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Combating Ebola- Vaccines and Interferons
- 1. LEADING RESEARCH AGAINST POTENTIALHEMORRHAGIC EPIDEMIC P R E S E N T E D B Y – B. SANDILYA 1 7 M S L S H G 0 7 CENTRAL UNIVERSITY OF PUNJAB C R E D I T S E M I N A R S E M - 2 Combating Ebola – Vaccines and Interferons
- 2. INTRODUCTION The Ebolavirus as been the topic of discussion since the severe outbreak of Ebola Hemorrhagic Fever in the African subcontinent recently. Identified in the 1970’s, Ebola has been seen to be infecting both the humans and non-human primates in a lethal way. Severe breakdown of immune system leading to multi-organ failure and death in a matter of few days, the world fears this to be potential agent for bio-wars. Combating Ebola in any way possible is the upcoming challenge to scientists all over the globe.`
- 3. HISTORY • First knownsubtype of Filoviruses family was Marburg virus appeared in 1967. Commercial laboratory workers - infected with a distinct clinical picture. Source of the virus? Green monkeys imported from Africa for research and vaccine production purposes. Epidemic - Quickly contained. • In the late 1970’s, The Democratic Republic of Congo, and Sudan. Outbreak of severe hemorrhagic fever. Causative agent initially thought to be another sub strain of Marburg virus. Later discovered to be a different virus; named Ebola.
- 4. Phylogenetic comparison withMarburg virus https://digitalworldbiology.com/archive/checking-out-new-ebola- virus-and-playing-some-tricks-blast
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- 7. CLASSIFICATION • Order -Mononegavirale Owing to their single stranded, negative sense RNA genome; • Family - Filoviridae Owing to the filament like morphological structure of the mature virus; • Genus - Ebolavirus Outbreak near the ‘Ebola’ river.
- 8. SUBSTRAINS OF Ebola •The various reported subspecies are: • Zaire Ebolavirus (EBOV-Z) : The first identified -1970’s. Most pathogenic with approximately 90% fatality rate in case of human infections and 100% lethality in case of research macaque models. • Sudan Ebolavirus (EBOV-S) : The second discovered substrain; in The Democratic Republic of Congo, and Sudan. Approximate of 50% fatality and stands to be the second most pathogenic strain of Ebola.
- 9. • Reston ebolavirus(EBOV-R) : The first strain of Ebola to break out in the U.S.; lethal infections only in non human primates; observable non lethal infections to humans. • Cote d’Ivoire or Ivory Coast ebolavirus (EBOV-CI) : Late 1990’s; Identified in Tai forest chimpanzee troupes with high mortality. Lethal infections in non human primates; Observable non lethal infections in case of humans. • Bundibugyo ebolavirus (EBOV-BE) : Discovered recently, Fifth identified strain . Associated with fatal human infections with approximately 40% fatality rate.
- 10. STRUCTURE OF Ebola Ebolavirus is a long filament like structure, its capsule crooked which allows it to evade the antibodies. The centre of the virus particle is an empty space in which this RNA resides, which can be anywhere from 20-80nm in width and is about 800-1000nm long. https://encrypted- tbn0.gstatic.com/images?q=tbn:ANd9GcRtAsY_mcgbLVczVyf2Yi0wcuZt -J2ALmCDxrzviZgBavvXWFMvpw
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- 12. GENETIC COMPOSITION • Theviral particles have a negative sense stranded, non-segmented, RNA genome. The genetic material approximately 19kb in size. The genome of Ebola virus encodes seven genes basically: NP (nucleoprotein), VP35, VP40, GP, VP30, VP24, RNA-dependent RNA polymerase (L) which code for a maximum of eight viral proteins. https://www.med.unc.edu/orfeome/projects/identification-of-new- genes-in-ebola-and-influenza-viruses
- 13. • Nucleoprotein(NP), VP35,VP30 and RNA-dependent RNA polymerase (L) genes are linked with viral replication and transactivation. • VP40 is the matrix protein and is involved in budding of the viral particle and its delivery. • VP24 is the minor matrix protein and is involved with with nucleocapsid formation. • Both the matrix proteins, VP40 and VP24 have been observed to obstruct interferon signaling. • The only surface protein is the GP, which is present as trimeric spikes comprises of GP1 and GP2. • The non-structural, soluble form of GP, ‘sGP’, is a unique product of ZEBOV GP gene, a splice varient, which gets secreted from infected cells and is speculated to act as decoy for the host immune system.
