Zika plasma viral dynamics in nonhuman primates provides insights into early infection and antiviral strategies.

The recent outbreak of Zika virus (ZIKV) has been associated with fetal abnormalities and neurological complications, prompting global concern. Here we present a mathematical analysis of the within-host dynamics of plasma ZIKV burden in a nonhuman primate model, allowing for characterization of the growth and clearance of ZIKV within individual macaques. We estimate that the eclipse phase for ZIKV, the time between cell infection and viral production, is most likely short (∼4 h), the median within-host basic reproductive number R0 is 10.7, the rate of viral production is rapid (>25,000 virions d-1), and the lifetime of an infected cell while producing virus is ∼5 h. We also estimate that the minimum number of virions produced by an infected cell over its lifetime is ∼5,500. We assess the potential effect of an antiviral treatment that blocks viral replication, showing that the median time to undetectable plasma viral load (VL) can be reduced from ∼5 d to ∼3 d with a drug concentration ∼15 times the drug's EC50 when treatment is given prophylactically starting at the time of infection. In the case of favipiravir, a polymerase inhibitor with activity against ZIKV, we predict a dose of 150 mg/kg given twice a day initiated at the time of infection can reduce the peak median VL by ∼3 logs and shorten the time to undetectable median VL by ∼2 d, whereas treatment given 2 d postinfection is mostly ineffective in accelerating plasma VL loss in macaques.

Antiviral efficacy of favipiravir against Ebola virus: A translational study in cynomolgus macaques.

BACKGROUND: Despite repeated outbreaks, in particular the devastating 2014-2016 epidemic, there is no effective treatment validated for patients with Ebola virus disease (EVD). Among the drug candidates is the broad-spectrum polymerase inhibitor favipiravir, which showed a good tolerance profile in patients with EVD (JIKI trial) but did not demonstrate a strong antiviral efficacy. In order to gain new insights into the antiviral efficacy of favipiravir and improve preparedness and public health management of future outbreaks, we assess the efficacy achieved by ascending doses of favipiravir in Ebola-virus-infected nonhuman primates (NHPs). METHODS AND FINDINGS: A total of 26 animals (Macaca fascicularis) were challenged intramuscularly at day 0 with 1,000 focus-forming units of Ebola virus Gabon 2001 strain and followed for 21 days (study termination). This included 13 animals left untreated and 13 treated with doses of 100, 150, and 180 mg/kg (N = 3, 5, and 5, respectively) favipiravir administered intravenously twice a day for 14 days, starting 2 days before infection. All animals left untreated or treated with 100 mg/kg died within 10 days post-infection, while animals receiving 150 and 180 mg/kg had extended survival (P < 0.001 and 0.001, respectively, compared to untreated animals), leading to a survival rate of 40% (2/5) and 60% (3/5), respectively, at day 21. Favipiravir inhibited viral replication (molecular and infectious viral loads) in a drug-concentration-dependent manner (P values < 0.001), and genomic deep sequencing analyses showed an increase in virus mutagenesis over time. These results allowed us to identify that plasma trough favipiravir concentrations greater than 70-80 µg/ml were associated with reduced viral loads, lower virus infectivity, and extended survival. These levels are higher than those found in the JIKI trial, where patients had median trough drug concentrations equal to 46 and 26 µg/ml at day 2 and day 4 post-treatment, respectively, and suggest that the dosing regimen in the JIKI trial was suboptimal. The environment of a biosafety level 4 laboratory introduces a number of limitations, in particular the difficulty of conducting blind studies and performing detailed pharmacological assessments. Further, the extrapolation of the results to patients with EVD is limited by the fact that the model is fully lethal and that treatment initiation in patients with EVD is most often initiated several days after infection, when symptoms and high levels of viral replication are already present. CONCLUSIONS: Our results suggest that favipiravir may be an effective antiviral drug against Ebola virus that relies on RNA chain termination and possibly error catastrophe. These results, together with previous data collected on tolerance and pharmacokinetics in both NHPs and humans, support a potential role for high doses of favipiravir for future human interventions.

Favipiravir Pharmacokinetics in Nonhuman Primates and Insights for Future Efficacy Studies of Hemorrhagic Fever Viruses.

