Protective MCMV immunity by vaccination of the salivary gland via Wharton's duct: replication-deficient recombinant adenovirus expressing individual MCMV genes elicits protection similar to that of MCMV

Abstract

Salivary glands, a major component of the mucosal immune system, confer antigen-specific immunity to mucosally acquired pathogens. We investigated whether a physiological route of inoculation and a subunit vaccine approach elicited MCMV-specific and protective immunity. Mice were inoculated by retrograde perfusion of the submandibular salivary glands via Wharton's duct with tcMCMV or MCMV proteins focused to the salivary gland via replication-deficient adenovirus expressing individual MCMV genes (gB, gH, IE1; controls: saline and replication deficient adenovirus without MCMV inserts). Mice were evaluated for MCMV-specific antibodies, T-cell responses, germinal center formation, and protection against a lethal MCMV challenge. Retrograde perfusion with tcMCMV or adenovirus expressed MCMV proteins induced a 2- to 6-fold increase in systemic and mucosal MCMV-specific antibodies, a 3- to 6-fold increase in GC marker expression, and protection against a lethal systemic challenge, as evidenced by up to 80% increased survival, decreased splenic pathology, and decreased viral titers from 10(6) pfu to undetectable levels. Thus, a focused salivary gland immunization via a physiological route with a protein antigen induced systemic and mucosal protective immune responses. Therefore, salivary gland immunization can serve as an alternative mucosal route for administering vaccines, which is directly applicable for use in humans.

Keywords: inflammation; mucosal immunity; vaccine development.

Figure 1.

Retrograde perfusion of the submandibular gland with tcMCMV stimulated MCMV-specific antibodies and increased lymphocyte yields. Balb/cByJ mice were immunized via cannulation with either saline (mock treatment) or 10⁵ pfu/mouse tcMCMV and evaluated on d 14. A, B) Serum (A) and saliva (B) samples were collected for detection of MCMV-specific IgM, IgG, and IgA antibodies via an MCMV-specific ELISA. C) Single-cell suspensions were prepared from the salivary glands (SG), PGLNs, and spleen, and total cell yields were assessed. Data are means ± sd from 5 mice/group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. saline.

Figure 2.

Key markers of GCs were induced after retrograde perfusion of the submandibular gland with tcMCMV. Balb/cByJ mice were immunized via cannulation with either saline (mock treatment) or 10⁵ pfu/mouse tcMCMV. RNA was prepared from whole salivary glands after removal of the associated lymph nodes on d 14 after inoculation. A) RNA was analyzed via RT-PCR for expression of AID, IμCα, PAX5, MCMV IE1, and β-actin. B) Histogram represents the densitometric data of the ratio of mock/average mock (open bars) and the ratio of tcMCMV/average mock (solid bars). Data are means ± sd from 3 mice/group. **P < 0.01, ***P < 0.001 vs. saline.

Figure 3.

Retrograde perfusion of the submandibular gland with tcMCMV increased MCMV antigen-specific T cells. Balb/cByJ mice were immunized via cannulation with either saline (mock treatment) or 10⁵ pfu/mouse tcMCMV. On d 21 after primary immunization, mice were boosted with either saline or 10⁵ pfu/mouse tcMCMV and evaluated 10 d after boosting. Single-cell suspensions were prepared from the salivary glands, PGLNs, and spleens. A) Cells were first stained with the B-cell marker, CD19, and then CD19⁻ cells were analyzed for expression of the T-cell surface marker, CD8 (x axis) and the MCMV IE1 pentamer (H-2L^d YPHFMPTNL; y axis). Percentage of dual-staining cells is shown in the top right quadrant. Data are representative of 5 individual mice/group demonstrating similar results. B) Histogram demonstrates the absolute numbers of CD8⁺ pentamer⁺ T cells from saline-treated (open bars) and tcMCMV-immunized (solid bar) mice. Data are means ± sd from 5 mice/group. *P < 0.05, **P < 0.01 vs. saline. C) For in situ pentamer staining, frozen sections from the salivary gland were stained with CD8α (green), H-2L^d YPHFMPTNL pentamer (red), and DAPI (blue). Images were then merged, and dual-positive CD8⁺/pentamer⁺ cells (yellow) were observed. View: ×40. Scale bars = 50 μM. Images are representative of 5 mice/group demonstrating similar results.

Figure 4.

