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BackgroundThe SARS-CoV-2 virus, responsible for the COVID-19 pandemic, has four structural proteins and sixteen non-structural proteins. The S-protein is one of the structural proteins exposed on the surface of the virus and is the main target for producing neutralizing antibodies and vaccines. The S-protein forms a trimer that can bind the angiotensin-converting enzyme 2 (ACE2) through its receptor binding domain (RBD) for cell entry.MethodsWe stably expressed in a constitutive manner in HEK293 cells a new recombinant protein containing a signal sequence of immunoglobulin to produce an extended C-terminal portion of the RBD followed by a region responsible for the trimerization inducer of the bacteriophage T4, and a sequence of 6 histidines. The protein was produced and released in the culture supernatant of cells and was purified by Ni-agarose column and exclusion chromatography. It was then characterized by SDS-polyacrylamide gel and used as antigen to generate protective antibodies to inhibit ACE2 receptor interaction and virus entry into Vero cells.ResultsThe purified protein displayed a molecular mass of 135 kDa and with a secondary structure like the monomeric RBD. Electrophoresis analysis in SDS-polyacrylamide gel with and without reducing agents, and in the presence of crosslinkers indicated that it forms a multimeric structure composed of trimers and hexamers. The purified protein was able to bind the ACE2 receptor and generated high antibody titers in mice (1:10000), capable of inhibiting the binding of biotin labeled ACE2 to the virus S1 subunit, and to neutralize the entry of the SARS-CoV-2 Wuhan strain into cells.ConclusionOur results characterize a new multimeric protein based on S1 subunit to combat COVID-19, as a possible immunogen or antigen for diagnosis.

Until universal influenza vaccines become available, pandemic preparedness should include developing classical vaccines against potential pandemic influenza subtypes. We here show that addition of SWE adjuvant, a squalene-in-water emulsion, to H7N9 split influenza vaccine clearly enhanced functional antibody responses in ferrets. These were cross-reactive against H7N9 strains from different lineages and newly emerged H7N9 variants. Both vaccine formulations protected in almost all cases against severe pneumonia induced by intratracheal infection of ferrets with H7N9 influenza; however, the SWE adjuvant enhanced protection against virus replication and disease. Correlation analysis and curve fitting showed that both VN- and NI-titers were better predictors for protection than HI-titers. Moreover, we show that novel algorithms can assist in better interpretation of large data sets generated in preclinical studies. Cluster analysis showed that the adjuvanted vaccine results in robust immunity and protection, whereas the response to the non-adjuvanted vaccine is heterogeneous, such that the protection balance may be more easily tipped toward severe disease. Finally, cluster analysis indicated that the dose-sparing capacity of the adjuvant is at least a factor six, which greatly increases vaccine availability in a pandemic situation.

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 (H

Interferons are innate and adaptive cytokines involved in many biological responses, in particular, viral infections. With the final response the result of the balance of the different types of Interferons. Cytokine storms are physiological reactions observed in humans and animals in which the innate immune system causes an uncontrolled and excessive release of pro-inflammatory signaling molecules. The excessive and prolonged presence of these cytokines can cause tissue damage, multisystem organ failure and death. The role of Interferons in virus clearance, tissue damage and cytokine storms are discussed, in view of COVID-19 caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The imbalance of Type I, Type II and Type III Interferons during a viral infection contribute to the clinical outcome, possibly together with other cytokines, in particular, TNFα, with clear implications for clinical interventions to restore their correct balance.

In March 2013 it was reported by the World Health Organization (WHO) the first cases of human infections with avian influenza virus A (H7N9). From 2013 to December 2019, 1568 cases have been reported with 616 deaths. H7N9 infection has been associated with high morbidity and mortality rates, and vaccination is currently the most effective way to prevent infections and consequently flu-related severe illness. Developing and producing vaccines against pandemic influenza viruses is the main strategy for a response to a possible pandemic. This study aims to present the production of three industrial lots under current Good Manufacturing Practices (cGMP) of the active antigen used to produce the pandemic influenza vaccine candidate against A(H7N9). These batches were characterized and evaluated for quality standards and tested for immunogenicity in mice. The average yield was 173.50 ± 7.88 μg/mL of hemagglutinin and all the preparations met all the required specifications. The formulated H7N9 vaccine is poorly immunogenic and needs to be adjuvanted with an oil in water emulsion adjuvant (IB160) to achieve a best immune response, in a prime and in a boost scheme. These data are important for initial production planning and preparedness in the case of a H7N9 pandemic.

