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OUTLINE

1. Highlights
2. Abstract
3. Keywords
4. Abbreviations
5. 1. Introduction
6. 2. Methods
7. 3. Results
8. 4. Discussion
9. Declaration of Competing Interest
10. Acknowledgements
11. Authors’ contributions
12. Funding
13. Trademark
14. Appendix A. Supplementary material
15. Data sharing statement
16. References

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1. Interactive Case InsightsSupplementary data 1
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4. Interactive Case InsightsSupplementary data 4
5. Interactive Case InsightsSupplementary data 5
6. Interactive Case InsightsSupplementary data 6

VACCINE

Available online 21 December 2022
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SYSTEMS ANALYSIS OF HUMAN RESPONSES TO AN ALUMINIUM HYDROXIDE-ADSORBED TLR7
AGONIST (AS37) ADJUVANTED VACCINE REVEALS A DOSE-DEPENDENT AND SPECIFIC
ACTIVATION OF THE INTERFERON-MEDIATED ANTIVIRAL RESPONSE

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HIGHLIGHTS

•

An AS37-adjuvanted vaccine induced immune responses which maintained for
6 months.

•

Extensive immune profiling was conducted on a subset of participants.

•

AS37 increased expression of interferon-inducible genes and serum CXCL10
(IP-10).

•

AS37 upregulated specific innate immune cells and Ag-specific B and T
lymphocytes.

•

The immune signature is consistent with toll-like receptor 7 engagement.

ABSTRACT

The candidate Adjuvant System AS37 contains a synthetic toll-like receptor
agonist (TLR7a) adsorbed to alum. In a phase I study (NCT02639351), healthy
adults were randomised to receive one dose of licensed alum-adjuvanted
meningococcal serogroup C (MenC-CRM197) conjugate vaccine (control) or
MenC-CRM197 conjugate vaccine adjuvanted with AS37 (TLR7a dose 12.5, 25, 50 or
100 µg). A subset of 66 participants consented to characterisation of peripheral
whole blood transcriptomic responses, systemic cytokine/chemokine responses and
multiple myeloid and lymphoid cell responses as exploratory study endpoints.
Blood samples were collected pre-vaccination, 6 and 24 h post-vaccination, and
3, 7, 28 and 180 days post-vaccination. The gene expression profile in whole
blood showed an early, AS37-specific transcriptome response that peaked at 24 h,
increased with TLR7a dose up to 50 µg and generally resolved within one week.
Five clusters of differentially expressed genes were identified, including those
involved in the interferon-mediated antiviral response. Evaluation of 30
cytokines/chemokines by multiplex assay showed an increased level of
interferon-induced chemokine CXCL10 (IP-10) at 24 h and 3 days post-vaccination
in the AS37-adjuvanted vaccine groups. Increases in activated plasmacytoid
dendritic cells (pDC) and intermediate monocytes were detected 3 days
post-vaccination in the AS37-adjuvanted vaccine groups. T follicular helper
(Tfh) cells increased 7 days post-vaccination and were maintained at 28 days
post-vaccination, particularly in the AS37-adjuvanted vaccine groups. Moreover,
most of the subjects that received vaccine containing 25, 50 and 100 µg TLR7a
showed an increased MenC-specific memory B cell responses versus baseline. These
data show that the adsorption of TLR7a to alum promotes an immune signature
consistent with TLR7 engagement, with up-regulation of interferon-inducible
genes, cytokines and frequency of activated pDC, intermediate monocytes,
MenC-specific memory B cells and Tfh cells. TLR7a 25–50 µg can be considered the
optimal dose for AS37, particularly for the adjuvanted MenC-CRM197 conjugate
vaccine.

KEYWORDS

Adjuvant System
Toll-like receptor 7
Immune response
Vaccine
Plasmacytoid dendritic cells
Type I interferon

ABBREVIATIONS

ANOVA
analysis of variance
APC
antigen-presenting cell
BTM
blood transcriptional module
cDNA
complementary DNA
D
study day
DC
dendritic cells
DEG
differentially-expressed gene
ELISpot
enzyme-linked immunospot
FACS
fluorescence-activated cell sorting
GCRMA
GC Robust Multiarray Average
GEO
Gene Expression Omnibus
GSEA
Gene Set Enrichment Analysis
HBV
hepatitis B virus
hSBA
human complement serum bactericidal assay
IFN
interferon
IgG
immunoglobulin G
IgM
immunoglobulin M
IL
interleukin
MBC
memory B cells
MenC
Neisseria meningitidis serogroup C
MFI
median fluorescence intensity
NHP
non-human primate
PBMC
peripheral blood mononuclear cells
PBS
phosphate-buffered saline
PCA
principal component analysis
pDC
plasmacytoid dendritic cells
SMIP
small molecule immune potentiator
Tfh
T follicular helper cells
TLR
toll-like receptor
TLRa
toll-like receptor agonist

1. INTRODUCTION

Adjuvants are molecular compounds, added into vaccine formulations, that are
capable of modulating the quality and magnitude of the immune response [1], [2],
[3]. Where protection relies on activating immune arms other than antibody
generation, adjuvants can be effective in shifting the balance towards a
cell-mediated immunity, as recently shown [4]. The first generation of adjuvants
to be used in human vaccines was based either on aluminium salts or oil-in-water
emulsions [2]. A new generation of adjuvants includes small molecule immune
potentiators (SMIPs) that target toll-like receptors (TLRs) [5].

