dendritic cells by enhancing antigen preservation and MHC class I antigen permanence on the dendritic cell surface.

ssRNA can interact with dendritic cells (DCs) through binding to TLR7, inducing secretion of proinflammatory cytokines and type I IFN. Triggering TLR7 enhances cross-priming of CD8(+) T cells, which requires cross-presentation of exogenous Ag to DCs. However, how TLR triggering can affect Ag cross-presentation is still not clear. Using OVA as an Ag model, we observed that stimulation of TLR7 in DCs by polyuridylic acid (polyU), a synthetic ssRNA analog, generates a strong specific cytotoxic response in C57BL/6 mice. PolyU stimulate CD8α(+) DCs to cross-prime naive CD8(+) T cells in a type I IFN-dependent fashion. This enhanced cross-priming is accompanied by a higher density of OVA(256-264)/H-2K(b) complexes on CD8α(+) DCs treated with polyU, as well as by upregulation of costimulatory molecules and increased secretion of proinflammatory cytokines by DCs. Cross-priming of CD8(+) T cells by DCs treated with polyU requires proteasome and Ag translocation to cytosol through the Sec61 channel in DCs. The observed enhancement in OVA cross-presentation with polyU in DCs could be mediated by a limited Ag degradation in endophagosomal compartments and a higher permanence of OVA peptide/MHC class I complexes on DCs. These observations clearly reveal that key steps of Ag processing for cross-presentation can be modulated by TLR ligands, opening new avenues for understanding their mechanisms as adjuvants of the immune response.

A double-blind, placebo-controlled, randomized, clinical trial of the TLR-3 agonist rintatolimod in severe cases of chronic fatigue syndrome.

BACKGROUND: Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a severely debilitating disease of unknown pathogenesis consisting of a variety of symptoms including severe fatigue. The objective of the study was to examine the efficacy and safety of a TLR-3 agonist, rintatolimod (Poly I: C(12)U), in patients with debilitating CFS/ME. METHODS AND FINDINGS: A Phase III prospective, double-blind, randomized, placebo-controlled trial comparing twice weekly IV rintatolimod versus placebo was conducted in 234 subjects with long-standing, debilitating CFS/ME at 12 sites. The primary endpoint was the intra-patient change from baseline at Week 40 in exercise tolerance (ET). Secondary endpoints included concomitant drug usage, the Karnofsky Performance Score (KPS), Activities of Daily Living (ADL), and Vitality Score (SF 36). Subjects receiving rintatolimod for 40 weeks improved intra-patient placebo-adjusted ET 21.3% (p = 0.047) from baseline in an intention-to-treat analysis. Correction for subjects with reduced dosing compliance increased placebo-adjusted ET improvement to 28% (p = 0.022). The improvement observed represents approximately twice the minimum considered medically significant by regulatory agencies. The rintatolimod cohort vs. placebo also reduced dependence on drugs commonly used by patients in an attempt to alleviate the symptoms of CFS/ME (p = 0.048). Placebo subjects crossed-over to receive rintatolimod demonstrated an intra-patient improvement in ET performance at 24 weeks of 39% (p = 0.04). Rintatolimod at 400 mg twice weekly was generally well-tolerated. CONCLUSIONS/SIGNIFICANCE: Rintatolimod produced objective improvement in ET and a reduction in CFS/ME related concomitant medication usage as well as other secondary outcomes. TRIAL REGISTRATION: ClinicalTrials.gov NCT00215800.

Rapid, non-invasive imaging of alphaviral brain infection: reducing animal numbers and morbidity to identify efficacy of potential vaccines and antivirals.

