streptococcus pneumoniae pan-genome microarray  (Antigen Discovery Inc)

Journal: eLife

Article Title: Panproteome-wide analysis of antibody responses to whole cell pneumococcal vaccination

doi: 10.7554/eLife.37015

Figure Lengend Snippet: In both panels, the RM200 contigs at the bottom are aligned to a reference genome at the top, with red bands between the two linking regions of sequence similarity identified by BLAST. The blue boxes marked in each sequence are predicted protein coding sequences. ( A ) Introduction of the serotype three capsule polysaccharide synthesis locus into the S. pneumoniae Rx1 background. The sequence of the serotype three strain S. pneumoniae OXC141 is shown at the top, with the extent of the cps gene cluster responsible for capsule synthesis marked. The alignment shows the entirety of this gene cluster is present in the unencapsulated RM200, which was derived from a serotype 3-expressing recombinant genotype. The indicated homologous recombination distinguished RM200 from its serotype two original progenitor, D39. This spanned the entire cps locus and likely indicates the import of DNA causing the switch in the expressed capsule type. ( B ) Alteration of the pneumolysin toxin-encoding gene ply and removal of the lytic amidase-encoding lytA . The ply sequence was modified though insertion-duplication mutagenesis using the E. coli-S. pneumoniae shuttle vector pDP28. This introduced three amino acid substitutions that eliminated the cytolytic activity of the protein. The lytA gene was replaced by the Janus cassette through allelic replacement. The homologous recombination distinguishing these two genomes through which this occurred is marked by the red bar underneath the RM200 sequence.

Article Snippet: The Streptococcus pneumoniae Pan-Genome Microarray, produced by Antigen Discovery, Inc. (ADI, Irvine, CA, USA), was designed and assayed as described previously ( ).

Techniques: Sequencing, Derivative Assay, Expressing, Recombinant, Homologous Recombination, Modification, Mutagenesis, Plasmid Preparation, Activity Assay

Journal: eLife

Article Title: Panproteome-wide analysis of antibody responses to whole cell pneumococcal vaccination

doi: 10.7554/eLife.37015

Figure Lengend Snippet: In a pilot experiment, the serum samples were applied to an array consisting of probes representing the proteome of Streptococcus pneumoniae TIGR4. These were also present on the panproteome array, but they were not included in subsequent analyses. These two independent experiments provided an estimate of the variation between technical replicates, although the differences in the array designs means there is substantial systematic variation between them. The Pearson correlation (R 2 ) was calculated between all pairwise comparisons from the two sets of replicates, and the overall distribution for different types of comparison shown as violin plots for ( A ) all probes and ( B ) the 1165 immunoreactive probes (a maximum IgG binding of at least one across the S. pneumoniae TIGR4 probes from the panproteome dataset). There was no significant difference between the correlations observed across all probes for technical replicates and other comparisons between samples from the same individual (Wilcoxon rank sum tests; N = 312, W = 13239, p = 0.073 for all probes). However, restricting the analysis to the immunoreactive probes found technical replicates to exhibit a significantly stronger correlation with one another, relative to samples from the same individual at other timepoints ( N = 312, W = 13480, p = 0.036 for immunoreactive probes), consistent with the WCV-induced changes associated with antibody-binding target being reproducibly detectable. This high similarity between technical replicates and samples from the same individual is necessary for the persistence of an immune fingerprint in longitudinal samples . Comparisons between these two categories were significantly more correlated than comparisons with the next most similar category, different individuals in the same cohort at the same timepoint (Wilcoxon rank sum tests; N = 601, W = 48170, p < 10 −16 for all probes; N = 601, W = 48738, p < 10 −16 for immunoreactive probes). As there was relatively little variation between technical replicates, but reproducibly high variation between trial participants, biological replicates were prioritised over technical replicates in designing the study.

Article Snippet: The Streptococcus pneumoniae Pan-Genome Microarray, produced by Antigen Discovery, Inc. (ADI, Irvine, CA, USA), was designed and assayed as described previously ( ).

Techniques: Comparison, Binding Assay

Journal: eLife

Article Title: Panproteome-wide analysis of antibody responses to whole cell pneumococcal vaccination

doi: 10.7554/eLife.37015

Figure Lengend Snippet: The t-SNE analysis shown in was repeated, but only using those probes corresponding to proteins on the array conserved with ≥90% sequence identity in S. mitis and S. pseudopneumoniae . Longitudinal samples from the same individual again cluster together, demonstrating the distinctiveness of individuals’ antibody repertoires was apparent even to proteins exhibiting little variation across S. pneumoniae isolates. The longer-range structure of the projection is stochastic, and these differences with are not biologically relevant.

Article Snippet: The Streptococcus pneumoniae Pan-Genome Microarray, produced by Antigen Discovery, Inc. (ADI, Irvine, CA, USA), was designed and assayed as described previously ( ).

