Effects of Antigen Dosage and Chitosan Micro/Nanoparticle Size on Immune Responses in Mice Immunized with H5N1 Influenza Vaccine

TL;DR

This study shows that chitosan nanoparticles improve immune responses to H5N1 vaccines at low antigen doses in mice.

Contribution

Chitosan nanoparticles outperform microparticles and traditional adjuvants in enhancing immune responses at low antigen doses.

Findings

Chitosan nanoparticles significantly increased IgG and HI titers at low antigen doses compared to controls.

Immune responses saturated at 1.5 µg antigen for nanoparticles and 3.0 µg for microparticles.

IgG subtype analysis indicated a balanced IgG1/IgG2a immune profile.

Abstract

Highly pathogenic avian influenza A/H5N1 remains a persistent threat to public health and poultry production. H5N1 antigens are typically poorly immunogenic and require effective adjuvants for antigen dose-sparing. Here, we evaluated chitosan microparticles (CSMs) and nanoparticles (CSNs) as polymeric nano-adjuvants for an H5N1 influenza vaccine, focusing on the roles of antigen dose and particle size. A purified hemagglutinin antigen was adsorbed onto chitosan particles at doses ranging from 0.15 to 5.0 µg. Both CSNs and CSMs showed consistently high loading efficiency (97–99%). BALB/c mice were immunized intramuscularly in a prime–boost schedule. Chitosan nanoparticles significantly enhanced IgG and hemagglutination inhibition (HI) titers at low antigen doses compared with aluminum hydroxide and antigen-only controls (p < 0.05). Immune responses reached saturation at a 1.5 µg dose of antigen for chitosan nanoparticles and 3.0 µg for chitosan microparticles. IgG subtype analysis suggested a balanced IgG1/IgG2a profile. Collectively, these findings support chitosan-based polymeric nanoparticles as promising adjuvants enabling dose-sparing H5N1 vaccination.

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Full text

1. Introduction

Avian influenza A/H5N1 has posed a serious zoonotic and economic threat since its first human outbreak in Hong Kong in 1997. Large-scale outbreaks during the 2003–2005 period affected more than 60 countries, resulting in substantial poultry losses and high case fatality rates in humans. Despite advances in vaccine development, H5N1 antigens are generally poorly immunogenic and often require high antigen doses or potent adjuvants to elicit protective immunity. Highly pathogenic avian influenza A/H5N1 has caused substantial poultry losses and severe human infections, underscoring the need for effective vaccines and adjuvants [1].

Inactivated H5N1 antigens often elicit weak immune responses and may require antigen-sparing strategies to support outbreak preparedness. Aluminum-based adjuvants such as aluminum hydroxide and aluminum phosphate remain the only adjuvants licensed for human influenza vaccines; however, they primarily promote Th2-biased humoral immunity and show limited efficacy in enhancing cellular immune responses [2,3,4,5]. Furthermore, aluminum adjuvants may cause persistent local inflammation and are less effective for antigen dose-sparing strategies [6].

Chitosan is a naturally derived cationic polysaccharide (a functional biopolymer) that can form micro- and nanoparticles [7,8,9,10,11,12]. These particulate systems can electrostatically associate with negatively charged antigens, increase local antigen retention, and facilitate uptake by antigen-presenting cells (APCs) [13,14,15]. In the field of vaccine adjuvant development, chitosan has attracted considerable attention due to its strong immuno-stimulatory properties. Numerous studies have demonstrated that chitosan effectively induces antigen-specific delayed-type hypersensitivity (DTH) responses and stimulates both B and T lymphocytes. In particular, chitosan has been reported to enhance the expression of the early activation marker CD69 on B cells and CD4^+^ T lymphocytes, while also promoting the secretion of key immunomodulatory cytokines, including granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin-12 (IL-12), which are critical for T-cell activation and differentiation [3,16,17].

Owing to their nanoscale size and positive surface charge, chitosan nanoparticles significantly enhance antigen uptake by antigen-presenting cells and prolong antigen retention within immune tissues, thereby leading to a stronger and more sustained immune response. Importantly, chitosan-based nanocarriers have been shown to induce both humoral immune responses and cell-mediated immunity, making them particularly attractive as multifunctional vaccine adjuvants for viral infections [4,18,19,20].

