Carbonate Apatite Nanoparticles Act as Potent Vaccine Adjuvant Delivery Vehicles by Enhancing Cytokine Production Induced by Encapsulated Cytosine-Phosphate-Guanine Oligodeoxynucleotides

K-type CpG ODNs (CpG K3: 5′-atcgactctcgagcgttctc-3′) and Alexa 488-labeled K-type CpG ODNs (Alexa 488-labeled CpG K3) were purchased from GeneDesign (Osaka, Japan). EndoGrade endotoxin-free ovalbumin (OVA) (<0.1 EU/mg protein) purchased from Hyglos GmbH (Bayern, Germany) was used for immunization. Grade V OVA purchased from Sigma-Aldrich (St. Louis, MO, USA) was used for enzyme-linked immunosorbent assays (ELISA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and IgG1 antibodies were purchased from Merck Millipore (Darmstadt, Germany). HRP-conjugated goat anti-mouse IgG2c was purchased from GeneTex, Inc. (Irvine, CA, USA). Alhydrogel adjuvant 2% (alum) was purchased from InvivoGen (San Diego, CA, USA).

C57BL/6J mice were purchased from SLC (Kyoto, Japan). IFN-α/β receptor 2 (Ifnar2)-Il-12 p40 double-deficient mice have been described previously (27) and were kindly provided by Dr. Ishii (National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka, Japan). Mice were used at 7 to 8 weeks of age and were housed in a room with a 12-h-light/12-h-dark cycle (lights on at 8:00 am, lights off at 8:00 pm) with access to food and water. All animal experiments were performed in accordance with institutional guidelines of Osaka University for the ethical treatment of animals (protocol number H26-11-0).

Carbonate apatite nanoparticles and CA-CpG were prepared as described previously (26). We incubated a 25 mL solution of NaHCO₃ (44 mM), NaH₂PO₄ (0.9 mM), and CaCl₂ (1.8 mM) (pH 7.5) without or with 1.9, 5.6, 16.7, 50, or 150 µg of K-type CpG ODNs at 37°C for 30 min. The solution was then centrifuged at 12,000 rpm for 3 min, and the pellet was dissolved in saline containing 0.5% mouse serum. The resulting solution of CA nanoparticles or CA-CpG was sonicated in a water bath for 10 min. For determination of the amount of endotoxin, the collected CA-CpG was dissolved in 1 mL of 0.02 M ethylenediaminetetraacetic acid and the amount of endotoxin was determined by means of a Toxicolor LS-50M kit (Seikagaku Corporation, Tokyo, Japan). CA-CpG was found to include <0.2 EU/mL of endotoxin.

For determination of the K-type CpG ODN encapsulation efficiency, the collected CA-CpG was dissolved in 1 mL of 0.02 M ethylenediaminetetraacetic acid and the amount of K-type CpG ODNs was determined with a Qubit ssDNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).

The size distributions of CA nanoparticles and CA-CpG were analyzed with a Zetasizer Nano ZS (Malvern Instruments, Malvern, Worcestershire, UK). Specifically, the mean diameters and particle size distributions of the nanoparticles in mouse serum albumin (Sigma-Aldrich) were measured by means of a dynamic light-scattering method using capillary cells. The size distributions of both types of nanoparticles were also analyzed by atomic force microscopy conducted with a scanning probe microscope (SPM-9500, Shimadzu, Kyoto, Japan) operated in dynamic mode and equipped with a microcantilever (OMCL-AC240TS-R3, Olympus, Tokyo, Japan).

