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VACCINES Copyright © 2020

The Authors, some
Transcriptional profiles of adjuvanted hepatitis B rights reserved;
exclusive licensee
vaccines display variable interindividual homogeneity American Association
for the Advancement
but a shared core signature of Science. No claim
to original U.S.
Government Works
Laurane De Mot1*†, Viviane Bechtold1*, Vanesa Bol1, Andrea Callegaro1, Margherita Coccia1,
Ahmed Essaghir1, Dicle Hasdemir2,3, Fernando Ulloa-Montoya1, Emilio Siena4, Age Smilde3,
Robert A. van den Berg5, Arnaud M. Didierlaurent1‡§, Wivine Burny1‡, Robbert G. van der Most1‡||

The current routine use of adjuvants in human vaccines provides a strong incentive to increase our understanding
of how adjuvants differ in their ability to stimulate innate immunity and consequently enhance vaccine immuno-
genicity. Here, we evaluated gene expression profiles in cells from whole blood elicited in naive subjects receiving

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the hepatitis B surface antigen formulated with different adjuvants. We identified a core innate gene signature
emerging 1 day after the second vaccination and that was shared by the recipients of vaccines formulated
with adjuvant systems AS01B, AS01E, or AS03. This core signature associated with the magnitude of the hepatitis
B surface-specific antibody response and was characterized by positive regulation of genes associated with
interferon-related responses or the innate cell compartment and by negative regulation of natural killer cell–­
associated genes. Analysis at the individual subject level revealed that the higher immunogenicity of AS01B-­
adjuvanted vaccine was linked to its ability to induce this signature in most vaccinees even after the first vaccination.
Therefore, our data suggest that adjuvanticity is not strictly defined by the nature of the receptors or signaling
pathways it activates but by the ability of the adjuvant to consistently induce a core inflammatory signature
across individuals.

INTRODUCTION quantities. AS03 contains -tocopherol in an oil-in-water emulsion,

Adjuvants in human vaccines can potentiate the immunogenicity of
vaccine antigens, particularly those derived from purified proteins,
through activation of a complex network of innate immune re-
sponses. This activation is thought to be a prerequisite for eliciting
adaptive immune responses (1, 2). Human system biology studies
have contributed to unraveling the transcriptional pathways acti-
vated by adjuvanted vaccines (3–8), but such studies have yet to
include a direct head-to-head comparison of different adjuvants. In
particular, the understanding of how the nature of these transcrip-
tional pathways and the intensity of their activation can influence
interindividual variability in the immune response to adjuvanted
vaccines remains elusive.
Here, we evaluated gene expression profiles in cells in whole
blood elicited in naive subjects receiving two doses of vaccines
containing the prototypic hepatitis B virus (HBV) surface antigen
(HBs) formulated with AS01B, AS01E, AS03, or AS04. These four
adjuvant systems (AS) were designed to combine various proin-
flammatory immunostimulants, each with their own distinctive fea-
tures (9). Their components include 3-O-desacyl-4′-monophosphoryl
lipid A (MPL) and saponin fraction 21 extracted from Quillaja
saponaria Molina (QS-21), both present in AS01B and AS01E. AS01E
is composed of a half-dose of AS01B with respect to MPL and QS-21
1
GSK, 1330 Rixensart, Belgium. 2Bioinformatics Laboratory, University of Amsterdam,
1012 WX Amsterdam, Netherlands. 3Biosystems Data Analysis Group, University
of Amsterdam, 1012 WX Amsterdam, Netherlands. 4GSK, 53100 Siena, Italy. 5GSK,
Rockville, MD 20850, USA.
*These authors contributed equally to this work as co-first authors.
†Present address: Clarivate Analytics, 2600 Berchem, Belgium.
‡These authors contributed equally to this work as co-senior authors.
§Present address: Faculty of Medicine, University of Geneva, 1205 Geneva, Switzerland.
||Corresponding author. Email: robbert.x.van-der-most@gsk.com
and AS04 contains MPL adsorbed on aluminum salt (Alum). Mecha-
nistic studies in mice treated with AS-containing vaccines have iden-
tified key mechanisms leading to efficient antigen presentation to
adaptive immune cells in the lymph node (10–13). Links between these
mechanisms identified in mice and the peripheral blood signatures
detected in nonhuman primates have been proposed (13–15).
AS-containing vaccines have demonstrated immunogenicity and
efficacy in diverse human populations, irrespective of age [reviewed in
(2, 9, 16)]. These populations include older or immunocompromised
adults (the licensed AS01B-adjuvanted herpes zoster and AS04-­
adjuvanted HBV vaccines), adolescents (the licensed AS04-adjuvanted
HPV-16/18 vaccine), and children (the AS01E-adjuvanted vaccines
against malaria and tuberculosis). Furthermore, several age ranges
are targeted by AS03-adjuvanted influenza vaccines. Although age
is an important determinant for an individual’s vaccine responsive-
ness, a host of other, mostly nonheritable, intrinsic factors are also
relevant (17). Their cumulative effect causes substantial variation in
prevaccination (baseline) immune status and vaccine responses be-
tween individuals, even within a population spanning the same age
segment (18, 19). For instance, baseline frequencies of monocytes and
natural killer (NK) cells in healthy adults can vary from negligible to
constituting about half of the total immune cell population (17, 20).
Accepted population-based thresholds of vaccine-induced protec-
tive immunity, such as those established for the hemagglutination
inhibition antibody titers used in influenza vaccine evaluations, do
not account for such variability. A better understanding of inter-
individual variation in vaccine responses could enable tailoring
these responses to specific needs, particularly with respect to low-­
responding or at-risk individuals.
In this study, we built on previous analyses of the innate and
adaptive responses detected (21, 22) by performing a deeper analysis

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of the blood transcriptional responses at various time points. We

focused on the heterogeneity of the early response across individuals
who received the same adjuvanted vaccine. Using this global gene
profiling approach, we expanded the targeted gene expression data
(22) by identifying additional functional pathways. Using the Alum-­
adjuvanted HBs vaccine as the comparator, previous studies reported
the most potent enhancement of adaptive and innate responses for
the AS01B-containing HBs vaccine, and an overall ranking (AS01B ≥
AS01E > AS03 > AS04 > Alum) emerged from these studies (21, 22).
We also previously detected rapid (within 3 to 6 or 24 hours) re-
sponses of inflammatory markers in recipients of any of these HBs/
AS vaccines, monocytes and neutrophils in recipients of the AS01B
or AS01E (AS01B/E) formulations, and cytokines implicated in innate
immunity in recipients of the AS01B/E or AS03 formulations (22).
Interferon (IFN)–related responses and increased C-reactive pro-

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tein (CRP) and interleukin-6 (IL-6) concentrations correlated with
adaptive responses and these markers were shared in recipients of
the AS01- or AS03-adjuvanted vaccines (22), despite their distinct
modes of action in mice (11, 12). This raised the question of whether
these commonalities reflected truly convergent response profiles
or a core inflammatory signature, with more nuanced distinctions
remaining undetected.
Here, we identified a distinct core gene expression signature
induced by hepatitis B vaccine adjuvants, irrespective of their com-
position, and investigated the association of this signature with
antibody response. Through an innovative data analysis approach to
study the interindividual heterogenicity of the response, we found
that this core signature was more consistently induced by some ad-
juvants, that is, some adjuvants induced this core response in most
recipients. The current study provides criteria to consider in adju-
vant selection for the development of future human vaccines.

