**1. Introduction**

Carbonaceous materials constitute an attractive group of adsorbents due to their properties, such as large surface area and thermal and chemical stability. In addition, activated carbons with different pore sizes can be obtained, thus adapting them to the functions they are designed for. On the other hand, functionalizing the surface of activated carbons is possible, giving them specific chemical properties adapted to a particular purpose. It is also of great importance that carbonaceous materials are considered safe, accessible, and affordable [1]. Various types of biomass residues, due to their low cost and abundance, have been evaluated to obtain functional carbonaceous materials. Many methods provide materials whose physicochemical properties can be tuned at will, thus obtaining materials that have shown promising results in different fields, such as adsorption or energy storage [2]. For this reason, they are considered suitable candidates for obtaining sustainable materials. These materials have been successfully applied in catalysis, water

**Citation:** Izquierdo, S.; Pacheco, N.; Durán-Valle, C.J.; López-Coca, I.M. From Waste to Resource: Utilizing Sweet Chestnut Waste to Produce Hydrothermal Carbon for Water Decontamination. *C* **2023**, *9*, 57. https://doi.org/10.3390/c9020057

Academic Editors: Indra Neel Pulidindi, Pankaj Sharma, Aharon Gedanken and Shu-Yuan Pan

Received: 30 March 2023 Revised: 8 May 2023 Accepted: 29 May 2023 Published: 1 June 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

purification, and soil remediation since they can remove heavy metals, agrochemicals, antibiotics, and other organic compounds. Excellent reviews on these aspects can be found in Soffian et al. [3], Adegoke et al. [4], G˛eca et al. [5], Borchardt et al. [6], and Ma et al. [2]. One such type of carbonaceous material is biochar, which can be produced from biomass waste through hydrothermal carbonization, chemical carbonization, gasification, torrefaction, and pyrolysis [3,7–9].

Sweet chestnut (*Castanea sativa*) is a deciduous tree of the Fagaceae family, native to humid temperate regions of the Northern Hemisphere and widely distributed throughout Europe. It can reach 35 m in height and 2 m (exceptionally more) in diameter. Sweet chestnut trees live to an age of 600 years or even more. By autumn, the female flowers develop into spiny cupules containing 3–7 brownish nuts, which are shed in October [10–12]. After the chestnuts are harvested, the spiny dome is disposed of as waste and used as fuel for fireplaces.

On the other hand, per- and poly-fluoroalkyl substances (PFASs) are a group of synthetic chemicals that have been used in a wide range of industrial and consumer products, such as firefighting foams, non-stick coatings, and water-repellent fabrics [13,14]. Due to their persistence, bioaccumulation, and toxicity, PFASs are considered Contaminants of Emerging Concern (CECs), with potentially adverse effects on human health and the environment [15–21]. These substances have been detected in various environmental media, including water, soil, air, and biota, and have attracted increasing attention from regulators, scientists, and the public worldwide [22]. Among the different methods for treating PFAS-contaminated water, adsorption has been recognized as a promising technology, given its effectiveness, versatility, and ease of implementation [23,24]. Carbonaceous materials are widely used as adsorbents for PFAS removal due to their high surface area, microporous structure, and affinity for hydrophobic compounds. One of the classic and best-known carbonaceous materials is activated carbon (AC) [25–27]. It is usually obtained at high temperatures in a double carbonization and activation process. This material is characterized by a highly developed porous structure, a generally alkaline character, and a predominantly hydrophobic surface [28,29]. It is well known that these properties can be modified by adjusting the conditions of the activation processes or employing suitable chemical or thermal treatment [30,31].

In recent years, numerous studies have investigated using these adsorbents for PFAS adsorption in water, aiming to optimize the adsorbent properties and conditions, understand the adsorption mechanisms, and evaluate the performance and feasibility of the technology. The effectiveness of AC as an adsorbent for PFAS removal depends on its physicochemical properties, which can be tailored by adjusting the activation process and conditions. The surface area, pore size, surface chemistry, and surface charge of AC determine its adsorption capacity, selectivity, and kinetics for PFAS removal. The larger the surface area and the more mesopores present, the higher the adsorption capacity; this is due to the increased availability of adsorption sites and the enhanced accessibility of PFAS molecules to the internal surface of the carbonaceous material. The surface chemistry can also affect PFAS adsorption, as hydrophobic interactions between PFASs and the nonpolar surface are favored over electrostatic interactions with the polar surface of water molecules. Therefore, adsorbents with a higher content of surface functional groups, such as oxygen-containing or nitrogen-containing groups, may have a lower adsorption capacity for PFASs than non-functionalized ones due to the reduced hydrophobicity and the increased competition for adsorption sites.

In the last decade or so, a new type of carbonaceous material, hydrothermal carbons (HC), has attracted considerable interest from researchers in this field [32,33]. It is obtained by treating carbon-rich materials with water at a moderate temperature in a closed vessel so that endogenous water vapor pressure acts on this material [34]. Generally, some biomass is used as raw material. This type of material is characterized by low pore development, acidity, and a hydrophilic surface, with many surface groups. As in the previous case, these properties can be modified by thermal or chemical treatments. This

has the advantage of being a more environmentally friendly manufacturing process, as it is carried out in hermetically sealed containers and at a lower temperature. This allows for easier management of possible waste and lower energy consumption. Additionally, despite its limited specific surface area, its high proportion of functional groups can give rise to adequate adsorption capacity. We have been working on the obtention and use of hydrothermal carbons from chestnut cupules for the last few years. Our preliminary results (unpublished) show that these carbons are suitable adsorbents in the decontamination of heavy metals, such as thorium, from water. Given these results, we thought it would be apt to check for the adsorption capacity of apolar organic molecules such as PFAS. Adsorption onto carbonaceous materials has been established as a promising technology for PFAS removal from water [20,35–42]. as it is carried out in hermetically sealed containers and at a lower temperature. This allows for easier management of possible waste and lower energy consumption. Additionally, despite its limited specific surface area, its high proportion of functional groups can give rise to adequate adsorption capacity. We have been working on the obtention and use of hydrothermal carbons from chestnut cupules for the last few years. Our preliminary results (unpublished) show that these carbons are suitable adsorbents in the decontamination of heavy metals, such as thorium, from water. Given these results, we thought it would be apt to check for the adsorption capacity of apolar organic molecules such as PFAS. Adsorption onto carbonaceous materials has been established as a promising technology for PFAS removal from water [20,35–42]. The objective of this work is to investigate the use of chestnut cupule waste as sustainable biomass raw material to obtain hydrothermal carbons—hence, adding value to

This has the advantage of being a more environmentally friendly manufacturing process,

*C* **2023**, *9*, x FOR PEER REVIEW 3 of 18

The objective of this work is to investigate the use of chestnut cupule waste as sustainable biomass raw material to obtain hydrothermal carbons—hence, adding value to it—and its use as adsorbent material for the removal from water of sodium perfluorooctanoate (PFONa), the anion of perfluorooctanoic acid, one of the most significant PFASs. it—and its use as adsorbent material for the removal from water of sodium perfluorooctanoate (PFONa), the anion of perfluorooctanoic acid, one of the most significant PFASs. **2. Materials and Methods**

#### **2. Materials and Methods** *2.1. Materials*

#### *2.1. Materials* Sodium perfluorooctanoate 97%, CAS 335-95-5 (Figure 1), was purchased from

Sodium perfluorooctanoate 97%, CAS 335-95-5 (Figure 1), was purchased from Thermo Fisher. Milli-Q water was used as a solvent in all determinations. Sweet chestnut (*Castanea sativa*) cupules were collected from chestnut groves in Valle del Jerte, in Cáceres, Spain. Thermo Fisher. Milli-Q water was used as a solvent in all determinations. Sweet chestnut (*Castanea sativa*) cupules were collected from chestnut groves in Valle del Jerte, in Cáceres, Spain.

**Figure 1.** Structural formula of sodium perfluorooctanoate. **Figure 1.** Structural formula of sodium perfluorooctanoate.

#### *2.2. Methods 2.2. Methods*

2.2.1. Synthesis of the Hydrothermal Carbons 2.2.1. Synthesis of the Hydrothermal Carbons

The chestnut cupules were allowed to dry naturally. They were subsequently ground and sieved, collecting three fractions: large size (1–2 mm), medium size (0.5–1 mm), and small size (less than 0.5 mm). Then, they were subjected to hydrothermal treatment in a The chestnut cupules were allowed to dry naturally. They were subsequently ground and sieved, collecting three fractions: large size (1–2 mm), medium size (0.5–1 mm), and small size (less than 0.5 mm). Then, they were subjected to hydrothermal treatment in a duralumin-coated Teflon™ reactor.

duralumin-coated Teflon™ reactor. To obtain HC-A, in a hydrothermal reactor, 100 mL of distilled water and 15 g of small-size sieved cupule were added. The reactor was closed, placed in an oven previously programmed at 180 °C, and kept for 24 h. The reactor was then removed from the oven and, once cold, opened. The final product was filtered and washed with plenty of distilled water (ca. 200 mL) on a filter. Subsequently, it was dried in an oven at 110 °C for To obtain HC-A, in a hydrothermal reactor, 100 mL of distilled water and 15 g of small-size sieved cupule were added. The reactor was closed, placed in an oven previously programmed at 180 ◦C, and kept for 24 h. The reactor was then removed from the oven and, once cold, opened. The final product was filtered and washed with plenty of distilled water (ca. 200 mL) on a filter. Subsequently, it was dried in an oven at 110 ◦C for 8 h. The other carbons were prepared similarly, according to the data in Table 1.

8 h. The other carbons were prepared similarly, according to the data in Table 1.

HC-C 1–2 15 200

**Table 1.** Operating conditions for the preparation of hydrothermal carbons.

#### 2.2.2. Characterization of the Hydrothermal Carbons

2.2.2. Characterization of the Hydrothermal Carbons Textural characterization was performed by N2 adsorption isotherms at 77 K in a Quantachrome Quadrasorb Evo (Quantachrome Ltd., Hook, UK), and the specific surface Textural characterization was performed by N2 adsorption isotherms at 77 K in a Quantachrome Quadrasorb Evo (Quantachrome Ltd., Hook, UK), and the specific surface area was calculated by applying the BET method [43]. Porosimetry was performed in a Quantachrome Poremaster 60 (Quantachrome, Hook, UK). Thermal analysis was carried out in an STA 449 F3 Jupiter thermobalance (Netzsch, Selb, Bavaria, Germany).

Fourier-transform infrared spectra were recorded in a Bruker Vertex70 spectrometer in the 400–4000 cm−1 range, with a DLaTGS detector and a 0.4 cm−1 spectral resolution. A KBr tablet was prepared with 1/250 dilution (Bruker, Billerica, Massachusetts, USA). Elemental analysis (C, H, N, S, O) was performed using a Leco CHMS-932 elemental analyzer (Leco, St. Joseph, MI, USA). C, H, N, and S were analyzed, and the difference was assigned to ash (measured from proximate analysis) and oxygen content. The point of zero charge (PZC) values were determined using the method proposed by Valente Nabais and Carrott [44]. To carry out this measurement, a 0.1 M solution of sodium nitrate was prepared, and 7% in weight of the carbon was added, followed by stirring for 48 h at 25 ◦C in a thermostatic bath. Subsequently, it was filtered, and the pH of the filtered solution was measured with a pH meter. This pH value corresponded to the point of zero charge. A field emission scanning electron microscope (SEM) Quanta 3D FEG (FEI Company, Hillsboro, OR, USA) was used to explore the surface morphological characteristics of all samples. The sample analysis was performed under high vacuum with a secondary electron. The surface of the samples was coated with gold due to their low conductivity. Energy dispersive X-ray analyses (EDX) were recorded with the same equipment.

