Introduction On 17 April 2009 a novel swine-origin influenza A (H1N1) virus was identified as the cause of two pediatric cases of febrile respiratory illness in California ,. Shortly thereafter, this virus was also identified as the cause of an outbreak of severe respiratory illness occurring among young people in Mexico in March and April ,. Subsequent spread throughout North America and elsewhere resulted in the declaration, on 11 June, of a phase 6 pandemic of the novel influenza A (H1N1) (now called pandemic influenza A (H1N1) [pH1N1]) by the World Health Organization (WHO) . Early surveillance and immunogenicity data and global summary by the WHO emphasized increased risk among young people 75% of community and hospitalized pH1N1 cases Household telephone survey of cases and controls conducted17 July to 10 August 2009 Medically attended pH1N1 test-positive non-hospitalized and hospitalized (>24 hours) patients with illness onset from 25 May to 1 July 2009 Population controls recruited through random-digit-dial to homes proportionate to number of cases per age group and region Non-hospitalized: 384 Hospitalized: 270 Community controls: 603 OR Age, comorbidity, sex, HCW status Hospitalized compared to nonhospitalized cases; 2007–08 TIV receipt; TIV receipt prior 5 y; age (50 y; 60 y) and comorbidity stratified. Ontario test-negative case-control Specimens submitted to Ontario Provincial Laboratory or Mount Sinai Hospital/University Health Network (Toronto) Household telephone survey of cases and controls conducted 1 August to 4 September 2009 Medically attended pH1N1 test-positive non-hospitalized and hospitalized patients with specimen submitted for influenza testing from 13 April to 20 July 2009 Medically attended pH1N1 test-negative non-hospitalized and hospitalized patients with specimen submitted for influenza testing from 13 April 1to 20 July 2009 Non-hospitalized: 250 Hospitalized: 136 Test-negative controls: 288 OR Age, comorbidity, sex, HCW status, specimen collection date (before/after 11 June), prior physician visits in past 12 mo. Number of children in household also explored. Hospitalized compared to nonhospitalized cases; 2007–08 TIV receipt; TIV receipt prior 5 y; age (50 y) and comorbidity stratified. Restriction based on test date within 4 d of symptom onset. Quebec household transmission study (prospective cohort) Quebec City, Quebec, Canada Study conducted 27 May to 10 July 2009 Secondary attack rates for respiratory symptoms, influenza-like illness and laboratory-confirmed pH1N1 in household members of confirmed pH1N1 case compared for vaccinated and unvaccinated children ( 50 years owing to small sample size, wide confidence intervals spanning 1, and further anticipated variation of effect with advancing age and immune status among older individuals ,. Estimates for crude and fully adjusted ORs for 2008–09 TIV effect on seasonal influenza for the period 17 April to 30 May 2009 remained 4 d) 0.34 (0.26–0.44) 1.07 (0.72–1.60) 0.47 (0.34–0.65) 1.91 (1.21–3.00) 0.34 (0.20–0.57) 0.42 (0.13–1.37) Age + chronic conditions 0.46 (0.35–0.62) 1.75 (1.10–2.79) 0.48 (0.34–0.67) 2.27 (1.37–3.76) 0.40 (0.23–0.71) 0.42 (0.13–1.39) Age+chronic conditions+province 0.44 (0.33–0.60) 1.62 (1.00–2.63) 0.47 (0.33–0.66) 2.16 (1.28–3.65) 0.34 (0.19–0.62) 0.41 (0.12–1.41) Age+chronic conditions+province+ interval 0.44 (0.33–0.59) 1.68 (1.03–2.74) 0.47 (0.33–0.66) 2.23 (1.31–3.79) 0.33 (0.18–0.61) 0.42 (0.12–1.46) Adjusted estimates by specification Restricted to period 17 May to 22 Julyb,c NA 1.69 (0.94–3.02) NA 2.45 (1.28–4.71) NA NSS Restricted to Quebec and Ontario onlyb,c 0.48 (0.32–0.72) 2.15 (1.14–4.04) 0.49 (0.30–0.80) 3.03 (1.54–5.97) NSS NSS Restricted to Quebec onlyb,c 0.49 (0.27–0.90) 2.66 (1.15–6.18) 0.61 (0.28–1.32) 4.50 (1.74–11.69) NSS NSS Restricted to Ontario onlyb,c 0.48 (0.27–0.83) 1.67 (0.65–4.31)† 0.47 (0.24–0.90) 2.04 (0.73–5.70) NSS NSS Restricted to adults 20–49 y onlyb,c NA NA 0.43 (0.28–0.68) 2.20 (1.16–4.18) NA NA Restricted only to those with no chronic conditionsb,c 0.43 (0.30–0.61) 1.48 (0.87–2.50) 0.42 (0.29–0.61) 2.39 (1.34–4.29) NSS NSS Vaccine status definition Immunized in 2007–08b,c,d (±2008–09) 0.72 (0.52–0.99) 1.58 (0.93–2.71) 0.79 (0.54–1.14) 1.89 (1.06–3.38) NSS NSS Immunized in 2007–08 but not 2008–09b,c,d 1.00 (0.63–1.60) 1.48 (0.63–3.44) 1.00 (0.60–1.66) 1.33 (0.54–3.23) NSS NSS Immunized in 2008–09 but not 2007–08b,c,d 0.23 (0.10–0.52) 1.91 (0.72–5.05) 0.17 (0.06–0.47) 2.06 (0.70–6.02) NSS NSS Immunized in 2007–08 and 2008–09 b,c,d 0.47 (0.32–0.69) 1.65 (0.92–2.95) 0.54 (0.34–0.86) 2.18 (1.15–4.14) NSS NSS a Specimens positive for seasonal influenza (29) or of unknown subtype (2) were excluded as controls from pH1N1 analysis period. b Adjusted for age as 1–8, 9–19, 20–49, 50–64, and ≥65 y, where possible, and 1–8, 9–49, ≥50 y where zero cells preclude adjustment with finer age categories (indicated by †); not further age adjusted for 20–49 years. Referent age category was the last age group included in each analysis, e.g., for overall analyses it was the category ≥65 y, and for the analysis restricted to those 4 d); d Includes data only from Alberta, Ontario and Quebec; BC did not collect information on 2007–08 immunization status. Children 1 for pH1N1 using the same methods during the same period to newly introduced bias. With its randomly selected community controls, the Quebec population case-control study likely provides the upper bound of a risk estimate for pH1N1 associated with TIV, since standardization of health care–seeking behavior between cases and controls was not afforded. Results from this design may be affected by differential characteristics among those who sought care or testing compared to community controls. The initial Quebec experience with the test-negative case-control design also provides an important caution regarding the potential for selection bias with this approach. This caution applies especially to test-negative designs when specimens are drawn from general laboratory submissions and/or when limitations are placed on testing, such as to individuals with chronic conditions or severe illness (see Text S2, Appendix B1a). Careful assessment of participant profiles showed that the test-negative controls in the initial Quebec study were not adequately representative of the source population from which the cases arose, although with appropriate adjustment for relevant covariates and restriction to patients with similar clinical presentation, the same increased risk of pH1N1 illness in association with 2008–09 TIV was found (Text S2, Appendix B1a–B1e). Ontario applied a later restriction on laboratory testing compared to Quebec (11 June versus 15 May), and the Ontario dataset showed test-negative controls to be representative of the case source population. Both cases and controls sought care and had similar health care–seeking behavior, as evidenced by the rate of physician visits in the previous 12 months and by the proportion with chronic conditions. Immunization rates among test-negative controls may have slightly exceeded population estimates for Ontario in young adults (tending to decrease the OR) but a statistically significant effect was still observed. Adjustment for other relevant covariates generally increased ORs among vaccinated participants. Sample sizes in Ontario were smaller than planned, especially for hospitalized cases, and confidence intervals surrounding ORs were wide, particularly among stratified analyses. Point estimates, however, remained consistent with other studies. A comparison between hospitalized and community cases in both the Quebec and the Ontario designs suggests that once pH1N1 illness was acquired, the risk of pH1N1 hospitalization was not further increased among TIV recipients. An increase in the absolute number of hospitalizations, however, would nevertheless still occur with this association, since that number is determined by the risk of becoming ill (increased) multiplied by the risk of hospitalization once ill (unchanged). Finally, with its active and prospective follow-up of all household members, the Quebec household transmission study provided corroborating evidence that was able to avert the potential for health care–seeking and other selection biases known to affect case-control studies. The results were consistent with other findings, and statistical significance was observed. A limitation of that study, however, is the small sample size that precluded further stratification. Confounding by indication may spuriously lower VE estimates in observational studies because influenza vaccine is administered to people at greater risk of clinical illness and more likely to seek care (such as those with chronic conditions) . More recent publications have also reported overestimation of influenza vaccine protection against serious outcomes among elderly persons as a result of better general health