Volume 71, Issue 6
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645


Three novel recombinant dengue type 3 (DEN3) virus vaccine candidates have been generated from a DEN3 virus isolated from a mild outbreak of dengue fever in the Sleman area of central Java in Indonesia in 1978. Antigenic chimeric viruses were prepared by replacing the membrane precursor and envelope (ME) proteins of recombinant DEN4 (rDEN4) virus with those from DEN3 Sleman/78 in the presence (rDEN3/4Δ30(ME)) and the absence (rDEN3/4(ME)) of the Δ30 mutation, a previously described 30-nucleotide deletion in the 3′ untranslated region. In addition, a full-length infectious cDNA clone was generated from the DEN3 isolate and used to produce rDEN3 virus and the vaccine candidate rDEN3Δ30. The chimeric viruses rDEN3/4(ME) and rDEN3/4Δ30(ME) appear to be acceptable vaccine candidates since they were restricted in replication in severe combined immune deficiency mice transplanted with human hepatoma cells, in rhesus monkeys, and in and mosquitoes, and each was protective in rhesus monkeys against DEN3 virus challenge. The rDEN3/4(ME) and rDEN3/4Δ30(ME) viruses were comparable in all parameters evaluated, indicating that antigenic chimerization resulted in the observed high level of attenuation. Surprisingly, rDEN3Δ30 was not attenuated in any model tested when compared with wild-type rDEN3 and therefore, is not a vaccine candidate at present. Thus, the rDEN3/4(ME) and rDEN3/4Δ30(ME) antigenic chimeric viruses can be considered for evaluation in humans and for inclusion in a tetravalent dengue vaccine.


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  1. Rice CM, 1996. Flaviviridae: the viruses and their replication. Fields BN, Knipe DM, Howley PM, Chanock RM, Melnick JL, Monath TP, Roizman B, Straus SE, eds. Fields Virology. Philadelphia: Lippincott-Raven Publishers, 931–959.
  2. Chang G-J, 1997. Molecular biology of dengue viruses. Gubler DJ, Kuno G, eds. Dengue and Dengue Hemorrhagic Fever. New York: CAB International, 175–198.
  3. Gubler DJ, 1998. Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 11 : 480–496. [Google Scholar]
  4. Gubler DJ, Meltzer M, 1999. Impact of dengue/dengue hemorrhagic fever on the developing world. Adv Virus Res 53 : 35–70. [Google Scholar]
  5. Innis BL, 1995. Dengue and dengue hemorrhagic fever. Porter-field JS, ed. Exotic Viral Infections. London: Chapman and Hall, 103–146.
  6. Almond J, Clemens J, Engers H, Halstead S, Khiem HB, Pablos-Mendez A, Pervikov Y, Tram T, 2002. Accelerating the development and introduction of a dengue vaccine for poor children, 5–8 December, 2001, Ho Chi Minh City, Vietnam. Vaccine 20 : 3043–3046. [Google Scholar]