#### 2.2.3. Adsorption Experiments

The conjugate base sodium salt (PFONa), instead of the perfluorooctanoic acid, was used to carry out the adsorption experiments, since the acid has a Ka ca. -1 and dissociates completely in water.

1. Adsorption kinetics

To determine the PFONa adsorption over time, the following procedure was used:

A 200 mg/L PFONa water solution was prepared by dissolving 10 mg of PFONa in a 50 mL volumetric flask. It was then transferred to an Erlenmeyer flask, and 50 mg of hydrothermal carbon (HC) was added, so the HC to PFONa ratio was 5:1. All flasks were thermostated at 25 ◦C and magnetically stirred at 400 rpm. Assays were performed in duplicate; 2 mL samples were taken at 2, 4, 24, 48, 72, and 96 h, then filtered with a 0.22 µm pore filter, and, finally, aliquots were analyzed for PFONa concentration.

#### 2. Adsorption isotherm

Different PFONa solutions of 40, 70, 100, 130, 165, and 200 mg/L were prepared in 10 mL volumetric flasks. They were then transferred to separate Erlenmeyer flasks, and 10 mg of hydrothermal carbon was added to each one, so the HC to PFONa mass quotient was 25.0, 14.3, 10.0, 7.7, 6.1, and 5.0, respectively. All flasks were thermostated at 25 ◦C and magnetically stirred at 400 rpm. Assays were performed in duplicate; 2 mL samples were taken at 96 h, then filtered with a 0.22 µm pore filter, and, finally, aliquots were analyzed for PFONa concentration.

#### 2.2.4. Analytical Determinations

The samples were analyzed using liquid chromatography (Agilent 1290 Infinity II LC (Agilent Technologies, Santa Clara, California, USA)) coupled to a triple quadrupole mass spectrometer (Agilent 6460 triple quad LC/MS (Agilent Technologies)). Aliquots of 3 µL were injected, and elution was carried out by operating in gradient mode at 0.4 mL/min with a reverse phase HPLC Zorbax Extend C18 column (3 µm, 100 mm × 1.8 mm) at 30 ◦C. As the mobile phase, 0.1% formic acid in ultrapure water (A) and 0.1% formic acid in acetonitrile (B) were used, as shown in Table 2.

Ionization was carried out in negative mode. The equipment parameters were gas temperature 225 ◦C, gas flow 10 L/min, nebulizer 45 psi, sheath gas temperature 350 ◦C, sheath gas flow 11 L/min, capillary voltage 3.6 kV, nozzle voltage 1500 V, and delta EMV 400. The MassHunter B.07.00 (Agilent) software was used for data acquisition and equipment control. Multiple Reaction Monitoring (MRM) was used, with the transitions sought to be 412.9 → 368.9 and 412.9 → 169, the former used in the quantification of the compound. The fragmentation and collision energy voltages were 86 V and 5 V, respectively.

**Table 2.** Mobile phase composition.

#### **3. Results and Discussion**

#### *3.1. Characterization of the Hydrothermal Carbons*

The nitrogen adsorption isotherms (Figure 2) corresponded to type III, according to the IUPAC classification [45,46]; therefore, the interaction between the N2 molecules and the surface must have been weak. The surface obtained by the BET method produced values that were higher than those obtained by the DFT method but in the same order of magnitude (Table 3) [43,47]. Nevertheless, since the isotherms were of Type III, the BET model was not to be considered highly reliable. All three carbons showed a minimal surface area, low microporosity, and a considerably greater volume of large pores, i.e., mesopores and macropores (Figures 3–5). The HC-A coal had the least-developed porosity. The DFT method was used to study the distribution of micropores and mesopores. The results show that the three carbons presented a similar pore distribution. A high volume of narrow mesopores was detected, whereas no micropores were present. The HC-B carbon had a larger volume of narrow mesopores, while the HC-C showed more volume when the pores widened. The HC-A carbon showed a more significant development of wide porosity than the other two adsorbents. It should be noted that HC-B and HC-C carbons showed a more developed narrow mesoporosity than HC-A. However, the mesopore and macropore structure was very similar in the three materials. *C* **2023**, *9*, x FOR PEER REVIEW 6 of 18

**Figure 2.** Isotherms of N2 adsorption at 77 K. **Figure 2.** Isotherms of N2 adsorption at 77 K.

Functional Theory [47].

**Material BET 1 DR 2 Porosimetry DFT 3**

porosimetry, is shown in Figure 5.

HC-B 53.2 0.007 0.061 39.13 0.122 HC-B 53.2 0.007 0.061 39.13 0.122

**Figure 3.** Distribution of porosity of the hydrothermal carbons.

1 Brunauer, Emmett, and Teller model [43]. 2 Dubinin and Radushkevich model [48,49]. 3 Density

The DFT method was used to study the distribution of porosity in micro and mesopores, and the results are shown in Figure 3. The cumulative volume plot obtained from porosimetry is shown in Figure 4, and the width pore distribution plot, also obtained from

**Table 3.** Porosity and specific surface area of carbons.

**Material BET 1 DR 2 Porosimetry DFT 3 S, m2 g**−**1 Vmicro, cm3 g**−**1 Vmeso, cm3 g**−**1 S, m2 g**−**1 Vtotal, cm3 g**−**1** HC-A 42.3 0.007 0.078 27.68 0.085 HC-B 53.2 0.007 0.061 39.13 0.122 HC-B 53.2 0.007 0.061 39.13 0.122 1 Brunauer, Emmett, and Teller model [43]. 2 Dubinin and Radushkevich model [48,49]. 3 Density Functional Theory [47]. The DFT method was used to study the distribution of porosity in micro and mesopores, and the results are shown in Figure 3. The cumulative volume plot obtained from

*C* **2023**, *9*, x FOR PEER REVIEW 6 of 18

**Table 3.** Porosity and specific surface area of carbons. HC-B 53.2 0.007 0.061 39.13 0.122

**Figure 2.** Isotherms of N2 adsorption at 77 K.

**Material BET 1 DR 2 Porosimetry DFT 3**

**Table 3.** Porosity and specific surface area of carbons.

HC-B 53.2 0.007 0.061 39.13 0.122

1 Brunauer, Emmett, and Teller model [43]. 2 Dubinin and Radushkevich model [48,49]. 3 Density Functional Theory [47]. porosimetry is shown in Figure 4, and the width pore distribution plot, also obtained from porosimetry, is shown in Figure 5.

**S, m2 g−1 Vmicro, cm3 g−1 Vmeso, cm3 g−1 S, m2 g−1 Vtotal, cm3 g−1**

**Figure 3. Figure 3.**  Distribution of porosity of the hydrothermal carbons. Distribution of porosity of the hydrothermal carbons.

**Figure 4.** Cumulative pore volume of the hydrothermal carbons. **Figure 4.** Cumulative pore volume of the hydrothermal carbons.

**Figure 5.** Pore width distribution of the hydrothermal carbons.

tribution of basicity by the aromatic structures.

**Table 4.** Point of zero charge of the carbons.

The measurements of the PZC show that all three materials were acidic (Table 4), as

**Material PZC**  HC-A 4.0 HC-B 3.8 HC-C 4.8

The Fourier-transform infrared (FTIR) spectra obtained are shown in Figure 6. Nu-

merous observable bands in the FTIR spectra belonging to many functional groups indicate that the degree of graphitization of these materials was not high. The most intense band at 3400 cm−1 corresponded to either alcohol or carboxylic O-H bonds. The band at 2935 cm−1 was assigned to C-H bonds, but its low wavenumber suggested they were

is usual in hydrothermal carbons. It must be considered that basic oxygenated functional groups (carbonyl) are more labile than those that provide acidic properties (alcohol and carboxylic), so it is to be expected that the former will degrade in a more significant proportion than the latter. In addition, as the degree of graphitization was low, as observed from the elemental composition with low carbon content, there was not a substantial con-

**Figure 4.** Cumulative pore volume of the hydrothermal carbons.

The measurements of the PZC show that all three materials were acidic (Table 4), as is usual in hydrothermal carbons. It must be considered that basic oxygenated functional groups (carbonyl) are more labile than those that provide acidic properties (alcohol and The DFT method was used to study the distribution of porosity in micro and mesopores, and the results are shown in Figure 3. The cumulative volume plot obtained from porosimetry is shown in Figure 4, and the width pore distribution plot, also obtained from porosimetry, is shown in Figure 5.

carboxylic), so it is to be expected that the former will degrade in a more significant proportion than the latter. In addition, as the degree of graphitization was low, as observed from the elemental composition with low carbon content, there was not a substantial contribution of basicity by the aromatic structures. **Table 4.** Point of zero charge of the carbons. The measurements of the PZC show that all three materials were acidic (Table 4), as is usual in hydrothermal carbons. It must be considered that basic oxygenated functional groups (carbonyl) are more labile than those that provide acidic properties (alcohol and carboxylic), so it is to be expected that the former will degrade in a more significant proportion than the latter. In addition, as the degree of graphitization was low, as observed from the elemental composition with low carbon content, there was not a substantial contribution of basicity by the aromatic structures.

**Material PZC Table 4.** Point of zero charge of the carbons.

merous observable bands in the FTIR spectra belonging to many functional groups indicate that the degree of graphitization of these materials was not high. The most intense band at 3400 cm−1 corresponded to either alcohol or carboxylic O-H bonds. The band at 2935 cm−1 was assigned to C-H bonds, but its low wavenumber suggested they were The Fourier-transform infrared (FTIR) spectra obtained are shown in Figure 6. Numerous observable bands in the FTIR spectra belonging to many functional groups indicate that the degree of graphitization of these materials was not high. The most intense band at 3400 cm−1 corresponded to either alcohol or carboxylic O-H bonds. The band at 2935 cm−1 was assigned to C-H bonds, but its low wavenumber suggested they were mainly aliphatic bonds, since the aromatic ones give a signal at higher values. The 1710 cm−1 band was due to C-O double bonds that can be carbonyl or carboxylic, whereas the neighboring band of 1610 cm−1 was characteristic of C-C conjugated double bonds. Several bands between 1550 cm−1 and 1300 cm−1 can be attributed to the carbon backbone of these materials, for instance, those at 1515 cm−1 and 1460 cm−1 ; however, the presence of O-H and C-O bands in this region made assigning them somewhat problematic. Between 1300 cm−1 and 100 cm−1, the bands were mainly due to oxygenated or nitrogenous functional groups (C-O or C-N bonds) [50].

groups (C-O or C-N bonds) [50].

**Figure 6.** Fourier-transform infrared spectra of the hydrothermal carbons. **Figure 6.** Fourier-transform infrared spectra of the hydrothermal carbons.

