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Beyond Antigenic Match: Possible Agent-Host and Immuno-epidemiological Influences on Influenza Vaccine Effectiveness During the 2015–2016 Season in Canada

Vaccine effectiveness estimates for 2015–2016 seasonal influenza vaccine are reported from Canada. Findings suggest that agent-host and immuno-epidemiologic factors beyond antigenic match—including viral genomic variation, birth (immunological) cohort effects, repeat vaccination, and potential within-season waning immunity—may influence vaccine performance.


SUPPLEMENT 1. Birth (immunological) cohorts defined by influenza A(H1N1) priming epochs SUMMARY
In their recent publication, Linderman et al. [1] emphasize amino acid position 166 (based on the A(H3) numbering scheme) located within pivotal antigenic site Sa atop the hemagglutinin (HA) surface protein of influenza A(H1N1) viruses as potentially immuno-dominant and influential on original antigenic sin (OAS) responses. Position 166 based on A(H3) numbering corresponds with position 163 based on the A(H1) numbering scheme [2] to which we instead preferentially refer throughout here for A(H1N1) viruses.
Authors hypothesize that first (priming) exposure to an A(H1N1) virus with a particular amino acid at position 163 (e.g. x163) induces a robust imprint that determines immunity to subsequent A(H1N1) viruses with a different amino acid at x163. Conversely, glycans (i.e. sugar residues) present nearby on the viral HA can mask that epitope, preventing antibody focusing or re-focusing toward x163 specificity and enabling a broader immune response to include other Sa epitopes or HA antigenic sites (such as Sb or Ca1). Authors predict immunological cohort effects based on priming epochs defined by exposure to a specific amino acid at x163 and driven by alterations in the gain or loss of N-linked glycosylation sites for which glycans may potentially shield or expose, respectively, x163 [1].
In 2012-13, a new clade of A(H1N1)pdm09 virus called clade 6B emerged that has the amino acid glutamine (Q) at position 163 (i.e. Q163) and that became the dominant circulating strain during the subsequent 2013-14 A(H1N1)pdm09 epidemic in the northern hemisphere. All previous A(H1N1) viruses since at least 1977 instead bore lysine (K) at that position (i.e. K163) [1].
Linderman et al. [1] hypothesize that individuals born between 1940 and 1984-notably between 1965 and 1979 (i.e. those aged between 37 and 51 years old in 2016)-and who were originally exposed to K163 viruses lacking glycan shielding would have mounted preferential K163-specific (but Q163-impaired) antibody responses. Authors also propose that annual administration of A/California/07/2009 A(H1N1)pdm09 vaccines that were consistently K163 since 2009 would have preferentially reinforced K163 specificity (as would A(H1N1)pdm09 infection prior to Q163 emergence in 2012-13).
In this Supplement, the priming epochs (or immunological cohort effects) proposed by Linderman et al. [1] are adapted for their potential effects on vaccine effectiveness (VE) during the 2015-16 season. Additional considerations related to lag time from birth or new variant emergence to priming exposure (ranging as much as 6-9 years [3][4][5]) as well as heterosubtypic exposures are also incorporated. Adjusted age groups (including children) are summarized in S1 Table 1 and explained in more detail in the ensuing pages: S1

x163 specificity
In 2012-13, A(H1N1)pdm09 viruses became Q163; prior to that, however, all A(H1N1) viruses were consistently non-Q at position 163 in so far as available archival sequences enable that determination. Influenza (H1N1) viruses had instead been consistently K163 since at least 1977 with more variability in the amino acid at that position (i.e. K/N/R/T/E) during the early to middle part of the last century (S1 Table 3).
In that context, preferential non-Q163 OAS responses could affect all birth cohorts prior to 2012-13, notably among those exposed to A(H1N1) viruses containing K163 after their reemergence in 1977. Variability by age in K163 specificity is hypothesized by Linderman et al. [1] to be driven by historic alterations in the location and efficiency of glycosylation sites potentially shielding K163.