  7. Monath TP, 1999. Yellow fever. Plotkin SA, Orenstein WA, eds. Vaccines. Philadelphia: W.B. Saunders Co., 815–879.
  8. Vaughn DW, Green S, Kalayanarooj S, Innis BL, Nimmannitya S, Suntayakorn S, Endy TP, Raengsakulrach B, Rothman AL, Ennis FA, Nisalak A, 2000. Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Infect Dis 181 : 2–9. [Google Scholar]
  9. Guzman MG, Kouri G, 2003. Dengue and dengue hemorrhagic fever in the Americas: lessons and challenges. J Clin Virol 27 : 1–13. [Google Scholar]
  10. Burke DS, Nisalak A, Johnson DE, Scott RM, 1988. A prospective study of dengue infections in Bangkok. Am J Trop Med Hyg 38 : 172–180. [Google Scholar]
  11. Thein S, Aung MM, Shwe TN, Aye M, Zaw A, Aye K, Aye KM, Aaskov J, 1997. Risk factors in dengue shock syndrome. Am J Trop Med Hyg 56 : 566–572. [Google Scholar]
  12. Halstead SB, 1988. Pathogenesis of dengue: challenges to molecular biology. Science 239 : 476–481. [Google Scholar]
  13. Eckels KH, Dubois DR, Putnak R, Vaughn DW, Innis BL, Henchal EA, Hoke CH Jr, 2003. Modification of dengue virus strains by passage in primary dog kidney cells: preparation of candidate vaccines and immunization of monkeys. Am J Trop Med Hyg 69 : 12–16. [Google Scholar]
  14. Kanesa-Thasan N, Edelman R, Tacket CO, Wasserman SS, Vaughn DW, Coster TS, Kim-Ahn GJ, Dubois DR, Putnak JR, King A, Summers PL, Innis BL, Eckels KH, Hoke CH Jr, 2003. Phase 1 studies of Walter Reed Army Institute of Research candidate attenuated dengue vaccines: selection of safe and immunogenic monovalent vaccines. Am J Trop Med Hyg 69 : 17–23. [Google Scholar]
  15. Kanesathasan N, Sun W, Kim-Ahn G, Van Albert S, Putnak JR, King A, Raengsakulsrach B, Christ-Schmidt H, Gilson K, Zahradnik JM, Vaughn DW, Innis BL, Saluzzo JF, Hoke CH Jr, 2001. Safety and immunogenicity of attenuated dengue virus vaccines (Aventis Pasteur) in human volunteers. Vaccine 19 : 3179–3188. [Google Scholar]
  16. Sabchareon A, Lang J, Chanthavanich P, Yoksan S, Forrat R, Attanath P, Sirivichayakul C, Pengsaa K, Pojjaroen-Anant C, Chokejindachai W, Jagsudee A, Saluzzo JF, Bhamarapravati N, 2002. Safety and immunogenicity of tetravalent live-attenuated dengue vaccines in Thai adult volunteers: role of serotype concentration, ratio, and multiple doses. Am J Trop Med Hyg 66 : 264–272. [Google Scholar]
  17. Guirakhoo F, Arroyo J, Pugachev KV, Miller C, Zhang ZX, Weltzin R, Georgakopoulos K, Catalan J, Ocran S, Soike K, Ratterree M, Monath TP, 2001. Construction, safety, and immunogenicity in nonhuman primates of a chimeric yellow fever-dengue virus tetravalent vaccine. J Virol 75 : 7290–7304. [Google Scholar]
  18. Guirakhoo F, Pugachev K, Arroyo J, Miller C, Zhang ZX, Weltzin R, Georgakopoulos K, Catalan J, Ocran S, Draper K, Monath TP, 2002. Viremia and immunogenicity in nonhuman primates of a tetravalent yellow fever-dengue chimeric vaccine: genetic reconstructions, dose adjustment, and antibody responses against wild-type dengue virus isolates. Virology 298 : 146–159. [Google Scholar]
  19. Sun W, Edelman R, Kanesa-Thasan N, Eckels KH, Putnak JR, King AD, Houng HS, Tang D, Scherer JM, Hoke CH Jr, Innis BL, 2003. Vaccination of human volunteers with monovalent and tetravalent live-attenuated dengue vaccine candidates. Am J Trop Med Hyg 69 : 24–31. [Google Scholar]
