Pandemrix® vaccination is not associated with increased risk of islet autoimmunity or type 1 diabetes in the TEDDY study children

Clinical diagnosis of type 1 diabetes is preceded by an autoimmune destructive process against the pancreatic islet beta cells; a prodromal period that may last a few months or several years. The risk of developing autoimmunity in genetically susceptible children is widely considered to be increased by perinatal or early childhood environmental exposures [1]. While genetic risk factors, such as HLA-DR-DQ [2] and non-HLA genetic factors [3], have been implicated, the environmental conditioning or trigger exposures have not yet been defined. In The Environmental Determinants of Diabetes in the Young (TEDDY) study, we have the opportunity to analyse triggers in relation to seroconversion to islet autoimmunity as a first primary endpoint, as over 70% of children with multiple islet autoantibodies develop type 1 diabetes over a 10-year period [4]. It has been speculated that vaccinations early in life may alter the immune response to infections, leading to a disturbed capacity to distinguish between self and non-self and thereby increasing the risk of autoimmune reactions. However, previous studies do not support the notion that type 1 diabetes can be triggered by vaccinations [5, 6].

During the H1N1 influenza pandemic in 2009–2010, mass vaccination of both children and adults took place in many countries. In Sweden and Finland, Pandemrix®, a vaccine containing the squalene-based adjuvant ASO3, was used while other countries used other types of vaccine. A few months after the Pandemrix® vaccination programme, the incidence of new narcolepsy diagnoses increased sharply in both countries. Additional investigations seeking an understanding of the potential mechanisms associated with Pandemrix® and narcolepsy suggested that the Pandemrix® vaccination might have resulted in the loss of orexin-producing neurons, leading to the development of narcolepsy in these individuals [7, 8, 9]. The mechanism that mediates this effect is not fully understood, but it seems that both the composite influenza virus vaccine and the squalene adjuvant could contribute to the induction of orexin-specific autoimmunity [10].

The aim of the present study was to investigate if exposure to the Pandemrix® vaccine affected the incidence of islet autoimmunity and type 1 diabetes in genetically susceptible children. This was an observational study carried out among high-risk children followed prospectively from birth [11].

Of the 3401 children considered at risk for type 1 diabetes as of 1 October 2009, 2413 (70.9%) were vaccinated with Pandemrix®. Of the children vaccinated, 98.8% (2385/2413) received their first vaccination between 1 October 2009 and 31 March 2010, five had their first vaccination in September 2009 and 22 had their vaccination between 1 April and 5 August 2010. Of the children vaccinated in Sweden, 72.9% (1010/1385) received a second Pandemrix® vaccination within a median 2.0 (interquartile range 1.5–3.0) months; however, only 0.6% (6/1028) of children from Finland received a second vaccination. The vaccination coverage by country and age of the child on 1 March 2010 (all children born) is shown in Fig. 2. In both Finland and Sweden, the coverage was low before the age of 6 months (all p < 0.001) and children were more likely to receive the vaccine if the mother had a college degree when the child was 9 months of age (see electronic supplementary material Table 1).

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There was no increased risk of any islet autoantibody (HR 0.76 [95% CI 0.57, 1.02]), multiple islet autoantibodies (HR 0.92 [95% CI 0.63, 1.35]) or type 1 diabetes (HR 0.68 [95% CI 0.43, 1.06]) among the vaccinated children. In contrast, the risk of any islet autoantibody (HR 0.50 [95% CI 0.32, 0.78]), multiple islet autoantibodies (HR 0.56 [95% CI 0.33, 0.96]) and type 1 diabetes (HR 0.41 [95% CI 0.23, 0.76]) was decreased among vaccinated children in Finland, but this was not seen in Sweden (HR 1.01 [95% CI 0.68, 1.50], HR 1.41 [95% CI 0.82, 2.46] and HR 1.10 [95% CI 0.53, 2.24], respectively) (Table 1). HLA genotype, age at vaccination, sex or family history of type 1 diabetes did not modify the association between H1N1 vaccination and outcomes (Table 1).

