The Nontoxic Cholera B Subunit Is a Potent Adjuvant for Intradermal DC-Targeted Vaccination

Abstract

CD4⁺ T cells are major players in the immune response against several diseases; including AIDS, leishmaniasis, tuberculosis, influenza and cancer. Their activation has been successfully achieved by administering antigen coupled with antibodies, against DC-specific receptors in combination with adjuvants. Unfortunately, most of the adjuvants used so far in experimental models are unsuitable for human use. Therefore, human DC-targeted vaccination awaits the description of potent, yet nontoxic adjuvants. The nontoxic cholera B subunit (CTB) can be safely used in humans and it has the potential to activate CD4⁺ T cell responses. However, it remains unclear whether CTB can promote DC activation and can act as an adjuvant for DC-targeted antigens. Here, we evaluated the CTB's capacity to activate DCs and CD4⁺ T cell responses, and to generate long-lasting protective immunity. Intradermal (i.d.) administration of CTB promoted late and prolonged activation and accumulation of skin and lymphoid-resident DCs. When CTB was co-administered with anti-DEC205-OVA, it promoted CD4⁺ T cell expansion, differentiation, and infiltration to peripheral nonlymphoid tissues, i.e., the skin, lungs and intestine. Indeed, CTB promoted a polyfunctional CD4⁺ T cell response, including the priming of Th1 and Th17 cells, as well as resident memory T (RM) cell differentiation in peripheral nonlymphoid tissues. It is worth noting that CTB together with a DC-targeted antigen promoted local and systemic protection against experimental melanoma and murine rotavirus. We conclude that CTB administered i.d. can be used as an adjuvant to DC-targeted antigens for the induction of broad CD4⁺ T cell responses as well as for promoting long-lasting protective immunity.

Keywords: CTB; T cells; adjuvant; anti-DEC205; dendritic cells; memory; skin.

Figure 1

Intradermal administration of CTB promotes recruitment and activation of DCs in the SDLN and the skin. C57BL6 mice received 10 μg of CTB or PBS i.d. in both ears, and they were sacrificed for skin and SDLN harvesting at the indicated times. (A) MHC-II⁺CD11c⁺ DCs were gated as in Supplementary Figure 2A. Graphs depicting the percentage, absolute cell numbers of DCs and geometric median fluorescence intensity (MFI) of CD86 on DCs in the skin. Mean ± SD, N = 4–6, data pooled from two independent experiments. One-way ANOVA with Tukey's multiple comparisons test (*P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001). (B) Migratory and (C) resident DCs from the SDLN were gated as in Supplementary Figure 2B. Graphs of percentage, total numbers of DCs and geometric MFI of CD86 on DCs. Mean ± SD, N = 4–6, data pooled from two independent experiments. One-way ANOVA with Tukey's multiple comparisons test (*P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001).

Figure 2

CTB co-administration with a DC-targeted or soluble antigen promotes expansion and differential activation of CD4⁺ T cells. C57BL6 mice were adoptively transferred with OT-II CD45.1⁺ cells, 24 h later they were immunized i.d. in both ears, as indicated, and 3 or 7 days later, they were sacrificed for SDLN and skin harvesting. (A) Representative dot plot of CFSE dilution and CD69 expression by SDLN OT-II cells 3 days after inoculation of anti-DEC205-OVA or soluble OVA and (B) total numbers of OT-II cells. (C) Geometric median fluorescence intensity (MFI) of CD69 by OT-II cells 3 days after anti-DEC205-OVA or soluble OVA ± CTB's i.d. administration. Mean ± SD, N = 4–6, data pooled from four independent experiments. One-way ANOVA with Turkey's multiple comparisons test (ns, P > 0.05, *P < 0.05, **P < 0.005, ***P < 0.0005). (D) Representative dot plots and total number of SDLN OT-II cells 7 days after anti-DEC205-OVA or soluble OVA ± CTB's i.d. administration. Mean ± SD, N = 5–8 data pooled from four independent experiments. One-way ANOVA with Tukey's multiple comparisons test (*P < 0.05, ****P < 0.0001). Transferred cells recovered from the SDLN were identified as viable CD4⁺CD45.2⁻TCRVβ 5.1, 5.2⁺ T cells (Supplementary Figure 3A). (E) Representative dot plot and total numbers of migrating OT-II cells identified as viable CD45⁺CD4⁺CD45.2⁻TCRVβ 5.1, 5.2⁺ (Supplementary Figure 3C). Mean ± SD, N = 4–6, data pooled from four independent experiments. One-way ANOVA with Tukey's multiple comparisons test (**P < 0.005, ****P < 0.0001).

Figure 3

CTB promotes Th1 and Th17 differentiation and recruitment to the skin after i.d. co-administration with a DC-targeted antigen. Mice were treated as in Figure 2, and 7 days after immunization, the SDLN and skin were collected to obtain cell suspensions for in vitro re-stimulation. (A) Cells from the SDLN were incubated for 48 h with OVA 323–339 peptide followed by 4 h with cell cocktail stimulation + protein transport inhibitor. Graphs of percentage and total numbers of IFNγ⁺ and IL-17⁺ OT-II cells (identified as in Figure 2A). (B) Ratio of SDLN Th17/Th1 cells. Mean ± SD, N = 6–8, data pooled from two independent experiments. Unpaired T-test (ns, P > 0.05, *P < 0.05, **P < 0.005). Skin cell suspensions were stimulated with cell cocktail stimulation + protein transport inhibitor for 4 h. (C) Graphs of percentage and total numbers of skin IFNγ⁺ and IL-17⁺ of OT-II cells (identified as in Figure 2B). (D) Ratio of skin Th17/Th1 cells. Mean ± SD, N = 6–8, data pooled from three independent experiments. Unpaired T-test (ns, P > 0.05, **P < 0.005, ***P = 0.0001).