Increasing pandemic influenza vaccine manufacturing capacity is considered strategic by WHO. Adjuvant use is key in this strategy in order to spare the vaccine doses and by increasing immune protection. We describe here the production and stability studies of a squalene based oil-in-water emulsion, adjuvant IB160, and the immune response of the H7N9 vaccine combined with IB160. To qualify the production of IB160 we produced 10 consistency lots of IB160 and the average results were: pH 6.4±0.05; squalene 48.8±.0.03 mg/ml; osmolality 47.6±6.9 mmol/kg; Z-average 157±2 nm, with polydispersity index (PDI) of 0.085±0.024 and endotoxin levels

Pathogenic spirochetes from genus Leptospira are etiologic agents of leptospirosis. Cellular vaccines against Leptospira infection often elicit mainly response against the LPS antigen of the serovars present in the formulation. There is no suitable protein candidate capable of replacing whole-cell vaccines, thus requiring new approaches on vaccine development to improve leptospirosis prevention. Our goal was to develop a whole-cell vaccine sorovar-independent based on LPS removal and conservation of protein antigens exposure, to evaluate the protective capacity of monovalent or bivalent vaccines against homologous and heterologous virulent Leptospira in hamster. Leptospire were subjected to heat inactivation, or to LPS extraction with butanol and in some cases further inactivation with formaldehyde. Hamsters were immunized and challenged with homologous or heterologous virulent serovars, blood and organs were collected from the survivors for bacterial quantification, chemokine evaluation, and analysis of sera antibody reactivity and cross-reactivity by Western blot. Immunization with either heated or low LPS vaccines with serovar Copenhageni or Canicola resulted in 100% protection of the animals challenged with homologous virulent bacteria. Notably, different from the whole-cell vaccine, the low LPS vaccines produced with serovar Canicola provided only partial protection in heterologous challenge with the virulent Copenhageni serovar. Immunization with bivalent formulation results in 100% protection of immunized animals challenged with virulent serovar Canicola. All vaccines produced were able to eliminate bacteria from the kidney of challenged animals. All the vaccines raised antibodies capable to recognize antigens of serovars not present in the vaccine formulation. Transcripts of IFNγ, CXCL16, CCL5, CXCL10, CXCR6, and CCR5, increased in all immunized animals. Conclusion: Our results showed that bivalent vaccines with reduced LPS may be an interesting strategy for protection against heterologous virulent serovars. Besides the desirable multivalent protection, the low LPS vaccines are specially promising due to the expected lower reatogenicity.

International audience; Pneumococcal Surface Protein A (PspA) has been successfully tested as vaccine candidate against Streptococcus pneumoniae infections. Vaccines able to induce PspA-specific antibodies and Th1 cytokines usually provide protection in mice. We have shown that the whole cell pertussis vaccine (wP) or components from acellular pertussis vaccines, such as Pertussis Toxin or Filamentous Hemagglutinin (FHA), are good adjuvants to PspA, suggesting that combined pertussis-PspA vaccines would be interesting strategies against the two infections. Here, we evaluated the potential of wP as a delivery vector to PspA. Bordetella pertussis strains producing a PspA from clade 4 (PspA4Pro) fused to the N-terminal region of FHA (Fha44) were constructed and inactivated with formaldehyde for the production of wPPspA4Pro. Subcutaneous immunization of mice with wPPspA4Pro induced low levels of anti-PspA4 IgG, even after 3 doses, and did not protect against a lethal pneumococcal challenge. Prime-boost strategies using wPPspA4Pro and PspA4Pro showed that there was no advantage in using the wPPspA4Pro vaccine. Immunization of mice with purified PspA4Pro induced higher levels of antibodies and protection against pneumococcal infection than the prime-boost strategies. Finally, purified Fha44:PspA4Pro induced high levels of anti-PspA4Pro IgG, but no protection, suggesting that the antibodies induced by the fusion protein were not directed to protective epitopes.

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.