The scanning electron microscopy images of the samples in which hydrothermal carbonization had been carried out (Figures 7–9) showed a more defined structure than in the original material (Figure 10), in which the cells of the lignocellulosic material were more clearly observed. The treatment, therefore, removes matter from the original material, increasing the porosity. In some images, small spheres on the surface could be seen; it is known that obtaining hydrothermal coals from carbohydrates gives rise to spherical particles, so they could have come from dissolved substances of the chestnut cupule that underwent hydrothermal carbonization. Another option is that they were wax deposited on the surface of the biochar; given the similarity of composition in both cases, it is difficult to determine which of the processes gave rise to these structures. The scanning electron microscopy images of the samples in which hydrothermal carbonization had been carried out (Figures 7–9) showed a more defined structure than in the original material (Figure 10), in which the cells of the lignocellulosic material were more clearly observed. The treatment, therefore, removes matter from the original material, increasing the porosity. In some images, small spheres on the surface could be seen; it is known that obtaining hydrothermal coals from carbohydrates gives rise to spherical particles, so they could have come from dissolved substances of the chestnut cupule that underwent hydrothermal carbonization. Another option is that they were wax deposited on the surface of the biochar; given the similarity of composition in both cases, it is difficult to determine which of the processes gave rise to these structures. *C* **2023**, *9*, x FOR PEER REVIEW 9 of 18

mainly aliphatic bonds, since the aromatic ones give a signal at higher values. The 1710 cm−1 band was due to C-O double bonds that can be carbonyl or carboxylic, whereas the neighboring band of 1610 cm−1 was characteristic of C-C conjugated double bonds. Several bands between 1550 cm−1 and 1300 cm−1 can be attributed to the carbon backbone of these materials, for instance, those at 1515 cm−1 and 1460 cm−1; however, the presence of O-H and C-O bands in this region made assigning them somewhat problematic. Between 1300 cm−1 and 100 cm−1, the bands were mainly due to oxygenated or nitrogenous functional

**Figure 7.** SEM image of HC-A. **Figure 7.** SEM image of HC-A.

**Figure 8.** SEM image of HC-B.

**Figure 8. Figure 8.**  SEM image of HC-B. SEM image of HC-B.

**Figure 7.** SEM image of HC-A.

**Figure 9.** SEM image of HC-C. ered simply as carbonaceous materials with a low content of mineral matter. **Figure 9.** SEM image of HC-C.

**Figure 10.** SEM image of raw chestnut cupule.

The thermal analysis program can be found in the Supplementary Material (Table S1). Thermograms (Figure 11) show that the moisture of these materials was low, as well as the amount of residual ash, which was less than 1% in the three carbons. Regarding the volatile matter (from 50 to 80 min approximately), it followed the order HC-A > HC-B > HC-C, and the fixed carbon followed the opposite order. Therefore, they can be consid-

**Figure 10.** SEM image of raw chestnut cupule. **Figure 10.** SEM image of raw chestnut cupule.

**Figure 9.** SEM image of HC-C.

The thermal analysis program can be found in the Supplementary Material (Table S1). Thermograms (Figure 11) show that the moisture of these materials was low, as well as the amount of residual ash, which was less than 1% in the three carbons. Regarding the volatile matter (from 50 to 80 min approximately), it followed the order HC-A > HC-B > HC-C, and the fixed carbon followed the opposite order. Therefore, they can be consid-The thermal analysis program can be found in the Supplementary Material (Table S1). Thermograms (Figure 11) show that the moisture of these materials was low, as well as the amount of residual ash, which was less than 1% in the three carbons. Regarding the volatile matter (from 50 to 80 min approximately), it followed the order HC-A > HC-B > HC-C, and the fixed carbon followed the opposite order. Therefore, they can be considered simply as carbonaceous materials with a low content of mineral matter. *C* **2023**, *9*, x FOR PEER REVIEW 11 of 18

**Figure 11.** Thermograms of the hydrothermal carbons. **Figure 11.** Thermograms of the hydrothermal carbons.

The results of the organic elemental analysis (C, H, N, S, and O) and the ash content (Table 5) show that the samples prepared at a higher temperature (HC-B and HC-C) presented more carbon, nitrogen, and ash, and less hydrogen and oxygen. This indicates that the carbonization process was more intense when the temperature was raised. The size did not seem to be crucial, though, since the smaller the particle size, the more intense the The results of the organic elemental analysis (C, H, N, S, and O) and the ash content (Table 5) show that the samples prepared at a higher temperature (HC-B and HC-C) presented more carbon, nitrogen, and ash, and less hydrogen and oxygen. This indicates that the carbonization process was more intense when the temperature was raised. The size did not seem to be crucial, though, since the smaller the particle size, the more intense

effect of pressurized water should have been, and this was not observed in the results. The data in the table agree with the fixed carbon content, which tended to increase with

**Sample C, % H, % N, % S, % O, % Ash, %**  HC-A 57.50 6.56 0.53 0.05 35.31 0.05 HC-B 61.70 5.93 0.58 0.05 31.55 0.19 HC-C 63.70 5.89 1.06 0.05 28.46 0.84

On the other hand, EDX analyses were performed on all three hydrothermal carbons and the raw chestnut cupule (Table 6). This determination, which studied a more superficial area than the elemental analysis, confirmed the same trend. All carbons had been enriched in carbon and depleted in oxygen. It is worth noticing that the HC-C sample contained the most significant mineral matter. This effect is larger than the one found in the elemental analysis, which indicates that the hydrothermal treatment affected the surface

C 54.2 69.6 72.5 72.0 O 43.6 30.0 27.4 26.9 Mg 0.1 0.0 0.0 0.0 Al 0.1 0.1 0.0 0.0 Si 0.2 0.1 0.0 0.0 P 0.1 0.0 0.0 0.2 K 1.0 0.0 0.1 0.2 Ca 0.7 0.1 0.0 0.5 Fe 0.1 0.0 0.0 0.0 S 0.0 0.1 0.1 0.1

**Raw Material HC-A HC-B HC-C**

**Table 5.** Elemental analyses of the hydrothermal carbons.

more than the interior of the material particles.

**Table 6.** EDX analyses of the hydrothermal carbons.

and hydrogen content.

the effect of pressurized water should have been, and this was not observed in the results. The data in the table agree with the fixed carbon content, which tended to increase with elemental carbon content, and the volatile matter, which tended to increase with oxygen and hydrogen content.

**Table 5.** Elemental analyses of the hydrothermal carbons.

On the other hand, EDX analyses were performed on all three hydrothermal carbons and the raw chestnut cupule (Table 6). This determination, which studied a more superficial area than the elemental analysis, confirmed the same trend. All carbons had been enriched in carbon and depleted in oxygen. It is worth noticing that the HC-C sample contained the most significant mineral matter. This effect is larger than the one found in the elemental analysis, which indicates that the hydrothermal treatment affected the surface more than the interior of the material particles.

**Table 6.** EDX analyses of the hydrothermal carbons.

### *3.2. Adsorption Experiments*

The kinetic study of adsorption processes makes it possible to relate the amount of adsorbate adsorbed per gram of adsorbent with the elapsed contact time between adsorbent and adsorbate. The variation in the excess PFONa concentration over time data is shown in Table 7. At 96 h, equilibrium can be considered established, having reached the maximum adsorption. Our preliminary studies show that HC-A and HC-B have adsorption of around 15%, while HC-C adsorbs 26%; therefore, further decontamination studies presented herein focused on the most promising adsorbent, HC-C.

**Table 7.** Adsorption kinetics results for HC-C.

1 Average value of duplicate runs.

The Lagergren adsorption kinetic model assumes that the adsorption rate is proportional to the number of unoccupied sites available for adsorption. However, when we

applied it, we found it to be inappropriate for our data. On the other hand, the pseudosecond-order adsorption kinetic model assumes that the rate-limiting step is the chemical adsorption process, and it can be expressed mathematically, as shown in Equation (1):

$$\frac{t}{q\_t} = \frac{1}{k \cdot q\_\varepsilon^2} + \frac{t}{q\_\varepsilon} \tag{1}$$

where *t* is the time, *qt* is the amount of solute adsorbed at time *t*, *qe* is the amount of solute adsorbed at equilibrium, and k is the rate constant. The data are shown in Table 8, whereas the graph of *t/qt* vs. *t* is plotted in Figure 12.

**Table 8.** Data for the pseudo-second-order kinetic model.

**Figure 12.** Plot for pseudo-second-order kinetics. **Figure 12.** Plot for pseudo-second-order kinetics.

The data points follow a straight line, suggesting that the pseudo-second-order adsorption kinetic model applies to this system. The slope was 9.7137 × 10−4, and the intercept was 1.1612 × 10−3, so we can calculate the rate constant (*k* = 8.126·10−4 h−1·mg−1·g) and the amount of solute adsorbed at equilibrium (*qe* = 1029.47 mg·g−1). The data points follow a straight line, suggesting that the pseudo-second-order adsorption kinetic model applies to this system. The slope was 9.7137 × 10−4 , and the intercept was 1.1612 × 10−3 , so we can calculate the rate constant (*k* = 8.126·10−4 h −1 ·mg−1 ·g) and the amount of solute adsorbed at equilibrium (*qe* = 1029.47 mg·g −1 ).

Experiments were conducted with different HC-C to PFONa ratios to determine the carbon adsorption isotherm. The values obtained are shown in Table 9 and plotted in Figure 13. Experiments were conducted with different HC-C to PFONa ratios to determine the carbon adsorption isotherm. The values obtained are shown in Table 9 and plotted in Figure 13.

We found that the data obtained did not mathematically fit the Langmuir adsorption model, i.e., the adjustment of the model to the experimental data was inconsistent. On the other hand, the Freundlich model fit these results. Data for the Freundlich model are

**Table 9.** Adsorption isotherm results for HC-C 1. **Table 9.** Adsorption isotherm results for HC-C 1 .

1 Time = 96 h. 1 Time = 96 h.

shown in Table 10.

**Figure 13.** Adsorption isotherm for PFONa onto HC-C.

**Figure 13.** Adsorption isotherm for PFONa onto HC-C. **Figure 13.** Adsorption isotherm for PFONa onto HC-C.

**Figure 12.** Plot for pseudo-second-order kinetics.

**Table 9.** Adsorption isotherm results for HC-C 1.

ure 13.

1 Time = 96 h.

amount of solute adsorbed at equilibrium (*qe* = 1029.47 mg·g−1).

We found that the data obtained did not mathematically fit the Langmuir adsorption model, i.e., the adjustment of the model to the experimental data was inconsistent. On the other hand, the Freundlich model fit these results. Data for the Freundlich model are shown in Table 10. We found that the data obtained did not mathematically fit the Langmuir adsorption model, i.e., the adjustment of the model to the experimental data was inconsistent. On the other hand, the Freundlich model fit these results. Data for the Freundlich model are shown in Table 10.

The data points follow a straight line, suggesting that the pseudo-second-order adsorption kinetic model applies to this system. The slope was 9.7137 × 10−4, and the intercept was 1.1612 × 10−3, so we can calculate the rate constant (*k* = 8.126·10−4 h−1·mg−1·g) and the

Experiments were conducted with different HC-C to PFONa ratios to determine the carbon adsorption isotherm. The values obtained are shown in Table 9 and plotted in Fig-

> *c0* **(mg/L)** *ce* **(mg/L)** *qe* **(mg/g)**  40 36.6 3.40 70 61.1 8.95 100 81.5 18.55 130 103.2 26.80 165 126.6 38.40 200 148.9 51.15

**Table 10.** Data points for the Freundlich model.

Using Equation (2) and the adjusted values, the Freundlich model constants can be calculated:

$$
ln q\_{\varepsilon} = \frac{1}{n} \ln c\_{\varepsilon} + \ln \mathcal{K}\_{F} \tag{2}
$$

The calculated values of Freundlich isotherm constants were *KF* = 12.3099; and *1/n* = 1.9513 (r2 = 0.9962).

