Ebola: Lessons Learned from the West African Epidemic and Looking Forward”

1. Ebola: Lessons Learned from the West African Epidemic and Looking Forward” Vijay AswaniMD, PhD, FACP, FAAP Associate Professor of Internal Medicine and Pediatrics Department of Medicine City-wide Grand Rounds October 5, 2017

2. Disclosure Statement I, Vijay Aswani, MD PhD, do not have any relevant financial interest or other relationships with a commercial entity producing health-care related product and or services.

3. Learning Objectives • To summarize the West African Ebola Epidemic • Describe lessons learned in ethics, response and public health • Summarize current state of diagnostics, treatment and vaccine development • Describe open questions in Ebola

4. Outline • The Past: • Summary of the West Africa Outbreak with contextual history of Ebola. • The Present: • Ebola Survivor syndrome • Immune privilege sites where Ebola continues to live • Ebola Diagnostics • Ebola Treatment • Ebola Vaccines • The Future: • What did we (could we) learn? • Open questions in Ebola Virus Disease • Coordinating global health response • Politics of Fear

5. Audience Response Questions Get out your smartphones! In your browser, go to: Live.voxvote.com Enter PIN: 26786

6. The Past Summary of the West Africa Outbreak with contextual history of Ebola.

7. Report of an International Commission (1978). Ebola haemorrhagic fever in Zaire, 1976.Bull World Health Organ. 1978; 56(2): 271–293.

8. The Ebola river was 60 miles away from Yambuku. Yambuku was so small, it is not on this map

11. 5 distinct Ebola species and associated mortality 29 epidemics of Ebola Case fatality rates: • Zaire (EBOV), 21-90% • Sudan (SUDV), ~ 50% • Bundibugyo (BDBV), 32% • Tai Forest (TAFV), 0% • Reston (RESTV), 0% 1976: simultaneous outbreaks in Sudan and Democratic Republic of Congo. 29 isolated outbreaks since then S Baize, et al. DOI: 10.1056/NEJMoa1404505

12. West Africa Ebola Epidemic • 12/26/2013: 18-month old boy in Meliandou, Guinea – fatal illness with fever, black stools, vomiting. The start of Zaire Ebola Virus Disease • 03/24/2014: WHO public announcement of 49 cases and 29 deaths • 08/08/2024: WHO declares Public Health Emergency of International Concern (PHEIC) – 1,711 reported cases and 932 deaths • 03/26/2016: PHEIC lifted: 28,646 confirmed, probably and suspected cases in Guinea, Liberia and Sierra Leone with 11,323 deaths and estimated 17,306 survivors • 881 healthcare workers were infected and 513 died • May 2017: DRC had 8 cases and 4 deaths

13. Ebola Primer Transmembrane glycoproteins (GP) Polymerase complex (VP35 & L) Nucleocapsid (VP30 & NP) Negative-sense single-stranded RNA Matrix (VP40 & VP24) http://www.nejm.org/doi/full/10.1056/NEJMp1410741

14. How does Ebola feel? • The following narrative comes from: • Dr, Kent Brantly • Nancy Writebol • 2 American aid workers with Samaritans Purse in Liberia who got Ebola from the work.

15. Insights from Clinical Research • Hemorrhagia – late finding; predominant finding of advanced disease was severe gastrointestinal disease • Sx: fever, fatigue, anorexia, vomiting, diarrhea, headache and abdominal pain • Volumes up to 10 L per day of stool output reported in repatriated patients. • Fever absent in 10% of cases (part of case definition) • Hiccups, confusion and conjunctivitis – good discriminatory importance

16. Epidemiological Findings • Median age 32 years (uncertain if report bias or true increased risk of exposure) • No marked gender difference – 48.8% probable or confirmed cases were men • Young age predictor of death: Odds Ratio per year of life 0.91 (CI: 0.85 – 0.97) • For children, median time of 3 days from admission to ETU to death (study of 300 children) • Mortality higher in men > 45 years • Previous Case Fatality Rates (CFR) reported were maternal 90% and neonatal 100% -- possibly over-estimated. • 7.5% of pts in a study of 187 individuals were antibody positive (anti-EBOV glycoprotein) but did not have EVD • CFR: 70% (CI 69 – 72) (WHO) • Mortality was lower in pts admitted to hospital (CFR 61% CI 59-62) compared to not admitted (88% CI 86-90) • CFR in Europe and USA was 19%

17. Rojek A, Horby P and Dunning J. 2017. Insight from clinical research completed during the west Africa Ebola virus disease epidemic. Lancet 17: e80 – 92. 2017.

