Author:
- INTRODUCTION
- MATERIALS AND METHODS
- DEN viruses, mammalian, and insect cells
- Microbial host strains and plasmid vector
- Others
- Creation of recombinant P. pastoris clone expressing the EDIII-based tetravalent gene
- Expression and purification of recombinant EDIII-T protein
- His-Sorb ELISA for the determination of rEDIII-T protein
- Protein blot assays
- Immunization of mice
- Detection of anti-DEN virus antibodies in immune sera
- Immunofluorescence assay
- Plaque reduction neutralization test
- Proliferation and cytokine release assays
- RESULTS
- Design of a novel tetravalent DEN antigen encoding the P. pastoris construct
- Purification and characterization of the tetravalent DEN antigen protein
- Tetravalent DEN antigen elicits antibodies that can recognize and neutralize all four serotypes of DEN viruses
- Tetravalent antigen induces T-cell responses specific to each DEN virus serotype
- DISCUSSION
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1
Lindenbach BD, Rice CM, 2001. Flaviviridae: the viruses and their replication. Knipe DM, Howley PM, eds. Field’s Virology. Fourth edition. Philadelphia, PA: Lippincott Williams & Wilkins, 991–1041.
-
2
Gubler DJ, 1998. Dengue and dengue haemorrhagic fever. Clin Microbiol Rev 11 :480–496.
-
3
Guzmán MG, Kouri G, 2002. Dengue: an update. Lancet Infect Dis 2 :33–42.
-
4
Gibbons RV, Vaughn DW, 2002. Dengue: an escalating problem. BMJ 324 :1563–1566.
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5
Innis BL, 1997. Antibody responses to dengue virus infection. Gubler DJ, Kuno G, eds. Dengue and Dengue Hemorrhagic Fever. New York: CAB International, 221–243.
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6
Halstead SB, Heinz FX, Barrett ADT, Roehrig JT, 2005. Conference report on Dengue virus: molecular basis of cell entry and pathogenesis, 25–27 June 2003, Vienna, Austria. Vaccine 23 :849–856.
-
7
Kurane I, Ennis FA, 1997. Immunopathogenesis of dengue virus infections. Gubler DJ, Kuno G, eds. Dengue and Dengue Hemorrhagic Fever. Wallingford: CAB International, 273–290.
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8
Hombach J, Barrett AD, Cardosa MJ, Deubel V, Guzman M, Kurane I, Roehrig JT, Sabchareon A, Kieny MP, 2005. Meeting report on Review on flavivirus vaccine development: proceedings of a meeting jointly organized by the World Health Organization and the Thai ministry of public health, 26–27 April 2004, Bangkok, Thailand. Vaccine 23 :2689–2695.
-
9
Kanesa-thasan 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 CHJr, 2001. Safety and immunogenicity of attenuated dengue virus vaccines (Aventis Pasteur) in human volunteers. Vaccine 19 :3179–3188.
-
10
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 (Suppl 6):48–60.
-
11
Kitchener S, Nissen M, Nasveld P, Forrat R, Yoksan S, Lang J, Saluzzo JF, 2006. Immunogenicity and safety of two live-attenuated tetravalent dengue vaccine formulations in healthy Australian adults. Vaccine 24 :1238–1241.
-
12
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 im-munogenicity in nonhuman primates of a chimeric yellow fever-dengue virus tetravalent vaccine. J Virol 75 :7290–7304.
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13
Guirakhoo F, Kitchener S, Morrison D, Forrat R, McCarthy K, Nichols R, Yoksan S, Duan X, Ermak TH, Kanesa-Thasan N, Bedford P, Lang J, Quentin-Millet MJ, Monath TP, 2006. Live attenuated chimeric yellow fever dengue type 2 (Chi-meriVaxTM-DEN2) vaccine: phase I clinical trial for safety and immunogenicity: effect of yellow fever prior immunity in induction of cross neutralizing antibody responses to all. Human Vaccines 2 :60–67.
