Self-assembling small-molecule adjuvants as antigen nano-carriers

概要

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Cite this: Chem. Commun., 2022,
58, 12228
Received 12th September 2022,
Accepted 10th October 2022

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Self-assembling small-molecule adjuvants
as antigen nano-carriers†
Shuyu Jin,ab Shao-hua Zhuo,c Yasushi Takemoto,b Yan-mei Li
Motonari Uesugi *bde

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and

DOI: 10.1039/d2cc05016a
rsc.li/chemcomm

The effective co-delivery of antigens and immune potentiators
(adjuvants) and the high degree of antigen presentation have been
two major challenges in the development of subunit vaccines. Here,
we address these issues by conjugating peptide antigens with
cholicamide, a self-assembling small molecule adjuvant. Coassemblies of the conjugates and cholicamide achieved high levels
of both cytokine induction and MHC class II peptide presentation.

Traditional vaccines are derived from live-attenuated or inactivated pathogens, thereby potentially bringing along with them
unexpected risks.1 Subunit vaccines consisting of isolated
protein antigens are a safer and cost-effective option. However,
they often lack sufficient immunogenicity and require the
inclusion of immune potentiators (termed adjuvants), which
trigger early innate immune responses to ensure the generation
of robust and long-lasting adaptive immune responses.2
The effective co-delivery of adjuvants and antigens to
antigen-presenting cells (APCs) has been a major challenge in
the development of subunit vaccines.3,4 To address this issue,
conjugate vaccines, in which adjuvant and antigen are covalently
attached, have been explored.5 Such self-adjuvanting vaccines
benefit the cytokine induction to boost early innate immune
responses of APCs and enhance the uptake of antigen through
receptor-mediated endocytosis.6 The adjuvant components often
used in conjugate vaccines are agonists of toll-like receptors
(TLRs). For instance, HIV gag protein has been conjugated with
a

Graduate School of Medicine, Kyoto University, Uji, Kyoto 611-0011, Japan
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
E-mail: uesugi@scl.kyoto-u.ac.jp
c
Key Lab of Bioorganic Phosphorus Chemistry & Chemical Biology, Department of
Chemistry, Tsinghua University, Beijing 100084, China
d
Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University,
Kyoto 606-8501, Japan
e
School of Pharmacy, Fudan University, Shanghai 201203, China
f
Beijing Institute for Brain Disorders, Beijing 100069, China
g
Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084,
China
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/
10.1039/d2cc05016a
b

12228 | Chem. Commun., 2022, 58, 12228–12231

a TLR7/8 agonist, a thiazoloquinolone derivative CL-075,7 and
tumor-associated carbohydrate antigen MUC1 has been combined with TLR1/2 agonists, Pam3Cys and Pam3CSK4.8
Another important component of subunit vaccines is an
antigen carrier that facilitates the delivery of antigens to
immune cells. The immune system has developed the ability
to recognize and respond to nano- and micron-sized particles
such as viruses and bacteria.9 Nano- and micron-sized antigen
carriers, including virus-like particles,10 viral protein cages,11
and de novo self-assembling peptides,12 hold great potential for
delivering and presenting antigens in APCs.13
The TLR-agonist adjuvants that simultaneously serve as
nano-carriers of antigens might be ideal constituents of minimalist conjugate vaccines. We previously discovered a small
molecule vaccine adjuvant, cholicamide (molecule 1), which
self-assembles to form a nanoparticle approximately 150 nm in
size and stimulates APCs through the activation of TLR7, an
endosomal Toll-like receptor.14 Activation of TLR7 has been
demonstrated to induce highly potent humoral and cellmediated immune responses. Taking advantage of the selfassembling property of cholicamide (1), we postulated that
cholicamide (1), when covalently conjugated with peptides,
serves simultaneously as a nano-carrier and an adjuvant for
peptide antigens (Fig. 1A).
Structure-activity relationship study of cholicamide (1) has
shown that the deoxycholate moiety is essential for both
immunopotentiation and self-assembly, suggesting the linker
as a potential conjugation site for peptide antigens. The original linker was replaced by lysine for the introduction of a
branched carboxylic acid functional group (Fig. S1A, ESI†).
The resulting molecule (molecule 2) was further functionalized
by the introduction of an alkyne group (molecules 3 and 4),
allowing subsequent copper-catalyzed azide-alkyne cycloaddition (CuAAC) with azide-peptides.
To examine the feasibility of our concept, we initially
selected molecule 3 for conjugation with an azide-Flag peptide
(N3-GGSDYKDDDDK) through CuAAC (Fig. 2A). The resulting
conjugate, 3-Flag, readily formed nanoparticles in water and

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Fig. 1 Schematic representation of nano-carrier adjuvants for peptide
antigens. (A) Nano-assemblies of a cholicamide-peptide conjugate.
(B) Formation of co-assemblies of cholicamide and its peptide conjugate
at different mixing ratios.

