Nanoformulated Single-Stranded RNA-Based Adjuvant with a Coordinative Amphiphile as an Effective Stabilizer: Inducing Humoral Immune Response by Activation of Antigen-Presenting Cells

Article abstract of DOI:10.1002/anie.202002979

As agonists of TLR7/8, single-stranded RNAs (ssRNAs) are safe and promising adjuvants that do not cause off-target effects or innate immune overactivation. However, low stability prevents them from mounting sufficient immune responses. This study evaluates the adjuvant effects of ssRNA derived from the cricket paralysis virus intergenic region internal ribosome entry site, formulated as nanoparticles with a coordinative amphiphile, containing a zinc/dipicolylamine complex moiety as a coordinative phosphate binder, as a stabilizer for RNA-based adjuvants. The nanoformulated ssRNA adjuvant was resistant to enzymatic degradation in vitro and in vivo, and that with a coordinative amphiphile bearing an oleyl group (CA-O) was approximately 100 nm, promoted effective recognition, and improved activation of antigen-presenting cells, leading to better induction of neutralizing antibodies following single immunization. Hence, CA-O may increase the efficacy of ssRNA-based adjuvants, proving useful to meet the urgent need for vaccines during pathogen outbreaks.

Full text of DOI:10.1002/anie.202002979

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Nanoformulated ssRNA-based Adjuvant with a Coordinative
Amphiphile as an Effective Stabilizer to Induce a Humoral
Immune Response by Activation of Antigen-presenting Cells
Authors: Hyo-Jung Park, Eun-Kyoung Bang, Jung Joo Hong, Sang-
Myeong Lee, Hae Li Ko, Hye Won Kwak, Hyelim Park, Kyung
Won Kang, Rhoon-Ho Kim, Seung Rok Ryu, Green Kim,
Hanseul Oh, Hye-Jung Kim, Kyuri Lee, Minjeong Kim, Soo
Young Kim, Jae-Ouk Kim, Karim El-Baz, Hyukjin Lee, Manki
Song, Dae Gwin Jeong, Gyochang Keum, and Jae-Hwan
Nam
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202002979
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10.1002/anie.202002979
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RESEARCH ARTICLE
Nanoformulated Single-stranded RNA-based Adjuvant with a
Coordinative Amphiphile as an Effective Stabilizer to Induce a
Humoral Immune Response by Activation of Antigen-presenting
Cells
Hyo-Jung Park[a],+, Eun-Kyoung Bang[b],+, Jung Joo Hong[c],+, Sang-Myeong Lee[d],+, Hae Li Ko[a],+, Hye
Won Kwak[a], Hyelim Park[a], Kyung Won Kang[d], Rhoon-Ho Kim[a], Seung Rok Ryu[d], Green Kim[c],
Hanseul Oh[c], Hye-Jung Kim[a], Kyuri Lee[e], Minjeong Kim[e], Soo Young Kim[e], Jae-Ouk Kim[f], Karim
El-Baz[b], Hyukjin Lee[e], Manki Song[f], Dae Gwin Jeong[g], Gyochang Keum[b],*, Jae-Hwan Nam[a],*
[a]
Dr. Hyo-Jung Park, Hae Li Ko[++], Hye Won Kwak, Dr. Hyelim Park, Rhoon-Ho Kim, Hye-Jung Kim, Prof. Dr. Jae-Hwan Nam
Department of Biotechnology
The Catholic University of Korea
Bucheon 14662, Republic of Korea
E-mail: jhnam@catholic.ac.kr
[b]
Dr. Eun-Kyoung Bang, Karim El-Baz, Dr. Gyochang Keum
Center for Neuro-Medicine, Brain Science Institute
Korea Institute of Science and Technology
Seoul 02792, Republic of Korea
E-mail: gkeum@kist.re.kr
[c]
[d]
[e]
[f]
Dr. Jung Joo Hong, Green Kim, Dr. Hanseul Oh
National Primate Research Center
Korea Research Institute of Bioscience and Biotechnology
Cheongju 28116, Republic of Korea
Prof. Dr. Sang-Myeong Lee, Kyung Won Kang, Seung Rok Ryu
Division of Biotechnology, College of Environmental and Bioresource Sciences
Chonbuk National University
Iksan 54596, Republic of Korea
Dr. Kyuri Lee, Minjeong Kim, Soo Young Kim, Prof. Dr. Hyukjin Lee
College of Pharmacy, Graduate School of Pharmaceutical Sciences
Ewha Womans University
Seoul, 03760, Republic of Korea
Dr. Jae-Ouk Kim, Dr. Manki Song
Clinical Research Lab
International Vaccine Institute
Seoul National University Research Park, Seoul 08826, Republic of Korea
Dr. Dae Gwin Jeong
[g]
Infectious Diseases Research Center
Korea Research Institute of Bioscience and Biotechnology
Daejeon 34141, Republic of Korea
[+]
These authors contributed equally to this work
[++] Present address: Scripps Korea Antibody Institute, Chuncheon 24341, Republic of Korea
Supporting information for this article is given via a link at the end of the document.
