A Novel Human Papillomavirus 16 L1 Pentamer-Loaded Hybrid

Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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A Novel Human Papillomavirus 16 L1 Pentamer-Loaded Hybrid Particles Vaccine System: Influence of Size on Immune Responses Chengcheng Jia,†,‡,1 Tingyuan Yang,† Yongjiang Liu,‡ Ali Zhu,† Fei Yin,‡ Yajun Wang,‡ Lan Xu,‡ Yan Wang,‡ Mei Yan,‡ Qingman Cai,§ Xiaoxu Liang,§ Ruijun Ju,§ Jianping Chen,*,‡ and Lianyan Wang*,† †

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State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, P. R. China ‡ Beijing Health Guard Biotechnology Co., LTD, Beijing 100176, P.R. China § Beijing Institute of Petrochemical Technology, Beijing 102617, P.R. China S Supporting Information *

ABSTRACT: Cervical cancer remains the second-most prevalent female malignancy around the world, leading to a great majority of cancer-related mortality that occurs mainly in developing countries. Developing an effective and low-cost vaccine against human papillomavirus (HPV) infection, especially in medically underfunded areas, is urgent. Compared with vaccines based on HPV L1 viruslike particles (VLPs) in the market, recombinant HPV L1 pentamer expressed in Escherichia coli represents a promising and potentially cost-effective vaccine for preventing HPV infection. Hybrid particles comprising a polymer core and lipid shell have shown great potential compared to conventional aluminum salts adjuvant and is urgently needed for HPV L1 pentamer vaccines. It is well-reported that particle sizes are crucial in regulating immune responses. Nevertheless, reports on the relationship between the particulate size and the resultant immune response have been in conflict, and there is no answer to how the size of particles regulates specific immune response for HPV L1 pentamer-based candidate vaccines. Here, we fabricated HPV 16 L1 pentamer-loaded poly(D,L-lactide-co-glycolide) (PLGA)/lecithin hybrid particles with uniform sizes (0.3, 1, and 3 μm) and investigated the particle size effects on antigen release, activation of lymphocytes, dendritic cells (DCs) activation and maturation, follicular helper CD4+ T (TFH) cells differentiation, and release of pro-inflammatory cytokines and chemokines. Compared with the other particle sizes, 1 μm particles induced more powerful antibody protection and yielded more persistent antibody responses, as well as more heightened anamnestic responses upon repeat vaccination. The superior immune responses might be attributed to sustainable antigen release and robust antigen uptake and transport and then further promoted a series of cascade reactions, including enhanced DCs maturation, increased lymphocytes activation, and augmented TFH cells differentiation in draining lymph nodes (DLNs). Here, a powerful and economical platform for HPV vaccine and a comprehensive understanding of particle size effect on immune responses for HPV L1 pentamer-based candidate vaccines are provided. KEYWORDS: human papillomavirus, pentamer, particle, size, adjuvant

1. INTRODUCTION Persistent infection with oncogenic HPV subtypes may develop into cervical cancer, the second leading cancer, causing a great majority of cancer mortality diagnosed in women globally.1,2 Around the world, about 49 300 new cases are diagnosed each year, and 274 000 die from cervical cancer. The mortality ratio among women in developing countries is more than 80%, and this ratio is predicted to grow to 90% by 2020.3,4 So far, a bivalent HPV vaccine with Adjuvant System 04 (AS04) as adjuvant (Cervarix, GlaxoSmithKline), and a quadrivalent HPV vaccine (Gardasil, Merck) and the latest nine-valent HPV vaccine (Gardasil 9, Merck), both with amorphous aluminum © XXXX American Chemical Society

hydroxyphosphate sulfate as adjuvant, have been licensed in many countries. These vaccines have been proved to be successful in protecting from HPV infection. However, undoubtedly, price remains one of the main obstacles to introducing the above vaccines into developing countries.3 It is especially necessary and urgent in developing countries to develop efficacious and cost-effective HPV vaccines, and pentameric capsomeres may become a promising alternative to Received: July 11, 2018 Accepted: October 1, 2018

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DOI: 10.1021/acsami.8b11556 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

biomaterials: (i) poly(D,L-lactide-co-glycolide) (PLGA) was selected as the hydrophobic polymer core for encapsulating antigens due to its steric stabilization and controlled release ability and (ii) soybean lecithin was chosen as the outer cationic lipid monolayer enveloping the polymer core to confer biocompatibility and biomimetic properties to the polymer core.32,33 Particle size is thought to have a major influence on particulateinduced immune responses.34−37 The particle size not only determines the magnitude but also dictates the persistence and type of immune response.34,37−39 Particle size plays a crucial role in multiple bioprocesses that modulate their immunogenicity, such as the particles uptake by immune cells at the injection site, transfer to peripheral lymphoid tissues and organs, antigen presentation, and activation of APCs.40 However, reports on the correlation between particle size and resultant immune response have been in conflict, with researchers at odds over forceful data supporting the effectiveness of submicron-sized particles over micron-sized particles and vice versa, while other researchers presented excellent data showing that particles with submicron and micron sizes are equally effective at inducing a powerful response.41−43 Further, if the antigens loaded by the particles are different, the size-dependent immune responses induced may also different. There is no answer to how the size of particles regulates specific immune response for HPV L1 pentamer-based candidate vaccines. Thus, we have attempted to comprehensively study the size effect of particle adjuvants on the immune responses and assist in achieving the optimal immune responses. Here, we designed a novel, powerful, and economical HPV16 L1 pentamer-loaded PLGA/lecithin hybrid particles platform for HPV vaccine and systemically investigated the size effect of particles on antibody response, antigen release, DCs activation and maturation, lymphocytes activation, TFH cells response, and release of pro-inflammatory cytokines and chemokines. We expected to find that the fundamental of particle size regulates the immune response in principle and to provide a superior and cost-effective HPV vaccine in this study.

viruslike particles (VLPs) because of their price advantage, since the pentameric capsomeres expressed in Escherichia coli are easy to purify5,6 and highly stable.7,8 Neutralizing antibodies are induced after immunization with papillomavirus L1 capsomeres to protect against infection.9,10 Additionally, subunit vaccines with limited immunogenicity usually require adjuvants and delivery systems to enhance their immunogenicity. Although aluminum salts effectively enhance immune responses, they have several drawbacks,11 including severe local tissue stimulus, inflammatory responses at the injection spot, biased T helper 2 (Th2) responses, poorly stimulated cellular adaptive response, and a tendency to induce unwanted immunoglobulin E responses.12 Furthermore, the antigens adsorbed onto aluminum release slowly only at the site of injection (depot effect), leading to few interaction with antigenpresenting cells (APCs) and then the low antigen uptake. Therefore, new adjuvants are needed to enhance the immunity against weak antigens. Recent studies have demonstrated that particle-based delivery platforms have great potential to improve the immunogenicity of vaccines.13−15 Over the past decades, biodegradable polymer particles16,17 and liposomes18 have come forth as dominant classes of drug carriers.19,20 They have also been demonstrated to have potential adjuvant activity, as evidenced by mounting quantities of clinical tests, research papers, and licensed drugs that are based on particle delivery.18,20−22 Particle adjuvants could act as a valid antigen-delivery carrier and readily reach APC regions and, hence, reinforce and/or promote antigen uptake by APCs, for instance, dendritic cells (DCs) or macrophages.23 In addition, an enhanced interaction between antigen and immunocytes may be seen, since particlebased antigen delivery systems act as a reservoir for long-term controlled antigen release.24 Moreover, particles could be capable of shielding the embedded antigen from degradation before being carried to the immunocytes.25 Generally, polymer particles exhibit excellent biocompatibility, high structural integrity, improved drug stability during storage, superior drug/ vaccine encapsulation, and controlled release profiles;16,22 however, they often have limitations of drug leakage, polymer cytotoxicity, and polymer degradation.26 Liposomes are characterized by good biocompatibility, nontoxicity or slight toxicity, flexibility, and nonimmunogenicity,18 but they suffer from a lack of structural integrity, resulting in content leakage, inherent instability, irreproducibility, and difficulty in manufacturing scale-up.27,28 Recently, a new generation of integrated lipid− polymer hybrid particles comprising a polymer core and lipid shell has been explored as versatile carriers and potentially more potent alternatives to polymeric or liposome particles. These hybrids combine merits of liposomal drug delivery (cell affinity and biomimetic characteristics) and polymeric particles (structural integrity, controlled release, and high serum stability).20,29,30 In this study, we fabricated antigen-loaded lipid−polymer hybrid particles for vaccine delivery by uniting the solvent evaporation approach and premix membrane emulsification technology, a simple, efficient, and controllable single-step method excluding the interference of the heterogeneity of particle size. Lipid−polymer hybrid particles, prepared by joining polymers and lipids together, feature tunable size and surface charge, advanced drug loading capacity, controlled and continuing drug release profile, and remarkable sera stability. Notably, the manufacturing procedure is simple and easy to amplify.20,31,32 In this study, the lipid−polymer hybrid particles (referred to as “PLGA/lecithin particles”) were fabricated using two

