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.

Formyl peptide receptors are involved in CTX-induced impairment of lymphocyte functions.

Crotoxin (CTX) is a neurotoxin that is isolated from the venom of Crotalus durissus terrificus, which displays immunomodulatory, anti-inflammatory, and anti-tumoral effects. Previous research has demonstrated that CTX promotes the adherence of leukocytes to the endothelial cells in blood microcirculation and the high endothelial venules of lymph nodes, which reduces the number of blood cells and lymphocytes. Studies have also shown that these effects are mediated by lipoxygenase-derived mediators. However, the exact lipoxygenase-derived eicosanoid involved in the CTX effect on lymphocytes is yet to be characterized. As CTX stimulates lipoxin-derived mediators from macrophages and lymphocyte effector functions could be modulated by activating formyl peptide receptors, we aimed to investigate whether these receptors were involved in CTX-induced redistribution and functions of lymphocytes in rats. We used male Wistar rats treated with CTX to demonstrate that Boc2 (butoxycarbonyl-Phe-Leu-Phe-Leu-Phe), an antagonist of formyl peptide receptors, prevented CTX-induced decrease in the number of circulating lymphocytes and increased the expression of the lymphocyte adhesion molecule LFA1. CTX reduced the T and B lymphocyte functions, such as lymphocyte proliferation in response to the mitogen Concanavalin A and antibody production in response to BSA immunization, respectively, which was prevented by the administration of Boc2. Importantly, mesenteric lymph node lymphocytes from CTX-treated rats showed an increased release of 15-epi-LXA4. These results indicate that formyl peptide receptors mediate CTX-induced redistribution of lymphocytes and that 15-epi-LXA4 is a key mediator of the immunosuppressive effects of CTX.

Evaluation of tumor growth remission in a murine model for subcutaneous solid tumors, benefits of associating the antitumor agent crotamine with mesoporous nanosilica particles to achieve improved dosing frequency and efficacy

Crotamine is a highly cationic polypeptide first isolated from South American rattlesnake venom, which exhibits affinity for acidic lysosomal vesicles and proliferating cells. This cationic nature is pivotal for its in vitro cytotoxicity and in vivo anticancer actions. This study aimed to enhance the antitumor efficacy of crotamine by associating it with the mesoporous SBA-15 silica, known for its controlled release of various chemical agents, including large proteins. This association aimed to mitigate the toxic effects while amplifying the pharmacological potency of several compounds. Comprehensive characterization, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential analysis, confirmed the successful association of crotamine with the non-toxic SBA-15 nanoparticles. The TEM imaging revealed nanoparticles with a nearly spherical shape and variations in uniformity upon crotamine association. Furthermore, DLS showed a narrow unimodal size distribution, emphasizing the formation of small aggregates. Zeta potential measurements indicated a distinct shift from negative to positive values upon crotamine association, underscoring its effective adsorption onto SBA-15. Intraperitoneal or oral administration of crotamine:SBA-15 in a murine melanoma model suggested the potential to reduce the frequency of crotamine doses without compromising efficacy. Interestingly, while the oral route enhanced the antitumor efficacy of crotamine, pH-dependent release from SBA-15 was observed. Thus, associating crotamine with SBA-15 could reduce the overall required dose to inhibit solid tumor growth, bolstering the prospect of crotamine as a potent anticancer agent.

Evaluation of tumor growth remission in a murine model for subcutaneous solid tumors, benefits of associating the antitumor agent crotamine with mesoporous nanosilica particles to achieve improved dosing frequency and efficacy

Crotamine is a highly cationic polypeptide first isolated from South American rattlesnake venom, which exhibits affinity for acidic lysosomal vesicles and proliferating cells. This cationic nature is pivotal for its in vitro cytotoxicity and in vivo anticancer actions. This study aimed to enhance the antitumor efficacy of crotamine by associating it with the mesoporous SBA-15 silica, known for its controlled release of various chemical agents, including large proteins. This association aimed to mitigate the toxic effects while amplifying the pharmacological potency of several compounds. Comprehensive characterization, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential analysis, confirmed the successful association of crotamine with the non-toxic SBA-15 nanoparticles. The TEM imaging revealed nanoparticles with a nearly spherical shape and variations in uniformity upon crotamine association. Furthermore, DLS showed a narrow unimodal size distribution, emphasizing the formation of small aggregates. Zeta potential measurements indicated a distinct shift from negative to positive values upon crotamine association, underscoring its effective adsorption onto SBA-15. Intraperitoneal or oral administration of crotamine:SBA-15 in a murine melanoma model suggested the potential to reduce the frequency of crotamine doses without compromising efficacy. Interestingly, while the oral route enhanced the antitumor efficacy of crotamine, pH-dependent release from SBA-15 was observed. Thus, associating crotamine with SBA-15 could reduce the overall required dose to inhibit solid tumor growth, bolstering the prospect of crotamine as a potent anticancer agent.

