TYPE Review

PUBLISHED 12 October 2022

DOI 10.3389/fimmu.2022.902840

Narcolepsy and H1N1 influenza

OPEN ACCESS immunology a decade later:
EDITED BY
Lindsay B Nicholson,
University of Bristol, United Kingdom
What have we learned?
REVIEWED BY
Rebeca Perez De Diego, Sofia M. Buonocore * and Robbert G. van der Most †
University Hospital La Paz Research
Institute (IdiPAZ), Spain GSK, Rixensart, Belgium
Maria Fernanda Naufel,
Federal University of São Paulo, Brazil

*CORRESPONDENCE
Sofia M. Buonocore In the wake of the A/California/7/2009 H1N1 influenza pandemic vaccination
sofi[email protected]
campaigns in 2009-2010, an increased incidence of the chronic sleep-wake
†
PRESENT ADDRESS
disorder narcolepsy was detected in children and adolescents in several
Robbert G. van der Most,
Translational Science, BioNTech, European countries. Over the last decade, in-depth epidemiological and
Mainz, Germany immunological studies have been conducted to investigate this association,
SPECIALTY SECTION which have advanced our understanding of the events underpinning the
This article was submitted to observed risk. Narcolepsy with cataplexy (defined as type-1 narcolepsy, NT1)
Autoimmune and Autoinflammatory
Disorders,
is characterized by an irreversible and chronic deficiency of hypocretin
a section of the journal peptides in the hypothalamus. The multifactorial etiology is thought to
Frontiers in Immunology include genetic predisposition, head trauma, environmental triggers, and/or
RECEIVED 23 March 2022 infections (including influenza virus infections), and an increased risk was
ACCEPTED 13 July 2022
observed following administration of the A/California/7/2009 H1N1 vaccine
PUBLISHED 12 October 2022
Pandemrix (GSK). An autoimmune origin of NT1 is broadly assumed. This is
CITATION
Buonocore SM and van der Most RG
based on its strong association with a predisposing allele (the human leucocyte
(2022) Narcolepsy and H1N1 antigen DQB1*0602) carried by the large majority of NT1 patients, and on links
influenza immunology a decade with other immune-related genetic markers affecting the risk of NT1. Presently,
later: What have we learned?
Front. Immunol. 13:902840. hypotheses on the underlying potential immunological mechanisms center on
doi: 10.3389/fimmu.2022.902840 molecular mimicry between hypocretin and peptides within the A/California/7/
COPYRIGHT 2009 H1N1 virus antigen. This molecular mimicry may instigate a cross-reactive
Sofia Buonocore and Robbert G. van autoimmune response targeting hypocretin-producing neurons. Local CD4+
der Most, © 2022 GSK. This is an open-
access article distributed under the T-cell responses recognizing peptides from hypocretin are thought to play a
terms of the Creative Commons central role in the response. In this model, cross-reactive DQB1*0602-
Attribution License (CC BY). The use,
restricted T cells from the periphery would be activated to cross the blood-
distribution or reproduction in other
forums is permitted, provided the brain barrier by rare, and possibly pathogen-instigated, inflammatory processes
original author(s) and the copyright in the brain. Current hypotheses suggest that activation and expansion of
owner(s) are credited and that the
original publication in this journal is cross-reactive T-cells by H1N1/09 influenza infection could have been
cited, in accordance with accepted amplified following the administration of the adjuvanted vaccine, giving rise
academic practice. No use,
to a “two-hit” hypothesis. The collective in silico, in vitro, and preclinical in vivo
distribution or reproduction is
permitted which does not comply with data from recent and ongoing research have progressively refined the
these terms. hypothetical model of sequential immunological events, and filled multiple
knowledge gaps. Though no definitive conclusions can be drawn, the
mechanistical model plausibly explains the increased risk of NT1 observed
following the 2009-2010 H1N1 pandemic and subsequent vaccination
campaign, as outlined in this review.

KEYWORDS

narcolepsy, H1N1 influenza, vaccine, hypocretin, Pandemrix, CD4+ T cells

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Buonocore and van der Most 10.3389/fimmu.2022.902840

