title: Enhancing biosynthesis and secretion of premembrane and envelope proteins by the chimeric plasmid of dengue virus type 2 and Japanese encephalitis virus.
abstract: We have constructed a series of plasmids encoding premembrane (prM) and envelope (E) protein genes of dengue virus type 2 (DEN-2). These plasmids included an authentic DEN-2 prM-E construct (pCBD2-14-6), and two chimeric constructs, 90% DEN-2 E-10% Japanese encephalitis (JE) virus E (pCB9D2-1J-4-3) and 80% DEN-2 E-20% JE E (pCB8D2-2J-2-9-1). Monoclonal antibody (MAb) reactivity indicated that all three plasmids expressed authentic DEN-2 virus E protein epitopes representative of flavivirus domains 1, 2, and 3. However, only the pCB8D2-2J-2-9-1 construct secreted high levels of prM, M (membrane), and E proteins into the culture fluid of plasmid-transformed COS-1 cells. The major portion of the prM and E proteins expressed by COS-1 cells transformed by pCBD2-14-6 or pCB9D2-4-3 plasmids remained membrane-bound. The results supported the notion that an unidentified membrane retention sequence is located between E-397 and E-436 of DEN-2 virus E protein. Replacing the carboxyl-terminal 20% of DEN-2 E (397-450) with the corresponding JE sequence had no effect on anti-DEN-2 MAb reactivity, indicating that this region is antigenically inert, although it is required for antigen secretion. Plasmid pCBD2-2J-2-9-1, which expressed secreted forms of prM/M and E that have the potential to form subviral particles, was superior to other constructs in stimulating an antibody response. Ninety percent neutralization titers ranging from 1:40 to >1:1000 were observed in seven of nine serum specimens from pCB8D2-2J-2-9-1-immunized mice. Eleven of twelve 2-day-old neonatal mice, derived from a pCB8D2-2J-2-9-1 immunized female mouse, survived intraperitoneal challenge of DEN-2 New Guinea C virus.
authors.affiliation: Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Service, Post Office Box 2087, Fort Collins, CO 80522, USA. gxc7@cdc.gov
authors.forename: Gwong-Jen J
authors.initials: GJ
authors.lastname: Chang
authors.forename: Ann R
authors.initials: AR
authors.lastname: Hunt
authors.forename: Derek A
authors.initials: DA
authors.lastname: Holmes
authors.forename: Tracy
authors.initials: T
authors.lastname: Springfield
authors.forename: Tzong-Shi
authors.initials: TS
authors.lastname: Chiueh
authors.forename: John T
authors.initials: JT
authors.lastname: Roehrig
authors.forename: Duane J
authors.initials: DJ
authors.lastname: Gubler
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003715:Dengue / Q000276:immunology / Q000517:prevention & control / Q000821:virology; D003716:Dengue Virus / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D004664:Encephalitis Virus, Japanese / Q000235:genetics* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D005260:Female; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D009500:Neutralization Tests; D010957:Plasmids / Q000235:genetics* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014611:Vaccination; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014765:Viral Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology
chemical_list: D000914:Antibodies, Viral; C051821:E-glycoprotein, Dengue virus type 2; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D014765:Viral Vaccines; C074932:prM protein, Flavivirus