Promoção de crescimento e controle de tombamento de plântulas de pepino por rizobactérias

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.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

H7N9 influenza split vaccine with SWE oil-in-water adjuvant greatly enhances cross-reactive humoral immunity and protection against severe pneumonia in ferrets

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.</p

IgG Induced by Vaccination With Ascaris suum Extracts Is Protective Against Infection

Human ascariasis has a global and cosmopolitan distribution, and has been characterized as the most prevalent neglected tropical disease worldwide. The development of a preventive vaccine is highly desirable to complement current measures required for this parasitic infection control and to reduce chronic childhood morbidities. In the present study, we describe the mechanism of protection elicited by a preventive vaccine against ascariasis. Vaccine efficacy was evaluated after immunization with three different Ascaris suum antigen extracts formulated with monophosphoryl lipid A (MPLA) as an adjuvant: crude extract of adult worm (ExAD); crude extract of adult worm cuticle (CUT); and crude extract of infective larvae (L3) (ExL3). Immunogenicity elicited by immunization was assessed by measuring antibody responses, cytokine production, and influx of tissue inflammatory cells. Vaccine efficacy was evaluated by measuring the reductions in the numbers of larvae in the lungs of immunized BALB/c mice that were challenged with A. suum eggs. Moreover, lung physiology and functionality were tested by spirometry to determine clinical efficacy. Finally, the role of host antibody mediated protection was determined by passive transfer of serum from immunized mice. Significant reductions in the total number of migrating larvae were observed in mice immunized with ExL3 61% (p &lt; 0.001), CUT 59% (p &lt; 0.001), and ExAD 51% (p &lt; 0.01) antigens in comparison with non-immunized mice. For the Ascaris antigen-specific IgG antibody levels, a significant and progressive increase was observed with each round of immunization, in association with a marked increase of IgG1 and IgG3 subclasses. Moreover, a significant increase in concentration of IL-5 and IL-10 (pre-challenge) in the blood and IL-10 in the lung tissue (post-challenge) was induced by CUT immunization. Finally, ExL3 and CUT-immunized mice showed a marked improvement in lung pathology and tissue fibrosis as well as reduced pulmonary dysfunction induced by Ascaris challenge, when compared to non-immunized mice. Moreover, the passive transfer of specific IgG antibodies from ExL3, CUT, and ExAD elicited a protective response in naïve mice, with significant reductions in parasite burdens in lungs of 65, 64, and 64%, respectively. Taken together, these studies indicated that IgG antibodies contribute to protective immunity

Industrial production development of a new whole cell Pertussis Vaccine with low reatogenicity.

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 &cong;21% do conteúdo de LPS, porém com aumento de &cong; 52% da atividade endotóxica; por TFFSW houve a redução de 46% do conteúdo de LPS e uma redução endotóxica média de &cong; 24%; por CT ocorreu à redução de &cong; 66% do conteúdo de LPS e de &cong; 73% da atividade endotóxica; com a CFC houve a redução de &cong; 92% do conteúdo de LPS e de &cong; 61% da atividade endotóxica. Os rendimentos de processo foram &cong; 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 &cong; 21% in LPS content, but with a &cong;52% increase in endotoxic activity; by TFFSW there was a reduction of &cong; 46% of the LPS content and an average reduction of endotoxic activity of &cong;24%; CT reduced &cong; 66% of LPS content and &cong; 73% of endotoxic activity; with CFC there was a reduction of &cong; 92% in LPS content and &cong;1% in endotoxic activity. The process yields were &cong; 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)

H7N9 influenza split vaccine with SWE oil-in-water adjuvant greatly enhances cross-reactive humoral immunity and protection against severe pneumonia in ferrets.

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

Preparedness against pandemic influenza: Production of an oil-in-water emulsion adjuvant in Brazil.

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 <0.5 EU/mL. The emulsion particle size was stable for at least six months at 25°C and 24 months at 4-8°C. Two doses of H7N9 vaccine formulated at 7.5 μg/dose or 15 μg/dose with adjuvant IB160 showed a significant increase of hemagglutination inhibition (HAI) titers in sera of immunized BALB/c mice when compared to control sera from animals immunized with the H7N9 antigens without adjuvant. Thus the antigen-sparing capacity of IB160 can potentially increase the production of the H7N9 pandemic vaccine and represents an important achievement for preparedness against pandemic influenza and a successful North (IDRI) to South (Butantan Institute) technology transfer for the production of the adjuvant emulsion IB160