World Health Organization Collaborating Centre for Research & Reference ( & VHFs) Porton Down 1978

Crimean – Congo Haemorrhagic fever virus – an of international concern?

Prof. Roger Hewson Virology & Pathogenesis Group Lead Head: WHO Collaborating Centre for Virus Reference and Research (Arboviruses & VHFs) Dangerous Infections: New Public Health England - Microbiology Services Solution – A Look into the Future

Porton Down, Oct 2-3 2019 Salisbury, UK Summary

o Background • Taxonomy • / Nairovirus / • CCHF disease / Transmission / Outbreaks • Historical perspective o Laboratory containment o Virus biology / Evolutionary potential o Human activity (increased zoonotic events) o Diagnostics o Vaccines o Conclusions Order Bunyavirales • Single stranded –ve sense RNA • Lipid enveloped • Segmented genome [Small, (Medium) Large] • borne (x Hanta x Arena) • Human disease (some) • 10 Families

Arenaviridae (3), Cruliviridae (1), Fimoviridae (1) Hantaviridae (3) Mypoviridae (1) (3) Peribunaviridae (4) Phasmaviridae (5) Phenuiviridae (12), Wupedeviridae (1)

Genus Mammarenavirus Orthohantavirus Orthobunyavirus Orthonairovirus Phlebovirus Banyangvirus

Species 34 35 49 14 9 1

Host / Res Rodents Rodents / rum / tick Ruminant Rum / tick

Vector Rodents Rodents Mosquito Ticks Mosquito Ticks

Disease e.g. Lassa / Junin HFRS / HPS Oropuche fever CCHF RVFV SFTS

3 Bunyavirales – medically (& veterinary, agriculturally) important order Family Nairovirus Genus Orthonairovirus

Dugbe virus orthonairovirus Qalyub orthonairovirus Kupe virus •Bandia virus •Qalyub virus Nairobi sheep disease orthonairovirus •Nairobi sheep disease virus Dera Ghazi Khan orthonairovirus •Ganjam virus •Dera Ghazi Khan •Abu Hammad virus Sakhalin orthonairovius •Clo Mor virus Hughs orthonairovirus •Sakhalin virus •Hughes virus Thiafora orthonairovirus •Puffin Island virus •Erve virus Hard ticks •Solado •Thiafora virus •Keterah orthonairovirus •Burana orthonairovirus •Issyk-Kul virus •Kasokero orthonairovirus •Burana virus •Tamdy virus Soft ticks Crimean-Congo haemorrhagic fever orthonairovirus

4 Orthonairovirus: Tick borne members some of which are medically / veterinary important

Crimean - Congo Haemorrhagic Fever • Severe human infection. • Fatality rate 30% (>80% nosocomial infections). • No FDA or European approved vaccine or treatment. • ACDP - Hazard Group 4 pathogen. • Reservoired in ticks & wild life mammals, amplified in cattle sheep, goat, camel [No disease in animals] • Transmission by tick bite or direct / indirect contact with infected blood/body fluids.

5 CCHF Preclinical – A Tick development borne VHF [ofPics the - PHESalih CCHFAhmeti vaccine Kosovo & Farida PHE Tishkova, pipeline Tajikistan] fund PLF 1516/108/MR

CCHF - Clinical Disease

• Incubation period 2-9 days

• Haemorrhagic state develops 3 - 5 days

• Petechial rash / ecchymoses in the skin

• Bleeding from the mucous membranes Epitaxsis, Haematuria, Haemoptysis • Loss of blood pressure - shock

• Death 7-9 days [massive bleeding / cardiac arrest]

6 [Pics - Salih Ahmeti Kosovo & Farida Tishkova, Tajikistan CCHF - Clinical Disease

7 [Pics - Salih Ahmeti Kosovo & Farida Tishkova, Tajikistan ….also subclinical infections Bordur et al 2012 >80% Tky] Transmission cycle & annual distribution of cases Drop off & Molt

Nymph Ungulates / livestock Adults Small mammals Larva (& birds)

Ovipositation & Hatching Tick activity

Humans

Nosocomial transmission

Inc. H2H

8 Transmission cycle modified from Bente et al., 2013 Annual cases – permission from Turkish MoH Human – Human transmission of CCHFV

