Interim estimates of 2013 / 14 vaccine effectiveness against influenza A ( H 1 N 1 ) pdm 09 from Canada ’ s sentinel surveillance network , January 2014

D M Skowronski (danuta.skowronski@bccdc.ca)1,2, C Chambers1, S Sabaiduc1, G De Serres3,4,5, J A Dickinson6, A L Winter7, K Fonseca6,8, J B Gubbay7,9, H Charest3, M Petric1,2, M Krajden1,2, S M Mahmud10,11, P Van Caeseele11,12, T L Kwindt1,2, A Eshaghi7, N Bastien13, Y Li11,13 1. British Columbia Centre for Disease Control, Vancouver, Canada 2. University of British Columbia, Vancouver, Canada 3. Institut National de Santé Publique du Québec (National Institute of Health of Quebec), Québec, Canada 4. Laval University, Quebec, Canada 5. Centre Hospitalier Universitaire de Québec (University Hospital Centre of Quebec), Québec, Canada 6. University of Calgary, Calgary, Canada 7. Public Health Ontario, Toronto, Canada 8. Alberta Provincial Laboratory, Calgary, Canada 9. University of Toronto, Toronto, Canada 10. Winnipeg Regional Health Authority, Winnipeg, Canada 11. University of Manitoba, Winnipeg, Canada 12. Cadham Provincial Laboratory, Winnipeg, Canada 13. National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Canada

The 2013/14 influenza season to date in Canada has been characterised by predominant (90%) A(H1N1) pdm09 activity.Vaccine effectiveness (VE) was assessed in January 2014 by Canada's sentinel surveillance network using a test-negative casecontrol design.Interim adjusted-VE against medicallyattended laboratory-confirmed influenza A(H1N1) pdm09 infection was 74% (95% CI: 58-83).Relative to vaccine, A(H1N1)pdm09 viruses were antigenically similar and genetically well conserved, with most showing just three mutations across the 50 amino acids comprising antigenic sites of the haemagglutinin protein.

Background
Since the 2009 pandemic, influenza A(H1N1)pfdm09 viruses have comprised a small proportion (<20%) of seasonal influenza virus detections each year in Canada [1].However, A(H1N1)pdm09 activity has recently resurged in North America, comprising more than 90% of detected influenza strains in both Canada and the United States (US) to mid-January of the 2013/14 season [1,2].This profile is in contrast to that of the same period last season in Canada, when 90% of detected strains instead belonged to the A(H3N2) subtype [3].
The 2013/14 trivalent influenza vaccine (TIV) for the northern hemisphere retains the same A(H1N1)pdm09 (A/California/07/2009-like) strain recommended since 2009 by the World Health Organization (WHO) [4].In response to substantial A(H1N1)pdm09 resurgence, interim 2013/14 vaccine effectiveness (VE) was assessed in January 2014 using Canada's sentinel surveillance network.VE estimates are discussed in the context of antigenic and genetic characterisation of circulating A(H1N1)pdm09 viruses.
Community-based practitioners at sentinel surveillance sites across participating provinces (British Columbia, Alberta, Manitoba, Ontario and Quebec) may offer nasal or nasopharyngeal swabbing to any patient presenting within seven days of symptom onset of ILI, defined as acute onset of respiratory illness with fever and cough and one or more of the following: sore throat, arthralgia, myalgia or prostration.
The analysis period included specimens collected from 1 November 2013 (week 44: 27 October 2013-2 November 2013) to 23 January 2014 (week 4: 19-25 January 2014), selected to account for influenza activity beginning in early November (Figure 1) and immunisation campaigns typically commencing in October.Epidemiological information was obtained from consenting patients or their parents/guardians using a standard questionnaire at specimen collection.Ethics review boards in participating provinces approved this study.
Specimens were tested for influenza A (by subtype) and B viruses at provincial reference laboratories using real-time RT-PCR.Odds ratios (OR) for medicallyattended, laboratory-confirmed influenza were estimated by multivariable logistic regression.VE was calculated as (1−OR)x100%.Patients for whom comorbidity status was unknown or for whom the timing of vaccination was unknown or less than two weeks before symptom onset were excluded from the primary analysis but explored in sensitivity analyses.Agestratified analysis and a study period beginning from week 49 (1-7 December 2013) to allow for additional vaccine uptake were also explored.

