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Simultaneous enhancement of cellular and humoral immunity by the high salt formulation of Al(OH) 3 adjuvant

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Adjuvants play essential roles in vaccination by enhancing and/or shaping antigen-specific immune responses... Moreover, cross-presentation of extracellular protein antigens by DCs contributes to the generation of cytotoxic T lymphocyte (CTL) responses... Sodium chloride (NaCl) is closely related to our daily lives and has recently been found to have potential effects on the development of autoimmune diseases and specific states of macrophages... Here, we used CD8 T cells from OT-I mice (specific for the H-2K/OVA complex) to test whether high salt-stimulated DCs can perform cross-presentation of soluble OVA... Next, we examined the in vivo antitumor effect of the OVA/Al/high salt vaccine using the E.G7-OVA tumor model in mice... In either the prophylactic or therapeutic vaccine model, immunization of the OVA/Al/high salt complex inhibited tumor growth more effectively compared with the other groups (Figure 1K)... The antitumor effect of the OVA/Al/3.6% NaCl vaccine was further studied in an adoptive cellular/serum therapy model... Lymphocytes from mice immunized with OVA/Al/3.6% NaCl effectively inhibited tumor growth, while the serum showed no apparent antitumor effect (Supplementary information, Figure S1M and S1N)... Only depletion of CD8 T lymphocytes and NK cells showed effective abrogation of the antitumor effect induced by the OVA/Al/3.6% NaCl vaccine (Supplementary information, Figure S1O)... All the mice were under good physiological conditions as shown by the monitoring of heart rate and mean arterial pressure, even when the NaCl concentration reached as high as 14.4% (16 times higher than the normal osmolality; Supplementary information, Table S1)... In conclusion, we discovered a novel and safe formulation (high salt formulation) of Al(OH)3 adjuvant, which, aside from maintaining the induction of humoral immune response by normal Al-adjuvant, could further enhance the cellular immune response; and this effect may be closely related to the activation of and antigen cross-presentation by DCs... Consequently, the OVA/Al/high salt formulation exhibited a significant antitumor effect against the E.G7-OVA tumor model in vivo, which might be largely due to CD8 CTL-mediated cellular immunity and independent of CD4 T cells... The most interesting and surprising finding of this study is that the adjuvant effects could be significantly improved simply by altering the NaCl concentration in vaccines and such formulation is safe, easy to prepare, and of low cost... This concept may assist in the design of broad vaccine formulations and the development of safer and more effective adjuvants for therapeutic uses.

