Concept: Western Red Colobus
Background Data from studies in nonhuman primates suggest that the triple monoclonal antibody cocktail ZMapp is a promising immune-based treatment for Ebola virus disease (EVD). Methods Beginning in March 2015, we conducted a randomized, controlled trial of ZMapp plus the current standard of care as compared with the current standard of care alone in patients with EVD that was diagnosed in West Africa by polymerase-chain-reaction (PCR) assay. Eligible patients of any age were randomly assigned in a 1:1 ratio to receive either the current standard of care or the current standard of care plus three intravenous infusions of ZMapp (50 mg per kilogram of body weight, administered every third day). Patients were stratified according to baseline PCR cycle-threshold value for the virus (≤22 vs. >22) and country of enrollment. Oral favipiravir was part of the current standard of care in Guinea. The primary end point was mortality at 28 days. Results A total of 72 patients were enrolled at sites in Liberia, Sierra Leone, Guinea, and the United States. Of the 71 patients who could be evaluated, 21 died, representing an overall case fatality rate of 30%. Death occurred in 13 of 35 patients (37%) who received the current standard of care alone and in 8 of 36 patients (22%) who received the current standard of care plus ZMapp. The observed posterior probability that ZMapp plus the current standard of care was superior to the current standard of care alone was 91.2%, falling short of the prespecified threshold of 97.5%. Frequentist analyses yielded similar results (absolute difference in mortality with ZMapp, -15 percentage points; 95% confidence interval, -36 to 7). Baseline viral load was strongly predictive of both mortality and duration of hospitalization in all age groups. Conclusions In this randomized, controlled trial of a putative therapeutic agent for EVD, although the estimated effect of ZMapp appeared to be beneficial, the result did not meet the prespecified statistical threshold for efficacy. (Funded by the National Institute of Allergy and Infectious Diseases and others; PREVAIL II ClinicalTrials.gov number, NCT02363322 .).
Background The unprecedented 2014 epidemic of Ebola virus disease (EVD) has prompted an international response to accelerate the availability of a preventive vaccine. A replication-defective recombinant chimpanzee adenovirus type 3-vectored ebolavirus vaccine (cAd3-EBO), encoding the glycoprotein from Zaire and Sudan species that offers protection in the nonhuman primate model, was rapidly advanced into phase 1 clinical evaluation. Methods We conducted a phase 1, dose-escalation, open-label trial of cAd3-EBO. Twenty healthy adults, in sequentially enrolled groups of 10 each, received vaccination intramuscularly in doses of 2×10(10) particle units or 2×10(11) particle units. Primary and secondary end points related to safety and immunogenicity were assessed throughout the first 4 weeks after vaccination. Results In this small study, no safety concerns were identified; however, transient fever developed within 1 day after vaccination in two participants who had received the 2×10(11) particle-unit dose. Glycoprotein-specific antibodies were induced in all 20 participants; the titers were of greater magnitude in the group that received the 2×10(11) particle-unit dose than in the group that received the 2×10(10) particle-unit dose (geometric mean titer against the Zaire antigen, 2037 vs. 331; P=0.001). Glycoprotein-specific T-cell responses were more frequent among those who received the 2x10(11) particle-unit dose than among those who received the 2×10(10) particle-unit dose, with a CD4 response in 10 of 10 participants versus 3 of 10 participants (P=0.004) and a CD8 response in 7 of 10 participants versus 2 of 10 participants (P=0.07). Conclusions Reactogenicity and immune responses to cAd3-EBO vaccine were dose-dependent. At the 2×10(11) particle-unit dose, glycoprotein Zaire-specific antibody responses were in the range reported to be associated with vaccine-induced protective immunity in challenge studies involving nonhuman primates. Clinical trials assessing cAd3-EBO are ongoing. (Funded by the Intramural Research Program of the National Institutes of Health; VRC 207 ClinicalTrials.gov number, NCT02231866 .).
Herein we describe production of purified equine IgG obtained from horses immunized with plasmid DNA followed by boosting with Kunjin replicon virus-like particles both encoding a modified Ebola glycoprotein. Administration of the equine IgG over 5 days to cynomolgus macaques infected 24 hours previously with a lethal dose of Ebola virus suppressed viral loads by more than 5 logs and protected animals from mortality. Animals generated their own Ebola glycoprotein-specific IgG responses 9-15 days after infection, with circulating virus undetectable by day 15-17. Such equine IgG may find utility as a post-exposure prophylactic for Ebola infection and provides a low cost, scalable alternative to monoclonal antibodies, with extensive human safety data and WHO-standardized international manufacturing capability available in both high and low income countries.