TLRs are transmembrane proteins consisting of leucine-rich ectodomains that are
expressed either on cell surfaces recognising extracellular pathogen components
(TLR1, TLR2, TLR4, TLR5, TLR6 and TLR11) or within intracellular compartments
and specialised in the recognition of exogenous nucleic acids (TLR3, TLR7, TLR8
and TLR9) [5]. Some TLR ligands are included in adjuvants that are used in
commercial vaccines [2]. For example, the Adjuvant System AS04, containing 50 µg
of monophosphoryl lipid A (a non-toxic TLR4 ligand) adsorbed on aluminium salt
(500 µg Al3+), was the first TLR agonist (TLRa) adjuvant to be used in humans
and is now a component of commercial vaccines against human papilloma virus and
hepatitis B virus (HBV) [6], [7]. Other examples include HEPLISAV-B, a HBV
vaccine containing as adjuvant a synthetic oligonucleotide that stimulates
innate immunity through TLR9 [8], and BBV152, a whole-virion inactivated
SARS-CoV-2 vaccine formulated with a TLR 7/8 agonist adsorbed to aluminium
hydroxide (alum) [9].

Synthetic molecules are designed to bind and trigger TLR7 and TLR8 that usually
detect viral single-stranded RNA molecules within endosomes [10]. They activate
nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) and
interferon (IFN) regulatory factor 7, via the adaptor protein myeloid
differentiation primary response 88 (MyD88), to induce inflammatory cytokines
and type I IFN [10]. In humans, TLR7 is mainly expressed by plasmacytoid
dendritic cells (pDC) and, to a lesser extent, by B cells and neutrophils [11],
and it is involved in the enhanced expression of type I IFN and pro-inflammatory
cytokines [10]. Engagement of TLR7 can shift the immune response toward a T
helper 1 (Th1)-type mediated phenotype, induce the expansion of memory B cells
(MBC) and increase specific immunoglobulin G (IgG2a) titres [12], [13], [14].

A new Adjuvant System, AS37, is based on a synthetic TLR7a molecule with a
benzonaphthyridine chemical scaffold, which has been adsorbed to alum to limit
systemic exposure [12], [15]. Pre-clinical studies of AS37 demonstrated a
superior effective immune response when compared to alum-adjuvanted controls in
animal models using several target pathogens, including Staphylococcus aureus
and Neisseria meningitidis serogroup C (MenC) [12], [13], [15], [16]. Adsorption
of TLR7a to alum significantly improved expansion of the MBC compartment in mice
[17].

A clinical dose-escalation study assessed the safety and immunogenicity of an
investigational AS37-adjuvanted MenC-CRM197 conjugate vaccine (TLR7a dose 12.5,
25, 50 or 100 µg) in healthy adults, as compared to a control group that
received a licensed alum-adjuvanted MenC-CRM197 conjugate vaccine [18]. A single
dose of the AS37-adjuvanted vaccine induced antigen-specific antibody responses
that were maintained at 6 months post-vaccination, with a trend for a TLR7a
dose-dependent increase in MenC polysaccharide binding antibodies. Only slight
differences were observed between groups in the ability to induce opsonising
antibodies (as detected by human complement serum bactericidal assay, hSBA). The
study also identified a clinically acceptable dose range for AS37 (TLR7a
12.5–50 µg).

Here, we present results from in-depth profiling of the immune response to the
AS37-adjuvanted vaccine in the same clinical dose-escalation study. This reveals
that AS37 promotes an upregulation of IFN-inducible genes, an immune signature
consistent with the engagement of TLR7 and induces activation of immune cells,
such as activated pDC, intermediate monocytes, T follicular helper (Tfh) cells
and antigen specific B cells.

2. METHODS

2.1. STUDY DESIGN AND PARTICIPANTS

The design of the phase 1 clinical study (NCT02639351), performed at a single
centre in Germany, was described previously [18]. Briefly, 80 healthy adults
aged 18–45 years were randomised in a 1:4 ratio to receive either
alum-adjuvanted MenC-CRM197 conjugate vaccine (Menjugate, GSK; control group) or
MenC-CRM197 vaccine formulated with AS37 adjuvant containing TLR7a (dose 12.5,
25, 50 or 100 µg) completely adsorbed to alum. Results on the primary and
secondary objectives of the study (assessment of safety and humoral
immunogenicity) have been reported [18].

A subset of 66 participants, distributed across all five treatment groups (Fig.
1; Supplementary File 1), consented to extensive immune profiling consisting of
the assessment of peripheral whole blood transcriptomic responses, systemic
cytokine/chemokine responses and detailed characterization of the responses of
multiple myeloid and lymphoid cell subpopulations. Additional blood samples were
collected for the assessment of these exploratory endpoints at baseline (before
vaccination) and at 6 and 24 h after vaccination (study Day 1), and 3, 7, 28 and
180 days after vaccination (Days 4, 8, 29 and 181, respectively).

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Fig. 1. Analyses conducted for in-depth profiling of immune responses to
vaccination in 66 participants randomly assigned to one of five treatment
groups. Day 1 refers to the day of vaccination. Filled squares indicate the
immune responses that were measured at each study visit. Human complement serum
bactericidal assay (hSBA) and enzyme-linked immunosorbent assay (ELISA) results
were reported previously [18]. MenC-CRM197 + Alum, group that received
alum-adjuvanted N. meningitidis serogroup C (MenC) CRM197-conjugated vaccine
(Menjugate, control); MenC-CRM197 + AS37, group that received
MenC-CRM197-conjugated vaccine with AS37 adjuvant containing Toll-like receptor
7 agonist (TLR7a dose 12.5, 25, 50 or 100 µg). ELISpot, enzyme-linked
immunospot; FACS, fluorescence-activated cell sorting; h, hours
post-vaccination; n, number of participants.

2.2. TRANSCRIPTOME DATA ANALYSIS

Whole blood gene expression profiles were assessed at baseline, 6 and 24 h, and
3 and 7 days after vaccination. The last two timepoints (Days 4 and 8) were
tagged as D4 and D8, respectively.

