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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.
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Background Pertussis cases have increased worldwide and knowledge on immune response and cytokine profile after Tdap vaccine in immunodeficient adolescents is scarce. Objective To evaluate the immune response after Tdap in HIV-infected (HIV) and in healthy adolescents (CONTROL). Methodology Thirty HIV adolescents with CD4 cell counts?>200 and 30 CONTROLs were immunized with Tdap, after a prior whole-cell DTP vaccine primary scheme. Blood samples were collected immediately before and after vaccine. Lymphocyte immunophenotyping was performed by flow cytometry; tetanus, diphtheria and pertussis toxin antibodies were assessed by ELISA; whole blood was stimulated with tetanus toxoid and Bordetella pertussis and supernatants were assessed for cytokines by xMAP. Results Mean age of HIV and CONTROL groups were 17.9 e 17.1?years, respectively. Pain at injection site was more intense in CONTROL group. HIV group had similar increase in tetanus antibodies at 28?days (geometric mean concentration, GMC, 15.6; 95% CI, 7.52–32.4) than CONTROL group (GMC, 23.1; 95% CI, 15.0–35.5), but lower diphtheria antibodies at 28?days (GMC, 2.3; 95% CI, 0.88–6.19) than CONTROL group (GMC, 16.4; 95% CI, 10.3–26.2); for pertussis, the percentage of individuals who seroconverted was lower in HIV than CONTROL group (HIV, 62.1% versus CONTROL, 100%; p?=?.002). Both groups built a cellular immune response to tetanus, with a Th2 (IL-4, IL-5 and IL-13) and Th1 (IFN-?) response, with lower cytokine levels in HIV than in CONTROL group. Especially for pertussis, cellular and humoral responses were less intense in HIV adolescents, with a lower Th1 and Th17 profile and higher IL-10 levels. HIV-infected adolescents on viral suppression showed an enhanced immune response to all the three vaccine antigens, although still at lower levels if compared to CONTROL group. Conclusions Both groups tolerated well and built an immune response after Tdap. However, HIV-infected adolescents would probably benefit from more frequent booster doses.
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An improved whole cell pertussis vaccine, designated as Plow, which is low in endotoxicity due to a chemical extraction of lipo-oligosaccharide (LOS) from the outer membrane, was evaluated for safety, immunogenicity and potency, comparatively to a traditional whole cell pertussis vaccine. Current whole cell pertussis vaccines are effective but contain large quantities of endotoxin and consequently display local and systemic adverse reactions after administration. Endotoxin is highly inflammatory and contributes considerably to the reactogenicity as well as the potency of these vaccines. In contrast, acellular pertussis vaccines hardly contain endotoxin and are significantly less reactogenic, but their elevated costs limit their global use, especially in developing countries. In this paper, bulk products of Plow and a traditional whole cell vaccine, formulated as plain monocomponents or combined with diphtheria and tetanus toxoids (DTPlow or DTP, respectively) were compared by in vitro and in vivo assays. Chemical extraction of LOS resulted in a significant decrease in endotoxin content (20%) and a striking decline in endotoxin related toxicity (up to 97%), depending on the used in vitro or in vivo test. The LOS extraction did not affect the integrity of the product and, more importantly, did not affect the potency and/or stability of DTPlow. Moreover, hardly any differences in antibody and T-cell responses were observed. The development of Plow is a significant improvement regarding the endotoxicity of whole cell pertussis vaccines and therefore a promising and affordable alternative to currently available whole cell or acellular pertussis vaccines for developing countries.
Assuntos
Endotoxinas/isolamento & purificação , Vacina contra Coqueluche/efeitos adversos , Vacina contra Coqueluche/imunologia , Potência de Vacina , Animais , Estabilidade de Medicamentos , Endotoxinas/análise , Feminino , Camundongos , Vacina contra Coqueluche/administração & dosagem , Vacina contra Coqueluche/química , CoelhosRESUMO
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.
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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
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 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...
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Humanos , Animais , Vacinas Pneumocócicas/classificaçãoRESUMO
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.
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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
Proteases were identified and characterized from the culture supernatant of the C. diphtheriae and B. pertussis bacteria. The proteases were secreted in the media and detected at the end of the exponential growth phase. Activity was detected in some fluorescent substrates, based on selected protein sequences such as insuline beta-chain, bradykinin, and synaptobrevin. The proteases were purified by means of gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified proteins indicated, for the main secreted proteins, an estimated molecular mass of 30 kDa in C. diphtheriae and 69 kDa in B. pertussis culture media. The proteases were stable and presented enzymatic activity at 37 degrees C. These proteases were not related to the main toxic compounds described in these two bacteria, but could represent good markers for the fermentation process when the enzyme activity was measured with the fluorescent substrates.