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14
Seligman SJ, Gould EA, 2004. Live flavivirus vaccines: reasons for caution. Lancet 363 :2073–2075.
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15
Putnak R, Feighny R, Burrous J, Cochran M, Hackett C, Smith G, Hoke C, 1991. Dengue-1 virus envelope glycoprotein gene expressed in recombinant baculovirus elicits virus-neutralizing antibody in mice and protects them from virus challenge. Am J Trop Med Hyg 45 :159–167.
-
16
Delenda C, Staropoli I, Frenkiel MP, Cabanié L, Deubel V, 1994. Analysis of C-terminally truncated dengue 2 and dengue 3 virus envelope glycoproteins: processing in insect cells and im-munogenic properties in mice. J Gen Virol 75 :1569–1578.
-
17
Simmons M, Nelson WM, Wu SJ, Hayes CG, 1998. Evaluation of the protective efficacy of a recombinant dengue envelope B domain fusion protein against dengue 2 virus infection in mice. Am J Trop Med Hyg 58 :655–662.
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18
Kelly EP, Greene JJ, King AD, Innis BL, 2000. Purified dengue 2 virus envelope glycoprotein aggregates produced by bacu-lovirus are immunogenic in mice. Vaccine 18 :2549–2559.
-
19
Konishi E, Yamaoka M, Kurane I, Mason PW, 2000. A DNA vaccine expressing dengue type 2 virus premembrane and envelope genes induces neutralizing antibody and memory B cells in mice. Vaccine 18 :1133–1139.
-
20
Men R, Wyatt L, Tokimatsu I, Arakaki S, Shameem G, Elkins R, Chanock R, Moss B, Lai CJ, 2000. Immunization of rhesus monkeys with a recombinant of modified vaccinia virus An-kara expressing a truncated envelope glycoprotein of dengue type 2 virus induced resistance to dengue type 2 virus challenge. Vaccine 18 :3113–3122.
-
21
Simmons M, Murphy GS, Hayes CG, 2001. Short report: antibody responses of mice immunized with a tetravalent dengue recombinant protein subunit vaccine. Am J Trop Med Hyg 65 :159–161.
-
22
Hermida L, Rodríguez R, Lazo L, Lopez C, Márquez G, Páez R, Suárez C, Espinosa R, Garcia J, Guzmán G, Guillén G, 2002. A recombinant envelope protein from dengue virus purified by IMAC is bioequivalent with its immune-affinity chromatography purified counterpart. J Biotechnol 94 :213–216.
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23
Muné M, Rodríguez R, Ramírez R, Soto Y, Sierra B, Roche RR, Marquez G, Garcia J, Guillén G, Guzmán MG, 2003. Carboxy-terminally truncated dengue 4 virus envelope glycoprotein expressed in Pichia pastoris induced neutralizing antibodies and resistance to dengue 4 virus challenge in mice. Arch Virol 148 :2267–2273.
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24
Wei HY, Jiang LF, Xue YH, Fang DY, Guo HY, 2003. Secreted expression of dengue virus type 2 full-length envelope glyco-protein in Pichia pastoris. J Virol Methods 109 :17–23.
-
25
Hermida L, Rodríguez R, Lazo L, Bernardo L, Silva R, Zulueta A, López C, Martín J, Valdes I, del Rosario D, Guillén G, Guzmán MG, 2004. A fragment of the envelope protein from dengue-1 virus, fused in two different sites of the meningococ-cal P64k protein carrier, induces a functional immune response in mice. Biotechnol Appl Biochem 39 :107–114.
-
26
Hermida L, Rodríguez R, Lazo L, Silva R, Zulueta A, Chinea G, López C, Guzmán MG, Guillén G, 2004. A dengue-2 envelope fragment inserted within the structure of the P64k meningo-coccal protein carrier enables a functional immune response against the virus in mice. J Virol Methods 115 :41–49.
