Viral recovery in lung tissue of mice challenged with pH1N1.

<p>^aGroups of 13 mice were intramuscularly immunized as described in Section 2.6.</p><p>^bMice were intranasally challenged with a 10x LD₅₀ dose of pH1N1 on 7 days post immunization. Mice were sacrificed and the lung samples in PBS group and in the rest of groups were collectedexamined on day 6 and 7, respectively.</p><p>Viral titers in lung homogenates were determined as described in Section 2.8.</p><p>Virus titers were determined by infection of MDCK cells, and are expressed as log₁₀ TCID₅₀/ml. Data were presented as means ± SD of titers of samples. The number of mice that shed virus is indicated in parentheses (number of mice shedding virus/number of mice tested).</p><p>Viral recovery in lung tissue of mice challenged with pH1N1.</p

Analysis of IFN-γ production in mice immunized with killed virus vaccine together with AcHERV-GmCSF.

<p>The number of IFN-γ spots from pH1N1-specific T cells in splenocytes (3/16) 2 weeks post immunization were analyzed using an ELISPOT assay. Statistical analysis showed that data were significant with <i>p</i><0.05 (one way ANOVA).</p

Humoral immune responses in mice immunized with killed vaccine alone and together with AcHERV-GmCSF.

<p>Sera from mice injected intramuscularly with PBS, AcHERV-GmCSF, killed vaccine alone, or killed vaccine together with AcHERV-GmCSF were collected 2 weeks after immunization and evaluated for humoral immune response. (A) Antigen-specific IgG antibody titers against pH1N1 (8 HAU) in mouse sera were determined by ELISA. (B) HAI titer in mouse sera. ELISA and HAI assays were performed using eight randomly selected samples from each group (8/16). Statistical analysis showed that data were significant with <i>p</i><0.05 (one way ANOVA).</p

Hematological analysis of changes in white blood cell composition.

<p>Two samples (2/2) of blood from mice immunized with AcMNPV or AcHERV-GmCSF, or control mice injected with PBS, were collected from the jugular vein at 5-day intervals, and hematological analyses were performed. Neutrophils, dark gray; lymphocytes, dotted gray; monocytes, black; eosinophils, gray. Data were presented as mean percentage of leukocyte ± SD and pie graphs are presented mean percentage of leukocyte.</p

Determination of an effective dose of virus for killed vaccine.

<p>A dose of killed vaccine that was effective when used in conjunction with GmCSF was determined by intramuscularly injecting BALB/c mice with 2 μg of killed vaccine (positive control), serially diluted (1–0.1 μg) killed vaccine together with AcHERV-GmCSF (1×10⁷ FFU), or AcHERV-GmCSF (1×10⁷ FFU) alone. (A) Antigen-specific IgG antibody titers against pH1N1 (8 HAU) in mouse sera were determined by ELISA (3/3). (B) HAI response in mouse sera (3/3). Statistical analysis showed that data were not significant with <i>p</i>>0.05 (one way ANOVA).</p

Histological lesions in lung sections from immunized mice after challenge with pH1N1.

<p>Separated mice (n = 4 mice/group) from each group were sacrificed 6 or 7 days post challenge, and their lungs were HE-stained for histological evaluation. (A) Non-infected BABL/c mice; (B) mice injected with PBS; (C) mice injected with AcHERV-GmCSF; (D) mice vaccinated with 0.2 μg of killed vaccine; (E) mice vaccinated with 0.2 μg of killed vaccine together with AcHERV-GmCSF; and (F) mice vaccinated with 2 μg of killed vaccine. Arrows indicated the infiltration of inflammatory cells, including the infiltration in the vessels, in the pulmonary parenchyma, and in the alveolar septa. Scale bar, 100 μm.</p

Diagram of the recombinant baculovirus, AcHERV-GmCSF, and its expression in mammalian cells.

<p>(A) Diagram of Bacmid DNA of AcHERV-GmCSF containing HERV envelope and GmCSF genes under the transcriptional control of the AcMNPV PolH and hEF1α promoters, respectively. (B) Detection of mRNAs for GmCSF and 18s rRNA in baculovirus-infected 293TT cells by RT-PCR. Lane 1: Control for RT-PCR; lane 2: mock infection with AcMNPV in 293TT cells; lane 3: AcHERV-GmCSF-infected 293TT cells. (C) Quantification of GmCSF expression in baculovirus-infected 293TT cell lysates and supernatants by ELISA. NTC, not treated control; Mock, AcMNPV baculovirus-infected cells; AcHERV-GmCSF, AcHERV-GmCSF-infected cells. (D) Fluorescence micrograph of baculovirus-infected 293TT cells. Seventy-two hours after infection, the cells were incubated with a monoclonal mouse antibody against GmCSF followed by incubation with a FITC-conjugated goat anti-mouse IgG antibody. Mock, AcMNPV-infected cells; AcHERV-GmCSF, AcHERV-GmCSF-infected cells; Merge, merged image.</p