The magnitude of the 2013-2016 Ebola virus disease (EVD) epidemic enabled an unprecedented number of viral mutations to occur over successive human-to-human transmission events, increasing the probability that adaptation to the human host occurred during the outbreak. We investigated one nonsynonymous mutation, Ebola virus (EBOV) glycoprotein (GP) mutant A82V, for its effect on viral infectivity. This mutation, located at the NPC1-binding site on EBOV GP, occurred early in the 2013-2016 outbreak and rose to high frequency. We found that GP-A82V had heightened ability to infect primate cells, including human dendritic cells. The increased infectivity was restricted to cells that have primate-specific NPC1 sequences at the EBOV interface, suggesting that this mutation was indeed an adaptation to the human host. GP-A82V was associated with increased mortality, consistent with the hypothesis that the heightened intrinsic infectivity of GP-A82V contributed to disease severity during the EVD epidemic.
MB-003, a plant-derived monoclonal antibody cocktail used effectively in treatment of Ebola virus infection in non-human primates, was unable to protect two of six animals when initiated 1 or 2 days post-infection. We characterized a mechanism of viral escape in one of the animals, after observation of two clusters of genomic mutations that resulted in five nonsynonymous mutations in the monoclonal antibody target sites. These mutations were linked to a reduction in antibody binding and later confirmed to be present in a viral isolate that was not neutralized in vitro. Retrospective evaluation of a second independent study allowed the identification of a similar case. Four SNPs in previously identified positions were found in this second fatality, suggesting that genetic drift could be a potential cause for treatment failure. These findings highlight the importance selecting different target domains for each component of the cocktail to minimize the potential for viral escape.
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).
To date there are no approved antiviral drugs for the treatment of Ebola virus disease (EVD). While a number of candidate drugs have shown limited efficacy in vitro and/or in non-human primate studies, differences in experimental methodologies make it difficult to compare their therapeutic effectiveness. Using an in vitro model of Ebola Zaire replication with transcription-competent virus like particles (trVLPs), requiring only level 2 biosafety containment, we compared the activities of the type I interferons (IFNs) IFN-α and IFN-ß, a panel of viral polymerase inhibitors (lamivudine (3TC), zidovudine (AZT) tenofovir (TFV), favipiravir (FPV), the active metabolite of brincidofovir, cidofovir (CDF)), and the estrogen receptor modulator, toremifene (TOR), in inhibiting viral replication in dose-response and time course studies. We also tested 28 two- and 56 three-drug combinations against Ebola replication. IFN-α and IFN-ß inhibited viral replication 24 hours post-infection (IC50 0.038μM and 0.016μM, respectively). 3TC, AZT and TFV inhibited Ebola replication when used alone (50-62%) or in combination (87%). They exhibited lower IC50 (0.98-6.2μM) compared with FPV (36.8μM), when administered 24 hours post-infection. Unexpectedly, CDF had a narrow therapeutic window (6.25-25μM). When dosed >50μM, CDF treatment enhanced viral infection. IFN-ß exhibited strong synergy with 3TC (97.3% inhibition) or in triple combination with 3TC and AZT (95.8% inhibition). This study demonstrates that IFNs and viral polymerase inhibitors may have utility in EVD. We identified several 2 and 3 drug combinations with strong anti-Ebola activity, confirmed in studies using fully infectious ZEBOV, providing a rationale for testing combination therapies in animal models of lethal Ebola challenge. These studies open up new possibilities for novel therapeutic options, in particular combination therapies, which could prevent and treat Ebola infection and potentially reduce drug resistance.
GS-5734 is a monophosphate prodrug of an adenosine nucleoside analog that showed therapeutic efficacy in a non-human primate model of Ebola virus infection. It has been administered under compassionate use to two Ebola patients, both of whom survived, and is currently in Phase 2 clinical development for treatment of Ebola virus disease. Here we report the antiviral activities of GS-5734 and the parent nucleoside analog across multiple virus families, providing evidence to support new indications for this compound against human viruses of significant public health concern.
Ebola virus causes hemorrhagic fever with a high mortality rate and for which there is no approved therapy. Two human monoclonal antibodies, mAb100 and mAb114, in combination, protect nonhuman primates against all signs of Ebola virus disease, including viremia. Here, we demonstrate that mAb100 recognizes the base of the Ebola virus glycoprotein (GP) trimer, occludes access to the cathepsin-cleavage loop, and prevents the proteolytic cleavage of GP that is required for virus entry. We show that mAb114 interacts with the glycan cap and inner chalice of GP, remains associated following proteolytic removal of the glycan cap, and inhibits binding of cleaved GP to its receptor. These results define the basis of neutralization for two protective antibodies and may facilitate development of therapies and vaccines.
Nonhuman primate (NHP) models of Ebola virus (EBOV) infection primarily use parenteral or aerosol routes of exposure. Uniform lethality can be achieved in these models at low doses of EBOV (≤100 plaque-forming units [PFU]). Here, we exposed NHPs to low doses of EBOV (Makona strain) by the oral or conjunctival routes. Surprisingly, animals exposed to 10 PFU by either route showed no signs of disease. Exposure to 100 PFU resulted in illness and/or lethal infection. These results suggest that these more natural routes require higher doses of EBOV to produce disease or that there may be differences between Makona and historical strains.