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.

reference: "Favipiravir is an inhibitor of viral RNA polymerase (27), we thus hypothesize that it decreases the viral load and limits the flood of PAMPs, resulting in lower inflammatory responses and reduced pathology as observed in patients that naturally present lower symptoms and recover (28)". The reference associated corresponds to McElroy, Current Opinion in Virology, 2018. Line 391.
-The control animals synthesized significantly higher levels of chemokines on day 7 than those that were treated, except for IL8. I was expecting to read some discussion about IL8, which was not strongly expressed in most of the controls. Statistical analyses were performed at day 7. Unfortunately, we had only one value at day 7 for control animals, we thus were unable to realize the statistical tests for this protein. This corresponds to the following sentence in the text : "Only three of ten animals showed high amounts of this protein, but only one sample was available on day 7 and thus included in the group comparison". Lines 203-204. We also added a discussion on the variability of cytokine expression in control animals. Lines 367-370. -I couldn't see any limitations to their study nor opportunities of future work, it is recommended to conclude the discussion with such remarks. These points have been added in the discussion. "Because of evident ethical reasons, the protocols conducted with NHPs comprise few animals and the mortality observed is to be considered with reservation because of the euthanasia of animals with acute syndrome. The results observed in studies such as the one we analyzed here is however very informative in the understanding of the parameters involved in the outcome and in the evaluation of treatment efficiency. Further studies would be needed to improve the dose regimen and the kinetic of treatment (6)".Lines 434-439 -Find another place for Fig S4,

not in the discussion
This figure that had been added in response to a reviewer's remark in the previous version has been changed to appear in the results section. Lines 237-241. -There have been multiple studies looking at EBOV-infection in NHP. I think it would be good for the authors to add some more language to put their findings into context with that already in the literature: what findings reported here are new, which agree or disagree with previous reports. Some efforts have already been made to put our study in the context as can be seen with some of the sentences in the discussion: "Previous studies showed that" lines 363-364, "We confirm that" line 368, "Similar results have been reported during severe EVD in humans" line 378, "This discrepancy between humans and NHP could be of interest to investigate" line 413, "It was thus suggested that the inhibition of HSP90 may reduce EBOV replication" lines 430-431. -In particular, please compare with the results described in the previous paper Madelain et al where some of the parameters were measured. Is this study with different samples or were they processed in a different way ? A sentence has been added in the material and methods section to clarify this point: "The data of viral load, IFNα, IL6 and TNFα presented in this article have been previously reported in Madelain et al, 2018 (6), but this study aims at studying the outcome of the disease ; we thus needed to present once more the data depending on our criteria of analysis". Lines 89-91. -It would be convenient to state the favipiravir treatment regimen the animals received to save the reader having to go back and look up these references This has been modified in the Materials and methods section. Lines 73-74 -"Cytokine expression is directly linked to viremia", please change to use "correlated". Line 229.
The modification has been made. Line 243 -"Cytokine levels are therefore dependent on viral replication", please change to "correlated. Line 241 This has been modified. Line 255 -"These results demonstrate that reduction of the viral load by favipiravir treatment resulted in decreased cytokine/chemokine release", suggest changing to "correlated with decreased cytokine/chemokine release".Line 363 This has been modified. Line 379.
-"By acting on viral replication, favipiravir decreases the viral load and limits the flood of PAMPs", suggest changing to "we hypothesize that…". Line 372 This has been modified. Line 394 -Unless I am misunderstanding what it meant here, the sentence starting "Similarly…" seems to actually be opposite of the previous sentence? Please clarify. Line 375 This sentence was actually poorly worded and was not useful in the paragraph to explain the purpose. We deleted it. Line 398. -Please change "directly results from" to "correlates with". Line380 This has been modified. Line 396. -"… this improvement", I think should be read "improving these" Line380 The sentence has been modified to be more easily understood. Lines 396-397. -"…the lack of efficacy…", change to "the incomplete efficacy". Line 388 This has been modified. Line 403 -A mAb-based treatment for EBOV has recently been approved by FDA. Please update to reflect this.
The FDA approval reached on October 2020, during the review process of the paper. We thus added this point in the discussion. Lines 431-432. -I would take issue with this statement, as the recently FDA-approved mAb therapy did indeed show clinical benefit "in the epidemic setting, when initiated after disease onset", albeit with incomplete efficacy. Please soften this statement. Line 437 We really think that the words used "to cure a HIGHLY acute infection such as EVD", line 455, and "Indeed, patients who came to seek care during the epidemics probably arrived too LATE after symptom onset" lines 440-441 do soften the statement. As described in the references cited that demonstrate this efficacy of the monoclonal antibody cocktail approved by the FDA, the mortality rate was lower in patients treated but the treatment benefited essentially to patients with moderate viral loads. The patients that were admitted for treatment with high viral load did not took so much advantage of the treatment. Lines 442-444. -I was wondering is this study a part of a clinical trials? Or is there a plan to conduct one ?
Favipiravir has been tested during EBOV epidemic and the results of this study are cited (references 1 and 2). However, this study was conducted independently from the clinical trial.