Genetic characterisation of sentinel influenza A(H1N1)pdm09 viruses
The haemagglutinin (HA) genes (HA1/HA2) from a convenience sample of sentinel influenza A(H1N1) pdm09 viruses from original patient specimens were sequenced for phylogenetic analysis and pair-wise amino acid (aa) identity based on antigenic maps spanning the 50 aa residues across HA1 antigenic sites Sa, Sb, Ca1, Ca2 and Cb [12,13].Findings were expressed as percentage identity to vaccine, calculated as (1− (number of aa substitutions in antigenic sites)/(total antigenic site aa residues))x100%.After removal of the signal peptide (residues 1-17), the approximate likelihood method was used to generate the phylogenetic tree of aligned nucleotide sequences in FastTree [14], visualised in FigTree [15], including reference HA sequences shown in Table 1.

Interim estimates of influenza vaccine effectiveness
A total of 1,091 specimens were submitted between 1 November 2013 and 23 January 2014.After exclusion ), we excluded from the epidemic curve specimens from the following patients: those failing to meet the influenza-like illness (ILI) case definition or for whom it was unknown (n=50), those whose specimens were collected more than seven days after symptom onset or for whom the interval was unknown (n=169), those whose age was unknown or less than one year (n=10), those with unknown comorbidity status (n=80), and those for whom influenza test results were unavailable or indeterminate (n=10).Specimens were included regardless of the patient's vaccination status or timing of vaccination.Excluded specimens may have more than one exclusion criterion that applies.Counts for each criterion will sum to more than the total number of specimens excluded.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.criteria were applied (Figure 2), 792 specimens were included in the primary analysis.

Virus characterisation
All A(H1N1)pdm09 isolates from Canada this season through week 4 (n=473, including 84 sentinel submissions) were identified by haemagglutination inhibition (HI) assay as antigenically similar to the A/California/07/2009 reference virus [1].Only two A(H1N1)pdm09 isolates and none of the tested sentinel viruses, showed eightfold or higher reduction in HI titres against the reference strain, signalling sporadic antigenic change in only a very small proportion (<0.5%) [1,19].
HA1/HA2 sequences of a subset of 76 of 287 (26%) sentinel A(H1N1)pdm09 viruses were also assessed, including four collected in November, 45 in December and 27 in January (Figure 3; Table 5).All 76 sequences clustered within the European Centre for Disease Prevention and Control (ECDC)-described clade 6B (Figure 3) [20], representing a switch from clade 6C viruses that predominated among A(H1N1)pdm09 viruses during the 2012/13 season, albeit at substantially lower levels than A(H3N2) viruses [21].a Excluded specimens may have more than one exclusion criterion that applies.Counts for each criterion will sum to more than the total number of specimens excluded.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.Median interval in days (range) 3 (0-7) 3 (0-7) 3 (0-7) 0.04 TIV: trivalent inactivated vaccine.
a Differences between cases and controls were compared using the chi-squared test, Fisher's Exact test, or Wilcoxon rank-sum test.b Patient's sex was missing for four specimens.c Chronic co-morbidities that place individuals at higher risk of serious complications from influenza as defined by Canada's National Advisory Committee on Immunization (NACI) [43], including heart, pulmonary (including asthma), renal, metabolic (such as diabetes), blood, cancer, immune comprising conditions or those that compromise the management of respiratory secretions and increase the risk of aspiration, or morbid obesity.Questionnaire was answered as 'yes,' 'no,' or 'unknown' to any of these conditions without specifying.d Vaccination status was based on self/parent/guardian report.Detail related to special paediatric dosing requirements was not sought.e Immunised participants were predominantly offered split (non-adjuvanted) 2013/14 trivalent inactivated influenza vaccine during the regular autumn immunisation campaign.In British Columbia and Quebec, influenza vaccine is provided free of charge to high-risk groups [43].Others are encouraged to receive vaccine but must purchase it.In Ontario, Alberta and Manitoba, the vaccine is provided free of charge to all residents aged six months or older. f In Canada, live-attenuated vaccine for nasal administration is approved for those aged two to 59 years [43] but its use remains infrequent.For the 2013/14 season (as of 23 January 2014), of 169 participants reporting vaccine receipt at least two weeks before symptom onset in this study, 149 reported this was given through muscular injection and five through nasal spray (of whom four were individuals younger than 20 years); route of administration was unspecified for 15 participants.g In Canada, MF59-adjuvanted vaccine is approved for people aged 65 years and older [43].For the 2013/14 season (as of 23 January 2014), of the 33 people aged 65 years and older who were immunised at least 2 weeks before symptom onset in this study, eight reported they had received the adjuvanted vaccine and 13 did not know, while 12 received the non-adjuvanted formulation.h Participants with unknown 2012/13 vaccine receipt and children younger than two years in 2013/14 were excluded from 2012/13 vaccine uptake analysis.Children younger than two years may not have been eligible for vaccination during the fall 2012/13 immunisation campaign on the basis of age under six months.Two egg-adapted A/California/07/2009 seed strains, NYMC X-179A and X-181, have been available to manufacturers for vaccine production since 2009, both identical in their antigenic site aa sequence to the WHO-recommended A/California/07/2009 reference strain (with a single substitution in a non-antigenic site (N129D) in X-181).Of the publicly supplied TIV in Canada, 70% was derived from X-179A and 30% from X-181.Sentinel viruses shared 90%-94% aa identity with the vaccine across antigenic sites, the majority showing 94% identity with the vaccine.All 76 sentinel sequences had the same three antigenic site mutations: K163Q (site Sa), a clade 6B marker, as well as S185T (site Sb) and S203T (site Ca1), both of which were also identified among dominant circulating A(H1N1) pdm09 viruses of the past two seasons [12,21].Five of 76 sequences bore a fourth aa substitution unique to each virus, and one Quebec sequence bore five substitutions (Table 5).Other than S185T, present in all 76 sequences, A186T, present in the single Quebec sequence, and possibly N156K and S157L [22], each present in a single and different Alberta sequence, none of the other substitutions were located within or adjacent to the receptor-binding site.With the exception of the single Quebec sequence, antigenic site mutations R205K, A141T, and A186T, which are located close to the receptor-binding site [22][23][24][25] and which occurred in 37%, 30% and 14%, respectively, of sentinel sequences during the 2012/13 season [21], were not evident in 2013/14.

