Background
In general, vaccination materials consist of a specific antigen (Ag) and an adjuvant capable of potentiating the immunogenicity of the Ag to achieve efficient Ag-specific adaptive immunity [
1]. Vaccines made up of live attenuated and/or killed whole pathogens usually contain endogenous adjuvants, such as bacterial cell wall components, genomic nucleic acids, and various pathogen-derived materials, that act as pathogen-associated molecular patterns and are sufficient to induce Ag-specific adaptive immunity by potentiating immunogenicity through the activation of innate immunity [
2,
3]. However, subunit vaccines that utilize recombinant and/or purified Ags usually lack these endogenous innate immune stimulators. Consequently, the addition of exogenous materials with adjuvant activity is required to mimic natural infection to draw effective pathogenic Ag-specific adaptive immunity [
4].
The innate immune response includes the production of interferons (IFNs), complements, and antimicrobial peptides (AMPs) and is crucial for controlling infectious diseases and inducing adaptive immunity [
5]. AMPs have been proposed as multifunctional peptides that participate in the elimination of pathogenic microorganisms, including bacteria, fungi, and viruses [
6]. In particular, defensins, one of the major AMP families in mammals, contribute to the antimicrobial innate immune response by disrupting the cell membranes of pathogens [
7]. Six α-defensins and 31 β-defensins are expressed in humans. Human β-defensins (HBDs), unlike α-defensins, are produced in a wide variety of epithelial tissues, including skin and mucosa, and cells, including phagocytic cells and mucosal epithelial cells [
7]. The expression of defense genes is tightly regulated by cytokines as part of the host defense and is suppressed by various virulent factors of pathogens [
6]. It is important to note that AMPs were recently reported to modulate adaptive immunity by triggering the recruitment and activation of immune cells via various pathways linked with innate immunity [
8]. For example, HBDs are chemotactic for immature dendritic cells (DCs) and memory T cells to the site of pathogen invasion by interacting with CCR6 and promote the adaptive immune response by recruiting immune cells [
9,
10]. However, their antiviral and immune-modulatory functions against viral infection have not been clearly elucidated in crucial innate immune cells, such as neutrophils and macrophages.
Here we investigated the use of AMPs as an adjuvant to stimulate the induction of not only antiviral innate immunity but also Ag-specific adaptive immune responses using HBD 2 and the receptor-binding domain (RBD) of Middle East respiratory syndrome-coronavirus (MERS-CoV) spike (S) protein (S RBD) as a model Ag. We assessed the immune-modulatory activity of HBD 2 in macrophage-like THP-1 cells to determine the active participation of HBD 2 in innate immunity against virus infection. In addition, we confirmed the adjuvant activity of HBD 2 in vivo by determining the level of Ag-specific immune response induction after the administration of HBD 2-conjugated S RBD.
Methods
Experimental animals and materials
Six- to eight-week-old female C57BL/6 mice were purchased from the Koatech Laboratory Animal Center (Pyeongtaek, Korea) and housed under specific pathogen-free conditions with water and food provided ad libitum. Animal experiments were approved by the Institutional Animal Care and Use Committee of Chonbuk National University (Approval No. CBNU 2017–0055) and followed the guidelines suggested by the committee.
THP-1 (ATCC® TIB-202™) and Vero E6 (ATCC® CRL-1586™) cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Huh-7 cells (KCLB No. 60104) were obtained from the Korean Cell Line Bank (Seoul, Korea). MERS-CoV (1–001-MER-IS-2015001) was obtained from the Korean Center for Disease Control and Prevention (KCDC). All experiments using MERS-CoV were performed in accordance with the World Health Organization’s recommendations under biosafety level 3 conditions in a biosafety level 3 facility in the Korea Zoonosis Research Institute at Chonbuk National University. Unless otherwise specified, the chemicals and laboratory wares used in this study were obtained from Sigma Chemical Co. (St. Louis, MO, USA) and SPL Life Sciences (Pocheon, Korea), respectively.