The consistency with the Freundlich model indicates some degree of heterogeneity on the surface (not all active sites are identical) and the possibility that more than one layer can be adsorbed. Since the material used as adsorbent was amorphous, this relative heterogeneity can be expected. Moreover, the value of n indicates that the interaction between adsorbent and sorbate is mainly of the chemisorption type. Our results are promising, with a *qe* = 1029.47 mg·g −1 ; interestingly, they suggest that the maximum adsorption capacity had not been achieved at high concentrations. To put these results in perspective, we can refer to the excellent review by Liang et al.; in it, the authors compared the different adsorption methods for removing PFAS from water [37]. Analyzing the results obtained in water at pH 7, we found that, for granular activated carbon (GAC), a value of *qe* = 2.34–102.7 mg·g −1 is reported; and for powdered activated carbon (PAC), *qe* values of 16.9–500 mg·g −1 are found. On the other hand, for biochar derived from hardwood sawdust, the value obtained for *qe* at pH 7.2 is 2.4 mg·g −1 , and for pinewood at the same pH, *qe* is 2.1 mg·g −1 . Liang et al. found that ion exchange resins (*qe* = 525–1500 mg·g −1 ) and carbon nanotubes (*qe* = 140–1110 mg·g −1 ) show the highest adsorption capacities, yet they interestingly concluded that low-cost adsorbents with high adsorptive capacity are highly recommended in place of commercial AC due to their local availability, technical feasibility, and cost-effectiveness.

The mechanisms affecting PFAS adsorption have been reviewed by Liang et al. in an excellent review [37]. Diffusion, electrostatic interaction, hydrophobic interaction, ion exchange, and hydrogen bonds all can play a role in the adsorption process and are affected by the physicochemical characteristics of the adsorbent, such as pore size, pore distribution, and functional groups. In our case, HC-C is the carbon with the largest volume of mesopores; since PFONa is a large PFAS, it is essential for diffusion and adsorption that there are large numbers of mesopores. It is also the most graphitized hydrothermal carbon, i.e., the least polar; this also plays a vital role in the PFONa adsorption mechanism onto HC-C. Because of the hydrophobic nature of PFAS chains, the sorption of anionic PFAS can be dominated by hydrophobic interactions between the negatively-charged hydrophobic fluorinated tail and the surface of hydrophobic adsorbents, regardless of the electrostatic repulsion [51,52]. Particularly at high pH, hydrophobic interaction, which is not sensitive to changes in pH, should be the primary PFAS adsorption mechanism [53,54].

Given these results, and due to the advantages of the hydrothermal carbon herein reported, further work is underway to gain insight into the full potential of this adsorbent in water decontamination.

#### **4. Conclusions**

The preparation conditions of the hydrothermal carbons greatly influence the result; this allows for obtention of coals with tuned properties by optimizing the obtention process.

Carbonaceous material obtained by hydrothermal carbonization of sweet chestnut (*Castanea sativa*) cupule residues is a cost-effective, environmentally benign adsorbent, which effectively adds value to this biomass waste.

The carbons thus obtained show a limited surface area and a low microporosity while having a large volume of mesopores and macropores. All of them are acidic, with a low graphitization index, minimal moisture, and slight residual ash content; they are carbonaceous materials with a low mineral matter content.

It is possible to purify water contaminated with perfluorooctanoate by adsorption on this type of hydrothermal carbon. The adsorption process follows pseudo-second-order kinetics. Additionally, it fits the Freundlich model, showing that more than one layer can be adsorbed, which is expected in such an amorphous adsorbent.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/c9020057/s1, Table S1. Thermal Analysis Program; Figure S1. SEM Images of HC-A; Figure S2. SEM Images of HC-B; Figure S3. SEM Images of HC-C; Figure S4. SEM Images of raw chestnut cupule.

**Author Contributions:** Conceptualization, I.M.L.-C. and S.I.; methodology, I.M.L.-C. and C.J.D.-V.; investigation, S.I., N.P., C.J.D.-V. and I.M.L.-C.; writing—original draft preparation, S.I., C.J.D.-V. and I.M.L.-C.; writing—review and editing, S.I., N.P., C.J.D.-V. and I.M.L.-C.; funding acquisition, C.J.D.-V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Spanish Regional Government 'Junta de Extremadura' and European Regional Development Fund (ERDF), grants IB20026 and GR21107.

**Data Availability Statement:** Data may be obtained from the authors under reasonable request.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.


**Author details**

• 'Not invented here' syndrome'7

16 Knowledge Management Strategies and Applications

among those who should use it.

**7. Conclusion**

7

;

plex structures, such as communities of practice and invisible colleges.

Miranda and Simeão's model [108] proposes the existence of elements, not so discernible (such as, technological communication systems), but of high value for the transfer of knowledge, such as, "a set of knowledge and practices"; the practice of "Granting specific know-how," or the sharing of "how-to-do". The authors point out to the irrevocability of human interaction in the transfer of tacit knowledge, setting up an 'extensive model of communication.' They argue that in TKS, the process occurs in an interpersonal way, developing increasingly com-

Tonet and Paz [106] consider the knowledge sharing process as composed of four phases: initiation, which are actions that seek to identify and analyze the needs of knowledge, the existing opportunities to employ different or new knowledge, and the tracking of sources where this knowledge can be found; implementation, actions that seek to promote integration between sources and recipients of knowledge; support, actions that seek to create opportunities to exercise shared knowledge and that promote the guidance of its practice with the objective of assimilating knowledge and the development of the necessary skills for a proficient use; and incorporation, actions aimed at making that shared knowledge can flow freely

Knowledge has been occupying the center of studies and research for some time, and this chapter seeks to contribute to the depth of this collection and aims to offer articles that deal

This work aimed to bring a review of the literature regarding TKS and to contribute with some definitions related to the topic. The observed understanding with this literature review is that organizations, and to a lesser degree, The Small Claims Federal Courts, are moving from a traditional model that is attached to physical and tangible assets to another, where knowledge, especially tacit, is treated as an organizational asset that, despite being immate-

In this trajectory and encouraged by initial questioning, this chapter depicts the search for different references in which the sharing of tacit knowledge was related to a range of organizational vectors covering definitions that go from understanding it as an innovation factor to a strategic component for leveraging organizational performance. Seen by some as difficult to transfer, tacit knowledge is accepted by others as a component of communication flows, where dialogues, social networks, communities of practice, invisible colleges, and even 'TKS

In the literature 'not invented here' (nih) is described as an idea or innovation that was not developed within the company's workplace environment or that was not conducted by their own internal members, so the solution does not have

the 'necessary characteristics or identity of the organization' and thus should not be considered.

• Intolerance toward mistakes or for needing assistance.

with tacit knowledge and the sharing of that knowledge.

rial, becomes valuable and a generator of wealth.

José Roberto Ferretti1 \* and Maria da Conceição L. Afonso2

\*Address all correspondence to<EMAIL_ADDRESS>
1 Brazilian Federal Court–1st Region, Brazil

2 National Confederation of Industry Brazil, Brazil


*Article* **Investigation of Avian Influenza H5N6 Virus-like Particles as a Broad-Spectrum Vaccine Candidate against H5Nx Viruses**

**Yu-Hsuan Yang 1 , Ching-Hui Tai 2 , Dayna Cheng 3 , Ya-Fang Wang 2 and Jen-Ren Wang 1,2,3,\***

**Abstract:** Highly pathogenic avian influenza (HPAI) clade <IP_ADDRESS> viruses have been reported to be the source of infections in several outbreaks in the past decades. In a previous study, we screened out a broad-spectrum virus strain, H5N6-Sichuan subtype, by using a lentiviral pseudovirus system. In this project, we aimed to investigate the potential of H5N6 virus-like particles (VLPs) serving as a broad-spectrum vaccine candidate against H5Nx viruses. We cloned the full-length M1 gene and H5, N6 genes derived from the H5N6-Sichuan into pFASTBac vector and generated the VLPs using the baculovirus-insect cell system. H5N6 VLPs were purified by sucrose gradient centrifugation, and the presence of H5, N6 and M1 proteins was verified by Western blot and SDS-PAGE. The hemagglutination titer of H5N6 VLPs after purification reached 5120 and the particle structure remained as viewed by electron microscopy. The H5N6 VLPs and 293T mammalian cell-expressed H5+N6 proteins were sent for mice immunization. Antisera against the H5+N6 protein showed 80 to 320 neutralizing antibody titers to various H5Nx pseudoviruses. In contrast, H5N6 VLPs not only elicited higher neutralizing antibody titers, ranging from 640 to 1280, but also induced higher IL-2, IL-4, IL-5, IFN-γ and TNF production, thus indicating that H5N6 VLPs may be a potential vaccine candidate for broad-spectrum H5Nx avian influenza vaccines.

**Keywords:** avian influenza virus; virus-like particles; vaccine; H5N6

## **1. Introduction**

Avian influenza virus (AIV) belongs to the *Orthomyxoviridae* family, *Alphainfluenzavirus* genus, and contains eight negative-sense, single-stranded RNA segments as its genome [1]. During viral replication, reassortant genes help the AIVs to overcome the species barrier to infect humans, thus causing a major threat to public health [2]. The highly pathogenic avian influenza (HPAI) virus A/goose/Guangdong/1/96 H5N1 (Gs/GD) lineage caused global epidemics not only in migratory birds and domestic poultry but also in the human population. Taiwan had previously encountered an outbreak of HPAI clade <IP_ADDRESS> derived from Gs/GD lineage in poultry [3]. Clade <IP_ADDRESS> has evolved since 2008 and had several reassortant neuraminidase (NA) genes with N2, N3, N5, N6, and N8 variants. Phylogenetic analysis based on the sequence of hemagglutinin (HA) and M gene reveals that HPAI H5Nx viruses in October 2014 were related to two HPAI H5N8 viruses identified from wild birds in Japan. Two subsequent outbreaks in late 2014 and 2016 resulted from seven H5Nx reassortant genes that spread through Taiwan and eight novel introduced internal genes mostly derived from the gene pool of avian influenza virus circulating in wild birds in Asia [3]. In several outbreaks in past decades, it has been reported that the sources of infection were novel HPAI clade <IP_ADDRESS> viruses. These newly emerging, highly pathogenic H5Nx avian influenza viruses, including H5N1, H5N2, H5N3, H5N6, and H5N8, have

**Citation:** Yang, Y.-H.; Tai, C.-H.; Cheng, D.; Wang, Y.-F.; Wang, J.-R. Investigation of Avian Influenza H5N6 Virus-like Particles as a Broad-Spectrum Vaccine Candidate against H5Nx Viruses. *Viruses* **2022**, *14*, 925. https://doi.org/10.3390/ v14050925

Academic Editor: Chi-Young Wang

Received: 7 March 2022 Accepted: 26 April 2022 Published: 28 April 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

raised great concern worldwide [4–6]. Taiwan also encountered the largest outbreak of avian influenza viruses in 2015, including H5N2, H5N6 and H5N8 [7]. Genetic analysis revealed that the HA and NA gene of H5N2 circulating in Taiwan before 2015 outbreaks were derived from American H5N2 (old Mexican-like lineage), and the other six gene segments were similar to local H6N1 in Taiwan [8,9]. The HA of the new epidemic strain H5N2 in 2015 belonged to clade <IP_ADDRESS> HPAI; therefore, the old vaccine might not be able to protect against new emerging H5N2, H5N3, H5N8 and H5N6 viruses. Thus, a new strategy of vaccine platform for rapid application is necessary.

