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Monday, March 13, 2017

THERAPEUTIC INTERVENTION OF EBOLA VIRUS INFECTION


The US government wouldn't admit the creation of the Ebola virus as a bio-warfare product but warns is a high-priority public health threat

The US government wouldn't admit the creation of the Ebola virus as a bio-warfare product but warns is a high-priority public health threat 



Ebola virus (EBOV) remains one of the most lethal transmissible infections and is responsible for high fatality rates and substantial morbidity during sporadic outbreaks. 


With increasing human incursions into endemic regions and the reported possibility of airborne transmission, EBOV is a high-priority public health threat for which no preventive or therapeutic options are currently available.


Recent studies have demonstrated that cocktails of monoclonal antibodies are effective at preventing morbidity and mortality in nonhuman primates (NHPs) when administered as a post-exposure prophylactic within 1 or 2 days of the challenge.


To test whether one of these cocktails (MB-003) demonstrates efficacy as a therapeutic (after the onset of symptoms), we challenged NHPs with EBOV and initiated treatment upon confirmation of infection according to a diagnostic protocol for U.S. Food and Drug Administration Emergency Use Authorization and observation of a documented fever. 


Of the treated animals, 43% survived the challenge, whereas both the controls and all historical controls with the same challenge stock succumbed to infection. These results represent successful therapy of EBOV infection in NHPs.


Since its discovery and initial characterization in the mid-1970's, Ebola virus (EBOV; formerly known as Zaire ebolavirus; genus: Ebolavirus, family: Filoviridae) has remained one of the most virulent and deadly pathogens known. With mortality rates approaching 90%, the virus quickly overwhelms the host, inducing a severe hemorrhagic fever and often death during sporadic outbreaks.


There are currently no licensed vaccines or therapeutics to prevent or treat infection with EBOV or any filovirus. With the increasing ease and speed of global travel and the potential for viral spread via the aerosol route, EBOV is a potential public health threat. Classification by the Centers for Disease Control as a category An agent also designates EBOV as a bio- terrorism threat, making this virus a biodefense research priority).


Research has identified phosphorodiamidate morpholino oligomers (PMOs), small interfering RNAs (siRNAs), and a vesicular stomatitis virus (VSV)–based vaccine as potential candidates for post-exposure treatment. 


These candidates have shown promising efficacy in reducing mortality when administered to nonhuman primates (NHPs)  up to 1 hour after exposure. More recently, antibodies were demonstrated to be highly effective in a post-exposure prophylaxis of NHPs against EBOV.


Passive transfer of macaque hyperimmune globulin was shown to protect rhesus macaques when dosing began 2 days after exposure. Similarly, a cocktail of three murine monoclonal antibodies (mAbs) provided 100 and 50% efficacy in cynomolgus macaques when dosing began 1 or 2 days after exposure, respectively. 


Finally, a cocktail of three mAbs with human constant regions (MB-003) manufactured in Nicotiana benthamiana provided 100 or 67% protection in the rhesus macaque model when treatment began 1 hour or 2 days after exposure, respectively, with no clinical indications of disease observed in survivors.


The development of MB-003 built upon previous work showing that mouse mAbs conferred protection up to 2 days after exposure in a murine challenge model. 


Three of these mAbs—13C6, 13F6, and 6D8—were protective individually, recognizing different non-overlapping EBOV glycoprotein epitopes, and these antibodies were subsequently deimmunized and/or chimerized with human constant regions to make them suitable for use in humans (that is, MB-003). 


When tested in mice and in initial NHP studies, we found that N. benthamiana–derived MB-003 provided superior protection compared to Chinese hamster ovary–derived MB-003. 


With efficacy in NHPs when administered 48 hours after exposure, these antibody post-exposure prophylactics may be useful for laboratory accidents where the agent and time of infection are known,  but from a public health standpoint,  treatment of the disease after the onset of symptoms and identification of the causative pathogen are priorities. 


Here, we present a concept study in the rhesus macaque model in which we observed significant protection using a therapeutic protocol where the administration of MB-003 was delayed until two “triggers,” a positive reverse transcription-polymerase chain reaction (RT-PCR) result and fever resulting from EBOV challenge, were met.


After intramuscular challenge with a target dose of 1000 plaque-forming units (PFU) (actual dose, 1067 PFU as determined by agarose plaque assay) of a well-characterized EBOV stock, animals were monitored via telemetry, and small samples of plasma were taken twice daily for RT-PCR analysis. 


Viremia was initially detected via the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) Diagnostic Services Division (DSD) EZ1 rRT-PCR pre-EUA Assay Kit and quantified using a separate RT-PCR assay. 


Fever was noted remotely in all animals at varying time points using Data Sciences International (DSI) telemetry implants and software. Upon a positive RT-PCR test and a documented fever of ≥1.5°C above baseline (established before challenge) for 1 hour (six consecutive time points), treatment of the animals with the MB-003 cocktail (50 mg/kg) was initiated. 


The limiting trigger in five of the seven animals was a fever. NHPs presented with a fever between 100 and 110 hours after exposure, and RT-PCR for detection resulted in the positive detection of virus in all NHPs by the 111-hour post-exposure sample collection time point. For all animals, the first treatment with MB-003 began by 120 hours after exposure, and the NHPs received two additional doses of MB-003 at about 170 and 250 hours after exposure.


Both the phosphate-buffered saline (PBS) control (NHP B) and the irrelevant mAb-treated control [N. benthamiana–derived human anti- HIV immunoglobulin G1 (IgG1) mAb; NHP G] succumbed to challenge on days 7 and 11, respectively. 


These animals had a time to death consistent with historical controls using the same viral stock and presented with symptoms consistent with a filoviral disease. Four of the seven NHPs treated with MB-003 did not survive the infection, succumbing to challenge between days 7 and 12 with symptoms consistent with the filoviral disease. 


In contrast, three of seven experimental animals survived challenge with varying levels of disease (Fig. 2, P = 0.029 by Mantel-Cox against historic controls challenged intramuscularly with the same stock under similar infusion regiments). 


One (NHP E) of the three survivors developed a moderate rash and moderate prostration, whereas the others (NHP C and I) presented with no apparent symptoms of the disease. All animals had positive titers via quantitative RT-PCR, although the survivors had noticeably lower levels of viremia at the time of treatment com- pared to the treated nonsurvivors. 


Virus was only detected by agarose plaque assay in the control animals; the lack of plaques in samples from nonsurvivors has been reported in previous studies in which samples from nonsurvivors has been reported in previous studies in which MB-003 was administered, suggesting not only that there was sufficient mAb present to inactivate the virus (in the presence of complement) but also that there were no escape mutants present in the serum .


Notable differences between survivors and nonsurvivors were observed in whole blood and in the chemical analysis of blood samples collected during the course of the study. 


Decreased platelet and red blood cell counts were observed in all NHPs succumbing to challenge, whereas nonsurvivors who received treatment exhibited elevated liver enzymes and substantial morbidity.


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