The cumulative incidence of each of the outcomes in the two countries after vaccination or while considered at risk after 1 October 2009 is shown in Fig. 3. In Finland, the decreased risk of islet autoimmunity and multiple islet autoantibodies for the vaccinated children was seen primarily between 6 months and 36 months after the first vaccination. The cause-specific risk of IAA as a first solitary autoantibody, before other islet autoantibodies appear, primarily occurred in younger children and tended to be more frequent in Finland than in Sweden [17]. Therefore, we examined whether the incidence of IAA or GADA as first appearing solitary autoantibody separately, or any islet autoantibody, in Finland and Sweden, in both younger and older children shortly after vaccination were influenced by the vaccination after adjusting for previously reported risk factors. Genetic risk factors previously found to be associated with islet autoimmunity, including INS-23Hph1 (rs689), PTPN22 R620W (rs2476601), CTLA4, T17A (rs231775), SNPs rs2292239 in ERBB3, rs3184504 in SH2B3, rs10517086 and rs12708716 in CLEC16A, and rs4948088 in COBL [18] were included in this analysis, as well as probiotic use before 90 days of age [19], maternal age at birth of child and maternal education (Table 2). After adjustment, the HR for islet autoantibodies (0.47 [95% CI 0.29, 0.75]), for multiple islet autoantibodies (0.50 [95% CI 0.28, 0.90]) and for type 1 diabetes (0.38 [95% CI 0.20, 0.72]) remained significantly decreased in Finland. Moreover, cause-specific risk of IAA as the first appearing islet autoantibody was decreased, particularly among children vaccinated at younger than 2 years of age (Table 2).

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Table 2

Association between H1N1 vaccination and type 1 diabetes outcomes before age 10 years adjusting for sex, family history of type 1 diabetes, probiotics before 90 days, maternal education, maternal age, HLA-DR-DQ high-risk genotypes, INS-23Hph1 (rs689), PTPN22 R620W (rs2476601), CTLA4 T17A (rs231775), SNPs rs2292239 in ERBB3, rs3184504 in SH2B3, rs10517086 and rs12708716 in CLEC16A, and rs4948088 in COBL

Variable	Multiple proportional hazard model of H1N1 on type 1 diabetes outcomes
	First appearing islet autoantibodies						Multiple islet autoantibodies		Type 1 diabetes
	IAA		GADA		Any
	HR	p value	HR	p value	HR	p value	HR	p value	HR	p value
Adjusteda	0.64	0.10	0.78	0.29	0.75	0.07	0.85	0.41	0.67	0.09
Finland
Adjusteda	0.38	0.01	0.55	0.12	0.47	0.002	0.50	0.02	0.38	0.003
< 2 yearsb	0.30	0.009	1.18	0.79	0.51	0.03	0.45	0.04	0.25	0.002
≥ 2 yearsb	0.52	0.35	0.51	0.14	0.51	0.06	0.58	0.21	0.62	0.34
Sweden
Adjusteda	0.88	0.70	0.98	0.96	1.03	0.89	1.33	0.34	1.19	0.67
< 2 yearsb	0.79	0.57	1.08	0.87	0.96	0.88	1.31	0.46	1.81	0.33
≥ 2 yearsb	0.32	0.15	1.12	0.81	0.97	0.94	1.33	0.57	0.82	0.70

^aVariable included in multivariate proportional hazards model; all variables available on 2966 children at risk of islet autoantibodies, of which 214 developed islet autoantibodies; 3037 observed at risk for multiple islet autoantibodies, of which 140 developed multiple islet autoantibodies; and 3103 observed at risk for type 1 diabetes, of which 91 developed type 1 diabetes

