E b o l a v i r u s | 1

21 September 2014: In search of a durable cure for the Ebola virus (EBOV)Zeena Nackerdien, PhD

Epidemiology Largest prior EBOV outbreak in 1976 had 318 cases (previous outbreaks

confined to remote regions of central Africa)1 To date, 2014 West-African outbreak reported in Guinea, Sierra Leone, Liberia,

Nigeria, and Senegal2

o Ebola transmission has also recently been identified in the Democratic Republic of the Congo3

As of September 18, 2014, 5335 EBOV cases have been identified3

To date, 2622 deaths have been associated with the disease3

In fact, 1 in 2 people infected during the current outbreak have died4

o This translated to more deaths than in all previous EBOV outbreaks combined

Description Enveloped, negative-RNA-strand virus of the Filoviridae family5

Known EBOV species6 o Zaire ebolavirus (ZEBOVo Sudan ebolavirus (SEBOV)o Cote d’Ivoire ebolavirus (CIEBOV)o Reston ebolavirus (REBOV) o Bundibugyo ebolavirus (BEBOV)

Phylogenetic comparisons suggest that the West African variant diverged from the Central African lineage around 20041

o Mutations include a notable variation in the RNA-editing site of a glycoprotein (GP) gene, 341 fixed substitutions, 55 single nucleotide polymorphisms (SNPs) and 263 intrahost single-nucleotide variant (iSNVs)1

o Epidemiological and genomic surveillance is ongoing to identify viral dynamics

Key EBOV disease (EVD) facts4

Natural hosts: fruit bats of the Pteropodidae family7 Infection caused by:7

o Direct contact with bodily fluids of an EBOV-infected patiento Contact with objects or surfaces7 contaminated with EBOV

E b o l a v i r u s | 2

o Contact with infected animals (contact with blood or fluids or infected meat)

Pre-symptomatic incubation period: 2 to 21 days7

Average EVD case fatality rate is around 50%o Case fatality rates have varied from 25% to 90% in past outbreaks7

Diagnosis7

Symptoms manifest as systemic, gastrointestinal, respiratory and neurologic abnormalities5

Laboratory findings of low white blood cell count and elevated liver enzymes Tests include virus isolation by cell culture, ELISA, RT-PCR, EM, antigen-

capture detection and serum neutralization tests Terminal stages include multiple organ failure and symptoms resembling the

onset of severe septic shock5

TreatmentsDrug candidates

BCX4430, a novel nucleoside analog designed to cause premature termination of transcription and replication of viral RNA8

Nilotinib or cAbl-1-tyrosine-kinase-specific small interfering RNAs reduced productive replication of ZEBOV by 4 orders of magnitude in vitro9

Antisense oligonucleotides Piperazine-modified phosphorodiamidate morpholino antisense oligos against specific EBOV transcripts10,11

o Phase 1 study of AVI-6002 completed (NCT01353027)o Phase 1 study of AVI-7537 withdrawn (NCT01593072)o Phase 1 study of AVI-7288 completed (condition: Marburg fever;

NCT01566877)

Vaccines in development12

Currently there is no licensed filovirus vaccineo Ideally an efficacious and safe vaccine should, with one shot, confer long-

term protection with little or no filovirus viremia against all EBOV strains pathogenic to humans as well as related species that can cause severe hemorraghic fever13

Promising vaccine candidateso Various recombinant Vesicular Stomatitis Virus (rVSV)-based EBOV

vaccines (Canada’s National Microbiology Laboratory and New Link Genetics Corporation)13

Characteristic: VSV glycoprotein (GP) is replaced by filovirus GP (single shot)

Complete protection as a preventive agent against all public-health-relevant filovirus species and partial to complete efficacy post-exposure achieved against homologous challenge of 3 strains of EBOV and related Marburg virus in non-human primates

E b o l a v i r u s | 3

Other rVSV-based vaccines: Prime-boost combination of plasmid DNA vaccine

expressing Ebola/Marburg surface antigens together with a rVSV-vector expressing same epitopes10

o Adenovirus-based (Ad) vaccines Chimpanzee-derived (Ch), replication-defective Ad (cAD3-EBO)

vaccine characteristic: Encodes wild-type GPs of ZEBOV and SEBOV (prime and boost shots)

Vaccine boosted with modified vaccinia Ankara provided durable protection against lethal EBOV challenge in non-human primates14

Safety, tolerability and immunogenicity to be evaluated in healthy subjects (NCT02231866)

Ad26 and Ad35 vectors derived from replication-incompetent, rAd5 vector used as platforms for non-human primate vaccines; contains EBOV GP from Zaire and Sudan/Gulu EBOV species8

A combination of Ad26/Ad35 vaccines achieved complete protection against Zaire EBOV challenge in macaques8