Abstract: As agonists of TLR7/8, single-stranded RNAs (ssRNAs)
are safe and promising adjuvants that do not cause off-target effects
or innate immune overactivation; however, low stability prevents them
from mounting sufficient immune responses. This study aimed to
evaluate the adjuvant effects of ssRNA derived from the cricket
paralysis virus intergenic region internal ribosome entry site,
ssRNA-based adjuvants, proving useful to meet the urgent need for
vaccines during pathogen outbreaks.
Introduction
formulated as nanoparticles with
a coordinative amphiphile,
In recent decades, the emergence of new viral strains, including
severe acute and Middle East respiratory syndrome coronavirus
(SARS- and MERS-CoV), has been reported regularly. Most
recently is the SARS-CoV-2 outbreak in 2019 and currently
ongoing in 2020. It induces coronavirus disease 2019 (COVID-
19), which causes severe pneumonia, and the ongoing pandemic
has been declared by the World Health Organization as a world
health emergency.[1-3]
containing a zinc/dipicolylamine complex moiety as a coordinative
phosphate binder. We applied our amphiphile system as a stabilizer
for RNA-based adjuvants. The nanoformulated ssRNA-based
adjuvant was resistant to enzymatic degradation in vitro and in vivo.
The ssRNA-based adjuvant formulated with a coordinative amphiphile
bearing an oleyl group (CA-O) was approximately100 nm, promoted
effective recognition and improved activation of antigen-presenting
cells, leading to better induction of neutralizing antibodies following
single immunization. Hence, CA-O may increase the efficacy of
Accordingly, the development of a fast, safe, and efficient vaccine
against emerging viruses is gaining increasing attention.
1
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10.1002/anie.202002979
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RESEARCH ARTICLE
Inactivated pathogens as vaccines to induce neutralizing
antibodies (NAbs) have proven highly effective in the prevention
of infectious diseases.[4,5] Although these vaccines have been
widely used in clinical practices, they exhibit certain drawbacks,
including the induction of a weak immune response, short-lived
immunity, and several safety issues, such as the development of
fever and rashes.[5,6] In recent years, researchers have focused
Generally, a combination of various classes of amphiphilic or
cationic lipids, polymers, or peptides have been used to improve
ssRNA stability and permeability. These nanocomplexes protect
ssRNAs against enzymatic degradation; thus, they have an
adjuvant effect in tumor vaccines and various subunit
vaccines.[30,36] We previously reported the adjuvant effects of
ssRNAs derived from the cricket paralysis virus (CrPV) intergenic
region (IGR) internal ribosome entry site (IRES),[43, 44]. Here, we
studied the adjuvant effect of these ssRNAs formulated with
coordinative amphiphiles (CAs).[45]
on
identifying
antigen
components
responsible
for
immunogenicity to generate safer and more effective vaccines.
However, purified recombinant antigens, known as subunit
vaccines, generally show poor immunogenicity.[5] To overcome
this drawback, vaccines have been formulated with
immunostimulatory agents, known as adjuvants.[7]
A CA is defined as an amphiphilic molecule consisting of a
phosphate-directing group (a phosphate-specific binder, zinc-
dipicolylamine (Zn/DPA) complex) and a membrane-directing
group.[45] It serves as a cationic lipid mimic, which are amphiphiles
with positively charged heads and are commonly used as nonviral
vectors for nucleic acids. The CA was developed to reduce the
toxicity of cationic lipids, which is determined primarily by the
cationic nature of their hydrophilic heads.[46] Owing to their
cationic character, cationic lipids can bind nonspecifically to
proteins in the bloodstream and their detergent-like structure can
cause cell lysis, while the cationic complexes cause systematic
toxicity in vivo.[47] By substituting the head group with Zn/DPA,
CAs interact with phosphate groups in the backbone of RNAs via
specific coordinative binding rather than electrostatic interaction,
resulting in low cytotoxicity as well as RNA stabilization.[45] In the
current study, we applied CAs as a stabilizer for ssRNA-based
adjuvants and evaluated whether they effectively stabilize
ssRNA-based adjuvants.
