Stability of poly(epsilon-caprolactone) microparticles containing Brucella ovis antigens as a vaccine delivery system against brucellosis.

In previous works, our research group has successfully proved the use of subcellular vaccines based on poly(epsilon-caprolactone) (PEC) microparticles containing an antigenic extract of Brucella ovis (HS) against experimental brucellosis in both mice and rams. However, the successful exploitation of pharmaceutical products, and therefore of this product as veterinary vaccine, requires preservation of both biological activity and native structure in all steps of development from purification to storage. In this context, we have carried out an accelerated stability study to evaluate the relative stability of HS when loading in PEC microparticles. For this purpose, freeze-dried microparticles were stored at 40 +/- 1 degrees C and 75% RH as a preliminary analysis of a stability testing. The results showed that both physico-chemical (size, morphology, antigen content, release profile) and biological (integrity and antigenicity of the HS) properties were preserved after 6 months of storage. On the contrary, after 1 year of storage, the HS release profile was dramatically affected probably due to a progressive loss of the polymer microstructure. In addition, the degradation and loss of the antigenicity of the HS components was also evident by SDS-PAGE and immunoblotting analysis. In fact, after 12 months of storage, only the integrity and antigenicity of two of the major protective proteins of the HS antigenic complex were preserved.

Encapsulation of antigenic extracts of Salmonella enterica serovar. Abortusovis into polymeric systems and efficacy as vaccines in mice.

The properties of drug-vaccine delivery systems based on the use of biodegradable polymers, and its application in the control of experimental infection by Salmonella enterica serovar. Abortusovis (SAO), are described in this manuscript. Micelles of major membrane antigens from SAO (HSao extract) can be encapsulated in microparticles of poly(epsilon-caprolactone) or in nanoparticles of Gantrez polymer. The encapsulation process was optimized by the combined use of cyclodextrins. The resulting particles contained unaltered significant amounts of the antigenic complex. To establish the protective value of these subunit vaccines, particles were injected in one single dose (20 microg of HSao) subcutaneously in BALB/c mice in order to observe the protection conferred against experimental infection with the virulent strains S. Abortusovis 15/5. Control non-immunized animals resulted infected, as well as the group that received unloaded or HSao loaded into microparticles. In contrast, nanoparticles conferred a significant protection when compared to unvaccinated controls, similar to that induced by the attenuated commercial vaccine Rv6. In conclusion, protection against experimental infection in mice after one single shoot, and its potential for mucosal vaccination suggest that HSao-nanoparticles may represent a serious alternative to the conventional attenuated vaccines against S. Abortusovis.

Optimization of the entrapment of bacterial cell envelope extracts into microparticles for vaccine delivery.

The encapsulation of a Brucella ovis extract (HS) in microparticles has been proved effective against experimental infections in mice. This work describes different strategies to increase the HS loading and prepare large batches as necessary to test this vaccine in ovine. The mixture of HS with beta-cyclodextrin was optimized in order to increase the HS loading in microparticles. On the other hand, TROMS ('Total Recirculation One-Machine System') led microparticles with a more homogeneous size than the laboratory or standard procedure. Moreover, the initial burst release of HS from the standard microparticles was higher than for the TROMS ones. In fact, standard microparticles displayed a higher amount of adsorbed HS. On the contrary, both preparative methods were found effective to preserve the integrity and anti-genicity of the loaded HS. In summary, beta-CD can be used to increase the loading of large hydrophobic materials and TROMS is a valid large production of antigen-loaded microparticles.

Experiments on a sub-unit vaccine encapsulated in microparticles and its efficacy against Brucella melitensis in mice.

The aim of this study was to evaluate the effect of the excipients used to facilitate the encapsulation of high hydrophobic antigenic complex extracted from Brucella ovis (HS) on the physico-chemical properties of the resulting microparticles. Poly(epsilon-caprolactone) (PEC) microparticles containing HS were prepared by the solvent extraction/evaporation method using total recirculation one-machine system (TROMS). Different excipients, beta-cyclodextrin (beta-CD), Pluronic F68, Tween 20 or Tween 80, were used in order to facilitate the encapsulation and conserve the bioactivity of the encapsulated antigenic complex. HS was efficiently loaded in all the different PEC-microparticle formulations, although the combined use of beta-cyclodextrin and Pluronic F68 permitted an increase in the amount of antigenic extract in the core of the resulting microparticles without loss of its antigenic properties. Finally, the protective ability of this F68-CD-MP formulation was evaluated against an experimental challenge with the virulent Brucella melitensis H38 strain in BALB/c mice. This innocuous subcellular vaccine induced a similar protection to that of the live attenuated Rev 1 vaccine; these are promising results that would merit further investigation in target animals.

Brucella outer membrane complex-loaded microparticles as a vaccine against Brucella ovis in rams.

Due to the important drawbacks of the Brucella melitensis Rev 1 vaccine, a safer vaccine based on an outer membrane complex from Brucella ovis encapsulated in poly-epsilon-caprolactone (PEC) microparticles (MP) was developed and tested in rams. Homogeneous batches of microparticles were prepared by a new double emulsion solvent evaporation method called "Total Recirculation One-Machine System" (TROMS). Such microparticles presented a mean diameter of 2 microm and displayed an antigen loading of about 13 microg HS per mg of microparticles. Subcutaneous vaccination of rams with 800 microg HS (hot saline antigenic extract of B. ovis) in PEC microparticles induced an adequate serological response against B. ovis antigens and conferred similar protection against challenge with B. ovis to that induced by the living attenuated B. melitensis Rev 1 reference vaccine. By contrast, lower doses (80 microg) of HS-PEC evoked reduced serological responses against B. ovis antigens and did not induce significant protection. The revaccination with 800 microg of HS-PEC increased the intensity and duration of the serological response against B. ovis antigens but did not improve the protection conferred by the single vaccination. Sample sera taken from any of the animals immunized with Rev 1 were seropositive in both Rose Bengal and the Complement Fixation tests (RBT, CFT) used for the diagnosis of smooth Brucella infections. By contrast, no positive reactors in both tests were recorded in the animals vaccinated with HS-PEC, being this a target objective of this study. HS-PEC microparticles can be used as a safe vaccine against brucellosis in rams, but further studies using higher doses of antigens are necessary to exploit their full potential for the prophylaxis of brucellosis in sheep.