Vaccine 37 (2019) 5191–5202

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Vaccinejournal homepage: www.elsevier.com/locate/vaccine

Cricket paralysis virus internal ribosome entry site-derived RNA
promotes conventional vaccine efficacy by enhancing a balanced
Th1/Th2 response
Hye Won Kwak a,1, Hyo-Jung Park a,1, Hae Li Ko a,1, Hyelim Park a,1, Min Ho Cha b,1, Sang-Myeong Lee c,1,
Kyung Won Kang c,1, Rhoon-Ho Kim a, Seung Rok Ryu c, Hye-Jung Kim a, Jae-Ouk Kim d, Manki Song d,
Hun Kim e, Dae Gwin Jeong f, Eui-Cheol Shin g, Jae-Hwan Nam a,⇑a

Department of Biotechnology, The Catholic University of Korea, Bucheon, Republic of Korea
KM Application Center, Korea Institute of Oriental Medicine, Daegu, Republic of Korea
c
Division of Biotechnology, The Chonbuk National University, Iksan, Republic of Korea
dClinical Research Lab, International Vaccine Institute, Seoul National University Research Park, Seoul, Republic of Korea
eLife Science Research Institute, SK Bioscience, Seongnam, Republic of Korea
f
Infectious Diseases Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea
g
Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Koreaba r t i c l e

i n f oArticle history:
Received 1 May 2019
Received in revised form 21 June 2019
Accepted 18 July 2019
Available online 29 July 2019
Keywords:
MERS
HPV
CrPV
IRES
RNA
Adjuvant
VaccineTh1/Th2Aluma b s t r a c t
An ideal adjuvant should increase vaccine efficacy through balanced Th1/Th2 responses and be safe touse. Recombinant protein-based vaccines are usually formulated with aluminum (alum)-based adjuvants
to ensure an adequate immune response. However, use of alum triggers a Th2-biased immune induction,and hence is not optimal. Although the adjuvanticity of RNA has been reported, a systematic and overall
investigation on its efficacy is lacking. We found that single strand RNA (termed RNA adjuvant) derived
from cricket paralysis virus intergenic region internal ribosome entry site induced the expression of various adjuvant-function-related genes, such as type 1 and 2 interferon (IFN) and toll-like receptor (TLR), T
cell activation, and leukocyte chemotaxis in human peripheral blood mononuclear cells; furthermore, its
innate and IFN transcriptome profile patterns were similar to those of a live-attenuated yellow fever vaccine. This suggests that protein-based vaccines formulated using RNA adjuvant function as liveattenuated vaccines. Application of the RNA adjuvant in mouse enhanced the efficacy of Middle East respiratory syndrome spike protein, a protein-subunit vaccine and human papillomavirus L1 protein, avirus-like particle vaccine, by activating innate immune response through TLR7 and enhancing pAPC
chemotaxis, leading to a balanced Th1/Th2 responses. Moreover, the combination of alum and the RNA
adjuvant synergistically induced humoral and cellular immune responses and endowed long-term immunity. Therefore, RNA adjuvants have broad applicability and can be used with all conventional vaccines toimprove vaccine efficacy qualitatively and quantitively.
Ó 2019 Elsevier Ltd. All rights reserved.

1. Introduction
Abbreviations: APCs, Antigen presenting cells; DCs, dendritic cells; Th2, T helper
2; TLR, Toll-like receptor; IFN, interferon; ssRNAs, single-stranded RNAs; IL,interleukin; Tfh, follicular helper T; RIG, retinoic acid-inducible gene; IRESs,
internal ribosome entry sites; CrPV, Cricket paralysis virus; IGR, intergenic region;
MERS, Middle East respiratory syndrome; CoV, coronavirus; HPV, human papillomavirus; dLN, draining lymph node; NAbs, neutralizing antibodies; VLP, virus-likeparticle; Tg, transgenic; WT, wild-type; ELISA, enzyme-linked immunosorbent
assay; PRNT, plaque-reduction neutralization test; PFUs, plaque-forming units;
ELISPOT, enzyme-linked immunospot; hPBMCs, human peripheral blood mononuclear cells; TNF, tumor necrosis factor; Myd88, myeloid differentiation primary
response 88.
⇑ Corresponding author at: Department of Biotechnology, The Catholic Universityof Korea, 43-1, Bucheon, Gyeonggi-do 14662, Republic of Korea.
E-mail address: jhnam@catholic.ac.kr (J.-H. Nam).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.vaccine.2019.07.070
0264-410X/Ó 2019 Elsevier Ltd. All rights reserved.

Although inactivated and protein-based vaccines have long
been used to prevent the spread of various pathogens [1] successfully, some of them elicit a weak immune response, short immune
duration, and have several safety issues [2,3]. Therefore, research
has focused on identifying antigen components responsible for
immunogenicity in order to generate safer and more effective vaccines. However, purified recombinant antigens generally showpoor immunogenicity [2]. To overcome this, immunostimulatory
agents (i.e., adjuvants) are added to vaccines to induce robust
immune responses against vaccine antigens [4–6].

