Enhancement of immune response of HBsAg loaded poly(L-lactic acid) microspheres against Hepatitis B through incorporation of alum and chitosan

Purpose: Poly (L-lactic acid) (PLA) microparticles encapsulating Hepatitis B surface antigen (HBsAg) with alum and chitosan were investigated for their potential as a vaccine delivery system. Methods: The microparticles, prepared using a water-in-oil-in-water (w/o/w) double emulsion solvent evaporation method with polyvinyl alcohol (PVA) or chitosan as the external phase stabilising agent showed a significant increase in the encapsulation efficiency of the antigen. Results: PLA-Alum and PLA-chitosan microparticles induced HBsAg serum specific IgG antibody responses significantly higher than PLA only microparticles and free antigen following subcutaneous administration. Chitosan not only imparted a positive charge to the surface of the microparticles but was also able to increase the serum specific IgG antibody responses significantly. Conclusions: The cytokine assays showed that the serum IgG antibody response induced is different according to the formulation, indicated by the differential levels of interleukin 4 (IL-4), interleukin 6 (IL-6) and interferon gamma (IFN-γ). The microparticles eliciting the highest IgG antibody response did not necessarily elicit the highest levels of the cytokines IL-4, IL-6 and IFN-γ

Particulate Carriers for Vaccines

Soluble antigens and antigens synthesized by recombinant DNA techniques are weakly immunogenic and need protection against passage through the mucosal epithelium. Encapsulation of antigens into polymeric carriers is one of the techniques developed for delivering these agents. However, this technology needs further improvements to enhance the immune responses and stabilization of the antigen within the carriers. The incorporation of adjuvants to enhance immune responses is well known, and therefore incorporation of adjuvants with the polymeric carriers could also be beneficial. The focus of this study, therefore, was to co-encapsulate known and unknown adjuvants with polymeric particles. Polymeric particulate carriers were prepared using the solvent evaporation or spray drying technique. The biodegradable polymers selected were polylactic acid and polycaprolactone. Diphtheria toxoid (DT) and hepatitis B surface antigen (HBsAg) were used as model antigens. The adjuvants used for co-encapsulation with antigens were penetration enhancers and mineral compounds. These adjuvants were either mixed with the polymer in the organic phase or in the internal phase during the preparation of the particles. The penetration enhancers used in the study were tocopherol acetate (TA), tocopherol nicotinate (TN), D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), cholic acid, lithocholic acid and melittin. Mineral compounds such as alum, zinc sulphate and zinc oxide were also co-encapsulated to evaluate their effect on the physicochemical properties and adjuvancy of the carriers. As surface charge plays an important role in the particle uptake, chitosan was used as an external phase stabilizer in some formulations to impart a positive charge on the particles. The penetration enhancers used here have not been used to co-encapsulate antigens in polymeric particles before, and there is not much data available for co-encapsulation of mineral compounds. The formulations were characterized with respect to size, surface charge, encapsulation efficiency, uptake in the cell lines and toxicity. These formulations were also evaluated in mice for antibody responses and cytokine analysis. Incorporation of tocopherol derivatives into the particulate formulations significantly increased the encapsulation efficiency of the antigen. The in vitro toxicity studies showed that these formulations were non-toxic. In vivo, the HBsAg-loaded particles containing TPGS significantly improved the serum antibody levels when compared with free HBsAg. TA and TN also improved the serum antibody levels when administered intramuscularly and intranasally. However, the intramuscular responses were higher than the intranasally administered formulations. DT-loaded particles containing TPGS significantly improved the antibody levels. Co-encapsulation of zinc sulphate and alum in nanoparticles improved the encapsulation efficiency of HBsAg. Sizes were in the range of 300 to 500nm for these formulations. The serum immune responses of particles containing zinc sulphate were equivalent to particles containing alum. However, the intranasal route elicited much lower antibody responses when compared with the intramuscular groups. Particles coated with chitosan significantly improved the antibody levels. The co-encapsulation of bile salts (cholic acid or lithocholic acid) and melittin along with HBsAg did not have any effect on the encapsulation efficiency. Particles containing bile salts had a higher cellular uptake compared with particles without bile salts. Particles containing bile salts or melittin significantly improved the antibody levels when compared with free antigen and antigen plus cholera toxin B

