Vaccination comes second in importance after introduction of clean water as a public health intervention which has largely contributed in the reduction of deaths from infectious diseases. Success in the development of a group A streptococcal (GAS) vaccine is expected to save 517 000 deaths per annum according to a recent independent review commissioned by the world health organization (WHO) and would offer an ideal means to prevent rheumatic heart disease (responsible for the greatest health burden) and other GAS-associated diseases which affect the health of 600 million. Traditional vaccine approaches (killed or live attenuated) have demonstrated great success against many bacterial and viral infectious diseases, crowned by the global eradication of smallpox announce by the WHO in 1980 and near-to-be announced eradication of polio viral disease. However, application of traditional techniques in many cases such as HIV/AIDS, malaria, GAS and Mycobacteria tuberculosis, has not shown the same success. Risk associated with the use of live–attenuated pathogens, such as recurrence of virulence (e.g. HIV), development of autoimmune diseases (e.g. GAS), and difficulties of manufacture hindered the use of such approaches. Other vaccine approaches such as subunit vaccines (recombinant proteins) and carrier conjugated vaccine are also hindered by the lack of suitable adjuvants, carriers and delivery systems. The current thesis focused on the design, synthesis and evaluation of novel adjuvants and vaccine delivery systems against GAS. The first chapter reviews recent approaches in the field of GAS vaccine design and new findings in immunology which represent the basis of our novel strategies. The second chapter describes the design, synthesis and evaluation of a novel library of lipopeptides as self-adjuvanting GAS vaccine candidates, composed of: (i) a universal helper T-cell epitope (P25), (ii) a target GAS B-cell epitope (J14), and (iii) a lipid moiety. Systemic J14-specific IgG antibodies were detected following subcutaneous immunization of BALB/c (H-2d) mice with each construct without the need for an additional adjuvant. The effect of changing the order of P25, J14, and lipid moiety attachment, or incorporation of P25 and J14 into a lipid-core peptide system (LCP) on antibody titers was assessed. The point of lipid moiety attachment had the greatest influence on systemic J14-specific IgG antibody titers. Overall, the best vaccines featured a C-terminal lipid moiety, conjugated through a lysine residue to P25 at the N-terminus, and J14 on the lysine side-chain. Mucosal surface of the nasal-oral route is a primary site of GAS infections. An ideal GAS vaccine would have to elicit both mucosal as well as systemic immune responses and hence would not only prevent the development of GAS-associated diseases but also would prevent primary GAS infections. Therefore, the nasal route is considered a highly promising route of vaccine administration to provide local as well as systemic immune responses against pathogens that utilize mucosal surface as site of infection. The third chapter includes immunological assessment of the lipopeptide vaccine library described in the second chapter following intranasal immunization of B10BR (H-2k) mice. The whole library was first investigated in a small scale experiment (5 mice per group) to select promising candidates which demonstrate the best local and systemic J14-specific antibodies. Four selected lipopeptides were further investigated in a larger scale experiment (15 mice per group) followed by intranasal challenge of vaccinated mice with a virulent GAS M1 strain. The best local and systemic immune responses were demonstrated by a lipopeptide featuring a lipid moiety consisting of two 16 carbon chains incorporated at the C-terminus of the lipopeptide. However, this candidate did not achieve protection against bacterial challenge. The best protection (100%) was shown by a lipopeptide candidate featuring a C-terminal J14, conjugated through a lysine residue to P25 at the N-terminus, and a lipid moiety on the lysine side-chain. A possible explanation for these results was investigated where antibodies elicited by the former candidate was found to better recognize the minimal B-cell epitope in the native p145 sequence of the M protein. Circular dichroism study of lipopeptides used in the previous experiment demonstrated that the former candidate features α-helical conformation which is required to produce protective J14-specific antibodies. Further studies are needed to explain structural features required to achieve both α-helicity and strong mucosal immune responses shown by the previously mentioned two lipopeptides. Signaling through toll-like receptors expressed by immune cells was recently shown to result in a robust immune response and was investigated as a possible mode of action for our novel lipopeptides. The fourth chapter introduces our lipopeptide vaccine approach as novel synthetic ligands targeting TLR2. A lipid moiety consisting of two alkyl chains of 16 carbons was found to achieve optimal TLR2 signaling regardless of the position of lipid attachment. Carbohydrates as polyhydroxy compounds provide an easily accessible class of compounds to design scaffolds (carriers) to attach lipids and peptide epitopes in different number and stereochemical positions which makes glycolipopeptides an attractive target for adjuvant research and structure-adjuvanticity relationships studies. The Fifth chapter reports immunological assessment of two series of glycolipopeptides as GAS vaccine candidates and novel vaccine delivery systems. The first series: lipid carbohydrate core peptide system (LCCP); represents a modification of the classical LCP system where polylysine dendrimer is replaced by different monosccharides as carriers for peptide antigens. LCCP analogues induced proper humoral immune responses against incorporated epitopes comparable to the LCP delivery system and as strong as the immune response elicited by CFA mixtures. Moreover, LCCP delivery system has been proved to be tolerant to the use of different epitopes as well as changing carbohydrate cores. Design of novel carbohydrate cores with different orthogonal protecting groups is needed to explore the potential advantage of various stereochemical arrangements provided by monosaccharides. The second series of glycolipopeptides incorporates various glycolipid moieties (self-adjuvanting activity) covalently coupled to the N-terminus of J8 (a model epitope). The new glycolipopeptide vaccine candidates (containing only one copy of J8) bear comparison with an LCP analogue (containing four copies of J8) which would improve the ease of synthesis, purification and cost of vaccine production. The slight difference in immunogenicity among these glycolipopeptides was difficult to be explained due to intervening effects of both the number and orientation of lipids on immunological activity. Further investigation is needed to determine the contribution of each factor.