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27
Mota J, Acosta M, Argotte R, Figueroa R, Méndez A, Ramos C, 2005. Induction of protective antibodies against dengue virus by tetravalent DNA immunization of mice with domain III of the envelope protein. Vaccine 23 :3469–3476.
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28
Apt D, Raviprakash K, Brinkman A, Semyonov A, Yang S, Skinner C, Diehl L, Lyons R, Porter K, Punnonen J, 2006. Tetravalent neutralizing antibody response against four dengue sero-types by a single chimeric dengue envelope antigen. Vaccine 24 :335–344.
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29
Konishi E, Kosugi S, Imoto JI, 2006. Dengue tetravalent DNA vaccine inducing neutralizing antibody and anamnestic responses to four serotypes in mice. Vaccine 24 :2200–2207.
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30
Hermida L, Bernardo L, Martín J, Alvarez M, Prado I, López C, Sierra B, Martínez R, Rodríguez R, Zulueta A, Pérez AB, Lazo L, Rosario D, Guillén G, Guzmán MG, 2006. A recombinant fusion protein containing the domain III of the dengue-2 envelope protein is immunogenic and protective in non-human primates. Vaccine 24 :3165–3171.
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31
Zulueta A, Martín J, Hermida L, Alvarez M, Valdés I, Prado I, Chinea G, Rosario D, Guillén G, Guzmán MG, 2006. Amino acid changes in the recombinant dengue 3 envelope domain III determine its antigenicity and immunogenicity in mice. Virus Res 121 :65–73.
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An Envelope Domain III–based Chimeric Antigen Produced in Pichia pastoris Elicits Neutralizing Antibodies Against All Four Dengue Virus Serotypes
Behzad Etemad
Behzad Etemad
Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India
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Gaurav Batra
Gaurav Batra
Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India
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Rajendra Raut
Rajendra Raut
Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India
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Satinder Dahiya
Satinder Dahiya
Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India
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Saima Khanam
Saima Khanam
Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India
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Sathyamangalam Swaminathan
Sathyamangalam Swaminathan
Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India
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Navin Khanna
Navin Khanna
Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India
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There is currently no vaccine to prevent dengue (DEN) virus infection, which is caused by any one of four closely related serotypes, DEN-1, DEN-2, DEN-3, or DEN-4. A DEN vaccine must be tetravalent, because immunity to a single serotype does not offer cross-protection against the other serotypes. We have developed a novel tetravalent chimeric protein by fusing the receptor-binding envelope domain III (EDIII) of the four DEN virus serotypes. This protein was expressed in the yeast, Pichia pastoris, and purified to near homogeneity in high yields. Antibodies induced in mice by the tetravalent protein, formulated in different adjuvants, neutralized the infectivity of all four serotypes. This, coupled with the high expression potential of the P. pastoris system and easy one-step purification, makes the EDIII-based recombinant protein a potentially promising candidate for the development of a safe, efficacious, and inexpensive, tetravalent DEN vaccine.
Author Notes
- INTRODUCTION
- MATERIALS AND METHODS
- DEN viruses, mammalian, and insect cells
- Microbial host strains and plasmid vector
- Others
- Creation of recombinant P. pastoris clone expressing the EDIII-based tetravalent gene
- Expression and purification of recombinant EDIII-T protein
- His-Sorb ELISA for the determination of rEDIII-T protein
- Protein blot assays
- Immunization of mice
- Detection of anti-DEN virus antibodies in immune sera
- Immunofluorescence assay
- Plaque reduction neutralization test
- Proliferation and cytokine release assays
- RESULTS
- Design of a novel tetravalent DEN antigen encoding the P. pastoris construct
- Purification and characterization of the tetravalent DEN antigen protein
- Tetravalent DEN antigen elicits antibodies that can recognize and neutralize all four serotypes of DEN viruses
- Tetravalent antigen induces T-cell responses specific to each DEN virus serotype
- DISCUSSION
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1
Lindenbach BD, Rice CM, 2001. Flaviviridae: the viruses and their replication. Knipe DM, Howley PM, eds. Field’s Virology. Fourth edition. Philadelphia, PA: Lippincott Williams & Wilkins, 991–1041.
