Targeted deletion of regions rich in immune-evasive genes from the cytomegalovirus genome as a novel vaccine strategy

Abstract

Human cytomegalovirus (CMV), a ubiquitous human pathogen, is a leading cause of congenital infections and represents a serious health risk for the immunosuppressed patient. A vaccine against CMV is currently not available. CMV is characterized by its large genome and by multiple genes modulating the immunity of the host, which cluster predominantly at genome termini. Here, we tested whether the deletion of gene blocks rich in immunomodulatory genes could be used as a novel concept in the generation of immunogenic but avirulent, herpesvirus vaccines. To generate an experimental CMV vaccine, we selectively deleted 32 genes from the mouse cytomegalovirus (MCMV) genome. The resulting mutant grew to titers similar to that of wild-type MCMV in vitro. In vivo, the mutant was 10,000-fold attenuated and well tolerated, even by highly susceptible mice deficient for B, T, and NK cells or for the interferon type I receptor. Equally relevant for safety concerns, immune suppression did not lead to the mutant's reactivation from latency. Immunization with the replication-competent mutant, but not with inactivated virus, resulted in protective immunity, which increased over time. Vaccination induced MCMV-specific antibodies and a strong T-cell response. We propose that a targeted and rational approach can improve future herpesvirus vaccines and vaccine vectors.

FIG. 1.

Analysis of the BAC-cloned genomes pSM3fr and Δm01-17+m144-158 and of the corresponding viral DNA after virus reconstitution. (a) The pΔm01-17+m144-158 (Δ) and the pSM3fr (WT) BACs were analyzed by restriction endonuclease digests using NdeI, NotI, and EcoRV. Filled arrowheads indicate NdeI fragments which are lost by the deletion of the regions comprising the genes m01 to m17 and m144 to m158. Open arrowheads indicate two new fragments generated by mutagenesis. Analysis of NotI and EcoRV restriction patterns excluded unwanted mutagenesis that occurred in other areas of the mutant genome. (b) Schematic representation of the m158 gene locus in the pSM3fr (WT-MCMV BAC) and pΔm01-17+m144-158-BAC plasmids and upon reconstitution of the respective virus (BAC and virus maps are connected with thin black arrows). The viral ORFs are indicated by thick black arrows; the BAC vector sequences are represented by a gray box; the repeat region, which is duplicated at both the BAC/viral DNA joints, are shown as black boxes (d and d′); the loxP sites at the ends of the BAC vector sequences are indicated by black dots. The open arrowhead indicates the site at which the region m144 to m158 was deleted from the mutant genome. The probe used for the Southern blotting shown in panel c was amplified on the pSN3fr template and is indicated in the uppermost map by the thick horizontal (h) bar. PciI restriction sites are indicated by the light gray vertical bars. PciI fragments recognized by the Southern probe and corresponding to the signals indicated in panel c are indicated with bold capital letters below each map (L, ΔL, R, B, B1, and B2). (c) Southern blotting analysis of the BAC vector sequence in pΔm01-17+m144-158 and in the Δm01-17+m144-158-MCMV genome. DNA isolated from M2-10B4 cells and infected either with WT (lane 2) or Δm01-17+m144-158-MCMV (lane 4) was cleaved by PciI, separated by electrophoresis, and hybridized with the probe “h,” indicated in panel b (lanes 2 and 4). pSM3fr and pΔm01-17+m144-158-BAC plasmid DNA was used for control (lanes 1 and 3). The specifically recognized fragments are indicated by the letters on the left side of the panel corresponding to the maps in panel b. To visualize the separation of fragments B1 and B2, only half of the pSM3fr BAC DNA was loaded (lane 1). Values shown are given in kilobases.

FIG. 2.

Replication of Δm01-17+m144-158-MCMV and WT-MCMV in vitro. NIH 3T3 fibroblasts (a), Ana-1 macrophages (b), SVEC4-10 endothelial cells (c), and C127 mammary epithelial cells (d) were infected in triplicate with Δm01-17+m144-158-MCMV or WT-MCMV at an MOI of 0.1. Virus in the supernatant was harvested every 24 h, from day 0 (input virus) to day 4 or 5 p.i., and infectious virus titers were determined by plaque assay with MEF. Values shown are means ± standard deviations.

FIG. 3.

