1. Ebola virus disease and vaccine

2. Definition
Ebola virus is a pathogenic virus that has an epidemic outbreak throughout Africa and worldwide in with high mortality rate reaches 40-90% near the outbreak onset.

3. Sign & Symptom
There is no specific sign and symptoms for EVD , mainly characterized by :
Fatigue , myalgia , arthralgia, headache
then after days patients show hemorrhagic fever
Anorexia , nausea , vomiting , diarrhea
Multiorgan dysfunction .
Uncontrolled bleeding and persistent elevated temperature are the main cause of death.

5. Transmission
The incubation time is 2-21 days , during this period Ebola is not contagious and pateint are asymptomatic.
This viruse is transmitted via contact with body fluids such as blood , breast milk, saliva , feces and tears .
Direct contact is not sufficient for transmission .

6. Intervention
By isolation of infected patients and offer supportive care to reduce symptoms associated with this disease involves rehydration and pain management.
Using PPE to prevent transmission.

8. Although some patient may undergo spontaneous remission with no explanation. And some of them who survive may exhibit Post-Ebola syndrome characterized by : 1-Mental health and cognitive sequelae 2-Chronic headache 3-Auditory disturbances and ocular effect

10. Scientists have poor prognosis for ebola virus disease because we lack the proper understanding of the basic biology for this virus and all drugs and vaccines that may treat or prevent this virus are still under development.

11. Clinical manifestation &Treatment

12. Treatment
No Standard treatment available, however, several experimental strategies have shown promise in treating Ebola disease.
Patients receive supportive therapy:
treating of  secondary bacterial infections and pre-existing comorbidities.
balancing electrolytes (body salts)and maintaining oxygen level and blood pressure status.
balancing patient’s fluids

13. Experimental drug therapy
Immunotherapeutic
Nucleic-acid
based inhibitors
Nucleoside/nucleotide viral polymerase inhibitors

14. ZMapp is a cocktail of monoclonal antibodies and is being able to revert advanced Ebola disease when administered up to five days post
The structure of antibodies bound to the Ebola virus glycoprotein and inhibit entry of the virus
into cells .
Immunotherapeutic:

15. Mortality in the ZMapp treated participants was 40 percent lower than the mortality in participants receiving standard of care alone ZMapp treated participants also had a more rapid elimination of virus from the bloodstream.

16. Nucleoside/nucleotide viral polymerase inhibitors
Only the nucleoside analogue favipiravir has been tested extensively in humans.. Besides activity against influenza virus infection, this drug also has documented activity against a wide variety of RNA viruses including Ebolaviruses.
Favipiravir prevented death in mice infected with EBOV when treatment was started six days post infection.

17. Nucleic-acid based inhibitors
Such as small-interfering RNAs (siRNAs ) .

18. Four siRNAs targeting the polymerase (L) gene of the Zaire species of EBOV (ZEBOV) were either complexed with polyethylenimine (PEI) or formulated in stable nucleic acid–lipid particles (SNALPs). Guinea pigs were treated with these siRNAs either before or after lethal ZEBOV challenge.

19. Replicating Ebola virus vaccines

20. 1. Recombinant Vesicular Stomatitis Virus-based vaccines
The first replicating Ebola virus vaccine shown to be protective in NHPs .
It was identified by the WHO expert panel as one of the most advanced candidates for use in the West Africa outbreak.
In this vaccine, the VSV GP was replaced with ZEBOV (Zaire Ebola virus) GP.
A transient rVSV viremia may occur in vaccinated animals and humans .

21. The recombinant virus was tested in NHPs infected with SHIV and it did not cause any clinical illness and the vaccine was able to protect 4 out of 6 NHPs.
This study is of particular importance because of the high prevalence of HIV in areas with ebolavirus outbreaks
A blended formulation of three rVSV encoding GP from ZEBOV , SEBOV ( Sudan Ebola virus) , and Marburg virus was able to protect against challenge with all of those viruses and also against challenge with CIEBOV (Cote d'Ivoire Ebola virus).

22. 2. Recombinant paramyxovirus-based vaccines
HPIV3 is a negative-sense RNA virus.
A transcription unit encoding the ZEBOV GP and/or ZEBOV-NP gene was introduced between the P and M genes of HPIV3.
this vaccine was given intranasally to Guinea pigs and it was 100%protective after a single vaccination . In rhesus macaques , two vaccine doses were required to achieve 100%protection.

24. One of the problems that may affect the efficacy of this vaccine is the pre-existing immunity .
So this vaccine was improved by deleting the HPIV3 F and HN genes, which are the main targets for the HPIV3-specific humoral immune response.
Bukreyev and colleague developed a new vector based on Newcastle disease virus (NDV) which is an avian paramyxovirus with no detectable pre- existing immunity in humans and a new recombinant vector expressing ZEBOV-GP was generated (rNDV/ZEBOV-GP).

25. First data suggest that the rNDV/ZEBOV-GP vector might be less immunogenic than the HPIV3-based vaccine.
The main advantages of the HPIV3-based vector are :
The potential for needle-free administration.
It is easy to be produced in large quantities
It induces a systemic and local immunity in the lungs which is beneficial against aerosol infection.

