Ebola Biotechnology Lecture Notes PDF

http://www.newsweek.com Biotechnology and Immunology http://searchpp.com/ebola-cure/ Teresa Johnson, MS PhD BMS 5308 Lecture #42 [email protected] Lecture Objectives For the therapies, technologies, advances, or diseases discussed: • Identify the immunologic principle or pathway that is uti...

http://www.newsweek.com Biotechnology and Immunology http://searchpp.com/ebola-cure/ Teresa Johnson, MS PhD BMS 5308 Lecture #42 [email protected] Lecture Objectives For the therapies, technologies, advances, or diseases discussed: • Identify the immunologic principle or pathway that is utilized by the technology. • Determine how the technology or therapy enhances the generation of an effective immune response or alters disease pathogenesis. • Define the change in knowledge or technology that facilitated this technological advance. • Identify potential applications of the technology. • Recognize potential challenges in implementation or use of the technology. Ebola Epidemiology CDC “Ebola 2014” Report “Ebola by the Numbers” • Total cases – 28,616 • Total deaths – 11,310 • 40% mortality • Number of healthcare workers trained by CDC in West Africa – 24,665 • Number cases diagnosed in US – 4 • Number of cases treated in US – 11 http://www.cdc.gov/about/ebola/index.html Recent History of African Ebola Outbreaks Source: CDC and WHO, 10/28/22 Ebola Virus Outbreaks in Africa, 1976 - present Year(s) Country subtype # cases # (%) deaths Uganda – 7 districts Sudan 137 53 (39%) Aug – Sept 2022 DRC, North Kivu Zaire? 1? At least 1 April – July 2022 DRC, Equateur Zaire? 2 2 Sept – Dec 2021 DRC, North Kivu Zaire February – June 2021 Guinea Zaire 23 12 (52%) February – May 2021 DRC, North Kivu Zaire 12 6 (50%) DRC, Equateur Zaire 130 55 (54.5%) DRC, North Kivu Zaire 3,470 2,287 (66%) Jan-July 2018 DRC, Bikoro Zaire 54 33 (61%) May-July 2017 DRC Zaire 8 4 (50%) Aug-Nov 2014 DRC Zaire 66 49 (74%) Mar 2014-2016 Multiple Zaire 28,616 11,310 (40%) Nov 2012 – Jan 2013 Uganda Sudan 6 3 (50%) June-Nov 2012 DRC Bundibugyo 36 13 (36%) June-Oct 2012 Uganda Sudan 11 4 (36%) Aug 2022 - ongoing June – Nov 2020 Aug 2018 – June 2020 https://www.cdc.gov/vhf/ebola/history/summaries.html 4? – EBV symptoms, 1 lab confirmed, all died The 2022 Ebolavirus Outbreak in Uganda Links to follow the current Ebolavirus outbreak in Uganda: • https://www.cdc.gov/vhf/ebola/outbreaks/uganda/202 2-sep.html • https://www.who.int/emergencies/disease-outbreaknews/1 https://www.who.int/emergencies/disease-outbreak-news/item/2022-DON421 Transmission of Ebolavirus • Zoonosis = reservoirs – Bats – Non-human primates – Bush meat – Other animals having contact with bats or contaminated fruits • Human-to-human infection – CLOSE contact – Blood and body fluids – Used needles, burial rituals – Sexual contact post-recovery – extended presence of virus in semen (101 days) and vaginal fluids (33 days) http://www.cdc.gov/vhf/ebola/resources/virus-ecology.html Ebola Virology and Pathogenesis http://ebolaviruspictures.com/images/Ebola-Hemorrhagic-Fever-Symptoms.jpg Ebolavirus Virology • Filovirus, negative-sense ssRNA genome, enveloped • A single glycoprotein, GP, in viral envelope with many functions • Natural production of secreted form of GP – Immune decoy – Immunopathogenic • Not a lytic infection, immune-mediated damage http://ars.els-cdn.com/content/image/1-s2.0-S0966842X01022016-gr1.jpg Ebolavirus Disease • Cellular target = macrophage and DCs • Incubation period = 2-21 days (average 8-10 days) • Progressive disease – generalized, nonspecific symptoms to hemorrhagic disease • https://www.youtube.com/watch?v=HML zEnfKP74 • https://www.youtube.com/watch?v=SC7SRMZwu8 • https://www.youtube.com/watch?v=UMM wgvLmN-M – EXCELLENT! http://www.md-health.com/images/10900011/ebola-symptoms.jpg Predictors of Disease Severity • Disease severity correlates with viral load • Non-fatal cases begin to recover 6-11 days after symptom onset • Complicating factors – malnutrition, pre-existing infections (malaria, parasites), secondary infection http://www.cdc.gov/vhf/ebola/transmission/human-transmission.html Symptoms