Discussion
To date, the 2013/14 influenza season in North America has been characterised by substantial A(H1N1)pdm09 activity.This dramatic resurgence after only low-level circulation in the years since the 2009 pandemic has raised questions about possible virus evolution (i.e.antigenic drift) and reduced VE (i.e.vaccine failure).Our interim 2013/14 virological and VE analysis provides timely reassurance against both of these concerns.We show that circulating A(H1N1)pdm09 viruses are well-conserved based on genotypic and phenotypic characterisation, and that vaccine protection is substantial, reducing the risk of medically-attended laboratory-confirmed A(H1N1)pdm09 illness by about three quarters.
Our point estimate of ca 75% VE for the 2013/14 nonadjuvanted TIV against influenza A(H1N1)pdm09 is comparable, if not exceeding, 2009 estimates for nonadjuvanted formulations of the monovalent pandemic vaccine used in the US (ca 60%) [26,27], albeit lower than the 93% VE estimated by our sentinel system for the 2009 AS03-adjuvanted pandemic vaccine used in Canada [10].The 2013/14 mid-season VE estimate against influenza A(H1N1)pdm09 of ca 75% is in the upper range of recent seasons' VE estimates for nonadjuvanted TIV against A(H1N1)pdm09 reported since 2010 from Canada [11,12,21], Europe [28][29][30][31][32] and the US [33][34][35], which span ca 60-80%.With several times more influenza A(H1N1)pdm09 cases already contributing thus far in 2013/14 than in previous seasons in Canada, we are likely to converge upon a more stable and accurate estimate of TIV protection against A(H1N1) pdm09 infection this season.
Although a switch from clade 6C to clade 6B* occurred between the 2012/13 and 2013/14 seasons [21], A(H1N1) pdm09 viruses remain genetically and antigenically similar to the A/California/07/2009 vaccine strain, a somewhat surprising finding given that this virus has circulated globally since 2009.Historically, however, H1N1 compared with H3N2 subtype viruses generally have shown a slower pace of HA antigenic change, judging at least by the recommended updates to vaccine composition made by the WHO between 1990/91 and 2008/09 (five H1N1 versus 11 H3N2 vaccine strain switches), with two H1N1 (but no H3N2) strains retained as TIV components for at least seven consecutive years during that period [4,36].Genetic conservation of A(H1N1)pdm09 viruses may also be surprising in the context of population-level immune pressure.A   The phylogenetic tree was created by aligning the 76 Canadian sentinel sequences (colour-coded green for British Columbia, blue for Alberta, purple for Ontario and red for Quebec) against sequences representative of emerging viral clades as described by the European Centre for Disease Prevention and Control (ECDC) [20] (n=9), a random selection of A(H1N1)pdm09 sequences collected globally between 1 October 2013 and 21 January 2014 and obtained from the Global Initiative on Sharing Avian Influenza Data (GISAID) (n=43), and recent vaccine reference and egg-adapted seed strains (n=3).recent serosurvey conducted in May 2013 in Canada showed that levels of seroprotective antibody to A/ California/07/2009 were high among school-aged children and the elderly; however, seroprotection was lower among very young children and adults between 20 and 69 years of age [37].These findings may explain why conserved A(H1N1)pdm09 viruses resurged in 2013/14 and why there has been an apparent shift in the age distribution toward 20-64 year-old adults among medically-attended laboratory-confirmed influenza cases identified through the sentinel surveillance network this season.Such a demographic shift in disease burden toward adults following the 2009 pandemic was previously predicted in mathematical models from Canada [38] and warrants further empiric evaluation in additional datasets.
Limitations of the Canadian sentinel surveillance network for VE estimation have been described previously [3,[5][6][7][8][9][10][11][12].Although the validity of VE estimates derived by the test-negative has been demonstrated theoretically and in relation to randomised clinical trial analysis [39,40], the design remains observational, and bias and confounding cannot be ruled out.VE estimates for 2013/14 may vary at the end of the season, particularly since A(H1N1)pdm09 activity is still peaking in some regions of Canada [1].However, end-of-season estimates for the 2012/13 VE differed by less than 5% from interim results presented in midseason, even though the number of contributing cases increased by more than one third [3,21].Ongoing monitoring is nevertheless warranted for changes in virus and/or VE with further time across the season.Variable

