Serum antibody off-rates to H5-Viet-rHA1 following prime-boost strongly correlates with the <i>in-vitro</i> cross-neutralizing capacity against diverse H5N1 strains.

<p>End-point virus neutralization titers of samples collected after the H5N1 subunit vaccine boost are plotted on the X-axis. Antibody off-rate constants that describe the fraction of antibody-antigen complexes decaying per second were determined directly from the serum sample interaction with rHA1 protein using SPR in the dissociation phase are shown on Y-axis. Serum samples from low-dose Ad4-H5-Vtn primed (red circles), high-dose Ad4-H5-Vtn primed (blue circles) or unprimed (green circles) individuals following H5N1 subunit vaccination are shown. Antibody affinity of post-H5N1 vaccinated human sera against HA1 of H5N1-A/Vietnam/1203/2004 strongly correlated with the <i>in-vitro</i> heterologous MN titers against H5N1 clade 2.1- A/Indonesia/5/2005 (A), clade 2.3.4- A/Anhui/1/2005 (B), clade 2.2- A/Turkey/15/2006 (C), and clade 2.2- A/Egypt/NO3072/2010 (D). Spearman correlations are reported for the calculation of correlations between off-rate and MN titers combined across all vaccine groups.</p

Binding of post-H5N1 vaccination polyclonal human serum to properly folded HA1 proteins from A/Vietnam/1203/2004 and A/Indonesia/05/2005.

<p>Steady-state equilibrium analysis of the total binding antibodies in the polyclonal human vaccine sera to properly folded functional H5N1-A/Vietnam/1203/2004 HA1-His₆ (panel A) or H5N1-A/Indonesia/05/2005-His₆ (Panel B) was measured by SPR. Each individual post-boost H5N1 serum sample, diluted ten-fold, was injected simultaneously onto HA1 immobilized on a sensor chip through the free amine group and onto a blank flow cell, free of peptide. Maximum resonance unit (Max RU) values for HA1 binding by serum antibodies obtained from multiple individuals from either low dose (10⁷ VP) Ad4-H5-Vtn primed (red circles), high dose Ad4-H5-Vtn (10¹¹VP) (blue circle), or unprimed (green circles) on day 0 (Pre), 28 days after the third prime (P-P), and 28 days post boost with 90 μg HA/dose of the Sanofi Pasteur (P-V). Differences between groups (p-values) were examined for statistical significance by the multiple comparison adjustment using Bonferroni method. A <i>p-value</i> less than 0.05 was considered to be significant. ‘ns’ represents non-significant (<i>p = >0.05</i>).</p

Serum antibody off-rates to H5-Viet-rHA1 (but not rHA2) following heterologous prime-boost strongly correlate with the <i>in-vitro</i> neutralizing capacity against the homologous H5 vaccine viruses.

<p>Antibody off-rate constants were determined directly from the serum sample interaction with H5N1 rHA1 or rHA2 proteins using SPR in the dissociation phase. SPR analysis of post-boost vaccination human sera from all 3 vaccine groups combined was performed with rHA1 (A) or rHA2 (B) of the H5N1-A/Vietnam/1203/2004 strain, or rHA1 of the heterologous H5N1-A/Indonesia/5/2005 (Clade 2.1) strain (C). Each symbol represents one individual. Serum samples on day 28 following single H5N1 booster vaccination with the subunit H5N1 vaccine (Sanofi Pasteur, 90 μg HA/dose) from the low-dose Ad4-H5-Vtn adjuvanted primed (red circles), high-dose Ad4-H5-Vtn primes (blue circles), or unprimed (green circles) are shown. Antibody affinity of post-H5N1 vaccinated human sera against HA1 (but not HA2) of H5N1-A/Vietnam/1203/2004 correlated with the homologous virus microneutralization titers (MN) against the A/Vietnam/1203/2004 (H5N1). Similarly, polyclonal antibody affinity of post-H5N1 vaccinated human sera against HA1 of the heterologous H5N1-A/Indonesia/5/2005 (Clade 2.1) strain correlated with the microneutralization titers (MN) against the A/Indonesia/5/2005 (H5N1) virus. Spearman correlations are reported for the calculation of correlations between off-rate and MN titers combined across all vaccine groups.</p

H5N1 prime-boost vaccine trial design.

<p>The vaccine study design representing the three groups that were used in the current study and the individual numbers (N) per group is indicated. For more details of the clinical study see Reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115476#pone.0115476.ref003" target="_blank">3</a>. Ad4-H5N1-Viet and MIV-H5N1-Viet refer to: Ad4-H5-Vtn is a recombinant, replication competent Ad4, encoding full-length haemagglutinin from influenza A H5N1 virus (A/Vietnam/1194/2004) given orally thrice either as 10⁷ virus particles (VP) or 10¹¹ virus particles per dose. MIV-H5N1-Viet is a formalin-inactivated, licensed inactivated H5N1 partially purified subunit vaccine (A/Vietnam, 90 μg HA/dose; Sanofi Pasteur).</p

Ad4-H5-Vtn priming enhances antibody affinity (slower off-rates) to H5N1-HA1 (but not HA2) following a single H5N1 subunit vaccine boost.