Use of nanostructured silica SBA-15 as an oral vaccine adjuvant to control mycoplasma hyopneumoniae in swine production

Mycoplasma hyopneumoniae is a difficult-to-control bacterium since commercial vaccines do not prevent colonization and excretion. The present study aimed to evaluate the performance of an orally administered vaccine composed of antigens extracted from Mycoplasma hyopneumoniae and incorporated into mesoporous silica (SBA-15), which has an adjuvant-carrier function, aiming to potentiate the action of the commercial intramuscular vaccine. A total of 60 piglets were divided into four groups (n = 15) submitted to different vaccination protocols as follows, Group 1: oral SBA15 + commercial vaccine at 24 days after weaning, G2: oral vaccine on the third day of life + vaccine commercial vaccine at 24 days, G3: commercial vaccine at 24 days, and G4: commercial vaccine + oral vaccine at 24 days. On the first day, the piglets were weighed and, from the third day onwards, submitted to blood collections for the detection and quantification of anti-Mycoplasma hyopneumoniae IgG. Nasal swabs were collected to monitor IgA by ELISA, and oropharyngeal swabs were used to assess the bacterial load by qPCR. Biological samples were collected periodically from the third day of life until the 73rd day. At 41 days of life, 15 individuals of the same age, experimentally challenged with an inoculum containing M. hyopneumoniae, were co-housed with the animals from groups (1 to 4) in a single pen to increase the infection pressure during the nursery period. At 73 days, all piglets were euthanized, and lungs were evaluated by collecting samples for estimation of bacterial load by qPCR. Quantitative data obtained from physical parameters and laboratory investigation were analyzed by performing parametric or non-parametric statistical tests. Results indicate that animals from G2 showed smaller affected lung areas compared to G3. Animals from G2 and G4 had a low prevalence of animals shedding M. hyopneumoniae at 61 days of age. Additionally, no correlation was observed between lung lesions and M. hyopneumoniae load in lung and BALF samples in animals that received the oral vaccine, while a strong correlation was observed in other groups. In the present study, evidence points to the effectiveness of the oral vaccine developed for controlling M. hyopneumoniae in pig production under field conditions.

Efficacy evaluation of a novel oral silica-based vaccine in inducing mucosal immunity against Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae, the main etiological agent of Porcine Enzootic Pneumonia, is widely spread in swine production worldwide. Its prevention is of great interest for the productive system, since its colonization in the lung tissue leads to intense production losses. This study aimed to compare the M. hyopneumoniae shedding and acute-phase response in 30 pigs submitted to different vaccination protocols: an experimental oral vaccine using a nanostructured mesoporous silica (SBA-15) as adjuvant (n = 10); an intramuscular commercially available vaccine at 24 days of age (n = 10); and a control group (n = 10) following experimental challenge with M. hyopneumoniae. Laryngeal and nasal swabs were collected weekly and oral fluids were collected at 7, 10, 14, 17, 23, 28, 35, 42, and 49 days post-infection to monitor pathogen excretion by qPCR. Nasal swabs were also used to detect anti-M. hyopneumoniae IgA by ELISA. Blood samples were collected for monitoring acute phase proteins. The antibody response was observed in both immunized groups seven days after vaccination, while the control group became positive for this immunoglobulin at 4 weeks after challenge. Lung lesion score was similar in the immunized groups, and lower than that observed in the control. SBA-15-adjuvanted oral vaccine provided immunological response, decreased shedding of M. hyopneumoniae and led to mucosal protection confirmed by the reduced pulmonary lesions. This study provides useful data for future development of vaccines against M. hyopneumoniae.

