Potentiating humoral and cellular immunity using a novel hybrid polymer-lipid nanoparticle adjuvant for HBsAg-VLP vaccine

doi:10.21203/rs.3.rs-3137881/v1

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Research Article

Potentiating humoral and cellular immunity using a novel hybrid polymer-lipid nanoparticle adjuvant for HBsAg-VLP vaccine

https://doi.org/10.21203/rs.3.rs-3137881/v1

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Journal Publication

published 22 Nov, 2023

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Aluminum adjuvants are commonly used in vaccines to stimulate the immune system, but they have limited ability to promote cellular immunity which is necessary for clearing viral infections like hepatitis B. Current adjuvants that do promote cellular immunity often have undesired side effects due to the immunostimulants they contain. In this study, a hybrid polymer lipid nanoparticle (HPLNP) was developed as an efficient adjuvant for the hepatitis B surface antigen (HBsAg) virus-like particle (VLP) vaccine to potentiate both humoral and cellular immunity. The HPLNP is composed of FDA approved polyethylene glycol-b-poly (L-lactic acid) (PEG-PLLA) polymer and cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and can be easily prepared by a one-step method. The cationic optimised vaccine formulation HBsAg/HPLNP (w/w = 1/600) can maximise the cell uptake of the antigen due to the electrostatic adsorption between the vaccine nanoparticle and the cell membrane of antigen-presenting cells. The HPLNP prolonged the retention of the antigen at the injection site and enhanced the lymph node drainage of antigen, resulting in a higher concentration of serum anti-HBsAg IgG compared to the HBsAg group or the HBsAg/Al group after the boost immunisation in mice. The HPLNP also promoted a strong Th1-driven immune response, as demonstrated by the significantly improved IgG2a/IgG1 ratio, increased production of IFN-γ, and activation of CD4 + and CD8 + T cells in the spleen and lymph nodes. Importantly, the HPLNP demonstrated no systemic toxicity during immunisation. The advantages of the HPLNP, including good biocompatibility, easy preparation, low cost, and its ability to enhance both humoral and cellular immune responses, suggest its suitability as an efficient adjuvant for protein-based vaccines such as HBsAg-VLP. These findings highlight the promising potential of the HPLNP as an HBV vaccine adjuvant, offering an alternative to aluminum adjuvants currently used in vaccines.

hybrid polymer-lipid nanoparticle

HBV

vaccine adjuvant

humoral immunity

cellular immunity

While combating the spread of infectious diseases, vaccination stands as a highly effective method for preventing hospitalization and fatality[1, 2]. Despite most marketed vaccines consisting of attenuated or inactivated pathogens, the trend is shifting towards the design of microbial subunit vaccines based on specific antigenic components from the pathogens. As microbial subunit vaccines do not contain live components of the pathogen, there is no risk of disease transmission, making them safer and more stable than vaccines containing whole pathogens[3]. Among subunit vaccines, virus-like particles (VLPs) formed by self-assembled viral proteins are highly desirable due to their exceptional immunogenicity[4, 5]. The Hepatitis B surface antigen (HBsAg) and human papillomavirus (HPV) vaccines are well-known examples of VLP-based vaccines[6–8]. However, VLP-based vaccines face several interrelated pharmacological challenges, including rapid degradation and clearance, low accumulation in secondary lymphoid organs, and inefficient delivery of antigen to the MHC class I antigen processing pathway, which is crucial for inducing a CD8 + T cell response[9–11]. As a result, VLP-based antigens alone may not be immunogenic enough, and often require the addition of appropriate adjuvants to enhance the immune response[12].

Adjuvants are immunomodulatory substances added to vaccines for several benefits, such as enhanced immunogenicity, antigen dose sparing, quicker immune response, prolonged duration of prophylaxis, and less booster vaccinations[13, 14]. Adjuvants act as an antigen reservoir and increase the duration of antigen presentation to immune cells. They also stimulate the recruitment and activation of antigen-presenting cells (APCs), which play a critical role in immunostimulation. APCs process vaccine antigens and transport them to draining lymph nodes, where they promote the proliferation and differentiation of T and B cells[15].

To date, most licensed VLP-based vaccines utilize the classic aluminum adjuvants, such as aluminum hydroxide or aluminum phosphate. These adjuvants are highly effective in enhancing antibody responses as they can adsorb antigens through electrostatic interaction, which helps to maintain the physiochemical properties and stability of the vaccine[16]. When injected, antigens that are bound with adjuvants diffuse more slowly from the site of administration, allowing antigen-presenting cells (APCs) to accumulate for better recognition, processing, and presentation of the antigens. Flach et al. demonstrated that aluminum adjuvant could perturb the DC membrane and induce cell membrane lipid reordering without entering the cell, which further lead to antigen uptake and upregulation of CD4 + T cells[17]. However, aluminum adjuvants are usually associated with adverse reactions such as localized inflammation, pain and tenderness at the injection site, and allergic reactions, immunosuppression, and even teratogenic, carcinogenic, and mutagenic risks [18–23]. In some cases, they have also been linked to neurodegeneration, renal dysfunction, and the increased production of eosinophils and immunoglobulin E due to the propensity of Th2 immune bias[24]. Furthermore, substantial amounts of mechanistic studies have demonstrated that the aluminum salt-based adjuvants stimulate the Th2 immune responses, with minimum or no Th1 responses[25]. It is not ideal for viral diseases that require a Th1-mediated immunity and activation of cytotoxic T cells for protection. Th1-type immune responses can be stimulated by using innate immunostimulants such as CpG oligodeoxynucleotides[26], monophosphoryl lipid A[27], and poly I:C[28], however, immunostimulants may produce adverse reactions such as autoimmune diseases while effectively inducing an immune response.

