Chikungunya Fever and the Development of Novel Vaccine

Candidates

Paige Adams, D.V.M., Ph.D.Sealy Center for Vaccine Development

University of Texas Medical BranchGalveston, Texas

Chikungunya virus (CHIKV)

• Mosquito-borne alphavirus (Togaviridae; Alphavirus)

• Derived from Makonde word that describes the posture of persons affected by severe joint pains

• First isolated in Tanzania in 1953 by Ross

• Recently emerged, causing major epidemics in India and islands off the east coast of Africa

• Chikungunya fever is often misdiagnosed and easily confused with other febrile-causing viruses.

Alphavirus genomeAlphavirus genome

•Single stranded, positive-sense RNA genome (11.5 kb)

nsP1 nsP2 nsP3 nsP4 C E2 E1

26S SG promoter

5’cap Poly[A]-3’

(-) strand RNA synthesis, RNA

capping

helicase,proteinase

RNA synthesis

RNA-dependent RNA polymerase

capsid envelope glycoproteins

E3 6K

CHIKV transmission cycles

Sylvatic(enzootic with occasional

epidemics)

Africa

Arboreal Aedes spp.

Asia, Indian Ocean

Ae. aegyptiAe. albopictus

Urban(sporadic outbreaks but

explosive)

Clinical disease• Acute, febrile illness with arthralgia and rash

• Typically self-limiting disease, but the highly debilitating arthritic symptoms can last from months-to-years

• No effective treatments or licensed vaccines

• CHIKV has rarely been associated with high case fatalities.

• 2005-2009: Unusual appearance of neurologic disease (encephalitis, encephalopathy, myelopathy, myeloneuropathy) with case fatality rate ca. 0.1%.

CHIKV outbreak: 2005-present

• Islands in the Indian Ocean (Spring 2005 - Fall 2006)– 250,000+ cases; attack rates of ~30%– 1000+ excess deaths associated with CHIKV outbreak in

La Réunion and Mauritius (Josseran 2006, Ramchurn 2008)• Estimated case fatality rate: ~1/1000 pop. (mainly elderly)

• Continental India (Winter 2005 - present)– Up to 6.5 million cases (Mavalankar 2008); attack rates of ~30%– 3000+ excess deaths associated with CHIKV outbreak in

Ahmedadbad, India alone (Mavalankar 2008)

• E1-A226V mutation (Schuffenecker 2006)

– Allows CHIKV to more efficiently infect Ae. albopictus (Vazeille et al., 2007; Tsetsarkin et al., 2008)

Exportation of CHIKV

1. Indian Ocean outbreak locations are popular travel destinations.– 1000+ importations into France alone (2005-2006)– >35 imported U.S. cases (2005-2006), most from India– Exported from India to Hong Kong, Taiwan, Singapore, and China

2. Most of Latin America supports endemic dengue virus transmission by Ae. aegypti.– CHIKV introduction would likely lead to an epidemic, followed by

endemic CHIK.

3. In the U.S. and Europe, Ae. albopictus is plentiful.– CHIKV imported from a traveler could become endemic (or at least

be transmitted locally). Italy, Summer 2007

CHIK epidemic (2004-2009)

S. C. Weaver, W. K. Reisen, Antiviral Res, (Oct 23, 2009).

Movement of 2004-2009 CHIK epidemic

Imported CHIK cases in travelers, 2005-2009

Sites of Asian endemic transmission, 1958-1996

Sites of enzootic CHIKV, East/Central/South African clade

Sites of enzootic CHIKV, West African clade

IND CHIK Vaccine

• TSI-GSD-218 IND vaccine developed by USAMRIID during the 1980s (Levitt et al., 1986)

– Attenuated live vaccine strain 181/clone 25– 18 plaque-to-plaque passages in human embryonic lung

cells (MRC-5)

• Phase II safety trials completed (Edelman et al., 2000)

– 98% developed neutralizing antibodies by day 28, 85% seropositive after 1 year

– 5/59 (9%) healthy adults experienced mild, transient arthralgia

• Potential for mosquito transmission (Turell & Malinoski, 1992)

Gene Nucleotide position

Amino acid position

Amino acid change

nsP1 978 301 Thr Ile

E2 8576 12 Thr Ile

E2 8785 82 Gly Arg

6K 9935 42 Cys Phe

E1 11204 404 Ala Val

Mutations in CHIKV Vaccine StrainTSI-GSD-218 (181/clone 25)

Gene Nucleotide position

Amino acid position

Amino acid change

nsP1 978 301 Thr Ile

E2 8576 12 Thr Ile

E2 8785 82 Gly Arg

6K 9935 42 Cys Phe

E1 11204 404 Ala Val

Mutations in CHIKV Vaccine StrainTSI-GSD-218 (181/clone 25)

nsP1 nsP2 nsP3 nsP4 C E2 E1

SGVEE/CHIKV

nsP1 nsP2 nsP3 nsP4 C E2 E1

nsP1 nsP2 nsP3 nsP4 C E2 E1

VEEV (TC-83), EEEV (BeAr436087), or SINV (AR339) backbone

CHIKV La Réunion strain

Chimeric alphavirus/CHIKV constructs

EEE/CHIKV

SIN/CHIKV

Chimeric CHIKV vaccine candidates are highly attenuated in 6-day-old NIH Swiss mice after IC infection

Logrank test: p=0.001

•Dose-response to the chimeric vaccine candidates.•All survived IN challenge with a virulent CHIKV strain.