2. MATERIALS AND METHODS 2.1. Mice, Reagents, and Materials. Female BALB/c mice were purchased from Vital River Laboratories (Beijing, China). All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals and were approved by the Experimental Animal Ethics Committee in Beijing. HPV16 L1 pentamer antigen and aluminum hydroxide were fabricated by Beijing Health Guard Biotechnology Inc. PLGA (75/25, molecular weight ≈ 13 kDa) was ordered from Lakeshore Biomaterials (Birmingham, AL). Egg yolk lecithin PC-98T was ordered from Q.P. Corp. Poly(vinyl alcohol) (PVA-217, 1700 polymerization degree, 88.5% hydrolysisdegree) was obtained from Kuraray (Tokyo, Japan). Premix membrane emulsification equipment (FMEM-500M) was provided by the National Engineering Research Center for Biotechnology (Beijing, China). Shirasu porous glass (SPG) membrane was bought from SPG Technology Co. Ltd. (Sadowara, Japan). Roswell Park Memorial Institute (RPMI) 1640, Dulbecco’s modified Eagle medium (DMEM), and fetal bovine serum (FBS) were supplied by Gibco (Carlsbad, CA). Phosphate-buffered saline (PBS) was supplied by Hyclone (Logan, UT). Concanavalin A was purchased from Roche. The 70 μm mesh cell strainers were ordered from BD Bioscience (San Diego, CA). Red blood lysis buffer was supplied by Solarbio (Beijing, China). Bovine serum albumin (BSA) was ordered from SigmaAldrich (St. Louis, MO). Mouse cytokine enzyme-linked immunosorbent assay (ELISA) kits were ordered from eBioscience (San Diego, CA). Fluorochrome-labeled anti-mouse antibodies targeting B


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was performed as follows: 100 μg of aluminum hydroxide gel was added to 0.1 mL of PBS buffer containing 10 μg of HPV16 L1 pentamer, and the resultant solution was placed on a thermostatic shaking table (25 °C, 200 rpm, 120 min). To examine antigen adsorbing capacity, the supernatants were harvested by a centrifugal force at 2000g for 5 min from the mixed solution after adsorption for 120 min. The antigen concentrations in the supernatants were measured by a Micro-BCA protein assay kit as the manufacturerʼs instructions (Pierce Biotechnology, Rockford, IL). 2.6. Immunization Studies. Mice (4−6 weeks old) (n = 6 mice/ group) were intramuscularly injected in the hind legs twice at a 2-week interval (Figure 2A) with 100 μL of suspensions of various vaccines containing 10 μg of HPV 16 L1 pentamer (5 μg in 50 μL buffer per hind leg), including 0.3227 μm HPV-loaded PLGA/lecithin particles in PBS, 0.9057 μm HPV-loaded PLGA/lecithin particles in PBS, 3.857 μm HPV-loaded PLGA/lecithin particles in PBS, PBS plus HPV, or aluminum hydroxide plus HPV. Blood samples were drawed for antibody analysis at days 0 (prior to priming immunity), 14 (prior to boosting), and 28 from the venous plexus of the posterior orbits of mice (Figure 3A). At day 28 after primary immunization, splenocytes were collected for immunological tests. For assessing the persistence of antibody response, 30 mice (4−6 weeks of age) were randomly assigned into three groups (n = 10) and intramuscularly administrated at the hind legs with 100 μL of a suspension of PLGA/lecithin particles with different sizes containing 10 μg of HPV 16 L1 pentamer (5 μg in 50 μL buffer per hind leg) on days 0, 28, and 142 (Figure 4A). Orbital blood samples were collected for serum antibody titer determination on days 0 (prior to priming immunity), 21, 42, 112, and 156 (Figure 4A). Mice (6−8 weeks old) were intramuscularly immunized in the hind legs with 100 μL of a suspension of the indicated vaccines containing 10 μg of HPV 16 L1 pentamer (5 μg in 50 μL of buffer per quadriceps muscle). At specific time points, the mice were euthanized, the DLNs were isolated, and then single-cell suspensions were collected from DLNs for flow cytometry assays. Additionally, muscle tissues were isolated at indicated time points and homogenized to evaluate cytokine and chemokine contents. 2.7. Determination of HPV-Specific IgG and IgG Subclasses Titers. Blood samples were treated by thermal inactivation for 30 min at 56 °C, and then serum was harvested by a centrifugal force at 3000g for 10 min from inactivated blood for antibody detection. The 96-well microplates were first coated with 3 μg (100 μL per well) of HPV 16 L1 pentamer in coating buffer (10 mM PBS, pH 7.0−7.2, Hyclone) at 4 °C overnight. Washing six times with PBS containing 0.05% (v/v) Tween 20 (PBST) was performed to remove unbound HPV 16 L1 pentamer. Subsequently, 250 μL of 1% (wt/vol) BSA in PBST was added to each well, and the microplates were incubated for 90 min at 37 °C to block them. After washing another four times, appropriate sera dilutions (100 μL per well) were added, serially diluted by 2-fold in dilution buffer [0.1% (wt/vol) BSA in PBST], and incubated for 30 min at 37 °C. Plates were then washed, 100 μL per well of horseradish peroxidase-conjugated anti-mouse antibodies (Abcam; IgG diluted by 20 000-fold; IgG1 and IgG2a diluted by 10000-fold) was added, and the plates were incubated for 30 min at 37 °C. Thenceforth, the ELISA plates were washed again with PBST. Next, 100 μL of 3,3′,5,5′-tetramethylbenzidine substrate was pipetted into the washed wells. After incubation for 20 min at room temperature, the reactions were stopped by adding 50 μL of 2 M H2SO4 to each well, and the optical densities (OD) at 450 nm were determined using an Infinite M200 microplate spectrophotometer (Tecan, Männedorf, Switzerland). End-point titers were calculated as the sample dilution corresponding to twice higher the OD450 of the negative serum. 2.8. Determination of HPV Neutralization Antibody Titers. In brief, human embryonic kidney cells (293FT cells) in the exponential phase were plated at 15 000 cells per well in 96-well flat-bottom plates in DMEM (Hyclone), complemented with 10% fetal calf serum (FCS) (Gibco, BRL) and 1% penicillin/streptomycin (Life Technologies). Hence, incubation for approximately 6 h at 37 °C with 5% CO2 was performed until the cells attached to the bottom of

