Vaccination of mice with bacteria carrying a cloned herpesvirus genome reconstituted in vivo

Abstract

Bacterial delivery systems are gaining increasing interest as potential vaccination vectors to deliver either proteins or nucleic acids for gene expression in the recipient. Bacterial delivery systems for gene expression in vivo usually contain small multicopy plasmids. We have shown before that bacteria containing a herpesvirus bacterial artificial chromosome (BAC) can reconstitute the virus replication cycle after cocultivation with fibroblasts in vitro. In this study we addressed the question of whether bacteria containing a single plasmid with a complete viral genome can also reconstitute the viral replication process in vivo. We used a natural mouse pathogen, the murine cytomegalovirus (MCMV), whose genome has previously been cloned as a BAC in Escherichia coli. In this study, we tested a new application for BAC-cloned herpesvirus genomes. We show that the MCMV BAC can be stably maintained in certain strains of Salmonella enterica serovar Typhimurium as well and that both serovar Typhimurium and E. coli harboring the single-copy MCMV BAC can reconstitute a virus infection upon injection into mice. By this procedure, a productive virus infection is regenerated only in immunocompromised mice. Virus reconstitution in vivo causes elevated titers of specific anti-MCMV antibodies, protection against lethal MCMV challenge, and strong expression of additional genes introduced into the viral genome. Thus, the reconstitution of infectious virus from live attenuated bacteria presents a novel concept for multivalent virus vaccines launched from bacterial vectors.

FIG. 1.

129SvEv IFN-γR^0/0 mice were infected with the indicated CFU of WB240 (six mice per group). Positive-control mice were infected with 5 × 10⁵ PFU of wild-type MCMV, and negative controls were mock infected. (A) Sera were collected at dpi 28, serially diluted up to 1:1,024, and assayed for anti-MCMV antibodies by ELISA. Median and quartile absorbance values at 492 nm are displayed. Pooled sera from a group of mice that received 5 × 10⁵ PFU of MCMV were used as positive controls. (B) On the same day, mice were challenged with 2 LD₅₀ of salivary gland-passaged MCMV and monitored for survival. Survival curves for the first 10 days following challenge are shown. Exclusively for presentation purposes, the survival curves are presented in two separate graphs. Error bars, quartiles.

FIG. 2.

129 SvEv IFN-γR^0/0 mice were i.p. infected with the indicated doses of WB240 or with 5 × 10⁵ PFU of MCMV. Mice were sacrificed on dpi 28, and lung homogenates were assayed on MEFs for infectious MCMV titers. Circles represent virus titers in the organs of individual mice. D.L., limit of detection.

FIG. 3.

(A) BALB/c or (B) 129 SvEv IFN-γR^−/− mice were i.p. infected with 10⁸ CFU of WB232 (○) or WB233 (•). T lymphocytes were depleted on dpi 6 and 13, and hydrocortisone was applied on alternate days from dpi 13 onwards. Mice were sacrificed on dpi 21, and organ homogenates were assayed on MEFs for infectious MCMV titers. Circles represent virus titer in organs of individual mice. D.L., detection limit.

FIG. 4.

129 SvEv IFN-γR^0/0 mice were i.p. infected with 10⁸ CFU of either Eco/M/inv or Eco/P/inv. T lymphocytes were depleted on dpi 6 and 13, and hydrocortisone was applied on alternate days from day 13 onwards. Sera were collected at days 12 and 20 and assayed for the presence of HBsAg by direct ELISA. At day 12, no HBsAg could be detected in any of the groups. At day 20, four out of five mice infected with Eco/M/inv and none of the mice infected with Eco/P/inv showed detectable amounts of HBs in their sera.

FIG. 5.

129 SvEv IFN-γR^0/0 mice were infected i.p. with 10⁸ CFU of indicated bacteria (five mice per group). Sera were obtained at 14, 24, and 42 dpi, serially diluted in PBS, and assayed for anti-MCMV antibodies by ELISA. Arithmetic means and standard deviations of absorbance values at 492 nm for the 1:64 dilution step are shown. Sera from mice infected with 10⁵ PFU of MCMV were obtained at dpi 70 and used as positive controls (▴). Sera from uninfected mice (▵) were used as negative controls.

FIG. 6.

129 SvEv IFN-γR^0/0 mice were i.m. or s.c. infected with 10⁹ CFU of Eco/M or Eco/M/inv bacteria. Control mice were infected with Eco/P/inv. At 28 dpi, mice were challenged with 2 LD₅₀ of SGD MCMV and monitored for survival. Survival curves for the first 14 days following challenge are shown.