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 fevershock syndrome (DHFDSS) 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 (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. In this past year, we have focus on the development of two back-up vaccines for dengue virus type 1 and dengue virus type 3.? ? Dengue serotype 1 vaccine development? ? Antigenic chimeric viruses have previously been generated in which the structural genes of recombinant dengue virus type 4 (rDEN4) have been replaced with those derived from DEN2 or DEN3. Two vaccine candidates were identified, rDEN2430(ME) and rDEN3430(ME), which contain the membrane (M) precursor and envelope (E) genes of DEN2 and DEN3, respectively, and a 30 nucleotide deletion (30) in the 3 untranslated region of the DEN4 backbone. Based on the promising preclinical phenotypes of these viruses and the safety and immunogenicity of rDEN2430(ME) in humans, we now describe the generation of a panel of four antigenic chimeric DEN4 viruses using either the capsid (C), M, and E (CME) or ME structural genes of DEN1 Puerto Rico94 strain. Four antigenic chimeric viruses were generated and found to replicate efficiently in Vero cells: rDEN14(CME), rDEN1430(CME), rDEN14(ME), and rDEN1430(ME). With the exception of rDEN14(ME), each chimeric virus was significantly attenuated in a SCID-HuH-7 mouse xenograft model with a 25-fold or greater reduction in replication compared to wild type DEN1. In rhesus monkeys, only chimeric viruses with the 30 mutation appeared to be attenuated as measured by duration and magnitude of viremia. rDEN1430(CME) appeared over-attenuated since it failed to induce detectable neutralizing antibody and did not confer protection from wild type DEN1 challenge. In contrast, rDEN1430(ME) induced 66% seroconversion and protection from DEN1 challenge. Presence of the 30 mutation conferred a significant restriction in mosquito infectivity upon rDEN1430(ME) which was shown to be non-infectious for Aedes aegypti fed an infectious bloodmeal. The attenuation phenotype in SCID-HuH-7 mice, rhesus monkeys, and mosquitoes and the protective immunity observed in rhesus monkeys suggest that rDEN1430(ME) should be considered for evaluation in a clinical trial.? ? Dengue serotype 3 vaccine development? ? The dengue virus type 3 (DEN3) vaccine candidate, rDEN330, was previously found to be under-attenuated in both SCID-HuH-7 mice and rhesus monkeys. Herein, 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), nine novel deletion mutant viruses were designed and found to be viable. Four of nine deletion mutants replicated efficiently in Vero cells and were genetically stable. Second, chimeric rDEN3 viruses were generated by replacement of the 3-UTR of the rDEN3 cDNA clone with that of rDEN4 or rDEN430 yielding the rDEN3-3D4 and rDEN3-3D430 viruses, respectively. Immunization of rhesus monkeys with either of two deletion mutant viruses, rDEN33031 and rDEN386, or with rDEN3-3D430 resulted in infection without detectable viremia, with each virus inducing a strong neutralizing antibody response capable of conferring protection from DEN3 challenge. The rDEN33031 virus showed a strong host range restriction phenotype with complete loss of replication in C636 mosquito cells despite robust replication in Vero cells. In addition, rDEN33031 had reduced replication in Toxorynchites mosquitoes following intrathoracic inoculation. The results are discussed in the context of vaccine development and the physical structure of the DEN virus 3-UTR.
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