Glycosylation: alterations and implications
A potential gain (or loss) of glycosylation is conferred to a protein whenever an amino acid change occurs that confers (or disrupts) the amino acid sequence "N-z-S/T", where N is the amino acid asparagine, z is any amino acid other than proline (non-P), and S/T is either serine or threonine [6][7][8]. The potential gain of a glycosylation site is often represented as +CHO; conversely loss of glycosylation is often represented as -CHO.
Glycosylation site alterations occur more often within the HA than the neuraminidase surface protein of influenza viruses and more frequently on the top of the HA head, proximal to the receptor binding site (RBS) and the pivotal antigenic site Sa of A(H1N1) viruses [6][7][8].
Few early A(H1N1) viral sequences exist, but historically, N-linked glycosylation sites affecting antigenic site Sa have involved amino acid positions 125 or 127, position 155, and positions 160 or 162 [6][7][8]. Potential glycosylation at position 155 (in the 1950s) has been debated but historic alterations and implications of N125/N127 and N160/N162 as emphasized by Linderman et al. [1] are outlined below: N125 or N127 These two glycosylation sites have been emphasized for their differential effects in shielding or exposing x163. N125 is located at the centre of antigenic site Sa and may shield it more effectively than N127 located instead adjacent to the Sa.
N125 and N127 were variously present in A(H1N1) viruses during the 1930s; N127 became more prominent in the 1940s and 1950s and from 1977 to 1985; whereas N125 replaced N127 from 1986 to 2008. A(H1N1)pdm09 viruses since 2009 have had neither N125 nor N127. Linderman et al. [1] propose that, through more effective K163 shielding, the gain of the N125 glycosylation from 1986 to 2008 may have blocked the induction of K163-specific priming during that period.
N160 or N162 Both N160 and N162 are located within antigenic site Sa, but N160 is in a more central Sa position, whereas N162 is located at its edge. Both glycosylation sites may shield not only the Sa of the same HA monomer but also part of antigenic sites Sb and Ca2 on the adjacent monomer of the HA trimer.

Glycan shielding of K163 between 1986 and 2008
As outlined above, glycan shielding of x163 likely became more efficient between 1986 and 2008. As per Linderman et al. [1] shift in the glycosylation site to position 125 located at the centre of the Sa, combined with the established glycosylation site at position 160 also at the Sa centre (i.e. N125 + N160), will have prominently masked the K163 epitope, preventing K163-specific priming. With the Sa site maximally shielded, antibodies might then also be directed toward other sites such as Sb.

Delay from birth to first influenza priming exposure
Potential x163-specific priming epochs determined by variation in the gain or loss of glycan shielding are shown in S1 Table 3 by birth cohort, translated into age (in years) for 2016. A lag from birth to first influenza priming exposure should also be taken into account in considering priming epochs and potential immunological cohort effects.
In a cross-sectional sero-prevalence study conducted in the Netherlands in 2006 and 2007 among children 1-7 years old, the proportion of children with detectable antibodies (titre ≥10) against influenza gradually increased with age until they reached 6-years old, when they all had antibodies to at least one influenza A virus [3]. However, sero-prevalence was higher for A(H3N2) than A(H1N1), consistent with A(H3N2) dominant epidemics between 1999 and 2006, with about three-quarters of children 7-years-old having detectable antibody to A(H1N1) strains. For children receiving influenza vaccine for the first time, expert recommendations in both Canada and the United States are to provide two spaced doses to children <9-years old but a single dose to previously unvaccinated children ≥9-years old for whom prior influenza priming is otherwise assumed [4,5]. The interval from birth to first influenza priming exposure may thus range by as much as 6-9 years around a particular birth annum and potentially longer in relation to a specific subtype or variant. We have chosen a 9-year lag to more clearly delineate distinct priming epochs as explained below.

Re-defining age groups based on potential K163-specificity, accounting for possible priming delay
Allowing as much as a 6-9-year lag to the first influenza infection, those within the birth cohort specified by the earliest YOB in a given column of S1 Variable K163-specificity is already predicted among those <8 years old (and >60 years old) owing to changes in glycan shielding or amino acid substitutions associated with those birth years. As per above, K163specificity is also variably anticipated among those 31-39 years old in 2016 (born 1977-1985) conditional upon the actual lag from birth to first influenza exposure, whereas those 40-59 years old will have had first A(H1N1) exposure with K163-specificity.
The choice of a 6-vs. 9-year lag interval is particularly relevant to the Linderman et al. [1] hypothesis in defining further age sub-strata among those born after 1985 (i.e. <31 years old), notably those born before 2009 (without K163-specificity) or since 2009 (variably with/without K163-specificity)(see S1 Table 3).
We used a 9-year (rather than 6-year or less) lag time in order to exclude those potentially with K163specificity (i.e. those <17 years old) from the group without such priming (i.e. those 17-30 years old) after 1985. We further explored based on a 6-year lag (i.e. using age categories <14 years old and 14-30 years old) although this may have increased misclassification by including individuals potentially with K163-specificity in the group otherwise predicted to be without K163-specificity (potentially lowering VE). Ultimately, however, we had limited precision to discern differences by choice of 9-or 6-year lag interval (see footnotes 7 and 8 in Supplement 15).
Accordingly, in 2016 1,2 (and as summarized in S1 Table 1 and detailed in S1 Table 3): 1. K163 priming specificity should not be evident in people born between 1986 and 1999 3 (i.e. 17-30 years old) because of effective glycan shielding by N125 + N160 among A(H1N1) viruses to which they were earlier primed in childhood. K163 specificity may be variously expressed in those <17 years old depending upon actual age of priming. Immunity to a broader array of A(H1N1) epitopes may be anticipated vs. older cohorts.
2. K163 priming specificity may be evident in people born before 1986 (i.e. ≥31 years old) due to absent or less effective glycan shielding among A(H1N1) viruses to which they were earlier primed in childhood. K163 specificity may be prominent in those born between 1957 and 1976 (i.e. middle-age approximately 40-59 years) but also variously expressed in those 31-39 years and in older adults depending upon the actual age of A(H1N1) priming.
These effects on age-related risk were originally proposed by Linderman et al. [1] for 2013-14 but may be more pronounced in 2015 if additional doses of annual K163-containing A/California/07/2009 vaccine further reinforced K163 specificity. The re-appearance of N162 glycosylation in clade 6B.1 viruses in 2015 may have also partially shielded the Sa, potentially reducing 2015-16 VE against A(H1N1)pdm09 overall but especially in adults ≥31 years old with a greater dependency on antibody directed toward that site. Immunity to other epitopes (non-x163 directed) in younger individuals may provide compensatory protection.

