Entrapped Antigen Research Articles

Novel anionic microparticles are a potent adjuvant for the induction of cytotoxic T lymphocytes against recombinant p55 gag from HIV-1

Microparticles with entrapped antigens have recently been shown to possess significant potential as vaccine delivery systems and adjuvants. However, the potential of microparticles as adjuvants has been seriously limited by the common problem of degradation and denaturation of antigens following encapsulation and release. To overcome these problems, we have developed a novel way to use microparticles as adjuvants, by the adsorption of proteins onto their surface. Anionic microparticles were prepared through the inclusion of an anionic detergent, sodium dodecyl sulphate (SDS), in the microparticle preparation process. The anionic microparticles were capable of the efficient and reproducible adsorption of recombinant p55 gag protein from HIV-1. Microparticles with adsorbed p55 were capable of inducing potent cytotoxic T lymphocyte responses in mice following intramuscular immunization. In addition, the microparticles also exhibited a potent adjuvant effect for antibody induction against p55.

Influence of various technological parameters on the preparation of spray-dried poly(epsilon-caprolactone) microparticles containing a model antigen

This work evaluates the efficacy of the spray-drying technique to prepare poly(epsilon-caprolactone) (PCL) microparticles containing an entrapped model antigen (bovine albumin, BSA). The presence of a stabiliser was found to be an important parameter when preparing PCL microparticles containing a hydrophilic antigen. The effect of various technological parameters (concentration of the polymer and protein solutions, organic/aqueous phases ratio, nature of solvents and emulsion parameters such as duration and speed of agitation) on microparticle morphology and size, BSA entrapment and encapsulation efficiency was studied. Microparticles were characterized by a mean size from 9.56+/- 0.25 to 24.31+/- 2.87mum and a BSA entrapment from 0.80+/- 0.02 to 24.21+/- 0.23% (w/w). SDS-PAGE electrophoresis and isoelectric focusing (IEF) confirmed the conservation of the physicochemical characteristics of the BSA entrapped within PCL microparticles produced by spray-drying. Together, these results showed that spray-drying is an efficient technique to overcome the key obstacle that represents the scaling-up of the manufacturing process to produce sufficient quantities of vaccine for clinical trials and, ultimately, commercialization.

Vaccine properties of antigens entrapped in microparticles produced by spray-drying technique and using various polyester polymers

The present study investigated the suitability of various microparticles produced by spray-drying technique to entrap and preserve the physiochemical and biological properties of an antigen. These microparticles were constituted either by poly(lactide) polymers characterized by various molecular weight or poly(lactide-co-glycolide) polymers. The recombinant 28 kDa glutathione S-transferase of Schistosoma mansoni (rSm28GST) characterized by major epitopes involved in the active site of this enzyme was selected as model antigen. The microparticles were characterized by a mean size ≤5 μm and an antigen loading of approximately 2% (w/w). The analysis by SDS-PAGE electrophoresis of the rSm28GST released from microparticles confirmed the conservation of its physicochemical characteristics. The conservation of the native structure of the entrapped antigen was confirmed by detecting its enzymatic activity after release from microparticles. A single intraperitoneal immunization of mice with rSm28GST entrapped in microparticles resulted in a specific antibody response, which remained high for at least 7 months. The analysis of the isotype profile indicated that immunized mice primarily produced anti-rSm28GST immunoglobulin (Ig) G1 with the coexistence of lower IgG2a and IgG2b levels. Finally, the recognition of the major epitopic regions and the neutralization of the enzymatic activity of the rSm28GST by the antisera confirmed the specificity of the response against the native structure of the antigen. These results confirmed the integrity of the entrapped antigen. Moreover, our results supported the hypothesis that the duration of antigen release is the limiting factor for the duration of antibody production. Indeed, the use of polymers characterized by different molecular weights allowed us to modify the duration of the immune response. Together, these results demonstrated that microencapsulation of an antigen by spray-drying preserved its crucial characteristics required to generate an effective humoral immune response after a single-dose administration.

Vaccine Entrapment in Liposomes

The use of liposomes as carriers of peptide, protein, and DNA vaccines requires simple, easy-to-scale-up technology capable of high-yield vaccine entrapment. Work from this laboratory has led to the development of techniques that can generate liposomes of various sizes, containing soluble antigens such as proteins and particulate antigens (e.g., killed or attenuated bacteria or viruses), as well as antigen-encoding DNA vaccines. Entrapment of vaccines is carried out by the dehydration–rehydration procedure which entails freeze-drying of a mixture of “empty” small unilamellar vesicles and free vaccines. On rehydration, the large multilamellar vesicles formed incorporate up to 90% or more of the vaccine used. When such liposomes are microfluidized in the presence of nonentrapped material, their size is reduced to about 100 nm in diameter, with much of the originally entrapped vaccine still associated with the vesicles. A similar technique applied for the entrapment of particulate antigens (e.g., Bacillus subtilis spores) consists of freeze-drying giant vesicles (4–5 μm in diameter) in the presence of spores. On rehydration and sucrose gradient fractionation of the suspension, up to 30% or more of the spores used are associated with generated giant liposomes of similar mean size.

