Ordered mesoporous silicas for potential applications in solid vaccine formulations

In an effort to develop efficient vaccine formulations, the use of ordered mesoporous silica (SBA-15) as an antigen carrier has been investigated. SBA-15 has required properties such as high surface area and pore volume, including narrow pore size distribution to protect antigens inside its matrix. This study aimed to examine the impact of solvent removal methods, specifically freeze-drying and evaporation on the intrinsic properties of an immunogenic complex. The immunogenic complexes, synthesized and incorporated with BSA, were characterized by various physicochemical techniques. Small Angle X-ray Scattering measurements revealed the characteristic reflections associated to pure SBA-15, indicating the preservation of the silica mesostructured following BSA incorporation and the formation of BSA aggregates within the macropore region. Nitrogen Adsorption Isotherm measurements demonstrated a decrease in surface area and pore volume for all samples, indicating that the BSA was incorporated into the SBA-15 matrix. Fluorescence spectroscopy evidenced that the tryptophan residues in BSA inside SBA-15 or in solution displayed similar spectra, showing the preservation of the aromatic residues’ environment. The Circular Dichroism spectra of BSA in both conditions suggest the preservation of its native secondary structure after the encapsulation process. The immunogenic analysis with the detection of anti-BSA IgG did not give any significant difference between the non-dried, freeze-dried or evaporated groups. However, all groups containing BSA and SBA-15 showed results almost three times higher than the groups with pure BSA (control group). These facts indicate that none of the BSA incorporation methods interfered with the immunogenicity of the complex. In particular, the freeze-dried process is regularly used in the pharmaceutical industry, therefore its adequacy to produce immunogenic complexes was proved Furthermore, the results showed that SBA-15 increased the immunogenic activity of BSA.

Adjuvant effect of mesoporous silica SBA-15 on anti-diphtheria and anti-tetanus humoral immune response.

Routine immunization against diphtheria and tetanus has drastically reduced the incidence of these diseases worldwide. Anti-diphtheria/tetanus vaccine has in general aluminum salt as adjuvant in its formulation that can produce several adverse effects. There is a growing interest in developing new adjuvants. In this study, we evaluated the efficiency of SBA-15 as an adjuvant in subcutaneous immunization in mice with diphtheria (dANA) and tetanus (tANA) anatoxins as well as with the mixture of them (dtANA). The tANA molecules and their encapsulation in SBA-15 were characterized using Small-Angle X-ray Scattering (SAXS), Dynamical Light Scattering (DLS), Nitrogen Adsorption Isotherm (NAI), Conventional Circular Dichroism (CD)/Synchrotron Radiation Circular Dichroism (SRCD) Spectroscopy, and Tryptophan Fluorescence Spectroscopy (FS). The primary and secondary antibody response elicited by subcutaneous immunization of High (HIII) and Low (LIII) antibody responder mice with dANA, tANA, or dtANA encapsulated in the SBA-15 were determined. We demonstrated that SBA-15 increases the immunogenicity of dANA and tANA antigens, especially when administered in combination. We also verified that SBA-15 modulates the antibody response of LIII mice, turning them into high antibody responder. Thus, these results suggest that SBA-15 may be an effective adjuvant for different vaccine formulations.

Using crystallography tools to improve vaccine formulations.

This article summarizes developments attained in oral vaccine formulations based on the encapsulation of antigen proteins inside porous silica matrices. These vaccine vehicles show great efficacy in protecting the proteins from the harsh acidic stomach medium, allowing the Peyer's patches in the small intestine to be reached and consequently enhancing immunity. Focusing on the pioneering research conducted at the Butantan Institute in Brazil, the optimization of the antigen encapsulation yield is reported, as well as their distribution inside the meso- and macroporous network of the porous silica. As the development of vaccines requires proper inclusion of antigens in the antibody cells, X-ray crystallography is one of the most commonly used techniques to unveil the structure of antibody-combining sites with protein antigens. Thus structural characterization and modelling of pure antigen structures, showing different dimensions, as well as their complexes, such as silica with encapsulated hepatitis B virus-like particles and diphtheria anatoxin, were performed using small-angle X-ray scattering, X-ray absorption spectroscopy, X-ray phase contrast tomography, and neutron and X-ray imaging. By combining crystallography with dynamic light scattering and transmission electron microscopy, a clearer picture of the proposed vaccine complexes is shown. Additionally, the stability of the immunogenic complex at different pH values and temperatures was checked and the efficacy of the proposed oral immunogenic complex was demonstrated. The latter was obtained by comparing the antibodies in mice with variable high and low antibody responses.