18. The present • Ebola Survivor syndrome • Immune privilege sites where Ebola continues to live • Ebola Diagnostics • Ebola Treatment • Ebola Vaccines

19. Post Ebola Virus Disease Sybdrome (PEVDS) • Ocular disease – uveitis the most common - 13 – 34 % of survivors • Arthritis, enthesitis, arthralgias • Hearing loss • Abdominal pain • Neuropsychiatric disorders, headaches, memory loss, sleep disorders • Virus persistence in immune-privileged organs • Fatigue • Mechanism: elevated inflammatory cytokines, molecular mimicry, damage from virus and viral persistence in immune-privileged sites

20. Eye Disease in Repatriated healthcare workers • Physician 1 • Unilateral anterior uveitis developed 40 days after initial EVD • Vision declined to 20/200 • Resolved completely with topical homoatropine and oral corticosteroids • Labs suggested broad but transient systemic immune activation • Physician 2 • Another physician had unilateral, hypertensive anterior uveitis that went to sight threatening panuveitis 10 weeks after illness • High levels of virus from anterior chamber paracentesis • Treatment: • Antivirals (one pt got favipiravir) • Steroids – topical and oral • More blindness in West African pts due to lack of timely intervention

21. Survivor complications • Neurological deficits important contributor to morbidity • Psychological distress → neurocognitive dysfunction • Poor social acceptance, stigmatization • Pathological mechanisms: not understood yet – possibly post-infectious inflammatory processes, viral replication in immune privileged sites • EBOV isolated from aqueous humor of survivor with panuveitis 14 weeks after diagnosis, total duration of viral sequestration unknown but less than 18 months • EBOV detected in CSF in a survivor with meningoencephalitis 9 months after acute illness • Follow up study of 151 survivors showed that late redescence (illness or death after full recovery) was estimated at 0.7% • EBOV detected in semen 18 months after discharge from ETC; one study reports prevalence in males at 49%

22. EBOV Persistence in immune-privileged organs • Cultured from aqueous humor during the uveitis • EVICT (Ebola Virus Persistence in Ocular Tissues and Fluids underway in Sierra Leone – evaluating prevalence of persistent EBOV in ocular fluids in survivors • Also found EBOV RNA in CNS, placenta and semen up to 565 days post acute EVD • Breastfeeding and sexual contact new transmission chains • Identified in eye, brain and testes in rhesus monkeys (Zheng et al, 2017) • Followed monkeys 43 days after EBOV exposure • 9.8% detectable RNA in eye • 1/11 in brain • 1/11 in testes • Capable of infecting human retinal pigment epithelial cells

23. Unlocking Ebola persistence Racine T and Konger GP. Unlocking Ebola Persistence. Nature Microbiology 2, 17124 (2017)

24. EBOV in Retinal epithelial cells • Anterior uveitis, painful, leads to glaucoma or cataract • Posterior leads to scarring in macular region of retina and visual disturbances • 75% of 277 cohort in Sierra Leone with uveitis developed visual loss and 26% went legally blind • Eye is an immune privileged site, i.e. limited inflammation – specifically the monolayers of pigment epithelial cells in posterior eye and iris and ciliary body in anterior eye • ocular pigment epithelial cells produce membrane-bound ligands and soluble factors that inhibit inflammatory activities of leukocytes. • Smith et al grew EBOV in ARPE-19 human retinal pigment epithelial cells • Increased viral antigen and recoverable viral particles from the cytoplasm of the cell lines • EBOV increased interferon production by the infected cells perhaps modulating immune response • Ultimately, uveitis presents because the immune response overwhelms the immune privilege

25. Eye lesions Shantha, JG, Crozier I, Yeh, S. 2017. An update on ocular complications of Ebola Virus Disease. Curr Opin Ophthalmol 28 (0). Sep 1. doi: 10.1097/ICU.0000000000000426

26. Ocular Disease in PEVDS • Impacts ADLs and quality of life • Most common finding is uveitis – eye pain, redness and photophobia and may lead to acute or chronic vision loss • Conjunctival injection was seen in 48-58% patients with acute EVD and in one study predictive of acute EVD infection • Treatment was first 1% atropine and corticosteroids in previous epidemics (Kitwit) • Uveitis: posterior (57%) and panuveitis (29%) and anterior (14%) • Unilateral more common, vision worse than 20/400 in 39% affected eye