Figure 4

Antigen targeting to DCs along with CTB promotes T EM, T CM and T RM cell differentiation. Mice were treated as in Figure 2 and the SDLN along with the ears were collected at the indicated times. (A) Representative contour plots of T EM (CD44⁺CD62L⁻) and of T CM (CD44⁺CD62L⁺) cells from OT-II CD45.1⁺ cells (identified as in Figure 2A), and graphs of the percentage of each population 7 days post-immunization. Mean ± SD, N = 4–6, data pooled from two independent experiments. One-way ANOVA with Tukey's multiple comparisons test (*P < 0.05, **P < 0.005, ***P < 0.0005). (B) Representative contour plots and a graph showing percentages of CD69⁺CCR7⁻ OT-II CD45.1⁺ cells (identified as in Figure 2B) from the inoculation site 7 days post-immunization. Mean ± SD, N = 5–6, data pooled from three independent experiments. (C) CD45.1⁺ mice received i.v. OT-II CD45.2⁺ cells and 1 day later were inoculated with 1 μg of anti-DEC205-OVA or with 30 μg of OVA, both in combination with CTB. Representative contour plots and a graph showing percentages of CD69⁺ OT-II CD45.2⁺ cells 30 days post-immunization. Mean ± SD, N = 3–5 data pooled from two independent experiments. Unpaired T-test.

Figure 5

Intradermal immunization with CTB, along with a DC-targeted antigen, provides local and systemic long-lasting immunity against melanoma. (A) Diagram showing the strategy followed for immunizations and a graph showing survival rate after MO4 s.c. challenge. N = 5 per group, data pooled from two independent experiments. Log-rank (Mantel-Cox) test. Naïve mice were i.d. immunized as indicated, and after 30 days i.v. challenged with MO4 cells. Mice immunized with anti-DEC-OVA+CTB received i.p. anti-CD4 or the control isotype Ab, before, during, and after the inoculation of MO4 cells. (B) Representative pictures of lungs and a graph of metastatic nodules per lung, 16 days after challenge. Mean ± SD, N = 5–10, data pooled from two independent experiments. One-way ANOVA with Tukey's multiple comparisons test (ns, P > 0.05, ****P < 0.0001).

Figure 6

A single dose of a DC-targeted antigen adjuvanted with CTB induces infiltration of antigen specific CD4⁺ T cells in the intestine and partial protection against rotavirus. Mice were treated as in Figure 2 and intestines were collected 7 days post-inoculation. (A) OT-II CD45.1⁺ transferred cells were identified as viable CD45⁺CD4⁺TCRVα2⁺CD45.1⁺ cells. (B) percentage and total numbers of OT-II CD45.1⁺ cells in the intestines. Mean ± SD, N = 5 per group, data pooled from two independent experiments. Unpaired T-test (*P < 0.05, **P < 0.005). (C) Percentage and total numbers of OT-II CD45.1⁺ cells expressing CD69. Mean ± SD, N = 5 per group, data pooled from two independent experiments. Unpaired T-test (*P < 0.05, **P < 0.005). (D) Diagram showing the immunization strategy followed for viral challenge with murine rotavirus. Stool samples were collected every day up to day 8 and viral load was determined by sandwich ELISA to calculate percentage of protection relative to control (vehicle) mice. Graph depicting percentage of protection after infection. Mean ± SD, N = 5 per group, data pooled from two independent experiments. One-way ANOVA with Tukey's multiple comparisons test (ns, P > 0.05, **P < 0.005, ***P < 0.0005).

Figure 7

Intradermal prime/i.p. boost immunization with a DC-targeted antigen + CTB induces functional CD4⁺ T cells in the intestine and provides CD4⁺ T cell dependent protection against rotavirus. C57BL6 mice were adoptively transferred with OT-II CD45.1⁺ cells 24 h before i.d. anti-DEC205-OVA or soluble OVA with CTB. Fifteen days later, immune mice received i.p. anti-DEC205-OVA or soluble OVA and after 5 days, mice were sacrificed, and intestines were collected. (A) Cells were gated as viable CD45⁺CD4⁺CD45.1⁺ cells to calculate percentage and total number of transferred cells present in the intestine. Mean ± SD, N = 6 per group, data pooled from two independent experiments. Unpaired T-test (*P < 0.05). (B) Freshly isolated cells were stimulated 4 h with cell cocktail stimulation + protein transport inhibitor. Graphs of percentage and total numbers of CD4⁺CD45.1⁺ cytokine producing cells (gated as in B). Boolean combinations were calculated using FlowJo software. Mean ± SD, N = 6 per group, data pooled from two independent experiments. Two-way ANOVA with Bonferroni's multiple comparison test (**P = 0.0017, ****P < 0.0001). (C) Strategy followed for oral viral challenge with murine rotavirus after i.d. immunizations and i.p. boost. Mice immunized with anti-DEC-VP6+CTB received i.p. anti-CD4 or the control isotype Ab, before, during and after the viral challenge. (D) Stool samples were collected every day up to day 8 and viral load was determined by sandwich ELISA. (E) Percentage of protection relative to control (vehicle) mice, calculated as area under the curve (From D). Mean ± SD, N = 5–8 per group, data pooled from two independent experiments. Two-way ANOVA with Tukey's multiple comparisons test.