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.
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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) • 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 genome 26S SG promoter 5’cap nsP1 nsP2 nsP3 nsP4 (-) strand RNA synthesis, RNA capping helicase, proteinase RNA synthesis RNA-dependent RNA polymerase C E2 E3 capsid E1 6K envelope glycoproteins •Single stranded, positive-sense RNA genome (11.5 kb) Poly[A]-3’ CHIKV transmission cycles Sylvatic Urban (enzootic with occasional epidemics) (sporadic outbreaks but explosive) Arboreal Aedes spp. Ae. aegypti Ae. albopictus Africa Asia, Indian Ocean Clinical disease • Acute, febrile illness with arthralgia and rash • Typically self-limiting disease, but the highly debilitating arthritic symptoms can last from months-toyears • 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) 2007 2006 2009 2004 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 S. C. Weaver, W. K. Reisen, Antiviral Res, (Oct 23, 2009). 2008 2005 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) Mutations in CHIKV Vaccine Strain TSI-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 Strain TSI-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 Chimeric alphavirus/CHIKV constructs SG VEE/CHIKV nsP1 nsP2 nsP3 nsP4 C E2 E1 nsP3 nsP4 C E2 E1 nsP3 nsP4 C E2 E1 EEE/CHIKV nsP1 nsP2 SIN/CHIKV nsP1 nsP2 VEEV (TC-83), EEEV (BeAr436087), or SINV (AR339) backbone CHIKV La Réunion strain Chimeric CHIKV vaccine candidates are highly attenuated in 6-day-old NIH Swiss mice after IC infection 100 80 % Survival TC-83/CHIKV 60 EEE/CHIKV SIN/CHIKV 40 181/clone 25 WT-CHIKV 20 Logrank test: p=0.001 0 0 2 4 6 8 10 Days after infection 12 14 16 •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, subcutaneous Detection 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 structural proteins under IRES control CHIKV-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 receptordeficient) 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 12 Log10 PFU/ml 10 8 La Reunion wt 6 181/25 Army vaccine CHIK/IRESv1 4 CHIK/IRESv2 2 0 0 1 2 Days after infection Infection with 106 PFU 3 4 Attenuation: Replication in the legs of 6-day-old outbred mice 9 8 Log10 PFU/g 7 6 5 La Reunion wt 4 181/25 Army vaccine 3 CHIK/IRESv1 CHIK/IRESv2 2 1 0 0 2 4 6 Days after infection Infection with 106 PFU 8 10 Attenuation: Brain titers in 6-day-old outbred mice 10 Log10 PFU/g 8 6 La Reunion wt 181/25 Army vaccine 4 IRESv1 IRESv2 2 0 0 Infection with 106 PFU 2 4 6 8 Days after infection 10 Survival of vaccinated mice after IN challenge with CHIKV Ross strain 100 Survival (%) 80 60 Sham-PBS CHIK-IRES1 40 CHIK-IRES2 181/25 Army vaccine 20 0 0 5 10 Days after IN challenge 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 CHIK/IRESv1 Army vaccine 181/25 Foot pad swelling (mm) 0.8 0.6 0.4 0.2 0 1 Sham wtCHIKV Protection of A129 mice from wt-CHIKV infection CHIK/IRESv1 Army vaccine 181/25 Sham 100 % Survival 80 60 40 20 0 1 2 3 4 5 6 7 8 9 Days post infection 10 11 12 13 14 Passive transfer of immune serum protects naïve A129 mice from wt-CHIKV challenge 100 % Survival 80 60 Neat 40 1:2 20 1:4 NMS 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Days post infection 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 •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. Acknowledgements UTMB – Scott Weaver Eryu Wang Kenneth Plante Rodion Gorchakov Justin Darwin Robert Seymour Naomi Forrester Grace Leal UAB, Birmingham – Ilya Frolov Eugenia Volkova Olga Petrakova Tulane National Primate Research Center Chad Roy Marcelo Kuroda CDC, Ft. Collins Ann Powers Inviragen, Ft. Collins Dan Stinchcomb Jill Livengood University of Wisconsin Harry Partidos Jorge Osorio Funding: NIH-NIAID Western Regional Center for Excellence (U54 AI057156), NIH-NIAID U01 AI082202, Merial