Vaccination comes second in importance after introduction of clean water as a public health intervention which has largely contributed in the reduction of deaths from infectious diseases. Success in the development of a group A streptococcal (GAS) vaccine is expected to save 517 000 deaths per annum according to a recent independent review commissioned by the world health organization (WHO) and would offer an ideal means to prevent rheumatic heart disease (responsible for the greatest health burden) and other GAS-associated diseases which affect the health of 600 million. Traditional vaccine approaches (killed or live attenuated) have demonstrated great success against many bacterial and viral infectious diseases, crowned by the global eradication of smallpox announce by the WHO in 1980 and near-to-be announced eradication of polio viral disease. However, application of traditional techniques in many cases such as HIV/AIDS, malaria, GAS and Mycobacteria tuberculosis, has not shown the same success. Risk associated with the use of live–attenuated pathogens, such as recurrence of virulence (e.g. HIV), development of autoimmune diseases (e.g. GAS), and difficulties of manufacture hindered the use of such approaches. Other vaccine approaches such as subunit vaccines (recombinant proteins) and carrier conjugated vaccine are also hindered by the lack of suitable adjuvants, carriers and delivery systems. The current thesis focused on the design, synthesis and evaluation of novel adjuvants and vaccine delivery systems against GAS. The first chapter reviews recent approaches in the field of GAS vaccine design and new findings in immunology which represent the basis of our novel strategies. The second chapter describes the design, synthesis and evaluation of a novel library of lipopeptides as self-adjuvanting GAS vaccine candidates, composed of: (i) a universal helper T-cell epitope (P25), (ii) a target GAS B-cell epitope (J14), and (iii) a lipid moiety. Systemic J14-specific IgG antibodies were detected following subcutaneous immunization of BALB/c (H-2d) mice with each construct without the need for an additional adjuvant. The effect of changing the order of P25, J14, and lipid moiety attachment, or incorporation of P25 and J14 into a lipid-core peptide system (LCP) on antibody titers was assessed. The point of lipid moiety attachment had the greatest influence on systemic J14-specific IgG antibody titers. Overall, the best vaccines featured a C-terminal lipid moiety, conjugated through a lysine residue to P25 at the N-terminus, and J14 on the lysine side-chain. Mucosal surface of the nasal-oral route is a primary site of GAS infections. An ideal GAS vaccine would have to elicit both mucosal as well as systemic immune responses and hence would not only prevent the development of GAS-associated diseases but also would prevent primary GAS infections. Therefore, the nasal route is considered a highly promising route of vaccine administration to provide local as well as systemic immune responses against pathogens that utilize mucosal surface as site of infection. The third chapter includes immunological assessment of the lipopeptide vaccine library described in the second chapter following intranasal immunization of B10BR (H-2k) mice. The whole library was first investigated in a small scale experiment (5 mice per group) to select promising candidates which demonstrate the best local and systemic J14-specific antibodies. Four selected lipopeptides were further investigated in a larger scale experiment (15 mice per group) followed by intranasal challenge of vaccinated mice with a virulent GAS M1 strain. The best local and systemic immune responses were demonstrated by a lipopeptide featuring a lipid moiety consisting of two 16 carbon chains incorporated at the C-terminus of the lipopeptide. However, this candidate did not achieve protection against bacterial challenge. The best protection (100%) was shown by a lipopeptide candidate featuring a C-terminal J14, conjugated through a lysine residue to P25 at the N-terminus, and a lipid moiety on the lysine side-chain. A possible explanation for these results was investigated where antibodies elicited by the former candidate was found to better recognize the minimal B-cell epitope in the native p145 sequence of the M protein. Circular dichroism study of lipopeptides used in the previous experiment demonstrated that the former candidate features α-helical conformation which is required to produce protective J14-specific antibodies. Further studies are needed to explain structural features required to achieve both α-helicity and strong mucosal immune responses shown by the previously mentioned two lipopeptides. Signaling through toll-like receptors expressed by immune cells was recently shown to result in a robust immune