Célia Sayoko Takata

Combination of Pneumococcal Surface Protein A (PspA) with Whole Cell Pertussis Vaccine Increases Protection Against Pneumococcal Challenge in Mice

Streptococcus pneumoniae is the leading cause of respiratory acute infections around the world. In Latin America, approximately 20,000 children under 5 years of age die of pneumococcal diseases annually. Pneumococcal surface protein A (PspA) is among the best-characterized pneumococcal antigens that confer protection in animal models of pneumococcal infections and, as such, is a good alternative for the currently available conjugated vaccines. Efficient immune responses directed to PspA in animal models have already been described. Nevertheless, few low cost adjuvants for a subunit pneumococcal vaccine have been proposed to date. Here, we have tested the adjuvant properties of the whole cell Bordetella pertussis vaccine (wP) that is currently part of the DTP (diphtheria-tetanus-pertussis) vaccine administrated to children in several countries, as an adjuvant to PspA. Nasal immunization of BALB/c mice with a combination of PspA5 and wP or wPlow – a new generation vaccine that contains low levels of B. pertussis LPS – conferred protection against a respiratory lethal challenge with S. pneumoniae. Both PspA5-wP and PspA5-wPlow vaccines induced high levels of systemic and mucosal antibodies against PspA5, with similar profile, indicating no essential requirement for B. pertussis LPS in the adjuvant properties of wP. Accordingly, nasal immunization of C3H/HeJ mice with PspA5-wP conferred protection against the pneumococcal challenge, thus ruling out a role for TLR4 responses in the adjuvant activity and the protection mechanisms triggered by the vaccines. The high levels of anti-PspA5 antibodies correlated with increased cross-reactivity against PspAs from different clades and also reflected in cross-protection. In addition, passive immunization experiments indicated that antibodies played an important role in protection in this model. Finally, subcutaneous immunization with a combination of PspA5 with DTPlow protected mice against challenge with two different pneumococcal strains, opening the possibility for the development of a combined infant vaccine composed of DTP and PspA.

Bordetella pertussis monophosphoryl lipid A as adjuvant for inactivated split virion influenza vaccine in mice

The world production capacity of influenza vaccines is a concern in face of the potential influenza pandemic. The use of adjuvants could increase several fold the current installed production capacity. Bordetella pertussis monophosphyl lipid A (MPLA) was produced by acid hydrolysis of LPS, obtained as a by-product of its removal from cellular pertussis vaccine, generating a product with 4 side chains. We have investigated different formulations including MPLA alone or combined with Al(OH)(3) as adjuvants for an inactivated split virion influenza vaccine. Our results demonstrate that MPLA at concentrations as low as 0.01 microg per dose of vaccine is effective, even with a 4-fold reduction of the regular vaccine dose, as measured by the induction of protective hemagglutination inhibition (HAI) titers. Al(OH)(3) can be combined with 0.01-10 microg MPLA, inducing even higher immune responses. Al(OH)(3) caused a drift of the immune response induced by the vaccine towards a Th2 profile, as evaluated by an increase in the IgG1:IgG2a ratio, while MPLA showed a more balanced response. Moreover, the use of MPLA and Al(OH)(3) combination led to the induction of the highest IgG levels together with the secretion of both IFN-gamma and IL-4. Although cell-mediated immune responses have not been usually taken into account for influenza vaccine formulations, they may be relevant for the induction of cross-protection as well as immunological memory for both inter-pandemic and pandemic influenza vaccines. Our results indicate that a more favorable profile of both humoral and cell-mediated immune responses may be obtained using the MPLA/Al(OH)(3) formulation.

Dressing liposomal particles with chitosan and poly(vinylic alcohol) for oral vaccine delivery

Liposomes have been used as adjuvants since 1974. One major limitation for the use of liposomes in oral vaccines is the lipid structure instability caused by enzyme activities. Our aim was to combine liposomes that could encapsulate antigens (i.e., Dtxd, diphtheria toxoid) with chitosan, which protects the particles and promotes mucoadhesibility. We employed physical techniques to understand the process by which liposomes (SPC: Cho, 3:1) can be sandwiched with chitosan (Chi) and stabilized by PVA (poly-vinylic alcohol), which are biodegradable, biocompatible polymers. Round, smooth-surfaced particles of REVs-Chi (reversed-phase vesicles sandwiched by Chi) stabilized by PVA were obtained. The REVs encapsulation efficiencies (Dtxd was used as the antigen) were directly dependent on the Chi and PVA present in the formulation. Chi adsorption on the REVs surface was accompanied by an increase of ζ−potential. In contrast, PVA adsorption on the REVs-Chi surface was accompanied by a decrease of ζ−potential. The presence of Dtxd increased the Chi surface-adsorption efficiency. The PVA affinity by mucine was 2,000 times higher than that observed with Chi alone and did not depend on the molecule being in solution or adsorbed on the liposomal surface. The liberation of encapsulated Dtxd was retarded by encapsulation within REVs-Chi-PVA. These results lead us to conclude that these new, stabilized particles were able to be adsorbed by intestinal surfaces, resisted degradation, and controlled antigen release. Therefore, REVs-Chi-PVA particles can be used as an oral delivery adjuvant.

