T follicular helper cells (T-FH) are a specialized subset of CD4 T cells that reside in B cell follicles and promote B cell maturation into plasma cells and long-lived memory B cells. During chronic infection prior to the development of AIDS, HIV-1 (HIV) replication is largely concentrated in T-FH. Paradoxically, T-FH numbers are increased in early and midstages of disease, thereby promoting HIV replication and disease progression. Despite increased T-FH numbers, numerous defects in humoral immunity are detected in HIV-infected individuals, including dysregulation of B cell maturation, impaired somatic hypermutation, and low quality of antibody production despite hypergammaglobulinemia. Clinically, these defects are manifested by increased vulnerability to bacterial infections and impaired vaccine responses, neither of which is fully reversed by antiretroviral therapy (ART). Deficits in T-FH function, including reduced HIV-specific IL-21 production and low levels of co-stimulatory receptor expression, have been linked to these immune impairments. Impairments in T-FH likely contribute as well to the ability of HIV to persist and evade humoral immunity, particularly the inability to develop broadly neutralizing antibodies. In addition to direct infection of T-FH, other mechanisms that have been linked to T-FH deficits in HIV infection include upregulation of PD-L1 on germinal center B cells and augmented follicular regulatory T cell responses. Challenges to development of strategies to enhance T-FH function in HIV infection include lack of an established phenotype for memory T-FH as well as limited understanding of the relationship between peripheral T-FH and lymphoid tissue T-FH. Interventions to augment T-FH function in HIV-infected individuals could enhance immune reconstitution during ART and potentially augment cure strategies.

Student Number : 9000987E - PhD thesis - School of Medicine - Faculty of Health Sciences / The generation of human monoclonal antibodies (mAbs) that are able to block HIV-1 infection in vitro would be useful reagents for studying virus neutralization, and assist in identifying neutralizing antibody (NAb) epitopes of HIV-1 envelope glycoprotein. This may provide important information for designing HIV-1 vaccine that aim to induce NAbs. HIV-1 subtype C individuals with high levels of NAb titres were identified, and peripheral blood mononuclear cells (PBMC) from these individuals were isolated and B-cells transformed with Epstein-Barr virus (EBV). Clones specific to HIV-1 gp120 using cell lysate preparations derived from HIV-1 subtype C infected cell lines were generated by performing limiting dilutions. Transformation efficiencies were estimated at over 80% by evaluating EBV-transformation cultures by microscopic visualization. Of these approximately 5% were HIV-1-specific. Five clones derived from the Du23 (1) sample secreting anti-HIV-1 antibodies were generated: 2.3C, 2.9D, 3.2C, 4.12E, and 1.5D. The 1.5D mAb could not be confirmed as anti-HIV-1 clone and it was probably lost during the process of subculturing. The remaining four Du23 mAbs were determined to be of IgG1 isotype lambda (λ) light chain. These mAbs bind to gp120, and 2.9D is probably a polyreactive clone. Clones 2.3C, 3.2C and 4.12E appear to be A32-like, but do not share the same epitope. We have determined that the binding sites for all four Du23 mAbs require at least the C1 region, and they also showed binding sites overlapping with F91 and 1.5E. All four Du23 mAbs required intact gp120 proteins for their binding, and soluble CD4 enhance their binding. Thus, their binding site is discontinuous and conformational. These mAbs are non-neutralizing as they showed limited activity of 30-59% when tested using T-cell line grown viruses or 0-30% when tested against pseudovirions. This activity is rather low when compared to over 80% shown by broadly neutralizing mAbs that have been described in the literature. The challenge in generating mAbs, in particular subtype C-derived, is to find those antibodies capable of suppressing viral replication in vivo and be capable of preventing infection. These reagents could be used to identify epitopes to guiding the design of HIV-1 subtype C envelope immunogens or vaccines. It is also envisaged that neutralizing antibodies used in therapeutic setting or in combination with antiviral drug therapy could reduce viral load and retard disease progression in infected people.

