Transmissible gastroenteritis virus (TGEV)-based vectors with engineered murine tropism express the rotavirus VP7 protein and immunize mice against rotavirus

Abstract

A coronavirus vector based on the genome of the porcine transmissible gastroenteritis virus (TGEV) expressing the rotavirus VP7 protein was constructed to immunize and protect against rotavirus infections in a murine model. The tropism of this TGEV-derived vector was modified by replacing the spike S protein with the homologous protein from mouse hepatitis virus (MHV). The rotavirus gene encoding the VP7 protein was cloned into the coronavirus cDNA. BALB/c and STAT1-deficient mice were inoculated with the recombinant viral vector rTGEV(S-MHV)-VP7, which replicates in the intestine and spreads to other organs such as liver, spleen and lungs. TGEV-specific antibodies were detected in all the inoculated BALB/c mice, while rotavirus-specific antibodies were found only after immunization by the intraperitoneal route. Partial protection against rotavirus-induced diarrhea was achieved in suckling BALB/c mice born to dams immunized with the recombinant virus expressing VP7 when they were orally challenged with the homotypic rotavirus strain.

Fig. 1

Rescue of rTGEV_S-MHV/C11 from cDNA in LR7 cells. (A) Genetic organization of the rTGEV_S-MHV/C11 virus. Restriction sites Pac I, Mlu I, Fse I, Pme I, and Asc I are indicated in the genome. Letters and numbers indicate the viral genes. TRS-SC11, transcription regulating sequence of TGEV-C11 S gene; SMHV-A59, MHV-A59 S gene; TM, transmembrane domain; SC11, TGEV S gene. (B) Genetic organization of the rTGEV_S-MHV/C11-VP7 virus. Restriction sites Pac I, Mlu I, Blp I, FseI, Pme I, and Asc I are indicated in the genome.

Fig. 2

Growth kinetics of rTGEV_S-MHV/C11 in murine and porcine cells. The viral infectivity of rTGEV_S-MHV/C11, TGEV, and MHV-A59 in murine LR7 (A) and porcine ST cells (B) was determined at different times postinfection by plaque assay. Mean values from four experiments are represented, and standard deviations are shown as error bars.

Fig. 3

Growth phenotype of rTGEV_S-MHV/C11 in LR7 murine and ST porcine cells. (A) Monolayers of murine LR7 and porcine ST cells were infected with rTGEV_S-MHV/C11, TGEV or MHV-A59. Cytopathic effect was analyzed by plaque assay and optical microscopy and visualized at 72 h postinfection. (B) Immunofluorescence analysis of rTGEV_S-MHV/C11-infected cells. LR7 and ST cells were infected with rTGEV_S-MHV/C11, TGEV or MHV-A59. Infections were visualized at 24 h.p.i. by immunofluorescence microscopy on permeabilized cells with specific MAbs against TGEV M protein (α-M-TGEV) and MHV-A59 S protein (α-S-MHV).

Fig. 4

Expression of VP7 protein in LR7 cells infected with rTGEV_S-MHV/C11-VP7 detected by immunofluorescence assay. LR7 cells were infected with rTGEV_S-MHV/C11-VP7 (MOI = 1), at 24 h.p.i. cells were fixed in metanol/acetone for 10 min and stained with a sheep polyclonal anti-rotavirus antibody (Silenus) and 3D.C10 MAb to recognize N protein of TGEV. An appropriate secondary fluorescent-conjugated antibody was applied (Cy5-labeled anti-sheep IgG to detect rotavirus protein and FITC-labeled anti-mouse IgG to reveal TGEV protein).

Fig. 5

Detection of rTGEV_S-MHV/C11 antigen in histological sections of tissue samples of rTGEV_S-MHV/C1-inoculated STAT1−/− mice. Mice were inoculated with 2 × 10⁶ FFU of rTGEV_S-MHV/C11. Two days later intestine (A), lungs (B), liver (C) and spleen (D) tissue samples were collected. Histological sections of tissues were prepared and stained with Mab 3D.C10 raised against the N protein. FITC-conjugated secondary antibody was then used to visualize viral protein by immunofluorescence.

Fig. 6

IgG antibody response elicited by immunization with rTGEV_S-MHV/C11-VP7 vector. Groups of five female BALB/c mice were immunized with three doses of 10⁶ FFU of rTGEV_S-MHV/C11-VP7 by either the intragastric, intranasal, or intraperitoneal routes three times at two-weekly intervals. Mice in the control group received three doses of 2 × 10⁶ FFU of rTGEV_S-MHV/C11 (void vector) by the intragastric route with the same schedule. Mice were bled at the indicated points. TGEV (A) and rotavirus (B)-specific serum IgG antibodies were determined by ELISA. *Significantly higher than levels of the negative control sera (p < 0.05 by the Mann–Whitney test).

Fig. 7

Serum IgA response to TGEV. Mice were immunized three times at two-weekly intervals with 2 × 10⁶ FFU/mL of rTGEV_S-MHV/C11 or LR7 cells lysates (negative control). Serum samples were collected from all mice at the indicated time points. Serum IgA to TGEV were assayed by ELISA. Data are means ± SD (six mice per group). *Significantly higher than levels of the preimmune sera (p < 0.05 by the Rang Wilcoxon test).

Fig. 8

Cytokine responses in BALB/c mice after immunization with rTGEV_S-MHV/C11 and rTGEV_S-MHV/C11-VP7. Mice were immunized intragastrically three times at two-weekly intervals with 2 × 10⁶ PFU of rTGEV_S-MHV/C11 or 1 × 10⁶ FFU of rTGEV_S-MHV/C11-VP7, and serum was obtained 15 days after the last inoculation. Serum samples were also obtained 2 days after the first inoculation from both mice groups (A). Two weeks after the last immunization, mice were sacrificed and their spleens were collected. After erythrocyte depletion, splenocytes were cultured in 24-well plates at a concentration of 2 × 10⁶ cells/mL. The cells were either stimulated with the mitogen ConA (5 μg/mL), TGEV coronavirus (2 × 10⁶ FFU), RF rotavirus (5 × 10⁶ FFU) or cultured without stimulation. Cytokine levels were measured in the culture supernatants at the 5th day poststimulation (B). The levels of cytokines were evaluated by a multiplex fluorescent bead immunoassay for quantitative detection of 10 mouse cytokines (FlowCytomix mouse Th1/Th2 10plex, Bender Medsystem) and flow cytometry (Beckman Coulter). Data are mean values ± SD. *Significantly higher than cytokine levels produced by preimmune sera or not stimulated splenocytes (p < 0.05 by the Mann–Whitney test or Wilcoxon test). Non S, nonstimulated.

Fig. 9

Passive protection of newborns against rotavirus-induced diarrhea. Female mice were immunized three times intragastrically or intraperitoneally with rTGEV_S-MHV/C11-VP7. Immunized females were mated with males. Four-day-old newborns were orally infected with 2 × 10⁶ ffu/mice of RF or 10 DD₅₀ of EDIM virus. Control groups of females were inoculated with RF and EDIM, and the offspring was challenged with RF or EDIM strains, respectively. Pups presenting diarrhea were considered unprotected. Fisher's exact test was used to determine significant differences in protection percentage between rotavirus-challenged pups born to rTGEV-VP7 and to rotavirus-inoculated dams. The symbols * and ** indicate significant differences with respect to the EDIM and RF control groups (p < 0.05), respectively. N.D., determined.