Immune-Enhancing Effect of Acanthopanax gracilistylus W. W. Smith Polysaccharides and Liposomes as Dilutions of Chicken Newcastle Disease Vaccine

TL;DR

This study shows that polysaccharides from Acanthopanax gracilistylus and their liposomes boost chicken immune responses to vaccines, especially when used as adjuvants.

Contribution

AGSPL liposomes are shown to be more effective than AGSP in enhancing vaccine immunogenicity in chickens.

Findings

AGSP and AGSPL significantly increased chicken antibody levels and cytokine concentrations after vaccination.

AGSPL outperformed AGSP in promoting immune cell proliferation and enhancing vaccine efficacy.

AGSPL has potential as a natural, effective vaccine adjuvant or diluent for poultry.

Abstract

This study aims to investigate the immunostimulatory effects of Acanthopanax gracilistylus W. W. Smith Polysaccharide and its liposomes on vaccine immunogenicity. Research has revealed that these two substances (particularly liposomes) effectively stimulate the proliferation of immune cells in chickens. Animal experiments demonstrated that following vaccination, they significantly elevate antibody levels and the content of multiple cytokines. This indicates that Acanthopanax gracilistylus W. W. Smith Polysaccharide and its liposomes can safely and effectively enhance vaccine immune responses, with liposomes exhibiting particularly pronounced effects. These findings provide crucial evidence for developing novel, highly effective adjuvants or diluents derived from natural plant sources. Future applications in poultry farming hold promise for enhancing vaccine efficacy and reducing disease incidence, carrying significant socioeconomic value.

Acanthopanax gracilistylus W. W. Smith is a traditional Chinese herbal medicine that contains a variety of bioactive components, including polysaccharides. In this study, a polysaccharide was extracted from A. gracilistylus (AGSP) using an aqueous alcohol precipitation method. The sugar and glucuronic acid contents of AGSP are 43% and 8.06%, respectively. It was consisted of seven monosaccharides, mannose (Man), rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal), and arabinose (Ara), with molar ratios of 0.32:0.15:0.04:0.12:5.12:2.50:0.85. AGSP liposomes (AGSPL) was prepared via the reverse-phase evaporation method. The encapsulation rate of AGSPL was 45.17% and its average particle size, polymer dispersity index (PDI), and zeta potential were 98.79 nm, 0.191, and −8.38 mV, respectively. The proliferation of chicken splenic T and B lymphocytes was significantly promoted in vitro at AGSP concentrations of 125–250 μg/mL and AGSPL concentrations of 15.63–31.25 μg/mL and 1.95–15.63 μg/mL, respectively. In vivo, the HI antibody titers and the concentrations of IFN-γ, IL-2, IL-4, and IL-6 in the AGSP and AGSPL groups were higher than those in the vaccine control (VC) and blank control (BC) groups. The efficacy of AGSPL was superior to that of AGSP. These findings indicate that AGSP and AGSPL markedly enhance the immunogenicity of ND vaccines, especially AGSPL, which is a promising candidate for polysaccharide-based vaccine diluents or adjuvants.

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1. Introduction

Newcastle Disease (ND) is a highly contagious disease caused by the Newcastle Disease Virus (NDV), which can lead to high mortality rates and serious economic losses in poultry [1]. Vaccination is a vital preventive measure against the disease, and live vaccines are widely used because they contain live attenuated viruses that are effective in stimulating an immune response [2]. At present, live vaccines are mostly stored in lyophilized form and require dilution with saline or phosphate buffer prior to administration. However, vaccines diluted with traditional diluents exhibit problems such as low antibody potency and increased susceptibility to stress and allergic reactions after vaccination, thereby affecting the effectiveness of immunization. Therefore, the development of a vaccine diluent that enhances the immune response is of great significance for improving the immunological effect of vaccines. It has been demonstrated that Glycyrrhiza polysaccharides have significant immune-enhancing effects and can strengthen chicken NDV vaccine immunity [3].