Earmarked for clinical trialso Synthetic polyvalent-filovirus DNA vaccine15

Characteristic: contains GP genes of Marburg marburgvirus (MARV), SEBOV or ZEBOV

Preclinical vaccination of guinea pigs and mice using rodent-adapted viruses was highly potent, eliciting robust neutralizing antibodies and protecting against MARV and ZEBOV challenges

Antibody (Ab) cocktailo ZMAPP: Three murine mAbs (13C6, 13F6 and 6D31) engineered with

human constant regions targeting non-overlapping Ebola virus epitopes (the mucin-like domain as well as the 6D31 and core epitopes of GP1) manufactured in transgenic N. benthamiana lacking plant-specific N-glycan residues10

o Post-exposure prophylaxis within 1 or 2 days of challenge with EBOV (Zaire Kikwit strain) was effective at preventing morbidity and mortality in non-human primates16

o Human studies are set to begin in early 2015

Prevention7

Reduce risk of wild-life-to-human transmission by thoroughly cooking meat and handling infected meat or fruit bats with appropriate protective gear

Reduce risk of human-to-human transmission from direct or close contact with EBOV-infected individuals by regular hand washing and wearing appropriate protective gear

Implement containment measures such as good hygiene, quarantine of sick individuals, monitoring the health of EBOV-exposed individuals and their contacts for 21 days and prompt and safe burial of the dead

E b o l a v i r u s | 4

Adhere to WHO-recommended guidelines for infection control in healthcare settings

References

1. Gire SK, Goba A, Andersen KG, et al. Genomic surveillance elucidates Ebola virus origin and transmission during the 2014 outbreak. Science. 2014;345(6202):1369-1372.

2. US Centers for Disease Control and Prevention. 2014 Ebola Outbreak in West Africa - Outbreak Distribution Map. 2014; http://www.cdc.gov/vhf/ebola/resources/distribution-map-guinea-outbreak.html#areas. Accessed September, 2014.

3. Baden LR, Kanapathipillai R, Campion EW, Morrissey S, Rubin EJ, Drazen JM. Ebola — An Ongoing Crisis. New England Journal of Medicine.0(0):null.

4. US Centers for Disease Control and Prevention. West Africa Ebola outbreak. 2014; http://www.cdc.gov/vhf/ebola/pdf/west-africa-outbreak-infographic.pdf. Accessed September, 2014.

5. Zampieri CA, Sullivan NJ, Nabel GJ. Immunopathology of highly virulent pathogens: insights from Ebola virus. Nat Immunol. 2007;8(11):1159-1164.

6. Falzarano D, Geisbert TW, Feldmann H. Progress in filovirus vaccine development: evaluating the potential for clinical use. Expert Rev Vaccines. 2011;10(1):63-77.

7. World Health Organization. Ebola virus disease. Fact sheet No. 103 (Updated September). 2014; http://www.who.int/mediacentre/factsheets/fs103/en/. Accessed September, 2014.

8. Warren TK, Wells J, Panchal RG, et al. Protection against filovirus diseases by a novel broad-spectrum nucleoside analogue BCX4430. Nature. 2014;508(7496):402-405.

9. García M, Cooper A, Shi W, et al. Productive Replication of Ebola Virus Is Regulated by the c-Abl1 Tyrosine Kinase. Science Translational Medicine. 2012;4(123):123ra124.

10. Bioentrepeneur (Trade Secrets). Table 1. Ebola treatments and vaccines in clinical development 2014; http://blogs.nature.com/tradesecrets/files/2014/08/Ebola-treatments.pdf. Accessed Nature blogs, 2014.

11. Iversen PL, Warren TK, Wells JB, et al. Discovery and early development of AVI-7537 and AVI-7288 for the treatment of Ebola virus and Marburg virus infections. Viruses. 2012;4(11):2806-2830.

12. Infographic: Ebola vaccine available by November. Aberdeen News2014.13. Geisbert TW, Feldmann H. Recombinant Vesicular Stomatitis Virus–Based

Vaccines Against Ebola and Marburg Virus Infections. Journal of Infectious Diseases. 2011;204(suppl 3):S1075-S1081.

14. Stanley DA, Honko AN, Asiedu C, et al. Chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge. Nat Med. 2014;advance online publication.

E b o l a v i r u s | 5

15. Shedlock DJ, Aviles J, Talbott KT, et al. Induction of broad cytotoxic T cells by protective DNA vaccination against Marburg and Ebola. Mol Ther. 2013;21(7):1432-1444.

16. Pettitt J, Zeitlin L, Kim do H, et al. Therapeutic intervention of Ebola virus infection in rhesus macaques with the MB-003 monoclonal antibody cocktail. Sci Transl Med. 2013;5(199):199ra113.