Adjuvant effect of mesoporous silica SBA-15 on anti-diphtheria and anti-tetanus humoral immune response

Routine immunization against diphtheria and tetanus has drastically reduced the incidence of these diseases worldwide. Anti-diphtheria/tetanus vaccine has in general aluminum salt as adjuvant in its formulation that can produce several adverse effects. There is a growing interest in developing new adjuvants. In this study, we evaluated the efficiency of SBA-15 as an adjuvant in subcutaneous immunization in mice with diphtheria (dANA) and tetanus (tANA) anatoxins as well as with the mixture of them (dtANA). The tANA molecules and their encapsulation in SBA-15 were characterized using Small-Angle X-ray Scattering (SAXS), Dynamical Light Scattering (DLS), Nitrogen Adsorption Isotherm (NAI), Conventional Circular Dichroism (CD)/Synchrotron Radiation Circular Dichroism (SRCD) Spectroscopy, and Tryptophan Fluorescence Spectroscopy (FS). The primary and secondary antibody response elicited by subcutaneous immunization of High (HIII) and Low (LIII) antibody responder mice with dANA, tANA, or dtANA encapsulated in the SBA-15 were determined. We demonstrated that SBA-15 increases the immunogenicity of dANA and tANA antigens, especially when administered in combination. We also verified that SBA-15 modulates the antibody response of LIII mice, turning them into high antibody responder. Thus, these results suggest that SBA-15 may be an effective adjuvant for different vaccine formulations.

Using crystallography tools to improve vaccine formulations

This article summarizes developments attained in oral vaccine formulations based on the encapsulation of antigen proteins inside porous silica matrices. These vaccine vehicles show great efficacy in protecting the proteins from the harsh acidic stomach medium, allowing the Peyer's patches in the small intestine to be reached and consequently enhancing immunity. Focusing on the pioneering research conducted at the Butantan Institute in Brazil, the optimization of the antigen encapsulation yield is reported, as well as their distribution inside the meso- and macroporous network of the porous silica. As the development of vaccines requires proper inclusion of antigens in the antibody cells, X-ray crystallography is one of the most commonly used techniques to unveil the structure of antibody-combining sites with protein antigens. Thus structural characterization and modelling of pure antigen structures, showing different dimensions, as well as their complexes, such as silica with encapsulated hepatitis B virus-like particles and diphtheria anatoxin, were performed using small-angle X-ray scattering, X-ray absorption spectroscopy, X-ray phase contrast tomography, and neutron and X-ray imaging. By combining crystallography with dynamic light scattering and transmission electron microscopy, a clearer picture of the proposed vaccine complexes is shown. Additionally, the stability of the immunogenic complex at different pH values and temperatures was checked and the efficacy of the proposed oral immunogenic complex was demonstrated. The latter was obtained by comparing the antibodies in mice with variable high and low antibody responses.

Assessing the efficiency of SBA-15 as a nanocarrier for diphtheria anatoxin

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.

Avaliação da resposta imune aos Antígenos S e Pré-S1-S2/S da Hepatite B em camundongos geneticamente selecionados (HIII E LIII) e BALB/C

