Pneumococcal Surface Protein A-Hybrid Nanoparticles Protect Mice from Lethal Challenge after Mucosal Immunization Targeting the Lungs.

Pneumococcal disease remains a global burden, with current conjugated vaccines offering protection against the common serotype strains. However, there are over 100 serotype strains, and serotype replacement is now being observed, which reduces the effectiveness of the current vaccines. Pneumococcal surface protein A (PspA) has been investigated as a candidate for new serotype-independent pneumococcal vaccines, but requires adjuvants and/or delivery systems to improve protection. Polymeric nanoparticles (NPs) are biocompatible and, besides the antigen, can incorporate mucoadhesive and adjuvant substances such as chitosans, which improve antigen presentation at mucosal surfaces. This work aimed to define the optimal NP formulation to deliver PspA into the lungs and protect mice against lethal challenge. We prepared poly(glycerol-adipate-co-ω-pentadecalactone) (PGA-co-PDL) and poly(lactic-co-glycolic acid) (PLGA) NPs using an emulsion/solvent evaporation method, incorporating chitosan hydrochloride (HCl-CS) or carboxymethyl chitosan (CM-CS) as hybrid NPs with encapsulated or adsorbed PspA. We investigated the physicochemical properties of NPs, together with the PspA integrity and biological activity. Furthermore, their ability to activate dendritic cells in vitro was evaluated, followed by mucosal immunization targeting mouse lungs. PGA-co-PDL/HCl-CS (291 nm) or CM-CS (281 nm) NPs produced smaller sizes compared to PLGA/HCl-CS (310 nm) or CM-CS (299 nm) NPs. Moreover, NPs formulated with HCl-CS possessed a positive charge (PGA-co-PDL +17 mV, PLGA + 13 mV) compared to those formulated with CM-CS (PGA-co-PDL -20 mV, PLGA -40 mV). PspA released from NPs formulated with HCl-CS preserved the integrity and biological activity, but CM-CS affected PspA binding to lactoferrin and antibody recognition. PspA adsorbed in PGA-co-PDL/HCl-CS NPs stimulated CD80+ and CD86+ cells, but this was lower compared to when PspA was encapsulated in PLGA/HCl-CS NPs, which also stimulated CD40+ and MHC II (I-A/I-E)+ cells. Despite no differences in IgG being observed between immunized animals, PGA-co-PDL/HCl-CS/adsorbed-PspA protected 83% of mice after lethal pneumococcal challenge, while 100% of mice immunized with PLGA/HCl-CS/encapsulated-PspA were protected. Therefore, this formulation is a promising vaccine strategy, which has beneficial properties for mucosal immunization and could potentially provide serotype-independent protection.

Combined Subcutaneous-Intranasal Immunization With Epitope-Based Antigens Elicits Binding and Neutralizing Antibody Responses in Serum and Mucosae Against PRRSV-2 and SARS-CoV-2.

New vaccine design approaches, platforms, and immunization strategies might foster antiviral mucosal effector and memory responses to reduce asymptomatic infection and transmission in vaccinated individuals. Here, we investigated a combined parenteral and mucosal immunization scheme to induce local and serum antibody responses, employing the epitope-based antigens 3BT and NG19m. These antigens target the important emerging and re-emerging viruses PRRSV-2 and SARS-CoV-2, respectively. We assessed two versions of the 3BT protein, which contains conserved epitopes from the GP5 envelope protein of PRRSV-2: soluble and expressed by the recombinant baculovirus BacDual-3BT. On the other hand, NG19m, comprising the receptor-binding motif of the S protein of SARS-CoV-2, was evaluated as a soluble recombinant protein only. Vietnamese mini-pigs were immunized employing different inoculation routes: subcutaneous, intranasal, or a combination of both (s.c.-i.n.). Animals produced antigen-binding and neut1ralizing antibodies in serum and mucosal fluids, with varying patterns of concentration and activity, depending on the antigen and the immunization schedule. Soluble 3BT was a potent immunogen to elicit binding and neutralizing antibodies in serum, nasal mucus, and vaginal swabs. The vectored immunogen BacDual-3BT induced binding antibodies in serum and mucosae, but PRRSV-2 neutralizing activity was found in nasal mucus exclusively when administered intranasally. NG19m promoted serum and mucosal binding antibodies, which showed differing neutralizing activity. Only serum samples from subcutaneously immunized animals inhibited RBD-ACE2 interaction, while mini-pigs inoculated intranasally or via the combined s.c.-i.n. scheme produced subtle neutralizing humoral responses in the upper and lower respiratory mucosae. Our results show that intranasal immunization, alone or combined with subcutaneous delivery of epitope-based antigens, generates local and systemic binding and neutralizing antibodies. Further investigation is needed to evaluate the capability of the induced responses to prevent infection and reduce transmission.