Recombinant protein production and cell culture
Gene recombination, expression, and the purification of recombinant MERS-CoV S RBD with or without HBD 2 at the C terminus of the RBD (residues 291–725) of the S1 domain were performed as described previously with minor modifications [
11]. Briefly, the gene encoding S RBD was synthesized with codon optimization based on the MERS-CoV S protein sequence (GenBank: AKL59401.1; GenScript, Piscataway, NJ, USA). The S RBD gene with the HBD 2 gene at its 3′ terminus was amplified by polymerase chain reaction (PCR) using the forward primer (5′-
GAG CTC AAG TAT TAT TCT ATC ATT CCT-3′, where the underlined letters represent the SacI restriction site) with the HBD 2 gene together with the reverse primer (5′-
TCT AGA TCA TGG CTT TTT GCA GCA TTT TGT TCC AGG GAG ACC ACA GGT GCC AAT TTG TTT ATA CCT TCT AGG GCA AAA GAC TGG ATG ACA TAT GGC TCC ACT CTT AAG GCA GGT AAC AGG ATC GCC TAT ACC CTC TAC GAA CAA AGA GGA-3′, where the underlined and italicized letters represent the XbaI restriction site and the HBD 2 sequence, respectively). Amplified genes were cloned into the pColdII
Escherichia coli expression vector (TaKaRa Bio, Shiga, Japan). Recombinant proteins were purified by Ni-NTA Superflow (Qiagen, Valencia, CA, USA) for proteins with an N-terminal His tag according to the manufacturer’s instructions. Any residual endotoxin contamination was filtered out using a Sartobind Q75 membrane chromatography system (Sartorius, Goettingen, Germany), such that the final endotoxin content of recombinant proteins was below 0.5 EU per μg proteins, determined using an LAL chromogenic endotoxin quantification kit (Thermo-Fischer Scientific, Rockford, IL, USA).
THP-1 cells were cultured in RPMI medium (Welgene, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) at 37 °C in a humidified CO
2 incubator. THP-1 cells were treated with phorbol-12-myristate-13-acetate (1 μg/mL for 1 × 10
6 cells) for 2–3 days to differentiate into monocyte-derived macrophage cells [
12]. The cells were replenished with fresh media and maintained for 3 days and then treated with recombinant protein (1 μg/mL per 1 × 10
6 cells). The cells were harvested 6 h and 24 h after recombinant protein treatment and subjected to quantitative real-time PCR (qRT-PCR) to assess the expression levels of the target genes. At the same time, cell culture supernatants were collected and subjected to expression profiling for cytokine and chemokine proteins, which are related to innate immunity, using a LEGENDplex human pro-inflammatory chemokine and Type I/II/III interferon panel (BioLegend, San Diego, CA, USA), according to the manufacturer’s protocol.
RNA extraction and qRT-PCR
We performed RNA extraction using TRIzol® reagent (Thermo-Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. We converted prepared RNA into cDNA using an MMLV Reverse Transcription Kit (Promega, Fitchburg, WI, USA). We quantified gene expression via qRT-PCR using the QuantiTect SYBR Green PCR Kit (Qiagen, Hilden, Germany) with an ABI 7500 system (Applied Biosystems, Foster City, CA, USA) using 50 ng first-strand cDNA under the following conditions: 95 °C for 5 min followed by 40 amplification cycles at 95 °C for 15 s, 55 °C for 30 s, and 72 °C for 30 s. We normalized the expression level of each gene to that of β-actin (hACTB) via a relative quantification method using 7500 FAST software version 2.0.6 (Applied Biosystems). The gene-specific primer sets used to amplify each gene are listed in Table
1.