Techniques: Sequencing

Journal: eLife

Article Title: Panproteome-wide analysis of antibody responses to whole cell pneumococcal vaccination

doi: 10.7554/eLife.37015

Figure Lengend Snippet: Each histogram shows the pairwise protein sequence identity between the proteins on the array, and the closest orthologues in the corresponding genome. Data are shown for ( A ) S. pneumoniae RM200, the strain on which the vaccine is based; ( B ) S. pseudopneumoniae IS7493, a representative of the species most closely-related to S. pneumoniae ; ( C ) S. mitis B6, a representative of the diverse species after which the mitis group streptococci (of which S. pneumoniae is one) is named; ( D ) S. mutans UA159, which was found to be too divergent for an informative analysis to be performed. The vertical red dashed line indicates the empirically-determined 90% sequence identity threshold for determining presence in, or absence from, RM200 in and . Altering this threshold to 95% did not substantially alter the results presented in these figures.

Article Snippet: The Streptococcus pneumoniae Pan-Genome Microarray, produced by Antigen Discovery, Inc. (ADI, Irvine, CA, USA), was designed and assayed as described previously ( ).

Techniques: Sequencing

Journal: eLife

Article Title: Panproteome-wide analysis of antibody responses to whole cell pneumococcal vaccination

doi: 10.7554/eLife.37015

Figure Lengend Snippet: Description of WCV antigenic proteins, represented by probes associated with significantly increased IgG binding following WCV administration by either the empirical Bayes or linear mixed effects analyses ( Supplementary file 2 ). Each protein is listed (either by common name, or COG assignation), alongside a functional annotation and the coding sequence for the orthologue in S. pneumoniae D39, where it could be identified. IgG-binding values were aggregated across all probes corresponding to the named protein that were associated with a significant change. Values are summarised as a median, with the interquartile range in parentheses. The columns show the median initial IgG binding to the probes across all cohorts, and the Δ 0→84 values for each of the four cohorts separately. The final column identifies in which analyses the set of probes corresponding to the named protein were found to be associated with elevated IgG binding.

Article Snippet: The Streptococcus pneumoniae Pan-Genome Microarray, produced by Antigen Discovery, Inc. (ADI, Irvine, CA, USA), was designed and assayed as described previously ( ).

Techniques: Binding Assay, Functional Assay, Sequencing, Membrane, Histone Deacetylase Assay

Journal: eLife

Article Title: Panproteome-wide analysis of antibody responses to whole cell pneumococcal vaccination

doi: 10.7554/eLife.37015

Figure Lengend Snippet: pneumoniae RM200 penicillin-binding proteins and orthologous variants on the proteome array. ( A ) Phylogeny demonstrating Pbp1A from RM200 is most similar to variant one on the array. ( B ) Phylogeny demonstrating Pbp2X from RM200 is most similar to variant one on the array. ( C ) Phylogeny demonstrating Pbp2B from RM200 is most similar to variant one on the array.

Article Snippet: The Streptococcus pneumoniae Pan-Genome Microarray, produced by Antigen Discovery, Inc. (ADI, Irvine, CA, USA), was designed and assayed as described previously ( ).

Techniques: Binding Assay, Variant Assay

Journal: eLife

Article Title: Panproteome-wide analysis of antibody responses to whole cell pneumococcal vaccination

doi: 10.7554/eLife.37015

Figure Lengend Snippet: pneumoniae RM200 PclA sequence and the orthologous proteins on the proteome array. The tree shows that the RM200 protein is most similar to CLS01333 and CLS03265, as well as the truncated non-functional variant CLS99466. The CLS03616 and CLS03178 variants are highly diverged from the variant expressed by RM200.

Article Snippet: The Streptococcus pneumoniae Pan-Genome Microarray, produced by Antigen Discovery, Inc. (ADI, Irvine, CA, USA), was designed and assayed as described previously ( ).

Techniques: Sequencing, Functional Assay, Variant Assay

Journal: eLife

Article Title: Panproteome-wide analysis of antibody responses to whole cell pneumococcal vaccination

doi: 10.7554/eLife.37015

Figure Lengend Snippet: Data are shown as in . The type one and two pili, and the pneumococcal serine-rich repeat protein (PsrP), are all absent from S. pneumoniae RM200, and therefore WCV is not expected to trigger an elevated IgG response to these structures. The PclA variants CLS01333 and CLS03265 show some evidence of increased IgG binding, indicating the S. pneumoniae RM200 PclA protein is being recognised by WCV-induced antibodies.

Article Snippet: The Streptococcus pneumoniae Pan-Genome Microarray, produced by Antigen Discovery, Inc. (ADI, Irvine, CA, USA), was designed and assayed as described previously ( ).

Techniques: Binding Assay