RESULTS
Expression profile after one AS01B dose resembles those
seen after two AS01E or AS03 doses
To assess the dynamic regulation of gene expression, we used
linear-mixed models and moderated t tests on datasets normalized
by GCRMA (guanine cytosine robust multi-array analysis) (fig. S1).
When applying a false discovery rate (FDR)–adjusted P value of
<0.05 for the gene expression observed for at least one adjuvant and
on at least one time point, we retained a primary gene set of 1791
unique, differentially expressed genes (DEGs). Consistent with pre-
vious findings (22), transcriptomic response (in terms of numbers
of DEGs) across all time points were highest for the AS01B, AS01E,
and AS03 groups (≤1050 DEGs); modest for the AS04 (≤102 DEGs)
group; and low for the Alum (≤4 DEGs) group (Fig. 1A). This was
in line with the more complex innate immune stimulation provided
by AS in comparison to Alum (22).
After the first vaccination, responses for the AS01B, AS01E, and
AS03 groups (in numbers of DEGs) were negligible after 3 to 6 hours
[day 0+ (D0+)] but prominent on D1 (24 hours after administration)
with the largest D1 response detected in the AS01B group. These
responses had decreased by D14 after the first vaccination and were
low in the AS01B group and absent in the AS01E and AS03 groups
at 1 month after vaccination (D30). A greater number of DEGs for
these three groups was seen after dose 2. In contrast to the responses
at D1, these three groups had similar numbers of DEGs at D31
(1 day after the second dose) with the subsequent responses at D33
Fig. 1. Kinetics and clustering profiles of gene expression in cells from whole
blood induced by adjuvanted vaccines. Baseline-corrected gene expression was
determined for the primary gene set (fig. S1) at 3 to 6 hours and 1, 14, and 30 days
after the first dose (D0+, D1, D14, and D30) and 3 to 6 hours and 1, 3, and 7 days
after the second dose (D30+, D31, D33, and D37) of hepatitis B virus surface antigen
(HBs) vaccines adjuvanted with AS01B, AS01E, AS03, AS04, or aluminum hydroxide
(Alum), administered to HBs-naive adults. (A) Effects on the whole blood transcrip-
tome, expressed for each adjuvant group as numbers of differentially expressed
genes [DEG versus baseline; false discovery rate (FDR) P value of <0.05], are pre-
sented. Increased expression and decreased expression (up and down) were de-
fined by the sign of the moderated t statistics (>0, up and <0, down, indicating
increased and decreased gene expression, respectively). (B) The dendrogram above
the heatmap presents the global similarities among the timed gene expression
profiles per adjuvant group and was obtained by hierarchical clustering of the
baseline-corrected gene expression presented in the heatmap (red/blue: up-/
down-regulated genes represented by positive/negative moderated t statistics)
for each group and time point (among D1, D31, D33, and D37).
and D37 following the hierarchy observed after the first dose:
AS01B > AS01E > AS03. Responses in the AS04 group peaked at D14
after dose 1, but the response kinetics after dose 2 were comparable
to that seen for the other AS groups.
To extend this analysis, we identified adjuvant and time point
conditions with similar expression profiles, focusing on the tran-
scriptionally most active time points. Hierarchical clustering by
adjuvant and time point revealed a dichotomy between “early” (D1

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SCIENCE TRANSLATIONAL MEDICINE | RESEARCH RESOURCE
and D31) and “late” (D33 and D37) DEG responses (Fig. 1B). The
predominant clustering patterns were mostly time point based for
the vaccines containing AS01B, AS01E, or AS03 but treatment-based
for the AS04- or Alum-containing vaccines. The D1 response for
the AS01B group clustered with the D31 response for the AS01B,
AS01E, or AS03 groups. This early branch also contained the D1,
D31, and D33 responses for the AS04 group. The late branch con-
tained mostly responses to the second dose (that is, D33 responses
for AS01B/E and AS03 and D37 responses for all four AS), as well as
a separate cluster for all time points in the Alum group.
Thus, similarities were detected in the D1 responses for AS01B
and the D31 responses for AS01B, AS01E, and AS03. The different
clustering patterns—temporal for AS01 or AS03 versus treatment-­
based for AS04 or Alum—possibly signified different modes of
action for the three sets of adjuvants, which could be grouped as
AS01B/E/AS03, AS04, and Alum on the basis of this analysis.
AS01B, AS01E, and AS03 induce similar functional patterns
after two doses
We characterized the functional pathways perturbed by each adju-
vant. To identify gene sets with similar expression kinetics, we per-
formed clustering analyses of the primary gene set of 1791 DEGs.
We clustered the DEGs based on the time profiles in each adjuvant
group (see fig. S1 and Materials and Methods for analysis details).
Using these gene clusters, we performed enrichment analysis using
blood transcriptional modules (BTMs) as defined in (23) and ob-
tained 88 modules (FDR q value of <0.05). We performed an addi-
tional clustering step, which yielded five functional clusters, referred
to as C1 to C5 (Fig. 2, table S1, and data file S1). Of these clusters,
two were related to regulation of early response parameters: C1 in-
cluded 35 BTMs, many of which related to innate immune cells, and
C2 included 8 BTMs, many of which related to the IFN pathway
and antiviral sensing. C3 included 10 BTMs, which corresponded
principally to NK cells. Late response clusters pertained to the acti-
vation of adaptive immune cells and cell division (C4 with 21 BTMs)
or to metabolism and involved cell cycling, heme biosynthesis, and
platelet and erythrocyte functions (C5 with 14 BTMs). Aligned with
the hierarchy between groups seen with the condition-based clus-
tering (Fig. 1B), the AS01B/E and AS03 groups had DEGs indicating
responses for each cluster, whereas the AS04 group had DEGs asso-
ciated with responses for just C1, and the Alum group had no DEGs
enriched in any cluster.
With these clustered data, we noted different kinetics of the
functional responses. Innate cell–related responses (C1) were mark-
edly up-regulated in the AS01B/E and AS03 groups 1 day after each
vaccination. In line with the trends seen in DEG responses across
the groups (Fig. 1A), the strongest up-regulation at D1 occurred in
the AS01B group. Responses in this group were mostly repressed at
D37. In the AS04 group, responses were moderate at D14 and ab-
sent at D30, but responses at D31 (1 day after the second dose) were
increased when compared to the response 1 day after the first dose.
IFN-related responses (C2) in the AS01B/E and AS03 groups were
robustly up-regulated after each dose. The magnitudes at D1, in terms
of percentages of DEGs in this functional cluster, were AS01B ≈
AS01E > AS03, but the magnitudes were similar among these groups
after dose 2. These kinetics resembled those of the STAT1 up-­
regulation for these groups previously identified by quantitative
polymerase chain reaction (qPCR) (22). NK cell gene signatures
(C3) were slightly down-regulated at D1, most prominently for the
De Mot et al., Sci. Transl. Med. 12, eaay8618 (2020) 11 November 2020
AS01E group, and more strongly down-regulated at D31 in the
AS01B/E and AS03 groups. Overall, the changes functional responses
represented by C1, C2, and C3 were transient: Responses decreased
to undetectable or balanced (similar numbers of DEGs showing
up-regulation as those showing down-regulation) at either D14 for
AS01B/E and AS03 or D30 for AS04 after the first dose and at D33
for the decrease in the NK cell response (C3) or D37 for the innate
and IFN pathway responses (C1 and C2) after the second dose. The
exception was the inverted response in C1 for AS01B at D37.
In contrast to the early response clusters (C1 and C2), the late
response clusters (C4 and C5) in the AS01B/E and AS03 groups
generally persisted at D37. The C4 response exhibited complex dy-
namics. Responses related to plasma cells and B and T lymphocytes
(C4) were mostly low and reduced at D1 (a low percentage of DEGs
in the cluster and most of those were down-regulated). However, at
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D30+ (3 to 6 hours after the second dose) in the AS01B and AS03
groups, the adaptive response cluster (C4) was activated, possibly
associated with the proliferation of these cells. This was followed at
D31 by primary repression in the AS01B/E groups or bidirectional
regulation in the AS03 group and at D37 by uniform up-regulation
in these three groups, with AS01B > AS01E ≈ AS03. A reason for the
greater response in the AS01B group at D37 was that several T cell–
related modules (M83, M97, M7.4, M19, and M7.0) were activated
only in this group.
Up-regulation of metabolism-related functions (C5) was weak at
D1 in the AS01B/E groups and modest at D14 in the AS01B group
but stronger after the second dose in the AS01B/E and AS03 groups,
persisting with a high percentage of DEGs up-regulated through
D37 in the AS01B/E groups. This is consistent with proliferation of
cycling adaptive cells and aligns with the trends observed for other
AS01-adjuvanted antigens or oil-in-water emulsion-adjuvanted
antigens (6, 8).
Together, responses to HBs/AS01B/E and HBs/AS03 were char-
acterized by a core signature comprising the activation of genes
regulating innate cellular or IFN responses or both, consistent with
previous data (22), and down-regulation of NK cell–associated genes.
The responses followed an intergroup gradient (AS01B > AS01E > AS03)
after the primary vaccination, which disappeared after the secondary
vaccination, because these three AS shared similar modular response
levels at peak (D31). In contrast, HBs/AS04 triggered exclusively
innate cell–related responses, which peaked at D14, rather than D1,
as was observed for the other AS-adjuvanted vaccines.
Interindividual variability in the early response depends
on AS adjuvanticity
We asked whether all individuals receiving the same adjuvant would
respond in the same fashion. Two approaches were followed to
define the interindividual variability in the transcriptional response.
First, individual response patterns were visualized using cumulative
distribution curves for each of the functional clusters. Second, syn-
chronization of gene expression at the group level was approached
by principal components (PC) analysis (PCA).
In the first approach, the number of genes that were significantly
up- or down-regulated in each BTM cluster was counted for each
individual. We set the threshold absolute fold change of >1.5 in gene
expression over its expression at D0. To quantify the homogeneity
in the numbers of perturbed genes across all subjects receiving the
same vaccine, the number of significantly up- or down-regulated genes
in each BTM cluster was then plotted as cumulative distribution
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Fig. 2. Functional charac-