-
2
Gubler DJ, 1998. Dengue and dengue haemorrhagic fever. Clin Microbiol Rev 11 :480–496.
-
3
Guzmán MG, Kouri G, 2002. Dengue: an update. Lancet Infect Dis 2 :33–42.
-
4
Gibbons RV, Vaughn DW, 2002. Dengue: an escalating problem. BMJ 324 :1563–1566.
-
5
Innis BL, 1997. Antibody responses to dengue virus infection. Gubler DJ, Kuno G, eds. Dengue and Dengue Hemorrhagic Fever. New York: CAB International, 221–243.
-
6
Halstead SB, Heinz FX, Barrett ADT, Roehrig JT, 2005. Conference report on Dengue virus: molecular basis of cell entry and pathogenesis, 25–27 June 2003, Vienna, Austria. Vaccine 23 :849–856.
-
7
Kurane I, Ennis FA, 1997. Immunopathogenesis of dengue virus infections. Gubler DJ, Kuno G, eds. Dengue and Dengue Hemorrhagic Fever. Wallingford: CAB International, 273–290.
-
8
Hombach J, Barrett AD, Cardosa MJ, Deubel V, Guzman M, Kurane I, Roehrig JT, Sabchareon A, Kieny MP, 2005. Meeting report on Review on flavivirus vaccine development: proceedings of a meeting jointly organized by the World Health Organization and the Thai ministry of public health, 26–27 April 2004, Bangkok, Thailand. Vaccine 23 :2689–2695.
-
9
Kanesa-thasan 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 CHJr, 2001. Safety and immunogenicity of attenuated dengue virus vaccines (Aventis Pasteur) in human volunteers. Vaccine 19 :3179–3188.
-
10
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 (Suppl 6):48–60.
-
11
Kitchener S, Nissen M, Nasveld P, Forrat R, Yoksan S, Lang J, Saluzzo JF, 2006. Immunogenicity and safety of two live-attenuated tetravalent dengue vaccine formulations in healthy Australian adults. Vaccine 24 :1238–1241.
-
12
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 im-munogenicity in nonhuman primates of a chimeric yellow fever-dengue virus tetravalent vaccine. J Virol 75 :7290–7304.
-
13
Guirakhoo F, Kitchener S, Morrison D, Forrat R, McCarthy K, Nichols R, Yoksan S, Duan X, Ermak TH, Kanesa-Thasan N, Bedford P, Lang J, Quentin-Millet MJ, Monath TP, 2006. Live attenuated chimeric yellow fever dengue type 2 (Chi-meriVaxTM-DEN2) vaccine: phase I clinical trial for safety and immunogenicity: effect of yellow fever prior immunity in induction of cross neutralizing antibody responses to all. Human Vaccines 2 :60–67.
-
14
Seligman SJ, Gould EA, 2004. Live flavivirus vaccines: reasons for caution. Lancet 363 :2073–2075.
-
15
Putnak R, Feighny R, Burrous J, Cochran M, Hackett C, Smith G, Hoke C, 1991. Dengue-1 virus envelope glycoprotein gene expressed in recombinant baculovirus elicits virus-neutralizing antibody in mice and protects them from virus challenge. Am J Trop Med Hyg 45 :159–167.
-
16
Delenda C, Staropoli I, Frenkiel MP, Cabanié L, Deubel V, 1994. Analysis of C-terminally truncated dengue 2 and dengue 3 virus envelope glycoproteins: processing in insect cells and im-munogenic properties in mice. J Gen Virol 75 :1569–1578.
-
17
Simmons M, Nelson WM, Wu SJ, Hayes CG, 1998. Evaluation of the protective efficacy of a recombinant dengue envelope B domain fusion protein against dengue 2 virus infection in mice. Am J Trop Med Hyg 58 :655–662.