To generate bone marrow-derived DCs, we isolated bone marrow cells from the femurs of C57BL/6J mice and cultured the cells for 7 days with 100 ng/mL human Flt-3L (PeproTech, Rocky Hill, NJ, USA). Cells were seeded at a density of 5 × 10⁵ cells/well in a 96-well flat-bottom culture plate (Nunc, Roskilde, Denmark) and were cultured in complete RPMI medium (RPMI 1640 supplemented with 10 vol% fetal calf serum (FCS), penicillin, and streptomycin). These cells were stimulated with K-type CpG ODNs or CA-CpG for 24 h. Supernatants were subjected to ELISA to determine the levels of IFN-α (PBL Assay Science, Piscataway, NJ, USA), IFN-γ (BioLegend, San Diego, CA, USA), and IL-12 p40 (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. Human peripheral blood mononuclear cells (PBMCs) were obtained from two healthy adult male Japanese volunteers (26 years of age) with informed consent. After the PBMCs were prepared with Ficoll, they were plated at a concentration of 1 × 10⁷ cells/mL in a 96-well flat-bottom culture plate and maintained in complete RPMI medium. They were stimulated with K-type CpG ODNs or CA-CpG for 24 h. Supernatants were subjected to ELISA to determine the levels of pan-IFN-α and IFN-γ according to the manufacturers' instructions. All experiments using human PBMCs were approved by the Institutional Review Board of the Research Institute for Microbial Diseases, Osaka University.

Mouse bone marrow-derived DCs (1 × 10⁷ cells/mL in a 96-well flat-bottom culture plate) were treated with Alexa 488-labeled K-type CpG ODNs (1.25 µg CpG ODNs/mL) or CA nanoparticles containing Alexa 488-labeled K-type CpG ODNs (CA-Alexa 488-CpG; 1.25 µg CpG ODNs/mL) for 10, 30, 90, 120, or 180 min. Cells were stained with 0.4 w/v% trypan blue solution (Wako, Osaka, Japan) to quench any Alexa 488-labeled K-type CpG ODNs bound to the cell surface, and then the cells were analyzed by means of flow cytometry (NovoCyte Flow Cytometer, ACEA Bioscience, San Diego, CA, USA). To separate various subsets of DCs from the collected cells, we incubated the cells with anti-mouse CD16/CD32 antibody (TONBO biosciences, San Diego, CA, USA), anti-mouse B220 antibody (BioLegend), anti-CD11c antibody (BioLegend), and anti-CD11b antibody (BioLegend) in the absence of trypan blue. In this way, the DCs were separated into the following subsets: B220⁺ CD11c⁺ plasmacytoid DCs, CD11b⁺ CD11c⁺ conventional DCs, and CD11b⁻ CD11c⁺ conventional DCs.

C57BL/6J mice were treated with saline, Alexa 488-labeled K-type CpG ODNs (8 or 40 µg CpG ODNs/mouse), or CA-Alexa 488-CpG (8 µg CpG ODNs/mouse) into the ear pinna. Twenty-four hours after administration, draining lymph nodes were collected. To prepare single-cell suspensions, we incubated the draining lymph nodes with 5 µg/mL collagenase D (Wako, Tokyo, Japan) for 60 min at 37°C. We used flow cytometry to analyze the uptake of CpG ODNs by cells in draining lymph nodes.

C57BL/6J mice were treated with K-type CpG ODNs (8 or 40 µg CpG ODNs/mouse) or CA-CpG (8 µg CpG ODNs/mouse) into the ear pinna. Twenty-four hours after administration, draining lymph nodes were collected. To prepare single-cell suspensions, we incubated the draining lymph nodes with collagenase D (5 µg/mL) for 60 min at 37°C. Prepared cells were incubated for 8 h, culture supernatants were collected, and cytokines were measured by means of ELISA.

C57BL/6J mice were treated with OVA (100 µg/mouse) without or with K-type CpG ODNs (10 or 50 µg CpG ODNs/mouse) or OVA with CA-CpG (10 µg CpG/mouse) subcutaneously at the base of the tail at days 0 and 14. Using hematocrit capillary tubes (Terumo, Tokyo, Japan), we obtained blood samples from the retro-orbital venous plexus at day 21 and centrifuged the samples at 3,000 × g at 4°C. The resulting plasma was stored at −80°C until analysis. To investigate the adjuvant effects of CA-CpG administered by another route, we treated C57BL/6J mice with OVA (80 µg/mouse) without or with K-type CpG ODNs (8 or 40 µg CpG ODNs/mouse) or OVA with CA-CpG (8 µg CpG/mouse) into the ear pinna at days 0 and 7, and we obtained blood samples at day 14.

