Ebola Outbreak 2014: An Overview of the Experimental Therapeutics
Shailendra K Saxena, CSIR-Centre for Cellular and Molecular Biology (CCMB)
Santosh Dahal, CSIR-Centre for Cellular and Molecular Biology (CCMB)

2014 outbreak of Ebola associated viral hemorrhagic fever has fatality rate of 55-60 per cent. The incubation period of Ebola is below 21 days. Once there is appearance of symptoms the person will be infective. Lack of specific vaccine, antiviral or drugs for treating Ebola is causing large number of deaths. Most of the recent outbreaks occurred in remote areas of West Africa. Poverty, lack of awareness, access to health centers, human habitats taking account in spreading the disease in large scale. Certain therapeutic molecules such as antibody, nucleoside analog, and siRNA have shown a promising role in treating Ebola.

Currently various countries in Africa, including Liberia, Sierra Leone, Guinea, Nigeria, are facing disaster due to Ebola virus disease (EVD) with at least 7,493 suspected cases and 3,439 deaths (including laboratory confirmed 4,108 cases and 2,078 deaths; see Figure. 1 on the next page), and more recently the cases have been reported in Senegal, the United States and Spain[1,2,3]. Ebola virus disease (EVD), Ebola hemorrhagic fever (EHF) or simply Ebola is a serious fatal illness caused by the Ebola virus. The virus causes the disease in the human and other primates. The first evidence of the Ebola was dated in 1976, in two places simultaneously, one in Nzara, Sudan, and the other in Yambuku, Democratic Republic of Congo. The latter one occurred in the village near the Ebola River, thus the name given to the virus. Since then there are the reported cases of the chronological outbreaks of the Ebola in the various African countries (CDC). Recent outbreak in 2014 is supposed to be the largest outbreak considering the highest number of cases and deaths compared to all previous outbreaks. There is no data for the natural reservoir of the virus; however it is believed to be animal-borne zoonotic infection[4].

Transmission: A Matter of Concern
The current Ebola outbreak is generally due to the human to human contact transmission of the virus. Transmission occurs via blood or bodily fluids from the infected person or by the contact with the objects contaminated by the virus, particularly needles and syringes[1,2,3]. The virus can enter into the body through nose, mouth, eyes, or open wounds, cuts and abrasions. As the contact with the fluids from the dead person can also transmit disease, there are many reported cases for the transmission to the mourners during the burial ceremony of the dead infected patient. Health care personnel may also get the disease during the treatment of the infected individual [1].

Clinical Features
The incubation period ranges between 2 to 21 days for the Ebola virus [1,2]. Human are non-infectious until they develop the clinical symptoms for the EVD [1]. The initial phase of the Ebola disease includes the flu like symptoms such as fatigue, fever, headaches, joint, muscle, and abdominal pain[5]. The initial phase is followed by the bleeding phase, which begins at 5-7 days after the first symptoms, which is characterised by internal and subcutaneous bleeding in the form of reddened eyes and hematemesis (blood vomiting), followed by heavy bleeding of the gastrointestinal tract etc. People infected with the Ebola generally shows symptoms related to the circulatory system and this includes the impair blood clotting.

Diagnosis
As there are no initial typical clinical symptoms for the EVD, thus it is difficult to distinguish the disease from the other infectious disease such as malaria, typhoid fever and meningitis. Isolating the virus (by cell culture), detecting its RNA or proteins (by PCR and ELISA), or detecting antibodies specific for viral antigens in a person’s blood are some of the methods applied to diagnose the disease. These techniques works best early and in those who have died from the disease, whereas detecting the antibodies work late in the disease and those who recovered from the disease. Sequencing method can be used for the efficient identification of the virus and differential diagnosis[6]. The EBV biomarkers such as miRNA and IgG (specific for the Ebola virus) may be efficiently used as a method for the molecular diagnosis and prognosis of the EBV infection [7,8].

Treatment and Vaccines: Challenge to Mankind
No vaccine/specific treatment for the EVD are approved by the Food and Drug Administration (FDA, USA) till now. Treatment of the Ebola is symptomatic and early supportive care with rehydration may increase the survival rate [9]. Several treatment measures such as clotting factors, blood products, inhibitors of fibrinolysis and regulators of coagulation have been tried to counteract hemorrhage. In contrast anticoagulants like heparin were administered in some patients to prevent disseminated intravascular coagulation and thrombosis[9]. However, there is no evidence that these methods are significant for the treatment. Recombinant vaccines have evoked good levels of immunity in case of knock-out mouse but also the antibody response in interferon knock-out mouse was similar to vaccinated wild-type mice [10]. A wide variety of investigational drugs (See figure 2 on the next page) are being tested for the treatment against the Ebola [11], but all of them are yet in the level of clinical trial in animals or further. A brief overview of the few experimental treatments is given below.