^bAge of child on 1 March 2010

In Sweden, 1.2% of children observed at risk for type 1 diabetes were vaccinated with the seasonal flu vaccine before (0.2%) or only after (1.0%) the mass vaccination for H1N1 had begun. The frequency of receiving the seasonal flu vaccine was similar between children who were vaccinated for H1N1 (1.3%) compared with children who were not (1.0%). In Finland, 58.7% of children who were vaccinated for H1N1 had a seasonal flu vaccine either before (36.1%) or only after (22.6%) the H1N1 vaccination. This was significantly higher compared with the children who were not vaccinated for H1N1 (28.3% received seasonal flu vaccine,11.7% before [p < 0.001] and 16.6% only after [p ≤ 0.01]). Mothers could also have received the seasonal flu vaccine during pregnancy; however, the percentage was similar between countries (Finland, 2.1%; Sweden 1.4%). Interestingly, of the 1.7% (58/3381) of mothers who did get the seasonal flu vaccine during pregnancy, 6.2% (46/743) were mothers of younger children (aged < 1 year on 1 March 2010) and the vaccine was given towards the end of the mass vaccination. Of these mothers, only 2/46 had their child vaccinated for H1N1. Seasonal flu vaccine administered to mothers during pregnancy or children during follow-up did not explain the association between H1N1 vaccination and type 1 diabetes outcomes. The seasonal flu vaccine did not have a modifying effect on the association between H1N1 vaccination and type 1 diabetes outcomes (data not shown). Furthermore, in Sweden, a second vaccination dose did not influence any of the type 1 diabetes outcomes (data not shown).

When analysing these results over time, we did not find any short- or long-term initiating or accelerating effects of the Pandemrix® vaccination on risk of islet autoimmunity, multiple islet autoantibodies or type 1 diabetes.

In this study, we investigated whether risk of islet autoimmunity and type 1 diabetes is increased in children who have been vaccinated with the squalene (ASO3)-containing H1N1 flu vaccine (Pandemrix®). Notably, Pandemrix® has been associated with autoimmunity to orexin-producing cells and the development of narcolepsy. As such, we hypothesised that this vaccine may not only promote autoimmunity to orexin-producing cells but also islet autoantigens. The results of our study do not support this hypothesis and argue against any connection between the H1N1 mass vaccination campaign and the risk of type 1 diabetes. In fact, we found that the incidence of type 1 diabetes outcomes in Finland was actually lower in children vaccinated against H1N1 than in non-vaccinated children. Theoretically, this finding could indicate that the vaccine may have reduced the risk of type 1 diabetes in higher risk populations by unknown mechanisms.

In the prospective cohort of children followed in the TEDDY study, we have the unique capacity to analyse environmental factors associated with the initiation of islet autoimmunity and development of type 1 diabetes. Type 1 diabetes is preceded by progressive autoimmune destruction of beta cells, which can last just a few months or up to many years. Since islet autoimmunity and multiple islet autoantibodies are known to confer over 70% risk to the development of type 1 diabetes within 10 years [4], it was important to consider these markers as surrogate endpoints when analysing a hypothetical increased risk of the disease during the first few years after the vaccination. By examining for risk of seroconversion to two or more islet autoantibodies as an endpoint along with type 1 diabetes, we have enabled an earlier detection of an increased or decreased risk of clinical type 1 diabetes.

Among a wide range of data currently being collected in TEDDY, vaccination records are prospectively documented in the study. Pandemrix® was used exclusively as the H1N1 vaccine in Sweden and Finland. Other types of vaccine were used in the USA as part of the H1N1 vaccination programme. In Germany, Pandemrix® was used as part of the vaccination programme, but it had low coverage. Due to heterogeneity and low numbers, we did not include the German TEDDY population in this analysis. Therefore, these analyses were limited to Swedish and Finnish children. A weakness in our study is that our population was selected for HLA genotypes associated with risk for type 1 diabetes [12]. One cannot exclude the possibility that the vaccine may increase the risk of islet autoimmunity in children with lower risk genotypes, who were screened but not enrolled for follow-up in TEDDY. This may be further emphasised by the observation that all children who developed narcolepsy after the Pandemrix® vaccination had HLA genotypes containing HLA-DQB1*06:02 [10]. Because the DQ-0602 genotype is actually protective against the development of type 1 diabetes, children with this genotype were excluded from the TEDDY study. As HLA plays such a critical role in the presentation of antigens to the immune system and how the immune system reacts immunologically to infections, individuals with different HLA genotypes are known to react differentially to vaccinations [20].