Chimeric CHIKV vaccine candidates are highly attenuated in 4-day-old mice after SC infection

Dose: 105 PFU, subcutaneousDetection limit = 0.9 Log10 PFU.

Chimeric CHIKV vaccine candidates are less competent than parental strains of being transmitted

by mosquito vectors

Internal Ribosome Entry Site (IRES)

• Discovered in 1988 in poliovirus and then in encephalomyocarditis virus (EMCV) (Sonenberg, 1988; Wimmer,1988)

• IRES allows 5’-cap independent translation.– Secondary structure of the RNA

elements allows for recruitment of ribosomes and internal initiation.

• EMCV IRES element is not translated in various insect cells (Finkelstein, 1999)

•No infectivity of C7/10 mosquito cells as measured by serial, blind passages using both infectious and RT-PCR assays.•CPE, RT-PCR assays were negative for Ae. aegypti mosquitoes after intrathoracic inoculation.•Fully protective in mice after challenge.

CHIKV recombinants with structuralCHIKV recombinants with structuralproteins under IRES controlproteins under IRES control

CHIKV-La Reunion strainCHIKV-La Reunion strain

Version 1 (CHIK/IRESv1)

Version 2 (CHIK/IRESv2)

Lack of CHIK/IRES infectivity for mosquitoes

• Lack of RT-PCR or CPE detection after 5 blind passages in C6/36 mosquito cells.

• Lack of RT-PCR or CPE detection after intrathoracic inoculation of 103 PFU into Aedes aegypti mosquitoes.

Animal models for CHIK vaccine testing

• Attenuation: Outbred 6-day-old CD1 mice (Ziegler & Tesh, 2008): develop viremia, myositis, and muscle necrosis

• Immunogenicity: 5-6-week-old outbred CD1 mice

• Attenuation and efficacy (challenge): A129 (IFN α/β receptor-deficient) mice (Couderc et al., 2008): fatal disease after wild-type CHIKV infection

• Efficacy (challenge): Outbred adult CD1 mice: fatal disease after IN infection with neuroadapted Ross CHIKV strain

Attenuation: Viremia in 6-day-old outbred mice

Infection with 106 PFU

Attenuation: Replication in the legs of 6-day-old outbred mice

Infection with 106 PFU

Attenuation: Brain titers in 6-day-old outbred mice

Infection with 106 PFU

Survival of vaccinated mice after IN challenge with CHIKV Ross strain

Vaccination with 105 PFU, single dose

Attenuation: Weight change in A129 mice after vaccination

Vaccination with 105 PFU, single dose

Attenuation: Febrile response to vaccination in A129 mice

Vaccination with 105 PFU, single dose

Foot pad swelling 48h after vaccination of A129 mice

Protection of A129 mice from wt-CHIKV infection

Passive transfer of immune serum protects naïve A129 mice from

wt-CHIKV challenge

•Chimeric CHIKV vaccine candidates are highly attenuated in newborn mice and elicit robust neutralizing antibody responses.

•A single dose of chimeric CHIKV fully protects mice against disease after challenge with wild-type CHIKV.

•Chimeric CHIKV vaccine candidates exhibit reduced infectivity for mosquito vectors when compared to parental strains.

•Initial results indicate that novel genetic strategies of placing one or more structural genes under the control of EMCV IRES eliminates mosquito infectivity and also mediates attenuation with retention of immunogenicity.

•CHIK/IRESv1 exhibits greater attenuation than the 181/25 U.S. Army vaccine (no viremia or muscle replication detected in young mice), yet is equally immunogenic and protects as well against wt-CHIKV challenge.

Summary

•Chimeric CHIKV vaccine candidates are highly attenuated in newborn mice and elicit robust neutralizing antibody responses.

•A single dose of chimeric CHIKV fully protects mice against disease after challenge with wild-type CHIKV.

•Chimeric CHIKV vaccine candidates exhibit reduced infectivity for mosquito vectors when compared to parental strains.

•Initial results indicate that novel genetic strategies of placing one or more structural genes under the control of EMCV IRES eliminates mosquito infectivity and also mediates attenuation with retention of immunogenicity.

•CHIK/IRESv1 exhibits greater attenuation than the 181/25 U.S. Army vaccine (no viremia or muscle replication detected in young mice), yet is equally immunogenic and protects as well against wt-CHIKV challenge.

Summary

Acknowledgements

UTMB – Scott WeaverEryu WangKenneth PlanteRodion GorchakovJustin DarwinRobert SeymourNaomi ForresterGrace Leal

UAB, Birmingham – Ilya FrolovEugenia VolkovaOlga PetrakovaOlga Petrakova

Tulane National Primate Research CenterChad RoyMarcelo KurodaMarcelo Kuroda

CDC, Ft. CollinsAnn Powers

Inviragen, Ft. CollinsDan StinchcombJill Livengood

University of WisconsinHarry PartidosJorge Osorio

Funding: NIH-NIAID Western Regional Center for Excellence (U54 AI057156), NIH-NIAID U01 AI082202, Merial