MHC II, CD86, CD11c, CCR7, CXCR5, PD-1, CD25, CD71, CD69, IFN-γ, IL-4, CD4, CD8, CD19, and 7-aminoactinomycin D and fixable viability dye for flow cytometric measurements were obtained from eBioscience. BD Cytofix/Cytoperm plus fixation/permeabilization kit with BD GolgiPlug protein transport inhibitor containing brefeldin A (BFA) was ordered from BD Bioscience (San Diego, CA). The ProcartaPlex multiplex immunoassay (Luminex) kit for cytokines was obtained from Affymetrix eBioscience. All reagents were of analytical grade. 2.2. Fabrication and Characterization of HPV16 L1 Pentamer-Loaded PLGA/Lecithin Particles. Uniform-sized HPV16 L1 pentamer-loaded PLGA/lecithin particles were fabricated by premix membrane emulsification techonology and solvent evaporation method. The exhaustive preparation procedure of PLGA/ lecithin particles was as follows: Add 1 mL of PBS buffer containing 15 mg of HPV (internal water phase, W1) into 10 mL of dichloromethane dissolving 280 mg of PLGA and 120 mg of egg yolk lecithin (oil phase, O). Sonication at 120 W (Digital Sonifier 450, Branson Ultrasonics Corp., Danbury, CT) over an ice bath for 1 min (4 s on and 2 s off) was employed to obtained the primary water-in-oil emulsion (W1/O). The external water phase (W2) was obtained by dissolving a certain amount of PVA into distilled water (1.5%, w/v). To prepare the coarse emulsions of W1/O/W2, 11 mL of primary emulsion was added slowly into 65 mL of external water phase and the mixture was magnetically stirred at 450 rpm for 120 s. Afterward, the obtained emulsion was poured into a premix reservoir, and extruded through the different pore sized SPG membranes (1.4, 7.2, and 15.2 μm) under a high nitrogen pressure five times. The resultant uniform-sized double-emulsion droplets were stirred overnight at 250 rpm to vapor the volatile organic solvent. The PLGA/lecithin particles with different sizes were collected by different centrifugal force at 10 000g, 5000g, and 2000g, respectively, for 5 min, and residual PVA was removed by washing three times with deionized water. Ultimately, PLGA/lecithin particles were dried by lyophilization and were then stored at −20 °C for future use. The hydrodynamic size of submicron-sized PLGA/lecithin particles was measured with a Nano-ZS Zeta Sizer (Malvern Instruments Ltd., Malvern, UK), and micron-sized PLGA/lecithin particles were measured with a Mastersizer 2000 (Malvern Instruments Ltd., Malvern, UK). The morphology of the PLGA/lecithin particles with different sizes was observed by scanning electron microscopy (JEM-6700F, JEOL Ltd., Tokyo, Japan). In addition, the ζ-potential of particles was determined by the Nano-ZS Zeta Sizer. The loading capacity of HPV 16 L1 pentamer in PLGA/lecithin particles was investigated by a Micro-BCA protein assay kit (Pierce Biotechnology, Rockford, IL). The protein concentrations in the solutions were measured by dissolving 5 mg of lyophilized PLGA/ lecithin particles into 1 mL of 0.1 M NaOH and incubating in a lowspeed shaker overnight. BSA prepared in 0.1 M NaOH was utilized to build a standard curve, and blank PLGA/lecithin particles were employed as control. 2.3. Evaluation of HPV 16 L1 Pentamer Release in vitro. To produce an antigen release curve, 10 mg of HPV 16 L1 pentamerloaded PLGA/lecithin particles was dissolved in 0.5 mL of PBS (10 mM, pH 7.0−7.2) and reacted under gentle shaking (120 rpm) at 37 °C. Supernatants were periodically harvested by a centrifugal force (5000g, 5 min) and substituted with fresh PBS buffer of the same volume. The amounts of HPV 16 L1 pentamer released from the particles were measured by a Micro-BCA protein assay kit as the manufacturerʼs instructions. All release experiments were performed in triplicate. 2.4. Serum Stability of PLGA/Lecithin Particles in vitro. PLGA/lecithin particles were incubated with 10 vol % FBS (Gibco) solutions under mild agitation in a thermotank (37 °C), at a concentration of 1 mg/mL. An aliquot of PLGA/lecithin particles solutions was gathered to investigate particles size with a Nano-ZS Zeta Sizer at a predetermined time point. The measurements were implemented in triplicate. 2.5. Fabrication and Characterization of Aluminum Hydroxide/HPV16 L1 Pentamer. The antigen adsorption assay C


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measured by flow cytometry using BD LSR Fortessa (BD Biosciences) and analyzed by Flowjo V.10.1 software. 2.13. Evaluation of Lymphocytes Late Activation. On day 3 after immunization, mice were euthanized and soaked in 75% ethanol for 5 min. The single-cell suspensions harvested from DLNs were prepared as described in section 2.10. After Fc receptors were blocked as mentioned above, the single-cell suspensions were labeled with anti-CD25, -CD71, -CD4 and -CD8 antibodies (eBioscience). Flow cytometry assays were performed to evaluate the expression of the late activation markers CD25 and CD71 in CD4+ and CD8+ T cells. The percentages of CD25+ and CD71+ in CD4+ and CD8+ T cells were measured using the BD LSR Fortessa (BD Biosciences) and analyzed by Flowjo V.10.1 software. 2.14. Evaluation of Antigen-Specific T Cells Response. Mice were euthanized on day 28 after the first vaccination and soaked in 75% ethanol for 5 min. The spleens were immediately isolated and rinsed in a Petri dish containing PBS to remove any remaining blood residue. Next, the spleens were gently ground in PBS to obtain singlecell suspensions. After that cells were filtrated via 70 μm mesh cell strainers (BD Falcon). Next, the cells were centrifuged at 500g for 5 min, the supernatant was aspirated off with care, and the cell pellets were resuspended in red blood lysis buffer for about 5 min. Then, the cells were spun at 500g for 5 min again, the supernatant was prudently discarded, and the cell pellets were resuspended in RMPI 1640 medium. Next the splenocytes collected from immunized mice were restimulated with 10 μg/mL HPV L1 pentamer and cultured in a 37 °C humidified incubator with 5% CO2 for 48 h to assess antigen-specific T cell responses. BFA was added during the last 6 h of incubation. After Fc receptors were blocked as stated above, each sample was specifically stained for surface expression of CD4 and CD8, fixed, permeabilized, and then stained with the indicated antibodies against IL-4 and IFN-γ (eBioscience). The percentages of IL-4- and IFN-γsecreting cells at the single-cell level within CD4+ and CD8+ T cells were measured using BD LSR Fortessa (BD Biosciences) and analyzed using Flowjo V.10.1 software. 2.15. Release of Cytokine and Chemokine at Injection Sites. Mice (n = 3) were vaccinated at both hind legs with 50 μL per quadriceps muscle of the different vaccine formulations or buffer control. Cytokine and chemokine concentrations at injection sites were measured at 3, 8, 16, 24, 48, and 72 h after injection. Mice were euthanized and soaked in 75% ethanol for 5 min, and muscle tissues were isolated. The muscles were rinsed in ice-cold PBS to remove any remaining blood residue. Then icy RIPA buffer (Thermo Scientific) containing Complete Mini Protease Inhibitor Cocktail tablets (1 tablet/10 mL, Roche Diagnostics) was employed to homogenize muscle using an Ultra-Turrax T25. Homogenates were centrifuged (15 000g, 10 min, 4 °C), the supernatant was aspirated off with care, and the total protein concentrations in the supernatant were measured with a Micro-BCA protein assay kit (Pierce). Muscle homogenates were diluted with RIPA buffer to a protein concentration of 1 mg/mL for subsequent cytokine and chemoattractant analysis. TNF-α, IP-10, IL-6, MIP-1α, MCP-1, MCP-3, MIP-2, MIP1β, Eotaxin, and Gro-α contents in muscle homogenates were assessed using ProcartaPlex multiplex immunoassays (Affymetrix, eBioscience). 2.16. Statistical Analysis. GraphPad Prism V.6.01 software (San Diego, CA) was used to perform statistical analysis. Differences among groups were evaluated with one-way ANOVA with the Newman−Keuls multiple comparisons test. Significant differences among groups are expressed as follows: *p < 0.05, **p < 0.01, and ***p < 0.001.