terization of the transcrip-
tomic vaccine responses.
The figure shows the amount
of activity in the 88 blood
transcriptional modules
(BTMs) enriched (FDR q
value of <0.05) using DEGs
induced by the indicated
adjuvanted vaccine. BTMs
were grouped in five data-­
driven clusters (C1 to C5)
using k-means clustering

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analyses. Gene sets included
in the modules have been
described (23). Sphere size
indicates the percentage
of the DEGs in the BTM,
calculated as the number
of DEGs (FDR P < 0.05)
among all genes in the
BTM × 100, over the total
number of genes present
in the microarray chip for
the given BTM. Sphere color
indicates the change in
activity of the BTM based
on the ratio of up-regulated
to number of DEGs in the
BTM (as determined by
moderated t statistics >0
for up-regulated and <0 for
down-regulated). Changes
in activity were considered
positive (red) if more DEGs
were up-regulated than
down-regulated, no change
(0) if the amount of up-
and down-regulated DEGs
were equal, or negative
(blue) if more DEGs were
down-regulated than up-­
regulated. Dashed vertical
lines separate the first and
second administrations of
the same adjuvanted vac-
cine; solid vertical lines
separate the different ad-
juvanted vaccines. Day of
sample collection is indi-
cated along the bottom
from early (D0+ to D37)
for each.

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SCIENCE TRANSLATIONAL MEDICINE | RESEARCH RESOURCE

curve for each vaccine group (Fig. 3A). This workflow was adapted interindividual heterogeneity between the adjuvant groups, with
from (24). Cumulative distribution curves of early (D1 and D31) patterns varying both by time point and by functional cluster
responses in the individual subjects revealed different degrees of (Fig. 3B). At D1, individual responses of genes related to the innate
response, IFN pathway, and NK cells
(C1 to C3) displayed a hierarchy among
the adjuvants of AS01B > AS01E > AS03,
except for NK cells in which AS01E >
AS01B. For instance, for expression of
genes related to the IFN pathway (C2),
changes in ≥25 genes were detected in
~70% of AS01B and 55% of AS01E re-
cipients but in only ~25% of the AS03
recipients. As expected, none of the AS04
or Alum recipients reached this high of
a response.

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At D31, a clear separation between
the groups emerged with respect to
innate cell responses (C1), adaptive
immune cell responses (C4), and IFN-­
mediated responses (C2). At this time
point, we detected relatively homogeneous
individual responses for AS01 recipients
for these clusters. For example, for C1,
>80% of individuals have between 160
and 180 genes for AS01 differentially
expressed at D31, whereas this was more
spread out for the other AS vaccines.
Heterogeneity among individual re-
sponses was higher for AS03 recipients
than for the AS01 recipients; however,
the amount of heterogeneity at D31 in
the AS03 group was closer to that of the
AS01 groups when compared to the
heterogeneity at D1. The most hetero-
geneous expression at D31 for innate
cell responses, adaptive immune cell re-
sponses, and IFN-mediated responses
was observed for the AS04 or Alum re-
cipients. Responses for NK cell–related
functions (C3) were stronger in terms of
numbers of genes perturbed for AS01B
recipients and more modest for AS01E
or AS03 recipients. The responses in
the metabolism-related cluster (C5) were
heterogeneous at both D1 and D31 with
a slight separation in the curves between
the Alum and AS04 recipients and the
AS01B/E and AS03 recipients.
In the second approach, we quantified
the interindividual synchronization of
each gene’s expression in subjects of the
same adjuvant group. For each gene
and each adjuvant group, we performed
Fig. 3. Relationship between the adjuvants and individual transcriptional responses 1 day after each vaccina-
PCA on datasets that contained expres-
tion. (A) Schematic overview of the workflow followed to generate the reverse cumulative distribution plots. (B) Reverse
cumulative distribution plots representing the numbers of responsive genes 1 day after primary (D1) or secondary
sion values over all time points and all of
(D31) vaccination (│FCD1/D0│ > 1.5 and │FCD31/D0│ > 1.5, respectively, where FC refers to fold change), detected in the group’s subjects (39,200 probe sets;
cells from whole blood from individuals who received two doses of vaccine with the indicated adjuvants. Results are fig. S1). We then used the percentage of
stratified by the five identified BTM clusters (C1 to C5; see Fig. 2). Genes included at D1 or D31 were those of the variation captured by PC1 as a metric to
primary gene set (fig. S1). quantify the amount of synchronization

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(Fig. 4A). The percent synchronization

was slightly higher in AS01B recipients
than in all other recipients (Fig. 4B).
We used the percent of synchronization
for the highly synchronized outlying
genes to compare the synchronization
among different groups. Using the max-
imum value of the outlying genes, we
found that the groups followed the hier-
archy we observed in other analyses:
~85% in the AS01B/E groups, ~70% in
the AS03 group, and ~55% in the AS04
and Alum groups (Fig. 4B).
To provide biological context, we clus-
tered the highly synchronized genes,

