1. Introduction
A vaccine requires the proper combination of pathogen-specific antigens and immunostimulating adjuvants to induce a protective adaptive immune response. NPs acting as both antigen carriers and adjuvants have been reported to improve the antigen stability and cellular uptake [
1], promote the humoral and/or cell-mediated immune response [
2,
3,
4], and thus enhance vaccine efficacy with antigen dosage reduction. For example, NPs loaded with detained toxins effectively trigger the formation of germinal centers and induce a highly neutralizing antibody titer [
5].
One of the major contributions of NP-associated adjuvanticity is ascribed to the increased availability of antigens when carried by NPs. On the one hand, specific forms of antigen loading (e.g., encapsulation) in the NP carrier was found to be effective in protecting the antigen (forms such as DNA or RNA [
6]) against decomposition in the biological environment [
7]. On the other hand, the promotion of antigen uptake also results from the rather easy cellular uptake of NPs at both the cellular and system level, especially when a special size range of the NPs is chosen [
8,
9] and/or they are equipped with surface-targeting functions (e.g., to lymph nodes [
10,
11,
12] or dendritic cells (DCs) [
13,
14]). A report is also available on liposome-encapsulated hen egg-white lysozyme (HEL) targeting early endosomes and thus entering the MHC class I and II presentation pathways [
15]. Though NPs could increase the availability of antigens by changing the antigen loading patterns and surface targeting modification, the underlying mechanism for how the physicochemical parameters of NPs affect the antigen availability is unclarified. Moreover, different vaccination routes have different requirements regarding the NPs to enhance the antigen availability. For example, ideal vaccines for the intravenous vaccination route require better stability of the NPs in order to avoid antigen release during the circulation, as well as long circulation properties for enhancing the immune cells’ opportunities for uptake. On the other hand, in the subcutaneous vaccination route, better decomposability is needed for faster antigen release, leading to faster antigen presentation and immune response.
As a matter of fact, the effects of some of the material parameters on NPs’ adjuvanticity have been studied in the literature, but in a rather scattered manner. For example, the geometrical configuration match was identified as an effective cue to enhance the antibody binding affinity and broaden its viral neutralizing spectrum [
16]. The degradability of a lipid NP carrier was observed to be a physical cue to direct antigens towards cross presentation [
15]. Mechanical forces are also important determinants in immune cell activation, such as regulating cell-surface receptor activation, cell migration, intracellular signaling and intercellular communication [
17,
18]. More specifically, NPs that retain their force-dependent deformability were found to stimulate both humoral and cellular adaptive responses, and thus increase the survival of mice upon a lethal dose of influenza virus [
18]. The stiffness of the NPs was also recognized recently as an important factor in affecting immune cell behaviors via modulating the FAK-NF-κB signaling pathway [
19].
In the present work, we took
S. aureus as a model system, due to its growing impact on public health resulting from the emergence and spread of methicillin-resistant
S. aureus (MRSA) strains. In fact, the emergence of drug-resistant strains urged the search for alternative treatments such as immunotherapeutic approaches. Here we fabricated both soft and hard PLGA-based NPs as carriers for an
S. aureus antigen (i.e., recombinant ess extracellular B (rEsxB)) [
20] and the medium for adjuvanticity (
Figure 1). We discovered that tuning the stiffness of the carrier materials by modulating the carrier composition promoted the cellular uptake of rEsxB-carrying NPs, but at the same time affected the decomposability of the NPs (and thus rEsxB release). In the mouse group receiving a S.C. vaccination, a significantly higher rEsxB-specific antibody titer was found in mice treated with a soft nano-vaccine, while similar results were obtained in mouse groups receiving a I.V. vaccination but with a hard nano-vaccine. For vaccination via S.C. and I.V. alike, the NP-based
S. aureus vaccines show excellent protection for mice against
S. aureus lethal challenge, and their survival rates are 100% after the inoculation of ATCC25923 strain, being far superior to the clinically used Alum adjuvant.
