Retention of structure, antigenicity, and biological function of pneumococcal surface protein A (PspA) released from polyanhydride nanoparticles
Introduction
The World Health Organization estimates that pneumonia causes 1.6 million deaths every year, with the majority occurring in children in developing countries [1], [2]. Streptococcus pneumoniae is the leading cause of bacterial pneumonia world wide, especially in children [3]. Current prophylactic options against S. pneumoniae include a 23-valent soluble polysaccharide vaccine (pneumovax) and 7- and 13-valent pneumococcal conjugate vaccines (PCV) [4]. The polysaccharide-based vaccine has been shown to induce humoral immunity in immune-competent patients, but fails to stimulate a cellular immune response, making the vaccine ineffective in high risk groups such as infants, the elderly, and immune-compromised individuals [5]. Following the introduction of a multivalent, polysaccharide–protein conjugate pneumococcal vaccine into childhood immunization regimens the incidence of community-acquired pneumonia in children was reduced by 18% [6]. Although the PCV has been shown to be effective in reducing cases of pneumonia, it has several limitations. The vaccine is expensive and complicated to manufacture, leading to limited availability in developing countries, it does not provide cross-protection across pneumococcal serotypes and, while it reduces capsular type-specific carriage, it has not been shown to reduce nasopharyngeal carriage of pneumococci in general. Additionally, the PCV requires a three dose vaccination regimen and the 7-valent vaccine leads to increased prevalence within the community of strains not included in the vaccine (i.e. serotype substitution) within several years of introduction [6], [7].
Subunit vaccines against pneumonia using non-capsular antigens, specifically protein-based vaccines, have been extensively studied in recent years [5], [8]. Of particular interest in this regard is pneumococcal surface protein A (PspA), which is a choline-binding protein found on the surface of all pneumococcal strains and a critical S. pneumoniae virulence factor [9]. PspA plays two different roles in invasive infection and nasopharyngeal carriage. During invasive systemic infections with S. pneumoniae PspA prevents the deposition of complement on the surface of the bacterium, thus inhibiting the opsonization and killing of S. pneumoniae [9], [10]. PspA also inhibits bactericidal activity medicated by apolactoferrin (ALF) found on mucosal surfaces and in sites of inflammation [9], [11], [12], [13]. Vaccination with PspA protects mice against a lethal challenge with S. pneumoniae via the generation of anti-PspA serum antibodies that are highly cross-reactive to other strains [14], [15], [16], [17], [18]. However, PspA is poorly immunogenic and not capable of inducing a productive immune response without the addition of an adjuvant [19], [20], [21]. In fact, a vaccine regimen based on the inclusion of aluminum hydroxide, a commonly used adjuvant, required three doses to provide protective immunity in a murine model [19], [20], [21]. Therefore, there is a need to design novel adjuvants and/or delivery vehicles for the formulation of efficacious vaccines that can protect against multiple strains of S. pneumoniae and enhance patient compliance by utilizing an acceptable dose regimen.
Because of the promise of PspA as a protective antigen against S. pneumoniae it has been the subject of numerous studies to evaluate novel vaccine delivery systems. Several research groups have shown the induction of immune responses through delivery of PspA with live attenuated bacteria such as Salmonella [22], [23] and through co-delivery with a whole cell pertussis vaccine [4]. Additionally, other novel nanoscale delivery systems containing PspA have been evaluated, including gold nanoparticles [24] and nanogel-based vaccine formulations [25]. In this work we demonstrate that biodegradable polyanhydride nanoparticles can successfully encapsulate and release stable antigenic PspA.
Polyanhydrides have a number of benefits compared with other vaccine delivery systems. Their tunable polymer chemistry can allow modulation of the immune response and enable tailoring of the antigen release kinetics [26], [27]. Additionally, encapsulation into polyanhydride particles has been shown to protect fragile protein antigens against degradation [28], [29]. Polyanhydrides can be fabricated into nanoparticles for administration via inhalation or injection and have shown promise as vaccine adjuvants and delivery vehicles [26], [30], [31], [32], [33]. These polymers exhibit excellent biocompatibility and have been shown to degrade into non-toxic, non-mutagenic products [34]. Polyanhydride particles have also been shown to stabilize fragile proteins throughout the manufacture, storage, and release steps and elicit immune responses in vitro and in vivo [28], [29], [30], [32], [35], [36]. In particular, amphiphilic polyanhydrides, which degrade through a combination of bulk and surface erosion, provide sustained release of protein while maintaining protein structure and function upon release [28], [37]. For example, encapsulation of the recombinant F1-V protein into nanoparticles made from a co-polymer of 1,6-bis(p-carboxyphenoxy)hexane (CPH) and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) stabilized the protein [28] and provided protective immunity against a lethal challenge with Yersinia pestis that persisted for at least 23 weeks post-immunization using a single dose vaccine regimen [32].
In this work we describe the encapsulation and release of stable PspA from polyanhydride nanoparticles. The released PspA retained its primary and secondary structure and preserved both its antigenicity and biological functionality. When the nanoparticle-based vaccine formulations consisting of soluble and encapsulated PspA were administered subcutaneously to mice the animals developed and sustained high anti-PspA IgG titers that were also characterized by high avidity. These studies provide a framework for the rational design of an anti-S. pneumoniae vaccine based on PspA-containing polyanhydride nanoparticles.
Section snippets
Materials
The materials used for monomer synthesis, including sodium hydroxide, hydrobenzoic acid, dibromohexane, 1-methyl-2-pyrrolidinone and triethylene glycol, were purchased from Sigma Aldrich (St. Louis, MO). Acetone, sulfuric acid, potassium carbonate, dimethyl formamide, toluene, acetonitrile, N,N-dimethylacetamide and acetic acid were purchased from Fisher Scientific (Fairlawn, NJ), and 4-p-fluorobenzonitrile was purchased from Apollo Scientific (Cheshire, UK). Sebacic acid monomer was purchased
Results and discussion
Several proteins, including F1-V, Bacillus anthracis protective antigen (PA), bovine serum albumin (BSA) and ovalbumin, have been shown to be stably released from polyanhydride particles [28], [29], [30], [36]. Each of these proteins has different mechanisms of instability and/or degradation. Herein the lessons learned from these previous studies were applied to determine the optimal polyanhydride nanoparticle formulations for stabilization and sustained delivery of functional PspA in order to
Conclusions
The primary and secondary structure, antigenicity and biological functionality of PspA protein were preserved during encapsulation and release from polyanhydride nanoparticles. This result is significant because the release of intact, functional protein increases the probability of preserving important, neutralizing epitopes and facilitates the development of an effective immune response. Herein we have demonstrated not only the release of functionally intact protein, but also how a PspA
Acknowledgements
The authors thank Dr David McPherson of the University of Alabama at Birmingham for producing the recombinant PspA used in this work. The authors acknowledge financial support from NIH-NIAID (R56-AI075026). They are grateful to Ms Adeola M. Olayiwola for her assistance in optimizing the killing assay. The present work was supported by the National Science Foundation (EEC 1156933). B.N. acknowledges the support of the Vlasta Klima Balloun Professorship.
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