1. Introduction
ND is a novel nanomaterial which belongs to the carbon family with excellent biocompatibility and optical properties [
1]. They are very small and versatile in surface or lattice features [
2]. ND has emerged as an exciting nanomaterial in recent years in many fields of research due to its unique characteristics [
3,
4,
5,
6]. Prior characterization has demonstrated NDs superior mechanical and physical characteristics, as well as its large surface area, biocompatibility, and tuneable surface structures [
7]. These characteristics make ND well-suited for biomedical applications.
NDs can be produced by several methods, namely chemical vapor deposition, HPHT synthesis, and detonation [
3,
5]. The different production methods generated various types of ND with distinct surface properties, structure, size, and shape [
8]. NDs generated by HPHT presses has broad size range (usually 30–100 nm), and are monocrystalline. These HPHT-NDs might be completely purified, and be surface oxidized by strong acid washes and air oxidation. In contrast, NDs are produced by the shock wave resulting in DNDs with narrow particle size distribution of 4–5 nm [
9].
NDs can be applied in vaccinology owing to the fact that bacteria and viruses have micro- and nano-sizes [
10]. They have the potential to act as an adjuvant to deliver antigens or a type of novel vaccine carrier system that can be recognized by the immune system [
11]. Based on the intrinsic hydrophobicity of strong acid-oxidized NDs, their surface can conjugate with native membrane protein, as well as soluble proteins [
4]. The remarkably high affinity of NDs and recombinant proteins against viruses, as well as the effect of NDs, have been demonstrated in several reports [
12,
13,
14]. In our previous study of Pham and colleagues, the nanoconjugate containing the viral protein HA/H7N9 attached on the surface of oxidized NDs with the size in the range from ~50 nm to ~500 nm in diameter. This resulted in an artificial virus-like particle vaccine suspension, which was subsequently tested in vitro (hemagglutination assay) and in vivo in a mouse model [
14].
The highly pathogenic avian influenza (A/H5N1) virus is emerging as a leading candidate for a worldwide disease outbreak despite of its speedy proliferation through avian hosts and the ability to directly transmit infection to humans [
15]. Haemagglutinin (HA) is the major surface protein of the A/H5N1 virus. HA contains virus-neutralizing epitopes and, therefore, it is the main antigen for vaccine development against A/H5N1 [
16]. Generation of a universal vaccine that induces broad immunity response and broad protective against several A/H5N1 virus strains from different clades is attracting goal for researchers. In a previous study carried out by Thi Pham and colleagues (2019), a novel artificial HA sequence (H5.c2) generated by a method, namely, computationally optimized broadly reactive antigen (COBRA) H5.c2 sequence was selected as a representative of the subclades 2.3.2.1, 2.3.2.1a, 2.3.2.1b, and 2.3.2.1c of A/H5N1 strains from 2005 to 2015 in Vietnam. The H5.c2 protein was successfully expressed in
Nicotiana benthamiana via agroinfiltration [
17]. However, the immunogenicity of the H5.c2 protein was not evaluated due to its low expression level.
To follow up on our previous initial work, in the current study, the effects of size and surface properties of NDs on the immunogenicity of the H5.c2 protein in mice were assessed. The plant based-purified H5.c2 protein was conjugated with different ratios of NDs in different sizes and surface properties. The optimized H5.c2:NDs were selected in vitro by hemagglutination assay, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis, and the changes in size and zeta-potential of the particles before and after protein coating. The optimal mixture of H5.c2:NDs was then validated in vivo in mice. Finally, the H5.c2-specific-antibody and neutralizing antibody responses elicited in mice vaccinated with the optimal H5.c2:NDs conjugates, free H5.c2 and NDs were evaluated by ELISA, Western blot, and hemagglutination inhibition assay.
2. Materials and Methods
2.1. Nanodiamond Preparation
Production of surface oxidized nanodiamonds (ND40 and ND100): The crude nanodiamond purchased from FNDbiotech (Taiwan) was first surface-functionalized with oxygen containing groups under microwave heating (~100 °C) in a mixture of H
2SO
4 and HNO
3 with the volume ratio of 3:1 (~100 °C). This harsh treatment was based on the protocol reported in a former publication [
4]. A microwave reactor (100 W, Model Discover, CEM, Cridersville, OH, USA) was used to heat the material for 2 h. Subsequently, the remaining acids were diluted carefully and the NDs then were collected. The microwave reactor was put in a chemical fume hood to shield the operator from NO
2 contamination during working process. Next, NaOH 1N was added into the mixture. ND was separated from the mixture by 15,000×
g centrifugation for 2 h. NDs were then resuspended with deionized water (DI-H
2O). The ND100 fraction was collected by centrifugation of the suspension at 10,000×
g for 5 min, and the remaining suspension was centrifuged at 15,000×
g for 2 h to collect ND40. The obtained NDs are washed with DI-H
2O twice and resuspended again in DI-H
2O for use.