The levels of OVA-specific antibodies in plasma were determined by means of ELISA. To detect OVA-specific IgG, IgG1, and IgG2c, we coated ELISA plates (Corning, Corning, NY, USA) with OVA in carbonate buffer (10 µg/mL) overnight at 4°C. The coated plates were incubated with PBS containing 10% FCS. Plasma samples were diluted with PBS containing 10% FCS, and the dilutions were added to the OVA-coated plates. After incubation with plasma for 2 h, the coated plates were incubated with an HRP-conjugated goat anti-mouse IgG, IgG1, or IgG2c solution for 1 h at room temperature. After incubation, the color was developed with tetramethyl benzidine (Moss, Pasadena, MD, USA), the reaction was stopped with 2 N H₂SO₄, and the OD_450–570 was measured on a microplate reader (Power Wave HT, BioTek, Winooski, VT, USA).

C57BL/6J mice were treated with OVA (80 µg/mouse) without or with K-type CpG ODNs (8 or 40 µg CpG ODNs/mouse) or CA-CpG (8 µg CpG ODNs/mouse) into the ear pinna. Draining lymph nodes were collected at day 7, and lymph node cells were prepared for determination of OVA_257–264-specific CD8⁺ CTL responses. Lymph node cells (1 × 10⁶ cells) were added to the wells of a 96 half-well plate and then stimulated with OVA_257–264 peptide (SIINFEKL, final concentration 5 µg/mL) for 24 h at 37°C. After the incubation, the concentration of IFN-γ in the supernatants was analyzed by means of ELISA. For the tetramer assay, lymph node cells (1 × 10⁶ cells) were stained with purified anti-mouse CD16/CD32 antibody and then with the phycoerythrin-labeled H-2K^b/OVA_257–264 tetramer (MBL, Aichi, Japan). Fluorescein isothiocyanate-conjugated CD8α (Abcam, Cambridge, UK), allophycocyanin-conjugated CD44 (BioLegend), and 7-amino-actinomycin D (BioLegend) were then added. The number of CD8α⁺ CD44⁺ tetramer⁺ cells was determined by flow cytometry.

C57BL/6J mice were treated with ether-treated hemagglutinin antigen-enriched virion-free split vaccine (SV; strain A/California/7/2009, H1N1, 0.5 µg/mouse) without or with K-type CpG ODNs (10 or 50 µg CpG ODNs/mouse), CA-CpG (10 µg CpG ODNs/mouse), CA nanoparticles, or alum at days 0 and 14. At day 21, we obtained blood samples from the retro-orbital venous plexus and centrifuged the samples at 3,000 × g at 4°C; the resulting plasma was stored at −80°C until analysis. To detect SV-specific IgG, IgG1, and IgG2c, we coated ELISA plates with SV in carbonate buffer (10 µg/mL) overnight at 4°C. The coated plates were incubated with PBS containing 10% FCS. Plasma samples were diluted with PBS containing 10% FCS, and the dilutions were added to the SV-coated plates. After incubation with plasma for 2 h, the coated plates were incubated with a solution of HRP-conjugated goat anti-mouse IgG, IgG1, or IgG2c for 1 h at room temperature. After incubation, the color was developed with tetramethyl benzidine, the reaction was stopped with 2 N H₂SO₄, and OD_450–570 was measured on a microplate reader. For analysis of hemagglutination inhibition (HI) titers, plasma samples were incubated with RDE (II) (DENKA SEIKEN, Tokyo, Japan) for 18 h at 37°C and then heated at 56°C for 30 min to deactivate the enzyme. To avoid nonspecific hemagglutination, plasma samples were treated with guinea pig red blood cells for 1 h at room temperature, and the supernatants were serially diluted 2-fold in microtiter plates. An equal volume of influenza virus (strain A/California/7/2009, H1N1) was added to each well, and the plates were incubated for 1 h at room temperature. Guinea pig red blood cells were added and allowed to settle for 1 h at 4°C. The HI titer was determined from the dilution of the last well that contained non-agglutinated guinea pig red blood cells. Moreover, immunized mice were challenged intranasally with 7.2 × 10³ pfu (10-LD50) of influenza virus (strain A/PR/8/34, H1N1) (28). Body weights and survival rates of challenged mice were monitored for 14 days. Ether-treated hemagglutinin antigen-enriched virion-free split vaccine and influenza virus were kindly provided by the Research Foundation for Microbial Diseases of Osaka University, Suita, Japan.