S1 Table 3. Potential N-linked glycosylation effects on x163 epitope priming by birth cohort and age in 2016
N= asparagine -conferring N-linked glycosylation through the amino acid codon "N-z-S/T "where "z" is any amino acid other than proline and S/T is either one of serine or threonine; K= lysine; R= arginine; T= threonine; E=glutamic acid; Q=glutamine

CAVEATS
Linderman et al. [1] report greater K163 specificity in sera from middle-aged adults born between 1940 and 1984 (notably 1965 to 1979) compared to adults born after 1985. This interpretation is based on two-fold differences in hemagglutination inhibition (HI) assay titres to K163 vs. Q163 viruses (reinforced through further mouse/ferret investigations). Heterotypic effects or other potential contributors to immunity, alone or in combination were not considered although, as authors highlight, other variation even in nearby positions may affect HI findings. Heterosubtypic effects, particularly among birth cohorts 1957-1976, were not considered at all. As also acknowledged, differences of twofold dilution may be considered within the margin of error for the HI assay, and only individuals with ≥2-fold differences on all three triplicate assay repeats were considered to have K163-specific responses. Authors indicated (but did not show) similar pattern based on 4-fold or 8-fold titre differences. More generally, however, the limitations of the HI assay in predicting immunity are well recognized. The human sera used in this study were collected from <200 adults across birth cohorts ranging from 1940 to 1997 as surrogates for possible x163 priming epochs; however, limited methodological details (e.g. sampling frame) or epidemiological characteristics (e.g. beyond age) were provided for interpretation and children were not included. Sequencing is available for only a small subset of viruses historically so that the timing of potential gain/loss of glycosylation or x163specificity are difficult to conclude. Viral sequences submitted to public databases are limited, and may not be representative, particularly in defining more distant epochs. Finally, influenza priming infection may vary several years-assumed by as much as 6-9 years around a birth annum but likely shorter but potentially longer in relation to a particular subtype or variant or in adults with less social contacts or exposure opportunities. Such lag may obscure the precise demarcation of priming epochs or immunological cohort effects within the population. We have explored the influence of K163specific priming epochs on A(H1N1)pdm09 VE estimates given the Linderman et al. [1] report and influence, but underscore this remains a hypothesis only.

Influenza diagnosis
Specimens were tested for influenza viruses using RT-PCR assays validated for use at participating provincial reference laboratories. This includes in-house assays in Alberta [1], and in Ontario where targets included the M gene of influenza A and NS1 gene of influenza B, with influenza A(H1N1)pdm09 and A(H3N2) subtyping by real-time RT-PCR targeting the HA gene (CDC protocol) or an in-house assay targeting the NA gene of A(H1N1)pdm09 [2][3][4]. In British Columbia a combination of in-house and commercial assays were used [5][6][7] and in Quebec a commercial assay was used supplemented by in-house assay for confirmatory testing when results were inconclusive by commercial assay [2,7].

Antigenic characterization
A subset of RT-PCR-positive specimens was inoculated into Madin-Darby Canine or Rhesus Monkey Kidney cell cultures for virus isolation by provincial public health reference laboratories. Aliquots of virus isolates were submitted to Canada's National Microbiology Laboratory (NML) for antigenic characterization by HI assay using turkey or guinea pig erythrocytes and post-infection ferret anti-sera raised against egg-and/or cell-passaged reference strains, as specified [8]. For A(H3N2) viruses, HI assays were conducted in the presence of 20nM oseltamivir following re-growth in SIAT cells [9][10][11]. A ≥8-fold reduction in post-infection ferret HI-antibody titre raised to a given reference strain and tested against a field isolate was interpreted as antigenic distinction [8].

