1. Etiology of Ebola and Existing Treatment
PHM Fall 2019
Instructor: Chesa Dojo Soeandy
Coordinator: Jeffrey Henderson
Etiology of Ebola and Existing Treatment
Annie Yung Ching Shi, Michelle (Eun Sun) Choi, Yuehan (Carina) Zhao, and Eulaine Ma
October 1st, 2019

2. What causes Ebola Virus Disease?
4 different viruses: Bundibugyo virus (BDBV), Sudan virus (SUDV), Tai Forest virus (TAFV) and Zaire ebola virus (EBOV)
EBOV is responsible for most outbreaks and most dangerous

3. EBOV (Zaire ebola virus)
Single stranded RNA (19000 nucleotides long)
On this RNA there are 7 genes for 7 proteins:
Nucleoprotein (NP)
Polymerase cofactor
VP35 GP
Transcription Activator (VP30)
VP24
RNA polymerase (L)

4. History of Ebola

5. History of Ebola
Initial appearance: 1976 in Sudan and Democratic Republic of Congo
outbreak in Sierra Leone, Liberia and Guinea was largest outbreak

6. Transmission From animals: Between person to person
Fruit bats are natural reservoirs of Ebola
Transmitted to humans through bodily fluids of infected animals
Between person to person
Bodily fluids or blood from people who are infected
Objects that have been contaminated (vomit, feces)
Burial ceremony of the diseased
Healthcare workers

7. Symptoms Clinical Outcome Incubation period of 2 to 21 days
Ebola hemorrhagic fever
Disseminated intravascular coagulation (DIC)
Shock
Death
Incubation period of 2 to 21 days
Initial symptoms
Fever, fatigue, muscle pains
Headaches
Sore throats
Later symptoms:
Vomiting, diarrhea
Impaired organ functions
Internal and external bleeding

8. The fatality rate for ebola virus disease is 25-90%.
A lot of unknowns: We do not know why some people survive

9. Virology Viral Cycle Entry Replication Release
Now we are gonna look at the viral cycle. So how the ebola virus enters the host cell, how it replicates within the cell, and how the virus progeny exits the host cell

10. Virology (entry of the virus)
Glycoproteins on the Ebola virus bind to receptors on human cells
C-type lectins (DC-SIGN)
Macrophages & dendritic cells
Integrin-like receptors
Binding triggers macropinocytosis
There are glycoproteins on the surface of Ebola virus which bind to receptors on human cells. One example of the receptors is C-type lectins. DC-SIGN is a C-type lectin receptor present on the surface of both macrophages and dendritic cells, which is why these two types of immune cells are usually the first cells that the virus attacks. Integrin-like receptors are another example of what the virus bind to, which are pretty prevalent receptors on immune cells as well. Binding of the virus to the cell receptor triggers macropinocytosis, so as it shows in this picture, the virus actually gets engulfed by a wave-like motion of the cell membrane.

11. Virology (inside the host cell)
Transported to endosomes & lysosomes
GP cleaved into GP2
Fusion between viral & endosomal membranes
Release of nucleocapsid
RNA polymerase uncoats the nucleocapsid
After being internalized by macropinocytosis, the virus then gets trafficked to endosomal compartments, where the glycoprotein on the viral surface is cleaved into another form called GP2, so glycoprotein2. The GP2 interacts with the cellular protein NPC1 allowing fusion between the viral and endosomal membranes. After fusion, the viral nucleocapsid in which the viral RNA is enclosed is released into the cytoplasm. After the nucleocapsid is released, the viral RNA polymerase partially uncoats it, and transcribes the genes using host cell’s machinery.

12. Virology (inside the host cell)
Here’s a flowchart of viral transcription, translation, and replication inside the host cell. You can see here is the negative strand parental Ebola RNA. When it gets transcribed, the resulting mRNA is positive. Then the mRNA is translated into structural & nonstructural proteins. Then the half of the chart is the replication process, first full-length, positive strand anti-genomes are produced from the negative parental RNA, then that gets transcribed back into negative-sense, so it has the same sense as the parental virus. And this, together with the proteins will self-assemble, and that would be the progeny virus.