RESUMO Proposta de um método prático para avaliação do poder de neutralização existente na cavidade oral.A prevenção da doença cárie ainda é uma das principais metas da Odontologia. Considerando a multifatoriedade de sua etiologia, torna-se necessário o conhecimento do maior número de informações possíveis a respeito do paciente para que o cirurgião-dentista possa estabelecer um plano de tratamento preventivo individualizado a seus pacientes. A saliva, devido a suas várias funções, apresenta grande importância no combate a patogênese da doença. A análise da capacidade tampão é de extrema importância para que se possa prever o risco do paciente ao desenvolvimento da doença cárie. Vários testes têm sido utilizados para a determinação da capacidade tampão salivar, sendo que alguns exigem equipamentos laboratoriais e outros, mais simplificados, que permitem a utilização no consultório odontológico. A proposta do presente trabalho foi desenvolver um método prático para determinação do poder neutralizante existente na cavidade oral. O método proposto consiste na realização de bochecho com 10 ml de Coca-cola® durante 30 segundos, por parte dos 50 participantes (crianças, adolescentes e adultos) e determinação da variação do pH entre a mistura saliva + Coca-cola® e o pH inicial do refrigerante. Foram realizados dois métodos de determinação da capacidade tampão salivar – titulação com ácido lático e o método simplificado Dentobuff Strip® -e, após teste de correlação entre o método de neutralização proposto e os dois testes descritos acima foi encontrada correlação estatisticamente significante entre o método proposto e a titulometria com ácido lático (Pearson=0,304;p=0,032). Porém em relação ao Dentobuff Strip® não foi verificada correlação estatisticamente significante. De acordo com a proposta da metodologia apresentada nesta pesquisa, foi encontrada uma média de neutralização da saliva após o bochecho com o refrigerante, de 23,8% (dp=16,5) até o pH crítico do esmalte (5,5) e considerando o pH fisiológico da saliva em torno de 7,0, a neutralização até este valor foi de 17% (dp=12,4). Diante dos resultados foi possível concluir que o método desenvolvido apresentou-se prático e satisfatório para avaliação da capacidade de neutralização existente na cavidade oral e pode ser utilizado como mais um recurso para predição do risco de cárie do paciente. / SUMMARY Caries prevention remains one of the main goals in dentistry. Since caries is a multifactorial disease, it becomes necessary to obtain all possible information about the patient during anamnesis. Hence, the professional is able to establish an individual preventive treatment for each patient. Saliva bears several functions in the oral cavity; consequently, it is an important host factor that modifies the caries process. Saliva buffering capacity is one of the important factors usually taken into account to predict the individual caries risk. Several tests have been applied to identify this saliva function. Some of them require laboratorial features, whereas others are easy to handle, and can be applied at dental offices. The purpose of the present study was to develop a practical method to assess the neutralizing power inherent of the oral cavity. The methodology was based on a rinse of Coke Ô for 30 seconds, performed by 50 subjects (including children, teenagers and adults), followed by the assessment of pH variation between the initial sample of soft drink and the final mixture (saliva + Coke Ô). Along with this method, two other well known buffer capacity tests were performed – titration with lactic acid and Dentobuff Strip®. We found statistically significant correlation between the proposed method and the titration with lactic acid (Pearson=0.304;p=0.032). On the other hand, there was no significant correlation between the proposed method and the test using Dentobuff Strip®. According to our results, the mean saliva neutralizing power after soft drink rinse, considering the cases of the critical enamel pH (5.5) and physiological saliva pH (7.0), were 23.8% (sd=16.5) and 17.0% (sd=12.4), respectively. The proposed method was practical and reliable to assess the neutralizing power of oral cavity and may be an additional technique to predict caries risk.

Foi investigada a existência de proteção cruzada entre bacterinas bivalentes formuladas com um de três representantes do Sorogrupo Sejroe: hardjo (bacterina A), wolffi (bacterina B) e guaricura (bacterina C), e uma estirpe do sorovar pomona, empregada por ser patogênica para hamsters (Mesocricetus auratus) e possibilitar a realização do teste de potência com desafio. Os ensaios foram efetuados em hamsters machos, comparando-se os níveis de anticorpos aglutinantes e neutralizantes, respectivamente obtidos nos testes de soroaglutinação microscópica (SAM) e inibição de leptospiras in vitro (ICL). Os animais receberam duas doses de vacinas via subcutânea; aos dez dias da segunda dose, foram inoculados com culturas não inativadas dos respectivos sorovares do Sorogrupo Sejroe. Aos 21 dias pós-infecção (d.p.i.), os animais foram sangrados, os soros (n=8) foram agrupados em pools e submetidos aos testes de SAM e ICL. O teste de potência com desafio para o sorovar pomona foi adaptado do protocolo preconizado pelo United States Department of Agriculture, mas