Polysaccharides are polymers consisting of 10 or more monosaccharides linked by glycosidic bonds, which offer the advantages of low toxicity, minimal side effects, and high safety [4]. As important biomolecules in nature, polysaccharides are widely distributed in plants, microorganisms, algae, and animals [5]. Studies have shown that Chinese medicine polysaccharides exhibit biological activities such as immunomodulation [6], anti-tumor [7,8], anti-oxidation [9], and anti-inflammation [10]. The 2010 edition of the Chinese Pharmacopeia includes the dried root bark of Acanthopanax gracilistylus W. W. Smith (AGS), a plant belonging to the family Araliaceae, which is a traditional Chinese herbal medicine with the effects of dispelling wind and dampness, strengthening tendons and bones, activating blood circulation, and removing blood stasis, as well as promoting diuresis to reduce edema [11]. Modern pharmacology has demonstrated that this genus contains a variety of bioactive ingredients, including triterpenoids, flavonoids, and polysaccharides [11,12,13]. In recent years, research on the active components of AGS has mainly focused on triterpenoids, with fewer studies concerning its polysaccharides. With the rapid progress in polysaccharide research, the polysaccharide of Acanthopanax gracilistylus W. W. Smith (AGSP) has gradually become a research hotspot, attracting widespread attention due to its potential biological activities and pharmacological effects.

Liposome (Lip) is a bilayer vesicle structure composed primarily of phospholipids and cholesterol, and represents a highly promising system for drug and vaccine delivery [14,15]. Liposomes are both hydrophilic and hydrophobic, capable of encapsulating hydrophilic or lipophilic drugs, and possess the advantages of low toxicity, significantly slow-release properties, and targeted delivery. These features enable liposomal drugs to demonstrate reduced toxicity while maintaining enhanced efficacy [16,17]. Many studies have shown that encapsulating polysaccharides in liposomes as vaccine adjuvants or immune enhancers can significantly enhance both cellular and humoral immune responses [18,19]. However, there are fewer studies on the use of AGSP and AGSP liposomes (AGSPL) as vaccine diluents. In this study, AGSP will be extracted from the traditional Chinese medicine Acanthopanax gracilistylus W. W. Smith, and AGSPL will be prepared by the reverse evaporation method. The aim of this study is to investigate the immune-enhancing effect of AGSP and AGSPL as a diluent of the chicken Newcastle disease vaccine.

In order to investigate the immune-boosting effects of AGSP and AGSPL as diluents of the chicken Newcastle disease vaccine, this study assessed their effects on the proliferation of chicken splenic lymphocytes in vitro, and subsequently evaluated serum antibody potency and the concentrations of related cytokines in vivo.

2. Materials and Methods

2.1. Materials and Reagents

The dried root bark of Acanthopanax gracilistylus W. W. Smith was obtained from Hubei Province. Petroleum ether, dimethyl sulfoxide (DMSO), absolute ethanol, trichloromethane, ether, and Tween-80 were sourced from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). RPMI 1640, D-arabinose, L-rhamnose, D-galactose, D-mannose, D-galacturonic acid, D-glucuronic acid, and dextran standards of varying molecular weight were purchased from Shanghai Yuan Ye Bio-Technology Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS) and phosphate-buffered saline (PBS) were acquired from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was obtained from Beyotime Biotechnology (Shanghai, China). Soybean phospholipids and cholesterol were purchased from Shanghai AVT Pharmaceutical Technology Co., Ltd. (Shanghai, China). Chicken NDV, La Sota strain, was sourced from Harbin Pharmaceutical Group Co., Ltd. (Harbin, China). Positive and negative serum for the ND hemagglutination inhibition (HI) test and chicken ND antigen were acquired from Qingdao Lijian Bio-Tech Co., Ltd. (Qingdao, China). Cytokine assay reagent kits were obtained from Quanzhou Rui Xin Biotechnology Co., Ltd. (Quanzhou, China).

2.2. Extraction of AGSP

2.2.1. Isolation of AGSP

Following pulverization of AGS, Soxhlet extraction was sequentially performed using petroleum ether and methanol as solvents in a water bath at 80 °C, with solvent evaporation to dryness. Fifty grams of the treated AGS powder was combined with ten times its volume of distilled water and extracted for 2 h at 90 °C in a water bath. The extract was filtered, and the residue was re-extracted under identical conditions. The filtrates were combined and concentrated, followed by the addition of anhydrous ethanol and incubation at 4 °C for 12 h to precipitate the polysaccharide. The resulting precipitate was collected by centrifugation and freeze-dried to yield AGSP.

2.2.2. Savage Method for Protein Removal

AGSP was re-dissolved in distilled water, and one quarter of its volume of Savage reagent (trichloromethane:n-butanol, 4:1) was added for deproteinization. The mixture was vigorously shaken and centrifuged at 4000 rpm for 10 min. The upper aqueous layer was collected, and the procedure was repeated until no white flocculent material appeared. The deproteinized polysaccharide solution was then concentrated and freeze-dried to obtain purified AGSP.