Total RNA was extracted and purified following procedures described by Burny et
al. [19]. RNA quality was assessed using Bioanalyzer-2100 (Agilent
Technologies); total RNA was used for complementary DNA (cDNA) amplification,
fragmentation and labelling using the Ovation Kit (Tecan Genomics, Inc.).
Fragmented cDNA was hybridised using hgu133 Plus2.0 GeneChip (Thermo Fisher
Scientific; 54,675 probe sets). Resulting probe sets’ fluorescence intensity
values were then GC Robust Multiarray Average (GCRMA)-normalised,
log2-transformed and filtered by removing probe sets not mapping to genes or not
showing variation across the entire dataset (log2 inter-quartile range ≤ 0.5)
[20]. Probe sets mapping to the same gene were collapsed by computing the
geometric mean of their fluorescence intensity, resulting in a final list of
7564 gene expression values. Non-human primate (NHP) transcriptome data were
derived from a previous study [21]. Briefly, microarrays were quantile
normalized using the preprocessCore Bioconductor package. Probe sets mapping to
the same gene were then collapsed by computing the geometric mean of their
fluorescence intensity. Genes showing low expression values across the dataset
(≤100 absolute fluorescence units in ≥ 90 % of samples) and low modulation at
24 h post immunization (log2 fold-change from baseline ≤ 0.1 in ≥ 90 % of
sample) were filtered out, leading to a working dataset of 13,255 genes.

Transcriptional modulation from baseline was assessed using R package limma [22]
and by fitting a mixed-effect model setting subjects’ identity as random effect
variable and treatments and timepoints as fixed effect explanatory variables.
P-values were adjusted for multiple testing using the Benjamini-Hochberg
procedure. Hierarchical clustering of differentially-expressed genes (DEGs) was
performed using the average response of each treatment group at each individual
timepoint. Clusters’ functional enrichment analysis was computed by applying a
Fischer’s exact test on the biological process gene ontology annotations. For
each cluster, the proportion of genes mapped to a specific biological process
was compared with that observed in the whole set of 7564 genes. Functional
enrichment analysis of the principal component analysis (PCA) components was
performed by ranking the genes based on their assigned weight and applying the
Gene Set Enrichment Analysis (GSEA) [23], [24], using the biological process
gene ontology annotation. For all tests, p-values were adjusted for multiple
testing through the Benjamini-Hochberg procedure.

Blood transcriptional module (BTM) responses were obtained based on the set of
modules proposed by Obermoser et al. [25]. Module-level responses were computed
by averaging the log2-scaled transcript abundance values of the mapped genes.

2.3. MULTIPLEX ANALYSIS OF CYTOKINES

Serum concentrations of a panel of 30 chemokines and cytokines were evaluated at
baseline, 6 and 24 h post-vaccination, and at D4 and D8 by multiplex
electrochemiluminescence-based assay, using the Mesoscale Discovery (MSD) V-PLEX
Human Cytokine 30-plex Kit (K15054-D) according to the manufacturer’s
instructions, as summarised in Supplementary File 2.

2.4. INNATE CELL PHENOTYPING BY FLOW CYTOMETRY

Innate cell phenotyping was analysed by flow cytometry (fluorescence-activated
cell sorting, FACS), evaluating the frequency and quality of antigen-presenting
cells (APCs), in particular plasmacytoid and myeloid dendritic cells and
monocyte subsets (classical, non-classical and intermediate), by a combination
of specific surface markers. Number and activation status of the cell
populations was assessed at baseline, 24 h after vaccination, and at D4 and D8
using frozen peripheral blood mononuclear cells (PBMC) from participants, which
were thawed and analysed by flow cytometry. Cell staining methods are described
in the Supplementary File 2. Each immune-identified subset was expressed as
number per million of total viable PBMC and activation markers in each subset
were expressed as median fluorescence intensity (MFI).

2.5. FLOW CYTOMETRIC CHARACTERISATION OF CD4+ T CELLS SPECIFIC FOR CRM197

The frequency and functional profile of CD4+ T cells specific for CRM197 were
analysed at baseline, D8 and Day 29 (D29) by flow cytometry (FACS) using
intracellular staining with a wide panel of cytokines and surface markers to
identify cell populations, including Th cell subsets, Th0, Th1, Th2, Th17 and
Tfh cells.

PBMC were stimulated for 18 h at 37 °C with anti-CD28 and anti-CD49d (1 µg/mL
each) and CRM197 (10 µg/mL) or Staphylococcus enterotoxin B (1 µg/mL) or with
medium alone as negative control, in the presence of Brefeldin A (5 µg/mL), as
previously described [26]. PBMC staining methods are described in the
Supplementary File 2. The frequency of total antigen-specific CD4+ T cells was
calculated by summing the frequency of CD4+ T cells producing all not
overlapping permutations of the cytokines tested combined to CD40L (Boolean and
logical gates with FlowJo). The response to medium was subtracted from each
subject and timepoint. Data were expressed as number of antigen-specific CD4+ T
cells per million of total CD4+ T cells.

2.6. B CELL RESPONSE ENZYME-LINKED IMMUNOSPOT ASSAY

The frequency of MenC polysaccharide-specific and CRM197-specific B cells was
assessed at baseline, and D8, D29, and Day 181 (D181) by enzyme-linked
immunospot (ELISpot). Ninety-six well ELISpot plates (Millipore MultiScreenHTS
HA Filter Plate) were coated with 100 µL/well of phosphate-buffered saline (PBS)
containing human serum albumin, CRM (5 µg/mL) or MenC (1 µg/mL) or 2.5 µg/mL
goat anti-human IgM + IgG (BD Pharmingen), for 16–20 h at 4 °C. The ELISpot
assay was performed as previously described [27].

2.7. INTEGRATED DATA ANALYSIS

For a comprehensive view of immune modulations triggered by AS37, an integrated
data analysis was performed. Flow cytometry, cytokine/chemokine, transcriptomic
(BTM responses) and serological data were combined and processed, as described
in the Supplementary File 2, leading to a final dataset of 335 response
variables.

The specific contribution of the TLR7a component on vaccine-induced responses
was evaluated by comparing the four TLR7a dose groups to the control group
through an analysis of variance (ANOVA). Early (collapsed 6 and 24 h
post-vaccination and D4) and late (D8, D29 and D181) responses were analysed
separately. After computing p-values for each, these were adjusted for multiple
testing using the Benjamini-Hochberg procedure.

