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, but not DEN2 or DEN3, 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. ? ? Last year we described the successful evaluation of three formulations of a tetravalent vaccine in rhesus monkeys. This year we have focused our efforts on bringing vaccine candidates for each of the serotypes to clinical trials in humans and on the further development of additional attenuating mutants for each serotype to serve as backup candidates in event that the current candidates do not achieve an acceptable balance between attenuation in humans. These efforts include:? ? Studies with DEN1 candidates:? Vaccines and INDs? Vaccine lot of DEN1del30 manufactured by Charles River Laboratories ? In progress.? IND to be filed for this vaccine lot in September 2006 ? New Vaccine Candidates ? Two additional attenuated vaccine candidates of DEN1 have been generated and are in the process of evaluation in experimental animals: one is the DEN1del30 virus containing two additional attenuating mutations and the other is an DEN1/4del30 antigenic chimeric virus.? ? Studies with DEN2 candidates:? Vaccines and INDs? Vaccine lot of DEN2/4del30 manufactured by Charles River Laboratories ? In progress.? IND to be filed for this vaccine lot in October 2006.? New Vaccine Candidates? Two additional attenuated vaccine candidates of DEN2 have been generated and are in the process of evaluation in experimental animals: one is the DEN2/4del30 virus containing two additional attenuating mutations and the other is an attenuated derivative of DEN2. These are currently in preclinical studies.? ? Studies with DEN3 candidates:? Vaccines and INDs? Vaccine lot of DEN3/4del30 manufactured by Charles River Laboratories - completed.? IND to be filed for this vaccine lot in August 2006.? New Vaccine Candidates ? Four additional attenuated vaccine candidates of DEN2 have been generated and are in the process of evaluation in experimental animals. These are attenuated derivatives of DEN3 possessing all proteins of DEN3, i.e., they are not chimeric viruses. These are currently in preclinical studies.? ? Studies with DEN4 candidates:? Vaccines and INDs? Two vaccine lots of DEN4del30 containing additional attenuating mutations have been manufactured by Charles River Laboratories.? Two INDs have been filed for these vaccine candidates and FDA approval for each have been received. A clinical trial with one has been initiated and a trial for the other is planned to initiate in September 2006.
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