  20. Durbin AP, Karron RA, Sun W, Vaughn DW, Reynolds MJ, Perreault JR, Thumar B, Men R, Lai CJ, Elkins WR, Chanock RM, Murphy BR, Whitehead SS, 2001. Attenuation and immunogenicity in humans of a live dengue virus type-4 vaccine candidate with a 30 nucleotide deletion in its 3′-untranslated region. Am J Trop Med Hyg 65 : 405–413. [Google Scholar]
  21. Whitehead SS, Falgout B, Hanley KA, Blaney JE Jr, Markoff L, Murphy BR, 2003. A live, attenuated dengue virus type 1 vaccine candidate with a 30-nucleotide deletion in the 3′untranslated region is highly attenuated and immunogenic in monkeys. J Virol 77 : 1653–1657. [Google Scholar]
  22. Whitehead SS, Hanley KA, Blaney JE, Gilmore LE, Elkins WR, Murphy BR, 2003. Substitution of the structural genes of dengue virus type 4 with those of type 2 results in chimeric vaccine candidates which are attenuated for mosquitoes, mice, and rhesus monkeys. Vaccine 21 : 4307–4316. [Google Scholar]
  23. Hanley KA, Lee JJ, Blaney JE Jr, Murphy BR, Whitehead SS, 2002. Paired charge-to-alanine mutagenesis of dengue virus type 4 NS5 generates mutants with temperature-sensitive, host range, and mouse attenuation phenotypes. J Virol 76 : 525–531. [Google Scholar]
  24. Blaney JE Jr, Johnson DH, Firestone CY, Hanson CT, Murphy BR, Whitehead SS, 2001. Chemical mutagenesis of dengue virus type 4 yields mutant viruses which are temperature sensitive in Vero cells or human liver cells and attenuated in mice. J Virol 75 : 9731–9740. [Google Scholar]
  25. Blaney JE Jr, Manipon GG, Murphy BR, Whitehead SS, 2003. Temperature sensitive mutations in the genes encoding the NS1, NS2A, NS3, and NS5 nonstructural proteins of dengue virus type 4 restrict replication in the brains of mice. Arch Virol 148 : 999–1006. [Google Scholar]
  26. Blaney JE Jr, Johnson DH, Manipon GG, Firestone CY, Hanson CT, Murphy BR, Whitehead SS, 2002. Genetic basis of attenuation of dengue virus type 4 small plaque mutants with restricted replication in suckling mice and in SCID mice transplanted with human liver cells. Virology 300 : 125–139. [Google Scholar]
  27. Hanley KA, Manlucu LR, Manipon GG, Hanson CT, Whitehead SS, Murphy BR, Blaney JE Jr, 2004. Introduction of mutations into the non-structural genes or 3′ntranslated region of an attenuated dengue virus type 4 vaccine candidate further decreases replication in rhesus monkeys while retaining protective immunity. Vaccine 22 : 3440–3448. [Google Scholar]
  28. Hanley KA, Manlucu LR, Gilmore LE, Blaney JE Jr, Hanson CT, Murphy BR, Whitehead SS, 2003. A trade-off in replication in mosquito versus mammalian systems conferred by a point mutation in the NS4B protein of dengue virus type 4. Virology 312 : 222–232. [Google Scholar]
  29. Gubler DJ, Suharyono W, Lubis I, Eram S, Gunarso S, 1981. Epidemic dengue 3 in central Java, associated with low viremia in man. Am J Trop Med Hyg 30 : 1094–1099. [Google Scholar]
  30. Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J, 1982. Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res 42 : 3858–3863. [Google Scholar]
  31. Lai CJ, Zhao BT, Hori H, Bray M, 1991. Infectious RNA transcribed from stably cloned full-length cDNA of dengue type 4 virus. Proc Natl Acad Sci USA 88 : 5139–5143. [Google Scholar]
  32. Men R, Bray M, Clark D, Chanock RM, Lai CJ, 1996. Dengue type 4 virus mutants containing deletions in the 3′noncoding region of the RNA genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in rhesus monkeys. J Virol 70 : 3930–3937. [Google Scholar]