Adjuvants are essential for the induction of robust immune
responses against vaccine antigens[8,9]. Ideal adjuvants induce
innate immune responses, which lead to the activation of adaptive
immune responses.[8,10] They also stimulate a balanced immune
response, including enhanced antibody production, cellular
immunity, and mucosal immunity.[11,12] Aluminum hydroxide
(alum) has been widely used as an adjuvant in human vaccines
for decades[13] as it stimulates the immune system by recruiting
antigen-presenting cells (APCs), such as dendritic cells (DCs)
and macrophages, and by activating inflammasome.[12] However,
alum is ineffective at inducing Th1 responses and long-term
immunity.[14] As alum induces only Th2 responses, new adjuvants
capable of promoting Th1 responses are required. Nonclassical
adjuvants, such as Toll-like receptor (TLR) ligands for pattern
recognition receptors (PRRs), can stimulate the production of
type I interferon (IFN) and proinflammatory cytokines.[15] For
example, lipopolysaccharide (LPS),[16] an endotoxin, acts on
TLR4; oligonucleotides containing CpG motifs[17-18] act as TLR9
agonists; imidazoquinoline[19-20] is a small molecule agonist of
TLR7/8; and highly immunogenic double-stranded (ds)RNAs[22–28]
act as TLR3 agonists. However, LPS and CpG oligonucleotides
are highly toxic with well-characterized side effects, including the
induction of inflammation in vivo. Monophosphoryl lipid A (MPLA)
is the detoxified version of LPS. AS04, developed by Glaxo-
SmithKlaine, is mixed with MPLA and alum.[29] The synthetic
dsRNA analog double-stranded polyriboinosinic:polyribocytidylic
acid [poly(I:C)] has strong adjuvanticity,[22–28] activating TLR3 and
inducing interleukin (IL)-12 and type I IFN.[9,17] Since its successful
application in a Japanese encephalitis virus (JEV) vaccine, it has
been tested in various other vaccines,[24–33] and has been shown
to exhibit potent immunostimulatory properties; however, specific
concerns have arisen regarding the induction of adverse effects
and excessive immune re23onses, including autoimmune
diseases, and the difficulty associated with standardization for
mass production.[26]
Results and Discussion
Screening of three CA-formulated ssRNA-based adjuvants
We synthesized three CAs and evaluated their RNA adjuvant-
stabilizing effects. All CAs had a Zn/DPA head for affinity to the
phosphates in the RNA backbone via specific coordination. The
membrane-directing part of the amphiphiles consisted of oleoyl,
biotinyl, or retinoyl groups, generating CA-O, CA-B, and CA-R,
respectively (Figure 1). The oleoyl group is a component of
phospholipids in the body, and is commonly used to establish
efficient, hydrophobic tails. Biotin reportedly affects cytokine
expression in immune cells.[48] Retinoic acid (RA), a metabolite of
vitamin A, was introduced because it induces Th1 and Th2
responses.[49] Thus, we prepared an amphiphilic derivative of
CR12C (Palm-CR12C, LP), which is a carrier/stabilizer for ssRNA
adjuvants[31,40, 42] through conjugation of the palmitoyl group to the
peptide via amide coupling, as a positive control.
As agonists of TLR7 and TLR8, single-stranded RNAs
(ssRNAs) have been recently highlighted as safe and promising
adjuvants.[30–38] The ssRNA-type adjuvants promote Th1
responses through the induction of localized IFN-α and IL-12
responses in DCs at the injection site, supporting their safe
use.[30,31,38] However, low stability and poor cellular uptake have
prevented their widespread clinical adoption. Thus, a stabilizer or
carrier is required to effectively increase the immune activity of
ssRNAs.
The effects of the three CA stabilizers (CA-O, CA-B, CA-R)
were evaluated by measuring IgG1 and IgG2a levels two weeks
after immunization of mice with a JEV envelope (E) protein
subunit vaccine candidate, which is supported by the International
Vaccine Institute (Figure S1). The ssRNA adjuvant-JEV, in which
the JEV E gene was inserted into the CrPV backbone (Figure
S1A), alum, JEV E protein, and stabilizers were formulated in
various ways (Figure S1B). In a previous study, an N/P ratio[50] of
0.9 between the RNA platform and CR12C peptide was used for
in vivo expression.[42] For the initial screening, we followed the
reported protocol and the stabilizers were formulated at an N/P
ratio of 0.9. LP and the CA-O-formulated ssRNA adjuvant induced
42]
39–42]
Cationic polymers,[40,
cationic peptides,[25,
dendrimer,[18] and lipidoid compounds,[26, 36] among others, have
demonstrated stabilizing effects on RNA adjuvants and mRNA
vaccines as individual or mixed nanoparticle (NP) formulations.
