Retention of structure, antigenicity, and biological function of pneumococcal surface protein A (PspA) released from polyanhydride nanoparticles

Pneumococcal surface protein A (PspA) is a choline-binding protein which is a virulence factor found on the surface of all Streptococcus pneumoniae strains. Vaccination with PspA has been shown to be protective against a lethal challenge with S. pneumoniae, making it a promising immunogen for use in vaccines. Herein, the design of a PspA-based subunit vaccine using polyanhydride nanoparticles as a delivery platform is described. Nanoparticles based on sebacic acid (SA), 1,6-bis-(p-carboxyphenoxy)hexane (CPH) and 1,8-bis-(p-carboxyphenoxy)-3,6- dioxaoctane (CPTEG), specifically 50:50 CPTEG:CPH and 20:80 CPH:SA, were used to encapsulate and release PspA. The protein released from the nanoparticle formulations retained its primary and secondary structure as well as its antigenicity. The released PspA was also biologically functional based on its ability to bind to apolactoferrin and prevent its bactericidal activity towards Escherichia coli. When the PspA nanoparticle formulations were administered subcutaneously to mice, the animals elicited a high titer and high avidity anti-PspA antibody response. Together, these studies provide a framework for the rational design of a vaccine against S. pneumoniae based on polyanhydride nanoparticles

Polyanhydride Nanoparticle Delivery Platform Dramatically Enhances Killing of Filarial Worms.

Filarial diseases represent a significant social and economic burden to over 120 million people worldwide and are caused by endoparasites that require the presence of symbiotic bacteria of the genus Wolbachia for fertility and viability of the host parasite. Targeting Wolbachia for elimination is a therapeutic approach that shows promise in the treatment of onchocerciasis and lymphatic filariasis. Here we demonstrate the use of a biodegradable polyanhydride nanoparticle-based platform for the co-delivery of the antibiotic doxycycline with the antiparasitic drug, ivermectin, to reduce microfilarial burden and rapidly kill adult worms. When doxycycline and ivermectin were co-delivered within polyanhydride nanoparticles, effective killing of adult female Brugia malayi filarial worms was achieved with approximately 4,000-fold reduction in the amount of drug used. Additionally the time to death of the macrofilaria was also significantly reduced (five-fold) when the anti-filarial drug cocktail was delivered within polyanhydride nanoparticles. We hypothesize that the mechanism behind this dramatically enhanced killing of the macrofilaria is the ability of the polyanhydride nanoparticles to behave as a Trojan horse and penetrate the cuticle, bypassing excretory pumps of B. malayi, and effectively deliver drug directly to both the worm and Wolbachia at high enough microenvironmental concentrations to cause death. These provocative findings may have significant consequences for the reduction in the amount of drug and the length of treatment required for filarial infections in terms of patient compliance and reduced cost of treatment

Room Temperature Stable PspA-Based Nanovaccine Induces Protective Immunity

Streptococcus pneumoniae is a major causative agent of pneumonia, a debilitating disease particularly in young and elderly populations, and is the leading worldwide cause of death in children under the age of five. While there are existing vaccines against S. pneumoniae, none are protective across all serotypes. Pneumococcal surface protein A (PspA), a key virulence factor of S. pneumoniae, is an antigen that may be incorporated into future vaccines to address the immunological challenges presented by the diversity of capsular antigens. PspA has been shown to be immunogenic and capable of initiating a humoral immune response that is reactive across approximately 94% of pneumococcal strains. Biodegradable polyanhydrides have been studied as a nanoparticle-based vaccine (i.e., nanovaccine) platform to stabilize labile proteins, to provide adjuvanticity, and enhance patient compliance by providing protective immunity in a single dose. In this study, we designed a room temperature stable PspA-based polyanhydride nanovaccine that eliminated the need for a free protein component (i.e., 100% encapsulated within the nanoparticles). Mice were immunized once with the lead nanovaccine and upon challenge, presented significantly higher survival rates than animals immunized with soluble protein alone, even with a 25-fold reduction in protein dose. This lead nanovaccine formulation performed similarly to protein adjuvanted with Alum, however, with much less tissue reactogenicity at the site of immunization. By eliminating the free PspA from the nanovaccine formulation, the lead nanovaccine was efficacious after being stored dry for 60 days at room temperature, breaking the need for maintaining the cold chain. Altogether, this study demonstrated that a single dose PspA-based nanovaccine against S. pneumoniae induced protective immunity and provided thermal stability when stored at room temperature for at least 60 days

