Main input parameter values for Greater Vancouver Area (GVA) contact network model.

<p>HI = hemagglutination inhibition assay; SPR = Sero-protection rate – defined as the proportion (%) considered sero-protected on the basis of having met or exceeded the specified antibody titre threshold.</p><p><b>pre-H1N1pdm09</b>: 2009 H1N1 pandemic virus; SPR based on PRE-pandemic antibody levels in 2009 or earlier.</p><p><b>post-H1N1pdm09</b>: 2009 H1N1 pandemic virus; SPR based on POST-pandemic antibody levels fall 2010.</p><p><b>H3N2v:</b> swine-origin H3N2 variant strain; SPR based on antibody levels in fall 2010.</p><p><b>post-Brisbane</b>: a seasonal human influenza H3N2 virus; SPR based on antibody levels fall 2010.</p>a<p>Statistics Canada 2006 Community Profiles [<a href="http://www12.statcan.ca/census-recensement/2006/dp-pd/prof/92-591/index.cfm?Lang=E" target="_blank">http://www12.statcan.ca/census-recensement/2006/dp-pd/prof/92-591/index.cfm?Lang=E</a>].</p>b<p>The same typical seasonal influenza parameter values were applied to each of the three influenza viruses assessed, recognizing their human influenza virus ancestral origins.</p>c<p>Based on 100% SPR defined at HI titre ≥40; ^dBased on 50%SPR defined at HI titre ≥40; ^e100%SPR defined at HI titre ≥80.</p><p>Note that a blended composite of sero-protection based on a gradient of immunity defined as <b>¼</b>SPR20, ½SPR40, ¾SPR80 and 100% SPR160 was also explored (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054015#pone.0054015.s003" target="_blank">Table S1</a> and narrative).</p

Age-stratified epidemic attack rates by scenario and assumed basic reproduction number (<i>R₀</i> = 1.40). SPR

<p> = Sero-protection rate – defined as the proportion (%) considered sero-protected on the basis of having met or exceeded the specified antibody titre threshold; <b>pre-H1N1pdm09:</b> 2009 H1N1 pandemic virus; SPR presented based on PRE-pandemic antibody levels measured in 2009 or earlier; <b>post-H1N1pdm09:</b> 2009 H1N1 pandemic virus; SPR presented based on POST-pandemic antibody levels measured in fall 2010; <b>H3N2v:</b> swine-origin H3N2 variant strain; SPR presented based on antibody levels measured in sera collected in fall 2010; <b>post-Brisbane:</b> a contemporary seasonal human influenza H3N2 virus; SPR presented based post-circulation antibody levels in sera collected in fall 2010; <b>SPR40:</b> the proportion considered sero-protected according to the standard hemagglutination inhibition (HI) titre threshold of 40; <b>½SPR40:</b> assumes half the individuals meeting SPR40 are considered sero-protected; <b>SPR80:</b> the proportion considered sero-protected according to a hemagglutination inhibition (HI) titre threshold of 80 Overall attack rates are indicated by the horizontal line. Based on simulations using age-specific parameters, these overall attack rates were derived as the total number of infections divided by the total population size.</p

Effective reproduction number (<i>R_eff</i>) according to various scenarios of population immunity by initial basic reproduction number (<i>R₀</i>) assumed.

<p><b>SPR</b> = Sero-protection rate – defined as the proportion (%) considered sero-protected on the basis of having met or exceeded the specified antibody titre threshold; <b>pre-H1N1pdm09:</b> 2009 H1N1 pandemic virus; SPR presented based on PRE-pandemic antibody levels measured in 2009 or earlier; <b>post-H1N1pdm09:</b> 2009 H1N1 pandemic virus; SPR presented based on POST-pandemic antibody levels measured in fall 2010; <b>H3N2v:</b> swine-origin H3N2 variant strain; SPR presented based on antibody levels measured in sera collected in fall 2010; <b>post-Brisbane:</b> a contemporary seasonal human influenza H3N2 virus; SPR presented based post-circulation antibody levels in sera collected in fall 2010; <b>SPR40:</b> the proportion considered sero-protected according to the standard hemagglutination inhibition (HI) titre threshold of 40; <b>½SPR40:</b> assumes half the individuals meeting SPR40 are considered sero-protected; <b>SPR80:</b> the proportion considered sero-protected according to a hemagglutination inhibition (HI) titre threshold of 80.</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

Study protocol.

<p>Randomized blinded placebo-controlled experiment of Canadian manufactured, commercially-available 2008–09 trivalent inactivated influenza vaccine (TIV: Fluviral) on A(H1N1)pdm09 disease risk in ferrets.</p

HA1 microarray serological values by study antigens, group and day.