Introduction AS03 is an oil-in-water Adjuvant System containing DL-a-

tocopherol and squalene oil dispersed in water (6, 7). The safety
In the aftermath of the A/California/7/2009 H1N1 (A/ profiles of the AS03-adjuvanted influenza vaccines observed in
H1N1pdm09) influenza pandemic in 2009-2010, an increased clinical trials conducted in adults and children were clinically
incidence of the sleep-wake disorder narcolepsy was observed in acceptable (8). Reactogenicity was increased compared to non-
several European countries, mostly in children and adolescents who adjuvanted influenza vaccines, but symptoms generally
received Pandemrix (GSK) (1–5). This AS03-adjuvanted (6, 7) remained transient and, mild-to-moderate in intensity and
inactivated split-virion A/H1N1/pdm09 vaccine was mostly localized at the site of injection (8). In children,
manufactured in Germany and predominantly used in Europe especially those younger than 3 years of age, increased
during the pandemic. Given the influenza strain’s persistent incidence of fever was observed particularly after the second
circulation, vaccination continued in the UK on a small scale dose (13–15).
(~148,000 individuals) through the end of the 2010-2011 season, The adjuvant’s defined mode of action includes increased
in situations when seasonal vaccine supply was limited (8). The innate cytokine expression and antigen uptake by monocytes,
narcolepsy vaccine safety signal derived from these rare events as seen in mice (6), and activation of interferon pathways
triggered intense research efforts to expand the epidemiological (measured through transcriptomics), as demonstrated in
knowledge, and to generate evidence informing a hypothetical humans (16–19). Combined with different antigens, the
pathogenesis mechanism, which centered on an autoimmune innate immune activation translated into increased antigen-
etiology (9, 10). The collective, multidisciplinary research has specific CD4 + (but not CD8 + ) T-cell responses, and
progressively enabled to refine and extend the initially posed quantitative/qualitative enhancement of specific antibody
hypotheses of the likely underlying mechanism. Though the responses (11, 20–22). Another defined feature of AS03
evidence does not suffice to draw definitive conclusions, the activity, noted when used in combination with H5N1
emerging mechanistical model offers a plausible and coherent antigen, is an epigenetic modification of innate cells, which
explanation for the association between Pandemrix vaccination augments the cells’ responsiveness upon subsequent viral
and narcolepsy. This review synthesizes the research conducted exposure (17). A Phase 3 trial, comparing seasonal influenza
over the last decade since the detection of the signal, with further vaccines with or without AS03 in older adults, although it
mechanistic research still ongoing. missed its primary endpoint of preventing any influenza
infection, has shown higher efficacy of the adjuvanted
vaccine in preventing A/H3N2 infection and all-cause
Context mortality or pneumonia (23). For (pre)pandemic H5N1 and
A/H1N1pdm09 vaccines, AS03 enabled enhanced
AS03-adjuvanted pandemic immunogenicity, antigen-sparing, persistence of immune
influenza vaccines response and cross-clade reactivity (20, 24).
Overall, the extensive experience gained with AS03-
Besides Pandemrix, a similar monovalent AS03-adjuvanted adjuvanted vaccines has yielded a substantial and
A/H1N1pdm09 vaccine (Arepanrix; GSK) was supplied during comprehensive safety and immunogenicity database (8), and
the pandemic. This vaccine was manufactured in Canada and suggests that vaccines formulated with this adjuvant could be
distributed mostly in the Americas (Brazil, Canada, Mexico), crucial to adequately address pandemic threats, not limited to
Malaysia and Turkey. Of the estimated 90 million doses influenza viruses (7).
administered in total (Pandemrix/Arepanrix: 31/59 million),
around 9.5 million were given to children (8). Single-dose
regimens were generally administered, due to the high NT1 immunopathogenesis
effectiveness of one dose of the vaccine, though in some
countries a two-dose schedule was used to vaccinate Narcolepsy is a chronic neurological disorder that is
immunocompromised adults and younger children (8). primarily characterized by a reduced ability of the brain to
The Pandemrix and Arepanrix vaccine antigens were derived control sleep-wake cycles and, in most individuals, cataplexy (an
from the NYMC X-179A reassortant influenza strain, where the abrupt loss of muscle tone). When presenting in conjunction
haemagglutinin (HA), neuraminidase (NA) and polymerase with cataplexy, the disorder is commonly referred to as type 1
basic 1 (PB1) originated from A/H1N1pdm09, and the narcolepsy (NT1). Though no cure is available, some symptoms,
remaining proteins from a 1934 A/H1N1 strain (PR8) (11). such as excessive daytime sleepiness and cataplexy, may be
Clinical trials demonstrated that the two AS03-adjuvanted controlled by medication (25, 26). NT1 is furthermore
pandemic influenza vaccines share similar reactogenicity and associated with obesity and a range of psychosocial and
immunogenicity profiles (12, 13). psychiatric problems (27, 28).

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Narcolepsy with cataplexy has a global prevalence of 25–50 Sweden, the ‘signaling countries’ (1, 39). The timeline and key
per 100,000 individuals, with the onset of symptoms usually in events are summarized in a publication by M. Sturkenboom
childhood or adolescence (25). While it can have a post- (39): “a suspicion of the association between Pandemrix® and
traumatic etiology, a causal role of infections such as narcolepsy in a child had already been noticed by Dr. Partinen in
Streptococcus or influenza has also been suggested (25). NT1 is December 2009 and this association was discussed among
diagnosed by the Multiple Sleep Latency Test, as summarized by neurologists in February 2010 in Finland”, and: “a safety signal
Bassetti et al. (25). Diagnosis can be supported by measuring around Pandemrix, an AS03 adjuvanted influenza A(H1N1)
levels of the neuropeptide hypocretin (HCRT) in the pdm09 vaccine potentially causing narcolepsy in children and
cerebrospinal fluid. For a more detailed overview of the NT1 adolescents became public in August 2010, long after cessation of
clinical spectrum, pathophysiology, diagnosis, and treatment, we the influenza A(H1N1) pdm09 campaigns in Europe”. Across
refer to the above-cited review by Bassetti et al. (25). Symptoms Europe, the accumulating reports prompted several single-
are likely caused by selective loss of the limited number country, retrospective pharmacoepidemiological studies, as
[approximately 60,000 (29)] of hypothalamic neurons well as an extensive multinational case-control study by the
producing HCRT, resulting in a permanent and irreversible ‘VAESCO’ consortium, which confirmed an association for 5–
lack of HCRT signaling in the brain. Upon its secretion, the 19-year-olds in the signaling countries (1–4, 39, 40). Subsequent
precursor hormone (prepro-HCRT) is processed into the European studies estimated relative risks (95% confidence
HCRT1 and HCRT2 peptides which then undergo amidation, intervals) ranging from 1.5–25.0 (0.3–48.5) for children/
a post-translational modification essential for biological activity adolescents and 1.1–18.8 (0.6–207.4) for adults (2, 8), and
(30, 31). These peptides can bind to the HCRT receptors types 1 attributable risks (excess cases/100,000 vaccinees) of 1.4–8 and
and 2 (HCRT-R1/R2), which are preferentially expressed in the 1.0, respectively (1, 2).
central nervous system (CNS), by different cell types located
throughout the brain—though most likely not by HCRT—
producing neurons themselves (32). The resulting interactions Other adjuvanted H1N1 vaccines
are involved in the regulation of cognitive and physiological
functions, including sleep/wake states. A specific study was launched in Quebec (Canada), to
The prevailing consensus is that NT1 has an autoimmune investigate whether any potential association would exist
origin, similar to type 1 diabetes which is caused by between Arepanrix and narcolepsy, based on validated cases of
autoimmunity to the secreted hormone prepro-insulin (33). A narcolepsy from sleep centers (41). In the primary analysis of
key feature of autoimmune diseases is a genetic association with this study (41), the relative risk was estimated at 4.3 (95%
specific human leukocyte antigen (HLA) alleles (34). The confidence interval: 1.5–11.1). This estimate is not
occurrence of NT1 is strongly linked to positivity for certain incompatible with the range of the relative risks observed for
HLA class II (HLA-II) alleles. HLA-II molecules are involved in Pandemrix (1). The authors of the Quebec study concluded by
immune system regulation, and expressed by cells presenting stating that their results were consistent with a risk of narcolepsy
antigenic peptides to CD4+ T cells (antigen-presenting cells). following administration of Arepanrix, but that the attributable
Indeed, while positivity for HLA-DPA1 or DQB1*0603 is risk was of small magnitude (approximately one case per
thought to have a protective effect, DQB1*0602 (‘DQ0602’) is million) (41). Attributable risks for Pandemrix were around 1
considered a genetic marker that strongly predisposes for NT1 per 20,000 (1). Furthermore, secondary analyses of the Quebec
(29, 35). The latter is based on the observation that 95-98% of study, intended to address the biases differently, have produced
NT1 patients are positive for this HLA-II allele, and that lower (and non-significant) relative risks (8, 41). Such a large
DQ0602 homozygosity (versus heterozygosity) significantly heterogeneity in relative risk estimates between different
increases susceptibility (30, 35–37), linking this allele tightly to analyses of the same study were also typical of the studies
the risk of NT1. However, the relative commonness of DQ0602- on Pandemrix.
positivity, found in 15–30% of the general population, also Other measures of relative risk were produced for Canada in
strongly suggests a role for environmental triggers in the the SOMNIA study (3), a global retrospective and observational
overall NT1 etiology (36, 38). study with data from three provinces (Ontario, Manitoba, and
Alberta), of which the case-control study in Ontario did not
detect any increased risk. In all three provinces, the overall
Pharmacoepidemiological evidence incidence rate of narcolepsy was not higher after versus before
the vaccination campaign. It should also be noted that this study
Safety signal with Pandemrix only detected such an increase in Sweden (the ‘signaling’ country
for narcolepsy) but not in any of the other six included countries.
The first reports of narcolepsy in children and adolescents In addition, no signal was observed in this study for the Focetria
vaccinated with Pandemrix emerged in 2010 in Finland and pandemic H1N1 vaccine (Novartis; adjuvanted with MF59 (7),

