Following publication of this [1] article, questions were raised about some of the
reported methods and results, and about differences between the findings reported
in this article and in a previous article published by another group [2]. The PLOS
ONE Editors reviewed this matter, and consider that the research reported in the PLOS
ONE article is scientifically valid and meets the journal’s publication criteria,
but that some items require clarification and additional controls. The authors address
these issues below:
“As noted in [1], we were unable to replicate some findings reported by Ahmed et al.
in [2]. We would like to provide some clarifications regarding some methodological
differences between the two studies.
First, a statement in the Discussion was inaccurate in relaying the differences between
the results of peptide binding affinity to DQ0602. The statement: ‘The fact that NP111-121
(YDKEEIRRIWR) (116I was underlined) and HCRTR234-45 (YDDEEFLRYLWR) did not appear
to bind to DQ0602 (unlike what was reported with the Proimmune® array by Ahmed et
al. [43]) (Fig 2, S19 Table) suggested this epitope was likely irrelevant to DQ0602-associated
narcolepsy or differential vaccine risk’ should instead read: The fact that NP111-121
(YDKEEIRRIWR) (116I was underlined) and HCRTR234-45 (YDDEEFLRYLWR) did not appear
to bind to DQ0602 (Competition binding results S23 Table this Correction) (Of note,
‘NP109-113 116I’ of Fig 2 in [1] should be corrected to ‘NP109-123 116I’) suggested
this HCRTR2 epitope was likely irrelevant to DQ0602-associated narcolepsy or differential
vaccine risk. Indeed, S3 Table of Ahmed and Steinman’s Proimmune REVEAL® data [2]
showed weak binding for RELILYDKEEIRRIWRQANNG (24.4 and 16.9 of REVEAL® score at first
measure and post 24h, respectively), little binding for RELILYDKEEMRRIWRQANNG (1.5
and 0.5 of REVEAL® score at first measure and post 24h, respectively) and no binding
for the HCRTR2 peptide (LNPTDYDDEEFLRYLWREYLH) (0.0 of REVEAL® score at both first
measure and post 24h). We overlooked these small differences. Our results show little
binding for RELILYDKEEIRRIWRQANNG (92.40±2.33% of Bio-EBV binding), very weak binding
for RELILYDKEEMRRIWRQANNG (77.84±1.10% of Bio-EBV binding) and no binding for the
HCRTR234-45 peptide (98.12±1.38% or 99.85±2.10% of Bio-EBV binding) (competition binding
results S23 Table this Correction). This does not change our interpretation.
Second, intrigued by the results reported by Ahmed et al. [2], we also conducted our
own peptide binding studies to DQ0602 using the Proimmune REVEAL® binding assay (See
S1 File of this Correction) and compared these results for 144 peptides with our own
competition assay that uses DQ0602 monomers bound to Bio-EBV (more than 1,446 peptides
have been tested using this platform). Results are shown in S12 Fig this Correction
S23 Table this Correction; underlying data and analyses are provided in S2 File of
this Correction. Overall, no significant correlation was found between our competition
binding assay using Bio-EBV and Proimmune results (r2 = 0.00795, p = 0.290 at 0h;
r2 = 0.00026, p = 0.848 at S12 Fig this Correction). Classifying our binders into
strong (high displacement, ≤25% of Bio-EBV binding) or weak binders (partial displacement,
25–50% of Bio-EBV binding) in our competition assay and positive versus negative in
the Proimmune assay also did not reveal any correlation using χ2 (all p values>0.56,
S2 File of this Correction). Further illustrating this, the known EBV binding epitope
of DQ0602 (EBV486-500) [6–8], which was used in our competition binding assay, and
HA273-287, a strong binder in our assay (8.10±1.53% of Bio-EBV S23 Table this Correction),
were found not to bind to DQ0602 using the Proimmune® assay (0.0 of REVEAL® score
at both 0h and post 24h, S1 File of this Correction). This led us to conclude that
the Proimmune DQ0602 binding assay was unreliable. However, as these results were
peripheral to the message of our manuscript and lack of replication of the presence
of anti-HCRTR2 antibodies, these were not included in our original publication.