Introdução: A vacina recombinante da hepatite B, atualmente utilizada, mostra alta variação de resposta de anticorpos, incluindo 5 a 10% de não-responsividade após a imunização. Desde 1998, o Instituto Butantan vem reproduzindo essa vacina a qual é composta pelo antigeno de superfície S e no intuito de superar o efeito de não-responsividade, está desenvolvendo uma nova vacina recombinante que expressa à proteína S e outros componentes de superfície do virus: pré-S1 e pré-S2, Objetivos: Analisar a produção de anticorpos e o perfil de ativação de células CD4/CD8/CD19 em camundongos Balb/C e em camundongos geneticamente selecionado para boa e má produção de anticorpos, em resposta à imunização com a vacina composta pelos antígenos pré-S1-S2/S, para avaliar parâmetros da resposta imunológicas á essas duas preparações vacinais e estabelecer um modelo animal que represente diferentes padrões de resposta aos antígenos de superfície do virus da hepatite. Métodos: Foram utilizadas duas linhagens de camundongos, obtidas por seleção genética bidirecional de acordo com alta (HIII) ou baixa (LIII) produção de anticorpos em resposta a antígenos flagelares de Salmonella. Além disso, camundongos Balb/C também foram utilizados, uma vez que está é uma linhagem mundialmente utilizada como modelo animal em testes de vacinas. Amostras de antigenos S e Pré-S1-S2/S purificados foram produzidas e fornecidas pelo Laboratório de Hepatite do Instituto Butantan. Os grupos de camundongos receberam duas doses dos antígenos S ou Pré-S1-S2/S absorvidos em hidróxido de alumínio pela via subcutânea com intervalo de 30 dias. O soro individual foi periodicamente coletado e testado por ELISA para titulação específica de anticorpos. Além disso, sete dias após a ultima coleta e a injeção dos antígenos por via intreperitoneal, isolou-se o baço e o linfonodo mesentérico desses animais, cujas células foram citometria de fluxo. Resultados e Discussão: Os antígenos S e Pré-S1-S2/S estimularam semelhntes níveis de resposta ao antígeno Pré-S1-S2/S apresentaram títulos de IgG específicos mais altos aos 15 dias (p<0,05) em camundongos LIII e níveis mais altos de CD19 em camundongos HIII quando comparados aos níveis de resposta ao antígeno Pré-S1-S2/S (p<0,01). Os camundongos Balb/C não mostraram diferenças na resposta de anticorpos e estimulação celular a ambos os antígenos. Estes resultados mostram que a resposta imunológica final frente às duas preparações vacinais é semelhante e que as modificações genéticas presentes nas linhagens HIII e LIII provocaram diferenças na resposta imune aos antígenos S e Pré-S1-S2/S da hepatite, indicando assim que este pode ser usado como modelo para estudos da resposta da vacina contra hepatite B.

Characterization of production processes for tetanus and diphtheria anatoxins.

Tetanus and diphtheria are diseases that still cause significant morbidity and mortality. Clostridium tetani produces the tetanus toxin, a 150-kDa protein. The diphtheria toxin is synthesized by Corynebacterium diphtheriae as a protein of 58 kDa. The objective of this study was to carry out a chemical characterization of the tetanus and diphtheria toxin forms in the several production process stages, and thus to establish an affordable alternative in vitro quality control to aggregate to the classical tests. The 150 kDa band of the tetanus toxin and approximately 58 kDa band of the diphtheria toxin were observed by electrophoresis similar as that described in the literature. The same band of 58 KDa was detected in Western blotting reactions. The results obtained for diphtheria toxin showed very similar protein profiles between distinct lots. For the tetanus toxin, the profiles of the initial stage showed some variability, but the ones of the following stages were similar. The similarity of the electrophoresis results indicated reproduction and consistency of the production processes in Butantan Institute and correlated with the yield and antigenic purity classical data. The establishment of alternative in vitro quality control tests can significantly contribute to achieve the consistency approach supported by WHO.

Lymphatic system as a path underlying the spread of lung and gut injury after intestinal ischemia/reperfusion in rats.

We investigated in rats the influence of the lymphatic system and of tumor necrosis factor (TNF) on the lung inflammation resulting from intestinal ischemia/reperfusion (I/R) performed by 45-min occlusion of the superior mesenteric artery followed by 2 h of reperfusion. A group of rats had the thoracic lymph duct ligated before I/R. In lungs, intestinal I/R evoked a significant neutrophil recruitment, and enhanced microvascular permeability, in addition to generation of TNF in serum. In the gut, there was lowered lactate dehydrogenase (LDH) activity and increased microvascular permeability. Upon lymph duct ligation, I/R rats had a significant reduction of pulmonary neutrophil recruitment and plasma extravasation, in addition to high amounts of TNF in the lymph, contrasting with undetectable levels in the serum. In addition, LDH gut levels in these animals were close to basal values; there was also some (yet significant) reduction of microvascular permeability, suggesting that the ligation of the lymphatic duct exerted some degree of protection against the intestinal injury caused by I/R. In I/R rats, the treatment with pentoxifylline (PTX) reduced TNF in serum and blunted other lung alterations. The gut alterations caused by intestinal I/R were largely blocked by PTX. On the other hand, in I/R rats with lymph duct ligation, PTX exacerbated the reduction of pulmonary neutrophil recruitment, but did not affect pulmonary and intestinal microvascular permeabilities. Similarly, intestinal LDH activity and serum TNF levels were unaffected. Overall, our data show that the pulmonary and gut injuries induced by intestinal I/R are partially dependent on TNF, which is conceivably generated in the injured gut tissue due to intestinal I/R and carried by the lymphatic system. Thus, the mesenteric lymphatic drainage seems to play a role as a path modulator of the pulmonary and intestinal dysfunctions that follow a gut trauma.