Table 1
Primer sequences used for qRT-PCR to measure the transcript levels of specific genes
hACTB | F: 5′-CCA ACC GCG AGA AGA TGA-3′ |
R: 5′-TCC ATC ACG ATG CCA GTG-3′ |
CXCL-1 | F: 5′-ATT CAC CCC AAG AAC ATC CA-3′ |
R: 5′-TGG ATT TGT CAC TGT TCA GCA-3′ |
CXCL-10 | F: 5′-AGT GGA TGT TCT GAC CCT GCT TCA-3′ |
R: 5′-TGG GCC CCT TGG GAG GAT GG-3′ |
IFN-β | F: 5′-TTT CAG TGT CAG AAG CTC CT-3′ |
R: 5′-TGG CCT TCA GGT AAT GCA GA-3′ |
IFN-γ | F: 5′-CCA ACG CAA AGC AAT ACA TGA-3′ |
R: 5′-CCT TTT TCG CTT CCC TGT TTT-3′ |
IL-1β | F: 5′-CCT GTC CTG CGT GTT GAA AGA-3′ |
R: 5′-GGG AAC TGG GCA GAC TCA AA-3′ |
IL-6 | F: 5′-TGG CTG AAA AAG ATG GAT GCT-3′ |
R: 5′-TCT GCA CAG CTC TGG CTT GT-3′ |
MCP-1 | F: 5′-ACT GAA GCT CGC ACT CTC-3′ |
R: 5′-CTT GGG TTG TGG AGT GAG-3′ |
MIP-1α | F: 5′-CAG CAG ACA GTG GTC AGT CC-3′ |
R: 5′-TTC TGA GCA GGT GAC GGA AT-3′ |
MxA | F: 5′-CTG TGG CCA TAC TGC CAG GA-3′ |
R: 5′-ACT CCT GAC AGT GCC TCC AA-3′ |
NOD2 | F: 5′-CGG CGT TCC TCA GGA AGT AC-3′ |
R: 5′-ACC CCG GGC TCA TGA TG-3′ |
Protein kinase R | F: 5′-CAG GCA CGA CAA GCA TAG AA-3′ |
R: 5′-CTA CTC CCT GCT TCT GAC GG-3′ |
RANTES | F: 5′-CCT CAT TGC TAC TGC CCT CT-3′ |
R: 5′-GGT GTG GTG TCC GAG GAA TAT-3′ |
RNase L | F: 5′-GCA GAA ATG CCT TGA TCC AT-3′ |
R: 5′-AGT CTT CAG CAG GAG GGT GA-3′ |
TNF-α | F: 5′-GGA GAA GGG TGA CCG ACT CA-3′ |
R: 5′-CTG CCC AGA CTC GGC AA-3′ |
upE | F: 5′-GCC TCT ACA CGG GAC CCA TA-3′ |
R: 5′-GCA ACG CGC GAT TCA GTT-3′ |
Vero E6 β-actin | F: 5′-ATC GTG CGT GAC ATT AAG GAG-3′ |
R: 5′-AGG AAG GAA GGC TGG AAG AG-3′ |
Immunization of mice and sample collection
C57BL/6 mice were immunized subcutaneously at the base of the tail and intramuscularly in the hind leg with 10 μg/mouse of each recombinant protein dissolved in 50 μL phosphate-buffered saline (PBS) emulsified with an equal volume of Freund’s complete adjuvant and boosted once with the same immunogen emulsified with Freund’s incomplete adjuvant 10 days after the first immunization. Control mice were immunized with the inoculum prepared identically but with PBS only. Sera were collected 3 days after boost immunization to assess the MERS-CoV S RBD-specific antibody (Ab) response.
Enzyme-linked immunosorbent assay (ELISA)
The level of the MERS-CoV S RBD-specific Ab in mouse sera was determined by ELISA. Briefly, a 96-well ELISA plate (Thermo-Fisher Scientific) was precoated with S RBD protein (2 μg/mL) overnight at 4 °C and blocked with 5% nonfat dried milk at 37 °C for 2 h. After the addition of serially diluted sera to each well, the plate was incubated at 37 °C for 1 h, followed by four washes with PBS containing Tween 20. Bound Abs were incubated with alkaline phosphate-conjugated anti-mouse IgG at 37 °C for 1 h and the reaction was visualized with the addition of p-nitrophenyl phosphate substrate. We measured color development using reading the absorbance at 405 nm on an ELISA plate reader (SPECTROstar Nano, BMG Labtech, Ortenberg, Germany).
