Vaccine candidates derived from a novel infectious cDNA clone of an American genotype dengue virus type 2

Abstract

Background: A dengue virus type 2 (DEN-2 Tonga/74) isolated from a 1974 epidemic was characterized by mild illness and belongs to the American genotype of DEN-2 viruses. To prepare a vaccine candidate, a previously described 30 nucleotide deletion (Delta30) in the 3' untranslated region of DEN-4 has been engineered into the DEN-2 isolate.

Methods: A full-length cDNA clone was generated from the DEN-2 virus and used to produce recombinant DEN-2 (rDEN-2) and rDEN2Delta30. Viruses were evaluated for replication in SCID mice transplanted with human hepatoma cells (SCID-HuH-7 mice), in mosquitoes, and in rhesus monkeys. Neutralizing antibody induction and protective efficacy were also assessed in rhesus monkeys.

Results: The rDEN2Delta30 virus was ten-fold reduced in replication in SCID-HuH-7 mice when compared to the parent virus. The rDEN-2 viruses were not infectious for Aedes mosquitoes, but both readily infected Toxorynchites mosquitoes. In rhesus monkeys, rDEN2Delta30 appeared to be slightly attenuated when compared to the parent virus as measured by duration and peak of viremia and neutralizing antibody induction. A derivative of rDEN2Delta30, designated rDEN2Delta30-4995, was generated by incorporation of a point mutation previously identified in the NS3 gene of DEN-4 and was found to be more attenuated than rDEN2Delta30 in SCID-HuH-7 mice.

Conclusions: The rDEN2Delta30 and rDEN2Delta30-4995 viruses can be considered for evaluation in humans and for inclusion in a tetravalent dengue vaccine.

Figure 1

Molecular construction of the DEN-2 full-length cDNA plasmids p2 and p2Δ30. A. Diagram of the complete full-length DEN-2 Tonga/74 cDNA plasmid p2 is shown annotated with the restriction enzyme and corresponding cleavage site locations used to assemble the subcloned RT-PCR fragments. Restriction enzyme cleavage sites are numbered relative to nucleotide position in the virus genome. The corresponding genomic regions encoded by each subcloned RT-PCR fragment, X, L, R, A, B, C, and Y, are shown above the plasmid diagram. Relative positions of the SP6 promoter and tetracycline resistance gene (Tet^r) are indicated. B. To generate plasmid p2Δ30, 30 nucleotides are removed from the 3'-UTR (Fragment Y). The nucleotide sequence encompassing the Δ30 region is shown for the p2 parent cDNA and the resulting p2Δ30 cDNA. Nucleotide positions in the virus genome are indicated for p2.