3. RESULTS

3.1. AS37 INDUCES SYSTEMIC, TRANSIENT AND DOSE-DEPENDENT ACTIVATION OF
IFN-MEDIATED ANTIVIRAL RESPONSE

The analysis of genes, whose abundance was significantly modulated (50 %
modulation, Benjamini-Hochberg adjusted p-value ≤ 0.05) compared to pre-immune
levels, identified 1439 DEGs throughout the study (Supplementary File 3). AS37
induced an early (6–72 h post-vaccination) transcriptome response that peaked at
24 h and increased with TLR7a dose up to 50 µg (Fig. 2A, Fig. S1 Supplementary
File 2). The transcriptome response with the highest dose, 100 µg, was
consistently lower than those observed with the 25 µg and 50 µg doses at the
6 h, 24 h and D4 timepoints (Fig. 2A, Fig. S1 Supplementary File 2). Most genes
modulated within the first 3 days after vaccination were upregulated compared to
baseline (Fig. S1 Supplementary File 2). At D8, transcriptome modulation did not
show any specific TLR7a association and the number of DEGs was comparable among
the control group and different TLR7a dose groups, except for the 25 µg dose
group, which showed a stronger response (Fig. S1 Supplementary File 2).

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Fig. 2. Peripheral blood transcriptome response to AS37- and alum-adjuvanted
MenC-CRM197 vaccines. (A) Volcano plots representing significantly modulated
genes 24 h after vaccination. Genes with more than 50 % modulation compared to
baseline value (absolute fold-change ≥ 1.5 on a log2 scale) and a
Benjamini-Hochberg corrected p-value ≤ 0.05 are shown in blue. (B) Heatmap
representing transcriptome modulation (geometric mean) across all groups and
timepoints. Heatmap colours represent the log2-fold change from baseline. Genes
annotated by the GEO, as involved in type I and type II interferon (IFN)
response, are shown by purple and turquoise lines, respectively (no Type III
interferon associated genes were found to be modulated in this study). Numbers
to the right of the heatmap indicate cluster identifiers. (C) Response profile
of representative genes from clusters #3 (STAT1, STAT2), #4 (CD38, TNFRSF17) and
#5 (CXCL10, IFIT1). Whiskers represent the 95 % confidence interval for the
mean. MenC-CRM197 + Alum, group that received alum-adjuvanted N. meningitidis
serogroup C (MenC) CRM197-conjugated vaccine (Menjugate, control);
MenC-CRM197 + AS37, group that received MenC-CRM197-conjugated vaccine with AS37
adjuvant containing Toll-like receptor 7 agonist (TLR7a dose 12.5, 25, 50 or
100 µg). D, study day. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)

Hierarchical clustering of the DEGs across all timepoints revealed five distinct
response clusters (Fig. 2B and Fig. S2). The first two clusters (from top to
bottom in Fig. 2B) included the majority of observed DEGs and consisted of genes
being either downregulated (cluster #1) or upregulated (cluster #2) at D4 and
D8. Gene Expression Omnibus (GEO) functional enrichment analysis revealed that
these two clusters are enriched for genes involved in metabolic processes
(cluster #1) and protein production (cluster #2; Supplementary File 4). A third
cluster (#3) included genes that were specifically modulated in the
AS37-adjuvanted vaccine groups, with a transient response, peaking at 24 h and
generally returning to baseline at D8 (Fig. 2B). This cluster was enriched for
IFN-inducible genes and genes involved in IFN signalling. Examination of key
genes involved in the IFN-mediated antiviral response, such as the two signal
transducers coding genes STAT1 and STAT2 [28], shows activation of this response
specific to the AS37-adjuvanted vaccine groups (Fig. 2C). A fourth cluster (#4)
of 11 genes, involved in B cell activation and proliferation, was upregulated at
D8 (Fig. 2B). As shown by the two B cell activation markers CD38 and TNFRSF17
(Fig. 2C), genes within this cluster showed a comparable response across the
different groups, regardless of the presence or dose of TLR7a. Finally, a fifth
cluster of 20 genes (#5), mainly involved in the IFN response, showed strong
upregulation at 24 h and D4 in all AS37-adjuvanted vaccine groups (Fig. 2C,
CXCL10 and IFIT1). For the TLR7a 25 µg, 50 µg and 100 µg dose groups,
transcripts abundance remained generally above baseline until D8 (Fig. 2B,
cluster #5).

3.2. THE PLASMABLASTS’ TRANSCRIPTIONAL SIGNATURE AT D8 IS NOT LINKED TO EARLY
ACTIVATION OF THE SYSTEMIC IFN RESPONSE

The set of 1439 DEGs was further analysed through PCA to assess the
transcriptome response evolution over time. Fig. 3 shows the different treatment
groups mapped on the first two principal components (PC1, PC2). The different
timepoints appear to be well separated, in line with the clustering results
shown in Fig. 2B. Specifically, the 6 h responses (Fig. 3A, blue) were closely
related and positioned orthogonally to the 24 h and D8 responses, consistent
with the fact that almost no DEGs were identified at this timepoint. The 24 h
responses, instead, were shifted along PC2 (Fig. 3A, yellow). The shift was
proportional to the TLR7a dose up to 50 µg and it partially folded back with
TLR7a 100 µg dose. Functional characterisation of the genes contributing to PC2
highlighted an enrichment of IFN-inducible genes participating in the antiviral
response (Supplementary File 5). Among those, the most influential were found to
be IFIT1, IFI44L, RSAD2 and SIGLEC1 (Fig. 3B). D4 responses (Fig. 3A, grey) were
positioned between the D1 and D8 clusters, suggesting a transitional stage in
which the D1 response is waning and the D8 response is emerging. D8 responses
were shifted toward the positive region of PC1 (Fig. 3A, red), representing
upregulation of genes involved in B cell activation and proliferation (File S5),
with no clear TLR7a dose-related trend. Among these, MZB1 and TFNRSF17 had the
highest weights on PC1 (Fig. 3B).

