Cosue Miyaki

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.

Production of H5N1 (NIBRG-14) inactivated whole virus and split virion influenza vaccines and analysis of immunogenicity in mice using different adjuvant formulations.

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.

Safety and immunogenicity of hepatitis b vaccine butang in adults

Recombinant yeast-derived hepatitis B vaccine manufactured by Instituto Butantan was administered in two groups of adult volunteers (I, II) following two different schedules of immunization. In the first trial (10 &mgr;g doses and 0, 1, 3 months vaccination schedule) 106 individuals completed the full immunization program. The results of seroconversion by age group varied from 70 to 100% and the GMT from 46.5 to 124.9 mIU mL-1. In the second trial with 68 individuals (for dosage comparison and 0, 1, 6 months vaccination schedule) indicated that the vaccine formulated in 20 &mgr;g was more effective than in 10 &mgr;g. The adverse reactions observed in the vaccinees were less frequent than the ones previously found since the introduction of similar vaccines.

Micoplasma como contaminante de culturas celulares mantidas em laboratórios de instituições particulares e oficiais

Mycoplasma is one of the most serious contaminants of cell cultures. Its detection is very important in virology, as well as its eradication. The aim of this study was to verify the incidence of mycoplasma in cell lines maintained in seven laboratories of private, government and college institutions of the State of São Paulo, Brazil, for the purposes of research, production of reagents for diagnosis and production of biologicals for human and animal use. Of the 29 cell lines, eight were derived from human tissues and 21 from other animal species (dog, rabbit, mouse, hamster, monkey, pig, chicken and ox). Using the direct method with specific liquid and solid media for detection of mycoplasma, 48 out of the 106 cell samples tested were positive, corresponding to a contamination index of 45.28%. The incidence of contamination among the 35 cell samples of human origin was 51.43% (18 positive). Of the 71 samples originated from other species, 30 were positive (42.25%). The high incidence of contamination found calls for the adoption of measures for the prevention of this hazard: the elimination of mouth pipetting, the use of aseptic techniques and a rigid control of trypsin, serum and other components of cell culture media. The substitution of mycoplasma-free cultures for all contaminated ones and the performance of periodical tests for mycoplasma detection must also be carried out to prevent and avoid the dissemination of these organisms. Data obtained showed that contamination appeared in the 2nd (72.92%), in the 3rd (20.83%) and in the 4th passage (6.25%).(ABSTRACT TRUNCATED AT 250 WORDS)Foi realizado estudo sobre a incidencia de contaminacao por micoplasma em 29 tipos de linhagens celulares pertencentes a sete laboratorios de instituicoes particulares, oficiais e de ensino superior. Utilizando o metodo de cultivo direto e oito passagens seriadas em meios especificos, liquido e solido, verificou-se que, do total de 106 amostras, 48 apresentaram-se contaminadas por micoplasma (45,28%), o que constitui elevado indice de contaminacao. O fato indica que testes periodicos para a determinacao da presenca de micoplasma nas culturas em utilizacao e recomendavel e que as culturas contaminadas devem ser eliminadas para evitar a disseminacao do microrganismo. Outras medidas preventivas devem ser adotadas, como a eliminacao da pipetagem bucal, execucao de tecnicas assepticas mais estritas no manuseio das celulas, controle dos soros de origem animal, da tripsina e de outros componentes dos meios de cultura utilizados em cultura celular. O estudo mostrou que, ao inves das oito passagens seriadas propostas inicialmente, cinco foram suficientes para a deteccao dos micoplasmas, o que representa economia de tempo e de materiais de custo elevado, reduzindo de 848 para 530 o numero de passagens e a duracao do teste, de oito para cinco semanas.

Influenza vaccine production for Brazil: A classic example of successful North–South bilateral technology transfer

Technology transfer is a promising approach to increase vaccine production at an affordable price in developing countries. In the case of influenza, it is imperative that developing countries acquire the technology to produce pandemic vaccines through the transfer of know-how, as this will be the only way for the majority of these countries to face the huge demand for vaccine created by influenza pandemics. Access to domestically produced influenza vaccine in such health crises is thus an important national defence strategy. However, technology transfer is not a simple undertaking. It requires a committed provider who is willing to transfer a complete production process, and not just the formulation and fill-finish parts of the process. It requires a recipient with established experience in vaccine production for human use and the ability to conduct research into new developments. In addition, the country of the recipient should preferably have sufficient financial resources to support the undertaking, and an internal market for the new vaccine. Technology transfer should create a solid partnership that results in the joint development of new competency, improvements to the product, and to further innovation. The Instituto Butantan-sanofi pasteur partnership can be seen as a model for successful technology transfer and has led to the technological independence of the Instituto Butantan in the use a strategic public health tool.

