Number of positive patients and percent positivity for EV-D68, PHOL/NML testing, September 1 to October 31, 2014.

<p>*Date when specimen was collected was used for this analysis and date when specimen was received in laboratory was used in the event the date of specimen collection was missing.</p

Geographical distribution of EV-D68 cases in Ontario, PHOL/NML, September 1 to October 31, 2014.

<p>A) Health unit reflects the jurisdiction in which the patient resides. In the event this was not available, health unit of the specimen submitter was used.</p

Acute and persisting antibody and memory T cell responses to pandemic H1N1 infection in one PCR case-confirmed donor.

<p>Longitudinal samples of unfractionated PBMC were challenged with influenza virus or controls for 18h. (A) Frequency of pandemic H1N1-responsive CD8 T cells out of total CD8 T cells as measured by IFNγ staining. IFNγ responsive CD8 T cells were also sub-divided by expression of other effector markers, granzyme B and CD107a. (B) Memory phenotypes of influenza-responsive CD8 T cells at various times post-onset of influenza symptoms. (C) Frequency and phenotypes of IFNγ⁺ CD4 T cells after pandemic H1N1 challenge. (D) Antibody titers in serum as detected by microneutralization (MN), hemagglutination inhibition (HAI), and a pandemic H1-specific ELISA assay. BLD  =  below the limits of detection.</p

T cell analysis in the Toronto seroprevalence and case/control cohorts.

<p>(A) Bin separation of IFNγ responses in CD8 and CD4 T cells specific to pH1N1 stimulation. Frequencies have been corrected for background IFNγ production in LCMV and unstimulated control cultures. (B) Spearman correlation between pH1N1-responding CD8 T cells and donor age. (C) Combinations of effector molecule expression of IFNγ⁺ CD8 T cells from the responder subset. P values above the bars indicate the level of statistical significance compared to all other bars as determined by ANOVA and Tukey test. (D) Spearman correlation between the CD8 T cell response to pH1N1 and the frequency of responding cells with multiple effector functions. (E) CD8 T cell response in case and control subjects. Groups were compared using a nonparametic Mann-Whitney test. (F) Spearman correlation for pH1N1 response and frequency of CD8 T cells with multiple effector functions in cases and controls.</p

Infection followed by vaccination boosts antibody but not T cell responses to pandemic H1N1.

<p>(A) Antibody titers against pH1N1 for vaccinated and unvaccinated donors in the entire cohort 8-10 months post-pandemic. Vaccinations were self-reported from October 2009 to January 2010. A non-parametric Mann-Whitney test was used for statistical significance. (B) CD8 and CD4 responses to pH1N1 for vaccinated and unvaccinated donors in the total Toronto cohort, measured 8-10 months post-pandemic. Groups were compared using a Mann-Whitney test. (C) IFNγ⁺ CD8 T cell responses in donors with both antibody and CD8 T cell responses, T cell responses only, antibodies only, or no antibody or T cell response to pH1N1. Data has been normalized using log transformation to represent Gaussian distribution; groups were compared using ANOVA and Tukey test. (D) Normalized CD8 T cell response in cases and controls with differing vaccination history for pH1N1. Groups were compared by ANOVA and Tukey test. PCR-confirmed infections were reported from April-November 2009; vaccination was self-reported from October 2009-January 2010. (E) Pandemic-specific antibody responses as measured by microneutralization in the case/control cohort, separated by self-reported vaccination history for the monovalent pH1N1 vaccine. PCR-confirmed infections were reported from April-November 2009; vaccination was self-reported from October 2009-January 2010. Nonparametric Kruskal-Wallis and Mann-Whitney tests were performed to determine statistical significance.</p

Detection of influenza-responsive CD8 T cells by multicolour flow cytometry.

<p>Total PBMC were stimulated for 18 hours with pH1N1 influenza, or as a control, with LCMV Armstrong, or left unstimulated and then assessed for IFNγ production by intracellular cytokine staining and flow cytometry. Gates are based on fluorescence minus one controls. (A) Representative gating used to identify IFNγ⁺ CD8 T cells from total PBMC. (B) Sample non-responder, weak responder, and strong responder to pH1N1 identified in the Toronto cohort 8-10 months post-pandemic; positive versus non-responder is defined in the results. A representative “weak” responder was arbitrarily chosen from the bottom third of positive responses whereas the “strong” responder was from the top third of responders.</p

Low 2012–13 Influenza Vaccine Effectiveness Associated with Mutation in the Egg-Adapted H3N2 Vaccine Strain Not Antigenic Drift in Circulating Viruses