Sequence analysis
Sequencing was attempted on HA1 genes for influenza A and HA1/HA2 genes for influenza B viruses from original patient specimens included in VE analysis. Phylogenetic analysis using the approximate-likelihood method determined clade distribution. Deduced amino acids of HA1 were aligned in FastTree [12] and visualized in FigTree [13]. Global reference sequences, including vaccine and clade reference viruses, were obtained from the Global Initiative on Sharing All Influenza Data (GISAID) (www.gisaid.org).
Amino acid differences from the egg-adapted vaccine strain at key antigenic sites were interpreted according to previously published maps labelled A-E for A(H3N2) and Sa, Sb, Ca1, Ca2, and Cb for A(H1N1)pdm09 [14][15][16][17]. The antigenic site map for influenza B is shown below in S2 Table 4 based on B(Victoria) numbering with the signal peptide removed [16]. Subtraction of one amino acid where appropriate is required for the corresponding B(Yamagata) antigenic site map (as previously published in [17]).
Amino acid substitutions close to the receptor-binding site (RBS) and involving antigenic site A and immunodominant site B of A(H3N2) viruses or their equivalents for H1 (Sa and Sb) and influenza B (160 loop and 190 helix) are considered most relevant to antigenicity or immunogenicity [9,14,17]. Substitutions associated with potential gain/loss of N-linked glycosylation are also emphasized for their potential effects in masking/uncovering antibody epitopes [18]. S2 Table 4. Influenza B(Victoria) map (n=73 residues) shown as amino acid number of the HA1 domain, with signal peptide (n=15 residues) removed [16] Antigenic site ( Table 5). vaccination was also explored-about 95% of monovalent A(H1N1)pdm09 doses distributed in Canada in 2009 were an AS03-adjuvanted formulation [1].

Stratified and exploratory cohort analyses
Stratified analyses were primarily conducted using an interaction term for vaccination status by the stratification variable, including standard age grouping (children: 1-19 years; non-elderly adults: 20-64 years; elderly adults: ≥65 years old), month (January-April), or epidemic-period defined as early (January-February) or late (March-April).
Age re-grouping was also undertaken for A(H1N1)pdm09 based on predicted priming epochs in order to explore potential immunological cohort effects, as defined in Supplement 1. Adjusted VE was explored for these regrouped age strata modeled using an interaction term for age group*vaccination status, and also by deriving separate adjusted VE estimates for subsets of data based on the stratification variable (i.e. age group).
To further explore these effects, VE by age in years was also modeled for A(H1N1)pdm09 with age smoothed as a restricted cubic-spline function using 5 knots based on percentiles (as per [2]) and an interaction term for single year of age by vaccination status.
The model with 5 knots had slightly improved fit (i.e. lower Akaike Information Criterion (AIC) values) than with 3 or 7 knots, the latter also presented in sensitivity analyses (see

Supplement 16).
Year of birth (YOB) was derived based on recorded age in years in 2016.
Owing to sparse data in the very old, additional age-based evaluations were subset to participants 1-76-years-old in 2016 (i.e. YOB=1940-2015).
All analyses were performed using SAS version 9.4 (SAS Inc., Cary, NC).

. Influenza detections by type/subtype and week of specimen collection, 2015-16 influenza vaccine effectiveness evaluation, Canadian Sentinel Practitioner Surveillance Network (SPSN)
Epidemic curve is based on the same exclusion criteria as the primary analysis. Study period for primary analysis from January 3 to April 30, 2016 (week 1-17) is indicated. Missing collection dates were imputed as the date the specimen was accessioned at the provincial laboratory minus two days, the average time between specimen collection and accession date among specimens with complete information for both values.

SUPPLEMENT 6. Phylogenetic tree -influenza A(H1N1)pdm09, 2015-16 season S6 Figure 2. Phylogenetic tree of sentinel influenza A(H1N1)pdm09 viruses contributing to 2015-16 vaccine effectiveness analysis by the Canadian Sentinel Practitioner Surveillance Network (SPSN)
Deduced amino acids of hemaggultinin (HA1) were aligned in FastTree 1 and visualized in FigTree 2 . Substitutions in bold are located in antigenic sites; those in italics are located in the receptor binding site. Vaccine virus and clade reference virus sequences were obtained from the Global Initiative on Sharing All Influenza Data (GISAID) (www.gisaid.org).      Exclusion criteria for both 2013-14 and 2015-16 are the same as specified for primary analysis in 2015-16 but subset to participants ≥9 years old and with complete information for current and prior season's vaccine receipt. Note that 2013-14 season's estimates span November-April whereas, owing to the delayed epidemic in 2015-16, estimates for the latter were subset to January-April.