-
18
Kelly EP, Greene JJ, King AD, Innis BL, 2000. Purified dengue 2 virus envelope glycoprotein aggregates produced by bacu-lovirus are immunogenic in mice. Vaccine 18 :2549–2559.
-
19
Konishi E, Yamaoka M, Kurane I, Mason PW, 2000. A DNA vaccine expressing dengue type 2 virus premembrane and envelope genes induces neutralizing antibody and memory B cells in mice. Vaccine 18 :1133–1139.
-
20
Men R, Wyatt L, Tokimatsu I, Arakaki S, Shameem G, Elkins R, Chanock R, Moss B, Lai CJ, 2000. Immunization of rhesus monkeys with a recombinant of modified vaccinia virus An-kara expressing a truncated envelope glycoprotein of dengue type 2 virus induced resistance to dengue type 2 virus challenge. Vaccine 18 :3113–3122.
-
21
Simmons M, Murphy GS, Hayes CG, 2001. Short report: antibody responses of mice immunized with a tetravalent dengue recombinant protein subunit vaccine. Am J Trop Med Hyg 65 :159–161.
-
22
Hermida L, Rodríguez R, Lazo L, Lopez C, Márquez G, Páez R, Suárez C, Espinosa R, Garcia J, Guzmán G, Guillén G, 2002. A recombinant envelope protein from dengue virus purified by IMAC is bioequivalent with its immune-affinity chromatography purified counterpart. J Biotechnol 94 :213–216.
-
23
Muné M, Rodríguez R, Ramírez R, Soto Y, Sierra B, Roche RR, Marquez G, Garcia J, Guillén G, Guzmán MG, 2003. Carboxy-terminally truncated dengue 4 virus envelope glycoprotein expressed in Pichia pastoris induced neutralizing antibodies and resistance to dengue 4 virus challenge in mice. Arch Virol 148 :2267–2273.
-
24
Wei HY, Jiang LF, Xue YH, Fang DY, Guo HY, 2003. Secreted expression of dengue virus type 2 full-length envelope glyco-protein in Pichia pastoris. J Virol Methods 109 :17–23.
-
25
Hermida L, Rodríguez R, Lazo L, Bernardo L, Silva R, Zulueta A, López C, Martín J, Valdes I, del Rosario D, Guillén G, Guzmán MG, 2004. A fragment of the envelope protein from dengue-1 virus, fused in two different sites of the meningococ-cal P64k protein carrier, induces a functional immune response in mice. Biotechnol Appl Biochem 39 :107–114.
-
26
Hermida L, Rodríguez R, Lazo L, Silva R, Zulueta A, Chinea G, López C, Guzmán MG, Guillén G, 2004. A dengue-2 envelope fragment inserted within the structure of the P64k meningo-coccal protein carrier enables a functional immune response against the virus in mice. J Virol Methods 115 :41–49.
-
27
Mota J, Acosta M, Argotte R, Figueroa R, Méndez A, Ramos C, 2005. Induction of protective antibodies against dengue virus by tetravalent DNA immunization of mice with domain III of the envelope protein. Vaccine 23 :3469–3476.
-
28
Apt D, Raviprakash K, Brinkman A, Semyonov A, Yang S, Skinner C, Diehl L, Lyons R, Porter K, Punnonen J, 2006. Tetravalent neutralizing antibody response against four dengue sero-types by a single chimeric dengue envelope antigen. Vaccine 24 :335–344.
-
29
Konishi E, Kosugi S, Imoto JI, 2006. Dengue tetravalent DNA vaccine inducing neutralizing antibody and anamnestic responses to four serotypes in mice. Vaccine 24 :2200–2207.
-
30
Hermida L, Bernardo L, Martín J, Alvarez M, Prado I, López C, Sierra B, Martínez R, Rodríguez R, Zulueta A, Pérez AB, Lazo L, Rosario D, Guillén G, Guzmán MG, 2006. A recombinant fusion protein containing the domain III of the dengue-2 envelope protein is immunogenic and protective in non-human primates. Vaccine 24 :3165–3171.
-
31
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