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 several distinct recombinant methods and found to be attenuated and immunogenic in mouse and rhesus monkey models. In one method, deletion of 30 contiguous nucleotides (del30) from the 3 prime UTR of wild type cDNA clones of DEN1-4 was used to generate vaccine candidates such as rDEN1del30 and rDEN4del30. Point mutations previously selected for their attenuation phenotype were also introduced into rDEN4del30 to yield vaccine candidates rDEN4del30-200,201 and rDEN4del30-4995. Because the del30 mutation failed to properly attenuate DEN2 and DEN3, alternative strategies were employed to create vaccine candidates for these serotypes. Antigenic chimeric viruses were generated by replacing wild type M and E structural genes of rDEN4del30 with those from DEN1, DEN2 or DEN3, and the resulting chimeric viruses, rDEN1/4del30, rDEN2/4del30, and rDEN3/4del30 were attenuated and immunogenic in rhesus monkeys. Two additional vaccine candidates for DEN3 were generated by 1) exchanging the 3 prime UTR of rDEN3 with that derived from attenuated rDEN4del30 to generate rDEN3-3D4del30, and 2) introducing a pair of 3 prime UTR deletions into DEN3 to create vaccine candidate rDEN3del30/31. Clinical lots of each of these vaccine candidates were manufactured in prior years, however, during the previous year, a clinical lot of rDEN1/4del30 was manufactured to ensure that a back-up candidate is available for DEN1. Although the dengue virus vaccine program is predominantly in a clinical mode at this time, considerable effort is currently devoted to support a number of vital functions, including, 1) manufacture, maintenance, and distribution of clinical lots of vaccines suitable for study in human subjects, 2) submission and laboratory support of IND applications, including the submission this year of an IND for the clinical evaluation of three tetravalent dengue vaccine formulations, 3) support of the five companies/foundations that have licensed our vaccine technology or virus products, which includes consultative visits, preparation and shipping of vaccine seed or clinical lot viruses, and sharing of IND/clinical trial data. Efforts to develop a live attenuated Japanese encephalitis virus (JEV) vaccine are ongoing. It is envisioned that a suitable live attenuated JEV vaccine could be combined with the live attenuated DEN virus vaccine to create a second generation vaccine for the control of these viruses in Southeast Asia. Toward this end, a fully virulent JEV isolate (India/78) was selected as the parent virus for vaccine development based on the pathogenicity of a number of wildtype JEV isolates tested in mice by intracerebral or peripheral inoculation. Genome sequencing of this virus provided the template for creating full-length recombinant cDNA clones of JEV and chimeric cDNA clones containing the JEV structural genes and the DEN4 nonstructural genes. The laboratory is currently in the process of recovering infectious virus from these cDNA constructs.

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