Evaluation of a Diphtheria and Tetanus PLGA Microencapsulated Vaccine Formulation without Stabilizers

Polymeric microspheres containing diphtheria and tetanus toxoids were prepared without protein stabilizers. A vaccine containing 2 Lf(tetanus) and 0.4 Lf(diphtheria) was injected either in BALB/c mice or in guinea-pigs. As control, a group received the alum-adsorbed unencapsulated toxoids. In mice, on day 44 one group and control received a booster and at day 111 the other group received the same booster dose. Before de booster, all groups had very low neutralizing antibodies, as determined by Toxin binding inhibition assay. One week after booster all groups had high antibody titers, especially those immunized with microencapsulated vaccine, which were at least 5 times higher than those immunized with alum vaccine for both antigens. Besides, guinea pigs receiving lower dose had antibodies titers as high as 60 UI/mL, and 30 times higher than those immunized with alum vaccine. Therefore by using an encapsulated vaccine without any kind of protein stabilizer we were able to induce in vivo protective responses irrespective of observed in vitro protein degradation by HPLC. Manipulating the vaccination schedule at the same time to the toxoids encapsulation does not only increase the antibody titers but also their specificity.

Fotossensibilidade e termoestabilidade de vacinas contra o sarampo (cepa Biken CAM-70) liofilizadas e/ou reconstituídas para administração

Three different lots of measles vaccines produced with the Biken CAM-70 virus strain were requested from the central cold store of the Public Health Department of the State of S. Paulo, Brazil, and assays on photosensitivity at 2-8 degrees C, and on stability at 28, 36.5 and 45 degrees C were carried out to find out for how long these vaccines would maintain their minimum potency, established as being 3.70 log10 or 5000 TCID50 (50% tissue culture infective dose) per human dose. The analysis of the adjusted straight regression lines indicated that, with the passage of time, the potency of lyophilized or reconstituted vaccines, as well as of vaccines exposed to or protected from light decreased. Light-exposed vaccines, however, became less potent than vaccines protected from the light. None of the vaccine lots studied, reconstituted and stored at 2-8 degrees C, exhibited homogeneity as to sensitivity to light. When freeze-dried vaccines had their photosensitivity studied at 2-8 degrees C, lots 1 and 2 presented greater thermal degradation when exposed to light than when protected from it. However, in both instances, it was found that potency fell below that taken as minimum for the Biken CAM-70 virus strain. At all other temperatures considered, even when protected from light, lots 1 and 2 did not retain the minimum potency. Lot 3 kept the expected stability for 60 days at 2-8 degrees C when protected from light and for 40 days when unprotected, but its thermal degradation at other temperatures was more intense (28 degrees C: 5 days; 36.5 degrees C: 2 days; 45 degrees C: 0.5 day).(ABSTRACT TRUNCATED AT 250 WORDS)Tres lotes de vacinas contra o sarampo, produzidos com a cepa de virus Biken CAM-70, sob as formas liofilizada e reconstituida, pertencentes ao estoque da rede de vacinacao da Secretaria de Estado da Saude de Sao Paulo, Brasil, foram submetidos a testes de sensibilidade a luz, a temperatura de 2 a 8°C, e de termoestabilidade (protegidos da luz) as temperaturas de 28, 36,5 e 45°C, objetivando verificar por quanto tempo retinham sua potencia, isto e, a concentracao ideal recomendada para a cepa de virus presente (3,70 log 10 DICT50 ou 5.000 doses infectantes de cultura de tecidos 50% por dose). A analise de retas de regressao ajustadas demonstrou que, de modo geral, tanto os lotes de vacinas liofilizados como os reconstituidos, mantidos as referidas temperaturas, expostos ou protegidos da luz, apresentaram queda de potencia no decorrer do experimento, a qual foi mais acentuada para vacinas expostas a luz. Reconstituidos e mantidos a 2 a 8°C, os lotes nao apresentaram homogeneidade no referente a sensibilidade a luz. Quando a fotossensibilidade de lotes de vacinas liofilizadas foi testada a 2 a 8°C eles mostraram-se mais sensiveis a degradacao termica quando expostos a luz do que quando protegidos dela. Entretanto, expostos ou protegidos, a potencia foi inferior a minima aceita para a cepa Biken CAM-70. As demais temperaturas, mesmo ao abrigo da luz, os dois lotes nao retiveram potencia minima. Quanto as vacinas do lote 3, conservadas a 2 a 8°C, mantiveram-se de acordo com os requerimentos minimos de potencia durante 60 dias quando protegidas da luz, e durante 40 dias quando expostas a ela. A degradacao termica as demais temperaturas foi mais acentuada (28°C: 5 dias; 36,5°C: 2 dias; 45°C: 0,5 dia). Considerando a concentracao viral minima que vacinas produzidas com a cepa Biken CAM-70 devem conter para induzir efetiva resposta imunologica, os lotes de vacinas pesquisados (sob a forma liofilizada ou reconstituidos para administracao) apresentaram, alem de baixa estabilidade ao calor, pouca homogeneidade com relacao a este parâmetro.

