Avaliação da resposta imunológica na febre amarela e infecção pelo HIV: importância da escolha do método e interpretação dos resultados
Data
2023-05-18
Autores
Munhoz, Luiz Gustavo Cano 
Orientadores
Pinto, Maria Isabel de Moraes
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Objetivos: Evidenciar os desafios na padronização, a escolha do método diagnóstico e a interpretação de resultados em diferentes situações laboratoriais. Métodos: Estudo 1: foram avaliadas duas variações de ensaios imunoenzimáticos (duplo antígeno e indireto) utilizando diferentes antígenos visando a detecção de anticorpos neutralizantes para febre amarela. Estudo 2: 27 indivíduos infectados verticalmente pelo HIV (19 com supressão virológica e 8 sem supressão virológica) e 16 indivíduos sem infecção pelo HIV foram acompanhados por 24 meses após receberem reforço com a vacina dTpa para avaliação dos níveis de anticorpos para tétano, difteria e coqueluche. Estudo 3: foram quantificadas 21 citocinas correspondentes aos perfis Th1, Th2 e Th17 de 29 indivíduos infectados verticalmente pelo HIV (17 com supressão virológica e 12 sem supressão virológica) e de 29 indivíduos sem infecção pelo HIV utilizando a técnica XMAP para comparar duas diferentes metodologias: ex vivo e in vitro após estimulação. Resultados: Estudo 1: o ensaio imunoenzimático duplo antígeno não produziu curvas compatíveis com a mensuração de anticorpos para febre amarela. No ensaio imunoenzimático indireto, embora tenha sido possível obter curvas de diluição adequadas, não foi possível diferenciar anticorpos para febre amarela de anticorpos para outros flavivírus. Estudo 2 – Independentemente de apresentarem ou não supressão virológica, alguns indivíduos infectados pelo HIV não mantiveram níveis de anticorpos protetores para tétano e difteria decorridos 2 anos do reforço com dTpa; apresentar nadir de linfócitos T CD4+ <200/mm3 foi um fator preditivo para a não manutenção dos níveis de anticorpos protetores para tétano e difteria. Estudo 3 – Quando comparados com o grupo controle, indivíduos infectados pelo HIV sem supressão virológica apresentaram menor expressão ex vivo de GM-CSF, MIP-3α, IL-1β, IL-23 e MIP-1β; na metodologia in vitro, os mesmos pacientes apresentaram menor expressão de IL-10, IL-4 e IL-6 que aqueles do grupo controle. Somente a avaliação de citocinas após estimulação in vitro permitiu identificar uma correlação inversa entre a expressão de citocinas inflamatórias e anti-inflamatórias e a expressão de marcadores de ativação e de exaustão em linfócitos T CD4+ e T CD8+. Conclusão: Estudo 1 – Fomos incapazes de padronizar o ELISA duplo antígeno para a detecção de anticorpos para febre amarela, sendo o maior desafio a conjugação adequada do vírus/antígeno a enzima ou molécula. No caso do ELISA indireto fomos incapazes de evitar a reatividade cruzada comum aos flavivírus. Estudo 2 - Entre indivíduos infectados verticalmente pelo HIV, o nadir de linfócitos T CD4+<200/mm3 é capaz de auxiliar na decisão do clínico na antecipação do reforço com dTpa ou mesmo da administração de imunoglobulina específica para tétano no caso de um ferimento tetanogênico grave. Estudo 3 - Demonstramos que diferentes metodologias analíticas para dosagem de citocinas produzem resultados diversos, sendo de vital importância o conhecimento do método utilizado para evitar interpretações equivocadas.
