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

Abstract

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.

Short report: Antibody responses of mice immunized with a tetravalent dengue recombinant protein subunit vaccine.

“…Furthermore, the titer of neutralizing antibody induced against each dengue virus serotype after immunization with the tetravalent recombinant vaccine is as high or higher than titers reported after immunization of mice with live dengue virus. 7,20 These encouraging results suggest that further investigation of the DEN-MBP tetravalent recombinant subunit vaccine is warranted. Studies of nonhuman primates are planned to assess the ability of this vaccine to prevent viremia after challenge with live virus and to determine the duration of protection.…”

“…7 Another report that used a baculovirus-expressed recombinant hybrid DEN-2 and DEN-3 envelope protein also showed that immunized mice produced antibody to both DEN-2 and DEN-3 virus by Western blot assay. 10 However, our study is the first to show that a dengue tetravalent recombinant protein vaccine can induce a strong neutralizing antibody response to all 4 dengue virus serotypes.…”

“…Most research efforts have focused on the development of live attenuated vaccines selected by serial passage in mice or mammalian cell cultures and recombinant protein vaccines. [3][4][5][6][7][8][9][10][11] Efforts to develop genetically modified and chimeric live attenuated vaccines or DNA vaccines also have emerged. [12][13][14][15][16] In most dengue vaccine efforts, the long-term goal has been to develop a tetravalent vaccine that would induce neutralizing antibody against all 4 viral serotypes.…”

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

“…Unfortunately, the current murine model used for dengue virus protection studies, i.e. challenge of very young mice by the intracerebral route, may be inappropriate for revealing the true potential of this and previously described subunit dengue virus vaccines (Aaskov et al, 1988 ;Delenda et al, 1994). Susceptibility of mice to intracerebral dengue virus challenge is age-dependent, and immunization must therefore be initiated at a time when the immune system is still immature (Sarzotti et al, 1996 ;Barrios et al, 1996).…”

“…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. Although an attenuated multivalent dengue virus vaccine is currently under evaluation (Bhamarapravati & Yoksan, 1989), a similar approach using multivalent recombinant E proteins has not been reported.…”