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 for developers to formulate a tetravalent vaccine with low reactogenicity that induces a broad neutralizing antibody response against each DEN serotype. Fourth, 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. Clinical lots of each of these vaccine candidates were manufactured in prior years and have been evaluated individually in Phase I clinical trials. During the past year, a replacement lot for our DEN3del30/31 vaccine candidate was manufactured and released for use. To date, tetravalent mixtures have been successfully tested in over 180 human subjects. 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 for the clinical evaluation of tetravalent dengue vaccine formulations, 3) support of the five companies/foundations that have licensed our vaccine technology or virus products, which includes consultative visits and clinical trial planning, development of manufacturing processes, preparation and shipping of vaccine seed or clinical lot viruses, help with sequence analysis, and sharing of IND/clinical trial data. Mosquito-borne Japanese encephalitis virus (JEV) causes the most important viral encephalitis in the AsiaPacific region, accounting for more than 20,000 reported cases and 6,000 deaths annually. The high fatality rate and frequent residual neuro-psychiatric sequelae in survivors make JEV infection a considerable health problem. Efforts to develop a JEV vaccine continue in our laboratory. It is envisioned that a suitable live attenuated JEV vaccine could be combined with our 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 previously recovered these engineered viruses and evaluated their pathogenicity in mice. The recombinant rJEV virus remains fully virulent in mice and thus provides a background for the evaluation of attenuating mutations. Sets of mutations derived from the attenuated SA-14-14-2 vaccine virus produced in China have been introduced into the rJEV virus clone in order to evaluate the attenuating potential of mutations found in both the structural and non-structural genes. A number of the rJEV/DEN4 chimeric viruses based on the DEN4del30 background have also been generated and are awaiting evaluation in mice. Unfortunately, we have been without an BSL3 select agent animal facility since July 2011 and hope to soon be able to return to the facility to complete our studies.

Project Start
Project End
Budget Start
Budget End
Support Year
12
Fiscal Year
2012
Total Cost
$183,413
Indirect Cost
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State
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Zip Code
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