Binding affinity of Hangzhou-H7 and H7N7 for human and avian receptors.

<p>Hangzhou-H7 and the closely related H7N7 virus (A/Netherlands/219/2003) were docked to human and avian sialic acid receptors and affinities calculated. (<b>A</b>) The predicted Hangzhou-H7 model receptor affinities are shown by the black bars and the A/Netherlands/219/2003 affinities by the white bars. (<b>B</b>) The highest affinity docking conformation of Hangzhou-H7 on the human (brown), and avian (purple), receptors. HA mutations that influence the receptor binding specificity are highlighted in red and other residues that interact with the receptor in yellow. Residues that form the receptor-binding pocket are highlighted in green.</p

Hangzhou-H7 neutralizing antibody binding.

<p>(<b>A</b>) The structures of neutralizing antibodies are shown either binding to the Hangzhou-H7 (shown in blue) globular head region (left figure) or stalk region (right figure). Different colours represent different antibodies. HA residues interacting with stalk-binding neutralizing antibodies are shown in the right hand figure highlighted in yellow. (B) HA-neutralizing antibodies binding the head or stalk regions were docked to the predicted Hangzhou-H7 structure and binding energies were calculated and presented as an absolute value. PDB IDs of antibodies against H1 are 3LZF, 3SM5, 4M5Z, 3GBN, 3ZTN, PDB IDs for H3 are 1EO8, 1KEN, 1QFU, 2VIR, 4FQR, 3SDY, 4NM8, H5 PDB IDs are 3FKU and 3GBM and H7 ID is 4FQV.</p

Advax adjuvant enhances plasmablast AID expression.

<p>Each subject’s 7dpv-plasmablasts were FACS-sorted into three pools per study group for RNA extraction and qPCR analysis of <i>AID</i> expression (A). To assess for a direct effect of Advax on B cells, PBMC were cultured with Advax adjuvant at 10μg/ml for 24h <i>in vitro</i> and then AID mRNA expression quantified by qPCR (B).</p

Delta Inulin Adjuvant Enhances Plasmablast Generation, Expression of Activation-Induced Cytidine Deaminase and B-Cell Affinity Maturation in Human Subjects Receiving Seasonal Influenza Vaccine

<div><p>There is a major need for new adjuvants to improve the efficacy of seasonal and pandemic influenza vaccines. Advax is a novel polysaccharide adjuvant based on delta inulin that has been shown to enhance the immunogenicity of influenza vaccine in animal models and human clinical trials. To better understand the mechanism for this enhancement, we sought to assess its effect on the plasmablast response in human subjects. This pilot study utilised cryopreserved 7 day post-vaccination (7dpv) peripheral blood mononuclear cell samples obtained from a subset of 25 adult subjects from the FLU006-12 trial who had been immunized intramuscularly with a standard dose of 2012 trivalent inactivated influenza vaccine (TIV) alone (n=9 subjects) or combined with 5mg (n=8) or 10mg (n=8) of Advax adjuvant. Subjects receiving Advax adjuvant had increased 7dpv plasmablasts, which in turn exhibited a 2-3 fold higher rate of non-silent mutations in the B-cell receptor CDR3 region associated with higher expression of activation-induced cytidine deaminase (AID), the major enzyme controlling BCR affinity maturation. Together, these data suggest that Advax adjuvant enhances influenza immunity in immunized subjects via multiple mechanisms including increased plasmablast generation, AID expression and CDR3 mutagenesis resulting in enhanced BCR affinity maturation and increased production of high avidity antibody. How Advax adjuvant achieves these beneficial effects on plasmablasts remains the subject of ongoing investigation.</p><p>Trial Registration</p><p>Australia New Zealand Clinical Trials Register ACTRN12612000709842 <a href="https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=362709" target="_blank">https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=362709</a></p></div

Correlation of plasmablast frequency 7dpv and HI titer increase 28dpv.

<p>Shown p-values represent the significance of the correlation between 7dpv-plasmablast frequency and 28dpv-HI titer increase within each group for each of the three vaccine antigens. No correction was made for multiple comparisons.</p><p>Correlation of plasmablast frequency 7dpv and HI titer increase 28dpv.</p

Advax adjuvant has no effect on CDR1 and CDR2 mutations.

<p>The IgG variable region cloned from sorted 7dpv plasmablasts from each subject were analysed with IMGT/HighV-Quest online tool for mutation rate analysis. A minimum of 20 IgG variable region clones was sequenced from each subject immunized with TIV alone (9 subjects), Advax 5mg (8 subjects) and Advax 10mg (7 subjects) subjects. Plots show non-silent and silent mutations for CDR1 (A-B) and CDR2 (C-D).</p

Subject Characteristics—FLU006-12 Substudy.

<p>¹ Differences were compared between groups using ANOVA, median test or chi-square test as appropriate.</p><p>Subject Characteristics—FLU006-12 Substudy.</p

Seroprotection, seroconversion and GMT fold increase 28 days post immunization for subjects in the FLU006-12 substudy.

<p>Seroprotection, seroconversion and GMT fold increase 28 days post immunization for subjects in the FLU006-12 substudy.</p

Effect of Advax adjuvant on post-immunization plasmablast response.

<p>The frequency of 7dpv TIV-specific B cells secreting IgG, IgM or IgA (mean ± SEM) was enumerated by ELISPOT (A-C). Plots show the correlation between plasmablast frequency 7pdv (measured by FACS) and influenza-specific IgG+, IgM+ or IgA+ plasmablasts 7dpv (measured by ELISPOT) (D-F).</p

Advax adjuvant is associated with enhanced BCR heavy chain CDR3 affinity maturation.

<p>Schematic of human <i>Igh</i> gene and VDJ recombination (A). The IgG variable region cloned from sorted 7dp plasmablasts from each subject were analysed with IMGT/HighV-Quest online tool for mutation rate analysis. A minimum of 20 IgG variable region clones was sequenced from each subject. Shown is the frequency of non-silent (B) or silent (C) V region mutations and non-silent (D) or silent (E) CDR3-specific mutations.</p