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Simultaneous enhancement of cellular and humoral immunity by the high-salt formulation of Al(OH)3 adjuvant. (A) High salt concentration enhances Al(OH)3 adjuvant-induced humoral immunity. C57BL/6 mice (n = 5 per group) were vaccinated subcutaneously three times with OVA/Al complex containing different concentrations of NaCl (5 μg OVA per mouse). Seven days after the third immunization, the mouse serum was collected and levels of the total IgG and IgG subclasses and IgG1 titer were determined by ELISA. (B-E) OVA/Al/high salt vaccine induces specific cellular immunity. C57BL/6 mice were immunized in the same way as described in A. Lymphocytes were isolated from the spleen and further incubated in vitro with CD8+-specific OVA257–264 peptides (10 μg/ml) for 3 days. The generation of CD8+ CTLs was determined by FCM using PE-conjugated H-2Kb/OVA257–264tetramer (B). The expression of IFN-γ was examined by FCM (C) and ELISA (D). The same lymphocytes were tested for CTL-mediated cytotoxicity against E.G7-OVA cells by a standard 6 h 51Cr release assay (E). (F) High concentrations of NaCl promote the maturation of DCs in vitro. Bone marrow-derived DCs isolated from C57BL/6 mice were cultured in medium with the indicated NaCl concentrations for 48 h and the expression of maturation markers was analyzed by FCM. (G) High concentrations of NaCl promote the antigen uptake of DCs in vitro. DCs were incubated with 2 μg/ml Alexa Fluor 488-labeled OVA for 1 h at 37 °C in medium with the indicated NaCl concentrations and analyzed under a fluorescent microscope. Scale bars, 20 μm. (H) High concentration of NaCl promotes secretion of the pro-inflammatory cytokines by DCs through the p38 MAPK pathway in vitro. DCs were pretreated with 10 μM SB203580 (SB) or DMSO (DM) for 2 h and then treated with 0.9% or 1.5% NaCl for 48 h in the presence of 5 μM SB or DM. The levels of pro-inflammatory cytokines in the supernatant were measured by ELISA. (I-J) High salt concentration induces antigen cross-presentation in DCs. DCs were cultured in medium with the indicated NaCl concentrations containing 10 μg/ml OVA. Then the stimulated DCs were co-cultured with CFSE-labeled CD8+ T cells from OT-I mice. The proliferation of T cells was assessed by FCM after 3 days of co-culture (I). DCs were treated with 2 μg/ml Alexa Fluor 488-labeled OVA in the presence of the indicated NaCl concentrations for 1 h, stained with rabbit anti-LMP2/Cy3 antibody and DAPI, and visualized under a confocal laser scanning microscope. Scale bars, 5 μm (J). (K) High-salt formulation potentiates the antitumor effect of OVA/Al vaccine in vivo. In a prophylactic model (left), C57BL/6 mice (n = 10 per group) were immunized with different vaccines for three times and then challenged subcutaneously with 3 × 106 E.G7-OVA cells 1 week after the third immunization. In a therapeutic model (right), mice (n = 7 per group) were treated by subcutaneous injection of different vaccines once a week for 3 weeks starting on day 3 after subcutaneously introduction of 3 × 106 E.G7-OVA cells. The tumor volume was measured every 3 days. (L) The high-salt formulation of OVA/Al diminished immune suppressive cells in tumor microenvironment. After the third treatment, a single-cell suspension of tumor tissues was prepared. The percentages of infiltrating CD8+ lymphocytes, MDSCs and M2 macrophages were analyzed by FCM.
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fig1: Simultaneous enhancement of cellular and humoral immunity by the high-salt formulation of Al(OH)3 adjuvant. (A) High salt concentration enhances Al(OH)3 adjuvant-induced humoral immunity. C57BL/6 mice (n = 5 per group) were vaccinated subcutaneously three times with OVA/Al complex containing different concentrations of NaCl (5 μg OVA per mouse). Seven days after the third immunization, the mouse serum was collected and levels of the total IgG and IgG subclasses and IgG1 titer were determined by ELISA. (B-E) OVA/Al/high salt vaccine induces specific cellular immunity. C57BL/6 mice were immunized in the same way as described in A. Lymphocytes were isolated from the spleen and further incubated in vitro with CD8+-specific OVA257–264 peptides (10 μg/ml) for 3 days. The generation of CD8+ CTLs was determined by FCM using PE-conjugated H-2Kb/OVA257–264tetramer (B). The expression of IFN-γ was examined by FCM (C) and ELISA (D). The same lymphocytes were tested for CTL-mediated cytotoxicity against E.G7-OVA cells by a standard 6 h 51Cr release assay (E). (F) High concentrations of NaCl promote the maturation of DCs in vitro. Bone marrow-derived DCs isolated from C57BL/6 mice were cultured in medium with the indicated NaCl concentrations for 48 h and the expression of maturation markers was analyzed by FCM. (G) High concentrations of NaCl promote the antigen uptake of DCs in vitro. DCs were incubated with 2 μg/ml Alexa Fluor 488-labeled OVA for 1 h at 37 °C in medium with the indicated NaCl concentrations and analyzed under a fluorescent microscope. Scale bars, 20 μm. (H) High concentration of NaCl promotes secretion of the pro-inflammatory cytokines by DCs through the p38 MAPK pathway in vitro. DCs were pretreated with 10 μM SB203580 (SB) or DMSO (DM) for 2 h and then treated with 0.9% or 1.5% NaCl for 48 h in the presence of 5 μM SB or DM. The levels of pro-inflammatory cytokines in the supernatant were measured by ELISA. (I-J) High salt concentration induces antigen cross-presentation in DCs. DCs were cultured in medium with the indicated NaCl concentrations containing 10 μg/ml OVA. Then the stimulated DCs were co-cultured with CFSE-labeled CD8+ T cells from OT-I mice. The proliferation of T cells was assessed by FCM after 3 days of co-culture (I). DCs were treated with 2 μg/ml Alexa Fluor 488-labeled OVA in the presence of the indicated NaCl concentrations for 1 h, stained with rabbit anti-LMP2/Cy3 antibody and DAPI, and visualized under a confocal laser scanning microscope. Scale bars, 5 μm (J). (K) High-salt formulation potentiates the antitumor effect of OVA/Al vaccine in vivo. In a prophylactic model (left), C57BL/6 mice (n = 10 per group) were immunized with different vaccines for three times and then challenged subcutaneously with 3 × 106 E.G7-OVA cells 1 week after the third immunization. In a therapeutic model (right), mice (n = 7 per group) were treated by subcutaneous injection of different vaccines once a week for 3 weeks starting on day 3 after subcutaneously introduction of 3 × 106 E.G7-OVA cells. The tumor volume was measured every 3 days. (L) The high-salt formulation of OVA/Al diminished immune suppressive cells in tumor microenvironment. After the third treatment, a single-cell suspension of tumor tissues was prepared. The percentages of infiltrating CD8+ lymphocytes, MDSCs and M2 macrophages were analyzed by FCM.

