Delivery of Polysaccharides Using Polymer Particles - American

Article pubs.acs.org/molecularpharmaceutics

Delivery of Polysaccharides Using Polymer Particles: Implications on Size-Dependent Immunogenicity, Opsonophagocytosis, and Protective Immunity Chakkumkal Anish,†,§ Naeem Khan,‡ Arun Kumar Upadhyay,†,∥ Devinder Sehgal,‡ and Amulya Kumar Panda*,† †

Product Development Cell, ‡Molecular Immunology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India S Supporting Information *

ABSTRACT: Bacterial capsular polysaccharides are components of many modern vaccines, but they are weakly immunogenic. Herein, we describe the delivery of pneumococcal capsular polysaccharide serotype-1 (PCP-1) in polylactide polymeric particles to enhance its immunogenicity. Immunization with PCP-1-entrapped particles elicited long-term memory antibody responses from a single intramuscular injection. PCP-1-entrapped nanoparticles (NPs) elicited significantly higher anti-PCP-1 IgG responses than that observed with soluble and microparticles (MPs) formulations. Delivering PCP-1 and pneumococcal proteins in same particles did not improve the IgG response. The sera of animals immunized with PCP-1-entrapped particles promoted efficient opsonophagocytosis of pneumococci by macrophages. Single-dose immunization with PCP-1-entrapped particles conferred a long-term serotype-specific protection against lethal pneumococcal challenge. The higher immunogenicity of PCP-1 nanoparticles showed correlation with enhanced uptake by antigen-presenting cells. The results highlight the potential of polymeric nanoparticles as an efficient means of presenting polysaccharide antigens to the immune system. KEYWORDS: pneumococcal capsular polysaccharides, antigen-delivery systems, polylactide nanoparticles, opsonophagocytic assay, memory antibody, pneumococcal surface antigen A (PsaA), pneumococcal surface protein A (PspA)

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INTRODUCTION Polymeric particle-based delivery systems have been very widely used to modulate immune responses against protein antigens.1−4 Particles made of biodegradable polylactide (PLA) polymer offer an attractive platform to deliver vaccine components owing to their excellent safety profile and immunomodulatory properties.1,3,5 These particles can be formulated with different surface chemistry and particle size as well as varied release characteristics. All of these parameters are important for eliciting the desired immune response against the entrapped vaccine components.6,7 Additionally, diverse targeting functionalities can be tethered to these polymeric particles to drive desired immunological outcomes.8,9 Optimizing the design principles involved in the formulation of antigenentrapped particles will help to meet the challenges involved in the development of better vaccine formulations. Routinely, the immune system interacts with a diverse set of natural particulate antigens, and the properties of these biological particles have a significant impact on the outcome of the immune responses against the antigens presented on them. © 2014 American Chemical Society

Antigen density, organization, repetitive structure, release kinetics, multivalency, and particle size are important characteristics that play a significant role in driving the antibody responses against the antigens displayed on biological particles such as viruses, bacteria, parasites, protein scaffolds, and so forth.5,10−13 Insights drawn from these reports can be applied for designing particulate vaccine-delivery systems to augment immune responses against weakly immunogenic vaccine components. In this context, polysaccharide-based antigens are very relevant because they are weakly immunogenic, elicit poor memory antibody responses, and promote limited isotype switching. Numerous pathogenic bacteria produce a unique polysaccharide, called the capsule, that attaches to the bacterial surface, preventing its clearance by the host. These capsular Received: Revised: Accepted: Published: 922

October 6, 2013 January 2, 2014 January 21, 2014 January 21, 2014 dx.doi.org/10.1021/mp400589q | Mol. Pharmaceutics 2014, 11, 922−937

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polymeric particles would preserve the zwitterionic character, and it can be presented in its native form. The current study describes the enhancement of anti-PCP-1 antibody responses using PLA particles. The polymer particle size and antigen density and release pattern were analyzed for their influence on improving the immunogenicity of PCP-1. A mouse pneumococcal infection model was used to evaluate the immunogenicity and functional implications of PCP-1entrapped particles. The results indicate that particle-based immunization generates memory antibody response and serotype-specific protective immunity.

polysaccharides are biochemically distinct and offer unique advantages to the encapsulated bacteria in their interaction with the host’s immune system. They are important components for bacterial virulence, and numerous vaccines composed of capsular polysaccharides are available in the clinic.14 Multivalent polysaccharide antigens induce multiple domains of highly cross-linked membrane (m)Ig, thereby promoting high levels of B-cell activation. Prolonged contact of the antigen with (m)Ig and subsequent persistent B-cell signaling are important for eliciting an anti-polysaccharide antibody response.15 This suggest that sustained-release particulate formulations may promote these (m)Ig polysaccharide interactions. Furthermore, nanoparticles (NPs) offer a higher contact surface area, thereby promoting multivalent interactions. NPs allow the antigen to be presented with different surface organization and epitope density, which are key attributes for generating robust IgG responses. There is ample evidence to suggest that soluble and partially aggregated antigens induce poor IgG responses in the absence of adjuvants, whereas highly organized antigens, such as bacterial or viral surface proteins and antigens in particulate form, can induce strong IgG responses under the same conditions.16 Moreover, the amount and molecular size of the capsular polysaccharide are directly related to the virulence of the encapsulated bacteria. This suggests that surface density and organization of capsular polysaccharides plays an important role in its interaction with the immune system. Encapsulated bacteria differentially express polysaccharides with varied thickness and organization. Bacteria expressing thicker capsular polysaccharides are more immunodominant, and those with the thinner capsule evade the immune recognition.17 Considering these factors, presenting polysaccharide antigens on biodegradable particles is potentially interesting, and it allows the possibility of presenting polysaccharides in the desired surface density and organization. Although capsular polysaccharide antigens are important components of commercial vaccines, they have inherent limitations because of their T-independent nature and are neither processed nor presented in the context of major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs).14,18 Recently, we reported that polymeric particle-based formulations can be used to improve the immunogenicity of anionic polysaccharides from Salmonella typhi.19 Although bacterial polysaccharides are generally anionic or without a net charge, a few, like pneumococcal capsular polysaccharide serotype-1 (PCP-1), are zwitterionic with repeating units of both positive and negative charges. The trisaccharide repeating unit of PCP-1 consists of carboxylic acids and amino groups that are crucial for its T-cell stimulatory properties.20 Although, like other zwitterionic polysaccharides (ZPS), PCP-1 interacts with MHC and promotes T-cell responses, it induces weak antibody responses and does not produce immunological memory and isotype switching. Chemical conjugation of PCP-1 to a carrier protein in current commercial vaccines addresses these limitations. This bioconjugation process compromises its zwitterionic nature, which is important for interaction with MHC and T-cells.21 Presenting PCP-1 entrapped in NPs would preserve the zwitterionic character, and it can be delivered in its unmodified natural form. To study the contribution of antigen density, organization, release pattern, and particle size on promoting antibody responses, the current study employed PCP-1 as the model polysaccharide antigen because of its T-cell activation properties and zwitterionic character. Entrapping PCP-1 in