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those exhibiting percent of synchroni-
zation of >55%, using synchronization
metric values over five adjuvants. This
cluster analysis of the highly synchro-
nized genes resulted in three main func-
tional synchronization clusters (SC1, SC2,
and SC3) (Fig. 4C). We observed that
both SC1 and SC3 displayed a highly
synchronized expression for the AS01B/E
or AS03 groups. Synchronization levels
in SC1 (of which the gene shown in
Fig. 4A is a representative) displayed
the intergroup hierarchy as expected
from the preceding analyses, whereas
SC3 displayed similar levels for both
AS01 groups. We functionally annotated
the SCs using BTM enrichment analy-
ses (see fig. S1). SC3 contained mainly
IFN-inducible genes, whereas the genes
in SC1 were not significantly enriched
in any BTM (table S2). SC2, by contrast,
displayed only a highly synchronized ex-
pression for AS01B recipients and in-
cluded genes mediating T cell activation,
inflammation, or antigen presentation.
Collectively, the results from analy-
ses at the individual level indicated that
the differences observed in group response
magnitudes in terms of numbers of DEGs
(Figs. 1A and 2) between the AS01B/E
and AS03 groups at D1 appeared linked
to differences in the proportions of re-
sponding subjects rather than to func-
tional differences (Fig. 3B). Vaccination
with HBs/AS01 B led to a high inter-
subject homogeneity even after the
primary vaccination, and this initial homo­ Fig. 4. Synchronization analysis workflow and results. Figure shows a schematic presentation of the workflow
followed to assess the synchronization of gene expression among subjects. (A) Plots represent the kinetics of the
geneous response was particularly evident
standardized expression profiles of a representative gene (for example, ApoL6, encoding apolipoprotein L6) over the
for the genes comprising the core signa-
nine time points. The values in each plot represent the percentages of variance captured by the first principal com-
ture of IFN-inducible genes and genes ponent (PC1). PCA, principal components analysis. (B) Percentages of variance captured by PC1 among subjects of
regulating innate and NK cell–related func- the same adjuvant group are presented. Boxplots represent the medians, and the lower and upper whiskers present
tions (Fig. 3B). This core signature was the first quartile − [1.5 × the interquartile range (IQR)] and the third quartile + (1.5 × IQR), respectively, based on a
identified on the basis of the group re- default option in the R program. (C) The percentage of variance explained by PC1 is presented for each functional
sponses to the AS01B/E- or AS03-adjuvanted synchronization cluster (SC) and for each adjuvant group. BTMs included in each SC are presented in table S2.

De Mot et al., Sci. Transl. Med. 12, eaay8618 (2020) 11 November 2020 6 of 13
SCIENCE TRANSLATIONAL MEDICINE | RESEARCH RESOURCE
vaccines (Fig. 2). The difference in heterogeneity between AS01B/E
and AS03 observed at D1 persisted after the second dose (D31)
(Fig. 3B), despite the apparent similarity of gene expression patterns,
especially those associated with the IFN pathway, identified at the
adjuvant-group level (Fig. 2).
Early activation of specific innate pathways is associated
with the adaptive response
The observation of the apparent convergence of the blood tran-
scriptomic signatures identified for AS01B/E and AS03 recipients
prompted us to assess whether these gene responses were associated
with the HBs-specific adaptive response at D44, as reported previ-
ously (21). To that aim, we performed PC regression of the log2 fold
change in the expression values of genes within the primary gene set
and used the adjuvant group, the first three PCs, and the technical
scaling factor as predictors. Expression at D1 or D31 versus D0 and
expression at D31 versus D30 were analyzed.
The gene loadings summarized in PC1 were associated with the
scaling factor values. Gene loadings of PC2 for D31 versus D30 were
associated with the antibody response at D44 (P = 0.001) but not
with any of the CD4+ T cell response parameters (Fig. 5, A and B).
As identified by pathway analysis, genes contributing to this associ-
ation (that is, those with the highest ranking PC2 loadings) were
mostly implicated in regulation or activation, or both, of the IFN
pathway (Fig. 5C). Previously, we identified an association between
early responses after the second dose—in particular, increased
serum IL-6 and CRP concentrations and IFN pathway activation—
and the humoral or cellular responses to these vaccines at D44 (22).
Here, consistent with the previous analysis (22), we found a strong
association (P < 0.001) between the PC2 that summarizes these early
responses for the current cohort and the PC2 for the D31 versus
D30 transcriptomic data using linear regression analysis (fig. S2).
To integrate the outcomes of the different analyses, we subjected
the 300 genes most highly ranked in PC2 loadings and therefore
most likely to be associated with the antibody titers to cluster analysis
using the five functional clusters defined in Fig. 2 (Fig. 6). These
genes were annotated mostly in the three clusters regulating early
and NK cell responses (C1, C2, and C3; see Fig. 2), with a small
fraction of the genes belonging in the late response clusters (C4 and C5).
However, ~60% (184 of 300) of the genes in the analysis were not
associated with any of the 88 BTMs contained in the five functional
clusters. When we subjected these 184 genes to Ingenuity Pathway
Analysis, we found that only few canonical pathways were signifi-
cantly enriched, of which four were related to the already identified
innate (TREM1, FcRIIB, CCR5, and salvage pathways of pyrimidine
ribonucleotides) and cell cycle responses (table S3).
The cluster analysis also visualized the individual heterogeneity
within the same treatment group. We demonstrated that the adjuvant
groups differed with respect to the numbers of “responders” after the
second dose (where responders are individuals expressing the specific
transcriptome signature identified as being associated with antibody
titers). Most AS01B or AS01E recipients responded, but only about
half of the AS03 recipients responded, with a marked separation
between nonresponders and responders. Proportions of responders
were low among AS04 and Alum recipients, although some of these
individuals mounted modest responses for specific genes. These re-
sults support our results from the reverse cumulative distribution
analysis (Fig. 3) that, compared to the response induced by two doses
of AS01B, substantial heterogeneity among individual responses
De Mot et al., Sci. Transl. Med. 12, eaay8618 (2020) 11 November 2020
Downloaded from http://stm.sciencemag.org/ at Cambridge University on November 21, 2020
Fig. 5. PCA results of the transcriptomic response at D31 compared to D30.
(A) Statistical associations between the (log 10) adaptive response parameters
(antibodies and CD4 T cells) at 14 days after dose 2 (see table S5) and the transcrip-
tional response at D31/D30 [represented by the scores of the second principal
component (PC2) for the 1791 genes of the primary gene set; fig. S1] were calculated
using linear regression models. Note that the PC1 was not used because it was
strongly associated with the microarray scaling factor. PCA was performed on the
log2 FCs in gene expression at D1 and D31 over baseline expression (D1/D0 and
D31/D0, respectively) or at D31 over expression on D30 (day of the second dose)
(D31/D30). P values were not adjusted for multiplicity; statistical significance
(P < 0.01) is indicated in bold. Polyfunctionality indices were calculated on
CD4 + T cells positive for at least CD40L alone or with an additional one, two,
or three markers among interleukin-2 (IL-2), tumor necrosis factor– (TNF-), or
interferon- (IFN-) (21). (B) D44 antibody (Ab) concentrations (in log10) are plotted
against the PC2 scores (D31/D30). Each point represents an individual subject,
color-coded by the adjuvant treatment. (C) Plots of loadings of expression values
(PC2 and PC3) (FC D31/D30) for the primary gene set are shown. For ease of
representation of the data, an arbitrary cutoff of 0.070 was applied for the PC2
loadings. Genes with loadings below this cutoff are shown as gray dots; genes with
a PC2 loading of ≥0.070 are labeled, red indicates an association with the IFN pathway,
and black indicates no association.
persisted after the second dose of AS03, regardless of the apparent
similarity in responses to the AS01B/E- or AS03-adjvuvanted vaccines
at the group level.
To assess whether these findings were unique to the HBs/AS
vaccines, we evaluated these same 300 genes and five BTM clusters
in 15 recipients of the malaria vaccine RTS,S/AS01E (4, 25) and ob-
served a similar pattern to the transcriptomic responses 1 day after
the second vaccination (D29 versus D28 data) (Fig. 6).
Collectively, our results showed that the transcriptomic responses
after immunization with HBs antigen formulated with AS01 B,
AS01E, or AS03 were described by five clusters of BTMs. The sta-
tistical association between transcripts relating to innate immunity
at D31 (comprising transcriptomic responses of innate-related,
IFN-related, and NK cell–related genes) and the antibody titers
at D44 is consistent with the previously identified association (22).
Responses to two doses of AS01B/E or AS03 shared a core signature;
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sponses (C3), which was followed by an

increase in adaptive cell-related responses
(C4). The last cluster related to metabo-
lism (C5) was most likely linked to both
innate and adaptive responses. C5 also
contained BTMs related to platelet acti-
vation, which may provide interesting
avenues for further research. The de-
scriptive analysis of the transcriptional
data revealed a dichotomy between the
AS01 B-, AS01 E-, or AS03-formulated
vaccines, which induced shared tran-
scriptional responses, and vaccines for-
mulated with AS04 or Alum, for which
responses were weaker (AS04) or nearly