9 Chronology of events during a CCHFV outbreak

10 CCHF – Historical Perspective (i) 1st Descriptions of Central Asian Haemorrhagic fever

• 1136 Tajikistan / (Zayn al-Din Sayyed) • 11th century A.D. Abu Ali Al-Husayn ibn Abd Allah ibn Sina

11 Historical perspective 1 CCHF – Historical Perspective (ii) 1st Descriptions of Crimean Haemorrhagic fever

• 1941 Crimea modern medical description • 1944 Viral origin - passage through human volunteers M.P Chumakov et al., Isolation / registration - Yale 1968 (Crimean HF)

12 Historical perspective 2 CCHF – Historical Perspective (iii) 1st Descriptions of Congo fever

Kisangani

Entebbe

Kinshasa

• Stanleyville March 1956: 13 year old presented with fever / bruising • Isolated / adapted to mice / maintained by passage - Unidentified • Sent to EAVRI / Entebbe 1957

13 Historical perspective 3 East Africa Virus Research Institute (EAVRI)

Dir: Sandy Haddow: John Woodall, David Simpson: New virus (1956) Woodall et al., 1962 Simpson et al., 1967 Registered - Arbovirus catalogue Yale 1961 - Congo Virus MP Chumakov: 1968 Isolation of Crimean HF virus

Registration at Yale

1969 - Identical to Congo

1973 – CCHF virus

14 CCHF – a political name coined in the cold war David Simpson: Director Virology Porton Down // 1960 - 82

• Name synonymous with CCHF virus

• WHO & future VHF outbreaks (inc. EVD)

Global distribution of CCHF

• Amassed wide range of different strains • Part of Special Pathogens Reference collection - WHO CC

15 DI Simposin @ Porton Down VHFs - High consequence pathogens

Hazard Group 4 • Virus Country Date Natural Mortality Highly infectious first host rate recognised (humans) • H2H transmission Arenaviridae Lassa Nigeria 1969 Rodent 15% Old & New world Lujo Zambia 2008 Rodent 80% • Hazard to employees arenaviruses Guanarito Venezuela 1989 Rodent 30% Junin Argentina 1957 Rodent 25% • Spread within community Machupo Bolivia 1962 Rodent 30% • No effective intervention Sabia Brazil 1990 Rodent 40% Chapare Bolivia 2003 Rodent 30% Nairoviridae Crimean – Congo Congo 1968 Ticks 30-80% European directive 2000/54/EC http_eur-lex.europa.pdf Haemorrhagic Hazard / Risk Group 4 fever Filoviridae Eboa Zaire Congo 1976 Bats 90% Ebola Ebola Sudan Sudan 1976 Bats 50% • Endemic in many regions of the world Taï Forest Ivory coast 1994 Bats 40%

Ebola Reston USA 1989 Bats Sub-clinical

Bundibugyo Uganda 2007 Bats 35% • Risk of acquisition to EU:  Imported in patients (Travel related) Marburg Marburg Germany 1967 Bats 50% Herpesviridae Herpes B virus Worldwide 1932 Macaques 80%  Imported animal products Paramyxoviridae Nipah Malaysia 1999 Bats 40%  Laboratory infections Hendra Australia 1994 Bats 60% Falviviridae Russian Spring Russia 1937 Ticks 30%  Climate change (vector / reservoir distributions) Summer Encephalitis  Other anthropogenic factors Kyasanur Forest India 1957 Ticks 10%  Viral emergence Omsk Russia 1990 Rats 20% Orthopoxviradae Variola Eradicated Humans 30%

16 Biosafety classification / sources of introduction into the EU High Containment CL3 & CL4 labs CL4 (in-vitro) CL3 (in-vitro)

CL4 (in-vivo) CL4 (in-vivo)

17 High containment facilities at PHE CCHF - Geographic Distribution 50 ° North limit for the geographic distribution of genus Hyalomma ticks

• Spain [2016] • Mongolia [2012] Country with low risk (presence of vector) • India [2011] Country at risk (serological evidence + vector) • Greece [2008] 5 to 15 cases per year • Turkey [2002] 20 to 49 cases per year 50 to 200 cases per year