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EXPERIMENTAL SECTION Expression and Purification of Pneumococcal Protein Antigens. For expression purposes, the construct were transformed into Escherichia coli expression strains SG13009 (Qiagen) for PsaA and PspA. Bacterial cultures were grown in Luria−Bertani medium containing ampicillin (100 μg/mL) and kanamycin (25 μg/mL). Isopropyl-β-D-thiogalactopyranoside (1 mM) was added to a mid-logarithmic-phase culture (A600 = 0.6) for 2 h to induce expression of recombinant PspA and PsaA. Bacteria were harvested by centrifugation at 4000g for 30 min and resuspended in lysis buffer (50 mM Tris buffer). Cell lysate preparation involved 10 sonication cycles of 20 s in duration with a 20 s time interval between pulses. The lysate was centrifuged at 20 000g for 30 min at 4 °C.The recombinant protein was purified from the supernatant using anion-exchange chromatography followed by Ni-NTA affinity chromatography as per the manufacturer’s instructions (Qiagen). Preparation of Poly(D,L-lactide) (PLA) Particles Entrapping Antigens. Preparation of poly(D,L-lactide) particles entrapping antigens employed a double-emulsion (w/o/w) solvent-evaporation method.22 Briefly, emulsification of the internal aqueous phase (IAP) containing PCP-1 (10 mg/mL) and 1% (v/v) Tween 20 to the organic phase (OP) (50 mg/mL PLA solution in dichloromethane) by sonication (20 W, 40% duty cycle, 20 cycles) (Bandelin probe Sonifier 450, USA) formed a primary emulsion (w/o). Dropwise addition of a primary emulsion to an external aqueous phase (EAP) containing 2% (w/v) PVP in deionized water and subsequent homogenization (10 000 rpm for 10 min Polytron, KINEMATICA, Switzerland) for MPs or sonication (20 W, 40% duty cycle, 20 cycles) resulted in a w/o/w double emulsion. Evaporation of dichloromethane from the final w/o/w double emulsion resulted in solidified particles. Nonentrapped PCP-1 was separated from particulate PCP-1 by washing and centrifuging the particle suspension. Centrifugation (24 336g, 20 min) followed by lyophilization produced free-flowing powder. The same process was carried out without adding antigen in IAP to produce dummy particles. Addition of either 50 μL of 6-coumarin dye (1 mg/mL in dichloromethane) to OP during primary emulsion step or FITC-BSA (10 mg/mL) in IAP yielded fluorescent particles. Both protein antigens (PspA or PsaA) and PCP-1 solubilized in the IAP produced particles co-entrapping both the antigens. IAP with different PCP-1/protein ratios yielded particles co-entrapping varying concentrations of PCP-1 and or PspA. Flow cytometry of the particles labeled with rabbit anti-PCP-1 and mouse anti-PspA polyclonal sera confirmed the presence of both antigens in the same polymeric matrix. A similar process with slight modifications generated particles with a higher surface density. Three different methods were explored for adsorbing PCP-1 on the surface of the particles: (a) incubation of preformed 923

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mixture of soluble PspA/PsaA and PCP-1 antigens in normal saline. Serum antibody responses analyzed by ELISA yielded both anti-PCP-1 and anti-PspA/PsaA antibody titers. To assess the effect of co-entrapment on improving the anti-PCP-1 immune response, we compared the formulations containing only PCP-1 and particles co-entrapping both PCP-1 and carrier proteins. PLA particles surface-coated with PCP-1 and protein antigens were evaluated using a similar protocol. Because these formulations entrapped protein and polysaccharides in the polymer core and dense polysaccharides on the surface, a physical mixture of soluble PCP-1 and protein antigens formed the control. We determined anti-PCP-1 antibody response by ELISA and compared the responses generated by particles with higher and lower surface-antigen densities. Evaluation of Anti-PCP-1 Memory Antibody Response upon Immunization with PCP-1 Loaded Particles. To evaluate the long-lasting memory effect of immunization with PCP-1-entrapped particles, we immunized mice using similar protocols as those given earlier. After 3 months of primary immunization, all groups received a fraction (1/5) of the primary dose. Serum antibody responses analyzed by ELISA yielded the secondary anti-PCP-1 antibody responses. All groups were compared for the generation of anti PCP-1 memory antibody responses. Challenge Studies Using Pathogenic Bacteria in Animals Immunized with PCP-1-Loaded Polymer Particles. To evaluate the protective role of anti-capsular PS antibodies, we challenged all immunized animals (each group contained n = 12 Balb/c mice) with pathogenic Streptococcus pneumoniae. All groups received immunizations as described earlier. We challenged the groups at different stages of immunization to reveal the immunoprotective effects of antibodies at each stage. When the primary antibody response subsided after 2 weeks of immunization, we boosted all mice (1 μg of PCP-1) via IM injection and challenged them with pathogenic S. pneumoniae. For the secondary stage, when the antibody response subsided after 3 months, we challenged all immunized mice. To neutralize the anti-phosphoryl choline antibodies, all mice received 100 μg of phosphorylcholine (PC) in 100 μL of saline 30 h before the pneumococcal challenge. One week after boosting, all groups were challenged intraperitoneally (IP) with 5 × 102 CFU pneumococci (serotype-1 (ATCC 6301); serotype 3 (ATCC 6303); serotype 2 (D39)). All animals were routinely observed in 12 h intervals for mortality, and the percentage survival was recorded as a sign of immunoprotection. In Vitro Phagocytic Uptake Studies Using Fluorescent Particles. In vitro phagocytic uptake studies were carried out in the murine macrophage cell line J774A.1 using 6-coumarinand FITC-BSA-labeled particles. Fluorescent particles were added (25 μL, 1 mg/mL) to 0.5 × 106 cells/3 mL of complete DMEM medium plated in sterile 6-well tissue culture plates (Falcon, Becton Dickinson, USA). The plates were incubated at 37 °C in an atmosphere of 5% CO2 for a defined time period. Cells were washed three times with sterile 50 mM PBS and analyzed by flow cytometry or microscopy. Confocal Laser Scanning Microscopy (CLSM). CLSM images were obtained by the simultaneous scanning of contrasting double-labeled specimens using a Zeiss confocal LSM 510 microscope equipped with an argon−krypton laser (Carl Zeiss Micro Imaging, Inc., USA). Macrophages were grown on coverslips inside sterile 6-well tissue culture grade plates and incubated with fluorescent NPs or MPs at 37 °C and

particles with PCP-1 at different temperatures, (b) addition of excess PCP-1 to EAP, and (c) direct spray-drying of the final w/o/w emulsion with an excess of PCP-1 to EAP. Anti-PCP-1 antibody labeling and zeta potential measurements confirmed the PCP-1 surface coating of the particles. Characterization of Size, Surface Morphology, Entrapment Efficiency, and in Vitro Release Profile of Particles. The size distribution of MPs and NPs was determined using Mastersizer Hydro 2000S particle-size analyzer and Zetasizer from Malvern (UK). Scanning electron microscopy (SEM) (JEOL, JSM 6100, Tokyo, Japan) was performed after coating the particle surface with gold−palladium over an aluminum stub, revealing the surface morphology of the particles. Transmission electron microscopy (TEM, CM 10, Philips, Holland) using AMT image-capture engine (version 5.42.391) after coating the particles with 1% uranyl acetate over a copper grid (Polysciences, USA) provided details on the internal structure of the particles. Zeta potential analysis using a Malvern Zetasizer reported the colloidal stability of the particles. Solubilizing accurately weighed particles in acetonitrile precipitated the encapsulated antigens and enabled estimation of the antigen content in the particles. A microBCA assay estimated the protein antigen content, and a colorimetric method based on a phenol sulphuric acid method yielded the PCP-1 content in the particles.23 The percent weight of antigen per unit weight of polymer represented the antigen load. Twenty milligrams of each formulation was suspended in 1 mL of 50 mM phosphate buffered saline (PBS) at pH 7.4 with 0.02% sodium azide in 1.5 mL microcentrifuge tubes. Then, samples were incubated in a shaker at 37 °C at 200 rpm to determine the in vitro antigen-release profile. Supernatants collected at different time intervals after centrifugation at 13 000 rpm for 10 min at room temperature were analyzed for antigen content. Fresh PBS was replenished to each pellet after every withdrawal, and incubation was continued over a period of 12 months. In Vivo Studies. To determine the immunogenicity of PCP-1-entrapped PLA particles, we immunized six female inbred Balb/c mice per group. Animals were maintained according to the guidelines of Animal Ethics Committee (IAEC) of the National Institute of Immunology (NII), New Delhi. The required dose of particles suspended in normal saline immediately before immunization comprised the final injected formulation. The final injected formulation contained only PCP-1 entrapped in particles and was free of nonentrapped antigen. As a control, mice received a single dose of soluble PCP-1 antigen (5 μg/animal). Through an intramuscular (IM) route, groups received an equivalent amount of PCP-1-entrapped MPs and NPs. Sera collected at different time intervals via the retro-orbital plexus were analyzed for serum antibody titers by ELISA.1 To neutralize anti-cell wall PS antibodies, all sera were preincubated with cell wall polysaccharides (Statens Serum Institute, Denmark) for 30 min prior to applying them to PCP-1-coated plates. Absorbance values at 490 nm at fixed dilutions based on the background absorbance from preimmunization sera are reported as the antiPCP-1 IgM and IgG antibody responses. Immunogenicity of PLA Particles Co-Entrapping PCP-1 and Pneumococcal Proteins. Immunization studies using particles co-entrapping PCP-1 antigen and PspA or PsaA were carried out as described before with slight modifications. All groups received an amount of particles equivalent to 5 μg of PCP-1 in normal saline. As a control, mice received a physical 924

dx.doi.org/10.1021/mp400589q | Mol. Pharmaceutics 2014, 11, 922−937

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Table 1. Details of the PCP-1 Particle Formulations Used for Immunization Studies formulations microparticles

nanoparticles

antigens PCP-1 PCP-1 PCP-1 PCP-1 PCP-1 PCP-1

+ PspA + PsaA + PspA + PsaA

PCP-1 load (μg/mg)

size distribution

entrapment efficiency (%)