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Aluminum hydroxide (alum) is widely used as an adjuvant inseveral human vaccines [7]. It has been reported that alum injection leads to the rapid accumulation of neutrophils and other cells
forming lumps composed of neutrophil extracellular traps. Thisfurther induces lysosome-mediated lysis to distort the plasma
membrane of antigen presenting cells (APCs) and/or rapidly
induces the synthesis cytokines/chemokines, finally activating
the NALP3 inflammasome and triggering the secretion of IL-1b.
This process might contribute to the adjuvant effect of alum. However, further confirmation is required [8]. In addition, alum induces
a profound T helper 2 (Th2) response along with antibody production. However, alum cannot induce Th1 responses and sometimes
stimulates autoimmunity by upregulating IgE presentation [9].
Since classical adjuvants solely induce Th2 responses, new adjuvants capable of promoting Th1 responses are required to increase
the cell-mediated response [8]. Non-classical adjuvants, such as
Toll-like receptor (TLR) ligands, can stimulate the production oftype I interferon (IFN) and proinflammatory cytokines [10]. TypeI IFN facilitates antigen presentation to major histocompatibility
complex class I/II, leading to an enhanced cytotoxic CD8+ T cell
response, as well as a CD4+ T cell response [11].
Since its first use as an adjuvant with the inactivated Japanese
encephalitis virus (JEV) vaccine to efficiently promote neutralizing
antibodies (NAbs), IFN production, and resistance to JEV infection,
poly I:C and its derivates have been widely used in the development of various vaccines [12,13]. In addition, RNase-resistant poly
I:C stabilized with poly-L-lysine (poly ICLC) was shown to havestrong adjuvanticity by microbial mimicking for inducing innate
immune responses in humans through TLR3 and MDA5 [14].
Despite the strong adjuvanticity of poly I:C, there are concerns
about its induction of adverse and excessive immune responses,
such as autoimmune diseases and difficulty in standardization
for mass production [15]. In previous studies, poly I:C-induced
massive type I IFNs were shown to be critical for the pathogenesisof lupus [16]. Moreover, repeated administration of poly I:C derivate, poly ICLC, and carboxymethylcellulose, showed severe
pathogenicity and resulted in the development of tuberculosis inmice [17].
Single-stranded RNAs (ssRNAs) function as vaccine adjuvantsand promote Th1 responses through the induction of IFN-a andinterleukin (IL)-12 in DCs [18]. Sendai virus-derived defective
interfering RNA activates the innate immune response, leading toincreased efficacy of inactivated influenza vaccine [19]. Additionally, nucleoside-modified influenza mRNA vaccines showed adjuvant effects by inducing antigen-specific follicular helper T (Tfh)cells and germinal center B cell responses [20]. Recently, ssRNAformulated with carriers showed immune-enhancement effects
through TLR and retinoic acid-inducible gene (RIG)-like helicase
signaling pathways [21,22]. Although these studies demonstrated
that ssRNAs could function as adjuvants, systematic investigationsof their effects and applicability to protein-based or inactivatedvaccines are lacking, thereby preventing their widespread clinicaladoption.
We previously developed novel ssRNA-expression platforms
from several viral internal ribosome entry sites (IRESs) [23]. In thisstudy, we investigated the adjuvant effects of ssRNAs (RNA adjuvant) derived from the Cricket paralysis virus (CrPV) intergenic
region (IGR) IRES [23]. We formulated the RNA adjuvant withprotein-subunit vaccine for Middle East Respiratory Syndrome(MERS)-coronavirus (CoV) and virus-like particle vaccine forhuman papillomavirus (HPV) and showed that the RNA adjuvant
induced a balanced Th1/Th2 response and innate immunity. Moreover, as expected, the RNA adjuvant worked through TLR7 andrecruited APCs to increase antigen uptake into the injected site
and their retention in the draining lymph node (dLN). Furthermore,
we showed that the RNA adjuvant enhanced humoral and cellularimmune responses by promoting antigen-specific neutralizing
antibodies (NAbs) and the Th1 response and ultimately endowing
long-term immunity. The results demonstrated that an RNA adjuvant could be developed as a general, safe, and effective adjuvant
applicable to protein-based vaccine types.
2. Material and methods
2.1. Mice
For immunization with MERS S soluble protein vaccine and HPV
L1 virus-like particle (VLP) vaccine, female BALB/c and C57BL/6
mice were purchased from Dae-Han Bio-Link. Female TLR7 KO
mice were kindly provided by Dr. Bumseok Kim, Chonbuk National
University, and hDPP4 Tg mice, stably expressing hDPP4 as the
MERS-CoV receptor, were kindly provided by Dr. Sungkyun Park,
Korea Research Institute of Bioscience & Biotechnology. Mice werehoused at the Catholic University of Korea under specificpathogen-free conditions with 12-h light/dark cycle and handledaccording to protocols approved by the Catholic University of
Korea. The animal facility at the Catholic University of Korea isfully accredited by the Korean Association for Laboratory Animals.
All mice experimental procedures conducted in this study followed
the guidelines of the Institutional Animal Care and Use Committeeof the Catholic University of Korea (CUK-IACUC-2016-039, 2016046, 2017-028, 2018-027, and 2018-028).
For MERS-CoV challenge, hDPP4-transgenic (Tg) mice (5–7weeks old) were maintained in a biosafety level 3 facility at the
Korea Zoonosis Research Institute of Chonbuk National University
and followed the guidelines of the Institutional Animal Care and
Use Committee (2018-045).
2.2. Vaccines
Soluble MERS spike (S) protein vaccine was provided by theInternational Vaccine Institute (Seoul, Korea). This MERS S protein
expressed in insect cells has no transmembrane domain and comprises the first 1296 amino acids of MERS-CoV EMC/2012 strain(GenBank #AFS88936.1). VLPs containing HPV L1 (a mixture of
10 types of L1 – 6, 11, 16, 18, 31, 33, 35, 45, 52, and 58 L1) wereobtained from SK Bioscience (Seoul, Korea) and expressed in insect
cells. Due to patent issues, detailed information on the HPV vaccine
was unavailable.
2.3. In vitro transcription and RNA purification
The DNA platform was designed using the IGR IRES and SV40
late-polyadenylation signal sequences (Supplementary Fig. 1)[23]. DNA templates were linearized with NotI. In vitro transcription was performed using the EZ T7 high yield in vitro transcription
kit (Enzynomics, Daejeon, Korea) and HiScribe T7 Quick high yield
RNA synthesis kit (New England Biolabs, Ipswich, MA, USA). See
Supplementary Methods for more details.
2.4. Analysis of transcriptome in human PBMC
Human peripheral blood mononuclear cells (hPBMCs) obtained
from Zen bio were cultured in RPMI 1640 medium (Hyclone Laboratories Inc, South Logan, UT, USA) supplemented with 10% heatinactivated FBS (Life Technologies, Carlsbad, CA, USA), 2.05 mM
L-glutamine (Hyclone Laboratories Inc, South Logan, UT, USA),and 1% Pen-Strep Glutamine (Gibco, Waltham, MA, USA). Cells
were maintained in a humidified atmosphere at 37 °C with 5%
CO2. PBMCs were stimulated with 10 lg/ml of poly I:C (Sigma

