RESUMO
In a recent vaccine trial performed with African children, immunization with a recombinant protein based on Plasmodium falciparum apical membrane antigen 1 (AMA-1) conferred a significant degree of strain-specific resistance against malaria. To contribute to the efforts of generating a vaccine against Plasmodium vivax malaria, we expressed the ectodomain of P. vivax AMA-1 (PvAMA-1) as a secreted soluble protein in the methylotrophic yeast Pichia pastoris. Recognized by a high percentage of sera from individuals infected by P. vivax, this recombinant protein was found to have maintained its antigenicity. The immunogenicity of this protein was evaluated in mice using immunization protocols that included homologous and heterologous prime-boost strategies with plasmid DNA and recombinant protein. We used the following formulations containing different adjuvants: aluminum salts (Alum), Bordetella pertussis monophosphoryl lipid A (MPLA), flagellin FliC from Salmonella enterica serovar Typhimurium, saponin Quil A, or incomplete Freund's adjuvant (IFA). The formulations containing the adjuvants Quil A or IFA elicited the highest IgG antibody titers. Significant antibody titers were also obtained using a formulation developed for human use containing MPLA or Alum plus MPLA. Recombinant PvAMA-1 produced under "conditions of good laboratory practice" provided a good yield, high purity, low endotoxin levels, and no microbial contaminants and reproduced the experimental immunizations. Most relevant for vaccine development was the fact that immunization with PvAMA-1 elicited invasion-inhibitory antibodies against different Asian isolates of P. vivax. Our results show that AMA-1 expressed in P. pastoris is a promising antigen for use in future preclinical and clinical studies.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Proteínas de Membrana/imunologia , Pichia/imunologia , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Leveduras/imunologia , Adjuvantes Imunológicos/genética , Animais , Formação de Anticorpos/imunologia , Antígenos de Protozoários/genética , Feminino , Humanos , Imunização/métodos , Imunoglobulina G/imunologia , Vacinas Antimaláricas/genética , Vacinas Antimaláricas/imunologia , Malária Vivax/genética , Malária Vivax/imunologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Pichia/genética , Plasmodium vivax/genética , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Leveduras/genéticaRESUMO
The production of a healthy cloned calf is dependent on a multitude of successful steps, including reprogramming mediated by the oocyte, the development of a functional placenta, adequate maternal-fetal interaction, the establishment of a physiological metabolic setting and the formation of a complete set of well-differentiated cells that will eventually result in well-characterised and fully competent tissues and organs. Although the efficiency of nuclear transfer has improved significantly since the first report of a somatic cell nuclear transfer-derived animal, there are many descriptions of anomalies concerning cloned calves leading to high perinatal morbidity and mortality. The present article discusses some our experience regarding perinatal and neonatal procedures for cloned Zebu cattle (B. indicus) that has led to improved survival rates in Nellore cloned calves following the application of such 'labour-intensive technology'.
Assuntos
Criação de Animais Domésticos/métodos , Bovinos/embriologia , Clonagem de Organismos/veterinária , Animais , Animais Recém-Nascidos , Clonagem de Organismos/métodos , Anormalidades Congênitas/mortalidade , Anormalidades Congênitas/veterinária , Feminino , Técnicas de Transferência Nuclear/veterinária , Gravidez , Taxa de SobrevidaRESUMO
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.
Assuntos
Perda do Embrião/induzido quimicamente , Feto/anormalidades , Leishmania braziliensis , Vacinas contra Leishmaniose/efeitos adversos , Exposição Materna/efeitos adversos , Modelos Biológicos , Peroxirredoxinas/efeitos adversos , Proteínas de Protozoários/efeitos adversos , Animais , Anticorpos Antiprotozoários/imunologia , Avaliação Pré-Clínica de Medicamentos , Feminino , Feto/imunologia , Imunoglobulina G/imunologia , Vacinas contra Leishmaniose/imunologia , Vacinas contra Leishmaniose/farmacologia , Peroxirredoxinas/imunologia , Peroxirredoxinas/farmacologia , Gravidez , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/farmacologia , RatosRESUMO
PURPOSE: To study the immunogenicity and the stability of the porcine pulmonary surfactant preparation produced by the Instituto Butantan. METHOD: Immunogenicity assay: Sixteen New-Zealand-White rabbits (1000 g body weight) were divided into 4 study groups. Each group was assigned to receive either a) Butantan surfactant, b) Survanta (Abbott Laboratories), c) Curosurf (Farmalab Chiesi), or d) no surfactant. The surfactants were administered intratracheally, and the animals were collected immediately before and 60 and 180 days after surfactant administration. Sera were assayed for the presence of antisurfactant antibodies by enzyme-linked immunosorbent assay (ELISA). Stability assay: The Butantan surfactant used in this assay had been stored for one year in the refrigerator (4 to 8 degrees C) and its stability was evaluated in distinct assay conditions using a premature rabbit model. RESULTS: Immunogenicity assay: None of the surfactants analyzed triggered antibody immune responses against their components in any of the animals. Stability assay: The results of this study demonstrate that Butantan surfactant was as effective as Curosurf when both were submitted to the adverse circumstance of short- and long-term storage at room temperature. A similar level of efficacy for the Butantan surfactant, as compared to Curosurf was demonstrated by the pulmonary dynamic compliance, ventilatory pressure, and pressure-volume curve results. CONCLUSION: The results of our study demonstrate that Butantan surfactant may be a suitable alternative for surfactant replacement therapy.