Favipiravir is an RNA polymerase inhibitor that showed strong antiviral efficacy in vitro and in small-animal models of several viruses responsible for hemorrhagic fever (HF), including Ebola virus. The aim of this work was to characterize the complex pharmacokinetics of favipiravir in nonhuman primates (NHPs) in order to guide future efficacy studies of favipiravir in large-animal models. Four different studies were conducted in 30 uninfected cynomolgus macaques of Chinese (n = 17) or Mauritian (n = 13) origin treated with intravenous favipiravir for 7 to 14 days with maintenance doses of 60 to 180 mg/kg of body weight twice a day (BID). A pharmacokinetic model was developed to predict the plasma concentrations obtained with different dosing regimens, and the model predictions were compared to the 50% effective concentration (EC50) of favipiravir against several viruses. Favipiravir pharmacokinetics were described by a model accounting for concentration-dependent aldehyde oxidase inhibition. The enzyme-dependent elimination rate increased over time and was higher in NHPs of Mauritian origin than in those of Chinese origin. Maintenance doses of 100 and 120 mg/kg BID in Chinese and Mauritian NHPs, respectively, are predicted to achieve median trough plasma free concentrations above the EC50 for Lassa and Marburg viruses until day 7. For Ebola virus, higher doses are required. After day 7, a 20% dose increase is needed to compensate for the increase in drug clearance over time. These results will help rationalize the choice of dosing regimens in future studies evaluating the antiviral effect of favipiravir in NHPs and support its development against a variety of HF viruses.

Impact of obesity on antiretroviral pharmacokinetics and immuno-virological response in HIV-infected patients: a case-control study.

Background: Obesity has high prevalence among HIV-infected patients. Increased adipose tissue mass affects the pharmacokinetics of numerous drugs, but few data are available for antiretroviral drugs. Objectives: In this study we aimed to explore the pharmacokinetics of antiretroviral drugs and the immuno-virological response in obese patients with HIV infection. Patients and methods: We examined data from 2009 to 2012 in our hospital's database for HIV-1-infected patients who received an antiretroviral drug (abacavir, emtricitabine, lamivudine, tenofovir, efavirenz, etravirine, nevirapine, atazanavir/ritonavir, darunavir/ritonavir, lopinavir/ritonavir or raltegravir). Obese patients were defined as those with BMI ≥30 kg/m 2 and normal-weight patients as those with BMI 19-25 kg/m 2 . Plasma concentrations ( C 12/24 ) were compared for each antiretroviral drug using a Mann-Whitney test. Suboptimal dosing and virological outcome were assessed by logistic regression, adjusting on covariates. Results: We enrolled 291 obese and 196 normal-weight patients. Among the 12 analysed antiretroviral drugs, tenofovir, efavirenz and lopinavir C 12 values were significantly lower in obese than normal-weight patients: 66 versus 86 ng/mL, 1498 versus 2034 ng/mL and 4595 versus 6420 ng/mL, respectively ( P < 0.001). Antiretroviral drug C 12/24 values were more frequently below efficacy thresholds for obese than for normal-weight patients after adjustment for other covariates ( P < 0.001). Although obese patients showed a higher CD4 count than normal-weight patients (510 versus 444 cells/mm 3 , P < 0.001), the groups did not differ in virological failure rate. Conclusions: This study highlights the impact of obesity on antiretroviral drug plasma exposure, but identifies no consequence of this suboptimal exposure on the immuno-virological control in this population.

Early control of viral load by favipiravir promotes survival to Ebola virus challenge and prevents cytokine storm in non-human primates.

Ebola virus has been responsible for two major epidemics over the last several years and there has been a strong effort to find potential treatments that can improve the disease outcome. Antiviral favipiravir was thus tested on non-human primates infected with Ebola virus. Half of the treated animals survived the Ebola virus challenge, whereas the infection was fully lethal for the untreated ones. Moreover, the treated animals that did not survive died later than the controls. We evaluated the hematological, virological, biochemical, and immunological parameters of the animals and performed proteomic analysis at various timepoints of the disease. The viral load strongly correlated with dysregulation of the biological functions involved in pathogenesis, notably the inflammatory response, hemostatic functions, and response to stress. Thus, the management of viral replication in Ebola virus disease is of crucial importance in preventing the immunopathogenic disorders and septic-like shock syndrome generally observed in Ebola virus-infected patients.

Ribavirin does not potentiate favipiravir antiviral activity against Ebola virus in non-human primates.