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Fig. 3. Principal component analysis of transcriptome responses to AS37- and
alum-adjuvanted MenC-CRM197 vaccines. (A) PC1-PC2 scatter plot for the different
groups clustered according to the time of sample collection. (B) Loadings plot
representing the 10 most influential genes in the PC1-PC2 space. Alum, group
that received alum-adjuvanted N. meningitidis serogroup C (MenC)
CRM197-conjugated vaccine (Menjugate, control); AS37-12.5/25/50/100, group that
received MenC-CRM197-conjugated vaccine with AS37 adjuvant containing Toll-like
receptor 7 agonist (TLR7a dose 12.5, 25, 50 or 100 µg). D, study day; h, hours
post-vaccination; PC, principal component.

To check the consistency of observed patterns among different study
participants, individual response profiles were analysed using a multilevel PCA.
Despite higher dispersion of the data points on the PCA space (Fig. S3), 24 h
and D8 responses still appeared to be well separated, confirming the observed
response evolution over time.

Fig. 3A shows a similar level of B cell-related genes activation at D8
(represented by the shift along PC1) between vaccine groups. The fact that the
alum-adjuvanted vaccine could induce a B-cell response signature comparable to
the AS37-containing formulations without inducing IFN-inducible genes at 24 h
(as indicated by the lack of shift along PC2) suggests that the early IFN
signature elicited by AS37 may be irrelevant for subsequent B cell activation,
at least in this specific experimental setting. To corroborate this hypothesis
and identify other potential transcriptional correlates of immunogenicity, we
checked for correlations between the first two PCA components and those adaptive
immune responses (cell-mediated and humoral immune response variables) measured
7 and 28 days post-vaccination using canonical correlation analysis. Twenty-four
hours and D8 transcriptome responses were analysed separately. The analysis did
not show any appreciable correlation (Fig. S4).

3.3. INNATE IMMUNE SYSTEM ACTIVATION FOLLOWING VACCINATION

The serum concentration of 30 different cytokines/chemokines was analysed to
evaluate biomarkers that may be predictive of innate immune system activation.
An increased level of interleukin (IL)-6 at 6 and 24 h post-vaccination was
observed in all treatment groups (Fig. 4A), suggesting this to be a
vaccination-induced effect rather than a response specific to AS37. Differently,
the CXCL10 (IP-10) chemokine was found to be upregulated at 24 h
post-vaccination in the AS37-adjuvanted vaccine groups (Fig. 4B). In the 25 µg
AS37 dose groups, such upregulation was statistically significant
(Benjamini-Hochberg adjusted p ≤ 0.01). No specific trends were observed in
relation to timepoint or TLR7a dose for the other chemokines/cytokines analysed.

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Fig. 4. Jitter plots representing the main modulated immune parameters: (A)
interleukin 6 (IL-6) and (B) CXCL10 (IP-10) chemokine serum concentrations, (C)
frequency of intermediate monocytes (defined as CD14++CD16+ monocyte subset),
(D) activation of CD40 in plasmacytoid dendritic cells
(CD14-HLA-DR+CD11c-CD123+), (E) frequency of CRM197-specific CD4+CD40L+IL-21+ T
cells (T follicular helper cells), (F) CRM197-specific IgG memory B cells and
(G) MenC-specific IgG memory B cells. Overlaid boxplots represent medians and
central interquartile ranges. * ≤ 0.1, ** ≤ 0.01, *** ≤ 0.001 (two-way ANOVA,
Benjamini-Hochberg adjusted p-value). Alum, group that received alum-adjuvanted
MenC-CRM197-conjugated vaccine (Menjugate, control); AS37-12.5/25/50/100, group
that received MenC-CRM197-conjugated vaccine with AS37 adjuvant containing
Toll-like receptor 7 agonist (TLR7a dose 12.5, 25, 50 or 100 µg). D, study day;
h, hours post-vaccination; IgG, immunoglobulin G; MBC, memory B cells; N,
number; PBMC, peripheral blood mononuclear cells.

Innate cell phenotyping by flow cytometry was investigated to define the impact
of AS37 adjuvant on different innate immunity cell populations and their
activation status comparing among pre- and post-vaccination status within each
group. The intermediate monocytes population, a highly phagocytic subset, showed
an increase after vaccination that peaked at D4 and decreased at D8. This effect
was AS37-specific and was more prominent in the highest AS37 dose groups of 50
and 100 µg, where the change reached statistical significance (Fig. 4C). A trend
towards an increased number of monocytes at D4 was observed in all vaccine
groups, irrespective of TLR7a dose (data not shown), which can be attributed to
a vaccination effect. (Fig. 4C).

Innate cell activation at each sampling timepoint for myeloid cells (DC and
monocytes), evaluated by measuring level of expression (by MFI) of activation
markers (HLA-DR, CD86, CD40, CD32 and CD64), showed no specific trends
associated with either TLR7a dose or sampling time point. The only appreciable,
albeit non-significant, effect was an increase of pDC expressing the
costimulatory molecule CD40, with a trend towards an increase at D4 in the
AS37-adjuvanted vaccine groups versus alum-adjuvanted vaccine group,
particularly for TLR7a dose 25 µg (Fig. 4D).

3.4. FREQUENCY AND QUALITY OF ANTIGEN-SPECIFIC CD4+ T CELLS AND MEMORY B CELLS

Analysis of the abundance and quality of CRM197-specific CD4+ T cells in each Th
cell subset showed a trend towards an increase across all groups at D8 and D29,
with no overall specific trend in relation to AS37 (data not shown). However,
the frequency of CRM197-specific CD4+CD40L+IL21+ Tfh cells showed a stronger
post-vaccination increase in the AS37-adjuvanted vaccine groups as compared to
the alum-adjuvanted control group (Fig. 4E). A similar response profile was also
observed with CRM-specific Th1 cells (IFN-γ producing cells), particularly for
TLR7a doses 12.5, 25 and 50 µg (Fig. S5). In this case, however, the
AS37-induced modulation did not reach statistical significance.