2.3. Quality Control of AGSP

2.3.1. Determination of Sugar Content and Glucuronic Acid Content

Sugar content and glucuronic acid of AGSP were detected by the phenol-sulfuric acid method and meso-hydroxybiphenyl method [20], respectively.

2.3.2. Determination of AGSP Infrared Spectra

Infrared spectroscopy of AGSP was performed with a Thermo Scientific™ Nicolet™ iS10 FTIR spectrometer and OMNIC software, using a Smart iTR diamond ATR (attenuated total reflectance) sampling accessory (Waltham, MA, USA). Scanning was conducted over a wavenumber range of 4000 to 400 cm^−1^ with a resolution of 4 cm^−1^ and 16 scans averaged per position.

2.3.3. Determination of the Monosaccharide Composition of AGSP

The monosaccharide composition of AGSP was determined by high-performance liquid chromatography (HPLC) [21]. AGSP was hydrolyzed with trifluoroacetic acid (2 mol/L), and the hydrolysis products were derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) prior to analysis. Seven monosaccharide standards were derivatized in the same manner. Analyses were conducted on a WondaSil C18-WR column (4.6 × 250 mm, 5 µm; Shimadzu Corporation, Takamatsu, Japan).

2.4. Preparation and Structural Characterization of AGSPL

2.4.1. Preparation of AGSPL and Determination of Encapsulation Rate

AGSPL were prepared by the reverse evaporation method, and the encapsulation rate was determined by ultracentrifugation, following Huang et al. [22]. The method of encapsulation rate determination was adopted from Huang [22] and Wu [23].

2.4.2. Determination of Polymer Dispersity Index (PDI) and Zeta Potential of AGSPL

AGSPL was diluted tenfold, and the average particle size, PDI, and Zeta potential were measured at room temperature using a particle size potentiostat.

2.5. In Vitro Immunogenicity of AGSP and AGSPL

The method of Gao et al. was adopted with minor modifications [24]. Chicken splenic lymphocytes were isolated, adjusted to a concentration of 1 × 10^7^/mL, and seeded in 96-well plates at 100 µL per well. AGSP and AGSPL were serially diluted (250–1.95 µg/mL) and added to the wells (100 µL per well). After incubation at 37 °C in a 5% CO_2_ incubator, 20 µL MTT (5 mg/mL) was added to each well, followed by further incubation for 4 h and centrifugation. DMSO (100 µL per well) was then added. Absorbance was measured at 570 nm using an enzyme-linked immunoassay reader as an indicator of lymphocyte proliferation. Value-added assays for T and B lymphocytes were performed as before. The difference is that after cell counting and plate laying, the addition of Concanavalin A (Con A; 5 µg/mL) or lipopolysaccharide (LPS; 10 µg/mL) is required to induce proliferation of T and B lymphocytes.

2.6. Immunization Tests of AGSP and AGSPL as Dilutions of NDV

2.6.1. Immunization and Grouping of Animals

Two hundred 14-day-old Dawu Golden Phoenix laying hens were randomly assigned to four groups (AGSP group, AGSPL group, vaccine control group [VC], and blank control group [BC]). There were 50 hens in each group, and each group had five replicate groups. All animal procedures were performed under the ethical guidelines of Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, and housed in temperature and humidity-controlled facilities (Ethics numbers: SV-20241227-07). The AGSP and AGSPL groups received NDV diluted in PBS containing 1600 μg/mL AGSP or AGSPL. The VC group received the vaccine diluted in PBS, and the BC group received PBS alone. The initial vaccination was administered at 14 days of age, with a booster administered two weeks later. At 7, 14, 21, 28, 35, and 42 days after the first immunization, blood samples were collected from 20 randomly selected chickens in each group through the subterminal vein, and serum was isolated. Serum antibody titers were assessed by the HI test, and serum cytokine (IL-2, IL-4, IL-6, and INF-γ) levels were determined by enzyme-linked immunosorbent assay (ELISA).

2.6.2. Determination of Serum Antibody Titers

Collected blood was incubated at 37 °C for 2 h and at 4 °C for 1 h. The serum layer was collected by centrifugation at 4000 rpm for 10 min. NDV-specific antibody titers in each group were determined by the HI test using 96-well V-bottom microplates and verified by a four-unit antigen hemagglutination test. The highest dilution at which erythrocyte precipitation occurs in a linear fashion was recorded as the HI titer and expressed as log_2_.

