A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine in the specialty of Virology, June 2018 / The discovery of broad and potent neutralizing HIV antibodies (bNAbs) has opened up new opportunities of passive immunization for HIV-1 prevention. In this study, we have engineered CAP256-VRC26.25, a V1V2 bNAb that neutralizes 70% of clade C viruses, as a single domain antibody (sdAb). These small antigen binding entities are derived from naturally occurring heavy chain only antibodies present in members of the dromedary families, and are characterized by the absence of a light chain, long complementarity-determining regions (CDR) heavy (H) chain 3 and high stability. Since CAP256.25 contains a highly charged and protruding CDR-H3 that binds mainly through its heavy chain, we hypothesized that it may function well as an sdAb. Multiple camelization approaches to engineer CAP256.25 as a sdAb were tested in silico utilising structural modelling software. Parameters such as germline sequence homology, hydrophobicity and solubility, folding energy, torsion angles and native conformation of CAP256.25 in complex with its binding epitope were major factors considered during the modelling process. Four CAP256.25 sdAb derivatives were generated from parental antibody, the mut_0 or a wild type (WT), which was used as a base line for downstream optimization. CAP256.25 mut_4 in which residues involved in LC interactions were replaced with residues strongly conserved in camel sdAbs, which minimize hydrophobic interface of the sdAb. Mut_8 variant, which included four additional substitutions to increase solubility and mut_9 contained a single additional mutation at the base of CDR-H3 to improve the energetic landscape of sdAb. All genes were synthesized and sub-cloned into a mammalian expression vector and recombinant proteins expressed in HEK293T cell line, and purified by Immobilized Metal Ion Affinity Chromatography (IMAC) and Fast Protein Liquid Chromatography (FPLC). CAP256.25_mut0 expression was below the detectable level and whilst mut_4 expressed at low levels, it showed no neutralization activity. CAP256.25 sdAb mut_8 and mut_9 expressed at significantly lower levels compared to m36, a previously described sdAb used a positive control. Nevertheless CAP256.25mut_8 sdAb showed neutralization capability although it lost significant potency in comparison to the parental antibody, yet still within the therapeutic window of the VRC01 bNAb. Importantly, CAP256.25 sdAb was unable to neutralize the K169E mutant confirming that it retained specificity for the V2 epitope. These data suggest that camelization of human antibodies is possible although further engineering is required to increase expression and improve stability. As such, sdAb engineering could be an encouraging step for the generation of small antigen binding fragments for future therapeutic purposes including topical delivery at mucosal surfaces, to interrupt or block sexual transmission of HIV. / XL2018

INTRODUÇÃO: A Febre Amarela (FA) é uma doença viral aguda endêmica em grande parte do Brasil. A principal medida de prevenção é a vacinação. O presente estudo avaliou a prevalência e os títulos de anticorpos neutralizantes em pessoas com 60 anos ou mais que haviam recebido anteriormente a vacina de FA 17DD, em comparação a adultos saudáveis com 18 a 59 anos. Além disso, foram avaliadas a correlação entre os títulos de anticorpos e o tempo decorrido desde a vacinação, nos participantes que receberam apenas uma dose da vacina, e a prevalência de anticorpos nos vacinados há menos e há mais de dez anos. MÉTODOS: Os participantes foram recrutados entre pessoas que procuraram o Centro de Referência para Imunobiológicos Especiais (CRIE) do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP) para receber diferentes vacinas e que referiam ter recebido a vacina de FA anteriormente. Os seguintes dados foram coletados: idade, etnia, sexo, número de doses da vacina de FA recebidas, data da última vacinação de FA. Foi realizada contagem de linfócitos TCD4+ usando citometria de fluxo. Os anticorpos neutralizantes contra FA foram dosados pelo teste de neutralização por redução de 50% das placas de lise (PRNT50). RESULTADOS: Foram incluídos 94 indivíduos, 46 com idade de 60 anos ou mais (Grupo 1) e 48 com 18 a 59 anos (Grupo 2). Não houve diferença significativa entre os dois grupos na distribuição de gênero, etnia, número de doses de vacina de FA recebidas anteriormente, tempo desde a última dose e contagem de linfócitos TCD4+. Não houve diferença na prevalência de anticorpos neutralizantes anti-FA entre os dois grupos (87% e 93,8% nos Grupos 1 e 2, respectivamente, p=0,263). O título médio geométrico (GMT) dos anticorpos neutralizantes foi maior no grupo mais jovem (3,77 log10mUI/mL) comparado ao grupo mais velho (3,64 log10mUI/mL) e essa diferença foi estatisticamente significante (p=0,022). Não foi encontrada correlação entre os títulos de anticorpos neutralizantes e o tempo decorrido desde a vacinação entre os participantes que receberam apenas uma dose de vacina, tendo sido analisados os dois grupos conjuntamente. Também não foi encontrada diferença estatisticamente significativa na prevalência de anticorpos neutralizantes entre os participantes que receberam apenas uma dose da vacina de FA há mais de 10 anos ou há menos de 10 anos. CONCLUSÕES: São necessários outros estudos de persistência de anticorpos na população idosa devido à possibilidade de resposta vacinal alterada pela imunosenescência / INTRODUCTION. Yellow Fever (YF) is an acute viral disease endemic in large parts of Brazil. The main preventive measure is vaccination. This study aimed to assess the prevalence and titers of neutralizing antibodies in persons aged 60 years and older who had previously received YF 17DD vaccine, in comparison to healthy adults aged 18 to 59 years. The study also evaluated the correlation between the antibodies titers and the time elapsed since vaccination, in participants who had received a single dose of the vaccine, and the prevalence of antibodies in participants vaccinated within ten years and more than ten years before enrollment. METHODS. Participants were recruited among persons who came to the Reference Center for Special Immunobiologicals (Centro de Referência para Imunobiológcos Especiais, CRIE) of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP) to receive any vaccine and who had previously received the YF vaccine. The following data were collected: age, ethnicity, gender, number of YF vaccine doses taken and date of last YF vaccination. CD4 T cells counts were performed using flow cytometry. YF neutralizing antibodies were measured using test of neutralization by 50% reduction of lysis plaques (PRNT50). RESULTS. Ninety-four subjects were enrolled: 46 persons aged 60 years and older (Group 1) and 48 persons aged 18 to 59 years (Group 2). There was no significant difference between the groups regarding gender, ethnicity, number of YF vaccine doses previously received, time since the last dose and CD4+T cells count. There was no difference in the prevalence of YF neutralizing antibodies between the groups (87% and 93.8% in Groups 1 and 2, respectively, p=0.263). The log-transformed Geometric Mean Titer (GMT) of YF neutralizing antibodies was higher in the younger group (3.77 log10mUI/mL) in comparison to the older group (3.64 log10mUI/mL), and this difference was statistically significant (p=0.022). There was no correlation between YF neutralizing antibodies titers and time elapsed since vaccination among the participants who had previously received a single dose of YF vaccine, with the two groups analyzed together. There was no significant difference in the prevalence of neutralizing antibodies among participants who received a single dose of YF vaccine within ten years or more than 10 years before enrollment. CONCLUSIONS. Further studies on antibodies persistence in the elderly are necessary, considering the possibility of compromised immune response to vaccines due to immunosenescence

