Fig 1.
OVA/DOTAP/D35/Alhydrogel immunization induces strong T cell and antibody responses.
C57BL/6J mice were immunized i.d. with OVA and the indicated adjuvant combinations at the tail base. OVA (10 μg), DOTAP (100 μg), D35 (10 μg), and Alhydrogel (40 μg) were mixed in a 1:1 mixture of 5% glucose and phosphate-buffered saline (PBS)(Glu/PBS buffer). After 7 days of immunization, splenocytes and serum were collected. The splenocytes were stimulated in vitro with OVA257-264 peptide that induces MHC class I restricted response (CD8+ T cell response), OVA323-339 peptide, or OVA protein that induces MHC class II restricted response (CD4+ T cell response). (a) After 24 h, the concentration of mouse IFN-γ in the culture supernatant was measured by ELISA. (b) The OVA-specific antibody titers of total IgG, IgG1, and IgG2c were determined by ELISA after 7 days of the single immunization. The bar graph indicates the mean + SD of cytokine concentration observed in six mice per group (a) and antibody titer calculated from three mice per group (b). Statistical significance compared with OVA only is shown by asterisks in the bar graph; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 2.
Type A CpG D35 and Alhydrogel are better components for the combination adjuvant to induce T cell responses.
C57BL/6J mice were immunized i.d. with OVA and a combination adjuvant, as indicated at the tail base. OVA (10 μg), DOTAP (100 μg), CpG (D35, K3, or P21889; each 10 μg), and aluminum salt (Alhydrogel or AdjuPhos; each 40 μg) in Glu/PBS buffer were used. Seven days after immunization, splenocytes were collected and stimulated with OVA257-264 peptide to induce CD8+ T cell response, and OVA323-339, or OVA protein to induce CD4+ T cell response. After 24 h, the concentration of mouse IFN-γ in the culture supernatant was measured by ELISA. (a) Three different CpG comparisons were performed. (b) Comparison of Alhydrogel and Adjuphos in Glu/PBS buffer. The bar graph indicates the mean + SD of cytokine concentrations derived from three mice per group. Statistical significances compared with OVA/DOTAP/D35 (without Alhydrogel or Adjuphos) are shown by asterisks in the bar graph; *p < 0.05.
Fig 3.
The interaction of the antigen and aluminum salt is important for the induction of T cell response with the combination adjuvant.
OVA (10 μg), OVA plus Alhydrogel (40 μg), and OVA plus AdjuPhos (40 μg) in Glu/PBS were mixed and centrifuged at 5000 rpm for 3 min. The protein concentration in the supernatant was measured using a Qubit Protein Assay Kit. HEL (10 μg) only, HEL plus Alhydrogel (40 μg), HEL plus AdjuPhos (40 μg) in 20 mM histidine buffer (pH 6.5)(Glu/His buffer) (a) or in 20 mM histidine with 150 mM NaCl buffer (pH 6.5)(Glu/His+NaCl buffer) (b) were assayed in a similar manner. The bar graph indicates the mean ± SD of triplicate experiments. Statistical significance is indicated by an asterisk in the bar graph; ***p < 0.001(parametric unpaired t-test). (c) BALB/c mice were immunized with the indicated combination adjuvant. HEL (10 μg), DOTAP (100 μg), D35 (10 μg), and Alhydrogel or AdjuPhos (40 μg) in a 1:1 mixture of 5% glucose and histidine buffer (Glu/His) were used. As a control, HEL (10 μg) was also immunized with the FCA emulsion (see Methods). The AdjuPhos group also used as another control with 0.15 M NaCl addition. Seven days after immunization, splenocytes were collected and stimulated with HEL107-116 peptide or HEL protein to induce CD4+ T cell response. After 24 h, the concentration of mouse IFN-γ in the culture supernatant was measured by ELISA. The bar graph indicates the mean + SD of cytokine concentrations derived from three mice per group. The data are representative of at least two independent experiments with similar results.
Fig 4.
Interactions between adjuvant components in different buffers.
The two-component interactions were examined using four different buffers. OVA (10 μg), DOTAP (100 μg), D35 (10 μg), and Alhydrogel (40 μg) were used. Two components were mixed in 100 μL of the total volume and examined as described in the Methods section. (a) DOTAP vs. Alhydrogel/AdjuPhos interaction (b) D35 vs. Alhycrogel/AdjuPhos interaction (c) OVA vs. Alhycrogel/AdjuPhos interaction (d) OVA vs. DOTAP interactions (e) D35 vs. DOTAP interaction. The bar graph indicates the mean ± SD of triplicates. Statistical significance is indicated by an asterisk in the bar graph; **p < 0.01, ***p < 0.001, ****p < 0.0001 (parametric unpaired t-test).
Fig 5.
Induction of T cell response by different aluminum salt and buffers.
C57BL/6J mice were immunized i.d. with OVA plus the indicated combination adjuvants and buffers at the tail base. OVA (10 μg), DOTAP (100 μg), D35 (10 μg), and Alhydrogel (40 μg) in the indicated buffers were used. Seven days after immunization, splenocytes were stimulated in vitro with OVA257-264 peptide that induced MHC class I restricted response (CD8+ T cell response), and OVA323-339 peptide, or OVA protein that induced MHC class II restricted response (CD4+ T cell response). After 24 h, the concentration of mouse IFN-γ in the culture supernatant was measured by ELISA. (a) Comparison of Glu/PBS and MES buffers (b) Comparison of Glu/His and Glu/His + NaCl solutions. (c) Comparison of Glu/PBS and Glu/His buffers. The bar graph indicates the mean + SD of cytokine concentrations derived from three mice per group. Statistical significances compared with Alhydrogel in Glu/PBS and AdjuPhos in MES (in panel (a)) were detected and are shown by asterisks in the bar graph; *p < 0.05. Other comparisons did not reach statistical significance (non-parametric test).
Fig 6.
Biodistribution of OVA antigen in vivo after injection of different formulation of adjuvants.
C57BL/6J mice were administered Alexa647-labeled OVA (10 μg) or the indicated adjuvant formulations via the tail base. For combination, Alexa647-OVA (10 μg), DOTAP (100 μg), D35 (10 μg), and Alhydrogel (40 μg) in the Glu/PBS buffer were used. The fluorescence intensity of Alexa647 was measured every 1–2 days. (a) Representative image of Alexa647-labeled OVA detection using the VISQUE® InVivo imager. The arrow indicates the signal at the injection site, and the arrowhead indicates the signal observed in the draining lymph node. DOTAP containing formulations were examined; (b)(d) None(Glu/PBS buffer only), OVA, OVA+Alhydrogel, OVA+D35. DOTAP containing formulations were examined; (c)(e) OVA+Alhydrogel+DOTAP+D35, OVA+DOTAP+D35, OVA+alhydrogel+DOTAP, OVA+DOTAP, OVA, None. The fluorescent intensities at the injection site (b)(c) and inguinal lymph node (d)(e) were measured using the VISQUE® InVivo imager.