In 2004, chikungunya virus (CHIKV) re-emerged from East Africa to cause devastating epidemics of debilitating and often chronic arthralgia that have affected millions of people in the Indian Ocean Basin and Asia. More limited epidemics initiated by travelers subsequently occurred in Italy and France, as well as human cases exported to most regions of the world, including the Americas where CHIKV could become endemic. Because CHIKV circulates during epidemics in an urban mosquito–human cycle, control of transmission relies on mosquito abatement, which is rarely effective. Furthermore, there is no antiviral treatment for CHIKV infection and no licensed vaccine to prevent disease. Here, we discuss the challenges to the development of a safe, effective and affordable chikungunya vaccine and recent progress toward this goal.

Abstract. Three tetravalent formulations of chimeric dengue (DENVax) viruses containing the pre-membrane and envelope genes of serotypes 1-4 expressed by the attenuated DENV-2 PDK-53 genome were tested for safety, immunogenicity, and efficacy in cynomolgus macaques ( Macaca fascicularis ). Subcutaneous injection of the DENVax formulations was well-tolerated. Low levels of viremia of only one of the four vaccine viruses were detected yet virus neutralizing antibody titers were induced against all four dengue virus serotypes after one or two administrations of vaccine. All animals immunized with the high-dose formulation were protected from viremia, and all immunized animals were completely protected from DENV-3 and DENV-4 challenge. A lower dose of DENVax formulation partially protected animals from DENV-1 or DENV-2 challenge. In contrast, all control animals developed high levels of viremia for multiple days after challenge with DENV 1-4. This study highlights the immunogenicity and efficacy of the tetravalent DENVax formulations in nonhuman primates.*Address correspondence to Jorge E. Osorio, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706. E-mail: osorio@svm.vetmed .wisc.edu †These authors contributed equally to this article. PROTECTION BY A DENGUE VACCINE IN MACAQUESderived (DENV-1 16007, DENV-2 16681, DEN-3 16562, and DENV-4 1036). 21 Virus plaque titration was performed under double agarose overlay in six-well plates of confluent Vero cells as described. 20,22 The second agarose overlay containing neutral red vital stain was added four or seven days after infection, depending on the virus plaque phenotypes. Plaques were counted for three consecutive days after the second agarose overlay.Tetravalent DENVax vaccine formulations. The construction and characterization of the four DENVax viruses has been reported. 21 To complete preclinical development of DENVax, new viral stocks were generated by introducing RNAs transcribed from infectious cDNA clones (pD2-PDK53 and chimeric pD2/1, /3, and /4 plasmids) into the certified vaccine production Vero cells by electroporation as described 21 under Good Manufacturing Practice (GMP) conditions at Shantha Biotechnics. The viruses were amplified, plaque purified, characterized, and sequenced for each of the four DENVax serotypes. On the basis of these analyses, a formal pre-master virus seed was chosen for each DENVax serotype and was then amplified to generate the master virus seed for each serotype (Huang, C. and others, unpublished data).For this study, individual pre-master seed viruses were used to make non-GMP surrogate master seeds in serum-free media as follows. Viruses diluted in DMEM to achieve a multiplicity of infection of 0.001 were adsorbed for 1.5 hours onto rinsed Vero cell monolayers at 37°C. After adsorption, the monolayers were rinsed three times with phosphate-buffered saline, and then fresh serum-free DMEM medium was added. The viruses were grown for 8-12 days in an atmosphere of 5% CO...

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Chikungunya virus (CHIKV) is a reemerging arbovirus capable of causing explosive outbreaks of febrile illness, polyarthritis, and polyarthralgia, inflicting severe morbidity on affected populations. CHIKV can be genetically classified into 3 major lineages: West African (WA); East, Central, and South African (ECSA); Indian Ocean (IOL); and Asian. Additionally, the Indian Ocean (IOL) sublineage emerged within the ECSA clade and the Asian/American sublineage emerged within the Asian clade. While differences in epidemiological and pathological characteristics among outbreaks involving different CHIKV lineages and sublineages have been suggested, few targeted investigations comparing lineage virulence levels have been reported. We compared the virulence levels of CHIKV isolates representing all major lineages and sublineages in the type I interferon receptor-knockout A129 mouse model and found lineage-specific differences in virulence. We also evaluated the cross-protective efficacy of the IOL-derived, live-attenuated vaccine strain CHIKV/IRESv1 against the Asian/American CHIKV isolate YO123223 in both murine and nonhuman primate models, as well as the WA strain SH2830 in a murine model. The CHIKV/IRES vaccine provided protection both in mice and in nonhuman primate cohorts against Caribbean strain challenge and protected mice against WA challenge. Taken together, our data suggest that Asian/American CHIKV strains are less virulent than those in the Asian, ECSA, and WA lineages and that despite differences in virulence, IOL-based vaccine strains offer robust cross-protection against strains from other lineages. Further research is needed to elucidate the genetic basis for variation in CHIKV virulence in the A129 mouse model and to corroborate this variation with human pathogenicity.

BackgroundWe have developed a manufacturing strategy that can improve the safety and genetic stability of recombinant live-attenuated chimeric dengue vaccine (DENVax) viruses. These viruses, containing the pre-membrane (prM) and envelope (E) genes of dengue serotypes 1–4 in the replicative background of the attenuated dengue-2 PDK-53 vaccine virus candidate, were manufactured under cGMP.Methodology/Principal FindingsAfter deriving vaccine viruses from RNA-transfected Vero cells, six plaque-purified viruses for each serotype were produced. The plaque-purified strains were then analyzed to select one stock for generation of the master seed. Full genetic and phenotypic characterizations of the master virus seeds were conducted to ensure these viruses retained the previously identified attenuating determinants and phenotypes of the vaccine viruses. We also assessed vector competence of the vaccine viruses in sympatric (Thai) Aedes aegypti mosquito vectors.Conclusion/SignificanceAll four serotypes of master vaccine seeds retained the previously defined safety features, including all three major genetic loci of attenuation, small plaques, temperature sensitivity in mammalian cells, reduced replication in mosquito cell cultures, and reduced neurovirulence in new-born mice. In addition, the candidate vaccine viruses demonstrated greatly reduced infection and dissemination in Aedes aegypti mosquitoes, and are not likely to be transmissible by these mosquitoes. This manufacturing strategy has successfully been used to produce the candidate tetravalent vaccine, which is currently being tested in human clinical trials in the United States, Central and South America, and Asia.