Dengue virus type 3 vaccine candidates generated by introduction of deletions in the 3' untranslated region (3'-UTR) or by exchange of the DENV-3 3'-UTR with that of DENV-4

Abstract

The dengue virus type 3 (DENV-3) vaccine candidate, rDEN3Delta30, 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 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 rDEN4Delta30 yielding the rDEN3-3'D4 and rDEN3-3'D4Delta30 viruses, respectively. Immunization of rhesus monkeys with either of two deletion mutant viruses, rDEN3Delta30/31 and rDEN3Delta86, or with rDEN3-3'D4Delta30 resulted in infection without detectable viremia, with each virus inducing a strong neutralizing antibody response capable of conferring protection from DENV-3 challenge. The rDEN3Delta30/31 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, rDEN3Delta30/31 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.

Figure 1

A. The predicted secondary structure for the 3’-UTR of wt DENV-3 Sleman/78. The sequence used for construction of the secondary structure model is the last 276 nucleotides of DENV-3 (nucleotides 10432 – 10707 from GenBank accession number AY648961). The M-fold program [42, 43] was used and nucleotides 267–276 and 95–104 were constrained to be single stranded in order to prevent circularization of the structure and loss of the conserved terminal 3’ hairpin stem loop (designated 3’ SL). Nucleotides are boxed at every 50 bases for reference to indicate position relative to the 3’ end. Circled nucleotides represent borders of the described deletions. The two stem-loop structural elements, designated SE-1 and SE-2, correspond approximately to the previously described TL2 and TL1, DB1 and DB2, and A3 and A2 structural elements [36, 37, 41]. B. Summary of the recovery and properties of the nine mutant viruses with deletions introduced into the 3’-UTR.

Figure 2

Chimerization of rDEN3 with the 3’-UTR of rDENV-4 or rDEN4Δ30. A. Recombinant 3’-UTR chimeric dengue viruses were constructed by replacing the 3’-UTR of rDENV-3 with regions derived from either rDENV-4 or rDEN4Δ30. The relative location of the Δ30 mutation in the 3’-UTR is indicated by an arrow. The junctions between the ORF and UTR for rDENV-3 and rDENV-4 are indicated as junctions 1 and 2, respectively. Intertypic junction 3 is also indicated for the resulting chimeric viruses. B. Nucleotide and amino acid sequence of the junction regions are shown. For junction 3, nucleotide substitutions used to introduce a unique HpaI restriction enzyme recognition site are shown in lower case.

Figure 3

Replication of rDENV-3 in Vero cells and C6/36 cells. Cells were infected at an MOI of 0.01. Virus titer of supernatants was determined by plaque assay in Vero cells for all samples. The limit of detection is 10¹ PFU/ml.