of Ebolavirus Infection • Non-specific, systemic symptoms • Not contagious until symptomatic • Highly indicative – persistent and uncontrollable hiccupping http://www.whatisebola.co/wp-content/uploads/2015/02/sym.jpg http://www.cdc.gov/vhf/ebola/symptoms/index.html The Immune System in Early Ebolavirus Infection http://www.medicalnewstoday.com/imag es/articles/280/280870/ebola-virus.jpg • Macs or DCs at site of virus entry are infected. • Cells migrate to regional lymph node, then to spleen, liver, adrenal gland. • Massive replication of virus and production of secreted Ebola GP • Cytokine production (e.g., IL-1, IL-6, TNF-α) cause prodrome (early symptoms) – fever, aches, headache, anorexia, nausea • Leads to increased vascular permeability – LEAKING!! http://aapsblog.aaps.org/2014/09/09/ebola-basics-its-resurgence-and-treatment/ Hemorrhagic Phase of Ebolavirus Disease • Though not infected, when virus or GP binds, cells undergo necrosis leading to disease symptoms: – Lymphocytes → lymphopenia – Hepatocytes → dysregulation of clotting factors, coagulopathy – Adrenocortical cells → hypotension, impaired steroid synthesis • Continued infection of macs and DCs → more virus and more soluble GP produced! • Virus and secreted GP bind to endothelium, causing massive production of pro-inflammatory cytokines → CYTOKINE STORM • Cytokine storm important in triggering hemorrhagic phase of disease – Alter adhesion expression on endothelial cells, causing vascular leak – Activate endothelium and platelets leading to microclot formation → depletes clotting factors which aren’t replaced because of hepatocellular necrosis. Leads to hemorrhage. – Multiorgan failure, shock, and death follow. http://microbewiki.kenyon.edu/index.php/Infection_Mechanism_of_Genus_Ebolavirus Manifestations of Hemorrhagic Disease Range of hemorrhagic responses – from mild, localized to severe, diffuse http://ebolaviruspictures.com/images/Ebolavirus-Pictures.jpg http://howpk.com/wp-content/uploads/2014/08/Deadly-Ebola-VirusSymptoms-disease-and-Treatment-howpk.com_.jpg Diagnosis of Ebolavirus Infection • Challenges to diagnosis: –No test is effective until after symptoms manifest – when patient becomes contagious –Requires blood – use of sharps increases risk to healthcare workers, draw on hemorrhagic patient! –Requires advanced technology, specialized training, and specialized space and equipment Timeline of Infection Within a few days after symptoms begin • Biotechnology needs: –Assays which may be performed early – soon after potential exposure and before onset of symptoms –Less invasive sampling – testing of something besides blood (e.g., saliva) –More simplified tests – can be performed on site without specialized equipment or space and with minimal to moderate training Later in disease course or after recovery Retrospectively in deceased patients Diagnostic tests available • Antigen-capture enzymelinked immunosorbent assay (ELISA) testing • IgM ELISA • Polymerase chain reaction (PCR) • Virus isolation • IgM and IgG antibodies • Immunohistochemistry testing • PCR • Virus isolation http://www.cdc.gov/vhf/ebola/diagnosis/index.html Review: Biotechnology Advance #1 Understanding the pathogenesis of Ebolavirus infection and disease – Construction of BSL-4 facilities – Development of tissue culture and animal model systems – Identification of cytokines and their role in this immune-mediated disease Ebola Treatment Treatment of Ebolavirus Infection Current Standard of Care = aggressive supportive care • Fluid replacement – preventing hypovolemic shock and balancing electrolytes • Oxygen • Antibiotics (secondary bacterial infection) http://i.cbc.ca/1.2865822.1418140707!