2
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10.1002/anie.202002979
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RESEARCH ARTICLE
-
N
Zn2+
2NO3
H
N
N
N
O
O
CA-O
CA-B
-
N
H
NH
S
2NO3
Zn2+
H
N
N
N
O
H
HN
Figure 2. Interaction between Zn/DPA and phosphates. (A) Gel retardation
assays for the complex of the RNA adjuvant, and each CA at various N/P ratios.
(B) Fluorescence intensity (I/Imax) after adding coumarin sulfate to a mixture of
CAs (40 μM) in the presence of various concentrations of RNAs. The
experiments were performed in 5 mM HEPES-NaOH buffer (pH 7.4); λex = 347
nm; λem = 480 nm. Imax represents the fluorescence intensity of the fluorescent
ligand alone (10 μM).
O
O
N
-
2NO3
Zn2+
N
H
N
N
O
O
CA-R
CAs, the Hill coefficient was larger than 1, indicating positive
cooperative binding. This result implies that once a CA molecule
binds to an ssRNA, it increases the binding affinity of the other
molecules. CA-O bound to ssRNA-based adjuvants the fastest,
as indicated by the fact that it had the smallest EC50 value.
However, the most effective stabilizer, CA-O, had a slightly higher
Kd (calculated as the n power of the EC50) than CA-R owing to the
high cooperativity of CA-O. The binding affinity was not fully
explained by the dissociation constant because cooperative
binding could be affected by the membrane-directing part of CAs.
The hydro-dynamic radius of CA-nanoformulated ssRNA
adjuvants was measured using a dynamic light scattering (DLS)
HS
HS
O
O
H
N
OH
N
N
H
H
O
O
12
NH
Palm-CR12C (LP)
HN
NH2
Figure 1. CA-O, CA-B, CA-R, and LP structures.
higher IgG1 and IgG2a titers than the other formulations (Figure
S1C). Among the CA series, vaccination with CA-O-formulated
ssRNA adjuvant resulted in the highest IgG2a production (Figure
S1C), indicating this adjuvant induced the highest Th1 response.
Unexpectedly, CA-R did not show a strong additional adjuvant
effect when compared with ssRNA adjuvants without stabilizers
(Figure S1C).
instrument (Table S1). CA-O-formulated ssRNA adjuvants were
124.7 ± 41.8 nm, whereas CA-R formulated ssRNA formed 222.4
± 29.1 nm and 52.7 ± 9.9 nm particles at a ratio of approximately
8:2 and CA-B did not form a stable enough particle to detect.
Considering the DLS data and the gel retardation assay results
(Figure 2A), we reasoned that CA-O could form more stable
particles than those formed by the other CAs. Based on these
results, CA-O was selected for further testing.
A gel retardation assay revealed that each CA formulated
with ssRNA adjuvants in a different way (Figure 2A). Although
the CA-R formulation showed bands for NPs with low mobility,
there were also small RNA/CA-R adducts, as well as small
RNA/CA-B adducts, even at the highest N/P ratios. The RNA/CA
adduct, of which the band was hardly retarded but prevented RNA
degradation, was characteristic pattern of CAs.[45] CA-O clearly
showed NP formation of RNA/CA-O complexes, which improved
with increasing N/P ratio (Figure 2A). When we formulated
ssRNAs with CA-O at various N/P ratios, the ssRNA bands
gradually disappeared with increasing N/P ratio, whereas new
bands, which represented large complexes, appeared at an N/P
ratio of 1.1. The smeared upper band adjacent to the ssRNA band
was interpreted to represent ssRNAs complexed with CA-O in
various forms. Combined with the highest IgG production results
of CA-O (Figure S1C), these results suggested that better
nanosized formulations of ssRNA adjuvants may induce a
stronger immune response.
Protection of ssRNA by CA-O in vitro and in vivo
CA-O has a Zn/DPA moiety as a phosphate-directing group and
an oleyl chain as a membrane-directing group (Figure 3). As a
mimic of phospholipids in the cell membrane, this amphiphile can
form a multilamellar vesicular structure.[45] Figure 3A shows a
schematic structure of their theoretical nanoformulation.
