This ppt tells you about Ebola virus; its transmission methods, how it affects the immune system and evades its action, its major symptoms, epidemiology and how to combat it. Main focus is given on vaccines and use of interferons
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Combating Ebola- Vaccines and Interferons
1. LEADING RESEARCH AGAINST
POTENTIAL HEMORRHAGIC 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 Ebola virus 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 known subtype 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 with Marburg virus
https://digitalworldbiology.com/archive/checking-out-new-ebola-
virus-and-playing-some-tricks-blast
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
Ebola virus 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.
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tbn0.gstatic.com/images?q=tbn:ANd9GcRtAsY_mcgbLVczVyf2Yi0wcuZt
-J2ALmCDxrzviZgBavvXWFMvpw
12. GENETIC COMPOSITION
• The viral 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 Ebola WITHIN THE
HOST
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15. 1. Attachment
The glycoproteins on 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 the Cell
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
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
25. • Ebola’s high pathogenicity 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
• The heat 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.
30. • This vaccine has 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 OF ANTI VIRAL RESPONSE
https://viralzone.expasy.org/resources/IFNsignaling.jpg
33. TYPE I INTERFERON therapy
• 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
• INF gamma 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 INFECTIONS IN 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 signals through 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 GAMMA AGAINST 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 in vivo 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 .