Chikungunya virus vaccines?

QIMR-B, >600 scientists students & staff

Group Leader; Inflammation Biology QIMR B-PRF NHMRC Australia-Prof Griffith University & James Cook University-Adj Ass Prof University of Queensland.-Member; Australian Infectious Disease Res. Centre.  TEL +61 7 3362 0415email Andreas.Suhrbier@qimrberghofer.edu.au

www.qimrberghofer.edu.au/page/Lab/Inflammation_Biology/

Prof Andreas Suhrbier,QIMR Berghofer

Medical Research Institute, Brisbane, Queensland, Australia

- CHIKV is a biosafety level 3 pathogen (PC2 in Singapore).

- Category C Priority Pathogen by the National Institute of Allergy and Infectious Disease (USA).

- US Army, CHIKV recognized as a potential bioweapon.

- Information export controlled under Defense Trade Controls Act, 2012 (Australia)

“chikungunya” derived from the Makonde language (Tanzania) means "that which bends up" referring to the severe joint-pain-induced posture of afflicted individuals

Vadilal Sarabhai Hospital, Ahmadabad, India

Virus OccurrenceChikungunya virus Large sporadic epidemics every 2-50 years

Ross River virus Mean of ≈4,000 cases per annum in Australia.Also an epidemic (1979/80) >60,000 cases

Barmah Forest virus Mean of ≈ 1000 cases per annum in AustraliaSindbis virus family Karelian fever Ockelbo virus Pogosta virus

Rare (Karelia, West Russia)Mean ≈30 cases per annum (Sweden)

Mean ≈140 (range 1-1282) cases p.a. (Finland) O'nyong-nyong Igbo Ora Rare epidemics, >2 million cases in 1959-61

Mayaro Small outbreaks (30-100 cases)

ALPHAVIRUSES THAT CAUSE ARTHRITIC DISEASE IN HUMANS

- Transmitted by mosquito (arboviruses)- Single stranded positive sense RNA virus, ≈12 kb genomes.- Symptomatic infections nearly always associated with weeks to months polyarthritis/polyarthralgia.

Suhrbier, A. et al. 2012. Nature Rev. Rheumatol. 8, 420–429.

Approximate geographical locations of diseases associated with arthritogenic alphaviruses . For CHIKV disease, locations of documented large outbreaks are shown; epidemics prior to 1952 are shown in dashed lines and were initially classified as outbreaks of dengue, but were likely to have been due to CHIKV. *Geographical locations of RRV and BFV diseases overlap, with BFV restricted to the Australian mainland. ‡Main location of diseases caused by the Sindbis virus family. § O’nyong-nyong virus disease outbreaks in 1959–1961 (East Africa), 1996–1997 (Uganda), 2003 (West Africa). Abbreviations: BFV, Barmah Forest virus; CHIKV, chikungunya virus; RRV, Ross River virus Suhrbier, A. et al. 2012. Nature Rev. Rheumatol.

Largest ever CHIKV epidemic started 2004; Estimated 1.4-6.5 million cases. (Imported cases in ≈ 40 countries – not shown)

CHIKV has been in

the USA, 1827/8

First ever CHIKV

transmission in Europe Reached

PNG2013

>1 million CHIKV cases in the Americas

2014

First chikungunya case locally acquired in the United States reported in Florida July 2014

CHIKV EPIDEMIC 2005/6, REUNION ISLAND (FRANCE)

>250 deaths – often elderly with comorbidities and very young

High attack rate -266,000 cases of CHIKV disease were reported (38% of the population). (50% Grande Comore Island in 2005)

Rapid rise in case numbers -increased to 130,000/month in 4 months-45,000 cases during the week of 29 Jan, 2006.

