A new methodology for polyvalent intravenous immunoglobulin solution production with a two-stage process of viral inactivation

Highly purified intravenous immunoglobulin G concentrate (IV IgG) was produced with the use of polyethylene glycol associated to a single-stage precipitation by ethanol, instead of the classic Cohn-Oncley process, which employs cold alcohol as the precipitating agent, in a three-stage process. Precipitation of crude fraction containing more than 95% of immunoglobulin G was performed by liquid chromatography with a cation exchanger, CM-Sepharose, as a stationary phase. During the process, the product was subjected to two-stage viral inactivation. The first stage was performed by the action of sodium caprylate, 30 mM at pH 5.1+/- 0.1, and the second stage was performed by the action of a solvent-detergent mixture. The finished product was formulated at 5% with 10% sucralose as the stabilizing agent. The process yields 3.3g of IgG/liter of plasma. The finished product analysis showed an anti-complementary activity lower than 1CH50. Polymer and aggregate percent levels were lower than 3% in the five batches studied. The analysis of neutralizing capacity showed the presence of antibacterial and antiviral antibodies in at least three times higher concentrations than the levels found in source plasma. The finished product fulfilled all purity requirements stated in the 4th edition of the European pharmacopeia.Obteve-se concentrado de imunoglobulina G intravenosa IgGIV, altamente purificado, utilizando-se polietilenoglicol associado a uma única etapa de precipitação por etanol, em substituição ao tradicional método descrito por Cohn-Oncley, que emprega, em três etapas, o mesmo álcool resfriado, como agente precipitante. A purificação da fração bruta contendo mais de 95% de imunoglobulina G foi realizada utilizando-se cromatografia líquida com um trocador de cátion, a CM-Sepharose, como fase estacionária. Durante o processamento o produto foi submetido a dupla inativação viral sendo a primeira pela ação do caprilato de sódio, 30 mM a pH 5,1+/- 0,1 e a segunda por ação de mistura de solvente/detergente. O produto acabado foi formulado a 5% utilizando-se sucralose 10% como estabilizante. O rendimento da metodologia foi de 3,3g de IgG/litro de plasma. A análise do produto acabado demonstrou atividade anti-complementar inferior a 1CH50. O valor percentual de polímeros e agregados em cinco lotes realizados foi inferior a 3%. O estudo da capacidade de neutralização demonstrou a presença de anticorpos anti-bacterianos e anti-virais em concentração pelo menos três vezes maior que o plasma de origem. O produto acabado apresentou conformidade com todos os requisitos de pureza dispostos na farmacopéia européia IV edição

Subversion of early innate antiviral responses during antibody-dependent enhancement of Dengue virus infection induces severe disease in immunocompetent mice

Dengue is a mosquito-borne disease caused by one of four serotypes of Dengue virus (DENV-1–4). Epidemiologic and observational studies demonstrate that the majority of severe dengue cases, dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS), occurs predominantly in either individuals with cross-reactive immunity following a secondary heterologous infection or in infants with primary DENV infections born from dengue-immune mothers, suggesting that B-cell-mediated and antibody responses impact on disease evolution. We demonstrate here that B cells play a pivotal role in host responses against primary DENV infection in mice. After infection, μMT[superscript −/−] mice showed increased viral loads followed by severe disease manifestation characterized by intense thrombocytopenia, hemoconcentration, cytokine production and massive liver damage that culminated in death. In addition, we show that poly and monoclonal anti-DENV-specific antibodies can sufficiently increase viral replication through a suppression of early innate antiviral responses and enhance disease manifestation, so that a mostly non-lethal illness becomes a fatal disease resembling human DHF/DSS. Finally, treatment with intravenous immunoglobulin containing anti-DENV antibodies confirmed the potential enhancing capacity of subneutralizing antibodies to mediate virus infection and replication and induce severe disease manifestation of DENV-infected mice. Thus, our results show that humoral responses unleashed during DENV infections can exert protective or pathological outcomes and provide insight into the pathogenesis of this important human pathogen

Logistic model for determining factors associated with HIV infection among blood donor candidates at the Fundação HEMOPE