2. Materials and Methods
2.1. Materials
BL21 (DE3)-competent Escherichia coli (E. coil) was purchased from TIANGEN BIOTECH (BEIJING) Co., Ltd. (Beijing, China). Ni-Sepharose was purchased from Cytiva (Shanghai, China). A High-Capacity Endotoxin Removal column was purchased from Xiamen Bioendo Technology Co., Ltd. (Xiamen, China). PLGA-PEG X% polymers (X = 0, 14, 20, 25, 33, PLGA with a lactic acid to glycolic acid ratio of 50:50) and fluorescein isothiocyanate (FITC)-PEG2000-NH2 were purchased from Xi’an Ruixi Biological Technology Co., Ltd. (Xi’an, China). Isopropylbeta-D-thiogalactopyranoside (IPTG), imidazole, polyvinyl alcohol (PVA), dichloromethane (DCM), acetone, 2-(N-morpholino) ethanesulfonic acid hydrate (MES), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-Hydroxysuccinimide (NHS), bovine serum albumin (BSA), Tween 20, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), horseradish peroxidase (HRP)-labeled goat anti-mouse IgG, and 0.22 μm and 0.45 μm sterile filters were purchased from Merck (Shanghai, China). Imject™ Alum adjuvant was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Ninety-six-well polystyrene plates for the enzyme-linked immunosorbent assay (ELISA) were purchased from Corning (New York, NY, USA). Enzyme-linked immunospot (ELISPOT) kits were purchased from Mabtech (Stockholm, Sweden). All other solvents are of analytical grade and were used without further purification.
2.2. Metheds
2.2.1. Expression and Purification of rEsxB Antigen
rEsxB was chosen as the model antigen to investigate the adjuvanticity of the NP carriers. His-tagged rEsxB was prepared using the standard IPTG induction protocol [
21]. Briefly, full-length wild-type EsxB DNA was subcloned into the pET-28a vector and expressed as a 6×His-tag fusion protein in BL21 (DE3)
E. coil. rEsxB expression was induced by IPTG, purified from bacterial cell lysates by means of Ni-Sepharose chromatography, and was gradient eluted from the Ni-Sepharose with 100 mmol/L, 300 mmol/L and 500 mmol/L imidazole. The sample was then dialyzed with phosphate-buffered saline (PBS) and endotoxin was removed using the High-Capacity Endotoxin Removal column. Finally, the rEsxB solution was filtered through a 0.22 µm sterile filter and the total protein concentration was quantified using a bicinchoninic acid (BCA) protein assay. The purity and specificity of the rEsxB antigen were analyzed by means of SDS-PAGE and Western Blot.
2.2.2. Preparation and Characterization of the PLGA-PEG NPs Conjugated with rEsxB
The apparent Young’s modulus of the PLGA-PEG X% (X = 0, 14, 20, 25, 33) polymers was measured using a atomic force microscope (AFM, Bruker BioScope Catalyst). Referring to the stiffness of the cell membrane [
22], two sets of PLGA-PEG X% polymers (PEG
5000-PLGA
30,000, PEG
5000-PLGA
15,000) were chosen for the synthesis of PLGA-PEG X% NPs (X = 14, 25). The PLGA-PEG X% NPs were prepared via the precipitation/solvent diffusion method according to our previous work with small modifications [
23,
24]. Briefly, 100 mg of PLGA-PEG was dissolved in a mixture of 1.5 mL of DCM and 1 mL acetone. The polymer solution was directly added to 10 mL of a 5% PVA (Mw 31,000~50,000 Da, 87~89% hydrolyzed) solution and stirred at 600 rpm to evaporate the DCM and acetone. The mixture was then homogenized for 2 min by using a probe sonicator at 20 W to generate an oil-in-water (O/W) emulsion. The formed emulsion was added to 50 mL of deionized (DI) water and stirred for 4 h at room temperature. After that, the PLGA-PEG X% NPs were collected and filtered using a 0.45 μm filter. The NP solution was centrifuged at 16,500×
g force at 4 °C for 1 h, washed with DI water 3 times, and lyophilized for storage.
The rEsxB was chemically linked to the PLGA-PEG X% NPs via a condensation reaction between the carboxyl group on the NPs and the amino group of the rEsxB. Then, a 10 mg sample of PLGA-PEG X% NPs was re-suspended in 10 mL of 25 mmol/L MES buffer, and 0.4 mL of 1 mol/L EDC and 0.25 mL of 1 mol/L NHS were added into the mixture and stirred for 4 h at the room temperature to activate the carboxyl groups on the NPs. After that, the NPs were centrifuged at 16,500× g force at 4 °C for 1 h and washed with DI water 3 times. The activated NPs were re-suspended in 1 mL of 1 mg/mL rEsxB in PBS (pH 8.0) overnight at 4 °C, then the PLGA-PEG X% NPs-rEsxB were centrifuged at 16,500× g force at 4 °C for 1 h, washed with DI water 3 times, and lyophilized for later use.
