Vaccination with Antigen Combined with αβ-ATP as a Vaccine Adjuvant Enhances Antigen-Specific Antibody Production via Dendritic Cell Activation

Abstract

Adjuvants are required to enhance antigen-specific immune responses by vaccines. Extracellular ATP serves as a danger signal to alert the immune system of tissue damage by acting on P2X and P2Y receptors and triggers the activation of dendritic cells (DCs). Here we investigated the in vivo adjuvant efficacy of α,β-methylene-ATP (αβ-ATP), a non-hydrolysable form of ATP. We found that intradermal injection of ovalbumin (OVA), as a model antigen, combined with αβ-ATP, as the adjuvant, enhanced OVA-specific immune responses more than OVA alone. Additionally, DCs in the skin of mice injected with OVA and αβ-ATP had increased expression of major histocompatibility complex class II and co-stimulator molecules, CD40, CD80, and CD86, suggesting that αβ-ATP activated DC. These findings indicate that αβ-ATP functions as a potent vaccine adjuvant.

Vaccination is the most effective way to protect against infection. Most vaccines require the use of adjuvants to induce a strong immune response. The development of new-type vaccine adjuvants is necessary to facilitate the development of new vaccine formulations.

Dendritic cells (DCs) are the most potent antigen-presenting cells known and have the ability to stimulate naïve immune cells and memory effector cells.¹⁾ DCs take up and process antigens in peripheral tissue, undergo maturation in response to inflammatory stimuli resulting in the upregulation of major histocompatibility complex (MHC) and costimulatory molecules and production of cytokines. Antigen-bearing DCs migrate to the T cell-rich areas of lymph node, where they efficiently stimulate antigen-specific T cells via MHC molecules. The first step toward including immune responsiveness by vaccines is the delivery of antigen to DCs. Recent studies suggest that activation signals are required to induce efficient immune responses^2–4); thus, antigen delivery to DCs must coincide with the delivery of an activation signal to DCs.

The intracellular adenine nucleotide ATP is involved in energy metabolism in biological system and represents a ubiquitous class of signaling molecules in many tissues, including the immune system. Extracellular ATP can be released by damaged cells, which can trigger inflammatory reactions, such as the release of interleukin (IL)-1β, enhanced phagocytosis, and chemotaxis.^5,6) DCs express the nucleotide receptors P2X and P2Y and extracellular ATP has been shown to affect the T cell stimulatory capacity of human monocyte-derived DCs.^7,8) P2X and P2Y were shown to be important in cytokine secretion and antigen presentation in DCs. Thus, extracellular ATP induces activation signals in DCs.

We hypothesized that ATP activates DC function to trigger immune responses; thus, we investigated the efficacy of using α,β-methylene-ATP (αβ-ATP), a non-hydrolysable form of ATP, as a vaccine adjuvant, in vivo.

MATERIALS AND METHODS

Animals

Female C57BL/6 mice (7 weeks old) were purchased from SLC Inc. (Hamamatsu, Japan). Three to five mice per each group were used for the experiments. Animals were handled in accordance with the Kindai University guidelines for experimental animal welfare. The research protocols described in this report were reviewed and approved by the Animal Care and Use Committee of Kindai University.

Vaccination Protocol

Ovalubumin (OVA; Sigma-Aldrich Inc., St. Louis, MO, U.S.A.) was used as a model antigen. C57BL/6 mice were intradermally injected with OVA (1 µg) alone or OVA (1 µg) mixed with αβ-ATP (10 µg; Sigma-Aldrich Inc.). This procedure was repeated three times every 2 weeks.

Antibody Titer Measurement

Serum was collected from immunized mice at the indicated time points, and the OVA-specific immunoglobulin (Ig)G titer was determined by enzyme-linked immunosorbent assay (ELISA) following the previously described protocols.⁹⁾ End-point titers of the OVA-specific IgG, IgG1, and IgG2c antibody were expressed as the reciprocal log₂ of the last dilution that had 0.1 absorbance units after subtracting the background.