OBJECTIVE: To determine factors associated with HIV infection in blood donor candidates in Recife, Brazil. METHODS:A transversal study was performed of 106,203 blood donor candidates found eligible by the routine clinical screening process in the blood bank in Recife of the Fundação de Hematologia e Hemoterapia de Pernambuco (HEMOPE) in the period from January 1998 to November 2003. Additional indirect immune and western blot confirmation tests for HIV infection were performed and the candidates were classified as HIV positive or negative. The Chi-squared test and stepwise multiple logistic regression were conducted to examine any association between HIV infection and age, gender, place of residence, schooling, number of donations and serological tests for core hepatitis B antigen virus (anti-HBc), the hepatitis C antivirus (anti-HCV), human T-type antivirus lymph cells (anti-HTLV 1 and 2), serological tests for syphilis (VDRL) and the hepatitis B virus surface antigen (HBsAg). RESULTS: In the observed sample, 0.204% of blood donor candidates were found to be HIV positive. Among the studied variables, the age, education level, residency, donation type, and serologic status for anti-HBc and VDRL tests were found to be associated with HIV infection. CONCLUSION: The younger, spontaneous donor candidates living in the Recife metropolitan area with a low level of education and positive for anti-HBc and VDRL have an higher risk of HIV infection than the other candidates. Data such as these are useful to understand the dynamics of infection and to guide healthcare policies.OBJETIVO: Determinar fatores associados à infecção por HIV em doadores de sangue no Recife, Brasil. MÉTODOS: Desenvolveu-se um estudo transversal com 106.203 doadores de sangue de 1ª doação, aptos na Triagem Clínica (TC) no Hemocentro Recife da Fundação de Hematologia e Hemoterapia de Pernambuco (HEMOPE), considerando-se os critérios rotineiros empregados pelo Hemocentro para seleção de candidatos no período de janeiro de 1998 a novembro de 2003. Os testes Imunofluorescência Indireta e Western Blot foram utilizados para confirmação da infecção HIV, e doadores foram classificados como HIV positivo ou negativo. O teste do qui-quadrado e método stepwise de regressão logística múltipla foram realizados para examinar associação entre a infecção pelo HIV e as variáveis: idade, sexo, área de residência, escolaridade, tipo de doação e testes sorológicos de triagem para Anti-HBc, HBsAg, Anti-HCV, Anti-HTLV 1/2 e VDRL. RESULTADOS: Na amostra observada, foram encontrados 0,204% doadores de sangue HIV positivo. Entre as variáveis estudadas, idade, escolaridade, residência, tipo de doação e status sorológico para Anti-HBc e VDRL foram associados com a infecção pelo HIV. CONCLUSÃO: Doadores com doação espontânea, da área metropolitana de Recife, de menor idade e nível de ensino, positivo para o Anti-HBc e VDRL, apresentam risco aumentado de infecção pelo HIV. Tais dados são necessários para compreender a dinâmica da infecção e para guiar políticas sanitárias

A new methodology for polyvalent intravenous immunoglobulin solution production with a two-stage process of viral inactivation

Highly purified intravenous immunoglobulin G concentrate (IV IgG) was produced with the use of polyethylene glycol associated to a single-stage precipitation by ethanol, instead of the classic Cohn-Oncley process, which employs cold alcohol as the precipitating agent, in a three-stage process. Precipitation of crude fraction containing more than 95% of immunoglobulin G was performed by liquid chromatography with a cation exchanger, CM-Sepharose, as a stationary phase. During the process, the product was subjected to two-stage viral inactivation. The first stage was performed by the action of sodium caprylate, 30 mM at pH 5.1+/- 0.1, and the second stage was performed by the action of a solvent-detergent mixture. The finished product was formulated at 5% with 10% sucralose as the stabilizing agent. The process yields 3.3g of IgG/liter of plasma. The finished product analysis showed an anti-complementary activity lower than 1CH50. Polymer and aggregate percent levels were lower than 3% in the five batches studied. The analysis of neutralizing capacity showed the presence of antibacterial and antiviral antibodies in at least three times higher concentrations than the levels found in source plasma. The finished product fulfilled all purity requirements stated in the 4th edition of the European pharmacopeia.Obteve-se concentrado de imunoglobulina G intravenosa IgGIV, altamente purificado, utilizando-se polietilenoglicol associado a uma única etapa de precipitação por etanol, em substituição ao tradicional método descrito por Cohn-Oncley, que emprega, em três etapas, o mesmo álcool resfriado, como agente precipitante. A purificação da fração bruta contendo mais de 95% de imunoglobulina G foi realizada utilizando-se cromatografia líquida com um trocador de cátion, a CM-Sepharose, como fase estacionária. Durante o processamento o produto foi submetido a dupla inativação viral sendo a primeira pela ação do caprilato de sódio, 30 mM a pH 5,1+/- 0,1 e a segunda por ação de mistura de solvente/detergente. O produto acabado foi formulado a 5% utilizando-se sucralose 10% como estabilizante. O rendimento da metodologia foi de 3,3g de IgG/litro de plasma. A análise do produto acabado demonstrou atividade anti-complementar inferior a 1CH50. O valor percentual de polímeros e agregados em cinco lotes realizados foi inferior a 3%. O estudo da capacidade de neutralização demonstrou a presença de anticorpos anti-bacterianos e anti-virais em concentração pelo menos três vezes maior que o plasma de origem. O produto acabado apresentou conformidade com todos os requisitos de pureza dispostos na farmacopéia européia IV edição

Contribution of the Retrovirus Epidemiology Donor Study (REDS) to research on blood transfusion safety in Brazil

The Retrovirus Epidemiology Donor Study (REDS) program was established in the United States in 1989 with the purpose of increasing blood transfusion safety in the context of the HIV/AIDS and human T-lymphotropic virus epidemics. REDS and its successor, REDS-II were at first conducted in the US, then expanded in 2006 to include international partnerships with Brazil and China. In 2011, a third wave of REDS renamed the Recipient Epidemiology and Donor Evaluation Study-III (REDS-III) was launched. This seven-year research program focuses on both blood banking and transfusion medicine research in the United States of America, Brazil, China, and South Africa. The main goal of the international programs is to reduce and prevent the transmission of HIV/AIDS and other known and emerging infectious agents through transfusion, and to address research questions aimed at understanding global issues related to the availability of safe blood. This article describes the contribution of REDS-II to transfusion safety in Brazil. Articles published from 2010 to 2013 are summarized, including database analyses to characterize blood donors, deferral rates, and prevalence, incidence and residual risk of the main blood-borne infections. Specific studies were developed to understand donor motivation, the impact of the deferral questions, risk factors and molecular surveillance among HIV-positive donors, and the natural history of Chagas disease. The purpose of this review is to disseminate the acquired knowledge and briefly summarize the findings of the REDS-II studies conducted in Brazil as well as to introduce the scope of the REDS-III program that is now in progress and will continue through 2018