Development of dengue virus replicons expressing HIV-1 gp120 and other heterologous genes: a potential future tool for dual vaccination against dengue virus and HIV

Abstract

Background: Toward the goals of providing an additional vector to add to the armamentarium available to HIV vaccinologists and of creating a bivalent vaccine effective against dengue virus and HIV, we have attempted to create vectors which express dengue virus non-structural proteins and HIV immunogens. Previously we reported the successful construction of dengue virus replicons which lack structural genes necessary for virion release and spreading infection in culture but which can replicate intracellularly and abundantly produce dengue non-structural proteins. Here we attempted to express heterologous genetic material from these replicons.

Results: We cloned into a Deltapre-M/E dengue virus replicon genes for either green fluorescent protein (GFP), HIV gp160 or HIV gp120 and tested the ability of these constructs to express dengue virus proteins as well as the heterologous proteins in tissue culture after transfection of replicon RNA.

Conclusions: Heterologous proteins were readily expressed from these constructs. GFP and gp120 demonstrated minimal or no toxicity. Gp160 expressing replicons were found to express proteins abundantly at 36 hours post transfection, but after 50 hrs of transfection, few replicon positive cells could be found despite the presence of cellular debris positive for replicon proteins. This suggested that gp160 expressed from dengue virus replicons is considerably more toxic than either GFP or gp120. The successful expression of heterologous proteins, including HIV gp120 for long periods in culture suggests this vector system may be useful as a vaccine vector, given appropriate delivery methods.

Figure 1

Construction of wild type dengue virus and dengue virus replicon vectors used in these studies. The diagram at the top represents the wild type dengue virus genome.

Figure 2

Expression of green fluorescent protein (GFP) by Δpre-M/E-GFP 48 hours post transfection.

Figure 3

Expression of proteins by Δpre-M/E-gp120 50 hours post transfection. Left and right frames are two independent fields. Anti-dengue serum was used in these experiments.

Figure 4

Expression of proteins by Δpre-M/E-gp120 48 hours post transfection. Left and right frames are two independent fields. Anti-HIV serum was used in these experiments.

Figure 5

Expression of gp160 by Δpre-M/E-gp160 48 hours post transfection. Left and right frames are independent fields. Anti-HIV serum was used in these experiments.

Figure 6

Expression of gp160 by Δpre-M/E-gp160 36 hours post transfection. Anti-dengue Serum was used in these experiments. Left and right panels are independent fields.

Figure 7

Expression of proteins by Δpre-M/E-gp120 9 days post transfection. Cells were trypsinized and replated on day 7 post transfection and harvested for immunofluorescence two days later. Left and right frames are two independent fields. Anti-dengue serum was used in these experiments.

Figure 8

Simultaneous expression of HIV and dengue proteins by Δpre-M/E-gp120-transfected cells 4 days post transfection. Left frame: FITC detection of HIV proteins. Right frame: Rhodamine detection of dengue proteins.

Figure 9

Simultaneous expression of HIV and dengue proteins by Δpre-M/E-gp120-transfected cells 7 days post transfection. Left frame: FITC detection of HIV proteins. Right frame: Rhodamine detection of dengue proteins.