It has been known that Class I MHC (MHC-I) molecules present primarily endogenous antigens, i.e. antigens that are present in the cytosol and are subject to the cytosolic processing mechanisms that comprise the conventional MHC-I processing pathway, which plays important roles in generating CD8 T cell responses[29]. After phagocytic or endocytic uptake, some exogenous antigens can escape the vacuolar system and penetrate into the cytosol, accessing the conventional MHC-I antigen processing mechanisms[30]. Thus, it is possible for adjuvants to deliver exogenous antigens to stimulate CD8 T cell responses by mechanisms that may contribute to the substitution of MHC-I processing pathways. The cationic delivery system with “sponge effect” has the potential to allow for the exogenous antigen to escape from the endosomes and enter the cytosol for inducing CD8 + T cell responses via the MHC-I pathway[31].

Over the past few decades, substantial research efforts have been dedicated to the development of alternative vaccine adjuvants. The development and synthesis of adjuvants to regulate the immune system and enhance vaccine efficacy have been widely studied for the improvement of vaccine design and treatment of infectious diseases. The properties of adjuvants depend largely on particle size[32], hydrophobicity[33], administration route[34], antigen release kinetics[35], and surface charge[36]. For example, positive-charged adjuvants like liposomes, cationic polymers, or emulsions interact well with negative-charged cell membranes of antigen-presenting cells (APCs), promoting the intracellular uptake of antigens [37, 38]. However, these adjuvants are often composed of multiple components and complicated to prepare, making overall quality control more complex. For instance, chitosan, a cationic polymer, acts as an adjuvant largely based on its structure, amphiphilicity, and surface charge of self-assembled structures[39]. However, the varying quality of chitosan in different batches due to its complicated synthesis can affect the immunogenicity of vaccines and limit its clinical use[40, 41]. There is a pressing need for the development of adjuvants which have a broad-spectrum of safety, good stability, ease of production and use, and can effectively activate humoral and cellular immune responses with no adverse reactions.

Based on our previous work, hybrid nanoparticles provide a convenient approach for multiple functionalities and regulating properties[42, 43]. The structures and properties of hybrid nanoparticles can be easily regulated by changing the mixing ratios of different components rather than complicated synthesis. Cationic hybrid nanoparticles may be an effective vaccine adjuvant to potentiate the immune response, as they can delivery antigens and enhance the cellular uptake of antigens by APCs through electrostatic absorption. In light of the challenges posed by current vaccine adjuvants, we aim to develop a cationic hybrid polymer lipid nanoparticle (HPLNP) as an efficient vaccine adjuvant with multifaceted immune responses (Fig. 1B). The HPLNP is composed of two FDA-approved materials: polyethylene glycol-b- poly (L-lactic acid) (PEG-PLLA) polymer and cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). As shown in Fig. 1(A), the HPLNP can be prepared by a simple one-step method based on several minutes of mixing, stirring and organic solvent evaporation. The physicochemical properties of HPLNPs can be preciously controlled by regulating the mixing ratios of polymer and lipid. The size distribution, PDI, and zeta potential of HPLNPs were analysed through dynamic light scattering, and the morphology was confirmed through transmission electron microscopy (TEM). The HBsAg-VLP was used as a model antigen and mixed with the HPLNP to create vaccine formulations. Flow cytometry was applied to optimise the vaccine formulation through analysing the intracellular uptake of antigen by APCs. Finally, the in vivo immunisation of the optimised vaccine formulation was carried out. The antigen depot effect and the lymph node drainage of the optimised vaccine formulation were studied by the small animal in vivo imaging. The humoral responses of different formulations were compared by analysing the serum anti-HBsAg IgG concentration at specific time points. The IgG2a/IgG1 ratio was calculated to estimate the Th1/Th2 immune response trend. The secretion of cytokines in serum was evaluated by Elisa kit. Meanwhile, the activation of B cells and T cells in the spleen or lymph nodes was tested by flow cytometry. For biosafety assessment, the immunohistochemical staining of different organs and intramuscular injection sites post prime immunisation were finally analysed and the mice body weights were monitored during the immunisation.

2.1 Materials

mPEG_5k-PLLA_10k was bought from Tansh Tec Co., LTD (Guangzhou, China), tetrahydrofuran (THF), 4% (w/v) paraformaldehyde solution, fluorescein isothiocyanate (FITC), 4’,6-diamidino-2-phenylindole (DAPI), Triton X-100 were purchased from Sigma-Aldrich. Cy5.5-labeled BSA was purchased from Shanghai Yuanye Biotechnology Co. LTD. 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) DOTAP was bought from Avanti Polar Lipids. CCK8 assay was obtained from Abcam (Cambridge, UK) Trypsin-EDTA (0.25%, w/v), fetal bovine serum (FBS) and penicillin-streptomycin (100×) were bought from Gibco (CA, USA). Phosphate buffered saline (PBS), RPMI-1640 medium and high glucose Dulbecco's modified eagle medium (DMEM) were obtained from Hyclone lab (UT, USA). Fixation/Permeabilization solution and Perm/Wash buffer were purchased from Nuowei Biotechnology Co. LTD (Beijing, China). Sulfo-Cyanine5.5 (Cy5.5) fluorescently labelled antibody protein/kit and fluorescein isothiocyanate (FITC) fluorescently labelled antibody protein/kit were purchased from Abcam (Cambridge, UK). Antibodies of PerCP/Cy5.5-CD3, ER780-CD8a were obtained from Elabscience Biotechnology Co. LTD. Antibodies of Ms CD4 FITC GK1.5, Ms CD8a PerCP-Cy5.5 (53 − 6.7), Ms CD19 APC-Cy7 1D3, Ms CD69 APC H1.2F3 and Ms CD178 PE MFL3 were brought from Becton, Dickinson and Company (New Jersey, USA). Mouse ELISA Kits of Anti-HBsAg IgG, Anti-HBsAg IgG1 and Anti-HBsAg IgG2a were obtained from Shanghai Runyu Biotechnology Co. LTD (Shanghai, China). Mouse ELISA Kits of IL-4, IL-6, IFN-γ and Gzms-B were purchased from Beyotime Biotechnology Co. LTD (Shanghai, China). RAW264.7 cells were obtained from the ATCC (American Type Culture Collection, Wesel, Germany). Sheep blood was purchased at TCS Biosciences (Buckinghamshire, UK). HBsAg-VLP antigen was both kindly gifted by AIM Honesty Biopharmaceutical Co., Ltd (AIM, Dalian, China). Al adjuvant was obtained from Miragen (Delaware, USA).