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another oil-in-water emulsion), of which 12 million doses were influenza infection and unexplained fevers were identified as
administered in Europe, to 8 million individuals across all ages increasing the risk of narcolepsy in a case-control study (53).
(5, 42). Finally, it was concluded in the SOMNIA study that no Further evidence is provided by the up to four-fold increase
changes in narcolepsy incidence were observed between the (compared with other years) in cases in 2010 in the
period after the initiation of the adjuvanted A/H1N1pdm09 abovementioned Chinese data from Han et al, in the absence
vaccination and the period before virus circulation, in any age of vaccination (44, 45, 54). More recently, a modelling study of
group or country (3), except for two countries where an de novo European cases concluded that the NT1 peak in children
increased risk was observed: Sweden, one of the signaling detected in 2013 (thus in the absence of recent Pandemrix
countries, and Taiwan, where the incidence increased vaccination), was most likely triggered by other risk factors,
concomitantly with influenza virus circulation, in the absence such as viral infections caused by H1N1 recirculation, or
of Pandemrix or Arepanrix vaccination (3, 43). circulation of other/new influenza strains, e.g., influenza B
In summary, the associations between Pandemrix or (55). A recent study by Stowe et al. shows that an increased
Arepanrix with narcolepsy were measured in different regions. risk of NT1 associated with Pandemrix vaccination was
The heterogeneity of relative risk estimates between the two “confined to those with onset within the first 12 months with
vaccines is not necessarily larger than the heterogeneity of the a return to baseline thereafter” (56). This limited (~1 year)
estimates between the different, more numerous studies timeframe in which the vaccine could be linked to NT1 onsets
performed for Pandemrix. thus suggests that thereafter other risk factors would have
become more likely causes.
Finally, the likelihood of an infectious etiology aligns with
Role of natural infection the relative chronology of pandemic waves and rises in
narcolepsy with cataplexy cases seen globally (41, 43, 46), as
A putative role of natural infection was initially postulated exemplified by the Quebec Spring wave in 2009. In several
by Han et al. in 2011, based on data from Beijing, China (44). European countries, the pandemic peak preceded the
Epidemiological studies of the Chinese Narcolepsy Cohort maximum capacity of the vaccination campaigns, with only a
identified a seasonal increase in narcolepsy onset during the short duration between these events (2). For example,
pandemic—in a context of very low vaccine coverage and no mathematical modelling using real-world evidence suggested
vaccination with Pandemrix—followed by a decrease in the two that most (66%) Norwegian children aged 10-20 years had
years after the pandemic (45). Furthermore, Huang et al. been asymptomatically or symptomatically exposed to the
reported an increased narcolepsy incidence in Taiwan during virus before vaccination (though separating peaks in
the pandemic that was not associated with pandemic vaccines, background illness from vaccine-induced effects proved
which in this region were either non-adjuvanted or MF59- difficult) (2, 57). A role of preceding A/H1N1pdm09 infection
adjuvanted vaccines (43). An etiological role for influenza in narcolepsy etiology was however called into question in a
infection may also explain the chronology of NT1 incidence serological survey of Finnish patients (58), though the assay
seen in Germany in the same period (46). Finally, whereas the characteristics and data interpretation were queried (59).
Quebec data did not suggest a strong evidence of a risk Nonetheless, the overall evidence indicates that a role for A/
associated with Arepanrix of a similar magnitude as that with H1N1pdm09 infection cannot be ruled out, and that infection
Pandemrix, the local case numbers did increase slightly during may act as a confounder in the apparent association between
the first (spring) pandemic wave in the absence of vaccination vaccination and NT1. This points again towards a potential role
(41). Overall, this body of evidence supports a putative role for for influenza antigen(s) impacting the interpretation of patient
natural infection as a risk factor for NT1. This is aligned with the data, both by complicating the attribution of cases to either the
outcomes of multiple systematic reviews, meta-analyses and vaccine or the infection, and by generating bias (2, 57). Indeed,
multi-bias modelling studies (1, 2, 4, 39–41, 47). Across these the vaccinated population, which may have been exposed to the
studies and other communications (48), the various identified virus and/or had symptoms before vaccination, was also more
confounders (e.g., faster diagnoses in individuals exposed versus prone to seek care and get diagnosed as a result of the signal, due
non-exposed to Pandemrix, geographical differences in the to widespread media and public health attention (2, 57).
H1N1 epidemic curve) consistently included the presence of The strong link of this rare immunopathologic disorder with
infection as a risk factor. a single predisposing HLA-II allele, combined with the increase
It has long been recognized that infectious pathogens can in NT1 incidence in non-vaccinated populations during A/
trigger or exacerbate autoimmune diseases [reviewed in (49)], H1N1pdm09 circulation (44–46, 54), pointed towards a
and for narcolepsy with cataplexy, a link with Streptococcus multifactorial yet highly specific disease etiology, in which T
infection has been reported (50–52). Narcolepsy etiology could cells (rather than potential autoantibodies) take center stage.
also be linked to natural A/H1N1pdm09 infection (5, 25, 50), as This key assumption informed the mechanistic research plan,
supported by several lines of published evidence. First, both performed as part of GSK’s commitment to the European