Mentions: Here we developed a new formulation of aluminum hydroxide (Al(OH)3) adjuvant with a high salt concentration and utilized the OVA model antigen and the HBsAg antigen to evaluate its adjuvant effect. The serum ELISA results showed that the increase of the total IgG level reached a peak at 3.6% NaCl (Figure 1A). Compared with the adjuvant prepared in regular NaCl concentration (0.9%), the high-salt formulation of Al(OH)3 mildly improved OVA-induced production of IgG1-associated Th2 responses rather than IgG2a- and IgG2b-associated Th1 responses (Figure 1A). Similar results were also observed in the HBsAg model (Supplementary information, Figure S1A). These data suggest that the high-salt formulation of Al(OH)3 enhances humoral immunity by stimulating the Th2 response.


Simultaneous enhancement of cellular and humoral immunity by the high salt formulation of Al(OH) 3 adjuvant
Simultaneous enhancement of cellular and humoral immunity by the high-salt formulation of Al(OH)3 adjuvant. (A) High salt concentration enhances Al(OH)3 adjuvant-induced humoral immunity. C57BL/6 mice (n = 5 per group) were vaccinated subcutaneously three times with OVA/Al complex containing different concentrations of NaCl (5 μg OVA per mouse). Seven days after the third immunization, the mouse serum was collected and levels of the total IgG and IgG subclasses and IgG1 titer were determined by ELISA. (B-E) OVA/Al/high salt vaccine induces specific cellular immunity. C57BL/6 mice were immunized in the same way as described in A. Lymphocytes were isolated from the spleen and further incubated in vitro with CD8+-specific OVA257–264 peptides (10 μg/ml) for 3 days. The generation of CD8+ CTLs was determined by FCM using PE-conjugated H-2Kb/OVA257–264tetramer (B). The expression of IFN-γ was examined by FCM (C) and ELISA (D). The same lymphocytes were tested for CTL-mediated cytotoxicity against E.G7-OVA cells by a standard 6 h 51Cr release assay (E). (F) High concentrations of NaCl promote the maturation of DCs in vitro. Bone marrow-derived DCs isolated from C57BL/6 mice were cultured in medium with the indicated NaCl concentrations for 48 h and the expression of maturation markers was analyzed by FCM. (G) High concentrations of NaCl promote the antigen uptake of DCs in vitro. DCs were incubated with 2 μg/ml Alexa Fluor 488-labeled OVA for 1 h at 37 °C in medium with the indicated NaCl concentrations and analyzed under a fluorescent microscope. Scale bars, 20 μm. (H) High concentration of NaCl promotes secretion of the pro-inflammatory cytokines by DCs through the p38 MAPK pathway in vitro. DCs were pretreated with 10 μM SB203580 (SB) or DMSO (DM) for 2 h and then treated with 0.9% or 1.5% NaCl for 48 h in the presence of 5 μM SB or DM. The levels of pro-inflammatory cytokines in the supernatant were measured by ELISA. (I-J) High salt concentration induces antigen cross-presentation in DCs. DCs were cultured in medium with the indicated NaCl concentrations containing 10 μg/ml OVA. Then the stimulated DCs were co-cultured with CFSE-labeled CD8+ T cells from OT-I mice. The proliferation of T cells was assessed by FCM after 3 days of co-culture (I). DCs were treated with 2 μg/ml Alexa Fluor 488-labeled OVA in the presence of the indicated NaCl concentrations for 1 h, stained with rabbit anti-LMP2/Cy3 antibody and DAPI, and visualized under a confocal laser scanning microscope. Scale bars, 5 μm (J). (K) High-salt formulation potentiates the antitumor effect of OVA/Al vaccine in vivo. In a prophylactic model (left), C57BL/6 mice (n = 10 per group) were immunized with different vaccines for three times and then challenged subcutaneously with 3 × 106 E.G7-OVA cells 1 week after the third immunization. In a therapeutic model (right), mice (n = 7 per group) were treated by subcutaneous injection of different vaccines once a week for 3 weeks starting on day 3 after subcutaneously introduction of 3 × 106 E.G7-OVA cells. The tumor volume was measured every 3 days. (L) The high-salt formulation of OVA/Al diminished immune suppressive cells in tumor microenvironment. After the third treatment, a single-cell suspension of tumor tissues was prepared. The percentages of infiltrating CD8+ lymphocytes, MDSCs and M2 macrophages were analyzed by FCM.
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Related In: Results  -  Collection