A coqueluche é uma doença respiratória contagiosa, causada pela bactéria Bordetella pertussis. Tendo um significativo impacto epidemiológico, esta doença sofreu uma expressiva redução após o uso disseminado de vacinas pertussis. Efeitos adversos na imunização com a Vacina de células inteiras Whole cell pertussis (wP), atribuídos a presença de lipopolissacarídeos (LPS), levou ao desenvolvimento da Vacina pertussis acelular (aP). Contudo, a imunização com aP não tem demonstrada a mesma eficiência que a imunização com wP. Diante desse cenário o objetivo deste trabalho é desenvolver o processo de produção industrial de uma nova Vacina Pertussis de células inteiras com reduzida quantidade de LPS e baixa reatogenicidade, a Vacina Pertussis Low (wPlow). Para alcançar o objetivo, foram produzidos em escala industrial 25 lotes de cultivos inativados, concentrados e submetidos à extração de LPS com solvente orgânico. Para extração de LPS foram avaliadas 4 diferentes metodologias: filtração de fluxo tangencial (TFF); filtração de fluxo tangencial com lavagem com solução com solvente orgânico (TFFSW); centrifuga tubular (CT); centrifugação de fluxo contínuo de pratos (CFC). A wPlow produzida em centrifuga de bancada e a wP foram usadas para comparação. O processo de bancada resultou na redução de LPS (método Purpald) em média de 75% do conteúdo de LPS e a redução de 81% da atividade endotóxica (dosagem de LAL). Nos processos industriais, por TFF houve a redução de ≅21% do conteúdo de LPS, porém com aumento de ≅ 52% da atividade endotóxica; por TFFSW houve a redução de 46% do conteúdo de LPS e uma redução endotóxica média de ≅ 24%; por CT ocorreu à redução de ≅ 66% do conteúdo de LPS e de ≅ 73% da atividade endotóxica; com a CFC houve a redução de ≅ 92% do conteúdo de LPS e de ≅ 61% da atividade endotóxica. Os rendimentos de processo foram ≅ 83%, 61%, 37% e 63% respectivamente para os processos de TFF, TFFSW, CT e CFC. Através de microscopia eletrônica, foi possível visualizar a integridade celular após o processamento por CFC, e o principal antígeno vacinal, a toxina pertussis foi detectada na preparação, por western-blot. Quanto à imunogenicidade, anticorpos IgG anti-pertussis foram detectados por ELISA e resultados preliminares não mostraram diferença significativa de redução de colonização pulmonar de B. pertussis em camundongos imunizados com a wPlow ou wP. Diante dos resultados obtidos, podemos concluir que os processos de TFF e TFFSW não foram eficientes na remoção da atividade endotóxica da wPlow, embora tenha ocorrido a redução do LPS. Com relação aos processos utilizando centrífugas industriais, eficientes tanto na remoção do LPS e na redução da atividade endotóxica, houve, contudo, um baixo rendimento no processo com CT. A wPlow produzida por CFC foi imunogênica, indicando a eficácia potencial desta vacina e que a produção em escala industrial é um processo viável. Como parte deste trabalho, a cepa vacinal de Bordetella pertussis foi também caracterizada pelo seu sequenciamento genômico completo (genbank número CP010323). Whooping cough is a contagious respiratory disease caused by Bordetella pertussis. In the past this infection had a high epidemiological impact, only reduced after the use of pertussis vaccine. The association of adverse events in immunization with whole cell Pertussis vaccine (wP), attributed to the presence of lipopolysaccharides (LPS), has led to the development of acellular Pertussis vaccine (aP). However, it is known that immunization with aP does not have the same efficiency as compared to immunization with wP vaccine. In this scenario, the objective of this work is to develop the industrial production process of a new whole cell pertussis vaccine with reduced amount of LPS and low reatogenicity, the whole cell Pertussis low vaccine (wPlow). To achieve this aim, we produced 25 lots of inactivated and concentrated cultures prepared on an industrial scale and subjected to LPS extraction with organic solvent. We evaluated four industrial processes to extract the LPS from the cells: tangential flow filtration (TFF), TFF with organic solvent washing (TFFSW), tubular centrifugation (CT) and continuous flow centrifugation (CFC). These methodologies were compared with wPlow produced at bench scale obtained by centrifugation and with traditional wP. The bench process resulted in the reduction of 75% of LPS content (Purpald method) and 81% reduction in endotoxic activity (LAL dosage) on average. In the industrial processes, TFF reduced ≅ 21% in LPS content, but with a ≅52% increase in endotoxic activity; by TFFSW there was a reduction of ≅ 46% of the LPS content and an average reduction of endotoxic activity of ≅24%; CT reduced ≅ 66% of LPS content and ≅ 73% of endotoxic activity; with CFC there was a reduction of ≅ 92% in LPS content and ≅1% in endotoxic activity. The process yields were ≅ 83%, 61%, 37% and 63% respectively for the TFF, TFFSW, CT and CFC processes. Through electron microscopy, it was possible to visualize cell integrity after CFC processing, and the major vaccine antigen, pertussis toxin, was detected in the preparation by western blot. As for immunogenicity, anti-pertussis IgG antibodies were detected by ELISA and preliminary results showed no differences in the B. pertussis colonization of lungs in mice immunized with wPlow or wP. We can conclude that the TFF and TFFSW processes were not efficient in removing the endotoxic activity of wPlow, although LPS reduction occurred. Although the processes using industrial centrifuges were efficacious in the removal of LPS and in the reduction of endotoxic activity, there was a low yield in the CT process. The wPlow produced by CFC was immunogenic indicating its potential as a vaccine and that this industrial scale production is a viable process. The characterization of the vaccine strain of Bordetella pertussis by complete genome sequencing was also presented here as part of this work (genbank - number CP010323).