2.2 Preparation and characterisation of HPLNP and HBsAg/HPLNP formulations

The HPLHNs were prepared by a simple one-step method presented in Fig. 2A[44]. Typically, 650 µg PEG_5k-PLLA_10k and 250 µg DOTAP were co-dissolved in 0.5 mL THF, followed by mixing with 1 mL of water. The mixture was kept stirring in a ventilation cabinet and the HPLNPs were obtained until THF was evaporated. The size distribution and zeta potentials of HPLNPs were tested by dynamic light scattering (DLS). TEM was used to observe the morphology of HPLNPs. Various HBsAg/HPLNP formulations were prepared by mixing the HBsAg and HPLNPs with different mass ratios directly, and their size distribution and zeta potentials were also evaluated by DLS.

2.3 In vitro antigen release

The Cy5.5-labeled Bovine Serum Albumin (Cy5.5-BSA) was utilized as the model antigen to study the in vitro antigen release profile. The BSA/HPLNP formulation with a weight ratio of 1/600 was prepared using the previous method described for the HBsAg/HPLNP (w/w = 1/600) formulation in section 2.2. Then the BSA/HPLNP (w/w = 1/600) formulation, containing 40 µg of BSA, was incubated in a 100 kDa molecular weight cut off dialysis bag (Yuanye Biotechnology Co., Ltd, Shanghai, China) in PBS (pH 7.4) at 37°C for 72 hours. The accumulated release of BSA from the BSA/HPLNP formulation was analysed at predetermined time intervals by measuring the fluorescence intensity of the release media using a fluorescence spectrophotometer (Yidian 970CRT, Shanghai, China), resulting in the generation of the corresponding release curve. The BSA/Al group, which was prepared by mixing BSA and aluminum adjuvant at a mass ratio of 1/25 as per the manufacturer’s instructions, was used as a control, along with the BSA group.

2.4 Cell culture

RAW264.7 cells were cultured in high glucose DMEM medium supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin (100×) in a humidified atmosphere with 5% CO₂ at 37^oC.

2.5 Cytotoxicity and haemolysis of HPLNPs

The cytotoxicity of HPLNPs with different concentrations on RAW264.7 cells was analysed using a CCK8 assay. RAW264.7 cells were seeded in a 96-well plate at a density of 5×10⁴ cells/well. After incubation for 24 h, cells were treated with various concentrations of HBsAg/HPLNPs (w/w = 1/600) formulation in a completed DMEM medium for 24 h. Then, 10 µL of CCK8 solution was added to each well. After further incubation for 4 h, the absorbance of each well at 450 nm was measured using a Spectrofluorometer (GloMax® Discover Microplate Reader, Promega, USA). The cytotoxic effect was determined from the absorbance readings.

The haemolysis of HPLNPs was also investigated. Briefly, defibrinated sheep erythrocytes (RBCs) were centrifuged at 1500 × g for 10 min at 4°C and washed with PBS for three times. The cell pellets were re-suspended into a 5% (v/v) erythrocyte suspension with PBS. The HPLNPs was added into the RBC suspension with a final concentration at 0.5 mg mL^− 1. Al adjuvant was separately added into the RBC suspension at the same concentration for comparison. After incubation at 37°C for 1 h, the RBC suspension was centrifuged, and 100 µL of the supernatant was transferred to a 96-well plate. The RBC suspension treated with deionized water or PBS was used as the positive or negative control, respectively. The absorbance (A) was measured at 540 nm using a spectrofluorometer (GloMax® Discover Microplate Reader, Promega, USA). The relative haemolysis was calculated according to the following equation:

$$\text{R}\text{e}\text{l}\text{a}\text{t}\text{i}\text{v}\text{e} \text{h}\text{a}\text{e}\text{m}\text{o}\text{l}\text{y}\text{s}\text{i}\text{s}\left(\text{\%}\right)= \frac{{\text{A}}_{\text{S}}-{\text{A}}_{\text{N}\text{C}}}{{\text{A}}_{\text{P}\text{C}}-{\text{A}}_{\text{N}\text{C}}}$$

Where the A_S, A_NC and A_PC represent the absorbance of the sample, the negative control and the positive control respectively.

2.6 Cellular uptake of HBsAg/HPLNP formulation by macrophages

FITC-labelled HBsAg was obtained according to the protocol of fluorescein isothiocyanate (FITC) fluorescently labelled antibody protein/kit. Flow cytometry was utilized to examine the cellular uptake of the HBsAg/HPLNP formulations. The HBsAg/HPLNP formulations were prepared by mixing the FITC-conjugated HBsAg and HPLNP in varying HBsAg/HPLNP mass ratios, ranging from 1/20 to 1/1600. The size distribution and zeta potential of these formulations were analysed using DLS.