Biodegradable microparticles of influenza viral vaccine: comparison of the effects of routes of administration on the in vivo immune response in mice

The objective of this study was to investigate the comparative immune response following administration of biodegradable microparticles loaded with influenza viral vaccine using subcutaneous and oral routes. Influenza viral vaccine was entrapped in poly( d,l-lactide-co-glycolide) (PLG) and poly(isobutylcyanoacrylate) (PIBCA) microparticles. Stability and immunogenicity of entrapped antigen were retained, as evaluated by SDS-PAGE and immunoblot. Microparticles in the size range of <11 μm were evaluated for protein loading and in vitro antigen release. The mice were immunized with microparticle loaded antigen and IgG levels in blood and IgA levels in saliva and gastric secretions were monitored by ELISA method. When the mice were immunized with microparticle suspensions, IgG levels were higher if administered by subcutaneous primed by oral route compared to oral primed by subcutaneous route or subcutaneous or oral route. The IgA level in saliva and gastric secretions were also found to be higher when subcutaneous immunization was given followed by oral booster than oral priming followed by subcutaneous booster. The polymer types of the microparticles had effects on both IgG and IgA levels. This study provided insights into the design of microparticles of influenza vaccine for subcutaneous administration followed by an unlimited oral boosting, which will have high cost-effectiveness and patient compliance.

Intranasal immunization against influenza virus using polymeric particles

The aim of this study was to evaluate the potential of poly(D,L-lactide-co-glycolide) nanoand microspheres, with a mean diameter of 220 nm and 8 μm, respectively, to enhance the nasal and systemic immune responses against influenza virus antigen. High encapsulation levels of antigen were achieved in all cases. Neither the molecular weight nor the antigenicity of the entrapped antigen were affected by the encapsulation procedure. Following nasal immunization, the nasal washes IgA and the serum IgG responses were evaluated. With the soluble antigen, relatively high immune responses were observed. With nanospheres, nasal washes IgA levels were significantly lower (p < 0.01) and serum IgG levels were not significantly different (p > 0.05) from those obtained with the soluble antigen. With microspheres, both nasal washes IgA and serum IgG levels were significantly lower (p < 0.01 and < 0.05, respectively) as compared to the levels found for the soluble antigen. In addition, fluorescent microspheres administered intranasally failed to reach the nasal-associated lymphoid tissue (NALT). This lack of particle uptake by NALT and the high immunogenicity of the antigen used in this study, could explain the absence of enhancement of the immune responses by the polymeric particles.

ChemInform Abstract: Synthesis of Neoglycolipids Containing Oligosaccharides Based on 3,6‐Branched‐α‐D‐mannopyranosides as the Carbohydrate Moieties.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Preparation and characterization of alginate microspheres containing a model antigen

The goal of this study was to evaluate the technological feasibility of delivering antigen using alginate microspheres. The microspheres were prepared by an emulsification technique and fully characterized as antigen delivery system. Selection of appropriate parameters enabled the preparation of alginate microspheres with a mean diameter of 8 μm. The encapsulation efficiency of bovine serum albumin (BSA), chosen as model antigen, as well as the BSA loading were very high (>90% and 10% w/w, respectively). The process of encapsulation did not affect the molecular weight or the antigenicity of the entrapped antigen. The in vitro release profile showed a fast release rate of encapsulated BSA, particularly in phosphate buffered saline solution. However, a decrease of the release rate was observed when alginate microspheres were coated with poly( l-lysine) or prepared with higher alginate molecular weight. Therefore, alginate microspheres appear, technologically, a promising antigen delivery system.

Synthesis of Neoglycolipids Containing Oligosaccharides Based on 3,6-Branched-α-D-Mannopyranosides as the Carbohydrate Moieties1

Several oligosaccharides containing 3,6-branched-α-D-mannopyranosides were obtained by selective glycosylation of 5-azido-3-oxapentyl α-D-mannopyranoside with acetobromosugars. After deacetylation and reduction of the spacer azido group, the oligosaccharides were coupled with the activated hemisuccinate derivatives of cholesterol and 1,2-di-O-alkylglycerol. The neoglycolipids so obtained were characterized by NMR spectroscopy and will be used as liposome coating molecules for targeting entrapped antigens. 1 Presented at the XlXIIth Infemutjonul Carbohydrate Symposium, Milan, Italy, July 21-26, 1996.