The frequency of circulating IgG and IgM MBC specific for MenC polysaccharide
and CRM197 antigen was analysed by ELISpot and expressed as percentage of
antigen-specific MBC/total IgG and MBC/total IgM. The CRM197-specific MBC showed
a significantly higher response at D29 versus baseline in the control vaccine
group and similarly in the groups that received the AS37-adjuvanted vaccines,
especially the 12.5 and 25 µg doses (Fig. 4F). Finally, the groups that received
vaccine containing 25, 50 and 100 µg TLR7a showed a greater MenC-specific MBC
response versus baseline as compared to the control group, in particular at
25 µg TLR7a dose. (Fig. 4G).

3.5. AS37 IMMUNE-MODULATION PROPERTIES ARE DRIVEN BY ITS TLR7A COMPONENT

We compared the four AS37-adjuvanted vaccine groups with the control group to
assess specific effects contributed by TLR7 stimulation.

After an initial normalisation and filtering step, the different types of
immunological data were analysed collectively. The 335 selected immune readouts
are summarised in Table 1. Modulations induced by AS37 within the first 3 days
following vaccination were almost exclusively represented by boosted IFN-related
signals (Fig. 5). All three IFN-related BTMs (M1.2, M3.4 and M5.12) were
significantly upregulated in at least one TLR7a dose group. Consistent with
observations from the gene level transcriptome analysis (Fig. 2), the 50 µg dose
had the strongest effect. The other IFN-related response to be boosted by TLR7a
was the serum concentration of CXCL10 (IP-10) chemokine (Fig. 5). TLR7a 25 and
50 µg doses also induced a higher frequency of activated APC subsets, such as
CD86+ or HLADR+ monocytes, CD64+CD1c+ DC and CD40+ pDC (Fig. 5). Finally, in
line with previous observations, the AS37 effect at D8, D29 and D181 was
substantially weaker as compared to earlier timepoints (Fig. S6).

Table 1. Summary of 335 selected immune readouts from the comparison of the four
AS37-adjuvanted vaccine groups with the control group. D1 refers to the day of
vaccination.

Empty CellEarly immune parametersLate immune parametersMaximum
(D1–D4)D8D29D181MenC hSBA and ELISA–222Blood transcriptional
modules99115––Cytokines/chemokines913––Myeloid/lymphoid cells3331––CD4+ T
cells–89–Memory B cells/plasmablasts–444

D, study day; ELISA, enzyme-linked immunosorbent assay; hSBA, human complement
serum bactericidal assay; MenC, N. meningitidis serogroup C.

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Fig. 5. Volcano plot of differential modulation for each immune parameter at 6
and 24 h post-vaccination and Day 4 in the four AS37-adjuvanted MenC-CRM197
vaccine groups as opposed to the alum-adjuvanted MenC-CRM197 vaccine group. The
highest response observed at the three timepoints was used for each parameter.
Solid coloured points indicate immune parameters with an absolute fold
change ≥ 0.5 (in log2 scale) and an associated p-value ≤ 0.05 (ANOVA test).
White-dotted symbols indicate a false discovery rate ≤ 5 %. Only parameters
significantly modulated (p ≤ 0.05) in at least one dose group are represented.
AS37-12.5/25/50/100, group that received MenC-CRM197-conjugated vaccine with
AS37 adjuvant containing Toll-like receptor 7 agonist (TLR7a dose 12.5, 25, 50
or 100 µg). ANOVA, analysis of variance; BTM, blood transcriptional module; CMo,
classical monocytes; cmDCs, conventional myeloid dendritic cells; IMo,
intermediate monocytes; MenC, N. meningitidis serogroup C; pDC, plasmacytoid
dendritic cells.

3.6. THE SAME AS37 IFN-RELATED SIGNATURE IS INDUCED IN NHP

In a NHP model in which animals were immunised with the human immunodeficiency
virus (HIV) gp140 glycoprotein [21], AS37 was reported to induce the
upregulation of antiviral and IFN genes, while not having any effect on the
modulation of pro-inflammatory genes, which could theoretically be induced by
the TRL7 engagement. We compared the peripheral blood transcriptome responses in
the NHP study with those obtained in our study. Fig. 6 shows the modulation of
10 BTMs, selected based on findings from the NHP study [21] and representing
clusters of genes involved in either IFN (M1.2, M3.4 and M5.12) or inflammatory
(M3.2, M4.2, M4.6, M4.13, M5.1, M5.7 and M7.1) responses, at 24 h
post-vaccination. The analysis shows that, in both humans and NHPs, AS37 induced
a significant (Benjamini-Hochberg corrected ANOVA p-value ≤ 0.05) upregulation
of IFN-related BTMs, while not interfering with the inflammation-related BTM
response (Supplementary File 6). The AS37 transcriptome response patterns were
remarkably similar between the two studies (Fig. 6).

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Fig. 6. Radar chart showing the effect of AS37 (containing TLR7a 50 µg) on
interferon- and inflammation-related blood transcriptional modules (BTMs),
including interferon-inducible and inflammation-related genes, 24 h after
vaccination in the present study and in a previous study of non-human primates
[21]. Results shown with alum-adjuvanted MenC-CRM197 vaccine and AS37-adjuvanted
MenC-CRM197 vaccine (Toll-like receptor 7 agonist dose 50 µg) administered to
humans and with AS37-adjuvanted HIV envelope glycoprotein 140 (Gp140) vaccine
administered to non-human primates. BTMs were originally described by Li et al
[42]. Solid lines show average BTM responses and shaded areas the 95 %
confidence intervals. Log2 fold-changes from baseline values are represented.