Formyl peptide receptors are involved in CTX-induced impairment of lymphocyte functions

Crotoxin (CTX) is a neurotoxin that is isolated from the venom of Crotalus durissus terrificus, which displays immunomodulatory, anti-inflammatory, and anti-tumoral effects. Previous research has demonstrated that CTX promotes the adherence of leukocytes to the endothelial cells in blood microcirculation and the high endothelial venules of lymph nodes, which reduces the number of blood cells and lymphocytes. Studies have also shown that these effects are mediated by lipoxygenase-derived mediators. However, the exact lipoxygenase-derived eicosanoid involved in the CTX effect on lymphocytes is yet to be characterized. As CTX stimulates lipoxin-derived mediators from macrophages and lymphocyte effector functions could be modulated by activating formyl peptide receptors, we aimed to investigate whether these receptors were involved in CTX-induced redistribution and functions of lymphocytes in rats. We used male Wistar rats treated with CTX to demonstrate that Boc2 (butoxycarbonyl-Phe-Leu-Phe-Leu-Phe), an antagonist of formyl peptide receptors, prevented CTX-induced decrease in the number of circulating lymphocytes and increased the expression of the lymphocyte adhesion molecule LFA1. CTX reduced the T and B lymphocyte functions, such as lymphocyte proliferation in response to the mitogen Concanavalin A and antibody production in response to BSA immunization, respectively, which was prevented by the administration of Boc2. Importantly, mesenteric lymph node lymphocytes from CTX-treated rats showed an increased release of 15-epi-LXA4. These results indicate that formyl peptide receptors mediate CTX-induced redistribution of lymphocytes and that 15-epi-LXA4 is a key mediator of the immunosuppressive effects of CTX.

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.

Antibodies as Snakebite Antivenoms: Past and Future.

Snakebite envenomation is considered a neglected tropical disease, affecting tens of thousands of people each year. The recommended treatment is the use of antivenom, which is composed of immunoglobulins or immunoglobulin fragments obtained from the plasma of animals hyperimmunized with one (monospecific) or several (polyspecific) venoms. In this review, the efforts made in the improvement of the already available antivenoms and the development of new antivenoms, focusing on snakes of medical importance from sub-Saharan Africa and Latin America, are described. Some antivenoms currently used are composed of whole IgGs, whereas others use F(ab')2 fragments. The classic methods of attaining snake antivenoms are presented, in addition to new strategies to improve their effectiveness. Punctual changes in immunization protocols, in addition to the use of cross-reactivity between venoms from different snakes for the manufacture of more potent and widely used antivenoms, are presented. It is known that venoms are a complex mixture of components; however, advances in the field of antivenoms have shown that there are key toxins that, if effectively blocked, are capable of reversing the condition of in vivo envenomation. These studies provide an opportunity for the use of monoclonal antibodies in the development of new-generation antivenoms. Thus, monoclonal antibodies and their fragments are described as a possible alternative for the production of antivenoms, regardless of the venom. This review also highlights the challenges associated with their development.

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.

Antibodies as snakebite antivenoms: past and future

Snakebite envenomation is considered a neglected tropical disease, affecting tens of thousands of people each year. The recommended treatment is the use of antivenom, which is composed of immunoglobulins or immunoglobulin fragments obtained from the plasma of animals hyperimmunized with one (monospecific) or several (polyspecific) venoms. In this review, the efforts made in the improvement of the already available antivenoms and the development of new antivenoms, focusing on snakes of medical importance from sub-Saharan Africa and Latin America, are described. Some antivenoms currently used are composed of whole IgGs, whereas others use F(ab’)2 fragments. The classic methods of attaining snake antivenoms are presented, in addition to new strategies to improve their effectiveness. Punctual changes in immunization protocols, in addition to the use of cross-reactivity between venoms from different snakes for the manufacture of more potent and widely used antivenoms, are presented. It is known that venoms are a complex mixture of components; however, advances in the field of antivenoms have shown that there are key toxins that, if effectively blocked, are capable of reversing the condition of in vivo envenomation. These studies provide an opportunity for the use of monoclonal antibodies in the development of new-generation antivenoms. Thus, monoclonal antibodies and their fragments are described as a possible alternative for the production of antivenoms, regardless of the venom. This review also highlights the challenges associated with their development.

