Sandra Diederich

Experimental Treatment with Favipiravir for Ebola Virus Disease (the JIKI Trial): A Historically Controlled, Single-Arm Proof-of-Concept Trial in Guinea

Background Ebola virus disease (EVD) is a highly lethal condition for which no specific treatment has proven efficacy. In September 2014, while the Ebola outbreak was at its peak, the World Health Organization released a short list of drugs suitable for EVD research. Favipiravir, an antiviral developed for the treatment of severe influenza, was one of these. In late 2014, the conditions for starting a randomized Ebola trial were not fulfilled for two reasons. One was the perception that, given the high number of patients presenting simultaneously and the very high mortality rate of the disease, it was ethically unacceptable to allocate patients from within the same family or village to receive or not receive an experimental drug, using a randomization process impossible to understand by very sick patients. The other was that, in the context of rumors and distrust of Ebola treatment centers, using a randomized design at the outset might lead even more patients to refuse to seek care. Therefore, we chose to conduct a multicenter non-randomized trial, in which all patients would receive favipiravir along with standardized care. The objectives of the trial were to test the feasibility and acceptability of an emergency trial in the context of a large Ebola outbreak, and to collect data on the safety and effectiveness of favipiravir in reducing mortality and viral load in patients with EVD. The trial was not aimed at directly informing future guidelines on Ebola treatment but at quickly gathering standardized preliminary data to optimize the design of future studies. Methods and Findings Inclusion criteria were positive Ebola virus reverse transcription PCR (RT-PCR) test, age ≥ 1 y, weight ≥ 10 kg, ability to take oral drugs, and informed consent. All participants received oral favipiravir (day 0: 6,000 mg; day 1 to day 9: 2,400 mg/d). Semi-quantitative Ebola virus RT-PCR (results expressed in “cycle threshold” [Ct]) and biochemistry tests were performed at day 0, day 2, day 4, end of symptoms, day 14, and day 30. Frozen samples were shipped to a reference biosafety level 4 laboratory for RNA viral load measurement using a quantitative reference technique (genome copies/milliliter). Outcomes were mortality, viral load evolution, and adverse events. The analysis was stratified by age and Ct value. A “target value” of mortality was defined a priori for each stratum, to guide the interpretation of interim and final analysis. Between 17 December 2014 and 8 April 2015, 126 patients were included, of whom 111 were analyzed (adults and adolescents, ≥13 y, n = 99; young children, ≤6 y, n = 12). Here we present the results obtained in the 99 adults and adolescents. Of these, 55 had a baseline Ct value ≥ 20 (Group A Ct ≥ 20), and 44 had a baseline Ct value < 20 (Group A Ct < 20). Ct values and RNA viral loads were well correlated, with Ct = 20 corresponding to RNA viral load = 7.7 log10 genome copies/ml. Mortality was 20% (95% CI 11.6%–32.4%) in Group A Ct ≥ 20 and 91% (95% CI 78.8%–91.1%) in Group A Ct < 20. Both mortality 95% CIs included the predefined target value (30% and 85%, respectively). Baseline serum creatinine was ≥110 μmol/l in 48% of patients in Group A Ct ≥ 20 (≥300 μmol/l in 14%) and in 90% of patients in Group A Ct < 20 (≥300 μmol/l in 44%). In Group A Ct ≥ 20, 17% of patients with baseline creatinine ≥110 μmol/l died, versus 97% in Group A Ct < 20. In patients who survived, the mean decrease in viral load was 0.33 log10 copies/ml per day of follow-up. RNA viral load values and mortality were not significantly different between adults starting favipiravir within <72 h of symptoms compared to others. Favipiravir was well tolerated. Conclusions In the context of an outbreak at its peak, with crowded care centers, randomizing patients to receive either standard care or standard care plus an experimental drug was not felt to be appropriate. We did a non-randomized trial. This trial reaches nuanced conclusions. On the one hand, we do not conclude on the efficacy of the drug, and our conclusions on tolerance, although encouraging, are not as firm as they could have been if we had used randomization. On the other hand, we learned about how to quickly set up and run an Ebola trial, in close relationship with the community and non-governmental organizations; we integrated research into care so that it improved care; and we generated knowledge on EVD that is useful to further research. Our data illustrate the frequency of renal dysfunction and the powerful prognostic value of low Ct values. They suggest that drug trials in EVD should systematically stratify analyses by baseline Ct value, as a surrogate of viral load. They also suggest that favipiravir monotherapy merits further study in patients with medium to high viremia, but not in those with very high viremia. Trial registration ClinicalTrials.gov NCT02329054

The nipah virus fusion protein is cleaved within the endosomal compartment.