Enhancement of immune response of HBsAg loaded poly (L-lactic acid) microspheres against Hepatitis B through incorporation of alum and chitosan

Purpose: Poly (L-lactic acid) (PLA) microparticles encapsulating Hepatitis B surface antigen (HBsAg) with alum and chitosan were investigated for their potential as a vaccine delivery system

Assessing the value of system theoretic process analysis in a pharmacovigilance process : an example using signal management

Introduction: To design and run an effective pharmacovigilance system, one must understand potential hazards that may cause the system to fail. System-Theoretic Process Analysis (STPA) is a hazard analysis technique that has been successfully applied in domains including aviation, nuclear power, and defence systems; however, it has not yet been applied to a critical pharmacovigilance process. Objectives: The objective of this project was to assess the value of STPA in pharmacovigilance by using the example of signal management, in order to identify process risks, areas for improvement, and applicable process metrics. Methods: Hierarchical control structure (HCS) is the starting point for STPA. The HCS models a process as a set of interacting feedback control systems, which maintain the system in a “safe state” where losses do not occur. “Losses” can be defined as harm to patients, regulatory non-compliance, or other negative consequences. The HCS viewpoint is a considerable departure from the usual consideration of a pharmacovigilance process as a series of sequential steps carried out by responsible individuals. Following the definition of the HCS, a two-step STPA process was carried out, leading to a set of unsafe actions and scenarios by which process failures could lead to loss. Recommendations were then made to prevent those scenarios that were not already covered by existing features of the process. Results: The signal management process was successfully modelled as a feedback control system, which dynamically ensures that an incoming stream of safety data is accurately reflected in the reference safety information for the product. After creating the HCS, we conducted the STPA itself. This yielded 215 scenarios through which the process could end up in an unsafe state where losses could occur. The scenarios included 91 that were covered by existing measures, 28 related to existing or planned metrics, and 25 were scenarios considered implausible, inconsequential, undetectable, or out-of-scope. The remaining 71 scenarios were consolidated into 8 proposed recommendations for enhancements to metrics, 10 proposed recommendations for enhancements to the process itself, and 8 proposed recommendations regarding infrastructure or related processes, including the associated quality management system. STPA considers “soft” factors such as cultural influences, and this was reflected in several recommendations, for instance regarding training. Conclusion: STPA is a labour-intensive and time-consuming process, most appropriate for high-risk, compliance-critical processes. STPA is a powerful technique for identification of risks in pharmacovigilance systems, and can contribute to process reliability with the potential to improve patient safety and the maintenance of regulatory compliance

Antibody and cytokine-associated immune responses to S. equi antigens entrapped in PLA nanospheres

Strangles is an infectious disease caused by Streptococcus equi subspecies equi that affects the upper respiratory tract of the Equidae. The control of this disease seems to be dependent on its earlier detection and prevention, but prolonged animal protection without development of strong and severe side effects has not yet been achieved. Convalescent horses exhibit a protective immune response, mainly against SeM (58 kDa), an antiphagocytic and opsonogenic S. equi M-like protein, known as the major protective antigen against strangles. Purified recombinant SeM and S. equi protein extract-entrapped poly(lactic acid) (PLA) nanospheres were developed and their adjuvant potential was studied via the intramuscular route. The effect including molecules with adjuvant properties such as spermine, oleic acid, alginate and glycol-chitosan was also evaluated. Spherical nanometric particles 500 nm containing the protein antigen were prepared by the solvent evaporation method and protein structure was not affected throughout preparation. The humoral immune response induced by nanospheres was markedly higher than that elicited by soluble antigens, isolated or co-admixed with CpG. The IgG and IgG subtypes, along with cytokine titres, indicated that nanospheres composed by glycolchitosan developed a more balanced Th1/Th2 response for both purified SeM and S. equi enzymatic extract proteins, although those induced by the pure antigen-entrapped particles were higher than the S. equi tested vaccines composed by total antigens entrapped in polymeric nanospheres. (C) 2009 Elsevier Ltd. All rights reserved.. - Portuguese Ministry of Science and Technology [SFRH/BD/14300/2003, POCI/BIO/59147/2004, PPCDT/BIO/59147/2006]. - work was supported by research grants awarded by the Portuguese Ministry of Science and Technology (SFRH/BD/14300/ 2003; POCI/BIO/59147/2004; PPCDT/BIO/59147/2006)