<p>Box plots display median (dash) and mean (dot) of log₁₀-transformed HA1 protein microarray signal values. The box extends to the 25^th/75^th percentiles and whiskers extend to minimum/maximum values. H1-07 indicates A/Brisbane/59/2007 (H1N1)-like; H3-07 indicates A/Brisbane/10/2007 (H3N2)-like; H1-09 indicates A/California/7/2009 (H1N1)pdm09-like (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086555#pone.0086555.s004" target="_blank">Table S3</a>;</b> grey-shaded). Sample size as follows: Pre-immunization Vaccine = 15, Placebo = 16 (3 ferrets each per group pre-shipment serum was substituted owing to insufficient day 0 available); Day 28 Vaccine = 14, Placebo = 15; Day 49 Vaccine = 12, Placebo = 11; Day 54 Vaccine = 2, Placebo = 4; Day 63 Vaccine = 9, Placebo = 8. **indicates statistical significance at p<0.01 and *indicates statistical significance at p<0.05 in comparing vaccine to placebo group at the designated time point. ΔΔ indicates statistical significance at p<0.01 and Δ indicates statistical significance at p<0.05 in comparing values within study groups at days 28, 49, 54 and 63 relative to pre-immunization, colour coded by vaccine (red) or placebo (blue). □□ indicates statistical significance at p<0.01 and □ indicates statistical significance at p<0.05 in comparing day 63 to day 49 within groups, colour coded per above by study group.</p

Clinical outcomes including weight loss, nasal wash and lung virus titers by study group and day.

<p>Clinical outcomes are displayed including: (A) Mean percentage weight relative to baseline by study group and day, with standard errors. (B) Nasal wash virus titers by study group and day. (C) Lung homogenate virus titers at day 5 post-challenge. Box plots (B and C) display mean (dot) and median (line) virus titres as log pfu/mL. Per usual, the box extends to the 25^th/75^th percentiles and whiskers extend to minimum/maximum values. Ch refers to challenge day and Ch+1, Ch+2 etc indicate day post-challenge (i.e. day one post-challenge, day two post-challenge etc). Ch+5 indicates day five post-challenge on which four animals per group were randomly selected for sacrifice. Statistically significant between-group differences are as specified.</p

Influenza A antibody results by study day and group based on ELISA assay.

<p><b>Competitive Nucleoprotein-based IDEXX ELISA (Influenza A antibody). Ratios <0.60 classified as positive; ratios ≥0.60 classified as negative.</b></p><p>ND = Not done; IDEXX Inc. = Commercial ELISA assay (note the lower the ratio, the greater the antibody detected); Ch = challenge.</p>a<p>Where numbers tested differ from the number randomized per group in parentheses at the specified time point it is because insufficient sera remained for testing of all animals.</p>b<p>Number of sera tested by group shown in adjacent columns.</p>c<p>One day 0 serum in each group was insufficient for ELISA testing and substituted with pre-shipment values for these ferrets. Excluding these ferrets (leaving n = 15 per group) gives ELISA ratios of 1.04 (0.99–1.09) and 1.05 (1.01–1.09) for vaccine and placebo groups, respectively.</p>d<p>Two sera belonging to placebo group close to serologic threshold for positivity with ratios of 0.61.</p

Haemagglutinin antigenic site differences in circulating H3N2 viruses relative to the 2012–13 egg-adapted A/Victoria/361/2011 IVR-165^a high growth reassortant vaccine strain.

<p>N = number of sentinel viruses with that sequence. Bold font signifies amino acid (AA) substitutions compared with IVR-165. Clade designation, number of antigenic site differences and percent antigenic site pairwise identity are also displayed. Only the 31/131 antigenic site positions showing differences between circulating H3N2 viruses and IVR-165 are displayed. AA sequences at those positions for other recent vaccine viruses are also displayed.</p>a<p>. IVR-165 is the egg-adapted high growth reassortant strain substituted by manufacturers for the MDCK-passaged A/Victoria/361/2011 (H3N2) prototype virus recommended as 2012–13 vaccine component by the World Health Organization (WHO) designated here as A/Victoria/361/2011 (MDCK).</p>b<p>. Number of antigenic site AA differences and percent antigenic site identity relative to IVR-165. Percent identity derived as per <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092153#pone.0092153.s009" target="_blank">Text S1</a></b>.</p>c<p>. A/Victoria/210/2009 (X-187) is the egg-adapted high growth reassortant strain used by manufacturers for the 2011–12 influenza vaccine for the northern hemisphere., shown for historic comparison.</p>d<p>. A/Texas/50/2012 (MDCK) and (X-223) are the WHO-recommended prototype and egg-adapted high growth reassortant strains, respectively, for the 2013–14 influenza vaccine for the northern hemisphere, shown for added comparison.</p

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