18 Global distribution of CCHF Crimean - Congo Haemorrhagic Fever virus X20,000

19 CCHFV Morphogenesis

Maturation and budding into the Golgi

HPA Golgi membranes fuse with plasma membrane releasing HPA particles

20 Budding into the Golig and release from the plasma membrane S Segments ML SegmentsSegments Hazara Hazara Hazara

AP92 Greece Europe 2 VLG- TI29414 Europe 1 30908 S Russia Asia 2 DAK 8194 Senegal Kosovo/9553/2001 M4 Africa 1 Europe 1 Semunya Uganda SR3 Africa 2 K229Europe-243 S. Russia 2 Drosdov Russia SPU41-84 Europe 1 HU29223 Russia IbAr10200 Baghdad Iraq 66019 China XinJiang AfricaSPU128 1 -84 Asia 1 88166 China XinJiang U2-2-002--U-6415 Matin Pakistan 8402 China XinJiang Asia 1 Baghdad M2

HY13 China XinJiang 75024 Semunya Uganda Africa 2 75024 China XinJiang 7803 7803 China XinJiang Asia 2 Hodzha Africa 2 C68031 China Hodzha Uzbekistan Tadj-HU8966 TADJ/HU8966 Asia 2 66019 Tajiki 2010 TADJ/HU8966Africa 3 Hodzha Uzbekistan Matin Evidence of 79121 China XinJiang IbAr10200 NigeriaHY13 segment 7001 China XinJiang M1 8402 7 CCHF S genotypes Baghdad-12 Iraq reassortment Tajiki 2010 SPU128/81/7 S. Africa Matin Pakistan Asia 1 SEMUNYA Africa 3 JD206 Pakistan IbAn10248 CONGONigeria SR3 Pakistan M5 UG3010 SPU4/81 S. Africa Africa 3 SPU4/8179121 S. Africa IbAr10200 Nigeria 0.1 0.10.1 7001 M3

21 Phylogeny of CCHF viruses CCHF Segment reassortment between strains

L L L

S S S

L S L L M S S + M M

• Co-replication of two strains in the same cell / organism L • Ticks are suitable hosts to support reassortment S L M S

 Global & dynamic reservoir of CCHF virus

22 Reassortment in CCHF viruses could lead to new viruses and new disease…

Budding reassortant L Exchange of M segments S viruses S L M influence host range

L

S S L M Envelope glycoproteins L S M influence M cellular tropism L S

N altered pathogenicity

23 Diagnostics should be based on strains isolated globally Alternative pathology observed in some forms of CCHF…. e.g.Tajikistan

“..alternative clinical presentation” visceral tropic form of CCHF

Prof F Tishkova Tajik Ministry of Health Institute of Preventive Medicine Dushanbe

24 Autochthonous CCHF in Spain Positive Hyalomma ticks identified in: 2011, 2013, 2014, 2015 Ávila 2016

Livestock districts

Positive municipality Negative +ve in previous years

• 1st case • Tick bite / hiking in Ávila

• 2nd case • Hospital transmission / Madrid • Genotype Africa 3

25 Two human cases (2016) - Ávila 2ndry hospital transmission (Madrid) 3rd case Badajoz 2018 July 2018 August 24th 31st 4th 7th 8th

Travel to Badajoz Fever Hospitalized PCR +ve Possible tick bite

Rare & Imported Fevers Lab

4 samples processed for confirmatory RT-PCR & virus isolation

Genomic analysis • Reassortant virus o L – Africa 3 o M – Africa 3 o S – Asia 1

26 Presentation title - edit in Header and Footer Transmission cycle & annual distribution of cases Drop off & Molt

Nymph Ungulates / livestock

Adults

Small mammals Larva (& birds)

Ovipositation & Hatching Tick activity

Humans

Nosocomial transmission

Inc. H2H

27 Transmission cycle modified from Bente et al., 2013 Annual cases – permission from Turkish MoH Direct zoonotic transmission “Backyard farming / slaughtering” of livestock is common in endemic countries

