Submitted by Haroudo Xavier Filho (haroudo.xavierfo@ufpe.br) on 2016-02-25T17:28:13Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação versão correta_Erika Trindade.pdf: 2153375 bytes, checksum: 27828150a490aba6be042d8498b9068e (MD5) / Made available in DSpace on 2016-02-25T17:28:13Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação versão correta_Erika Trindade.pdf: 2153375 bytes, checksum: 27828150a490aba6be042d8498b9068e (MD5) Previous issue date: 2015-02-26 / FACEPE / A infecção com o Vírus da Hepatite B (HBV) é uma das principais causas de morbidade e mortalidade em todo o mundo. Estima-se que 350 milhões de pessoas no mundo têm infecção crônica pelo HBV, o que representa aproximadamente 5% da população mundial. Levando-se em conta que a transmissão do HBV ocorre principalmente pelas vias parenteral e sexual, bolsas de sangue devem ser rigorosamente controladas. Um teste rápido antes da doação para identificar o marcador do HBV é desejável para reduzir os custos com o descarte desnecessário de bolsas de sangue. Atualmente, os anticorpos produzidos contra os antígenos do core do vírus da hepatite B (anti-HBc IgG) têm sido relatados como o marcador mais prevalente para o HBV devido a persistir por toda a vida e indicam infecção passada. Imunossensores são dispositivos analíticos que utilizam antígenos ou anticorpos e um transdutor para detecção rápida e quantificação de analitos e também podem ser portáteis. Diferentes tipos de transdutores para imunossensores são utilizados, tais como eletroquímico, óptico e piezoeléctrico. As técnicas eletroquímicas têm como princípio básico a detecção de espécies eletroativas presentes no processo de interação do elemento biológico com o transdutor. Transdutores eletroquímicos amperométricos estão entre os mais utilizados, devido à sua facilidade para portabilização. Os nanomateriais e os polímeros funcionalizados têm atraído grande interesse no desenvolvimento de matrizes de imobilização para biossensores devido a proporcionarem maior estabilidade e maior área para ancoramento de biomoléculas. Quando os nanotubos de carbono carboxilados são associados com politiramina, eles formam ligações amida que permitem que esta superfície seja muito mais estável, aumentando a precisão analítica. Este trabalho teve como objetivo o desenvolvimento de um imunossensor para detecção do anti-HBc através da imobilização do antígeno do HBc (HBcAg) em um eletrodo de ouro nanoestruturado formado por nanotubos de carbono carboxilados e politiramina. As modificações no eletrodo foram acompanhadas por voltametria cíclica e voltametria de onda quadrada. Foi obtida uma matriz estável, com processo de interação reversível e com possibilidade de portabilização. A faixa linear de resposta foi de 1,0 a 5,0 ng/mL, com limite inferior de detecção de 0,1ng/mL, mostrando grande sensibilidade para o anticorpo. Para uso clínico, o protótipo precisa ser validado em mais amostras sorológicas. / Infection with Hepatitis B Virus (HBV) is a major cause of morbidity and mortality worldwide. An estimated 350 million people have chronic HBV infection, which represents approximately 5% of the world population. Taking into account that transmission of HBV occurs mostly by via parenteral and sexual, blood bags should be tightly controlled. A quick test before donating to identify the marker of HBV is desirable to reduce unnecessary costs to dispose of blood bags. Currently, antibodies against the core antigen of hepatitis B (anti-HBc IgG) have been reported as the most prevalent marker for HBV virus due to persist throughout life, indicating past infection. Immunosensors are analytical devices that use antigens or antibodies, and a transducer for rapid detection and quantification of analytes and can also be portable. Different types of transducers for immunosensors are used, such as electrochemical, optical and piezoelectric. Electrochemical techniques have as basic principle the detection of electroactive species present in the interaction process of the biological element with the transducer. Amperometric electrochemical transducers are among the most widely used due to be easily portabilized. And nanomaterials functionalized polymers have attracted great interest in the development of immobilization matrices for biosensors owing to provide greater stability and larger area for biomolecule anchoring. When the carboxylated carbon nanotubes are associated with polytyramine they form amide bonds that allow the surface to be much more stable, enhancing the analytical precision. This work aimed at the development of an immunosensor for the detection of anti-HBc by immobilizing the HBc antigen (HBcAg) in a nanostructured gold electrode formed by carboxylated carbon nanotubes and polytyramine. The changes in the electrode were followed by cyclic voltammetry and square wave voltammetry. A stable matrix was obtained, with reversible interaction process and the possibility of portabilization. The linear response range was 1.0 to 5.0 ng/ml, with a lower detection limit of 0.1 ng/mL, showing great sensitivity to the antibody. For clinical use, the prototype must be validated in more serum samples.