the plates. HPV-16 pseudovirions (Psv) were generated with three plasmids encoding HPV L1, HPV L2, or green fluorescent protein (GFP) cotransfected into 293FT cells. Serial diluted by 4-fold, serum samples with an initial dilution factor of 1:20 were precultured with HPV pseudovirions (at 0.2 multiplicity of infection) at 2−8 °C for 1 h, subsequently transferred to the plates seeded by a monolayer of 239FT cells, and incubated at 37 °C and 5% CO2 for 72 h. Cells alone, cells cultured with pseudovirions, and cells cultured with pseudovirions and positive serum containing neutralizing antibodies were employed as controls. Following this incubation, the medium was removed. Then cells were washed three times with 10 mM PBS, and the fresh medium was replenished to harvest 293FT cells. Detection of GFP expression of 293FT cells harvested with BD FACS Calibur was analyzed using Flowjo V.10.1 software (Tree Star Inc.). The neutralization activity was calculated by the following formula: % neutralization = (Psvalone − Psvserum)/(Psvalone − PsvAb) × 100%. Serum with at least 50% neutralizing activity was regarded as neutralizing. 2.9. Determination of Cytokine Secretion and Granzyme B Expression by ELISA. Mice were euthanized at day 28 after primary immunization and soaked in 75% ethanol for 5 min. The spleens were immediately isolated and rinsed in a Petri dish containing PBS to remove any remaining blood residue. Next, the spleens were gently ground in PBS to obtain single-cell suspensions. After that, the cells were filtrated with 70 μm mesh cell strainers (BD Falcon). Next, the cells were pelleted by centrifugation (500g, 5 min), the supernatant was aspirated off with caution, and the cell pellets were resuspended in red blood lysis buffer for about 5 min. Then, the cells were centrifuged at 500g for 5 min, the supernatant was discarded deliberately, and the cell pellets were resuspended in RMPI 1640 medium. Then the harvested splenocytes were restimulated with HPV L1 pentamer (10 μg/mL) in a 37 °C humidified incubator with 5% CO2 for 60 h. The supernatants were obtained by centrifugation (500g, 5 min) for detection, and ELISA kits (eBioscience, San Diego, CA) were used to measure the concentrations of granzyme B and IL-4 cytokines according to the manufacturer’s instructions. 2.10. Expression of MHC II and Costimulatory Molecules CD86 on DCs in DLNs. Mice were euthanized at 6, 24, and 96 h after administration and soaked in 75% ethanol for 5 min. The popliteal lymph nodes were immediately extracted and rinsed in a Petri dish containing PBS to remove any remaining blood residue. Next, the popliteal lymph nodes were gently ground in PBS to obtain cell suspensions. After that cells were passed across 70 μm mesh cell strainers (BD Falcon). At last, cells were pelleted by centrifugation at 500g for 5 min, the supernatant was aspirated off with care, and the cell pellets were resuspended for further assays. Then the harvested single-cell suspensions were incubated with purified rat anti-mouse CD16/CD32 clone 2.4G2 (BD Pharmingen) to block Fc receptors and stained with fluorescent-labeled anti-mouse antibodies (antiCD11c antibody, anti-CD86 antibody, and anti-MHC II antibody; eBioscience). The expression of the costimulatory molecule CD86 and MHC II on DCs was measured using the BD LSR Fortessa (BD Biosciences) and analyzed using Flowjo V.10.1 software (Tree Star Inc.). 2.11. Evaluation of Lymphocytes Early Activation in DLNs. Mice were euthanized at 24 h after administration and soaked in 75% ethanol for 5 min. The single-cell suspensions harvested from DLNs were prepared and Fc receptors were blocked the same way as those in section 2.10. Hence, the single-cell suspensions were labeled with anti-mouse CD4, CD8, CD19, and CD69 antibodies (eBioscience). The percentages of activated B cells (CD69+CD19+), activated CD4+ T cells (CD69+CD4+), and activated CD8+ T cells (CD69+CD8+) were evaluated by flow cytometry assay using the BD LSR Fortessa (BD Biosciences) and analyzed using Flowjo V.10.1 software. 2.12. Determination of TFH Cells in DLNs. On day 7 after immunization, mice were euthanized and soaked in 75% ethanol for 5 min. The single-cell suspensions harvested from DLNs were prepared as described in section 2.10. After Fc receptors were blocked as stated above, the single-cell suspensions were labeled with antiCD4, -CCR7, -CXCR5, and -PD-1 antibodies (eBioscience). The frequencies of TFH cells (CD4+CCR7+CXCR5hiPD-1hi) were

3. RESULTS 3.1. Fabrication and Characterization of HPV 16Loaded PLGA/Lecithin Particles and Aluminum Hydroxide-HPV16 L1 Pentamer. Uniform-sized PLGA/lecithin particles with three different sizes were prepared by premix membrane emulsification in combination with the solvent evaporation method. Scanning electron micrographs (Figure 1A−C) revealed that all PLGA/lecithin particles were smooth and spherical D


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Figure 1. Characteristics of different vaccine formulations. (A−C) Scanning electron microscope images of HPV 16 L1 pentamer-loaded PLGA/ lecithin particles of various size (A, 0.3 μm; B, 1 μm; and C, 3 μm). Scale bars represent 1 μm. (D−F) Size distributions of HPV 16 L1 pentamerloaded PLGA/lecithin particles of various sizes (D, 0.3 μm; E, 1 μm; and F, 3 μm). (G) Transmission electron microscope image of aluminum hydroxide. (H) Size distribution of aluminum hydroxide. (I) HPV 16 L1 pentamer release profiles for PLGA/lecithin particles. Curves show release at 37 °C in PBS. Release was assessed in triplicate over 80 days. Data are presented as mean ± SD.