Table 5
Amino acid changes in the haemagglutinin (HA1) genes (antigenic regions) a of a subset of 2013/14 Canadian sentinel influenza A(H1N1)pdm09 strains relative to vaccine reference strains b , Canada, a Antigenic regions Sa, Sb, Ca1, Ca2 and Cb comprise 50 amino acid residues [12,13].Only the nine positions in those 50 residues showing mutations in the present study are displayed.
b The northern hemisphere influenza A(H1N1)pdm09 vaccine reference strain since 2009, including the current 2013/14 season, is A/ California/07/2009.The two egg-adapted seed strains available to manufacturers for vaccine production (NYMC X-179A and NYMC X-181) are both identical in their antigenic site amino acid sequences to the A/California/07/2009 reference strain recommended by the World Health Organization.
Bold font signifies amino acid substitution compared with the 2013/14 northern hemisphere vaccine reference strain.
efficacy of repeated immunisation has previously been described, with differential effects depending upon the antigenic distance between successive vaccine components and circulating strains [41].In that context, as in previous years, we emphasise that a substantial proportion of our immunised participants are repeat recipients of unchanged A(H1N1)pdm09 vaccine antigen.Generalisability to regions with a different profile of vaccine uptake may be limited on that basis.In recent analyses, we [12] and others [29,30,42] have noted a trend toward improved VE with recurrent receipt of the A(H1N1)pdm09 antigen, although other studies have reported contrary findings [28,31,35].Assessment of these effects may benefit from the additional power available in end-of-season analysis.
In summary, our interim findings indicate that the 2013/14 TIV provides substantial protection against resurgent but conserved A(H1N1)pdm09 viruses circulating in Canada during the 2013/14 season, reducing the risk of medically-attended laboratory-confirmed A(H1N1)pdm09 illness by about three quarters.Neither antigenic drift nor homologous vaccine failure can account for resurgent A(H1N1)pdm09 activity this season in Canada.Other factors involved in agent-host interaction, including pre-existing antibody, should be considered in explaining the current epidemiology of this virus.

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ILI case definition not met or unknown (n=45) -Specimen collection date >7 days since ILI onset or ILI onset date unknown (n=156) -Vaccination <2 weeks before symptom onset or vaccination timing unknown (n=39) -Vaccination status unknown (n=24) -Age unknown or age <1 year (n=10) -Co-morbidity status unknown (n=74) -PCR results indeterminate (n=9) Specimens collected between 1 Nov 2013 and 23 Jan 2014 with data for primary analysis of vaccine effectiveness Specimens collected between 1 Nov 2013 and 23 Jan 2014

i
Participants with unknown 2011/12 vaccine receipt and children younger than three years in 2013/14 were excluded from 2011/12 vaccine uptake analysis.Children younger than three years may not have been eligible for vaccination during the fall 2011/12 immunisation campaign on the basis of age under six months.j Participants with unknown 2009 vaccine receipt and children younger than five years in 2013/14 were excluded from monovalent A(H1N1)pdm09 vaccine uptake analysis.Children younger than five years may not have been eligible for vaccination during the fall 2009 immunisation campaign on the basis of age under six months.More than 95% of the monovalent A(H1N1)pdm09 vaccine administered in Canada during the 2009 campaign was AS03-adjuvanted product[10].

Table 1a
Reference haemagglutinin sequences obtained from the EpiFlu database of the Global Initiative on Sharing Avian Influenza Data (GISAID) and used in phylogenetic analysis, 2013/14 sentinel surveillance network, Canada We acknowledge the authors, originating and submitting laboratories of the sequences from GISAID's EpiFlu Database on which this research is based.All submitters of data may be contacted directly via the GISAID website: www.gisaid.org.