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...
Cryopreserved peripheral blood mononuclear cells (PBMC) constitute an important component of immune monitoring studies as they allow for efficient batch- testing of samples as well as for the validation and extension of original studies in the future. In this study, we systematically test the permutations of PBMC thawing practices commonly employed in the field and identify conditions that are high and low risk for the viability of PBMC and their functionality in downstream ELISPOT assays. The study identifies the addition of ice-chilled washing media to thawed cells at the same temperature as being a high risk practice, as it yields significantly lower viability and functionality of recovered PBMC when compared to warming the cryovials to 37 °C and adding a warm washing medium. We found thawed PBMC in cryovials could be kept up to 30 minutes at 37 °C in the presence of DMSO before commencement of washing, which surprisingly identifies exposure to DMSO as a low risk step during the thawing process. This latter finding is of considerable practical relevance since it permits batch-thawing of PBMC in high-throughput immune monitoring environments.
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