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

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

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

Microbial Translocation and Inflammation Occur in Hyperacute Immunodeficiency Virus Infection and Compromise Host Control of Virus Replication

<div><p>Within the first three weeks of human immunodeficiency virus (HIV) infection, virus replication peaks in peripheral blood. Despite the critical, causal role of virus replication in determining transmissibility and kinetics of progression to acquired immune deficiency syndrome (AIDS), there is limited understanding of the conditions required to transform the small localized transmitted founder virus population into a large and heterogeneous systemic infection. Here we show that during the hyperacute “pre-peak” phase of simian immunodeficiency virus (SIV) infection in macaques, high levels of microbial DNA transiently translocate into peripheral blood. This, heretofore unappreciated, hyperacute-phase microbial translocation was accompanied by sustained reduction of lipopolysaccharide (LPS)-specific antibody titer, intestinal permeability, increased abundance of CD4+CCR5+ T cell targets of virus replication, and T cell activation. To test whether increasing gastrointestinal permeability to cause microbial translocation would amplify viremia, we treated two SIV-infected macaque ‘elite controllers’ with a short-course of dextran sulfate sodium (DSS)–stimulating a transient increase in microbial translocation and a prolonged recrudescent viremia. Altogether, our data implicates translocating microbes as amplifiers of immunodeficiency virus replication that effectively undermine the host’s capacity to contain infection.</p></div

Longitudinal levels of SIV RNA and bacterial rDNA in plasma.

<p>Eight MHC-identical cynomolgus macaques became infected following intrarectal inoculation with SIVmac239. (<b>A</b>) The number of SIV RNA copies/ml of plasma was enumerated using qRT-PCR. Values are Log₁₀-transformed and plotted longitudinally. (<b>B</b>) 16S sequencing data was used to correct raw 16S rDNA qPCR data by removing the proportion of 16S rDNA copies that corresponded to taxa detected in matched water controls. Corrected 16S rDNA copy data was Log₁₀-transformed and plotted longitudinally. By Bonferroni-corrected one-way ANOVA, plasma levels of 16S rDNA did not change significantly between -42 and 0 DPI. Plasma levels of 16S rDNA increased significantly (P<0.0005) from both -42 to 8 DPI and 0 to 8 DPI. In both plots, the vertical checkered box positioned between 14 and 18 DPI corresponds to the acute-phase peak of SIV replication as detected by our sampling resolution.</p

Chemically inducing microbial translocation stimulates multiple host inflammatory processes and increases plasma viremia and levels of bacterial rDNA.

<p>(<b>A</b>) Log₁₀-transformed plasma SIV load. (<b>B</b>) Linear plasma 16S rDNA load. The bacteria-specific host response was assessed by monitoring plasma levels of (<b>C</b>) EndoCAb and (<b>D</b>) sCD14. Plasma IFABP levels (<b>E</b>) were used to monitor changes to the integrity of the gastrointestinal epithelium. Generalized inflammation was monitored using plama levels of (<b>F</b>) MCP-1. In all panels, 5 black vertical lines indicate the 5-day period of once-daily treatment with dextran sulfate sodium (DSS).</p

Plasma markers of intestinal breach and host response to microbial translocation.

<p>(<b>A</b>) Longitudinal proportion (%) of plasma genera detected in contemporaneous stool. Each line corresponds to a single animal. Bonferroni-corrected one-way ANOVA was used to calculate statistical significance. (<b>B</b>) Longitudinal plasma levels of IFABP, a marker of enterocyte loss and generalized damage to the intestinal epithelium. The host response to microbial translocation was measured using plasma levels of (<b>C</b>) EndoCAb, and (<b>D</b>) sCD14. By linear regression analysis, plasma levels of sCD14 at (<b>E</b>) 8 DPI, and (<b>F</b>) 21 DPI correlated positively with chronic-phase set-point viral loads. Acute inflammation was measured using plasma levels of (<b>G</b>) MCP-1, and (<b>H</b>) SAA1. For B, C, D, G, and H, differences between 0–8 DPI were evaluated for statistical significance by two-tailed Wilcoxon signed rank testing.</p

Taxonomic characterization of translocating microbial products.

<p>(<b>A</b>) Longitudinal relative abundance (%) of major phyla detected in blood plasma. (<b>B</b>) Number of unique bacterial genera for which genomic DNA was detected in blood plasma throughout the period of observation. Each line corresponds to a single animal. (<b>C</b>) Longitudinal relative abundance (%) of major genera detected in blood plasma. For (A and C), vertical bars within a given cluster (time-point) correspond to each individual animal, and colored segments correspond to the proportion of specific taxa. Owing to sample limitations, relative abundance of microbial taxa could not be determined for all animals at all time-points.</p