<i>H</i>. <i>pylori</i> infection promotes the genes and proteins expression in gastric cancer tissues.

<p>(A) Western blot analysis of the indicated proteins in the control cells (1), <i>H</i>.<i>pylori</i>-infected SGC-7901 cells (2) and <i>cagA</i>-overexpressed SGC-7901 cells (3). GAPDH served as the loading control. The data are representative of three independent experiments. (B) Quantitative RT-PCR analysis of the indicated genes in 30 gastric cancer tissues. Values are represented as average Ct fold compared to peri-cancerous tissues, where peri-cancerous tissues were set to 1. (C) Western blot analysis of the indicated proteins in 30 gastric cancer tissues. 200 mg tissues were homogenized and total proteins were collected. A total of 50 μg protein extracts were subjected to SDS-PAGE gel electrophoresis. GAPDH served as the loading control. (D) Detection of the <i>H</i>. <i>pylori</i> 16S <i>rRNA</i> gene and <i>cagA</i> gene in gastric cancer tissues by PCR (Upper). M represents the DNA molecular weight marker. Lane 1 is the positive control. Lane 2 is the negative control. Lanes 3, 4 and 6 are positive samples. Lanes 5 are negative samples. Quantitative RT-PCR analysis of the indicated genes in gastric cancer tissues with and without <i>H</i>. <i>pylori</i> infection (Lower). Values are presented as the average Ct fold compared to the group without <i>H</i>. <i>pylori</i> infection, which was set to 1. The figure presents the average of 30 samples. Data are presented as the means ± SD. Error bars represent standard deviations. LDH, L-lactate dehydrogenase. DLD, Dihydrolipoamide dehydrogenase. PRPF19, pre-mRNA processing factor 19 homolog. RanGAP, Ran-specific GTPase-activating protein. CaM, calmodulin. p64 CLCP, nuclear chloride ion channel protein. *, <i>P</i><0.05 compared to peri-cancerous tissue (B and C) and tissues without <i>H</i>. <i>pylori</i> infection (D).</p

Introduction of CagA into gastric cancer cells.

<p>(A and B) Western blot analysis of CagA and phosphorylated CagA in <i>H</i>. <i>pylori</i>-infected SGC-7901(A) and AGS (B) cells. The cells infected with the indicated ratio of cells to <i>H</i>. <i>pylori</i> for the indicated time were collected and lysed, and the proteins were separated by SDS-PAGE. Cells infected with <i>H</i>. <i>pylori</i> boiled for 15 min at a MOI of 1:1000 were used as a control. (C and D) Detection of CagA mRNA and protein in <i>cagA</i>-overexpressing SGC-7901 cells by RT-PCR (C) and western blot (D). GAPDH served as the loading control. The data are representative of three independent experiments. <i>Hp</i>, <i>H</i>. <i>pylori</i>; P-CagA, phosphorylated CagA; GAPDH, Glyceraldehyde-3-phosphate- dehydrogenase.</p

Robust Immunity and Heterologous Protection against Influenza in Mice Elicited by a Novel Recombinant NP-M2e Fusion Protein Expressed in <em>E. coli</em>

<div><h3>Background</h3><p>The 23-amino acid extracellular domain of matrix 2 protein (M2e) and the internal nucleoprotein (NP) of influenza are highly conserved among viruses and thus are promising candidate antigens for the development of a universal influenza vaccine. Various M2e- or NP-based DNA or viral vector vaccines have been shown to have high immunogenicity; however, high cost, complicated immunization procedures, and vector-specific antibody responses have restricted their applications. Immunization with an NP–M2e fusion protein expressed in <em>Escherichia coli</em> may represent an alternative strategy for the development of a universal influenza vaccine.</p> <h3>Methodology/Principal Findings</h3><p>cDNA encoding M2e was fused to the 3′ end of NP cDNA from influenza virus A/Beijing/30/95 (H3N2). The fusion protein (NM2e) was expressed in E. coli and isolated with 90% purity. Mice were immunized with recombinant NM2e protein along with aluminum hydroxide gel and/or CpG as adjuvant. NM2e plus aluminum hydroxide gel almost completely protected the mice against a lethal (20 LD₅₀) challenge of heterologous influenza virus A/PR/8/34.</p> <h3>Conclusions/Significance</h3><p>The NM2e fusion protein expressed in <em>E. coli</em> was highly immunogenic in mice. Immunization with NM2e formulated with aluminum hydroxide gel protected mice against a lethal dose of a heterologous influenza virus. Vaccination with recombinant NM2e fusion protein is a promising strategy for the development of a universal influenza vaccine.</p> </div

NM2e protein immunization schedule.

