Transcutaneous immunization induces mucosal CTLs and protective immunity by migration of primed skin dendritic cells

Abstract

Transcutaneous immunization (TCI), the application of vaccines on the skin, induces robust systemic and mucosal antibodies in animal models and in humans. The means by which mucosal immune responses to vaccine antigens are elicited by TCI has not been well characterized. We examined the effect of TCI with an HIV peptide vaccine on the induction of mucosal and systemic CTL responses and protective immunity against mucosal challenge with live virus in mice. Robust HIV-specific CTL responses in the spleen and in the gut mucosa were detected after TCI. The responses were dependent upon the addition of an adjuvant and resulted in protection against mucosal challenge with recombinant vaccinia virus encoding HIV gp160. Although it is clear that adjuvant-activated DCs migrated mainly to draining lymph nodes, coculture with specific T cells and flow cytometry studies with DCs isolated from Peyer's patches after TCI suggested that activated DCs carrying skin-derived antigen also migrated from the skin to immune-inductive sites in gut mucosa and presented antigen directly to resident lymphocytes. These results and previous clinical trial results support the observation that TCI is a safe and effective strategy for inducing strong mucosal antibody and CTL responses.

Figure 1

Induction of CTL responses in SP and PPs after TCI with HIV peptide and adjuvant. BALB/c mice (n = 5) were immunized twice 3 weeks apart with 50 μg of PCLUS3-18IIIB peptide alone (triangles), with 50 μg CT as an adjuvant (circles), or with 50 μg CT and 50 μg CpG as adjuvants (squares). SP (A) and PP (B) cells from immunized mice were incubated in vitro with 1 μM P18-I10 peptide for 7 days before cytolytic activity was measured in a 4-hour ⁵¹Cr-release assay against P815 target cells alone (open symbols) or pulsed with P18-I10 peptide (filled symbols). E/T, effector-to-target ratio.

Figure 2

(A and B) TCI at multiple anatomic skin sites elicits systemic (A) and mucosal (B) CTL responses. BALB/c mice (n = 5) were skin-immunized on the back (squares), abdomen (diamonds), or ear (triangles) with 100 μg PCLUS3-18IIIB peptide with 50 μg CT and 50 μg CpG as adjuvants. Immunizations occurred on weeks 0, 1, 3, and 5. SP (A) and PP (B) cytolytic activity was measured in a ⁵¹Cr-release assay against P815 target cells alone (open symbols) or pulsed with P18-I10 peptide (filled symbols).

Figure 3

TCI with peptide and adjuvant induces protective immunity against intrarectal recombinant vaccinia challenge. BALB/c mice (n = 5) were skin-immunized three times at 2-week intervals with 100 μg PCLUS3-IIIB peptide alone or with 25 μg CT or 25 μg LT as an adjuvant. Two weeks after the final immunization, mice were challenged intrarectally with 1 × 10⁷ PFU of vaccinia virus carrying the gene encoding HIV-1 gp160 from the IIIB isolate (vPE16). Six days after the challenge with recombinant vaccinia virus, the mice were euthanized and their ovaries were removed, homogenized, sonicated, and assayed for vPE16 by plating of serial tenfold dilutions on monolayers of BSC-2 indicator cells (12). Cells were stained with crystal violet and the resulting plaques were counted for each dilution. The minimal detectable level of virus was 100 PFU. Bars: SEM of five mice per group. The difference between unvaccinated and vaccinated with either adjuvant is significant at P < 0.01 by Student’s t test.

Figure 4

Prime and boost strategy using skin and intrarectal immunization regimens induces systemic and mucosal CTLs. BALB/c mice (n = 5) were immunized four times at 2-week intervals with no treatment or with 50 μg PCLUS3-IIIB peptide, 50 μg CT, and 50 μg CpG by the indicated route of administration: one intrarectal and then three transcutaneous immunizations (IR/TCI/TCI/TCI); three transcutaneous and then one intrarectal immunization (TCI/TCI/TCI/IR); four transcutaneous immunizations (TCI/TCI/TCI/TCI); or no treatment during the first three immunization intervals and an intrarectal immunization at the fourth interval (–/–/–/IR). On day 4 after the cell culture was established, CD8⁺ CTLs were purified by gradient centrifugation. Cytolytic activity of pooled SP (A) and PP (B) cells was measured in a ⁵¹Cr-release assay against P815 target cells alone (open symbols) or pulsed with P18-I10 peptide (filled symbols).