Synthesis and Characterization of Innovative Microgels Based on Polyacrylic Acid and Microalgae Cell Wall and Their Potential as Antigen Delivery Vehicles.

In this study, hybrid polyacrylic acid and Schizochytrium sp. microalgae (PAA/Schizo) microgels were synthesized by inverse emulsion assisted by ultrasound using the cell wall fraction as crosslinker. Physicochemical characterization of PAA/Schizo microgels revealed polymeric spherical particles (288 ± 39 nm) and were deemed stable and negatively charged. The produced microgels are not inherently toxic as cell viability was sustained above 80% when mice splenocytes were exposed to concentrations ranging 10-900 µg/mL. PAA/Schizo microgels were evaluated as antigen delivery nanovehicle by adsorbing bovine serum albumin (BSA); with a loading efficiency of 72% and loading capacity of 362 µg/mg. Overall, intranasally-immunized BALB/c mice showed null IgG or IgA responses against PAA/Schizo microgel-BSA, whereas soluble BSA induced significant humoral responses in systemic and mucosal compartments. Splenocytes proliferation assay upon BSA stimulus revealed positive CD4+ T cells-proliferation response in PAA/Schizo microgels-BSA group. Thus, PAA/Schizo microgels constitute functional antigen delivery vehicles of simple and ecofriendly synthesis. Moreover, the use of cell wall fraction as cross-linker agent provides an alternative use for the generation of high-value products using residual algae biomass from the oil industry. Our data suggests that the PAA/Schizo microgels are potential antigen delivery vehicles for immunotherapy development.

Pneumococcal surface protein a-hybrid nanoparticles protect mice from lethal challenge after mucosal immunization targeting the lungs

Pneumococcal disease remains a global burden, with current conjugated vaccines offering protection against the common serotype strains. However, there are over 100 serotype strains, and serotype replacement is now being observed, which reduces the effectiveness of the current vaccines. Pneumococcal surface protein A (PspA) has been investigated as a candidate for new serotype-independent pneumococcal vaccines, but requires adjuvants and/or delivery systems to improve protection. Polymeric nanoparticles (NPs) are biocompatible and, besides the antigen, can incorporate mucoadhesive and adjuvant substances such as chitosans, which improve antigen presentation at mucosal surfaces. This work aimed to define the optimal NP formulation to deliver PspA into the lungs and protect mice against lethal challenge. We prepared poly(glycerol-adipate-co-ω-pentadecalactone) (PGA-co-PDL) and poly(lactic-co-glycolic acid) (PLGA) NPs using an emulsion/solvent evaporation method, incorporating chitosan hydrochloride (HCl-CS) or carboxymethyl chitosan (CM-CS) as hybrid NPs with encapsulated or adsorbed PspA. We investigated the physicochemical properties of NPs, together with the PspA integrity and biological activity. Furthermore, their ability to activate dendritic cells in vitro was evaluated, followed by mucosal immunization targeting mouse lungs. PGA-co-PDL/HCl-CS (291 nm) or CM-CS (281 nm) NPs produced smaller sizes compared to PLGA/HCl-CS (310 nm) or CM-CS (299 nm) NPs. Moreover, NPs formulated with HCl-CS possessed a positive charge (PGA-co-PDL +17 mV, PLGA + 13 mV) compared to those formulated with CM-CS (PGA-co-PDL −20 mV, PLGA −40 mV). PspA released from NPs formulated with HCl-CS preserved the integrity and biological activity, but CM-CS affected PspA binding to lactoferrin and antibody recognition. PspA adsorbed in PGA-co-PDL/HCl-CS NPs stimulated CD80+ and CD86+ cells, but this was lower compared to when PspA was encapsulated in PLGA/HCl-CS NPs, which also stimulated CD40+ and MHC II (I-A/I-E)+ cells. Despite no differences in IgG being observed between immunized animals, PGA-co-PDL/HCl-CS/adsorbed-PspA protected 83% of mice after lethal pneumococcal challenge, while 100% of mice immunized with PLGA/HCl-CS/encapsulated-PspA were protected. Therefore, this formulation is a promising vaccine strategy, which has beneficial properties for mucosal immunization and could potentially provide serotype-independent protection.

