Protection of ZIKVLPs in AG129 mice.

<p>3A: Neutralizing antibody titers (+/- SD) of vaccinated AG129 mice pre boost and pre challenge. 3B: Average weight loss (+/- SD) of AG129 after ID challenge with 200 PFU ZIKV over a 14 day period. 3C: Survival of 11 week old AG129 after ID challenge with 200 PFU ZIKV over a 14 day period. 3D: Viremia (+/- SD) in serum samples from mice two days post challenge by qRT-PCR. Values are total RNA copies per reaction. 3E. Viremia (+/- SD) in serum samples from mice two days post challenge by TCID₅₀. 3F: PRNT₅₀ values (+/- SD) of serum samples taken from ZIKVLP vaccinated AG129 mice post challenge, and pre challenge serum from PBS/alum mice.</p

LD50 of ZIKV in AG129 mice.

<p>Survival of AG129 after ZIKV over a 14 day period.</p

Dose response of ZIKVLPs in AG129 mice.

<p>5AB: PRNT₅₀ values (+/- SD) of serum samples taken from AG129 mice administered a prime and boost of 0.45 μg (5A) or a prime only of 3.0 μg (5B) ZIKVLPs pre and post challenge. 5C: Survival of 11 week old AG129 after ID challenge with 200 PFU ZIKV over a 14 day period.</p

<i>In vitro</i> characterization of Zika virus like particles.

<p>1A: Schematic of pCMV-prM/E expression cassette. 1B: Western blot analysis of Zika virus like particles. Lanes are, 1) Bio-rad precision plus kaleidoscope protein standards. 2): pCMV-prM/E transfection pre sucrose cushion purification supe. 3) 3.5x10⁴ PFU ZIKV positive control. 4) pCMV-prM/E transfection post sucrose cushion purification pt. 5) pCMV-GFP transfection post sucrose cushion purification pt. 1C-E: Sucrose cushion purified Zika VLPs observed using transmission electron microscopy. 1C: VLPs stained with Tungsten. Diameter is indicated. Background protein staining also apparent. 1D: VLP stained with Tungsten. Membrane proteins visible on the surface of VLP are indicated with arrow. Background protein staining apparent. 1E: VLP stained with Uranyl acetate. Membrane proteins visible on the surface of VLP are indicated with an arrow.</p

ZIKVLP serum transfer to naïve AG129 mice.

<p>4A: Average weight loss (+/- SD) of 8 week AG129 transferred serum from mice vaccinated with ZIKVLPs after ID challenge with 20 PFU of ZIKV over a 14 day period. 4B: Survival of AG129 after challenge with ZIKV over a 14 day period.</p

Protection of ZIKVLPs in BALB/c mice.

<p>6A: PRNT₅₀ values (+/- SD) of serum samples taken from BALB/c mice administered a prime only of 3.0 μg ZIKVLPs post challenge. 6B: Viremia (+/- SD) in serum samples from mice two days post challenge by qRT-PCR. Values are total RNA copies per reaction. 6C: Average weight loss (+/- SD) of BALB/c mice after ID challenge with 200 PFU ZIKV over a 14 day period.</p

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

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

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

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