Table 1. Characteristics of PLGA/Lecithin-HPV 16 Particlesa formulation 0.3 μm PLGA/lecithin-HPV 16 1 μm PLGA/lecithin-HPV 16 3 μm PLGA/lecithin-HPV 16 aluminum hydroxide

diameter (nm) 303.53 926.53 3857.00 7038.5

± ± ± ±

3.35 18.22 15.12 164.76

size distribution (PDIb/spanc) 0.11 0.11 0.34 1.20

± ± ± ±

0.002b 0.082b 0.018c 0.375c

ζ-potential (mV)

HPV content (μg/mg)

loading efficiency (%)

−17.17 ± 0.35 −22.23 ± 1.99 −20.17 ± 1.17

21.767 ± 2.33 23.774 ± 2.26 21.843 ± 3.16 100

81.626 89.153 81.911 99.900

a Data are displayed as mean ± SD. bPDI from dynamic light scattering (Nano-ZS Zeta Sizer). cSpan = (Dv,90% − Dv,10%)/Dv,50%, where Dv,90%, Dv,50%, and Dv,10% are volume size diameters at 90%, 50%, and 10% of the cumulative volume (Mastersizer 2000).

modestly uniform size with low span value (0.34 ± 0.018) (Table 1). Furthermore, as shown in Table 1, the HPV 16 L1 pentamer contents measured by Micro-BCA kits for the three batches of particles were consistent [21.767 ± 2.33 μg/mg for 0.3035 μm particles (loading efficiency 81.626%); 23.774 ± 2.26 μg/mg for 0.9265 μm particles (loading efficiency 89.153%), and 21.843 ± 3.16 μg/mg for 3.857 μm particles (loading efficiency 81.911%)]. In addition, the ζ-potentials of different sized PLGA/lecithin particles were similar (about −20 mV) (Table 1). The preparation of aluminum hydroxide-HPV16 L1 pentamer used direct adsorption. Transmission electron micrographs revealed that the aluminum hydroxide exhibited fiberous structures with average diameters of 7038.5 nm (Figure 1G,H and Table 1) and a slightly higher span value (1.20 ± 0.375).

with narrow size distributions. The core−shell structure of PLGA/lecithin particles prepared by the same premix membrane emulsification had been reported by Ma et al. previously.44 The size distribution data showed that their respective average diameters were 303.53 ± 3.35, 926.53 ± 18.22, and 3857.00 ± 15.12 nm (Figure 1D−F, Table 1). For the submicron size particles (∼0.3 and ∼1.0 μm), the size analysis was performed by dynamic light scattering, including average size and polydispersity index (PDI); for the micron size particles (∼3.0 μm), the size analysis was determined with laser diffraction, including average diameter as well as span value. The PLGA/lecithin particles (0.3 and 1.0 μm) displayed relatively uniform size with low PDI value (0.11 ± 0.002 for the 0.3035 μm particles, 0.11 ± 0.082 for the 0.9265 μm particles), and the PLGA/lecithin particles (3.857 μm) exhibited E


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swelling behavior upon exposure to the aqueous medium, and after that the particles retained size stability throughout the 24 h study (diameter = 337.02 ± 11.64 nm, PDI = 0.137 ± 0.042, mean ± SD). Consistent with a previous report, the adequate lecithin coverage of the hydrophobic PLGA core might prevent protein adsorption on the particle surface. 3.4. Systemic Antibody Responses. To study the adjuvant effect of HPV 16 L1-loaded PLGA/lecithin particles, mice were immunized intramuscularly with particles of 0.3227 μm (0.3 μm PLGA/lecithin-HPV16), 0.9265 μm (1 μm PLGA/ lecithin-HPV 16), or 3.857 μm (3 μm PLGA/lecithin-HPV 16). Aluminum hydroxide-based HPV 16 vaccine (Alum-HPV 16) and HPV 16 alone (HPV 16) were used as controls. HPV 16 L1 pentamer-specific IgG, IgG1, and IgG2a antibody titers in serum collected at days 14 and 28 were evaluated by ELISA (Figure 3A). At day 14 after the first injection, all vaccines elicited similar serum IgG titers. Furthermore, boost immunization with the same vaccine formulations elicited markedly increased IgG titers in the PLGA/lecithin particle-adjuvanted vaccine groups on day 28 (Figure 3B). Comparable specific antibody was elicited by particles of 1 μm (anti-HPV16 IgG titers >103 in 50% of immunized mice) and 0.3 μm (anti-HPV16 IgG titers >103 in 33.33% of vaccinated mice), which was significantly higher than the titers elicited by 3 μm particles and other control groups. The IgG titers were not significantly different among the mice vaccinated with 3 μm PLGA/lecithinHPV 16 particles, alum-HPV 16, and HPV16 (anti-HPV 16 IgG titers 105 in 100% of mice injected with 1 μm particles, 90% of mice immunized with 0.3 μm particles, and 80% of mice vaccinated

Furthermore, assessment of HPV 16 L1 pentamer contents with a Micro-BCA kit revealed that the aluminum hydroxide almost completely adsorbed HPV16 L1 pentamer. 3.2. Antigen Release in vitro. To examine the antigen release kinetics of different-sized particles, we evaluated the cumulative release of HPV 16 L1 pentamer. As shown in Figure 1I, dramatic differences in the release profiles in vitro of the different-sized particles were observed. The 0.3 μm PLGA/ lecithin particles showed a larger extent of initial release (nearly 50%) and a higher release rate, with 85% release between 30 and 80 days; however, 1 μm particles showed obviously lower initial release (around 25%), followed by sustained release of up to 85% between 70 and 80 days. The 3 μm particles exhibited the lowest burst release (about 20%), followed by incomplete and slow release of up to 70% between 60 and 80 days. These results suggested that the extent of the burst release was decreased with increasing diameter, which is consistent with a previous report. Consistent with previous reports,3,4,10 our results indicated that hybrid particle size affected the antigen release profiles, which had a critical impact on the immune response against HPV infection. Generally, antigen situated at the top layer of particles referred to initial burst,11 and antigen entrapped inside the particles tended to be exposed and released until the particles were degraded.12 Here we found that 1 μm particles showed a lower initial burst than 0.3 μm particles and a higher initial burst than 3 μm particles. This might be ascribed to the decrease in surface area/volume ratio with increasing size.13,14,56 The proteins loaded in smaller sized particles release rapidly due to the shorter diffusion path to the surface and more rapid erosion and disintegration of particles, while the slow release with larger sized particles depends on the longer path of diffusion to the surface of particles and the longer time to be degraded. On the basis of these findings, the 1 μm particles would be predicted to elicit the most favorable immune response due to their sustained release of encapsulated antigen and high overall extent of release. 3.3. Serum Stability of PLGA/Lecithin Particles in vitro. Serum stability of particles is critical for their application as drug carriers in vivo. We evaluated the serum stability of 0.3 μm PLGA/lecithin particles, employing the size change in the presence of serum over time. As shown in Figure 2, the

Figure 2. Serum stability of PLGA/lecithin particles in vitro. The 0.3 μm PLGA/lecithin particles were incubated with 10 vol % FBS at 37 °C under mild agitation. At the appointed time point, an aliquot of particles suspension was collected to determine the size of particles with a Nano-ZS Zeta Sizer. Data are displayed as mean ± SD (n = 3).

hybrid particles had sufficient stability in 10 vol % FBS solution. In the beginning, the size of particles was 319.74 ± 9.01 nm (PDI = 0.143 ± 0.032, mean ± SD). An initial ∼20 nm increase in size was observed, which might be due to the very slight F


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Figure 3. Antigen-specific antibodies in serum. (A) Scheme of immunization (arrows) and sera sampling (circles). Mice (n = 6) were intramuscularly injected twice over a 2-week interval. ELISA assays were performed to determine HPV-specific antibody titers in the sera. HPV pseudovirus neutralization assays were performed with pooled serum samples to define the 50% neutralization end points. HPV-specific serological IgG (B), IgG1 (C), and IgG2a (D) titers on days 14 and 28 after primary immunization. (E) HPV neutralization titers on days 14 and 28 after the first dosing. Data are displayed as geometric mean with 95% CI (n = 6).