Studies have reported the successful adsorption of influenza vaccines onto chitosan nanoparticles, resulting in enhanced mucosal and systemic immune responses. Similarly, influenza vaccines formulated with N-trimethyl chitosan nanoparticles and mannose–chitosan nanoparticles exhibited improved antigen stability and significantly increased immunogenicity compared with non-adjuvanted formulations [19,21,22]. Two Egyptian avian strains acting as Local Egyptian AIV H5N1 strains, namely strains S1 (A/Chicken/Egypt/Q1995D/2010) and S2 (A/duck/Egypt/M 2583A/2010), were encapsulated on chitosan nanoparticles to formulate vaccines [14]. Chitosan nanoparticles of various sizes, namely 165.6 nm, 272.5 nm, 343.2 nm, and 443.5 nm, were used to load the H9N2 influenza vaccine [23]. However, despite extensive research on chitosan-based nano-adjuvants for seasonal influenza vaccines, to the best of our knowledge, no studies have reported the use of nano- or micro-sized chitosan particles as adjuvants for influenza A/H5N1 vaccines. This lack of investigation represents a significant research gap and highlights the novelty of exploring chitosan nano- and microparticle-based adjuvant systems for highly pathogenic avian influenza vaccines.

Previously, we demonstrated that chitosan nanoparticles had a molecular weight of approximately 465 kDa and sizes ranging from 100 to 3000 nm, and we found that a particle size in the range of 300–500 nm significantly enhanced immune responses to H5N1 antigens in mice at an H5N1 antigen dose of 0.375 µg [24]. However, the optimal antigen dose at the nano-size (300–500 nm) and micro-size (1000–1200 nm) scales for achieving maximal immunogenicity while ensuring safety and economic feasibility remains unclear.

Therefore, this study aimed (i) to determine an optimal H5N1 antigen dose when loaded on chitosan nanoparticles (CSNs) and chitosan microparticles (CSMs) and (ii) to compare immunogenicity and safety between chitosan nanoparticles and microparticles under intramuscular administration.

2. Materials and Methods

2.1. Preparation and Characterization of Chitosan Nanoparticles

Chitosan was purchased from Taehoon Bio, Seoul, Republic of Korea. The degree of deacetylation of chitosan was approximately 90%, as determined through Fourier Transform Infrared Spectroscopy (FT-IR) (Alpha, Brucker, Billerica, MA, USA). Viscosity molecular weights (M_v_) of the chitosan were ~465 kDa [24]. The viscosity average molecular weight (M_v_) of chitosan was determined by measuring its intrinsic viscosity (Ubbelohde viscometer) and using the Mark–Houwink–Sakurada equation as follows [25]:

$$[eqn]$$

where η = intrinsic viscosity; K = 1.81 × 10^−3^; a = 0.93; and M_v_ = viscosity-derived average molecular weight.

Chitosan microparticles (CSMs) and nanoparticles (CSNs) were prepared using a Nano B90 spray dryer (Büchi, Flawil, Switzerland), as previously reported [26]. In brief, chitosan solutions of different concentrations (0.1% w/v) were prepared by dissolving chitosan powder in 0.5% acetic acid solution and leaving the solutions overnight. Afterward, a spray dryer with different sized spray nozzles (4.0 µm and 7.0 µm) was used to prepare chitosan micro- and nanoparticles. The spraying process was conducted under the following conditions: flow rate of 2 mL·min^−1^; drying gas flow rate of 1.3 m^3^·min^−1^, inlet temperature of 120 °C, and outlet temperature of 80 °C. After the spraying process, chitosan micro- and nanoparticles were collected and placed in glass tubes and then stored at 5 °C until use.

Morphological characteristics of chitosan micro- and nanoparticles were examined using a transmission electron microscope (TEM; JEM-1400, JEOL, Tokyo, Japan) and a scanning electron microscope (SEM, S 4800; Hitachi, Tokyo, Japan). The mean size and zeta potential of the particles were determined using a Nanosizer Nano ZS (Malvern Instruments, Malvern, UK) at 25 °C in PBS (pH 7.0). Measurements were performed in triplicate, and the sample concentration was adjusted to 1 mg/mL.

2.2. Antigen and Reagents

Standard Vero cells (ECAAC No. 88020401) and the H5N1 viral strain NIBRG-14: A/Vietnam/1194/2004 × PR8 (WHO) were employed for vaccine preparation. The H5N1 antigen (A/Vietnam/1194/2004, NIBSC code 09/184) and its corresponding antibody (NIBSC code 04/214) were used in all experiments.

Culture media included MEM0643, MEM1018, Ex-Cell Vero, DMEM F12 (Sigma, St. Louis, MO, USA), VP-SFM, and L15 (Gibco, Grand Island, NY, USA). Other reagents included β-propiolactone, formalin, L-glutamine, penicillin-streptomycin, trypsin-EDTA, TPCK-trypsin, dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), HEPES, Tween 80, methylcellulose, phosphate-buffered saline (PBS), NaHCO_3_, bovine serum albumin (BSA), citric acid, trypan blue, and trypsin soybean inhibitor (all purchased from Sigma or Gibco).