100’s of imported case in FranceEurosurveillance, Volume 11, Issue 34, 24 August 2006

Reunion Island, French holiday destination

Reun

ion

case

s

Impo

rted

cas

es

The Indian Ocean/ Reunion Island epidemic was

associated with a new clade of CHIK viruses

Mutation allowed efficient CHIK virus

transmission by A. albopictus

East-,Central-, and South-African (ECSA) phylogroup

Schuffenecker et al Plos Med July 2006 3 ( 7 ) e263

Normal vector A. aegypti

An aggressive biter – the BBQ stopper

© Landcare Research

Aedes albopictus; a global invader Tiger mosquito

Intercepted

Native

Introduced

Virus%

asympto-matic

Fever Rash Myalgia Arthralgiaarthritis

CHIKV 5-18% 90% 40-50% 90% >95%

RRV 55-75% 20-60% 40-60% 40-80% 80-100%3-6 months

BFV ? 50% 50-100% 50-80% 70-95%

SINV Very common 15-40% 90% 50% 95%

ONNV - 80-100% 70-90% 70% 60-100%

MAYV 8% 100% 30-50% 75% 50-90%

A Suhrbier, MC Jaffar-Bandjee, P Gasque. 2012. Arthritogenic alphaviruses - an overview. Nat Rev Rheumatol. 8(7):420-9.

Disease characterised by acute and chronic

symmetrical polyarthritis-

polyarthralgia.

ELISA based

serology

CHRONIC DISEASE

CHIKV SEVERE MANIFESTATIONS (RARE)

Severe disease manifestation more prominent in the elderly, the very young & in patients with co-morbidities e.g. hypertension, lupus or cardiac disorders.

Mother to child transmission. Reunion experience: 19 children born to viraemic mothers in 7,504 pregnancies

• About half the children borne to viraemic mothers became infected. • About half the infected neonates developed serious disease; haemorrhage, DIC and/or cardiac and neurological manifestations (often leading to permanent disabilities)

Prior to the recent epidemic, chikungunya was not generally considered to be a fatal illness

Reunion Island 260,000 CHIK cases 260 deaths (0.1%)

Mostly >75 years old

Ahmedabad (2006) 60,777 suspected CHIK cases

2944 more deaths than in same period of previous year (4.8%)Mavalankar et al 2008

For 610 atypical cases of CHIK: 222 were severe, 65 died.

Economopoulou et al 2009

Developed country mortality 0.01% – 0.1%

similar to influenza

Haemorrhage & shock (occasional)Rudd et al J Virol. 2012. 86(18):9888-98.

Chronic disease

The main burden of CHIKV disease is chronic polyarthritis/polyarthralgia

Often several months occasionally > 1 year (Hoarau et al. 2010. J Immunol 184:5914-27)

Viral arthritis is likely due to presence or persistence in joints of virus or viral products that stimulate innate & cognate immune responses

Molecular mimicry – an attractive hypothesis, but

little/no evidence.

Virus, viral antigens and/or viral RNA/DNA found in arthritides caused by

Chikungunya virus (Hoarau et al. 2010)Ross River virusRubellaEchovirusParvovirus B19 Caprine arthritis encephalitis virus Avian reovirus AdenovirusCMV EBV Varicella

Virus infections can cause auto-immune responses butno good evidence these are

responsible for arthritic disease or result in

autoimmune disease

A. Suhrbier, S. Mahalingham. The immunobiology of viral arthritides. 2009. Pharmacol Ther. 124(3):301-8.

Rash – anti-histamine, calamine lotions (pruritis) Indian J Dermatol. 2010 55(1): 64–67

Rheumatic symptoms/fever NSAIDS/paracetomol - can provide relief, but often inadequate.

Rectal NSIADs (suppository)

Injectable NSAIDs in emergency settings? Drug Saf. 1993 Nov;9(5):380-93.

NSAIDS plus steroids - some benefit - J. Rheumatol. 4, 94–101. (2009).but must consider side effects of steriods

J Clin Rheumatol.10:326-30 (2004)

Chloroquine - ineffective - J. Med. Virol. 83, 1058–1059 (2011).- Arthritis Rheumatol. 2014 Feb;66(2):319-26.

