The number of glycosylation sites present on HA determines the virus infection rates in DC-SIGN expressing cells.

<p>MDCK (A) and Vero (B) cells, transfected with the DC-SIGN gene (black bars) or not (white bars) were treated with neuraminidase from vibrio cholerae and GolgiStop for 30 minutes to remove sialic acids from the cell surface. These cells were subsequently inoculated with A/Netherlands/26/07, A/Netherlands/26/07-Δ125, A/Netherlands/602/09, A/Netherlands/602/09-Δ276 or A/Netherlands/602/09-VN54 N125 N160. The percentage of infected cells relative to the positive control (untreated cells still possessing sialic acid) was assessed after detecting infected cells using a FITC-labeled antibody to the viral nucleoprotein and flow-cytometry.</p

DC-SIGN expression in stably transfected MDCK DC-SIGN and Vero DC-SIGN cells.

<p>MDCK cells (A) and Vero cells (B) without (dotted line) and transfected with the gene encoding DC-SIGN (solid line) were analyzed for DC-SIGN expression after staining with a PE-labeled antibody to DC-SIGN and flow cytometry.</p

Schematic representation of the passage history and infection experiments with influenza viruses GFP-H1 and L194AY195F-GFP-H1.

<p>Schematic representation of the passage history and infection experiments with influenza viruses GFP-H1 and L194AY195F-GFP-H1.</p

Replication kinetics of viruses GFP-H1 and L194AY195F-GFP-H1 in MDCK and DC-SIGN-expressing MDCK cells.

<p>After transfection of 293T cells with reverse genetics plasmids, culture supernatants of influenza viruses GFP-H1 (A, C, E, G) and L194AY195F-GFP-H1 (B, D, F, H) virus passaged in MDCK (A–D) and MDCK-DC-SIGN (E–H) cells were obtained and used to inoculate MDCK (solid symbols) or MDCK DC-SIGN cells (open symbols) at a moi of 0.01. At the indicated time points post inoculation culture supernatant were tested for the infectious virus titers to determine the replication kinetics. Virus L194AY195F-GFP-H1 could not be rescued in MDCK cells.</p

Expression of DC-SIGN supports replication of influenza A viruses in the absence of sialic acids.

<p>MDCK (A and C) and Vero cells (B and D) transfected with the DC-SIGN gene (black bars) or not (white bars), were treated with neuraminidase from vibrio cholerae and GolgiStop for 30 minutes to remove sialic acids from the cell surface. These cells were subsequently inoculated with five different A/H1N1 viruses (A and B) and four A/H3N2 viruses (C and D). The percentage infected cells relative to the untreated control cells, still possessing sialic acid, was assessed after detecting infected cells using a FITC-labelled antibody to the viral nucleoprotein and flow-cytometry. To confirm that the entry was mediated via DC-SIGN, Vero and Vero DC-SIGN were treated with neuraminidase from vibrio cholerae for 30 minutes to remove sialic acids from the cell surface and incubated with or without antibodies to DC-SIGN or an isotype control antibody as indicated (E and F). These cells were subsequently inoculated with influenza viruses. NL/312/03 and USSR/90/77. The percentage of infected cells compared to the positive control (untreated cells, still possessing sialic acid) was assessed as described above.</p

DC-SIGN expression on DC supports replication of influenza virus in absence of sialic acids.

<p>DC were treated with neuraminidase from vibrio cholerae for 30 minutes to remove sialic acids from the cell surface and incubated with or without antibodies to DC-SIGN or an isotype control antibody as indicated. These cells were subsequently inoculated with two A/H3N2 influenza viruses. The percentage of infected cells compared to the positive control (untreated cells, still possessing of sialic acid) was assessed as described above.</p

Number of putative N linked glycosylation sites present in HA1 and in HA2 of the viruses used in this study.

<p>The software NetNGlyc (<a href="http://www.cbs.dtu.dk/services/NetNGlyc/" target="_blank">http://www.cbs.dtu.dk/services/NetNGlyc/</a>)was used to predict the number of glycosylation sites that will be utilized.</p

GFP expression after infection with L194AY195F-GFP and GFP-H1 in MDCK and DC-SIGN-expressing MDCK cells.

<p>MDCK and MDCK DC-SIGN cells were inoculated with influenza viruses GFP-H1 (A) and L194AY195F-GFP-H1 (B) at a MOI of 0.01 (solid lines). Both viruses were passaged in MDCK-DC-SIGN cells two or three times as indicated. Twenty-four hours post inoculation the cells were tested for GFP expression by flow cytometry. Uninfected cells were included as negative controls (dotted lines). Infection experiments with GFP-H1 virus passaged in MDCK cells essentially gave the same results as the virus passaged in MDCK DC-SIGN cells (data not shown).</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

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