2.3. Determination of HA Titer of the Virus Strain

The influenza virus strain A/H5N1 NIBRG-14 was stored at −70 °C, thawed, and kept on ice prior to use. The hemagglutination (HA) titer was determined using a 96-well V-bottom microtiter plate. Briefly, 50 µL of cold PBS was dispensed into wells of columns 2–12. The virus suspension was then serially diluted two-fold across columns 2–12. Subsequently, 50 µL of 1% horse red blood cells was added to each well. The plate was incubated at room temperature for 30 min. Results were read by tilting the plate vertically at an angle of 45–60°. A positive reaction (+) was indicated by hemagglutination, in which erythrocytes formed a diffuse layer and did not flow down the well. A negative reaction (−) was identified when erythrocytes rapidly settled at the bottom of the well, forming a characteristic teardrop shape. The HA titer was defined as the reciprocal of the highest virus dilution showing complete hemagglutination and was expressed as HAU/50 µL. The virus stock was subsequently diluted in cold PBS to obtain a working virus solution with a final titer of 8 HAU/50 µL

2.4. Antigen Adsorption and Quantification

Chitosan micro- and nanoparticles (2 mg/mL) were mixed with purified H5N1 antigen (0.15–5.0 µg per dose) in phosphate buffer (pH 7.2) containing 0.5% (w/w) Tween 80 and incubated for 15 min at room temperature. After centrifugation (12,000 rpm, 4 °C, 10 min), HA activity in the supernatant was quantified by hemagglutination. Loading efficiency (LE) and loading capacity (LC) were calculated as described in the source dataset.

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2.5. Animals and Immunization

BALB/c mice (6–8 weeks old; n = 10 per group) were immunized intramuscularly with a prime–boost schedule at weeks 0 and 2. Formulations included antigen-only control, Alum-adjuvanted control, and antigen adsorbed to chitosan nanoparticles or microparticles (Table 1). Sera were collected at weeks 2 and 4.

2.6. Collection of Serum Samples

Two weeks after each immunization, mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg). Blood samples were collected by cardiac puncture, left at 37 °C for 1 h to clot, and centrifuged at 500× g for 5 min at 4 °C. Serum was separated and stored at −20 °C until further analysis.

2.7. Hemagglutination Inhibition (HI) Assay

Antibody titers were determined using the standard HI assay with horse red blood cells. Briefly, sera were serially diluted two-fold in 96-well V-shaped plates. Twenty-five microliters of H5N1 virus (8 HAU) were added to each well and incubated at room temperature for 30 min. Then, 50 µL of 1% horse red blood cells was added, and plates were incubated for 1 h. The HI titer was expressed as the reciprocal of the highest serum dilution inhibiting hemagglutination.

2.8. ELISA for IgG Antibody Titers

ELISA plates were coated overnight at 4 °C with 100 µL of H5N1 antigen (0.4 µg/mL) in each well. After blocking with 5% skim milk, serially diluted mouse sera were added and incubated at 37 °C for 2 h. Following three washes, HRP-conjugated anti-mouse IgG antibody was added, incubated for 1 h, and developed using p-nitrophenyl phosphate (pNPP). Absorbance was measured at 405 nm. Antibody titers were expressed as the reciprocal of the highest dilution, with OD values exceeding the cutoff.

Total IgG and IgG subtypes (IgG1 and IgG2a) were measured using an ELISA. Statistical significance was defined as p < 0.05.

2.9. General Safety Evaluation

The general safety evaluation was conducted in accordance with the Standard Operating Procedures (SOPs) of the Pasteur Institute, using healthy white mice weighing 18–22 g. Three vaccine formulations containing the H5N1 influenza antigen were prepared with different adjuvants: (i) 0.2 mg of chitosan nanoparticles + 1.5 µg of H5N1 antigen, (ii) 0.2 mg of chitosan microparticles + 1.5 µg of H5N1 antigen, and (iii) 0.06 mg of aluminum hydroxide + H5N1 antigen as control. Each formulation was prepared in three independent batches to ensure reproducibility and consistency. Each group consisted of 10 mice, immunized intraperitoneally at weeks 0 and 2 with a 0.1 mL dose of vaccine formulations. Body weight and general health conditions of the mice were monitored daily over a 14-day period to assess the safety profile of the vaccines (Table 2). The animals were housed under standard laboratory conditions with free access to food and water. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and complied with relevant ethical guidelines for animal research.

2.10. Vaccine Endotoxin Testing

Vaccine formulations consisted of 0.2 mg of CSNs + 1.5 µg of HA (H5N1), 0.2 mg of CSMs + 3.0 µg of HA (H5N1), and 0.6 mg of aluminum hydroxide (Alum) + 1.5 µg of HA (H5N1). Endotoxin levels in the vaccine samples were determined using a semi-quantitative Limulus Amebocyte Lysate (LAL) assay (Pyrogent™ Plus, Multi-Test kit N283-125; US License No. 1701, Cambrex Bio Science, Walkersville, MD, USA). The assay is based on the gel-clot reaction of Limulus Amebocyte Lysate with bacterial endotoxin. Standard endotoxin solutions were prepared at concentrations ranging from 0.06 to 0.5 EU/mL. Vaccine samples were diluted to a ratio of 1:600 prior to analysis. All procedures were performed in accordance with the European Pharmacopoeia 2005. Each formulation was tested in triplicate.

