Attenuation of vaccinia virus by the expression of human Flt3 ligand

Abstract

Background: Vaccinia virus, one of the best known members of poxvirus family, has a wide host range both in vivo and in vitro. The expression of Flt3 ligand (FL) by recombinant vaccinia virus (rVACV) highly influenced properties of the virus in dependence on the level of expression.

Results: High production of FL driven by the strong synthetic promoter decreased the growth of rVACV in macrophage cell line J774.G8 in vitro as well as its multiplication in vivo when inoculated in mice. The inhibition of replication in vivo was mirrored in low levels of antibodies against vaccinia virus (anti-VACV) which nearly approached to the negative serum level in non-infected mice. Strong FL expression changed not only the host range of the recombinant but also the basic protein contents of virions. The major proteins - H3L and D8L - which are responsible for the virus binding to the cells, and 28 K protein that serves as a virulence factor, were changed in the membrane portion of P13-E/L-FL viral particles. The core virion fraction contained multiple larger, uncleaved proteins and a higher amount of cellular proteins compared to the control virus. The overexpression of FL also resulted in its incorporation into the viral core of P13-E/L-FL IMV particles. In contrary to the equimolar ratio of glycosylated and nonglycosylated FL forms found in cells transfected with the expression plasmid, the recombinant virus incorporated mainly the smaller, nonglycosylated FL.

Conclusions: It has been shown that the overexpression of the Flt3L gene in VACV results in the attenuation of the virus in vivo.

Figure 1

In vitro multiplication and sFL production by rVACV. Confluent CV-1cell cultures were infected with purified virus at a MOI of2.5 at 37°C for 1 hour, washed with PBS and the fresh medium was added. The medium and cells were collected, frozen and thawed, and cell debris was removed. The titers of the infectious virus (A) were determined in the medium and in the cell lysate. Total FL production was determined by ELISA (B). The columns in all graphs represent the mean ± s.d. The intracellular location of sFL (C) in 293T cells (a, b) 48 h after transfection or in infected CV1 (c, d) and HeLa cells (e, f) 9 h after infection or in J774.G8 cells (g, h) 3 h after infection with P13-H5-FL (c, e, g) or P13-E/L-FL (d, f, h) as visualized by an immunofluorescent microscope at a magnification of 1000×. The colocalisation of sFL (D) with endoplasmic reticulum marker calreticulin or with cis-Golgi marker GM130 in HeLa cells 3 h after infection as visualized by an immunofluorescent microscope at a magnification of 1000×.

Figure 2

In vivo multiplication, sFL production and antibody response to rVACV. Groups (n = 4) of C57Bl/6 female mice were inoculated i.p. with 1 × 10⁶ pfu of P13-E/L-FL, P13-H5-FL or P13-E7. The ovaries and sera were collected at various intervals after infection. Replication of rVACV in the ovaries was determined by plaque assay (A) or by Q-PCR (B). The in vivo production of FL was measured in mouse sera by ELISA (C). To determine the immunogenicity of recombinants, groups of mice (n = 5) were i.p. inoculated with 1 × 10⁶ pfu of rVACV. Five weeks later, the animals were anesthesized and the serum was collected. The levels of VACV specific IgG1 or IgG2a antibodies were quantified by ELISA (D). The data were analyzed by the two-way ANOVA.

Figure 3

Multiplication of rVACV and sFL production in macrophage cell line J774.G8. Culture of J774.G8 cells was infected with the purified virus at MOI of 2.5 (A, C, D) or at MOI of 0.1 (B) at 37°C for 1 hour, washed with PBS and the fresh medium with or without cytosine arabinoside (40 μg/ml) was added. Multiplication of virus was determined as beta-galactosidase activity (A, C) or by Q-PCR (B) at indicated intervals (A, B) or 24 hours after infection (C). Total FL production was determined by ELISA (D).

Figure 4

Incorporation of sFL into viral particles. Aliquots of the indicated sucrose purified virions (A) or their solubilized and separated fractions (B) were analyzed by western blot using the FL specific BAF308 monoclonal antibody. The sample prepared from the culture medium of 293T cells transfected with pBSC-FL expression plasmid served as a positive control. To neutralize virus infectivity, the saccharose purified particles were incubated at 37°C for 1 hour with PBS, anti-FL antibody or anti-VACV serum. The first aliquot of samples was used for infection of cell cultures (C). Cytosine arabinoside (40 μg/ml) was added for inhibition of viral replication as the positive control. The beta-galactosidase assay was performed after 24 hours of cultivation. The second aliquot of samples was applied to a formvar membrane coated copper grid (D). Negatively stained particles were examined under an electron-microscope at a magnification of 50 000×.

Figure 5

Analysis of virion protein composition. Purified P13, P13-H5-FL or P13-E/L-FL viruses were fractionated into enriched membrane and core fractions. Aliquots were separated by 12% or 8-15% gradient SDS-PAGE. The gel was processed by western blotting and stained with anti-VACV mouse serum (A) or stained with Coomassie blue (B). The proteins of differing bands were identified by MALDI.