Correlation of survival of passively immunized mice with neutralizing antibody titer following challenge with wild-type H5N1 virus.

<p>Data points represent the mean % of surviving animals 14 days following lethal challenge of CD1 mice which had received immune sera from (<b>A</b>) CD1 mice, (<b>B</b>) guinea pigs or (<b>C</b>) humans. Reciprocal neutralizing antibody titers shown are extrapolated from titers measured 2 h prior to challenge. All individual mice receiving mouse immune sera of titer ≥1∶18 or guinea pig immune sera of titer ≥1∶37 were protected from disease; these animals are omitted from the figure to allow better resolution of lower titers.</p

Dose-dependent protective efficacy of H5N1 vaccine-induced immune sera in mice.

<p>Shown are reciprocal MN titers:</p>a<p>expected titer based on the volume and titer of injected immune sera;</p>b<p>measured circulating titer 2 h prior to challenge;</p>c<p>extrapolated from titers measured 2 h prior to challengea.</p>d<p>Mice were challenged intranasally with 10⁴ TCID₅₀ wild-type H5N1 virus. Animals surviving for ≥14 days are considered protected.</p>e<p>Immune serum administered on 3 successive days. n.a., not applicable.</p

Survival after vaccination with recombinant MVAs and challenge with different H5N1 strains.

<p>For single dose vaccinations recombinant MVA vaccines expressing the HA of VN/1203 (A), of IN5/05 (B), of CE3/06 (C), of TT01/05 (D) and AH1/2005 (E) were used. As controls, mice were vaccinated with wt MVA (F) or were treated with PBS (G). After challenge with wild-type H5N1 strains of the different clades, mice were monitored for 14 days. The data represent two separate experiments with six animals per group.</p

Protection of mice from death, clinical score and serology results after single dose vaccination with MVA H5 recombinants.

(1)<p>Mice were vaccinated once with 10⁶ pfu of recombinant MVA or wt MVA; for abbreviations see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016247#pone-0016247-t002" target="_blank">Tables 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016247#pone-0016247-t003" target="_blank">3</a>;</p>(2)<p>challenge dose, 1×10⁵ TCID₅₀; for abbreviations see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016247#pone-0016247-t001" target="_blank">Table 1</a>;</p>(3)<p>n/nt, number of survivors/total animals of two separate experiments with 6 animals per group;</p>(4)<p>clinical score at day 8 (peak)/day 14 (the end of experiment) after challenge. Each number represents the arithmetic mean from 12 animals;</p>(5)<p>final serum dilutions in µNT assay were 1∶20 (dl 14), in all other sera 1∶10 (detection limit <7).</p

Amino acid sequence identities of influenza HA proteins of strains used in this study.

(1)<p>VN/1203, A/Vietnam/1203/2004(H5N1);</p>(2)<p>IN5/05, A/Indonesia/5/05(H5N1);</p>(3)<p>TT1/05, A/turkey/Turkey/1/2005(H5N1);</p>(4)<p>CE3/06, A/Chicken/Egypt/3/2006(H5N1);</p>(5)<p>AH1/05, A/Anhui/1/2005(H5N1).</p

Amino acid sequences of the CD4 T cell-specific 15mer peptide HA140-154 (located in the HA1 subunit) in the different hemagglutinins.

<p>Amino acid sequences of the CD4 T cell-specific 15mer peptide HA140-154 (located in the HA1 subunit) in the different hemagglutinins.</p

Recombinant MVA viruses used for immunizations.

(1)<p>All HA genes are controlled by the vaccinia virus mH5 promoter;</p>(2)<p>titers of sucrose-purified virus preparations from CEC cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016247#s2" target="_blank">methods</a>);</p>(3)<p>particle infectivity ratio, ratio of genomic equivalents determined by qPCR to infectious virus particles (pfu);</p>(4)<p>HA expression as determined by immunostaining (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016247#s2" target="_blank">methods</a>).</p

Induction of influenza-specific T cells by the hemagglutinin expressing live vaccines.

<p>Frequencies of antigen specific IFN-γ+ CD4 T cells (A) or CD8 T-cells (B) after immunizing twice with various hemagglutinin MVA-H5 vaccines or with the MVA wild-type control, and stimulation with protein antigens and peptides (shown on x-axis). Splenocytes were stimulated with buffer or with formalin-inactivated whole viral antigens of H5N1 Vietnam (H5N1-VN), H5N1 Indonesia (H5N1-IN) or H1N1/California (H1N1-CA), CD4 T cell-reactive peptide HA140-154 and the CD8 T cell-reactive peptide HA189-197. Background medium responses were subtracted, and the means +/− standard deviation of two separate experiments is shown.</p

Western blot of chicken cell lysates tested for influenza virus HA expression.

<p>Lane 1, protein ladder, size in kDa; lane 2, positive control, 0.5 µg of formalin inactivated purified influenza virus A/Vietnam/1203/2004 H5N1. Lane 3, MVA-HA-VN. Lane 4, MVA-HA-IN. Lane 5, MVA-HA-TT. Lane 6, MVA-HA-CE. Lane 7, MVA-HA-AN. Lane 8, negative control, empty vector MVA wt. For abbreviations see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016247#pone-0016247-t002" target="_blank">Table 2</a>. All recombinant MVAs (lanes 3–7) express the HA0 (band around 80 kDa), the HA1 (band around 55 kDa, and the HA2 (band around 25 kDa). The specific HA bands co-migrate with the ones of the positive control (lane 2) and are absent in the negative control (lane 8).</p

Differences in the HA amino acid sequence compared to the VN/1203 master sequence.

(1)<p>HA1 numbering starts with the first amino acid downstream of the signal peptide with D as the first residue (Aichi numbering);</p>(2)<p>amino acids exposed on the surface are printed in bold (equivalent to yellow marked molecules in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016247#pone-0016247-g005" target="_blank">Fig. 5</a>);</p>(3)<p>VN, A/Vietnam/1203/2004; IN, A/Indonesia/5/05; AN, A/Anhui/1/2005; CE, A/Chicken/Egypt/3/2006; TT, A/turkey/Turkey/1/2005;</p>(4)<p>first R residue of polybasic cleavage site;</p>(5)<p>numbering of the HA2 starts downstream of the polybasic cleavage site with G as the first residue.</p