2.11. Statistical Analysis

Data were presented as mean ± standard deviation (SD). Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparison test (GraphPad Prism 9.0). p-value < 0.05 was considered statistically significant.

3. Results and Discussion

3.1. Morphology and Characterization of Chitosan Micro- and Nanoparticles

The shape of nanoparticles can have a significant influence on their physical, chemical and biological properties, e.g., surface area, packing properties, flow behavior, mechanical properties, drug release kinetics and efficiency. The morphology and physicochemical properties of chitosan nanoparticles (CSNs) and chitosan microparticles (CSMs) were systematically investigated using TEM, FE-SEM, dynamic light scattering (DLS), and zeta potential analysis.

3.1.1. Morphological Analysis by TEM and FE-SEM

As shown in the TEM images (Figure 1), CSNs exhibited a nearly spherical morphology with nanoscale dimensions and relatively uniform particle distribution, whereas CSMs displayed significantly larger sizes and less homogeneous structures. The formation of well-defined spherical nanoparticles indicates effective control of particle nucleation and growth during the nanoparticle fabrication process.

FE-SEM micrographs (Figure 2) further confirmed these observations, revealing that CSNs possessed smoother surfaces with minimal aggregation, while CSMs showed rougher surfaces and a higher tendency toward particle agglomeration. This behavior can be attributed to the reduced surface area-to-volume ratio and weaker electrostatic stabilization in microparticles, which promotes interparticle adhesion.

Similar morphological features have been widely reported for chitosan-based nano-systems prepared by spray drying [26,27] and ionic gelation or spray-drying techniques [8,28]. Previous studies demonstrated that chitosan nanoparticles typically exhibit spherical morphology and improved dispersibility compared to their microparticle counterparts, which often suffer from irregular shapes and aggregation phenomena.

3.1.2. Particle Size

The particle size distribution analysis (Figure 3) revealed that CSNs had a markedly smaller average size (mean size 334.6 nm, ranged from 300 to 500 nm) with a PDI of 0.467 and a narrower size distribution compared with CSMs (mean size 1015 nm), which has a polydispersity index (PDI) of 0.781. A narrow size distribution is a critical parameter indicating high uniformity and reproducibility of the nanoparticle system, which is essential for biomedical applications such as drug and antigen delivery.

In contrast, CSMs exhibited a broader size distribution, as supported by the data in Figure 2 and Figure 3, reflecting less controlled particle formation at the microscale. This observation is consistent with previous reports showing that chitosan microparticles often display higher polydispersity due to diffusion-limited aggregation and heterogeneous nucleation during particle formation.

Notably, the nanoscale size range obtained for CSNs in this study falls within the optimal window reported for enhanced cellular uptake, mucosal penetration, and intracellular delivery in drug delivery systems [29]. For example, nanoparticles formed from 540 kDa, 102 kDa, and 49 kDa chitosan exhibited mean diameters of ~480 nm, 240 nm, and 140 nm, respectively [30]. Chitosan nanoparticles prepared by ionic gelation with a size in the range of 150–397 nm and a zeta potential values in the range of 11.5–16.0 mV were encapsulated in inactivated influenza avian vaccine [14]. This is similar to the results obtained when a lentogenic Newcastle live virus vaccine was entrapped in chitosan nanoparticles with a size of 371 nm [31]. Chitosan nanoparticles prepared from chitosan in the range of 465 kDa to 732 kDa, obtained via spray drying, had sizes ranging from 95.4 nm 335.9 nm [24].

Previous studies have reported notable differences in the particle size and morphology of chitosan-based microparticles depending on molecular weight and fabrication method. Chitosan microcapsules were produced by using a spray-drying system (L-117, Beijing, China) from 200 and 350 kDa chitosan to evaluate lipid adsorption capacity in mice fed a high-fat diet. The resulting microspheres exhibited smooth surfaces and relatively large sizes ranging from 1.5 to 7.21 μm [32]. Similarly, chitosan microspheres were prepared via an emulsification technique. Depending on the glutaraldehyde concentration, the mean particle size remained large, approximately in the range of 12.80–15.34 μm, confirming that chemical crosslinking tends to generate micro-scale particles [33].

3.1.3. Zeta Potential

Zeta potential measurements (Figure 4) demonstrated that CSNs possessed a higher positive surface charge (+59 mV) compared to CSMs (+51 mV). The positive zeta potential arises from the protonation of amino groups (–NH_2_) on the chitosan backbone and serves as an important indicator of colloidal stability.