Controlled release microparticles as a single dose diphtheria toxoid vaccine: immunogenicity in small animal models

Diphtheria toxoid (DT) was encapsulated in microparticles prepared from polylactide-co-glycolide (PLG) polymers using a solvent evaporation technique. Combinations of small and large sized microparticles with controlled release characteristics were used to immunize Sprague Dawley rats and the antibody responses were monitored for one year. For comparison, control groups of rats were immunized at 0, 1 and 2 months with DT adsorbed to alum. The antibody responses generated by the microparticles were comparable to the alum immunized control groups from 32 weeks. Microparticles with a single entrapped antigen (DT) induced better antibody responses than microparticles with two antigens entrapped simultaneously (DT+TT). Microparticles prepared from a single polymer were less effective for long term antibody induction than a combination of microparticles prepared from three different polymers. A combination vaccine consisting of antigen adsorbed to alum and also entrapped in microparticles gave the best response. In an inhibition assay designed to determine the relative binding of antisera to the antigen, the sera from the microparticle and the alum immunized animals showed comparable binding. An intradermal challenge study was performed in rabbits, which showed similar levels for the alum and the microparticle immunized animals at 4, 12 and 32 weeks after immunization.

Biodegradable nanoparticles as a sustained release system for the antigens/allergens of Aspergillus fumigatus: preparation and characterisation

Ultrafine monodisperse polyvinylpyrrolidone nanoparticles (PVP np) entrapping allergens/antigens of A. fumigatus were prepared by reverse micelles method. The entrapment efficiency of various formulations of PVP np varied from 74 to 92%. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), enzyme linked immunosorbent assay (ELISA) and western blot analysis showed intact integrity and immunoreactivity of the allergens/antigens after entrapment in nanoparticles. The effect of various factors such as size of the particles, ratio of monomer to antigen concentration, degree of cross-linking and schedule of immunisation were examined on the in vitro release rate and production of specific IgG and IgE antibodies in the mice. The studies showed that the formulation with 1% cross-linking agent with a mean particle size of 100 nm and a monomer: antigen ratio of 0.09:1 released encapsulated antigens in 9 weeks. The same formulation showed a sustained IgG level (>0.400 Abs) for approximately 12 weeks in comparison to IgG level (>0.400 Abs) for 7 days with free antigen. Increase in particle size or change in monomer concentration resulted in accelerated release rate. A sustained level of IgG antibodies achieved with three divided doses of entrapped antigen could also be achieved with same amount of entrapped antigens administered in single dose. The A. fumigatus specific IgE levels of nanoparticle entrapped allergens/antigens were lower than obtained with free allergens/antigens. It is concluded that PVP np provide a promising delivery system for proteins as it is biocompatible, conserves the integrity and biological activities of proteins, augments antibody response and provide a sustained antibody level with single step immunisation. The formulation may also be useful for hyposensitisation therapy for various allergens.

Immunogenicity and protection in small-animal models with controlled-release tetanus toxoid microparticles as a single-dose vaccine.

Tetanus toxoid (TT) was encapsulated in microparticles prepared from polylactide-co-glycolide polymers by a solvent-evaporation technique. Combinations of small- and large-sized microparticles with controlled-release characteristics were used to immunize Sprague-Dawley rats, and the antibody responses were monitored for 1 year. For comparison, control groups of rats were immunized at 0, 1, and 2 months with TT adsorbed to alum. The antibody responses generated by the TT entrapped in microparticles were comparable to those generated by TT adsorbed to alum in control groups from 32 weeks onwards. Microparticles with a single entrapped antigen (TT) induced better antibody responses than microparticles with two antigens (TT and diphtheria toxoid) entrapped simultaneously. A combination vaccine consisting of TT adsorbed to alum and also entrapped in microparticles gave the best antibody responses. In an inhibition assay designed to determine the relative levels of binding of antisera to the antigens, the sera from the microparticle- and the alum-immunized animals showed comparable levels of binding. In addition, in a passive-challenge study with mice, TT adsorbed to alum and TT entrapped in microparticles provided equal levels of protection against a lethal challenge with tetanus toxin. An intradermal-challenge study was also performed with rabbits, which showed similar levels of protection in sera from alum- and microparticle-immunized animals at 4, 12, and 32 weeks after immunization.