Hoffmeister Series Ions Protect Diphtheria Toxoid from Structural Damages at Solvent/Water Interface

During the W1/O phase (in the W1/O/W2 process) of protein microencapsulation within poly-lactide-co-glycolide (PLGA), hydrophobic interfaces are expanded where interfacial adsorption occurs followed by protein unfolding and aggregation. Spectroscopic and immunological techniques were used to ascertain the effects of the Hoffmeister series ions on Diphtheria toxoid (Dtxd) stability during the W1/O phase. A correlation was established between salts used in aqueous solutions and the changes in Dtxd solubility and conformation. The Dtxd α-helical content was quite stable thus leading to the conclusion that encapsulation was followed by protein aggregation, with minor exposition of hydrophobic residues and a small change at the S-S dihedral angle. Dtxd aggregation is 95% avoided by the chaotropic SCN-. This was used to prepare a stable Dtxd and immunologically recognized/PLGA formulation in the presence of 30 mM SNC-. The recovery increased by 10.42% or 23.2% when microencapsulation was within the -COOMe or -COOH (12kDa) PLGA, respectively. In conclusion, the aim of this work was achieved, which was to obtain the maximum of Dtxd stability after contact with CH2Cl2 to begin its PLGA microencapsulation within ideal conditions. This was a technological breakthrough because a simple solution like salt addition avoided heterologous proteins usage.

Enhanced liposomal vaccine formulation and performance: simple physicochemical and immunological approaches

The Dtxd (Diphtheria toxoid) was the first antigen encapsulated within liposomes, their adjuvant properties were discovered (their capacity to enhance the vaccine immunogenicity). The point here is not to propose a new method to prepare this lipossomal vaccine. The central idea is to give new dresses for old vaccines by using classical and well-established liposome preparation method changing only the encapsulation pH and the immunization protocol. The most appropriate method of Dtxd encapsulation within liposome was based on lipid film hydration in 100 mM citrate buffer, pH 4.0. This was accompanied by changes on protein hydrophobicity, observed by CD and fluorescence spectroscopies. Whenever the Dtxd exposed its hydrophobic residues at pH 4.0, it interacted better with the lipossomal (observed by electrophoretic mobility) film than when its hydrophobic residues were buried (pH 9.0). The Dtxd partition coefficient in Triton-X114 and the acrylamide fluorescence quenching were also pH dependent. Both were bigger at pH 4.0 than at pH 9.0. The relationship protein structure and lipid interaction was pH dependent and now it can be easily maximized to enhance encapsulation of antigens in vaccine development. Mice were primed with formulations containing 5 μg of Dtxd within liposomes prepared in pH 4.0 or 7.0 or 9.0. The boosters were done 38 or 138 days after the first immunization. The IgM produced by immediate response of all lipossomal formulations were higher than the control (free protein). The response patterns and the immune maturity were measured by IgG1 and IgG2a titrations. The IgG1 titers produced by both formulations at pH 4.0 and 7.0 were at least 22 higher than those produced by mice injected lipossomal formulation at pH 9.0. When the boosters were done, 138 days after priming the mice produced a IgG2a titer of 29 and the group that received the booster 30 days after priming produced a titer of 25. The strongest antibody production was the neutralizing antibody (245 higher than the control) produced by those mice injected with lipossomal formulation at pH 4.0 with the booster done 138 days after priming. The simple change on lipossomal pH formulation and timing of the booster enhanced both antibody production and selectivity.

