The D30 deletion mutation, originally created in dengue virus type 4 (DEN4) by the removal of nucleotides 172-143 from the 3?-UTR, was introduced into a homologous region of wild type dengue virus type 1 (DEN1). The resulting virus, rDEN1del30, was attenuated in rhesus monkeys to a level similar to that of the rDEN4del30 vaccine candidate. rDEN1del30 was more attenuated in rhesus monkeys than the previously described vaccine candidate, rDEN1mutF, which also contains mutations in the 3?-UTR, and both vaccines were highly protective against challenge with wild type DEN1. Both rDEN1del30 and rDEN1mutF were also attenuated in HuH-7-SCID mice. However, neither rDEN1del30 nor rDEN1mutF showed restricted replication following intrathoracic administration in the mosquito Toxorhynchites splendens. The ability of the D30 mutation to attenuate both DEN1 and DEN4 viruses suggests that a tetravalent DEN vaccine could be generated by introduction of the del30 mutation into wild type DEN viruses belonging to each of the four serotypes. DEN2 Antigenic chimeric viruses in which the structural genes of dengue virus type 4 (DEN4) have been replaced with those derived from dengue virus type 2 (DEN2) have been created and evaluated as a first step in generating a live attenuated tetravalent dengue virus vaccine. Specifically, the capsid, membrane precursor, and envelope (CME) or the membrane precursor and envelope (ME) gene regions of DEN2 were substituted for the corresponding genes of wild-type rDEN4 or vaccine candidate rDEN4del30 which contains a 30 nucleotide deletion (del30) in the 3? untranslated region. The two DEN2/4 chimeric viruses lacking the del30 mutation were highly attenuated in tumor-bearing SCID-HuH-7 mice, mosquitoes, and rhesus monkeys, indicating chimerization with either the CME or ME regions lead to attenuation. In mosquitoes and SCID-HuH-7 mice, addition of the del30 mutation to the chimeric viruses resulted in comparable or only slightly increased levels of attenuation. In rhesus monkeys, addition of the D30 mutation rendered the CME chimeric virus non-infectious, indicating that the attenuation resulting from chimerization and the D30 mutation were additive for these animals. In contrast, the attenuation in rhesus monkeys of ME chimeric virus was not significantly modified by the addition of the D30 mutation. The satisfactory level of attenuation and immunogenicity achieved by the ME containing DEN2/4D30 chimeric virus, as well as its very low infectivity for mosquitoes, make it a vaccine candidate suitable for evaluation in phase I clinical trials. DEN3 Three novel recombinant DEN virus type 3 (DEN3) vaccine candidates have been generated from a DEN3 virus isolated from a mild outbreak of dengue fever in Sleman, Central Java, Indonesia in 1978. Antigenic chimeric viruses were prepared by replacing the membrane precursor and envelope proteins (ME) of recombinant DEN4 (rDEN4) virus with those from DEN3 Sleman/78 in the presence (rDEN3/4del30(ME)) and absence (rDEN3/4(ME)) of the del30 mutation, a previously described 30 nucleotide deletion in the 3? untranslated region. In addition, a full-length infectious cDNA clone was generated from the DEN3 isolate and used to produce rDEN3 virus and the vaccine candidate, rDEN3del30. rDEN3/4(ME) and rDEN3/4del30(ME) appear to be acceptable vaccine candidates since they were restricted in replication in SCID mice transplanted with human hepatoma cells, in rhesus monkeys, and in Aedes and Toxorynchites mosquitoes, and each was protective in rhesus monkeys against DEN3 virus challenge. The rDEN3/4(ME) and rDEN3/4del30(ME) viruses were comparable in all parameters evaluated, indicating that antigenic chimerization resulted in the observed high level of attenuation. Surprisingly, rDEN3del30 was not attenuated in any model tested when compared to wild type rDEN3 and therefore, is not a vaccine candidate at present. Thus, the rDEN3/4(ME) and rDEN3/4del30(ME) antigenic chimeric viruses can be considered for evaluation in humans and for inclusion in a tetravalent dengue vaccine. DEN4 A dengue virus vaccine candidate, rDEN4del30, has been previously reported to be safe and immunogenic in humans, but a subset of vaccinees developed asymptomatic rash, elevation of liver enzymes and/or mild neutropenia. In the current study, mutations that had previously been shown to reduce replication of DEN4 virus in suckling mice and/or in SCID mice engrafted with human liver cells (SCID-HuH-7 mice) were introduced into rDEN4del30 in an attempt to further attenuate this virus. Three of the five resulting modified rDEN4del30 viruses showed decreased replication in SCID-HuH-7 mice relative to rDEN4del30. Moreover, in rhesus monkeys, two of the modified rDEN4del30 viruses showed a decrease in replication relative to rDEN4del30 while generating levels of neutralizing antibody similar to rDEN4del30 virus. All of the modified rDEN4?30 viruses completely protected immunized rhesus monkeys from challenge with wild-type DEN4 virus. Based on their attenuation for both human liver cells and rhesus monkeys, two of the modified rDEN4del30 vaccine candidates are currently being prepared for use in clinical trials. The application of these attenuating mutations to flavivirus vaccine development is discussed. Mutations which increase the replication of dengue viruses in cell culture would greatly facilitate the manufacture of both a live attenuated or inactivated dengue virus vaccine. We have identified eight missense mutations in dengue virus type 4 (DEN4) that increase the plaque size and kinetics of replication of recombinant DEN4 (rDEN4) virus in Vero cells. rDEN4 viruses bearing these Vero cell adaptation mutations were also evaluated for the level of replication in the brains of mice. Two recombinant viruses expressing distinct mutations in NS3 were both restricted in replication in the brains of suckling mouse. In contrast, six recombinant viruses, each encoding individual mutations in NS4B (five) or in NS5 (one), were not attenuated in mouse brain. Recombinant viruses encoding various combinations of these Vero cell adaptation mutations did not demonstrate enhanced replication in Vero cells over that exhibited by the single mutations. Finally, addition of a subset of the above non-attenuating, adaptation mutations to a DEN2/4 chimeric vaccine candidate was found to increase the virus yield in Vero cells by up to 500-fold. The importance of these Vero cell adaptation mutations in flavivirus vaccine design and development is discussed.
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