Protective role of immune sera.

<p>Naive sera from unimmunized mice or immune sera from vaccinated mice (10 µg VLP single dose) were serially diluted (1X, 50X, 100X, 500X, and 2500X or naïve sera). These diluted serum samples (20 µl) were mixed with 40 µl of A/California/04/2009 virus (10 LD₅₀) and incubated for 30 min at 30°C. Mice (n = 4 BALB/c mice per each diluted serum-virus group) were intranasally infected with an in vitro incubated mixture of naïve or immune sera and A/California/04/2009 virus (10 LD₅₀), and monitored daily for 14 days for body weight changes (A, B). Survival rates (C). The numbers in the parenthesis indicate survival rates in each infected group.</p

Antibody secreting cells (ASC).

<p>Antibody secreting cells (ASC) from spleen and bone marrow. A: Mouse spleen monolayer cells were prepared at day 4 post challenge. ASC were determined after 2 or 6 days of in vitro culture specific to A/California/04/2009 virus. B: Antibody secreting cells (ASC) from bone marrow. Cells from mouse bone marrow at day 4 post challenge were prepared in vitro. ASC for IgG was determined after cultured in vitro for 2 or 6 days to A/California/04/2009 virus.</p

IgG antibody responses with low dose (0.1 µg) of VLPs.

<p>IgG serum antibodies specific to A/California/04/2009 influenza virus were determined at week 1, 3, 5 in the group of mice immunized with low dose 1 µg of VLPs. Titers are expressed as the highest dilution of serum having a mean optical density at 450 nm greater than the mean plus 2 standard deviations of naive serum samples. Significant higher IgG titers against new H1N1 were detected at week 3 compared to week 1 (P<0.01); and at week 5 compared to week 3 (P<0.001).</p

Protection of mice from lethal influenza virus challenge.

<p>A–B: Protection against A/California/04/2009 virus challenge. Mice intramuscularly immunized with a single dose of VLPs (10 µg) were challenged with a lethal dose (100 LD₅₀) of A/California/04/2009 virus at week 6 post immunization. Mice (n = 12) were monitored daily for 14 days for body weight changes (A) and survival rates (B). C–D: Protection against the antigenically distant A/PR8/1934 virus (10 LD₅₀). Body weight changes (C) and survival rates (D) are shown.</p

Lung virus titer and inflammatory cytokine IFN-gamma.

<p>(A) Lung virus titers. Lung samples from individual mice immunized with 10 µg VLPs in each group (n = 6) were collected on day 4 post-challenge with a lethal dose of A/California/04/2009 or A/PR8/1934 virus. Each lung sample from a mouse was suspended in 1 ml with Dulbecco's modified Eagle's medium. Statistical significance is indicated between groups of mice challenged with A/California/04/2009 (P<0.001) or A/PR8/34 (P<0.01) compared to naive mice challenged with the same lethal dose. (B) Lung inflammatory cytokine IFN-γ after A/California/04/2009 challenge. (C) Lung inflammatory cytokine IFN-gamma after A/PR8/1934 challenge. H1N1 Cha, VLP immunized mice after A/California/04/2009 challenge, N+H1N1 Cha: Naïve mice after A/California/04/2009 challenge, PR8 Cha: VLP immunized mice after A/PR8/1934 challenge, N+PR8 cha: Naive mice after A/PR8/1934 challenge. Naïve: Untreated mice.</p

Silver stained SDS-PAGE, western blot and electron microscopy examination.

<p>(A) Silver stained gel showing HA and M1 bands in A/California/04/2009 H1 VLPs. M: a standard molecular size marker, Lane 1∶2.5 µg of purified influenza VLP protein, Lane 2∶1 µg of purified influenza VLP protein. (B) The incorporation of A/California/04//2009 H1N1 influenza HA or M1 into VLPs (10, 2, and 0.4 µg of total protein) was determined by Western blot using mouse anti-2009 H1N1 sera or anti-M1 IgG antibody. (C) Cleavage of A/California/04/2009 virus HA in VLPs. VLPs containing HA (10 µg of total protein) were incubated for 5 min at 37°C with different concentrations of TPCK treated trypsin, resolved by SDS-PAGE, and probed by Western blotting. The thicker bands of the HA2 subunit are commonly observed after trypsin treatment due to the more effective transfer of HA2 during western blot. Lanes from left to right represent 0, 0.5, 2.5 and 10 µg/ml trypsin respectively. (D) Electron microscopy of influenza H1N1 VLPs.</p

Recall antibody responses in lung and serum.

<p>Lung IgG (A) and IgA (B), and serum IgG (C) and IgA (D) antibody responses to A/California/04/2009 virus were determined before (week 6.5 post immunization) and after challenge (day 4 post challenge) with the homologous virus A/California/04/2009. Lung and serum samples before and after challenge were collected at the same time (n = 6) and analyzed under the same assay condition (week 6.5 post-vaccination). Lung IgA (B) before and after challenge: P<0.05. Serum IgG (C) and IgA (D) responses before and after challenge from the A/California/04/2009 virus challenge: P<0.001. Low and moderate naïve backgrounds were observed in the serum and lung samples respectively and these values have been subtracted from the immune samples.</p

Humoral responses.

<p>A–B: IgG serum antibodies specific to A/California/04/2009 (A) or A/PR8/34 (B) H1N1 influenza virus were determined at week 1, 3, 5 in the group of mice that were intramuscularly immunized with 10 µg of VLPs. Titers are expressed as the highest dilution of serum having a mean optical density at 450 nm greater than the mean plus 2 standard deviations above naive serum samples. Significantly higher IgG titers against A/California/04/2009 or PR8 viruses were detected at week 3 compared to week 1 (P<0.01); and at week 5 compared to week 3 (P<0.001). C–D: IgG2a and IgG1 responses. Serum was serially diluted and ELISA was performed for serum antibodies specific to A/California/04/2009 (C) or PR8 viruses (D). E–F: HAI titers. HAI titers against A/California/04/2009 (E) or A/PR8/34 (F) viruses at week 0, 1, 3 and 5 after a single immunization were determined. A/California/04/2009 infected sera at 5 weeks after infection were used as control. Significant HAI titers against A/California/04/2009 viruses were determined at week 5 compared to week 3 or week 1 (P<0.01). Significant HAI titers against A/PR8 viruses were also determined at week 5 compared to week 1 (P<0.01).</p

Protection of mice immunized with a low dose of VLPs.

<p>Mice were intramuscularly immunized with 0.1 µg of VLPs once or twice, and were challenged with a lethal dose of A/California/04/2009 (10 LD₅₀) (n = 6) day 10 post immunization. Mice were observed daily to monitor changes in body weight and to record mortality (25% loss in body weight as the IACUC endpoint).</p

Changes in GSH/GSSG redox potential (E_hGSSG) in Tg and WT following H1N1 influenza virus infection.

<p>The same lung samples described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018918#pone-0018918-g005" target="_blank">Fig. 5</a> were examined for GSH and GSSG concentrations by HPLC, and E_hGSSG was calculated using the Nernst equation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018918#pone.0018918-Jones2" target="_blank">[60]</a>. No differences in concentrations or E_hGSSG oxidation were observed in lung tissues of Tg or WT following infection, but more oxidation of plasma E_hGSSG was observed in Tg compared to WT. Three mice for each Tg and WT without infection were analyzed as control (0 in A). Plasma GSH, GSSG, and E_hGSSG in Tg and WT before and 3 d post infection are shown in B. * p<0.05.</p