The dengue virus type 3 (DENV-3) vaccine candidate, rDEN3Δ30, was previously found to be underattenuated in both SCID-HuH-7 mice and rhesus monkeys. Herein, two strategies have been employed to generate attenuated rDEN3 vaccine candidates which retain the full complement of structural and nonstructural proteins of DENV-3 and thus are able to induce humoral or cellular immunity to each of the DENV-3 proteins. First, using the predicted secondary structure of the 3' untranslated region (3'-UTR) of DENV-3 to design novel deletions, nine deletion mutant viruses were engineered and found to be viable. Four of nine deletion mutants replicated efficiently in Vero cells and were genetically stable. Second, chimeric rDENV-3 viruses were generated by replacement of the 3'-UTR of the rDENV-3 cDNA clone with that of rDENV-4 or rDEN4Δ30 yielding the rDEN3-3'D4 and rDEN3-3'D4Δ30 viruses, respectively. Immunization of rhesus monkeys with either of two deletion mutant viruses, rDEN3Δ30/31 and rDEN3Δ86, or with rDEN3-3'D4Δ30 resulted in infection without detectable viremia, with each virus inducing a strong neutralizing antibody response capable of conferring protection from DENV-3 challenge. The rDEN3Δ30/31 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, rDEN3Δ30/31 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 DENV 3'-UTR.
SummaryTo develop a live attenuated virus vaccine against St. Louis encephalitis virus (SLE), two antigenic chimeric viruses were generated by replacing the membrane precursor and envelope protein genes of dengue virus type 4 (DEN4) with those from SLE with or without a 30 nucleotide deletion in the DEN4 3′ untranslated region of the chimeric genome. Chimeric viruses were compared with parental wild-type SLE for level of neurovirulence and neuroinvasiveness in mice and for safety, immunogenicity, and protective efficacy in rhesus monkeys. The resulting viruses, SLE/DEN4 and SLE/DEN4Δ30, had greatly reduced neuroinvasiveness in immunodeficient mice but retained neurovirulence in suckling mice. Chimerization of SLE with DEN4 resulted in only moderate restriction in replication in rhesus monkeys, whereas the presence of the Δ30 mutation led to overattenuation. Introduction of previously described attenuating paired charge-to-alanine mutations in the DEN4 NS5 protein of SLE/DEN4 reduced neurovirulence in mice and replication in rhesus monkeys. Two modified SLE/DEN4 viruses, SLE/DEN4-436,437 clone 41 and SLE/DEN4-654,655 clone 46, have significantly reduced neurovirulence in mice and conferred protective immunity in monkeys against SLE challenge. These viruses may be considered for use as SLE vaccine candidates and for use as diagnostic reagents with reduced virulence.
Background: 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, rDEN2/4Δ30(ME) and rDEN3/4Δ30(ME), which contain the membrane (M) precursor and envelope (E) genes of DEN2 and DEN3, respectively, and a 30 nucleotide deletion (Δ30) in the 3' untranslated region of the DEN4 backbone. Based on the promising preclinical phenotypes of these viruses and the safety and immunogenicity of rDEN2/4Δ30(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 Rico/94 strain.
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