RAW264.7 cells were seeded in the 6-well plate at the density of 4×10⁵ per well for 24 h. After replacing the culture medium into fresh serum-free medium, the HBsAg or various HBsAg/HPLNP formulations with different mass ratios were added in at a concentration of 1 µg mL^− 1 of HBsAg. Following a 4-hour incubation period, the cells were washed with PBS for three times and collected for analysing by flow cytometry. The percentage of FITC-positive cells and the mean fluorescence intensity (MFI) were both evaluated. Meanwhile, 2×10⁵ of RAW264.7 cells were also seeded in the confocal dish for 24 h. The HBsAg or HBsAg/HPLNP (w/w = 1/600) formulation was added at the concentration of 1 µg mL^− 1 of HBsAg and incubated for another 4 h. Then the cells were washed with PBS for three times and incubated with 5 µg mL^− 1 of DAPI for 10 min. After washing with PBS and fixed with 4% paraformaldehyde, the cells were observed under a fluorescence confocal microscope (Leica SP8 Inverted) to investigate the intracellular uptake of HBsAg.

2.7 Animals

6 ~ 8 weeks-old female C57BL/6 mice used for animal experiments were purchased from SPF (Beijing) Biotechnology Co., Ltd. The mice were maintained under specific pathogen-free condition at the animal facility in the Key Lab, Shen Zhen University General Hospital.

2.8 Injection site antigen depot and lymph nodes drainage

The Cy 5.5-labelled HBsAg was prepared according to the protocol of Cy5.5 fluorescently labelled antibody protein/kit for analysing the antigen depot at the injection site. 6 ~ 8 weeks-old female C57BL/6 mice were grouped (n = 4) and intramuscularly injected at the right back leg with HBsAg, HBsAg/Al or HBsAg/HPLNP (w/w = 1/600) formulations at the dose of 1 µg HBsAg per mouse. After intramuscular administration, the fluorescence graphs of the injection site were collected through in vivo imaging system FX Pro (Kodak), at 12, 24, 48 and 72 hours. The mean fluorescence intensity graph of mice at the injection site in each group was obtained at different time points. After intramuscular administration, the fluorescence signals of mesenteric lymph nodes were also collected at 12, 24, 48 and 72 hours. The mean fluorescence intensity at the injection sites and mesenteric lymph nodes was calculated for different groups to compare the efficacy of various vaccine formulations in terms of antigen depot effect and lymph nodes drainage.

2.9 Activation of lymphocytes in lymph nodes

The mice were divided into HBsAg, HBsAg/Al and HBsAg/HPLNP (w/w = 1/600) groups (n = 5) and intramuscularly injected with various formulations at the dose of 1 µg HBsAg per leg. After 18 h, the mice were sacrificed and the mesenteric lymph nodes were collected. The lymphoid tissue suspension can be prepared by initially cleaning the tissue with PBS and subsequently cutting it into small pieces (1 ~ 2 mm³), followed by using a tissue grinder equipped with a sandpaper mesh to grind the lymph nodes until complete disintegration. This process is repeated until all the lymph nodes are uniformly homogenised. The resulting homogenate is then transferred into a centrifuge tube, and the serum-free RPMI-1640 medium is added to prepare the lymphoid tissue suspension. To obtain a single-cell suspension, the lymphoid tissue suspension is aspirated using a 5 mL syringe with a 21G needle, filtered through a 40 µm-cell strainer, and collected into a 15 mL centrifuge tube. The desired single-cell suspension was applied for further downstream applications. To prepare the cells for intracellular staining, the cell pellet is resuspended in 250 µL of fixation/permeabilization solution per tube and incubated at 4°C for 30 minutes in the dark. After fixation and permeabilization, the cells are centrifuged at 450 g for 5 minutes, and 1 mL of 1× Perm/Wash™ buffer is added, followed by incubation for 15 minutes. Next, the cells are centrifuged at 450 g for 5 minutes at 4°C, and the fixed and permeabilized cells are resuspended in 100 µL of 1× Perm/Wash™ buffer. Subsequently, the desired fluorescently conjugated antibody including PerCP/Cy5.5-CD3, ER780-CD8a, Ms CD4 FITC GK1.5, Ms CD19 APC-Cy7 1D3 and Ms CD69 APC H1.2F3 were added at a predetermined optimal concentration, and the mixture is vortexed and incubated for 40 ~ 45 minutes at 4°C. The cells were analysed by flow cytometry for detecting of activation of B cells and T cells in lymph nodes.

2.10 In vivo immunisation

The immunisation schedule was shown in Fig. 6A. The study involved testing various vaccine formulations using groups of six mice each. The HBsAg/Al formulation was prepared by mixing with HBsAg and Al adjuvant at a mass ratio of 1/25, following the manufacturer’s guidelines. The optimal mass ratio for the HBsAg/HPLNP formulation was determined to be 1/600. Mice were randomly grouped into four groups (PBS, HBsAg, HBsAg/Al, HBsAg/HPLNP). Each mouse was administered a 50 µL injection of the formulation, at a dose of 1 µg of HBsAg per leg. Boost injections were carried out on Day 14 and Day 28, respectively. The blood was collected at predetermined time points from the orbit venous plexus and was stationarily placed at room temperature for 4 h, before being centrifuged at 10,000 g for 10 minutes to obtain the serum. The serum anti-HBsAg IgG concentration was tested on Day 21, Day 35 and Day 42 by ELISA Kits according to the manufacturer’s protocol. The concentrations of serum anti-HBsAg IgG1 and anti-HBsAg IgG2a were also studied on Day 42. Cytokines including IL-4, IL-6, IFN-γ and Gzms-B in serum on Day 42 were also measured by ELISA Kits. Meanwhile the body weights of the mice were monitored throughout the immunisation period.