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Medicines Agency, to further investigate the signal (9). proteins. This approach was based on mapping DQ0602-
Interestingly, an extensive 2014 dataset spanning most of the restricted epitopes of the four target proteins (A/H1N1pdm09
Swedish population (61%; ~6 million persons including 3.3 HA/NA, HCRT1/2), using overlapping 15-mer peptides
million Pandemrix vaccinees), showed that apart from NT1, spanning their sequences. Thus, the central tenets guiding the
no neurological or autoimmune disease was reported at an identification of potentially cross-reactive CD4 + T cells
increased incidence (60). This observation supported the initial (Figure 1) were:
assumption on the specificity of the etiology of the NT1 cases
reported after the pandemic. 1. to identify DQ0602-restricted A/H1N1pdm09 and
HCRT peptides, and assess potential sequence
homology,
Clinical and translational 2. to visualize CD4+ T cells recognizing these epitopes, and
mechanistic research use their TCR sequences as unique identifiers to search
for potential HA/HCRT cross-reactivity,
The T-cell etiology hypothesis 3. to analyze the resulting single-cell TCR sequence
databases, with a specific interest in the known NT1-
The tight association of NT1 with the HLA-II DQ0602 allele associated SNP,
and its function in CD4+ T-cell antigen presentation, suggested a 4. to assess the peptides’ conformational homology when
disease mechanism involving CD4+ T cells with T-cell receptors bound to the DQ0602 groove, to complement the
(TCRs) specifically recognizing peptides from HCRT-secreting sequential homology data from step 1, and confirm
neurons, bound to the endogenous DQ0602 allele. The essential that molecular mimicry (i.e., sequence or structural
role of HCRT deficiency in the etiology of NT1 led to the similarities shared between the viral antigens and self-
assumption that this signature protein of HCRT neurons itself antigen(s) as T-cell targets), underpins their recognition
could be the autoantigen. HCRT neurons would then act as the by cross-reactive T cells.
prime autoimmune targets for direct and/or cytokine-mediated
attacks by CD4+ or CD8+ T cells. A putative T-cell-mediated The binding to DQ0602 of the individual peptides was
etiology of NT1 was further supported by several NT1- measured, and the binding peptides were then inspected for
associated gene signatures identified in the last decade, which, their uniqueness to the H1N1 virus and for putative HA/HCRT
jointly with DQ0602, formed the basis of the genetic sequence similarities (31, 64–67).
predisposition. These findings included a single nucleotide Next (step 2 above), the peptides were assayed for T-cell
polymorphism (SNP) in a gene segment of the TCR’s a-chain activation/recognition using DQ0602-positive donor CD4+ T
(TRAJ24) serving as a predisposing marker, and other mutations cells. To ensure DQ0602 specificity, two approaches were
in immune-regulating genes (e.g. CTSH, P2RY11 and IFNAR1) followed: stimulating purified CD4+ T cells using antigen-
which are thought to affect T-cell activation (35, 61–63). presenting cells expressing solely DQ0602 (9, 31), or,
employing HLAII-DQ0602–peptide tetramers to directly
visualize/isolate cognate CD4+ T cells (31, 64). While this
Immunological research to evaluate the initially allowed the detection of CD4+ T cells with some
CD4+ T-cell etiology hypothesis evidence of cross-reactivity (9), refinement was achieved by
subjecting isolated CD4+ T cells to single-cell TCR sequence
Several publications that emerged over the last decade have analysis, a technology first reported in 2014 (73). This allowed
begun to shed light on the potential role of T-cell responses in researchers to directly compare the TCRs recognizing HA or
the context of NT1 (31, 64–71). These studies demonstrated HCRT epitopes, and to define cross-reactivity as TCR sequence
amongst others that HCRT can be a target for both CD4+ and identity. In silico interrogation of the TCR-sequence databases
CD8+ T cells, and provided some indications for cross-reactivity. for HA/HCRT epitope homologies (step 3) led to the
An overview of the mechanistic insights from this research on identification of TCR-identical, DQ0602-restricted CD4+ T
NT1-related T-cell immunology, which was performed by cells isolated with the HA and HCRT tetramers. These cells
multiple research groups (31, 64–71), is discussed below. The were detected in most of the investigated patients with NT1,
seminal data, identifying HCRT as a potential T-cell target in but, also in around half of the DQ0602-matched controls (9,
NT1, were published by Latorre and coworkers in 2018 (70). 31). Given the predisposing role of the TRAJ24 SNP (35, 62),
These data led several authors to propose a role of autoimmune the presence/absence of this ‘risk’ allele received particular
T cells in NT1 (36, 72). Investigations into a potential T-cell– attention. The mutation in TRAJ24 was identified in two
mediated etiology for NT1 continued by evaluating potential T- patient cases and in one control (31), and matched the
cell cross-reactivity between HCRT and H1N1 influenza phenotype of a patient TCR (TCR27) carrying this mutation

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FIGURE 1
Research workflow [see refs (31, 64) for details]. Figure represents the four workflow steps and tenets. Step 1: Pools of overlapping 15-mer
peptides spanning the A/H1N1pdm09 haemagglutinin (HA)/neuraminidase (NA), hypocretin (HCRT)1 and HCRT2 sequences were used to
measure binding to DQB1*0602 (DQ0602). Then, the peptides’ uniqueness for A/H1N1pdm09 and putative similarities between the HA and
HCRT sequences were determined, with peptide sequence homology considered a first indication for putative molecular mimicry. Step 2: using
DQ0602-positive donor CD4+ T cells and the human leukocyte antigen class II (HLA-II) tetramers, the peptides from Step 1 were assayed for
their abilities to promote T-cell activation and recognition. Isolated tetramer-binding cells were subjected to single-cell T-cell receptor (TCR)
sequencing. Step 3: TCR databases were inspected for cross-reactivity and the presence of type-1 narcolepsy (NT1)-associated mutations. The
inset [from ref (31)] shows the clustering and overlap of TCR sequences found using tetramers with peptides from nucleoprotein control (NP;
blue), HCRT (orange), or HA (grey), allowing to identify cross-reactive TCR families. In Step 4, X-ray diffraction and crystallography analyses were
used to investigate structural/conformational similarities between the peptides bound to the DQ0602 groove, to complement the sequential
homology data from Step 1. PBMCs, peripheral blood mononuclear cells. FACS, fluorescence-activated cell sorting. Data are shown for
illustrative purposes only.