Objective: To emphasize the challenges in standardization, the choice of diagnostic method and the interpretation of results in different laboratory situations. Methods: Study 1: two variations of enzymelinked immunosorbent assays (double and indirect antigen) were evaluated using different antigens, aiming at the detection of neutralizing antibodies to yellow fever. Study 2: 27 vertically infected HIVinfected individuals (19 with virological suppression and 8 without virological suppression) and 16 individuals without HIV infection were followed for 24 months after receiving a Tdap vaccine booster to assess antibody levels for tetanus, diphtheria, and pertussis. Study 3: 21 cytokines corresponding to the Th1, Th2 and Th17 profiles of 29 vertically infected HIVinfected individuals (17 with virological suppression and 12 without virological suppression) and of 29 individuals without HIV infection were quantified using the XMAP technique to compare 2 different methodologies: ex vivo and in vitro after stimulation. Results: Study 1: the double antigen enzymelinked immunosorbent assay did not produce curves compatible with the measurement of antibodies to yellow fever. In the indirect enzymelinked immunosorbent assay, although it was possible to obtain adequate dilution curves, it was impossible to differentiate antibodies to yellow fever from antibodies to other flaviviruses. Study 2 – Regardless of whether they had virological suppression, some HIVinfected individuals did not maintain levels of protective antibodies to tetanus and diphtheria after 2 years of Tdap booster; presenting CD4+ T lymphocyte nadir <200/mm3 was a predictive factor for the nonmaintenance of protective antibody levels for tetanus and diphtheria. Study 3 – Compared to the control group, HIVinfected individuals without virological suppression had lower ex vivo expression of GMCSF, MIP3α, IL1β, IL23, and MIP1β; in the in vitro methodology, the same patients presented lower expression of IL10, IL4, and IL6 than those of the control group. Only the evaluation of cytokines after in vitro stimulation allowed the identification of an inverse correlation between the expression of inflammatory and antiinflammatory cytokines and the expression of activation and exhaustion markers in CD4+ T and CD8+ T lymphocytes. Conclusion: Study 1: the double antigen enzymelinked immunosorbent assay did not produce curves compatible with the measurement of antibodies to yellow fever. In the indirect enzymelinked immunosorbent assay, although it was possible to obtain adequate dilution curves, it was not possible to differentiate antibodies to yellow fever from antibodies to other flaviviruses. Study 2 – Regardless of whether they had virological suppression, some HIVinfected individuals did not maintain levels of protective antibodies to tetanus and diphtheria after 2 years of Tdap booster; presenting CD4+ T lymphocyte nadir <200/mm3 was a predictive factor for the nonmaintenance of protective antibody levels for tetanus and diphtheria. Study 3 – Compared the control group, HIVinfected individuals without virological suppression had lower ex vivo expression of GMCSF, MIP3α, IL1β, IL23 and MIP1β; in the in vitro methodology, the same patients presented lower expression of IL10, IL4 and IL6 than those of the control group. Only the evaluation of cytokines after in vitro stimulation allowed the identification of an inverse correlation between the expression of inflammatory and antiinflammatory cytokines and the expression of activation and exhaustion markers in CD4+ T and CD8+ T lymphocytes.
Objective: To emphasize the challenges in standardization, the choice of diagnostic method and the interpretation of results in different laboratory situations. Methods: Study 1: two variations of enzymelinked immunosorbent assays (double and indirect antigen) were evaluated using different antigens, aiming at the detection of neutralizing antibodies to yellow fever. Study 2: 27 vertically infected HIVinfected individuals (19 with virological suppression and 8 without virological suppression) and 16 individuals without HIV infection were followed for 24 months after receiving a Tdap vaccine booster to assess antibody levels for tetanus, diphtheria, and pertussis. Study 3: 21 cytokines corresponding to the Th1, Th2 and Th17 profiles of 29 vertically infected HIVinfected individuals (17 with virological suppression and 12 without virological suppression) and of 29 individuals without HIV infection were quantified using the XMAP technique to compare 2 different methodologies: ex vivo and in vitro after stimulation. Results: Study 1: the double antigen enzymelinked immunosorbent assay did not produce curves compatible with the measurement of antibodies to yellow fever. In the indirect enzymelinked immunosorbent assay, although it was possible to obtain adequate dilution curves, it was impossible to differentiate antibodies to yellow fever from antibodies to other flaviviruses. Study 2 – Regardless of whether they had virological suppression, some HIVinfected individuals did not maintain levels of protective antibodies to tetanus and diphtheria after 2 years of Tdap booster; presenting CD4+ T lymphocyte nadir <200/mm3 was a predictive factor for the nonmaintenance of protective antibody levels for tetanus and diphtheria. Study 3 – Compared to the control group, HIVinfected individuals without virological suppression had lower ex vivo expression of GMCSF, MIP3α, IL1β, IL23, and MIP1β; in the in vitro methodology, the same patients presented lower expression of IL10, IL4, and IL6 than those of the control group. Only the evaluation of cytokines after in vitro stimulation allowed the identification of an inverse correlation between the expression of inflammatory and antiinflammatory cytokines and the expression of activation and exhaustion markers in CD4+ T and CD8+ T lymphocytes. Conclusion: Study 1: the double antigen enzymelinked immunosorbent assay did not produce curves compatible with the measurement of antibodies to yellow fever. In the indirect enzymelinked immunosorbent assay, although it was possible to obtain adequate dilution curves, it was not possible to differentiate antibodies to yellow fever from antibodies to other flaviviruses. Study 2 – Regardless of whether they had virological suppression, some HIVinfected individuals did not maintain levels of protective antibodies to tetanus and diphtheria after 2 years of Tdap booster; presenting CD4+ T lymphocyte nadir <200/mm3 was a predictive factor for the nonmaintenance of protective antibody levels for tetanus and diphtheria. Study 3 – Compared the control group, HIVinfected individuals without virological suppression had lower ex vivo expression of GMCSF, MIP3α, IL1β, IL23 and MIP1β; in the in vitro methodology, the same patients presented lower expression of IL10, IL4 and IL6 than those of the control group. Only the evaluation of cytokines after in vitro stimulation allowed the identification of an inverse correlation between the expression of inflammatory and antiinflammatory cytokines and the expression of activation and exhaustion markers in CD4+ T and CD8+ T lymphocytes.