- 14. REPRODUCTION OF EbolaWITHIN THE HOST https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcRqnXSIGUjzua5Xii3RuGuJiIxl3uj4IEwbNOgxGCFyXRahCgok
- 15. 1. Attachment The glycoproteinson the envelope of Ebola are capable of binding to various receptors on the cell surface like lectins, integrins, cathepsins and others like NPC1’s, TIM’s and TAM’s depending on the cell type. http://www.sinobiologicalcdn.com/styles/default/images/ebolavirus/e bov-replication.png
- 16. 2. Entering theCell To initiate the entry, Ebola stimulates the cells apoptotic pathway by stimulating particular lipids with the help of tyrosine kinase receptors and glycoproteins of the viral envelope, which results in macropinocytosis, a normal phenomenon in apoptosis. http://www.mdpi.com/1999-4915/4/12/3336/htm
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- 18. 4. Viral replication,assembly and budding https://encrypted- tbn0.gstatic.com/images?q=tbn:ANd9GcTOD8xpZVv8aXa0AeVaD3u6M BU7_qCSFk7HuaTf3sTM5ze5I8U7
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- 20. Ebola Virus disease •Hemorrhagic fever. • Symptoms are abrupt, in about 2-21 days of infection, mimicking those of other viruses since the hemorrhagic fever is not quickly diagnosed. • The time span between early symptoms and death falls between 6-16 days of infection. • By the second week, the patient either begins recovery or undergoes multi organ failure systemically. https://upload.wikimedia.org/wikipedia/commons/thumb/4/4a/Symptoms_ of_ebola.png/800px-Symptoms_of_ebola.png
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- 23. BIOCHEMICAL AFFECTS ONTHE BODY https:/reddit.com
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- 25. • Ebola’s highpathogenicity is seen as a potential to become a bio war agent. • Threatening the people worldwide, Ebola needs to be combated, leading to stream of research ideas. • The major research is based either on finding a vaccine, or on Interferon therapies.
- 26. Ebola vaccines • Theheat killed strains of wild type EBOV-Z : Not that effective; only 29% of the non vaccinated control animals succumbed to infection. • DNA vaccines : Found to be protective in Guinea pigs and mice models. This, combined with an adenoviral delivery system was found to be fairly effective in NHP’s leading to phase one clinical trials of the vaccine. • Recombinant adenoviral strains : The adenoviral particle consisting of the EBOV surface proteins. Found to be moderately effective. The only problem with adenoviral vaccines is the pre-exisiting immunity seen in large number of individuals tried. The adenoviral particles are immunogenic in nature in about 60-90%of the exposed population which leads to questioning the vaccines’ efficiency.
- 27. • Subunit vaccines: Not yet been evaluated in NHP’s. • Replication deficient Ebola virus : The Ebola virus particles are engineered to have a deletion of VP30, an important transcriptional activator in EVD. Growing this strain on a cell line stably expressing VP30, to infect the target cells; does not produce infectious progeny in its absence. • Concerns exist since the ‘vaccine’ still consists of more than 95% of the original EBOV genome even if the the current understanding of the filoviral replication doesn’t give any possibility for recombination or recombination like events to occur.
- 28. • rVSV/EBOV-GP vaccine •Recombinant VSV is the leading and most trending vaccine; first self replicating recombinant Ebola vaccine. • VSV is Vesicular stomatitis virus, cattle virus. • A recombinant VSV includes the gene coding for Ebola surface glycoprotein (GP) incorporated into an attenuated VSV particle. Viral particles so produced are not pathogenic to humans while they are immunogenic. • Theoretically, the potential of the immune system to identify the surface glycoprotein and not the soluble glycoprotein should increase.
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- 30. • This vaccinehas proved to be effective in eliciting 100% immune response in challenged mice and hamsters. • Survival rates in guinea pigs and NHP’s is lower, at 83 and 50% respectively. • Incomplete understanding of the mechanisms as post-exposure vaccination . • However, strategies devised for an outbreak or a bioterrorism attack scenario suggest that rVSV vaccinations would be much helpful when compared to the non replicating recombinant adenoviral particles where a single injection of rVSV would be paramount when compared to the multiple doses of Ad5 required for complete effectiveness.
- 31. SPECULATION for TREATMENTS •The Ebola virus infection: Disregulates the antigen presenting cells, namely the macrophages and dendritic cells. Increases the production and release of pro-inflammatory molecules – which recruit various other target cells at the site of infection providing additional cells for viral infection. Leads to an uncontrolled amplification of infection in the host body Cytokines accumulating excessively, exponential infection and circulatory collapse. • Induction of an innate immune response, possibly prior to Ebola virus infection is speculated to lead into a systemic control of Ebola virus replication.
- 32. NORMAL FUNCTIONING OFANTI VIRAL RESPONSE https://viralzone.expasy.org/resources/IFNsignaling.jpg
- 33. TYPE I INTERFERONtherapy • Administration of IFN-beta to rhesus macaques has shown to prolong survival within a combination off type 1 IFN with monoclonal antibodies against the EBOV-GP . Produced maximum response together while neither of the monoclonal antibodies or the IFN alone was effective enough. TYPE II INTERFERON therapy • Administration of type 2 INF, INF-gamma, has inhibited the EBOV infections of the macrophages in in vivo murine peritoneal macrophages. In mice macrophages, it has been observed that the IFN-gamma treatments also work as late as being administered 24 hours after the infection
- 34. IFN-gamma therapy • INFgamma directly stimulated the expression of number of interferon-stimulated genes (ISG’s) which have antiviral activity. • Since the EBOV infecting macrophages and producing large amounts of cytokines is speculated, IFN-gamma has been seen to potentially activate the T-cell responses by enhancing the antigen presentation and phagocytosis.