response and was investigated as a possible mode of action for our novel lipopeptides. The fourth chapter introduces our lipopeptide vaccine approach as novel synthetic ligands targeting TLR2. A lipid moiety consisting of two alkyl chains of 16 carbons was found to achieve optimal TLR2 signaling regardless of the position of lipid attachment. Carbohydrates as polyhydroxy compounds provide an easily accessible class of compounds to design scaffolds (carriers) to attach lipids and peptide epitopes in different number and stereochemical positions which makes glycolipopeptides an attractive target for adjuvant research and structure-adjuvanticity relationships studies. The Fifth chapter reports immunological assessment of two series of glycolipopeptides as GAS vaccine candidates and novel vaccine delivery systems. The first series: lipid carbohydrate core peptide system (LCCP); represents a modification of the classical LCP system where polylysine dendrimer is replaced by different monosccharides as carriers for peptide antigens. LCCP analogues induced proper humoral immune responses against incorporated epitopes comparable to the LCP delivery system and as strong as the immune response elicited by CFA mixtures. Moreover, LCCP delivery system has been proved to be tolerant to the use of different epitopes as well as changing carbohydrate cores. Design of novel carbohydrate cores with different orthogonal protecting groups is needed to explore the potential advantage of various stereochemical arrangements provided by monosaccharides. The second series of glycolipopeptides incorporates various glycolipid moieties (self-adjuvanting activity) covalently coupled to the N-terminus of J8 (a model epitope). The new glycolipopeptide vaccine candidates (containing only one copy of J8) bear comparison with an LCP analogue (containing four copies of J8) which would improve the ease of synthesis, purification and cost of vaccine production. The slight difference in immunogenicity among these glycolipopeptides was difficult to be explained due to intervening effects of both the number and orientation of lipids on immunological activity. Further investigation is needed to determine the contribution of each factor.

Orientador: Hélio José Montassier / Banca: Antonio Carlos Alessi / Banca: Ricardo Luiz Moro de Souza / Banca: Luis Francisco Prata / Banca: Antonio Carlos Paulillo / Resumo: O vírus da bronquite infecciosa aviária (VBIG) permanece como um dos principais problemas para a avicultura industrial. Para a imunoprofilaxia dessa virose existem vacinas "vivas" atenuadas ou inativadas, mas elas não são efetivas para o controle dessa doença infecciosa a longo prazo, especialmente contra estirpes variantes desse vírus. A função primordial das vacinas usuais contra o VBIG são estimular as respostas imunes sistêmicas e locais, mediadas por anticorpos ou por células T efetoras. Com a finalidade de investigar os mecanismos envolvidos na imunidade protetora, os níveis de anticorpos locais e sistêmicos e a expressão de genes associados a respostas de linfócitos T citotóxicos, tais como o CD8 e a granzima A foi avaliada na mucosa traqueal, após a imunização primária de pintinhos de 1 dia de idade com vacinas atenuadas contra o VBIG seguida do desafio 42 dias depois. Proteção ao desafio foi avaliada por meio da ciliostase traqueal, histopatologia e detecção de vírus pela técnica de RT-PCR em tempo real na traquéia. Os níveis de anticorpos no soro e na secreção lacrimal foram mensurados pelo Sandwich- Concanavalina A - ELISA. A expressão dos genes de CD8 e de granzima A foram avaliadas pela técnica de RT-PCR em tempo real. Os resultados demonstraram que a proteção contra a infecção pelo VBIG em aves vacinadas se correlacionou com os níveis de anticorpos anti-virais específicos dos isótipos IgG, IgM e IgA na secreção lacrimal e com os níveis de expressão de CD8 e de granzima A. Concluindo, os resultados possibilataram definir o perfil cinético do desenvolvimento das respostas imunes de memória contra o VBIG na mucosa que é o sítio primário de replicação viral e indicaram que os mecanismos de imunidade humoral e celular podem ser muito importantes para a proteção de hospedeiros naturais contra a infecção pelo VBIG. / Abstract: Avian infectious bronchitis (IBV) remains a major problem in the poultry industry. Live and inactivated vaccines are available, but they are not effective long term in controlling IBV infection, specially against variant strains. The essential function of existing IBV vaccines is to elicit, ideally, local and systemic specific antibodies, as well as cell-mediated immunity to this virus. To investigate the mechanisms of protective immunity, the levels of systemic and local specific antibodies and the expression of genes responsible for cytotoxic T cell killing