K-type CpG ODNs (10 or 50 µg CpG ODNs/mouse) or CA-CpG (10 µg CpG ODNs/mouse) was administered to C57BL/6J mice subcutaneously at the base of the tail at days 0, 2, and 4. Twenty-four hours after the final administration, the entire spleen, the entire liver, and draining lymph nodes were isolated and weighed.

Statistical analyses were performed with GraphPad Prism (GraphPad Software, San Diego, CA, USA). All data are presented as mean with SD or SEM. Significant differences between control groups and experimental groups were determined by means of Dunnett's test, Tukey's test, or Student's t-test. A p value of <0.05 was considered to indicate statistical significance.

We evaluated the usefulness of CA nanoparticles as CpG ODN delivery vehicles. We began by determining the amount of K-type CpG ODNs encapsulated in CA-CpG. We found that the amount of K-type CpG ODNs used to generate CA-CpG affected the efficiency with which the K-type CpG ODNs was encapsulated in CA-CpG (Figure 1A). When we used 50 µg K-type CpG ODNs, the encapsulation efficiency was high (58%). Therefore, we used CA-CpG prepared with 50 µg K-type CpG ODNs for subsequent experiments. Dynamic light scattering showed that both the empty CA nanoparticles and CA-CpG were uniform and monodispersed, with hydrodynamic diameters ranging from 30 to 40 nm, respectively (Figure 1B). In addition, we confirmed that CA-Alexa 488-CpG was also uniform and monodispersed, with hydrodynamic diameters ranging from 30 to 40 nm, as was the case for both the empty CA nanoparticles and CA-CpG (data not shown). Atomic force microscopy showed that the sizes of the CA nanoparticles and CA-CpG ranged from 13 to 25 nm (Figure 1C). These results suggest that we successfully generated nanosize CA-CpG with high encapsulation efficiency for K-type CpG ODNs.

To determine the immunostimulatory effects of CA-CpG on DCs, we treated mouse-derived DCs with CA-CpG or K-type CpG ODNs in vitro and measured cytokine levels in the supernatants by means of ELISA (Figure 2A). DCs stimulated with CA-CpG produced substantial amounts of not only IFN-γ and IL-12 p40 but also IFN-α, whereas K-type CpG ODNs did not induce cytokine production at the tested doses. In human PBMCs, higher levels of IFN-α and IFN-γ were produced in response to CA-CpG than in response to K-type CpG ODNs (Figure 2B). These data suggest that CA-CpG might be superior to K-type CpG ODNs as an adjuvant. In addition, they suggest that CA-CpG has some characteristics of D-type CpG ODNs, because IFN-α is known to be a D-type CpG ODN-specific cytokine.