Rapid and accurate identification of disease progression are key factors in testing novel vaccines and antivirals against encephalitic alphaviruses. Typical efficacy studies utilize a large number of animals and severe morbidity or mortality as an endpoint. New technologies provide a means to reduce and refine the animal use as proposed in Hume's 3Rs (replacement, reduction, refinement) described by Russel and Burch. In vivo imaging systems (IVIS) and bioluminescent enzyme technologies accomplish the reduction of animal requirements while shortening the experimental time and improving the accuracy in localizing active virus replication. In the case of murine models of viral encephalitis in which central nervous system (CNS) viral invasion occurs rapidly but the disease development is relatively slow, we visualized the initial brain infection and enhance the data collection process required for efficacy studies on antivirals or vaccines that are aimed at preventing brain infection. Accordingly, we infected mice through intranasal inoculation with the genetically modified pathogen, Venezuelan equine encephalitis, which expresses a luciferase gene. In this study, we were able to identify the invasion of the CNS at least 3 days before any clinical signs of disease, allowing for reduction of animal morbidity providing a humane means of disease and vaccine research while obtaining scientific data accurately and more rapidly. Based on our data from the imaging model, we confirmed the usefulness of this technology in preclinical research by demonstrating the efficacy of Ampligen, a TLR-3 agonist, in preventing CNS invasion.

Toll-like receptor 7 agonists are potent and rapid bronchodilators in guinea pigs.

BACKGROUND: Respiratory tract viral infections result in asthma exacerbations. Toll-like receptor (TLR) 7 is a receptor for viral single-stranded RNA and is expressed at high levels in the lungs. OBJECTIVE: Because TLR7 polymorphisms are associated with asthma, we examined the effects of TLR7 agonists in guinea pig airways. METHODS: We induced bronchoconstriction in guinea pigs in vivo by means of electrical stimulation of the vagus nerve or intravenous administration of acetylcholine and measured the effect of a TLR7 agonist administered intravenously. We induced contraction of airway smooth muscle in segments of isolated guinea pig tracheas in vitro and measured the effect of TLR7 agonists, antagonists, and pharmacologic inhibitors of associated signaling pathways administered directly to the bath. RESULTS: TLR7 agonists acutely inhibited bronchoconstriction in vivo and relaxed contraction of airway smooth muscle in vitro within minutes of administration. Airway relaxation induced by the TLR7 agonist R837 (imiquimod) was partially blocked with a TLR7 antagonist and was also blocked by inhibitors of large-conductance, calcium-activated potassium channels; prostaglandin synthesis; and nitric oxide generation. Another TLR7 agonist, 21-mer single-stranded phosphorothioated polyuridylic acid (PolyUs), mediated relaxation that was completely blocked by a TLR7 antagonist. CONCLUSIONS: These data demonstrate a novel protective mechanism to limit bronchoconstriction and maintain airflow during respiratory tract viral infections. The fast time frame is inconsistent with canonical TLR7 signaling. R837 mediates bronchodilation by means of TLR7-dependent and TLR7-independent mechanisms, whereas PolyUs does so through only the TLR7-dependent mechanism. TLR7-independent mechanisms involve prostaglandins and large-conductance, calcium-activated potassium channels, whereas TLR7-dependent mechanisms involve nitric oxide. TLR7 is an attractive therapeutic target for its ability to reverse bronchoconstriction within minutes.

Antitumor activity of polyuridylic acid in human soft tissue and bone sarcomas.

BACKGROUND: The immunomodulatory properties of polyuridylic acid (PolyU) make it a promising agent in cancer immunotherapy. However, there is limited information on its direct effects on tumor cells. MATERIALS AND METHODS: TLR8 mRNA and protein expression in soft tissue sarcoma (STS) and bone sarcoma (BS) cell lines were determined by PCR and flow cytometry, respectively. Apoptosis and proliferation assays were performed using annexin V staining and BrdU incorporation assays, respectively. A relative cell enumeration was evaluated with WST-1 reagent. Expression levels of apoptotic proteins were evaluated by Western blotting. RESULTS: We demonstrate that PolyU treatment resulted in a significant decrease in STS and BS cell count by inducing apoptosis and inhibition of cell proliferation. All cell lines examined expressed TLR8 and the effect of PolyU was partially mediated through TLR8. Several apoptotic proteins including caspases were activated or increased in STS cells after treatment with PolyU. Administration PolyU resulted in significant growth inhibition of STS without any observable adverse effects in mouse xenograft tumor models. CONCLUSIONS: These results elucidate the effect of PolyU in STS and BS cells and demonstrate that PolyU may be a potential therapeutic agent for STS and BS.

