Analysis of a recombinant dengue-2 virus-dengue-3 virus hybrid envelope protein expressed in a secretory baculovirus system.
In a step towards a tetravalent dengue virus subunit vaccine which is economical to produce, highly immunogenic and stable, a hybrid dengue virus envelope (E) protein molecule has been constructed. It consists of 36 amino acids from the membrane protein, the N-terminal 288 amino acids of the dengue-2 virus E protein plus amino acids 289-424 of the dengue-3 virus E protein. It has been engineered for secretory expression by fusion to a mellitin secretory signal sequence and truncation of the hydrophobic transmembrane segment. Using the baculovirus expression system and serum-free conditions, more than 95% of recombinant dengue-2 virus-dengue-3 virus hybrid E protein (rD2D3E) was secreted into the cell culture supernatant in a stable form with multiple features indicative of preserved conformation. The hybrid molecule reacted with a panel of dengue virus- and flavivirus-specific MAbs which recognize linear or conformational epitopes on dengue virions. Human dengue virus-specific antisera also reacted with the protein. The hybrid rD2D3E protein was able to inhibit the in vitro binding of dengue-2 and dengue-3 viruses to human myelomonocytic cells, suggesting that the receptor-binding epitope(s) was preserved. Adjuvant-free immunization with the hybrid protein induced an antibody response to both dengue-2 and dengue-3 virus in outbred mice, comparable in strength to that of individual rD2E and rD3E proteins. Notably, these antibody responses were primarily of the IgG2a and IgG2b isotype. A strong dengue virus cross-reactive T cell response was also induced in the mice, suggesting that dengue virus hybrid E proteins could form the basis of an efficacious multivalent dengue virus vaccine.
Bielefeldt-Ohmann
H
H
Centre for Molecular Biotechnology, School of Life Science, Queensland University of Technology, Brisbane, Australia. helle@biosci.uq.edu.au
Beasley
D W
DW
Fitzpatrick
D R
DR
Aaskov
J G
JG
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D003716:Dengue Virus / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D051379:Mice; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013601:T-Lymphocytes / Q000276:immunology / Q000821:virology*; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins