ABSTRACT
Neisseria meningitidis is a major cause of meningitis and fulminant septicemia and is also a major reason for mortality associated with epidemics that occur periodically in sub-Saharan Africa, also known as the “meningitis belt” [1]. Capsular polysaccharides (CPS) are a major virulence factor in meningococcal infections and form the basis for serogroup designation and protective vaccines. CPS is anchored in the outer membrane through a 1,2-diacylglycerol moiety [2] and functions to protect the bacteria from complement-mediated killing while also inhibiting phagocytosis by professional phagocytes [1,3]. The serogroup A CPS is composed of a homopolymer of a16-linked ManNAc 1-phosphate that is distinct from the capsular structures of the other meningococcal
Rikhav P. Gala,a Ruhi V. Ubale,b Martin J. D’Souza,a and Susu M. ZughaiercaMercer University, Atlanta, GA, USAbLECOM School of Pharmacy, Bradenton, FL, USAcDepartment of Microbiology & Immunology, Emory University School of Medicine, Atlanta, GA, USA
disease-causing serogroups, B, C, Y, and W-135, which typically contain sialic acid derivatives [4]. N. meningitidis is a leading cause of bacterial meningitis and sepsis in young children and young adults in the United States and is associated with a high mortality rate [5,6]. Childhood vaccination
has been shown to induce a herd immunity effect by reducing nasopharyngeal carriage [4,7]. The current available polysaccharide-based meningococcal vaccines are licensed for use in adolescents and adults but are expensive. These vaccines are Menactra™ (Sanofi Pasteur quadrivalent meningococcal conjugate MCV4 vaccine), Menomune® (Sanofi Pasteur unconjugated quadrivalent meningococcal polysaccharide MPSV4 vaccine), and Menveo® (Novartis quadrivalent conjugate meningococcal vaccine). All of these vaccines contain meningococcal serogroup A, C, W135, and Y. Meningococcal vaccines are poorly immunogenic in infants due to their under-developed immune responses [8]. Since the incidence of meningococcal disease is highest in very young children under the age of two, the FDA very recently approved the use of meningococcal conjugate vaccines Menatcra™ and Menveo®in toddles and infants as young as 9 months old. However, the high cost and limitation of producing and distributing conjugate vaccines is inherent to all currently licensed vaccines. Therefore, utilizing micro/nanotechnology to explore novel meningococcal vaccine formulations that boost innate and adaptive immunity and offer protection to our most vulnerable population is very important. The aim of this study was to formulate a microparticulate meningococcal vaccine that serves as a sustained release system, obviating the need for added adjuvants that are currently present in most vaccines. The proposed vaccine formulation consists of meningococcal CPS polymers encapsulated in albumin-based biodegradable matrix microparticles that mimic the chemical conjugation process of CPS to a protein carrier, which enhance antigen uptake via albumin receptors and elicit a T-cell-dependent immune response [9-11]. Immune responses to polysaccharide antigens are inherently thymus independent, therefore do not induce memory and hence require conversion to thymus dependent immune responses [12]. Such conversion is usually achieved by coupling the polysaccharide with an immunogenic carrier protein using chemical conjugation processes
[13,12]. Two of the commercially available vaccines Menatcra™ and Menveo® involve chemical conjugation with the Diphtheria toxoid to potentiate the immune response as well as induce a T-cell-dependent immune response [13]. The conjugation-free vaccine formulation has previously been successfully tested with other polysaccharide-based vaccines by D’Souza and co-workers with typhoid [6], TB, and Streptococcus pneumonia, in addition to melanoma [11], and influenza vaccines designed with similar polymer matrices. The microparticles are prepared by a well-established process using bovine serum albumin (BSA) as the protein matrix by a one-step spray drying process using the Buchi mini spray dryer B-191 [11].We have report the novel formulation of meningococcal CPS-loaded microparticles with or without the non-toxic meningococcal unglycosylated lipid A (kdtA) [14] as adjuvant. Here we show that the CPS-loaded microparticles but not the empty microparticles, induced innate immune responses consequent to their uptake and recognition by macrophages. We have attempted to describe a novel microparticle-based delivery system for meningococcal vaccine. 6.2 MethodsEudragit S was a generous gift from Evonik Degussa Corporation Parsippany, NJ 07054, USA. Trehalose was purchased from Sigma Aldrich. Bicinchoninic Acid (BCA) protein assay kit was purchased from Thermo Scientific, chicken red blood cells (RBC) cells for hemagglutination assay from Lampire Biologicals, and ELISA flat bottom plates from NUNC. BALB/c mice (6-8 weeks old) were purchased from Harlan (Indianapolis, IN) and used in this study. 6.2.1 Preparation of MicroparticlesPreparation of meningococcal polysaccharide microparticles, a 1% solution of sterile BSA in sterile water was prepared and kept for overnight cross-linking with glutaraldehyde. Excess glutaraldehyde was neutralized with sodium bisulphate the following day.Polysaccharide antigens and or adjuvant were added to the solution
as per the concentrations mentioned in Table 6.1 and spray dried using the Buchi Mini Spray Dryer B-191. The solution was passed through a 0.5 mm nozzle at a flow rate of 20 mL/h to obtain the microparticles. Table 6.1 Preparation of albumin (BSA)-based microparticles containing meningococcal CPS, adjuvant, or CPS + adjuvant Formulation
Note: The concentrations listed are representative of the percentage of the constituent in the formulation. BSA was cross-linked using glutaraldehyde following which the antigen and/or the adjuvant was added to deionized water and the mixture was then spray dried. BSA: sterile and endotoxin-free tissue culture grade bovine serum albumin.Polysaccharide content in the microparticles was measured by initially breaking down the particle using surfactants and protease enzymes. Briefly, 200 µL of Tris-EDTA buffer was added to 5 mg microparticles and the mixture was incubated at 37°C for 4 h. Following this, 200 µL of 1% SDS in 0.2 N NaOH was added and the mixture was vortexed repeatedly, after which 50 µg of proteinase K was added followed by incubation overnight at 37°C. The mixture was then centrifuged at 14,000 rpm for 15 min and the supernatant analyzed to determine the extracted polysaccharide content by the resorcinol-hydrochloric acid assay as described earlier [1]. Briefly, 1 mL of resorcinol-HCl reagent (10 mL of 2% w/v aq. resorcinol solution added to 30 mL concentrated HCl and 0.25 mL of 0.1 M CuSO4, volume adjusted to 100 mL with water) was added to 1 mL of extracted polysaccharide sample, which was then heated in a boiling water bath for 15 min. After cooling to room temperature, 5 mL of amyl alcohol was added and the mixture was centrifuged at 1000 rpm for 1 min; the absorbance of the upper phase was read at 580 nm.