Retention of structure, antigenicity, and biological function of pneumococcal surface protein A (PspA) released from polyanhydride nanoparticles

Pneumococcal surface protein A (PspA) is a choline-binding protein which is a virulence factor found on the surface of all Streptococcus pneumoniae strains. Vaccination with PspA has been shown to be protective against a lethal challenge with S. pneumoniae, making it a promising immunogen for use in vaccines. Herein, the design of a PspA-based subunit vaccine using polyanhydride nanoparticles as a delivery platform is described. Nanoparticles based on sebacic acid (SA), 1,6-bis-(p-carboxyphenoxy)hexane (CPH) and 1,8-bis-(p-carboxyphenoxy)-3,6- dioxaoctane (CPTEG), specifically 50:50 CPTEG:CPH and 20:80 CPH:SA, were used to encapsulate and release PspA. The protein released from the nanoparticle formulations retained its primary and secondary structure as well as its antigenicity. The released PspA was also biologically functional based on its ability to bind to apolactoferrin and prevent its bactericidal activity towards Escherichia coli. When the PspA nanoparticle formulations were administered subcutaneously to mice, the animals elicited a high titer and high avidity anti-PspA antibody response. Together, these studies provide a framework for the rational design of a vaccine against S. pneumoniae based on polyanhydride nanoparticles

Panel A: Scanning electron photomicrograph of 20:80 CPTEG:CPH nanoparticles loaded with 5% IVM, 5% DOX, and 2% rhodamine.

<p>Scale bar: 500 nm. Panel B: Release kinetics of ivermectin (IVM) and doxycycline (DOX) from 20:80 CPTEG:CPH nanoparticles quantified using HPLC.</p

Panel A. Table outlining survival of <i>B</i>. <i>malayi</i> females after treatment with 195, 49, 10, 5, 1.95, or 0.049 μM concentrations of ivermectin (IVM) and doxycycline (DOX) delivered solubly or encapsulated into 20:80 CPTEG:CPH nanoparticles.

<p>Panel B. Average number of days to death of <i>B</i>. <i>malayi</i> females after administration of soluble or encapsulated IVM/DOX treatments and comparison to control worms. Significance was determined at p<0.05, 0.01, or 0.001 as noted using a Student’s T test. Panel C. Average motility scores of <i>B</i>. <i>malayi</i> females after IVM/DOX treatments scored using a 2X objective on a Nikon Microscope following a 0–5 scoring system, as described in the Methods. Panel D. Average number of microfilaria shed by <i>B</i>. <i>malayi</i> females after administration of soluble or encapsulated IVM/DOX treatments and comparison to control worms. The NP only group contains comparable amount of rhodamine and the total amount of particle in this group corresponds to that of the highest drug concentration of 195 μM. At 14 days, all worms treated with NP only with a motility score of 0 remained viable based on the MTT assay and recovery of motility upon transferring to fresh medium^†. Significance was determined at p<0.05, 0.01, or 0.001 as noted using a Student’s T test.</p

The motility of <i>B</i>. <i>malayi</i> MF treated with decreasing doses of either soluble or encapsulated ivermectin (IVM)/doxycycline (DOX) was recorded for 14 days post treatment.

<p>The recorded motility scores (Panel B) were used to calculate an average time to death for each treatment group and dose (Panel A). To calculate the average motility score for each time, dose and treatment group, triplicate wells containing a minimum of 200 MF in each well were treated as indicated. A motility score of 0 was equated with death and the average time to death was plotted along with standard error. Data presented are from one of two experiments with similar results. Significance was determined at p<0.05, 0.01, or 0.001 as noted using a Student’s T test.</p

Confocal microscopy of female <i>B</i>. <i>malayi</i> with nanoparticles.

<p>Worms were incubated for 96 h with either soluble controls (panel A) or 20:80 CPTEG:CPH nanoparticles containing ivermectin (IVM), doxycycline (DOX), and rhodamine B (panel B), fixed and imaged by LSCM. Controls contained 195 μM each of IVM and DOX, and 3.9 μM rhodamine B, while the nanoparticles contained 5 μM of each drug and 0.1 μM of rhodamine B. Left panels are DNA (blue), rhodamine (red) and bright field image overlays and right panels are the respective individual rhodamine images collected using identical image acquisition settings. Inset box within the nanoparticle-treated worm outlines the area selected for side view rendering (C). Representative images shown demonstrate the accumulation of nanoparticles within tissues throughout the worm (B) as compared to the higher amount of soluble rhodamine that was not detected within the body of the worms.</p