/fileImage/httpImage/image.jpg_gen/deriva tives/16x9_460/ebola-treatment-in-sierra-leone.jpg Treatment – Convalescent Sera http://blogs-images.forbes.com/davidkroll/files/2014/09/Blood-to-serumImupro-International.jpg • Survival rates – 10-60% • Survival dependent upon species of infecting virus, good supportive care, patient’s immune system WAIT FOR IT! • Survivors can maintain Ebola-specific antibody titers for 20+ years • Direct transfusion of plasma or of enriched IgG from survivor can be an effective treatment • May need to be given early in disease process http://www.infowars.com/wp-content/uploads/2014/10/Table.jpg Review: Biotechnology Advance #2 • Development of apheresis equipment – Can remove plasma and re-infuse cells into donor • Development of methods to characterize immunoglobulin molecules – High-throughput screening – can identify Abs with the highest binding affinity – Can define Ab binding specificity and function – neutralization, complement fixation, ADCC potential Ebola Experimental Vaccines and Treatments EXPERIMENTAL TREATMENTS http://3.bp.blogspot.com/-W8Ui_gBN3t0/VHdTJLJ8tI/AAAAAAAAOc0/NOAzl0h-jb0/s1600/MRK-Race-Ebola3-2014.png http://media.npr.org/assets/img/2014/10/10/zmapp-plants_slide3293b465538afea8c4642b9b2510f3b57cf6f37f.jpg Spreading a Wide Net http://www.independent.co.uk/incoming/article9787261.ece/ alternates/w460/EbolaDrugs1.jpg http://3.bp.blogspot.com/XSOVd5XJwiA/VAFvgnKn5YI/AAAAAAAAAVE/KIJMZF5ZUIY/s1600/ebola%2Bmedicine%2Bchart2barnews.jpg Experimental Therapy #1: Zmapp • Cocktail of 3 humanized monoclonal antibodies • Each is specific for the Ebolavirus Zaire GP glycoprotein • Neutralizes both virus and secreted GP glycoprotein • https://www.youtube.com/watch?v=Mbiy0wS9lhY http://graphics8.nytimes.com/images/2014/10/30/learning/antibodyLN/antibodyLN-blog480.jpg http://www.mtbeurope.info/news/images/ZMappCocktail_Image-s.jpg Zmapp Synthesis and Production • Mouse monoclonal Abs produced, then humanized • Protective capacity of Abs – alone and as cocktails – evaluated in vitro and in vivo • Genes for each Ab of optimized mix cloned into vesicular stomatitis virus (VSV) vector • Recombinant VSV vector injected into tobacco plants • Antibody extracted from tobacco leaves • Production capacity = 40-100 doses/month • A number of passive Ab therapy trials conducted (clinicaltrials.gov) • Neat factoid – virus (Zaire strain) used for mouse immunization was from outbreak >20 years earlier. Gene sequence was sufficiently conserved to protect against 2014 outbreak strain! Zmapp Efficacy • Passive Ab normally effective only in early stages of infection or disease → neutralization of pathogen or toxin before disease state is well-established • Zmapp Ab can be effective at later stages of Ebolavirus disease – during hemorrhagic phase • Why? Neutralization of secreted GP to inhibit “cytokine storm” • But not effective in all patients. Why? http://www.infowars.com/wp-content/uploads/2014/10/Table.jpg Review: Biotechnology Advance #3 • Construction of antibody molecules – Generation of monoclonal antibodies – Humanization of mouse or rat monoclonal antibodies • Construction of recombinant viral vectors – Animal versus plant viral vector systems • Development of plant-based expression systems http://www.ourprg.com/wp-content/uploads/2014/08/ebolacure.jpg Traditional Viral Vaccines • Viruses must be grown in eukaryotic host cells • Viruses are then killed or weakened (“attenuated”) • OR recombinant vaccines (protein, viral) are made • Requires highly specialized space (sterile tissue culture facility, biosafety cabinet), costly reagents (cells, fetal calf serum), and advanced training https://www.labconco.com/images/cms/widelarge/49628225761_e57ed93439_o.jpg https://www.corning.com/content/dam/corning/media/worldwide/cls/images/cellculturecategory/ cls-lab-cell-falcon-flask-group-hood-square-us.jpg.transform/tablet-469/img.jpg http://www.looklocal.co.za/article/hollow_tree_in_guinea_was_possible_site_of__ebola's_ground_zero.html Vaccine Development I • NewLink in conjunction with Dept of Defense • Gene for Ebolavirus GP glycoprotein (EBOV-GP) is cloned into vesicular stomatitis virus vector • Result – replication-defective virus-like particles (VLP) of VSV encoding EBOV-GP • Produce vaccine virus in tissue culture • If construct proven to be immunogenic, then produce a replication-competent VSV vector that can be grown in plants. • Result – elicited strong Ab response in animal models http://www.ebola.org.za/list-of-experimental-treatments/ http://humanviruses.org/viruses-in-the-news/ebola-virus-vaccines-begin-clinial-trials/ Vaccine Development II • NIAID/VRC in partnership with GlaxoSmithKline • Ebolavirus Zaire GP glycoprotein gene inserted into replication-defective chimpanzee adenovirus 3 (chAd3) vector • Vaccine virus produced in tissue culture with helper viruses • Use of chAd3 backbone avoids pre-existing Ab generated by natural human adenovirus infection • Elicits strong T cell response along with strong antibody response • Vaccines using human adenovirus-based vectors tested in many clinical trials • See notes and slides 46 and 47 in suppl. section for extra info on experimental vaccines and therapies http://abcnews.go.com/Health/human-trial-ebola-vaccine-begin-week/story?id=25204379 Shots in the Dark • Many therapeutic interventions were tried in attempts to reduce transmission and mortality in order to slow and stop the outbreak – some with a scientific rationale and some with a minimal “testable hypothesis” • Some may have had an impact, and we have no idea why. (Recall slide 9 where it states Ebolavirus has an RNA-based genome.) – An antiviral medication for DNA virus (suppl. slide 48) – An antiviral medication for retrovirus Review: Biotechnology Advance #4 • Construction of recombinant viral vectors – Replication-competent versus replication-defective – Identification of new vector systems • Characterization of vaccine-induced immunity – Antibody versus CTL Updates and Advances: 2020-2023 The 2018-2020 Eastern DRC Outbreak • Policy – updates and new – https://www.who.int/publications/i/item/WHO-EVD-OHEPED-16.1 – WHO guidance “Clinical Care for Survivors of Ebola Virus Disease”, 1/2016 – https://apps.who.int/iris/bitstream/handle/10665/325000/ 9789241515894-eng.pdf?sequence=1 – WHO manual “Optimized Supportive Care for Ebola Virus Disease”, 5/2019 • 2018-2020 outbreak – Prevention and Containment https://www.statnews.com/2019/05/07/who-broadens-eligibility-ebola-vaccine/ In 2018 eastern DRC outbreak: • 250,292 contacts registered • 303,867 individuals vaccinated (rVZV-GP) ‒ Handwashing and PPE ‒ Disinfection of space (hospital or home) ‒ “Safe and dignified” vs “traditional and ritual” burials ‒ Community involvement – for ring vaccination, contact tracing, education ‒ Ring vaccination o rVSV-ZEBOV-GP – one dose (Merck), prequalified o chAd3-MVA – two dose (Johnson and Johnson) ‒ Immunotherapy – passive antibody to GP o ZMAPP, MAb114, or REGN-EB3 • Prevention – Ervebo o o o o Advances in Prevention and Clinical Management of EVD Pseudotyped to express Ebola GP instead of VSV GP rVSV-ZEBOV FDA approved December 19, 2019 Single dose, IM injection https://www.iavi.org/iavi-report/vol-23-no-2-2019/proven-against-ebola-avector-shows-its-broader-potential – Additional vaccines used under WHO emergency use (“clearance”), but not FDA-approved yet; EU marketing authorization – 5/2020 o Ad26.ZEBOV immunization with MVA-BN-Filo boost • Therapeutics – Inmazeb™ (atoltivimab, maftivimab, and odesivimab-EBGN) o Combination of 3 monoclonal antibodies (MAb or MoAb) o FDA approved October 2020 – Ebanga™ (ansuvimab-zykl) o Single MoAb o FDA approved December 2020 • Efficacy evaluated only against Ebolavirus Zaire to-date (i.e., not against other Ebolavirus strains or against Marburg virus) • https://www.cdc.gov/vhf/ebola/index.html Ebola GP bound by Inmazeb Mortality ± Ebanga therapy Review: Advance #5 – Policy and Regulatory • New and improved policies – Updated case description and standard of care guidelines – Care of Ebola survivors – Involvement of community members for contact tracing and ring vaccination • Regulatory advances – authorization and approvals – A VSV-based vaccine (FDA and WHO) – “first in kind” – Ad/MVA vaccine combo (EU, compassionate use WHO) – 2 MoAb therapies X X X X X X X http://lsconnect.thomsonreuters.com/focus-ebola-new-strategies-discovery-design-therapies-lethal-disease/ Ways to Block Ebola’s Replication and Disease Processes Some Perspective? http://www.dailymail.co.uk/wires/afp/article-2866926/Malaria-deaths-halved-2000-Ebola-risksgains-WHO.html http://beforeitsnews.com/travel/2014/08/dont-worrytoo-much-about-ebola-2-2465444.html