4. DISCUSSION

We conducted in-depth profiling, in healthy adults, of immune responses to a
MenC conjugate vaccine formulated with a novel SMIP adjuvant component (AS37),
containing different doses of TLR7a adsorbed to alum, and compared them to a
control group that received a licensed alum-adjuvanted MenC conjugate vaccine.
Results from the clinical study showed that the AS37-adjuvanted MenC-CRM197
vaccine was safe and well tolerated, with a trend towards dose-dependent
increase of MenC polysaccharide-binding antibodies in the groups that received
AS37-adjuvanted vaccine, but no appreciable difference in functional antibody
titres, although the study was not powered to compare immune responses [18]. In
a sub-cohort of 66 participants, additional exploratory analyses were conducted
to further characterise the immune response.

Overall, the peripheral blood transcriptome responses indicated that AS37
induces systemic, transient activation of the IFN-mediated antiviral response,
which peaks 24 h after vaccination and generally resolves within one week. This
response was dose-dependent up to the 50 µg TLR7a dose, while the 100 µg dose
was less effective than the 50 µg and 25 µg doses. Hierarchical clustering
revealed five clusters of DEGs, segregating genes involved in metabolic
processes and protein production (clusters #1 and #2), IFN-inducible genes and
genes involved in IFN response (clusters #3 and #5) and B cell activation and
proliferation (cluster #4). The B cell transcriptional signature observed in
peripheral blood 7 days after vaccination is indicative of vaccine-specific
lymphocytes that differentiate into plasmablasts and start producing antibodies.
There was also evidence that the observed plasmablasts’ transcriptional
signature may be decoupled from early activation of the systemic IFN response as
suggested by the alum-adjuvanted vaccine group, which showed a similar level of
activation of B cell-related genes as the AS37-adjuvanted vaccine groups 7 days
post-vaccination, with no appreciable IFN response at 24 h.

In addition, analysis of the serum expression of cytokines/chemokines showed an
increased level of CXCL10 (IP-10) chemokine at 24 h and 3 days post-vaccination
in AS37-adjuvanted vaccine groups as compared to baseline. This is also
supported by CXCL10 responses detected at mRNA level. CXCL10 (C–X–C motif
chemokine 10), also known as IFN γ-induced protein 10 kDa (IP-10) [29], binds
CXCR3 receptor to induce chemotaxis, which can be produced by monocytes and
lymphocytes in response to IFN responses. The increased level of CXCL10 in
AS37-adjuvanted vaccine groups may indicate local production at the injection
site or draining lymph nodes that might in turn trigger recruitment of immune
cells, creating the appropriate microenvironment for an optimal immune response
to the vaccine antigens.

Regarding APCs response, there was evidence of an increase in intermediate
monocyte, a phagocytic subset of innate immune cells, that peaked 3 days
post-vaccination and waned 7 days post-vaccination, with highest increases in
the TLR7a 25 and 50 µg dose groups. Moreover, a trend for augmentation of
activated pDC expressing CD40 was observed in the AS37-adjuvanted vaccine groups
3 days post-vaccination. Overall, these findings combined with the CXCL10
increase suggest that AS37 may have a positive effect on the activation and
recruitment of innate immune cells.

As previously shown [26], [30], a post-vaccination increase of vaccine-specific
CD4+ T cells correlated in a predictive manner with the rise and long-term
maintenance of protective antibody titres. Here, in particular, we observed an
increased frequency of CRM-specific Tfh cells (CD4+CD40L+IL21+ T cells) at
7 days post-vaccination that was maintained at 4 weeks post-vaccination, with
highest increases in the AS37-adjuvanted vaccine groups (Fig. 4E). Tfh cells
have been identified as the CD4+ T cells population specialised in providing
help to B cells [31], [32], [33]. Analysis of their phenotype in lymph nodes and
tonsils has pointed to a central role of IL-21, despite the absence of specific
markers for this lymphocyte subset. A migratory counterpart of antigen-specific
Tfh cells has been identified in peripheral blood with similar functional
properties and their presence was proposed to be associated with effective
vaccination [34], [35], [36]. As TLR7 is expressed by DCs and B cells, which
interact directly with Tfh and can influence their activation and
differentiation, observation of an enhanced Tfh response in the AS37-adjuvanted
vaccine groups could reflect an indirect effect on Tfh by the TLR7a molecule via
crosstalk with the DC and B cell population.

An integrated data analysis was conducted to assess specific effects contributed
by TLR7 stimulation, as the main difference between study vaccines was the
presence of the TLR7a molecule in the investigational vaccine. This confirmed
that the TLR7a component elicited an IFN-mediated immune response and increased
activation of specific APCs within the first 3 days after vaccination. The
post-vaccination increase in IFN-related genes with TLR7a (Fig. 2B, cluster #5)
seems in agreement with the increase in activated pDC detected through the
analysis of CD40 expression on their surface. pDC are indeed the main APC subset
producing type I IFN cytokine after TLR7 engagement. These early responses to
the vaccine, however, did not translate into a strong improvement in the
adaptive immune response compartment (e.g. functional antibody) over that
observed with the alum-adjuvanted vaccine and it may be due to pre-existing
immunity to the MenC antigen in the participants. Indeed, MenC is the second
most common meningococcal serogroup after serogroup B in Germany [37], where the
clinical study was conducted [18]. Moreover, as previously discussed [18],
interpretation of the immunogenicity results from this clinical study should
take into account that both the control MenC-CRM197 vaccine and the
investigational formulation contain alum which has immunogenic properties by
itself. Therefore, considering the fact that the study was conducted in a young
healthy population, that usually have a strong robust immune response to the
MenC vaccine, this could account for the fact that it was difficult to detect a
dose–response relationship with increasing TLR7a dose and explain why the early
signatures described did not translate into a strong rise in antibody
functionality as compared to the control vaccine. Moreover, we must consider
that antibodies can confer protection through many mechanisms beyond
neutralisation and further investigations could be done to assess the impact of
AS37 on other functional characteristics of vaccination-induced antibodies,
assessable through complementary approaches such as systems serology [38]. Also,
further clinical study immunising with vaccines in which AS37 will be formulated
with antigens for which most individuals are still naïve, could help to better
identify the advantages of this adjuvant as compared to other immunostimulatory
molecules. Indeed, previous study comparing early responses to HBV surface
antigen combined with different Adjuvant Systems in healthy HBV-naive adults
helped in clarifying the role of adjuvants on vaccine immunogenicity,
reactogenicity and innate immune response activation [19], [39]. Future studies
could be conducted in a most naïve population like paediatric, to evaluate the
added value of this adjuvant in an immune setting with no pre-existing immunity
and better appreciate changes of the immune response following vaccination as
compared to baseline. However, the innate immune stimulation induced by AS37,
that has been shown by these results, is also likely to enhance the
immunogenicity to vaccines of individuals that are already primed for selected
antigens. Therefore, further studies, aimed to evaluate the immune response of
adult populations with different experienced vaccine antigens, will be also
carried in the future.