Controlled release microparticles as a single dose hepatitis B vaccine: evaluation of immunogenicity in mice

Hepatitis B surface antigen (HBsAg) was encapsulated in microparticles prepared from polylactide-co-glycolide (PLG) and polylactide (PLA) polymers using a solvent evaporation process. The immunoreactivity of the entrapped antigen was investigated by SDS-PAGE and Western blot. The microencapsulation process was modified to obtain both small (<10 μm) and large microparticles (10–100< μm), 80% of the antigen was encapsulated. Various combinations of small and large microparticles with controlled release characteristics were investigated in CD1 mice. Groups of animals were immunized with 30 μg equivalent of HBsAg in microparticles per animal. The control group received three injections of 10 μg of HBsAg on alum at 0, 1 and 6 months. Results indicated that a single injection of HBsAg in microparticles could maintain the antibody response at a level comparable to the three-injection alum schedule for at least 1 year. An in vitro inhibition assay was developed to demonstrate that antigen-antibody reactivity were comparable for the microparticle immunized mice and the alum immunized mice. A competition assay with a monoclonal antibody specific for the neutralizing epitope of HBsAg demonstrated comparable binding for the sera from the microparticle and alum immunized mice.

Sensitized Liposomes as an Antigen Delivery System for the Stimulation of Mucosal Immunity

This study was designed to exploit the ability of Peyer's patch M cells to recognize antigen-antibody complexes in the targeted delivery of a model antigen for the induction of mucosal immunity. Sensitized liposomes consisted of an entrapped model antigen, ovalbumin (OVA), and coated with unrelated antigen-antibody complexes. Sensitized liposomes were administered intrajejunally to mice either with or without monophosphoryl lipid A (MLA). Humoral immune responses were monitored in saliva, feces, serum, and bile. Mice which received sensitized liposomes showed up to 4-fold amounts of specific IgA in saliva, feces, and bile compared to controls. Transient increases in anti-OVA IgA and IgG were observed in serum. Formulations including MLA generated positive anti-OVA IgG responses in both serum and bile. In separate experiments, cell proliferation studies were performed with Peyer's patch lymphocytes harvested from mice immunized with OVA in either standard or sensitized liposomes. Lymphocytes from test mice receiving only sensitized liposomes proliferated in the presence of OVA, but not an unrelated antigen. Taken together, these results support the potential application of antigen-antibody complexes in the stimulation of mucosal immune responses and that MLA may play an important role in overcoming OVA tolerogenicity.

Liposomes as Immunoadjuvants and Vaccine Carriers: Antigen Entrapment

Successful use of liposomes as immunological adjuvants in vaccines requires simple, easy to scale up technology capable of high-yield antigen entrapment. Recent work from this laboratory has led to the development of techniques that can generate liposomes of various sizes containing soluble antigens such as proteins or particulate antigens such as whole, live, or attenuated bacteria or viruses. Entrapment of proteins is carried out by the dehydration-rehydration procedure, which entails freeze-drying of a mixture of "empty" small unilamellar vesicles and free antigens. Upon rehydration, the large multilamellar vesicles that are formed incorporate up to 80% of the antigen used. When such liposomes are microfluidized in the presence of nonentrapped material, their size is reduced to about 100 nm in diameter, with much of the originally entrapped antigen still associated with the vesicles. A similar technique applied to the entrapment of particulate antigens (e.g., Bacillus subtilis spores) consists of freeze-drying giant vesicles (4-5 μm in diameter) in the presence of spores. On rehydration and sucrose gradient fractionation of the suspension, up to 27% of the spores used are associated with generated giant liposomes of similar mean size.

Protective oral immunization of chickens against Eimeria tenella with sporozoite surface antigens

Antigens were extracted from the surface of Eimeria tenella sporozoites with a solution containing Triton X 100 (1%), sodium dodecyl sulphate (0.5%) Na deoxycholate (1%) and EDTA (1 mM). After removal of the detergents, these surface antigen preparations conferred an immunity that protected chickens against a subsequent infection (104 sporulated oocysts). The best results were obtained after two 250 μg injections of Al(OH)3 adsorbed antigens (oocyst output per g caecal material on Day 7 post infection: 2.39 × 107 ± 0.32 × 107 oocysts for controls and 7.37 × 106 ± 3.19 × 106 oocysts for vaccinated birds) and after four gastric intubations of liposome entrapped antigens (oocyst output on Day 7 postinfection: 2.75 × 106 ± 2.02 × 106 g−1 caecal material). These results represented respectively 70 and 88% protection indexes.Studies on the systemic and local antibody response after one or several infections of chickens with the parasite indicated at least 20 different molecules in the detergent antigens which are classified after immunoblotting according to their properties.