Hepatitis C virus single-stranded RNA induces innate immunity via Toll-like receptor 7.

BACKGROUND/AIMS: Innate immune responses to HCV infection are triggered through host recognition of pathogen-associated molecular patterns. Interferons are critical for the protection against HCV infection. However, the pathways linking virus recognition to IFN induction remain poorly understood. METHODS: Immune cells and Huh-7 cells were infected with HCV cell culture (HCVcc) or transfected with HCV-derived immunostimulatory RNA oligonucleotides (ORNs), and immune activation was assessed. RESULTS: We found that HCVcc suppressed immune responses because the HCVcc protein impaired the PBMC and pDC responses. However, HCVcc genomic RNA had an immunostimulatory effect. HCV encodes G/U-rich ssRNA TLR7 ligands that significantly activate innate immunity, and induced IFN-alpha production. Moreover, HCV-derived ORNs also activated IRF7 and NF-kappaB in Huh-7 cells. In particular, the HCV 3'-UTR strongly induced cytokine production. Different lengths of polyuridine tract in the 3'-UTR of different HCV strains induced IFN-alpha production. These data demonstrate that the HCV-specific G/U fragment is a motif sequence, and is recognized by TLR7 as a PAMP. The requirement for TLR7 to recognize HCV RNA was confirmed using specific inhibitors, RNAi and by TLR7overexpression. CONCLUSION: These results provide an insight into the development of immune adjuvant for vaccines and for the production of new antiviral drugs.

Ampligen: a potential toll-like 3 receptor adjuvant for immunotherapy of cancer.

Therapeutic vaccination against cancer-associated antigens represents an attractive option for cancer therapy in view of its potential efficacy, the possibility of long-lasting immunity against the cancer, and safety profile. Cancer patients are however usually immunosuppressed and most cancer-associated antigens are 'self-antigens', making it a significant challenge to vaccinate patients against a cancer-associated antigen. Various immunostimulant adjuvants are therefore under investigation in an effort to boost the immune system to overcome the tolerance to tumour associated self-antigens and the ambient immunosuppressory influence of cytokines and regulatory T-cells (Tregs). Many adjuvants appear to be specific ligands for toll-like receptors (TLR) which are potent activators of innate immune responses [Kwissa M, Kasturi SP, Pulendran B. Expert Rev Vaccines. The Science of Adjuvants 2007 6(October(5)):673-84] [1], activating dendritic cell (DC) maturation and inflammatory cytokine secretion, inducing an increase in the cross talk between the innate and adaptive immune systems, and thereby driving the expansion of CTLs that can destroy cancer cells. However, recently some TLR agonists such as CpG have been shown to generate parallel immunosuppressive and inflammatory responses in innate immune cells capable of induction and expansion of Tregs [Conroy H, Marshall NA, Mills KH. TLR ligand suppression or enhancement of Treg cells? A double-edged sword in immunity to tumours. Oncogene 2008 27(January 7(2)):168-80 [2]; Huang B, Zhao J, Unkeless JC, Feng ZH, Xiong H TLR signaling by tumor and immune cells: a double-edged sword. Oncogene 2008;27(2):218-24] [3]. In this context it is noteworthy that poly(I:C), a synthetic double-stranded RNA polymer and a TLR3 agonist, has been shown in mouse models to be capable of enhancing the T cell response to nonimmunogenic self-antigen linked dendritic cell based vaccine strategy, and stimulating diabetic insulitis in the presence of Tregs [Hornum L, Lundsgaard D, Markholst H. PolyI:C induction of diabetes is controlled by Iddm4 in rats with a full regulatory T cell pool. Ann N Y Acad Sci 2007;1110:65-72] [4]. Ampligen poly(I:C(12)U) is a GMP-grade synthetic analogue of poly(I:C) and therefore suitable for human use. Its effects in a preclinical setting have been recently tested to be similar to poly(I:C) [Hornum L, Lundsgaard D, Markholst H. PolyI:C induction of diabetes is controlled by Iddm4 in rats with a full regulatory T cell pool. Ann N Y Acad Sci 2007;1110:65-72]. In particular, it potently induces in vitro maturation of human monocyte derived DC with sustained bioactive IL12 production. In addition human monocyte derived DC primed with tumour lysate and matured with Ampligen are capable of generating Th1 specific anti-cancer responses in peripheral blood T-cells derived from cancer patients in the presence of ascites medium containing immunosuppressory cytokines. In this review the key findings are summarised and discussed with a view to offering a rationale for the use of Ampligen as a potentially safe adjuvant capable of overcoming the tumour-related immune tolerance mechanisms in a clinical setting.