26. 3. Recombinant Rabies-virus based vaccine
Rabies virus is a negative-sense RNA virus of the Rhabdoviridae family.
It causes more than 24,000 deaths per year in Africa so an effective bivalent RABV/ebolavirus vaccine would be a valuable vaccine in that region .

28. BNSP333-GP which retains rabies G .
SAD B19 rabies virus is the vaccine that is already used for rabies wildlife vaccination in Europe .
This strain was further attenuated by introducing a point mutation in the rabies glycoprotein G gene(particularly at amino acid 333) to reduce neurovirulence.
ZEBOV GP was introduced as an additional transcriptional unit between the N and P genes , and two varients were generated:
BNSP333-GP which retains rabies G .
A replication-deficient derivative , the BNSPΔG-GP, in which rabies G gene was deleted .

29. The BNSPΔG-GP did not cause any clinical signs or lethality after intramuscular, intranasal or intraperitoneal infection of mice, even after intracerebral inoculation of sucking mice.
Recently this vaccine have been evaluated in the rhesus macaque model in which one dose of the vaccine was 100%protective .
The BNSP333-GP remained neurovirulent in the sucking mice model which is considered a very stringent model.

30. 4.Recombinant Cytomegalovirus-based
It is a DNA virus. It was genetically engineered to express a CTL epitope located on ZEBOV-NP.
This vaccine can be used as a disseminating vaccine to target wildlife involved in the initial transmission of Ebola virus to humans because of its unique potential to re-infect and disseminate through target populations regardless of prior CMV immunity.

31. Mice were vaccinated with two doses of recombinant murine CMV/ZEBOV-NP and challenged with a lethal dose of MA-ZEBOV.
the vaccinated mice survived the challenge but were not protected from MA-ZEBOV replication.
but clearly the protective efficacy of CMV- based vaccines should be evaluated in larger animal models such as NHPs.

32. Non-replicating ebola virus vaccine

33. 1.Inactivated vaccine: 2.VLPs:
Ebola virus inactivated by heat , formalin or gamma irradiation
Not effective due to:
[1] the potential risk of reversion to virulence of conventional inactivated vaccine
[2] ineffectiveness ot this vaccine in protecting NHPs
2.VLPs:
subunits based vaccine produced from mammalian cell transfected with plasmid encoding matrix protein of filoviruse , VP40 , GP and NP.
Efficacy studies represent VLPs as a promising filoviruse vaccine for the use in human ,100% protection Against ZEBOV with VLPs consisting of VP40 and GP.
researchers switch (293T) mammalian cell to bacloviruse based expression system using insect cell , in order to increase VLPs production.

34. 3.Recombinant adeno-virus:
Highly promising and the most studied approach
developed by GP or NP antigen of EBOV or ZEBOV introduction into the rAd5 plasmid which has the ability to generate robust T and B cell responses to viral antigenes
safe and immunogenic in phase  clinical trail using 1010 IFU rAd5 based vector expressing ZEBOV-GP as an antigen against ZEBOV.
Problems:
[1] High vaccination dose required
[2] preexisting Immunity to Ad5 which may represent (60-90)% of a population.
In order to overcome these problems researchs find that delivering of the vaccine via the oral , nasal or intratracheal rout circumvent pre-existing immunity without affecting efficacy against lethal challenge and improve T cell response , and changing the rAd5 vector to a different serotype

35. 4.DNA VACCINS DNA vaccination was discover in last years to
treat many viruses including Ebola virus, Particularly in regard to emerging and re-emerging pathogens.

36. Advantages of using DNA vaccine
rapidly adapted as pathogens.
The plasmids are
Noninfectious.
Easy to produce in large quantities.

38. DNA vaccine induce cellular and humeral immune responses, but this require administration of several doses to achieve the desired immunity.

39. For ZEBOV, the first successful immunization strategy using DNA was described in 1998 .
RESULTS :
100% of the vaccinated mice can be protected from lethal disease when given four doses of plasmid DNA encoding either ZEBOV-GP or ZEBOV-NP .
Partial protective efficacy with three doses of plasmid DNA
But 50% of the surviving animals developed viremia.
Clinical trial phase 1 showing that three doses of a DNA vaccine-encoding ZEBOV-GP, -NP and SEBOV-GP are immunogenic in humans.

40.   5.Alphavirus replicons  
Venezuelan equine encephalitis virus (VEEV) is an alphavirus and was used early on in the EBOV vaccine development as a potential platform .

41. 5.Alphaviruses
VEE replicon was achieved by replacing the structural genes of an attenuated VEEV strain with ZEBOV-GP, -NP, -VP24, -VP30, -VP35 or - VP40 and expressed from an RNA expression vector; then coverting to particle by providing the structural VEEV genes in trans.
All the vectors were immunogenic in mice, only the one expressing ZEBOV-NP conferred 100% protection in the mouse model.
Combination of the vectors expressing ZEBOV- GP and ZEBOV-NP resulted in 100% survival in mice.

42. The result (differed from the data obtained in mice )
Protective efficacy of these two promising VEEV/ZEBOV vaccine vectors was further investigated using strain 13 guinea pigs.
The result (differed from the data obtained in mice )
The VEEV/ZEBOV-GP vector alone or in combination with
the vector-expressing ZEBOV-NP showed 100% protection.
Passive transfer of serum from vaccinated animals into naïve strain 13 guinea pigs resulted in no protection from lethal infection.