Despite the reduction in incidence after vaccination, pertussis disease is still considered a public health problem worldwide, mainly due to recent and potential new outbreaks. We report here the complete genome of the Bordetella pertussis Butantan strain used in the Brazilian National Immunization Program as a whole-cell pertussis antigen to compose vaccines such as DTwP (diphtheria, tetanus, and whole-cell pertussis).

While the immunogenic potential of the vaccination against infectious diseases was extensively shown, data on the safety assessment of recombinant proteins in vaccine formulations administered during pregnancy are still scarce. In the current study, the antigenicity of a vaccine against leishmaniasis (based on Leishmania braziliensis recombinant protein peroxidoxin) during pregnancy and possible maternal reproductive outcomes and fetal anomalies after immunization with a leishmanial vaccine or adjuvant alone (Bordetella pertussis derived MPLA adjuvant) were assessed. Rats were mated and allocated in three groups: Control—rats received saline; Adjuvant—rats received the adjuvant MPLA, and Vaccine—rats received the combination of MPLA and peroxidoxin. The administration was subcutaneously at the dorsal region, three times (days 0, 7, 14 of pregnancy). On day 21 of pregnancy, all rats were bled for biochemical and immunological measurements. The gravid uterus was weighed with its contents, and the fetuses were analyzed. The immunization with peroxidoxin induced a significant production of circulating IgG levels compared to other groups but caused a significant in post-implantation loss (14.7%) when compared to Control (5.0%) and Adjuvant (4.4%) groups. Furthermore, a significantly high rate of fetal visceral anomalies, such as hydronephrosis and convoluted ureter, was also observed in animals that received vaccine when compared to Control or Adjuvant groups. These data indicate the importance of safety evaluation of vaccines during pregnancy and the limited use of peroxidoxin administration during pregnancy. More importantly, the safety monitoring of immunization with MPLA derived from Bordetella pertussis demonstrated no reproductive outcomes associated with adjuvant administration, suggesting its safe use during pregnancy.

O objetivo deste trabalho foi avaliar o efeito de rizobactérias, no crescimento de plântulas de pepino e no controle de tombamento, causado por Pythium aphanidermatum. Foram realizados em laboratório ensaios de: degradação de 1-aminociclopropano-1-carboxilato (ACC); colonização das raízes de plântulas de pepino; e pareamento de culturas. A identificação dos melhores isolados foi feita pela determinação das seqüências do gene 16S rDNA. Trinta e sete isolados, dos 165 testados, aumentaram a massa de matéria seca das plantas de pepino em até 63%. Desses, somente um isolado (N13 - Pseudomonas fluorescens) reduziu o tombamento de plântulas em 25%; 21 isolados inibiram o crescimento micelial de P. aphanidermatum, colonizaram o sistema radicular das plantas de pepino e cresceram em presença de ACC como única fonte de nitrogênio. Dos dez isolados que apresentaram resultados satisfatórios, cinco foram identificados como pertencentes aos gêneros Bacillus, quatro Pseudomonas e um Stenotrophomonas. Dos 165 isolados de rizobactérias testados, sete possuem potencial para promover o crescimento de plantas de pepino e um para controlar o tombamento causado por P. aphanidermatum. The aim of this work was to evaluate the rhizobacteria effect on cucumber seedling growth and in the control of damping-off disease Pythium aphanidermatum. In laboratory, the following tests were carried out: degradation of 1-aminocyclopropane-1-carboxylate (ACC); colonization of cucumber root system; and, the capacity to antagonize the pathogen. The best isolates were identified through determination of 16S rDNA gene sequences. Thirty-seven isolates, among 165 tested, enhanced the dry weight of cucumber plants in up to 63%. From these 37, only one isolate (N13 - Pseudomonas fluorescens) reduced the incidence of pre-emergence damping-off disease by 25%; 21 isolates antagonized P. aphanidermatum in vitro, colonized the cucumber roots, and degradated ACC, as the only source of nitrogen. Out of ten most effective isolates, five were identified as members of the genus Bacillus, four of Pseudomonas and one of Stenotrophomonas. Out of the 165 tested rhizobacteria isolates, seven have potential to promote cucumber plant growth, and one to control P. aphanidermatum damping-off disease.

