RESUMO
Ordered mesoporous silica was proved to be an efficient oral adjuvant capable to deliver a wide in size variety of different antigens, promoting efficient immunogenicity. This material can be used in single or polivalent vaccines, which have been developed by a group of Brazilian scientists. The experiments performed with the model protein Bovine Serum Albumin (BSA) gave the first promissing results, that were also achieved by testing the virus like particle surface antigen of hepatitis B (HBsAg) and diphtheria anatoxin (dANA). Nanostructured ordered mesoporous silica, SBA-15 type, with bi-dimensional hexagonal porous symmetry was used to encapsulate the antigens either in the mesoporous (pore diameter ~10 nm) or macroporous ( pore diameter > 50 nm) regions. This silica vehicle proved to be capable to create an inflammatory response, did not exhibit toxicity, being effective to induce immunity in high and low responder mice towards antibody production. The silica particles are in the range of micrometer size, leaving no trace in mice organs due to its easy expulsion by faeces. The methods of Physics, usually employed to characterize the structure, composition and morphology of materials are of fundamental importance to develop proper oral vaccines in order to state the ideal antigen load to avoid clustering and to determine the rate of antigen release in different media mimicking body fluids.
RESUMO
SBA-15 ordered mesoporous silica can be considered a promising inorganic nanocarrier with emerging potential as an oral vaccine adjuvant. In this study, we investigated its application in the encapsulation of the diphtheria anatoxin (dANA). We observed a considerable preservation of dANA secondary and tertiary structures, even after the drying process by means of Circular Dichroism (CD) and fluorescence spectroscopies. Antigen loading was assessed at a number of different ratios of adjuvant-to-antigen using a combination of nitrogen adsorption porosimetry and Small Angle X-ray Scattering (SAXS). Our data showed that the mass ratio of 1:10 (dANA:SBA-15) is recommended for total encapsulation of dANA in the mesopores, considering that at this relative mass concentration antigen clustering was avoided, which is deleterious effect for immunization purposes. dANA release in mimetic intestine fluid, at a pH equal to 6.8, was followed by in-situ SAXS measurements and shown to be slow, being more pronounced after 6 h and continuous up to 35 h. Finally, the immunogenic complex was tested in isogenic Balb C mice by oral and subcutaneous immunization routes, including a comparison with the only permitted adjuvant for human use, aluminum hydroxide. A higher antibody titer was obtained by subcutaneous and oral administration routes using SBA-15 as the vehicle of dANA, compared with the conventional aluminum hydroxide, demonstrating the viability to use this ordered mesoporous silica in the formulation of oral vaccines, as already proved for the Virus Like Particles (VLP) Hepatitis B (HBsAg) case.
RESUMO
Developing a technology that enables oral vaccines to work efficiently remains a considerable effort since a number of difficulties must be addressed. The key objective being to ensure the safe passage through the harsh conditions within the gastrointestinal tract, promoting delivery that induces enhanced immune response. In the particular case of hepatitis B, the oral formulation in the nanostructured silica SBA-15 is a viable approach. As a result of its porous structure, low toxicity and structural stability, SBA-15 is capable to protect and release the hepatitis B surface antigen (HBsAg), used in the vaccination scheme, at the desired destination. Furthermore, when compared to the currently used injection based delivery method, better or similar antibody response has been observed. However, information about the organisation of the antigen protein remains unknown. For instance, HBsAg is too large to enter the 10?nm ordered mesopores of SBA-15 and has a tendency to agglomerate when protected by the delivery system. Here we report on the pH dependence of HBsAg aggregation in saline solution investigated using small angle X-rays scattering that resulted in an optimisation of the encapsulation conditions. Additionally, X-ray microscopy combined with neutron and X-ray tomography provided full 3D information of the HBsAg clustering (i.e. agglomeration) inside the SBA-15 macropores. This method enables the visualisation of the organisation of the antigen in the interior of the delivery system, where agglomerated HBsAg coexists with its immunological effective uniformly distributed counterpart. This new approach, to be taken into account while preparing the formulation, can greatly help in the understanding of clinical studies and advance new formulations.
RESUMO
As a consequence of its ordered pore architecture, mesoporous SBA-15 offers new possibilities for incorporating biological agents. Considering its applicability in oral vaccination, which shows more beneficial features when compared with parenteral vaccines, SBA-15 is also seen as a very promising adjuvant to carry, protect, and deliver entrapped antigens. Recent studies have shown several remarkable features in the immunization of hepatitis B, a viral disease transmitted mainly through blood or serum transfer. However, the surface antigen of the hepatitis B virus, HBsAg, is too large to fit inside the SBA-15 matrix with mean pore diameter around 10 nm, thus raising the question of how SBA-15 can protect the antigen. In this work, thermal analysis combined with neutron spectroscopy allowed us to shed light on the interactions between HBsAg and SBA-15 as well as on the role that these interactions play in the efficiency of this promising oral vaccination method. This information was obtained by verifying how the dynamic behaviour of the antigen is modified under confinement in SBA-15, thus also establishing an experimental method for verifying molecular dynamics simulations.
RESUMO
Developing a technology that enables oral vaccines to work efficiently remains a considerable effort since a number of difficulties must be addressed. The key objective being to ensure the safe passage through the harsh conditions within the gastrointestinal tract, promoting delivery that induces enhanced immune response. In the particular case of hepatitis B, the oral formulation in the nanostructured silica SBA-15 is a viable approach. As a result of its porous structure, low toxicity and structural stability, SBA-15 is capable to protect and release the hepatitis B surface antigen (HBsAg), used in the vaccination scheme, at the desired destination. Furthermore, when compared to the currently used injection based delivery method, better or similar antibody response has been observed. However, information about the organisation of the antigen protein remains unknown. For instance, HBsAg is too large to enter the 10?nm ordered mesopores of SBA-15 and has a tendency to agglomerate when protected by the delivery system. Here we report on the pH dependence of HBsAg aggregation in saline solution investigated using small angle X-rays scattering that resulted in an optimisation of the encapsulation conditions. Additionally, X-ray microscopy combined with neutron and X-ray tomography provided full 3D information of the HBsAg clustering (i.e. agglomeration) inside the SBA-15 macropores. This method enables the visualisation of the organisation of the antigen in the interior of the delivery system, where agglomerated HBsAg coexists with its immunological effective uniformly distributed counterpart. This new approach, to be taken into account while preparing the formulation, can greatly help in the understanding of clinical studies and advance new formulations.
RESUMO
As a consequence of its ordered pore architecture, mesoporous SBA-15 offers new possibilities for incorporating biological agents. Considering its applicability in oral vaccination, which shows more beneficial features when compared with parenteral vaccines, SBA-15 is also seen as a very promising adjuvant to carry, protect, and deliver entrapped antigens. Recent studies have shown several remarkable features in the immunization of hepatitis B, a viral disease transmitted mainly through blood or serum transfer. However, the surface antigen of the hepatitis B virus, HBsAg, is too large to fit inside the SBA-15 matrix with mean pore diameter around 10 nm, thus raising the question of how SBA-15 can protect the antigen. In this work, thermal analysis combined with neutron spectroscopy allowed us to shed light on the interactions between HBsAg and SBA-15 as well as on the role that these interactions play in the efficiency of this promising oral vaccination method. This information was obtained by verifying how the dynamic behaviour of the antigen is modified under confinement in SBA-15, thus also establishing an experimental method for verifying molecular dynamics simulations.