Comparative Evaluation of Immune Responses and Protection of Chitosan Nanoparticles and Oil-Emulsion Adjuvants in Avian Coronavirus Inactivated Vaccines in Chickens.

Efficient vaccines are the main strategy to control the avian coronavirus (AvCoV), although several drawbacks related to traditional attenuated and inactivated vaccines have been reported. These counterpoints highlight the importance of developing new alternative vaccines against AvCoV, especially those able to induce long-lasting immune responses. This study evaluated and compared two inactivated vaccines formulated with AvCoV BR-I variants, one composed of chitosan nanoparticles (AvCoV-CS) and the second by Montanide oily adjuvant (AvCoV-O). Both developed vaccines were administered in a single dose or associated with the traditional Mass attenuated vaccine. The AvCoV-CS vaccine administered alone or associated with the Mass vaccine was able to induce strong humoral and cell-mediated immune (CMI) responses and complete protection against IBV virulent infection, wherein single administration was characterized by high IgA antibody levels in the mucosa, whereas when associated with the Mass vaccine, the serum IgG antibody was predominantly observed. On the other hand, single administration of the oily vaccine presented poor humoral and CMI responses and consequently incomplete protection against virulent challenge, but when associated with the Mass vaccine, immune responses were developed, and complete protection against infection was observed. Both of our experimental vaccines were able to induce full protection against virulent IBV challenge. A single dose of AvCoV-CS vaccine was sufficient to achieve complete protection, while AvCoV-O required a previous priming by a Mass strain to complete the protection.

Stability Studies of the Vaccine Adjuvant U-Omp19.

Unlipidated outer membrane protein 19 (U-Omp19) is a novel mucosal adjuvant in preclinical development to be used in vaccine formulations. U-Omp19 holds two main properties, it is capable of inhibiting gastrointestinal and lysosomal peptidases, increasing the amount of co-administered antigen that reaches the immune inductive sites and its half-life inside cells, and it is able to stimulate antigen presenting cells in vivo. These activities enable U-Omp19 to enhance the adaptive immune response to co-administrated antigens. To characterize the stability of U-Omp19 we have performed an extensive analysis of its physicochemical and biological properties in a 3-year long-term stability study, and under potentially damaging freeze-thawing and lyophilization stress processes. Results revealed that U-Omp19 retains its full protease inhibitor activity, its monomeric state and its secondary structure even when stored in solution for 36 months or after multiple freeze-thawing cycles. Non-enzymatic hydrolysis resulted the major degradation pathway for storage in solution at 4 °C or room temperature which can be abrogated by lyophilization yet increasing protein tendency to form aggregates. This information will play a key role in the development of a stable formulation of U-Omp19, allowing an extended shelf-life during manufacturing, storage, and shipping of a future vaccine containing this pioneering adjuvant.

Proof of Concept for Prevention of Natural Colonization by Oral Needle-Free Administration of a Microparticle Vaccine.