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Aldrich, St. Louis, MO, USA) and 20 lg/ml of RNA adjuvant for 6 and24 h. See Supplementary Methods for more details.
2.5. Immunization
For MERS S protein vaccine studies, C57BL/6 WT and hDPP4-Tg
mice (6-weeks old) were inoculated intramuscularly into the upper
thigh twice a week at 2-week intervals with the following formulations: (1) 1 mg MERS S protein vaccine with/without 20 mg RNA
adjuvant or 500 mg alum (Thermo Fisher Scientific, Waltham, MA,
USA) for wild-type (WT) mice; and (2) 1 mg MERS S protein vaccine
with/without 20 mg RNA adjuvant or 24 mg alum (Brentanne, Frederikssund, Denmark) for hDPP4-Tg mice. For VLP-HPV-L1 vaccine
studies, BALB/c mice (6-weeks old) were inoculated by intramuscular injection into the upper thigh three times a week at 2-weekintervals with 6 mg VLP-HPV-L1 with/without 20 mg protamineformulated RNA adjuvant or 5 mg alum.

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stained with anti-CD3-BV510, anti-CD4-APCH7, and anti-CD8-PEcy7 (Clone 145-2C11, Clone GK1.5, and Clone 53-6.7) (BD Biosciences). The stained cells were permeabilized using
Cytofix/Cytoperm kit (eBioscience) and then stained with antiIFN-c-APC, anti-TNF-a-FITC, and anti-IL-2-PE (Clone XMG1.2, BD
Biosciences; Clone MP6-XT22, Invitrogen; Clone JES6-5H4, eBioscience). Cells were fixed with 1% paraformaldehyde, analyzed
using an LSRII flow cytometer (BD Biosciences), and T cells positive
for the various combinations of cytokines and degranulation wereanalyzed and quantified using a Boolean gating function in FlowJo(TreeStar).
2.10. In vivo imaging and image processingSerum from MERS-CoV infected hDPP4-Tg mice were seriallydiluted from 10- to 5120-fold with serum-free medium, and thevirus-serum mixture was prepared by mixing 100 plaqueforming units (PFUs) of MERS-CoV using the diluted serum samples and incubated at 37 °C for 1-h. HPV-specific NAb titration
was performed as described previously [24]. See Supplementary
Methods for more details.

A laser-scanning intravital confocal microscope (IVM-C, IVIM
Technology) was used to visualize dendritic cells and monocyte
recruitment. CX3CR1-GFP mice (Jackson Laboratory, Bar Harbor,
ME, USA, stock #005582) that endogenously express GFP in dendritic cells and monocytes were used. Mice were deeply anesthetized with intraperitoneal injections of zoletil (30 mg/kg) andrompun (10 mg/kg). During imaging, mouse body temperature
was maintained at 37 °C with a homeothermic controller (PhysioSuite; RightTemp; Kent Scientific). A mixture of Alexa Fluor 647(A20006, Thermo Fisher Scientific) and either RNA adjuvant or
PBS was subcutaneously injected into the mouse ear skin using a
31G microinjector (1700 series syringe, Hamilton). To label vasculatures, an anti-CD31 antibody (553369, BD Biosciences) conjugated with Alexa Flour 555 (A20009, Thermo Fisher Scientific)
was injected intravascularly 1 h before imaging. Using the distinctive vasculature and the fluorescence signal of Alexa Fluor 647 as alandmark for the repetitive imaging of the injected site, the recruitment of CX3CR1-GFP+ dendritic cells (irregular outlines) andmonocytes (round outlines) was imaged in vivo at 20 min and
24 h after the injection. The recruitment of dendritic cells andmonocytes was quantified by calculating the area occupied by
CX3CR1-GFP+ cells using ImageJ plugins (NIH).

2.8. Enzyme-linked immunospot (ELISPOT)

2.11. Multiplex cytokine assay

Splenocytes from immunized mice and GPs at the end of theexperiments were stimulated with 0.125–1 lg/well of antigens
for 48 h at 37 °C. ELISPOT for the detection of IFN-c-secreting T
cells was performed according to manufacturer instructions (Mabtech, Stockholm, Sweden).

After stimulating the human peripheral blood mononuclear
cells (hPBMCs) with the RNA adjuvant for 24 h, concentrations oftumor necrosis factor (TNF)-a, IFN-c, IL-2, IL-12, IL-6, and IL-10
were analyzed using the Magnetic Luminex screening assay kit(R&D Systems, Minneapolis, MN, USA) according to manufacturer
instructions.

2.6. Enzyme-linked immunosorbent assay (ELISA)
Antigen-specific IgG1, IgG2a, and IgG2c in mouse serum were
measured by ELISA. The 96-well plates (Corning, Corning, YN,
USA) were coated with 50 ng/well MERS S protein and 100 ng/well
VLP-HPV-L1 vaccine and incubated overnight at 4 °C. See Supplementary Methods for more details.
2.7. Plaque-reduction neutralization test (PRNT) for MERS-CoV and
HPV