13. Virology (leaving the host cell)
Progeny virions accumulate near the cell membrane
Bud off from the cell
Infect other cells & repeat the cycle
Those newly self-assembled progeny virus accumulate near the inside of the cell membrane. Then they bud off from the cell, gaining their envelopes from the cellular membrane. Here in this EM photo, you can actually see the virus budding off from the host cell. The mature progeny particles then infect other cells to repeat the cycle.

14. Ebola Virus Disease (EVD) Pathogenesis
Clinical Outcome
Immune dysregulation
Ebola Hemorrhagic Fever
Disseminated Intravascular Coagulation
Shock
Death
Infection
Now that we’ve talked about the virology of the Ebola virus, I’m going to go into how infection leads to ebola virus disease. Specifically, I’ll talk about the immune dysregulation that leads to the clinical outcomes we mentioned before.

15. EVD Pathogenesis: Immune dysregulation
Interferons (IFNs) are the first line of defense against viral infection
Inhibition of viral replication
MHC class I molecules
Proinflammatory cytokines
IFN
To begin, the immune system’s first line of defense against viral pathogens is the interferon system. In a typical scenario, a virus infected cell would produce and release IFNs that signal to other uninfected cells to enter an antiviral state. This includes inhibiting viral replication and increasing immune function through increased MHC molecules and proinflammatory cytokines.
Infected cell
Un-infected cell

16. EVD Pathogenesis: Immune dysregulation
EBOV evades the immune system through viral proteins VP24, VP30, VP35
VP35 inhibits IFN production
VP24 disrupts IFN signalling
Inhibition of viral replication
MHC class I molecules
Proinflammatory cytokines
IFN
One of the reasons that ebola infection is so hard to fight is because it evades the immune system through 3 viral proteins, VP24, 30, and 35. These proteins are expressed in Ebola-infected cells, and disrupt normal IFN function by suppressing its production and signalling, for example
Infected cell
Un-infected cell

17. In addition to the inhibition of IFN antiviral activity, the infection of innate immune cells like monocytes, macrophages and dendritic cells has a whole host of effects on the immune response (next slide).

19. Viral replication leads to necrosis of infected cells
Less immune cells to fight infection
Tissue injury and organ failure
To start, viral replication leads to necrosis of infected cells. The effect is two-fold, where less immune cells are available to fight the infection, but also the infection of other cells such as endothelial cells or hepatocytes can lead to tissue injury and organ failure.

20. Infected cells release proinflammatory cytokines, chemokines, nitric oxide
Recruitment of more immune cells
More cytokines → cytokine storm
Systemic blood vessel permeability → shock
More infection of target cells
Even though IFN function is disabled, infected cells can still release proinflammatory cytokines, chemokines and nitric oxide. This leads to a chain of immune cells being recruited leading to more and more release of cytokines, ultimately putting the body in a systemic inflammation state called cytokine storm. Blood vessels throughout the body become leaky and the host experiences shock. To add onto all of this, recruitment of more immune cells is essentially recruiting more target cells for Ebola to infect, leading to quicker dissemination of the virus.

21. Overexpression of tissue factor (TF) in infected macrophages
Overactivation of extrinsic pathway
Leads to disseminated intravascular coagulation (DIC)
Lastly, the overexpression of tissue factor in infected macrophages over activates the extrinsic pathway, leading to the development of small blood clots all throughout the bloodstream in a condition called disseminated intravascular coagulation.