Methotrexate - benefit in some chronic patients (RA-like disease) Expert Rev. Anti Infect. Ther. 8(9), 987–996 (2010). J Assoc Physicians India. 59:83-6 (2011)

No benefit in acute RRV mouse model PLoS One. 2013. 8(8):e71146

Treatments

Entity Vaccine type Pre-

Clinical

Phase

I

Phase

II

Phase

III

USAMRIID Live, attenuated (TSI-GSD-218, MRC-5 attunated strain of 15561) (1998) (2000)Valnova/Karolinska Inst. Live, attenuated (CHIKV-Δ5nsP3)

Takeda/UTMB Live, attenuated (CHIKV/IRES)

Arbovax/NC State Uni Live, attenuated (transmembrane deletion)

UTMB Live, attenuated chimeric (various alphavirus backbones)

Themis Bioscience/Inst Pasteur Live, vectored (measles virus) (2015)

Profectus/Yale/UTMB Live, vectored (VSVΔG-CHIKV) 2016?

Karolinska Inst/CSIC Madrid Live, vectored (MVA-CHIKV E1E226KE3)

Uni Wisconsin/Takeda Live, vectored (MVA-CHIKV E2E3)

NIAID/Leidos Biomed Virus-like particles (HEK293 production process) (2014)

TI Pharma/Wageningen Uni Virus-like particles (Baculovirus/insect cell production process)

Merck Virus-like particles (Baculovirus/insect cell production process)

Bharat Biotech Inactivated (various strains, various methods)

Indian Immunological Inactivated (formalin-treated 181/25 from US Army) 2016?

DRDE India Inactivated (formalin-treated India 2006 isolate)

Nanotherapeutic Inc (from Baxter) Inactivated (proprietrary adjuvant formulation)

Medigen DNA (plasmid-launched 181/25 live attenuated)

DRDE India Recombinant subunit (E coli expressed E1/E2)

National Inst. Virology, India Recombinant subunit (E coli expressed E2)

Sementis (Australia) Recombinant SCV (live recombinant attenuated vaccina)

Candidate CHIKV vaccines in developmentVACCINES

Early CHIKV vaccines• Formalin-inactivate vaccine; human study (Harrison et al 1971 J Immunol 107; 643-647).

• Live-attenuated vaccine. Phase II human trial (Edelman et al., 2000).

Side effects included arthralgia

• 25 participants were enrolled 10 μg (n=5), 20 μg (n=10), and 40 μg (n=10).

• Neutralising antibodies were detected in all dose groups after the second vaccination

• All injections were well tolerated, with no serious adverse events reported.

Lancet 2014; 384: 2046–52

Manufacturing costs? Memory? Adjuvant not used but could provide benefits.

Lancet Infect Dis 2015; 15: 519–27

• 42 participants to receive the low dose (n=12), the medium dose (n=12), or the high dose (n=12)

• Second vaccination resulted in a 100% seroconversion for all participants

• No vaccination-related serious adverse events were recorded.

Overcoming measles immunity especially in recently vaccinated children requires dose increases increasing risk of adverse events

• Antibodies are protective (T cells have a minor role)

• T cells also associated with arthritis (induction of CD4 T cells in the absence of good antibodies in mouse models promotes arthritis upon challenge – Poo et al Plos NTD 2014)

Market for CHIKV vaccines?

• A single shot, rapid onset of protective immunity.

• Capacity for rapid manufacture and deployment

• Low cost, highly stable (afflicted countries often resource poor)

Protective correlate

CHIKV vaccines – some considerations

Tourists? Army? General population?

Desirable for an epidemic setting

Sporadic epidemics (2-50 years), usually self limiting disease, mortality low and usually restricted to elderly and patients with comorbidities.

7 suite Biosafety level 3

floor with equipment

animal houses & insectaries.

Large PC2 mouse breeding facility 14,000 cage capacity

Research Innovation 2012; Battling viral rheumatism

>140 genetically modified mouse strains

QIMR Berghofer

Reunion

Control

Days post CHIKV inoculation

7

8

9

10

0 2 4 6 8 10 12 14Foot

wid

th x

bre

adth

, m

m2 ±

SE

Adult wild-type mouse model of chikungunya virus infection (viraemia) and disease (foot swelling/arthritis)

Control Reunion Islandisolate

Vira

emia

, log

10CC

ID50

/ml ±

SE

0

1

2

3

4

5

6

0 1 2 3 4 5 6Days post CHIKV inoculation

ViraemiaArthritis

Female C57BL/6 mice > 6 weeks old

Dominance of monocytes/macrophages and NK cells in the swollen feet of CHIKV infected mice