2.21 1.87 1.54 2.14 1.74 1.4

2−6 μm 1.12−7.1 μm 2.4−6.7 μm 200−600 nm 212−380 nm 233−366 nm

49.23 ± 1 51.5 ± 0.5 56.55 ± 1 60 ± 1 58.2 ± 2.5 54.8 ± 1.5

Table 2. Formulation Components Used for Making Final PCP-1 Particle Formulations for the Immunization Studies formulations microparticles and nanoparticles

internal aqueous phase (IAP)

organic phase (OP)

external aqueous phase (EAP)

phase/volume ratio (OP/EAP)

1% w/v antigens, 10% w/v NaHCO3, and 0.1% v/v Tween 20

poly(D,L-lactide) 45 kDa 50 mg/mL in 4 mL of dichloromethane

1% w/v polyvinyl pyrrolidone (PVP) K30 in 16 mL of water

1:4

Figure 1. Electron microscopy images of polymeric particles entrapping PCP-1: (A) scanning electron microscope image of MPs and (B) transmission electron microscope image of NPs.

propidium iodide and analyzing cells that were propidium iodide-negative. A rat IgG2b anti-mouse CD16/CD32 monoclonal antibody was used to block Fc-mediated uptake. Phagocytic uptake studies were also carried out with fluorescent dummy particles in the presence and absence of PCP-1. Statistical Analysis. All immunization experiments were carried out three times. Antibody titers were determined by ELISA and expressed as the optical density at 490 nm (OD490nm) at fixed dilutions. Antibody titers of individual animals (n = 6) were estimated in duplicates, and their OD490nm values were expressed as a group mean. At any given time point, the comparisons for statistical significance among the group mean and standard deviation (SD) values were generated by one-way analysis of variance (one-way ANOVA) along with the Tukey−Kramer multiple comparisons post test using GraphPad InStat Software Inc. The post test is recommended when comparisons are made for groups ≥3 to compare pairs of group means. A Tukey−Kramer multiple comparisons post test was performed only if p < 0.05. All tests were performed at 95% confidence intervals.

5% CO2 for various amounts of time. At defined time points, cells were washed with 50 mM sterile PBS, labeled with 50 nM rhodaminephalloidin at 37 °C for 30 min, and washed again three times with 50 mM sterile PBS (pH 7.4). The coverslip was then placed on the stage of a confocal microscope. A representative cell was selected at random, and a series of optical sections (Z sections) was taken in dual-filter mode. Images captured in RITC, FITC, and dual mode were overlaid to determine localization and colocalization of fluorescent particles. Flow Cytometric Analysis of Fluorescent Particle Uptake. Flow cytometric acquisition of fluorescent particle uptake was performed using a BD-LSR flow cytometer (BD Biosciences, USA) with Cell Quest software. The data were analyzed using WinMDI 2.8 (Joseph Trotter, Scripps Institute, USA) or FlowJo 7.6.5. Macrophage cell suspensions (0.5 × 106 cells) preincubated with fluorescent NPs (25 μg) or MPs (25 μg) for various time points (at 37 °C, 5% CO2) were analyzed for the presence or absence of internalized fluorescent particles. Extracellular fluorescence was quenched using an appropriate dye (0.1% (w/v) crystal violet or 0.4% (w/v) trypan blue). Dead cells were excluded in the analysis by staining with 925

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RESULTS AND DISCUSSION Preparation of Polylactide (PLA) Particles Entrapping Pneumococcal Antigens. A modified double-emulsion (w/ o/w) solvent-evaporation method yielded polymeric particles entrapping pneumococcal antigens.22 To study the effect of particle size variations on the immune response, we prepared both MPs and NPs. Formulation parameters, such as phase volume ratio, polymer concentration, and surfactant concentration in external aqueous phase (EAP), and process parameters, such as homogenization speed and sonication energy, were systematically optimized to obtain particles of the desired size distribution, entrapment efficiency, and antigenrelease rate. MPs prepared using the optimized parameters showed a volume-average diameter (Vd) of 2−6 μm, NPs showed a 200−600 nm hydrodynamic size, and they both showed a good entrapment efficiency (MPs = 49.6% and NPs = 60%, Tables 1 and 2) and smooth surface morphology (Figure 1). Optimizing the formulation parameters yielded an antigen load of approximately 2 μg of PCP-1 per milligram of particles. Using a higher amount of PCP-1 in the IAP resulted in a higher antigen loading per milligram of particles but a lower entrapment efficiency. We also prepared formulations loaded with both protein and polysaccharide antigens using the purified carrier proteins PspA or PsaA along with PCP-1. Both recombinant PspA and PsaA were purified to homogeneity from E. coli cell lysate supernatants using anion-exchange and Ni-NTA affinity chromatography. SDS-PAGE analysis confirmed the homogeneity of the purified proteins, and the results are shown in the Supporting Information (Figure S1 for PspA and Figure S2 for PsaA). Co-entrapping protein and PCP-1 in the same particle did not result in significant changes in the particle-size distribution (PSD). The entrapment efficiency was in the range observed with MPs entrapping only PCP-1 (PCP-1−PspA MPs = 51.5 ± 0.5% and PCP-1 MPs = 49.23 ± 1%) and NPs (PCP-1−PspA NPs = 58.2 ± 2.5% and PCP-1 NPs = 60 ± 1%) (Table 1). The same amount of PCP-1 was used in IAP in both cases. The presence of protein antigen in IAP did not reduce the loading of PCP-1 compared to formulations containing only PCP-1. Proteins components in IAP can stabilize the primary emulsion and promote encapsulation efficiency. This may explain the higher PCP-1 loading observed in co-entrapped MPs compared to MPs containing only PCP-1. Flow cytometry after immunolabeling the particles with the respective antibodies confirmed the presence of both protein and PCP-1 antigens in the same polymeric particle. Although the percentage of particles co-entrapping both antigens varied between 70 and 55% from batch to batch, the overall mean was about 60 ± 2% (Supporting Information Figure S3), with 90 ± 0.5% of particles showing immunolabeling for protein antigens and 60 ± 2% of particles showing the presence of polysaccharide antigens, suggesting the preferential entrapment of protein antigens (Supporting Information Figure S3). We employed three different formulation strategies to enhance the surface density of the polysaccharide antigens on the particles: (1) adding a large excess (10-fold more than that used in IAP) of PCP-1 antigen to the EAP of the w/o/w emulsion before particle solidification, (2) physical adsorption to already solidified particles, and (3) spray-drying a physical mixture of prepared particles and polysaccharide antigens. Adding a higher excess to the EAP and spray-drying were the most efficient for producing particles with higher polysaccharide surface densities

(Table 3). Adding a higher excess of PCP-1 to the EAP enabled slow adsorption and incorporation of polysaccharide to the Table 3. Formulation Details of Microparticles Prepared with Higher Surface Antigen Densities formulation

method

PCP-1-PspA co-encapsulated microparticles with surface-coated PCP-1

overnight surface adsorption to MPs addition of PCP-1 to EAP spray-drying W/O/W emulsion with polysaccharide in EAP

size distribution (μm)

antigen load (μg/mg)