(64). Collectively, this work identified HCRT peptides as likely Pedersen et al. (71), using DNA barcode-labeled HLA-
T-cell targets and autoantigens, but importantly, only in their multimers to track the antigen specificities of the investigated
bioactive amidated form (31). The short half-times of amidated T cells.
peptides could then explain why T cells recognizing these Finally, the notion of peptide mimicry was supported by
peptides might escape tolerance induction in the thymus. studying three-dimensional (3D) structures of the HA and
Whereas these data provide interesting insights pointing HCRT peptides in the DQ0602 groove, using X-ray
towards T-cell cross-reactivity, it was clear that more crystallography (step 4). Structural analysis of the crystals
research was needed. synthesized of DQ0602 complexes with HA or HCRT peptides
Detection of rare and/or cross-reactive T cells was facilitated in their binding grooves, revealed similarities that were not
by combining DQ0602-tetramers with single-cell TCR obvious from the sequences (64). Indeed, whereas the HA275-
sequencing. Single-cell analysis was important because bulk 287, HCRT56-68 (HCRT1) and HCRT87-99 (HCRT2) sequences
stimulation assays using the same peptides failed to detect displayed differences in five of their nine core residues,
cross-reactivity on the basis of TCR sequence analysis (67). conformational homologies were more remarkable at the 3D-
The latter was likely due to a low frequency of cross-reactive T level (67). Nevertheless and in apparent contrast, Latorre et al.
cells in the pool of cognate cells (69). Nevertheless, a potential identified HCRT-specific CD4+ and CD8+ T cells which did not
role of cross-reactivity for DQ0602-restricted epitopes was display any cross-reactivity with influenza antigens (70). Using
further supported by a study comparing HCRT-specific T-cell different detection methods, these authors found that the
responses between pediatric patients with NT1 and healthy HCRT-specific CD4+ T cells were HLA-DR–restricted, as well
control children, using peptide stimulation and intracellular as undetectable in healthy DQ0602-positive controls and thus
cytokine staining (68). This analysis revealed that the NT1 disease-specific.
patients displayed higher HCRT-specific CD4+/CD8+ T-cell Collectively, the T-cell data are consistent with a model in
frequencies, which were amplified by priming with influenza which a DQ0602-restricted and cross-reactive CD4+ T-cell
virus peptides. Higher HCRT-specific CD8+ T-cell responses in response may act as the initiator of the autoimmune response.
patients versus DQ0602-matched controls were also found by This implies that phenotypic differences between patients and

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controls may be important, and that cross-reactive CD4+ T cells the CNS) were used by Bernard-Valnet et al, to demonstrate a role for
per se do not represent a biomarker for disease. Hence, the HA-specific T cells in the immune attack of HCRT neurons (74).
critical event would likely be the loss of peripheral tolerance While hypothalamic inflammation was seen following injection of
mechanisms, leading to the presence of unchecked activated HA-specific CD4+ and/or CD8+ T cells, narcolepsy-like symptoms
autoreactive CD4+ T cells in the brain. It was hypothesized that, were only observed when activated CD8+ T cells were administered
subsequently, local immune activation in the hypothalamus to the mice, pointing towards a role for CD8+ T-cell–mediated
would (due to a phenomenon known as ‘epitope spreading’) cytotoxicity. The researchers then went on to evaluate the role of
lead to involvement of other T cells with different targets, Pandemrix vaccination in this model, and their results confirm the
including HLA-DR–restricted CD4+ and CD8+ T cells (36). data from the original work: induction of HA-specific CD4+ and
Because these responses would be activated after disease CD8+ T cells in the HA-transgenic mouse model led to an
initiation by cross-reactive T cells, it is expected that such “immunopathological process mimicking narcolepsy” (76).
secondary immune responses would then be more disease- Whereas this mouse model was not designed to study T-cell cross-
specific and not necessarily cross-reactive. This would be reactivity, the results support a role for self-recognizing CD4+ and
supported by the peak response of cells with the DQ0602- CD8+ T cells in the pathology of NT1. The key role of CD8+ T cells in
based specificity detected at onset of the disease (31). Finally, the mouse model is consistent with the abovementioned discovery of
involvement of HCRT-specific CD8+ T cells in the response may HCRT-specific CD8+ T cells (70, 71), and with the expectation that
provide an explanation for how exactly CD4+ T cells could CD4+ T cells do not kill HCRT neurons, which only express HLA-I
orchestrate an autoimmune response resulting in the loss of and therefore would be recognized by CD8+ rather than CD4+ T cells.
HCRT neurons, given that these neurons do not express HLA-II. Given the pathogenic role of CD4+ T cells suggested by the human
In summary, rare cross-reactive DQ0602-restricted CD4+ T data, possibly CD8+ T cells are only engaged once an immune
cells could play a pivotal role as a trigger in disease onset under response is triggered by cross-reactive CD4+ T cells. However, it
specific conditions, most likely involving local inflammation should be noted that vaccine evaluations have failed to detect the
facilitating access across the blood-brain barrier. Once induction of any CD8+ T-cells in the blood of human recipients of
initiated, other cells, such as CD8+ and CD4+ T cells with AS03-adjuvanted vaccines (11, 77, 78).
different HLA restrictions and specificities, could become Tesoriero et al, on the other hand, used Recombinant
involved. It then follows that there would be neither an activating gene 1 (RAG1)-knockout (T-cell–lacking) mice to
immunological need, nor a plausible mechanism, for these demonstrate that intranasal infection with a neuro-adapted A/
cells to be cross-reactive. H1N1 influenza strain can trigger hypothalamic neuronal
infection (75). In these mice, the virus travelled via the
trigeminal nerve through the blood-brain barrier into the
Extending the CD4+ T-cell etiology hypothalamus where it infected HCRT neurons, which in turn
hypothesis: non-clinical data triggered NT1-like symptoms. While it has been argued that this
mechanism supports a virus-based rather than a T-cell–
The human data generated so far provided potential mediated auto-immune etiology (79), both are not necessarily
evidence for the T-cell recognition of HCRT, as well as mutually exclusive, because infection would serve as a strong T-
indications for cross-reactivity with HA, supporting a plausible cell attractant, as explained above.
immunological mechanism underlying the pathogenicity. Still, The model that emerged from the combined murine data
they do not connect the peripheral infectious events with the explained that (i) local brain inflammation is plausible and can
hypothetical local autoimmune responses in the human brain. be localized to HCRT-secreting neurons—an event that is likely
Specifically, insight was needed into which CNS events would be to attract a T-cell response that originated at the vaccine
required to first activate peripheral HA/HCRT-cross-reactive injection site, infected airways and/or draining lymph nodes,
CD4+ T cells to cross the blood-brain barrier, and then, instigate into the brain; and that (ii), if mimicry exists [as modeled by HA
the targeting of HCRT neurons in the CNS. This was where in the mouse study (74)], the involved T cells can instigate NT1-
animal models came into play. However, the models created like symptoms. However, cotton rat data have indicated that a
since the discovery of HCRT and HCRT-Rs, such as mice trigger for such T-cell migration is unlikely to come from an
deficient for these components or narcoleptic dog models, did intramuscular adjuvanted vaccine alone. Indeed, repeated
not fit the purpose because they do not allow to study disease injections of either AS03-adjuvanted vaccine or AS03 alone
etiology. To address this, novel mouse models were generated, as did not cause any inflammation in the CNS (80). Moreover,
reported by two independent teams in 2016 (74, 75). no changes in the blood-brain barrier integrity were observed in
Transgenic mice artificially expressing HA by HCRT neurons these animals (80). The collective animal data then informed a
under control of the HCRT promoter (thus bypassing the need for second hypothesis refining the mechanistic model, and
peptide mimicry, since it forces expression of an influenza epitope in confirmed/extended the earlier suggested necessity of an

