H7N9 pandemic preparedness: A large-scale production of a split inactivated vaccine.

In March 2013 it was reported by the World Health Organization (WHO) the first cases of human infections with avian influenza virus A (H7N9). From 2013 to December 2019, 1568 cases have been reported with 616 deaths. H7N9 infection has been associated with high morbidity and mortality rates, and vaccination is currently the most effective way to prevent infections and consequently flu-related severe illness. Developing and producing vaccines against pandemic influenza viruses is the main strategy for a response to a possible pandemic. This study aims to present the production of three industrial lots under current Good Manufacturing Practices (cGMP) of the active antigen used to produce the pandemic influenza vaccine candidate against A(H7N9). These batches were characterized and evaluated for quality standards and tested for immunogenicity in mice. The average yield was 173.50 ± 7.88 µg/mL of hemagglutinin and all the preparations met all the required specifications. The formulated H7N9 vaccine is poorly immunogenic and needs to be adjuvanted with an oil in water emulsion adjuvant (IB160) to achieve a best immune response, in a prime and in a boost scheme. These data are important for initial production planning and preparedness in the case of a H7N9 pandemic.

H7N9 pandemic preparedness: a large-scale production of a split inactivated vaccine

In March 2013 it was reported by the World Health Organization (WHO) the first cases of human infections with avian influenza virus A (H7N9). From 2013 to December 2019, 1568 cases have been reported with 616 deaths. H7N9 infection has been associated with high morbidity and mortality rates, and vaccination is currently the most effective way to prevent infections and consequently flu-related severe illness. Developing and producing vaccines against pandemic influenza viruses is the main strategy for a response to a possible pandemic. This study aims to present the production of three industrial lots under current Good Manufacturing Practices (cGMP) of the active antigen used to produce the pandemic influenza vaccine candidate against A(H7N9). These batches were characterized and evaluated for quality standards and tested for immunogenicity in mice. The average yield was 173.50 ± 7.88 μg/mL of hemagglutinin and all the preparations met all the required specifications. The formulated H7N9 vaccine is poorly immunogenic and needs to be adjuvanted with an oil in water emulsion adjuvant (IB160) to achieve a best immune response, in a prime and in a boost scheme. These data are important for initial production planning and preparedness in the case of a H7N9 pandemic.

Vitamins as influenza vaccine adjuvant components.

A number of adjuvant formulations were assayed in mice immunized with 3.75 µg of A/California/7/2009 (H1N1) pdm09 influenza vaccine with vitamins A, D and/or E in emulsions or B2 and/or B9 combined with Bordetella pertussis MPLA and/or alum as adjuvants. Squalene was used as positive control, as well as MPLA with alum. The immune response was evaluated by a panel of tests, including a hemagglutination inhibition (HAI) test, ELISA for IgG, IgG1, and IgG2a and IFN-γ, IL-2, IL-6 and IL-10 quantification in splenocyte culture supernatant after stimulus with influenza antigen. Immunological memory was evaluated using a 1/10 dose booster 60 days after the first immunization followed by assessment of the response by HAI, IgG ELISA, and determination of the antibody affinity index. The highest increases in HAI, IgG1 and IgG2a titers were obtained with the adjuvant combinations containing vitamin E, or the hydrophilic combinations containing MPLA and alum or B2 and alum. The IgG1/IgG2a ratio indicates that the response to the combination of B2 with alum would have more Th2 character than the combination of MPLA with alum. In an assay to investigate the memory response, a significant increase in HAI titer was observed with a booster vaccine dose at 60 days after immunization with vaccines containing MPLA with alum or B2 with alum. Overall, of the 27 adjuvant combinations, MPLA with alum and B2 with alum were the most promising adjuvants to be evaluated in humans.

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.

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.

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.

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.

Vero-cell rabies vaccine produced using serum-free medium.

A new rabies vaccine was developed from Vero cells adhered to microcarriers, cultivated in a bioreactor in serum-free medium and infected with the PV/VERO-Paris rabies virus strain. The viral suspensions were concentrated by tangential filtration, purified by chromatography and inactivated with beta-propiolactone. In immunogenicity studies performed in mice immunized with three doses of the new vaccine (seven batches) and the commercial Verorab and HDCV, mean titers of neutralizing antibodies of 10.3-34.6, 6.54 and 9.36 IU/ml were found, respectively. The vaccine presented stability during 14 months at 2-8 degrees C, 30 days at 37 degrees C and 8 h at 45 degrees C. The use of serum-free medium facilitated the downstream process leading to residual cellular DNA values <22.8 pg per dose of vaccine in all produced batches. The effective immunogenicity induced in mice by this vaccine, the degree of purity of the product, the high antigen yield and the reduction of the cost of the product due to the virus production and purification processes, makes this technology very important for countries where rabies presents a great public health problem.