Nipah virus (NiV) is a recently emerged and highly pathogenic paramyxovirus that causes a systemic infection in animals and humans and can infect a wide range of cultured cells. Interestingly, the NiV fusion (F) protein has a single arginine at the cleavage site similar to paramyxoviruses that are activated by exogenous trypsin-like enzymes only present in specific cells and tissues and therefore only cause localized infections. We show here that NiV F activation is not mediated by an exogenous serum protease but by an endogenous ubiquitous cellular protease after endocytosis of the protein. In addition to endocytosis, acidification of the endosome is a prerequisite for F cleavage. These results show that activation of the NiV F protein depends on a type of proteolytic cleavage that is clearly different from what is known for other paramyxoviral and orthomyxoviral fusion proteins. To our knowledge, this is the first example of a viral class I fusion protein whose activation depends on clathrin-mediated constitutive endocytosis.

Ubiquitous activation of the Nipah virus fusion protein does not require a basic amino acid at the cleavage site.

ABSTRACT Nipah virus (NiV), a highly pathogenic paramyxovirus, causes a systemic infection in vivo and is able to replicate in cultured cells of many species and organs. Such pantropic paramyxoviruses generally encode fusion (F) proteins with multibasic cleavage sites activated by furin or other ubiquitous intracellular host cell proteases. In contrast, NiV has an F protein with a single arginine (R109) at the cleavage site, as is the case with paramyxoviruses that are activated by trypsin-like proteases only present in specific cells or tissues and therefore only cause localized infections. Unlike these viruses, cleavage of the NiV F protein is ubiquitous and does not require the addition of exogenous proteases in cell culture. To determine the importance of the amino acid sequence at the NiV F protein cleavage site for ubiquitous activation, we generated NiV F proteins with mutations around R109. Surprisingly, neither the exchange of amino acids upstream of R109 nor replacement of the basic residue itself interfered with F cleavage. Thus, R109 is not essential for F cleavage and activation. Our data demonstrate that NiV F-protein activation depends on a novel type of proteolytic cleavage that has not yet been described for any other paramyxovirus F protein. NiV F activation is mediated by a ubiquitous protease that requires neither a monobasic nor a multibasic cleavage site and therefore differs from the furin- or trypsin-like proteases known to activate other ortho- and paramyxovirus fusion proteins.

Role of endocytosis and cathepsin-mediated activation in Nipah virus entry

Abstract The recent discovery that the Nipah virus (NiV) fusion protein (F) is activated by endosomal cathepsin L raised the question if NiV utilize pH- and protease-dependent mechanisms of entry. We show here that the NiV receptor ephrin B2, virus-like particles and infectious NiV are internalized from the cell surface. However, endocytosis, acidic pH and cathepsin-mediated cleavage are not necessary for the initiation of infection of new host cells. Our data clearly demonstrate that proteolytic activation of the NiV F protein is required before incorporation into budding virions but not after virus entry.

Endocytosis of the Nipah Virus Glycoproteins

ABSTRACT Nipah virus (NiV), a highly pathogenic member of the family Paramyxoviridae, encodes the surface glycoproteins F and G. Since internalization of the NiV envelope proteins from the cell surface might be of functional importance for viral pathogenesis either by regulating cytopathogenicity or by modulating recognition of infected cells by the immune system, we analyzed the endocytosis of the NiV F and G proteins. Interestingly, we found both glycoproteins to be internalized in infected and transfected cells. As endocytosis is normally mediated by tyrosine- or dileucine-dependent signals in the cytoplasmic tails of transmembrane proteins, all potential internalization signals in the NiV glycoproteins were mutated. Whereas the G protein appeared to be constitutively internalized with the bulk flow during membrane turnover, uptake of the F protein was found to be signal mediated. F endocytosis clearly depended on a membrane-proximal YXXΦ motif and was found to be of functional importance for the biological activity of the protein.

Activation of the Nipah Virus Fusion Protein in MDCK Cells Is Mediated by Cathepsin B within the Endosome-Recycling Compartment

ABSTRACT Proteolytic activation of the fusion protein of the highly pathogenic Nipah virus (NiV F) is a prerequisite for the production of infectious particles and for virus spread via cell-to-cell fusion. Unlike other paramyxoviral fusion proteins, functional NiV F activation requires endocytosis and pH-dependent cleavage at a monobasic cleavage site by endosomal proteases. Using prototype Vero cells, cathepsin L was previously identified to be a cleavage enzyme. Compared to Vero cells, MDCK cells showed substantially higher F cleavage rates in both NiV-infected and NiV F-transfected cells. Surprisingly, this could not be explained either by an increased F endocytosis rate or by elevated cathepsin L activities. On the contrary, MDCK cells did not display any detectable cathepsin L activity. Though we could confirm cathepsin L to be responsible for F activation in Vero cells, inhibitor studies revealed that in MDCK cells, cathepsin B was required for F-protein cleavage and productive replication of pathogenic NiV. Supporting the idea of an efficient F cleavage in early and recycling endosomes of MDCK cells, endocytosed F proteins and cathepsin B colocalized markedly with the endosomal marker proteins early endosomal antigen 1 (EEA-1), Rab4, and Rab11, while NiV F trafficking through late endosomal compartments was not needed for F activation. In summary, this study shows for the first time that endosomal cathepsin B can play a functional role in the activation of highly pathogenic NiV.