... less regulation during Eid-al-Adha festival

28 Blood – blood contact an efficient way to spread virus infection Tick activity coincides with human cases

Tick activity

…& viremic lifestock CCHFV Viremic animals

Slaughter of viremic lifestock > transmission opportunities

Dates of Eid-al-Adha

shift 10 days each year Leblebicioglu et al., 2015 CCHFV Diagnostics

30 Diagnostics… Nosocomial transmission is very common for CCHF

An early diagnosis helps to mitigate further transmission: • Patient isolation • Barrier nursing

… before more highly infectious haemorrhagic stages develop

31 Basic research has an important role CCHFV is genetically diverse over all 3 segments

Reassortment is very common

S segment data most commonly available

S segment phylogeny 7 genotypes

Diagnostic tests need to work against multiple strains RT-PCR tools based on circulating strains / international collaboration …

32 e.g. Universal CCHFV RT-PCR assay [Atkinson et al., 2012] Rapid nucleic acid detection Isothermal detection @ 37°C Replicase Polymerase Amplification (RPA)

• Simple set up & suitable for use with low power devices • Rapid amplification at a single temperature • Crude samples (serum, urine, saliva, tick peps..)

Rapid results to HC workers

33 10 minute RPAassay [Bonney et al., 2017] Serology tools Several commercial kits IgG & IgM CCHF ELISA available but varied using rec - NP as antigen / target sensitivity specificity…. Qualified for use on clinician samples Point of care assays:

Converting ELISA into a ‘dip-stick’ Lateral flow immuno-assay

CCHF Sero assay

IgG +VE IgG -VE IgM +VE IgM -VE Screening of test patient Field validation Specific, Sensitivesera Robust, Repeatable.

[Dowall et al., 2012]

34 Surveillance Iran : Afghanistan : Pakistan

Collections of +ve /-ve sera For CCHF assay validation …

• RT-PCR • •CCHF •RT-PCR •Dengue Helmand fever…• Antigen cature •Sand fly fever •Antibody•Sand fly fever •CCHF •West Nile •Antigen•Hantavirus •JapEV •CCHF •CHK •Q fever •Ricettsia HPA – NIH, Pak AF Inst, PHE – Inst Pasture … roll out Aga Khan & Quetta Uni collaboration Sero survey: UK MoD / HPA collaboration

35 DIAGNOSTICS PHE – Detect & Diagnose emerging disease (high consequence pathogens like VHFs) CCHFV Vaccine interventions

36 Vaccine interventions…

Lack of treatments against CCHF No vaccines or antiviral drugs are approved for CCHF by FDA or EMA.

Current vaccine…

Soviet / Bulgarian vaccine candidate major disadvantages: • Requires live CCHF virus • Crude preparation (non-standardised homogenisation of mouse brain) • No efficacy studies, no regulatory data package (since 70s) • Is not acceptable to FDA/MHRA/EMA approval

Alternative approach badly needed for a modern CCHF vaccine that can meet regulatory approval and is proven to be effective.

37 Available vaccine 1970:Tkachenko Recombinant approach based on MVA

Modified Vaccinia virus Ankara (MVA)

• Attenuated strain of Vaccinia virus, developed as a smallpox vaccine • Human safety history: >100,000 doses, no adverse effects reported • Thermostable – no cold chain required • Industrial GMP established • Recombinant MVA in advanced clinical trials (TB, cancer)

Recombinant MVA capability at PHE Porton Down • Induction of humoral & cellular immunity • Genome capacity sufficient for large CCHF genes • Mammalian glycosylation patterns

McFadden (2005) Nat Rev Microbiol

38 Use of MVA L S L MVA-GP vaccine S M Surface glycoprotein antigens Glycoprotein gene

p11 mH5 L MVA flank GFP GP V5 R MVA flank GFP for selection of C-terminal V5 for in vitro recombinant viruses antibody recognition

L R

L R MVA-infected cell MVA genome GFP+ plaque MVA L R purification

39 Construction of vaccine candidate [Transfer plasmid derived from pLW44 (B. Moss, NIH)] Vaccine Immunogenicity Prime Boost Analysis

MVA-GP MVA-empty Saline control Day 0 7 14 21 28

107 pfu of MVA-GP, intramuscular injection. Tested in 3 strains of mice.