The zeta potential values obtained in the present study were notably higher than those reported in a previous publication by Ha et al. (2019) [34]. In their work, the chitosan nanoparticles exhibited sizes ranging from 300 to 750 nm with zeta potentials of 40–50 mV. Similarly, chitosan nanoparticles produced by spray-drying methods typically show average sizes of 100–400 nm and zeta potentials of +39 to +45 mV [8,26]. Chitosan nanoparticles generally possessed mean diameters of 150–250 nm and zeta potential values of around +30 mV [35].

Higher zeta potential values for CSNs suggest stronger electrostatic repulsion between particles, resulting in enhanced dispersion stability and reduced aggregation. In contrast, the lower zeta potential observed for CSMs correlates well with their increased aggregation tendency observed in FE-SEM images.

Overall, the TEM, FE-SEM, particle size distribution, and zeta potential results clearly demonstrate that CSNs exhibit superior physicochemical characteristics compared with CSMs, including:

• ▪Smaller and more uniform particle size;
• ▪Spherical morphology with smooth surfaces;
• ▪Higher positive surface charge and improved colloidal stability.

3.2. Antigen Adsorption on Chitosan Micro- and Nanoparticles

Across all antigen doses tested (0.15–5.0 µg per dose), both chitosan nanoparticles and microparticles exhibited very high loading efficiency (LE) 97–99% (Table 3). This indicates robust antigen association under the chosen formulation conditions. From a formulation standpoint, consistently high LE is advantageous because it minimizes free antigen in the supernatant, supports dose accuracy, and can reduce batch-to-batch variability.

Loading capacity (LC) increased proportionally with antigen dose, reaching 33,759 ± 1453 HAU/mg for nanoparticles and 33,077 ± 1574 HAU/mg for microparticles at the dose of 5.0 µg. The close LC values between particle sizes at lower doses (0.15–3.0 µg) suggest that antigen availability—rather than carrier saturation—dominates in this range. The slightly higher LC for nanoparticles at 5.0 µg is consistent with their higher specific surface area and potentially greater density of binding sites. Similar high adsorption of influenza antigens on chitosan-based carriers has been reported, and particle surface engineering (e.g., mannose coating) can further modulate antigen display and immune outcomes [22]. An amount of 20 mg of chitosan nanoparticles and mannose–chitosan nanoparticles were prepared through ionic gelation with mannose and encapsulated with an antigen (KAg) of swine influenza A virus (SwIAV) at a dose of 2 mg. Both vaccines had ∼80% antigen encapsulation efficiency and lower EE% compared to those in this study (97–99%).

Yahyaei et al., 2025 [15], also reported the efficacy of intranasal mRNA vaccination in generating protective mucosal and systemic immunity against influenza A (H1N1). mRNA (H1N1) was encapsulated in mannose–histidine-conjugated chitosan lipid nanoparticles (MHCS-LNPs) as a vaccine against influenza A (H1N1) in BALB/c. The size of the nanoparticles was around 200 nm, and the EE% value was similar to that in this study, which reached 95%.

3.3. Antigen Dose-Dependent Immunogenicity in Mice

3.3.1. HI Titers as Functional Correlates of Vaccine Activity

HI titers represent functional antibodies that block influenza hemagglutinin-mediated receptor binding and are widely used as correlates of vaccine activity. Consistent with many influenza vaccine studies, the source dataset notes modest differences after the first dose but clear separation after boosting (Figure 5).

For chitosan nanoparticles (CSNs), HI titers were markedly higher at doses of 1.5–5.0 µg (512–640) compared with 0.15–0.375 µg (160–480), while differences among doses of 1.5, 3.0, and 5.0 µg were minimal—again indicating a plateau. For microparticles, low antigen doses (0.15–0.75 µg) produced limited HI responses, while doses of 3.0–5.0 µg produced clearly superior HI titers (p < 0.05). When compared across controls, both Alum and chitosan formulations improved HI titers relative to antigen-only controls at all doses, whereas differences between Alum and nano-chitosan were generally small at higher doses. This phenomenon has also been reported in a previous study evaluating chitosan-based adjuvants for H5N2 influenza vaccines, conducted using antigen doses of 0.375 µg, 0.75 µg, and 1.5 µg [36].