Coupling of ligands to liposomes independently of solute entrapment: observations on the formed vesicles

Bovine serum albumin (BSA), employed as a model ligand, was covalently linked (about 16% of the amount used) to small unilamellar vesicles (SUV) composed of phospholipid, cholesterol and N-(p- aminophenyl) stearylamide (APSA) (molar ratios 1:1:0.05). SUV with bound BSA were then used to generate dehydration-rehydration vesicles (DRV) in the presence of tetanus toxoid and/or carboxyfluorescein (CF). Nearly all of the SUV-bound BSA (about 15% of the original amount) was recovered in the multilamellar DRV formed, with a considerable proportion (42–62%) of the ligand becoming available on the outer bilayers. This apparent spatial reorientation of BSA within DRV also caused the entrapped toxoid to shift to some extent to the liposomal surface. There was no significant difference in the z average mean size between DRV with and without coupled BSA (543 and 555 nm diameter, respectively). Percent number diameter distribution data revealed that 71.2 (BSA-free) and 76.4% (BSA-containing DRV) of the vesicles had diameters of about 300–440 and 330–420 nm, respectively. However, in terms of percent mass diameter distribution, 69.5% (BSA-free) and 65.2% (BSA-containing DRV) of the mass was in vesicles with corresponding ranges of diameter of 1381–2975 and 1086–2840 nm. Vesicle size heterogeneity in both preparations was confirmed by freeze-fracture electron microscopy which also indicated that structures with or without bound BSA, were mostly vesicular of the multilamellar type. Judging from CF latency values, ligand-bearing DRV were stable on incubation with blood plasma at 37°C for 24 h. Stability was, however, reduced significantly when the amount of ligand bound was excessive. The present approach allows for the coupling of ligands to and the entrapment of antigens and other labile solutes in liposomes independently, thus avoiding potential damage of such solutes by the coupling reagents.

The preparation and characterization of poly(lactide-co-glycolide) microparticles. II. The entrapment of a model protein using a (water-in-oil)-in-water emulsion solvent evaporation technique.

Poly(lactide-co-glycolide) (PLG) microparticles with entrapped antigens have recently been investigated as controlled-release vaccines. This paper describes the preparation of PLG microparticles with an entrapped model antigen, ovalbumin (OVA), using a (water-in-oil)-in-water emulsion solvent evaporation technique. In a series of experiments, the effects of process parameters on particle size and OVA entrapment were investigated. It was found that smooth, spherical microparticles 1-2 microns in diameter containing up to 10% (w/w) OVA could be produced using a small volume of external aqueous phase containing a high concentration of emulsion stabilizer and a 1:5 antigen:polymer ratio. PAGE analysis, isoelectric focusing, and Western blotting of OVA released from the microparticles in vitro confirmed that the molecular weight and antigenicity of the protein remained largely unaltered by the entrapment procedure.

Ascaris suum — vaccination of mice with liposome encapsulated antigen

Vaccination with liposome encapsulated adult crude antigen with and without coencapsulated immunomodulator (levamisole) in a mice/larval Ascaris suum model provided protection against a challenge infection (2000 eggs) in mice immunised by immobilised antigen. The best results (88.9% protection) were obtained with a combination of two doses of liposome entrapped antigen with levamisole. Vaccination with liposome vaccine without modulator was slightly less effective (78.7% protection). A single dose of vaccine was ineffective (14.3% protection). Application of the soluble antigen without any adjuvants led to the enhancement of worm yield in lungs and liver.

The intestinal and serum humoral immune response of mice to orally administered antigens in liposomes: II. The response to liposome-entrapped bacterial proteins

Effective oral adjuvants are needed to improve the intestinal immune responses to oral vaccines that are based on relatively low molecular weight antigens refined from veterinary pathogens. Liposomes prepared by different methods and composed of phospholipids of varying transition temperature were used to entrap cholera toxin (CT) and fed to mice. No significant increase in the intestinal antibody nor the serum IgA antibody response was detected but levels of serum IgG anti-CT antibody were significantly elevated in the group fed CT in phosphatidylcholine-based liposomes. Levels of antibody were significantly reduced in the groups fed CT in dipalmitoylphosphatidylcholine liposomes. Escherichia coli wall extract (ECWE) entrapped in certain liposome types and fed to mice elicited significantly increased serum anti-ECWE antibody responses but intestinal antibody responses were insignificantly different from the controls. These results suggest that orally administered liposomes fail to act as potent intestinal adjuvants for the entrapped antigens of bacterial origin used in this study.