2−6

2.215

2−6

8.69

2−6

9.29

solidifying polymeric matrix. We used the first method for making the final formulations for the immunization studies because of the convenience of working on the milligram scale. Flow cytometry after immunolabeling with rabbit anti-PCP-1 polyclonal sera revealed a higher PCP-1 density on the surface of these particles compared to particles made by conventional methods. Particles with a higher surface density showed higher antigen-associated fluorescence (Supporting Information Figure S4). The increase in the mean fluorescence intensity (MFI) and antigen load, which correspond to PCP-1 surface attachment, showed a correlation with the concentration of PCP-1 in the EAP. A higher amount PCP-1 in the EAP resulted in a higher adsorption that reached saturation at very high concentrations (Figure 2). Changes in the zeta potential of these particles at different pH’s further confirmed the higher PCP-1 surface density relative to particles made by conventional methods. Particles with a higher PCP-1 density showed higher positive charges under acidic pH because of the protonation of amino groups of PCP-1 (Figure 2). Compared to the standard MPs prepared with PCP-1 only in IAP, these particles showed a higher PCP-1 load per milligram of particles (Table 3). This further confirmed the higher surface density earlier observed with the zeta potential and flow cytometry studies. Anti-Polysaccharide Antibody Responses from Polymer Particles Entrapping PCP-1. To generate antibody responses against PCP-1, we immunized 6−8 week old Balb/c mice with particles equivalent to 5 μg of PCP-1. All groups received a boosting dose of 1 μg of soluble PCP-1 on day 90. (Details of the immunization protocol used in the study are shown in Supporting Information Figure S5.) Figure 3 shows the anti-PCP-1 IgG responses evaluated by ELISA. The results indicate that immunizing with PCP-1-entrapped MPs and NPs elicited significantly higher antibody responses in comparison with that produced in response to soluble PCP-1. Presenting PCP-1 in particulate form enhanced both anti-PCP-1 IgM (data not shown) as well as IgG responses. For IgM, a heightened antibody response was observed only at the primary immunization stage. No significant IgM responses were observed on day 90, so comparison of the formulations was difficult, whereas significantly higher IgG responses elicited by PCP-1-loaded particles compared to soluble PCP-1 were observed after boosting the animals with soluble antigens. This effect was prominent at the secondary immune response stage (mean peak OD490nm at day 97: soluble PCP-1 = 0.181 ± 0.007, PCP-1 MPs = 0.365 ± 0.085, and PCP-1 NPs = 0.49 ± 0.032, p < 0.001). This enhanced memory antibody response elicited by particle-based immunizations is very important considering the inability to augment anti-polysaccharide 926

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Figure 2. (A) Graphical representation of increasing antigen density on the surface of PCP-1 on MPs by slow adsorption of PCP-1 from the external aqueous phase (EAP) of the w/o/w emulsion. PCP-1-associated fluorescence in particles, measured by flow cytometry and represented as the mean fluorescent intensity (MFI), and the antigen load per milligram of MPs are plotted against the concentration of PCP-1 in the EAP. Error bars represent the mean ± standard deviation. (B) Changes in the zeta potential with respect to pH for MPs with different surface densities of PCP-1.

presented on NPs elicited significantly higher IgG responses than PCP-1-entrapped MPs (mean peak OD490nm at day 97: PCP-1 MPs = 0.365 ± 0.085 and PCP-1 NPs = 0.49 ± 0.032, p < 0.001). This observation is in contrast to what has been reported earlier using protein-loaded particles.1,5,22,25 In the case of proteins that are T-cell-dependent antigens, MPs elicit significantly higher antibody responses compared to antigens entrapped in NPs.1,22,26,27 The general assumption is that NPs generate weak IgG responses. Contrary to this, the current results show that PCP-1 entrapped in NPs elicited significantly higher antibody responses. The higher response elicited by particulate formulations can be attributed to many factors such as efficient and prolonged priming induced by antigens immobilized on polymer particles. Compared to soluble antigens, particulate antigens efficiently present cognate ligands to cells of the immune system and evoke robust responses.28,29 This was also evident in the current study, where both MPs and NPs elicited higher IgG responses than soluble PCP-1. In addition, the sustained-release profile of the antigens from particulate formulations may have also contributed to the effective priming of the immune system. The sustained release of antigens ensures prolonged exposure of the antigen to B-cells and allows for re-exposure in the draining lymph node. This reexposure induces effective priming and prolongs the survival of polysaccharide-specific B-cells. The higher and sustained secondary antibody responses elicited by PCP-1 particles compared to other formulations can be attributed to this effect. Immunizations with a physical mixture of blank particles and soluble PCP-1 further confirmed this. The animals immunized with a physical mixture of soluble PCP-1 and blank particles elicited low-anti-PCP-1 IgG responses compared to those immunized with PCP-1 particles. In particular, memory IgG response associated with PCP-1-loaded particles were not observed (data not shown). The differences in the IgG response elicited by NPs and MPs can be due to the fundamental differences involved in their interactions with cells of the immune system. Many groups have reported that APCs phagocytose antigen-entrapped NPs much more efficiently than microparticles.1,7,30,31 In vivo, micrometer-sized particles are preferentially processed by macrophages, whereas NPs in the size range of 100 nm are processed by both dendritic cells (DCs) and macrophages.29 Earlier reports of in vivo trafficking studies with NPs showed their targeting to different sets of DCs.32 Moreover, NPs and MPs have different intracellular

Figure 3. Comparison of anti-PCP-1 IgG responses elicited upon immunization of mice with different PCP-1 formulations. Balb/c mice in different groups (n = 6) were immunized with the indicated formulations, and all groups were boosted on day 90 with 1/5 of the priming dose of soluble PCP-1. Antibody levels are represented as OD490nm at 400× serum dilution (the dilution at which OD490nm is 3× the standard deviation of the mean pre-immune OD values). Error bars represent the mean ± standard error. (Mean peak OD490nm at day 97: soluble PCP-1 = 0.181 vs PCP-1 MPs = 0.365, p < 0.001 and soluble PCP-1 = 0.181 vs PCP-1 NPs = 0.49, p < 0.001. ) ***, p < 0.001. Results are representative of three independent experiments with similar trends.

antibody responses by conventional adjuvants such as alum.24 In general, unconjugated polysaccharide antigens induce limited memory antibody response upon boosting. The observed enhancement in memory antibody response suggests that presentation of polysaccharide antigens on particles efficiently primes the immune system and promotes robust antibody responses. We observed this effect mainly at the secondary immunization stage. Particle-based immunization also promoted isotype switching. Anti-polysaccharide IgG responses are protective because of their higher opsonophagocytic potential and because maternal IgG transfers better across the placenta to neonates. Therefore, inducing a long-lasting IgG response against polysaccharide antigens is the main objective of carbohydrate-based vaccines. In light of this, the current results are very important in the context of capsular polysaccharide-based vaccinations. The IgG response shown in Figure 3 indicates size-dependent differences. PCP-1 927

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Figure 4. Comparison of anti-PCP-1 IgG responses elicited by immunizing animals with PCP-1 and protein co-entrapped formulations. Balb/c mice in different groups (n = 6) were immunized with the indicated formulations, and all groups were boosted on day 90 with 1/5 of the priming dose of soluble PCP-1. (A, B) Comparison of antibody responses from co-entrapped NP formulations (PCP-1−PspA NPs and PCP-1−PsaA NPs) with NPs entrapping only PCP-1 (PCP-1 NPs). (C, D) Comparison of antibody responses from co-entrapped MP formulations (PCP-1−PspA MPs and PCP1−PsaA MPs) with MPs entrapping only PCP-1 (PCP-1 MPs). Antibody levels were represented as OD490nm at 400× serum dilution (dilution at which OD490nm is 3× the standard deviation of the mean pre-immune OD values). Error bars represent the mean ± standard error. ***, p < 0.001; **, p < 0.01; and ##, p > 0.05. (Mean peak OD490nm at day 97 or day 7 of respective co-entrapped formulations was compared to the mean peak OD490 nm of PCP-1 only loaded formulations.)