The hydrodynamic diameter, polydispersity index (PDI), and zeta potential of the PLGA-PEG X% NPs and PLGA-PEG X% NPs-rEsxB were measured via dynamic light scattering (DLS) (Delsa™ Nano, Beckman-Coulter, High Wycombe, UK). The successful synthesis of the PLGA-PEG X% NPs-rEsxB was determined by means of Fourier Transform infrared spectroscopy (FTIR, Thermo Nicolet IS50, Thermo Fisher Scientific, Waltham, MA, USA). The BCA protein quantification assay was used to quantify the total rEsxB concentration loaded. The loading efficiency was calculated using the following formula:
2.2.3. Evaluations of the Cellular Uptake and Load Release of NPs-rEsxB
Quantitative evaluation of the cellular uptake of NPs was carried out by means of the analysis of the intracellular fluorescein intensity using FITC-loaded NPs (NPs-FITC), with FITC as the florescence label. The PLGA-PEG X% NPs (X = 14, 25) were conjugated with FITC-PEG2000-NH2 via the condensation reaction between the carboxyl groups on NPs and the amino groups on FITC-PEG2000-NH2 via sulfo-NHS-catalyzed EDC activation of carboxyl groups. To simulate different administration routes of S.C. and I.V. injection, DCs (5 × 104 cells/well) were fed with the 2.5 mL NPs-FITC solution at two different concentrations—2 mg/mL, for simulating a local high concentration after S.C. injection, and 10 μg/mL, for simulating a diluted environment after I.V. injection. At specific time intervals (0.5 h, 1 h, 2 h, 4 h, 6 h and 8 h), DCs were collected by means of centrifugation (250× g for 5 min) then re-suspended in 5 mL of RPMI 1640 medium without fetal bovine serum (FBS). The cell suspensions were frozen and thawed 4~6 times repeatedly then centrifuged at 1500× g for 10 min. Emission of the supernatants was measured at 528/20 nm with an excitation wavelength of 485/20 nm (Biotek SYNERGY LX Instruments, Santa Clara, CA, USA).
The load release experiment was carried out using the fluorescence method. FITC was chosen as a fluorescence tag and conjugated to the NPs in a similar manner to that of the rEsxB, then the NPs-FITC was used to examine the payload release profile of all NP samples. For the payload release studies, 50% FBS simulating the humoral environment was used. The experiments were carried out at 37 °C, and data were collected for a 12 h duration. In detail, 10 mg of PLGA-PEG X% NPs-FITC was re-suspended in 10 mL of 50% FBS at 37 °C and 1 mL of each NPs-FITC sample was collected at specific time points (1 h, 2 h, 4 h, 8 h and 12 h after release) then centrifuged at 16,500× g at 4 °C for 1 h. The emission of the supernatants was measured at 528/20 nm with an excitation wavelength at 485/20 nm (Biotek SYNERGY LX Instruments, Santa Clara, CA, USA).
2.2.4. Evaluation of Biocompatibility of PLGA-PEG X% NPs-rEsxB In Vitro and In Vivo
We first examined the cytotoxicity of the NPs-rEsxB conjugates in vitro using fibroblast L929 cell line using the cell counting kit-8 (CCK-8) assay. Cells were seeded in a 96-well plate (5 × 10
3 cells/well) for overnight attachment. In this process, 100 μL of the NPs-rEsxB medium solution with specific concentrations (10 ng/mL, 100 ng/mL, 1 μg/mL, 5 μg/mL, 10 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL, 500 μg/mL and 1 mg/mL) was added to the cell solution, respectively. Each sample was tested using 6 replicated wells. Then, the cells were incubated for another 24 h. Then,20 μL of the CCK-8 solution was added to each well and incubated at 37 °C for 1~4 h. The absorbance of cells was then measured with a microplate reader at a wavelength of 450 nm (Biotek SYNERGY LX Instruments, Santa Clara, CA, USA). The cell viabilities were calculated using the classic cytotoxicity equation:
Further evaluation of the biocompatibility of the NPs-rEsxB was carried out in vivo using BALB/c mice. All animals were monitored for their activity, physical condition, and body weight. The body weight of each mouse was measured and recorded every other day until 30 days after the first vaccination.