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) are two of the most prevalent swine pathogens that have impacted the global swine industry for decades. Both are RNA viruses with increasing heterogeneity over the years, making a vaccine solution ever so challenging. Modified live-attenuated vaccines (MLVs) have been the most common approach, but the long-term safety regarding their potential for pathogenic reversion still needs to be addressed. Subunit based vaccines have been the focus of numerous development studies around the world with renewed interest in their promising prospects in both safety and efficacy. Our lab has developed a unique approach to use hepatitis B virus core capsid protein (HBcAg) as a vaccine delivery vehicle for either PRRSV or PEDV viral epitope antigens. Recombinantly produced HBcAg forms an icosahedral capsid virus-like particle (VLP) that has 240 repeats in a single assembled particle. By inserting different epitope antigens from these porcine pathogens into the particle, we can achieve repetitive antigen presentation to the host's immune system by taking advantage of the polymeric nature of VLP. The first animal study evaluated the efficacy of 4 VLP based vaccine candidates against PRRSV in mice. These 4 vaccines incorporated 2 B-cell epitopes (61QAAIEVYEPGRS72 and 89ELGFVVPPGLSS100) and 2 T-cell epitopes (117LAALICFVIRLAKNC131 and 149KGRLYRWRSPVIIEK163) from PRRSV structural proteins GP3 and GP5 respectively. Candidate GP3-4 was able to stimulate a significant viral neutralizing response in mouse sera against two PRRSV strains, one being heterologous, demonstrating its potential of cross-protection against PRRSV. The second animal study took an optimized VLP vaccine candidate against PEDV from previous development studies in mice, and assessed its efficacy through a comprehensive pregnant gilt vaccination and neonatal piglet challenge model. The vaccine candidate incorporated B-cell epitope 748YSNIGVCK755 from the PEDV spike protein. It was able to elicit significant viral neutralization antibody titer in gilt milk at 3 days post-farrowing (DPF), and provided nursing piglets with clinical relief in terms of morbidity, viral shedding, small intestinal lesions, and 10 days post-challenge (DPC) survival rate. / Doctor of Philosophy / Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) are two pathogens that infect pigs, resulting in immense economic losses to the global pork production industry every year. Both viruses have large diversity with various strains due to mutations that have occurred over the years. This makes vaccine development that aims at combating the pathogens even more challenging. One common vaccine strategy has been immunizing animals with modified live viruses with decreased pathogenicity. Naturally, long term safety of this option has been a concern. A much safer vaccine approach that is purely protein based has attracted renewed interest around the world. Protein based vaccines lack genetic materials from the viruses and are not able to replicate inside the host. Our lab has developed a platform that uses protein-based particles (VLPs) originated from the hepatitis B virus (HBV), and incorporates short pieces of proteins from either PRRSV or PEDV to train host's immune system to recognize these pathogens, and hopefully to prevent future infection. For the first animal study, we tested 4 VLP vaccine candidates against PRRSV in mice and discovered that mouse serum from one candidate GP3-4 was able to prevent infection of 2 distinct PRRSV strains in petri dishes, paving the way for further development. For the second animal study, we took an optimized VLP vaccine candidate against PEDV from previous mouse studies, and evaluated its performance in pigs. We immunized pregnant mother pigs with the vaccine before they gave birth, then experimentally infected newborn piglets with the virus. Piglets from the vaccinated mothers showed improved clinical signs and faster recovery from the infection.