These patterns align with the broader literature where chitosan often matches Alum for systemic immunization but can outperform Alum in specific dose ranges or formulation formats that favor antigen presentation. The antigen from local Egyptian AIV H5N1 strains, namely the S1 strain (A/Chicken/Egypt/Q1995D/2010) and S2 strain (A/duck/Egypt/M 2583A/2010), was encapsulated and loaded on chitosan nanoparticles with a size of 397 nm and a zeta potential of 11.5 mV. The results show that the EE% was 100% and the concentration of HI antibody in the antigen-loaded chitosan nanoparticles was 8.8 higher than that of the antigen-encapsulated chitosan nanoparticles (6.1) and inactivated avian virus vaccine (7.5) 3 weeks after vaccine immunization in chicken. Mannose–chitosan nanoparticle intranasal influenza platforms have shown improved immune readouts (including HI) in pig models [22]. Chitosan nanoparticles (CSNs) and mannose–chitosan nanoparticles (mCSNs) were prepared by ionic gelation with mannose and encapsulated with a very high dose (2 mg) of an antigen (KAg) of swine influenza A virus (SwIAV). Both the vaccines had ∼80% antigen encapsulation efficiency, which is a lower EE% compared to those of this study (97–99%). The results of this study are similar, as both the CSNs-KAg and CSNs-KAg vaccines in MDA-positive pigs increased vaccine (H1N2-OH10) virus-specific serum HI titers. Chitosan-based intranasal delivery has repeatedly been associated with increased HI responses in multiple influenza settings [22]. A recent study also highlighted that particle size can modulate immuno-protective outcomes for chitosan nanoparticle adjuvants for the H5N1 influenza vaccine. The HI value showed significant differences between chitosan nanoparticles ranging from 300 to 500 nm in size (p < 0.05) and no significant differences between those ranging from 100 to 300 and from 500 to 1000 nm compared to the Alum adjuvant (p > 0.05) [24].

For chitosan microparticles (Figure 5), the HI assay indicated that formulations containing 0.15, 0.375, and 0.75 µg of antigen elicited comparable immune responses (176–192), with no significant differences among these groups. In contrast, higher antigen doses of 3.0 and 5.0 µg induced markedly stronger immune responses (448–512) compared with the lower-dose groups (p < 0.05).

When compared with the control groups receiving aluminum adjuvant or no adjuvant, we observed that at all antigen doses, the nano-chitosan adjuvant group and the aluminum adjuvant group exhibited no significant differences in HI titers. However, both adjuvanted groups showed markedly higher HI responses than the non-adjuvanted antigen group (p < 0.05).

As shown in Figure 5, comparing the CSN and CSM groups across antigen doses, chitosan nanoparticles (CSNs) elicited markedly stronger immune responses than chitosan microparticles (CSMs), particularly at antigen doses ranging from 1.5 to 5.0 µg. These differences were statistically significant (p < 0.05). The findings are consistent with previous reports demonstrating that nano-sized chitosan formulations induce stronger HI antibody responses than their micro-sized counterparts. Nano-sized chitosan particles induce stronger HI antibody responses than micro-sized chitosan carriers, primarily due to enhanced mucosal adhesion, improved antigen uptake, and more effective activation of antigen-presenting cells ([24,36,37,38,39]).

3.3.2. Serum IgG Responses and Dose-Sparing

The results presented in Figure 6 show that, after the second immunization, chitosan nanoparticles produced higher IgG titers (768–1024) than Alum (460–665) and antigen-only controls (211–179) at the lowest antigen doses of 0.15 and 0.375 µg (p < 0.05). This is a key finding because it indicates that the nano-sized polymeric adjuvant can compensate for limited antigen availability and thereby enable antigen dose-sparing.

At higher doses (0.75–5.0 µg), IgG titers in the nanoparticle group remained higher than antigen-only controls (p < 0.05) but were not consistently different from Alum, suggesting that both Alum and chitosan can achieve near-maximal humoral responses once the antigen is no longer limiting. Importantly, the dataset describes saturation behavior: IgG titers increased at a dose of 1.5 µg with nanoparticles (2048) but did not increase meaningfully at a dose of 3.0 (2457) or 5.0 µg (2662). This plateau is practically important for formulation optimization because it defines an effective ceiling and avoids unnecessary antigen use.

For chitosan microparticles, IgG titers increased at 1.5 µg (3276) and were reported to reach saturation at around 3.0 µg (4915). This shift in the saturation point (1.5 µg of nanoparticles vs. 3.0 µg of microparticles) supports the concept that particle size impacts dose efficiency. A plausible explanation is that nanoparticles provide greater surface area for antigen display and are more readily internalized by APCs, improving antigen processing and B-cell priming. These results are similar to those of other works. Chang et al. (2010) [40] reported that a noticeable difference between chitosan and aluminum adjuvants was observed only at an extremely low antigen dose of 0.1 μg, at which chitosan elicited higher IgG antibody titers compared with aluminum-based adjuvants.

The particle size of the chitosan nanoparticles used as vaccine adjuvants significantly influenced the immune response in mice immunized with an H5N1 antigen dose of 0.375 µg. The IgG titers in groups receiving CSNs with particle sizes between 100 and 1000 nm ranged from 1536 to 1843, approximately twofold higher than those induced by the conventional Al(OH)3 adjuvant (768) and the antigen alone (154) (p < 0.05) [24].