BACKGROUND: In spite of recurrent and dramatic outbreaks, there are no therapeutics approved against Ebola virus disease. Favipiravir, a RNA polymerase inhibitor active against several RNA viruses, recently demonstrated significant but not complete protection in a non-human primate model of Ebola virus disease. In this study, we assessed the benefit of the combination of favipiravir and ribavirin, another broad spectrum antiviral agent, in the same model. METHODS: 15 female cynomolgus macaques were challenged intramuscularly with 1,000 FFU of Ebola virus Gabon 2001 strain and followed for 21 days. All animals received favipiravir 180 mg/kg twice a day (BID), either as monotherapy (n = 5) or in combination with ribavirin (n = 10). Ribavirin was given either at the dose 10 mg/kg BID (n = 5) or 5 mg/kg BID (n = 5). Favipiravir and ribavirin were initiated two and one days before viral challenge respectively and treatment were continued for 14 days. Treatment effects on viral and hematological markers were assessed using a mathematical model. Survival rate of 0% and 20% were obtained in macaques receiving favipiravir plus ribavirin 10 and 5 mg/kg BID, respectively, compared to 40% in the favipiravir monotherapy group (P = 0.061 when comparing monotherapy and bitherapy, log rank). Viral dynamic modeling analysis did not identify an association between plasma concentrations of ribavirin and viral load levels. Using a model of erythropoiesis, plasma concentrations of ribavirin were strongly associated with a hemoglobin drop (p = 0.0015). CONCLUSION: Ribavirin plus favipiravir did not extend survival rates and did not lower viral replication rate compared to favipiravir monotherapy in this animal model. Patients receiving this combination in other indications, such as Lassa fever, should be closely monitored to prevent potential toxicity associated with anemia.

Modeling Favipiravir Antiviral Efficacy Against Emerging Viruses: From Animal Studies to Clinical Trials.

In 2014, our research network was involved in the evaluation of favipiravir, an anti-influenza polymerase inhibitor, against Ebola virus. In this review, we discuss how mathematical modeling was used, first to propose a relevant dosing regimen in humans, and then to optimize its antiviral efficacy in a nonhuman primate (NHP) model. The data collected in NHPs were finally used to develop a model of Ebola pathogenesis integrating the interactions among the virus, the innate and adaptive immune response, and the action of favipiravir. We conclude the review of this work by discussing how these results are of relevance for future human studies in the context of Ebola virus, but also for other emerging viral diseases for which no therapeutics are available.

Optimal Maturation of the SIV-Specific CD8+ T Cell Response after Primary Infection Is Associated with Natural Control of SIV: ANRS SIC Study.

Highly efficient CD8+ T cells are associated with natural HIV control, but it has remained unclear how these cells are generated and maintained. We have used a macaque model of spontaneous SIVmac251 control to monitor the development of efficient CD8+ T cell responses. Our results show that SIV-specific CD8+ T cells emerge during primary infection in all animals. The ability of CD8+ T cells to suppress SIV is suboptimal in the acute phase but increases progressively in controller macaques before the establishment of sustained low-level viremia. Controller macaques develop optimal memory-like SIV-specific CD8+ T cells early after infection. In contrast, a persistently skewed differentiation phenotype characterizes memory SIV-specific CD8+ T cells in non-controller macaques. Accordingly, the phenotype of SIV-specific CD8+ T cells defined early after infection appears to favor the development of protective immunity in controllers, whereas SIV-specific CD8+ T cells in non-controllers fail to gain antiviral potency, feasibly as a consequence of early defects imprinted in the memory pool.

Ebola viral dynamics in nonhuman primates provides insights into virus immuno-pathogenesis and antiviral strategies.

Despite several clinical trials implemented, no antiviral drug could demonstrate efficacy against Ebola virus. In non-human primates, early initiation of polymerase inhibitors favipiravir and remdesivir improves survival, but whether they could be effective in patients is unknown. Here we analyze the impact of antiviral therapy by using a mathematical model that integrates virological and immunological data of 44 cynomolgus macaques, left untreated or treated with favipiravir. We estimate that favipiravir has a ~50% efficacy in blocking viral production, which results in reducing virus growth and cytokine storm while IFNα reduces cell susceptibility to infection. Simulating the effect of delayed initiations of treatment, our model predicts survival rates of 60% for favipiravir and 100% for remdesivir when treatment is initiated within 3 and 4 days post infection, respectively. These results improve the understanding of Ebola immuno-pathogenesis and can help optimize antiviral evaluation in future outbreaks.

Favipiravir pharmacokinetics in Ebola-Infected patients of the JIKI trial reveals concentrations lower than targeted.