There has been substantial interest in the development of needle-free vaccine administration that has led to a variety of approaches for delivery through the skin for induction of a systemic immune response. The mucosal administration of vaccines has inherently been needle-free, but the simple application of vaccines on the mucosal surface by itself does not lead to mucosal immunity. Since many important bacterial infections develop after initial colonization of the upper respiratory tract of the host, prevention of colonization could not only prevent infection but also eliminate the reservoir of pathogens that reside exclusively in that ecologic niche. This study was designed to provide proof of concept for a needle-free immunization approach that would reduce or eliminate colonization and prevent infection. In order to accomplish this a microparticle vaccine preparation was delivered just below the oral mucosal epithelial cell layer where it would lead to a robust immune response. A vaccine antigen (mutant transferrin binding protein B) shown to be capable of preventing infection in pigs was incorporated into a polyphosphazene microparticle preparation and delivered by a needle-free device to the oral sub-epithelial space of pigs. This vaccination regimen not only provided complete protection from infection after intranasal challenge by Glaesserella parasuis but also eliminated natural colonization by this bacterium. Notably, the complete prevention of natural colonization was dependent upon delivery of the microparticle preparation below the epithelial layer in the oral mucosa as intradermal or intramuscular delivery was not as effective at preventing natural colonization. This study also demonstrated that a primary immunization in the presence of maternal antibody limited the resulting antibody response but a robust antibody response after the second immunization indicated that maternal antibody did not prevent induction of B-cell memory.

What Does Plant-Based Vaccine Technology Offer to the Fight against COVID-19?

The emergence of new pathogenic viral strains is a constant threat to global health, with the new coronavirus strain COVID-19 as the latest example. COVID-19, caused by the SARS-CoV-2 virus has quickly spread around the globe. This pandemic demands rapid development of drugs and vaccines. Plant-based vaccines are a technology with proven viability, which have led to promising results for candidates evaluated at the clinical level, meaning this technology could contribute towards the fight against COVID-19. Herein, a perspective in how plant-based vaccines can be developed against COVID-19 is presented. Injectable vaccines could be generated by using transient expression systems, which offer the highest protein yields and are already adopted at the industrial level to produce VLPs-vaccines and other biopharmaceuticals under GMPC-processes. Stably-transformed plants are another option, but this approach requires more time for the development of antigen-producing lines. Nonetheless, this approach offers the possibility of developing oral vaccines in which the plant cell could act as the antigen delivery agent. Therefore, this is the most attractive approach in terms of cost, easy delivery, and mucosal immunity induction. The development of multiepitope, rationally-designed vaccines is also discussed regarding the experience gained in expression of chimeric immunogenic proteins in plant systems.

Chitosan and carboxymethylated derivative nanoparticles as delivery systems for biological products: preparation, characterization, stability and in vitro/in vivo evaluation / Nanopartículas de quitosana e derivado carboximetilado como sistemas de fornecimento (delivery) de produtos biológicos: preparo, caracterização, estabilidade e avaliação in vitro/in vivo

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

Polissacarídeo capsular do Streptococcus agalactiae como antígeno vacinal: desenvolvimento de um modelo vacinal para mucosas com Nanopartícula de quitosana / Capsular polysaccharide of Streptococcus agalactiae as vaccine antigen: development of a mucosal vaccine model with chitosan nanoparticle