Suscetibilidade da linhagem de células Vero a cepas vacinais do vírus do sarampo / Susceptibility of Vero cell line to vaccine strains of the measles vírus

A suscetibilidade da linhagem de células Vero ao vírus do sarampo é bem conhecida e sua utilizaçäo no controle da potência da vacina contra o sarampo é amplamente difundida. Com o objetivo de comparar a suscetibilidade de células Vero empregadas em titulaçöes, amostras provenientes de dois laboratórios controladores (Vero IB e Vero INCQS), foram testadas frente a três cepas vacinais: Moraten Schwarz e Biken CAM-70. Foram titulados 72 lotes de vacinas contra o sarampo, sendo 25 produzidos com a cepa Moraten, 24 com a cepa Schwarz e 23 com a cepa Biken CAM-70. A análise estatística dos resultados obtidos nas titulaçöes, feita através dos testes Limites para uma Média e "t" de Student, mostrou que para as cepas Moraten e Biken CAM-70, as diferenças de títulos näo foram estatisticamente significantes, o mesmo näo ocorrendo com a cepa Schwarz, para a qual as células Vero IB se mostraram mais sensíveis

Identificaçäo de micoplasmas pela inibiçäo de crescimento de amostras isoladas de culturas celulares / Identification of mycoplasthe growth inhibition of samples isolated from cell cultures

As culturas celulares devem ser continuamente monitoradas quanto à presença de micoplasmas, pois, embora às vezes eles passem despercebidos, podem causar alteraçöes cromossômicas, interferir na replicaçäo viral, na produçäo de anticorpos e interferon. A Organizaçäo Internacional em Micoplasmologia (IOM) recomenda o isolamento e a identificaçäo de micoplasmas, visando detectar as prováveis origens da infecçäo e melhorar a qualidade das culturas. Assim, foram analisadas pela inibiçäo de crescimento, 37 amostras pertencentes a 27 linhagens celulares contaminadas por micoplasmas. Em nenhuma amostra foi observada a ocorrência de duas espécies. Foram identificados 18 (48,65%) Mycoplasma arginini, 15 (40,55%) Acholeplasma laidlawii, dois (5,40%) Mycoplasma orale, sendo que duas amostras (5,40%) näo foram identificadas. Considerando as espécies caracterizadas na pesquisa, os autores sugerem: a) a adoçäo do teste de isolamento de micoplasmas em caráter de rotina; b) o aprimoramento das técnicas de assepsia e desinfecçao; c) a eliminaçäo da pipetagem bucal; d) a utilizaçäo de soros e de outros componentes de meios de cultura de qualidade certificada; e) o questionamento da presença de micoplasmas quando linhagens celulares säo permutadas pelas instituiçöes; f) a avaliaçäo cautelosa de resultados obtidos quando se utilizam culturas infectadas por esse microrganismo

Micoplasma como contaminante de culturas celulares mantidas em laboratòrios de instituiçöes particulares e oficiais / Mycoplasma contamination of cell cultures maintained in laboratories of private, government and college institutions

Foi realizado estudo sobre a incidência de contaminaçäo por micoplasma em 29 tipos de linhagens celulares pertencentes a sete laboratòrios de instituiçöes particulares, oficiais e de ensino superior. Utilizando o método de cultivo direto e oito passagens seriadas em meios específicos, líquido e sòlido, verificou-se que, do total de 106 amostras, 48 apresentaram-se contaminadas por micoplasma (45,28%), o que constitui elevado índice de contaminaçäo. O fato indica que testes periòdicos para a determinaçäo da presença de micoplasma nas culturas em utilizaçäo é recomendável e que as culturas contaminadas devem ser eliminadas para evitar a disseminaçäo do microorganismo. Outras medidas preventivas devem ser adotadas, como a eliminaçäo da pipetagem bucal, execuçäo de técnicas assépticas mais estritas no manuseio das células, 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 invés das oito passagens seriadas propostas inicialmente, cinco foram suficientes para a detecçäo dos micoplasmas, o que representa economia de tempo e de materiais de custo elevado, reduzindo de 848 para 530 o número de passagens e a duraçäo do teste, de oito para cinco semanas .

