Post-exposure immunotherapy for two ebolaviruses and Marburg virus in nonhuman primates

The 2013–2016 Ebola virus (EBOV) disease epidemic demonstrated the grave consequences of filovirus epidemics in the absence of effective therapeutics. Besides EBOV, two additional ebolaviruses, Sudan (SUDV) and Bundibugyo (BDBV) viruses, as well as multiple variants of Marburg virus (MARV), have also caused high fatality epidemics. Current experimental EBOV monoclonal antibodies (mAbs) are ineffective against SUDV, BDBV, or MARV. Here, we report that a cocktail of two broadly neutralizing ebolavirus mAbs, FVM04 and CA45, protects nonhuman primates (NHPs) against EBOV and SUDV infection when delivered four days post infection. This cocktail when supplemented by the anti-MARV mAb MR191 exhibited 100% efficacy in MARV-infected NHPs. These findings provide a solid foundation for clinical development of broadly protective immunotherapeutics for use in future filovirus epidemics.

A full description of the statistics including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted

Data analysis
GraphPad Prism 6.0 Excel Office 365 FlowJo X was used to analyze the flow cytometry data Pristima® Suite (Version 7.3.0 Build 23) used for pathology data management SAS version 9.4 was used for the randomization For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

April 2018
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Sample size
The reported studies are pilot studies. For Ebola and Marburg a sample size of 4 treated animals (4 used for Marburg, 5 for Ebola) provides adequate (>80%) power to detect a difference in response rates assuming at least 100% response in treated groups vs. 0% response in controls (2 experimental control + historical controls) at the 95% confidence level (1-tailed Fisher exact test). Because historical data using the identical Marburg or Ebola strains are available, the number of animals in the control groups was reduced to two. The use of two animals as controls relies solely on the assumption of a uniform, stable response of control animals over time. Uniform lethality has been observed experimentally in untreated rhesus NHPs at 1000 pfu IM for MARV-Angola (n ≥ 4) and for Ebola Makona (N>10). IACUC committees generally do not allow use of more than 2 controls for NHP filovirus models. For Sudan study in Rhesus, this was the first time an immunotherapeutic was being tested in this model. As a pilot study we used n of 3 with 2 controls. However one of the controls survived, although this animal got very sick. Efficacy could be established based on lack of symptoms in two treated groups (total 6 animals). However due to partial lethality in the controls statistical significance in terms of protection could not be achieved. For all guinea pig studies with n-6 were powered to detect significant survival difference above 50% between control and treatment.

Replication
All ELISA assays were performed in intra-assay duplicates and data are presented as average. The error bars are too tight to show in the bar graphs. For Sudan and Marburg animal studies: Plaque assays are averaged from two replicates, and PCR has three replicates. For Ebola animal studies: TCID assays was performed with single point. PCR was performed in triplicate. Flow cytometry raw data are mean fluorescence intensity averaged from 10,000 cells. Biolayer interferometry data are from single measurements over a concentration range. Animal studies in guinea pigs are replicated in various designs. In general at least for one of the dose levels more than 3 experiments were performed. NHP proof of concept studies were single experiments as replication of such experiments is not ethically acceptable.
Randomization NHPs were randomized to groups stratified by sex. Randomization was performed using PROC PLAN in SAS Version 9.4 Blinding Study directors were not blinded to the group allocations, however, the technical staff performing daily animal care, data collection, and making decision about euthanasia were blinded to group allocations until each study was completed.
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