Third, in anti-HCRTR2 antibody testing experiments using serum samples presented in
Luo et al. [1], when we wrote we tested ‘similar’ samples to Ahmed et al. [2], we
did not mean to indicate we tested the exact same sera as Ahmed et al. [2]. We meant
to write that we tested ‘similar’ post-Pandemrix® narcolepsy and control samples but
collected in other countries (Ireland, primarily) (instead of Finland for Ahmed et
al. [2]). Our results with these samples (Fig 4 in [1]) differ from what is reported
in Ahmed et al. (Fig 2 in [2]). Unfortunately, we could not repeat testing of post-Focetria®
control (non-narcolepsy) samples, as these come from a clinical trial and are proprietary
to Novartis®. In Ahmed et al. [2], none of the post-Focetria® samples were reported
to have anti-HCRTR2 antibodies, unlike narcolepsy and control and samples from Finland
which are positive. We found that Focetria® and Pandemrix® do not differ significantly
in their NP sequence [1] suggested to be homologous to HCRTR2.
To further clarify the similarities in samples used in the two studies and enable
future efforts toward replicating these results, we provide the following details
regarding participant selection for the study reported in [1]. Inclusion criteria
for type 1 narcolepsy were narcolepsy with cataplexy and HLA-DQ0602 positivity, and/or
documented low CSF hypocretin-1. These criteria are based on the ICSD-3 and DSM-5
guidelines for diagnosing narcolepsy [4]. Controls were either healthy spouse or friends
of patients with narcolepsy or with other sleep disorders. These were screened for
the absence of narcolepsy through interviews and using a questionnaire [5] surveying
symptoms for all sleep disorders that contains questions about narcolepsy symptoms.
The patients selected for the Ahmed et al. [2] study were selected on the basis of
similar clinical criteria.
Fourth, regarding S4 Fig in [1], it was raised that the two lanes had substantial
differences in the number and weight of background bands, and that the presented western
blot did not clearly show that the transfected cell line had a strong specific band
at the molecular weight expected for HCRTR2. S13 Fig this Correction, we now present
a repeat of this experiment that also includes a gel image showing the amount of protein
extract used in this western blot experiment, plus a higher magnification view of
the region involved.
The experiment aimed to provide evidence that the commercial antibody recognized HCRTR2,
validating this antibody as positive controls for other assays. Of note, non-specific
bands are still observed outside of the focus area. In our opinion, these non-specific
bands we have observed are likely due to cross reactivity, post-translation modification
of HCRTR2, or fragment, although a monoclonal antibody was used. It is notably difficult
to obtain specific anti-G-protein coupled receptor (GPCR) antibodies and the western
blot was meant to show that the antibody may have off-target effects but was still
targeting HCRTR2, which was sufficient as a positive control for our experiments using
FACS [1].
Fifth, due to difficulty obtaining a proprietary cell line, we used alternative cell
lines in efforts to replicate the experiments reported in Ahmed et al. [2] and obtained
different results as reported in Luo et al. [1]. CHO-K1 cells were purchased in March
2016 from ATCC (https://www.atcc.org/products/all/CCL-61.aspx), and the CHO-HCRTR2
cell line, which was derived from CHO-K1, was purchased prior to 2008 from GenScript
(TM0508, GenScript, CHO-K1/OX2). It should be noted that the Chem-1 cell line, which
was used in Ahmed et al. [2], is described by the supplier Eurofins Discovery Services
as lacking endogenous expression of most GPCRs [3], which may not be the case with
CHO-K1. This could explain a very faint band at that location in our control cell
line.