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Buonocore and van der Most 10.3389/fimmu.2022.902840

environmental trigger preceding or acting in parallel with the (82). The potential inflammation triggers include brain injuries,
vaccination (5, 81). or the crossing of pathogens (e.g., A/H1N1pdm09,
Streptococcus) through the blood-brain barrier during
infection, but likely not the adjuvanted A/H1N1pdm09
Connecting the dots: the “two-hit” NT1 vaccination itself (80). Then, due to natural immune
etiology hypothesis surveillance, the local inflammation could result in T-cell
expansion and migration from the periphery into the brain.
Based on the collective immunological and epidemiological Importantly, in some DQ0602-positive predisposed individuals,
evidence, a hypothetical mechanistic model is proposed that such a response could include the rare DQ0602-restricted HA/
connects CD4+ T-cells targeting HCRT peptide sequences, with HCRT cross-reactive CD4+ T cells (“first hit”), which could then
T cells recognizing putative cross-reactive epitopes in H1N1 be amplified by an independent event at the time of, or soon after
antigens, such as the HA and NA antigens, given that these two infection (“second hit”; Figure 2) (5, 81). It is tempting to
antigens were present in both the H1N1 virus and the vaccine. speculate that the existence of an immune regulatory network
This model was based on assumed sequence or structural in the CNS (82) could necessitate a third hit, i.e., failure of the
similarities shared between the viral antigens and self-antigen immune tolerance mechanism. We hypothesize, based on the
(s) as T-cell targets (called ‘molecular mimicry’), such that CD4+ accumulated evidence, that in a very small proportion of
T cells responding to the influenza HA peptides could predisposed individuals, vaccination may have amplified the
conceivably cross-react with peptide sequences from HCRT (9, already triggered immune cascade, because administration of
31). The projected sequence of events would then be that the adjuvanted vaccines has been shown to stimulate CD4+ (but
secreted HCRT peptides would be picked up by antigen- not CD8+) T-cell responses (11, 77, 78). Due to the combined
presenting cells surrounding the HCRT neurons, such as action of both factors, and supported by the hypothesis of
microglia, making HCRT ‘visible’ to cognate CD4+ T cells. epitope spreading (36), local autoimmune recognition of
Consequently, in a predisposed host, these HLA-II–positive epitopes from hypothalamic HCRT neurons may then trigger
cells expressing DQ0602, would then present DQ0602-binding the activation of HCRT-specific CD4+ and CD8+ T cells. This
peptides to CD4+ T cells. In parallel, peptides from influenza might lead to the progressive loss of HCRT neurons, and,
antigens, such as those in the HA protein, are presented to ultimately, to NT1 symptoms in the affected individuals.
influenza-specific CD4+ T cells in peripheral lymph nodes In summary, if the A/H1N1pdm09 viral preexposure
following infection and/or vaccination. Such ‘mimicry suggested by some pharmaco-epidemiological studies is
peptides’ can then be recognized by HA/HCRT cross-reactive confirmed, a plausible hypothesis is that the ‘priming’ of the
CD4+ T cells. Any inflammation in the brain, caused by, for cross-reactive CD4+ T-cell response by natural infection was
example, infection or other trauma, could entice these T cells to further activated following pandemic influenza vaccination. In
cross the blood-brain barrier, in order to survey the local addition, if, as hypothesized, the putative mimicry peptide is
inflammation. These activated cross-reactive CD4+ T cells indeed specific to A/H1N1pdm09 protein, this would explain
could then recognize the mimicry HCRT peptide presented by several epidemiological observations demonstrating the
microglia, and trigger autoimmunity (36). association between an increased risk of NT1 and A/
To explain the high specificity of the peptide-DQ0602 H1N1pdm09 infection, including (i) the absence of a risk
binding, it was also assumed that the mimicry peptide, when increase prior to 2009, when the mimicry peptide was absent;
presented by DQ0602-expressing microglia, displays a unique (ii) the 2010 peak in China detected in the absence of Pandemrix
conformation. In this model, a slightly different way of binding vaccination (44, 45, 54); and, possibly, (iii) the European
of the same peptide to, for example, a protective allele such as incidence peak observed in 2013 (55).
DQ0603 (35), would prevent cross-reactive T cells from
becoming activated, because the peptide appears different from
the perspective of the TCR. Alternative hypotheses
The available evidence demonstrates that HA/HCRT peptide
similarity can occur independently from NT1 symptoms, as both Besides the T-cell hypothesis, several other possible scientific
healthy subjects and patients displayed CD4+ T cells recognizing explanations based on in silico or in vivo data have been
the presented DQ0602-HA/HCRT peptide complexes (31). It proposed, guided by purported differences between Pandemrix,
follows therefore that these cells may be required but are, by Arepanrix and Focetria. Table 1 presents an overview of these
themselves, insufficient to cause narcolepsy (NT1) symptoms. studies and their limitations, in several cases pertaining to the
Several relatively rare conditions can independently trigger local absence of plausible mechanistic links and/or confirming data
CNS inflammation, whereas the existence of a regulatory self- (32, 84, 85, 88). The scientific arguments centered mostly on
reactive immune network in the CNS should also be considered potential variations in the HA or nucleoprotein (NP) vaccine

