Vectors Based on Modified Vaccinia Ankara Expressing Influenza H5N1 Hemagglutinin Induce Substantial Cross-Clade Protective Immunity

New highly pathogenic H5N1 influenza viruses are continuing to evolve with a potential threat for an influenza pandemic. So far, the H5N1 influenza viruses have not widely circulated in humans and therefore constitute a high risk for the non immune population. The aim of this study was to evaluate the cross-protective potential of the hemagglutinins of five H5N1 strains of divergent clades using a live attenuated modified vaccinia Ankara (MVA) vector vaccine.The replication-deficient MVA virus was used to express influenza hemagglutinin (HA) proteins. Specifically, recombinant MVA viruses expressing the HA genes of the clade 1 virus A/Vietnam/1203/2004 (VN/1203), the clade 2.1.3 virus A/Indonesia/5/2005 (IN5/05), the clade 2.2 viruses A/turkey/Turkey/1/2005 (TT01/05) and A/chicken/Egypt/3/2006 (CE/06), and the clade 2.3.4 virus A/Anhui/1/2005 (AH1/05) were constructed. These experimental live vaccines were assessed in a lethal mouse model. Mice vaccinated with the VN/1203 hemagglutinin-expressing MVA induced excellent protection against all the above mentioned clades. Also mice vaccinated with the IN5/05 HA expressing MVA induced substantial protection against homologous and heterologous AH1/05 challenge. After vaccination with the CE/06 HA expressing MVA, mice were fully protected against clade 2.2 challenge and partially protected against challenge of other clades. Mice vaccinated with AH1/05 HA expressing MVA vectors were only partially protected against homologous and heterologous challenge. The live vaccines induced substantial amounts of neutralizing antibodies, mainly directed against the homologous challenge virus, and high levels of HA-specific IFN-γ secreting CD4 and CD8 T-cells against epitopes conserved among the H5 clades and subclades.The highest level of cross-protection was induced by the HA derived from the VN/1203 strain, suggesting that pandemic H5 vaccines utilizing MVA vector technology, should be based on the VN/1203 hemagglutinin. Furthermore, the recombinant MVA-HA-VN, as characterized in the present study, would be a promising candidate for such a vaccine

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

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

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

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

Clinical disease scoring after vaccination with recombinant MVAs and challenge with different H5N1 strains.

<p>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 for vaccination. Controls included wt MVA (F) or PBS (G). After challenge with wild-type H5N1 strains of the different clades, mice were clinically monitored for 14 days. The parameters used to evaluate the clinical score were ruffled fur (1 point), arched posture (2 points), apathy (3 points) and death (4 points). Each data point represents the arithmetic mean of two separate experiments with six animals per group.</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