O envenenamento por picada de cobra é considerado uma doença tropical negligenciada, afetando dezenas de milhares de pessoas a cada ano. O tratamento recomendado é o uso de antiveneno, que é composto por imunoglobulinas ou fragmentos de imunoglobulinas obtidos do plasma de animais hiperimunizados com um (monoespecífico) ou vários (poliespecíficos) venenos. Nesta revisão, são descritos os esforços realizados no aprimoramento dos antivenenos já disponíveis e no desenvolvimento de novos antivenenos, com foco em serpentes de importância médica da África Subsaariana e América Latina. Alguns antivenenos atualmente utilizados são compostos por IgGs inteiros, enquanto outros usam fragmentos F(ab’)2. São apresentados os métodos clássicos de obtenção de soros de serpentes, além de novas estratégias para melhorar sua eficácia. São apresentadas mudanças pontuais nos protocolos de imunização, além do uso de reatividade cruzada entre venenos de diferentes serpentes para a fabricação de antivenenos mais potentes e amplamente utilizados. Sabe-se que os venenos são uma mistura complexa de componentes; no entanto, avanços na área de antivenenos mostraram que existem toxinas-chave que, se efetivamente bloqueadas, são capazes de reverter a condição de envenenamento in vivo. Esses estudos oferecem uma oportunidade para o uso de anticorpos monoclonais no desenvolvimento de antivenenos de nova geração. Assim, anticorpos monoclonais e seus fragmentos são descritos como uma possível alternativa para a produção de antivenenos, independente do veneno. Esta revisão também destaca os desafios associados ao seu desenvolvimento.

The development of new oral vaccines using porous silica

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.

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.

Oral Tolerance Induction by Bothrops jararaca Venom in a Murine Model and Cross-Reactivity with Toxins of Other Snake Venoms.

Oral tolerance is defined as a specific suppression of cellular and humoral immune responses to a particular antigen through prior oral administration of an antigen. It has unique immunological importance since it is a natural and continuous event driven by external antigens. It is characterized by low levels of IgG in the serum of animals after immunization with the antigen. There is no report of induction of oral tolerance to Bothrops jararaca venom. Here, we induced oral tolerance to B. jararaca venom in BALB/c mice and evaluated the specific tolerance and cross-reactivity with the toxins of other Bothrops species after immunization with the snake venoms adsorbed to/encapsulated in nanostructured SBA-15 silica. Animals that received a high dose of B. jararaca venom (1.8 mg) orally responded by showing antibody titers similar to those of immunized animals. On the other hand, mice tolerized orally with three doses of 1 µg of B. jararaca venom showed low antibody titers. In animals that received a low dose of B. jararaca venom and were immunized with B. atrox or B. jararacussu venom, tolerance was null or only partial. Immunoblot analysis against the venom of different Bothrops species provided details about the main tolerogenic epitopes and clearly showed a difference compared to antiserum of immunized animals.

Oral vaccination of piglets against Mycoplasma hyopneumoniae using silica SBA-15 as an adjuvant effectively reduced consolidation lung lesions at slaughter

Mycoplasma (M.) hyopneumoniae is the main pathogen of porcine enzootic pneumonia (PEP). Its controlling is challenging, and requires alternative strategies. This study aimed to develop an oral vaccine against M. hyopneumoniae using a nanostructured mesoporous silica (SBA-15) as an adjuvant, and compare its effect with an intramuscular (IM) commercial vaccine (CV). Fifty 24 day-old M. hyopneumoniae-free piglets composed five equal groups for different immunization protocols, consisting of a CV and/or oral immunization (OI). Control piglets did not receive any form of immunization. All piglets were challenged with M. hyopneumoniae strain 232 on D49 by tracheal route. IgA antibody response in the respiratory tract, bacterial shedding and serum IgG were evaluated. The piglets were euthanized on 28 (D77) and 56 (D105) days post-infection. Lung lesions were macroscopically evaluated; lung fragments and bronchoalveolar fluid (BALF) were collected for estimation of bacterial loads by qPCR and/or histopathology examination. All immunization protocols induced reduction on Mycoplasma-like macroscopic lung lesions. IgA Ab responses anti-M. hyopneumoniae, the expression of IL-4 cytokine and a lower expression of IL-8 were induced by CV and OI vaccines, while IgG was induced only by CV. Oral immunization using silica as a carrier-adjuvant can be viable in controlling M. hyopneumoniae infection.

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.

Alphastatin-C a new inhibitor of endothelial cell activation is a pro-arteriogenic agent in vivo and retards B16-F10 melanoma growth in a preclinical model.