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fabrication endorses the ability to present antigens to APCs, it
would also mask the adjuvant nanoparticles from TLR7, limiting
its immunopotentiation activity. One potential solution is the
preparation of co-assemblies by mixing cholicamide (1) and its
peptide conjugate in an appropriate ratio (Fig. 1B). To analyze such
co-assembly formation, we prepared a cholicamide-rhodamine
conjugate (4-PEG-rhodamine) (Fig. 3A). This fluorescent conjugate
permits the detection of the co-assembly under a confocal microscope. While cholicamide (1) alone displayed non-fluorescent
particles, the mixture samples at a fixed total concentration (50
mM) but varied molar ratios (10–30% of 4-PEG-rhodamine) showed
fluorescent nanoparticles (Fig. 3B). Comparison with the brightfield images indicates that all the observed nanoparticles exhibit
fluorescence, suggesting co-assembly formation.
As a model peptide antigen, we initially used OVA257–264
(SIINFEKL), an ovalbumin epitope peptide presented by MHC I
molecule H-2Kb.15 Molecule 4 was coupled with OVA257–264
through two different linkers to yield 4-OVA257–264 and 4-PEGOVA257–264 (Fig. S2A, ESI†). Dynamic light scattering (DLS)
measurement confirmed the formation of B130 nm particles
at 30 mM irrespective of mixing ratios with cholicamide (1)
(Fig. S2B, ESI†), suggesting the ability of these peptide conjugates to form co-assemblies with cholicamide (1). Each
mixture was also added to the culture of RAW264.7 macrophage
cells for examining its ability to elicit IL-6 and TNF-a production. As anticipated, peptide conjugates alone exerted little, if
any, cytokine induction perhaps due to the masked adjuvant
surface. In contrast, the cytokine-inducing ability of cholicamide (1) was maintained at mixing ratios up to 30% (Fig. S2C,
ESI†). Encouraged by the result, we evaluated the antigenpresenting ability of the 20% and 30% co-assemblies in bone
marrow-derived dendritic cells (BMDCs) using an antibody
against H-2Kb-bound SIINFEKL (Fig. S2D, ESI†). To our

Fig. 2 Characterization of 3-Flag. (A) Chemical structure of 3-Flag.
(B) Self-assemblies of 3-Flag in H2O (left) stained with Nile Red (15 mM)
and PBS (right) recognized by Flag-antibody under a confocal microscope.
Scale bar: 25 mm.

PBS, as observed under a confocal microscope when stained
with Nile Red, an environment-sensitive fluorescent probe that
was previously used for detecting cholicamide assemblies14
(Fig. 2B). To assess the surface exposure of the hydrophilic
Flag peptide, we examined whether the particles of 3-Flag can
be recognized by a fluorescent Flag antibody, whose large size
often limits its access to the inside of the nanoparticles.
Confocal imaging revealed fluorescent dots whose sizes and
numbers are consistent with those of Nile Red-stained particles
of 3-Flag (Fig. 2B). The antibody’s recognition of the particles
suggests that 3-Flag forms nanoparticles in which the Flag
peptide segments are, at least in part, accessible to a large
macromolecule.
The surface fabrication of the adjuvant nanoparticles with
antigens can be a double-edged sword. While the surface

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Fig. 3 Co-assembly approach confirmed by 4-PEG-rhodamine. (A) Schematic illustration of 4-PEG-rhodamine co-assemblies with cholicamide
(1). (B) Co-assemblies of 4-PEG-rhodamine with cholicamide (1) at various
mixing ratios under a confocal microscope. Scale bar: 100 mm.

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disappointment, flow cytometric analysis of the cells detected
no significant enhancement of antigen presentation on the cell
surface by the co-assemblies. In fact, their antigen presentation
activities were even lower than that of OVA257–264 per se.
The antigen presentation in APCs is governed by two classes
of major histocompatibility complex (MHC): classes I and II.
Although both classes share the task of presenting antigen
peptides on the cell surface for recognition by T cells, they
capture the peptides from distinct sources. While MHC class I
molecules bind usually to cytosolic peptides, MHC class II
molecules bind selectively to peptide antigens generated by
proteolysis of proteins in endosomes and lysosomes for subsequent presentation on the cell surface.16 OVA257–264 is an
antigenic peptide that is known to be recognized by MHC class
I molecules, but not by class II. ...