particles presents a repetitive array of epitopes to B-cells and ensures prolonged contact of the antigen with (m)Ig. Thus, particles may promote cross-linking of BCRs and persistent Bcell signaling. The higher surface area associated with the lower size of NPs would improve this cross-linking and prolonged contact. Moreover, among polysaccharide antigens, PCP-1 is an exception because it is zwitterionic and is processed and presented on MHC to PCP-1-specific T-cells.35 The efficient uptake of smaller size NPs may promote the delivery of PCP-1 into MHC-loading compartments. This also favors generating efficient immune responses to PCP-1. T-cells in draining lymph nodes can promote an anti-polysaccharide antibody response without involving an MHC−T-cell receptor interaction through ancillary help induced by cytokines. Specifically, IFN-γsecreting cells promote T-independent antibody responses.36 Antigen-entrapped NPs and MPs promote differential expression of cytokines upon interaction with cells of the immune system, and this may explain the size-dependent differences in the immune responses that they elicit.1 Additional studies were performed to confirm the contribution of these factors further, and the results are described in later sections. Anti-Polysaccharide Antibody Responses from Polylactide Particles Co-Entrapping PCP-1 and Recombinant Pneumococcal Proteins. The surface proteins of encapsulated bacteria play a major role in modulating responses against

fates, and their uptake mechanisms vary in a size-dependent manner.33,34 Clathrin-mediated endocytosis is used to engulf the NPs, whereas MPs rely on caveolae-mediated internalization.34 These fundamental differences in the size-dependent interaction of the particles with the immune system also influence the fate of the entrapped antigens. It is welldocumented that antigens entrapped in NPs elicit lower antibody responses compared to MPs. However, the superior in vitro and in vivo attributes of NPs did not translate to vaccinedelivery applications, as indicated by the lower antibody responses elicited against entrapped proteins.1,27 The results of the current study suggest that NPs could be an optimal platform for delivering polysaccharide antigens. With their lower geometric size, NPs also offer higher surface area/volume ratios. The higher surface area per particle would be an ideal characteristic for presenting immunological ligands that mainly depend on multivalent interactions and subsequent crosslinking of receptors. Compared to protein antigens, which depend heavily on antigen processing and presentation, most of polysaccharide antigens, including PCP-1, are large multivalent macromolecules that predominantly cross-link with B-cell receptors (BCRs) to promote antibody responses.14,15 This multivalency enables them to induce multiple domains of highly cross-linked membrane (m)Ig, which has been shown to promote high levels of B-cell activation.15 Entrapping PCP-1 in 928

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antigens. So, in glycoconjugate vaccines, the ratio of carrier protein and polysaccharide antigen is a crucial parameter that drives anti-polysaccharide antibody responses.38 Moreover, some of the capsular polysaccharides are antiphagocytic, and their presence in the same particle may hamper the antibody response against the protein antigen.40 We have recently shown that co-delivering tetanus toxoid with Vi capsular polysaccharide dramatically lowered the antibody response against the carrier protein.19 To recruit CD-4 T-cell help for the polysaccharide antigen, a robust immune response against the co-delivered protein is inevitable. To evaluate the role of the polysaccharide antigen in regulating the response against codelivered protein antigens, we prepared co-entrapped formulations with a different carrier protein/PCP-1 ratio, immunized the mice, and analyzed the anti-PspA antibody responses. We prepared particles with different PCP-1/PspA ratios by changing the amount of PspA in IAP while keeping the amount of PCP-1 the same. The anti-PspA antibody response elicited by PspA only loaded MPs was compared to particles containing PspA and PCP-1. Varying PspA in IAP resulted in MPs with different PspA content but did not significantly change the PCP-1 loading. All co-entrapped formulations showed comparable PCP-1 loading per milligram of particles. The anti-PspA IgG responses in Figure 5 show that

polysaccharide antigens. To mimic the antigen organization and arrangement found on the pathogen, we formulated and tested particles containing pneumococcal protein antigens (PspA and PsaA) and PCP-1. The differences observed between PCP-1 particles and earlier reports on protein-loaded particles could be due to formulation or immunological differences. By loading both protein and polysaccharide antigens in the same polymeric matrix, we can study the effect of particle size on the antibody response to each antigen. This may delineate whether the observed differences in the antibody response between protein and polysaccharide antigens are due to formulation differences. Furthermore, protein and polysaccharide co-entrapped formulations mimic the covalent linkage of polysaccharide− protein in glycoconjugate vaccines. In a glycoconjugate vaccine, the carrier proteins recruit CD4 T-cell help for the polysaccharide antigen, which enhances the immunogenicity of the polysaccharide, induces immunological memory, and promotes isotype switching.14 Co-entrapping protein and PCP1 antigen in the same matrix could recruit CD-4 T-cell help against polysaccharide antigens. In the context of carbohydrate vaccines, this would be a great improvement in solving the limitations of bioconjugation-based processes. This would also help to recruit T-cell help for PCP-1, as in the case of classical conjugate vaccines, without chemically modifying the structure. The results from these experiments will give valuable mechanistic insights into the immunobiology of glycoconjugate vaccines. PspA and PsaA are two pneumococcal surface antigens in advanced stages of vaccine development as protein-based vaccines against S. pneumoniae infections. The findings of the current study may facilitate the development of combination vaccines against S. pneumoniae. The anti-PCP-1 IgG responses shown in Figure 4 indicate that co-entrapping a carrier protein along with PCP-1 does not improve the IgG responses. This effect was observed irrespective of the entrapped carrier proteins used or the size distribution of the formulations. NPs loaded with PspA and PCP-1 elicited lower antibody responses than NPs containing only PCP-1 (mean peak OD490nm at day 97: PCP-1 NPs = 0.49 ± 0.032 and PCP1−PspA NPs = 0.350 ± 0.043, p < 0.001). MPs also showed the same trend (PCP-1 MPs = 0.365 ± 0.085 and PCP-1−PspA MPs = 0.379 ± 0.03, p > 0.05). This trend was also true in the case of PCP-1−PsaA particles. (PCP-1 MPs = 0.365 ± 0.085 and PCP-1−PsaA MPs = 0.29 ± 0.093, p < 0.01, PCP-1 NPs = 0.49 ± 0.032 and PCP-1−PsaA NPs = 0.39 ± 0.042, p < 0.001). Although the general trend in which the particulate formulations exhibited higher IgG responses than soluble PCP-1-based immunizations was reproduced, the presence of an additional carrier protein in the same polymeric matrix did not provide any benefit. Co-entrapped formulations reproduced the higher IgG response elicited by PCP-1-entrapped NPs compared to MPs. Even in the presence of protein antigens, anti-PCP-1 responses showed a size-dependent enhancement, suggesting the importance of nanosized formulations for polysaccharide antigens and confirming the results observed earlier. However, NPs co-entrapping PCP-1 and carrier proteins showed lower IgG responses than NPs entrapping only PCP-1. These lower IgG responses may be due to carriermediated suppression induced by the co-entrapped proteins.37−39 Because protein antigens are more immunogenic than carbohydrates, they elicit stronger responses and can suppress anti-polysaccharide antibody responses. Upon codelivery of carbohydrates and proteins, the immune system reacts better to proteins and often neglects the polysaccharide

Figure 5. Comparison of anti-PspA IgG responses elicited from animals immunized with PCP-1 and protein co-entrapped formulations. Balb/c mice in different groups (n = 6) were immunized with the indicated formulations, and all groups were boosted on day 90 with 1 /5 of the priming dose of soluble PspA. Antibody levels were represented as OD490nm at 1000× serum dilution (dilution at which OD490nm is 3× the standard deviation of the mean pre-immune OD values). Error bars represent the mean ± standard error. All groups except for the soluble PspA immunized group showed a significantly higher anti-PspA response than they did at the pre-immunization stage. (Mean peak OD490nm at day 30 of all formulations was compared to the mean peak OD490nm at day 0.) ***, p < 0.001.

compared to formulations entrapping only PspA the anti-PspA responses from co-entrapped formulations are not significantly affected by the presence of PCP-1. Only at very high PspA/ PCP-1 ratios we did observe a decrease in the IgG responses. This may be due to a low PspA loading in these formulations. The antigen load per particle is an important parameter driving antiprotein antibody responses to microparticulate formulations.22 This suggested that the presence of PCP-1 in the particles did not affect the immune response against the carrier proteins but the stronger IgG responses against the proteins would have resulted in the carrier-mediated suppression of responses against the polysaccharide antigens. It is interesting 929

