Chimeric dengue type 2 (vaccine strain PDK-53)/dengue type 1 virus as a potential candidate dengue type 1 virus vaccine

Abstract

We constructed chimeric dengue type 2/type 1 (DEN-2/DEN-1) viruses containing the nonstructural genes of DEN-2 16681 virus or its vaccine derivative, strain PDK-53, and the structural genes (encoding capsid protein, premembrane protein, and envelope glycoprotein) of DEN-1 16007 virus or its vaccine derivative, strain PDK-13. We previously reported that attenuation markers of DEN-2 PDK-53 virus were encoded by genetic loci located outside the structural gene region of the PDK-53 virus genome. Chimeric viruses containing the nonstructural genes of DEN-2 PDK-53 virus and the structural genes of the parental DEN-1 16007 virus retained the attenuation markers of small plaque size and temperature sensitivity in LLC-MK(2) cells, less efficient replication in C6/36 cells, and attenuation for mice. These chimeric viruses elicited higher mouse neutralizing antibody titers against DEN-1 virus than did the candidate DEN-1 PDK-13 vaccine virus or chimeric DEN-2/DEN-1 viruses containing the structural genes of the PDK-13 virus. Mutations in the envelope protein of DEN-1 PDK-13 virus affected in vitro phenotype and immunogenicity in mice. The current PDK-13 vaccine is the least efficient of the four Mahidol candidate DEN virus vaccines in human trials. The chimeric DEN-2/DEN-1 virus might be a potential DEN-1 virus vaccine candidate. This study indicated that the infectious clones derived from the candidate DEN-2 PDK-53 vaccine are promising attenuated vectors for development of chimeric flavivirus vaccines.

FIG. 1

Genomic organization of the chimeric DEN-2/DEN-1 viruses. Designations of the chimeras are based on the DEN-2 virus-specific infectious clone backbones and the structural genes (C-prM-E) insert of DEN-1 viruses. Underlined letters of the backbone and insert viruses are used in the designations. Designations: D2/1-xy, where x represents the infectious DEN-2 clone background (P, parental 16881 clone; E, PDK53-E variant; V, PDK53-V variant) and y denotes DEN-1 virus-specific C-prM-E insert (P, parental 16007 strain; V, PDK-13 vaccine candidate).

FIG. 2

Growth characteristics of the chimeric DEN-2/DEN-1 viruses in LLC-MK₂ cells. Stippled bars indicate DEN-1 16007 virus and the chimeric viruses expressing the structural proteins of DEN-1 16007 virus; gray bars indicate DEN-1 PDK-13 virus and the chimeric viruses expressing structural proteins of PDK-13 virus; blank bars indicate the three DEN-2 backbone viruses derived from infectious clones of DEN-2 16681 virus (P48) and the two variants (PDK53-E and PDK53-V; E48 and V48, respectively). (A) Mean (± standard deviation) plaque diameters. Values were calculated from 10 individual plaques of each virus on day 10 after infection. pp, pinpoint-size plaques less than 1 mm. (B) Temperature sensitivity (ts) and peak titers of chimeric viruses on day 8 or 10 after infection. The ts scores were based on the reduction of the virus titers at 38.7°C versus those at 37°C (−, +, 2+, and 3+ indicate titer reduction of ≤60, 61 to 90, 91 to 99, and >99%, respectively, calculated from at least three experiments). The graph bar heights represent the log₁₀ titers of the viruses at 37°C. The MOI was approximately 0.001 PFU/cell.

FIG. 3

Growth curves of DEN-1 16007, DEN-1 PDK-13, DEN-2 16681-P48, DEN-2 PDK53-E48, DEN-2 PDK53-V48, and chimeric DEN-2/DEN-1 viruses in C6/36 cells. Cells were infected at an approximate MOI of 0.001 PFU/ml.

FIG. 4

Mean weights of mice inoculated intracranially with 5,000 PFU of DEN-1 16007, DEN-1 PDK-13, DEN-2 16681, or chimeric DEN-2/DEN-1 virus. No mice inoculated with DEN-2 16681 virus survived more than 18 days after challenge (average survival time of 14.1 ± 1.6 days), and one mouse died 21 days after inoculation with DEN-1 16007 virus.