Preparation of ND4: The aqueous DND colloids (NanoAmando) were provided from New Metals and Chemicals (Tokyo, Japan). NanoAmando was used as obtained, containing scattered DND particles dissolved in water (size: 3.7 nm in diameter, concentration: 5% DND) after optimization of the deagglomeration. The ND4 was characterized with positive surface charge and Zeta potential of +37 mV [
9].
2.2. Production of Recombinant the H5.c2 Antigen
In the previous study, Thi Pham and colleagues (2019) designed a novel artificial H5 sequence based on selection for the most common, conserved, consensus, or recent epitope after comparing all of the HA sequences of A/H5N1 subclade 2.3.2.1, 2.3.2.1a, 2.3.2.1b, and 2.3.2.1c recorded in Vietnamese poultry farm from 2005 to 2015 [
17]. The amino acid sequence of H5.c2 protein was provided in the
Supplementary 1. Recombinant H5.c2 protein fused with GCN4pII at its C-terminator was expressed transiently in
N. benthamiana, purified via immobilized affinity chromatography (IMAC) and size exclusion chromatography (SEC) according to the previous protocols described by Thi Pham and colleagues [
17]. The purity of the H5.c2 protein after purification was over 95%, as shown by SDS-PAGE (
Supplementary 2). The purified H5.c2 protein was stored at –20 °C before use. The physical characterization of H5.c2 protein was predicted by using the Bachem’s peptide calculator (
https://www.bachem.com/knowledge-center/peptide-calculator/, accessed on 20 May 2021).
2.3. Conjugation of H5.c2 Antigen onto the Surface of Nanodiamonds
H5.c2:ND conjugates were synthesized by the physical adsorption method. Various H5.c2:ND conjugate (w/w) of 1:1, 1:3, 1:6, 1:12, 1:24, and 1:48 were diluted in Phosphate Buffer Saline buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4, PBS). During coating, sonication was applied for 30 min. Centrifugation at 13,000× g for 5 min was used to collect the H5.c2:NDs conjugates. The non-binding complexes were then removed by washing twice in PBS buffer.
2.4. H5.c2:ND Conjugate Characterization
2.4.1. Hemagglutination Assay
Hemagglutination assays were carried out to evaluate the ability to agglutinate chicken red blood cells (RBC) of HA antigen. The hemagglutination assays were carried out according to the guideline of the World Organization for Animal Health [
18]. Briefly, all wells of a plastic V-bottom microtiter plate were introduced with 50 µL of PBS. Next, in the first well of plate, antigens (50 µL) were placed. Across the entire rows, a total 50 µL of two-fold serial dilution was performed. Following the introducing with 50 µL of 1% RBCs into each well of the microtiter plate, the incubation at 25 °C was conducted. After 30 min of incubation, the results were visualized. One hemagglutination unit was described as the endpoint dilution that results in complete hemagglutination (HAU).
2.4.2. Size and Zeta-Potential Measurements
To evaluation the change in size and Zeta potentials of NDs before and after coating with H5.c2 protein, a nanoparticle size analyzer (Delsa@NanoC, Beckman Coulter Inc., Fullerton, CA, USA) was utilized. Before measurement process, NDs and H5.c2:NDs were completely diluted to the concentration of 100 µg/mL in DI-H2O.
2.4.3. SDS-PAGE and Western Blot of H5.c2-NDs
The H5.c2 protein (100 ng/µL) was conjugated with ND4, ND40, and ND100 at different ratios (1:12, 1:3, 1:12, w/w), respectively. To remove non-binding products, the resulting mixtures were washed twice in PBS. The same volume of the H5.c2 protein (1.5 µg) and H5.c2:ND conjugates were diluted in SDS-sample buffer 1X (3% SDS, 1.5% DTT, 0.1% bromophenol blue, 0.5 M Tris-HCl pH 6.8, 10% glycerol), then separated by 4–10% SDS-PAGE gel. Casting and running of protein gels were performed via BioRad’s Miniprotean II (Laemmli systerm). Proteins on the gel were stained by Coomassie brilliant blue G-250 (Bio Basic Inc., Markham, ON, Canada).