2.9. Flow cytometry
2.12. Depletion of CD4+ and/or CD8+ T cells
For surface staining, splenocytes and isolated immune cells
from muscle and lymph nodes were stained with the following
antibodies for 15 min at room temperature; CD4 (Clone GK1.5,
eBioscience; Clone H129.19, Bio Legend), CD8 (Clone 53-6.7, BD
Biosciences; Clone 53-6.7, Invitrogen), CD69 (Clone H1.2F3, BD
Biosciences), CD44 (Clone IM7, Invitrogen), CD62L (Clone MEL 14,
BD Biosciences), CD11b (Clone M1/70, Bio Legend), F4/80 (Clone
BM8, Invitrogen), CD86 (Clone GL1, BD Biosciences), and CD11c(Clone N48, eBioscience). Cells were fixed with 1% paraformaldehyde, analyzed using a FACS Canto II flow cytometer (BD Biosciences), and the data were analyzed using FlowJo (TreeStar).
For determining polyfunctional T cells, isolated splenocytes werere-stimulated with 1 lg/well MERS spike protein or 100 ng/wellof the peptide mixture. To evaluate the cytotoxic degranulation
activity of T cells, anti-CD107a-BV421 (Clone 1D4B, BD Biosciences) was added into the culture medium. Brefeldin A (GolgiPlug, BD Biosciences) and monensin (GolgiStop, BD Biosciences)were added 2 h later. After another 10 h incubation, splenocytes
were first stained with ethidium monoazide (Sigma) and then

To examine the effects on T cells, C57BL/6 mice (6-weeks old)
were inoculated by intramuscular injection twice at 6-day intervals with 1 mg MERS S protein vaccine with 20 mg RNA adjuvant,followed by inoculation by intraperitoneal injection with 10 mg/
kg anti-mouse CD4 (YTS 191; BioXcell, West Lebanon, NH, USA)
and/or 10 mg/kg anti-mouse CD8 (YTS 169.4; BioXcell) every 3days after the first immunization.
2.13. Statistical analyses
One-way analysis of variance was used to assess significant differences among treatment groups. For each significant treatment
effect, the Tukey HSD test was used to compare multiple groupmeans. In addition, all histomorphometrical values are expressed
as means ± standard deviation (SD). Multiple comparison tests forthe different treatment groups were conducted. If the Levene test
indicated no significant deviations from variance homogeneity,
the data obtained were analyzed by a least-significant differences

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Fig. 1. The RNA adjuvant induces transcriptional profiles similar to those induced by poly(I:C) in hPBMCs. hPBMCs were stimulated with RNA adjuvant or poly(I:C) for 6-h
and 24-h. Nil indicates PBS treatment. (A) Th1- and Th2-related cytokine production in the hPBMC supernatant was measured by multiplex analyses at 24-h post-stimulation.
The data were statistically analyzed by ANOVA. The significance of differences between groups is indicated by different letters of the alphabet. p < 0.05. Data are presented asmean ± standard deviation (n = 3). (B) Principal component analysis plots for all samples (three samples per group at 6- and 24-h post-treatment) (left) and scatter plots(right) of RNA-seq data (fold ratio  0.7). Nil indicates PBS-treated hPBMCs. (C) Heat maps showing clustering based on the signaling pathways associated with type 1 and 2
IFNs, TLR-Myd88), T cell activation, and leukocyte chemotaxis. (D) Simulated dynamics of the transcriptional network representing differentially expressed genes (DEGs).
Interaction of proteins involved in the TLR signaling pathway, T cell activation, and proteins interacting with STAT1 and STAT3 (each square indicates a transcriptional factor,
and each circle indicates a gene affected by a transcriptional factor). Red nodes are upregulated, and blue nodes are downregulated DEGs (fold-change ratio > 1). DEGs:
differentially expressed genes, TLR: Toll-like receptor, Myd-88: myeloid differentiation primary response 88, Th: T helper cell, PBS: phosphate-buffered saline, hPBMC: humanperipheral blood mononuclear cells, STAT: signal transducer and activator of transcription. (For interpretation of the references to color in this figure legend, the reader isreferred to the web version of this article.)

H.W. Kwak et al. / Vaccine 37 (2019) 5191–5202

multi-comparison test. If significant deviations from variancehomogeneity were detected using the Levene test, the nonparametric Kruskal–Wallis H-test was conducted. When a significant difference was detected by the Kruskal–Wallis H-test, a
Mann–Whitney U-test was conducted as a post hoc analysis. Statistical analyses were conducted using SPSS for Windows (Release
14.0K, SPSS Inc.). To assess significant differences between two
groups, Student’s t-test was used. Differences were considered significant at P < 0.05.
3. Results
3.1. The RNA adjuvant affects genes related to the innate immune
responses, T cell activation, and chemotaxis similar to poly(I:C) in
hPBMCsTo investigate the adjuvant effect, we treated hPBMCs with the
RNA adjuvant and used poly(I:C) as a positive control, finding that
the RNA adjuvant induced Th1- and Th2-related cytokine
release similar to poly(I:C) (Fig. 1A). We then compared the transcriptome profiles of RNA adjuvant- and poly(I:C)-treated hPBMCs
by RNA sequencing. Control, poly(I:C), and RNA adjuvant treatment

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formed a separate cluster without any bias (Fig. 1B), with scatterplots of gene profiling confirming that changes in geneexpression between the RNA adjuvant and poly(I:C) groups were
not greater than those between the control and RNA adjuvantgroups at 6- and 24-h post-treatment (p  0.05; fold-change  2)
(Fig. 1B).
To investigate the biological pathways associated with genes
affected by the RNA adjuvant, we analyzed gene-expression patterns using the Kyoto Encyclopedia of Genes and Genomes database (https://www.genome.jp/kegg/mapper.html) (Fig. 1C) andthe network of genes involved in each pathway based on the
STRING database (Fig. 1D). After 6-h, similar biological pathways
were affected by RNA adjuvant and poly(I:C) treatment (Supplementary Fig. 2), and heat maps of innate-immune-related genes[type 1 and 2 IFNs and TLR-myeloid differentiation primary
response 88 (Myd88)], as well as T cell activation and leukocyte
chemotaxis, indicated similar expression patterns between thetwo groups (Fig. 1C). TLR2, TLR3, TLR7, IRF7, and NF-jB among the
TLR related genes were induced, and IL-6 and IL-15 among T cell
activation related genes and STAT 1 and 3 among multiple transcriptional factors function as key players in the RNA adjuvantgroup (Fig. 1D).

