Recombinant, live-attenuated tetravalent dengue virus vaccine formulations induce a balanced, broad, and protective neutralizing antibody response against each of the four serotypes in rhesus monkeys.

The mosquito-borne dengue (DEN) viruses, members of the Flaviviridae family, contain a single-stranded positive-sense RNA genome (36). A single polypeptide is cotranslationally processed by viral and cellular proteases generating three structural proteins (C, M, and E) and at least 7 nonstructural 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 DEN virus serotypes (DEN1, DEN2, DEN3, and DEN4) which circulate in tropical and subtropical regions of the world inhabited by more than 2.5 billion people (12). Annually, there are an estimated 50 to 100 million DEN virus infections and hundreds of thousands of cases of the more severe and potentially lethal DEN hemorrhagic fever/shock syndrome, with children bearing much of the disease burden (13). 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 (45). Unfortunately, many countries affected by DEN viruses have very limited financial resources for healthcare, and the economic burden of DEN disease is considerable (1, 45). 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 (31). However, the development of a live-attenuated DEN virus 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 (25, 26). Second, an effective live-attenuated DEN virus vaccine must consist of a tetravalent formulation of components representing each serotype because multiple serotypes typically cocirculate in a region, each DEN serotype is capable of causing disease, and the introduction of additional serotypes is common (18, 37, 42). In addition, the association of increased disease severity (DEN hemorrhagic fever/shock syndrome) in previously infected persons undergoing an infection by a different DEN virus serotype necessitates a vaccine that will confer long-term protection against all four serotypes (19). Third, it has been difficult to formulate a tetravalent vaccine (TV) with low reactogenicity that induces a broad neutralizing antibody response against each DEN virus serotype (16, 26, 39, 41). Fourth, a DEN vaccine must confer protection against a wide range of genetically diverse subtypes which are dispersed around the world and can be readily introduced into a new region by international travel (18, 37). Fifth, a DEN virus 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 DEN virus vaccine. To maximize the likelihood that suitable vaccine candidates would be identified, monovalent vaccine candidates for DEN1 to -4 were generated by two distinct recombinant methods and found to be attenuated and immunogenic in mouse and rhesus monkey models (2, 3, 9, 43, 44). In one method, deletion of 30 contiguous nucleotides from the 3′ UTR of wild-type cDNA clones of DEN1 to -4 was used to generate vaccine candidates. Specifically, the deletion of nucleotides 10478 to 10507 of the 3′ UTR (Δ30) of recombinant wild-type DEN4 yielded a vaccine candidate, rDEN4Δ30, which is safe, attenuated, and immunogenic in rhesus monkeys and humans (9). Incorporation of the Δ30 mutation into infectious cDNA clones of DEN1 and DEN2, but not DEN3, wild-type virus at a site homologous to that in DEN4 attenuated these viruses for rhesus monkeys (2, 3, 43). Using a second method, antigenic chimeric viruses were generated by replacing wild-type M and E structural genes of rDEN4Δ30 with those from DEN2 or DEN3, and the resulting chimeric viruses were attenuated and immunogenic in rhesus monkeys (2, 44). 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. We have also described a set of point mutations which may be used to further attenuate vaccine candidates if evaluation as a monovalent vaccine or a component of a tetravalent formulation demonstrates that further attenuation is required. Such mutations are capable of attenuating wild-type rDEN4 for suckling mice (4, 6, 20), for SCID mice transplanted with human liver cells (SCID-HuH-7) (5), for rhesus monkeys (22), or for mosquitoes (21). Since these mutations are in the nonstructural gene regions of DEN4, they can also be used to modify the attenuation phenotype of antigenic chimeric viruses with the DEN4 background. In addition, mutations identified in rDEN4 might be imported into conserved sites of cDNA clones for other DEN serotypes in an attempt to transfer desired phenotypes, as has been demonstrated for rDEN2Δ30 (3). Finally, to enhance replication in Vero cells and minimize the cost of manufacture, a panel of Vero cell adaptation mutations have been identified and incorporated into vaccine candidates (7). Thus, using the Δ30 mutation, intertypic chimerization, and the set of modifying point mutations, we have been able to generate and characterize attenuated DEN vaccine candidates for each serotype that can now be combined as tetravalent formulations. In the present study, three TV formulations, TV-1, -2, and -3, of the aforementioned monovalent DEN virus vaccine candidates were compared to a tetravalent formulation of wild-type DEN viruses, T-wt, for replication in SCID-HuH-7 mice or for replication and immunogenicity in rhesus monkeys. Attenuation and virus interference were assessed in SCID-HuH-7 mice for TV-1, TV-2, and T-wt. In rhesus monkeys, attenuation, virus interference, and neutralizing antibody responses were evaluated for each formulation and compared to T-wt. In addition, the timing and effect of booster vaccinations were determined, and the breadth of the neutralizing antibody response was tested against divergent DEN subtypes. Analysis of these results and protection studies with rhesus monkeys indicates that two tetravalent formulations can be considered for evaluation in humans.