Type 1 Narcolepsy (T1N) is a disabling disorder characterized by excessive daytime sleepiness, irresistible daytime sleep attacks, and sudden episodes of loss of muscle tone following emotions such as laughter, a symptom known as cataplexy [1
]. Genetic and immunological studies have shown that the disorder is autoimmune, and likely mediated by T cell attacks targeting hypocretin producing neurons, a population of 20,000 neurons located in the posterior hypothalamus [2
]. Hypocretins are critical regulators of wakefulness and Rapid Eye Movement sleep (REM sleep), and lack of hypocretin transmission is causal to the symptoms of the disorder [4
Until recently, the rationale for an autoimmune basis for narcolepsy was based mainly on epidemiological and genetic evidence. First, there is a uniquely strong association between narcolepsy and 6p21.3, a region of the genome, including the human leukocyte antigen HLA locus, more specifically, 97% of narcoleptic patients carry at least one copy of HLA DQB1*06:02 (DQ0602) across ethnicities, an HLA class II allele found in 25% of the general population [6
]. Additional weak effects in HLA-A*11:01 [9
] and an impact of DQB1*03:01 on the age of onset were also observed [10
]. Genome-wide association studies have found that narcolepsy is associated with T-cell receptor loci TRA & TRB, and immune genes such as CTSH, P2RY11, ZNF265, IFNAR1, and TNSF4 [10
]. As all these loci are involved in immune regulation and other autoimmune diseases, an autoimmune mediation of hypocretin cell death has long been proposed as the cause of narcolepsy. Of notable interest is the fact the TCR loci associated with narcolepsy are modulators of TRAJ24 and TRBV4-2, TCR segments only involved in 0.8% and 0.7% of the total TCR repertoire, respectively.
Narcolepsy studies have described environmental triggers in addition to the genetic susceptibilities; specifically, while some studies have noted increased humoral (IgG) and cellular (IFNγ) responses to streptococcus pyogenes infection [13
] others have reported no differences in Chinese narcolepsy patients [15
]. Similarly, epidemiological studies have suggested increased frequency of strep infections and flu-like illness in patients before developing narcolepsy [16
]. Most recently, the data has most strongly implicated influenza-A infection and vaccination. Following the 2009–2010 H1N1 “swine flu” influenza pandemic infection in China, increased T1N onsets were observed [10
]. In European countries, a significant 4–16-fold increase in the risk of developing narcolepsy in children was observed a few months following an aggressive pandemic H1N1 (pH1N1) flu vaccination campaign with the vaccine Pandemrix [17
]. In these cases, Pandemrix increased the incidence of narcolepsy by a factor of 2–15, from around 1/150,000 to 1/15,000 cases per year in children. The risk was mostly increased in younger children, but there was still a significant, albeit weaker effect in adults [20
]. Similar increases in narcolepsy incidence were not observed in countries where other pandemic vaccines, notably Arepanrix in Canada, were used, thereby elucidating the impression that Pandemrix uniquely triggered narcolepsy [25
Starting in May 2009 in response to the pandemic H1N1, vaccine manufacturers began to plan the production of a specialized vaccine targeting the new strain for vaccination the following winter, a concise timeline. The creation of vaccine strains involves growing strains derived from pathogenic strains reassorted with PR8 (an old 1918-H1N1-like strain 08/35 from Puerto Rico) in eggs. The reassortant strain is typically constructed by the New York Medical Center (NYMC), which is then distributed to manufacturers for growing millions of doses in eggs in specialized factories. The NYMC H1N1-like vaccine strains produced for the 2009–2010 swine flu campaign used A/H1N1/California/7/2009 as the pathogenic strain, so that only Hemagglutinin (HA), Neuraminidase (NA), and polymerase PB1 are derived from A/H1N1/California/7/2009, while other proteins are PR8 derived. In close succession, NYMC-X-179A and NYMC X-181, a higher growth reassortant derived from X-179A were created, with the former strain having been used more widely (X-181 was only used toward the end of the season in some cases) [26
]. Once distributed, vaccine manufacturers used their own patented process to produce vaccines using X-179A and X-181. In egg-based vaccine production processes, candidate vaccine viruses are grown in eggs per current FDA regulatory requirements. To do so, X-179A is injected into fertilized hen’s eggs and incubated for several days to allow viruses to replicate. The virus-containing allantoic fluid is then harvested from the eggs, viruses inactivated (killed), and virus antigens purified, with the general goal of preferentially isolating viral surface proteins HA and NA, which are most important for protective antibody responses sought with vaccine administration [27
Using this process, the manufacturer GlaxoSmithKline created Pandemrix and Arepanrix, both AS03-adjuvanted vaccines; the AS03 adjuvant is an immunological agent added to the vaccine to boost the immune system’s response to the target antigen while reducing the dosage of the viral antigen (antigen sparing, a property that was desirable considering short time of production) [28