Downloaded from http://stm.sciencemag.org/ at Cambridge University on November 21, 2020

absent (Alum). Second, transcriptional
responses for several clusters, C3 and
C4 for AS01B/E and C1 to C4 for AS03,
were stronger after the second dose than
after the first dose. Third, the nature of
the early response was similar between
the groups receiving AS01B/E and the
group receiving AS03, although these AS
contain different adjuvants with no
common immunostimulants. We de-
termined that these three AS shared a
core signature that was characterized by
a strong IFN-related (C2) response, which
is consistent with previous data (22), and
by innate responses and down-regulation
of NK cell–associated responses. Despite
Fig. 6. Interindividual heterogeneity in transcriptomic responses linked to antibody responses. Linear regres- this overall similarity in the responses
sion models were used to assess the association between the transcriptomic response at D31 and the antibody re- induced by these three AS, compared to
sponse at D44. Data of the five leftmost heatmaps represent the 300 genes (data file S1) (presented in rows) with the the first dose of AS01E or AS03, the first
highest loadings (absolute values) in PC2 from the PCA performed on the log2 FC D31/D30 matrix (see Fig. 5). The dose of AS01 B appeared to induce a
vertical colored bar shows the corresponding BTM cluster number(s) for the genes (see Fig. 2). Each column corre-
strong innate signal in a higher number
sponds to an individual subject. The rightmost heatmap represents the data generated for these 300 genes, using
samples from 15 healthy malaria-naive participants in a clinical trial evaluating the AS01E-adjuvanted RTS,S malaria
of individuals, coinciding with a lower
vaccine (4, 25). The rightmost heatmap was generated from a Pearson correlation–based hierarchical clustering anal- degree of interindividual heterogeneity.
ysis performed on the log2 FC D29/D28 data, with D29 corresponding to D1 after the second dose of RTS,S/AS01E. Although this apparent difference be-
tween the three groups was reduced
after the second dose, a considerable
however, the responses differed in the amount of interindividual degree of heterogeneity persisted in the AS03 cohort. Last, the pre-
heterogeneity. viously identified association between early transcriptional responses
and D44 antibody titers (22) was confirmed for the full transcrip-
tomic dataset.
DISCUSSION Collectively, the data painted a diverse picture of functional
A better understanding of the mechanisms underpinning adjuvant-­ responses, which were quantitatively or qualitatively different be-
induced innate immunity, including its interaction with adaptive tween the two doses and sometimes both quantitatively and qualita-
immunity, should inform the development and licensure of novel tively different. Activation of BTMs C1 to C3—linked to myeloid
vaccines and adjuvants for various human populations. For the four cells, dendritic cells (DCs), Toll-like receptor (TLR) and IFN signal-
AS evaluated here, we previously unraveled mechanisms of action ing (through a positive regulation), and to NK cells (through a
in animal models (10–13) and characterized the innate response in negative regulation)—was already detected after the first injection
human whole blood (22). To further our understanding, here, we of the AS01B/E- and AS03-formulated vaccines. This reflected a
used a systems biology approach and performed transcriptomics data direct activation of innate immunity, which also occurs in animal
analyses, both at the group and individual subject level. models (11–13). BTMs linked to adaptive responses (C4), compris-
Several conclusions can be drawn. First, four of the five identi- ing genes associated with B cells and CD4+ T cells, were observed
fied BTM clusters collectively captured the evolution from innate to later, at D37, in line with the higher HBs-specific responses mea-
adaptive immunity—early stimulation of innate responses and the sured after a second dose. The low and heterogeneous transcrip-
IFN pathway (C1 and C2), along with suppression of NK cell re- tomic response to the vaccine adjuvanted with AS04 suggested that,