A bactéria gram-positiva Streptococcus agalactiae do grupo B (GBS) faz parte da microbiota normal do trato geniturinário humano, sendo um organismo comensal do corpo da mulher. No entanto, em mulheres grávidas, quando há alterações na composição microbiana do canal vaginal, pode ocorrer a proliferação e a infecção pelo GBS. Este microrganismo, em sua forma patogênica oportunista, pode infectar o neonato durante o parto natural, assim como contribuir para infecções urinárias e uterinas durante a gestação. O GBS já foi identificado como um dos responsáveis pela alta taxa de mortalidade neonatal, sendo um dos principais agentes de infecção em recém-nascidos no mundo. Ele também pode ser a causa de infecções nas gestantes, levando a várias complicações, como corioamnionite, endometrite e infecções do trato urinário e do sítio cirúrgico. Pode haver comprometimento da gestação e do feto, com abortamento, morte fetal intrauterina e ruptura da membrana coriônica, levando a parto prematuro - que pode resultar em outras consequências graves. Este trabalho foi desenvolver um modelo vacinal para mucosa sublingual, utilizando o polissacarídeo capsular do Streptococcus agalactiae como antígeno, encapsulado em Nanopartículas de quitosana. Para o estudo de otimização dos parâmetros de fermentação, para aumentar a produtividade de cápsula polissacarídica (PS) presente na superfície celular, utilizou-se o Banco de Dados Kegg (Kyoto Encyclopedia of Genes and Genomes). A adição do suplemento L-Prolina foi o que propiciou a principio, maior relação entre crescimento bacteriano e formação de cápsula polissacarídica. A purificação e extração da cápsula polissacarídica foi realizada com etapas sucessivas de ultra filtração tangencial e precipitação alcóolica dos contaminantes. As caracterizações físico-químicas: difração de raios-X (DRX), cromatografia gasosa (CGMS), ressonância magnética (NMR) e determinação de açúcares pelo método fenol-sulfúrico, foram realizadas para identificação da composição e estrutura monossacarídica de açucares. O PS isolado apresenta ramificações de fucose, manose, glicose, galactose e N-acetil-glucosamina, apresentando estrutura amorfa. A liofilização do polissacarídeo foi realizada para fins de concentração e conservação. A encapsulação do polissacarídeo acoplada quimicamente com OVA, em uma Nanopartícula de quitosana, teve como finalidade aumentar a mucoadesividade e possibilitar maior absorção do antígeno entre as células da junção epitelial das mucosas sublinguais. A partir da análise de DLS (Espalhamento dinâmico de luz), as Nanopartículas apresentaram dimensões entre 200 a 400 nm e o Potencial Zeta acima de 20. O índice de polidispersão (PDI) está dentro do esperado (abaixo de 0.3). A capacidade de encapsulamento em relação à OVA foi de 92,8% dos grupos que continham PS. O teste IgG sérica total mostrou que o grupo G2 (Nanopartícula com Polissacarídeo e Proteína acoplados) foi o que teve maior reatividade no teste de ELISA, pela Análise de Variância (ANOVA) com ferramenta de Bonferrone. O teste sIgA mostrou que o grupo G2 (Nanopartícula com Polissacarídeo e Proteína acoplados) foi o que teve maior concentração de anticorpo sIgA total. Como resultado e conclusão, o polissacarídeo capsular do Streptococcus agalactiae é um bom candidato a antígeno vacinal

Gram-positive bacteria Streptococcus agalactiae group B (GBS) is part of the normal microbiota of the human genitourinary tract, being a commensal organism of the female body. However, in pregnant women, when there are changes in the microbial composition of the vaginal canal, GBS proliferation and infection may occur. This microorganism, in its opportunistic pathogenic form, can infect the neonate during natural childbirth, as well as contribute to urinary and uterine infections during pregnancy. The GBS has already been identified as one of the responsible for the high neonatal mortality rate, being one of the main agents of infection in newborns in the world. It can also be the cause of infections in pregnant women, leading to various complications such as chorioamnionitis, endometritis, and urinary tract and surgical site infections. There may be pregnancy and fetal impairment, with abortion, fetal intrauterine death, and rupture of the chorionic membrane, leading to premature labor - which can result in other serious consequences. This work was to develop a vaccine model for sublingual mucosa using the capsular polysaccharide of Streptococcus agalactiae as antigen, encapsulated in chitosan nano particles. For the study of optimization of the fermentation parameters, the Kegg (Kyoto Encyclopedia of Genes and Genomes) database was used to increase the productivity of polysaccharide capsule (PS) present on the cell surface. The addition of the L-Proline supplement gave rise to a higher ratio between bacterial growth and polysaccharide capsule formation. The purification and extraction of the polysaccharide capsule was performed with successive stages of tangential ultrafiltration and alcoholic precipitation of the contaminants. The physicochemical characterization of X-ray diffraction (XRD), gas chromatography (CGMS), magnetic resonance (NMR) and determination of sugars by the phenol-sulfuric method were performed to identify the composition and monosaccharide structure of sugars. The isolated PS presents branches of fucose, mannose, glucose, galactose and N-acetyl-glucosamine, presenting amorphous structure. Lyophilization of the polysaccharide was performed for concentration and conservation purposes. The encapsulation of the polysaccharide coupled chemically with OVA in a chitosan nano particle was aimed at increasing mucoadhesiveness and allowing greater absorption of the antigen between the cells of the sublingual mucosal epithelial junction. From the analysis of DLS (dynamic light scattering), the nanoparticles presented dimensions between 200 to 400 nm and the Zeta potential above 20. The polydispersity index (PDI) is within the expected range (below 0.3). The encapsulation capacity for OVA was 92.8% of the groups containing PS. The total serum IgG test showed that the G2 group (Nano particle with Polysaccharide and Protein coupled) was the one that had the highest reactivity in the ELISA test, by Analysis of Variance (ANOVA) with Bonferrone tool. The sIgA test showed that the G2 group (Nanoparticle with Polysaccharide and Protein coupled) had the highest concentration of total sIgA antibody. As a result and conclusion, the capsular polysaccharide of Streptococcus agalactiae is a good candidate for vaccine antigen