Coordination between a Zn/DPA head and a phosphate in the
RNA backbone results in the recruitment of ssRNA-based
adjuvants to the vesicles of CAs, forming NPs. Figure 3B shows
the structure of the CrPV-IGR-IRES-derived ssRNA-based
Table 1. Calculated values of Kd, EC50, and Hill coefficient, n, of each CA
We monitored the interactions between CAs and ssRNAs
using a competitive assay with the Zn/DPA binder, 6,7-dihydroxy-
4-sulfomethyl coumarin (Figure S2).[45] In the absence of ssRNAs,
the fluorophore bound to the Zn/DPA head and was effectively
quenched. In the presence of ssRNAs, the quenched
fluorescence was recovered through interaction of CAs with
ssRNAs (Figure 2B). The dissociation constant (Kd), which
represents a half-occupied concentration of RNAs (EC50), and the
Hill coefficient (n) were calculated from the plots (Table 1). For all
Stabilizer
CA-O
Kd (ng/μL)
65.6 ± 3.1
1282 ± 97
47.1 ± 3.4
EC50 (ng/μL)
13.8 ± 1.1
112.0 ± 7.0
29.3 ± 1.3
n
1.59 ± 0.18
1.72 ± 0.15
1.14 ±0.04
CA-B
CA-R
3
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Figure 3. (A) Schematic illustration of the nanoformulation of CA-O and RNA adjuvants. (B) Structure of ssRNA-based adjuvants. (C) Enzymatic stability tests for
complexes of the RNAs with CA-O (C) in vitro and (D) in vivo. Each CA-O-formulated ssRNA-based adjuvant complex was loaded into a gel after incubation in the
presence (+)/absence (–) of RNase A (0.1 U) at 37 °C for 5 min followed by typical RNA prcipitation. (D) ssRNA-based adjuvant and CA-O-formulated ssRNA-
based adjuvant complexes (ssRNA-based adjuvant + CA-O) at N/P 1.8 was injected intramuscularly. The amount of remaining RNA adjuvant in mouse muscle was
measured by real-time PCR. Data represent mean ± SD and were statistically analyzed by the t-test. (**p < 0.01 vs. ssRNA-based adjuvant).
complex can be used as a safe adjuvant because it rapidly
degrades after immune stimulation for a certain period of time.
adjuvant (Figure S1A). The structure was illustrated by combining
CrPV-IGR-IRES at 5’ untranslated region (UTR)[58,59] and the
SV40 polyadenylation sequence at 3’ UTR.[60] To demonstrate the
protective effect of CA-O on the ssRNA adjuvant, RNase A was
added to each formulation of CA-O–ssRNA adjuvants, and the
residual RNA was extracted and electrophoresed (Figure 3C). As
expected, CA-O effectively protected ssRNAs at all N/P ratios
tested. The protective effect was also observed in vivo; Figure 3D
shows the amount of ssRNA-based adjuvant in mouse muscle as
measured by real-time PCR over time, after intramuscular
injection of the CA-O-formulated ssRNA complex. CA-O
effectively protected the ssRNA adjuvant in mouse muscle for up
to 3 h. After this time point, the stability of the complexed ssRNA
became similar to that of the ssRNA. This result suggested that
the protective effect of CA-O is concentrated in the period early
after injection, and that the immune response thus was promoted
soon after injection. This finding further implies that although CA-
O is bound tightly to ssRNA outside of cells, the complexes may
rapidly dissociate inside cells. This result not only solves the
problem of low stability of ssRNAs, but also suggests that the
Charge and size of the CA-O-formulated ssRNA-based
adjuvant, and its activation of APCs
We previously reported that ssRNA-based adjuvants can induce
certain immune-related cytokines in human peripheral blood
mononuclear cells (hPBMCs).[40] In this study, we confirmed that
ssRNA-based adjuvant treatment induced cytokines in hPBMCs,
whereas CA-O treatment alone did not (Figure S3), indicating
that CA-O has no adjuvant effect. Inspired by these results, we
next measured the zeta potential of the CA-O-formulated ssRNA
complexes with various N/P ratios, which revealed a zeta potential
phase change with increasing N/P ratio (Figure 4A). This result
indicated that the complex particle became more compact with
increasing amount of the positively charged CA-O. Considering
the result shown in Figure 2A, the phase of the zeta potential
changed positively upon the formation of a complex of N/P 2.0,
with no free RNAs. A lipid-linked cationic peptide (Palm-CR12C,
LP), used as a positive control,[31–42] showed a similar zeta
4
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Figure 4. (A) Zeta potentials of stabilizer/ssRNA adjuvant nanocomplexes with various N/P ratios. The data represent the mean ± SD. n = 3. The left graph indicates
the N/P ratio of the LP-formulated ssRNA adjuvant nanocomplex, and the right graph indicates the N/P ratio of the CA-O-formulated ssRNA adjuvant nanocomplex.