Sixth, it was mentioned that in S11 Fig in [1], microscopy revealed HEK293 cells expressing
HCRTR2 in green (green fluorescent protein, GFP), mouse monoclonal antibody staining
of human HCRTR2 in red (Alexa Fluor 555), and nuclear staining in blue (DAPI) but
that the antibody staining against HCRTR2 is not demonstrating the expected “punctate”
staining pattern that would be typical for a GPCR such as HCRTR2. This was primarily
due to the poor quality of the microscope used. To complement this figure, we have
now repeated the experiment using a confocal microscope and show clear punctuate colocalized
staining (S14 Fig of this Correction). We are also showing similar punctate staining
of HCRTR2 with Alexa Fluor 555 on CHO-HCRTR2 cell line constitutionally expressing
HCRTR2, but not in control CHO-K1 (S15 Fig of this Correction).”
The Competing Interests statement for this article is incorrect. The correct statement
is: EM currently receives funding from Jazz Pharmaceutical and EM previously received
funding from GlaxoSmithKline for the study of the immunological basis of post-Pandemrix®-narcolepsy.
Conduct for these studies were supervised by and reported to the European Medical
Agency. GlaxoSmithKline holds the patent for Pandemrix®. Funding from these two sources
did not support the research reported in this article [1]. In addition, a provisional
patent on a potential DQ0602 hemagglutinin flu epitope sequence cross-reactive with
hypocretin was filed by GSK and Stanford with EM as one of the inventors, but the
patent was subsequently abandoned when the publication of De la Herran-Arita et al.
[9] was retracted [10].
Supporting information
S12 Fig
Lack of correlation of results obtained for DQ0602 binding using our in-house Bio-EBV
competition binding assay and the Proimmune REVEAL® assay.
144 peptides were tested using both assays. Note that the Bio-EBV competition results
were inverted (100%-displacement) so that higher value indicate higher binding for
easier comparison with the Proimmune assay.
(TIF)
Click here for additional data file.
S13 Fig
Repeat western blot of HCRTR2 over-expressing cell line (CHO-HCRTR2) versus control
host cell line (CHO-K1).
A. Coomassie blue staining of whole cell lysates used in S4 Fig of Luo et al. [1].
Note equal amount of protein in both lanes. B. Repeat western blot of the same protein
lysates stained with monoclonal anti-HCRTR2 antibody (Cat# WH0003062M1-100UG, Sigma),
with focus on the area of HCRTR2 molecular weight size.
(TIF)
Click here for additional data file.
S14 Fig
HEK293T and HCK293T-HCRTR2-GFP cells staining with monoclonal anti-HCRTR2 antibody
(Cat# WH0003062M1-100UG, Sigma).
Cells were cultured and stained as described in [1] (see “Anti-HCRTR2 autoantibody
detection with flow cytometry”). Images were taken using Leica TCS SP8 confocal microscope.
Contrast and brightness of the digital image of only DAPI channel from HEK293T-HCRTR2-GFP
were slightly adjusted for easier viewing. AF555, Alexa Fluor 555.
(TIF)
Click here for additional data file.
S15 Fig
CHO-K1 and CHO-HCRTR2 cell lines staining with polyclonal anti-HCRTR2 antibody (Cat#
ab65093, Abcam).
Cells were cultured and stained as described in [1] (see “Anti-HCRTR2 autoantibody
detection using in-cell ELISA”). Images were taken using Leica TCS SP8 confocal microscope.
These results complement our in-cell ELISA results obtained with this cell line. AF555,
Alexa Fluor 555.
(TIF)
Click here for additional data file.
S1 File
DQ0602 binding of Proimmune REVEAL® assay.
(XLSX)
Click here for additional data file.
S2 File
Raw data and analyses underlying S12 Fig this Correction.
(XLSX)
Click here for additional data file.
S3 File
Original gel image supporting results in panel A of S13 Fig.
(TIF)
Click here for additional data file.
S4 File
Original blot images supporting results in panel B of S13 Fig.
Note that the blot on the left, probed with the monoclonal antibody, was used in preparing
S13 Fig.
(TIF)
Click here for additional data file.
S23 Table
NP and HCRTR2 peptides binding to DQ0602 using Bio-EBV competition assay and Proimmune
REVEAL® assay.
(XLSX)
Click here for additional data file.