Raw starch microparticles have immunostimulant activity in mice vaccinated with BCG and challenged with Mycobacterium tuberculosis.

The main challenge for vaccine development or improvement is the lack of safe adjuvants or immunostimulants that induce protective immune responses and can be used for mucosal immunization, which is a highly desirable strategy for vaccination against infectious diseases acquired by oral or intranasal routes. One promising alternative is the use of biodegradable and biocompatible polymeric microparticles. Recently, we developed an immobilization and delivery system with starch microparticles (SMPs) and a starch-binding domain (SBDtag) suitable for the mucosal administration of antigens and the induction of antigen-specific immune responses. Here, we explore the immunostimulant and reinforcing potential of the system using BALB/c mice with progressive pulmonary tuberculosis (PPT). The heat shock protein alpha-crystallin from Mycobacterium tuberculosis immobilized on SMPs (µAcr-SBDtag) or SMPs alone were administered nasally as boosters to BCG-vaccinated mice without any extra adjuvant. The mice were challenged intratracheally with either moderately virulent or highly virulent M. tuberculosis strains. Our results showed that the administration of either the immobilized antigen or SMPs asa booster for the BCG vaccination induced a significant reduction of bacterial loads in the lungs of mice, even more than in mice that received the BCG vaccination alone. Since no difference was observed in pulmonary bacillary burdens between the two reinforced groups, the obtained effect was most likely primarily caused by the starch. As determined by histological study, the administration of boosters did not contribute to the progress of pneumonia, which diminishes the safety concerns related to the administration of SMPs intranasally. Taken together, our findings suggest that this system may be considered asa new carbohydrate-based adjuvant suitable for mucosal vaccines against tuberculosis and other infectious diseases, and more generally, they highlight the potential of particulate α-glucans as immune response modifiers.

A Perspective on the Development of Plant-Made Vaccines in the Fight against Ebola Virus.

The Ebola virus (EBOV) epidemic indicated a great need for prophylactic and therapeutic strategies. The use of plants for the production of biopharmaceuticals is a concept being adopted by the pharmaceutical industry, with an enzyme for human use currently commercialized since 2012 and some plant-based vaccines close to being commercialized. Although plant-based antibodies against EBOV are under clinical evaluation, the development of plant-based vaccines against EBOV essentially remains an unexplored area. The current technologies for the production of plant-based vaccines include stable nuclear expression, transient expression mediated by viral vectors, and chloroplast expression. Specific perspectives on how these technologies can be applied for developing anti-EBOV vaccines are provided, including possibilities for the design of immunogens as well as the potential of the distinct expression modalities to produce the most relevant EBOV antigens in plants considering yields, posttranslational modifications, production time, and downstream processing.

Mucosal and systemic immune responses induced by a single time vaccination strategy in mice.