<div><p>Background</p><p>Influenza vaccine effectiveness (VE) is generally interpreted in the context of vaccine match/mismatch to circulating strains with evolutionary drift in the latter invoked to explain reduced protection. During the 2012–13 season, however, detailed genotypic and phenotypic characterization shows that low VE was instead related to mutations in the egg-adapted H3N2 vaccine strain rather than antigenic drift in circulating viruses.</p><p>Methods/Findings</p><p>Component-specific VE against medically-attended, PCR-confirmed influenza was estimated in Canada by test-negative case-control design. Influenza A viruses were characterized genotypically by amino acid (AA) sequencing of established haemagglutinin (HA) antigenic sites and phenotypically through haemagglutination inhibition (HI) assay. H3N2 viruses were characterized in relation to the WHO-recommended, cell-passaged vaccine prototype (A/Victoria/361/2011) as well as the egg-adapted strain as per actually used in vaccine production. Among the total of 1501 participants, influenza virus was detected in 652 (43%). Nearly two-thirds of viruses typed/subtyped were A(H3N2) (394/626; 63%); the remainder were A(H1N1)pdm09 (79/626; 13%), B/Yamagata (98/626; 16%) or B/Victoria (54/626; 9%). Suboptimal VE of 50% (95%CI: 33–63%) overall was driven by predominant H3N2 activity for which VE was 41% (95%CI: 17–59%). All H3N2 field isolates were HI-characterized as well-matched to the WHO-recommended A/Victoria/361/2011 prototype whereas all but one were antigenically distinct from the egg-adapted strain as per actually used in vaccine production. The egg-adapted strain was itself antigenically distinct from the WHO-recommended prototype, and bore three AA mutations at antigenic sites B [H156Q, G186V] and D [S219Y]. Conversely, circulating viruses were identical to the WHO-recommended prototype at these positions with other genetic variation that did not affect antigenicity. VE was 59% (95%CI:16–80%) against A(H1N1)pdm09, 67% (95%CI: 30–85%) against B/Yamagata (vaccine-lineage) and 75% (95%CI: 29–91%) against B/Victoria (non-vaccine-lineage) viruses.</p><p>Conclusions</p><p>These findings underscore the need to monitor vaccine viruses as well as circulating strains to explain vaccine performance. Evolutionary drift in circulating viruses cannot be regulated, but influential mutations introduced as part of egg-based vaccine production may be amenable to improvements.</p></div

Three-dimensional model of antigenic-site differences between circulating H3N2 viruses and the 2012–13 egg-adapted A/Victoria/361/2011 IVR-165 high growth reassortant vaccine strain.

<p>One HA1 monomer is shown with five previously defined antigenic site residues of A–E colored in light green, dark green, light blue, dark blue and purple, respectively, mapped onto a related crystal structure (A/X-31(H3N2), PDB, 1HGG) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092153#pone.0092153-Sauter1" target="_blank">[27]</a> using PyMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092153#pone.0092153-The1" target="_blank">[28]</a>. The most prevalent antigenic site amino acid differences between circulating clade 3C viruses in Canada relative to the egg-adapted A/Victoria/361/2011 IVR-165 vaccine reassortant strain are shown in red and labelled with coloured font representing their antigenic sites, viewed from the front (A) or side (B). Three amino acid differences (Q156H, V186G and Y219S) are owing to mutation in the egg-adapted IVR-165 vaccine strain rather than circulating viruses which instead share identity with the MDCK-passaged WHO reference prototype at these positions. RBS indicates approximate location of the receptor-binding site.</p

Influenza specimens by week and subtype, 2012–13 sentinel surveillance period (N = 1682).

<p>NOTE: excludes specimens from patients failing to meet the influenza-like illness case definition or unknown; specimens collected >7 days after influenza-like illness onset or interval unknown; comorbidity unknown; age unknown or <1 year and influenza test results unavailable or inconclusive on typing. Missing collection dates were imputed as the laboratory accession date minus two days, the average time period between collection date and laboratory accession date for records with valid data for both fields. One specimen diagnosed with both A/H3N2 and A(H1N1)pdm09 in week 2 is not presented in the graph. Vaccine effectiveness analysis spans week 44 to week 18.</p

Specimen exclusion for influenza vaccine effectiveness analysis, Canada, 2012–13 sentinel surveillance system.

<p>NOTE: exclusions shown here in stepwise fashion to arrive at total case and control tally (i.e. those meeting multiple exclusion criteria are counted on the basis of the first exclusion criterion met in the list shown). Missing collection dates were imputed as the laboratory accession date minus two days, the average time period between collection date and laboratory accession date for records with valid data for both fields.</p