status among vaccinated compared to unvaccinated people (healthy user bias) ,. Influences on immunization status can thus skew estimates in either direction. It may be that in young people chronic conditions exert a greater influence in decreasing VE estimates (increasing ORs) than the counterforce of healthy user bias. Our national sentinel study population, however, included few participants with chronic conditions, and to further address this possible influence we applied recognized analysis techniques of restriction, stratification, and adjustment to all studies. We cannot rule out that these efforts were incomplete and that an unmeasured bias or residual confounding remains. Several studies from Australia , Mexico ,, and the US , have instead reported null or protective effects of 2008–09 TIV against pH1N1 illness based on test-negative case-control –, case-cohort , or ILI outbreak investigations . One other test-negative case-control analysis from a US outbreak found a statistically significant increased risk, with an unadjusted OR of 2.9 (95% CI 1.8–4.69) for pH1N1 illness among military beneficiaries who received influenza vaccine within the previous 12 months . Unlike in Canada, however, this association was driven primarily by receipt of live attenuated vaccine. Discrepant results across studies may reflect either differences in methods or real variation in the effect of specific vaccines, immunization programs, or population immunity. Most of the studies published to date, however, have not presented sufficient participant characteristics to properly assess methodological issues, sources of bias or confounding, or the validity of results. As previously highlighted, the importance of the public health and scientific implications requires that analyses of TIV effect on pH1N1 risk be more rigorous ,. At a minimum, a detailed profile of cases and controls specifically included in vaccine effectiveness analyses should be displayed by vaccine status, age, and chronic conditions, as well as timeliness of specimen collection and other recognized influences. Where participant characteristics have been presented by investigators, conspicuous evidence of selection bias can be seen to explain opposite findings of TIV protection against pH1N1 ,. In the detailed participant profiles we display for each of the four studies we report, evidence for this type of bias is not obvious, but that does not rule it out. We also cannot rule out the possibility that the increased risk of pH1N1 found in Canada was an effect specific to the Canadian vaccine: it is noteworthy that ORs were highest in Quebec, where a greater proportion of domestically produced vaccine is distributed than in the rest of Canada. However, even if our findings are considered a “Canadian problem,” if causal in nature they would still have wider implications for our understanding of influenza immunopathogenesis. In the event that our findings are valid and reflect a causal association by which prior receipt of TIV increased the risk of PCR-positive pH1N1 illness, several biological mechanisms have been considered to explain them. These proposed mechanisms must each be viewed as speculative since our epidemiologic studies were not designed to assess them. One hypothesis is that repeat immunization effectively blocks the more robust, complex, and cross-protective immunity afforded by prior infection. This mechanism was suggested by Hoskins et al. in the mid-1970s in evaluating the risk of A/H3N2 drift variants among previously infected versus immunized boys during successive boarding school outbreaks, with the consideration of other factors (such as viral neuraminidase [NA]) in addition to HA antibody . Bodewes et al. have more recently shown in mice that effective vaccination against human influenza A/H3N2 virus prevents virus-induced (cell-mediated) heterosubtypic immunity against severe (lethal) infection with avian influenza of a different subtype (A/H5N1) ,. That one influenza subtype may influence the risk of infection by another is also suggested by the subtype replacement that followed the pandemics of 1918, 1957, and 1968, although the mechanism for that is unknown . A BLAST sequence analysis demonstrates that pH1N1 and human influenza strains (A/H1N1 and A/H3N2, recent [since 2000] and historical [since 1974]) are nearly identical (95%–98%) with respect to their polymerase proteins, at both the overall and the antigenic levels, and other