Anti-CCHF Anti-GP T-cells GP Positive Balb/c 129 Sv/Ev A129 Media peptides control

6000 A129 129 Sv/Ev 4000 cells 6

2000 SFU/10

0 Response to GP Saline MVA-GP specific peptides MVA-empty

40 Immunogenicity: GP-Specific antibodies and INF-γ cellular response X2 Lethal Dose [intra-dermal] CCHF Prime Boost Challenge Analysis MVA-GP MVA-empty Saline control

Day 0 7 14 21 28 35 42 14d 4d Vaccine efficacy PB 14d 107 pfu of MVA-GP, intramuscular injection. Tested in A129 CCHF-susceptible mice.

Survival proportions Clinical Score mean + SD 100 4

75 Saline 3 Saline MVA-empty MVA-empty 50 MVA-GP MVA-GP 2 Control groups showed : 25 • Rise in temperature Percent survival

Signs of Illness 1 • Weight Loss

0 0 0 5 10 15 0 5 10 15 Days post-challenge Day post challenge

41 Vaccine efficacy after challenge with 2x lethal dose [100 ul 200 TCID50 intradermally upper medial / back] Vaccine protection Spleen (H&E) Liver (H&E) Liver (Immuno - HC)

Hepatocyte necrosis MVA-empty Lymphocyte loss apoptotic bodies & multiple infiltration of macrophages

Occasional staining MVA-GP

..inflammatory response to antigen • Little / no pathology in liver or spleen of vaccinated mice. • Pathology in vaccinated liver appears to be an immune response to cells positive for viral protein.

42 Protection: Gross histopathological disease changes in un-vaccinated animals. Phase I Study of a MVA based vaccine

o Bio-equivalence of original and new MVA-GP constructs in mouse immunogenicity study

o Manufacture and release of acceptable GMP MVA-GP material

o Development of clinical immune assays

o Phase I Clinical Trial

43 Innovate UK / BBSRC …2015>> Alternative vaccine approaches

DNA-based vaccines expressing the CCHFv M segment Spik K, et al., (2006) Vaccine 24: 4657–66.

Recombinant tobacco leaves expressing GN and GC Ghiasi et al., (2011). Clin Vaccine Immunol 18: 2031–7.

Inactivated virus from cell culture Canakoglu et al., (2015). PLOS NTD.

CCHF Virus Like Particles:

Anti Tick vaccines: Cement & midgut antigen (Bm86) partially protective Labuda et al 2006 PLOS one 2 (4) e24

44 Alternative vaccine approaches for CCHF 45 Outbreak response & control 100 Rapid Domestic animal epizootic Control Benefits Response 90 Human outbreak to animals 80 Wildlife Control Anticipate Benefit to 70 Early New Trends & Humans 60 Forecasting Detection Vector

50 Control

40

30

20 Number of Cases 10

0 -120-115-110 … -30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30 35 40 1. Preparedness / surveillance 2. Alert 3. Control 4. Recovery

46 Conclusions

• Highly flexible genome that can recombine and re-assort leading to new many virus possibilities

• Changing climate affects distribution of disease vectors and reservoirs

• Human interactions may increase transmission opportunities

Research programmes to o Keep track of virus variants and pathogenic strains . International collaboration essential o Map tick vectors / vector control strategies o Development of therapeutics and vaccines

47 Conclusions… Thanks for listening Babak Afrough Barry Atkinson Laura Bonnie Karen Buttigieg Andrew Bosworth Miles Carroll John Chamberlain Stuart Dowall Victoria Graham Liana Kafetzopoulou

Sali Ahmeti, David Bukbuk, Giorgi Babuadze, Bushra Jamil, Hakan Leblebicioglu, Yper Hall Talgat Nuranov, Mike McKenna, Bernard Moss, Masaayki Shimojima, Farida Tishkova Sarah Lumley Gillian Slack Rebecca Surtees Irene Taylor Anitha Varghese Robert Watson Deborah Wright

48 Local lab (Virology & Pathogenesis) international collaborators & funding