In the comparison of peripheral blood transcriptome responses in our study with
those observed in a NHP study [21], which evaluated cross-species AS37
immunomodulatory properties when formulated with different antigens, AS37
activated IFN-inducible genes in both humans and the NHP preclinical model. This
strengthens the conclusions drawn from our study, while also confirming the NHP
as a reliable model for investigational studies on the immune modulation of
TLRa-based adjuvants [40].

Taken together, our results are in line with what was expected from an adjuvant
candidate containing TLR7a ligand, given that TLR7 is mainly expressed by pDC
and is involved in the enhanced expression of type I IFN [12], [41]. This study
also provides further validation, in terms of increased gene expression and
production of CXCL10, of 25–50 µg as the optimal TLR7a dose in AS37. No safety
concerns were identified at these doses level in the clinical study [18].
Although an enhanced humoral immune response from AS37 inclusion was not
detected in the clinical study, apart from a trend for higher MenC-specific
antibody concentrations [18], the observations from this detailed analysis are
promising, in terms of the upregulation of IFN-mediated genes and immune
signature consistent with TLR7 engagement, with an increase in the innate cell
population after vaccination and antigen-specific MBC and Tfh cells at later
timepoints.

DECLARATION OF COMPETING INTEREST

The authors declare the following financial interests/personal relationships
which may be considered as potential competing interests: Emilio Siena reports
financial support was provided by GSK. Francesca Schiavetti reports financial
support was provided by GSK group of companies. Erica Borgogni reports financial
support was provided by GSK. Marianna Taccone reports financial support was
provided by GSK. Elisa Faenzi reports financial support was provided by GSK.
Michela Brazzoli reports financial support was provided by GSK. Susanna Aprea
reports financial support was provided by GSK. Monia Bardelli reports financial
support was provided by GSK group of companies. Gianfranco Volpini reports
financial support was provided by GSK. Francesca Buricchi reports financial
support was provided by GSK. Chiara Sammicheli reports financial support was
provided by GSK. Simona Tavarini reports financial support was provided by GSK.
Viviane Bechtold reports financial support was provided by GSK. Christoph J.
Blohmke reports financial support was provided by GSK. Carlo De Intinis reports
financial support was provided by GSK. Antonio Gonzalez-Lopez reports financial
support was provided by GSK. Derek T. O Hagan reports financial support was
provided by GSK. Sandra Nuti reports financial support was provided by GSK.
Claudia Seidl reports financial support was provided by GSK. Arnaud M
Didierlaurent reports financial support was provided by GSK. Sylvie Bertholet
reports financial support was provided by GSK. Ugo D Oro reports financial
support was provided by GSK. Duccio Medini reports financial support was
provided by GSK. Oretta Finco reports financial support was provided by GSK. Ugo
D Oro reports a relationship with GSK that includes: equity or stocks. Christoph
J. Blohmke reports a relationship with GSK that includes: equity or stocks.
Derek T. O Hagan reports a relationship with GSK that includes: equity or
stocks. Sylvie Bertholet reports a relationship with GSK that includes: equity
or stocks. Derek T O Hagan has patent issued to Alum/TLR7. Ugo D Oro and Sylvie
Bertholet are listed as inventor on patents owned by the GSK. Arnaud M
Didierlaurent reports personal fees from Speranza and Lubrizol for consultancy,
outside the submitted work. All authors declare no other financial or
non-financial relationships and activities.

ACKNOWLEDGEMENTS

The authors thank Sherryl Baker (GSK at the time of the study), Lisa Bedell (GSK
at the time of the study), Albertina Fanelli (GSK), Darren Kelly (GSK), Barry
Kunnel (GSK), Rob Mulder (GSK), Jaap Oostendorp (GSK at the time of the study),
Robert Seder (GSK at the time of the study) and Jakub Skrobanek (GSK) for their
contributions to the study. The authors also thank Business & Decision Life
Sciences platform for editorial assistance, manuscript coordination and writing
support, on behalf of GSK. Joanne Knowles (independent medical writer, on behalf
of Business & Decision Life Sciences) provided medical writing support.

AUTHORS’ CONTRIBUTIONS

All authors were involved in the study conception and design and/or the
acquisition and generation of data and/or the analysis and interpretation of the
data. All authors had full access to the data and contributed substantially to
the development of the manuscript and gave final approval before submission. All
authors attest they meet the International Committee of Medical Journal Editors
criteria for authorship.

FUNDING

This study (NCT02639351) was funded by GlaxoSmithKline Biologicals SA, which was
involved in all stages of study conduct, including analysis of the data.
GlaxoSmithKline Biologicals SA also took in charge all costs associated with the
development and publication of this manuscript.

TRADEMARK

Menjugate is a trademark owned by or licensed to the GSK group of companies.

APPENDIX A. SUPPLEMENTARY MATERIAL

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Form analysis 5 forms found in the DOM

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