For detection of protein by Western blots, proteins were transferred to PVDF membrane (Thermo Scientific). Proteins were then executed via the procedure reported by Gahrtz and Conrad [
19]. Briefly, the membrane was blocked with a PBS buffer containing 5% (
w/
v) fat-free milk for 2 h at room temperature. Next, the membrane was incubated for 2 h in PBS buffer including 5% (
w/
v) fat-free milk and monoclonal anti-c-myc antibody that was diluted 1:50 times. The membrane was washed three times with PBS buffer before incubation for 1 h at room temperature with the goat anti-mouse IgG secondary conjugated HRP at the dilution of 1:2000 times in 5% (
w/
v) fat-free milk dissolved in PBS buffer. Signals were visualized by incubation of membrane for 15 min in the dark with 3,3-diaminobenzidine (DAB, Thermo Scientific, Rockford, IL, USA) that was dissolved in 0.05 M Tris–HCl and 0.04% hydrogen peroxide. The intensity of H5.c2 band signals were analyzed by ImageJ software.
2.5. Mouse Experiment
The 6–8-week-old female BALB/C mice (five per group) were vaccinated with 2.5 µg of purified H5.c2 (group 1), a combination of 2.5 µg of purified H5.c2 and 7.5 µg of ND40 [1:3 (w/w), group 2], and another mixture of 2.5 µg of purified H5.c2 and 30 µg of ND100 [1:12 (w/w), group 3] on a schedule of 1, 14, 28 days via subcutaneous route. The mice of the negative control group were immunized with a mixture of PBS and 30 µg of NDs (group 4). In addition, to evaluate the effect of ND4 on the immunogenicity of H5.c2, a mixture of 2.5 µg of purified H5.c2 and 30 µg of ND4 [1:12 (w/w), group 5] was used for immunization in mice. In the first immunization, complete Freund’s adjuvant was used for the formulation of antigens. The incomplete Freund’s adjuvant was mixed with antigens in the second and the third immunizations. Seven days after the booster vaccination, mouse sera were obtained for ELISA tests, Western blot and hemagglutination inhibition assay.
2.6. ELISA
To evaluate H5.c2-specific-IgG antibody responses in mouse sera, 100 µL of 100 ng H5.c2 (purified by SEC) was applied in microtiter plates (ImmunoPlate Maxisorp, Nalgene Nunc International, Roskilde, Denmark), and the plates were incubated overnight at 4 °C. Next, the plates were blocked with PBS buffer containing 3% (w/v) BSA, 0.05% (v/v) Tween20 for 2 h. The plates were then added with 100 µL of PBS buffer including 1% (w/v) BSA, 0.05% (v/v) Tween 20 and the mouse sera with the dilution of 1:16,000, followed by plate incubations for 2 h at 25 °C. Each mouse serum was applied 3 times in the plate. A goat anti-mouse IgG conjugated horseradish peroxidase (HRP) diluted 2000 times in PBS buffer containing 1% (w/v) BSA, 0.05% (v/v) Tween 20 was applied in the plates for 1 h at 25 °C after washing the plates with PBS buffer containing 0.05% (v/v) Tween 20. The signals were detected by adding the 1-Step™ Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific, Vilnius, Lithuania). The plates were then incubated for 20 min before the addition of 1 M HCl to stop reaction. The microplate reader (Biorad Laboratories Inc., Hercules, CA, USA) was used for measurement of the absorbance signal at 450 nm. The values of all mouse sera in the ELISA plates were normalized after subtracting the background values for BSA.
2.7. Detection of H5.c2-Specific-IgG Antibody Responses by Western Blot
To detect the H5.c2-specific-IgG mouse antibodies, each 500 ng of H5.c2 protein (purified by SEC) was applied on a 4–10% SDS-PAGE gel. A semidry Quick Blotter was used to switch proteins to a PVDF membrane (Thermo Scientific, Rockford, IL, USA). The membrane was blocked with PBS buffer containing 5% (w/v) fat-free milk for 2 h at room temperature. The membrane was cut into five lanes that were then incubated with mouse serum mixture of each group at the dilution of 1:500 for 2 h. The membrane was washed three times for 15 min before incubation with goat anti-mouse IgG conjugated HRP (Thermo Fisher Scientific, Rockford, IL, USA) at the dilution of 1:2000 for 1 h. Before detection of specific signals, the membrane was washed three times for 15 min, then were incubated in the dark for 10 min with 0.05 M Tris-HCl buffer containing DAB (Thermo Scientific, Rockford, IL, USA) and 0.04% H2O2.