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SCIENCE TRANSLATIONAL MEDICINE | RESEARCH RESOURCE
in humans, this AS is a less potent stimulator of innate immunity
than are AS01B/E or AS03, with the caveats that (i) the described
responses are systemic, thus not fully reflecting any local responses;
and (ii) the absence of the identified core transcriptomic signature
does not necessarily imply the absence of involvement of other
pathways. We detected activation of modules in the C1 cluster reg-
ulating cells of the myeloid, but not lymphoid, lineages after each
dose of HBs/AS04, aligning with the changes in the frequencies of
innate cells and the increased IL-6 concentrations previously reported
in recipients of this adjuvanted vaccine (22). A potential concern
with our analysis is the timing of data collection, which could have
missed peak responses. However, in mice, a TLR4-dependent, tran-
sient inflammatory response and increased numbers of activated
antigen-loaded monocytes and DCs were observed within 1 day
after administration of AS04 (10), aligning with the activation of
human DCs in response to MPL observed ex vivo (26). Thus, we
think it unlikely that our selected time points missed detection of
the peak response. Overall, the consistency in our gene-based and
BTM-based analyses (IFN-related signals and cell cycle–associated
and lymphocyte proliferation–associated responses) with previous
observations in human vaccinees (22) and in animal studies (13)
supports the ability of our study to further translational research
efforts linking human and animal model data.
Another key observation was the difference between the BTM
responses measured after the first and second doses of AS01B/E- or
AS03-adjuvanted vaccines. Responses in the clusters containing innate
and IFN-associated BTMs (C1 and C2) were more prominent after
the second dose, particularly for AS03. Moreover, the down-regulation
of NK responses (C3) was stronger at D31 compared to that at D1
for the AS01B/E or AS03 groups. Similar observations were made for
AS01E-adjuvanted malaria or tuberculosis vaccines (4, 6). Because
the current subjects were naive before immunization, an obvious
difference between the first and second dose responses was the
presence of immunological memory at the time of the second dose.
As proposed (22), and given the key role of early IFN- in the mode
of action of adjuvants (13, 27), a mechanism could be envisaged by
which CD4+ T cell memory elicited after the first dose enhances the
subsequent down-regulation of NK cell responses and up-regulation
of IFN- responses, possibly through IL-2–mediated NK cell activation
and migration (28, 29), which then leads to an enhanced innate re-
sponse. Consistent with such a mechanism are the peripheral IFN-­–
producing NK cells observed in human recipients of AS01-adjuvanted
tuberculosis and malaria vaccines (29, 30) and the increased serum
IFN- concentrations in human or nonhuman primate recipients of
AS01-adjuvanted HBs or malaria vaccines (4, 13, 22, 31), which were
all detected mostly after the second dose. In contrast, AS03-adjuvanted
H1N1 or H5N1 split influenza vaccines induce up-regulation of the
IFN signaling pathway after the primary vaccination (3, 8, 32). A pos-
sible explanation for this response to the influenza vaccines is that
primed participants, who had received influenza vaccines or experienced
influenza infection previously, had cross-reactive nucleoprotein-­
specific memory CD4+ T cells that provided the innate-stimulating
memory signal at the time of the first dose. Alternatively, differences
in early transcriptomics responses could reflect trained innate
immunity (22).
The similarities in gene signatures in cells in whole blood be-
tween the vaccines adjuvanted with AS01B/E or AS03 is remarkable,
given that these AS are thought to have different mechanisms of
action. In the absence of the TLR4 ligand MPL, AS03 adjuvanticity
De Mot et al., Sci. Transl. Med. 12, eaay8618 (2020) 11 November 2020
is likely not mediated by TLR stimulation. Instead, the adjuvant
property of AS03 is, at least partially, mediated by inducing endo-
plasmic reticulum stress (12, 33). Data from mice show that adju-
vants, irrespective of their composition and the innate pathways that
they trigger, can induce a shared inflammatory response characterized
by common antigen presentation and cytokine-related pathways
(34, 35). It is therefore conceivable that the innate pathways activated
by AS01B/E and AS03 in humans also eventually converge toward a
shared innate response that is detectable in blood.
However, AS01B/E- and AS03-induced responses also exhibited
differences, particularly after the first dose. Compared to HBs/AS03,
the magnitude of the response (the number of DEGs) induced by
the first dose was notably higher for HBs/AS01B, with an overall
expression profile (Fig. 1B) that resembled those induced by two
doses of AS01E- or AS03-containing vaccines. Furthermore, our
Downloaded from http://stm.sciencemag.org/ at Cambridge University on November 21, 2020
data indicated that AS03 differs from AS01B/E after the second dose
in terms of interindividual response heterogeneity. The differences
in numbers of DEG and degrees of heterogeneity might reflect dif-
ferences not only in the composition but also in the type of interac-
tions between components of the AS01 and AS03 adjuvants. The
interaction between squalene and tocopherol in AS03 results mainly
in additive effects, whereas MPL and QS-21 in AS01 show molecular
and cellular synergy, resulting in stronger activation of the innate
immune response (12, 13). One key feature of this synergy is IFN-
production by lymph node–resident lymphoid cells, such as NK
cells, as well as an overall increase in innate responses. This may
explain the more prominent activation of innate (C1)– and IFN
(C2)–associated BTMs that we detected after the first dose of AS01B/E.
The higher level of innate stimulation by AS01B/E then resulted in a
more homogeneous response than was observed for AS03. Although
interindividual variability in baseline gene expression was not as-
sessed, we postulate that the stronger innate response mitigated the
effects of any baseline variability on the immune response. This hy-
pothesis is supported by data showing that the inflammatory immune
status before vaccination can negatively influence the response to
an Alum-adjuvanted HBs vaccine, presumably due to the weaker
adjuvanticity elicited by this vaccine (36).
Using PCA, we detected a correlation between antibody re-
sponses and 300 genes captured in the PC2 of genes regulated at
D31 versus D30. From the PC2 loadings, we concluded that the
up-regulation of transcripts related to IFN signaling and innate
cells observed 1 day after dose 2 correlated with the adaptive re-
sponses 13 days later (D44) and that there was an inverse correla-
tion between these adapt­ive responses and expression of transcripts
related to NK cells. The positive correlation is consistent with
the previously described correlation of antibody titers with D31
interferon-gamma (IFN-) and induced protein 10 (IP-10, also
known as CXCL 10) production (22). The presence of this positive
correlation is also consistent with several observations made in hu-
mans and mice: in human or murine recipients of AS01-adjuvanted
vaccines (4, 5, 13), in human recipients of a single dose of either
AS03- or MF59-adjuvanted H5N1 or H1N1 vaccines (3, 8, 32, 37)
or nonadjuvanted seasonal influenza vaccine (38), and in virus-­
infected mice (39). To investigate this further, we analyzed the ex-
pression profiles of these 300 genes in blood from participants of a
clinical malaria study collected 1 day after the second dose of RTS,S/
AS01E vaccine (4, 25). The gene profile induced by this vaccine was
similar to the one observed in specific individuals in the AS01B/E or
AS03 groups. These results confirmed the individual variability of
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the response to another AS01E-adjuvanted vaccine in a different
clinical setting.
Together, the human blood cell gene expression signatures seen
after two doses of the HBs/AS01B/E or HBs/AS01 vaccines studied
here or other AS01- or AS03-adjuvanted vaccines (3–6, 22), com-
bined with the local immune cell recruitment and pathway activa-
tion inferred from preclinical models (10–13), support our models
for the modes of action of AS01 and AS03 adjuvanticity. At D31, the
peak in up-regulation of innate cell– and IFN-associated functions
coincided with depletion of signatures regulated to NK cell pheno-
types, other lymphoid cells, such as nonspecific B and T cells. This
depletion may indicate local recruitment of the circulating cells out
of the primary lymphoid organs and blood, into the peripheral lym-
phoid organs, as occurs in human recipients of AS01-adjuvanted
malaria and tuberculosis vaccines (4, 6). Early cytokines produced
in the muscle and draining lymph node could enter the bloodstream
and instigate the activation of, among others, IFN-inducible genes
(15, 40). At D37, HBs-specific T and B cells would then migrate to
the bloodstream, aligning with the observed up-regulation of corre-
sponding BTMs, while blood monocyte and neutrophil cell counts