Chitosan is a biocompatible and biodegradable mucoadhesive polymer with unique advantages, such as the distinct trait of opening the junctions to allow paracellular transport of antigen and good tolerability. However, the poor solubility of chitosan in neutral or alkalinized media has restricted its applications in the pharmaceutical field. Chitosan can be easily carboxymethylated to improve its solubility in aqueous media, while its biodegradability and biocompatibility are preserved. Apart from this, carboxymethyl chitosan (CMCS) can be easily processed into nanoparticles which highlight its suitability and extensive usage for preparing different drug delivery formulations. The present study deals with the development and characterization of a delivery system based on CMCS nanoparticles using ovalbumin as model protein. We demonstrated that ovalbumin loaded nanoparticles were successfully synthetized using calcium chloride as a cross-linker by ionic gelation. The nanoparticles exhibited an average size of approximately 169 nm and presented a pseudo-spherical shape. The nanoparticles size increased according to the addition of CaCl2 due to the strong electrostatic attraction. During storage the nanoparticles size increased was attributed to swelling and aggregation. The loading efficiency of ovalbumin was found to be 17%. Confocal microscopy clearly showed the association between ovalbumin and CMCS chains into nanoparticles. Therefore, we suggest these nanoparticles can be considered as an attractive and promising carrier candidate for proteins and antigens. The major challenge that limits the use of such carriers is their instability in an aqueous medium. Thus, the next step of this work was to determine the robustness of several formulations using distinct freeze-drying protocols. This study demonstrated that mannitol in concentration of 10% (w/v) is well suited to preserve ovalbumin loaded CMCS nanocapsules from aggregation during lyophilization and subsequent reconstitution. Importantly, the results showed that an annealing step has a huge impact on porosity of freeze-dried cake by nearly complete crystallization of mannitol, once the crystalline matrix prevents the partial collapse and the formation of larger pores observed without annealing. Therefore, the usual observation that annealing increases the pore size due to growth of ice crystal size does not always apply, at least when crystallization of solute is involved. Since all characterizations and stability studies had been performed, the main purpose of this study was to develop a stable antigen delivery system for oral immunization using CMCS and inactivated rabies virus (RV) as the antigen. RV loaded nanoparticles was found to enhance both systemic (IgG) and local (IgA) immune responses against RV after oral delivery in mice. The effective doses 50% were 50-times higher than the negative controls, indicating that the immune response started only after the third boosting dose. Furthermore, enough neutralizing antibodies was produced to be protected against the harmful effects of the rabies virus. It is therefore concluded, that the CMCS nanoparticles formulated in this study, are suitable for oral vaccine delivery, and can be suggested as a promising delivery system for a diverse range of antigens as well as a gene/protein delivery system, especially for those positively charged. Since several approaches show that effective intervention in airway allergic inflammation can be achieved with allergen-activated interleukin-10-secreting cells, the final part of this work was dedicated to assessing whether IL-10 loaded chitosan nanoparticles (IL10-CSNPs) could be used as a possible inhalable therapeutic tool for preventing exacerbations in asthmatic patients. As positive controls, we also assess whether interleukin 17A and interleukin 9 have the ability to stimulate human airway smooth muscle (HASM) cell contractility using magnetic twisting cytometry (MTC). Significant decreased baseline cell stiffness was observed in HASM cells pre-treated with IL-10, but not with IL10-CSNPs, whereas treatment with IL-17A significantly enhanced baseline cell stiffening. Our findings reveal a previously unknown mechanism underlying immunotherapy for prevention and treatment of asthma. A quitosana é um polímero mucoadesivo biocompatível e biodegradável, com vantagens únicas, tais como a característica distinta de abrir as junções que permitim o transporte paracelular de antígenos e boa tolerabilidade. No