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Buonocore and van der Most 10.3389/fimmu.2022.902840

FIGURE 2
NT1 immunopathogenesis model: T-cell cross-reactivity and “two-hit” hypotheses. The hypothetical model connects peripheral (left) and
hypothalamic (right) events. In this model, rare conditions, including brain injuries or pathogens (A/H1N1pdm09 virus, Streptococcus) crossing
the blood-brain barrier (BBB), prime for local brain inflammation and, due to immune surveillance, the expansion and migration of T cells from
the periphery into the brain. In DQ0602-positive persons, these T-cell responses could include DQB1*0602 (DQ0602)-restricted,
haemagglutinin (HA)/hypocretin (HCRT)–cross-reactive CD4+ T cells, as a “first hit”. These low-frequency immune responses could then be
amplified by an independent event (“second hit”), which in highly rare occasions might have been a vaccination that stimulates CD4+ T-cell
responses. Further response amplification due to ‘epitope spreading’ and autoimmune recognition of epitopes in HCRT peptides from the
hypothalamic HCRT neurons, may then activate the HCRT-specific T cells to launch immune attacks on these neurons. These attacks are
mediated by cytokine release and cytotoxic (CD8+) T lymphocyte (CTLs) that are ‘licensed to kill’ by the CD4+ T cells. The subsequently released
cytotoxic granules, containing perforin and granzymes, directly attack the virus-infected neurons upon HLA-I mediated antigen recognition. The
progressive loss of the HCRT neurons ultimately results in type-1 narcolepsy (NT1) symptoms in the affected person. Note that failure of
peripheral tolerance may constitute a third parameter that could affect risk. APC, antigen-presenting cell. TCR, T-cell receptor. HLA, human
leukocyte antigen. CD40L, CD40 ligand.

antigens (86, 87, 89, 92, 94) rather than on adjuvant differences, Overall, the field has moved to a stronger emphasis on a
which led the European Medicines Agency to conclude that mechanistic role for T cells in the etiology of NT1, as exemplified
“based on the evidence generated so far, a hypothesis that takes by recent clinical data (96). Confirmation of the T-cell cross-
into account the potential role of antigen is more likely to explain reactivity was not only provided by the mechanistic murine data
the increased risk of narcolepsy observed with Pandemrix than (74, 75) (§ Extending the CD4+ T-cell etiology hypothesis: non-
hypotheses that are based on a direct role for the AS03 adjuvant” clinical data), but also by a recent publication postulating
(9). The latter (adjuvant-based) hypotheses, focusing on a- mimicry between a DQ0602-restricted epitope in A/
tocopherol (contained only in AS03) were the topic of only H1N1pdm09 NA and a self-epitope from a protein (protein-
one report (83). O-mannosyl-transferase; POMT1) expressed in the CNS (97).
Rather than a T-cell–based etiology, several of the studies The authors concluded that the data identified POMT1 as a
proposing alternative models suggested a central role for potential autoantigen for B and T cells, though it has no known
antibodies, including cross-reactive anti-HCRT-R2/NP links to NT1.
antibodies, antibodies against NP itself, and anti-ganglioside or
other autoantibodies (87, 89, 92, 94). In particular, the
hypothesized causal role of NP/HCRT-R2-cross-reactive Discussion
antibodies has been challenged in later studies, which all failed
to reproduce these cross-reactive antibody profiles (31, 90, 91, From the in-depth pharmacoepidemiological and
93). It is therefore not straightforward to translate the collective immunological studies that were triggered by the increased NT1
antibody data into a plausible mechanistic model (94). incidence in Europe immediately following the A/H1N1pdm09
Furthermore, recent data by Lind et al. suggested that HA influenza pandemic, a model has gradually emerged which
antibody responses are both qualitatively and quantitatively plausibly explains the sequential immunological events
comparable between NT1 cases and controls, leading these underpinning the increased risk. In brief, the hypothetical
authors to conclude that HA antibodies are likely not involved mechanism centers on a possible molecular mimicry between
in the pathogenesis (95). antigens in the A/H1N1pdm09 virus and vaccines and in HCRT.

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TABLE 1 Alternative hypotheses.

