Supplemental Figure 1

Tissue:

Colorectum Lungs Vaginal wall

Route: ICR IN IVAG IVAG (Depo)Dose: 2x109 5x108 1x109

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Supplemental Figure 2

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Supplemental Figure 3

Unprotected Protected after AdgB ICR

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Perineal pathology Vaginal virus shedding Mortality

Supplemental Figure 6

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Supplemental Figure 1 Local gene transfer by different mucosal routes. At two

days after AdLuc administration via ICR, IN and IVAG (with or without prior Depo-

Provera treatment), luciferase activities were determined by measuring

luminescence in each corresponding tissue as RLU per tw (density) or whole

organ tissue (total mass). At specified doses used for immune function studies,

there was no statistical difference in the density or total mass of gene expression

among different locations (P > 0.05) except for the vaginal tissue after IVAG

without Depo-Provera and the mass after IVAG with Depo-Provera. Averaged

density of gene expression in the colorectum did not exceed that in the lung or

vaginal tract. The values are the mean ±s.d. of 3 mice in each group. Asterisks

indicate significantly lower RLU compared to other groups.

Supplemental Figure 2 Perianal inflammation (pathology) and hindlimb paralysis

developed in the HSV-2 rectal challenge model. Doses of HSV-2 for rectal

challenge were titrated (N=10/group except for the groups of 2x102 and 2x103,

N=5). The absolute lethal dose (LD100) of HSV-2 was determined as 1x105 PFU,

at and above which animals developed 100% hindlimb paralysis (left panel; 100%

mortality) and the disease never resolved. Similar to vaginal HSV-2 infection

(76;80), rectally infected mice having received LD doses displayed uncontrolled

virus replication (middle panel; relatively constant from day 1 till death, with a

tendency to increase at day 2-3), increasingly severe perianal inflammation and

ulcerative lesions (right panel), and paralysis of hindlimbs developed within 7-10

days.

Supplemental Figure 3 Protection from HSV-2 rectal challenge in mice

immunized ICR with AdgB. Left panel: at day 7 after rectal challenge of 2x105

PFU of HSV-2, unimmunized mice developed the disease, severe perianal lesion

(white arrow) and paralyzed hindlimb (black arrow). Right panel: in contrast, AdgB

ICR immunized mice survived with diminished pathology and the disease was

completely resolved by day 7 (no lesion or paralysis developed).

Supplemental Figure 4 Protection at the large intestinal mucosa against HSV-2

depends on cellular immunity. Fourteen days after AdgB ICR immunization of

gene knockout mice as indicated, mice were challenged rectally with 2x105 PFU

of HSV-2. Pathology score (left panel) and mortality (right panel) were measured.

At least 5 mice in each group.

Supplemental Figure 5 Mucosal prime/boost vaccination requires large intestine

immunization to induce full protection against herpes infection. Intranasal

prime/boost (IN/IN) is often used to enhance mucosal immune responses. This

regimen was compared with priming/boosting of mice by ICR/IN with a 2-week

interval. Two weeks after the boost, animals were challenged with HSV-2 at a

dose of 2x106 PFU at colorectal mucosa (a) or 2x105 PFU at vaginal mucosa (b)

(N=5/group). Parianal pathology (left panels), virus shedding (middle panels) and

mortality (right panels) were compared. Asterisks in left panels and right panels

indicate a statistical difference of the group in comparison with ICR/IN group.

Although IN/IN was statistically different from unimmunized in pathology (left

panel, p<0.05) and mortality (right panel, p<0.01), ICR/IN provided more potent

mucosal protection than IN/IN. *, ** and *** indicate p<0.05, 0.01 and 0.001,

respectively.

Supplemental Figure 6 IVAG immunization fails to protect from HSV-2

challenge. Mice were immunized IVAG with 1x109 PFU of AdgB with or without

prior Depo-Provera (33) and 3 weeks later challenged with 2x105 PFU of HSV-2

at either vaginal (a) or colorectal mucosa (b). Pathology (left panels) and mortality

(right panels) were examined. N=5/group. No protection was seen after IVAG

immunization.