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

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

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

The evolution of lung cancer and impact of subclonal selection in TRACERx.

Lung cancer is the leading cause of cancer-associated mortality worldwide1. Here we analysed 1,644 tumour regions sampled at surgery or during follow-up from the first 421 patients with non-small cell lung cancer prospectively enrolled into the TRACERx study. This project aims to decipher lung cancer evolution and address the primary study endpoint: determining the relationship between intratumour heterogeneity and clinical outcome. In lung adenocarcinoma, mutations in 22 out of 40 common cancer genes were under significant subclonal selection, including classical tumour initiators such as TP53 and KRAS. We defined evolutionary dependencies between drivers, mutational processes and whole genome doubling (WGD) events. Despite patients having a history of smoking, 8% of lung adenocarcinomas lacked evidence of tobacco-induced mutagenesis. These tumours also had similar detection rates for EGFR mutations and for RET, ROS1, ALK and MET oncogenic isoforms compared with tumours in never-smokers, which suggests that they have a similar aetiology and pathogenesis. Large subclonal expansions were associated with positive subclonal selection. Patients with tumours harbouring recent subclonal expansions, on the terminus of a phylogenetic branch, had significantly shorter disease-free survival. Subclonal WGD was detected in 19% of tumours, and 10% of tumours harboured multiple subclonal WGDs in parallel. Subclonal, but not truncal, WGD was associated with shorter disease-free survival. Copy number heterogeneity was associated with extrathoracic relapse within 1 year after surgery. These data demonstrate the importance of clonal expansion, WGD and copy number instability in determining the timing and patterns of relapse in non-small cell lung cancer and provide a comprehensive clinical cancer evolutionary data resource