]. Arepanrix was produced and used in Canada around the same time that Pandemrix was deployed in Europe, but it did not sharply increase narcolepsy risk in Quebec [25
]. This is notable as Arepanrix is almost identical to Pandemrix with a same adjuvant from the same geographic origin as well as similar viral composition. However, different viral antigen purification techniques were used for either vaccine—Fluarix for Pandemrix and Flulaval for Arepanrix [30
]. Why Pandemrix in Europe and not Arepanrix in Canada triggered narcolepsy cases is unknown. One possibility may be the differential composition of the vaccine, notably due to the fact the viral antigens were extracted using different manufacturing processes. Another possibility involves the presence of other factors differentiating Canada and Europe at the time of vaccination. After all, it is essential to note that even with Pandemrix, only 1/16,000 vaccinated children (or 1/4000 DQB0602 positive subjects) developed narcolepsy [19
], so that almost undoubtedly other environmental or stochastic factors are involved in addition to vaccine trigger and genetic background. As Canadian and northern European populations are similar in term of DQ0602 frequency (and other narcolepsy-associated genetic factors), a possibility could be a differential immune history of both population regarding past flu or the fact that in Northern Europe vaccination occurred shortly before or exactly when the pandemic H1N1 infection affected the population. At the same time, in Canada the bulk of vaccination occurred immediately after the pandemic flu started to change the population [23
Our understanding of narcolepsy immunology changed significantly a few months ago, thanks to two studies [31
]. Latorre et al. used an ultrasensitive technique involving polyclonal expansion and cloning of CD45RA−
memory T cells, found strong line reactivity to HCRT in all patients versus no or limited responses in 12 DQ0602 controls, with significantly higher reactivity in T1N. These authors found responses to seasonal influenza vaccine to be comparable in patients and controls, concluding that flu antigen mimicry could not be detected [31
]. On other hand, Luo et al. [32
] screened peptides derived from HCRT and flu strains including pH1N1 for DQ0602 binding and presence of cognate tetramer-peptide specific CD4+
T cells in 35 T1N cases and 22 DQ0602 controls finding higher reactivity to influenza pHA273–287
(pH1N1 specific) and C-amidated but not native version of HCRT54–66
sequences (two homologous sequences we denoted HCRTNH2
) in T1N when presented by DQ0602.
Relatively few studies have examined composition differences across flu vaccines such as Pandemrix and Arepanrix. Comparative studies of antibody reactivity to Pandemrix and Arepanrix antigens in both post-Pandemrix patients and control individuals found that post-Pandemrix-vaccinated children had poorer antibody reactivity to Arepanrix, suggesting antigenic differences in antibody determinants [33
]. This could be important, although one would expect that differential susceptibility would be more likely due to T cell response differences. High-resolution gel electrophoresis quantitation and Mass Spectrometry (MS) identification analyses revealed higher amounts of structurally altered viral nucleoprotein (NP) in Pandemrix versus Arepanrix, a finding that can also be noted in a 2-D gel study by Jacob et al. [30
]. These results suggest complex protein aggregate conformation differences that could be relevant to differential activity of these vaccines, most notably in terms of antibody response to NP. Jacob et al. [30
] comparing single batches of Pandemrix and Arepanrix found that viral proteins NP and NA as well as selected non-viral chicken proteins (PDCD6IP, TSPAN8, H-FABP, HSP, and TUB) were more abundant in Pandemrix compared to Arepanrix. The study also found an accumulation of a specific mutation in Arepanrix, 146N to D in Arepanrix, but as the batch studied had only been synthesized in 2010 and had never been used for vaccination, it was hard to be sure if this mutation had been present in earlier batches. Finally, Ahmed et al. [34
] found an increased quantity of cross-reactive antibodies to structurally altered NP epitope NP116I with an epitope from HCRTR2 protein in narcolepsy cases. Although the proportion of NP116I mutations in both Arepanrix and Pandemrix was similar and, therefore, unlikely to be a causal effect, increased cross-reactive antibodies between NP116I and HCRTR2 were not observed in other studies [35
In the current study, we extended the previous findings from Jacob et al. [30
] by characterizing the protein content and mutational burden in 6 Pandemrix lots and 5 Arepanrix lots in relation to X-179A, the influenza strain from which both vaccines were derived. Data shows batch diversity in mutation burden that could be relevant to vaccine responses in some cases but not a distinct difference that could explain why Pandemrix was more associated with narcolepsy onsets than Arepanrix. Mutational drift and low-level mutations are present in some vaccine batches (as occurring in wild type virus), and this should be taken into consideration when studying the effects of these vaccines in the population.