INTRODUÇÃO: A Febre Amarela (FA) é uma doença viral aguda endêmica em grande parte do Brasil. A principal medida de prevenção é a vacinação. O presente estudo avaliou a prevalência e os títulos de anticorpos neutralizantes em pessoas com 60 anos ou mais que haviam recebido anteriormente a vacina de FA 17DD, em comparação a adultos saudáveis com 18 a 59 anos. Além disso, foram avaliadas a correlação entre os títulos de anticorpos e o tempo decorrido desde a vacinação, nos participantes que receberam apenas uma dose da vacina, e a prevalência de anticorpos nos vacinados há menos e há mais de dez anos. MÉTODOS: Os participantes foram recrutados entre pessoas que procuraram o Centro de Referência para Imunobiológicos Especiais (CRIE) do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP) para receber diferentes vacinas e que referiam ter recebido a vacina de FA anteriormente. Os seguintes dados foram coletados: idade, etnia, sexo, número de doses da vacina de FA recebidas, data da última vacinação de FA. Foi realizada contagem de linfócitos TCD4+ usando citometria de fluxo. Os anticorpos neutralizantes contra FA foram dosados pelo teste de neutralização por redução de 50% das placas de lise (PRNT50). RESULTADOS: Foram incluídos 94 indivíduos, 46 com idade de 60 anos ou mais (Grupo 1) e 48 com 18 a 59 anos (Grupo 2). Não houve diferença significativa entre os dois grupos na distribuição de gênero, etnia, número de doses de vacina de FA recebidas anteriormente, tempo desde a última dose e contagem de linfócitos TCD4+. Não houve diferença na prevalência de anticorpos neutralizantes anti-FA entre os dois grupos (87% e 93,8% nos Grupos 1 e 2, respectivamente, p=0,263). O título médio geométrico (GMT) dos anticorpos neutralizantes foi maior no grupo mais jovem (3,77 log10mUI/mL) comparado ao grupo mais velho (3,64 log10mUI/mL) e essa diferença foi estatisticamente significante (p=0,022). Não foi encontrada correlação entre os títulos de anticorpos neutralizantes e o tempo decorrido desde a vacinação entre os participantes que receberam apenas uma dose de vacina, tendo sido analisados os dois grupos conjuntamente. Também não foi encontrada diferença estatisticamente significativa na prevalência de anticorpos neutralizantes entre os participantes que receberam apenas uma dose da vacina de FA há mais de 10 anos ou há menos de 10 anos. CONCLUSÕES: São necessários outros estudos de persistência de anticorpos na população idosa devido à possibilidade de resposta vacinal alterada pela imunosenescência / INTRODUCTION. Yellow Fever (YF) is an acute viral disease endemic in large parts of Brazil. The main preventive measure is vaccination. This study aimed to assess the prevalence and titers of neutralizing antibodies in persons aged 60 years and older who had previously received YF 17DD vaccine, in comparison to healthy adults aged 18 to 59 years. The study also evaluated the correlation between the antibodies titers and the time elapsed since vaccination, in participants who had received a single dose of the vaccine, and the prevalence of antibodies in participants vaccinated within ten years and more than ten years before enrollment. METHODS. Participants were recruited among persons who came to the Reference Center for Special Immunobiologicals (Centro de Referência para Imunobiológcos Especiais, CRIE) of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP) to receive any vaccine and who had previously received the YF vaccine. The following data were collected: age, ethnicity, gender, number of YF vaccine doses taken and date of last YF vaccination. CD4 T cells counts were performed using flow cytometry. YF neutralizing antibodies were measured using test of neutralization by 50% reduction of lysis plaques (PRNT50). RESULTS. Ninety-four subjects were enrolled: 46 persons aged 60 years and older (Group 1) and 48 persons aged 18 to 59 years (Group 2). There was no significant difference between the groups regarding gender, ethnicity, number of YF vaccine doses previously received, time since the last dose and CD4+T cells count. There was no difference in the prevalence of YF neutralizing antibodies between the groups (87% and 93.8% in Groups 1 and 2, respectively, p=0.263). The log-transformed Geometric Mean Titer (GMT) of YF neutralizing antibodies was higher in the younger group (3.77 log10mUI/mL) in comparison to the older group (3.64 log10mUI/mL), and this difference was statistically significant (p=0.022). There was no correlation between YF neutralizing antibodies titers and time elapsed since vaccination among the participants who had previously received a single dose of YF vaccine, with the two groups analyzed together. There was no significant difference in the prevalence of neutralizing antibodies among participants who received a single dose of YF vaccine within ten years or more than 10 years before enrollment. CONCLUSIONS. Further studies on antibodies persistence in the elderly are necessary, considering the possibility of compromised immune response to vaccines due to immunosenescence