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fig1: Simultaneous enhancement of cellular and humoral immunity by the high-salt formulation of Al(OH)3 adjuvant. (A) High salt concentration enhances Al(OH)3 adjuvant-induced humoral immunity. C57BL/6 mice (n = 5 per group) were vaccinated subcutaneously three times with OVA/Al complex containing different concentrations of NaCl (5 μg OVA per mouse). Seven days after the third immunization, the mouse serum was collected and levels of the total IgG and IgG subclasses and IgG1 titer were determined by ELISA. (B-E) OVA/Al/high salt vaccine induces specific cellular immunity. C57BL/6 mice were immunized in the same way as described in A. Lymphocytes were isolated from the spleen and further incubated in vitro with CD8+-specific OVA257–264 peptides (10 μg/ml) for 3 days. The generation of CD8+ CTLs was determined by FCM using PE-conjugated H-2Kb/OVA257–264tetramer (B). The expression of IFN-γ was examined by FCM (C) and ELISA (D). The same lymphocytes were tested for CTL-mediated cytotoxicity against E.G7-OVA cells by a standard 6 h 51Cr release assay (E). (F) High concentrations of NaCl promote the maturation of DCs in vitro. Bone marrow-derived DCs isolated from C57BL/6 mice were cultured in medium with the indicated NaCl concentrations for 48 h and the expression of maturation markers was analyzed by FCM. (G) High concentrations of NaCl promote the antigen uptake of DCs in vitro. DCs were incubated with 2 μg/ml Alexa Fluor 488-labeled OVA for 1 h at 37 °C in medium with the indicated NaCl concentrations and analyzed under a fluorescent microscope. Scale bars, 20 μm. (H) High concentration of NaCl promotes secretion of the pro-inflammatory cytokines by DCs through the p38 MAPK pathway in vitro. DCs were pretreated with 10 μM SB203580 (SB) or DMSO (DM) for 2 h and then treated with 0.9% or 1.5% NaCl for 48 h in the presence of 5 μM SB or DM. The levels of pro-inflammatory cytokines in the supernatant were measured by ELISA. (I-J) High salt concentration induces antigen cross-presentation in DCs. DCs were cultured in medium with the indicated NaCl concentrations containing 10 μg/ml OVA. Then the stimulated DCs were co-cultured with CFSE-labeled CD8+ T cells from OT-I mice. The proliferation of T cells was assessed by FCM after 3 days of co-culture (I). DCs were treated with 2 μg/ml Alexa Fluor 488-labeled OVA in the presence of the indicated NaCl concentrations for 1 h, stained with rabbit anti-LMP2/Cy3 antibody and DAPI, and visualized under a confocal laser scanning microscope. Scale bars, 5 μm (J). (K) High-salt formulation potentiates the antitumor effect of OVA/Al vaccine in vivo. In a prophylactic model (left), C57BL/6 mice (n = 10 per group) were immunized with different vaccines for three times and then challenged subcutaneously with 3 × 106 E.G7-OVA cells 1 week after the third immunization. In a therapeutic model (right), mice (n = 7 per group) were treated by subcutaneous injection of different vaccines once a week for 3 weeks starting on day 3 after subcutaneously introduction of 3 × 106 E.G7-OVA cells. The tumor volume was measured every 3 days. (L) The high-salt formulation of OVA/Al diminished immune suppressive cells in tumor microenvironment. After the third treatment, a single-cell suspension of tumor tissues was prepared. The percentages of infiltrating CD8+ lymphocytes, MDSCs and M2 macrophages were analyzed by FCM.
Mentions: Here we developed a new formulation of aluminum hydroxide (Al(OH)3) adjuvant with a high salt concentration and utilized the OVA model antigen and the HBsAg antigen to evaluate its adjuvant effect. The serum ELISA results showed that the increase of the total IgG level reached a peak at 3.6% NaCl (Figure 1A). Compared with the adjuvant prepared in regular NaCl concentration (0.9%), the high-salt formulation of Al(OH)3 mildly improved OVA-induced production of IgG1-associated Th2 responses rather than IgG2a- and IgG2b-associated Th1 responses (Figure 1A). Similar results were also observed in the HBsAg model (Supplementary information, Figure S1A). These data suggest that the high-salt formulation of Al(OH)3 enhances humoral immunity by stimulating the Th2 response.