To elucidate the mechanism of the immunostimulatory effects of CA-CpG, we compared the cellular internalization of CA-CpG and K-type CpG ODNs in mouse DCs by using Alexa 488-labeled K-type CpG ODNs. DCs were treated with CA-Alexa 488-CpG or with Alexa 488-labeled K-type CpG ODNs, and cells were stained with trypan blue solution to quench any Alexa 488-labeled K-type CpG ODNs bound to the cell surface, so that only internalized K-type CpG ODNs were detected during flow cytometry (Figures 3A–C). The amount of Alexa 488-labeled K-type CpG ODNs in DCs treated with CA-Alexa 488-CpG or Alexa 488-labeled K-type CpG ODNs increased over time, and at 90, 120, and 180 min after treatment the groups treated with Alexa 488-labeled K-type CpG ODNs had more cells that were positive for K-type CpG ODNs than did the groups treated with CA-Alexa 488-CpG (Figures 3A,B). For more precise analysis, the fractions that were positive for CpG ODNs were divided into two fractions, one with low fluorescence and one with high fluorescence; then we again compared the groups treated with CA-Alexa 488-CpG and the groups treated with Alexa 488-labeled K-type CpG ODNs (Figures 3A,C). In the low-fluorescence fraction, at 90, 120, and 180 min after treatment, the groups treated with Alexa 488-labeled K-type CpG ODNs had more cells that were positive for K-type CpG ODNs than did the CA-Alexa 488-CpG-treated groups. In contrast, in the high-fluorescence fraction at all time points, the CA-Alexa 488-CpG-treated groups had significantly more cells that were positive for K-type CpG ODNs than did the groups treated with Alexa 488-labeled K-type CpG ODNs. On the basis of these results, we speculated that certain subsets of DCs might have taken up CA-CpG more efficiently than others. To evaluate this possibility, we categorized DCs into the following subsets: B220⁺ CD11c⁺ plasmacytoid DCs, CD11b⁺ CD11c⁺ conventional DCs, and CD11b⁻ CD11c⁺ conventional DCs. Then we analyzed the CpG ODN-positive cells in each subset at both 37°C and 4°C, so that we could evaluate nonspecific binding of CA-Alexa 488-CpG or Alexa 488-labeled K-type CpG ODNs to the cell surface (Figure 3D). There were fewer CpG ODN-positive cells at 4°C than at 37°C, indicating that the binding of CA-CpG or K-type CpG ODNs on the cell surface could be ignored in this assay. At 37°C, in all the subsets of DCs, the number of CpG ODN-positive cells in the CA-CpG-treated groups was lower than that in the K-type CpG ODN-treated groups (Figure 3D), indicating that CA-CpG was taken up into all the subsets of DCs equally, as was the case for the K-type CpG ODNs.

Generally, the delivery of an adjuvant to lymph nodes, and specifically to DCs in lymph nodes, is important for the efficient induction of adaptive immunity. However, the efficiency with which CA-CpG delivers CpG ODNs to DCs in lymph nodes is unknown. Therefore, to assess the distribution and uptake of CA-CpG into lymph nodes, we measured the quantity of CpG ODN-positive cells in draining lymph nodes by flow cytometry after administration of CA-Alexa 488-CpG or Alexa 488-labeled K-type CpG ODNs to mice (Figure 4A). Whereas in the group treated with Alexa 488-labeled K-type CpG ODNs at 40 µg CpG ODNs/mouse (the positive control), large proportions of the lymph node cells were CpG ODN-positive, there were no significant differences between the proportions of CpG ODN-positive cells in the groups treated with CA-Alexa 488-CpG at 8 µg CpG ODNs/mouse and the groups treated with Alexa 488-labeled K-type CpG ODNs at 8 µg CpG ODNs/mouse at any time point. These results suggest that the CA nanoparticles did not increase the number of CpG ODNs taken up by DCs in lymph nodes.

Next, to determine the immunostimulatory effects of CA-CpG in vivo, we measured cytokine production in draining lymph nodes after CA-CpG administration (Figure 4B). We found that production of the cytokines IL-6, IL-12 p40, and IFN-γ in draining lymph nodes was substantially higher after treatment with CA-CpG than after treatment with K-type CpG ODNs (Figure 4B). These data suggest that CA-CpG markedly enhanced innate immune activation responses, such as cytokine production, relative to K-type CpG ODNs both in vitro and in vivo.