Zmapp: Zmapp is a therapeutic combination of monoclonal antibodies for EVD. Treatment with Zmapp includes three monoclonal antibodies, which includes ZMab (consisting of murine mAbs m1H3, m2G4 and m4G7), c2G4 and c4G7, which attack proteins on the surface of the virus. Zmapp contains the neutralising antibodies which directly and specifically reacts with the viral proteins and provide passive immunity. During the experiment on the rhesus monkey the drug efficiently protected the animal when injected with the live Ebola virus[12]. Research in the non-human Primates (NHP’s) showed that the Zmapp provides protection against EVOD even after the 5 days post infection [12]. However, it is too soon to say whether ZMapp is effective, since it is undergoing an experimental stage.

ZMapp is manufactured in the Nicotiana benthamiana (tobacco plant), due to its capability of expressing foreign (non-tobacco) proteins using indoor cultivation under tightly controlled conditions. Monoclonal antibodies (mAbs) were first developed in mice by treating them with antigens from Ebola virus, harvesting their spleens, and hybridoma cell line is created by fusion of the B-cell specific for the viral antigens and the cancer cell lines. After the best antibody was selected, the gene encoding the antibody was extracted, and tagged with the gene portions encoding human protein, the process called humanisation. After the extraction of the gene, the gene is transferred to the tobacco plant using the viral vectors. Finally the antibody is extracted from the tobacco plant. The process of the production of the antibody takes nearly a month after the extraction of the gene encoding the antibody. The resultant antibody is tested for purity and potency before being formulated into the drug.

TKM-Ebola: TKM-Ebola is another experimental drug targeted to the genetic material of the virus (RNA). It is a RNA interference drug constructed in Stable Nucleic Acid-Lipid Particles (SNALPs) and developed by Tekmira Pharmaceuticals, and works by shutting off the genes of the virus. The drug is a combination of small interfering RNAs (siRNA) which targets three of the seven proteins in Ebola virus such as Zaire Ebola membrane-associated protein (VP24), Zaire Ebola L polymerase, and Zaire Ebola polymerase complex protein (VP35). The SNALP constructs were actively taken up by the cell targets of the EBOV such as reticulo-endothelial cell population [13]. Two (66 per cent) out of three rhesus monkey which were given four doses of the pooled anti-ZEBOV siRNAs post exposure were protected from the lethal dose of the ZEBOV (Zaire Ebola virus) whereas all the macaques which were given seven doses were protected [13].

Flavipiravir: Flavipiravir is a drug used to treat influenza, but the drug also appears to be useful in treating the Ebola infected mouse. T-705 (pyrazinecarboxamide derivative) has been found to suppress the replication of Zaire EBOV in cell culture by 4 log units constituting an IC90 of 110µM [14]. Administrations of the drug after 6 days of the infection with Zaire Ebola virus (EBOV) induced rapid virus clearance, decrease in biochemical parameters of disease severity, and prevented a fatal outcome in 100 per cent of the animals [14]. Immune deficient mice were protected from the 14 days of twice daily dosing, post-infection of aerosol Ebola virus E718 infection [15].

BCX4430: BCX4430 is an adenosine nucleoside analog, broad spectrum antiviral drug manufactured by BioCryst Pharmaceuticals. The drug is a viral RNA dependent-RNA polymerase inhibitor. The drug causes premature termination of the replication process by binding with the polymerase active site and gets incorporated in the growing chain. BCX4430 can be administered by oral route, Intravenous or Intramuscular injection. BCX4430 could stop post exposure Ebola and related Marburg infections from taking hold in rodents with the intramuscular administration[16]

Brincidofovir: Brincidofovir is another oral drug, which is a nucleoside analog and a prodrug of Cidofovir, has shown broad spectrum antiviral activity in vitro against five families of the DNA virus including herpes virus family and adenovirus. Brincidofovir is formulated by the conjugation of the Cidofovir with the lipid, and is designed to liberate Cidofovir intracellularly. It has subsequently been used to treat the first patient diagnosed with Ebola in the USA, after he had recently returned from Liberia. Recently (Oct 6, 2014), FDA authorised Chimerix for emergency investigational new drug applications of Brincidofovir for the treatment of Ebola virus disease [17].

Future Perspectives: Can Humans Outrun Nature?
Since the first evidence of Ebola in 1976, various research works has been conducted to prevent and cure the threat of Ebola. But there is no any specific vaccine or the therapeutic drugs against the Ebola virus yet. The outbreak of the 2014 has proposed great risk, since it has been described as the largest outbreak till date. After more than 30 years of the first Ebola outbreak, humans are still fighting to tackle with the virus. This directs towards the need of the specific therapeutic strategies to constrain the recent Ebola outbreak and minimise the fatality. The contact tracing should be efficiently monitored to prevent the spread of the Ebola from infected person, which is the only means of preventing the spread of outbreak. Prime focus must be given on the vaccine development. Health education must be provided in the areas having the outbreak to prevent further spreading of the virus. If optimum precautions are not taken then this outbreak might result into another historic epidemic in the history of mankind. This feature can be concluded from the epidemic potential and the fatality rate of the disease. In a nutshell there is an acute necessity of the development of the rapid and reliable diagnostic tools, vaccine and therapeutic drugs specific for Ebola.

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Contact: shailen@ccmb.res.in