Induction of pulmonary mucosal immune responses with a protein vaccine targeted to the DEC-205/CD205 receptor

Abstract

It is of great interest to develop a pneumonic plague vaccine that would induce combined humoral and cellular immunity in the lung. Here we investigate a novel approach based on targeting of dendritic cells using the DEC-205/CD205 receptor (DEC) via the intranasal route as way to improve mucosal cellular immunity to the vaccine. Intranasal administration of Yersinia pestis LcrV (V) protein fused to anti-DEC antibody together with poly IC as an adjuvant induced high frequencies of IFN-γ secreting CD4(+) T cells in the airway and lung as well as pulmonary IgG and IgA antibodies. Anti-DEC:LcrV was more efficient to induce IFN-γ/TNF-α/IL-2 secreting polyfunctional CD4(+) T cells when compared to non-targeted soluble protein vaccine. In addition, the intranasal route of immunization with anti-DEC:LcrV was associated with improved survival upon pulmonary challenge with the virulent CO92 Y. pestis. Taken together, these data indicate that targeting dendritic cells via the mucosal route is a potential new avenue for the development of a mucosal vaccine against pneumonic plague.

Figure 1

Immune responses at mucosal surfaces after αDEC:LcrV immunization in the presence of poly IC and αCD40 as adjuvants. (A) BALB/c mice were immunized with PBS, F1-V (10 μg) adsorbed in alum, αDEC:LcrV (10 μg), or αDEC:empty (10 μg) in the presence of adjuvants (50 μg of poly IC and 25 μg of αCD40 mAb) either i.n. or s.c. Ten days later, lungs were collected, restimulated with the LcrV reactive peptide LKIYSVIQAEINKHL, aa164-178, and ICS was performed. The frequency of IFN-γ⁺ (upper) or IL-2⁺ (lower) secreting CD3⁺/CD4⁺ pulmonary T cells was shown as percentage. (B) Anti-LcrV antibody titers for individual IgG isotypes in BAL. Immunization and injection routes were same as described in (A). Data are mean values from 3 independent experiments with similar results, using 3 mice per group. *p<0.05; **p<0.005; ***p<0.001

Figure 2

CD4⁺IFN-γ⁺ secreting pulmonary T cells following αDEC:LcrV immunization in three different strains of mice. (A) C57BL/6, BALB/c, and C3H/HeJ mice were primed and boosted i.n. with αDEC:LcrV (10 μg) or control Ig:LcrV (10 μg) in the presence of poly IC (50 μg). Six to 10 days later, lung cells were restimulated with peptide pools 1-8 and IFN-γ secreting CD4⁺ T cells were assessed by ICS (thick-lined boxes). This is one of several representative data sets having similar results (not shown). (B) C57BL/6 control and DEC-205^−/− mice were primed and boosted with αDEC:LcrV (10 μg) in the presence of poly IC (50 μg) i.n., and 6-10 days later, lung cells were collected, restimulated, and IFN-γ secreting CD4⁺ T cells were assessed by ICS. Data are mean values from 2 independent experiments, using 2 mice per group. ***p<0.001

Figure 3

Cellular immunity at mucosal surfaces after αDEC:LcrV immunization in the presence of poly IC alone as an adjuvant in a prime/boost strategy. BALB/c mice were s.c. priming, and the mice were boosted either s.c. (A) or i.n. (B) as described at the left with antigen and adjuvants. Six to 10 days after the boost, lungs were collected, restimulated, and ICS was performed. The frequency of IFN-γ secreting CD3⁺/CD4⁺ pulmonary T cells are shown as percentages. The doses of LcrV protein were either 100 μg in case of s.c. (A) or 25 μg in case of i.n. (B) route of boost. Data are mean values from 3 independent experiments with similar results. (C) BALB/c mice were primed and boosted both by the i.n. route, as described at the bottom of each graph. Six to 10 days after the boost, T cells from lungs, mediastinal lymph nodes (Med LNs), BAL fluid, or spleens were restimulated, and IFN-γ secreting CD4⁺ T cells were assessed by ICS. Data in (C) is one of three similar experiments (tabulated in Table 1), using 3 mice per group. **p<0.005; ***p<0.001

Figure 4

The targeting efficacy of the induction of LcrV-specific pulmonary cellular immunity. BALB/c were primed and boosted i.n. with different forms of LcrV protein of various dosages as shown at the bottom of the graphs. Six to 10 days later, lung cells were collected, restimulated, and IFN-γ or IL-2 or TNF-α secreting CD4⁺ T cells were assessed by ICS. Data are mean values from 2 independent experiments, using 2 mice per group. **p<0.005; ***p<0.001

Figure 5

Humoral immunity at mucosal surfaces, the lung, following αDEC:LcrV immunization. (A) BALB/c mice were primed and boosted s.c. as shown at the left side of the graphs. Six to 10 days after the boost, anti-LcrV antibody titers for individual IgG isotypes and IgA in BAL fluid were determined by ELISA. Data are mean values from one of three similar experiments. (B-C) BALB/c mice were primed and boosted i.n. as described at the left. Six to 10 days after the boost, anti-LcrV antibody titers for individual IgG isotypes and IgA were measured in BAL fluid (B) (C), and in nasal washes and serum (C) by ELISA. Data shown are mean values from three similar experiments, using 3 mice per group. *p<0.05; **p<0.005; ***p<0.001

Figure 6

Survival rates after challenge with 100 LD₅₀ of CO92 Y. pestis. BALB/c mice (10/group) primed and boosted i.n. or s.c. as described at the right with antigen and adjuvants. Six weeks after the boost, CO92 Y. pestis was given i.n. and the survival of the mice monitored daily for 14 days. ***p<0.001