Lung microvascular permeability and neutrophil recruitment are differently regulated by nitric oxide in a rat model of intestinal ischemia-reperfusion.

We investigated the effect of two inhibitors of nitric oxide (NO) synthesis, N(w)-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine, on lung inflammation caused by intestinal ischemia/reperfusion in rats. Relative to the sham-operated rats, intestinal ischemia/reperfusion (ischemia: 45 min; reperfusion: 30 min, 2 and 4 h) induced neutrophil recruitment (increased myeloperoxidase activity) and increased microvascular permeability (Evans blue dye extravasation) in the lungs and increased tumor necrosis factor (TNF) levels in the serum (L-929 cytotoxicity assay). L-NAME given before the ischemia exacerbated neutrophil accumulation, plasma extravasation, serum TNF and caused death of the animals, which was prevented by concomitant injection of L-arginine. Lung and systemic effects of intestinal ischemia/reperfusion were not modified when L-NAME was given just before reperfusion. Treatment with aminoguanidine inhibited plasma extravasation without affecting the other parameters evaluated. Dexamethasone reduced all the parameters. Our results indicate that during intestinal ischemia/reperfusion both constitutive and inducible NO synthases are called to exert a differential modulatory effect on lung inflammation and that maintenance of adequate levels of NO during ischemia is essential for the animals survival.

Differential regulation of the release of tumor necrosis factor-alpha and of eicosanoids by mast cells in rat airways after antigen challenge.

BACKGROUND: Rat trachea display a differential topographical distribution of connective tissue mast cells (CTMC) and mucosal mast cells (MMC) that may imply regional differences in the release of allergic mediators such as tumor necrosis factor-alpha (TNF-alpha) and eicosanoids. AIM: To evaluate the role of CTMC and MMC for release of TNF-alpha and eicosanoids after allergenic challenge in distinct segments of rat trachea. MATERIALS AND METHODS: Proximal trachea (PT) and distal trachea (DT) from ovalbumin (OVA)-sensitized rats, treated or not with compound 48/80 (48/80) or dexamethasone, were incubated in culture medium. After OVA challenge, aliquots were collected to study release of TNF-alpha and eicosanoids. RESULTS: Release of TNF-alpha by PT upon OVA challenge peaked at 90 min and decayed at 6 and 24 h. Release from DT peaked at 30-90 min and decayed 6 and 24 h later. When CTMC were depleted with 48/80, OVA challenge exacerbated the TNF-alpha release by PT at all time intervals, while DT exacerbated TNF-alpha levels 6 and 24 h later only. Dexamethasone reduced TNF-alpha production after 90 min of OVA challenge in PT and at 3 and 6h in DT. OVA challenge increased prostaglandin D2) in DT and leukotriene B4 in both segments but did not modify prostaglandin E2 and leukotriene C4 release. CONCLUSION: OVA challenge induces TNF-alpha release from MMC, which is negatively regulated by CTMC. The profile of TNF-alpha and eicosanoids depends on the time after OVA challenge and of the tracheal segment considered.

Co-localization of quantitative trait loci regulating resistance to Salmonella typhimurium infection and specific antibody production phenotypes.

Salmonella enterica serotype typhimurium is a facultative intracellular bacteria that induces systemic infection in mice. Resistance to this pathogen is under polygenic control in which Nramp1 is the major gene involved. Lines of mice obtained by selective breeding for high (HIII) or low (LIII) antibody response to flagellar antigens of salmonellae showed significant susceptibility differences, although both the lines display Nramp1(R) alleles. The HIII line was extremely susceptible to infection, while the LIII line was resistant. In order to examine the cellular and genetic mechanisms involved in this distinct pattern of resistance, HIII and LIII mice were analyzed for IFNgamma and IL4 production and screened for quantitative trait loci involved in S. typhimurium infection, using several polymorphic microsatellites. In the present work, HIII mice showed an IFNgamma downregulation in the early phase of infection when compared with LIII animals. No interline differences in IL4 production were verified. The loci screening was performed on immunized F2 intercrosses obtained from HIII and LIII mice. Three antibody-controlling chromosomal regions were coincident, and another was mapped near one of the four loci known to affect susceptibility to S. typhimurium. These results indicate a major role of IFNgamma in our model, and suggest the co-localization of quantitative trait loci modulating both infection and antibody production phenotypes.