Retrograde perfusion of the submandibular gland with tcMCMV protected mice from a lethal systemic challenge with MCMV. Balb/cByJ mice were immunized via cannulation with saline (mock treatment) or 10⁵ pfu/mouse tcMCMV. A, B) On d 28 after immunization, mice inoculated with saline via cannulation were challenged i.p. with either saline (mock-mock) or 5 × 10⁴ pfu/mouse MCMV (mock-MCMV). Mice immunized with 10⁵ pfu/mouse tcMCMV via cannulation were challenged i.p. with 5 × 10⁴ pfu/mouse MCMV (tcMCMV-MCMV). C, D) On d 28 after immunization, mice inoculated with saline via cannulation were challenged i.p. with either saline (mock-mock) or 2 × 10⁴ pfu/mouse MCMV (mock-MCMV). Mice immunized with 10⁵ pfu/mouse tcMCMV via cannulation were challenged i.p. with 5 × 10⁴ pfu/mouse MCMV (tcMCMV-MCMV). Weight loss (A, C) and survival (B, D) were monitored for 14 d postchallenge. Weight loss data are means ± sd from 5 mice/group. Survival data are from 5 individual mice/group.

Figure 5.

Retrograde perfusion of the submandibular gland with tcMCMV limited systemic pathology, prevented diminished salivary output, and lowered viral titers after a lethal challenge with MCMV. Balb/cByJ mice were immunized with saline (mock treatment) or 10⁵ pfu/mouse tcMCMV via cannulation. On d 28 after immunization, mice were challenged i.p. with 2 × 10⁴ pfu/mouse MCMV for saline-inoculated mice and 5 × 10⁴ pfu/mouse MCMV for tcMCMV-immunized mice. A) Spleens were harvested on d 14 postchallenge for assessment of pathological changes on H&E stain. Images are representative of 2 experiments with 3 mice/group demonstrating similar results. View: ×4. Scale bars = 100 μm. B) Saliva was collected and measured at the indicated time points after i.p. MCMV challenge. Data are means ± sd from 5 mice/group/time point. **P < 0.01, ***P < 0.001 vs. saline. C) Viral titers from the lung, spleen, salivary gland, and PGLNs were determined 14 d postchallenge by standard plaque assay. Data are means ± sd from 5 mice/group. Dashed lines represent limit of detection for each tissue. Statistical differences were not available since no detectable virus was found from lung, spleen, and PGLN, and only 1 of 5 salivary glands had a very low viral titer.

Figure 6.

Retrograde perfusion of the submandibular gland with tcMCMV induced MCMV specific antibodies after a lethal challenge with MCMV. Balb/cByJ mice were immunized with saline (mock treatment) or 10⁵ pfu/mouse tcMCMV via cannulation. On d 28 after immunization, saline-inoculated mice were challenged i.p. with 2 × 10⁴ pfu/mouse MCMV, and tcMCMV-immunized mice were challenged i.p. with 5 × 10⁴ pfu/mouse MCMV. Serum (A) and saliva (B) samples were collected on d 7 and 14 postchallenge. MCMV-specific IgM, IgG, and IgA antibodies were determined via an MCMV-specific ELISA. Data are means ± sd of 5 mice/group/time point. **P < 0.01, ***P < 0.001 vs. saline.

Figure 7.

Individual MCMV genes are expressed in the salivary glands after retrograde perfusion of the submandibular gland with replication-deficient recombinant adenovirus. Balb/cByJ mice were immunized (d 0) and boosted (d 30) with either saline (mock) or 10⁶ pfu/mouse of FG140 (negative control), Ad-gB, Ad-gH, Ad-IE1, or a combination of Ad-gB, Ad-gH, and Ad-IE1. A) RNA was prepared from salivary glands after removal of the associated lymph nodes on d 30 after boost immunization and analyzed via RT-PCR for expression of MCMV gB (top panel), gH (middle panel), or IE1 (bottom panel). RNA from MCMV infected salivary glands served as a positive control. Data are representative of 2 experiments with 3 mice/group. B) Frozen sections of salivary glands were stained with monoclonal antibodies specific for MCMV gB, gH, or IE1 (FITC, green) and DAPI (blue). Salivary glands from saline (mock)-infected mice served as negative controls. View: ×40. Images are representative of 2 experiments with 3 mice/group demonstrating similar results.

Figure 8.

Retrograde perfusion of the submandibular gland with replication-deficient recombinant adenoviruses expressing individual MCMV genes elicited mucosal and humoral antibodies. Balb/cByJ mice were immunized (d 0) and boosted (d 30) with 10⁶ pfu/mouse of FG140 (negative control), Ad-gB, Ad-gH, Ad-IE1, or a combination of Ad-gB, Ad-gH, and Ad-IE1. Saliva, vaginal wash, and feces (A) and serum samples (B) were collected on d 30 after boost immunization for detection of MCMV-specific IgA antibodies (A) and IgG antibodies (B) via an MCMV-specific ELISA. Data are means ± sd of 5 mice/group. *P < 0.05, ***P < 0.001, ****P < 0.0001 vs. FG140.