Fig. 2. The RNA adjuvant mimics live-attenuated vaccine YF17D in transcription profiles associated with the innate immune response. Transcription profiles in response to
live-attenuated vaccine YF17D in hPBMCs isolated from an immunized patient 7-days after vaccination (fold-change  2; p < 0.05; n = 30) and in response to the RNA
adjuvant from hPBMCs at 6-h post-treatment. Induction and reduction profiles of genes related to innate immune responses (A) and type 1 IFN (B) from the RNA adjuvant(red) and YF17D (blue). (C) Comparison of IRF7 and STAT1 induction between RNA adjuvant (red) and YF17D (blue) treatment. Fold-change (log2) in expression in response
treatment indicating the change in expression of each gene at 6-h post-treatment with the RNA adjuvant as compared PBS (RNA/Nil) and at 7-days post-immunization with
YF17D as compared with that before immunization (YF17D/Nil). PBS: phosphate-buffered saline, hPBMC: human peripheral blood mononuclear cells, STAT: signal transducer
and activator of transcription, IRF: interferon regulatory factor. (For interpretation of the references to color in this figure legend, the reader is referred to the web version ofthis article.)5196

H.W. Kwak et al. / Vaccine 37 (2019) 5191–5202

3.2. The RNA adjuvant induces a transcriptional profile similar to thatby a live-attenuated vaccine

3.3. The RNA adjuvant recruits APCs to the injection site and increases
their retention in the dLN

Yellow fever vaccine (YF17D) is the most successful liveattenuated vaccine, as its duration of protective immunity after
single immunization is >35 years [25]. We compared the transcription profile induced in RNA adjuvant-treated hPBMCs (6-h posttreatment) with that of PBMCs obtained from a YF17Dimmunized patient (at 7-days post-immunization) [26]. Approximately 80% of the innate immune response and 100% of type I
IFN-related genes, which are key factors contributing to a superior
vaccine [14,27], showed similar changes in expression pattern
between the two samples (Fig. 2A and B). The inductions of IRF7
and STAT1 are important upstream immune transcription factors
targeting different effectors of the immune response to the
YF17D vaccine [26]. Interestingly, increases in IRF7 and STAT1
levels in response to the RNA adjuvant were the same as those in
response to YF17D (Fig. 2C), indicating that the RNA adjuvant
affected important transcription factors that play key roles in
YF17D-vaccine efficacy.

The RNA adjuvant increased APC retention in the dLN to promote selection of antigen-specific T and B cells by epitopepresenting APCs in a process similar to that observed following
treatment with poly(I:C) (Fig. 3A). Furthermore, in vivo timelapse imaging clearly showed that the RNA adjuvant summoned
immune cells, mostly DCs (irregular outlines), to the injection site
(Fig. 3B and C), which was consistent with the heat map describing
upregulated leukocyte chemotaxis (Fig. 1C) and indicating that the
RNA adjuvant enhanced antigen uptake by DCs at injection sites
and promoted epitope presentation in the dLN.
3.4. The RNA adjuvant activates the Th1 response, synergisticallyincreases the level of NAbs, and protects against MERS-CoV challenge
To confirm adjuvant effects in vivo, we formulated it with the
MERS spike (S) protein and used it to immunize WT C57BL/6 mice
with/without alum (Fig. 4A). At 2-weeks after first immunization,

Fig. 3. The RNA adjuvant recruits DCs and macrophages to the injection site and increases their retention in the dLN at 24 h post-injection. BALB/c mice were intramuscularly
injected into the quadriceps muscle, and CX3CR1-GFP mice were subcutaneously injected into the ear skin with RNA adjuvant or poly(I:C). (A) Activated DCs (CD11c+CD11b+CD86+) and macrophages (CD11b+F4/80+Cd86+) in dLNs were counted by flow cytometry. The data were statistically analyzed by ANOVA. The significance ofdifferences between groups is indicated by different letters of the alphabet. p < 0.05. Data represent the mean ± standard deviation (n = 5/group). (B) Infiltration of CX3CR1+
cells (green; white arrows) by PBS and RNA adjuvant was monitored by in vivo imaging. Red color indicates Alexa Fluor 647 at injection sites, which spread to the surrounding
space. (C) The frequency of DC infiltration into mouse ear skin. Data represent the mean ± standard deviation (n = 10 image sites). Data were statistically analyzed byStudent’s t-test. DC: dendritic cell, PBS: phosphate-buffered saline, dLN: draining lymph node. (For interpretation of the references to color in this figure legend, the reader isreferred to the web version of this article.)

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Fig. 4. RNA adjuvant formulated with MERS S soluble protein vaccine induces Th1 response, as well as an increased number of NAbs. C57BL/6 mice were intramuscularly
immunized at 2-week intervals with two doses of MERS S soluble protein vaccine with/without RNA adjuvant or alum. (A) Overall study design. (B) MERS S protein-specific
IgG1 and IgG2c levels measured by ELISA at 2- and 4-weeks after the first immunization. Data represent the mean ± standard deviation (n = 10 mice). MERS-CoV-specific NAblevels of immunized mice were determined by PRNT at 2 weeks after the second immunization. Serum from the same group was pooled (n = 10) and diluted 2-fold. (C)Splenocytes were harvested at 2 weeks after second immunization and re-stimulated for 2 days with/without the MERS S protein. The frequency of MERS S-specific IFN-cproducing cells determined by ELISPOT. Data represent the mean ± standard deviation (n = 3) (triplicate results acquired in one pooled sample per group). The data were
statistically analyzed by ANOVA for (B) and (C). The significance of differences between groups is indicated by different letters of the alphabet. p < 0.05. (D) Frequencies of IFNc, IL-2, and TNF-a-producing polyfunctional CD4 + T cells assessed by flow cytometry. Data represent the mean ± standard deviation (n = 3). *p < 0.05. Data were statisticallyanalyzed by Student’s t-test for (D). ELISPOT: enzyme-linked immunospot, S: spike, IFN: interferon, ELISA: enzyme-linked immunosorbent assay, PRNT: plaque-reduction
neutralization test, MERS: Middle East Respiratory Virus, CoV: coronavirus, NAb: neutralizing antibody.