(B) AFM images of stabilizer-formulated ssRNA adjuvants. The red arrows indicate the stabilizer and ssRNA adjuvant nanocomplexes. (C) Activated DCs (CD11c+
CD86+) and macrophages (F4/80+Cd86+) in the injected muscle and the drain lymph node were counted by flow cytometry. Data represent the mean ± SD and
were analysed by the t-test (*p < 0.05, **p < 0.01 vs. Nil.). (D) mRNA levels of APC activation markers were measured in the injected muscle by real-time PCR.
(“fold” indicates ssRNA adjuvant/Nil or CA-O-formulated ssRNA adjuvant nanocomplex/Nil). “ssRNA-based adjuvant + CA-O” indicates CA-O-formulated ssRNA
adjuvant nanocomplex. Data represent the mean ± SD and were analysed by the t-test (**p < 0.01 or *p < 0.05 vs. ssRNA-based adjuvant).
potential pattern. Both complexes have similar N/P ratio
thresholds (1.8 and 2.0) for zeta potential phase change (Figure
4A). At N/P ratios of 1.8 and 2.0, LP and CA-O-formulated ssRNA
adjuvant induced similar IgG1 and IgG2a titers (data not shown).
Negatively charged particles are more easily taken up into
phagocytic cells than positively charged particles.[51] Therefore, in
subsequent experiments, the amount of ssRNA adjuvant was
fixed to 20 μg, according to our previous report,[44] and we
prepared formulation at an N/P ratio of 1.8, the maximum N/P ratio
that retained a negative surface potential and was high enough to
protect ssRNAs (Figure 3C, 3D). Successful complexation
between the RNA stabilizers (LP or CA-O) and ssRNA-based
adjuvant (Figure 4B) was verified by atomic force microscopy
(AFM). The CA-O-formulated ssRNA-based adjuvant existed as
large spherical structures with a diameter of approximately 100
nm, the LP-formulated ssRNA adjuvant was observed as
spherical structures of approximately 50 nm in diameter, whereas
the free ssRNA adjuvant formed smaller spheres with a diameter
of approximately 25 nm and a random coil of RNA was also
observed (Figure 4B). The presence of protein and/or alum did
not affect to the nanoformulation of ssRNAs and stabilizers (Table
S1 and Figure S5); thus, we showed AFM images only with
formulated ssRNAs without protein and alum to avoid the
interference of aluminum salt.
Interestingly, the CA-O-formulated ssRNA-based adjuvant
exhibited the strongest DC- and macrophage-activating effects in
injected muscle and draining lymph nodes of mice (Figure 4C).
mRNA levels of several APC activation markers, including MHC
class II, CD11c, CD86, F4/80, and IL-1β, at the injection site
(muscle) were measured to confirm DC and macrophage
activation. Both the ssRNA-based and the CA-O-formulated
5
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Figure 5. A single immunization with MERS S protein vaccine, alum, and CA-O-formulated RNA adjuvant can protect against MERS-CoV challenge. C57BL/6 WT
and hDPP4 transgenic (Tg) mice were intramuscularly immunized with MERS protein vaccine with/without adjuvants, LP- or CA-O-formulated ssRNA adjuvant and
alum (Brentanne). (A) Overall study design for WT mice. (B) MERS S protein-specific IgG1 and IgG2c levels were measured by ELISA in the second week after the
first immunization. Data represent the mean ± SD. n = 10 mice. *p < 0.05, **p < 0.01 vs. Nil. (C) MERS-CoV-specific NAb levels of immunized mice were determined
by Plaque reduction neutralization test (PRNT) in the third week after the first immunization. The serum from the same group (n = 9–11) was pooled and diluted
two-fold. (D) Percentage body weight and survival of hDPP4 Tg mice after challenged with 5 × 103 PFU of MERS-CoV/mouse at 3 weeks after the first immunization.
n = 9–11 mice. (E) Survival of female (left) and male (right) hDPP4 Tg mice after challenged with MERS-CoV (survival # / total #).