Figure 9.

Key markers of GCs were induced after retrograde perfusion of the submandibular gland with replication-deficient recombinant adenoviruses expressing individual MCMV genes. Balb/cByJ mice were immunized (d 0) and boosted (d 30) with 10⁶ pfu/mouse of FG140 (negative control), Ad-gB, Ad-gH, Ad-IE1, or a combination of Ad-gB, Ad-gH, and Ad-IE1. RNA was prepared from whole salivary glands after removal of the associated lymph nodes on d 30 after boost immunization. A) RNA was analyzed via RT-PCR for expression of AID, IμCα, PAX5, and β-actin. Images are representative of 5 mice/group demonstrating similar results. B) Histogram represents the densitometric data of the ratio of mock/average mock (solid bars) and the ratio of individual MCMV genes/average mock. Data are means ± sd of 5 mice/group. *P < 0.05, **P < 0.01 vs. mock treatment.

Figure 10.

Retrograde perfusion of the submandibular gland with replication-deficient recombinant adenoviruses expressing individual MCMV genes protected mice from a lethal systemic challenge with MCMV. Balb/cByJ mice were immunized (d 0) and boosted (d 30) with either saline (mock treatment) or 10⁶ pfu/mouse of FG140 (negative control), Ad-gB, Ad-gH, Ad-IE1, or a combination of Ad-gB, Ad-gH, and Ad-IE1. On d 30 after boost immunization, mice inoculated with saline were challenged i.p. with either saline (mock-mock) or 5 × 10⁴ pfu/mouse MCMV (mock-MCMV). Mice immunized with replication-deficient recombinant adenovirus were challenged i.p. with 5 × 10⁴ pfu/mouse MCMV (Ad-MCMV). Weight loss (A) and survival (B) were monitored for 14 d after lethal challenge. Data are means ± sd from 5 mice/group.

Figure 11.

Retrograde perfusion of the submandibular gland with replication-deficient recombinant adenoviruses expressing individual MCMV genes limited systemic pathology and lowered viral titers after a lethal challenge with MCMV. Balb/cByJ mice were immunized (d 0) and boosted (d 30) with either saline (mock treatment) or 10⁶ pfu/mouse of FG140 (negative control), Ad-gB, Ad-gH, Ad-IE1, or a combination of Ad-gB, Ad-gH, and Ad-IE1. On d 30 after boost immunization, mice inoculated with saline were challenged i.p. with either saline (mock-mock) or 5 × 10⁴ pfu/mouse MCMV (mock-MCMV). Mice immunized with replication-deficient recombinant adenovirus were challenged i.p. with 5 × 10⁴ pfu/mouse MCMV (Ad-MCMV). A) Spleens were harvested on d 14 postchallenge for assessment of pathological changes on H&E stain. Images are representative of 5 mice/group demonstrating similar results. View: ×4. Scale bars = 100 μm. B) Viral titers from the salivary gland, PGLNs, lung, and spleen were determined 14 d postchallenge by standard plaque assay. Dashed lines represent the limit of detection for each tissue. Data are from 5 mice/group. Statistical differences were not available (N/A) since there were no surviving animals in the mock and FG140 control groups.

Figure 12.

Retrograde perfusion of the submandibular gland with replication-deficient recombinant adenoviruses expressing individual MCMV genes induced MCMV-specific antibodies after a lethal challenge with MCMV. Balb/cByJ mice were immunized (d 0) and boosted (d 30) with either saline (mock treatment) or 10⁶ pfu/mouse of FG140 (negative control), Ad-gB, Ad-gH, Ad-IE1, or a combination of Ad-gB, Ad-gH, and Ad-IE1. On d 30 after boost immunization, all mice were challenged i.p. with 5 × 10⁴ pfu/mouse MCMV. Samples were collected from surviving mice for analysis of MCMV-specific antibodies at 14 d postchallenge. All mice immunized with FG140 and 5/6 mice immunized with Ad-gB died after lethal challenge. A) MCMV-specific IgG was measured from the serum and vaginal wash. B) MCMV-specific IgA was measured from saliva, vaginal wash, and feces. Data are means ± sd of 5 mice/group. *P < 0.05, **P < 0.01, ****P < 0.0001 vs. saline.