Surface Phenotype Analysis of DCs after Immunization

OVA (1 µg) alone or OVA (1 µg) combined with αβ-ATP (10 µg) was intradermally injected into mice. Three or six hours later, cells were isolated from the skin or 6, 24, 48 or 72 h after injection, cells were isolated from the regional lymph nodes. Isolated cells were incubated with anti-CD16/32 antibody (clone 2.4G2) to minimize nonspecific staining by the following immune reagents: fluorescein isothiocyanate (FITC)-labeled anti-CD11c (clone N418), phycoerythrin (PE)-labeled anti-CD11c (clone N418), FITC-labeled anti-CD40 (clone HM40-3), PE-labeled anti-CD86 (clone GL-1), APC-labeled anti-IA/IE (clone M5/114.15.2), or biotinylated anti-CD80 (clone 16-10A1), and PE-conjugated streptavidin. All immune reagents were purchased from BioLegend (San Diego, CA, U.S.A.). Cells were analyzed for their surface phenotype using BD LSRFortessa (BD Biosciences, San Diego, CA, U.S.A.) with FlowJo software (Tree Starm Inc., Ashland, OR, U.S.A.).

Cell Isolation from the Skin

To isolate skin cells, the skin was removed from mice and were incubated for 60 min at 37°C in RPMI1640 supplemented with 10% fetal bovine serum (FBS), 0.24 mg/mL collagenase A (Roche; Basel, Switzerland), and 40 U/mL DNase I (Thermo Fisher Scientific Inc.). After shaking vigorously for 10 s, the resulting suspension was filtered through a 70-µm cell strainer.

RESULTS AND DISCUSSION

To investigate the in vivo adjuvant efficacy of αβ-ATP, we first measured the antigen-specific IgG antibody titer of mice intradermally immunized with OVA combined with αβ-ATP as an adjuvant. In mice injected with OVA and αβ-ATP, the anti-OVA IgG titer increased after the first vaccination and reached maximum levels (reciprocal log 2 titer; approximately 19) by the second vaccination (Fig. 1a). Conversely, the anti-OVA IgG titer of mice injected with OVA alone was also increased after the first vaccination; however, the levels were lower than OVA combined with αβ-ATP and at least three vaccinations were required to reach maximum levels.

Fig. 1. Anti-OVA IgG Production in Mice Immunized with OVA and αβ-ATP

C57BL/6 mice were intradermally vaccinated with OVA (1 µg) alone or OVA (1 µg) and αβ-ATP (10 µg) three times at 2-week intervals. Serum was collected at indicated times and anti-OVA IgG (a), IgG1 (b), and IgG2c (c) titers (IgG at indicated points, IgG1 and IgG2c at 5 weeks) were measured by ELISA. Data are expressed as the mean±S.E. of results from five mice. Statistical significance was established using Student’s t-test. * p<0.01 vs. OVA alone group.

To evaluate the T-helper (Th)1/Th2 balance in the immune responses induced by the immunization of OVA and αβ-ATP as an adjuvant, we analyzed OVA-specific IgG subclasses. In mice, IgG1 is classified as Th2-dependent, and IgG2c as Th1-dependent.¹⁰⁾ Mice injected with OVA and αβ-ATP showed higher anti-OVA IgG1 titers than those injected with OVA alone (Fig. 1b). There were little significant differences in anti-OVA IgG2c titers between the two groups (Fig. 1c). Immunization with OVA and αβ-ATP did not induce OVA-specific cytotoxic T cell responses (data not shown). Further investigation of cytokine production is required, however, these data suggest that αβ-ATP enhanced mainly Th2-type humoral immune responses rather than Th1-type cellular immune responses by the intradermal route. These results indicated that αβ-ATP showed potent adjuvant efficacy, specifically increasing antigen-specific IgG production.

Next, we analyzed the surface phenotype of DCs after in vivo OVA and αβ-ATP injection. In normal state (immature state), CD11c⁺ DCs in skin possess a high endocytic/phagocytic activity, intermediate levels of surface MHC class II (MHC class II^int), and low levels of surface co-stimulatory molecules. αβ-ATP is known to enhance DC maturation through the activation of purinergic P2X and P2Y receptors.^7,8) DCs are also known to mature in response to stimulation with bacteria-derived products marked by an increase in the expression of co-stimulatory molecules such as CD40, CD80, and CD86 as cofactors for antigen presentation and T cell activation.^11,12) We, therefore, examined the surface expression of CD40, CD80, CD86, and MHC class II on CD11c⁺ DCs isolated from the skin, to investigate whether co-injection of αβ-ATP influenced DC maturation compared with OVA injected alone. Co-injection of αβ-ATP with OVA increased CD80, CD86, and MHC class II expression levels on CD11c⁺ DCs in the skin compared with OVA injected alone 6 h after injection (Fig. 2a). The interaction of CD80/CD86 with promotional receptors on T cells leads to enhanced T cell expansion and prolonged expression of the CD40 ligand.¹³⁾