http://www.bancodasaude.com/noticias/ebola-pode-fazer-alastrar-malaria-na-africa-ocidental/ SARS-CoV-2 Vaccines https://www.youtube.com/watch?v=lFjIVIIcCvc – whole virus, subunit, viral vector, mRNA vaccines – from GAVI Summary I • What immunologic pathways or methods are the basis of new and experimental therapies for Ebolavirus? – Viral immune-mediated disease or immunopathology – infection of macs/DCs, cytokine storm – Antibody-mediated neutralization of virus and viral GP protein – Active and passive immunization – Activation of innate and adaptive immunity – Viral replication, transcription, and/or translation • How is the efficacy of Ebolavirus therapies increased by application of these immunologic principles? – More targeted therapies – Ebolavirus-specific versus generalized supportive therapies – Targeted activation of the immune response – Targeted inhibition of the viral life cycle Summary II • What changes in the field of immunology allowed development of these technologies? – Discovery of the multitude of cytokines and chemokines that are produced in response to infection – B cell analytic and molecular technologies – monoclonal Ab production, humanization of mouse Abs, characterization of Abs (affinity, binding specificity, effector function) – Vaccine technologies – identification and development of new vectors (replication-incompetent, plant-based, non-human viruses) – Understanding the contribution of T cells versus B cells in vaccine-induced immunity – Identification of PRR and PAMPs – discovery of TLRs, RLRs, NLRs (used in these specific therapies) • What lessons were learned and advances made as a result of the 2014 outbreak that allowed more improved control of the 2018-2020 outbreak? – New policies – detailed/standardized case description, community involvement in tracing and vaccination – Development and regulatory approval of 1-2 vaccines and 2 passive antibody-based therapies Supplemental Figures and References Case Distribution of the 2014-2016 Outbreak Countries with Widespread Transmission and Current, Established Control Measures1 Previously Affected Countries5 Country Total Cases (Suspected, Probable, and Confirmed) LaboratoryConfirmed Cases Total Deaths Total Deaths Nigeria 20 19 8 3355 2543 Senegal 1 1 0 14124 8706 3956 Spain 1 1 0 Liberia 10675 3160 4809 United States 4 4 1 Total 28610 15221 11308 Mali 8 7 6 4/13/2016 1/28/2018 28,652 28,616 15,261 15,261 11,325 11,310 United Kingdom 1 1 0 Italy 1 1 0 Total 36 34 15 Country Guinea 2 Sierra Leone 4 3 Total Cases (Suspected, Probable, and Confirmed) LaboratoryConfirmed Cases 3811 http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/case-counts.html#modalIdString_cases-localized-transmission Viral Life Cycle Note: not a lytic infection! http://lsconnect.thomsonreuters.com/focus-ebola-new-strategies-discovery-design-therapies-lethal-disease/ Ebola on Clinicaltrials.gov I Y F 84 (78) (74) studies Status # 2019 (2018) (2017) Completed 48 (43) (28) Active, but not recruiting 10 (10) (19) Recruiting 13 (15) (16) Not yet recruiting 0 (1) (4) Withdrawn, terminated, suspended, or unknown rAd5 rVSV 8 (9) (7) Subject – January 2019 # Convalescent sera or Zmapp tx; Ab response in survivors or vaccinees 8 Vaccines 58 VSV 13 chAd3 12 Other AdV (mostly Ad26 ± MVA) 20 Other vaccines 13 Epidemiology 0 Treatments (antivirals, immunostim., siRNA) 13 Diagnostic testing 1 Screening of volunteers for vaccine/drug trials 0 Antiviral treatment and Virus in semen 1 Survivors – long-term immunity, host genetics, complications 3 I Y F Update on Ebola Experimental Therapies/Research • 1082 papers published on Ebola in 2017 and 883 in 2018 (from PubMed search of “Ebola 2017” and “Ebola 2018”) • No new drugs or vaccines advanced to human trials over the past ~2.5 years. More about optimizing lead candidates. • Several treatments (e.g., favipiravir, herbal products, equine-derived Abs) and 2 vaccines tested in animals • Diagnostic test development – rapid point of care tests, less invasive sampling • Analysis of STRIVE studies – LOTS done with Sierre Leone population (epi, vaccines, treatments, etc.) • Protective