Outcome of infection with IAV IND/05 (H5N1).

<p>Mice were inoculated with IAV IND/05 (groups 2 (▴), 3 (○), 4 (▿), 5 (⋄), 6 (▪) and 7 (×)) or PBS (group 1 (). (A) Body weight after infection was determined daily and expressed as the percentage of the original body weight before infection. (B) Survival rates after infection with IAV IND/05. The proportion of mice from the indicated groups that survived infection is shown in a Kaplan-Meier plot. Moribund animals were euthanized when they reached pre-fixed criteria regarding weight loss (>20%) and disease severity score, which was used to determine mortality rates. (C) Lung virus titers measured on 7 days p.i. in mice from the indicated groups. Horizontal bars represent the average of 2–6 mice. The difference in virus titers between mice of group 2 and group 3 was statistically significant (p = 0.025). N.S.: not significant. (D) Virus-specific CD8+ T cell responses on day 7 p.i.. The frequency of CD3+ CD8+ splenocytes specific for peptide NP_366–374 and PA_224–233 derived from IAV IND/05 was determined by intracellular IFN-γ staining. The horizontal bars represent the average frequency of IFN-γ+ cells in the CD8+ T cell population of 2–7 mice in the indicated groups. Differences between group 2 and group 3 were statistically significant for both peptides.</p

Induction of serum antibodies against IAV HK/68 (H3N2) by vaccination.

<p>Serum antibody levels were determined before and at the indicated time points after vaccination of mice with PBS (groups 1, 3 and 4; ○), subunit vaccine with alum (groups 2 and 5; ▴), subunit vaccine only (group 6; ▪) and alum only (group 7; ×) by HI assay (A) and VN assay (B).</p

Outcome of infection with IAV HK/68 (H3N2).

<p>Mice were inoculated with IAV HK/68 (groups 2 (▴), 3 (○), 6 (▪) and 7 (×)) or PBS (groups 1 (), 4 (▿) and 5 (⋄)). (A) Body weight after infection was determined daily and expressed as the percentage of the original body weight before infection. (B) Lung virus titers measured on day 4 p.i. in mice from the indicated experimental groups. Horizontal bars represent the average titers of five mice. The dotted line represents the cut-off value for obtaining a positive result. *This mouse from group 6 had before infection an HI antibody titer of 40. (C) Vaccination prevented the induction of iBALT after infection. Twenty-eight days post infection with IAV HK/68 iBALT was detected in mice from group 3, but not in mice from group 2. Lung tissue sections were stained with HE. (D) Virus-specific CD8+ T cell responses detected 28 days post infection. Splenocytes of mice from the indicated experimental groups were tested for the presence of CD8+ T cells that bound the H2-Db NP_HK Tetramer. Horizontal bars represent the average of 2–4 mice. The difference in %CD8+ Tm+ T cells between groups 2 and 3 was statistically significant (<i>P</i> = 0.030).</p

Experimental groups and design of the study.

<p>Mice were divided over seven groups and were either vaccinated twice with subunit vaccine with or without adjuvant (Alum), PBS, or adjuvant only as indicated. Four weeks after the second vaccination, mice were infected with IAV HK/68 (H3N2) or mock-infected. Twenty-nine days after the infection with IAV HK/68, mice were challenged with IAV IND/05 (H5N1).</p

Histopathological analysis and immunohistochemistry of the lungs of mice infected with IAV IND/05.

<p>Mouse lung sections were stained for influenza A virus nucleoprotein. Cytoplasm of infected cells stain red, the nuclei of infected cells stain deep red. In the groups without a history of productive A/H3N2 infection, including group 2 (A,B), infection with IAV IND/05 led to severe histopathological changes and to viral antigen expression in cells of the bronchiolar walls and in the alveoli (group 4: E,F and group 5: G,H). In mice of groups 3 (C,D) and 7 (I,J) that had experienced a productive infection with IAV HK/68 only moderate histopathological changes were observed and virus infected cells were detected sporadically (see insert in panel D). For more information please see text.</p

MVA-HA-VN/04 vaccination reduces histopathological changes induced by infection.

<p>Histopathological changes and the detection of virus-infected cells by immunohistochemistry in the lungs 4 days after infection with influenza A/H5N1 viruses A/VN/1194/04 and A/IND/5/05. Representative pictures were selected for the different classifications. Magnification: overview (10×), bronchiole (20×), alveoli (40×).</p

MVA-HA-VN/04 vaccination reduces virus replication in the lung.

<p>The data represent lung virus titers on day 4 post infection with influenza virus A/Vietnam/1194/04 and A/Indonesia/5/05 in mice that received one (A, B) or two (C, D) immunization(s) of: PBS, wtMVA or 10³, 10⁴, 10⁵, 10⁶ or 10⁸ (single shot only) pfu of MVA-HA-VN/04 as indicated. (* indicates a statistical significant difference with the PBS immunized group (p<0.05) (** indicates a significant difference with both the PBS and wtMVA immunized group (p<0.05)(*** indicates a statistical significant difference with the wtMVA immunized group).</p

Induction of antibodies to A/VN/1194/04 and A/IND/5/05 after immunization with MVA-HA-VN/04.

(1)<p>titers are expressed as GMT (Standard deviation).</p

MVA-HA-VN/04 vaccination prevents loss of body weight caused by infection.

<p>The bodyweight after infection with influenza virus A/Vietnam/1194/04 (A, C) and influenza virus A/Indonesia/5/05 (B, D) is shown relative to the body weight before infection(100%). Animals were infected four weeks after a single immunization (A, B) with: PBS, wtMVA, 10³, 10⁴, 10⁵, 10⁶ or 10⁸ pfu of MVA-HA-VN/04. A second group of animals was infected four weeks after two immunizations (C, D) with PBS, wtMVA, 10³, 10⁴, 10⁵ or 10⁶ pfu of MVA-HA-VN/04. (*Indicates weight loss of the proportion of animals in this group that survived post day 6 infection).</p

Survival after infection with A/VN/1194/04 or A/IND/5/05.

(1)<p>Dose of MVA-HA-VN/04 in pfu (immunization in a total volume of 100 µl).</p>(2)<p>One animal had to be euthanized before infection due to a complication unrelated to the experiment.</p