There are three major types of influenza vaccine approved by the Food and Drug Administration in the United States, including inactivated vaccine, live-attenuated vaccine and recombinant vaccine. The manufacture of inactivated vaccine or live-attenuated vaccine is derived from a specific strain of AIV that probably confers only strain-specific immunity. In addition, inactivated vaccines and live-attenuated vaccines seldom elicit cross-protective immunity against different subtypes of AIVs. A mismatch between the available vaccines and the circulating AIV strain may contribute to high mortality and wild transmission in the avian and human population.

Virus-like particles (VLPs) are non-infectious nanoparticles made up of assembled viral proteins without viral genetic materials. In comparison to whole inactivated vaccines, VLP vaccines consisting of different combinations of antigen have the advantage to induce a broader immune response [10]. At present, highly conserved ectodomain of M2 protein (M2e) or the stalk of the HA protein have been used as the antigen for a VLP vaccine candidate for influenza viruses [11,12]. Animals vaccinated with M2e VLP have lower morbidity and viral titers, while the viral clearance has not been observed. VLP with stalk HA elicits cross-protective humoral immunity against various strains of AIVs; however, it is less immunogenic [11,13]. Several studies also posed different strategies. For example, Wu et al. fused a conserved M2e protein at the N-terminus of HA, or at the N-terminus of NA in the H5N1 VLP, to protect against heterosubtypic H1N1 and H5N1 [13]. Kang et al. have expressed two HAs derived from a different clade of AIVs to enhance robust immune response [14]; however, expression of multiple genes in one vector may also contribute to assembly instability due to the tandem repetition of similar sequences [15]. In this study, we supposed that the conformation of VLP presenting the HA and NA may be similar to the native virions that induced antibodies and will be able to target the HA, which can effectively inhibit viral replication. VLPs can be produced in several expression systems, including mammalian cells, plants, insect cells, yeast and bacteria. Since the VLPs generated from a baculovirus-infected insect cell line have advantages of high expression yield, good protein folding with glycosylation modification and ease for scale-up production, we aimed to develop a potential VLP-based vaccine by using the baculovirus-insect cell expression system.

In our previous study, we analyzed genetic evolution of HA from H5Nx viruses circulating in Taiwan. The phylogenetic analysis performed by PAUP showed that all the new H5Nx viruses isolated in Taiwan belonged to clade <IP_ADDRESS>, except the old H5N2 circulating prior to 2015. HA and NA gene sequences derived from different avian influenza viruses, including A/Northern pintail/Washington/40964/2014 H5N2 (clade <IP_ADDRESS>b), A/duck/Taiwan/1702004/2017 H5N6 (clade <IP_ADDRESS>e), A/duck/Hyogo/1/2016 H5N6 (clade <IP_ADDRESS>e), A/Sichuan/26221/2014 H5N6 (clade <IP_ADDRESS>a), A/gyrfalcon/Washington/ 41088-6/2014 (H5N8) (clade <IP_ADDRESS>b) and A/chicken/Taiwan/x37/2016 H5N8 (clade <IP_ADDRESS>b), were used for lentiviral pseudovirus production, as described previously [16]. We further identified an H5N6-Sichuan subtype that exhibited high neutralizing titers against different subtypes [16]. A lentiviral vector-based vaccine is able to directly express the antigen of interest in target cells in vivo, which can effectively induce antigen-specific CD8+ T cell immune response [17]. Although several studies have demonstrated the protective efficacy in preclinical and clinical trials, it was still not approved to be used in humans to date. The main concern is the stability of viral integration in the host genome, which may activate the cellular protooncogene by integrated long terminal repeats (LTRs) or the transcriptional

interference and suppression by LTRs [18]. Therefore, we developed a potential vaccine candidate using a virus-like particles expression system that is non-infectious and had been approved to use in humans. In order to further investigate whether H5N6 Sichuan subtype would be a potential vaccine candidate for humans, we aimed to establish a virus-like particle expression system and further evaluate the immune response elicited by VLP and 293T mammalian cell-expressed protein in mice models.

#### **2. Materials and Methods**

#### *2.1. Cell Culture*

Insect cell lines, Spodoptera frugiperda ovarian cells (Sf21) and Trichoplusia ni ovarian cells (High FiveTM) were cultured at 28 ◦C in an incubator, without CO2 and water supplement. Sf21 cells were maintained in Grace's Insect medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Hyclone, Thermo Fisher Scientific, Carlsbad, CA, USA) for baculovirus production. For H5N6-Sichuan VLP production, High Five™ cells were cultured in serum-free HyClone™ SFM4 Insect medium (Hyclone) with 1% penicillin streptomycin mixtures (P/S).

293T cells and MDCK cells were cultured at 37 ◦C in an incubator with 5% CO2. 293T cells were maintained in DMEM supplemented with 10% FBS, 2% P/S, and 10 µM sodium pyruvate for the production of H5Nx pseudoviruses and 293T expressed H5+N6 proteins. MDCK cells were maintained in Eagle's MEM with 1 µg/mL trypsin, 1mM sodium pyruvate, 100 U/mL penicillin and 0.1 mg/mL streptomycin for titration of pseudoviruses and neutralization assay of VLP or mammalian cell-expressed proteins immunized mice serum against H5Nx pseudoviruses.

## *2.2. Generation of Virus-like Particle Expression Plasmid*

PCR products of NA were cloned into pBac-YCCRG3 plasmid containing an M1 sequence from an H7N9 subtype (pFastBac vector, gift from Dr. Yung-Chih Hu, NHRI) using KpnI and NheI restriction enzyme sites, named as pBac-YCCH7SCN6. By using BssHII and RsrII restriction enzyme sites, the amplified HA genes were cloned into the pBac-YCCH7SCN6, named as pBac3-SC. The inserted HA, NA sequences from H5N6 Sichaun subtype and M1 sequence from H7N9 subtype pBac3-SC plasmid were confirmed by Sanger sequencing. The pBac3-SC plasmid was transformed into a DH 10BacTM competent cell (Thermo Fisher Scientific) for transposition into bacmid. The colonies that contain the desired recombinant bacmid were identified by blue-white screening with 50 µg/mL kanamycin, 10 µg/mL tetracycline, 7 µg/mL gentamycin, 100 µg/mL Bluo-gal and 40 µg/mL IPTG. Sf21 cells were seeded in 6-well plates (8 × 105 cells/mL) and transfected with 2.5 µg/well of pBac3-SC plasmid by TransIT-Insect transfection reagent (Mirus Bio, Madison, WI, USA), and the supernatants were collected at 72 h posttransfection. The P1 viruses were immediately amplified in confluent 6-well plates of Sf21 cells and titrated by plaque assay for further experiments. The viruses were stored at −80 ◦C and protected from light.

### *2.3. Production of H5N6 VLPs*

The cultures were performed in 250 mL shake flasks. Each flask was inoculated with 2 × 105 cells/mL with a working volume of 100 mL. After three days, when the density of High Five™ cells reached 1.6 × 106 cells/mL, the cells were infected with 1 × 108 to 1 × 109 pfu/mL of recombinant baculovirus. The H5N6 VLPs were harvested at 3 days post-infection by centrifugation at 500× *g* (Kubota TA3780) at 4 ◦C for 10 min, and the yield of H5N6 VLPs was further determined by hemagglutination assay.

#### *2.4. Sucrose Density Gradient for Vlp Purification*

For VLPs concentration and purification, VLPs were first UV inactivated for 30 min. It was filtered with 0.45 µm filters to remove cell debris and precipitated by ultracentrifugation at 360,000× *g* (Beckman L90-K ultracentrifuge, 70Ti rotor) at 4 ◦C for 4 h with a

20% sucrose cushion and a drop of glycerol at the bottom of the tube. The pellets were resuspended with PBS and loaded on the top of a 20–60% sucrose gradient. After ultracentrifugation at 285,000× *g* for 2.5 h, the visible band at 45% sucrose was collected (Beckman L90-K ultracentrifuge, 40Ti rotor).

#### *2.5. SDS-PAGE and Western Blot Analysis for Protein Composition of VLP*

Purified H5N6 VLP samples were quantified by Qubit protein assay. Approximately 2 µg of purified H5N6 VLPs were separated on 12% SDS-PAGE. To confirm the purification efficiency of the sucrose density gradient, the SDS-PAGE gel was stained using silver staining. To detect the presence of target HA, the separated proteins on the SDS-PADE were transferred onto the PVDF membrane, blocked with 5% non-fat milk, and incubated with primary antibody (1:5000, avian flu HA antibody, Mab, Sino Biological, Beijing, China) at 4 ◦C overnight. The blots were then incubated with secondary antibody (1:5000, goat anti-mouse IgG, HRP-labeled, KPL) and developed using enhanced chemiluminescence (ECL, Bio-Rad, Hercules, CA, USA) substrate. All the images were captured by Amersham Imager 600.

#### *2.6. Transmission Electron Microscopy for Analysis of VLP Morphology*

Purified VLPs were absorbed onto a plasma-discharged copper grid. After washing with PBS, 2% phosphotungstic acid was applied for negative staining. The particles were observed under a transmission electron microscope (JEOL JEM-1400), performed by Core Facility Center, National Cheng Kung University, Taiwan.

#### *2.7. Hemagglutination Assay for HA Titer of VLP*

Turkey RBCs were washed with ice-cold PBS several times until the PBS remained clear after centrifugation at 500× *g* (Kubota TA3780) for 5 min. A 0.75% of turkey RBC suspension with PBS was then prepared. To measure the hemagglutination titers of VLP, VLPs were first diluted 10-fold, and a 2-fold serial dilution with PBS was performed on a 96-well plate at a total volume of 50 µL/well. Afterwards, 50 µL of 0.75 % turkey RBC suspension was added to each well and incubated at room temperature for 30 min to 1 h [19]. In the presence of VLPs, the HA on the surface of VLPs will bind to the sialic acid on the RBC, showing hemagglutination. The highest dilution titer that had shown hemagglutination was regarded as the hemagglutination titer.

#### *2.8. Expression of H5-HA and N6-NA Protein in 293T Mammalian Cell System*

293T cells were seeded in 15 cm dishes (0.75–0.9 × 106 /mL, 20 mL/dish). After 16 h of incubation, plasmids with pTT5 vector (pTT5-SCH5, pTT5-SCN6, Leadgene Biomedical, Inc., Tainan, Taiwan) were transfected with 15 µg of H5 or N6 plasmid DNA per dish, respectively, by Hyfect transfection reagent (Leadgene). Dishes were refreshed with cell medium (FreeStyle 293 expression medium) 24 h later. At 72 h post-transfection, supernatant was collected and centrifuged at 500× *g* to remove the cell debris. The supernatant was further precipitated via ammonium sulfate. The desired HA and NA proteins were visualized by Western blot with anti-His tag mAb (1:5000, Leadgene). The blots were then incubated with secondary antibody (1:5000, goat anti-mouse IgG, HRP-labeled, KPL) and developed using enhanced chemiluminescence (ECL, Bio-Rad, Hercules, CA, USA) substrate. All the images were captured by Amersham Imager 600.