- 35. 1. COMBATING INFECTIONSIN BSL2 TYPE Ebola • Apart from this, IFN-gamma exposure has also been noted to inhibit infections in: BSL2 model virus of EBOV rVSV/EBOV-GP vaccine treated cells Interferon alpha/beta receptor knock out mice. • Possibility that the IFN-gamma treatments might also work against other mononegavirales. • The ability of IFN-gamma to inhibit viral infection in humans was also seen in 24 hour IFN-gamma treated human monocyte derived macrophages following which they were able to evade the subjected infection of rVSV/EBOV-GP
- 36. • It signalsthrough the type2 IFN receptors on the cell surface by activating the JAK-STAT pathway. • In treating the infected IFNAR-/- cells with IFN gamma resulted in the discovery that functional type I IFN receptors are not required to be present for IFN-gamma signalling of JAK-STAT pathways. • When IFN-gamma receptor knock out cells were tested with IFN-gamma treatment, it was found that there was no change in the viral titre levels of rVSV/EBOV-GP in the IFN-gamma R-/- cells. • This proved that the interactions were taking place through the IFN- gamma receptor to activate the ISG’s.
- 37. 2. INTERFERON GAMMAAGAINST VIRAL RNA • IFN-gamma against RNA levels did not prove to be much effective since the control sample RNA levels and IFN-gamma treated samples RNA level seemed to be quite similar after qRT-PCR. • However the addition of CHX – cycloheximide, potential inhibitor of protein synthesis, to narrow down the steps effected by IFN-gamma leads to the finding that IFN-gamma can only interfere with RNA synthesis which is dependent upon protein synthesis. 3. UPREGULATION OF ISG’S • Extreme IFN-Gamma levels were seen to activate and up regulate many novel ISG’s such as various chemokines CXCL9, CXCL10,CCL8 and Complement components like C1s and C1r.
- 38. 3. LETHAL invivo rVSV/EBOV-GP challenge • In vivo IFN-gamma treatments of IFNAR-/- mice proves to be highly effective against the lethal rVSV/EBOV-GP challenge. • Varying dosage proves to be a useful strategy to combat EBOV strains but somehow seems to be less effective against the wild type VSV strains. • It has also been proved that IFN-gamma is completely functional in protecting mice against a lethal dose of EBOV, in both pre EBOV IFN- gamma exposure and post EBOV IFN-gamma exposure, with greater efficiency observed in case of post infection treatment groups.
- 39. DISCUSSION AND CONCLUSION •Since its discovery in the late 1970’s, Ebola virus’s infection techniques have the ability to strip down its host of any chance of survival unless a very strong immune response could be elicited. • Since its immune system evasion techniques are too severe to comprehend and escape, its high pathogenicity poses a threat as a potential bio war agent, a combating technique needs to be developed. • The vaccine development for Ebola has brought us ‘rVSV/EBOV-GP’ vaccine, an attenuated and recombined form of vesticular stomatitis virus, which is the most intelligent choice in case of massive outbreaks. • Interferon Gamma treatments on (a) murine peritonial macrophages, both IFN receptor recombinant and non recombinant versions, (b) human monocyte derived macrophages; when infected by various strains and biosafety level variants of the Ebola virus, have been successful in inducing and innate immune response, both before and after exposure to the virus. • IFN-gamma treatments are now awaiting human and non human primate trials.
- 40. REFERENCES • Bethany A.Rhein, L. S. (2015). Interferon-γ Inhibits Ebola Virus Infection. PLoS Pathogens . • Dong-Shan Yu1, 2. T.-H.-X.-Y.-B.-P.-J.-P. (2017). The lifecycle of the Ebola virus in host cells. Oncotarget . • Hussein Sweiti, O. E. (2017). Repurposed Therapeutic Agents Targeting the Ebola Virus: A Systematic review. • Mandy Kader Konde, D. P. (2017). Interferon β-1a for the treatment of Ebola virus disease: A historically controlled, single-arm proof-of-concept trial. PLos One . • Steve Jones, H. F. (2003). Ebola virus: from discovery to vaccine. Nature Reviews Immunology . • Thomas Hoenen, A. G. (2013). Current Ebola vaccines. Expert Opinion Biol Therapy . • Yitades Gebre, T. G. (2014). The Ebola virus: a review of progress and development in research. Asian Pacific Journal of Tropical Biomedicine .