Hematoxylin and eosin (H&E) staining of major organs by means of the standard method published in our previous paper [
25] was then carried out after the animals were sacrificed at day 42 after the first vaccination.
2.2.5. Animal Immunization
All of the BALB/c mice were purchased from the Animal Experiment Center of Air Force Medical University. All animal experiments complied with the National Research Council’s Guide for the Care and Use of Laboratory Animals and the animal experiments were approved by the Animal Care and Ethic Committee of Fourth Military Medical University (Approval NO. KY20213144-1). Female 6–8-week-old BALB/c mice were divided randomly into 5 groups (n = 6 or 10 each) and immunized with PBS and rEsxB as negative control groups, clinically used Alum mixed with rEsxB (Alum-rEsxB) as the positive control group and PLGA-PEG X% NPs-rEsxB (X = 14, 25) as the nano-vaccine groups. Four groups (except PBS) were immunized either S.C. or I.V. with the same rEsxB concentration of 50 µg/mouse. To determine humoral and cell mediated response all groups were boosted with the same formulation (rEsxB concentration of 25 µg/mice) 14 and 28 days after the first immunization. Mouse serum samples were collected at specific time points.
2.2.6. ELISA Assay
The rEsxB-specific antibody titer was determined through indirect ELISA on days 35 and 208 after the first immunization. Briefly, a 96-well ELISA plate was coated with rEsxB (500 ng/well) followed by blocking with 3% BSA on the next day. The plate was then washed 5 times with PBS containing 0.1% Tween 20 (PBST) and incubated with serially diluted immunized mouse serum ranging from 1:200 to 1:204,800 (100 μL/well) at 37 °C for 1 h. followed by five washes with PBST. HRP-labeled goat anti-mouse IgG (1:5000, 100 μL/well) was added and incubated at 37 °C for 45 min. This was followed by washing (as above) and development with ABTS and H
2O
2 as substrate chromogens. Finally, the color development for 30 min at room temperature in the dark was read at 405 nm by a microplate reader (Biotek SYNERGY LX Instruments, Santa Clara, CA, USA). The cut-off value was calculated as 2.1 times the absorbance from the negative control serum assayed [
26]. In the sample group comparisons of the ELISA titer results, the area under the curve (AUC) method had similar power to the absorbance summation (AS) method, and better power than the endpoint titer (ET) method. The rEsxB-specific antibody titer results were represented by the mean serum ELISA AUC [
27,
28] with the S.D. of 6 mice in each group.
2.2.7. ELISPOT Assay
The splenocytes of immunized animals 7 days after the third immunization (both S.C. and I.V. vaccination groups) were analyzed for IL-4 (representative of humoral immunity), IFN-γ (representative of cell-mediated immunity), IL-12 (representative of DC activation) and IL-17A (representative of TH17 T cell activation) production by means of ELISPOT assays using standard protocols [
29]. The ELISPOT plates (capture antibody precoated) were washed 4 times with sterile PBS and incubated with a complete culture medium (RPMI-1640, 10% FBS) for at least 30 min at room temperature. After removing the complete culture medium, splenocytes from the immunized mice (2.5 × 10
6 cells/well) and stimuli (4 μg/mL rEsxB) was mixed, added in the plate and incubated at 37 °C for 24 h. Phytohemagglutinin (PHA, 10 μg/mL) and the media alone served as positive and negative controls, respectively. Then, the splenocytes were removed and the plates were washed 5 times with PBS, followed by adding the detection antibodies into PBS containing 0.5% FBS (PBS-0.5% FBS) and incubation for 2 h at room temperature. The plates were washed as above and a TMB substrate solution was added to the plates until distinct spots emerged. The spots’ development was stopped by washing the plates extensively in DI water. The plates were dried in the dark at room temperature, and the spots were counted in an ELISPOT reader (Cellular Technology Limited, Cleveland, OH, USA).
2.2.8. Lethal Challenge
On the 7th day after the third immunization, 100 μL of S. aureus (ATCC 25923) at a concentration of 1.67 × 108 colony-forming units (CFU)/mL was administered to the mice in each of the groups via I.V. injection. Mice were then monitored daily for mortality and clinical signs. At the end point of the experiment, all remaining animals were euthanized.
