Dengue-1 Virus Envelope Glycoprotein Gene Expressed in Recombinant Baculovirus Elicits Virus-Neutralizing Antibody in Mice and Protects them from Virus Challenge

Plasma leakage is one of the characteristic features of dengue haemorrhagic fever. The interaction among peripheral blood mononuclear cells (PBMCs), dengue virus and endothelial cells was analysed in vitro. Human umbilical vein endothelial cells (HUVECs) were infected with dengue-2 virus (DV-2) at an m.o.i. of 0.5 p.f.u. per cell. PBMCs were added to DV-2-infected HUVECs, and transendothelial electrical resistance (TEER) and transalbumin permeability were assessed. Dengue virus infection at an m.o.i. of 0.5 p.f.u. per cell alone did not decrease the TEER, but addition of PBMCs decreased the TEER, increased the albumin permeability and induced morphological changes of HUVECs. The extent of the decrease was more profound with adherent PBMCs than with non-adherent PBMCs. The expression of vascular endothelial cadherin (VE-cadherin) was examined using real-time RT-PCR and immunofluorescence. Addition of PBMCs to DV-2-infected HUVECs decreased the levels of mRNA transcripts and cell-surface expression of VE-cadherin. The results indicate that PBMCs increased the permeability of DV-2-infected HUVECs and that the increased permeability was concomitant with morphological change and the decrease in VE-cadherin expression. The results suggest that functional impairment of the DV-2-infected HUVEC monolayer was caused by interaction with PBMCs.

Section: Methodsmentioning

confidence: 99%

Peripheral blood mononuclear cells increase the permeability of dengue virus-infected endothelial cells in association with downregulation of vascular endothelial cadherin

Dewi

Takasaki

Kurane

2008

“…Deubel et al, 1991). The protocol was similar to that used previously for immunization studies using baculovirus-expressed DEN-1 E protein (Putnak et al, 1991). Lysates of baculovirus recombinant-infected cells were administered without Freund's adjuvant because it did not appear to be of any value in similar experiments with recombinant DEN-4 proteins (Zhang et al, 1988).…”

Section: Expression and Biochemical Features Of D2erec And D3erec Promentioning

confidence: 99%

Analysis of C-terminally truncated dengue 2 and dengue 3 virus envelope glycoproteins: processing in insect cells and immunogenic properties in mice

Delenda¹,

Staropoli²,

Frenkiel³

et al. 1994

We constructed two recombinant Autographa californica nuclear polyhedrosis baculoviruses. Spodoptera frugiperda (Sf9) cells containing these constructs produce carboxy-terminally truncated envelope E proteins representing dengue (DEN) virus serotypes 2 and 3. The two recombinant proteins contained their homologous signal sequences at the N terminus and were truncated by 71 and 74 amino acids at the C terminus, respectively. This allowed the translocation of the recombinant proteins to the endoplasmic reticulum followed by glycosylation processing and secretion into the extracellular medium. An additional unglycosylated form which was not secreted was detected inside the infected Sf9 cells. Sera from Swiss mice immunized with the infected St9 cell lysates gave a DEN cross-reactive response in ELISA and substantial amounts of neutralizing antibodies to the homologous virus. Similar antibody titres were obtained when the two recombinant proteins were inoculated concomitantly. BALB/c mice were vaccinated with three doses of the recombinant E proteins, taken as monovalent or bivalent immunogens, and challenged with mouse-adapted DEN-2 virus. DEN-2 E protein induced a good protection (90 %) against lethal encephalitis and recombinant DEN-3 E protein gave a substantial cross-protection (54%). Eighty-two percent of the mice immunized with a mixture of both recombinant E proteins survived the DEN-2 virus challenge.

“…The system has also been employed to produce recombinant dengue virus E protein and NS-1, which, to varying degrees, induced protective immune responses in mice (Putnak et al, 1991 ;Deubel et al, 1991 ;Delenda et al, 1994 ;Smucny et al, 1995). Of these, only Delenda et al (1994) described elicitation of dengue virus serotype cross-reactive antibody responses.…”

Section: Introductionmentioning

confidence: 99%

Analysis of a recombinant dengue-2 virus-dengue-3 virus hybrid envelope protein expressed in a secretory baculovirus system.

Bielefeldt‐Ohmann

Beasley

FitzPatrick

et al. 1997

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 serumfree 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