APCs can be activated by enhancing the uptake of sufficiently
large spherical NPs of CA-O-formulated ssRNA adjuvant.
ssRNA-based adjuvant increased the mRNA levels of all these
markers, but the latter was the most effective in doing so (Figure
4D). In addition, the frequency of MHC class II on APCs in injected
muscle was increased by CA-O-formulated ssRNA-based
Single immunization with the CA-O-formulated ssRNA-based
adjuvant protects mice against MERS virus challenge
adjuvant (Figure S6). Adjuvant-nanocomplexes
of
Based on the above findings, we investigated the immunization
effect of ssRNA-based adjuvants stabilized with CA-O and LP in
approximately 100 nm are the most effective for the stimulation of
57]
APCs, especially DCs.[56,
Therefore, the data suggest that
6
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Figure 6. ssRNA-based adjuvant formulated with MERS S soluble protein vaccine induces antigen-specific immune responses in nonhuman primates. Cynomolgus
macaques intramuscularly immunized with MERS S protein vaccine with/without adjuvants, CA-O-formulated ssRNA adjuvant and alum (Brentanne). (A) Overall
study design and the schedules for immunization and bleeding. (n = 3/group) (B) Representative ELISPOT data of MERS S protein-specific IgM- (blue) and IgG-
secreting cells (red) of three individuals were analyzed at 0, 2 weeks after the first immunization as the mean spot number ± SD per 1 × 106 cells. (C) MERS-CoV-
specific NAb levels of immunized macaques were determined using a plaque reduction neutralization test 2, 8, and 24 weeks after the first immunization. The
plasma was diluted two-fold. The plaque reduction is 80%. *p < 0.05, ** p < 0.01 vs. G1; #p < 0.05, ## vs. G3.
mice. The adjuvants were injected with MERS S protein and alum
(Figure 5A). Two weeks after immunization with the various
combinations, the levels of IgG1 (indicating a Th2 response)
against the MERS S protein were not significantly different
between the alum and ssRNA-based adjuvant groups (G3 to G7
in Figure 5B). However, ssRNA-based adjuvants (G4 to G7 in
Figure 5B) induced higher IgG2c levels (indicating a Th1
response) than did antigen protein only or combined with alum
(G2 and G3 in Figure 5B). In particular, the CA-O-formulated
ssRNA-based adjuvant (G7) induced the highest IgG2c level
(Figure 5B). Because CA-O itself did not show any adjuvant effect
in hPBMCs (Figure S3) and mice (data not shown), this dramatic
increase in IgG2c suggested a strong Th1 response caused by
the CA-O-formulated ssRNA-based adjuvant. Although the CA-
O-formulated ssRNA-based adjuvant in the absence of alum
(Figure S4A) also effectively induced both IgG1 and IgG2c and
MERS S-specific NAb 2 weeks after the single immunization
(Figure S4B, S4C), we added alum to the CA-O-formulated
ssRNA-based adjuvant to synegistically induce Th1 and Th2
responses.
To investigate the protective effects of the MERS S protein
formulated with the ssRNA-based adjuvant, alum, and the RNA
stabilizers CA-O and LP, we tested various combinations (Figure
5A) and immunized hDPP4-transgenic mice, a surrogate mouse
model for MERS challenge.[52] Because the plaque reduction
neutralization test (PRNT) is the gold standard to represent
vaccine protection efficacy,[53,54] we measured NAb titer by a 2-
fold serum dilution, which showed the protection % compared to
the negative control (only virus infection). G7 showed the highest
MERS S-specific NAb levels among all groups (Figure 5C). At
three weeks after immunization, all immunized mice were
challenged with MERS virus (5,000 plaque-forming units
(PFU)/mouse). In G7, we did not observe a decrease in weight,
and survival was 100%, compared to ∼90% in G5 and G6, and
7
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10.1002/anie.202002979
Angewandte Chemie International Edition
RESEARCH ARTICLE
~70% in G3 and G4 (Figure 5D). Interestingly, the protective
efficacy was higher in females than in males in G4–G6, and only
in G7, males were 100% protected (Figure 5E). These results
indicated that sex-based immunological differences result in
variation in susceptibility against infectious agents and response
to vaccines.[53] These data suggested that the CA-O-formulated
ssRNA-based adjuvant induced the most efficient protective
immunity, and that a single vaccination with an appropriate
combination of a ssRNA-based adjuvant, CA-O, alum, and
antigen is sufficient to protect against viral infection.