have returned to steady-state levels, as evident from previous data
(22). Rather than an absolute decrease of innate cell counts, the re-
pression of innate cell transcripts seen for HBs/AS01B at D37 may
reflect changes in the relative proportions of innate and adaptive
cells. Correspondingly, in human recipients of AS03-adjuvanted
vaccine, frequencies of blood innate cells were significantly reduced
by D7 (32). However, because blood-derived transcriptional signa-
tures reflect an aggregate of changes expressed in discrete cell popula-
tions and are affected by the relative cell type proportions and absolute
cell counts, verification of these concepts in humans requires a cell-
based systems approach and investigation of local tissue responses,
including those of muscle and peripheral lymphoid organs.
This study is limited by a small sample size. The previous study
of recipients of the same vaccine preparations identified an associa-
tion between the CD4+ T cell response and the IFN-signaling path-
way (22). We did not detect this association here possibly because
the previous study had less parameters for testing as compared to
the current study. A limitation of the data analysis is that, because
our interpretation of BTM regulation was based on the numbers of
perturbed genes within the BTMs, rather than on fold changes in
expression, functional differences between the adjuvants with re-
spect to the intensity of the regulation were not considered in that
evaluation.
Last, it will be of interest to extend the current transcriptomics
analysis of adjuvanted HBs vaccines in antigen-naive young adults
to analysis of other AS-containing vaccines in different populations.
Of interest are AS01-adjuvanted herpes zoster or tuberculosis vac-
cines in populations of older adults or primed individuals (41, 42),
respectively, or AS03-adjuvanted H5N1 influenza vaccines in influenza-­
primed adults (43). The recent consideration of AS03 as an adjuvant
for COVID-19 (coronavirus disease 2019) vaccine development (44)
further emphasizes the relevance of in-depth analysis of the mode
of action of vaccine adjuvants in clinical research.
MATERIALS AND METHODS
Study design
The protocol for the previously reported, observer-blinded, multi-
center, randomized controlled trial (NCT00805389) was approved
De Mot et al., Sci. Transl. Med. 12, eaay8618 (2020) 11 November 2020
by all institutional Ethics Committees and conducted in accordance
with the Helsinki Declaration and Good Clinical Practice guidelines
(21, 22). Written informed consent was obtained from each subject
before trial participation. Healthy HBV-naive men or women 18 to
45 years of age received two intramuscular injections, 1 month apart,
of vaccines containing HBs antigen (20 g of dose) adjuvanted with
AS01B, AS01E, AS03A, or with AS04 (Fendrix, GSK) or with Alum as
Al(OH)3 (Engerix-B, GSK).
Evaluations of adaptive and innate immunogenicity (primary and
secondary endpoints, respectively) were previously performed for
their respective according-to-protocol (ATP) cohorts (21, 22). The
current analyses were conducted for the 112 subjects included in
the adaptive immunogenicity ATP cohort, as well as in subset 1 or 2
of the innate immunogenicity ATP cohort recruited at the Immune
Health Centre, La Louvière, Belgium (n = 18 for AS01B, 23 for
Downloaded from http://stm.sciencemag.org/ at Cambridge University on November 21, 2020
AS01E, 28 for AS03, 22 for AS04, and 21 for Alum). The demo-
graphic profile of subjects across groups was balanced in terms of
age (mean age, 33.9 years; SD, 6.6 years) and gender (49.1% females),
and most subjects (97.3%) were white Caucasian (table S4). For the
current study cohort, an overview of the innate and adaptive im-
mune response features (serum concentrations of CRP and key cyto-
kines, frequencies of different CD4+ T cell phenotypes, and antibody
concentrations) is presented in data file S2.
Whole blood samples for microarray analyses were collected be-
fore vaccination (D0 and D30), 3 to 6 hours after each dose (D0+
and D30+) or 1 day after each dose (D1 and D31), 14 days after dose 1
(D14), and 3 and 7 days after dose 2 (D33 and D37). Adaptive re-
sponses were previously evaluated at 2 weeks after dose 2 (D44) (22).
RNA extraction, microarray hybridization,
and data preprocessing
Total RNA was extracted and purified as described (22). RNA quality
was assessed using a Bioanalyzer-2100 (Agilent Technologies); 50 ng
of total RNA was used for complementary DNA (cDNA) amplifica-
tion, fragmentation, and labeling using the Ovation Kit (NuGEN).
Fragmented cDNA was hybridized using hgu133 Plus2.0 GeneChip
(Affymetrix; 54,675 probe sets). After screening of arrays by standard
quality control (QC), 84 of 1008 samples were missing or excluded.
The data were then GCRMA-normalized, log2-transformed, and fil-
tered by interquartile range (IQR > 0.75), retaining 3770 probe sets.
Data were submitted to the National Center for Biotechnology Infor-
mation Gene Expression Omnibus (GEO) (GSE116975).
Probe set annotation was performed using Bioconductor package
hgu133plus2.db. Probe sets with no available annotation were re-
moved. Data for genes mapping to multiple probe sets were collapsed
using PCA, without centering or scaling, of a matrix containing all
data corresponding to the gene. The probe set with the highest
Pearson correlation with PC1 was used; this probe set interrogated
2194 unique gene symbols.
Per-gene analysis
A linear-mixed model [R package; limma (45, 46)] was fitted to the
entire transcriptomic dataset. Moderated t statistics of the expres-
sion relative to D0 (with up- or down-regulation defined as values
>0 or <0, respectively) were calculated per gene, per time point after
vaccination, and per adjuvant group, along with the corresponding
P values and Benjamini-Hochberg FDR-adjusted P values. In total,
1791 genes were identified as differentially expressed (FDR P < 0.05)
at ≥1 time point and in ≥1 adjuvant group (the “primary gene set”;
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SCIENCE TRANSLATIONAL MEDICINE | RESEARCH RESOURCE
fig. S1 and data file S1). For selected time points of this gene set (D1,
D31, D33, and D37), the moderated t statistics were organized in a
matrix, with DEG in rows and adjuvant group and time point con-
ditions in columns, and subjected to hierarchical clustering analysis
based on Euclidean distance. Data were scaled by dividing each data
point by the maximal value within the same matrix row.
BTM enrichment analysis
BTM enrichment analysis was performed for the primary gene set
using previously annotated BTMs (23). Partitioning clustering analysis
was performed by adjuvant group on a matrix containing moderated
t statistics, with the genes (differentially expressed in ≥1 time point
for the same adjuvant) in rows and time points in columns, using
the k-means function (Rcmdr package, R) with ≤100 iterations and
10 seeds. The appropriate number of clusters (k = 6 for AS01B, 5 for
AS01E, 5 for AS03, and 4 for AS04) was determined using a graphical
test (a standard statistical approach referred to as the “elbow” method).
BTM enrichment (FDR q value of <0.05) was assessed by cluster,
using hypergeometric tests (t-mod package, R), with the unfiltered
list of genes of the microarray chip set as background. The 88 BTMs
enriched in ≥1 cluster and in ≥1 group (see “primary BTM set”; fig. S1)
were grouped using k-means partitioning clustering (k = 5; elbow
method) of a matrix containing the percentages of DEG (with their
sign depending on the behavior of the majority of genes), with BTMs
in rows and adjuvant group and time point conditions in columns.
The amount of BTM activation was visualized in a bubble chart as
described in the legend of Fig. 2.
Transcriptome synchronization analyses
The GCRMA-normalized transcriptomics dataset was filtered to
remove subjects not adhering to cohort criteria and then subjected
to within subject autoscaling. Samples from subjects with probe sets
exhibiting no change in expression over time and probe sets for
which ≥40% of the data (considering all time points and subjects)
were absent were excluded, leaving 39,200 probe sets in the analysis.
Data from each probe set were stored by the adjuvant group in a
matrix containing all nine time points (rows) and subjects (columns).
Missing data were imputed using the missMDA (47) package in R.
The amount of gene expression synchronization, across subjects and
time points, was expressed as the percentage of variance captured
by the first PC (VARPC1) of a PCA performed on each matrix. On
the basis of the data for the AS04 and Alum groups, only highly
synchronized data points (VARPC1 of >55% in ≥1 group) were re-
tained, leaving 1529 probe sets in the analysis. After excluding
probe sets not mapping to any gene and additional probe sets map-
ping to the same gene, 922 probe sets were retained. Hierarchical
clustering analysis based on Euclidean distance was performed on
the VARPC1 data, yielding seven SCs. Three SCs included a signifi-
cant number of genes and were subjected to BTM (23) enrichment
analysis (FDR q value of <0.05) using a hypergeometric test developed
in-house.
PCA and regression models
For genes in the primary gene set, PCA was performed on a matrix
containing the individual unscaled log2-transformed fold-change
