The mosquito-borne dengue (DEN) viruses, members of the Flaviviridae family, contain a single-stranded positive-sense RNA genome. A single polypeptide is co-translationally processed by viral and cellular proteases generating three structural proteins (C, M, and E) and at least seven non-structural proteins. The genome organization of the DEN viruses is 5?-UTR-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-UTR-3? (UTR ? untranslated region, C ? capsid, prM ? membrane precursor, E ? envelope, NS ? nonstructural). There are four dengue virus serotypes (DEN1, DEN2, DEN3, and DEN4) that circulate in tropical and subtropical regions of the world inhabited by more than 2.5 billion people. Annually, there are an estimated 50-100 million dengue infections and hundreds of thousands of cases of the more severe and potentially lethal dengue hemorrhagic fever/shock syndrome (DHF/DSS) with children bearing much of the disease burden. DEN viruses are endemic in at least 100 countries and cause more human disease than any other mosquito-borne virus. In at least eight Asian countries, the DEN viruses are a leading cause of hospitalization and death in children. Unfortunately, many countries affected by DEN viruses have very limited financial resources for healthcare, and the economic burden of DEN disease is considerable. As such, an economical vaccine that prevents disease caused by the DEN viruses is a global public health priority. The cost-effectiveness, safety, long-term immunity, and efficacy associated with the live attenuated vaccine against yellow fever virus, another mosquito-borne flavivirus, serves as a model for the feasibility of a live attenuated DEN virus vaccine. However, the development of a live attenuated dengue vaccine has been complicated by several factors. First, it has been difficult to develop monovalent vaccines against each of the four serotypes that exhibit a satisfactory balance between attenuation and immunogenicity. Second, an effective live attenuated dengue virus vaccine must consist of a tetravalent formulation of components representing each serotype because multiple serotypes typically co-circulate in a region, each DEN serotype is capable of causing disease, and the introduction of additional serotypes is common. In addition, the association of increased disease severity (DHF/DSS) in previously infected persons undergoing an infection by a different dengue serotype necessitates a vaccine that will confer long-term protection against all four serotypes. Third, it has been difficult to formulate a tetravalent vaccine with low reactogenicity that induces a broad neutralizing antibody response against each DEN serotype. Fourth, a dengue vaccine must confer protection against a wide range of genetically diverse subtypes that are dispersed around the world and can be readily introduced into a new region by international travel. Fifth, a dengue vaccine must be produced economically so that it can be made available to populations that need it most. We have tried to address these issues as part of a program to generate a live attenuated tetravalent dengue virus vaccine. To maximize the likelihood that suitable vaccine candidates would be identified, monovalent vaccine candidates for DEN1-4 were generated by two distinct recombinant methods and found to be attenuated and immunogenic in mouse and rhesus monkey models. In one method, deletion of 30 contiguous nucleotides from the 3? UTR of wild type cDNA clones of DEN1-4 was used to generate vaccine candidates. Specifically, the deletion of nucleotides 10478-10507 of the 3? UTR (del30) of recombinant wild type DEN4 yielded a vaccine candidate, rDEN4del30, which is safe, attenuated, and immunogenic in rhesus monkeys and humans (9). Incorporation of the del30 mutation into infectious cDNA clones of DEN1 and DEN2, but not DEN3, wild type virus at a site homologous to that in DEN4 attenuated these viruses for rhesus monkeys. Using a second method, antigenic chimeric viruses were generated by replacing wild type M and E structural genes of rDEN4del30 with those from DEN2 or DEN3, and the resulting chimeric viruses were attenuated and immunogenic in rhesus monkeys. Importantly, these vaccine candidates retain wild type structural proteins to maximize infectivity, thereby decreasing the potential for virus interference. In addition, immunity is induced by an authentic wild type E protein that will likely increase the magnitude and breadth of the neutralizing antibody response. In the present study, three tetravalent vaccine formulations, TV-1, -2, and -3, of the afore-mentioned monovalent DEN virus vaccine candidates were compared to a tetravalent formulation of wild type DEN viruses, T-wt, for replication in SCID-HuH-7 mice or for replication and immunogenicity in rhesus monkeys. TV-1 consists of recombinant DEN1, -2, -3, and -4, each with a 30 nucleotide deletion in the 3? untranslated region (del30). TV-2 consists of rDEN1del30, rDEN4del30, and two antigenic chimeric viruses, rDEN2/4del30 and rDEN3/4del30, both also bearing the del30 mutation. TV-3 consists of rDEN1del30, rDEN2del30, rDEN4del30, and a tenfold higher dose of rDEN3/4del30. Attenuation and virus interference were assessed in SCID-HuH-7 mice for TV-1, TV-2, and T-wt. TV-1 and TV-2 were attenuated in SCID-HuH-7 mice with minimal interference in replication among the virus components. In rhesus monkeys, attenuation, virus interference, and neutralizing antibody responses were evaluated for each formulation and compared to T-wt. TV-1, -2, and -3 were attenuated in rhesus monkeys as measured by duration and peak of viremia. Each monkey immunized with TV-1 and TV-3 seroconverted to the four DEN components by day 28 with neutralization titers ranging from 1:52 to 1:273 and 1:59 to 1:144 for TV-1 and TV-3, respectively. In addition, the timing and effect of booster vaccinations was determined. TV-2 induced low antibody titers to DEN2 and DEN3, but a booster immunization after 4 months increased the neutralizing antibody titers to greater than 1:100 against each serotype, and elicited broad neutralizing activity against 19 of 20 DEN subtypes. A single dose of TV-2 induced protection against wild type DEN1, DEN3 and DEN4 challenge, but not DEN2. However, two doses of TV-2 or TV-3 induced protection against DEN2 challenge. Analysis of these results and protection studies in rhesus monkeys indicates that two tetravalent formulations, TV-2 and TV-3, possess properties of a successful DEN vaccine and can be considered for evaluation in clinical trials. Efforts are underway to make clinical lots of each of the four dengue virus serotypes.