<p>The indicated mice were immunized intramuscularly with NM2e protein with or without adjuvant, three times at 2-week intervals. Blood was collected on days 14, 28, and 38, respectively. The immunized mice were challenged with influenza A virus PR8 at 20-fold the LD₅₀ on day 38. Body weight and survival were monitored for 3 weeks, until day 59.</p

Summary of mouse groups immunized with NM2e.

<p>NS, normal saline.</p>a<p>mice in G1 and G2 were mock-immunized with NS instead of NM2e protein.</p>b<p>mice in G1 and G3 were immunized without adjuvant.</p

Protective efficacy of immunization with NM2e formulated with Al(OH)₃ and CpG in mice.

<p>Groups of 15 mice were immunized with NM2e protein or NM2e formulated with adjuvant and were challenged with 20 LD₅₀ of influenza virus PR8. Mice immunized with normal saline or adjuvant alone were challenged as negative controls. Mice were monitored daily for 21 days after PR8 challenge. Mice were weighed daily to detect morbidity (A). Average weights in each treatment group were followed for the duration of the study, and the percentage of the original body weight was calculated based on the average starting weight for each group at day 0. Survival rates (B) following the challenge within each experimental group were calculated. Tables above the graph compare the results for groups 3, 4, 5, and 6. *, <i>p</i>≤0.05; **, <i>p</i>≤0.01; ***, <i>p</i>≤0.001; ns, not significant.</p

Antibody response trend and long-term humoral immune response induced by NM2e protein in mice.

<p>(A and B) Mice were immunized intramuscularly with 10 µg of NM2e protein three times at 2-week intervals. Al(OH)₃ and/or CpG 1826 were used as adjuvants. Mice immunized with normal saline (NS) or adjuvant alone was used as negative controls. Serum was obtained from each mouse on days 14, 28, and 38, respectively, and analyzed for the presence of IgG antibodies specific for NP (left) or M2e (right), in an ELISA, as described in the Materials and Methods. Antibody response trends after three immunizations are presented in A, and the comparison of results on day 38 are presented in B. Columns show geometric mean antibody titers, and bars indicate the 95% confidence interval in each group. Plots in B show the NP- and M2e-specific IgG titers of all of the mice in each treatment group on day 38, and bars indicate the geometric mean antibody titers of each treatment group (<i>n</i> = 6 mice per experimental group, except <i>n</i> = 5 mice in the NS group). Lines above two or more groups indicate that they have the same comparative results. *, <i>p</i>≤0.05; **, <i>p</i>≤0.01; ***, <i>p</i>≤0.001 by one-way ANOVA. (C) Mice were immunized intramuscularly with 10 µg of NM2e protein formulated with Al(OH)₃ three times at 2-week intervals or immunized with a single dose of 10 µg of NM2e formulated with Al(OH)₃. Serum was prepared from each mouse at the indicated times, and NP- and M2e-specific IgG antibodies were analyzed by ELISA, as described in the Materials and Methods.</p

Representative 2-DE maps and magnified image of differential spots in three cell lines.

<p>SGC-7901 and AGS cells infected with <i>H</i>. <i>pylori</i> for 6 h at a MOI of 1:1000 (cell to <i>H</i>. <i>pylori</i>) and SGC-7901 cells transfected with pcDNA3.1/<i>cagA</i> for 48 h were collected and lysed, and the protein concentrations were determined using Bradford colorimetry. A total of 800 μg of protein was loaded for two-dimensional electrophoresis. Cells infected with boiled <i>H</i>. <i>pylori</i> or transfected with empty vector served as controls for the infected or transfected cells, respectively. (A) SGC-7901 cells infected with <i>H</i>. <i>pylori</i>. (B) AGS cells infected with <i>H</i>. <i>pylori</i>. (C) SGC-7901 cells transfected with the <i>cagA</i>-vector. (D) Magnified image of 10 differential spots. B4, B11, C6, C7, C8 and C9 spots were up-regulated, whereas D12, D16, E2 and E11 spots were down-regulated. These spots are identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146521#pone.0146521.t003" target="_blank">Table 3</a>.</p

Correlations between survival percentage and immune responses in mice.

<p>A, Correlation analysis was conducted to determine the relationships of the survival percentage data from Fig. 6 with the NP-, M2e-specific IgG (left) ELISA data from Fig. 3, IgG1 (middle) and IgG2a (right) ELISA data in Fig. 4. Log conversion was performed for the murine serum antibody titers. B, Correlation analysis was conducted to determine the relationships of the survival percentage data in Fig. 6 with the IFN-γ- (left) IL-4- (middle), and IL-10-secreting (right) SMNCs stimulated with NP147-155, NP55-69, or M2e peptide pool based on the ELISPOT data in Fig. 5.</p