Figure 4. Antibody persistence in serum. (A) Scheme of immunization (arrows) and of sera sampling (circles). Mice (n = 10) were injected at days 0, 28 and 142, and serum was collected at days 21, 42, 112, and 156. (B) HPV-specific serological IgG titers at days 21, 42, 112, and 156 after the first immunization. (C) HPV neutralization antibody titers at days 21, 42, 112, and 156 after first dosing. Data are presented as the geometric mean with 95% CI (n = 10). ##p < 0.01, ###p < 0.001, 1 μm HPV 16-loaded PLGA/lecithin particles versus 3 μm HPV 16-loaded PLGA/lecithin particles; ††† p < 0.001, 1 μm HPV 16-loaded PLGA/lecithin particles versus 0.3 μm HPV 16-loaded PLGA/lecithin particles.

with 3 μm particles. At 112 days, little decline in antibody titers was observed for mice injected with 1 μm PLGA/lecithin-HPV 16 particles (average 7.69% decrease; 100% of mice with antiHPV 16 titers >105), though sharp decreases were observed for 3 μm particles (average 33.33% decrease; only 40% of mice with anti-HPV 16 titers >105) and 0.3 μm particles (average 62.82% decrease; barely 50% of mice with anti-HPV 16 titers >105). Similar results were observed just 6 days after the third injection (on day 156) with average anti-HPV16 IgG titers

>105 in 100% of mice injected with 1 μm particles (average titer 1:736 000), in 100% of mice immunized with 0.3 μm particles (average titer 1:352 000), and in 80% of mice vaccinated by 3 μm particles) (average titer 1:192 000). These results suggested that the vaccines elicited a high-level booster response and a rapid recall response. Analysis with the Newman− Keuls multiple comparisons test confirmed that 1 μm PLGA/ lecithin-HPV 16 particle-generated antibody responses were significantly different from those achieved by 3 μm particles G


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Figure 5. Activation of DCs in DLNs at 6 h after intramuscular vaccination. Mice (n = 3) were euthanized at 6 h after vaccination, and the popliteal lymph nodes were extracted to assess DCs maturation by flow cytometry. Single-cell suspensions from DLNs were labeled with anti-CD86, antiMHC II, and anti-CD11c antibodies. Typical flow cytometry plot of CD86+ (A) and MHC II+ (B) in CD11c+ DCs. Gray-filled histograms indicate naive mice, and black-line histograms stand for mice immunized with different vaccine formulations. (C) Typical flow cytometry plot of CD86+ MHC II+ in CD11c +MHC II+ cells. (D) Percentage of CD86+ in CD11c+ DCs. (E) Percentage of MHC II+ in CD11c+ DCs. (F) Percentage of CD86+ MHC II+ in CD11c+MHC II+ DCs. Data are displayed as mean + SEM (n = 3).

on day 42 and by 0.3 and 3 μm PLGA/lecithin-HPV 16 particles on days 112 and 142 (Figure 4B). Further observation demonstrated that the PLGA/lecithinHPV 16 particle-based vaccines induced continuously increased neutralization titers over time after the primary immunization (Figure 3C). While 1 μm PLGA/lecithin-HPV 16 particles induced modestly higher neutralization titers (peak average titer 1:24 320) in comparison with the 0.3 μm particles (peak average titer 1:2560) and the 3 μm particles (peak average titer 1:1012), the difference was not significantly significant. On the basis of these observations, the HPV neutralization titers may not be elevated as fast as binding IgG after immunization. 3.5. Expression of MHCII and Costimulatory Molecules CD86 on DCs in DLNs. To determine the activity of PLGA/lecithin particle-based vaccination on DCs, we evaluated the ex vivo expression of CD86 and MHCII on DCs. The expression of the costimulatory marker CD86 at 6 h postinjection

was significantly up-regulated after injection with 1 μm PLGA/ lecithin particles, 0.3 μm PLGA/lecithin particles, or aluminum hydroxide-adjuvanted vaccine compared to antigen alone, though the expression was not significantly upregulated after injection with 3 μm PLGA/lecithin particles (Figure 5A,D). Consistent results were identified for MHCII expression (Figure 5B,E). Furthermore, the proportion of mature DCs (CD86+MHCII+) was enhanced after immunization with PLGA/lecithin particle-based vaccine with 1 and 0.3 μm particles and the aluminum hydroxide-adjuvanted formulation compared with HPV antigen alone (Figure 5C,F). As shown in Figure S5 of the Supporting Information (SI), after 24 h the particle vaccines and aluminum hydroxide vaccine induced comparable CD86 expression, all markedly higher than antigen alone, while almost similar MHCII expression was observed in all vaccines. After 96 h, all vaccines induced comparable CD86 and MHC II expression. Over all, immunization with 1 μm PLGA/lecithin H


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Figure 6. Early activation of CD4+ T, CD8+ T, and B cells in DLNs after intramuscular vaccination with different formulations. Mice (n = 3) were euthanized at 24 h after vaccination. Single-cell suspensions harvested from isolated popliteal lymph nodes were dyed with anti-CD69, -CD4, -CD8, and -CD19 antibodies for flow cytometry assay. (A) Typical flow cytometry plots of CD69 expression on CD4+ T, CD8+ T, and B cells. (B) Percentage of CD69+ among CD4+ T cells, CD8+ T cells, and CD19+ B cells. Data are represented as the mean + SEM (n = 3).

PLGA/lecithin-HDV 16 particles, 0.3 μm PLGA/lecithinHDV 16 particles, Alum-HPV 16, or antigen alone, caused significantly enhanced upregulation of CD69 on CD4+ and CD8+ T cells (Figure 6). No remarkable enhancement of CD69 on CD19+ B cells was observed by any of the vaccine formulations over antigen alone, suggesting that lymphocyte activation might be primarily restricted to T cells. We further investigated the activity of the vaccine formulations on the expression of CD71 (lymphocytes’ early phase activation marker) and CD25 (lymphocytes’ late-phase activation marker) in DLNs obtained from mice 72 h after immunization. Significant up-regulation of CD71 was noticed for both CD4+ and CD8+ T cells in mice injected with 1 μm PLGA/lecithinHPV 16 particles (Figure S1, SI). There was no remarkable

particles elicited the highest percentage of mature DCs among the groups, followed by 0.3 μm PLGA/lecithin particles and aluminum hydroxide-adjuvanted vaccine, especially at the early stage of DCs maturation. These results indicated that PLGA/ lecithin particles could raise the expression of critical surface markers involved in DCs maturation, with the greatest enhancement by 1 μm PLGA/lecithin particles. 3.6. Lymphocytes Activation in vivo. To evaluate whether the vaccines can also enhance the activity of lymphocytes in the early stage, we performed a series of functional experiments. First, cells from DLNs were analyzed for the expression of CD69 (lymphocytes’ early phase activation marker45) by flow cytometry 24 h after vaccination of mice. The results show that administration with 1 μm PLGA/lecithin-HPV 16 particles, but not 3 μm I


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Figure 7. Cytokine secretion of splenocytes restimulated ex vivo with antigen. Mice (n = 6) were injected twice over a 2-week interval. At day 28 after the first dosing, splenocytes were collected from mice and restimulated with HPV (10 μg/mL) for 60 h at 37 °C with 5% CO2. The concentrations of IL-4 (A) and granzyme B (B) were determined by ELISA. Data are represented as mean + SEM (n = 6).