equipment – cleaning/re-use of emergency care facilities, optimization of PPE use for medical personnel, methods for improving compliance • Vaccines – phase I-III testing, durability of antibody responses, different prime-boost timelines (see next slide) • Treatment – focus on antibody-based protection due to successful use of immune patient sera – – – – Efficacy and durability (in treated humans and in experimental animal models) Timing post-exposure (in animal models only) Isolation high-affinity antibodies for in vitro production Development/testing of non-human antibodies (like ZMapp) • Basic science – viral pathogenesis, identification of ways to inhibit viral replication and disease • Policy, legal, social – impact and proposed changes • Influence of malaria on Ebola virus disease I Y F Anti-Virals – Brincidofovir • Developed by Chimerix for treatment of cytomegalovirus (CMV), adenovirus (AdV), herpes simplex virus (HSV), and smallpox virus • A pro-drug of cidofovir – conjugated to lipid to facilitate uptake of the drug across the cell membrane which allows lower dosing • Mechanism of action – binds to DNA polymerase and inhibits viral replication • Some limited, preliminary data of efficacy against Ebolavirus. • But Ebolavirus is an RNA virus, so how does this work? I Y F Anti-Virals – Lamivudine • Licensed for treatment in HIV- and HBV-infected patients (manufactured by GSK) • A nucleoside analog reverse transcriptase inhibitor (nRTI) • Is incorporated into DNA strand under synthesis and blocks further elongation by reverse transcriptase • Used “off-label” by Dr. Gobee Logan in Liberia • Administered to 15 Ebolavirus-infected patients, and 13 survived • Not a controlled study, but if effective, how? http://www.ebola.org.za/tag/lamivudine/ http://www.scq.ubc.ca/antisense-rna/ I Y F Anti-Virals – siRNA • siRNA = small interfering RNA or silencing RNA • Sequence-specific short RNA molecules are designed • Encapsidated in a lipid bilayer –To facilitate delivery to cell cytoplasm –To protect dsRNA molecule until delivered to cell cytoplasm http://www.cheminst.ca/sites/default/files/images/ACCN/NovemberDecember2014/ebola_figure.jpg I Y F TekMira Ebola siRNA Therapy • Protein targets – Ebolavirus polymerase, VP24 (matrix protein), and VP35 (nucleoprotein) • In vitro – found to be immunostimulatory • Liberian trial stopped because in vivo therapy appeared to be immunosuppressive • NOTE: dsRNA can bind and activate cytoplasmic PRR (TLR, NLR, RLR) and lipids of delivery nanoparticle can activate surface TLR • https://www.youtube.com/watch?v=SWku4VxeE8g http://www.cantechletter.com/2012/05/byron-capitals-douglas-loe-raises-target-ontekmira/rnai-interference/ https://gallery.mailchimp.com/f3b960e67245bbd7b734e7889/images/20462546-5d10-4dec-850e-eac3964f40d5.png Review: Biotechnology Advance #5 • Development of siRNA technology – Understanding Ebola virology and pathogenesis to understand which genes to target – Elucidating mechanism of action of siRNA therapy – Development of lipid nanoparticle technology to facilitate dsRNA delivery I Y F Biotechnology #6 – Other Considerations • Breastfeeding? – Benefits of nutrients (in malnourished population) versus transmission of virus in potentially infected mother • Zmapp protection? – Time of administration – outcome tied closely to virus load – Cytokine storm – genetic polymorphism, amount of GP secreted – Underlying factors – illness, age, nutritional status, other supportive care • siRNA immunostimulation versus immunomodulation? http://www.infowars.com/wpcontent/uploads/2014/10/Table.jpg – PRR stimulation by siRNA therapy (dsRNA molecule) – Time of administration, dosing versus virus dissemination and GP secretion • Can enough siRNA lipid particles get into the infected cells before they are “soaked up” by other host cells? • siRNA has no impact on GP production – either before production or after secretion. If administered late in infection, how much impact will this have on GP-induced cytokine storm? • Use of untested drug therapies in susceptible populations (i.e., no safety, efficacy, immunogenicity data).