Claudio Thomasin

A single administration of tetanus toxoid in biodegradable microspheres elicits T cell and antibody responses similar or superior to those obtained with aluminum hydroxide

The use of biodegradable polymer microspheres as a single dose vaccine delivery system was investigated by using tetanus toxoid (TT). In order to compare the immunogenicity of TT-microspheres (TT-MS) with aluminum hydroxide (alum)-based TT, BALB/c mice were immunized with TT in different formulations including individual or mixtures of MS and TT-alum. All TT-MS formulations elicited high proliferative and antibody responses comparable to those obtained with TT-alum formulation. Antibody levels remained elevated over a long period of time. Certain individual MS preparations elicited lower antibody titers than the MS mixtures. More importantly, the proliferative and antibody responses induced by a single injection of three TT-MS mixtures with different particle sizes and degradation rates were similar to those obtained with three injections of TT-alum. In addition, TT-MS induce similar isotypesubclass antibodies to those TT-alum induced. These results raise the possibility to obtain optimal and long-lasting immune responses by single administration of the three TT-MS mixture formulations alone.

Tetanus toxoid and synthetic malaria antigen containing poly(lactide)/poly(lactide-co-glycolide) microspheres: importance of polymer degradation and antigen release for immune response

Abstract The importance of in vitro degradation of poly(lactide)/poly(lactide-co-glycolide) (PLA/PLGA) microspheres and of the concomitant in vitro release of a natural and a synthetic antigen for eliciting immune response was studied in mice. A variety of PLAs and PLGAs differing in molecular weight ( M w of 14–130 kDa) and in polymer composition (lactic/glycolic acid ratio of 100:00, 75:25, and 50:50) were examined for their in vitro degradation, which ranged from approximately 4 to 20 weeks. Three specific polymers were then selected for microencapsulation of the two antigens tetanus toxoid (TT) and a weakly immunogenic synthetic branched malaria peptide (P30B2). The in vitro release data showed that antigen delivery correlates fairly well with polymer degradation giving rise to a distinct burst release during the first 24 h and an additional release pulse towards the end of polymer degradation. After single subcutaneous administration in mice, long lasting high antibody titers were obtained with the antigen containing microspheres, as compared to TT adsorbed on alum or to P30B2 in Incomplete Freunds Adjuvant. Moreover, the immune responses induced by microspheres were clearly influenced by the antigen release kinetics, the polymer type and the size of the microspheres. The results demonstrate the immunopotentiating properties of the biodegradable microspheres and their potential to elicit long-lasting immune responses after single administration when tailoring in vitro release characteristics and particle size.

MHC class I- and class II-restricted processing and presentation of microencapsulated antigens

Macrophages were found of having a strong capacity of phagocytosing small size microcapsules (MS) and presenting microencapsulated antigens to either CD4+ and CD8- T cells. The class I-restricted presentation of microencapsulated tetanus toxoid by macrophages requires an intracellular processing which might follow the phagosome-to-cytosol route to enter the classical MHC class I presentation pathway. In contrast, presentation of microencapsulated cytotoxic peptide PbCS252-260 to specific CD8+ T cells has been observed with different APC and is not blocked by cytochalasin D, suggesting that peptide released from MS may directly bind to MHC class I molecules on the cell surface. In the case of MHC class II-restricted T cells, prefixation or treatment of macrophages with chloroquine, brefeldin A and cycloheximide inhibits the presentation of microencapsulated and soluble tetanus toxoid. These findings illustrate the capacity of microencapsulated antigens to enter different presentation pathways and should facilitate the development of subunit vaccines.

Physico-chemical parameters governing protein microencapsulation into biodegradable polyesters by coacervation

Coacervation of various PLAs (poly(d,1,-lactic acid)) and PLGAs (poly(d,1-lactic-co-glycolic acid)) was studied for microencapsulation of BSA powder and aqueous BSA solution, focusing on the interfacial properties between coacervate, continuous liquid and solid or aqueous BSA and on selected process parameters. From interfacial tensions and contact angles, the necessary conditions for successful entrapment of both solid and aqueous BSA were given in a coacervation system using either dichloromethane or ethyl acetate as polymer solvents and silicone oil as the coacervating agent. Spreading coefficients were positive at the coacervate/aqueous BSA interface (0.2–2.6 mN m−1), but negative between continuous liquid and aqueous BSA. For solid BSA, work of adhesion to the coacervate was substantially higher (41–52 mN m−1) than to the continuous liquid (20–35 mN m−1). The process parameters, physical state and particle size of the protein, amount and viscosity of the coacervating agent, type and obvar|Mw, of polymer, nominal protein loading and amount of aqueous protein solution were the most critical with respect to encapsulation efficiency and burst release. Highest encapsulation efficiency (60–80%) and acceptable burst release ( < 20%) were found with micronized and aqueous BSA at low (1%) nominal loading, using a hydrophilic low ovbar|Mw polymer and a coacervating agent of moderate viscosity (100–1000 mPa s).