differentiation of B cells into plasma cells and memory cells that promote high-affinity antibody. 3.10. Release Profile of Pro-Inflammatory Cytokines and Chemokines at the Injection Spot. We also assessed a panel of pro-inflammatory cytokines and chemokines in muscle homogenate at a serial of time points after intramuscular administration of antigen alone or in combination with adjuvants (PLGA/lecithin particles or aluminum hydroxide). The particlebased vaccine groups displayed a transient release of multiple cytokines at the injection site (Figure S2, SI). The levels of TNF-α and IP-10 were induced more highly for the particlebased vaccines than for the aluminum hydroxide-adjuvanted vaccine. Furthermore, 1 μm PLGA/lecithin-HPV 16 particles induced slightly stronger secretion than other groups for a broader panel of factors. The levels of MIP-1β and MIP-2 at 8 h were significantly higher for 1 μm PLGA/lecithin particles versus the other groups. On the basis of these findings, we speculate that 1 μm PLGA/lecithin-HPV 16 particles may efficiently induce immune response by activating inflammatory cytokines and chemokines.

difference in CD25 expression among the vaccine formulations, although 1 μm PLGA/lecithin-HPV 16 particles produced a slightly higher expression of CD25 than the other particles. Overall, these results suggest that 1 μm PLGA/lecithin particles might enhance the activation of T lymphocytes, primarily in the early phase of lymphocytes activation. 3.7. Cytokine Level Secreted by ex vivo Restimulated Splenocytes. To further investigate whether immunization enhances cytokine secretion, we evaluated the ex vivo cytokines contents of restimulated splenocytes. Data demonstrated that the IL-4 secretion was elevated for splenocytes from mice that were immunized with 1 μm PLGA/lecithin-HPV 16 particles or 0.3 μm PLGA/lecithin-HPV 16 particles, but not with the other formulations (Figure 7A). Furthermore, the secretion of granzyme B was elevated for both the 1 and 0.3 μm PLGA/lecithin-HPV 16 particles, but the difference was only significant for the 1 μm particles (Figure 7B). These results support our other data suggesting that the 1 μm PLGA/lecithin particle formulation induced a strong immune response. 3.8. Antigen-Specific Memory T Cells Response. Next, we determined whether particle-based vaccines also enhanced antigen-specific memory T cells responses. A remarkable enhancement of HPV-specific IL-4-secreting CD4+ and CD8+ T cells was induced by 1 or 0.3 μm PLGA/lecithin-HPV 16 particles on day 156 after primary immunization, with relatively less enhancement by 3 μm particles (Figure 8). Similar results were observed for HPV-specific IFN-γ-secreting CD8+ T helper cells and CD4+ T cells. Thus, 1 μm PLGA/lecithin-HPV 16 particles are likely to strengthen the magnitude and quality of antigen-specific memory CD4+ and CD8+ T cells. 3.9. TFH Cells Response. To determine whether 1 μm PLGA/lecithin-HPV 16 particle vaccination facilitates the generation of long-lasting antibody responses by improving TFH cells activation, we measured the size effects on TFH cells located in the DLNs. A marked enhancement of CD4+CXCR5+CCR7loPD-1hi TFH cells in the DLNs of mice dosed with 1 μm PLGA/lecithin-HPV 16 particle formulations was observed compared to other formulations (Figure 9), which is consistent with a fundamental role for TFH cells in mediating the

4. DISCUSSION Fulfilling critical parameters of adaptive immunity to mount robust, sufficient, and sustained antibody responses remains a major challenge in vaccinology. Compared with HPV L1 VLPbased vaccines on market, recombinant HPV L1 pentamer expressed in E. coli represents a promising and potentially costeffective vaccine for preventing HPV infection. Hybrid particles comprising a polymer core and lipid shell have been explored as versatile carriers and potentially more potent alternatives to conventional aluminum salts adjuvant. Particle size is thought to have a major influence on particulate-induced immune responses.34−37 The particle sizes not only have correlation with the magnitude of immune reaction but also dictate the persistence and type of immune response.34,37−39 However, while the size of particles regulates specific immune response for HPV L1 pentamer-based candidate vaccines, many influencing factors are involved, including antigen release, antigen uptake, antigen presentation by APCs, and lymphocytes J


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Figure 8. Expression of intracellular cytokines. Mice (n = 6) were injected at days 0, 14, and 142 and euthanized on day 156 after injection. The splenocytes (106 cells/well) harvested from mice were restimulated in vitro with 10 μg/mL HPV, and IFN-γ-secreting CD4+ T or CD8+ T cells and IL-4-secreting CD4+ T or CD8+ T cells were identified. (A) The flow cytometry histograms are representative of six cultures of pooled splenic cells for each independent group. (B) The percentage of IL-4-secreting memory CD4+ T or CD8+ T cells and IFN-γ-secreting memory CD4+ T or CD8+ T cells among memory CD4+ T or CD8+ T cells. Data are presented as mean + SEM (n = 6).

particles containing 10 μg of HPV 16 L1 pentamer programmed the strongest and most lasting antigen-specific response among any of the formulations tested. Overall, in comparison with other particle sizes (0.3 and 3 μm), 1 μm PLGA/lecithin particles significantly enhanced the immunogenicity of naked HPV 16 L1 pentamer according to several parameters: extended antigen release, increased APCs activation and maturation, lymphocytes activation, and TFH cells differentiation in DLNs, enhanced cytokines secretion by Th cells in spleens, facilitated

activation. In this study, we developed a novel, promising, and potentially cost-effective HPV vaccine system to protect against HPV infection that employed recombinant HPV 16 L1 pentamer expressed in E. coli as candidate antigen and PLGA/lecithin hybrid particles as vaccine adjuvant and investigated the size effect on antigen release profiles, activation of lymphocytes, DCs activation and maturation, TFH cells differentiation, and release of pro-inflammatory cytokines and chemokines. Overall, our findings indicated that 1 μm PLGA/lecithin K


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Figure 9. The TFH cells in DLNs. Mice (n = 3) were euthanized at 7 days after dosing. Single-cell suspensions harvested from popliteal lymph nodes were labeled with anti-CD4, -CCR7, -CXCR5 and -PD-1 antibodies to assess the percentages of TFH cells (CD4+CCR7+CXCR5hiPD-1hi) by flow cytometry assay. (A) Typical flow cytometry plots and (B) frequency of TFH cells (CD4+CCR7+CXCR5hiPD-1hi) among CD4+ T cells. Data are displayed as mean + SEM (n = 3).

stability of hybrid particles for 24 h in vitro, and excellent serum stability was observed. To monitor antigen persistence over time, we assessed the antigen release profile of PLGA/ lecithin particles with varying sizes in vitro. Consistent with previous reports,34,37−39 our results indicated that hybrid particle size affected the antigen release profiles, which had a critical impact on the immune response against HPV infection. In general, antigen situated at the top layer of particles corresponded to the initial burst release,53 and antigen encapsulated inside the particles tended to emerge and be released as the particles were degraded.54 Here we found that 1 μm particles showed a lower initial burst than 0.3 μm particles and a higher initial burst than 3 μm particles. This might be ascribed to the decrease in surface area/volume ratio with increasing size.55,56 The proteins loaded in smaller sized particles released rapidly due to the shorter diffusion path to the surface and the more rapid erosion and disintegration of particles. While larger sized particles with slow release depended on the longer path of diffusion to the surface of particles and took a longer time to be degraded. The 1 μm PLGA/lecithin-HPV 16 particles exhibited a modest burst release, ensuring the start of the immune response and complete antigen release of the entrapped antigen for antigen processing and presentation, which might help to mount antigen-specific immune responses with the largest magnitude and longest duration among the particle formulations tested. After sustainable antigen release, efficient antigen uptake and transport by APCs is the critical step for the immune response. Particles uptake and traffic to the DLNs might also be in a sizedependent manner36,56 Jia et al. revealed that in comparison with other particle sizes (500 nm, 2.1 μm, and 4.9 μm), 900 nm particles, also prepared by premix membrane emulsification,