entanto, sua baixa solubilidade em meios neutros ou alcalinizados tem restringido suas aplicações no campo farmacêutico. A quitosana pode ser facilmente carboximetilada para melhorar de sua solubilidade em meios aquosos, enquanto sua biodegradabilidade e biocompatibilidade são preservadas. Além disso, a carboximetilquitosana (CMCS) pode ser facilmente processada na forma de nanopartículas, o que destaca sua adequabilidade para uso extensivo no preparo de sistemas de delivery de medicamentos. O presente estudo trata do desenvolvimento e caracterização de um sistema de delivery baseado em nanopartículas de CMCS utilizando ovalbumina como proteína modelo. Nós demonstramos que as nanopartículas carregadas com ovalbumina foram sintetizadas com sucesso utilizando cloreto de cálcio como agente de reticulação por gelificação iônica. As nanopartículas exibiram um tamanho médio de aproximadamente 169 nm e apresentaram uma forma pseudo-esférica. O tamanho das nanopartículas aumentou de acordo com a adição de CaCl2 devido à forte atração eletrostática. Durante o armazenamento, o tamanho aumentado das nanopartículas foi atribuído a incorporação de água e agregação. A eficiência de encapsulamento da ovalbumina foi de aproximadamente 17%. A microscopia confocal mostrou claramente a associação entre ovalbumina e a cadeias de CMCS nas nanopartículas. Sugerimos, portanto, que tal sistema pode ser considerado como candidato atraente e promissor para o carreamento de proteínas e antígenos. O principal desafio que limita o uso desses carreadores consiste na instabilidade em meio aquoso. Assim, o próximo passo deste trabalho foi determinar a robustez de várias formulações utilizandose diferentes protocolos de liofilização. Este estudo demonstrou que o manitol em uma concentração de 10% (p/v) é adequado para preservar da agregação as nanocápsulas de CMCS carregadas com ovalbumina durante a liofilização e subsequente reconstituição. Mais importante, os resultados mostraram que uma etapa de annealing tem um enorme impacto sobre a porosidade da amostra liofilizada devido a quase completa cristalização do manitol, uma vez que a matriz cristalina evita o colapso parcial e a formação de poros maiores observados na ausência do annealing. Portanto, a observação comum de que o annealing aumenta o tamanho doporos devido ao crescimento dos cristais de gelo nem sempre se aplica, pelo menos quando a cristalização de um soluto está envolvida. Uma vez que todas as caracterizações e estudos de estabilidade foram realizados, o principal objetivo deste estudo foi desenvolver um sistema estável de delivery de antígeno para imunização oral utilizando CMCS e vírus rábico inativado (RV) como antígeno. Verificou-se que as nanopartículas carregadas com RV aumentam as respostas imune sistêmica (IgG) e local (IgA) contra o RV após administração oral em camundongos. As doses efetivas 50% foram 50 vezes maiores que os controles negativos, indicando que a resposta imune foi iniciada apenas após a terceira dose da vacina. Além disso, foram produzidos anticorpos neutralizantes suficientes para proteção contra os efeitos nocivos do vírus rábico. Conclui-se, portanto, que as nanopartículas de CMCS formuladas neste estudo, são adequadas para o delivery oral de vacinas, e podem ser sugeridas como um sistema promissor de delivery para uma gama diversa de antígenos, bem como para o delivery de genes/proteínas, especialmente para aqueles carregados positivamente. Uma vez que diversas abordagens mostram que uma intervenção efetiva em casos de inflamação alérgica de vias aéreas pode ser conseguida por meio de células secretoras de interleucina 10 (IL-10) mediante ativação por alergenos, a parte final deste trabalho esteve dedicada a avaliação de nanopartículas de quitosana carregadas com IL-10 (IL10-CSNPs) como possível ferramenta terapêutica inalável para prevenção de exacerbações em pacientes asmáticos. Como controles positivos, avaliou-se adicionalmente se as interleucinas 17A (IL-17A) e 9 (IL-9) possuem a capacidade de estimular a contratilidade de células humanas de músculo liso de vias aéreas humanas (HASM) por meio de citometria de torção magnética (MTC). Uma diminuição significativa da rigidez celular basal foi observada em células HASM pré-tratadas com IL-10, mas não com IL10-CSNPs, enquanto que o tratamento com IL-17A aumentou significativamente a magnitude rigidez celular basal. Nossos resultados revelam um mecanismo previamente desconhecido subjacente à imunoterapia para prevenção e tratamento da asma.