Figure 5

TCI generated CTL responses in the PPs, SP, and lung. BALB/c mice (n = 5) were immunized twice 2 weeks apart with 50 μg PCLUS3-IIIB peptide, 10 μg CT, and 50 μg CpG by the indicated route of administration for each group as follows: group 1, transcutaneous prime and intrarectal boost; group 2, transcutaneous prime and boost; group 3, subcutaneous (SC) prime and intrarectal boost; group 4, intraperitoneal (IP) prime (peptide and CpG given intraperitoneally and CT administered intrarectally because CT is toxic given intraperitoneally) and intrarectal boost; group 5, intrarectal prime and intrarectal boost; and group 6, no treatment during the first immunization and an intrarectal immunization at the second immunization interval (Single IR). Four weeks after the final immunization, PPs (A), SP (B), and lung (C) were removed, processed, stimulated in vitro with P18-I10 peptide (1 μM) for 7 days, and assayed for cytolytic activity in a ⁵¹Cr-release assay.

Figure 6

CD11c-enriched cells isolated from inguinal LNs and PPs of skin-immunized mice have acquired antigen that activates high- or low-avidity CTLs ex vivo. (A–D) Mice were immunized transcutaneously with 50 μg LT or with 50 μg LT and 50 μg PCLUSS3-18IIIB peptide and were sacrificed 24 hours after immunization. Pooled inguinal LN and PP CD11c⁺ cells (APCs in figure) were enriched by MACS (enrichment of CD11c⁺ cells to about 70% by FACScan analysis). APCs (5 × 10⁴, 1 × 10⁴, or 1 × 10³) were cocultured in 96-well U-bottomed plates with 2 × 10⁴ cells/well high-avidity (0.001 μM; A and C) or low-avidity (10 μM; B and D) P18-I10-specific CTLs. Negative controls: CTL + Contr APCs, 5 × 10⁴ CD11c⁺ APCs isolated from the inguinal LNs or PPs of mice immunized with LT alone were cocultured with 2 × 10⁴ high-avidity (A and C) or low-avidity (B and D) P18-I10–specific CTLs; CTL alone, 2 × 10⁴ high-avidity (A and C) or low-avidity (B and D) P18-I10–specific CTLs were cultured alone; ING APC alone, 5 × 10⁴ CD11c-enriched cells isolated from the inguinal LNs of mice immunized with LT and peptide were cultured alone; 89.6 CTL + APC, 5 × 10⁴ CD11c⁺ APCs isolated from the inguinal LNs of LT- and peptide-immunized mice were cocultured with 2 × 10⁴ high-avidity (0.001 μM) P18-89.6A9–specific CTLs (89.6 strain of HIV), which do not cross-react with P18-I10–specific CTLs (IIIB strain of HIV). The level of IFN-γ produced by the CTL lines in the supernatant after a 48-hour coculture with CD11c-enriched APCs was measured by semiquantitative ELISA.

Figure 7

APCs in the PPs have increased cell surface levels of CD40 after TCI with LT. (A–D) C57BL/6 mice were immunized with 50 μg of LT and were sacrificed 24 hours later. Inguinal LNs (A and B) and PPs (C and D) were isolated from naive (A and C) or immunized (B and D) mice. Single-cell suspensions were produced and analyzed for levels of CD11c and CD40. Dead cells, B cells (CD19⁺), and T cells (CD3⁺) were gated out from the analysis. The percent of cells present in each quadrant are listed.

Figure 8

APCs in the PPs contain skin-derived fluorescent LT after TCI. (A–F) BALB/c mice were left untreated (A and D) or were immunized with 50 μg of Alexa Fluor 488–conjugated LT (AF-LT; B, C, E, and F). Forty-eight hours later, inguinal LNs and PPs were isolated and analyzed for cell populations containing AF-LT (A, B, D, and E). The CD11c, MHC class II (H-2 I-A^d) surface expression of AF-LT–positive cells was determined for both inguinal LN (C) and PP (F) cells. Similar results were observed 24 hours after application of AF-LT. The percent of AF-LT–positive cells are listed in A, B, D, and E. The percent of cells present in each quadrant are listed in C and F.