internal components, including M1 (94%–97%) and NP (85%–92%) are also well-conserved (Text S6, Appendix F). Conversely, both the HA and the NA surface proteins of recently circulating seasonal H1N1 influenza viruses and pH1N1 are more divergent, particularly with respect to their antigenic regions (Text S6, Appendix F). With a greater likelihood of seasonal influenza infection, unvaccinated people may have had a greater opportunity to develop cross-protective cell-mediated immunity to the more conserved internal viral components of pH1N1. Since those immunized in a given season may have been repeatedly immunized over several seasons, they may have lost multiple opportunities for infection-induced cross-immunity. TIV recipients may have boosted antibody to HA/NA (the only TIV components), effectively protecting against seasonal influenza, but without a cross-protective effect against the markedly different surface antigens of pH1N1. If this hypothesis explains our results, it also reassuringly implies that the TIV effect we have observed on pH1N1 risk would be induced again only if seasonal influenza circulates before pH1N1 and TIV blocks the potential cross-protection of that heterologous infection. There are, however, at least two considerations that oppose this hypothesis. First, the risk of laboratory-confirmed influenza illness per se—the outcome reported by our studies—is believed to be determined primarily by protective antibodies to viral surface proteins. As indicated above, the antigenic distance between the HA and NA surface proteins of pH1N1 and recently circulating human H1N1 strains is large, making cross-protection on that basis less likely. Differences in severe outcomes may be explained by cross-protective cell-mediated immunity, as shown by Bodewes et al. to be induced by prior infection with heterologous virus, but we did not detect differences in severe outcomes by vaccine status and that is not what we are trying to explain. The role of cell-mediated or other immune correlates of cross-protection against influenza illness per se likely warrants further study. Second, in order to show a 2-fold increase in pH1N1 illness, this hypothesis would require implausible assumptions of unreasonably high prior seasonal influenza attack rates, cross-protection against pH1N1 illness, and/or the effective block of that cross-protection by TIV (see Text S7 Appendix G). We have also considered the possibility of a direct immune mechanism to explain our results. One such mechanism is based on the induction of low-affinity/non-neutralizing but cross-reactive antibodies by TIV to the major surface proteins. Such antibodies are typically identified within an “original antigenic sin” response –, the relevance of which has long been debated for influenza, but for which Kim and colleagues have recently provided evidence in the mouse model . In a related concept, antibody-dependent enhancement (ADE) of virus replication may occur when preexisting low levels of weakly heterotypic antibodies (cross-reactive but not cross-protective) bind to virus but instead of neutralizing the virus, form bridging complexes that facilitate enhanced uptake and increased virus production by the target cell –. Original antigenic sin and ADE of virus replication have been described for secondary heterotypic dengue infections, but also in vitro for other viruses such as respiratory syncytial virus, Ebola, HIV, etc. –. ADE has also been shown in vitro for influenza uptake by macrophages, although these are not generally considered the main target cell for influenza replication –. Interestingly, vaccine-potentiated pneumonia has been reported following heterologous swine influenza challenge in pigs, and enhanced pneumonia due to heterologous swine influenza has also been reported in piglets vaccinated in the presence of maternal antibody, with ADE invoked by authors to possibly explain these findings ,. In further support of a direct vaccine-induced effect to explain our findings, a recent pH1N1 challenge study of influenza-naïve ferrets has shown that, compared to a nonimmunized control group, prior receipt of 2008–09 seasonal vaccine (notably live attenuated vaccine) was associated with slight worsening of day 3 upper airway viral loads, clinical disease, and mortality, with ADE also invoked by authors as a possible explanation . We did not find increased pH1N1 severity associated with seasonal vaccine in our epidemiologic