Fig. 2. The Maturation of DCs and Their Migration to the Regional Lymph Nodes Following Immunization

C57BL/6 mice were intradermally vaccinated with OVA (1 µg) alone or OVA (1 µg) and αβ-ATP (10 µg). Three or six hours later cells were isolated from skin and 6, 24, 48 or 72 h later cells were isolated from the regional lymph nodes. (a) The expression of CD40, CD80, CD86, and MHC class II were analyzed by flow cytometry. CD11c⁺ DCs were gated as indicated in SSC/CD11c dot-blot. The data are shown as histograms representing the expression of each molecule in the CD11c⁺ DC fraction of the skin from immunized mice (gray histogram) and non-treated mice (open histogram). Representative data are shown from three independent experiments. (b) The percentage of MHC class II^high CD11c^int migratory DCs in the regional lymph nodes was analyzed by flow cytometry. The representative MHC class II/CD11c dot blot at 0 and 48 h in OVA and αβ-ATP group were shown. MHC classII^high CD11c^int migratory DCs were gated as indicated in MHC class II/CD11c bot-blot. Data are expressed as the mean±S.E. of results from three mice. Statistical significance was determined by Student’s t-test. * p<0.01 vs. OVA alone group.

Activated antigen-bearing DCs up-regulate surface expression of co-stimulatory molecules and antigen-MHC class II complexes (MHC class II^high), and down-regulate surface expression of CD11c molecule (CD11c^int). These DCs migrate to draining lymph nodes and terminally differentiate into mature states which show increased antigen-presentation activity and reduced endocytic/phagocytic activity. The migratory DCs (MHC class II^high and CD11c^int) present antigens to T cells for induction of antigen-specific immune responses. In the regional lymph node after injection, the number of MHC class II^high, CD11c^int migratory DCs in mice injected OVA and αβ-ATP was higher than in mice injected with OVA alone after 48 h (Fig. 2b), leading to increased induction of antigen-specific immune responses. These findings suggested that administration of OVA and αβ-ATP increased the antigen-presentation ability of DCs and enhanced antigen-specific immune responses by enhancing the expression of co-stimulatory molecules.

It was previously reported that keratinocytes can also express purinergic receptors and ATP regulated their cell growth, differentiation, terminal differentiation, and cell-to-cell communication.^14–18) Additionally, extracellular ATP had a stimulatory effect on the release of IL-6, suggesting a role of keratinocytes in the induction of antigen-specific immune responses by ATP via purinergic receptor engagement.^19,20) Moreover, the release of cytokines can be regulated through P2 receptors in a variety of cells including macrophages.^19,20) Further studies of the roles of other cells, such as keratinocytes and macrophages are required to understand the adjuvant potential of αβ-ATP.

P2X receptors and P2Y receptors have seven subtypes (P2X_1–7) and eight subtypes (P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y_11–14), respectively and form heteromeric or homomeric channel assemblies. In mice, the P2X7 receptor was shown to be involved in antigen presentation²¹⁾; however, in human monocyte-derived DCs, there was no role for P2X7 receptors in antigen presentation.^7,22–24) Furthermore, P2Y11, P2X1, and P2X3 receptors are candidate targets for DC activation. Detailed roles of P2 receptor subtypes in enhancing antigen-specific immune responses may aid development of efficient adjuvant candidates and understand the immune adjuvant effects of αβ-ATP.

In conclusion, the findings of the present study demonstrate that αβ-ATP, as an adjuvant, enhanced antigen-specific antibody production via DC activation. Further studies are aimed at assessing the efficacy in combination with clinical antigens, such as hemagglutinin. Our results suggest that αβ-ATP is a potent vaccine adjuvant and will be broadly applicable to a number of vaccines designed to increase antibody production for preventing infectious diseases.

Conflict of Interest

The authors declare no conflict of interest.

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