Results 3.1. B virus surface antigen and influenza subunit vaccine [10, 11]. CIA07 also exhibited potent immunostimulating activity, which suggests its potential as a cancer immunotherapy [8]. A combination of dLOS and aluminum hydroxide (designated as CIA06) was also evaluated for adjuvant effects against human papillomavirus (HPV) L1 virus-like particles (VLPs) and anthrax protective antigen (PA) [12C15]. The results indicated that CIA06 effectively increased antibody titers to both vaccines and that the induced antibodies were effective in neutralizing HPV pseudovirus and anthrax lethal toxin, respectively [13C15]. Further, the enhancement of IgG and toxin-neutralizing antibody titers allowed the dose of PA antigen required for immunization to be reduced, which suggested a potential antigen sparing effect [14]. The CIA06 adjuvant was most effective in inducing immune responses to HPV L1 VLPs at a ratio of 1 1?:?50 between dLOS and alum, as evidenced by serum antibody titers, splenic IFN-secretion, and antigen-specific memory B cell responses [13]. These results indicated that CIA06 is capable of inducing both Th1- and Th2-type immune responses that persist for an extended period. Subsequently, the safety and immunogenicity of the CIA06-adjuvanted HPV vaccine were confirmed in a human trial (unpublished data). In this study, we investigated the adjuvant activity of CIA06 on a commercial H1N1 pandemic influenza vaccine in mice. The results showed that CIA06 significantly increased the immunogenicity of the vaccine and enhanced the protection of mice against a lethal influenza virus challenge more than 20-fold, which demonstrated that the addition of CIA06 as an adjuvant could promote the protective efficacy of influenza vaccine and allow us to reduce influenza vaccine doses during an influenza pandemic. 2. Materials and Methods 2.1. Materials Influenza virus A/California/07/2009 (H1N1) and mouse-adapted A/California/04/2009 (H1N1) virus strains were obtained from the Korea Centers for Disease Control and Prevention (Seoul, Korea) and the International Vaccine Institute (Seoul, Korea), respectively. The viruses were cultured in the allantoic cavity of embryonated eggs, harvested, and stored at ?80C until analysis. Madin-Darby canine kidney (MDCK) cells were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA). Cell culture media and antibiotics were purchased from Welgene (Daegu, Korea), and fetal bovine serum (FBS) was purchased from Gibco/Invitrogen (Grand Island, NY, USA). Aluminum Centrinone-B hydroxide (alum, Alhydrogel?) was purchased from Superfos Biosector (Frederikssund, Denmark), whereas squalene-based oil-in-water adjuvant AddaVax? was obtained from InvivoGen (San Diego, CA, USA). Goat anti-mouse IgG antibody was obtained from Jackson Immuno Research Labs (West Grove, PA, USA) and Serotec (Kidlington, Oxford, UK), Centrinone-B whereas purified anti-mouse CD4 and CD8 antibodies were purchased from BioLegend (San Diego, CA, USA). Mouse anti-influenza A nucleoprotein (NP) monoclonal antibody (mAb) and FITC-conjugated rat anti-myeloperoxidase (MPO) antibody were obtained from Millipore (Billerica, MA, USA) and Abcam (Cambridge, UK), respectively. Recombinant mouse IL-2 was acquired from R&D Systems (Minneapolis, MN, USA), and IFN-and IL-5 cytokine ELISA kits were obtained from BD Biosciences (San Diego, CA, USA) and R&D Systems. 2.2. Influenza Vaccine and Adjuvants Greenflu-S (Green Cross, Yong In, Korea), the pandemic influenza A/California/07/2009 (H1N1) virus split vaccine, was used in this study. dLOS was prepared from anE. coliLPS mutant strain as previously described [9], quantified using the 2-keto-3-deoxyoctonate assay as previously described [16], and visualized on a silver-stained SDS-polyacrylamide gel. The adjuvant system CIA06 was prepared by mixing dLOS and alum in hSPRY2 a 1?:?50 ratio [13]. 2.3. Immunization of Mice Specific pathogen-free 6-week-old female BALB/c mice were purchased from SLC (Hamamatsu, Japan) and randomly assigned into experimental groups consisting of three to six mice. The animals Centrinone-B were immunized twice at a 2- or 3-week interval via intramuscular injection with Greenflu-S alone or in combination with alum (25?and IL-5 cytokine levels were measured by sandwich ELISA. Results are expressed as the means SD of values obtained from triplicate cultures that used two spleens each. To determine the contribution of CD4+ and CD8+ T cells to IFN-production, T cell coreceptors were blocked by incubating cells with 1?values of less than 0.05 were considered statistically significant. 3. Results 3.1. CIA06 Increased Serum Antibody Titers to Influenza Vaccine Greenflu-S The adjuvant effect of CIA06 on the influenza vaccine was evaluated using the pandemic influenza A/California/07/2009 (H1N1) virus split vaccine Greenflu-S. Mice were immunized with 0.2, 0.1, or 0.05?< 0.001), which eliminated the vaccine dose-dependency. The adjuvant activity of dLOS alone was similar to that of alum (data not shown). Open in a separate window Figure 1 Increased serum antibody.