such as CD8-marker and granzyme-A at tracheal mucosa was evaluated after the primary immunization of 1- day-old chicks with an attenuated avian infectious bronchitis virus (IBV) and challenge 42 days later. Challenge protection was evaluated by tracheal ciliostasis, histopathology and virus detection by real time RT-PCR. The serum and lachrymal anti-IBV antibody levels of IgG, IgM and IgM isotypes were measured with a Sandwich-ELISA Concanavalina-A method. The expression of CD8 and granzyme A genes were evaluated by real time RT-PCR. The results showed protection against challenge with the M-41. Lachrymal IgG, IgM and IgA anti-IBV specific antibodies and the levels of expression of CD8 and granzyme A genes correlated significantly with the protection to challenge. Overall, the results provided the kinetics on the development of memory mucosal immune responses against IBV at the primary replication site and indicate that tracheal humoral and cellular immune mechanisms may be very important in protecting natural hosts against IBV infection. / Mestre

Made available in DSpace on 2014-06-11T19:27:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-14Bitstream added on 2014-06-13T20:36:40Z : No. of bitstreams: 1 santos_il_me_jabo.pdf: 445425 bytes, checksum: 8eabdcdd56ae9af7bcf160cad345bd86 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O vírus da bronquite infecciosa aviária (VBIG) permanece como um dos principais problemas para a avicultura industrial. Para a imunoprofilaxia dessa virose existem vacinas “vivas” atenuadas ou inativadas, mas elas não são efetivas para o controle dessa doença infecciosa a longo prazo, especialmente contra estirpes variantes desse vírus. A função primordial das vacinas usuais contra o VBIG são estimular as respostas imunes sistêmicas e locais, mediadas por anticorpos ou por células T efetoras. Com a finalidade de investigar os mecanismos envolvidos na imunidade protetora, os níveis de anticorpos locais e sistêmicos e a expressão de genes associados a respostas de linfócitos T citotóxicos, tais como o CD8 e a granzima A foi avaliada na mucosa traqueal, após a imunização primária de pintinhos de 1 dia de idade com vacinas atenuadas contra o VBIG seguida do desafio 42 dias depois. Proteção ao desafio foi avaliada por meio da ciliostase traqueal, histopatologia e detecção de vírus pela técnica de RT-PCR em tempo real na traquéia. Os níveis de anticorpos no soro e na secreção lacrimal foram mensurados pelo Sandwich- Concanavalina A – ELISA. A expressão dos genes de CD8 e de granzima A foram avaliadas pela técnica de RT-PCR em tempo real. Os resultados demonstraram que a proteção contra a infecção pelo VBIG em aves vacinadas se correlacionou com os níveis de anticorpos anti-virais específicos dos isótipos IgG, IgM e IgA na secreção lacrimal e com os níveis de expressão de CD8 e de granzima A. Concluindo, os resultados possibilataram definir o perfil cinético do desenvolvimento das respostas imunes de memória contra o VBIG na mucosa que é o sítio primário de replicação viral e indicaram que os mecanismos de imunidade humoral e celular podem ser muito importantes para a proteção de hospedeiros naturais contra a infecção pelo VBIG. / Avian infectious bronchitis (IBV) remains a major problem in the poultry industry. Live and inactivated vaccines are available, but they are not effective long term in controlling IBV infection, specially against variant strains. The essential function of existing IBV vaccines is to elicit, ideally, local and systemic specific antibodies, as well as cell-mediated immunity to this virus. To investigate the mechanisms of protective immunity, the levels of systemic and local specific antibodies and the expression of genes responsible for cytotoxic T cell killing such as CD8-marker and granzyme-A at tracheal mucosa was evaluated after the primary immunization of 1- day-old chicks with an attenuated avian infectious bronchitis virus (IBV) and challenge 42 days later. Challenge protection was evaluated by tracheal ciliostasis, histopathology and virus detection by real time RT-PCR. The serum and lachrymal anti-IBV antibody levels of IgG, IgM and IgM isotypes were measured with a Sandwich-ELISA Concanavalina-A method. The expression of CD8 and granzyme A genes were evaluated by real time RT-PCR. The results showed protection against challenge with the M-41. Lachrymal IgG, IgM and IgA anti-IBV specific antibodies and the levels of expression of CD8 and granzyme A genes correlated significantly with the protection to challenge. Overall, the results provided the kinetics on the development of memory mucosal immune responses against IBV at the primary replication site and indicate that tracheal humoral and cellular immune mechanisms may be very important in protecting natural hosts against IBV infection.