#### *2.9. Mouse Immunization*

For H5N6 VLPs and H5+N6 protein antisera, 6- to 8-week-old BALB/c mice (4 mice per group) were initially immunized with the purified H5N6 VLPs and 293T cell expressed H5+N6 proteins with adjuvant (Freund's complete adjuvant), which was performed by Leadgene Biomedical, Inc, Taiwan (IACUC-11006003). Ten mg of VLP in PBS or recombinant proteins emulsified in Complete Freund's Adjuvant (CFA, Sigma) for priming and Incomplete Freund's Adjuvant (IFA, Sigma) for the boost in a total of 100 µL were

immunized three times at one-week intervals. Serum samples were collected by submandibular blood collection at three time-points: pre-immunization, day 14 and day 35 post-immunization (antiserum of H5+N6 proteins were not collected at day 14, since these two immunizations were performed at separate times). At day 35, mice were sacrificed and the cardiac chambers punctured for the serum sample collection. The end-point serum samples were used for neutralization assay, and the serums collected at different time-points were used to measure the induction of cytokine levels. Each serum was collected from 4 BALB/c mice and pooled. All the serum samples were aliquoted and stored at −80 ◦C until further use.

#### *2.10. Enzyme-Linked Immunosorbent Assay (ELISA) for Specific IgG Titer*

The 96-well ELISA plates were coated with 50 µL of PBS containing 1 µg/mL of H5N6 VLP or 293T expressed H5+N6 proteins, respectively. The plates were incubated at room temperature for an hour and blocked with 3% skim milk in 0.05% PBS-T. Serum samples were 100-fold and then two-fold serially diluted with blocking buffer. After being washed several times with 0.05% PBS-T, the coated plates were then incubated with 50 µL of serially diluted serum, followed by incubating with HRP-conjugated anti-mouse IgG antibodies (1:5000, Leadgene) at 37 ◦C for 30 min. Lastly, 50 µL of tetramethyl benzidine dihydrochloride (TMB) substrate was added in each well and stopped with 50 µL/well of 1 N H2SO4 two minutes later. The absorbance at 450 nm was measured using an ELISA plate reader (Meter-tech M965+).

#### *2.11. Neutralization Assay for Neutralizing Antibody Titer*

To determine the neutralizing antibody titer of H5N6 VLPs and 293T expressed H5+N6 protein immunized mouse serum against H5Nx pseudoviruses, neutralization assay was performed as previously reported [16]. MDCK cells were seeded in 24-well plates with 7.5 × 104 cells/well and incubated at 37 ◦C for 16 h. Mouse antisera were complement inactivated at 56 ◦C for 30 min before neutralization assay. H5Nx pseudoviruses (5 × 106–1 × 107 TU/mL) were co-incubated with 2-fold serially diluted mouse serum in a 37 ◦C incubator for 30 min. MDCK cells were then infected with the mixture and adsorbed for 1.5 h. The serum-free DMEM medium was added after adsorption. The medium was changed to MDCK medium (Eagle's MEM with 1 µg/mL trypsin, 1mM sodium pyruvate, 100 U/mL penicillin and 0.1 mg/mL streptomycin) after 24 h. Infected cells with fluorescence were fixed with 2% paraformaldehyde at 4 days post-infection, and the percentage of fluorescence of infected cells was measured by flow cytometer. The neutralization titers (NT50) were defined as 50% reduction of transduction unit (TU) in both duplications compared with the transduction unit of virus control.

#### *2.12. Multiplex Analysis of Mouse Cytokine Productions*

Th1/ Th2-related multiple cytokines were measured by BD cytometric bead array. Five beads with distinct fluorescence intensities had been coated with capture antibodies specific for IL-2, IL-4, IL-5, TNF, and IFN-γ. Antisera against H5N6 VLPs and 293T cellexpressed H5+N6 proteins were collected at different time-points and were diluted threefold with assay diluent. Standard beads were prepared following the instruction manual. The captured beads were mixed with the serum samples and the PE detection reagents and incubated at room temperature for two hours. After washing with 1 mL of wash buffer for each tube, the tubes were centrifuged at 200× *g* for 5 min. Lastly, the pellet was resuspended with 300 µL of wash buffer, and the fluorescence was analyzed by FACS Calibur flow cytometer.

### **3. Results**

*3.1. Establishment of the H5N6 Virus-like Particles Expression System Using Baculovirus-Insect Cell System*

3.1.1. Construction of the VLP-Expressing Plasmid

In our previous study, we established a lentiviral pseudovirus system, which is able to rapidly screen out the broad-spectrum virus strain during outbreaks and examine vaccine immunogenicity. Through the neutralization assay of the generated avian influenza H5 subtypes pseudoviruses antisera, we found that H5N6-Sichuan subtype antisera exhibited better cross-protectivity among H5N2, H5N6 and H5N8 subtypes [16]. In order to further investigate whether H5N6-Sichuan strain could be a potential vaccine candidate, we first generated the virus-like particles derived from H5N6-Sichuan strain by using a Bac-to-Bac baculovirus expression system. The full-length HA and NA derived from H5N6- Sichuan were PCR amplified and cloned into pFASTBac vector. The plasmid was then transformed into DH 10BacTM competent cells, which contain a bacmid DNA. Using miniattTn7 site-specific transposition, the inserted foreign genes will disrupt the LacZα gene, and the recombinant bacmid containing the desired insert can be identified by blue-white selection (Figure 1A). *Viruses* **2022**, *14*, 925 7 of 15

type was generated by mini attTn7 transposition in DH10bac competent cells. (**B**) Recombinant baculovirus collected from Sf21 cells were used to infect High Five cells for VLP production. (**C**) The expression of the HA, NA and M1 proteins on the VLPs was visualized by SDS-PAGE with silver

**Figure 2.** Purification of H5N6 VLPs by sucrose gradient. (**A**) Purification steps for H5N6 VLPs purification. The H5N6 VLPs were precipitated by a 20% sucrose cushion first and further purified by a 20%–60% sucrose density gradient. (**B**) A visible band at 45% of sucrose fraction indicated the

purified H5N6 VLPs.

was generated by mini attTn7 transposition in DH10bac competent cells. (**B**) Recombinant baculovirus collected from Sf21 cells were used to infect High Five cells for VLP production. (**C**) The expression of the HA, NA and M1 proteins on the VLPs was visualized by SDS-PAGE with silver staining (lane 1) and confirmed by Western blot using avian flu HA antibody (Sino Biological, 11048-MM06) (lane 2) and Lenti-H5N6-SC mouse antisera (lane 3) (indicated by red arrow).

#### 3.1.2. Generation of the H5N6 Virus-like Particles from Sf21 and High Five Cells

*Viruses* **2022**, *14*, 925 7 of 15

After we confirmed the inserted gene in the recombinant bacmid by sequencing, the recombinant bacmid were amplified in LB broth and extracted with Qiagen plasmid midi kit. Previously, Lai et al. had demonstrated that Sf21 cells are suitable for a large amount of baculovirus production, while the VLPs are more easily generated by High Five™ cells [20]. Therefore, we transfected the recombinant bacmid with TransIT transfection reagent into Sf21 cells cultured in an adherent flask to generate the recombinant baculovirus. After three days of culture, the recombinant baculoviruses budded into the supernatant and were immediately propagated by infecting High Five™ cells. High Five™ cells were infected with MOI 1 of recombinant baculovirus from the above culture of Sf21 cells (Figure 1B). SDS-PAGE was first performed to analyze the production of the H5N6 VLPs. The results showed the presence of HA (64.08 kDa) and NA (52.4 kDa) proteins, followed by confirmation by Western blot with specific antibodies of avian flu H5N1 HA antibody and mouse antisera against lenti-H5N6 Sichuan pseudoviruses (Figure 1C).

#### 3.1.3. Precipitation and Purification of H5N6 VLP by Sucrose Density Gradient

In order to investigate whether the H5N6 VLPs induced immunogenicity, purification and concentration of H5N6 VLPs is needed for mice immunization. Previous studies have shown that norovirus capsid VLPs purified by sucrose density gradient obtained high yield and were more stable compared to other methods [21]. At day three post-infection, 250 mL of culture supernatant was clarified and precipitated by low-speed centrifugation with a 20% sucrose cushion and a drop of glycerol at the bottom of the tube. The pellet was resuspended with 1.5 mL of PBS and loaded onto the 20–60% sucrose density gradient. After ultracentrifuge, a visible band at 45% sucrose was observed, which indicated the purified H5N6 VLPs. Fractions from 15%, 25%, 35% and 45% sucrose were collected from the bottom for further analysis of the purification efficiency (Figure 2A,B). **Figure 1.** Production of H5N6 VLPs in the baculovirus expression vector system. (**A**) A schematic diagram of H5N6 recombinant bacmid construction. The recombinant bacmid carrying the hemagglutinin (HA), neuraminidase (NA) and matrix protein 1 (M1) genes derived from the H5N6 subtype was generated by mini attTn7 transposition in DH10bac competent cells. (**B**) Recombinant baculovirus collected from Sf21 cells were used to infect High Five cells for VLP production. (**C**) The expression of the HA, NA and M1 proteins on the VLPs was visualized by SDS-PAGE with silver staining (lane 1) and confirmed by Western blot using avian flu HA antibody (Sino Biological, 11048- MM06) (lane 2) and Lenti-H5N6-SC mouse antisera (lane 3) (indicated by red arrow).

**Figure 2.** Purification of H5N6 VLPs by sucrose gradient. (**A**) Purification steps for H5N6 VLPs purification. The H5N6 VLPs were precipitated by a 20% sucrose cushion first and further purified by a 20%–60% sucrose density gradient. (**B**) A visible band at 45% of sucrose fraction indicated the purified H5N6 VLPs. **Figure 2.** Purification of H5N6 VLPs by sucrose gradient. (**A**) Purification steps for H5N6 VLPs purification. The H5N6 VLPs were precipitated by a 20% sucrose cushion first and further purified by a 20–60% sucrose density gradient. (**B**) A visible band at 45% of sucrose fraction indicated the purified H5N6 VLPs.

#### *3.2. Characterization of H5N6 VLPs Properties*

3.2.1. The Protein Composition of the H5N6 VLPs

In order to verify whether the major immune epitope, HA proteins, still remained in the H5N6 VLPs after purification, we detected the proteins by Western blot using avian flu anti-HA antibody. SDS-PAGE with silver stain was also performed to examine the purity of

H5N6 VLPs. The results showed that the fraction of 45% sucrose not only had the majority of the HA but also the NA and M1 proteins; furthermore, other proteins derived from the High Five cells were mostly removed after purification (Figure 3A). *Viruses* **2022**, *14*, 925 9 of 15

**Figure 3.** Protein expression, hemagglutination and structure of H5N6 VLP after sucrose gradient purification. (**A**) The expression of the HA, NA and M1 proteins on the VLPs at different percentages of sucrose fraction was visualized by SDS-PAGE with silver staining and confirmed by Western blot using avian flu HA antibody, Mab (Sino Biological, 11048-MM06). The pellet of H5N6 VLPs was precipitated by a 20% sucrose cushion. (**B**) Comparison of hemagglutination activity between unpurified and purified VLPs (at 45% sucrose fraction) by hemagglutination assay. The serum sample started from a 10-fold dilution. (**C**) The H5N6 VLPs after sucrose gradient purification were imaged using transmission electron microscopy by negative staining. Scale bars, Left: 200 nm, Right: 100 nm. **Figure 3.** Protein expression, hemagglutination and structure of H5N6 VLP after sucrose gradient purification. (**A**) The expression of the HA, NA and M1 proteins on the VLPs at different percentages of sucrose fraction was visualized by SDS-PAGE with silver staining and confirmed by Western blot using avian flu HA antibody, Mab (Sino Biological, 11048-MM06). The pellet of H5N6 VLPs was precipitated by a 20% sucrose cushion. (**B**) Comparison of hemagglutination activity between unpurified and purified VLPs (at 45% sucrose fraction) by hemagglutination assay. The serum sample started from a 10-fold dilution. (**C**) The H5N6 VLPs after sucrose gradient purification were imaged using transmission electron microscopy by negative staining. Scale bars, Left: 200 nm, Right: 100 nm.