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Figure 6. Comparison of anti-PCP-1 IgG responses elicited from animals immunized with MPs with a higher PCP-1 surface density. Balb/c mice in different groups (n = 6) were immunized with the indicated formulations, and all groups were boosted on day 90 with 1/5 of the priming dose of soluble PCP-1. (A) Antibody responses from mice immunized with PsaA−PCP MPs in comparison to other formulations. (B) Antibody responses from mice immunized with PsaA−PCP MPs with other formulations. Antibody levels were represented as OD490nm at 400× serum dilution (the dilution at which the OD490nm is 3× the standard deviation of the mean pre-immune OD values). Error bars represent the mean ± standard error. ***, p < 0.001; ##, p > 0.05. (Mean peak OD490nm at day 97 or day 7 of each PCP-1 high surface density formulation was compared to the mean peak OD490nm of PCP-1 only loaded formulations.) The results are representative of two independent experiments for each carrier protein formulation with similar trends.

tested MPs with higher surface densities of PCP-1. We compared higher antigen-density formulations to MPs with a lower density formulation and PCP-1 NPs. All groups received the same PCP-1 dose, but the antigen loading per particle varied. To standardize this, we normalized the amount particles immunized in each group to 5 μg of PCP-1. NPs with a higher PCP-1 surface density aggregated upon lyophilization, leading to an increase in their size distribution. Therefore, this was not used for the immunization experiments. The anti-PCP-1 IgG responses shown in Figure 6 indicate that MPs with a higher surface density of PCP-1 elicit higher antibody titers than standard PCP-1 NPs and PCP-1 MPs. In this study, we used the MPs co-entrapping carrier proteins, which earlier showed insignificant antibody responses, because of carrier-mediated suppression. The lower immunogenicity observed earlier could be reversed by using particles with a higher surface density. Both of the formulations, irrespective of the carrier proteins coentrapped with PCP-1, showed this effect (mean peak OD490nm at day 97: PCP-1 MPs = 0.365, PCP-1 NPs = 0.49, PCP-1− PspA MPs with a higher surface density =0.853, and PCP-1− PsaA MPs with a higher surface density = 0.821, p < 0.001). This was true with MPs with a higher surface density without carrier proteins (data not shown). The higher surface density of antigen would have promoted multivalent interactions of the polysaccharide antigens with BCRs and resulted in efficient priming, leading to higher IgG responses. The results confirmed the role of surface antigen density and contact surface area for improving the immunogenicity of a polysaccharide antigen. Functional Evaluation of the Opsonophagocytic Role of Anti-PCP-1 Antibodies Generated by PCP-1-Loaded Polymer Particles. Host defense against encapsulated bacteria, such as S. pneumoniae, depends on the presence of opsonic antibodies specific for capsular polysaccharide antigens, and it is the basis of polysaccharide vaccines that offer protection against S. pneumoniae infections. Opsonophagocytosis is the primary mechanism for clearance of pneumococci from the host, and the measurement of opsonophagocytic antibodies appears to correlate with vaccine-induced protection.42 Immunization studies showed that PCP-1 particles

to note that upon immunization with co-entrapped formulations protein antigens elicit stronger antibody responses when delivered in MPs, whereas polysaccharide antigens show this effect when delivered in NPs. This suggests that the sizedependent enhancements of the antibody responses relates to its immunological nature and differs in its interaction with the immune system. The results confirmed the size-dependent enhancement of antibody responses elicited by particles entrapping PCP-1. The results also indicated that the coentrapment of carrier protein and polysaccharide antigen may not be sufficient to promote anti-polysaccharide antibody responses. In the context of glycoconjugate vaccines, this is a very important finding, and so far, very few studies have explored the co-delivery of carrier protein and polysaccharide antigens. These findings indicate that the covalent linkage of polysaccharide and carrier proteins would inevitably recruit CD4 T-cell help for polysaccharide antigens. Covalent linkage of polysaccharides to T-cell epitopes was recently shown to promote their presentation to cognate T-cells. This was suggested to be an alternate mechanism involved in the immunological efficacy of glycoconjugate vaccines.18 Anti-Polysaccharide Antibody Responses from Immunizations Using Microparticles with a Higher Surface Density of PCP-1. The higher IgG responses observed with particulate formulations, particularly the higher antibody responses demonstrated by the NP formulation, can be attributed to the higher surface area/volume ratios of the NP formulations. During the w/o/w double-emulsion method for preparing the particles, part of the emulsified hydrophilic antigens in IAP diffuses into the EAP because of the osmotic gradient, and this gradually adsorbs to the surface.29 The surface-adsorbed antigens get trapped in the outer PVA layer upon final lyophilization. Thus, a polymer particle mimics the natural particulate pathogen surrounded by a capsule made of polysaccharide antigens. Antigen surface organizations and geometric size are two important pathogen-associated features that the immune system uses as cues for activating effector functions.41 Because of their higher surface area, NPs may offer better cross-linking of BCRs, thus leading to the effective priming of B-cells. To test this hypothesis, we prepared and 930

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Figure 7. Comparison of the opsonophagocytic activities (OPA) of anti-PCP-1Abs elicited from animals immunized with different PCP-1 formulations. Flow cytometric analysis of phagocytic uptake of fluorescent bacteria by macrophages. Bacteria preopsonized with hyperimmune sera collected from immunized groups were used for the uptake experiments. FL1-H on the x axis of the histogram represents the fluorescence resulting from the phagocytosis of FITC-labeled bacteria. The blue curve on the left showed the highest bacteria-associated MFI, indicating higher OPA. The bar graph represents the percentage of cells that internalized bacteria. The percentage cells were significantly higher in samples incubated with serum of PCP-NPs-immunized groups compared to serum from mice immunized with PCP-MPs and soluble PCP-1 as well as normal control sera (the difference of PCP-NPs vs PCP-MPs, soluble PCP-1, and normal serum is statistically significant; ***, p < 0.001). Error bars represent the mean ± standard error.

induced strong anti-PCP-1 IgG responses. However, ELISA titers may not adequately reflect the presence of functional antibodies capable of triggering leukocyte effector functions. Determining serum opsonins that facilitate phagocytic killing is a superior read out for evaluating in vivo protective efficacy against pneumococcal infections.42 Therefore, we tested the opsonophagocytic activity of anti-PCP-1 antibodies generated by immunization using different polymeric formulations. S. pneumoniae serotype-1 was fluorescent-labeled with fluorescein isothiocyanate (FITC). Incubating fluorescent-labeled bacteria with murine macrophages in the presence of anti-PCP-1 serum from immunized mice yielded the opsonophagocytic activity. Murine cell line J774A.1 was the source of the macrophages. We employed sera from nonimmunized mice and mice immunized with soluble PCP-1 as controls. After the incubation, we isolated the cells by centrifugation and removed the unbound bacteria by washing with 1% BSA in PBS. Flow cytometry on isolated cells yielded the intracellular fluorescence associated with macrophages. Treating cells with 1% trypan blue prior to flow cytometric analysis quenched the extracellular fluorescence associated with surface-bound bacteria. FITC fluorescence was measured on the FITC (FL-1) channel of a BD-LSR flow cytometer. Later, fluorescence microscopy confirmed the correlation between fluorescence with phagocytosis (Supporting Information Figure S6). The results, shown in Figure 7, indicate a higher opsonophagocytosis induced by serum from PCP-1 NPs-immunized mice compared to other sera. The higher mean fluorescence intensity and number of FITC-positive cells confirmed the higher opsonophagocytosis potential of serum from PCP-1-NPsimmunized mice. The results suggested that the antibodies elicited by particulate formulations were specific to PCP-1 and

that particulate formulations are functionally competent for promoting the clearance of bacteria in case of a possible infection. These results support the vaccine-delivery potential of polylactide particles in augmenting antibody responses against polysaccharide antigens. Challenge Studies to Evaluate the Immunoprotective Effects of Primary Antibodies Elicited by Immunization with PCP-1-Entrapped Polymer Particles. To evaluate the immunoprotective function of antibodies elicited by PCP-1loaded polymer particles, we challenged immunized groups with virulent S. pneumoniae serotype-1. We immunized BALB/c mice in different groups (n = 12) with PCP-1-loaded particles as described earlier. For PCP-1 MPs, particles with a higher PCP-1 surface density were prepared and used in the challenge studies. Earlier, this formulation elicited strong anti-PCP-1 IgG responses. Mice immunized with normal saline and blank particles formed the control groups. ELISA on sera at different time points determined the antibody responses. To evaluate stage-specific protection, we challenged all groups separately at both the primary and secondary immune response stages. The primary antibody response subsided after 2 weeks of immunization. We boosted the mice with 1 μg of soluble PCP-1 and then challenged the mice at this stage. After 1 week of boosting, animals were challenged IP with 5 × 102 CFU pneumococci (the detailed immunization protocol is described in Supporting Information Figure S7). The survival percentage of infected animals was recorded as the degree of the immunoprotective effect. All animals immunized with PCP-1loaded NPs survived better than mice immunized with soluble PCP-1 (Figure 8). PCP-1-entrapped MPs and co-entrapped formulations showed a lower survival index and therefore a lower immunoprotection than mice immunized with PCP-1 931