<p>(A-C) SPR analysis of post-H5N1 vaccinated human sera after the H5N1-A/Vietnam/1203/2004 subunit vaccine boost in three vaccine groups was performed with properly folded HA1 (A) or HA2 (B) from H5N1 A/Vietnam/1203/2004 strain, or rHA1 of the heterologous H5N1-A/Indonesia/5/2005 (Clade 2.1) strain (C). Off-rates of polyclonal serum antibodies before (open symbols) or 28 days (filled symbols) after a single H5N1 (A/Vietnam) booster vaccination from low dose Ad4-H5-Vtn primed (red circles), high dose Ad4-H5-Vtn primed (blue circles) or unprimed (green circles) individuals are shown from either 28 days after the third prime (P-P), and 28 days post boost with 90 μg HA/dose of the Sanofi Pasteur vaccine (P-V). Antibody off-rate constants that describe the fraction of antibody-antigen complexes decaying per second were determined directly from the serum sample interaction with rHA1 (1–320) protein or rHA2 (331–480) using SPR in the dissociation phase. Serum antibody off-rate constants were determined as described in Materials and Methods. Differences between groups (<i>p-values</i>) were examined for statistical significance by the multiple comparison adjustment using Bonferroni method. <i>p-values</i> of less than 0.05 were considered significant.</p

Identification of pathways dysregulated in PD-brain regions, and the overlap of differentially expressed genes (DEGs) between the PD-affected brain regions.

<p>The 10 key pathways most significantly enriched for DEGs in substantia nigra (<b>a</b>), striatum (<b>b</b>) and cortex (<b>c</b>) of PD brains compared to control as measured by microarray. Enrichments for upregulated genes are shown on the left, for downregulated genes on the right. The numbers of DEGs populating each pathway are denoted in the right columns (#DEGs). (<b>d</b>) Overlap between DEGs in PD cortex and striatum as measured by microarrays. The overlap of upregulated genes is shown on the left, the overlap of downregulated genes on the right. (<b>e</b>) Overlap between DEGs in striatum and substantia nigra (S.nigra) as measured by microarrays. The overlap of upregulated genes is shown on the left, the overlap of downregulated genes on the right. (<b>f</b>) Overlap between DEGs in PD cortex and substantia nigra (S. nigra) as measured by microarrays. The overlap of upregulated genes is shown on the left, the overlap of downregulated genes on the right. (<b>g</b>) Overlap between DEGs in PD cortex, striatum and substantia nigra (S. nigra) as measured by microarrays. The overlap of upregulated genes is shown on the left, the overlap of downregulated genes on the right.</p

Protein alterations in PD brain are dominated by mitochondrial and lipid transport defects, and are largely independent of transcriptional changes.

<p>(<b>a</b>) Overlap between proteins and differentially expressed genes in striatum, as measured by mass spectrometry and microarray technologies. The overlap of upregulated proteins/genes is shown on the left, the overlap of downregulated proteins/genes on the right. (<b>b</b>) Overlap between proteins and differentially expressed genes in PD cortex, as measured by proteomics and microarray technologies. The overlap of upregulated proteins/genes is shown on the left, the overlap of downregulated proteins/genes on the right. (<b>c</b>) Oxidative phosphorylation pathway. The most significantly enriched upregulated pathway for PD cortex based on proteomics data. Red thermometers represent proteins with increased abundance. (<b>d</b>) Regulation of CDK5 in presynaptic signaling. The most significantly enriched downregulated pathway for PD cortex based on proteomics data. Blue thermometers represent proteins with decreased abundance.</p

Tracking peripheral biomarkers identified from the causal mapping of PD brain.

<p>(<b>a</b>) High confidence biomarkers consistently identified for PD cortex, striatum, and substantia nigra (S. nigra) using microarray analysis. Upregulated biomarkers are shown on the left together with fold changes in the three brain regions, downregulated biomarkers on the right. (<b>b</b>) Functional biomarker panel that combines DEGs, high confidence biomarkers shown in (a) and genes from the causal map. (<b>c</b>) Assessment of biomarkers in brain (top panels)(substantia nigra, SN) and blood (bottom panels) from age-matched control and PD patients using QuantiGene technology from Panomics. * <i>p</i>≤0.05 and ** <i>p</i>≤0.01 as determined using a two-tailed unpaired t-test with Welch's correction.</p

Overlap between pathways significantly enriched with differentially expressed genes identified by microarray and RNA-sequencing.

<p>(<b>a</b>) Overlap of pathways enriched with upregulated genes together with the five most significant overlapping pathways. (<b>b</b>) Overlap of pathways enriched with downregulated genes together with the five most significant overlapping pathways.</p

Overview of brain regions and methodology used in this study.

<p>(<b>a, b</b>) Overview of workflow for functional overview and focused analysis. (<b>a</b>) Expression data for healthy and diseased brain regions are statistically analyzed to obtain differentially expressed genes (DEGs). In the first part, the functional overview, the DEGs are used to identify expression regulators as well as pathways that are significantly enriched with DEGs. (<b>b</b>) In the second part, the focused analysis, pathways that are significantly enriched with expression regulators are combined with pathways that are significantly enriched with DEGs. Combining the two-pathway enrichment results leads to the identification of key pathways, which are the basis for the reconstruction of causal networks. (<b>c</b>) Cartoon representation of different brain regions used in the study, and the associated disease severity of each region denoted by gradations of red. Also shown is connectivity between the substantia nigra, striatum and cortex and the three methods used to interrogate the brain regions (microarray, RNAseq and proteomics).</p