22. Clinical Outcome Ebola Hemorrhagic Fever
IFN
Clinical Outcome
Ebola Hemorrhagic Fever
Disseminated Intravascular Coagulation
Shock
Death
To put this altogether, ebola’s ability to disable antiviral IFN activity makes it difficult for the host to overcome the infection, leading to dysregulated immune system and ultimately death

23. Current Treatments for Ebola
Basic supportive care:
IV fluids and electrolytes
Oxygen therapy
Stabilizing blood pressure with medication
Managing vomiting, diarrhea, fever, pain
Treating other infections as they occur
Currently, no FDA-approved antiviral drugs
But...
Recovery from Ebola greatly depends on having good basic supportive care as well as the patient’s immune response.
Care involves providing fluids and electrolytes intravenously, oxygen therapy, blood pressure, and managing other adverse symptoms that may appear.
Currently, there are no antiviral drugs approved by the FDA but there are many being developed and tested.
Two drugs, in particular, that have shown considerable promise in ongoing studies.

24. 2018 PALM Study
Following 2018 East DRC Outbreak, PALM launched to study 4 investigational treatments:
ZMapp, remdesivir, mAb114, REGN-EB3
mAb114 and REGN-EB3 showed most promising results in preliminary report; are in active use
PALM was launched last November in the Democratic Republic of the Congo as part of the emergency response to an ongoing Ebola outbreak in the Eastern provinces.
PALM is a randomized, controlled trial of four investigational agents: ZMapp, remdesivir, mAb114, and REGN-EB3.
Two of the drugs, mAb114 and REGN-EB3 outperformed the other two agents by a significant margin.
The survival rate for people who received either drug shortly after infection when virus counts were low, was 90%
The preliminary results were decisive enough that the independent board responsible for monitoring the trial recommended that this phase of the study be stopped and that all future patients be randomized to receive either REGN-EB3 or mAb114.

25. mAb114
mAb114 is a human IgG1 antibody that targets the receptor-binding domain of EBOV glycoprotein
Isolated from survivor of 1995 Kikwit outbreak
Neutralizing effect: prevents interaction of EBOV glycoprotein with NPC1 receptor on human cells
Impedes virus from entering & infecting target cell
mAb114 is composed of a single monoclonal antibody, IgG1, that targets the EBOV glycoprotein.
It was first isolated from a survivor of the 1995 Kikwit Ebola outbreak.
It produces a neutralizing effect by binding to a highly conserved region of amino acids in the receptor-binding domain of the virus, blocking the glycoprotein from interacting with the NPC1 receptor on human cells.
The virus is thus impeded from entering and infecting the target cell.

26. REGN-EB3
Co-formulated cocktail composed of 3 human monoclonal antibodies that target 3 non-overlapping epitopes on EBOV and can bind simultaneously
Goal is to reduce generation & selection of resistant virus strains during treatment
REGN-EB3 is the other drug singled out in the PALM study.
It is a co-formulated cocktail of 3 human monoclonal antibodies that target 3 non- overlapping epitopes on the ebola virus and can bind simultaneously.
The three mAbs are designed to have different biological properties, with the goal of reducing the generation and selection of resistant ebola virus strains during treatment.

27. Summary
Of 4 Ebola viruses, EBOV is responsible for most outbreaks and most dangerous
Transmitted through bodily fluids, blood, contaminated objects
Glycoproteins on the virus surface bind to receptors on host cells, then it’s engulfed by macropinocytosis
The virus then transcribes, translates, and replicates using the host cell’s machinery
Progeny virus exits cell by budding off the host cell, gaining their envelopes from cell membrane
Ebola virus disease results from infection of monocytes, macrophages and dendritic cells and subsequent immune dysregulation
EBOV genome contains viral proteins that inhibit production and signalling of antiviral cytokine IFN
Leads to organ failure, cytokine storm, shock, and disseminated intravascular coagulation (small blood clots throughout bloodstream)
2 promising Ebola drugs: mAb114 and REGN-EB3
mAb114 - single monoclonal antibody (mAb); targets receptor-binding domain of EBOV
REGN-EB3 - co-formulated cocktail of 3 mAbs; target 3 different epitopes on EBOV

28. References
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29. Supplementary Slides