2.11 Evaluation of intracellular IFN-γ secretion by splenocytes

After immunisation, the mice were sacrificed on Day 42, and the spleens were extracted and homogenized using a 40 µm-cell strainer. The splenocytes were then separated from the erythrocytes and washed with RPMI 1640 before being resuspended in a complete medium for further experiments. Antibody was coated onto pre-wet PVDF plates under sterile conditions and incubated overnight at 4°C. The plates were washed for five times, and 200 µL of complete medium was added and incubated for 30 minutes. Afterwards, 5×10⁵ splenocytes were added to each well, and HBsAg (5 mg mL^− 1) was added to re-stimulate the splenocytes for 24 h at 37^oC. The process involved washing the plates and then incubating them with the detection antibody for a period of 2 h. Following this, the plates were washed again and incubated with streptavidin-ALP for 1 h. Subsequently, substrate (BCIP/NBT) was added to initiate spot development in the dark. After 10 minutes of development, tap water was added to halt colour development, and the plates were left to dry. Finally, the spots were analysed using the ChampSpot Elispot II system.

2.12 Splenocyte proliferation in vitro

The splenocyte proliferation in vitro was assessed using the CCK8 assay, following the manufacturer’s instructions. In brief, 5×10⁵ splenocytes were seeded into a 96-well plate with or without stimulation of 5 µg mL^− 1 HBsAg for 24 or 48 h. After incubation, 10 µL of CCK8 solution in fresh culture medium was added and incubated at 37^oC for another 4 h. The absorbance was measured at 450 nm. The splenocyte proliferation was determined by dividing the absorbance of stimulated cultures by that of non-stimulated cultures, while the absorbance of the medium was used as the background.

2.13 Detection of the splenocyte activation

The splenocytes were collected as previously described. Following restimulation with 5 µg mL^− 1 HBsAg in vitro for 48 h, the splenocytes were washed and labelled with Ms CD4 FITC GK1.5, Ms CD8a PerCP-Cy5.5 (53 − 6.7) and Ms CD19 APC-Cy7 1D3 to distinguish T, B cells, respectively. Additionally, Ms CD69 APC H1.2F3 and Ms CD178 PE MFL3 were used to detect the activation of B and T cells, respectively. After a 30-minute incubation at 4°C, the cells were washed and suspended in 500 µL of PBS buffer before being analysed by flow cytometry.

2.14 Histologic analysis

Mice were euthanized 24 hours after the first intramuscular injection. Samples of the injection site muscle and key organs were collected and processed. They were fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin wax, and sliced into 8-mm sections for Hematoxylin and Eosin (H&E) staining. The sections were then examined using a Leica DM3000 optical microscope and photographed.

3.1 Preparation and characterisation of HPLNPs

The HPLNPs were prepared by a simple one-step method based on several minutes of mixing, stirring and organic solvent evaporation as shown in Fig. 2A. The size distribution and zeta potential of HBsAg, HPLNP and HBsAg/HPLNP (w/w = 1/600) have been shown in Fig. 2B and 2C. The average size of the HPLNPs was 53.3 ± 0.2 nm in diameter, with a low polydispersity index (PDI) of 0.32 ± 0.01 in Table S1. The HPLNPs had a positive surface charge, with an average zeta potential of 27.9 ± 1.7 mV. The morphology of the HPLNPs was observed through transmission electron microscopy (TEM) in Fig. 2D, which revealed a uniform size distribution around 50 ~ 60 nm, consistent with the DLS data. After mixing with HBsAg antigen (35.9 ± 0.8 nm, -17.7 ± 1.6 mV), HBsAg/HPLNP formulations with different HBsAg to HPLNP mass ratios showed increased size distribution (around 57 ~ 75 nm) and positive surface charge (around 17 ~ 37 mV) in Table S2. The HBsAg/HPLNP (w/w = 1/600) formulation exhibited an average size distribution around 58.5 ± 0.3 nm, which was slightly larger than that of the HPLNPs. The zeta potential of the HBsAg/HPLNP (w/w = 1/600) formulation was measured to be 22.9 ± 0.5 mV as indicated in Table S1. This value was lower than the zeta potential of the HPLNPs alone, but still positively charged, which is beneficial for the uptake of the formulation by APCs.

3.2 In vitro antigen release

One of the mechanisms by which adjuvants improve immune response is by adsorbing the antigen and creating a depot effect at the injection site. The in vitro antigen release result, which used a dialysis bag to simulate antigen release at the injection site, have been shown in Fig. 2E. The cumulative release of BSA in the BSA group without adjuvants increased over time and reached equilibrium at 33.8% after 24 hours. In contrast, the groups with Al or HPLNP adjuvants exhibited much slower antigen release rates than the BSA group, and equilibrium was not reached until 48 hours. Furthermore, the maximum cumulative release of BSA at 72 hours in the BSA/Al group and the BSA/HPLNP group was about 17.4% and 8.6%, respectively, which was significantly lower than that in the BSA group (**** p < 0.0001). Meanwhile, the release of BSA in the BSA/HPLNP group was significantly lower than that in the BSA/Al group (**** p < 0.0001). These results demonstrated the effective antigen absorption and depot effect of the HPLNPs. Consequently, the HPLNP showed a slower antigen release rate, increasing the likelihood of antigen presentation and subsequent cellular uptake by APCs. These findings suggest that the HPLNP may offer advantages over the Al adjuvant in terms of its ability to facilitate antigen presentation and cellular uptake.