This study extends the Jacob et al. report [30
] where only single batches of Arepanrix and Pandemrix were analyzed and presents a detailed characterization of the mutational burden and protein content of 5 Arepanrix and 6 Pandemrix batches. Mean coverage of the mass spectrometric characterization of influenza proteins, while still at high 71.1% in Arepanrix and 80.5% in Pandemrix, was slightly less than what Jacob et al. [30
]. The sampled lots were actual vaccine doses used during the 2009 pandemic influenza vaccination campaign in Northern Europe and Canada, except for two lots that were monovalent bulks and sourced directly from GSK (see Table 1
). Not surprisingly, considering that both Arepanrix and Pandemrix were derived from NYMC X-179A [26
], the mean global proportions of Influenza, chicken and bovine proteins were comparable between the two vaccines (Figure 1
). This finding agrees with Jacob et al. [30
], where similar global proportions were observed. The four main influenza proteins in order of abundance were NP, HA, M1, NA, and PB2 (Table 2
), which is consistent with other studie [26
], while PB1, NS1 and nuclear export protein (NEP) were only present at low concentrations (<1%) in both the vaccines.
We found that NA was significantly enriched three-fold in Pandemrix as compared to Arepanrix confirming the trend identified previously in Jacob et al. [30
]. In contrast, NP was underrepresented in Pandemrix compared to Arepanrix, although this difference was not statistically significant. This finding conflicts with the reports of Jacob et al. [30
], Vaarala et al. [33
] and Ahmed et al. [34
], all of which observed an overrepresentation of NP content in Pandemrix. The main limitation in these studies mentioned earlier is however that only one representative batch of each vaccine or the monovalent antigen bulk [30
]. In addition, the protein content was characterized by various techniques including western blots, PAGE gels and mass spectrometry in these studies. In this study, we have characterized 6 different Pandemrix lots and 5 different Arepanrix lots, with the majority of them being actual vaccine doses. Further, using both trypsin and chymotrypsin protein digests to increased our protein coverage thus the finding may be more reliable. However, we did not perform any enrichment before MS characterization, which may have influenced our current results.
As recently reported, we found that vaccine strains, like wild type virion infecting hosts, mutate in culture, and this leads to divergences in vaccine viral sequences in different vaccines or across vaccine batches. Differences may thus depend on how often the manufacturer reuses the primary NYMC strain versus continuing to amplify isolates from their own egg cultures for future propagation. As an example, Skowronski et al. found that H3N2 reassortant vaccine strains had mutated in key antigenic residues, likely contributing to reduced efficacy in 2012–2013 [37
]. Similarly, Jacob et al., conducting Mass Spectrometry (MS) characterization of X-179A derived pH1N1 vaccines, 2009 Pandemrix and 2010 Arepanrix, discovering that a specific HA mutation, N146D, had accumulated in Arepanrix, distinguishing the two antigens [30
] (limitation in the Jacob et al. study was that only a representative batch of actual Pandemrix vaccine (batch DFLSA014A) and bulk Arepanrix (batch SF1B0454Cl) was studied and compared). Further, the Arepanrix lot was a lot that had never been used and had been prepared one year after the pandemic (2010). In this study, we could confirm the dominance of N146D in Arepanrix but not Pandemrix across all lots. Interestingly, this mutation conferred higher growth and was selected in subsequent pH1N1 strains X-181 [26
]. It may thus be that the mutation accumulated in Arepanrix but not Pandemrix because of differences in Arepanrix culture procedures.
As it was conceivable that the N146 sequence found in Pandemrix and wild type H1N1 but not Arepanrix was essential to explain narcolepsy susceptibility, we further examined binding of both N146 and D146 peptides on DQ0602 molecules, confirming in vitro prediction indicating that 146 binds with lower affinity to DQ0602, another factor that could contribute to different susceptibility. Using DQ0602 tetramers for sequences; however, we found that very few T cells recognized these peptide sequences in both narcolepsy and control subjects, making it unlikely to be of significance in narcolepsy pathophysiology. Similar to the study of HA N146D, we also studied tetramers for NP T424I, NP T423I, and NP T257A, three other mutations that are much more abundant in Pandemrix versus Arepanrix (Table 1
) and were predicted to bind DQ0602 with an equivalent affinity (supplementary Table S1
). In vitro binding studies indeed found that all these peptides bound DQ0602 with high affinity. However, tetramer studies in narcolepsy and controls did not support abundance for T cells recognizing these epitopes making it unlikely to be functionally important.
With recent results suggesting that a potential mimic of HCRTNH2
, we also carefully examined frequency and sequence variation within this segment, present in both wild type pH1N1, X-179A and X181, but could not find any mutation or difference in frequency across vaccines, making it unlikely composition difference at this level explain differential narcolepsy risk. It is nonetheless interesting to note that pHA273–287
contains N at position 273 (predicted to bind DQ0602 in P1) and that this residue is partially glycosylated [30
], a modification that could make a difference in B cell reactivity and perhaps epitope processing and presentation to T cells. A future direction would be to profile in detail the post-translational modifications in these motifs across the two vaccines. For instance, possible deamidation in the MHC binding pocket could alter the DQ0602 binding register and glycosylation in or flanking the pHA273–287
or in other cases, PTMs such as N-linked or O-linked glycosylation have also been shown to protect epitope cleavage sites, prevent efficient antigen processing, or influence recognition by cognate T-cell receptors [38
]. Glycosylation patterns in key residues across vaccines are, therefore, also of interest in the context of DQ0602 binding and narcolepsy susceptibility.