Suscetibilidade da linhagem de células Vero a cepas vacinais do vírus do sarampo

Vero cells used by distinct measles vaccine control laboratories had their susceptibility to Moraten, Schwarz and Biken CAM-70 vaccine strains assayed. Of a total of 72 lots of measles vaccine whose potency was titrated by microtechnique in two Vero cell samples (Vero IB and Vero INCQS), 25 had been produced with Moraten strain, 24 with Schwarz and 23 with Biken CAM-70. The statistical analysis of the results demonstrated that both Vero cells assayed presented comparable susceptibility to Moraten and Biken CAM-70 strains. As to the Schwarz strain, Vero IB cells were more susceptible than the other cell sample tested, thus confirming the existence of different sensitivities of Vero cells to some measles vaccine strains, or even to viruses derived from the same strain but with different passage histories. An altered cell susceptibility to virus replication may significantly alter the results in potency testing. Such alteration may be caused not only by the adoption of distinct protocols for the maintenance of cell cultures by different control laboratories but also by the methodology followed in the vaccine titration. In order to minimize the differences existing among the results obtained in the potency testing, it is suggested that all control laboratories should use the same protocols for cell culture maintenance as well as for vaccine potency testing.

Diphtheria toxoid conformation in the context of its nanoencapsulation within liposomal particles sandwiched by chitosan

Chitosan (α-(1-4)-amino-2-deoxy-β-D-glucan) is a deacetylated form of chitin, a polysaccharide from crustacean shells. Its unique characteristics, such as positive charge, biodegradability, biocompatibility, nontoxicity, and rigid structure, make this macromolecule ideal for an oral vaccine delivery system. We prepared reverse-phase evaporation vesicles (REVs) sandwiched by chitosan (Chi) and polyvinylic alcohol (PVA). However, in this method, there are still some problems to be circumvented related to protein stabilization. During the inverted micelle phase of protein nanoencapsulation, hydrophobic interfaces are expanded, leading to interfacial adsorption, followed by protein unfolding and aggregation. Here, spectroscopic and immunological techniques were used to ascertain the effects of the Hoffmeister series ions on diphtheria toxoid (Dtxd) stability during the inverted micelle phase. A correlation was established between the salts used in aqueous solutions and the changes in Dtxd solubility and conformation. Dtxd α-helical content was quite stable, which led us to conclude that encapsulation occurred without protein aggregation or without exposition of hydrophobic residues. Dtxd aggregation was 98% avoided by the kosmotropic, PO2–4. This ion was used to prepare a stable Dtxd and immunologically recognized REV-Chi-PVA formulation in the presence of 50 mM of PO2–4. Under these conditions, the Dtxd retained its immunological identity. Therefore, we could obtain the maximum Dtxd solubility and stability after contact with CH3CO2C2H5 to begin its nanoencapsulation within ideal conditions. This was a technological breakthrough, because a simple solution, such as salt, addition avoided heterologous protein use.

Seringas hipodérmicas descartáveis versus reutilizáveis: estudo de possíveis efeitos sobre o vírus da vacina viva, atenuada contra o sarampo

Objetivou-se verificar entre seringas hipodermicas descartaveis e reutilizaveis qual interfere mais com o virus vivo presente na vacina contra o sarampo. Vacinas pertencentes a dois lotes foram reconstituidas com os dois tipos de seringas, de modo a formarem dois pools distintos, mantidos a temperatura de +2 a+8°C e protegidos da luz. De cada lote foram realizadas, no minimo, seis titulacoes em paralelo, com amostragem a cada hora, de zero a seis horas apos reconstituicao. A analise estatistica dos resultados obtidos nas titulacoes, feita pelo sistema de retas de regressao demonstrou que embora as vacinas manipuladas com ambos os tipos de seringas apresentassem um decrescimo de titulo estatisticamente significativo com o decorrer das horas, ele foi bem mais acentuado para as vacinas reconstituidas com seringas reutilizaveis. A menor interferencia das descartaveis no titulo da vacina viva, atenuada contra o sarampo, demonstrou que a preconizacao e uso desse tipo de seringas pela Secretaria de Estado da Saude de Sao Paulo e o ideal e recomendavel, por preservar mais a vacina desde a reconstituicao ate sua administracao e, consequentemente, a sua eficacia na prevencao dessa infeccao.The study was performed in the State of S.Paulo, SP, Brazil, with the purpose of finding out whether reusable (glass) and disposable hypodermic syringes used for administration of live attenuated measles vaccines would interfere with their virus. At least six different experiments using two distinct lots of vaccines were carried out. Each time, a lot was reconstituted with reusable and disposable syringes in parallel to form separate pools from which samples were collected hourly from zero to the sixtieth hour after reconstitution for virus titration in monolayers of Vero cells. The straight line regression system chosen for the analysis of the results demonstrated that although vaccines reconstituted with both types of syringes presented a statistically significative titer decrease as time went by, there was a more pronounced decrease for vaccines manipulated with the glass syringes. The fact that the disposable syringes affected the titer of the virus present in the live, attenuated measles vaccine less confirmed that the preconization and routine usage of this type of syringe by the Health Department of the State of S.Paulo, Brazil, is ideal and highly recommended because it preserves the vaccine from reconstitution to administration better, and thus, its efficacy in preventing the infection.