Vaccine candidates for dengue virus type 1 (DEN1) generated by replacement of the structural genes of rDEN4 and rDEN4Delta30 with those of DEN1

Abstract

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, rDEN2/4Delta30(ME) and rDEN3/4Delta30(ME), which contain the membrane (M) precursor and envelope (E) genes of DEN2 and DEN3, respectively, and a 30 nucleotide deletion (Delta30) in the 3' untranslated region of the DEN4 backbone. Based on the promising preclinical phenotypes of these viruses and the safety and immunogenicity of rDEN2/4Delta30(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.

Results: Four antigenic chimeric viruses were generated and found to replicate efficiently in Vero cells: rDEN1/4(CME), rDEN1/4Delta30(CME), rDEN1/4(ME), and rDEN1/4Delta30(ME). With the exception of rDEN1/4(ME), each chimeric virus was significantly attenuated in a SCID-HuH-7 mouse xenograft model with a 25-fold or greater reduction in replication compared to wild type DEN1. In rhesus monkeys, only chimeric viruses with the Delta30 mutation appeared to be attenuated as measured by duration and magnitude of viremia. rDEN1/4Delta30(CME) appeared over-attenuated since it failed to induce detectable neutralizing antibody and did not confer protection from wild type DEN1 challenge. In contrast, rDEN1/4Delta30(ME) induced 66% seroconversion and protection from DEN1 challenge. Presence of the Delta30 mutation conferred a significant restriction in mosquito infectivity upon rDEN1/4Delta30(ME) which was shown to be non-infectious for Aedes aegypti fed an infectious bloodmeal.

Conclusion: The attenuation phenotype in SCID-HuH-7 mice, rhesus monkeys, and mosquitoes and the protective immunity observed in rhesus monkeys suggest that rDEN1/4Delta30(ME) should be considered for evaluation in a clinical trial.

Figures

Figure 1

Molecular construction of the DEN1 chimeric cDNA plasmids. A. The CME structural protein coding region of the DEN4 cDNA plasmid p4 was replaced with the corresponding region from DEN1 (Puerto Rico/94) CME sub-clone pUCBXR-init. Following introduction of a PstI site near the C-M junction of pUCBXR-init, the ME region of p4 was replaced with the corresponding region from DEN1. Restriction sites used to facilitate the construction are indicated. The virus genomic regions for each of the resulting full-length chimeric cDNA plasmids are shown and the presence of the Δ30 mutation is indicated in the 3'-UTR. DEN1 sequence is shaded. The resulting cloning junctions are numbered 1 – 5 and the inserted linker sequence is indicated as a hatched box. The location of the linker is indicated. B. The nucleotide sequences surrounding the cloning junctions identified above are shown along with the predicted amino acid sequence corresponding to the virus polyprotein. Lower case letters indicate nucleotide substitutions that differ from wild type. For junction 3, the amino acid sequence is shown as predicted for the virus genome (with the linker sequence removed prior to transcription) and as predicted in E. coli where cryptic expression of the virus polyprotein with the linker sequence intact would result in translational termination. The termination codon in the polyprotein open reading frame that is located in the linker is indicated by * * *. Termination codons exist in the linker sequence for each of the additional forward and reverse open reading frames. For junction 4, the introduced PstI site is indicated. C = capsid protein; M = membrane protein (with precursor region), E = envelope protein; NS = nonstructural.

Figure 2

Virus replication in Vero cells. Virus titer in supernatants from infected cells was determined by plaque assay in Vero cells. The lower limit of detection was 0.7 log10 PFU/ml.