3.3 Cytotoxicity and haemolysis of HPLNPs

To evaluate the biocompatibility of HPLNPs, cell cytotoxicity and haemolysis, which are typical issues associated with cationic carriers due to their strong interaction with negatively charged cell membranes, were tested. The results indicated that the cell viability of RAW264.7 cells decreased as the concentration of HBsAg/HPLNP formulation increase. The increased cytotoxicity was due to the positive charge accompanied with the HPLNPs. It is encouraging that HPLNPs had a negligible haemolysis rate of less than 5%, which was significantly lower than that of Al adjuvant (*p < 0.05). These results demonstrated that the HPLNP had low cytotoxicity and favourable biocompatibility, making it possible for further in vivo studies.

3.4 Cellular uptake of vaccine formulations

Antigen cellular uptake by APCs is the basis of the protective immune response mechanism in vaccination. Furthermore, cytoplasmic delivery of antigens is crucial for the induction of antigen-specific immune responses. It is well known that soluble antigens alone are poorly effective in protective immunity, mainly because of quick degradation and insufficient cellular uptake by APCs. Thus, adjuvants can make the vaccine more immunogenic by protecting antigen and promoting its cell uptake by APCs (Fig. 3A). Herein, we formulated vaccine formulations using FITC-HBsAg adsorbed to HPLNPs and investigated the cellular uptake of formulations with different components in RAW264.7 cells using flow cytometry. Due to the positive charge of HPLNPs, HBsAg/HPLNPs formulations had a stronger electrostatic attraction to the negatively charged cell membrane, leading to a significantly higher cellular uptake compared to HBsAg alone. In Fig. 3B, only ~ 10.4% of cells in the HBsAg group were FITC-positive, while nearly all of the cells (~ 98.9%) in the HBsAg/HPLNP (w/w) = 1/600 group were FITC-positive. When the mass ratio of HPLNP to HBsAg reached 600, a significantly higher percentage of FITC-positive cells (*** p < 0.001) and intracellular MFI (**** p < 0.0001) were observed compared with the HBsAg/HPLNP (w/w = 1/400) group. Meanwhile, the percentage of FITC-positive cells and the intracellular MFI were kept stationary when the mass ratio of HPLNP to HBsAg exceeded 600, indicating the saturation of the adjuvant effect of HPLNP. It convincingly demonstrated that the cellular uptake of HBsAg increases with an increase in the amount of HPLNPs in Fig. 3C ~ 3D. The fluorescence confocal microscope images also confirmed that the HPLNP significantly enhanced the cellular uptake of HBsAg when the mass ratio of HPLNP to HBsAg reached 600. This was evident from the higher intracellular fluorescence intensity observed in the HBsAg/HPLNP group as compared to the HBsAg group in Fig. 3E. We postulated that the maximum cellular uptake of the antigen could be achieved using the HPLNP formulation with an optimised adjuvant/antigen mass ratio of around 600. Henceforth, the terms “HBsAg/HPLNP” formulations mentioned hereafter will pertain to the adjuvant/antigen mass ratio of 600, unless explicitly stated otherwise.

3.5 Injection site antigen depot and lymph node drainage

One possible mechanism by which adjuvants improve the immune response is by prolonging the retention of antigen at the injection site and enhancing lymph node drainage. Images obtained showed varying rates of antigen diffusion at the injection site among the HBsAg, HBsAg/Al, and HBsAg/HPLNP groups (Fig. 4A). The HBsAg group exhibited faster antigen diffusion compared to the HBsAg/Al and HBsAg/HPLNP groups, as evidenced by a larger fluorescence area after injection for 12 h. The HBsAg/HPLNP group retained the antigen at the injection site for up to 72 hours with a much lower diffusion speed than that in HBsAg group, suggesting that HPLNPs effectively prolonged the antigen depot and reduced antigen diffusion. While fluorescence signals were still detectable at the injection site of HBsAg and HBsAg/Al groups 72 hours post-injection, and their mean fluorescence intensities were significantly lower compared to the HBsAg/HPLNP group (as shown in Fig. 4B). Within 12 to 72 hours after the injection, the MFI of the HBsAg group decreased slowly (from 1.45×10⁸ to 1.17×10⁸) and the HBsAg/Al group showed little change (around 1.31 ~ 1.45×10⁸). While the MFI of the HBsAg/HPLNP group at 72 h (2.08×10⁸) was even higher than that at 12 h (1.72×10⁸). This finding may be attributed to the strong antigen depot effect of HPLNPs, which efficiently condensed the antigen at the injection site and released it slowly over time. The observed increase in fluorescence was likely due to the gradual release of the antigen from the injected muscle site, further indicating that HPLNPs enhance and prolong the antigen depot effect, which is beneficial for enhancing the antigen uptake by APCs and the following immune response.

Meanwhile, the fluorescence of the lymph node in HBsAg/HPLNP group at 12 h was obviously higher than that of the HBsAg (** p < 0.01) or HBsAg/Al (*** p < 0.001) group, indicating a faster and stronger antigen drainage to the mesenteric lymph node (Fig. 4C). On the other hand, there was a reduction in the signal observed at the lymph nodes after 48 hours, which was most likely due to the absorption of the vaccine nanoparticles into the lymphatic capillaries in the subcutaneous tissue. These nanoparticles were then transported into the lymph nodes, where the HBsAg could have been taken up by lymphocytes and broken down by the APCs. Over time, the antigen would be distributed throughout the circulatory and lymphatic system, resulting in a decrease in the signal observed at the lymph nodes (Fig. 4D). Despite a decrease in fluorescence intensity over time, the HBsAg/HPLNP group exhibited much higher fluorescence intensity in the lymph nodes compared to the HBsAg/Al group after 72 hours (* p < 0.05). These results provide evidence for the advantages of HPLNP as an adjuvant, which include prolonging the antigen depot at the injection site, enhancing antigen lymph node drainage, and decreasing antigen diffusion or degradation.