To evaluate the in vivo effects of the use of CA-CpG as a vaccine adjuvant in mice, we assessed antibody responses and CD8⁺ CTL responses following co-administration of CA-CpG or K-type CpG ODNs with OVA, a model protein antigen. Mice were immunized with OVA plus CA-CpG (10 µg CpG ODNs/mouse) or OVA plus K-type CpG ODNs (10 or 50 µg CpG ODNs/mouse) subcutaneously at the base of the tail. As a positive control, we used alum, which is widely used throughout the world as an adjuvant for human vaccines. Seven days after the final immunization, the levels of OVA-specific total IgG, IgG1, and IgG2c antibodies in plasma were analyzed by ELISA (Figure 5A). Levels of antigen-specific IgG subclasses reflect the subset of CD4⁺ T cells that are induced by vaccination, with production of IgG1 and IgG2c corresponding to Th2- and Th1-type responses, respectively. Although mice immunized with OVA plus alum showed high levels of total IgG and IgG1 production, mice immunized with OVA plus CA-CpG or OVA plus K-type CpG ODNs produced high levels not only of total IgG and IgG1 but also of IgG2c. Mice immunized with OVA plus CA-CpG (10 µg CpG ODNs/mouse) produced significantly higher levels of OVA-specific total IgG, IgG1, and IgG2c than did mice immunized with OVA plus K-type CpG ODNs (10 µg CpG ODNs/mouse). Antibody production in groups treated with OVA plus CA-CpG (10 µg CpG ODNs/mouse) was comparable to that in groups treated with OVA plus K-type CpG ODNs (50 µg CpG ODNs/mouse).

Next, to evaluate the adjuvant effects of CA-CpG when administered by a different route, we immunized mice with OVA plus CA-CpG or OVA plus K-type CpG ODNs into the ear pinna and then measured OVA-specific antibody responses (Figure 5B). As was the case for immunization subcutaneously at the base of the tail, the level of OVA-specific total IgG induced by immunization with OVA plus CA-CpG (8 µg CpG ODNs/mouse) was significantly higher than the level induced by immunization with OVA plus K-type CpG ODNs (8 µg CpG ODNs/mouse) and was comparable to the level induced by administration of OVA plus K-type CpG ODNs (40 µg CpG ODNs/mouse).

Clearance of viruses is known to require not only antibody responses but also strong CD8⁺ CTL responses, which are characterized by IFN-γ production by CD8⁺ T cells. To investigate the ability of CA-CpG to induce OVA-specific CD8⁺ CTL responses, we vaccinated mice with OVA plus CA-CpG and then examined the frequency of H-2K^b/OVA_257–264 tetramer⁺ CD8⁺ T cells (Figure 5C) and measured the production of H-2K^b/OVA_257–264-specific IFN-γ (Figure 5D). The frequency of H-2K^b/OVA_257–264 tetramer⁺ CD8⁺ T cells induced by OVA plus CA-CpG was significantly higher than the frequency induced by OVA plus K-type CpG ODNs (Figure 5C). Furthermore, the production of OVA_257–264-specific IFN-γ induced by OVA plus CA-CpG was significantly higher than that induced by immunization with OVA plus K-type CpG ODNs (Figure 5D). Taken together, these results suggest that, in vivo, CA-CpG stimulated antigen-specific antibody responses and CD8⁺ CTL responses more effectively than did K-type CpG ODNs.