G5 and G4 showed the highest IgG1 level (indicating a Th2
response). After boosting (at 2 weeks after second immunization),G2 to G5 showed similar IgG1 levels. Conversely, IgG2c (indicatinga Th1 response) was only induced in G4 and G5 (Fig. 4B), suggesting that the RNA adjuvant induced a Th1 response. Moreover, G5
showed the highest NAb level (indicating a strong Th2 response),and G3 and G4 showed similar levels (Fig. 4B). Interestingly, G5
and G4 showed higher numbers of IFN-c secreting cells following
stimulation with the MERS S protein as compared with G2 andG3 (Fig. 4C), indicating that the RNA adjuvant induced MERS S
protein-specific Th1 responses. Moreover, we observed elevationsin the number of polyfunctional CD4+ T cells in G5 (Fig. 4D). These5198

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results suggested that the RNA adjuvant promoted CD4+ T cell
responses, especially Th1 responses, consequently inducing
antigen-specific cellular immune responses. Furthermore, administering alum along with the RNA adjuvant generated a synergistic
effect, leading to an increase in NAb levels by stimulating balanced
Th1/Th2 responses.
To investigate the protective effect of immunization with the
MERS S protein formulated with the RNA adjuvant plus alum
(Fig. 5A), we immunized hDPP4-Tg mice, a surrogate mouse model
for MERS challenge [28]. Although the body weights of G2 and G3
were immediately reduced (but quickly recovered) after challenge(Fig. 5B), we observed 100% survival (Fig. 5C), possibly due to a sufficient NAb level derived from boosting immunization, although
G2 showed lower NAb levels relative to G3 in hDPP4-Tg mice (Supplementary Fig. 3).

3.5. The RNA adjuvant endows long-term immunity in VLP vaccine
To investigate the long-term immune effect of the RNA adjuvant, we immunized mice with a 10-value HPV vaccine as a VLP
vaccine, an RNA adjuvant-containing 16 HPV-L1 gene (RNAadjuvant-HPV; Supplementary Fig. 1), protamine (used as a condensing component of RNA-cationic peptide complex [29]), andalum (Fig. 6A). At 2-weeks after the first immunization, the IgG1
levels of G2 and G3 were similar, whereas the IgG2a level of G3
was higher than that of G2 (Fig. 6B). After three doses (7–27 weeks
after the first immunization), G2 and G3 showed similar IgG1 andIgG2a levels (Fig. 6B); however, G3 showed higher NAb levels
against 11, 16, 18, 35, 45, 52, and 58 HPVs, similar levels against
6 and 33 HPVs, and lower levels against 31 HPVs than G2(Fig. 6C). Furthermore, the frequency of IFN-c-secreting cells
among splenocytes from G3 after stimulation with 16, 18, or all
HPV-L1 proteins was significantly higher than that of G2, suggesting that G3 maintained an active HPV-L1-specific Th1 response,even at 27-weeks post-immunization (Fig. 6D). This indicated that
the RNA adjuvant-HPV helped maintain long-lasting humoral andcellular immune responses.

3.6. The RNA adjuvant-induced Th1 response is dependent upon both
CD4+ and CD8+ T cells and TLR7
Our results indicated that the RNA adjuvant induced T cell
activation and expression of TLR7-related genes, as well as a balanced Th1/Th2 response. To investigate this process, Tlr7-KO andWT mice were immunized with the MERS S protein, RNA adjuvant, and alum. As expected, IgG1 levels of in the KO mice werelower than those of WT mice at 2-weeks after first immunization (Fig. 7A). However, IgG1 was increased, while IgG2c wasnot, in KO mice at 2-weeks after second immunization(Fig. 7A), suggesting that the RNA adjuvant might have partiallyovercome the loss of TLR7 for Th2 induction but not Th1 via
boosting. Mouse embryonic fibroblasts treated with the RNA
adjuvant or a positive control (R848 as TLR7/8 agonist andpoly I:C) showed the induction of Myd88, RIG-I, melanoma
differentiation-associated protein 5, phosphorylated interferon
regulatory factor 3, and TANK-binding kinase 1/NF-jB activating
kinase, which are the downstream molecules of TLR7 and RIG-Ilike receptor pathways [30] (Supplementary Fig. 4), suggesting
that, while Th1 activation by RNA adjuvant may be criticallydependent on the TLR7 pathway, Th2 activation by RNA adjuvant may be induced by multiple mechanisms that bypass the
TLR7 signaling pathway, such as the pathway involving RIG-Ilike receptor, which is one of other pattern recognition receptors(PRRs).
To investigate the necessity of CD4+, CD8+, or both T cells toactivate the Th1 response, these were respectively depleted in
the mouse spleen (Fig. 7B and Supplementary Fig. 5). CD4depleted mice (G3) showed significant reductions in IgG1 andIgG2c levels, whereas CD8-depleted mice (G4) showed no reduction in IgG1 levels but had reduced IgG2c levels. Moreover, micelacking both CD4 and CD8 (G5) showed reductions in both IgG1
and IgG2c levels (Fig. 7C), indicating that IgG1 was dependent
upon CD4+ T cell activation alone, whereas IgG2c was dependent
on both CD4+ and CD8+ T cell activation. Moreover, IFN-csecreting cells (representing the Th1 response) after treatment
with mixed CD8+ T cell-epitope peptides [31] were not induced

Fig. 5. Immunization with RNA adjuvant-formulated MERS S soluble protein vaccine and/or alum protects against MERS-CoV challenge. hDPP4-Tg mice were intramuscularly
immunized with MERS S soluble protein vaccine with/without RNA adjuvant and/or alum. (A) Overall study design for hDPP4-Tg mice. (B) Percentage body weight andsurvival of hDPP4-Tg mice after challenge with 5  103 PFU of MERS-CoV/mouse at 3 weeks after second immunization (n = 4–5 mice). (C) Survival of hDPP4-Tg mice after
challenge with MERS-CoV. (survival # / total #). Tg: transgenic, MERS: Middle East Respiratory Virus, CoV: coronavirus, Nab: neutralizing antibody, S: spike, PFU: plaqueforming unit.

