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 (DHFDSS) 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. 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 this virus 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. The dengue virus vaccine program is predominantly in a clinical mode at this time. The preclinical phase of this program is currently devoted largely to support the manufacture of clinical lots of vaccines suitable for study in volunteers. Multiple clinical lots of dengue 1-4 have been manufactured over the past year, focusing largely on the DEN3 vaccine candidates and a new lot of DEN4del30. Dengue serotype 1 and -2 vaccine development. The attenuation phenotype of rDEN1del30 and rDEN2/4∆30 in SCID-HuH-7 mice, rhesus monkeys, and mosquitoes and the protective immunity observed in rhesus monkeys formed the basis for evaluation of virus in clinical trials. Further pre-clinical research with DEN1 and DEN2 vaccine candidates are on hold pending outcome of ongoing clinical studies with the lead rDEN1del30 and rDEN2/4∆30 candidates that continue to look good in clinical trials with up to 90 vaccinees receiving either vaccine. Dengue serotype 3 vaccine development: The dengue virus type 3 (DEN3) vaccine candidate, rDEN3/4del30, was previously found to be infectious, attenuated, and immunogenic in SCID-HuH-7 mice or rhesus monkeys, but was found to have decreased infectivity for humans. An additional, similarly designed DEN3 chimeric virus, based on a DEN3 wild type virus of greater virulence (a Sri Lanka strain of DEN3) than the donor (Slemons strain) of the M and E genes in clinically tested chimeric rDEN3/4del30 virus, was generated. The Sri Lanka rDEN3/4del30 was found to have a monkey infectious dose 50 similar to that of the Slemonds rDEN3/4del30. Therefore rDEN3/4del30 Sri Lnaka was not considered sufficiently more infectious to be tested in humans. As reported last year, two strategies have been employed to generate attenuated rDEN3 vaccine candidates that retain the full complement of structural and nonstructural proteins of DEN3 and thus are able to induce humoral or cellular immunity to each of the DEN3 proteins. First, using the predicted secondary structure of the DEN3 3 untranslated region (3-UTR), a novel deletion mutant was designed containing both the del 30 mutation and an additional del31 mutation and was found to be highly attenuated yet immunogenic in monkeys. Second, chimeric rDEN3 viruses were generated by replacement of the 3-UTR of the rDEN3 cDNA clone with that of rDEN4 or rDEN4del30 yielding the rDEN3-3D4 and rDEN3-3D4del30 viruses, respectively. Immunization of rhesus monkeys with either of two mutant viruses, rDEN3del30/31 or rDEN3-3D4del30, resulted in infection without detectable viremia, with each virus inducing a strong neutralizing antibody response capable of conferring protection from DEN3 challenge. The rDEN3del30/31 virus showed a strong host range restriction phenotype with complete loss of replication in C6/36 mosquito cells despite robust replication in Vero cells. In addition, rDEN3del30/31 had reduced replication in Toxorynchites mosquitoes following intrathoracic inoculation. Clinical lots of the rDEN3del30/31 and rDEN3-3D4del30 have been manufactured, and INDs have submitted. Phase 1 clinical trials with rDEN3-3D4del30 have begun and those with rDEN3del30/31 should start within four months. Efforts to develop a live attenuated Japanese encephalitis virus vaccine have been initiated.
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