Virus neutralization assay
Vero E6 cells were used to propagate MERS-CoV and were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Welgene) supplemented with 10% FBS (Gibco) at 37 °C in a humidified CO2 incubator. MERS-CoV was passed six times in Vero E6 cells and used to assess the neutralizing potential of each recombinant protein. Briefly, sera obtained from mice immunized with each recombinant protein were diluted 50-fold and incubated for 1 h at room temperature with 1 μg S RBD protein before being transferred to Huh-7 cells grown in confocal dishes. For the immunofluorescence assay, Huh-7 cell monolayers were fixed with 4% paraformaldehyde and treated with a premixture of sera and S RBD protein. Penta-His Ab coupled with Alexa Fluor® 488 (Qiagen) was used as a secondary Ab. Cells were covered with SlowFade Gold Antifade Reagent (Invitrogen, Grand Island, NY, USA), and slides were observed under a confocal laser scanning microscope (CLSM, LSM 510, Carl Zeiss, Thornwood, NY, USA). Additionally, a receptor binding inhibition assay was performed with MERS-CoV-susceptible Vero E6 cells as described above, and median fluorescence intensity was measured using a CytoFLEX flow cytometer (Beckman-Coulter, Indianapolis, IN, USA).
We assessed the neutralizing capacity of each individual serum sample by determining the viral loads in MERS-CoV-infected cells by quantifying the level of upstream E (upE) gene expression using qRT-PCR [
13,
14]. Briefly, 100-fold diluted mouse immune sera were incubated for 1 h at room temperature with 10
3 plaque-forming units (PFUs) of MERS-CoV and transferred to Vero E6 cells (1 × 10
6 cells/well) grown in a 6-well tissue culture plate. After 24 h of incubation, we extracted total RNA and performed qRT-PCR as described previously using the primer set listed in Table
1 to measure the level of MERS-CoV upE gene expression.
Statistical analyses
Statistical analyses were performed using Prism 7 (GraphPad, San Diego, CA, USA). Data are presented as means ± standard deviations (SDs). The statistical significance of numerical data was analyzed using two-way analysis of variance (ANOVA), and p < 0.05 was considered statistically significant.
Discussion
Human defensins are effector peptides produced by various cell types and have broad antibacterial, antiviral, and antifungal activity [
20]. Defensins are small cysteine-rich cationic and amphipathic proteins. Their antiviral and antibacterial activity was originally attributed to their lipid perturbation activity because the disruption of viral glycoprotein function by membrane lipid perturbation leads to the inhibition of receptor binding and fusion of the enveloped virus to host cells. However, the observation that several classes of non-enveloped viruses are also sensitive to defensins led to the discovery of additional antiviral mechanisms of defensins [
7]. Although additional mechanisms include receptor downregulation and the disruption of early events in viral infection, a universal mechanism for the neutralization of non-enveloped viruses remains elusive. In addition to their antibacterial and antiviral activity, defensins are one of the strongest central and peripheral defenders against pathogen infection, particularly in linking innate and adaptive immunity against pathogen infection through leukocytes, such as DCs and T cells [
9]. In addition, a potent adjuvant is needed to enhance the immunogenicity and protective efficacy of subunit vaccines against pathogens [
21,
22]. We thus investigated the adjuvant effects of defensins on antiviral innate and pathogenic Ag-specific adaptive immune responses using HBD 2 to analyze its potential use in the development of a subunit vaccine with self-adjuvant activity.