A clinical grade poly I:C-analogue (Ampligen) promotes optimal DC maturation and Th1-type T cell responses of healthy donors and cancer patients in vitro.

Maturation of dendritic cells (DC) can be triggered in vitro by inflammatory cytokines or Toll-like receptor (TLR) ligands such as CpG or polyI:C. Corresponding, well-characterized agents which can be applied in clinical settings are sparse. We have evaluated a clinical grade, non-toxic analogue of polyI:C, poly(I:C12U) (Ampligen), as a potential adjuvant for cancer immunotherapy, for its ability to drive maturation of human myeloid DC. Our results provide evidence that poly(I:C12U) is effective in inducing optimal phenotypic (elevated levels of MHC-Class I/Class II, CD83, CCR7, CD86 and CD40 molecules) and functional maturation of human DC in vitro, capable of promoting the production of the inflammatory (Th1-type) cytokine IL-12, with significantly lower levels of IL-10 production, compared to that induced by the parent compound polyI:C. Importantly, poly(I:C12U) has a comparable effect on the maturation and function of DC derived either from healthy donors or cancer patients indicating that it is able to overcome any immune suppressive factors associated with the tumour bearing state. These characteristics make poly(I:C12U) a suitable agent for use as an adjuvant in cancer directed immunotherapeutic regimes.

Cross-protection against H5N1 influenza virus infection is afforded by intranasal inoculation with seasonal trivalent inactivated influenza vaccine.

BACKGROUND: Avian H5N1 influenza A virus is an emerging pathogen with the potential to cause substantial human morbidity and mortality. We evaluated the ability of currently licensed seasonal influenza vaccine to confer cross-protection against highly pathogenic H5N1 influenza virus in mice. METHODS: BALB/c mice were inoculated 3 times, either intranasally or subcutaneously, with the trivalent inactivated influenza vaccine licensed in Japan for the 2005-2006 season. The vaccine included A/NewCaledonia/20/99 (H1N1), A/NewYork/55/2004 (H3N2), and B/Shanghai/361/2002 viral strains and was administered together with poly(I):poly(C(12)U) (Ampligen) as an adjuvant. At 14 days after the final inoculation, the inoculated mice were challenged with either the A/HongKong/483/97, the A/Vietnam/1194/04, or the A/Indonesia/6/05 strain of H5N1 influenza virus. RESULTS: Compared with noninoculated mice, those inoculated intranasally manifested cross-reactivity of mucosal IgA and serum IgG with H5N1 virus, as well as both a reduced H5N1 virus titer in nasal-wash samples and increased survival, after challenge with H5N1 virus. Subcutaneous inoculation did not induce a cross-reactive IgA response and did not afford protection against H5N1 viral infection. CONCLUSIONS: Intranasal inoculation with annual influenza vaccine plus the Toll-like receptor-3 agonist, poly(I):poly(C(12)U), may overcome the problem of a limited supply of H5N1 virus vaccine by providing cross-protective mucosal immunity against H5N1 viruses with pandemic potential.

Mismatched double-stranded RNA: polyI:polyC12U.

Ampligen [polyI:polyC12U] is a mismatched double-stranded RNA that acts by inducing interferon production (immunomodulator) and by activating an intracellular enzyme (RNase-L) against viral RNA transcripts (antiviral). Ampligen, currently under development by Hemispherx Biopharma in the US, acts on the immunological system through T-lymphocyte stimulation and is indicated for the treatment of chronic fatigue syndrome and acquired immunodeficiency deficiency syndrome (AIDS), as part of the combined therapy. Ampligen is available for licensing worldwide. In February 2004, Fujisawa Deutschland GmbH, a subsidiary of Fujisawa Pharmaceutical Co., entered into an option agreement with Hemispherx Biopharma with the intent of becoming a distributor for Ampligen for the potential treatment of chronic fatigue syndrome in Germany, Switzerland and Austria. An option fee of 400,000 euros was paid pursuant to the terms of the option agreement and upon execution of the Distribution Agreement, Fujisawa will pay Hemispherx fees and milestone payments with a potential worth of several millions of dollars. In September 2003, Hemispherx Biopharma Inc. entered into an agreement with Guangdong Medicine Group Corporation to organise clinical trials, marketing, sales and distribution for both of its lead compounds, Ampligen and Alferon N in the People's Republic of China. The agreement stipulates that the Guangdong Medicine Group Corporation (GMC) will conduct clinical trials with Ampligen for the treatment of HIV. All costs related to the trials are to be covered by GMC. Additionally, GMC has to develop and implement marketing and promotional programmes. In May 2003, Hemispherx Biopharma and the Center for Cell and Gene Therapy entered into a research project agreement that will see Ampligen implemented in a protocol used in patients with relapsed EBV-positive Hodgkin's Lymphoma. In March 2002, Esteve and Hemispherx Biopharma entered into a collaborative agreement under which Esteve will be the sole distributor of Ampligen in Spain, Portugal and Andorra for the treatment of chronic fatigue syndrome. Under this agreement, in addition to other terms, Esteve will also collaborate in the drug product development by conducting clinical studies in Spain in patients coinfected with HIV/HCV. In July 2001 Hemispherx Biopharma announced that it had formed a strategic alliance with Empire Health Resources for clinical trials of Ampligen in the treatment of HIV and hepatitis C virus infections. Empire Health Resources, a healthcare management firm, will be responsible for accrual and retention of patients for HIV trials, and protocols for trials in patients with hepatitis C or both HIV and hepatitis C infections. Hemispherx has entered into a collaboration with RED Laboratories, and RED Laboratories NV expects that this will facilitate the continued development of Ampligen. Hemispherx has also entered into an agreement with Schering Plough to use a Schering facility as its principal manufacturing platform in the US. This agreement may be expanded to include other territories. Hemispherx and AOP Orphan Pharmaceuticals have signed a marketing agreement for Ampligen for the treatment of chronic fatigue syndrome for Austria, the Czech Republic, Poland and Hungary. In an arrangement between Hemispherx and Bioclones, Bioclones has certain marketing rights for Ampligen in the Southern Hemisphere, UK and Ireland. In the US, Ampligen has been granted orphan drug status for the treatment of AIDS, renal cell carcinoma (phase II, completed), chronic fatigue syndrome (phase III) and invasive/metastatic malignant melanoma (phase II). In August 2004, Hemispherx announced that it intends to use the proceeds from the private placement of company stock to complete the clinical work for its immunotherapeutics/ antivirals Ampligen and Oragens. Previously, Hemispherx submitted an application to the EMEA for the approval of Ampligen for the treatment of chronic fatigue syndrome; the first stage of th;) for the treatment of chronic fatigue syndrome; the first stage of the regulatory review has been cleared. In 2000, Hemispherx Europe (Hemispherx) obtained orphan drug status for Ampligen for the treatment of chronic fatigue syndrome in the EU, providing Hemispherx with 10 years of marketing exclusivity following the launch of the drug, as well as potential financial research benefits for the agent. In February 2000, Crystaal Corporation (now Biovail Pharmaceuticals Canada) acquired exclusive marketing rights to Ampligen in Canada, where it submitted an NDA for the agent for the treatment of chronic fatigue syndrome. In the meantime, Ampligen has been available since May 1996 under the Canadian Emergency Drug Release Programme for the treatment of chronic fatigue syndrome and immune dysfunction syndrome by Rivex Pharma (Helix BioPharma). Bioclones has initiated clinical studies with Ampligen for the treatment of chronic fatigue syndrome in Australia. The active substance for Ampligen is manufactured by F.H. Faulding Ltd. Clinical treatment programmes for chronic fatigue syndrome in other Pacific Rim countries are planned. Ampligen is available for severe chronic fatigue syndrome on a named patient, cost-recovery basis in South Africa. Hemispherx has developed a 'ready-to-use' liquid formulation of the drug and has begun treating patients with chronic fatigue syndrome in ongoing clinical trials. Hemispherx has also developed an oral version of the drug (Oragen), which is undergoing preclinical evaluation. In February 2001, Hemispherx Biopharma announced that it was initiating phase II/III trials of Ampligen in the treatment of late-stage, multidrug-resistant strains of HIV in the European Union. Patients treated in these studies will have exhausted all other treatment options. In July 2001, Hemispherx stated that Ampligen was being evaluated in a phase IIb trial in patients with HIV in the US. The trial, comprising two studies, REARMI and REARMII (Research/Evaluation of Ampligen for Retroviral Mutations I and II), will evaluate the ability of Ampligen to prevent the emergence of mutated, drug-resistant strains of the virus. 'Several hundred' patients currently on antiretroviral therapy and at risk of viral relapse will be enrolled at centres in Connecticut, New York, Florida and California. A second phase IIb study evaluating the effect of Ampligen on structured treatment interruptions (STI) is also underway. Final results from this study were reported in December 2002. NIH sponsored studies of potential therapies for SARS have identified Ampligen as having unusually high and consistent antiviral activity against human coronavirus, the pathogen implicated as the causative agent of the disease. Ampligen demonstrated very high potency at very low concentrations (0.4 microg/mL) and had a favourable safety profile. In October 2003, Hemispherx announced that, based on these promising new results, the company will stockpile injectible and/or oral formats of Ampligen and Alferon N. Independent researchers have demonstrated the antiviral activity of Ampligen against flaviviruses (West Nile virus, Equine Encephalitis virus, Dengue fever virus and Japanese Encephalitis virus) as well as virus classes associated with bioterrorism. In an animal study, Ampligen was shown to prevent destruction of nerve cells, reduce virus concentrations in the brain and blood stream and increase survival rates. Researchers at the Rega Institute in Belgium have published results from an animal study demonstrating that Ampligen was superior at protecting mice against coxsackie B3 virus-induced myocarditis compared with pegylated interferon. In May 2004 Hemispherx announced that it had filed an expanded US patent application covering the use of Ampligen for the potential treatment and prevention of severe acute respiratory syndrome (SARS) and dreaded emerging viruses.