#### 3.2.2. The Hemagglutination Property of the H5N6 VLPs Remained

Hemagglutinin glycoprotein was reported as the receptor-binding protein of the influenza virus. In order to examine whether sucrose gradient purification affects the hemagglutination property, hemagglutination assay was performed. Approximately 250 mL of VLPs in the culture supernatant were precipitated and resuspended with 4 mL of PBS. Then, 4 mL of concentrated VLP was purified and the final volume of purified VLP was about 1 mL. The result indicates that the hemagglutination property of H5N6 VLPs purified from

sucrose gradient ultracentrifugation can reach 5120 HAU/ 50 µL. In contrast, the HA titer of H5N6 VLPs before concentration and purification was only 80 HAU/ 50 µL (Figure 3B).

#### 3.2.3. The Morphology of the H5N6 VLPs Remained Structurally Intact after Purification

To examine the formation of the H5N6 VLP, we directly visualized the particles using negative stain by transmission electron microscopy. The results showed that H5N6 VLPs were successfully prepared with morphology that resembled the native virions without genetic materials. The particles were homogenous and approximately 110 nm in size (Figure 3C). These data suggested that VLPs derived from H5N6-Sichuan strain can be successfully generated through a baculovirus expression system. After being concentrated and purified by a sucrose cushion and density gradient, H5N6 VLPs maintained a functional hemagglutination property and were structurally intact.

#### *3.3. Generation of 293T Mammalian Cell Expressed H5+N6 Protein*

*Viruses* **2022**, *14*, 925 9 of 15

To investigate whether the virus-like structure of VLPs is helpful to induce superior immunogenicity, we also used 293T mammalian cells to express H5 HA and N6 NA genes derived from the H5N6 Sichuan strain for comparison. The clones of HA and NA constructed in pTT5 expression vector were performed by Leadgene Biomedical, Inc. The plasmids were separately transfected into a different dish of 293T cells, the supernatant containing the desired H5 HA and N6 NA proteins was collected at day 4 post-transfection and the proteins were precipitated by ammonium sulfate (Figure 4A). The H5 and N6 proteins were observed by Western blotting with anti-His tag antibody and pooled for mice immunization (Figure 4B). **Figure 3.** Protein expression, hemagglutination and structure of H5N6 VLP after sucrose gradient purification. (**A**) The expression of the HA, NA and M1 proteins on the VLPs at different percentages of sucrose fraction was visualized by SDS-PAGE with silver staining and confirmed by Western blot using avian flu HA antibody, Mab (Sino Biological, 11048-MM06). The pellet of H5N6 VLPs was precipitated by a 20% sucrose cushion. (**B**) Comparison of hemagglutination activity between unpurified and purified VLPs (at 45% sucrose fraction) by hemagglutination assay. The serum sample started from a 10-fold dilution. (**C**) The H5N6 VLPs after sucrose gradient purification were imaged using transmission electron microscopy by negative staining. Scale bars, Left: 200 nm, Right: 100 nm.

**Figure 4.** Expression of HA and NA proteins derived from H5N6 Sichuan strain in 293T mammalian cells. (**A**) Schematic diagram of mammalian cell expressed H5-HA and N6-NA proteins. The HA and NA genes were cloned into a pTT5 protein expression vector and transfected into 293T cells. After 4 days of culture, the H5 and N6 proteins were collected from the culture supernatant. (**B**) The presence of H5 (62 kDa) and N6 (56.4 kDa) proteins was verified by anti-His tag antibody in Western blot.

#### *3.4. Investigation of the Immune Response Elicited by the H5N6 VLPs and 293T Cell-Expressed H5+N6 Protein*

3.4.1. Specific Total IgG Antibodies Were Produced in Immunized Mice

Groups of BALB/c mice were immunized by intraperitoneal route (i.p) with purified H5N6 VLPs or 293T cell expressed H5+N6 proteins. Serum samples were collected at pre-immunization and day 35 post-immunization (Figure 5A). The level of total IgG was measured by ELISA. The results showed that pre-immunized antisera had no specific IgG antibody against H5N6 VLPs and 293T cell-expressed H5+N6 proteins (Figure 5B,C). At day 35 post-immunization, IgG antibody end-point titers against their immunized antigens were both 100 × 2 10 in H5N6 VLPs and 293T cell-expressed H5+N6 proteins

blot.

*H5+N6 Protein*

antisera, suggesting that mice immunized with H5N6 VLPs and 293T cell-expressed H5+N6 proteins successfully generated the IgG antibodies against the antigens (Figure 5B,C). suggesting that mice immunized with H5N6 VLPs and 293T cell-expressed H5+N6 proteins successfully generated the IgG antibodies against the antigens (Figure 5B,C).

**Figure 4.** Expression of HA and NA proteins derived from H5N6 Sichuan strain in 293T mammalian cells. (**A**) Schematic diagram of mammalian cell expressed H5-HA and N6-NA proteins. The HA and NA genes were cloned into a pTT5 protein expression vector and transfected into 293T cells. After 4 days of culture, the H5 and N6 proteins were collected from the culture supernatant. (**B**) The presence of H5 (62 kDa) and N6 (56.4 kDa) proteins was verified by anti-His tag antibody in Western

*3.4. Investigation of the Immune Response Elicited by the H5N6 VLPs and 293T Cell-Expressed*

Groups of BALB/c mice were immunized by intraperitoneal route (i.p) with purified H5N6 VLPs or 293T cell expressed H5+N6 proteins. Serum samples were collected at preimmunization and day 35 post-immunization (Figure 5A). The level of total IgG was measured by ELISA. The results showed that pre-immunized antisera had no specific IgG antibody against H5N6 VLPs and 293T cell-expressed H5+N6 proteins (Figure 5B,C). At day 35 post-immunization, IgG antibody end-point titers against their immunized antigens were both 100 × 210 in H5N6 VLPs and 293T cell-expressed H5+N6 proteins antisera,

3.4.1. Specific Total IgG antibodies were Produced in Immunized Mice

*Viruses* **2022**, *14*, 925 10 of 15

**Figure 5.** Specific IgG antibody responses in mice immunized with H5N6 VLP or 293T cell-expressed H5+N6 protein. (**A**) Experimental design for mice immunization (n = 4 per group). The mice were immunized and boosted at one-week intervals after 14 days post-immunization (d.p.i). Serum samples were collected by submandibular blood collection at pre-immunization (Day 0), day 14 and day 35 post-immunization (Day 35). The total specific IgG level at Day 0 and Day 35 in H5N6 VLP antiserum (**B**) and H5+N6 protein antiserum (**C**) against their immunized antigens were analyzed by ELISA. **Figure 5.** Specific IgG antibody responses in mice immunized with H5N6 VLP or 293T cell-expressed H5+N6 protein. (**A**) Experimental design for mice immunization (n = 4 per group). The mice were immunized and boosted at one-week intervals after 14 days post-immunization (d.p.i). Serum samples were collected by submandibular blood collection at pre-immunization (Day 0), day 14 and day 35 post-immunization (Day 35). The total specific IgG level at Day 0 and Day 35 in H5N6 VLP antiserum (**B**) and H5+N6 protein antiserum (**C**) against their immunized antigens were analyzed by ELISA.

#### 3.4.2. Neutralization Antibodies Were Higher in VLP Immunized Mice

To compare the immunogenicity induced by H5N6 VLPs and 293T cell-expressed H5+N6 protein in mice, after three rounds of immunization with each immunogen formulated with adjuvant, mice were sacrificed and serum samples were collected by submandibular blood collection. Neutralization titers of antisera were distinguished by over 50% reduction of virus transduction in duplicate. The results demonstrated that H5+N6 protein antisera only elicited partial cross-reactive antibodies, ranging from 80 to 320 against different subtypes of avian influenza pseudoviruses, including H5N2, H5N6 and H5N8. In contrast, H5N6 VLPs antisera exhibited higher neutralizing antibody titers, between 640 to 1280 (Table 1). These findings indicated that H5N6 VLP can induce higher neutralizing antibodies than 293T cell-expressed H5+N6 proteins, suggesting that the structure of VLP is helpful in displaying the immune epitope on the surface, which might result in better cross-reactive neutralizing antibodies.

**Antisera**

better cross-reactive neutralizing antibodies.

*Viruses* **2022**, *14*, 925 11 of 15

3.4.2. Neutralization Antibodies were Higher in VLP Immunized Mice

To compare the immunogenicity induced by H5N6 VLPs and 293T cell-expressed H5+N6 protein in mice, after three rounds of immunization with each immunogen formulated with adjuvant, mice were sacrificed and serum samples were collected by submandibular blood collection. Neutralization titers of antisera were distinguished by over 50% reduction of virus transduction in duplicate. The results demonstrated that H5+N6 protein antisera only elicited partial cross-reactive antibodies, ranging from 80 to 320 against different subtypes of avian influenza pseudoviruses, including H5N2, H5N6 and H5N8. In contrast, H5N6 VLPs antisera exhibited higher neutralizing antibody titers, between 640 to 1280 (Table 1). These findings indicated that H5N6 VLP can induce higher neutralizing antibodies than 293T cell-expressed H5+N6 proteins, suggesting that the structure of VLP is helpful in displaying the immune epitope on the surface, which might result in

**Table 1.** Neutralizing antibody titers of H5N6 VLP-induced antisera and 293T cell expressed H5+N6 protein-induced antisera against various H5Nx pseudoviruses. **Lenti-H5N2 Washington Lenti-H5N6 TW17 Lenti-H5N6 Hyogo Lenti-H5N6 Sichuan Lenti-H5N8 Washington Lenti-H5N8 TWX37**

**Pseudoviruses Tested (Neutralizing Antibody Titer)**

pressed H5+N6 protein-induced antisera against various H5Nx pseudoviruses.