Solucoes de sorbitol-gelatina e de acido glutamico-lactose na estabilizacao de preparacoes referencia do virus do sarampo / Sorbitol-gelatin and glutamic acid-lactose solutions as stabilizers of reference measles virus preparations

The study described in this article was carried out for the purpose of evaluating the protective effects of two stabilizing solutions- sorbitol-gelatin and glutamic acid-lactose- on freeze-dried measles virus (Schwartz strain) with a view to the production of reference preparations in working lots. The effect of storage at -20 C and -70 C on the potency of stabilized virus suspensions, whether or not freeze-dried, was evaluated over a period of 21 months; the samples were concurrently titered for potency with a standard virus obtained from a laboratory authorized by the World Health Organization. After comparing and commenting on the different phases of the study, the types of cells and stabilizing media used, how the suspensions were stored, the titering of the stabilized viruses, etc., the authors conclude that they have demostrated a more satisfactory stabilization (r= -0.01) of freeze-dried virus suspensions stored at -20 C by sorbitol-gelatin, which is therefore regarded as an effective stabilizer for the preparation of freeze-dried reference measles virus in working lots

Seringas hipodérmicas descartáveis versus reutilizáveis: estudo de possíveis efeitos sobre o vírus da vacina viva, atenuada contra o sarampo / Disposable vs. reusable hypodermic syringes: study of feasible effects upon the virus of the live, attenuated measles vaccine

Objetivou-se verificar entre seringas hipodérmicas descartáveis e reutilizáveis qual interfere mais com o vírus vivo presente na vacina contra o sarampo. Vacinas pertencentes a dois lotes foram reconstituídas com os dois tipos de seringas, de modo a formarem dois pools distintos, mantidos à temperatura de +2 a+8-C e protegidos da luz. De cada lote foram realizadas, no mínimo, seis titulaçöes em paralelo, com amostragem a cada hora, de zero a seis horas após reconstituiçäo. A análise estatística dos resultados obtidos nas titulaçöes, feita pelo sistema de retas de regressäo demonstrou que embora as vacinas manipuladas com ambos os tipos de seringas apresentassem um decréscimo de título estatisticamente significativo com o decorrer das horas, ele foi bem mais acentuado para as vacinas reconstituídas com seringas reutilizáveis. A menor interferência das descartáveis no título da vacina viva, atenuada contra o sarampo, demonstrou que a preconizaçäo e uso desse tipo de seringas pela Secretaria de Estado da Saúde de Säo Paulo é o ideal e recomendável, por preservar mais a vacina desde a reconstituiçäo até sua administraçäo e, conseqüentemente, a sua eficácia na prevençäo dessa infecçäo

Avaliaçäo das condiçöes de estocagem de vacinas vivas, atenuadas contra o sarampo, em postos de vacinaçäo credenciados e em centros de saúde do Estado de Säo Paulo (Brasil) / Evaluation of storage conditions of live, attenuated vaccines against measles, in authorized vaccination centers and health services of the State of Säo Paulo (Brazil)

Para avaliar as condiçöes de estocagem de vacinas vivas, atenuadas contra o sarampo, da rede de vacinaçäo do Estado de Säo Paulo (Brasil), foram visitados 71 Postos de Vacinaçäo Credenciados particulares (PVC), assim como 117 Centros de Saúde 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) título das vacinas conservadas nestas geladeiras, avaliado pela inoculaçäo de diluiçöes das amostras de vacinas em células Vero; d) proteçäo à luz. dos CS pesquisados, 85,33% apresentaram geladeiras com temperatura de acordo com a recomendada e 100% das vacinas neles estocadas com título e validade satisfatórios. Nos PVC foram encontrados, com maior frequência, lotes de vacina fora do prazo de validade (14,49%), com títulos abaixo do mínimo requerido (3,53%) e geladeiras de uso e de estoque com temperaturas inadequadas (33,80%). Necessário se faz que as condiçöes de estocagem das vacinas contra o sarampo (temperatura e proteçäo à luz), prevalentes no momento, sejam melhoradas e que as bulas passem a acompanhar o produto a eles entregue, para que os responsáveis pela vacinaçäo obedeçam as recomendaçöes do laboratório producto com relaçäo às condiçöes de estocagem, validade e administraçäo do imunobiológico, uma vez que a pesquisa revelou que estas näo säo observadas com o rigor necessário

Solucoes de sorbitol-gelatina e de acido glutamico-lactose na estabilizacao de preparacoes referencia do virus do sarampo / Sorbitol-gelatin and glutamic acid-lactose solutions as stabilizers of reference measles virus preparations

The study described in this article was carried out for the purpose of evaluating the protective effects of two stabilizing solutions- sorbitol-gelatin and glutamic acid-lactose- on freeze-dried measles virus (Schwartz strain) with a view to the production of reference preparations in working lots. The effect of storage at -20 C and -70 C on the potency of stabilized virus suspensions, whether or not freeze-dried, was evaluated over a period of 21 months; the samples were concurrently titered for potency with a standard virus obtained from a laboratory authorized by the World Health Organization. After comparing and commenting on the different phases of the study, the types of cells and stabilizing media used, how the suspensions were stored, the titering of the stabilized viruses, etc., the authors conclude that they have demostrated a more satisfactory stabilization (r= -0.01) of freeze-dried virus suspensions stored at -20 C by sorbitol-gelatin, which is therefore regarded as an effective stabilizer for the preparation of freeze-dried reference measles virus in working lots