Vaccination is considered by the World Health Organization as the most cost-effective strategy for controlling infectious diseases. In spite of great successes with vaccines, many infectious diseases are still leading killers, because of the inadequate coverage of many vaccines. Several factors have been responsible: number of doses, high vaccine reactogenicity, vaccine costs, vaccination policy, among others. Contradictorily, few vaccines are of single dose and even less of mucosal administration. However, more common infections occur via mucosa, where secretory immunoglobulin A plays an essential role. As an alternative, we proposed a novel protocol of vaccination called Single Time Vaccination Strategy (SinTimVaS) by immunizing 2 priming doses at the same time: one by mucosal route and the other by parenteral route. Here, the mucosal and systemic responses induced by Finlay adjuvants (AF Proteoliposome 1 and AF Cochleate 1) implementing SinTimVaS in BALB/c mice were evaluated. One intranasal dose of AF Cochleate 1 and an intramuscular dose of AF Proteoliposome 1 adsorbed onto aluminum hydroxide, with bovine serum albumin or tetanus toxoid as model antigens, administrated at the same time, induced potent specific mucosal and systemic immune responses. Also, we demonstrated that SinTimVaS using other mucosal routes like oral and sublingual, in combination with the subcutaneous route elicits immune responses. SinTimVaS, as a new immunization strategy, could increase vaccination coverage and reduce time-cost vaccines campaigns, adding the benefits of immune response in mucosa.

A novel antigen-carrier system: the Mycobacterium tuberculosis Acr protein carried by raw starch microparticles.

Microparticles have been used as promising carriers for in vivo vaccine delivery. However, the processes for immobilizing peptides or proteins on microparticles usually require the use of undesirable compounds and complex protocols. In this work, we propose a new immobilization and delivery system with raw starch microparticles and a starch binding domain (SBD) tag fusion protein. The heat shock protein alpha crystallin from Mycobacterium tuberculosis was used as model. The immunogenicity of the system was investigated in BALB/c mice inoculated with purified Acr-SBDtag protein (pAcr-SBDtag) and starch immobilized Acr-SBDtag protein (µAcr-SBDtag) by oral and intranasal routes. We demonstrated mucosal immunization with the µAcr-SBDtag protein induced systemic antibodies that were predominantly immunoglobulin G2a (IgG2a). An analysis of the cytokines from spleen cells and lung homogenates revealed that loaded microparticles induced the secretion of interferon-γ (INF-γ), suggesting an adjuvant effect from the immobilization. The immune responses induced by immobilized protein were primarily affected by the route of administration. These results demonstrate that the system exhibits the necessary characteristics to improve antigen release and presentation to antigen presenting cells (APCs) in the mucosae. Because no extra adjuvants were used, we posit that the system may be suitable for delivery and presentation to the field of subunit vaccine development.

Adjuvant requirement for successful immunization with recombinant derivatives of Plasmodium vivax merozoite surface protein-1 delivered via the intranasal route

Recently, we generated two bacterial recombinant proteins expressing 89 amino acids of the C-terminal domain of the Plasmodium vivax merozoite surface protein-1 and the hexa-histidine tag (His6MSP1(19)). One of these recombinant proteins contained also the amino acid sequence of the universal pan allelic T-cell epitope (His6MSP1(19)-PADRE). In the present study, we evaluated the immunogenic properties of these antigens when administered via the intra-nasal route in the presence of distinct adjuvant formulations. We found that C57BL/6 mice immunized with either recombinant proteins in the presence of the adjuvants cholera toxin (CT) or the Escherichia coli heat labile toxin (LT) developed high and long lasting titers of specific serum antibodies. The induced immune responses reached maximum levels after three immunizing doses with a prevailing IgG1 subclass response. In contrast, mice immunized by intranasal route with His6MSP1(19)-PADRE in the presence of the synthetic oligonucleotides adjuvant CpG ODN 1826 developed lower antibody titers but when combined to CT, CpG addition resulted in enhanced IgG responses characterized by lower IgG1 levels. Considering the limitations of antigens formulations that can be used in humans, mucosal adjuvants can be a reliable alternative for the development of new strategies of immunization using recombinant proteins of P. vivax.