Assuntos
Surfactantes Pulmonares/química , Surfactantes Pulmonares/imunologia , Animais , Animais Recém-Nascidos , Feminino , Modelos Animais , Gravidez , Proteínas Associadas a Surfactantes Pulmonares/imunologia , Surfactantes Pulmonares/uso terapêutico , Coelhos , Doenças Respiratórias/tratamento farmacológico , Suínos , Fatores de TempoRESUMO
Consecutive lots of H5N1 (A/Vietnam/1194/2004 - NIBRG-14) split virion and whole virus vaccines were produced in a pilot-scale laboratory. The average yields of vaccine doses (15 microg HA) per egg were 0.57 doses for H5N1 split virion vaccine and 1.12 for H5N1 whole virus vaccine, compared to 2.09 doses for the seasonal H3N2 split virion vaccine. H5N1 split virion vaccine lots complied with WHO protein content criteria, while some lots of the H5N1 whole virus vaccine showed protein content per dose higher than the limit established. All lots of both vaccines showed ovalbumin (OVA) concentration below the recommended limit. Dose sparing strategies using adjuvant formulations using aluminum hydroxide (Al(OH)(3)) and monophosphoryl lipid A (MPLA) from Bordetella pertussis were tested in mice. Both 3.75 microg HA and 7.5 microg HA of H5N1 split virion vaccine with Al(OH)(3) or Al(OH)(3) plus MPLA in aqueous suspension showed higher hemagglutination-inhibition (HAI) titers when compared to the same vaccine dose without any adjuvant. Immunization with the H5N1 inactivated whole virus vaccine was also performed using 3.75 microg HA and HAI titers were higher than those induced by the split virion vaccine. Moreover, the use of Al(OH)(3) with MPLA as an emulsion induced a further increase in HAI titers.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Hidróxido de Alumínio/administração & dosagem , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/análise , Bordetella pertussis/química , Feminino , Testes de Inibição da Hemaglutinação , Vacinas contra Influenza/química , Lipídeo A/administração & dosagem , Lipídeo A/análogos & derivados , Lipídeo A/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/análise , Vacinas de Produtos Inativados/química , Vacinas de Produtos Inativados/imunologia , Vacinas de Subunidades Antigênicas/química , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
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 wP(low)--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-wP(low) 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 DTP(low) 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.
Assuntos
Proteínas de Bactérias/imunologia , Vacina contra Coqueluche/imunologia , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Administração Intranasal , Animais , Anticorpos Antibacterianos/imunologia , Proteção Cruzada/imunologia , Reações Cruzadas/imunologia , Imunização , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Mucosa Nasal/imunologia , Mucosa Nasal/microbiologia , Infecções Pneumocócicas/sangue , Doenças Respiratórias/sangue , Doenças Respiratórias/imunologia , Doenças Respiratórias/microbiologia , Análise de Sobrevida , Receptor 4 Toll-Like/metabolismoRESUMO
OBJECTIVE: to discuss the current PAHO recommendation that does not support the substitution of traditional cellular DTP vaccine by acellular DTP, and the role of mutations, in humans, as the main cause of rare adverse events, such as epileptic-like convulsions, triggered by pertussis vaccine. DATA REVIEW: the main components related to toxic effects of cellular pertussis vaccines are the lipopolysaccharide of bacterial cell wall and pertussis toxin. The removal of part of lipopolysaccharide layer has allowed the creation of a safer cellular pertussis vaccine, with costs comparable to the traditional cellular vaccine, and which may be a substitute for the acellular vaccine. CONCLUSION: The new methodology introduced by Instituto Butantan allows for the development of a new safer pertussis vaccine with low LPS content (Plow), and the use of the lipopolysaccharide obtained in the process in the production of monophosphoryl lipid A. This component has shown potent adjuvant effect when administered together with influenza inactivated vaccine, making possible to reduce the antigen dose, enhancing the production capacity and lowering costs.