**Table 1.** Neutralizing antibody titers of H5N6 VLP-induced antisera and 293T cell ex-

Th1 and Th2 cells play an important role in adaptive immunity. After immunization,

#### 3.4.3. Cytokine Production Th1 cells secrete IFN-γ, TNF and IL-2 to mediate cytotoxic T cell and opsonizing antibod-

Th1 and Th2 cells play an important role in adaptive immunity. After immunization, Th1 cells secrete IFN-γ, TNF and IL-2 to mediate cytotoxic T cell and opsonizing antibodies. Th2 cells stimulate the humoral immune response by secreted IL-4 and IL-5, which can regulate B cell proliferation and induce antibody production. We measured the levels of multiple cytokines to investigate the 293T cell-expressed H5+N6 protein or H5N6 VLPs induced immune response. H5+N6 protein and H5N6 VLPs antisera were collected at preimmunization, day 14 (H5N6 VLP only) and day 35 post-immunization. The results demonstrated that H5N6 VLPs can induce a higher production of Th1- and Th2-related cytokines (Figure 6). These data suggest that H5N6 VLPs have the advantage of particle formation that can display HA protein on the surface, which stimulated higher levels of cytokine production to eventually help to generate more neutralizing antibodies. ies. Th2 cells stimulate the humoral immune response by secreted IL-4 and IL-5, which can regulate B cell proliferation and induce antibody production. We measured the levels of multiple cytokines to investigate the 293T cell-expressed H5+N6 protein or H5N6 VLPs induced immune response. H5+N6 protein and H5N6 VLPs antisera were collected at preimmunization, day 14 (H5N6 VLP only) and day 35 post-immunization. The results demonstrated that H5N6 VLPs can induce a higher production of Th1- and Th2-related cytokines (Figure 6). These data suggest that H5N6 VLPs have the advantage of particle formation that can display HA protein on the surface, which stimulated higher levels of cytokine production to eventually help to generate more neutralizing antibodies.

**Figure 6.** Cytokine profiles of mice immunized with H5N6 VLPs or 293T cell-expressed H5+N6 proteins. Antisera of vaccinated mice were collected at different timepoints: pre-immunization, day 14 and day 35 post-immunization The cytokines (IL-2, IL-4, IL-5, IFN-γ, and TNF) in antisera were measured by cytometric bead array (CBA) multiplex assay. Serum samples collected at each time-point were measured in duplicates.

#### **4. Discussion**

Highly pathogenic avian influenza epidemics pose serious threats to socioeconomic and animal health. In the past decades, multiple outbreaks of H5 HPAI viruses have occurred, especially in Asian countries, including H5N1 in China and Vietnam, H5N2 in North America and Taiwan, H5N6 in China, Laos and Vietnam, and H5N8 in China, Europe, Japan, North America, South Korea and Taiwan [22]. Recently, reinvasion of H5N8 subtype viruses in Japan resulted in the cull of 1.9 million birds in October 2020 [23]. In order to develop an effective vaccine with robust protectivity for epidemic prevention, we established a VLP expression system and generated VLPs derived from a broad-spectrum virus

strain, H5N6-Sichuan subtype, as a vaccine candidate. We compared the immune response elicited by H5N6 VLPs and 293T mammalian cell-expressed H5+N6 proteins. The results showed that Th1- and Th2-related cytokines were induced by both antigens, while the level of cytokines was higher in H5N6 VLPs antisera, which resulted in a higher titer of cross-reactive neutralizing antibodies. Combined with the lentiviral–pseudovirus system, we can efficiently produce a large amount of VLPs carrying HA from a broad-spectrum virus strain during outbreaks, which may be helpful to the poultry industry and public health in the human population.

Evaluation of humoral and cellular immunity is an essential aspect to examine the effectiveness of vaccination. CD4+ T cells are activated via the secretion of Th2-related cytokines (IL-4, IL-5) to stimulate B cells for the production of antibodies to prevent viral infection. CD8+ T cells, stimulated by Th1-related cytokines, are responsible for killing the infected cells. ELISA was used to detect all the binding antibodies in the antisera. In our results, H5N6 VLPs induced a higher production of Th1- and Th2-related cytokines that lead to higher titers of neutralizing antibody in equal amounts of specific IgG level, measured by ELISA, in H5N6 VLPs and 293T cell-expressed H5+N6 protein antisera. We suggested that H5N6 VLP, with the advantage of displaying the immune epitope on the particle, exhibited a higher titer of neutralizing antibody. A large portion of specific IgG antibodies detected in 293T cell-expressed H5+N6 protein antisera may be the nonneutralizing antibody. Although non-neutralizing antibody is not able to prevent viral infection, it may still contribute to antiviral protection through other antibody-mediated effects, such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or antibody-mediated complement-dependent cytotoxicity (CDC) [24].

In our findings, antisera against H5N6 VLP exhibited a neutralizing antibody titer of 1280 against both Lenti-H5N6-Sichuan and Lenti-H5N8-Washington subtypes, which can be correlated to their close proximity on the antigenic cartography of avian influenza virus H5Nx pseudoviruses [16]. In addition to generating broad-spectrum VLPs with single HA as vaccine candidate, scientists had also proposed some other strategies for VLP-based vaccine development. Kang et al. exploited multi-clade VLPs carrying two HAs derived from two clades, clade <IP_ADDRESS>c and clade <IP_ADDRESS>c, within a single vector, and found that multi-clade VLPs not only show high productivity but also induced effective antibodies [14]. In contrast to variable surface antigens of HA and NA, the ion channel protein, Matrix protein 2 ectodomain (M2e), represents an alternative antigen target for AVI vaccine, since M2e protein has a relatively conserved sequence and plays a critical role in virus assembly and budding during replication. Mice intranasally immunized with a pandemic live attenuated vaccine supplemented with M2e-VLPs were prevented from weight loss and had low viral titers when challenged with heterosubtypic virus, indicating that the combination of split vaccine with M2e-VLPs provides cross protection by eliciting IgG, IgA antibodies as well as M2e-induced T cell response [25]. Apart from targeting the conserved epitope, Carter et al. have reported a new method, computationally optimized broadly reactive antigens (COBRA), in order to overcome the deficiency of weak protectivity induced by M2e or NP antigens when challenged with high-dose viruses. They provided a consensus HA sequence using multiple rounds of consensus building to generate a candidate antigen and expressed the COBRA HA proteins on VLP for broadly reactive, functional antibody responses [26].

Although vaccination is helpful to control the spread of AIV, even highly immunogenic AIV strains were not able to elicit sufficient immune response without appropriate adjuvants. Previous studies had evaluated the immune response between different adjuvants for AIV vaccine in poultry [27,28]. In this study, we vaccinated the mice model with addition of complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) to gain a strong and long-lasting immune response. Both of them are a water-in-oil emulsions mixture prepared at a ratio of 85% mineral oil and 15% emulsifier, as described by Stone et al. [29]. The difference between IFA and CFA is that CFA contains additional

heat-killed mycobacteria (Mycobacterium tuberculosis), which dramatically enhanced the immune responses by trafficking the macrophage and other immune cells to the inoculation site. In general, CFA was used with initial injection for the potentiation of T helper cells that leads to the production of immunoglobulins and effector T cells. IFA is for subsequent immunization following a strong Th2 immune response through the formation of a depot at the injection site and the stimulation of antibody producing plasma cells. The cytokine responses to H5N6-VLPs and H5N6 protein were increased to a certain degree when induced by CFA and IFA adjuvants. Since the immune response to CFA, IFA and commercial oil-based adjuvants such as Montanide ISA 70VG, 71VG, 760VG, GEL01, etc., were in a large variation [28], the use of CFA and IFA might not be able to exactly mimic the clinical situations. We used CFA and IFA for the purpose of evaluating immune response between H5N6-VLPs and H5N6 protein, while ISA 71 VG and Montanide ISA VG70 oil adjuvant might be a better choice to compare the results with others [14,30].

Taiwan has not approved the use of AIV vaccine to date. The main concerns are that a higher virus mutation rate may result in gene reassortment triggered by inactivated and live-attenuated vaccine, and there may be asymptomatic transmission between vaccinated animals. The distinct host range of AIVs also makes it difficult to raise the population immunity. Although there is still a wide spectrum of opinions about the issue of whether vaccination can actually control the spread of virus, Walker et al. have used the Bayesian Markov Chain Monte Carlo (MCMC) data augmentation technique to evaluate the impact of the vaccination in northern Vietnam. The results demonstrated a significant decrease in the transmissibility of infection following vaccination and the wave of outbreaks were less intense [31]. They suggested that vaccination is helpful to reduce the transmission of infection. In conclusion, based on our results, we suggest that H5N6-VLPs might be a potential vaccine candidate, since they induced potent humoral and cellular immune response to produce high levels of cross-reactive neutralizing antibodies and cytokines. The strategy of generating a broad-spectrum VLP with single HA as a vaccine candidate may be feasible during urgent AIV outbreaks.

**Author Contributions:** Y.-H.Y., C.-H.T. and J.-R.W. designed the experiments. Y.-H.Y. and C.-H.T. performed the experiments. Y.-H.Y., C.-H.T. and J.-R.W. analyzed the data. Y.-H.Y., D.C. and J.-R.W. wrote the manuscript. Y.-F.W. and J.-R.W. supervised the project and were in charge of conceptualization, funding acquisition and resources. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was financially supported by grants from the National Health Research Institutes (09AI-IVGP09-038, IV-109-PP-13, and IV-110-PP-115).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data that support the findings of this study are available within the article.

**Acknowledgments:** The authors would like to thank Yung-Chih Hu and Yu-Chieh Cheng of NHRI for providing the plasmids and their advice for VLP generation.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**4. Conclusions**

In this work, the hydrothermal conversion of CO2 captured as sodium bicarbonate and ammonium carbamate was studied. Glucose was used as a reducing agent, and metal and metal oxides (Cu, Ni, Pd/C 5%, Ru/C 5%, Fe2O3 and Fe3O4), as well as activated carbon (C), were used as catalysts. The main products of the reaction with ammonium carbamate were formic acid, acetic acid and lactic acid.

The yields of formic acid, acetic acid and lactic acid obtained by the reduction of ammonium carbamate were much lower (less than 25%) than those observed when sodium bicarbonate was used as the carbon source (less than 53%).

For ammonium carbamate experiments, C and Fe3O4 promoted higher yields of FA over AA and LA in comparison to the rest of the catalysts and improved the yield of FA in comparison to the sample without catalyst.

In the experiments with sodium bicarbonate, C and Fe3O4 appeared to be the most promising catalysts for improving the yield of formic acid. The origin of the carbon forming formic acid was investigated by using NaH13CO3. It was found that although C and Fe3O4 achieved the highest total formic acid yield, they seem to favor the oxidation of glucose instead of the reduction of CO2. However, it should be noted that even though Pd/C 5%, Ni and Ru/C 5% yields of total formic acid were lower, they were shown to be more selective in producing formic acid from CO2 than the other catalysts. This aspect is important when considering the selection of a catalyst for making a process that primarily promotes a higher conversion of the carbon source.

**Author Contributions:** Conceptualization, M.D.B., F.A.M., Á.M. and M.I.C.; methodology, M.D.B. and M.I.C.; formal analysis, M.I.C.; investigation, M.D.B. and M.I.C.; resources, M.D.B., F.A.M., Á.M. and M.I.C.; writing—original draft preparation, M.D.B. and M.I.C.; supervision, M.D.B., F.A.M. and Á.M.; funding acquisition, M.D.B. and Á.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Ministerio de Ciencia y Universidades by project RTI2018- 097456-B-I00 and by the Regional Government of Castilla y León and the EU-FEDER program (CLU-2019-04).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** M.I.C. acknowledges Universidad de Valladolid and Banco de Santander for the predoctoral grant. The authors acknowledge Laboratorio de Técnicas Instrumentals UVa for their assistance in the NMR and SEM analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.