A modified vaccinia Ankara vaccine vector expressing a mosaic H5 hemagglutinin reduces viral shedding in rhesus macaques

<div><p>The rapid antigenic evolution of influenza viruses requires frequent vaccine reformulations. Due to the economic burden of continuous vaccine reformulation and the threat of new pandemics, there is intense interest in developing vaccines capable of eliciting broadly cross-reactive immunity to influenza viruses. We recently constructed a “mosaic” hemagglutinin (HA) based on subtype 5 HA (H5) and designed to stimulate cellular and humoral immunity to multiple influenza virus subtypes. Modified vaccinia Ankara (MVA) expressing this H5 mosaic (MVA-H5M) protected mice against multiple homosubtypic H5N1 strains and a heterosubtypic H1N1 virus. To assess its potential as a human vaccine we evaluated the ability of MVA-H5M to provide heterosubtypic immunity to influenza viruses in a non-human primate model. Rhesus macaques received an initial dose of either MVA-H5M or plasmid DNA encoding H5M, followed by a boost of MVA-H5M, and then were challenged, together with naïve controls, with the heterosubtypic virus A/California/04/2009 (H1N1pdm). Macaques receiving either vaccine regimen cleared H1N1pdm challenge faster than naïve controls. Vaccination with H5M elicited antibodies that bound H1N1pdm HA, but did not neutralize the H1N1pdm challenge virus. Plasma from vaccinated macaques activated NK cells in the presence of H1N1pdm HA, suggesting that vaccination elicited cross-reactive antibodies capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Although HA-specific T cell responses to the MVA-H5M vaccine were weak, responses after challenge were stronger in vaccinated macaques than in control animals. Together these data suggest that mosaic HA antigens may provide a means for inducing broadly cross-reactive immunity to influenza viruses.</p></div

Early viral clearance from lower respiratory tract in monkeys vaccinated with an H5 mosaic MVA vaccine.

<p>Macaques were given a mosaic H5 HA antigen expressed on either plasmid DNA or in MVA and then boosted with MVA-H5M. All animals were challenged with the heterosubtypic virus A/California/04/2009 (H1N1pdm). Viral titers in (<b>A</b>) bronchoalveolar lavage (BAL) and nasal wash (<b>B</b>) were determined by standard plaque assays on MDCK cells. Control group includes 6 additional historical controls we previously published that were challenged with the same dose of the same viral stock by the same route [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181738#pone.0181738.ref029" target="_blank">29</a>]. The 2 control animals inoculated during this study are indicated by a star symbol. At day 4 post infection there was a statistically significant difference between DNA prime vaccinated and control animals as determined by a Kruskal-Wallis test combined with permutation. Bars indicate geometric mean and error bars indicate the 95% confidence interval of the geometric mean.</p

Vaccination with mosaic H5 MVA stimulates neutralizing and HI antibodies against H5N1 only.

<p>HI antibody titer was determined using a standard HI assay against both H1N1pdm A/California/04/2009 and H5N1 A/Vietnam/1203/2004. HI antibodies against H1N1pdm were undetectable until 30 days after challenge (<b>A</b>). HI antibodies against H5N1 were detected as early as 7 days after prime (<b>B</b>). Neutralization against the challenge H1N1pdm A/California/04/2009 virus was measured using plaque reduction neutralization test, where neutralization was only detected 14 days post challenge (<b>C</b>). Data points represent individual monkeys and report the IgG concentration where a 50% reduction in plaque formation is observed, bars indicate geometric mean and error bars indicated the 95% confidence interval of the geometric mean.</p

Pair-wise percent nucleotide identities among arteriviruses, including two new SHFV variants from Kibale red colobus (SHFV-krc1 and SHFV-krc2) and prototype <i>Arterivirus</i> strains^a.

a<p>Values are uncorrected pair-wise percent nucleotide identities of aligned, concatenated ORFs 1a, 1b, 2a, 2b, and 3–7, with reference to the EAV genome (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019056#pone-0019056-g001" target="_blank">Figure 1</a>). Other prototypical viruses included in the analysis are (GenBank accession numbers in parentheses): SHFV-LVR, the SHFV type strain LVR 42-0/M6941 (NC_003092.1); LDV-Plagemann strain (U15146.1); PRRSV-Lelystad strain (M96262.2); and EAV-Bucyrus strain (NC_002532.2).</p

Genome organization of novel simian hemorrhagic fever viruses from a Ugandan red colobus monkey.