2.6.3. Determination of Cytokine Levels in Serum

Following the procedures described in Section 2.6.2, cytokine concentrations (IL-2, IL-4, IL-6, and INF-γ) were determined using ELISA kits, according to the manufacturer’s instructions. All reagents were equilibrated to room temperature prior to use. Standard curve data were analyzed by logistic four-parameter curve fitting. Following curve fitting, the sample data were analyzed.

2.7. Statistical Analysis

Statistical analysis was conducted using one-way and two-way analysis of variance (ANOVA) in GraphPad Prism 10.0 software. Results were corrected using Tukey’s statistical method. All results are expressed as mean ± standard deviation. p-values < 0.05 were considered statistically significant.

3. Results and Analysis

3.1. Determination of Sugar Content and Glucuronic Acid Content of AGSP

The sugar content and glucuronic acid content of AGSP were determined as 43% and 8.06% using the phenol–sulfuric acid method and the m-hydroxybiphenyl method, respectively.

3.2. AGSP Infrared Spectral Analysis

As shown in Figure 1, AGSP exhibits clear absorption peaks characteristic of sugars. The absorption peak at 3292 cm^−1^ corresponded to O-H stretching vibrations, whereas the peak at 2933 cm^−1^ was attributable to C-H stretching vibrations in the sugar ring. These were typical infrared absorption peaks of sugars. The peak at 1692 cm^−1^ indicated the stretching vibration of a free carboxyl group (COO^−^), suggesting the presence of residual protein. Peaks at 1595 cm^−1^ and 1511 cm^−1^ were associated with the symmetric stretching vibration of C=O in carboxyl groups, indicating the presence of glyoxylate. Absorption in the 1300–800 cm^−1^ region corresponds to the polysaccharide fingerprint region. Peaks at 1378 cm^−1^ and 1257 cm^−1^ confirm the presence of C-H bonds. Peaks at 1154 cm^−1^, 1078 cm^−1^, and 1026 cm^−1^ were attributed to the stretching vibration of C-OH groups in the polysaccharide chain and the glycosidic bond (C-O-C). The 1154 cm^−1^ peak indicated the presence of pyranose, whereas the 1078 cm^−1^ and 1026 cm^−1^ peaks indicated glucose and galactose. The absorption at 852 cm^−1^ confirms that AGSP contained an α-glycosidic bond, and combined with the peaks at 916 cm^−1^ and 764 cm^−1^, suggests that AGSP was a D-glucopyranose derivative. The peak at 764 cm^−1^ is characteristic of Se=O.

3.3. Analysis of the Monosaccharide Composition of AGSP

The monosaccharide composition of AGSP was presented in Table 1 and Figure 2. AGSP was primarily composed of mannose (Man), rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal), and arabinose (Ara), with a molar ratio of 0.32:0.15:0.04:0.12:5.12:2.50:0.85.

3.4. Encapsulation Rate, Particle Size Distribution, PDI, and Zeta Potential of AGSP

The encapsulation rate of AGSPL was measured as 45.17% by ultracentrifugation. AGSPL samples were appropriately diluted, and the particle size, PDI, and zeta potential were determined using a zeta potential analyzer, with each sample measured in triplicate. As shown in Figure 3 and Table 2, the particle size distribution was narrow and normal, with an average particle size of 97.80 nm, a PDI of 0.199, and a zeta potential of –8.60 mV. Overall, AGSPL exhibited excellent dispersion and stability.

3.5. Effect of AGSP and AGSPL on the Proliferation of Chicken Splenic Lymphocytes

3.5.1. Effect of AGSP on the Proliferation of Chicken Splenic Lymphocytes

As shown in Figure 4A, the A_570_ values for AGSP were significantly higher than those for the corresponding cell control group at a concentration of 250 μg/mL (p < 0.01), indicating that AGSP significantly promotes the proliferation of chicken splenic lymphocytes. Figure 4B,C show that when AGSP was combined with LPS or Con A, the A_570_ values were significantly higher than those of the corresponding cell control group within the concentration range of 125–250 μg/mL (p < 0.01), indicating that AGSP synergized with LPS to promote B lymphocyte proliferation and with Con A to promote T lymphocyte proliferation in chicken spleen.