priming immunization. Moreover, upon administration of a MERS
vaccine together with alum, CA-O, and the ssRNA-based
adjuvant, MERS S-specific NAb production was synergistically
increased, and most notably, even after single immunization, the
MERS-specific NAb level was sufficiently high to ensure 100%
protection, whereas mice immunized with alum without the CA-O-
formulated ssRNA-based adjuvant showed only partial protection
after MERS-CoV challenge. We obtained similar results in
immunized macaques, in which the CA-O-formulated ssRNA-
based adjuvant with the MERS S protein and alum induced a high
humoral immune response against MERS S protein. The CA-O-
formulated ssRNA-based adjuvant showed similar results
regardless of the kind of vaccine used (data not shown). Based
on the promising humoral immune response induction, using
Zn/DPA-derived effective stabilizers of ssRNA-based vaccine
adjuvants, we have been continuously optimizing the formulation
conditions and structures of CAs to develop vaccine systems with
ideal APC activation and balanced Th1/Th2 responses. Our data
indicate that a single immunization with the abovementioned
combination, including CA-O as an RNA stabilizer, is suitable to
protect against outbreak and spread of hazardous pathogens.
This vaccine formulated platform may be applied to develop a
SARS-CoV2 vaccine to prevent COVID-19.
Immunization effect of the CA-O-formulated ssRNA-based
adjuvant in nonhuman primates
To evaluate the immunization effect of the ssRNA-based adjuvant
in nonhuman primates, we immunized macaques with the MERS
S protein with or without alum, the ssRNA-based adjuvant, and
CA-O through the intramuscular route, administering two doses
at 4 and 23 weeks after the initial dose (week 0) (Figure 6A).
Specific IgG-secreting cells, representing protein-specific
antibody-secreting cells (ASCs), were quantified by an enzyme-
linked immunospot (ELISPOT) assay 2 weeks after the first
immunization. G5 showed the highest number of ASCs (68 ±
47.9/million cells), whereas relatively low numbers were observed
in G2 (27 ± 24.2/million cells). A very weak protein-specific IgM-
secreting cell response was observed in all groups (Figure 6B). In
addition, MERS S-specific NAb levels in G2, G4, and G5 were
higher than those in G1 and G3 at 2 and 8 weeks after the first
immunization (Figure 6C). Furthermore, the NAb levels at 8
weeks, i.e., 4 weeks after the second immunization, were
increased compared to those at 2 weeks in all groups (Figure 6C),
indicating that the second immunization induced a boosting effect.
In contrast to mouse NAb results (Figure 5C), which showed that
CA-O-formulated ssRNA-based adjuvant induced the highest
NAb levels (in G7, Figure 5A), in macaques, the NAb levels in G5
were similar to those in G2 and G4 (Figure 6C), probably because
the amount of immunized MERS S protein (100 µg/macaque) was
sufficient to induce neutralizing immunity and masked differences
between the groups. To analyze the memory effect of immune
cells, we administered a second “boost” dose 23 weeks after the
initial immunization (Figure 6A). Comparing the NAb levels at 24
weeks, those of G4 and G5, in which ssRNAs were added as
adjuvant, were higher than those of G2 with alum alone. The Nab
level in G5, in which CA-O was added as a stabilizer for ssRNAs,
was slightly increased compared to that in G4 (Figure 6A). These
results demonstrated that the CA-O-formulated ssRNA-based
adjuvant has immune-stimulating effects, especially in the
induction of a humoral immune response via the activation of
immune memory effects in nonhuman primates.
Acknowledgements
J.-H. Nam received grants from the Korean Health Technology
R&D Project through the Korea Health Industry Development
Institute (KHIDI), funded by the Ministry of Health & Welfare,
Republic of Korea (HI15C2955); the Basic Science Research
Program through the National Research Foundation (NRF)
funded by the Ministry of Science, ICT & Future Planning (NRF-
2015M3A9B5030157); and the Catholic University of Korea,
Research Fund, 2019. G. Keum was supported by the KIST
institutional project (2E30180). E.-K. Bang received a grant from
the Basic Science Research Program through the NRF of Korea
(2014R1A6A3A04059719). M. Song received a grant from the
Ministry of Health & Welfare, Republic of Korea (HI15C2971). J.J.
Hong received grants from the Korea Research Institute of
Bioscience and Biotechnology Research Initiative Program (KGM
4571922) and KHIDI, funded by the Ministry of Health & Welfare,
Republic of Korea (BGC1691811).
Keywords: adjuvant • single-stranded RNA • stabilizer •
coordinative amphiphile • antigen-presenting cell
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RESEARCH ARTICLE
Entry for the Table of Contents
An ssRNA adjuvant formulated with coordinative amphiphiles, bearing a zinc/dipicolyamine complex moiety (Zn/DPA) as a
coordinative phosphate binder, promoted effective recognition and antigen-presenting cell activation.
10
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