Display Settings:

Items per page
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 8

1.
Figure 2

Figure 2. NM2e protein immunization schedule.. From: Robust Immunity and Heterologous Protection against Influenza in Mice Elicited by a Novel Recombinant NP-M2e Fusion Protein Expressed in E. coli.

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.

Wenling Wang, et al. PLoS One. 2012;7(12):e52488.
2.
Figure 8

Figure 8. Correlations between survival percentage and immune responses in mice.. From: Robust Immunity and Heterologous Protection against Influenza in Mice Elicited by a Novel Recombinant NP-M2e Fusion Protein Expressed in E. coli.

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.

Wenling Wang, et al. PLoS One. 2012;7(12):e52488.
3.
Figure 4

Figure 4. IgG1 and IgG2a isotypes in serum from NM2e-immunized mice.. From: Robust Immunity and Heterologous Protection against Influenza in Mice Elicited by a Novel Recombinant NP-M2e Fusion Protein Expressed in E. coli.

Mice were treated as described in Fig. 3. NP- and M2e-specific IgG isotypes in mouse serum were analyzed by ELISA. The plots show the (A) NP- (left) and M2e-specific (right) IgG1 isotypes and (B) the respective IgG2a isotypes. The scatter dot plots show the results for every mouse in each group, and the bars show the geometric mean of each group. The plots in (C) present the NP-(left) and M2e-specific (right) IgG1/IgG2a ratios, and the bars show the means with SD. Lines above two or more groups indicate that they have the same comparative results. *, p≤0.05; **, p≤0.01; ***, p≤0.001 by one-way ANOVA.

Wenling Wang, et al. PLoS One. 2012;7(12):e52488.
4.
Figure 6

Figure 6. Protective efficacy of immunization with NM2e formulated with Al(OH)3 and CpG in mice.. From: Robust Immunity and Heterologous Protection against Influenza in Mice Elicited by a Novel Recombinant NP-M2e Fusion Protein Expressed in E. coli.

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. *, p≤0.05; **, p≤0.01; ***, p≤0.001; ns, not significant.

Wenling Wang, et al. PLoS One. 2012;7(12):e52488.
5.
Figure 5

Figure 5. Cellular immune response in NM2e-immunized mice.. From: Robust Immunity and Heterologous Protection against Influenza in Mice Elicited by a Novel Recombinant NP-M2e Fusion Protein Expressed in E. coli.

SMNCs secreting IFN-γ, IL-4, or IL-10 upon stimulation were detected by ELISPOT assay. Groups of six mice were immunized intramuscularly three times at 2-week intervals using 10 µg of NM2e protein in normal saline (NS) or 10 µg of NM2e formulated with Al(OH)3, CpG 1826, or Al(OH)3 plus CpG 1826. Mice immunized with NS or Al(OH)3 plus CpG 1826 were treated as negative controls. All mice in each treatment group were sacrificed on day 38. SMNCs were separated from mouse spleen samples, and 5 µg/ml NP147–155, NP55–69, and M2e peptide pool were used as stimulants in the ELISPOT assays. The numbers of SMNCs producing IFN-γ (A), IL-4 (B), or IL-10 (C) after stimulation for 40 h with NP147–155 (left), NP55–69 (middle), or M2e peptides (right) are presented as spot-forming cells (SFCs)/106 SMNCs. Columns show the average SFCs/106 SMNCs, and bars indicate the standard deviation of each group. Lines above two or more groups indicate that they have the same comparative results. *, p≤0.05; **, p≤0.01; ***, p≤0.001 by one-way ANOVA.

Wenling Wang, et al. PLoS One. 2012;7(12):e52488.
6.
Figure 1

Figure 1. Construction, expression, and characterization of NM2e fusion protein.. From: Robust Immunity and Heterologous Protection against Influenza in Mice Elicited by a Novel Recombinant NP-M2e Fusion Protein Expressed in E. coli.