3.5.2. Effect of AGSPL on the Proliferation of Chicken Splenic Lymphocytes

As shown in Figure 5A, the A_570_ values for AGSPL were significantly higher than those for the corresponding cellular controls at concentrations of 15.63–31.25 μg/mL (p < 0.05 or p < 0.01), indicating that AGSPL also significantly promotes the proliferation of chicken splenic lymphocytes. However, at a concentration of 62.5 μg/mL, AGSPL alone significantly inhibited lymphocyte proliferation. Figure 5B,C demonstrate that when AGSPL was combined with LPS or Con A, the A_570_ values were significantly higher than those of the cellular controls at concentrations of 15.63–31.25 μg/mL and 1.95–15.63 μg/mL, respectively, suggesting that AGSPL synergistically enhances B lymphocyte proliferation with LPS and T lymphocyte proliferation with Con A.

3.6. Immunization Tests of AGSP and AGSPL as Dilutions of Chicken NDV

3.6.1. Analysis of Serum NDV Antibody Levels

Blood samples were collected from 20 randomly selected chickens in each group, and the serum was separated to determine the serum antibody titer. As shown in Figure 6, the serum antibody titers of both AGSP and AGSPL groups were higher than those of the VC and BC groups throughout the study period. At 7, 14, 21, 28, and 35 days after immunization, the AGSPL group exhibited significantly higher serum antibody titers compared with the VC group (p < 0.05 or p < 0.01). The antibody titers of the AGSP group were significantly higher than those of the VC group only at 35 d after immunization (p < 0.01). And at 7 d after immunization, the antibody titer of the AGSPL group was significantly higher than that of the AGSP group (p < 0.01). In conclusion, the immune-enhancing effect of AGSPL was superior to that of AGSP.

3.6.2. Analysis of Serum IL-2, IL-4, IL-6 and INF-γ Concentrations

The effects of AGSP and AGSPL on serum cytokines IL-2, IL-4, IL-6, and IFN-γ are shown in Figure 7. The concentrations of these cytokines in the AGSP and AGSPL groups were significantly higher than in the VC and BC. Compared to the VC group, the concentrations of IL-2, IL-4, IL-6, and IFN-γ in the AGSP group increased by 67.74%, 48.19%, and 18.37%, respectively. Whereas they were ascended by 60.25%, 82.10%, 22.27% and 61.47% in the AGSPL group, respectively, when compared with the VC group. In contrast to the AGSP group, IL-2 decreased by 4.47%, and IL-4, IL-6, and IFN-γ increased by 22.88%, 3.30%, and 21.80%, respectively. Overall, AGSPL was more effective than AGSP in increasing serum cytokine concentrations.

4. Discussion

ND remains one of the most serious diseases affecting poultry health, and vaccination is the most effective measure to prevent this disease [25]. The NDV (La Sota strain) is the most widely used on the Chinese market. However, suboptimal immunity after vaccination, attributed to factors such as poultry house environment and individual variation, necessitates the development of natural active substances to enhance immunity as vaccine diluents. In this study, AGSP was extracted using an aqueous alcoholic precipitation method and deproteinized using the Savage method, and then its quality was examined by a series of methods. AGSP liposomes were prepared by reverse evaporation. These agents were used in the dilution of a weakly virulent NDV in chickens to evaluate their immune-enhancing effect.

The spleen is a central immune organ in which T and B lymphocytes accumulate. Upon activation, lymphocytes can perform various immune functions, including rapid proliferation and migration [26]. Lymphocyte proliferation is a key indicator of the cellular immune response and is commonly used to detect the in vitro activity of biologically active substances [27]. It is well established that Con A stimulates T cells to proliferate, whereas LPS stimulates B cell proliferation [24]. In this study, splenic lymphocytes were isolated from chicken spleens for in vitro culture, and lymphocyte proliferation was evaluated by adding different concentrations of AGSP or AGSPL together with Con A or LPS. The results show that AGSP and AGSPL significantly promote the proliferation of splenic T and B lymphocytes within a specific concentration range, and AGSPL promotes cellular value-added at a lower concentration.

The spleen is a major component of the immune system, and splenic lymphocytes are integral to the immune response. Previous research has demonstrated that polysaccharides and polysaccharide liposomes can promote lymphocyte proliferation within a defined concentration range, such as Acanthopanax senticosus polysaccharide [28], Yu-Ping-Feng polysaccharide [29], and yam polysaccharide liposomes [30], which is consistent with the present findings.