Cells FACS markers% of cells

7 days post infection± SE

Monocytes CD11b+, F4/80lo/- 45 ± 9.9Macrophages F4/80+, CD11b+ 21 ± 4

NK cells NK1.1+ 16.3 ± 6CD4 T cells CD3+, CD4+ 5.2 ± 1.3CD8 T cells CD3+, CD8+ 1.2 ± 0.2

B cells CD19+ 4.6 ± 2

Conventional DC

CD11c+, F480-, NK1.1-

B220-, PDCA1-

6.4 ± 1.1

Plasmacytoid DC

CD11c+, B220+, PDCA1+ 3.8 ± 2.1

FACS

Virus strain LR2006-OPY1injected s.c. into the foot

Air hole

J. Virol. Meth. 1995. 52:51-54Many virus

isolates contaminated

with mycoplasmas

MYCOPLASMA NO MYCOPLASMAHela and Hoechst staining

MycoAlert™ Mycoplasma Detection Kit

Results in < 20 min

Grow virus prep on LCLs (non permissive for alphavirus infection for a week) prior to testing

RAW264-HIV-LTR-LUC cell lineDetection sensitivity 5-10 pg/ml E. coli LPS

“High sensitivity” endotoxin assay detection limit

Clear inhibition of alphavirus infection in macrophage cell lines

ENDOTOXIN CONTAMINATION (often in FCS, glassware, trypsin, sucrose etc)

Johnson.et al 2005. J Biol Chem. 280(6):4037-47

65C 30 mins to inactivate virus

Examples of the evaluation of vaccines in the adult wild-type CHIKV mouse model

1. Simple inactivated whole virus vaccineGardner et al. J Virol. 2010 84(16):8021-32

2. rBaculovirus VLP vaccine Metz et al PLoS NTD 2013;7:e2124A collaboration with Wageningen University, The Netherlands.

3. rAdenovirus vaccine Wang et al 2011 Vaccine 29;2803–2809A commercial collaboration with GenPhar Inc., USA.

4. rSementis Copenhagen Vector (SCV) A commercial collaboration with Sementis, Australia

4. Foroderm transcutaneous immunisation A collaboration with University Queensland, Australia

Model adopted by EU Integrated CHIKV Research Program, e.g.• rMVA vaccine (García-Arriaza et al. J Virol. 2014. 88(6):3527-47)• Attenuated vaccine (Hallengärd et al. J Virol. 2014 88(5):2858-66)

Rapid high volume production

CHIK

V VL

P pr

oduc

tion

in S

f21

inse

ct c

ells

Metz et al, 2013

Single shot:Complete protection

against viraemia

Complete protection

against arthritic disease

VIRAEMIA

ARTHRITIS

Gorben Pijlman, Wageningen University

The Sementis Copenhagen Vector (SCV) is a genetically attenuated, live virus vaccine

vector, based on the Copenhagen strain of the Vaccinia Virus

COI: A Suhrbier is on the SAB of Sementis

0123456789

0 1 2 3 4 5

SCV -CHIKControl vectorPBS

Log 10

CCI

D50

/ml ±

SE

Day post challenge

0

10

20

30

40

50

60

70

80

0 2 4 6 8 10 12 14 16Day post challenge%

incr

ease

in fo

ot s

wel

ling

(art

hriti

s) +

SE

SCV -CHIKControl vectorPBS

Complete protection from viraemia and arthritis by single vaccination with rSCV-CHIKV vaccine

SCV can be produced from a biotechnology “friendly” custom cell line under serum and protein free conditions in bioreactors

ForodermTM : Novel Drug Delivery TechnologyDr Tarl Prow et al University Queensland, Australia

Topical inactivated CHIKV+QuilATopical inactivated CHIKV

Foroderm plus QuilA

Sub. cut. inactivated CHIKV

NEGATIVE CONTROLS

POSITIVE CONTROL

Foroderm + inactivated CHIKV

0

10

20

30

40

50

60

70

80

90

100

110

120

0 5 10 15 20 25 30 35

Days post challenge% in

crea

se in

foot

sw

ellin

g (a

rthr

itis)