<p>The novel variants SHFV-krc1 and SHFV-krc2 are shown in comparison to the SHFV type strain LVR 42-0/M6941 and the prototype <i>Arterivirus</i>, equine arteritis virus (EAV-Bucyrus strain). Boxes represent open reading frames and are drawn to scale. Shaded boxes indicate ORFs unique to SHFV, with dashed lines indicating the location of putative insertion relative to the EAV genome.</p

Phylogenetic tree of newly discovered simian hemorrhagic fever viruses and other arteriviruses.

<p>The novel variants SHFV-krc1 and SHFV-krc2 are highlighted. Other viruses included in the analysis were chosen to represent the diversity within each viral species based on available full-genome sequences (GenBank accession numbers in parentheses): SHFV-LVR, the SHFV type strain LVR 42-0/M6941 (NC_003092.1); PRRSV-Lelystad, the European (type 1) type strain (M96262.2); PRRSV-VR2332, the North American (type 2) type strain (U87392.3); EAV-Bucyrus strain (NC_002532.2); EAV-s3685 strain (GQ903794.1); LDV-P, the Plagemann strain (U15146.1); and LDV-C, the neuro-virulent strain (L13298.1). This unrooted tree was the likeliest of all trees (−ln L 41,541) found during a maximum likelihood branch-and-bound search with the computer program PAUP* 4.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019056#pone.0019056-Swofford1" target="_blank">[17]</a>. Numbers beside internal nodes indicate statistical support for individual clades (percent), based on 1000 bootstrap replicates of the data. The scale bar indicates genetic distance (nucleotide substitutions per site).</p

Animals mount T cell responses against the H1N1pdm challenge after vaccination with H5 mosaic MVA vaccine.

<p>We detected T cell responses in peripheral blood mononuclear cells (PBMC) using an IFN-γ elispot assay. PBMCs were stimulated with 7 pools of overlapping synthetic peptides from HA. We detected no responses 7 days after prime (<b>A</b>) or 14 days after boost (<b>B</b>). T cell responses were detected 7 days after challenge, with most responses detected in PBMCs stimulated with peptide pool 6 (<b>C</b>). The H5 mosaic HA used in vaccination had the most amino acid sequence identity shared with the challenge strain A/California/04/2009 (H1N1pdm) in the HA 2 domain, with the highest sequence identity of 82.4% occurring within peptide pool 6 (identical amino acid residues indicated by a black stripe; <b>D</b>). Error bars in A, B, and C indicate standard deviation.</p

Within- and between-host patterns of selection.

<p>(A) Mapping of the distributions of synonymous (S) and nonsynonymous (N) single nucleotide polymorphisms along the ORF for 13 of 28 red colobus samples, for which coverage-depth was greater than 100 reads across most (≥96%) of the ORF. (B) Distribution of average substitution rates at non-synonymous (dN) and synonymous (dS) sites, and their ratio (dN/dS), along a sliding window (100 aa window, 20 aa step) for the comparison of selection pressures among the Kibale SPgVs (accession no. KF234523, KF234526 and KF234530).</p

Neighbor-joining amino acid phylogeny of the NS3 helicase comprising 43 pegivirus and 25 hepacivirus sequences.

<p>This 97-aa segment (polyprotein positions 1221 to 1317 relative to HPgV, NC_001710) is highly conserved among the <i>Flaviviridae</i> and has been targeted extensively for virus discovery and phylogenetic characterization. The sequences included in this analysis encompass the full genetic diversity of identified pegiviruses within each clade, minus those for which NS3 sequence data were unavailable, namely pegiviruses infecting the common marmoset, <i>Callithrix jacchus </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098569#pone.0098569-Bukh1" target="_blank">[7]</a> and several recently identified viruses infecting bats <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098569#pone.0098569-Quan1" target="_blank">[12]</a>. Inclusion of the two most diverse variants of both SPgV_krc and SPgV_krtg demonstrated the relatively high within-host similarity of these viruses within the study population. GenBank accession numbers for the included taxa are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098569#pone.0098569.s005" target="_blank">Tables S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098569#pone.0098569.s006" target="_blank">S2</a>.</p

Detection frequency, TaqMan qRT-PCR titer and genome statistics for Kibale SPgVs.

a<p>Ranges for positive animals; assays were sensitive to between 10 and 100 genome copies (g.c.) per 20-µL qRT-PCR reaction.</p