BACKGROUND: In 2014-2015, we assessed favipiravir tolerance and efficacy in patients with Ebola virus (EBOV) disease (EVD) in Guinea (JIKI trial). Because the drug had never been used before for this indication and that high concentrations of the drugs were needed to achieve antiviral efficacy against EBOV, a pharmacokinetic model had been used to propose relevant dosing regimen. Here we report the favipiravir plasma concentrations that were achieved in participants in the JIKI trial and put them in perspective with the model-based targeted concentrations. METHODS AND FINDINGS: Pre-dose drug concentrations were collected at Day-2 and Day-4 of treatment in 66 patients of the JIKI trial and compared to those predicted by the model taking into account patient's individual characteristics. At Day-2, the observed concentrations were slightly lower than the model predictions adjusted for patient's characteristics (median value of 46.1 versus 54.3 µg/mL for observed and predicted concentrations, respectively, p = 0.012). However, the concentrations dropped at Day-4, which was not anticipated by the model (median values of 25.9 and 64.4 µg/mL for observed and predicted concentrations, respectively, p<10-6). There was no significant relationship between favipiravir concentrations and EBOV viral kinetics or mortality. CONCLUSIONS: Favipiravir plasma concentrations in the JIKI trial failed to achieve the target exposure defined before the trial. Furthermore, the drug concentration experienced an unanticipated drop between Day-2 and Day-4. The origin of this drop could be due to severe sepsis conditions and/or to intrinsic properties of favipiravir metabolism. Dose-ranging studies should be performed in healthy volunteers to assess the concentrations and the tolerance that could be achieved with high doses. TRIAL REGISTRATION: ClinicalTrials.gov NCT02329054.

Ebola Virus Infection: Review of the Pharmacokinetic and Pharmacodynamic Properties of Drugs Considered for Testing in Human Efficacy Trials.

The 2014-2015 outbreak of Ebola virus disease is the largest epidemic to date in terms of the number of cases, deaths, and affected areas. In October 2015, no antiviral agents had proven antiviral efficacy in patients. However, in September 2014, the World Health Organization inventoried and has since regularly updated a list of potential drug candidates with demonstrated antiviral efficacy in in vitro or animal models. This includes agents belonging to various therapeutic classes, namely direct antiviral agents (favipiravir and BCX4430), a combination of antibodies (ZMapp), type I interferons, RNA interference-based drugs (TKM-Ebola and AVI-7537), and anticoagulant drugs (rNAPc2). Here, we review the pharmacokinetic and pharmacodynamic information presently available for these drugs, using data obtained in healthy volunteers for pharmacokinetics and data obtained in human clinical trials or animal models for pharmacodynamics. Future studies evaluating these drugs in clinical trials are critical to confirm their efficacy in humans, propose appropriate doses, and evaluate the possibility of treatment combinations.

Ebola virus dynamics in mice treated with favipiravir.

The polymerase inhibitor favipiravir is a candidate for the treatment of Ebola virus disease. Here, we designed a mathematical model to characterize the viral dynamics in 20 mice experimentally infected with Ebola virus, which were either left untreated or treated with favipiravir at 6 or 8days post infection. This approach provided estimates of kinetic parameters of Ebola virus reproduction, such as the half-life of productively infected cells, of about 6h, and the basic reproductive number which indicates that virus produced by a single infected cell productively infects about 9 new cells. Furthermore, the model predicted that favipiravir efficiently blocks viral production, reaching an antiviral effectiveness of 95% and 99.6% at 2 and 6days after initiation of treatment, respectively. The model could be particularly helpful to guide future studies evaluating favipiravir in larger animals.

[Development of a laboratory test on dried blood spots for facilitating early diagnosis of alpha-1-antitrypsin deficiency]. / Diagnostic biologique du déficit en alpha-1-antitrypsine : développement d'un test simplifié sur goutte de sang recueillie sur papier-filtre.

Alpha- 1-antitrypsin (A1AT) deficiency is a hereditary autosomal codominant genetic disorder resulting in low circulating levels of A1AT and leading to lung and/or liver disease. It remains underdiagnosed and only 5 to 10% of PIZZ patients, the most common form of severe A1AT deficiency, would be actually identified in France. Facilitating early diagnosis of A1AT deficiency would allow a better management of this disease; therefore we have developed and standardized in three laboratories involved in this study, a diagnostic test on dried blood spots (DBS) including quantitative A1AT measurement, phenotyping by IEF electrophoresis and, if necessary, genotyping by SERPINA1 gene sequencing. We performed a quantitative assay on 90 DBS samples by immunoturbidimetric or immunonephelometric methods. We demonstrated that both methods were suitable for this type of sampling and the results obtained were highly correlated (R(2)>0.9) between the three laboratories: for a target value of 1.00 g/L, the results obtained from the three laboratories were between 1.00 and 1.02 g/L. Phenotyping and genotyping were performed under redefined operating conditions and adapted to the analysis of DBS samples. The results were comparable with those obtained for venous blood samples. Following this work, it becomes possible to provide pulmonologists with a reliable kit to perform a capillary blood sampling on filter paper which would allow a large-scale screening of A1AT deficiency in the population particularly affected by this genetic condition.