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Bordetella pertussis/enzimologia , Corynebacterium diphtheriae/enzimologia , Meios de Cultura/química , Peptídeo Hidrolases/análise , Peptídeos/metabolismo , Sequência de Aminoácidos , Toxinas Bacterianas/análise , Bordetella pertussis/crescimento & desenvolvimento , Soluções Tampão , Cromatografia em Gel , Corynebacterium diphtheriae/crescimento & desenvolvimento , Eletroforese em Gel de Poliacrilamida , Filtração , Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Hidrólise , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Peptídeo Hidrolases/isolamento & purificação , Peptídeos/química , Toxina Pertussis/análise , Cloreto de Sódio/química , Especificidade por Substrato , Trometamina/químicaRESUMO
Filamentous hemagglutinin adhesin (FHA) is important for the adherence of Bordetella pertussis to the host ciliary epithelial cells of the respiratory tract. Several binding domains have been characterized in the FHA molecule. For example, an putative heparin-binding domain of FHA was previously located in the FHA(442-863) region. In this work, the HEP fragment, corresponding to FHA(430-873) was amplified by PCR and subcloned in an Escherichia coli expression plasmid. Purified recombinant HEP was used to produce polyclonal antibodies in mice that were able to recognize HEP and FHA in ELISA and in Western-blot assays. Although recombinant HEP displayed low ability to bind heparin and no hemagglutination activity, the anti-HEP antibodies were able to inhibit FHA mediated hemagglutination activity in goose erythrocytes. These results indicate that other amino acid residues that are not present in the FHA(430-873) fragment may be necessary for heparin binding. Further studies to address the immunogenic response against HEP are also required.
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Adesinas Bacterianas/genética , Adesinas Bacterianas/imunologia , Anticorpos Antibacterianos/imunologia , Bordetella pertussis/imunologia , Hemaglutinação/imunologia , Hemaglutininas/genética , Hemaglutininas/imunologia , Fatores de Virulência de Bordetella/genética , Fatores de Virulência de Bordetella/imunologia , Adesinas Bacterianas/química , Animais , Especificidade de Anticorpos , Clonagem Molecular , Eritrócitos/imunologia , Eritrócitos/microbiologia , Feminino , Hemaglutininas/química , Heparina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Fatores de Virulência de Bordetella/química , Coqueluche/prevenção & controleRESUMO
Cultivos de 24 horas de Bordetella pertussis em fermentadores Biolafitte 50L, obtidos em meios de cultura de STAINER & SCHOLTE (SS) e COHEN & WHEELER (CW), foram inativados por formalina 0,1% a 35 C por 48 horas e a 25 C por 24 horas, ou pelo calor a 56 C por 30 minutos. As suspencoes foram, entao, concentradas por centrifugacao. A utilizacao do meio CW para o cultivo de B.pertussis seguido de inativacao por formalina 0,1%, demonstrou ser o metodo de producao da Vacina Pertussis mais adequado, tendo em vista o rendimento global, o custo de producao e o tempo necessario a destoxificacao. O meio de CW apresentou uma economia da ordem de 41,28% em relacao ao meio SS, quando avaliados os custos de producao. Apotencia das vacinas foram superiores a 4UI/dose e demonstraram uma correlacao significante entre atividade imunogenica e Fator Promotor da Leucocitose (LPF).
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Bordetella pertussis/imunologia , Vacina contra Coqueluche/imunologia , Meios de Cultura , Toxina Pertussis/imunologia , Vacinas SintéticasRESUMO
O tempo de destoxificaçäo, o rendimento e a potência da vacina Pertussis foram avaliados em cultivos de Bordetella pertussis isentos e adicionados com diferentes concentraçöes de anti-espumante em fermentadores (Biolafitte 50L). Foram realizadas modificaçöes nos fermentadores e no procedimento da fermentaçäo, com a finalidade de evitar a formaçäo de espuma nos cultivos. Suspensöes de B. pertussis com anti-espumante apresentaram maior tempo de destoxificaçäo e menor rendimento quando comparadas com aquelas isentas do agente químico. Em todos os processos, a potência foi superior a 8 UI/ml, estando de acordo com as recomendaçöes da Organizaçäo Mundial da Saúde