3.6 Activation of lymphocytes in lymph node

Encouraged by the antigen depot and lymph node drainage outcome, we further explored the adjuvant effects of HPLNPs vaccines on aspects of in vivo lymphocytes activation. After the first immunisation with different formulations for 18 h, the lymphocytes in mesenteric lymph nodes were analysed by flow cytometry (Fig. 5A). It revealed that the HBsAg/HPLNP group induced 26.1% of CD69 + CD8 + T cells, which was significantly higher than the HBsAg (~ 13.02%, *p < 0.05) or HBsAg/Al (~ 5.87%, **p < 0.01) groups (Fig. 5B). Meanwhile, similar trend can be found in CD69 + CD4 + T cells among these groups (Fig. 5C). There was no significant difference in activation of B cells among these groups (Fig. 5D). It’s encouraging that the HPLNPs showed superiority in activating T cells in lymph nodes compared with commercial Al adjuvant.

3.7 In vivo immunisation

Based on the promising results of cellular uptake and lymph node drainage, an in vivo immunization study was conducted using the schematic procedure shown in Fig. 6A. The production of anti-HBsAg IgG tended to increase over time after the three intramuscular injections. The HBsAg/HPLNP group had considerably higher anti-HBsAg IgG concentration than the HBsAg and HBsAg/Al groups after the prime immunisation on Day 14 (Fig. 6B). After the first boost, the HBsAg/HPLNP group induced significantly higher anti-HBsAg IgG level compared to the HBsAg alone (*p < 0.05) or HBsAg/Al group (*p < 0.05) on Day 21 (Fig. 6C). After the second boost, the anti-HBsAg IgG concentration of the HBsAg/HPLNP group was still significantly higher than that of the HBsAg group on Day 35 (**p < 0.01, Fig. 6D) and Day 42 (* p < 0.05, Fig. 6E). These results indicated that the HPLNP adjuvant can induce a quicker and stronger humoral immune response (Th2 type) than the pure HBsAg group and Al adjuvant.

Furthermore, a preferred adjuvant should promote both Th1 and Th2 responses. The Th1-type immune response is particularly important especially in chronic virus-related diseases like Hepatitis B virus infection. However, the frequently used Al adjuvants are biased towards a Th2-driven response. Then we analysed the anti-HBsAg IgG2a/IgG1 ratio to evaluate the ability of HPLNPs adjuvant for inducing a Th1-driven response. In Fig. 6F, the concentrations of anti-HBsAg IgG1 isotype related to the humoral response (Th2) were similar in all groups after three injections on Day 42. However, the anti-HBsAg IgG2a reflecting the T cell activation (Th1) in HBsAg/HPLNPs group was significantly higher than that in HBsAg (** p < 0.01) or HBsAg/Al group (** p < 0.01) (Fig. 6G). The anti-HBsAg IgG2a/IgG1 ratio of HBsAg/HPLNP group was much higher than HBsAg group or HBsAg/Al group (**p < 0.01), while the HBsAg/Al group even reduced the IgG2a/IgG1 ratio compared to the pure HBsAg in Fig. 6H (**p < 0.01). These results prove that the HPLNP is effective than Al adjuvant in stimulating the Th1-driven immune response, which correlates to the cytotoxic T lymphocyte (CTL) reactions.

Except for the antibody analysis, we also tested the serum cytokine secretion. In Fig. 7A, the secretion of IL-6 in HBsAg/HPLNP group was obviously higher than that in HBsAg/Al group (* p < 0.05), which confirmed the promotion of humoral immunity by HPLNPs, as IL-6 plays an important role in B cell proliferation, differentiation and antibody production. Also, both the HBsAg/HPLNP and HBsAg/Al groups had a much higher IL-4 production than the HBsAg group, which further confirmed the HPLNPs can induce a Th2-driven response as effectively as Al adjuvant (Fig. 7B). Meanwhile, the HPLNPs significantly enhanced the serum concentration of IFN-γ than the HBsAg or HBsAg/Al groups, emphasizing the strong Th1 driving effect (Fig. 7C). It was because IFN-γ was the typical cytokine secreted by Th1 or CD8 + T cells. This result made a good correlation with the IgG2a/IgG1 results in Fig. 6H. Besides, the cytokine of Gzms-B was also analysed (Fig. 7D). Taken together, the HPLNP adjuvant can effectively improve the Th2 immune responses but also has a strong propensity to induce the Th1 immune response, which was more advantageous than the aluminum adjuvant.

3.8 Intracellular cytokines secretion, activation and proliferation of splenocytes

As the spleen is the most significant immune organ, we evaluated the activation of splenocytes and the intracellular cytokine secretion. After restimulated with HBsAg for 48 h, the number of IFN-γ secreting CD8 + T splenocytes in HBsAg/HPLNP group was significantly higher than that of the HBsAg or HBsAg/Al groups, indicating a preference for cellular immune response (Fig. 8A). For instance, a maximum of 1.5-fold or 1.67-fold of spots number could be observed for the HBsAg/HPLNP group, in comparison to the HBsAg group (*p < 0.05) or HBsAg/Al group (**p < 0.01), respectively (Fig. 8B). Furthermore, we observed a significantly higher antigen-specific proliferation of splenocytes in the HBsAg/HPLNP group than that in the HBsAg group at both 24 h (*p < 0.05) and 48 h (**p < 0.01) post-re-stimulation with HBsAg antigen (Fig. 8C). Meanwhile, the activation of splenocytes including B cell and T cell were also analysed. The percentage of CD69 + CD19 + B cells in HBsAg/HPLNP group was obviously higher than that of Al adjuvant in Fig. 8D (* p < 0.05). No significant difference was observed in the CD69+/CD4 + T cells among these groups (Fig. 8E). However, both the CD69+/CD8 + T and FasL+/CD8 + T cells in the HBsAg/HPLNP group were significantly increased compared to the HBsAg or the HBsAg/Al group, respectively (Fig. 8F, 8G). It confirmed that the HBsAg/HPLNP group had great advantages in activating the cytotoxic T cells. Taken together, these results demonstrated the superior ability of HPLNP in potentiating both humoral immunity and cellular immunity.