2.2.9. Serum Bactericidal Test
Standard Neisser–Wechsberg bacteriolysis evaluation [
30] was employed to investigate the bacteriolysis effects of the antibody in all nano-vaccines, namely the Alum-rEsxB, rEsxB and PBS groups, with some modifications. The
S. aureus ATCC 25923 strain was cultured until reaching the mid-exponential phase (OD
600 = 0.5), then the
S. aureus concentrations were adjusted to 3 × 10
6 CFU/mL with PBS (pH 7.4). The serum of immunized mice and normal guinea pig serum were added to the
S. aureus at a final concentration of 2% (
v/
v), respectively. The mixture was incubated at 37 °C for 0.5 h, plated on LB agar plates and incubated overnight at 37 °C. Finally, the number of colony-forming units of
S. aureus was counted and the percentage of lytic bacteria was calculated using the following formula:
2.2.10. Statistical Analysis
All results were expressed as mean ± standard deviation (SD) and statistical analysis was performed using the software of GraphPad Prism 9.0. Analysis of variance (students’ t test) was employed to determine the statistical significance of differences, and log-rank (Mantel–Cox) analysis was employed to determine the statistical significance of the survival rate. The statistical differences were defined as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, and p < 0.05 were considered significant.
4. Discussion and Conclusions
In order to enhance the protective efficacy of vaccines, including the induction of rapid and long-lasting immunity, new adjuvants, especially nanoadjuvants, have been studied as a promising platform with rational designs. By rationally selecting/designing polymers based on their physicochemical properties, and considering the antigen and vaccine regimen, it is possible to modulate appropriate immune responses for specific diseases.
In this study, by introducing PEG to the PLGA, we manipulated the mechanical properties (stiffness) of the NP adjuvants, which also served as carriers for the antigen of
S. aureus (EsxB). Two typical stiffness values were chosen for preparing the NP adjuvants—PLGA-PEG 14% NPs for the hard stiffness value and PLGA-PEG 25% NPs for the soft stiffness value. Two nanovaccine systems, PLGA-PEG 14% NPs-EsxB and PLGA-PEG 25% NPs-EsxB, were then obtained for immune efficacy evaluation using two separate vaccination routes (I.V. and S.C.). As shown in the Results section, a significantly higher level of protection against
S. aureus infection was always achieved with both hard and soft NP formulations (vs. the clinically used Alum adjuvant), as suggested by the lethal challenge experiments, with all animals being kept alive after the administration of a lethal dose of
S. aureus. This was mainly attributed to the production of neutralized antibodies induced by NP vaccines. Also, the higher protection efficacy results of NP vaccines were consistent with the antibody titer and the cytokine analysis. Moreover, the enhanced vaccine efficacy results from improved antigen accessibility when delivered by the PLGA-PEG nanocarriers, which were also found to serve as excellent adjuvants to effectively stimulate both humoral and cellular immune responses in mice. On the other hand, a higher PEG content led to softer NPs that promoted cellular uptake, but itself was also prone to faster decomposition (and thus faster release of the antigen payload). The differences in the decomposition speed between PLGA-PEG nanocarriers may be attributed to two reasons—the molecular weight of the polymer and the existence of enzymes in the surrounding environment. The molecular weight of the PLGA chain mostly affects its degradation in indirect ways, e.g., by determining its morphology and porosity [
31]. Moreover, the pancreatic lipase, which is the main component of serum lipase and subcutaneous tissue fluid, played a key role in PLGA degradation in the circulation [
32,
33]. The molecular weight of PLGA in PLGA-PEG 14% NPs is 30 KD, while that in PLGA-PEG 25% NPs is just 15 KD. These differences may affect the degradation of PLGA-based NPs by pancreatic lipase, so that the two PLGA-based NPs are subject to differential degradation. The interplay between these factors then determined the actual antigen uptake efficiency at the system level when different vaccination routes are utilized—that is, S.C. or I.V. injection. This provides a possible mechanism explanation for the observed higher antibody/cytokine levels induced by the rEsxB-loaded soft (hard) NPs in the case of S.C. (I.V.) vaccination. The present work provides a guideline for the vaccination-route-specific design of nanoparticle-based vaccines with maximized potency and minimized side effects.