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

GP42-H1 vector induces expression of HA in infected cells <i>in vitro</i>.

<p>A) CV-1 cells were cultured on glass slides and infected with the indicated viruses (MOI = 10). At 20 hours post-infection, the infected cells were fixed in acetone and evaluated for HA expression by staining with mouse PR/8 anti-sera followed by TRITC conjugated anti-mouse IgG. Slides were mounted with Prolong-Antifade containing DAPI stain and visualized by immunofluorescence microscopy. B) PR/8, GP42-GFP, GP42-H1 or mock infected CV-1 cells were stained with the mouse PR/8 anti-sera. PR/8 infected cell were also stained with pre-immune sera (grey filled histogram) as a control for binding of nonspecific serum proteins. Surface bound mouse antibodies were detected with anti-mouse IgG-PE and analyzed by flow cytometry. C) Eggs were inoculated with PR/8 virus (P), GP42-GFP (G) or GP42-H1 (H) at 1 HA unit per egg. Sucrose purified recombinant viruses and allantoic fluid from PR/8 infected eggs were resolved on SDS-PAGE. SDS-PAGE gel was stained with gel-code blue (left), anti-GFP western blot (middle) and anti-PR8 western blot (right).</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

NL/602 virus exhibits more rapid clearance from the lungs than CA/07 virus.

<p>Balb/c mice were infected with 800 PFU of CA/07 or NL/602 virus (i.n.) and lungs were harvested at the indicated times (A). Infectious viruses from lung homogenates were evaluated by plaque titration on MDCK monolayers (B). Viral RNA in the lungs was quantitated by viral M gene specific Real-time PCR. * P<0.05 indicates time had a significant effect on virus titers, **P<0.01 Infectious particles and viral transcripts were not detected at day 0 (N/A).</p

Closely Related Influenza Viruses Induce Contrasting Respiratory Tract Immunopathology

<div><p>The swine-origin H1N1 virus which emerged in 2009 resulted in the first influenza pandemic of the 21^st century. Although the majority of infections were moderate, a significant proportion of infections were severe and characterized by acute respiratory distress syndrome and pulmonary edema. We compared two isolates from the 2009 H1N1 pandemic; A/California/07/09 (CA/07) and A/Netherlands/602/09 (NL/602) viruses that share greater than 99% sequence identity. Though genetically similar, these viruses exhibit contrasting pathological effects. Mice that were infected with 800 plaque forming unit (PFU) of CA/07 virus rapidly lost weight, which was concurrent with detection of high pulmonary concentrations of MCP-1, MIG, IP-10 and TIMP-1. Initially, severe bronchiolar epithelial necrosis and acute respiratory distress was observed, followed by marked bronchiolar epithelial hyperplasia. Mononuclear cell infiltration was initially localized to perivascular and peribronchiolar interstitium and then spread to adjacent alveoli. Infiltrating cells were phenotypically CD11b^hi, F4/80^lo. In contrast, when mice were infected with 800 PFU of NL/602 virus, minimal weight loss was observed, and concentrations of cytokines in the lung were significantly lower. Inflammation was primarily restricted to the bronchioles and perivascular interstitium with minimal spread to alveoli. Infiltrating cells include foamy macrophages and surface markers were characterized as CD11b^lo/-, F4/80^hi. These two genetically similar viruses can be useful strains with which to investigate immune-regulatory determinants of pathogenesis of influenza virus.</p> </div

CA/07 and NL/602 induced contrasting peribronchiolar and perivascular inflammation and hyaline membrane formation.

<p>Lungs from naïve (A), NL/602 (B-C), and CA/07 (D-F) infected mice were collected 4 days (B and E) and 6 day post infection (D, C, and F) and stained by H&E (A-C, E-F). At 6 days post infection, hyaline membranes were present in the lungs of CA/07 infect mice (D) as indicated by PAS stain (arrow head). Inflammation of alveolar spaces was observed following infection with CA/07 virus (arrow). Shown are representative lungs from of each group. N=12.</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

GP42-H1 protects mice from lethal challenge with PR/8 virus.

<p>Mice that were administered AF, infected with 1LD₅₀ of PR/8 virus or immunized with 1,000 pfu of GP42-GFP, or 1,000 pfu GP42-H1 (i.n.) were challenged (seven weeks post-immunization) with 5LD₅₀ of PR/8 virus (i.n.) A) Body weight was recorded over 14 days and the percent of the body weight at the time of challenge is shown. B) Animals were monitored for 14 days post challenge and morbidity is recorded. Depicted are the percentages of mice that survived the virus challenge.</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

Profile of pulmonary cytokines at two days post-inoculation with influenza viruses.

<p>BAL was obtained two days post inoculation with the indicated viruses and levels of cytokines were compared to BAL from naïve animals. *N.D. cytokines were below the threshold of detection and not detected. † Values indicate integrated density values ± standard error of the means and obtained using FluorChem FCS2 software. Two-way ANOVA with Bonferroni post-tests were performed to evaluate differences in the levels of cytokines from BAL of CA/07 and NL/602 at two days post-infection. ** P-value < 0.01 ***P-value < 0.001.</p