Mismatched double-stranded RNA (polyI-polyC(12)U) is synergistic with multiple anti-HIV drugs and is active against drug-sensitive and drug-resistant HIV-1 in vitro.

Although highly active anti-retroviral therapy (HAART) is successful in the treatment of HIV infection, problems with toxicity, drug-resistant variants, and therapeutic failures have compromised the long-term utility of existing combination regimens. Mismatched double-stranded RNA (polyI-polyC(12)U) is an immune modulator with inherent anti-HIV activity. Cell toxicities and anti-HIV activities of fourteen anti-HIV agents were determined alone and in combination with polyI-polyC(12)U. Combination analyses for anti-HIV activity were performed at three drug ratios. Using Mixed Dose Effect analyses and the CalcuSyn for Windows software package, combination indeces were determined for all drug combinations. In general, polyI-polyC(12)U was synergistic in combination with abacavir, zidovudine, zalcitabine, didanosine, stavudine, efavirenz, indinavir, ritonavir, nelfinavir, and amprenavir. It was synergistic to antagonistic with lamivudine, delavirdine, nevirapine, and saquinavir. Thus, polyI-polyC(12)U is synergistic with most anti-HIV agents at most drug ratios and across most effective concentrations in vitro, although, certain members of each class were exceptions. PolyI-polyC(12)U alone was equally active against wild-type HIV and HIV resistant to nevirapine, protease inhibitors, or nucleoside analogue reverse transcriptase inhibitors. These results suggest that polyI-polyC(12)U should be re-evaluated as a potential adjunct therapy in patients who have failed current anti-retroviral therapeutic regimens.