generation of memory T cells, and augmented antibodies with high avidity. Importantly, compared with the other particle sizes that were tested (0.3 and 3 μm), there was a more remarkable magnitude of IgG antibody response mounted by 1 μm PLGA/lecithin particles. Moreover, 1 μm PLGA/lecithin particles yielded more long-lasting IgG antibody titers and more heightened and potent anamnestic reaction upon repeated dosing. We also analyzed the IgG1/IgG2a ratio, and mice immunized with 1 and 3 μm PLGA/lecithin-HPV 16 particles showed a minor predominance of IgG1 (an indicator of Th2 immunity), while 0.3 μm PLGA/lecithin-HPV 16 particles displayed a modest predominance of IgG2a (an indicator of Th1 immunity). Yet, as a whole, a balanced IgG1/IgG2a ratio was observed for all the PLGA/lecithin-HPV 16 particles tested. Sustained antigen release was generally desirable to induce a “depot” effect, which could extend antigen exposure to immune system, accordingly increasing the access to immune cells, and then resulted in a more potent and persistent immune response.46−50 An enhancement not only to the degree but also to the quality of immune responses by the action of extended antigen release might be presented, as reported.34−37 Kanchan et al. showed that antigen release characterized by a slow and sustained process had an important part in mounting anamnestic response by a single dose.51 Demento et al. revealed that a long-term memory T cell response was promoted by continuous antigen release from particles.49 Moreover, not only was the sustainability of antigen exposure to immune system crucial but incipient antigen burst also played a key role in eliciting effective immune responses.52 PLGA/ lecithin hybrid particles encapsulating antigen were characterized by a controlled and continuous antigen release profile and remarkable serum stabilities.20,31 We studied the serum L


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5. CONCLUSION We developed a novel, promising, and potentially cost-effective HPV vaccine system to protect against HPV infection that employed recombinant HPV 16 L1 pentamer expressed in E. coli as candidate antigen and PLGA/lecithin hybrid particles as vaccine adjuvant, and we investigated the size effect of HPV 16 L1 pentamer-loaded PLGA/lecithin particle formulations on the immune response to obtain the optimal vaccine potency. Our findings demonstrated that compared with the other sizes of particles tested (0.3 and 3 μm), the intermediate size range (1 μm) PLGA/lecithin particles encapsulating HPV 16 L1 pentamer induced antigen-specific immune responses with greater magnitude and persistence and also yielded more heightened and potent recall or anamnestic responses upon repeat vaccination. The superior response may be ascribed to controlled and sustained antigen release and robust antigen uptake and transport, promoting a series of cascade reaction, including facilitated cytokine secretion by splenocytes, enhanced DCs maturation, increased lymphocytes activation, and augmented TFH cells differentiation in DLNs.

exhibited more convenient as well as rapid internalization by APCs, stronger antibody protection, and higher cytokine secretion, such as IFN-γ and IL-4.57 Smaller sized (0.3 μm) particles might mostly be taken up by receptor-mediated endocytosis. While the particles with middle size (1 μm) might be taken up by phagocytosis, resulting in easier uptake. For particles with larger size (3 μm), uptake and presentation would be likely low due to the larger size relative to APCs.58 Zhang et al. reported that antigen-loaded nanoparticles exhibited remarkably extended antigen retention at the injection site and long-lasting antigen transfer into the periphery DLNs.59 These reports further supported our research that the uptake of 1 μm PLGA/lecithin particle-encapsulating antigen by APCs could be efficiently increased and transported to DLNs after persistent antigen release. Following efficient antigen uptake, antigen transport to lymphoid tissue and maturation and activation of DCs in DLNs were prerequisites for effective antigen presentation and subsequent lymphocytes activation. We demonstrated that the 1 μm PLGA/lecithin particle formulations elicited significantly augmented DCs maturation than the other particle sizes. Subsequently, the activated DCs could promote the activation of T/B lymphocytes. Our research exhibited that 1 μm PLGA/ lecithin particles enhanced lymphocytes activation compared with particles of other sizes. Next, the activated T cells could increase the cytokines secretion. The 1 μm PLGA/lecithin particles had enhanced secretion levels of IL-4 and granzyme B. As TFH cells are specialized in offering assitance to B cells, priming B cells differentiation into long-lived plasma cells,60−62 producing sustained high-affinity antibody responses,63−66 and activating memory B cells response,67−70 we also examined the percentages of TFH cells in the DLNs at day 7 after dose. Consistent with the demonstrated antibody persistence and recall response, mice immunized with 1 μm PLGA/lecithinHPV 16 particles displayed prominently increased percentages of TFH cells in the DLNs. Therefore, the extended antigen release and more efficient antigen uptake and transport might trigger a serial cascade of reactions on DCs, T/B lymphocytes, and TFH cells, which could provide a mechanism to explain why the PLGA/lecithin-HPV 16 particle vaccine formulations with intermediate size (1 μm) induced more strong and sustained immune response, compared to the other sizes of particles. Hence, on the basis of our results, we put forward the following model to reveal the potential functional mechanism of PLGA/lecithin particles as vaccine adjuvants to facilitate immune response (Figure S3, SI). First, particle size affected antigen release, uptake, and traffic to the DLNs. The 1 μm PLGA/lecithin particle vaccine formulations could initiate immune response by sufficient antigen released at the beginning stage and continuously stimulate immune system by persistent antigen released at the later stage due to the antigen depot effect. Subsequently, increasing interaction of antigen with APCs might be met, resulting in the expression of MHC II molecules and CD86 on DCs, lymphocytes activation, and TFH cells response in DLNs, which contributed to the immune response. TFH cells helped B cells divide into longlived plasma cells that produced antibodies with high affinity, ensuring persistent and strong immune protection, and memory B cells that elicit rapid and robust memory responses. Therefore, by optimizing the size and the resultant antigen release profile of PLGA/lecithin particle vaccine formulations, powerful and persistent responses can be mounted to obtain better protection against infectious virus.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b11556. Late activation of CD4+ T and CD8+ T cells in DLNs, cytokine and chemokine release at injection sites in response to injection with different vaccine formulations, schematic diagram of the presented functional mechanism of the different-sized PLGA/lecithin particle vaccine formulations, representative flow cytometric plots exhibiting the gating scheme, activation of DCs in the DLNs (popliteal lymph nodes) of mice at 24 and 96 h after intramuscular vaccination with different formulations (Figures S1−S5) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Lianyan Wang: 0000-0002-4421-0128 Author Contributions

C.J., T.Y., J.C., and L.W. designed the experiments; C.J. performed the experiments with the assistance of A.Z., L.X., and Y.W.; F.Y., Y.W., and M.Y. performed the determination of serum antibody titers; Y.W. generated the HPV 16 L1 pentamer antigen used in this study; C.J. fulfilled the other assays, carried out all statistical analysis, and wrote the manuscript. All authors contributed to scientific planning and discussions. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Science and Technology Major Project of China (Grant No. 2016ZX10004001-005 and 2014ZX09102045-008), the National Science Foundation of China (Grant No. 21476243), and the Special Innovation Fund for Beijing Economic− Technological Development Area (Grant No. JSYF2012658). M


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