Δm01-17+m144-158-MCMV is severely attenuated in BALB/c mice. Groups of five BALB/c mice were infected i.v. with 5 × 10⁵ PFU of the indicated viruses, and virus titers in lungs, spleen, liver, and salivary glands were determined at days 5 and 14 p.i. by plaque assay with MEFs. Virus titers in the salivary gland (SG), spleen, and lungs are shown per organ, and virus titers in the liver are shown per gram. Each circle represents a mouse; horizontal lines show median values; DL, detection limit.

FIG. 4.

No reactivation of Δm01-17+m144-158-MCMV. BALB/c mice were i.p. infected with 5 × 10⁵ PFU of WT-MCMV (n = 9) or Δm01-17+m144-158-MCMV (n = 4). Nine months later, mice were immune suppressed by CD8 and CD4 depletion and whole-body gamma irradiation with a dose of 7 Gy. Eleven days after immune suppression, spleen explants were placed on MEFs. Cells were kept for 3 weeks, and virus reactivation in individual animals (y axis) was monitored daily by the occurrence of MCMV-specific cytopathic effects.

FIG. 5.

Immunity to MCMV elicited by Δm01-17+m144-158-MCMV. (a) Blood CD8 T cells from mice infected with 5 × 10⁵ PFU of the indicated virus were analyzed for specificity of the immune response to the immunodominant YPHFMPTNL peptide. Cells from individual mice were split into two aliquots, which were subjected to tetramer staining (○) or to peptide stimulation, followed by ICCS for TNF-α and IFN-γ induction (•). Symbols represent percentage values of tetramer⁺ (○) or IFNg⁺ TNFa⁺ (•) cells in CD8⁺-gated lymphocytes of individual mice. Horizontal bars indicate average values, error bars indicate standard errors of the means. (b) Groups of mice (n = 4) were primed and 14 days later were boosted by s.c. injection of 10⁶ PFU of the indicated virus or of sterile PBS (MOCK). At week 16 p.i., sera were collected, and titers of antibodies specific for MCMV were determined by ELISA. (c) Blood cells from mice immunized with 5 × 10⁵ PFU of the indicated MCMV taken at 2 weeks (white bars) or 6 months (gray bars) p.i. were stained with antiCD8-FITC and YPHFMPTNL-MHC-I^Kd tetramers. CD8⁺ lymphocytes were gated, and the percentage of tetramer-positive cells was determined. Histograms indicate the means and standard deviations from at least four mice. OD, optical density.

FIG. 6.

Immune protection upon Δm01-17+m144-158-MCMV infection. (a) Mice that were s.c. primed and boosted with 10⁶ PFU of Δm01-17+m144-158-MCMV (white circles) or WT-MCMV (gray circles) or mock immunized (black circles) were challenged at 20 weeks postpriming with 10⁶ PFU of i.p.-delivered WT-MCMV. Five days later, infectious virus titers in lungs, spleen, and livers were determined by plaque assay on MEFs. (b) Mice were immunized with 10⁵ PFU of Δm01-17+m144-158-MCMV (white circles) or WT-MCMV (gray circles) or were mock immunized with sterile PBS (black circles). Where indicated, an equal amount of UV-inactivated virus was used. Challenge with 10⁶ PFU of WT-MCMV was performed i.p. at 4 weeks p.i., and virus was quantified 5 days later. (c) Mice were immunized with 10⁵ PFU of Δm01-17+m144-158-MCMV (white circles) or PBS (black circles). At 4 and 20 weeks p.i., mice were challenged, and virus was titrated in the indicated organs, as described for panel a. Short horizontal bars indicate medians. Circles represent individual mice, and horizontal bars mark the median values. DL, detection limit.

FIG. 7.

Δm01-17+m144-158-MCMV is severely attenuated in immunodeficient mice. (a) Groups of SCID mice were infected i.p. with 10⁴ PFU of the indicated virus. Where indicated, NK cells were depleted with α-asialo antibody. Virus titers in lungs and spleen at day 14 p.i. are shown. Each circle represents the value of an individual mouse; short horizontal bars indicate median values. DL, detection limit. (b) Groups of SCID/bg mice were s.c. infected with 10⁴ PFU of the indicated virus. Virus titers in lungs, spleen, livers, and salivary glands (SG) at 7 and 21 days p.i. are shown. Virus titers in the spleen and lungs are shown per organ, and virus titers in the liver are shown per gram.

FIG. 8.

Δm01-17+m144-158-MCMV is avirulent in immunodeficient mice. Groups (n = 10) of IFN-αβR^−/− mice were infected i.v. with 5 × 10⁴ PFU of Δm01-17+m144-158-MCMV (white circles) or WT-MCMV (gray circles), and the survival rate was monitored daily.