2.8. Hemagglutination Inhibition Assay
The hemagglutination inhibition (HI) assay was similarly performed using a standard procedure [
18]. The inactivated virus strain (IBT-RG02) was used for the HI assay. The IBT-RG02 strain was produced by reverse genetic technology using HA and neuraminidase (NA) proteins of the HPAI H5N1 (A/duck/Vietnam/HT2/2014(H5N1) of the clade 2.3.2.1c in the period 2012–2014 in Vietnam [
20]. The HA amino acid sequence similarities between the synthetic H5.c2 and native H5 of A/duck/Vietnam/HT2/2014(H5N1) strains is 99.4%. Firstly, all wells of a plastic V-bottom microtiter plate were placed 25 µL of PBS. Next, in the first well of plate, antigens (25 µL) were placed. The eight-well rows were then subjected to two-fold serial dilutions. A 25 µL of the last dilution was discarded. The 4 HAU of the inactivated IBT-RG02 strain (25 µL) was subsequently placed in each well. The reaction was incubated for 30 min at 25 °C. Finally, each well received 25 µL of 1% RBCs. The results were read after incubating for 30 min at 25 °C. The reciprocal of the highest dilution of serum that could fully inhibit hemagglutination was identified as the HI titer.
2.9. Statistical Analysis
The ELISA’s results were statistically analyzed by t-test of the Sigma Plot software (Chicago, IL, USA). The X ± standard deviation (SD) was shown for the mean difference of the sample’s data. The statistical differences were identified if p values < 0.05.
4. Discussion
A/H5N1 virus not only poses a serious danger to avian hosts, but also has the potential to directly infect humans [
15]. Due to viral antigenic drift, it is urgently needed to develop universal influenza vaccines that induce protective immunity for a long-term against distant influenza virus strains [
21]. Previous reports showed that the polyvalent VLP vaccine based on HA COBRA that represented different clades of A/H5N1 elicited a strong immunity response against different clades of A/H5N1 [
22,
23]. However, production of a native VLP vaccine based on native HA is time-consuming and complex. Nanoparticle displaying antigen-forming synthetic VLP is considered to be an alternative approach to conventional vaccines, since it supports antigen stabilization. Previous studies showed that a synthetic-VLP formed by conjugating proteins with nanoparticles enhanced immune response [
14,
24]. H5.c1 appeared for the A/H5N1 subclades 1.1, 1.1.1, and 1.1.2 induced strong immune responses against different A/H5N1 strains that belong to distinct clades [
17]. Based on previous results, in order to improve immune responses via enhanced antigen size, as in the case of synthetic-VLP formation, H5.c2 protein appeared for the HA protein of A/H5N1 subclades 2.3.2.1, 2.3.2.1.a, 2.3.2.1.b, 2.3.2.1.c was used to conjugate NDs of different surface properties (positive and negative) and sizes.
In the present study, the H5.c2:ND40 (1:3,
w/
w) and H5.c2:ND100 (1:12,
w/
w) conjugates agglutinated chicken RBCs at equal HA titers of 64, which are 16 times greater than that of free H5.c2 (
Figure 1,
Table 1). These data indicate a greater capacity for H5.c2 conjugation and the preservation of its antigenic properties and antigen bio-function with negatively charged ND particles. Our synthetic H5.c2:ND40 and H5.c2:ND100 exhibited the potential for agglutination RBCs at a minimum total protein amount of 0.04 μg which is 31.25 times lower than that of H5.c2 free protein of 1.25 μg. The differentiated HA titer is modulated mainly by charge and not size, raising questions about the mechanism of action in vitro experiments. Several reports have demonstrated that higher HA titers are obtained in lower HA protein amounts when the HA antigen size was increased via H5 oligomeric or H5-VLP formation [
14,
25,
26]. Thus, H5.c2 antigen size increased after coating negatively charged ND40 and ND100.
Moreover, we provided supporting evidence for the notion that charged-specific interactions can indeed mediate H5.c2 protein adsorption onto ND’s surface. Our data confirmed that the mixture of H5.c2 was strongly conjugated on the surface of negatively charged ND40 and ND100 but not with positively charged ND4. These results were almost identical to a previous study of Lin and co-workers. When compare the attachment of proteins (myoglobin, bovine serum albumin, and insulin) to these two types of NDs (DNDs and HPHT-NDs), the results showed that the HPHT-NDs (negative charged NDs) had strong protein-surface interaction with all three proteins [
9]. In contrast, DNDs had a tiny protein-surface interaction with the proteins [
9]. In addition, several previous studies indicated that oxidized HPHT-NDs are negative charged, can bind non-covalently but strongly with proteins through hydrogen bonding, the combination of electrostatic force and hydrophobic interactions [
12,
14].