We examined the vaccine adjuvant effects of CA-CpG by using clinically relevant influenza vaccination models in mice. Mice were immunized with A/California/7/2009 (H1N1) SV plus CA-CpG (10 µg CpG ODNs/mouse), K-type CpG ODNs (10 or 50 µg/mouse), CA nanoparticles, or Alum. The levels of SV-specific total IgG, IgG1, and IgG2c antibodies in plasma were analyzed by ELISA after the final immunization (Figures 6A,B). Mice immunized with SV plus CA-CpG (10 µg CpG ODNs/mouse) showed significantly higher levels of SV-specific total IgG, IgG1, and IgG2c than did mice immunized with OVA plus K-type CpG ODNs (10 and 50 µg CpG ODNs/mouse). The levels of SV-specific total IgG and IgG2c in mice treated with SV plus CA-CpG were significantly higher than those in mice treated with SV plus alum, whereas there was no difference in SV-specific IgG1 levels between these two groups of mice. The levels of total IgG and IgG1 in mice treated with SV plus CA nanoparticles were higher than the levels in mice treated with SV alone but were significantly lower than the levels in CA-CpG-treated mice. Next, we examined the neutralizing potential of these antibodies by using the HI assay against influenza virus A/California/7/2009 (Figure 6C), because this assay is widely used to evaluate anti-influenza virus neutralizing and protective antibodies. The HI titers showed the same trends as the levels of SV-specific total IgG, and mice immunized with SV plus CA-CpG had significantly higher HI titers than did mice immunized with SV plus K-type CpG ODNs (10 µg CpG ODNs/mouse). After the final immunization, we challenged the immunized mice with mouse-adapted influenza virus A/PR/8/34 (H1N1), which is a heterologous influenza virus from the one used as a vaccine antigen. Weights and survival rates of the infected mice were observed every day for 2 weeks (Figure 6D). Much greater pathogenesis was observed in the mice immunized without an adjuvant or with alum than in the mice co-immunized with CA-CpG or K-type CpG ODNs (10 or 50 µg CpG ODNs/mouse), as indicated by survival rate and weight loss. All mice died within 10 days after viral infection in the groups that received SV alone or SV plus alum, and 80% of the mice immunized with SV plus CA nanoparticles died within 7 days. Although only 40% of the mice immunized with SV plus K-type CpG ODNs (10 µg CpG ODNs/mouse) survived 14 days after challenge, 80% of mice immunized with SV plus CA-CpG (10 µg CpG ODNs/mouse) or K-type CpG ODNs (50 µg/mouse) survived, and they regained body weight more rapidly. These results indicate that CA-CpG is a potent vaccine adjuvant that protects against viral infection better than K-type CpG ODNs or alum.

Activation of CD8⁺ CTL responses requires T-cell activation by DCs via costimulatory signals and cytokine-mediated signals. For example, IFN-α and IL-12 work as critical survival signals that expand and differentiate effector CD8⁺ T cells (29 –31). Therefore, to elucidate the involvement of IFN-α and IL-12 in antibody responses and CD8⁺ CTL responses induced by CA-CpG, we treated mice deficient in both Ifnar2 and Il-12 p40 with OVA plus CA-CpG (Figure 7). The levels of OVA-specific total IgG, IgG1, and IgG2c in these double-deficient mice immunized with OVA plus CA-CpG tended to be lower than the levels in wild-type mice (Figure 7A). In addition, the frequency of H-2K^b/OVA_257–264 tetramer⁺ CD8⁺ T cells in the double-deficient mice tended to be lower than that in the wild-type mice (Figure 7B). These results suggest that CA-CpG enhanced both Th1-type humoral and cellular immune responses via the IFN-α pathway, the IL-12 pathway, or both.

Cytosine-phosphate-guanine ODNs are known to cause splenomegaly and to disrupt splenic microarchitecture in mice after repeated administration (32). To assess the adverse effects of CA-CpG, we treated mice three times every other day with CA-CpG or K-type CpG ODNs and then measured tissue weights (Figure 8). No significant differences in liver weight were observed between the groups. In contrast, spleen weights and draining lymph node weights in mice treated with K-type CpG ODNs at 50 µg CpG ODNs/mouse were higher than those in mice treated with CA-CpG (10 µg CpG ODNs/mouse), although the difference was statistically significant only for spleen weights. The effects of CA-CpG (10 µg CpG ODNs/mouse) were almost the same as those of K-type CpG ODNs at 10 µg CpG ODNs/mouse. These results suggest that CA-CpG improved the efficacy of CpG ODNs without enhancing their adverse effects.