27. PREVAIL (Partnership for Research on Ebola Virus in Liberia) III: • NIH-sponsored study in Liberia longitudinally following EVD survivors and first degree contacts for eye disease. • 24% have uveitis • Younger age and more time in ETU greater risk • In the nurse at Royal Free hospital in London, EBOV was recovered from CSFZ at higher levels than blood, indicating replication in CSF • PREVAIL in Liberia. 165 EBD survivors were evaluated. • Those with neurological symptoms (coma, delirium, meningitis during acute EVD or new neurological symptoms (n=22) were selected for LP. 18 consented to proceed. • Eligibility: 18 to 60 years, positive EBV antibody, no neurological findings at time of LP, negative for HIV, no recent trauma or seizure, no NSAIDs, no papilledema and normal coag studies. N=8 met these criteria • None of them had EBV in the CSF. Median time from discharge from ETC to LP was 414 days.

28. Novel Retinal Lesions • Case-Control study in Freetown, Sierra Leone of 82 EVD survivors with 105 asymptomatic controls. • Novel retinal lesion found in 14.6% of EVD survivors and no controls CI (7.1 – 25.6%) that followed the anatomical distribution of the optic nerve axons, suggesting neuronal transmission as a route of ocular entry • Ocular disease occurs in 14 to 60% of EVD survivors • Acute uveitis 18 – 58% • Retinal lesion did not affect vision • Visual impairment was due white cataract in 7.3% of EVD survivors • No isolation of EBOV in aqueous humor in this study. • Uveitis accounts for 24% of blindness in Sierra Leone – 2nd most common cause, 1st being cataracts

29. Steptoe PJ, Scott JT, Baxter, JM et al. 2017. Novel retina Lesion in Ebola Survivors, Sierra Leone, 2016. Emerging Infectious Diseases 23(7), July 2017, 1102-1109

30. Ebola Lab Diagnostics • Diagnostic assay play a vital role in confirming or excluding suspect cases • EBOV categorized as a high-hazard pathogen handled in BSL 4 facilities only • Preferred diagnostic method is by direct detection of viral RNA using a Nucleic Acid amplication test (NAAT) – See table 1 and 2 • Negative tests are repeated in 72 h • Presenting viral loads predict mortality • BiofireFilmArray uses a real-time nested NAAT to detect EBOV in ~ 2h. • Malaria Rapid diagnosis Test was used in W. Africa • New rapid handheld lateral flow assays that detect viral antigens in blood and body fluids; 100% sensitivity – rule out test

31. The rainbow unicorn • 2012: purified polyclonal antibodies from sera of NHPs against EBOV GP was used: treatment 2 days post-challenge resulted in 100% survival • Also in 2012, 2 labs identified monoclonal antibody combinations: ZMab (2G4, 4G7 and 1H3) and MB-003 (6D8, 13F6 and 13C6) which showed efficacy. These were combined in ZMapp (2G4, 4G7 and 13F6). • ZMApp completely protected NHPs as late as 5 days after exposure. • Desire an antiviral or vaccine that is efficacious against minimally EOV, SUDV and BDBV – the mythical ‘rainbow unicorn’. • Ebolaviruses diverge by 32 – 41% at nucleotide and amino acid level in GP. • GP is cleaved to GPCL which then goes through a fusion loop stage for insertion into the host-cell endosomal membrane • In figure, CA45 and ADI15878 158742 bind to more conserved regions shared between the Ebolavirus species • Has been protective in small animal models: mouse and guinea pigs (EBOV), mouse (SUDV) and ferret (BDBV)

32. Shorten RJ, Brown CS, Jacobs M et al. 2016. Diagnostics in Ebola Virus Disease in Resource-Rich and Resource-Limited Settings. PLOS Neglected Tropical Diseases 10 (10): e0004948

33. Clinical Features of Ebolavirus infection • Incubation period: 2 – 21 days • Sx: flu-like-illness, fever, headache, malaise, diarrhea • Animal-to-human transmission direct contact or consumption – animals: chimpanzees, gorillas, fruit bats, duikers • Exposure to body fluids: blood, saliva, sweat, urine, breast milk, feces, semen • Mucosal surfaces and open wounds • Macrophages and dendritic cells are infected →fibroblast, endothelial cells, hepatocytes, adrenal cortical cells, epithelial cells • Suppressed dendritic cell activation → ↓cytokine release and T-cell activation • Leads to upregulation of macrophages with activation and release of cytokines to cytokine storm • Downregulates interferons • Clinically lead to increased vascular permeability, hypovolemic shock, multisystem failure, DIC, diffuse hemorrhage, maculopapular rash and death • Diagnosed in BSL 4 lab by RT-PCR or immunoglobulin M and G antibodies or specific antigen detection