H.W. Kwak et al. / Vaccine 37 (2019) 5191–5202

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Fig. 6. RNA adjuvant-HPV-formulated VLP vaccine induces antigen-specific long-term immunity. BALB/c mice were intramuscularly immunized at 2-week intervals withthree doses of 10-value VLP-HPV-L1 with alum and with/without protamine-formulated RNA adjuvant-HPV. (A) Overall study design. (B) HPV-L1-specific IgG1 and IgG2alevels were measured by ELISA from 2-weeks to 27-weeks after the first immunization. Data represent the mean ± standard deviation (n = 5 mice). (C) HPV-L1 NAb levels
against different types of VLPs in the immunized mice were determined by pseudovirus PRNT at 27 weeks after the first immunization. Serum from the same group (n = 5)
was pooled. (D) Splenocytes were harvested at 27 weeks after the first immunization and re-stimulated for 2 days with/without type 16 or 18 HPV-L1 protein or 10 types of
VLP-HPV-L1. The frequency of HPV-specific IFN-c-producing cells was determined by ELISPOT. Data represent the mean ± standard deviation (n = 3) (triplicate results
acquired from one pooled sample per group). The data were statistically analyzed by ANOVA for (B) and (D). The significance of differences between groups is indicated bydifferent letters of the alphabet. p < 0.05. ELISPOT: enzyme-linked immunospot, IFN: interferon, VLP: virus-like particle, HPV: human papillomavirus, ELISA: enzyme-linked
immunosorbent assay, PRNT: plaque-reduction neutralization test, NAb: neutralizing antibody.

in G4 and G5 (Fig. 7D). These data showed that the RNA adjuvant
enhanced an antigen-specific Th1 response via the TLR7 signaling
pathway and CD4+/CD8+ T cell activation.
4. Discussion
In this study, we demonstrated the adjuvant effect of CrPV-IGR
IRES-derived ssRNA [23] using protein-based vaccines. The RNA
adjuvant induced the expression of genes related to the innate
immune response (IFN and TLR-Myd88), T cell activation, andleukocyte chemotaxis similar to poly(I:C) in hPBMCs; however,
the induction levels were lower than those by poly(I:C), and mostgenes returned to normal levels within 24 h (Fig. 1). This transientinduction of genes by the RNA adjuvant may be evidence of vaccine
safety. Similarly, a previous report demonstrated the rapid disappearance of the RNA platform within 24 h after injection [23].
We did not observe any adverse effects, such as autoimmune
symptoms, changes in liver function, or organ damage, in the
RNA adjuvant-immunized mice, even at a higher dose (200 mg/-mouse) (data not shown). Although poly(I:C) can be a superior
adjuvant, safety concerns, such as an excessive immune response,
represents a significant drawback [15]. However, our RNA adjuvant
appeared to overcome this safety issue without decreasing theimmune-stimulation effect.
Among the key factors induced in RNA adjuvant-treated
hPBMCs, intercellular adhesion molecule-1, IL-6, IL-15, CD80, andSTAT1/3 were induced (Fig. 1). In particular, IL-6, which is highly
expressed in CD4+ T cells, is partially responsible for Th2 cytokine
production in Th1-differentiated cells via protein kinase C theta
and NF-jB signaling pathways [32]. Additionally, IL-15 stimulates
T and natural killer cell immunity and promotes the generation of
long-lived memory T cells [33]. Furthermore, STAT1 functions as acentral regulator of innate and adaptive immunity, and STAT3 isessential for DC, Tfh cell, and memory CD8+ T cell development[34,35]. Given that the RNA adjuvant activated the Th1 response
and induced long-term immunity (Figs. 4–6), induction of thesegenes might have played an important role in this immune
response.

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Fig. 7. The RNA adjuvant induces a Th1 response through TLR7 and CD4+/CD8+ T cells. Tlr7-KO mice and C57BL/6 (WT) mice were intramuscularly immunized at 2-weekintervals with two doses of 1 mg MERS S soluble protein vaccine, 20 mg formulated RNA adjuvant or alum. (A) MERS S protein-specific IgG1 and IgG2c levels were measured by
ELISA at 2 weeks after the first and second immunizations. Data represent the mean ± standard deviation (n = 3–5 mice). (B) Overall study design and mouse schedule for
immunization and anti-CD4 or CD8 treatment. (C) MERS S protein-specific IgG1 and IgG2c levels were measured by ELISA at 2 weeks after the second immunization. Data
represent the mean ± standard deviation (n = 3–5 mice). (D) The frequency of MERS S protein-specific IFN-c-producing cells was determined by ELISPOT after treatment witha CD8+ T cell-epitope peptide mixture (7 peptides for C57BL/6, as described previously [30]) or MERS S protein. Data represent the mean ± standard deviation (n = 3)
(triplicate results acquired from one pooled sample per group). The data were statistically analyzed by ANOVA for (A), (C), and (D). The significance of differences between
groups is indicated by different letters of the alphabet. p < 0.05. Th: T helper cell, ELISPOT: enzyme-linked immunospot, S: spike, IFN: interferon, ELISA: enzyme-linked
immunosorbent assay, PRNT: plaque-reduction neutralization test, MERS: Middle East Respiratory Virus, TLR: Toll-like receptor, KO: knockout, WT: wild-type.

