Efficacy of a Tetravalent Chimeric Dengue Vaccine (DENVax) in Cynomolgus Macaques

Abstract

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...

Development of DENVax: A chimeric dengue-2 PDK-53-based tetravalent vaccine for protection against dengue fever

“…The individual monovalent vaccines and mixtures of all four DENVax viruses induce neutralizing antibody responses to all four DEN viruses in mice [41] and in monkeys [46]. Although there currently is no DEN-3 or DEN-4 challenge model in adult mice, AG-129 mice immunized with tetravalent DENVax formulations are protected from infection by wild-type DEN-1 or DEN-2 viruses ( [41]and Huang et al, in preparation).…”

“…Although there currently is no DEN-3 or DEN-4 challenge model in adult mice, AG-129 mice immunized with tetravalent DENVax formulations are protected from infection by wild-type DEN-1 or DEN-2 viruses ( [41]and Huang et al, in preparation). Monkeys immunized with tetravalent DENVax formulations are protected from challenge with wild- type DEN-1, DEN-2, DEN-3 or DEN-4 virus [46]. In this study, animals were challenged 30 days post second vaccination and the short timeframe from vaccination to challenge would be considered by some to not be optimal when testing vaccine protective efficacy.…”

“…Transient, low level viremia of only the DENVax-2 component was detected after the primary immunization (≤2.4 log PFU/mL for no more than five consecutive days), yet virus neutralizing antibody titers were induced against all four dengue virus serotypes after one or two administrations of vaccine (see Fig. 3, from table 1 in [46]). Significant neutralizing antibody titers were induced to DEN-1, DEN-2 and DEN-3 by one or two administrations of the tetravalent formulations.…”

“…Early immunogenicity studies in monkeys with the ChiDEN-V viruses were conducted at Mahidol University [47]. More recently, we evaluated the safety and efficacy of three tetravalent formulations containing different ratios of DENVax viruses in cynomolgus macaques (Macaca fascicularis) [46]. A formulation termed 3:3:3:3 contained 10 3 PFU per dose of each of the four DENVax viruses.…”

“…Tetravalent DENVax formulations were tested in preclinical models for safety (Huang et al, in preparation). The DENVax formulations also were tested for efficacy in non-human primates [46].…”