Summary Background Dengue virus is the most serious mosquito-borne viral threat to public health and no vaccines or antiviral therapies are approved for dengue fever. The tetravalent DENVax vaccine contains a molecularly characterised live attenuated dengue serotype-2 virus (DENVax-2) and three recombinant vaccine viruses expressing the prM and E structural genes for serotypes 1, 3, and 4 in the DENVax-2 genetic backbone. We aimed to assess the safety and immunogenicity of tetravalent DENVax formulations. Methods We undertook a randomised, double-blind, phase 1, dose-escalation trial between Oct 11, 2011, and Nov 9, 2011, in the Rionegro, Antioquia, Colombia. The first cohort of participants (aged 18–45 years) were randomly assigned centrally, via block randomisation, to receive a low-dose formulation of DENvax, or placebo, by either subcutaneous or intradermal administration. After a safety assessment, participants were randomly assigned to receive a high-dose DENVax formulation, or placebo, by subcutaneous or intradermal administration. Group assignment was not masked from study pharmacists, but allocation was concealed from participants, nurses, and investigators. Primary endpoints were frequency and severity of injection-site and systemic reactions within 28 days of each vaccination. Secondary endpoints were the immunogenicity of DENVax against all four dengue virus serotypes, and the viraemia due to each of the four vaccine components after immunisation. Analysis was by intention to treat for safety and per protocol for immunogenicity. Because of the small sample size, no detailed comparison of adverse event rates were warranted. The trial is registered with ClinicalTrials.gov, number NCT01224639. Findings We randomly assigned 96 patients to one of the four study groups: 40 participants (42%) received low-dose vaccine and eight participants (8%) received placebo in the low-dose groups; 39 participants (41%) received high-dose vaccine, with nine (9%) participants assigned to receive placebo. Both formulations were well tolerated with mostly mild and transient local or systemic reactions. No clinically meaningful differences were recorded in the overall incidence of local and systemic adverse events between patients in the vaccine and placebo groups; 68 (86%) of 79 participants in the vaccine groups had solicited systemic adverse events compared with 13 (76%) of 17 of those in the placebo groups. By contrast, 67 participants (85%) in the vaccine group had local solicited reactions compared with five (29%) participants in the placebo group. Immunisation with either high-dose or low-dose DENVax formulations induced neutralising antibody responses to all four dengue virus serotypes; 30 days after the second dose, 47 (62%) of 76 participants given vaccine seroconverted to all four serotypes and 73 (96%) participants seroconverted to three or more dengue viruses. Infectious DENVax viruses were detected in only ten (25%) of 40 participants in the low-dose group and 13 (33%) of 39 participants in the high-dose ...

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...

West Nile (WN) virus, a member of the Flavivirus genus, is a mosquito-borne virus of the Japanese encephalitis (JE) serocomplex. The JE serocomplex contains viruses that cause central nervous system infections, such as JE virus in Asia; St. Louis encephalitis virus in the Americas; Rocio virus in Brazil; Murray Valley encephalitis virus in Australia, New Guinea, and New Zealand; and Kunjin virus (reclassified as subtype WN recently) in Australia (44). Before the mid 1990s, WN virus caused sporadic outbreaks of illness, ranging from fever to occasional encephalitis, in Africa, the Middle East, and Western Asia. However, since 1996, WN encephalitis in humans has been reported more frequently in Europe, the Middle East, northern and western Africa, and Russia (33). In 1999, WN virus first emerged in the western hemisphere in New York City and surrounding areas, where the virus caused the deaths of seven humans and numerous birds and horses (15,33). Since then, WN virus has spread throughout most of the continental United States, with more than 4,156 reported human cases and 284 deaths in 2002 (37) and 9,862 cases with 264 deaths in 2003 (12). WN virus activity in humans, birds, and horses has been documented in Canada, the Caribbean, and Central America. The rapid spread of WN virus suggests it may pose a significant public health problem in future years (15). There is no licensed human WN vaccine available to protect at-risk populations from WN illness.Dengue (DEN) viruses are also human pathogens that are transmitted by mosquitoes. These viruses cause illness in millions of people every year throughout tropical regions of the world. Flaviviruses of the DEN serocomplex are classified into four serotypes, DEN 1 to DEN 4 (D1 to D4). Flaviviruses contain a single-stranded positive-sense genomic RNA of approximately 11 kb with the genomic organization 5ЈNCR-CprM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3ЈNCR (where NCR is noncoding region, C is capsid, prM is premembrane, E is envelope, and NS is nonstructural protein). One of the most promising D2 vaccine candidates, strain PDK-53, was derived by passage of the wild-type D2 16681 virus 53 times in primary dog kidney (PDK) cells (48). To study the attenuation loci of the candidate D2 PDK-53 vaccine virus, as well as to develop effective tetravalent DEN vaccines against all four DEN serotypes, we constructed infectious cDNA clones of the D2 viruses (9, 26) and used them to engineer chimeric DEN viruses containing the prM-E genes of D1, D3, or D4 virus in the D2 genetic backbones (18,19). The uncloned D2 PDK-53 vaccine virus contains a mixture of two genotypic variants (26), designated 19) in this report. The PDK53-V variant contains all nine PDK-53 virus vaccine-specific nucleotide mutations, including the Glu-to-Val mutation at amino acid position NS3-250 (26). The PDK53-E variant contains eight of the nine mutations of the PDK-53 vaccine, and the NS3-250-Glu of its parental D2 16681 virus. Our results showed that the phenotypic markers associated with the attenuation of PDK...