To test the immunogenicity of the H5.c2 protein conjugate with negatively charged NDs compared to the free H5.c2 protein, the optimized mixture of H5:ND40 and H5:ND100 were immunized in a mouse model (in vivo experiments). As expected, the mixture of H5.c2:ND40 and H5.c2:ND100 elicited levels of H5.c2-specific-IgG antibodies that were statistically significantly higher than those of the free H5.c2 protein (as shown in Western blot and ELISA results,
Figure 4a,b) and statistically significant stronger neutralizing antibodies against A/H5N1 virus clade 2.3.2.1c (as shown by hemagglutination inhibition assay,
Figure 5) which suggests a potential effect inherent to ND40 and ND100 (with negative charged surface). The elicitation of this strong and specific immune response might be explained by several reasons. A previous study showed that the HPHT-NDs with negatively charged NDs had significant surface associations with proteins and created a thin layer on surface of the particles [
9]. The synthetic HA-VLPs formation or particles enhances HA-specific-IgG immune responses by improving the presentation of HA antigen to the immune system which are then delivered to draining lymph nodes [
14]. Moreover, Garcia-Bennett and colleagues reported that NDs can interact with proteins in a biological milieu, forming protein corona by electrostatic attractions between proteins and NDs [
27]. In addition, VLPs can act as adjuvants by improving antigen processing pathways [
28]. Although the H5.c2-specific-IgG antibodies and neutralizing antibodies induced in the mouse group immunized with the H5.c2:ND40 conjugate was higher than that of the H5.c2:ND100 conjugate, there was no significant difference between the two groups. It can be explained by the fact that there was no difference in the physical characteristics of ND40 and ND100 after conjugating with the H5.c2 protein.
These results indicate the good effects of oxidative NDs as antigen carriers for H5.c2 antigen. NDs are used instead of other nanomaterials because of some unique and notable benefits. Firstly, antigens can be conjugated onto the surface of NDs very fast and economically. The formation of antigen-NDs conjugates is completed in less than 10 min and no chemical reactions are needed to link antigens to NDs. The conjugate formation process through the physical adsorption method is a green chemistry technology. Many other substances (e.g., magnetic nanomaterials, gold nanoparticles, polymeric nanoparticles) require chemical reactions or labor-intensive to carry protein antigens [
14]. Secondly, the oxidative NDs have a unique surface that has been characterized to have both hydrophobic area and area of functional oxygen groups (–COOH, –CO, –OH) [
4,
29]. Thanks to the ND unique surface chemistry, our NDs can conjugate many different types of antigen proteins, both soluble proteins, and micelle-solubilized membrane proteins [
4,
13]. Earlier studies also showed that proteins but not short peptides bind strongly to the surface of oxidative NDs [
4,
13,
30]. Almost proteins (MW > 10 KDa) do not dissociate from NDs under non-destructive conditions. In contrast, short peptides (e.g., tryptic peptide digest) are easily detached from NDs surface in pH 7.0 or basic pH. This may also explain why H5.c2:NDs are stable and effectively processed by the immune system to boost the immune response in comparison with free H5.c2 antigen. Thirdly, NDs can be produced to be fluorescent with the many [N-V] centers inside its lattice, or fluorescent ND (FND). A previous publication demonstrated the usage of FND conjugated with A27 vaccinia surface antigen to study the interaction of the viral recombinant with cultured mammalian cells to mimic virus host cells [
30]. As [N-V] centers of FNDs have unique photochemical properties such as no photo-bleaching, no photo-blinking, highly bright and inert to the environment, and thus FNDs can be used to track the antigen trafficking both in vitro and in vivo. Consequently, further study will perform to use FNDs for evaluation of the interaction of H5.c2:NDs with the immune system and the excretion of NDs from the body of animals.
Several publications reported about the excretion of NDs from our body after use. Rats were safe after vaccination with a single high dose of FDNP-(NV)-Z~800 (60 mg/kg) that were principally distributed in the liver, spleen, and the clearance (50%) happened after 33 min following the end of particle infusion [
31]. Furthermore, another publication indicated that amino-functionalized NDs labeled with F-18 were excreted into the urinary tract after accumulation mainly in the lung, spleen, and liver [
32]. In addition, the accumulation of FND was non-toxic even when a large quantity, up to 75 mg/kg body weight which is a far-excessive amount of NDs needed to prepare vaccines [
33]. Taken together, the excretion of the NDs from the animal’s body is dependent on size, surface modification, and injection routes. Most previous studies indicate that NDs are safe and very biocompatible, especially oxidative NDs.