  33. Troyer JM, Hanley KA, Whitehead SS, Strickman D, Karron RA, Durbin AP, Murphy BR, 2001. A live attenuated recombinant dengue-4 virus vaccine candidate with restricted capacity for dissemination in mosquitoes and lack of transmission from vaccinees to mosquitoes. Am J Trop Med Hyg 65 : 414–419. [Google Scholar]
  34. Reed LJ, Muench H, 1938. A simple method of estimating fifty percent endpoints. Am J Hyg 27 : 493–497. [Google Scholar]
  35. Pletnev AG, Putnak R, Speicher J, Wagar EJ, Vaughn DW, 2002. West Nile virus/dengue type 4 virus chimeras that are reduced in neurovirulence and peripheral virulence without loss of immunogenicity or protective efficacy. Proc Natl Acad Sci USA 99 : 3036–3041. [Google Scholar]
  36. Pletnev AG, Bray M, Huggins J, Lai CJ, 1992. Construction and characterization of chimeric tick-borne encephalitis/dengue type 4 viruses. Proc Natl Acad Sci USA 89 : 10532–10536. [Google Scholar]
  37. Blaney JE, Manipon GG, Firestone CY, Johnson DH, Hanson CT, Murphy BR, Whitehead SS, 2003. Mutations which enhance the replication of dengue virus type 4 and an antigenic chimeric dengue virus type 2/4 vaccine candidate in Vero cells. Vaccine 21 : 4317–4327. [Google Scholar]
  38. Sabchareon A, Lang J, Chanthavanich P, Yoksan S, Forrat R, Attanath P, Sirivichayakul C, Pengsaa K, Pojjaroen-Anant C, Chambonneau L, Saluzzo JF, Bhamarapravati N, 2004. Safety and immunogenicity of a three dose regimen of two tetravalent live-attenuated dengue vaccines in five- to twelve-year-old Thai children. Pediatr Infect Dis J 23 : 99–109. [Google Scholar]
  39. Edelman R, Wasserman SS, Bodison SA, Putnak RJ, Eckels KH, Tang D, Kanesa-Thasan N, Vaughn DW, Innis BL, Sun W, 2003. Phase I trial of 16 formulations of a tetravalent live-attenuated dengue vaccine. Am J Trop Med Hyg 69 : 48–60. [Google Scholar]
  40. Pletnev AG, Claire MS, Elkins R, Speicher J, Murphy BR, Chanock RM, 2003. Molecularly engineered live-attenuated chimeric West Nile/dengue virus vaccines protect rhesus monkeys from West Nile virus. Virology 314 : 190–195. [Google Scholar]
  41. Chen W, Kawano H, Men R, Clark D, Lai CJ, 1995. Construction of intertypic chimeric dengue viruses exhibiting type 3 antigenicity and neurovirulence for mice. J Virol 69 : 5186–5190. [Google Scholar]
  42. Huang CY, Butrapet S, Tsuchiya KR, Bhamarapravati N, Gubler DJ, Kinney RM, 2003. Dengue 2 PDK-53 virus as a chimeric carrier for tetravalent dengue vaccine development. J Virol 77 : 11436–11447. [Google Scholar]
  43. Sabin A, Boulger L, 1973. History of Sabin attenuated poliovirus oral live vaccine strains. J Biol Stand 1 : 115–118. [Google Scholar]
  44. Murphy BR, Chanock RM, 2001. Immunization against viral diseases. Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Strauss SE, eds. Fields Virology. Philadelphia: Lippincott Williams & Wilkins, 435–468.
  45. Proutski V, Gould EA, Holmes EC, 1997. Secondary structure of the 3′untranslated region of flaviviruses: similarities and differences. Nucleic Acids Res 25 : 1194–1202. [Google Scholar]
  46. Leitmeyer KC, Vaughn DW, Watts DM, Salas R, Villalobos I, de Chacon Ramos C, Rico-Hesse R, 1999. Dengue virus structural differences that correlate with pathogenesis. J Virol 73 : 4738–4747. [Google Scholar]
  47. Worobey M, Rambaut A, Holmes EC, 1999. Widespread intraserotype recombination in natural populations of dengue virus. Proc Natl Acad Sci USA 96 : 7352–7357. [Google Scholar]

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  • Received : 19 Apr 2004
  • Accepted : 22 Jun 2004

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