3.9 Safety assessment

To evaluate the biocompatibility of the HBsAg/HPLNP formulation, the immunohistochemistry of important organs and the intramuscular injection site were evaluated after the first immunisation for 24 h (Fig. 9A). Compared with the PBS group, there was no significant tissue damage on the main organs and the injected muscle site among the HBsAg, HBsAg/Al and HBsAg/HPLNP groups. It revealed that HPLNPs have good biocompatibility and limited side effects as a novel vaccine adjuvant. During the immunisation, the mice body weights of all these groups were also analysed to evaluate the safety of vaccine formulations. The negligible body weight change in HBsAg/HPLNP group compared to the PBS group further confirmed the satisfying biosafety of HPLNPs (Fig. 9B).

Here we developed a novel hybrid polymer lipid nanoparticle (HPLNP) as the HBsAg-VLP vaccine adjuvant. The HPLNP was composed of two FDA-approved materials, and can be prepared by a simple one-pot method based on several minutes of mixing, stirring and organic solvent evaporation. The optimised formulation with the mass ratio of HBsAg/HPLNP = 1/600 significantly improved the antigen cellular uptake by APCs. The optimised HBsAg/HPLNP formulation largely enhanced the antigen depot effect and prolonged the duration of antigen at the injection site. The lymph node drainage of antigen assisted by HPLNPs was obviously faster and higher compared to the use of Al adjuvant. Therefore, the serum anti-HBsAg IgG concentration of HBsAg/HPLNP group was much higher than the pure HBsAg group or HBsAg/Al group after the boost immunisation in mice. Even after the second boost, the final anti-HBsAg IgG production of the HBsAg/HPLNP group was obviously higher than the HBsAg group. Except for the effective humoral immunity, the markedly improved IgG2a/IgG1 of HPLNP group indicated a strong propensity for HPLNP to induce the Th1-driven immune response. Meanwhile, the elevated IL-4 and IL-6 secretion of HBsAg/HPLNP group further confirmed the improved humoral immunity as they were important in the B cell proliferation, differentiation and antibody production. Furthermore, the HPLNP enhanced the production of IFN-γ and FasL+/CD8⁺ T cells, which was obviously higher than the commercial Al adjuvant, emphasising the strong cellular immune stimulating effect. Last but not the least, there was limited systematic toxicity during the whole immunization in the HPLNPs group, demonstrated by the immunohistochemistry and mice body weights evaluation. Therefore, our newly developed HPLNP adjuvant, which offers advantages including good biocompatibility, easy preparation, low cost and the ability to improve both humoral and cellular immunity, would be a potential alternative to the aluminum adjuvant.

Ethics approval and consent to participate

All animal experiments were performed in strict accordance with the Regulations for the Care and Use of Laboratory Animals and Guideline for Ethical Review of Animals (China, GB/T 35892-2018).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author contributions

The project was conceived by XL, while QZ and WH provided guidance and supervision. XL designed the experiments, prepared the formulations, conducted cell studies, and performed data analysis. AY, HS and QL provided assistances with polymer synthesis and drug release, while QM conducted the animal study. XL wrote the paper, and all authors have read, provided comments on, and approved the final version of this paper.

Acknowledgements

This research was financially supported by Sanming Project of Medicine in Shenzhen (No：SZSM201911007), Shenzhen High-end Talent Scientific Research Start-up Foundation (827-000743), National Natural Science Foundation of China (82071465, 81571283, 81901713), Natural Science Foundation of Shandong Province (ZR2020MH154), Research Project of the First People’s Hospital of Yunnan Province (KHBS-2020-011) and Shenzhen Key Laboratory Foundation (ZDSYS20200811143757022).

Availability of data and materials

All data generated or analysed during this study are included in this published article and its supplementary information files, while the source data can be obtained from the corresponding author upon a reasonable request.

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Potentiating humoral and cellular immunity using a novel hybrid polymer-lipid nanoparticle adjuvant for HBsAg-VLP vaccine

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Materials

2.2 Preparation and characterisation of HPLNP and HBsAg/HPLNP formulations

2.3 In vitro antigen release

2.4 Cell culture

2.5 Cytotoxicity and haemolysis of HPLNPs

2.6 Cellular uptake of HBsAg/HPLNP formulation by macrophages

2.7 Animals

2.8 Injection site antigen depot and lymph nodes drainage

2.9 Activation of lymphocytes in lymph nodes

2.10 In vivo immunisation

2.11 Evaluation of intracellular IFN-γ secretion by splenocytes

2.12 Splenocyte proliferation in vitro

2.13 Detection of the splenocyte activation

2.14 Histologic analysis

3. Results and discussion

3.1 Preparation and characterisation of HPLNPs

3.2 In vitro antigen release

3.3 Cytotoxicity and haemolysis of HPLNPs

3.4 Cellular uptake of vaccine formulations

3.5 Injection site antigen depot and lymph node drainage

3.6 Activation of lymphocytes in lymph node

3.7 In vivo immunisation

3.8 Intracellular cytokines secretion, activation and proliferation of splenocytes

3.9 Safety assessment

4. Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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