+ S

E

Arthritis

0

1

2

3

4

5

6

0 1 2 3 4 5 6

Days post challenge

Vira

emia

, log

10 C

CID

50 m

l + S

EViraemia

Single application of inactivated CHIKV vaccine

with Foroderm provides complete protect against

Arthritis and Viraemia

Potential for self administration

Epidemics Year No. of casesTanzania 1952 ?Central East Africa 1959-62 ≈2 m (O'nyong'nyong)Thailand 1962 ≈40,000India 1963/4 >100,000Vietnam Myanmar 1975 ?Indonesia 1982 ?Reunion Island 2005-2006 >250,000 India/Asia/Caribbean/S America 2006-2015 1.4-6.5 m

Sporadic epidemics (2-50 years)

USA FDA unlikely to give approval on animal data alone

Phase II/III testing?

Outbreak size and location

unpredictable

By the time ethics approval given, trial infrastructure set up and volunteers vaccinated. Epidemic has passed or does not arrive.

CAN EMERGING ECONOMIES DO THEIR OWN APPROVAL ?E.g. Brazil ? India ?

• Use real insect repellent (eg DEET)• Wear long sleeved clothes• Uses mats, coils, screens indoors• Use impregnated bed nets• Spraying

•Remove peri-domestic breeding sites

•Add fish to ponds and water tubs

Mosquito control

Release of Wolbachia

infected mosquitos into

the wild population results

in spread of the Wolbachia

infection into the population Hoffmann et al 2011. Nature. 2011;476:454-7.

Wolbachia infected

A . aegyptimosquitos replicate

dengue virus, chikungunya

virus & yellow fever virus less

efficiently

Hurk et al 2012 PLoS Negl Trop Dis 6(11): e1892.

Walker et al., 2011. Nature 476, 450–453.

Scot ONeil

+ Wol - Wol

RT PCR

Wolbachia is a genus of bacteria

which infects insects

Make mosquitos less able to transmit CHIKV with Wolbachia

Surprising overlap in inflammatory signature

between CHIKV and rheumatoid arthritis.

Drugs being used/developed for RA may find application for

treatment of CHIKV.

(Nakaya et al, 2012. Arth Rheum 64 (11):3553-63)

Heat map showing the relative expression levels of the 282 up-regulated genes in CHIKV infected mouse feet et that were enriched in RA patients. Columns represent the 5 control, and 5 RA samples in the study and their respective inflammation scores.

-2 20

SD from mean

Healthy RA28

2 ge

nes

UP

regu

late

d in

CH

IKV

arth

ritis

and

UP

regu

late

d in

RA

patie

nts

-

4.5 5 8 7 7.5RA inflammation score

Improving treatment options

Thanks to • Luis Mateo and Rebecca Pawliw (Alere, Brisbane, Australia) for supply of purified inactivated CHIKV.• Clay Winterford (QIMR) for histology/immunohistochemistry

Funding National Health & Medical Research Council, AustraliaAustralian Infectious Disease Research CenterQueensland Tropical Health Alliance

QIMR-BJoy GardnerPenny RuddItaru Anraku

Thuy T Le Lee Major

Wayne A. Schroder

Ecole Nationale Vétérinaire, Nantes, FranceThibaut Larcher et al

Commissariat à l'Énergie Atomique (CEA), ParisKarine Labadie, Pierre Roques et al

University of Texas Medical BranchStephen Higgs et al

University of QueenslandTarl Prow Roy Hall

Alex Khromykh University of Wageningen

Gorben Piljman et al

Griffith UniversitySuresh Mahalingham

Nestor Rulli

Emory Vaccine CenterHelder Nakaya, Bali Pulendran

Université de la Réunion Hôpital Félix Guyon La Réunion, France Marie-Christine Jaffar-Bandjee Philippe Gasque

French guidelines for the management of chikungunya (acute and persistent presentations). November 2014.Simon et al. Med Mal Infect. 2015 Jul;45(7):243-63.

Queensland chikungunya management plan 2014–2019. https://www.health.qld.gov.au/cdcg/documents/chikungunya-management-plan.pdf (accessed 14/7/15).

Chikungunya virus. Centers for Disease Control and Prevention. http://www.cdc.gov/chikungunya/fact/index.html (accessed 17/6/15).

Information resources