Other studies and reviews similarly conclude that chitosan-based nanoparticle platforms can enhance antibody responses and support antigen sparing, especially when the antigen is adsorbed or displayed on the particle surface, to maximize immune recognition [35,38,39,40,41,42].

3.3.3. IgG1/IgG2a Responses and Th1/Th2 Balance

To evaluate immune polarization, IgG1 (Th2-associated) and IgG2a (Th1-associated) levels were analyzed (Figure 7). Both subclasses increased after priming and rose sharply after boosting, with significantly higher titers in chitosan-adjuvanted groups compared with antigen-only groups (p < 0.05). This coordinated rise indicates that chitosan enhances humoral immunity without strongly altering the underlying Th1/Th2 direction.

The IgG2a/IgG1 ratio did not differ among groups after the first immunization. However, after the booster dose, this ratio was significantly higher in the chitosan and antigen-only groups than in the Alum group (p < 0.05) (Figure 8). These results suggest that Alum tends to shift responses toward a Th2-biased profile, whereas chitosan maintains a more balanced Th1/Th2 response. Chitosan caused only minimal changes in the IgG2a/IgG1 ratio, indicating that its main effect is increasing antibody magnitude rather than modifying antibody quality—likely due to improved antigen presentation mechanisms (Zaharoff, 2007) [17].

Our data agree with previous reports showing that whole-virus A/H1N1/PR/8/34 vaccines elicit stronger IgG2a responses [43]. Similarly, live attenuated or whole-virus influenza vaccines generally promote Th1-skewed IgG2a responses, whereas subunit or inactivated formulations preferentially induce IgG1 [44,45]. These patterns highlight the influence of antigen type and adjuvant selection on antibody subclass distribution.

In mice, IgG1 plays a key role in neutralization through Th2 pathways, while IgG2a contributes to complement activation, Fc-mediated effector functions, and cellular immunity. IgG2a is often more protective against viral infection, and a combined IgG1–IgG2a response is essential for limiting viral replication. Therefore, the balanced induction observed in this study represents a favorable profile for influenza vaccine development.

Comparisons between chitosan nanoparticles and Alum showed no significant differences in overall antibody titers, consistent with findings in ovalbumin vaccines and in A/H5N1 formulations [40,46]. Although Alum remains the most widely used human vaccine adjuvant, its limitations include strong local inflammation and weak induction of T-cell responses.

In contrast, chitosan is a natural, biocompatible polymer with strong cationic charge that enhances interaction with negatively charged antigens and cell membranes. Chitosan can prolong antigen retention, improve uptake by antigen-presenting cells, and enhance both humoral and cellular immune activation. Prior studies have shown that chitosan nanoparticles facilitate antigen storage, slow release, and more efficient APC targeting, supporting balanced Th1/Th2 responses [37,38,39,40,41,42].

Overall, CSN formulations significantly enhanced IgG1 and IgG2a titers compared with antigen alone, confirming their strong adjuvant effect for the H5N1 antigen. The concurrent elevation of both subclasses and the balanced IgG2a/IgG1 ratio highlight chitosan as a promising next-generation adjuvant, offering advantages over Alum particularly for mucosal and nanoparticle-based influenza vaccines.

3.4. Safety and Stability Considerations

The dataset in Table 4 includes general safety testing of vaccine lots (CSNs, CSMs, and Alum) in mice undergoing weight monitoring. No adverse clinical signs were reported, and body weight changes were comparable, supporting tolerability of both chitosan particle sizes.

After more than 14 days of observation, the results (Table 4) demonstrated that all experimental BALB/c mice remained healthy and developed normally, with no clinical signs of toxicity observed. The average body weight of mice in both experimental and control groups reached approximately 25 g. These findings indicate that immunization with the H5N1 antigen formulated with chitosan nanoparticles or chitosan microparticles did not induce toxic effects in the tested animals. These results confirm the biocompatibility and nontoxic nature of chitosan nano- and microparticle-based vaccine formulations and are similar to the results of some recent works [47,48,49].

3.5. Vaccine Endotoxin Testing

Vaccine endotoxin testing, as part of quality control, was performed to ensure that vaccine lots with minimal endotoxin were selected to reduce side effects. The results of endotoxin testing of the vaccine formulations comprising CSNs, CSMs, and Alum with the H5N1 antigen are shown in Table 5. The results (Table 5) show that the endotoxin in three vaccine formulations ranged from 87.0 to 89.1 EU/mL. According to WHO recommendations, endotoxin levels in vaccines should not exceed 100 EU/mL. In the present study, all vaccine formulations—including antigen admixed with chitosan nanoparticles, chitosan microparticles, and aluminum hydroxide—exhibited endotoxin levels below 90 EU/mL, thereby meeting the recommended safety threshold.

Overall, the novelty of this work lies in systematically mapping antigen doses (0.15–5.0 µg) against two chitosan particle size regimes (nano vs. micro) under the same antigen adsorption workflow and intramuscular immunization schedule. Three specific advances emerge:

• (i)Demonstration of near-quantitative adsorption (97–99%) across the full dose range for both particle sizes, with dose-dependent LC quantified in HAU/mg (Table 3), supporting robust manufacturing feasibility;
• (ii)Identification of size-dependent optimal antigen doses, with immune response plateaus at 1.5 µg (nanoparticles) versus 3.0 µg (microparticles), informing rational antigen-sparing formulation design;
• (iii)Evidence that chitosan adjuvants can achieve Alum-comparable functional HI responses while preserving a more balanced IgG subtype profile.

The differential immunogenicity between chitosan nanoparticles and microparticles was discussed. The observation that chitosan nanoparticles (NPs) induced significantly higher HI titers, whereas chitosan microparticles (MPs) generated higher total IgG responses at antigen doses >3 µg, can be explained by fundamental size-dependent differences in antigen trafficking, cellular uptake, and immune polarization.

Particle size critically determines lymphatic transport and dendritic cell (DC) targeting. Nanoparticles within the 20–300 nm range can efficiently be drained via lymphatics to regional lymph nodes, where they directly interact with resident DC populations and promote germinal center reactions [50,51]. In contrast, larger microparticles (>1 µm) are primarily retained at the injection site and rely on cell-mediated transport, resulting in delayed antigen delivery [51].

Moreover, nanoparticles are more efficiently internalized by DCs via endocytosis and can promote cross-presentation pathways, enhancing Th1-biased immune responses and the generation of functional neutralizing antibodies [51,52]. Functional antibody quality, rather than total IgG quantity, is the major determinant of HI activity. The ability of nanoparticle-based vaccines to enhance neutralizing antibody titers (HI) has been well documented in nano-vaccine systems [53].

Additionally, chitosan nanoparticles have been shown to activate innate immune pathways, including the cGAS–STING signaling axis, thereby enhancing type I interferon responses and promoting stronger antiviral immunity [54]. Collectively, these mechanisms likely explain why nanoparticles achieved superior HI titers even at lower antigen doses.

In contrast, microparticles exhibit a stronger depot effect due to their larger size and slower clearance from the injection site. Prolonged antigen retention allows for sustained stimulation of antigen-presenting cells and promotes humoral responses, particularly Th2-skewed IgG production [55].

Size-dependent immune polarization has been demonstrated previously: particles >1 µm tend to favor phagocytosis-driven responses and humoral immunity, whereas nanoparticles more efficiently stimulate cellular and functional immune responses [51,55]. Sustained antigen exposure associated with larger particles may enhance total IgG titers at higher antigen loads, even if neutralizing efficiency (HI activity) does not proportionally increase [53]. Thus, while nanoparticles enhance antibody quality (HI), microparticles at high antigen doses may preferentially increase antibody quantity (IgG).

The data demonstrate a typical antigen dose–response saturation pattern. IgG titers increased markedly at a dose of 1.5 µg in the nanoparticle group but showed only marginal enhancement at 3.0 and 5.0 µg, indicating the establishment of a functional humoral ceiling. This plateau reflects classical immune kinetics in which antibody production increases proportionally only within an antigen-limited range before reaching biological saturation [56].

At low antigen doses, chitosan nanoparticles enhance dendritic cell uptake and antigen presentation, thereby amplifying B-cell activation and antibody production [17,44]. However, once the antigen threshold required for full recruitment of antigen-specific B cells and germinal center responses is reached, further antigen increases yield diminishing returns. At this stage, the response becomes constrained by intrinsic biological limits such as finite B-cell precursor frequency and T follicular helper cell support (Victora & Nussenzweig, 2012) [57]. The convergence of IgG titers between nanoparticle and Alum groups at higher doses suggests that when the antigen is no longer limiting, differences in delivery efficiency become less critical. Similar dose-sparing and saturation effects have been reported in influenza vaccination studies [36]. These findings support the identification of 1.5 µg as an optimal dose for achieving maximal humoral responses while avoiding unnecessary antigen use.

4. Conclusions

Chitosan micro- and nanoparticles adsorbed the H5N1 antigen with high efficiency across a wide dose range. Chitosan nanoparticles showed a pronounced antigen dose-sparing effect, achieving strong IgG and HI responses at low antigen doses and reaching an optimal plateau near a dose of 1.5 µg, whereas microparticles plateaued near 3.0 µg. Chitosan nanoparticles supported balanced IgG subtype responses and showed good tolerability and refrigerated stability. These results support chitosan-based polymeric particles as promising adjuvants for optimized H5N1 influenza vaccines.