(A) Schematic of influenza A virus NP and M2, and diagram for the construction of recombinant NM2e fusion protein. The entire sequence of NM2e is shown. The cDNA sequences encoding residues 1–498 of NP and residues 2–24 of M2 (extracellular domain of M2, M2e) from influenza A virus A/Beijing/30/95 (H3N2) were fused directly, with no linker, and this was cloned as pET30a-NM2e for expression in Escherichia coli. (B) Protein profile of cell lysates from induction experiments in E. coli BL21 (DE3) transformed with pET30a-NM2e at 25 and 37°C. Lane 1, Mid-range protein molecular weight marker; lane 2, whole-cell lysate of transformed E. coli before induction; lanes 3–5, whole-cell lysate, soluble supernatant, and insoluble fraction after 4-h induction with 0.1 mM IPTG at 25°C; lanes 6–8, whole-cell lysate, soluble supernatant, and insoluble fraction after 4-h induction with 0.1 mM IPTG at 37°C; lane 9, whole-cell lysate after 4 h of cultivation without IPTG at 37°C. The arrow indicates the 58-kDa band corresponding to NM2e. (C) SDS-PAGE (left) showing the purified NM2e fusion protein, NP of influenza A virus A/Beijing/30/95(H3N2), and lysates of E. coli transformed with pET30a(+). NM2e fusion protein was detected on Western blots probed with NP-immunized mouse serum (middle) and mouse anti-M2e monoclonal antibody (right). Lanes 1–3, purified influenza A virus NP, recombinant NM2e fusion protein expressed in E. coli, cell lysates of E. coli transformed with pET30a(+). M, protein molecular weight marker.

Wenling Wang, et al. PLoS One. 2012;7(12):e52488.
7.
Figure 7

Figure 7. Comparison of the immunogenicity and protection efficacy induced by NM2e and NP.. From: Robust Immunity and Heterologous Protection against Influenza in Mice Elicited by a Novel Recombinant NP-M2e Fusion Protein Expressed in E. coli.

Mice were immunized intramuscularly with 10 µg of NM2e (g2)or NP protein (g3) formulated with Al(OH)3 according to the time schedule in Fig. 2. Mice immunized with adjuvant alone were used as negative controls (g1). Serum, SMNC was prepared from each mouse and analyzed at the indicated times as described in the Materials and Methods. A), Ab and subtypes against NP (left) and M2e (right) on day 38. Columns show geometric mean antibody titers, and bars indicate the 95% confidence interval in each group (n = 6 mice per experimental group). B), SMNCs secreting IFN-γ, IL-4, or IL-10 upon stimulation were detected by ELISPOT assay. Six mice in each treatment group were sacrificed on day 38. The numbers of SMNCs producing IFN-γ (left), IL-4 (middle), or IL-10 (right) after stimulation for 40 h with NP147–155, NP55–69, or M2e peptides are presented as spot-forming cells (SFCs)/106 SMNCs. Columns show the average SFCs/106 SMNCs, and bars indicate the standard deviation of each group. *, p≤0.05; **, p≤0.01; ***, p≤0.001 by one-way ANOVA. C), Protective efficacy of immunization with NM2e or NP formulated with Al(OH)3 in mice. Three mice group were challenged with 20 LD50 of influenza virus PR8 (n = 15). Mice were monitored daily to detect morbidity (left) and mortality (right). *, p≤0.05; **, p≤0.01; ***, p≤0.001.

Wenling Wang, et al. PLoS One. 2012;7(12):e52488.
8.
Figure 3

Figure 3. Antibody response trend and long-term humoral immune response induced by NM2e protein in mice.. From: Robust Immunity and Heterologous Protection against Influenza in Mice Elicited by a Novel Recombinant NP-M2e Fusion Protein Expressed in E. coli.

(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 (n = 6 mice per experimental group, except n = 5 mice in the NS group). Lines above two or more groups indicate that they have the same comparative results. *, p≤0.05; **, p≤0.01; ***, p≤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.

Wenling Wang, et al. PLoS One. 2012;7(12):e52488.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk