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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 Branch Galveston, Texas. Chikungunya virus (CHIKV). Mosquito-borne alphavirus ( Togaviridae; Alphavirus ) - PowerPoint PPT Presentation
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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