Avaliação das condições de estocagem de vacinas vivas, atenuadas contra o sarampo, em postos de vacinação credenciados e em centros de saúde do Estado de São Paulo (Brasil)

In the State of S. Paulo, Brazil, health centers sponsored by the State, as well as private health services, located in throughout large districts, are in charge of the vaccination against the various diseases affecting children, including measles. In the present study three of the above mentioned districts, covering 385 State Health Centers (SHC) and 200 Private Health Services (PHS) were surveyed. From these totals 117 SHC and 71 PHS were chosen for the evaluation of: a) quality of the existing cold chain for measles vaccine storage (temperatura); b) vaccine titer; c) validity; d) protection from light. Of the SHC inspected, 85.33% kept vaccines at the recommended temperature, 100% presented vaccines with titers above the minimum required and within expiration date. Deficiency relating to protection from light, was found in 83.06%. Inspection of the PHS revealed that whereas 66.20% kept vaccine within the right temperature range, 76.06% did not protect them against light, only 96.47% had vaccines with potency above the level required and only 85.51%, observed the expiration date. The study led to the conclusion that in the State of S. Paulo, measles vaccines are better stored in the State Health Centers than in the Private Health Services surveyed indicating that the slight preference that parents show towards having their children vaccinated in PHS is not justified. It was suggested that some conditions of measles vaccine storage montly, temperature and protection from light should be improved and that instruction leaflets must always accompany immunobiological materials distributed to all health centers, to be consulted when doubts about the proper storage and administration of these vaccines arise.Para avaliar as condicoes de estocagem de vacinas vivas, atenuadas contra o sarampo, da rede de vacinacao do Estado de Sao Paulo (Brasil), foram visitados 71 Postos de Vacinacao Credenciados particulares (PVC), assim como 117 Centros de Saude oficiais (CS), sobre os quais interessava saber a respeito da qualidade da estocagem a frio. Os parâmetros adotados foram: a) temperatura das geladeiras de uso (+2 a +8°C) e de estoque (+ 8°C); b) validade do produto; c) titulo das vacinas conservadas nestas geladeiras, avaliado pela inoculacao de diluicoes das amostras de vacinas em celulas Vero; d) protecao a luz. Dos CS pesquisados, 85,33% apresentaram geladeiras com temperatura de acordo com a recomendada e 100% das vacinas neles estocadas com titulo e validade satisfatorios. Nos PVC foram encontrados, com maior frequencia, lotes de vacina fora do prazo de validade (14,49%), com titulos abaixo do minimo requerido (3,53%) e geladeiras de uso e de estoque com temperaturas inadequadas (33,80%). Necessario se faz que as condicoes de estocagem das vacinas contra o sarampo (temperatura e protecao a luz), prevalentes no momento, sejam melhoradas e que as bulas passem a acompanhar o produto a eles entregue, para que os responsaveis pela vacinacao obedecam as recomendacoes do laboratorio produtor com relacao as condicoes de estocagem, validade e administracao do imunobiologico, uma vez que a pesquisa revelou que estas nao sao observadas com o rigor necessario.

Identificação de micoplasmas pela inibição de crescimento de amostras isoladas de culturas celulares

As culturas celulares devem ser continuamente monitoradas quanto a presenca de micoplasmas, pois, embora as vezes eles passem despercebidos, podem causar alteracoes cromossomicas, interferir na replicacao viral, na producao de anticorpos e interferon. A Organizacao Internacional em Micoplasmologia (IOM) recomenda o isolamento e a identificacao de micoplasmas, visando detectar as provaveis origens da infeccao e melhorar a qualidade das culturas. Assim, foram analisadas pela inibicao de crescimento, 37 amostras pertencentes a 27 linhagens celulares contaminadas por micoplasmas. Em nenhuma amostra foi observada a ocorrencia de duas especies. Foram identificados 18 (48,65%) Mycoplasma arginini, 15 (40,55%) Acholeplasma laidlawii, dois (5,40%) Mycoplasma orale, sendo que duas amostras (5,40%) nao foram identificadas. Considerando as especies caracterizadas na pesquisa, os autores sugerem: a) a adocao do teste de isolamento de micoplasmas em carater de rotina; b) o aprimoramento das tecnicas de assepsia e desinfeccao; c) a eliminacao da pipetagem bucal; d) a utilizacao de soros e de outros componentes de meios de cultura de qualidade certificada; e) o questionamento da presenca de micoplasmas quando linhagens celulares sao permutadas pelas instituicoes; f) a avaliacao cautelosa de resultados obtidos quando se utilizam culturas infectadas por esse microrganismo.

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.

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.