To confirm the in vivo immune-enhancing effect of AGSP and AGSPL, both were used to dilute the weakly virulent NDV (La Sota strain). Fourteen-day-old chicks were immunized via nose-drops and eye-drops, and the immunization effect was evaluated by measuring serum antibody titers using the HI test. Antibody titers reflect the degree of humoral immunity [31]. Humoral immunity, mediated by B lymphocytes, is a major component of the immune response to infectious diseases. The HI test is based on the ability of the haemagglutinin (HA) on the virus surface to bind to the sialic acid receptor on the erythrocyte membrane, resulting in agglutination. This agglutination is inhibited when antibodies in the sample bind to the antigen-binding site of HA [32]. The antibody titers in the serum of immunized chickens were determined using the HI test. The results show that after the first immunization, antibody titers in the AGSP and AGSPL groups were higher than those in the VC and BC groups, indicating that both AGSP and AGSPL can enhance humoral immunity. The antibody titer in the AGSPL group was higher than that in the AGSP group, suggesting that AGSPL exerts a stronger effect in enhancing humoral immunity compared with AGSP. This finding is in line with previous research. For instance, encapsulation of Angelica sinensis polysaccharide in PLGA nanoparticles as a vaccine delivery and adjuvant system for ovalbumin induces strong and sustained immune responses and mixed Th1/Th2 responses [33]. Liu’s study demonstrated that Ganoderma lucidum polysaccharide liposomes (Lip-PS) used as a vaccine adjuvant induce a more effective specific immune response [18].

The activation of helper T cells (Th cells) is a central aspect of both humoral and cellular immunity. Th1 and Th2 cells are two key subpopulations of Th cells that maintain a relative balance through the secretion of cytokines, which regulate their respective immune responses [34]. Cytokines IL-4 and IL-6, secreted by Th2 cells, mediate humoral immunity [35]. IL-6, as a pro-inflammatory cytokine, also recruits immune cells. In this study, the concentrations of IL-4 and IL-6 in the serum of immunized chickens at 35 d were measured by ELISA. The results show that IL-4 and IL-6 concentrations in the AGSP and AGSPL groups were significantly higher than those in the VC group, and the efficacy of AGSPL was better than that of AGSP. This indicates that AGSP and AGSPL stimulate the immune response in a pro-inflammatory manner and enhance the humoral immune response to the ND vaccine, and that the enhancement of humoral immunity by AGSPL is more effective. Supporting this, it has been reported that Ophiopogon polysaccharide liposome(OPL) was superior to Ophiopogon polysaccharide (OP) in promoting the production of cytokines IFN-γ, IL-2, IL-4, and IL-6 [36].

IL-2 and IFN-γ are secreted by Th1 cells and mediate cellular immune responses [34]. IL-2 regulates cell proliferation, differentiation, and survival [37]. IFN-γ, as a critical Th1-associated cytokine, reflects the status of cellular immunity in the organism [38]. In this study, concentrations of IL-2 and IFN-γ in the AGSP and AGSPL groups were significantly higher than in the VC and BC groups, suggesting that AGSP and AGSPL promote secretion of IL-2 and IFN-γ and thus enhance cellular immune responses. Although the concentration of IL-2 in the AGSPL group was slightly lower than that of AGSP (4.47% lower), the concentration of IFN-γ was higher than that of AGSP (21.80% higher). Overall, AGSPL had a greater ability to enhance cellular immune responses. This observation is consistent with previous studies [19,39,40]. Liu’s work demonstrated that iLPSM enhances both Th1 and Th2 responses, resulting in a relatively balanced immune state [41]. Therefore, the balance between Th1 and Th2 cells is important in maintaining normal immune function. The serum concentrations of cytokines IL-4, IL-6, and IFN-γ were higher in the AGSPL group than in the AGSP group in this study, and only the concentration of IL-2 was slightly lower than in the AGSP group. This suggests that both AGSP and AGSPL are capable of stimulating both Th1 and Th2 immune responses, and that AGSPL is more capable of doing so. Consequently, when Th1 and Th2 immune responses are required after vaccination, AGSPL in ND vaccines can complement this requirement.

5. Conclusions

In summary, the in vitro MTT assay demonstrated that AGSP and AGSPL significantly promote the proliferation of chicken splenic lymphocytes. Use of these agents as ND vaccine diluents increased the serum antibody titer in vaccinated chickens and promoted production of IL-2, IL-4, IL-6, and IFN-γ in the serum, and the efficacy of AGSPL is better than that of AGSP, which can significantly improve the immunological effect of the ND vaccine. However, the specific mechanism of action of AGSP and AGSPL requires further investigation. This study provides theoretical and practical support for the development of future vaccine diluents or adjuvants.