Tyrosine Residues in the Cytoplasmic Domains Affect Sorting and Fusion Activity of the Nipah Virus Glycoproteins in Polarized Epithelial Cells

ABSTRACT The highly pathogenic Nipah virus (NiV) is aerially transmitted and causes a systemic infection after entering the respiratory tract. Airway epithelia are thus important targets in primary infection. Furthermore, virus replication in the mucosal surfaces of the respiratory or urinary tract in later phases of infection is essential for virus shedding and transmission. So far, the mechanisms of NiV replication in epithelial cells are poorly elucidated. In the present study, we provide evidence that bipolar targeting of the two NiV surface glycoproteins G and F is of biological importance for fusion in polarized epithelia. We demonstrate that infection of polarized cells induces focus formation, with both glycoproteins located at lateral membranes of infected cells adjacent to uninfected cells. Supporting the idea of a direct spread of infection via lateral cell-to-cell fusion, we could identify basolateral targeting signals in the cytoplasmic domains of both NiV glycoproteins. Tyrosine 525 in the F protein is part of an endocytosis signal and is also responsible for basolateral sorting. Surprisingly, we identified a dityrosine motif at position 28/29 in the G protein, which mediates polarized targeting. A dileucine motif predicted to function as sorting signal is not involved. Mutation of the targeting signal in one of the NiV glycoproteins prevented the fusion of polarized cells, suggesting that basolateral or bipolar F and G expression facilitates the spread of NiV within epithelial cell monolayers, thereby contributing to efficient virus spread in mucosal surfaces in early and late phases of infection.

A small-molecule inhibitor of Nipah virus envelope protein-mediated membrane fusion.

Nipah virus (NiV), a highly pathogenic paramyxovirus, causes respiratory disease in pigs and severe febrile encephalitis in humans with high mortality rates. On the basis of the structural similarity of viral fusion (F) proteins within the family Paramyxoviridae, we designed and tested 18 quinolone derivatives in a NiV and measles virus (MV) envelope protein-based fusion assay beside evaluation of cytotoxicity. We found five compounds successfully inhibiting NiV envelope protein-induced cell fusion. The most active molecules (19 and 20), which also inhibit the syncytium formation induced by infectious NiV and show a low cytotoxicity in Vero cells, represent a promising lead quinolone-type compound structure. Molecular modeling indicated that compound 19 fits well into a particular protein cavity present on the NiV F protein that is important for the fusion process.

Selective receptor expression restricts Nipah virus infection of endothelial cells

Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes severe diseases in animals and humans. Endothelial cell (EC) infection is an established hallmark of NiV infection in vivo. Despite systemic virus spread via the vascular system, EC in brain and lung are preferentially infected whereas EC in other organs are less affected. As in vivo, we found differences in the infection of EC in cell culture. Only brain-derived primary or immortalized EC were found to be permissive to NiV infection. Using a replication-independent fusion assay, we could show that the lack of infection in non-brain EC was due to a lack of receptor expression. The NiV entry receptors ephrinB2 (EB2) or ephrinB3 were only expressed in brain endothelia. The finding that EB2 expression in previously non-permissive aortic EC rendered the cells permissive to infection then demonstrated that EB2 is not only necessary but also sufficient to allow the establishment of a productive NiV infection. This strongly suggests that limitations in receptor expression restrict virus entry in certain EC subsets in vivo, and are thus responsible for the differences in EC tropism observed in human and animal NiV infections.

Ephrin-B2 expression critically influences Nipah virus infection independent of its cytoplasmic tail

BackgroundCell entry and cell-to-cell spread of the highly pathogenic Nipah virus (NiV) requires binding of the NiV G protein to cellular ephrin receptors and subsequent NiV F-mediated fusion. Since expression levels of the main NiV entry receptor ephrin-B2 (EB2) are highly regulated in vivo to fulfill the physiological functions in axon guidance and angiogenesis, the goal of this study was to determine if changes in the EB2 expression influence NiV infection.ResultsSurprisingly, transfection of increasing EB2 plasmid concentrations reduced cell-to-cell fusion both in cells expressing the NiV glycoproteins and in cells infected with NiV. This effect was attributed to the downregulation of the NiV glycoproteins from the cell surface. In addition to the influence on cell-to-cell fusion, increased EB2 expression significantly reduced the total amount of NiV-infected cells, thus interfered with virus entry. To determine if the negative effect of elevated EB2 expression on virus entry is a result of an increased EB2 signaling, receptor function of a tail-truncated and therefore signaling-defective ΔcEB2 was tested. Interestingly, ΔcEB2 fully functioned as NiV entry and fusion receptor, and overexpression also interfered with virus replication.ConclusionOur findings clearly show that EB2 signaling does not account for the striking negative impact of elevated receptor expression on NiV infection, but rather that the ratio between the NiV envelope glycoproteins and surface receptors critically influence cell-to-cell fusion and virus entry.