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-12-10T13:43:12Z No. of bitstreams: 1 marisol-simoes.pdf: 2202112 bytes, checksum: 3f9f53685909ddd3fdd381f8f3692f14 (MD5) / Made available in DSpace on 2012-12-10T13:43:12Z (GMT). No. of bitstreams: 1 marisol-simoes.pdf: 2202112 bytes, checksum: 3f9f53685909ddd3fdd381f8f3692f14 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil. / A febre amarela é causada pelo vírus protótipo do gênero Flavivirus e é transmitida ao homem através da picada de mosquitos hematófagos infectados com o agente viral. A doença permanece endêmica em regiões das florestas tropicais da África e América do Sul, apesar da disponibilidade de vacinas eficazes contra o vírus da FA – 17D-204 e 17DD – consideradas seguras e altamente imunogênicas, capazes de induzir uma rápida resposta imune específica, com a formação de anticorpos neutralizantes que aparecem precocemente, são protetores e de longa duração. O teste de neutralização por redução de placas de lise (PRNT) é considerado o teste mais sensível e mais específico para a detecção e quantificação dos anticorpos neutralizantes, sendo o método de referência para a avaliação da resposta imune protetora após a vacinação. Este estudo avaliou a confiabilidade e a acurácia do micro PRNT50 (aplicado em placas de 96 poços) tomando como referência o PRNT 50 (aplicado em placas de 6 poços) e comparou o desempenho do micro PRNT50 com o micro PRNT90 (redução de 50% e 90% das placas de lise, respectivamente). Foram selecionados 200 soros de indivíduos brasileiros que participaram de um estudo clínico de dose-resposta da vacina 17DD produzida em Bio-Manguinhos. A repetibilidade foi aferida a partir de três repetições independentes do teste para cada um dos 200 soros por uma dupla operadora e o mesmo procedimento foi realizado pelas outras duas duplas operadoras, com a finalidade de determinar a reprodutibilidade do micro PRNT50 . Foram determinados o Coeficiente de Correlação Intraclasse (CCI) e o Coeficiente de Correlação de Pearson. As medidas de acurácia determinadas neste estudo foram sensibilidade, especificidade, acurácia global, prevalência e valores preditivos positivo (VPP) e negativo (VPN). A curva ROC também foi utilizada para determinar o melhor ponto de corte do micro PRNT50 . Na avaliação da repetibilidade, os CCIs das duplas operadoras variaram entre 0,62 e 0,76; e os coeficientes de correlação de Pearson apresentaram-se entre 0,62 e 0,78. Para a reprodutibilidade, o CCI obtido foi de 0,72 e os coeficientes de correlação de Pearson variaram entre 0,70 e 0,76. As melhores medidas de acurácia foram alcançadas considerando o ponto de corte para o micro PRNT50 de 2,9 Log 10 mUI/mL, o qual apresentou sensibilidade de 91,1%, especificidade de 72,9% e acurácia global de 78%. O melhor desempenho do micro PRNT50 com este ponto de corte foi ratificado pela análise da curva ROC. Considerando um cenário hipotético de surto de FA, a prevalência seria de 28%, com VPP de 56,7% e VPN de 95,5%. O micro PRNT50 apresentou níveis satisfatórios de confiabilidade e de acurácia, mas o micro PRNT90 mostrou desempenho superior com sensibilidade de 100%, especificidade de 94,7% e acurácia global de 95%. Modificações na metodologia do teste e alterações nos critérios de classificação nas leituras dos resultados obtidos serão importantes para melhorar a acurácia do micro PRNT. / Yellow fever is caused by the prototype virus of the genus Flavivirus and it is transmitted to humans through the bite of bloodsucking mosquitoes infected with the viral agent. The disease remains endemic in tropical forest regions from Africa and South America, despite the availability of effective vaccines against yellow fever virus - 17D-204 and 17DD - considered safe and highly immunogenic, capable of inducing a rapid specific immune response, with the formation of neutralizing antibodies that appear early, are protective and long lasting. The plaque reduction neutralization test (PRNT) is considered the most sensitive and most specific test for detection and quantification of neutralizing antibodies, and the reference method for assessing the protective immune response after vaccination. This study evaluated the reliability and accuracy of micro PRNT50 (applied in 96-well plates) with reference to the PRNT50 (applied in 6-well plates) and compared the performance of the micro PRNT50 with the micro PRNT90. Two-hundred serum samples from Brazilian individuals who participated in a clinical study of dose-response of 17DD vaccine produced in Bio-Manguinhos were selected. The repeatability was measured from three independent repetitions of the test for each of the 200 sera by a dual operator and the same procedure was performed by the other two dual operators, in order to determine the reproducibility of the micro PRNT50. The intraclass correlation coefficient (ICC) and Pearson correlation coefficient were determined. Accuracy measures determined in this study were sensitivity, specificity, overall accuracy, prevalence and positive predictive values (PPV) and negative (NPV). The ROC curve was also used to determine the best cut-off point of micro PRNT50 . In the assessment of repeatability, ICCs for dual operators ranged from 0.62 to 0.76, and Pearson correlation coefficients were between 0.62 and 0.78. For reproducibility, the ICC obtained was 0.72 and the Pearson correlation coefficients ranged between 0.70 and 0.76. The best measures of accuracy were achieved considering the cut-off point for the micro PRNT50 of 2.9 log10 mIU/mL, which had a sensitivity of 91.1%, specificity of 72.9% and overall accuracy of 78%. The best performance of micro PRNT50 with this cut-off point was ratified by the ROC curve analysis. Considering a hypothetical scenario of an outbreak of YF, the prevalence would be 28%, with PPV of 56.7% and NPV of 95.5%. The micro PRNT50 showed satisfactory levels of reliability and accuracy, however the micro PRNT90 showed higher performance with sensitivity of 100%, specificity of 94.7% and overall accuracy of 95%. Modifications in the test methodology and changes in the classification criteria in the readings of the results obtained will be important to improve the accuracy of micro PRNT.

Antiviral drug resistance is a major problem in the treatment of viral infections, including influenza and hepatitis C virus (HCV). Influenza neuraminidase (NA) is a viral sialidase on the surface of the influenza virion and a primary antiviral target in influenza. Two subtypes of NA predominate in humans, N1 and N2, but different patterns of drug resistance have emerged in each subtype. To provide a framework for understanding the structural basis of subtype specific drug resistance mutations in NA, we used molecular dynamics simulations to define dynamic substrate envelopes for NA to determine how different patterns of drug resistance have emerged in N1 and N2 NA. Furthermore, we used the substrate envelope to analyze HCV NS3/4A protease inhibitors in clinical development. In addition, influenza hemagglutinin (HA) is a primary target of neutralizing antibodies against influenza. Novel broadly neutralizing antibodies (BnAbs) against the stem region of HA have been described and inhibit several influenza viral subtypes, but antibody neutralization escape mutations have emerged. We identified potential escape mutations in broadly neutralizing antibody F10 that may impact protein dynamics in HA that are critical for function. We also solved crystal structures of antibody fragments that are important for understanding the structural basis of antibody binding for influenza BnAbs. These studies can inform the design of improved therapeutic strategies against viruses by incorporating an understanding of structural elements that are critical for function, such as substrate processing and protein dynamics, into the development of novel therapeutics that are robust against resistance.