HBD 2-conjugated Ag treatment of macrophage-like THP-1 cells enhanced the transcript levels of antiviral molecules, including IFN-β, IFN-γ, MxA, PKR, RNaseL, and NOD2, and primary immune response-regulating cytokines and chemokines, although the induction level varied (Figs.
1,
2 and
3). IFNs are divided into type I and type II IFNs. Like IFN-γ, a type II IFN, type I IFNs, such as IFN-β, are induced by viral infection and confer antiviral activity to the host [
23,
24]. IFNs exert their antiviral activity by enhancing the expression of numerous genes with antiviral activity, including MxA, a cytoplasmic GTPase that inhibits the replication of several RNA viruses [
25,
26]. Double-stranded RNA-dependent protein kinase PKR and endogenous ribonuclease RNaseL are also antiviral effector proteins [
25]. In contrast to MxA, the activation of PKR requires its binding to double-stranded RNA, and the activation of PKR leads to translational arrest via the phosphorylation of an essential translation initiation factor. This in turn activates the endogenous ribonuclease RNase L, which subsequently cleaves single-stranded RNAs, including mRNAs of viral and host cell origin [
25].
We found that NOD2 expression was activated by HBD 2-conjugated Ag treatment of THP-1 cells (Fig.
3c). NOD2 was recently identified as a bacterial receptor that contributes to crosstalk between innate and adaptive immune systems in the digestive tract [
27,
28]. NOD2 also functions as a cytoplasmic viral pattern recognition receptor by triggering the activation of IFN-regulatory factor 3 (IRF3) and the production of IFN-β [
29]. NOD2 is expressed in immune cells, such as monocytes/macrophages, T cells, granulocytes, and DCs, as well as in colon epithelial cells [
30,
31] and is required for the expression of a subgroup of intestinal antimicrobial peptides, such as cryptdins [
28]. It is interesting that NOD2-deficient mice are susceptible to pathogen infection via the oral route but not intravenous or peritoneal routes. These reports indicate that NOD2 is essential for activating adaptive immunity by acting as an adjuvant receptor for Ab production, either directly or by enhancing the production of defensins [
32,
33] or other immunostimulatory molecules. In addition, β-defensins have been reported to function as chemoattractants for various antigen-presenting cells (APCs), including immature DCs and macrophages, through chemokine receptors CCR6 and CCR2 [
9,
34,
35]. Besides recruiting APCs to the inflammation site, β-defensins modulate the adaptive immune response by activating signal transduction through pattern recognition receptors for APC maturation, leading to the production of Th1-polarized cytokines and other important immunomodulatory factors. β-defensins also modulate the adaptive immune response by self-destructive signaling to eliminate activated APCs and prevent harmful effects of long-activated DCs and macrophages [
36,
37]. Therefore, some β-defensins may counter suppressive pathogen-derived factors by generating robust host primary immune responses, thereby inducing an efficient protective immune response. Collectively, we presume that HBD 2 is able to stimulate not only antiviral innate immune responses, but also specific adaptive immune responses through enhanced delivery of fused antigens to APCs in vivo. Consequently, we assessed the ability of HBD 2 to promote Ag-specific immune response induction using the HBD 2-conjugated S RBD of MERS-CoV.
To confirm the adjuvant ability of HBD 2, we measured the inducing level of Ag-specific IgG after immunization with S RBD alone or with the HBD 2-conjugate and performed receptor-binding and viral inhibition assays to detect and quantify serum-neutralizing Abs to MERS-CoV using a viral receptor-expressing host cell-based assay. As expected, similar to the results of Ag-specific Ab within the respective immune sera, higher virus-neutralized activity was observed in sera obtained from HBD 2-conjugated S RBD-immunized mice compared to mice treated with S RBD alone or PBS only (Fig.
5). We also noted that the magnitude of the inhibitory effects of sera from HBD 2-conjugated Ag-immunized mice was generally higher, similar to the pattern of enhanced induction of antiviral innate immunity in HBD 2-conjugated Ag-treated THP-1 cells.