Most characterized angiogenic modulators are proteolytic fragments of structural plasma and/or matrix components. Herein, we have identified a novel anti-angiogenic peptide generated by the in vitro hydrolysis of the C-terminal moiety of the fibrinogen alpha chain, produced by the snake venom metalloprotease bothropasin (SVMP), a hemorrhagic proteinase in Bothrops jararaca venom. The 14-amino acids peptide (alphastatin-C) is a potent antagonist of basic fibroblast growth factor, induced endothelial cell (HUVEC-CS) proliferation, migration and capillary tube formation in matrigel. It also inhibits cell adhesion to fibronectin. The basis of the antagonism between bFGF and alphastatin-C is elucidated by the inhibition of various bFGF induced signaling pathways and their molecular components modification, whenever the combination of the stimuli is provided, in comparison to the treatment with bFGF only. To corroborate to the potential therapeutic use of alphastatin-C, we have chosen to perform in vivo assays in two distinct angiogenic settings. In chick model, alphastatin-C inhibits chorioallantoic membrane angiogenesis. In mouse, it efficiently reduces tumor number and volume in a melanoma model, due to the impairment of tumor neovascularization in treated mice. In contrast, we show that the alphastatin-C peptide induces arteriogenesis, increasing pial collateral density in neonate mice. alphastatin-C is an efficient new antiangiogenic FGF-associated agent in vitro, it is an inhibitor of embryonic and tumor vascularization in vivo while, it is an arteriogenic agent. The results also suggest that SVMPs can be used as in vitro biochemical tools to process plasma and/or matrix macromolecular components unraveling new angiostatic peptides.

Alphastatin-C a new inhibitor of endothelial cell activation is a pro-arteriogenic agent in vivo and retards B16-F10 melanoma growth in a preclinical model

Most characterized angiogenic modulators are proteolytic fragments of structural plasma and/or matrix components. Herein, we have identified a novel anti-angiogenic peptide generated by the in vitro hydrolysis of the C-terminal moiety of the fibrinogen alpha chain, produced by the snake venom metalloprotease bothropasin (SVMP), a hemorrhagic proteinase in Bothrops jararaca venom. The 14-amino acids peptide (alphastatin-C) is a potent antagonist of basic fibroblast growth factor, induced endothelial cell (HUVEC-CS) proliferation, migration and capillary tube formation in matrigel. It also inhibits cell adhesion to fibronectin. The basis of the antagonism between bFGF and alphastatin-C is elucidated by the inhibition of various bFGF induced signaling pathways and their molecular components modification, whenever the combination of the stimuli is provided, in comparison to the treatment with bFGF only. To corroborate to the potential therapeutic use of alphastatin-C, we have chosen to perform in vivo assays in two distinct angiogenic settings. In chick model, alphastatin-C inhibits chorioallantoic membrane angiogenesis. In mouse, it efficiently reduces tumor number and volume in a melanoma model, due to the impairment of tumor neovascularization in treated mice. In contrast, we show that the alphastatin-C peptide induces arteriogenesis, increasing pial collateral density in neonate mice. alphastatin-C is an efficient new antiangiogenic FGF-associated agent in vitro, it is an inhibitor of embryonic and tumor vascularization in vivo while, it is an arteriogenic agent. The results also suggest that SVMPs can be used as in vitro biochemical tools to process plasma and/or matrix macromolecular components unraveling new angiostatic peptides.

The crotoxin: SBA-15 complex down-regulates the Incidence and Intensity of experimental autoimmune encephalomyelitis through peripheral and central actions

Crotoxin (CTX), the main neurotoxin from Crotalus durissus terrificus snake venom, has anti-inflammatory, immunomodulatory and antinociceptive activities. However, the CTX-induced toxicity may compromise its use. Under this scenario, the use of nanoparticle such as nanostructured mesoporous silica (SBA-15) as a carrier might become a feasible approach to improve CTX safety. Here, we determined the benefits of SBA-15 on CTX-related neuroinflammatory and immunomodulatory properties during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis that replicates several histopathological and immunological features observed in humans. We showed that a single administration of CTX:SBA-15 (54 μg/kg) was more effective in reducing pain and ameliorated the clinical score (motor impairment) in EAE animals compared to the CTX-treated EAE group; therefore, improving the disease outcome. Of interest, CTX:SBA-15, but not unconjugated CTX, prevented EAE-induced atrophy and loss of muscle function. Further supporting an immune mechanism, CTX:SBA-15 treatment reduced both recruitment and proliferation of peripheral Th17 cells as well as diminished IL-17 expression and glial cells activation in the spinal cord in EAE animals when compared with CTX-treated EAE group. Finally, CTX:SBA-15, but not unconjugated CTX, prevented the EAE-induced cell infiltration in the CNS. These results provide evidence that SBA-15 maximizes the immunomodulatory and anti-inflammatory effects of CTX in an EAE model; therefore, suggesting that SBA-15 has the potential to improve CTX effectiveness in the treatment of MS.