View Article: PubMed Central - PubMed

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Adjuvants play essential roles in vaccination by enhancing and/or shaping antigen-specific immune responses... Moreover, cross-presentation of extracellular protein antigens by DCs contributes to the generation of cytotoxic T lymphocyte (CTL) responses... Sodium chloride (NaCl) is closely related to our daily lives and has recently been found to have potential effects on the development of autoimmune diseases and specific states of macrophages... Here, we used CD8 T cells from OT-I mice (specific for the H-2K/OVA complex) to test whether high salt-stimulated DCs can perform cross-presentation of soluble OVA... Next, we examined the in vivo antitumor effect of the OVA/Al/high salt vaccine using the E.G7-OVA tumor model in mice... In either the prophylactic or therapeutic vaccine model, immunization of the OVA/Al/high salt complex inhibited tumor growth more effectively compared with the other groups (Figure 1K)... The antitumor effect of the OVA/Al/3.6% NaCl vaccine was further studied in an adoptive cellular/serum therapy model... Lymphocytes from mice immunized with OVA/Al/3.6% NaCl effectively inhibited tumor growth, while the serum showed no apparent antitumor effect (Supplementary information, Figure S1M and S1N)... Only depletion of CD8 T lymphocytes and NK cells showed effective abrogation of the antitumor effect induced by the OVA/Al/3.6% NaCl vaccine (Supplementary information, Figure S1O)... All the mice were under good physiological conditions as shown by the monitoring of heart rate and mean arterial pressure, even when the NaCl concentration reached as high as 14.4% (16 times higher than the normal osmolality; Supplementary information, Table S1)... In conclusion, we discovered a novel and safe formulation (high salt formulation) of Al(OH)3 adjuvant, which, aside from maintaining the induction of humoral immune response by normal Al-adjuvant, could further enhance the cellular immune response; and this effect may be closely related to the activation of and antigen cross-presentation by DCs... Consequently, the OVA/Al/high salt formulation exhibited a significant antitumor effect against the E.G7-OVA tumor model in vivo, which might be largely due to CD8 CTL-mediated cellular immunity and independent of CD4 T cells... The most interesting and surprising finding of this study is that the adjuvant effects could be significantly improved simply by altering the NaCl concentration in vaccines and such formulation is safe, easy to prepare, and of low cost... This concept may assist in the design of broad vaccine formulations and the development of safer and more effective adjuvants for therapeutic uses.

No MeSH data available.


Related in: MedlinePlus