34. Treatment conclusions • Treatment: Supportive Care • Oral and IV fluids, analgesia, antiemetics, antidiarrheal medications, empiric antibiotics and antimalarials • Loperamide contraindicated due to paralytic ileus • Gap in knowledge: an optimal package of supportive care measures to be used

35. Rojek A, Horby P and Dunning J. 2017. Insight from clinical research completed during the west Africa Ebola virus disease epidemic. Lancet 17: e80 – 92. 2017.

36. ZMapp • in 2012, 2 labs identified monoclonal antibody combinations: ZMab (2G4, 4G7 and 1H3) and MB-003 (6D8, 13F6 and 13C6) which showed efficacy. These were combined in ZMapp (2G4, 4G7 and 13F6). • ZMApp completely protected NHPs as late as 5 days after exposure. • ZMapp treated patient showed reversal of viral load, also got convalescent plasma, brincidofovir – a nurse treated at Royal Free Hospital in London; 9 months later, resurgence of virus in CNS and systemic circulation ((Jacobs et al, 2016) • Multicenter, randomized trial of ZMapp lowered CFR from 37% (supportive care) to 22%, did not meet specified statistical threshold, posterior survival probability was 91% • Discrepancy: 100% survival seen in NHP not replicated in the clinical trials. Why?

37. Ebola Drugs • EBOV pathogenesis: evades the type 1 interferon response through viral proteins, VP30, VP35 and VP24. • Early cellular targets are macrophages and dendritic cells • Dendritic cell maturation suppressed, fails to secrete proinflammatory cotkines and leads to impairment in antigen presentation to T cells.

38. Ebola Drugs • Current therapeutics candidates: • Small molecule inhibitors • Inhibit EBOV through suppression of viral transcription and replication. • Brincidofovir, BCZ4430, favipiravir, GS-5734 and AVI-6002 • Nucleotide analogs, siRNAs nonrandomized clinical trials • Favipiravir was studied in mid-November in Sierra Leone on 39 patients and n=17 in favipiravir treatment group. Higher survival rate (64.8% versus 27.8% and > 100-fold decrease in viral load. • Second trial in December 2014 did not show benefit, although in patients with lower viral load, possible benefit • Antisense phosphorodiamidate morpholino oligomers and siRNA; • These target L polymerase (transcription enzyme in virus) or VP35 and VP24 – viral proteins that cause immune suppresion • Immune-based therapeutics, including IFNS, plasma transfusion, mAbs

39. Liu G, Wong G, Su S et al. 2017. Clinical Evaluation of Ebola Virus Disease Therapeutics. Trends in Molecular Medicine 23(9): 820 – 830

40. Ebola Vaccines • GP contains 2 subunits as trimeric spikes on viral surface. Pivotal role in cell attachment, fusion and cell entry – key antigenic target Mire CE and Giesbert TM. Neutralizing the Threat: Pan-Ebolavirus Antibodies Close the Loop. Trends in Molecular Medicine. 23(8): 669 – 671. August 2017.

41. History of Vaccine Development • 1st attempts: inactivated whole virus. No clinical trials -- safety concerns, failure to demonstrate efficacy in non-human primate (NHP) models. • 1990s: pre-clinical studies expressing envelope GP or nucleocapsid (NP) genes demonstrated efficacy in ‘gold-standard’ model of cynomolgus macaques • 2003: 1st ever human clinical trial: 3-plasmid DNA vaccine encoding transmembrane deleted GP from the Zaire and Sudan viruses and NP, showed that s 3-dose schedule was safe and immunogenic. • 2010: replication-defective recombinant human adenovirus serotype 5 vaccine (rAd5) encoding GP genes with a point-mutation. Single dose – safety trial. Problem: pre-existing immunity again Ad5 • 2015: 2nd human clinical trial assessing safety and immunogenicity of constructs encoding wild type GP from Ebolavirus Zaire (EBOV), Sudan (SUDV) and Marburgvirus Angola strain. Result: vaccine safe but multiple doses required and immune response waned at 32 weeks • Work-around pre-existing immunity to Ad5 – use chimpanzee Ad3 (ChAd3): 100% efficacious with zero viremia against both EBOV and SUDV. Booster with replication deficient Modified Vaccinia Ankara (MVA) 8 weeks later, enhanced durable protection. • Recombinant, replication competent vesicular stomatitis virus (rVSV)-based vaccine encoding EBOV GP showed encouraging results in NHP. Given to 1 patient on compassionate basis after needle-stick injury (2011)