Assuntos
Vacina contra Difteria, Tétano e Coqueluche/efeitos adversos , Vacinas contra Difteria, Tétano e Coqueluche Acelular/efeitos adversos , Lipopolissacarídeos/imunologia , Mutação , Análise Custo-Benefício , Vacina contra Difteria, Tétano e Coqueluche/genética , Vacina contra Difteria, Tétano e Coqueluche/imunologia , Vacinas contra Difteria, Tétano e Coqueluche Acelular/genética , Vacinas contra Difteria, Tétano e Coqueluche Acelular/imunologia , Humanos , Lipopolissacarídeos/efeitos adversos , Organização Mundial da SaúdeRESUMO
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.
Assuntos
Adjuvantes Imunológicos/farmacologia , Bordetella pertussis/química , Vacinas contra Influenza/imunologia , Lipídeo A/análogos & derivados , Adjuvantes Imunológicos/isolamento & purificação , Hidróxido de Alumínio/farmacologia , Animais , Anticorpos Antivirais/sangue , Testes de Inibição da Hemaglutinação , Imunoglobulina G/sangue , Interferon gama/metabolismo , Interleucina-4/metabolismo , Lipídeo A/isolamento & purificação , Lipídeo A/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Objective: to discuss the current PAHO recommendation that does not support the substitution of traditional cellular DTP vaccine by acellular DTP, and the role of mutations, in humans, as the main cause of rare adverse events, such as epileptic-like convulsions, triggered by pertussis vaccine. Data review: the main components related to toxic effects of cellular pertussis vaccines are the lipopolysaccharide of bacterial cell wall and pertussis toxin. The removal of part of lipopolysaccharide layer has allowed the creation of a safer cellular pertussis vaccine, with costs comparable to the traditional cellular vaccine, and which may be a substitute for the acellular vaccine. Conclusion: The new methodology introduced by Instituto Butantan allows for the development of a new safer pertussis vaccine with low LPS content (Plow), and the use of the lipopolysaccharide obtained in the process in the production of monophosphoryl lipid A. This component has shown potent adjuvant effect when administered together with influenza inactivated vaccine, making possible to reduce the antigen dose, enhancing the production capacity and lowering costs.
Objetivo: Discutir as recomendações da WHO-OPAS que não consideram indicada a substituição da vacina DTP celular clássica pela DTP acelular e o papel de mutações, em humanos, como principal causa dos raros eventos de convulsões epileptiformes desencadeadas pela vacina pertussis. Revisão dos dados: Os principais componentes relacionados aos efeitos tóxicos da vacina pertussis celular são o lipopolissacarídio da parede celular da bactéria e a toxina pertussis. A remoção de parte da camada lipopolissacarídica permitiu a criação de uma vacina pertussis celular, mais segura e de custo comparável ao da vacina celular tradicional, podendo substituir a vacina pertussis acelular. Conclusão: A nova vacina pertussis, com baixo teor de LPS (Plow) desenvolvida pelo Instituto Butantan, além de oferecer uma vacina mais segura, permite o aproveitamento do lipopolissacarídeo para a produção de monofosforil lipídeo A. Esse componente mostrou-se potente como adjuvante e altamente eficiente quando administrado com a vacina de influenza, levando à possibilidade de se reduzir a dose de antígeno, aumentando a capacidade de produção e redução dos custos.
Assuntos
Humanos , Vacina contra Difteria, Tétano e Coqueluche/efeitos adversos , Vacinas contra Difteria, Tétano e Coqueluche Acelular/efeitos adversos , Lipopolissacarídeos/imunologia , Mutação , Análise Custo-Benefício , Vacina contra Difteria, Tétano e Coqueluche/genética , Vacina contra Difteria, Tétano e Coqueluche/imunologia , Vacinas contra Difteria, Tétano e Coqueluche Acelular/genética , Vacinas contra Difteria, Tétano e Coqueluche Acelular/imunologia , Lipopolissacarídeos/efeitos adversos , Organização Mundial da SaúdeRESUMO
Intranasal challenge of C57BL/6 mice with Streptococcus pneumoniae serotypes 6B, 14, and 23F produced colonization of the middle ear and NP. Intranasal vaccination with ethanol-killed nonencapsulated cells with adjuvant protected both sites. Of four nontoxic adjuvants tested, the cholera toxin B subunit was most effective and least nonspecifically protective.