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Challenge Studies to Evaluate the Immunoprotective Effects of Memory Antibodies Elicited by Immunization with PCP-1-Entrapped Polymer Particles. Immunological memory is the hallmark of vaccine-mediated protection. The results from earlier experiments proved that polysaccharide antigens entrapped in polymeric particles enhance memory antibody responses. To evaluate the long-term immunoprotection conferred by PCP-1-loaded particles, we challenged immunized animals at the secondary antibody response stage (more than 3 months after primary immunization). BALB/c mice received particles equivalent to 5 μg of PCP-1. Groups immunized with soluble PCP-1 and normal saline formed the control groups. Three months after immunization, all animal groups were boosted with 1/5 of the priming dose of soluble PCP-1. One week after boosting at the time when the secondary antibody response peaked, all mice were challenged IP with 5 × 102 CFU of pneumococci. (Detailed protocols of the challenge experiment are described in Supporting Information Figure S8.) The survival plot in Figure 9A indicates that all immunized animals survived better than unimmunized animals, suggesting the importance of the antibody response in protection. All immunized animal showed complete survival in this experiment, but groups immunized with soluble PCP-1 showed symptoms of infections, like higher body temperature, loss of weight, and abstinence from food, and recovered over a period of 2 weeks. A few animals did not survive the infection, and the others that survived were accounted for in the survival counts. The recovery from infection in soluble PCP-1-immunized animals could be due to the secondary antibody response elicited by challenging with a live particulate antigen (S. pneumoniae). No such symptoms were observed in animals immunized with PCP-1 particulate formulations. To check the quality of the memory antibody response and the single-dose vaccination potential of PCP-1entrapped particles, we challenged all immunized groups on day 90 without boosting (Figure 9B). Groups immunized with PCP-1-entrapped particles protected the mice from a lethal challenge even without booster immunizations, whereas groups immunized with soluble PCP-1 showed relatively lower survival rates (Figure 9B). The sustained release of antigen from the particles would have presented antigens slowly and repeatedly

Figure 8. Percentage survival plots from challenge studies. Balb/c mice in different groups (n = 12) were immunized with the indicated formulations, and all groups were challenged on day 14 with live S. pneumoniae serotype-1. Groups immunized with PCP-1-entrapped polymer particles were protected upon challenge at the primary immune response stage. PCP-1 NPs-immunized mice were protected significantly better than saline- or PCP−PspA MPs-immunized mice. ***, p < 0.001. The results are representative of three independent experiments with similar trends.

NPs. However, the percentage of survival was higher in all immunized groups in comparison to that observed with the saline-immunized group. The particle-size-dependent antibody responses elicited by PCP-1-entrapped particles were also reflected in the immunoprotective effects. This indicated that immunization with PCP-1 entrapped in NPs elicits stronger anti-PCP-1 responses that help in clearing the pathogenic pneumococci. This also correlated with the higher antibody responses generated by PCP-1 NPs. Thus, the anti-PCP-1 antibody responses generated by PCP-1 NPs are functionally competent and help in fighting a postvaccination infection. Although PCP-1 MPs with a higher surface density elicited stronger anti-PCP-1 IgG responses, they showed a lower protection than PCP-1 NPs. This may be due to nonspecific serum-independent protective effects of NPs. NPs can induce secretion of cytokines such as IFN-γ that promote protection against pneumococci.

Figure 9. Results of immunoprotection studies at the secondary immune response stage. Balb/c mice in different groups (n = 12) were immunized with the indicated formulations, and all groups were challenged on day 97 with live S. pneumoniae serotype-1. (A) Survival plots after challenging all animals with live S. pneumoniae on day 97 after boosting with soluble PCP-1 on day 90. No significant differences in survival rates were observed between mice given the different formulations. (B) Survival plots after challenging all animals with live S. pneumoniae on day 97 without boosting on day 90. No significant differences in survival rates were observed between mice given the different formulations. 932

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Figure 10. Immunization with PCP-1-entrapped polymer particles elicited a serotype-specific immunoprotective antibody response. (A) Survival plots comparing the percentage of survival of groups immunized with blank particles vs PCP-1-entrapped polymer particles. Immunized groups were challenged with live S. pneumoniae on day 97 without boosting on day 90. Groups immunized with PCP-1-loaded particles showed significantly higher survival rates than groups immunized with unloaded blank particles (***, p < 0.001). Each group contained n = 12 Balb/c mice. (B) Plots comparing the survival rates of immunized groups challenged with nonrelated serotypes (ATCC 6303 and D39) vs S. pneumoniae serotype-1 (ATCC 6301). All groups were immunized with PCP-1 NPs and challenged on day 14 with different serotypes. Groups immunized with soluble PCP-1 formed the positive control, and groups immunized with normal saline were included as negative controls. Immunized groups challenged with ATCC 6301 showed a significantly higher survival rate than that challenged with D39 or ATCC 6303, indicating protection against the PCP-1specific serotype (***, p < 0.001). Each group contained n = 12 Balb/c mice.

to the immune system. This mimics the classical boost−prime regimen of vaccinations. Therefore, with a single-dose administration, PCP-1-entrapped polymer particles could elicit protective antibody responses. It is also interesting to note in contrast to the primary immune response stage all animals immunized with polymer particle-entrapped PCP-1 showed better survival rates upon challenge with S. pneumoniae on day 90. This was true for both MPs and PCP-1−PspA MPs, which showed lower antibody responses than PCP-1 NPs. This indicated that immunization with polymer particle-entrapped PCP-1 induced immunological memory. When compared to soluble PCP-1 immunizations, vaccination with particulate formulations induced strong immunological memory, and those animals efficiently cleared the pathogen (Figure 9B). We did not observe significant differences in the survival rates among various groups immunized with particles, although they showed distinct differences in antibody levels. This may be due to either nonspecific immunomodulatory roles of particles or limitations of the current infection model to elucidate the differences. Infection experiments with varying challenge doses of bacteria also did not reveal conclusive results. S. pneumoniae serotype 1 is a very virulent microbe, and titrating the lethal dose given through a systemic route to control the infection is challenging. Instillation through alternate routes, such as intranasal, is important to have better control of the infection. Infection experiments are currently underway to address this issue. To rule out the contribution of antigen-independent immunomodulation of particles to the observed protection, we immunized the mice with dummy particles and challenged these mice with virulent bacteria. The very low survival rates observed in mice immunized with blank NPs and MPs confirmed the specificity of the anti-PCP-1 antibody response (Figure 10A). The results indicated the role of anti-PCP-1 antibodies in immunoprotection and ruled out the possibility of protection resulting from nonspecific effects (Figure 10A). To confirm the antibody-dependent immunoprotection, we challenged all immunized groups with pneumococci from nonrelated serotypes such as ATCC-6303 and D39. The results in Figure 10B showed low survival rates in mice challenged with