42. Vaccine Trials • Chimpanzee adenovirus 3 vectored vaccine (ChAd3): • given to humans in UK, Europe, US and in Mali (2016) • Large phase III trial in Liberia amended to a phase II because of waning cases • Being assessed in children ages 1 to 17 years in Nigeria, Mali and Senegal (ongoing) • Vesicular stomatitis virus vectored vaccine (rVSV): • Phase 1 clinical trials started in Europe and Africa shortly after ChAd3) trial • 10 – 20 % of healthy volunteers suffered severe adverse effects: arthralgia, arthritis with entry of VSV into CSF and 25 – 30% suffered fever. • Chosen by WHO for Phase III trial Ebola Ca Suffit!) in Guinea in a ring vaccination strategy – it worked 100% protection • Modified vaccinia Ankara vectored vaccine (MVA): • MVA-Bn-Filo – quadrivalent vaccine encoding GPs from EBOV and SUDV and GP from Marburg and NP from the Tai forest strain was tested with a 1-2 week booster dose (rather than 8 week)

43. Adenovirus vectored Vaccines • rAd26 along with MVA-Bn-Filo was trialed as a prime and booster vaccination at intervals of 2, 4 and 8 weeks. Responses were noted up to 8 months. • Ad26 tested with MVA in a large phase 3 trial in Sierra Leone (in progress) • rAd5 vaccine containing the GP of 2014 strain of EBOV was safe and immunogenic in healthy adults in China and Sierra Leone.

44. Ebola Ca Suffit! (Ebola, That’s Enough!) • developed in 1960s for smallpox eradication • vaccination of contacts and people at risk (contacts of contacts) • used for MMR outbreaks • Eboa ca Suffit! (Ebola, that’s enough!) trial (2017) showed 100% protection (CI 75 – 100%) up to 10 days after vaccination (trial limitation)

45. The future • What did we (could we) learn? • Open questions in Ebola Virus Disease • Coordinating global health response • Politics of Fear

46. Ebola still here? • DRC detected an Ebola outbreak on May 11, 2017. It was detected using the GeneXpert Ebola assay • Outbreak declared ended on July 2, 2017by WHO – 4 deaths • April 2017: 39 year old man had vomiting and bleeding after handling a carcass of a dead wild boar in a forest in Likati in Bas Uele in DRC. • He died en route and 2 transporters fell ill and their contacts. 2 of these tested positive for Ebola and ultimately 5 tested positive • Genetic analysis showed the strain was similar to the 1995 Kitwit epidemic strain

47. New data analysis shows Ebola is changing • Li et al analyzed the spatial-temporal and haplotype pattern of 514 EBOV genomes and clinical outcomes in Sierra Leone and identified 11 different lineages with 372 unique haplotypes that had different CFRs. They showed that certain SNPs in noncoding regions that correlated with different CFRs

49. Vaccines – Questions and Lessons Learned • Correlates of protection – a marker • None yet! Looking at GP antibody titers • What titer level determines protection? • Survivors show persistent virus in semen, breast milk and immune privilege sites (eye, brain): this may lead to recurrence • We may see further outbreaks – spill-over events • Accelerated development of vaccine candidates during the epidemic was remarkable and demonstrated in the epidemic • Ring vaccination strategy was a innovative trial design implemented • Gained great insight into the role of open communication and research collaboration when doing clinical trials in resource-poor countries with limited infrastructure. • There is still no licensed vaccine that can be manufactured, stockpiled and rapidly deployed

50. Public Health Challenge • There were more deaths in West Africa from other causes than from Ebola during the epidemic! • Major killers were lower respiratory tract diseases, malaria, HIV and TB. Ebola ranked 18th as cause of death in Sierra Leone • “public health paradox”: If the epidemic is successfully controlled, then it is highly likely that the eventual impact of the epidemic disease will have been less than the opportunity cost of the resources allocated to it from other health areas. • There was a Politics of Fear – fear of containment, securitization of the epidemic, political posing between governments, NGOs and countries.