Assuntos
Otopatias/microbiologia , Doenças Nasofaríngeas/microbiologia , Infecções Pneumocócicas/imunologia , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Otopatias/imunologia , Otopatias/prevenção & controle , Orelha Média/imunologia , Orelha Média/microbiologia , Camundongos , Doenças Nasofaríngeas/imunologia , Doenças Nasofaríngeas/prevenção & controle , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/farmacologia , Sorotipagem , Streptococcus pneumoniae/efeitos dos fármacosRESUMO
OBJETIVO: Estudar a imunogenicidade e a estabilidade do surfactante de origem porcina produzido pelo Instituto Butantan. MÉTODO: Experimento imunogenicidade: 16 coelhos da raça New-Zealand-White (Peso de 1000g) foram divididos em grupos de 4 animais. Cada grupo foi designado para receber: a) Surfactante do Butantan, b) Survanta® (Abbott Laboratories), c) Curosurf (Farmalab Chiesi) e d) nenhum tratamento com surfactante. Os surfactantes foram administrados via intratraqueal e o sangue dos animais foi coletado antes, 60 e 180 dias após a administração do surfactante. O soro obtido foi analisado quanto a presença de anticorpos anti-surfactante pelo método ELISA (enzyme-linked immunosorbent assay). Experimento estabilidade: O surfactante do Butantan usado neste experimento tinha sido armazenado por um ano em refrigerador (4 a 8°C) e sua estabilidade foi analisada em condições distintas de experimentação, usando o modelo de coelho prematuro. RESULTADOS: Experimento imunogenicidade: Nenhum dos surfactantes analisados determinou a produção de anticorpos contra seus constituintes. Experimento estabilidade: Os resultados deste estudo demonstraram que o surfactante do Instituto Butantan mostrou eficácia semelhante a do Curosurf após ter sido submetido à condições adversas ao longo do tempo. A eficácia foi demonstrada através da complacência pulmonar dinâmica, pressão ventilatória e da curva pressão-volume. CONCLUSÃO: Os resultados deste estudo demonstraram que o surfactante do Instituto Butantan pode representar um tratamento alternativo de reposição de surfactante.
Assuntos
Animais , Feminino , Gravidez , Coelhos , Surfactantes Pulmonares/química , Surfactantes Pulmonares/imunologia , Modelos Animais , Animais Recém-Nascidos , Doenças Respiratórias/tratamento farmacológico , Fatores de Tempo , Proteínas Associadas a Surfactantes Pulmonares/imunologia , Surfactantes Pulmonares/uso terapêutico , SuínosRESUMO
Susceptible insect cell lines to specific pathogens of agricultural insect pests could be directly applied to special programs of their biological control; or this could be also used in the propagation of vectors of expression, such as baculovirus for genetic enginnering. In this paper, several attempts of establishing cell lines from different tissues of two Lepidoptera, Dione juno, an injurious pest of the fruit-bearing plants Passifloraceae and Methona themisto, a pest of an urban ornamental tree, Brunfelsia sp., are described. Two cell cultures could be obtained from D. juno: One, Dj-0387, from macerated neonate larvae and the other, Dj3-0687, from minced ovaries of 5th instar larvae. Their morphological characteristics, viability and populational growth rate were similar. Approximately after 60 passages in vitro for Dj-0387 and 40 passages for Dj3-0687, these cell cultures became inviable. Apparently, these numbers of passages were their critical age limit to establish in vitro. Otherwise, at the 30th passage, mainly in the case of Dj-0387, the origin of the "larval beings"was observed. This phenomenon could be explained by the presence of the imaginal discs of the newborn larvae, that could resume development in vitro.
Assuntos
Animais , Lepidópteros/genética , Controle Biológico de Vetores , Pragas da Agricultura , Linhagem CelularRESUMO
A determinação espectrofotométrica na região do ultravioleta para quantificação de detergentes foi baseada na formação de complexo não estequiométrico entre o detergente e os reagentes (cloreto de bário e ácido fosfomolíbdico). A quantidade de complexo dissolvido com 2-metoxietanol foi quantitativamente medida em 310 nm. A metodologia foi reprodutível na faixa de 1-50 µg/mL para os três detergentes testados: Tween 20, Brij 35 e Triton X-100. Detectou-se interferência da proteína. Para superar tal interferência, brancos diferentes foram usados para as curvas padrão dos detergentes e para as amostras de proteínas purificadas