studies; this observation may reflect intrinsically less virulent virus or, as described above, other intact viral clearance mechanisms . In considering the ADE hypothesis, it should be recognized that this proposed mechanism has not been previously linked to epidemiologic observations for influenza in humans. ADE may require a precise balance of antigenic distance and cross-reactive versus neutralizing antibody to be manifest. The 2009 pandemic differs from other influenza epidemics or pandemics in that it has been caused by a novel virus distantly related to but nevertheless within the same HA/NA subtype as recently circulating H1N1 viruses. The role of antibody concentrations in ADE is under debate, so it is difficult to know whether our findings during spring–summer 2009 in Canada, if explained in this way, would have been observed during fall–winter 2009–10 with (or without) repeat administration of TIV, or whether seasonal vaccines may differ in the relative proportion of cross-reactive versus cross-protective antibody induced to pH1N1. In the end, our results may seem counterintuitive, but they cannot be dismissed on the basis that no biological mechanism can plausibly explain them. The mechanism may be a combination of the above or an as-yet unknown pathway. Further empiric evidence would be necessary to support a specific mechanism. After the substantial autumn 2009 wave of pH1N1 and the mass pH1N1 vaccination campaign, seasonal influenza viruses remain a possible threat to consider for the rest of the 2009–10 winter. The complex benefit–risk analysis for receipt of 2009–10 TIV will ultimately depend upon the extent to which seasonal A/H1N1, A/H3N2, or B viruses contribute to TIV-preventable morbidity this season. It remains uncertain whether the influenza A subtype replacement observed with previous pandemics will also occur with this pandemic. Thus far (to 1 March 2010), seasonal strains have not comprised a substantial proportion of influenza detections in the northern hemisphere's 2009–10 season, with the exception of a recent increase in influenza B in China . For the next season, WHO has recommended that pH1N1 be included in seasonal vaccine formulations, thereby providing direct pH1N1 protection and obviating the possible risk we identified in association with the seasonal vaccine in 2009 ,. The possible scientific implications of our findings, however, will remain important to consider over the long term. These include questions about influenza immunopathogenesis, the interaction between seasonal and novel pandemic strains, the complex immunoepidemiologic aspects of influenza prevention and control, and how best to assess these issues experimentally and epidemiologically. In summary, we report findings from four epidemiologic studies in Canada showing that prior receipt of 2008–09 TIV was associated with increased risk of medically attended pH1N1 illness during the spring–summer 2009. Bias cannot be ruled out in observational studies, and therefore these findings cannot be considered conclusive. If these observations do reflect a real biological effect, however, they raise important questions that warrant further scientific investigation. Supporting Information Text S1 Appendix A: Sentinel test-negative case-control study - Additional details. (0.08 MB PDF) Click here for additional data file. Text S2 Appendix B: Quebec test-negative case-control study - Additional details. (0.08 MB PDF) Click here for additional data file. Text S3 Appendix C: TIV coverage by age category estimated from Canadian Community Health Survey (2007–08 season). (0.03 MB PDF) Click here for additional data file. Text S4 Appendix D: Quebec population case-control study - Additional details. (0.05 MB PDF) Click here for additional data file. Text S5 Appendix E: Ontario test-negative case-control study - Additional details. (0.05 MB PDF) Click here for additional data file. Text S6 Appendix F: Overall and antigenic region identity of 2009 pandemic H1N1 (pH1N1) proteins compared to representative recently circulating and vaccine strains of A/H1N1 and A/H3N2. (0.03 MB PDF) Click here for additional data file. Text S7 Appendix G: Theoretical analysis of whether a 2-fold increased risk of pH1N1 associated with prior seasonal influenza vaccination could be explained by TIV effectively blocking the heterosubtypic cross-immunity provided by prior seasonal influenza infection. (0.06 MB PDF) Click here for additional data file.