After the reports of the increased incidence of narcolepsy, studies were established to examine for a possible influence of the Pandemrix® vaccination on the incidence of other autoimmune diseases. In one of these studies investigating the incidence of type 1 diabetes, along with other immunological diseases, the authors found a non-significant increased incidence of type 1 diabetes [21]. This finding was further discussed in a number of letters to the journal, indicating that missing data and the exclusion of various participants would have resulted in the lack of significance [22, 23, 24]. In our current study, we do not find any indication of increased incidence of islet autoimmunity or type 1 diabetes after vaccination with Pandemrix®. It is also important to emphasise that our prospective cohort study did not suffer from the same kind of missing data issues that affected the results of the previous study. Similar to our study, in 355 Swedish children who were diagnosed with type 1 diabetes during the vaccination campaign, younger individuals (< 3 years) with HLA DQ2/2 or 2/X were low responders to Pandemrix®, measured as antibodies against A/H1N1 haemagglutinin [25]. As the proportion of children < 3 years of age with the high-risk HLA DQ2/8 decreased after the vaccination, it was suggested that the vaccine affected clinical onset of type 1 diabetes by delaying onset in children with this genotype [25]. However, these studies were performed shortly after the vaccination and included only clinical type 1 diabetes and not islet autoimmunity as an endpoint. Therefore, an increase in type 1 diabetes could have been missed, since it would not be obvious until long after an increase in islet autoimmunity. Our current study is the first to investigate islet autoimmunity and multiple islet autoantibodies in relation to the vaccination. Another possibility would be to investigate if Pandemrix® affected progression from islet autoantibodies to clinical type 1 diabetes. Since autoantibodies appear early, while progression to type 1 diabetes may take many years, more time is needed to investigate progression to type 1 diabetes properly in our prospective study as the peak incidence has not yet been reached. The TEDDY children will be followed to age 15 years, and additional analyses will be done later on in the study.

Interestingly, when separating Swedish and Finnish data, we found that Finnish children had a decreased risk of islet autoimmunity (primarily IAA as first autoantibody), multiple islet autoantibodies and type 1 diabetes after vaccination. Incidence of type 1 diabetes outcomes is known to be higher in Finland compared with Sweden [26]; however, it is unclear as to why there was no increased risk among the vaccinated population. We noted that the majority (58.7%) of the H1N1-vaccinated Finnish children and only 27.6% of those not H1N1-vaccinated had received a prior seasonal flu vaccination, while only 1.2% of Swedish children had received previous seasonal flu vaccination with no difference between H1N1 vaccination rates. One could speculate that the immunological memory induced by previous flu vaccinations, regardless of the adjuvant in those vaccines, in Finnish children led to improved immune responses to H1N1 virus (a priming effect). After adjusting for seasonal flu vaccination, during and after pregnancy, the associations with type 1 diabetes outcomes in Finland remained.

Nevertheless, it is possible that influenza virus infections may increase the risk of type 1 diabetes, as reported by previous studies [27, 28], while others could not confirm this [29]. It is interesting to note that repeated doses of Pandemrix® were more frequent in Sweden than in Finland. One could speculate that these booster vaccinations may have led to better protection against H1N1 infections in Sweden. This, in turn, could have reduced the circulation of the virus and also partially protected non-vaccinated children in Sweden. While preliminary, these findings generate additional questions that will be further examined in TEDDY, in which analyses of virome data will be performed in the near future, and warrants study in larger cohorts to evaluate possible protective associations between influenza infections and vaccinations and islet autoimmunity.

In conclusion, our analyses did not find any increased risk of islet autoimmunity, multiple islet autoantibodies or type 1 diabetes in children who were given the ASO3-containing Pandemrix® flu vaccination during the H1N1 pandemic in 2009–2010. Additional studies are needed to further explore the potential protective effects of influenza vaccinations.