Article pubs.acs.org/Biomac
Engineering Biomaterial-Associated Complement Activation to Improve Vaccine Efficacy Yuan Liu,†,‡,∥ Ying Yin,§,∥ Lianyan Wang,*,† Weifeng Zhang,†,‡ Xiaoming Chen,†,‡ Xiaoxiao Yang,† Junjie Xu,*,§ and Guanghui Ma*,† †
National Key Laboratory of Biochemical Engineering, PLA Key Laboratory of Biopharmaceutical Production & Formulation Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China ‡ University of Chinese Academy of Sciences, Beijing 100049, PR China § Laboratory of Vaccine and Antibody Engineering, Beijing Institute of Biotechnology, Beijing 100071, PR China ABSTRACT: The complement system plays an important role in innate and adaptive immunity, which suggests that complement activation could be exploited as a potential strategy for vaccine adjuvants. Here we explored the potential of chitosan-based microparticles (CS-NH2 MPs) as a vaccine adjuvant with an active surface for complement activation due to the abundance of amino groups. In vaccination studies, using recombinant anthrax protective antigen as a model antigen, compared with the control microparticles (amino-cross-linked MPs), we found that microparticles (MPs) with abundant amino groups significantly enhanced higher antigen-specific IgG titers in vivo and enhanced the production of IL-4 and IFN-γ with ex vivo restimulation. Furthermore, proliferative responses of splenocytes to ex vivo antigen restimulation were enhanced following immunization with MPs with amino groups. Overall, these results indicated that CS-NH2 MPs with a high surface density of amino groups were favorable for complement activation and immune responses. Our data provide further design principles for studies on complement-activating MPs as a vaccine platform.
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INTRODUCTION
The complement system is a group of over 40 soluble and cell-surface proteins presented in animal body fluids and on cell surfaces and serving as a first line of defense against pathogens. Initially, complement was thought to play a major role in innate immunity, where a robust and rapid response is mounted against invading pathogens. However, recently, it is becoming increasingly evident that complement also plays an important role in adaptive immunity involving T and B cells that help in elimination of pathogens, maintaining immunologic memory, and preventing pathogenic reinvasion.12,13 There are three complement activation pathways. The classical pathway is triggered primarily by immune complexes (containing antigen and IgG or IgM); the lectin pathway is initiated by mannan-binding lectin/protein (MBL) and mannan-binding lectin-associated serine proteases (MASP and MASP2); and the alternative pathway is initiated spontaneously but is activated by contact with a variety of surfaces, such as carbohydrate structures on microorganisms and other surfaces. Solid biomaterials (e.g., microparticles, implants, and nanowires), depending on composition and other physicochemical properties, can activate complement via the alternative pathway.14
Vaccination is the most effective method to prevent infectious diseases. In view of safety issues, more defined vaccines based on partially purified preparations from the organism or recombinant subunit proteins have recently been developed.1 However, by themselves, these types of vaccines are often not sufficiently immunogenic due to the lack of an innate immune stimulus.2 Hence, adjuvants generally need to be added to these types of vaccines to facilitate the immune response.3 In experimental vaccine formulations, most adjuvants, such as monophosphoryl lipid A,4 the cytokine CD40 ligand,5 CpG ODN,6 and interferon (IFN)-γ,7 are danger signals exerting their functions through the activation of Toll-like receptors (TLRs) and inflammatory cytokine receptors. Despite the fact that some promising results were achieved, many complexities of these adjuvants remain unclear, such as toxicity of CpG ODN, high cost of cytokines, and so on. As an alternative adjuvant strategy, Reddy et al. explored the possibility of using the complement cascade as a danger signal for innate immunity, designing nanoparticles with a surface chemistry that spontaneously induces complement activation in situ. 8 Biomaterials scientists have typically sought to avoid complement activation to minimize effects such as implant rejection and clearance of systemic drug-delivery vehicles.9−11 However, for vaccine, complement activation is encouraged and it could generate a molecular adjuvant danger signal in situ.8 © 2013 American Chemical Society
Received: June 26, 2013 Revised: July 19, 2013 Published: July 22, 2013 3321
dx.doi.org/10.1021/bm400930k | Biomacromolecules 2013, 14, 3321−3328
Biomacromolecules
Article
The shape and surface features of microspheres were observed by a JEM-6700F scanning electron microscope (SEM; JEOL, Japan). Microspheres were suspended in distilled water, and the dispersion was dropped on aluminum foil and dried at ambient atmosphere. The sample was placed on a metal stub and coated with platinum under vacuum by an ion sputter (JFC-1600, JEOL). The size distribution and zeta potential of MPs were measured using the ZetaPlus apparatus (Brookhaven Instruments) by dynamic (DLS) and electrophoretic (ELS) light scattering techniques. The wavelength of the laser light was 670 nm. Size distributions were determined at 90° of the scattering angle of the laser beam and at 15° for zeta potential determination. The experiments were carried out at 20 °C. Endotoxin Levels. The endotoxin level in the final formulation was determined by the LAL assay method with a commercially available endotoxin assay kit (Pyrosate 0.25 EU/mL) from Associates of Cape Cod (Falmouth, MA) according to the manufacturer’s instructions. All formulations were tested and used only when the endotoxin levels were