Interestingly, we found that the expression of genes associatedwith the innate immune response, especially type 1 IFN-related
genes, was similar between the RNA adjuvant-treated hPBMCs
and PBMCs from a live-attenuated YF17D-immunized patient(Fig. 2). Moreover, it is also important that IRF7 expression was
induced in RNA adjuvant-treated hPBMCs similar to that observed
following immunization with YF17D because IRF7 is an essential
molecule for antigen-specific IFN production promoting T and B
cell activation [36]. These data assumed that the RNA adjuvant
may make inactivated and protein-based vaccines function like alive attenuated vaccine. However, further studies are required toconfirm this hypothesis.
Furthermore, Tlr7-KO mice and CD4+/CD8+ T cell-depleted mice
did not show any RNA adjuvant effects (Fig. 7), consistent with previous results [22,37,38]. These data indicated that this RNA adjuvant might be efficacious for inducing a broad immune response
mediated through TLR7 signaling and T cell activation. However,the PRR for the RNA adjuvant cannot be confined to TLR7 alone,as the RNA adjuvant could induce RIG-I-like receptor pathwayrelated molecules similar to R848 and poly I:C (Supplementary
Fig. 4). This indicated that the RNA adjuvant is recognized by
RIG-I-like receptor in addition to TLR7 to activate an immune
response. These data are accordance with a previous study, whichshowed that RNA based adjuvants triggered TLR- and RIG-I-likehelicase-dependent effect [22]. Therefore, the boosting with RNA
adjuvant may partially induce an IgG1 (Th2) response in Tlr7-KO
mice through the RIG-I-like pathway. However, we do not know
why this pathway cannot induce IgG2c (Th1). Further studies are
required to understand the detailed underlying mechanism.
In addition, although the RNA adjuvant mediated its effects
through the TLR7 signaling pathway, we did not use TLR agonists,such as imiquimod (TLR7 agonist) and resiquimod (TLR7/8 agonist), which are chemical adjuvants [39], as a controls in this study,as this study focused on RNA-based adjuvants, such as poly I:C, but
not chemical agonist-adjuvants. Poly I:C is a dsRNA that shows
strong adjuvanticity in inactivated and protein-based vaccines[12–14]; therefore, it was used as a control in this study.
Upon immunization with the MERS S protein vaccine together
with alum and the RNA adjuvant, MERS S-specific NAb production
increased synergistically (Fig. 4B). Our data suggest that T cell activation of G5 may also show a synergistic increase, however, moredata are needed to confirm this (Fig. 4D). Additionally, given thestrong immune-stimulating effect of the RNA adjuvant, it was
unsurprising that the RNA adjuvant-HPV group was able to maintain HPV-specific NAb levels and the Th1 response, even at 27weeks after first immunization (Fig. 6). These results indicated that
the RNA adjuvant triggered long-term immunity, including
humoral and cellular immune responses. This is an important fea-

H.W. Kwak et al. / Vaccine 37 (2019) 5191–5202

ture, given that HPV should have a long-term protective effect after
vaccination.
Here, we used one RNA molecule derived from the CrPV-IGR
IRES in the absence of target genes as the adjuvant and demonstrated its adjuvanticity for the MERS S protein vaccine. The RNA
adjuvant-containing target gene HPV-L1 also showed an
immune-stimulating effect. Therefore, although we previously
showed that an RNA platform could express a coding gene [23],RNA adjuvant including target gene is not critical to ensure adjuvanticity. However, should this RNA platform be used as an RNA
vaccine, it needs to encode target genes. Further, improvementsin the delivery strategy to increase the expression efficiency inthe inoculated animal muscle are warranted. Interestingly, we
compared the adjuvanticity of CrPV-IGR IRES-derived RNA withthose of other RNA virus-derived adjuvants, including coxsackievirus B3 (CVB3) and encephalomyocarditis virus (EMCV) IRESsderived RNAs. CrPV and CVB3 IRES-derived RNAs showed higher
adjuvant effects compared to that of the EMCV IRES-derived RNA(data not shown). Studies are underway on the effect of the viral
IRES type on the immune effect.
5. Conclusion
Use of the RNA adjuvant as an immune stimulator, regardless ofthe presence of antigen-encoding genes, has many advantages,
including its applicability for protein-based vaccines, simple manufacturing procedures, and easy standardization. Moreover, it canpotentially overcome challenges associated with protein-subunit
vaccines, such as weak T cell activation. The associated immune
responses consequently promote high NAb levels and balanced
Th1 and Th2 responses, resulting in long-term immunity. Therefore, since the RNA adjuvant exhibited immune-enhancing capacity, it might be valuable not only as a first-line vaccine in an
epidemic emergency but also to improve protein-based vaccine
efficacy.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgments
The authors thank Drs Jung Joo Hong and Hanseul Oh for technicalassistance, Hae-Yung Ahn for assistance with animal care, and Dr.
Jin-Seul Kim for assistance with HPV vaccine.
Financial support
J.-H. Nam was supported by the Korean Health Technology R&D
Project through the Korea Health Industry Development Institute(KHIDI) funded by the Ministry of Health & Welfare, Republic ofKorea (HI15C2955), the Basic Science Research Program throughthe NRF funded by the Ministry of Science, ICT & Future Planning(NRF-2015M3A9B5030157), and the Catholic University of Korea,
Research Fund 2018. M. Song was supported by the Ministry of
Health & Welfare, Republic of Korea (HI15C2971).
Author contributions
J.-H. Nam and H.-J. Park designed all studies. J.-H. Nam and S.M. Lee analyzed the results. S.-M. Lee, K.W. Kang, and S.R. Ryu measured MERS NAb levels and challenged with MERS-CoV. H.W.
Kwak, H.-J. Park, H.L. Ko, H.-J. Kim, and R.-H. Kim did the MERS,

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and the HPV related experiments. M.H. Cha and H.W. Kwak analyzed the RNA-seq data. J.O. Kim, M. Song, D.G. Jeong, and H. Kim
supplied the vaccines, the vaccine candidates, and hDPP4-Tg mice.
E.-C. Shin did the polyfunctional T cell assay. J.-H. Nam and H. Park
wrote the manuscript with help from the coauthors.
Conflicts of interest
There are no conflicts of interests to declare.
Appendix A. Supplementary material
Supplementary data to this article can be found online athttps://doi.org/10.1016/j.vaccine.2019.07.070.
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