data for D1 over D0, D31 over D0, and D31 over D30, with subjects
in rows and genes in columns, using the prcomp function in R. Linear
regression models were used to assess the association between the
PC scores and the adaptive immune parameter per individual (Ai),
De Mot et al., Sci. Transl. Med. 12, eaay8618 (2020) 11 November 2020
for the log10 values of either HBs antigen; antibody concentrations;
frequencies of HBsAg-specific CD4+ T cells expressing at least one
of IL-2, CD40L, or IFN- and TNF- (tumor necrosis factor–), as
measured by flow cytometry; or polyfunctional HBsAg-specific CD4+
T cells on the CD4+ T cell polyfunctionality index (Fig. 5A) (21, 48).
Ai was modeled as a function of regression coefficients  of the scores
of the first three PCs and the treatment effect for each AS group
relative to that of the Alum group, corrected for the technical scal-
ing factor, and with  of the Alum group intercept (0) considered
as baseline
​​A​ i​​  = ​​  0​​  + ​​  1​​ × adjuvant group + ​​  2​​  × PC1 [ ​EX​  Dx/Dy​​ ] + ​​  3​​  ×
PC2 [ ​EX​  Dx/Dy​​ ] + ​​  4​​ × PC3 [ ​EX​  Dx/Dy​​ ] + ​​  5​​ × ​SF​  Dx/Dy​​​
Generated P values were not adjusted for multiplicity. This ex-
Downloaded from http://stm.sciencemag.org/ at Cambridge University on November 21, 2020
ploratory study was not powered to evaluate multiple regression
models. PC2 loadings were inverted to keep the correlated genes
and the positive correlation with the antibody titers on the right-
hand side of the figures (Fig. 5, B and C). Similar linear regression
analyses were performed to assess the association between gene ex-
pression and previously assessed (22) innate immune response data
after the second dose. Input parameters included the PC2 of hema-
tological parameters, CRP and cytokines, and the PC2 of the gene
expression data, both measured after the second dose and expressed
in fold changes of the amounts at D30+, D31, D33, or D37 over
D30. Innate parameters included CRP and the cytokines IL-1, IL-6,
TNF-, IL-5, IL-10, IP-10, IFN-, and MCP-1. Innate hemato-
logical parameters included the cell subsets lymphocytes, monocytes,
eosinophils, basophils, neutrophils, and white blood cells.
To generate Fig. 6, the above models were used to identify the
300 genes with the highest absolute loadings in the PC2 from the
PCA performed on the log2 fold change D31 over D30 matrix.
Where possible, the genes were assigned to the BTM clusters (C1 to
C5). Genes that could not be assigned in one of the five BTM clus-
ters were analyzed using the Ingenuity Pathway Analysis algorithm
(QIAGEN).
We also compared the core gene expression profile obtained for
the subjects from the current study with that obtained for subjects
from a previously described observer-blind, randomized phase 2a
study of the RTS,S/AS01E malaria vaccine conducted in healthy,
malaria-naive adults (4, 25). One cohort in this study received three
doses of RTS,S/AS01E at 28-day intervals (“RRR” group; N = 21; %
female/male ratio, 37/63; mean age, 30 years). Transcriptional
responses to vaccination as compared to prevaccination baseline
(D0) were described previously (4). To enable comparison with the
current dataset generated for fold changes in gene expression of
D31 over D30, we analyzed the samples from 15 subjects of the RRR
group collected at D29, 1 day after their second vaccination. We
then analyzed the log2-transformed fold changes in the expression
of the 300 genes identified in PC2 by comparing D29 to D28. We
performed a Pearson correlation–based hierarchical clustering
analysis to generate a heatmap and assigned the genes to the five
BTM clusters.
SUPPLEMENTARY MATERIALS
stm.sciencemag.org/cgi/content/full/12/569/eaay8618/DC1
Fig. S1. Analysis pipelines.
Fig. S2. Early cytokine and hematology responses are associated with gene expression after
dose 2.
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Fig. S3. Temporal regulation of responses after the second dose of HBs/AS01B or HBs/AS03.
Table S1. Activated BTMs listed by cluster.
Table S2. BTM enrichment in clusters of highly synchronized genes.
Table S3. Canonical pathway enrichment analysis for genes not annotated in the BTMs.
Table S4. Demography of the study cohort.
Data file S1. List of DEGs.
Data file S2. Summary of immune response parameters.
View/request a protocol for this paper from Bio-protocol.
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Acknowledgments: We are grateful to the study participants and staff members of the
different study sites for contribution to the study. We thank N. Garçon, G. Leroux-Roels, and
A. Marchant for expert review of the data, helpful discussions, and contributions to this study.
We also thank E. Oe (GSK) for providing scientific writing services in the manuscript’s
development and G. Drevon (GSK) and J. Ghesquière (Business & Decision Life Sciences) for
editorial support and manuscript coordination. Editorial services were provided by
N. R. Gough (BioSerendipity). The ECR02 study group includes the following authors:
Viviane Bechtold, Wivine Burny, Isabelle Carletti, Frédéric Clément, Arnaud M. Didierlaurent,
Meral Esen, Laurence Fissette, Julian Gabor, Nathalie Garçon, Edwige Haelterman, Caroline Hervé,
Yves Horsmans, Michel Janssens, Peter Kremsner, Geert Leroux-Roels, Arnaud Marchant,
Downloaded from http://stm.sciencemag.org/ at Cambridge University on November 21, 2020
Philippe Moris, Tino F. Schwarz, Fernanda Tavares da Silva, Pierre Van Damme,
Robert A. van den Berg, Robbert G. van der Most, and Dirk Zuchner. The Business & Decision
Life Sciences platform provided editorial assistance and manuscript coordination, on behalf of
GSK. Funding: GlaxoSmithKline Biologicals SA funded the study, was involved in all stages of
the original studies conduct and analysis (ClinicalTrials.gov Identifiers: NCT00805389), and
funded the manuscript’s development. This work was supported by the Bill & Melinda Gates
Foundation (grant number OPP1120977 to R.G.v.d.M. and R.A.v.d.B.). Author contributions:
L.D.M., V. Bechtold, F.U.-M., E.S., R.A.v.d.B., A.M.D., W.B., and R.G.v.d.M. contributed substantially
to the conception and design of the studies. L.D.M., V. Bechtold, V. Bol, A.C., M.C., A.E., D.H.,
F.U.-M., E.S., A.S., and R.A.v.d.B. participated in the collection and analysis of the data. L.D.M.,
V. Bechtold, A.M.D., W.B., and R.G.v.d.M. contributed to the interpretation of the results. All
authors had full access to the data and were involved in critical revision of the manuscript for
important intellectual content. The corresponding author was responsible for manuscript
submission. Competing interests: V. Bechtold, V. Bol, A.C., M.C., A.E., F.U.-M., E.S., R.A.v.d.B.,
A.M.D., W.B., and R.G.v.d.M. are employees of the GSK group of companies. V. Bechtold, A.C.,
F.U.-M., E.S., R.A.v.d.B., A.M.D., W.B., and R.G.v.d.M. hold shares in the GSK group of companies
as part of their employee remuneration. A.M.D. and R.G.v.d.M. own patents related to AS01
(Methods for inducing an immune response, US10624961B2). L.D.M. was an employee of the
GSK group of companies at the time of the study. D.H. received a grant from GSK to conduct
the study. A.S. reports no conflict of interest. Fendrix and Engerix-B are trademarks owned by
or licensed to the GSK group of companies. Data and materials availability: Anonymized
individual participant data and study documents can be requested for further research at
www.clinicalstudydatarequest.com. Transcriptomic data are available from the NCBI GEO
(accession no. GSE116975).
Submitted 24 July 2019
Accepted 23 October 2020
Published 11 November 2020
10.1126/scitranslmed.aay8618
Citation: L. De Mot, V. Bechtold, V. Bol, A. Callegaro, M. Coccia, A. Essaghir, D. Hasdemir,
F. Ulloa-Montoya, E. Siena, A. Smilde, R. A. van den Berg, A. M. Didierlaurent, W. Burny, R. G. van der Most,
Transcriptional profiles of adjuvanted hepatitis B vaccines display variable interindividual
homogeneity but a shared core signature. Sci. Transl. Med. 12, eaay8618 (2020).
13 of 13
Transcriptional profiles of adjuvanted hepatitis B vaccines display variable
interindividual homogeneity but a shared core signature
Laurane De Mot, Viviane Bechtold, Vanesa Bol, Andrea Callegaro, Margherita Coccia, Ahmed Essaghir, Dicle
Hasdemir, Fernando Ulloa-Montoya, Emilio Siena, Age Smilde, Robert A. van den Berg, Arnaud M. Didierlaurent,
Wivine Burny and Robbert G. van der Most

Sci Transl Med 12, eaay8618.

Downloaded from http://stm.sciencemag.org/ at Cambridge University on November 21, 2020

DOI: 10.1126/scitranslmed.aay8618

Patterns of adjuvanticity
Understanding how adjuvants improve vaccine-induced immune responses is important for the
development of successful vaccines. De Mot et al. performed transcriptome analysis on whole blood taken from
the participants of a previously reported clinical study that trialed different adjuvants alongside hepatitis B vaccine
antigen. Differential expression of a core gene signature related to innate immune response and natural killer cells
correlated with the antibody response observed in vaccine recipients after two doses regardless of the adjuvant
administered, suggesting that these responses are important for vaccine immunogenicity.

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