Functional implications of ribosomal protein L2 in protein biosynthesis as shown by in vivo replacement studies.

The translational apparatus is a highly complex structure containing three to four RNA molecules and more than 50 different proteins. In recent years considerable evidence has accumulated to indicate that the RNA participates intensively in the catalysis of peptide-bond formation, whereas a direct involvement of the ribosomal proteins has yet to be demonstrated. Here we report the functional and structural conservation of a peptidyltransferase centre protein in all three phylogenetic domains. In vivo replacement studies show that the Escherichia coli L2 protein can be replaced by its homologous proteins from human and archaebacterial ribosomes. These hybrid ribosomes are active in protein biosynthesis, as proven by polysome analysis and poly(U)-dependent polyphenylalanine synthesis. Furthermore, we demonstrate that a specific, highly conserved, histidine residue in the C-terminal region of L2 is essential for the function of the translational apparatus. Replacement of this histidine residue in the human and archaebacterial proteins by glycine, arginine or alanine had no effect on ribosome assembly, but strongly reduced the translational activity of ribosomes containing these mutants.

SUG1, a component of the 26 S proteasome, is an ATPase stimulated by specific RNAs.

SUG1 is an integral component of the 26 S proteasome. Belonging to a novel putative ATPase family, it shares four conserved motifs characteristic of ATP-dependent DNA/RNA helicases. Recombinant rat SUG1 (rSUG1) produced in Escherichia coli was highly purified and characterized in terms of its biochemical properties. The rSUG1 exhibited a Mg2+-dependent ATPase activity. The Km for ATP and Vmax of rSUG1 were 35 microM and 7 pmol of ATP/min/microg of protein, respectively. Both ATPase activity to release [32P]monophosphate and [32P]ATP-labeling activity were coordinately affected by cold ATP severely, GTP and UTP moderately, and CTP little. Interestingly, the rSUG1 ATPase activity was stimulated by poly(U) and poly(C), but not by poly(A), poly(G), or by any forms of DNAs tested. A UV cross-linking assay also indicated poly(U)- and poly(C)-stimulated labeling of rSUG1 with [alpha-32P]ATP. Moreover, the ATPase activity was facilitated by cellular poly(A)+ RNA, but not by poly(A)- RNA. RNA transcribed in vitro from cDNA encoding a b-Zip protein could stimulate the ATPase activity. This is the first report to demonstrate a specific RNA requirement for ATPase with respect to the proteasomal ATPases. Our present work suggests that SUG1 can specifically interact with protein-coding RNA (mRNA) and play some roles in mRNA metabolism.

Polynucleotide modulation of the protease, nucleoside triphosphatase, and helicase activities of a hepatitis C virus NS3-NS4A complex isolated from transfected COS cells.

The hepatitis C virus (HCV) nonstructural 3 protein (NS3) is a 70-kDa multifunctional enzyme with three known catalytic activities segregated in two somewhat independent domains. The essential machinery of a serine protease is localized in the N-terminal one-third of the protein, and nucleoside triphosphatase (NTPase) and helicase activities reside in the remaining C-terminal region. NS4A is a 54-residue protein expressed immediately downstream of NS3 in the viral polyprotein, and a central stretch of hydrophobic residues in NS4A form an integral structural component of the NS3 serine protease domain. There is no evidence to suggest that the two domains of NS3 are separated by proteolytic processing in vivo. This may reflect economical packaging of essential viral replicative components, but it could also mean that there is functional interdependence between the two domains. In this study, a full-length NS3-NS4A complex was isolated after expression and autoprocessing in transiently transfected COS cells. The protein was used to examine the effects of polynucleotides on the NTPase, helicase, and protease activities. Unlike the previously reported behavior of a separately expressed NS3 helicase domain, the full NS3-NS4A complex demonstrated optimal NTPase activity between pH 7.5 and 8.5. All three NS3-NS4A activities were modulated by polynucleotides, with poly(U) having the most remarkable effect. These findings suggest that the domains within NS3 may influence the activity of one another and that the interplay of HCV genomic elements may regulate the enzyme activities of this complex HCV replicase component.