Focus Observations and Hypotheses Counter argument

a-tocopherol in Neuronal cells exposed in vitro to free a-tocopherol displayed increased HCRT expression, presumably leading • Different behavior a-
adjuvant to higher neuron sensitivity to apoptosis (83) tocopherol in vivo vs in vitro
(84)
• No migration of formulated
a-tocopherol to CNS (85)
Change in HA of The increased frequency of a single amino-acid change in Pandemrix and wild-type virus compared to No mechanistic link between
vaccine Ag Arepanrix or other H1N1 vaccines was hypothesized to have altered the predisposition to NT1, by changing the reduced binding to DQ0602 and
binding to DQ0602 (86) NT1 pathogenesis
Change in NP of Polysorbate detergent used in Pandemrix production allegedly induces structural changes in the NP of the • Pandemrix and Arepanrix
vaccine Ag vaccine Ag, which increases anti-NP Ab responses cross-reacting with DQ0602 (87) NP sequences originate from
enhances cross- PR8 strain rather than from A/
reactive Ab H1N1pdm09 (88)
responses • No mechanistic link between
anti-NP Abs and NT1
development
Alleged similarity in NP and HCRT-R2 sequences (dubbed the “mimicry sequence”) is a target of cross-reactive • No clear mechanistic
anti-NP/HCRT-R2 Ab responses, which were claimed to be more abundant in vaccinees of Pandemrix vs involvement of such cross-
Focetria due to the relative enrichment of NP in Pandemrix. This would link Pandemrix vaccination with reactive Abs in the loss of HCRT
increased NT1 risk (89) neurons (88):
– HCRT neurons do not
express HCRT-R2
– No need for DQ0602
binding of “mimicry peptide” to
induce cross-reactive anti-
HCRT-R2 Abs (32)
• Mimicry sequence not
confirmed by independent
analyses or GenBank database
(entry KJ_942731) (88)
• Cross-reactive Abs were
detected in vaccinees both with
and without NT1 symptoms (89)
• No cross-reactive Abs
detected in later studies (31, 90,
91)
Vaccine Ag target Anti-GM3 auto-Abs found more often in vaccinated vs unvaccinated persons (18% vs 7%; P=0.035), and in • Key results are borderline
of Abs against Pandemrix-vaccinated NT1 patients vs healthy controls (15% vs 4%; p = 0.047). This led to the claims that anti- statistically significant (92)
ganglioside GM3 GM3 Ab responses are associated with DQ0602 (p = 0.016) in both vaccinated patients and controls, collectively • The relationship, if any,
linking Pandemrix to NT1 risk (92) between the Ab responses and
any A/H1N1pdm09 protein is
unclear
• The hypothesis was discarded
in earlier research (93)
Other auto-Ab Using microarray screening technology, other Ab targets were found to be recognized differently in NT1 • Unclear relationship, if any,
responses patients and healthy controls (94) between these proteins and any
A/H1N1pdm09 proteins
• Unclear if the proteins are
indeed autoimmune targets

Ag, antigen; Ab, antibody; CNS, central nervous system; HA, haemagglutinin; HCRT-R2, hypocretin receptor type 2; NP, nucleoprotein; NT1, narcolepsy type I; DQ0602, DQB1*0602; A/
H1N1pdm09, A/California/7/2009 H1N1.

The consequence of this mimicry may be to trigger an autoimmune vaccination, effectuating a “two-hit” hypothesis. Failure of
response targeting HCRT-producing neurons, activated by local peripheral tolerance could be considered a third hit.
responses of CD4+ T cells recognizing peptides from HCRT. The The collective in silico, in vitro, and preclinical in vivo data from
reaction could be primed by rare pathogen-instigated brain the research efforts—which are still continuing today—have
inflammation, or by genetically driven loss of peripheral progressively refined the initial hypothetical model of sequential
tolerance, attracting DQB1*0602-restricted, cross-reactive T cells immunological events, and have filled multiple knowledge gaps.
from the periphery. The frequencies of these T cells in the The scientific debate over the last decade has also demonstrated the
circulation, and the numbers of these cells subsequently migrating value of combining immunology research (focusing on T-cell cross-
to the brain, could then be amplified by a subsequent event, such as reactivity) with the pharmacoepidemiological evidence. Indeed, this

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Buonocore and van der Most 10.3389/fimmu.2022.902840

combination has offered plausible explanations within the “two-hit” Funding

hypothesis mechanistic framework, and the current review aimed to
provide an interpretation of the T-cell data that emerged over the This study received funding from GlaxoSmithKline
last decade, which fits within this framework. Future research, using Biologicals SA, which funded this literature review and took in
single-cell analysis and HLA tetramers, could focus on further charge all costs associated with the development and the
characterization of HCRT-specific T cells, including their fine- publication of this manuscript.
specificities, phenotypes (effector versus regulatory), and cross-
reactivity profiles. Further refinement of mouse models, e.g.,
DQ0602-transgenic mice, to recapitulate antigen-specificity and Acknowledgments
mimicry of disease development, would also be of value.
Understanding the immunological mechanism(s) underlying the The authors thank Fernanda Tavares Da Silva, Vincent
observed increased risk of NT1 is important from the perspectives Bauchau, Maria de los Angeles Ceregido Perez, Margherita
of public health and the patients, and can ultimately inform future Coccia, Marguerite Koutsoukos, Derek O’Hagan (all GSK) and
research on NT1 and, potentially, on other autoimmune diseases. Norman Begg (independent vaccine consultant) for their critical
Altogether, though no definitive conclusions can be drawn, the review of the manuscript. They also thank Ellen Oe (GSK) for
hypothesis of possible molecular mimicry remains plausible to providing medical writing support, and Business & Decision Life
explain the increased risk of NT1 observed following infection Sciences platform for editorial assistance and manuscript
and/or vaccination against A/H1N1pdm09 pandemic influenza. A coordination, on behalf of GSK. Gé raldine Drevon (GSK) and
better understanding of the narcolepsy disease mechanisms and of Nathalie Arts (Business & Decision Life Sciences) coordinated
the potential role of cross-reactive CD4+ T cells has brought novel publication development and editorial support.
insights and refinements of the proposed hypothetical model. In
this context, several lines of evidence converge on the notion that
the presence of such cross-reactive CD4+ T cells alone is not a Conflict of interest
predictor for the narcolepsy pathogenesis.
SB is employed by the GSK group of companies. RvdM was
Trademarks employed by the GSK group of companies. SB and RvdM hold
shares in the GSK group of companies. GlaxoSmithKline
Pandemrix and Arepanrix are trademarks owned by or
Biologicals SA funded this literature review and took in charge
licensed to the GSK group of companies. Focetria is a
all costs associated with the development and the publication of
trademark of Novartis. All these products are now withdrawn
this manuscript.
from use in the European Union. The marketing authorisation
The funder was involved in the study design, collection,
for Pandemrix and Focetria expired on 13 August 2015 and the
analysis, interpretation of data, the writing of this article and the
marketing authorisation for Arepanrix expired on 13
decision to submit it for publication.
December 2010.

Author contributions Publisher’s note

All authors contributed to the research, reviewed the All claims expressed in this article are solely those of the
literature, provided substantial input and reviewed the authors and do not necessarily represent those of their affiliated
paper. All authors approved the final article and are organizations, or those of the publisher, the editors and the
accountable for all aspects of the work. For RvdM, it is reviewers. Any product that may be evaluated in this article, or
noted that the views and opinions in this publication are his claim that may be made by its manufacturer, is not guaranteed
personal views and opinions. or endorsed by the publisher.

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