nonspecific serotypes. The protection was conferred only to the mice both immunized and challenged with PCP-1 formulations (Figure 10B). The results confirmed that immunization with PCP-1 entrapped in polymer particles offers a better immunization mode than soluble immunizations. It augmented the anti-PCP-1 antibody responses and considerably improved the inherent limitations of polysaccharides vaccines. The memory antibody response induced by PCP-1 particles is important because T-independent antigens generally induce poor immunological memory. These challenge studies proved that the polymer particles have potential as an effective singledose vaccination mode for polysaccharide antigens. Phagocytic Uptake of Fluorescent Labeled Polymer Particles by Murine Antigen-Presenting Cells. Immune responses to polysaccharide antigens were, until recently, assumed to be completely independent of APCs. However, a growing body of evidence suggests a role for APCs in TI-2 responses. A subset of myeloid DCs, termed as plasmablastsassociated DCs, supports the differentiation of plasmablasts into antibody-producing plasma cells in response to a soluble TI-2 antigen.43 APCs promote survival of polysaccharide antigen-specific B-cells and their differentiation into immunoglobulin M (IgM)-secreting plasmablasts. Macrophages pulsed with S. pneumoniae elicit a T-cell-dependent antibody response upon transfer into naive mice.44 APCs are capable of priming the B-cells with the internalized antigens.45 Antigen-entrapped particles can interact with APCs at the site of immunization, deliver the antigens to APCs, and promote antigen internalization.1 This may promote effective APC-mediated priming of B-cells, leading to robust antibody responses. Considering this, we evaluated the interaction of polymeric particles with APCs. We incubated murine macrophages with fluorescent-labeled particles for different amounts of time and analyzed the cells by confocal laser microscopy and flow cytometry. Flow cytometric analyses of macrophages after incubation for defined time points determined the particle-associated fluorescence. Particleassociated fluorescence analyzed of 30 000 cells on the FL-1 channel (coumarin-6 excitation falls on the FL-1 channel of the BD-LSR) represented the percentage of cells that phagocytosed 933

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images (Figure 13A,B) clearly show particle-associated fluorescence only at the middle focal plains. No particleassociated fluorescence was observed in the initial sections of the Z stack that reveal the cell surface. This confirmed the particle internalization and ruled out adhesion to the cell surface. This was true for both MPs and NPs. The results corroborated the findings from the uptake studies carried out using flow cytometry.

the particles. Figure 11 represents time-dependent changes in the percentage of FL-1-positive cells as an average of three

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CONCLUSIONS Entrapping the capsular polysaccharide of S. pneumoniae (PCP1) in biodegradable polymer particles improved its immunogenicity and protected mice from lethal challenge of virulent S. pneumoniae. The enhanced antibody response from particulate formulations correlated with the size-dependent increase in internalization of antigen-loaded particles by APCs, higher contact surface area, and repetitive stimulation of B-cells through the sustained presence of released antigens. The results provide valuable insights into the design of these delivery systems, highlighting the importance of the surface area− volume ratio, antigen surface density, and particle size in inducing an antibody response. Because both the nanoparticleand microparticle-based delivery systems worked for the carbohydrate antigens, they may be working through two different mechanisms to elicit an immune response. The microparticles may be helping by promoting more interaction with the BCR through surface immunoglobulin, whereas nanoparticles may be delivering the carbohydrate intracellularly, thus promoting better processing and presentation of antigens. On the basis of various in vivo and in vitro studies using polymer particle-entrapped PCP-1 formulations, the following conclusions are made: (1) Pneumococcal polysaccharide antigens entrapped in polymer particles elicited a higher memory antibody response from a single-dose immunization. (2) Particle-based immunization generated anti-PCP-1 antibodies in serum, resulting in significantly higher opsonophagocytic activity. (3) The antibody response generated by different sized polymer particles correlated with their uptake by APCs. (4) Both nanoparticle- and microparticle-based delivery systems elicited protective antibodies for carbohydrate-based candidate antigens, and this was the opposite of that observed

Figure 11. Analyses of phagocytic uptake of fluorescent polymeric particles by murine macrophage cell line J774A. Flow cytometric analyses of macrophages after incubation for defined amounts of time were carried out to evaluate the particle-associated fluorescence. Fluorescent cells were counted and represented as the percentage of cells that phagocytosed the particles. Coumarin-6-labeled fluorescent nanoparticles were phagocytosed significantly better than microparticles (***, p < 0.001). Error bars represent the mean ± standard deviation.

representative experiments. We observed a more efficient phagocytosis of NPs compared to MPs (Figure 11). After 24 h of incubation, 81.4% of macrophages phagocytosed NPs, whereas only 33.9% of cells phagocytosed MPs. Both the rate and extent of phagocytic uptake were significantly higher for NPs compared to MPs. This efficient uptake of NPs would promote the delivery of antigens to the APCs, which helps in draining the administered antigens to the lymph node. The results suggested that particle uptake increases and saturates over time. Also, polymeric particles can interact actively and can deliver the antigen to APCs. Confocal laser microscopy analysis of cells incubated with polymer particles confirmed the phagocytosis (Figure 12A,B). As evident from the image, particles were within the cell boundary, indicating the phagocytosis of the particles. Z-stack imaging of representative cells further confirmed the phagocytosis of the particles. Z-stack

Figure 12. Confocal laser microscope images of phagocytosis of antigen-entrapped particles: (A) MPs and (B) NPs by murine macrophage cell line J774A at 24 h. Particles (coumarin, green) are clearly surrounded by the cell boundary (rhodamine phalloidin (F-actin), red), indicating internalization by cells. Spectral bleeding of coumarin fluorescence into the DAPI channel (blue) was also observed. This blue signal overlapped with DAPI signals from the nucleus, making intracellular localization of the particles difficult. 934

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Figure 13. Z-stacking confocal laser microscope images of the phagocytosis of antigen-entrapped particles: (A) MPs and (B) NPs by murine macrophage cell line J774A at 24 h. Blue, DAPI; green, coumarin (MPs and NPs); and red, rhodamine phalloidin (F-actin). ∥

with the particle-size-based antibody response from protein antigen. (5) Higher IgG responses correlated with a higher surface-to-volume ratio of particles as well with a higher surface antigen density. (6) Combined entrapment of both protein and carbohydrate in a single particle did not improve antipolysaccharide antibody titer. (7) The polymeric particlesbased delivery system improved the immunogenicity of PCP-1 without the need to conjugate them to a helper T-cell peptide. These findings should help in formulating better delivery systems for improving the immunogenicity of polysaccharidebased vaccines.

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University of Colorado, Anschutz Medical Campus, Department of Pharmaceutical Sciences, 12850 East Montview Boulevard, M/S C238, Room V20-4460D-N, Aurora, Colorado 80045, United States. Author Contributions

C.A. and A.K.P. developed the concept; C.A, N.K., D.S., and A.K.P. designed the experimental approach; C.A., N.K., and A.K.U. performed the experiments; C.A., N.K., A.K.U., D.S., and A.K.P. analyzed the data; and C.A. and A.K.P. wrote the manuscript. Notes

The authors declare no competing financial interest

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ASSOCIATED CONTENT

S Supporting Information *

ACKNOWLEDGMENTS The work was supported by a core grant from the National Institute of Immunology (NII), New Delhi, and by a project grant (BT/PR4411/PID/06/190/2003) received from the Department of Biotechnology, Government of India. We thank Drs. Ayub Qadri and Pramod Upadhyay, National Institute of Immunology, New Delhi, India, for valuable comments and discussions. We also thank Mr. Ajay, Cell Biology Laboratory, NII, for his assistance in the microscopic studies. A.K.P. is partly supported by a Tata Innovation Fellowship, Department of Biotechnology, Government of India.

Purification of pneumococcal surface protein A and pneumococcal surface adhesin A; flow cytometry analyses of polylactide particles co-entrapping protein antigens and polysaccharide antigens; flow cytometry analyses of polylactide particles with higher polysaccharide density on surface; diagrams of the immunization protocols; fluorescent microscope images from the opsonophagocytic assay; and additional experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.

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*E-mail: [email protected]; Tel.: +91 11 26703509; Fax: +91 11 2674212.

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Present Addresses §

Max-Planck Institute of Colloids and Interfaces, Department of Biomolecular Systems, 22-Arnimallee, 14195 Berlin, Germany. 935

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