as vacinas não foram diluídas e o esquema de imunização empregou duas aplicações de 1,0mL pela via subcutânea em intervalo de dez dias; o desafio foi efetuado aos dez dias da segunda aplicação; os óbitos por leptospirose foram registrados e, aos 21 d.p.i., os sobreviventes foram sacrificados e a condição de portadores renais foi investigada através de cultivos de tecido renal para isolamento de leptospiras. No teste de potência com o sorovar pomona, o número de doses infectantes empregado para desafio (100) situou-se dentro da faixa preconizada (10 a 100); respectivamente para as bacterinas A, B e C, as proporções de mortes por leptospirose entre os animais vacinados foram de 1/10, 0/10 e 3/10, e as de portadores renais de leptospiras entre os sobreviventes foram 2/9, 1/10 e 2/7. Os resultados do teste SAM revelaram que a bacterina A induziu reações para os sorovares hardjo e wolffi; a bacterina B, para hardjo, wolffi e guaricura, e a bacterina C, apenas para a guaricura, e do teste de ICL, que animais vacinados com as bacterinas B ou C apresentaram proteção para hardjo, wolffi e guaricura; entretanto, a bacterina A conferiu proteção apenas para wolffi. Apesar das variações no poder imunogênico segundo a estirpe de leptospira empregada para a produção das bacterinas, houve proteção cruzada entre os sorovares hardjo, wolffi e guaricura. / The existence of cross-protection among bivalent bacterins, produced with one of three leptospires belonging to Serogroup Sejroe: hardjo (bacterin A), wolffi (bacterin B) and guaricura (bacterin C), and a strain of serovar pomona (included because of its pathogenicity to hamsters and the possibility of performing potency assay with challenge), was investigated in male hamsters (Mesocricetus auratus) by comparison of agglutinating and neutralizing antibodies titers, respectively measured by microscopic agglutination (MAT) and in vitro growth inhibition (GIT) tests. All animals received two doses of bacterins by subcutaneous route; after ten days from the second dose, they were inoculated with non-inactivated cultures of respective serovars of Serogroup Sejroe. At 21 post-challenge day (p.c.d.), all animals were bled and their sera (n=8) were joined in pools and tested by MAT and GIT. The potency assay with challenge performed only with serovar pomona was modified from protocol of USDA, but vaccines were not diluted and the immunization schedule employed two 1.0 mL vaccine doses by subcutaneous route with 10?day interval; the challenge was performed after ten days from the second dose; the number of deaths due to leptospirosis was registered; at 21 p.c.d., the survivors were sacrificed and their renal carrier state was investigated by culture of renal tissue for leptospires isolation. In the potency assay with serovar pomona, the number of infectious doses employed for challenge (100 infective units) was in accordance with the recommended range (10-1,000 infective units); respectively to bacterins A, B and C, proportions of deaths due to leptospirosis among vaccinated animals were 1/10, 0/10 and 3/10, and proportions of leptospires renal carrier among survivors were 2/9, 1/10 and 2/7. Results of MAT showed that bacterin A induced reactions against serovars hardjo and wolffi; bacterin B, against hardjo, wolffi and guaricura, and bacterin C, against guaricura; results of GIT revealed that vaccinated animals with bacterins B or C presented protection against serovar hardjo, wolffi and guaricura; however, bacterin A induced protection only against wolffi. A cross?protection was observed among serovars hardjo, wolffi and guaricura, although variations exist in the immunogenic capacity according to the strain of leptospires used for the bacterins production.

RESUMO Proposta de um método prático para avaliação do poder de neutralização existente na cavidade oral.A prevenção da doença cárie ainda é uma das principais metas da Odontologia. Considerando a multifatoriedade de sua etiologia, torna-se necessário o conhecimento do maior número de informações possíveis a respeito do paciente para que o cirurgião-dentista possa estabelecer um plano de tratamento preventivo individualizado a seus pacientes. A saliva, devido a suas várias funções, apresenta grande importância no combate a patogênese da doença. A análise da capacidade tampão é de extrema importância para que se possa prever o risco do paciente ao desenvolvimento da doença cárie. Vários testes têm sido utilizados para a determinação da capacidade tampão salivar, sendo que alguns exigem equipamentos laboratoriais e outros, mais simplificados, que permitem a utilização no consultório odontológico. A proposta do presente trabalho foi desenvolver um método prático para determinação do poder neutralizante existente na cavidade oral. O método proposto consiste na realização de bochecho com 10 ml de Coca-cola® durante 30 segundos, por parte dos 50 participantes (crianças, adolescentes e adultos) e determinação da variação do pH entre a mistura saliva + Coca-cola® e o pH inicial do refrigerante. Foram realizados dois métodos de determinação da capacidade tampão salivar – titulação com ácido lático e o método simplificado Dentobuff Strip® -e, após teste de correlação entre o método de neutralização proposto e os dois testes descritos acima foi encontrada correlação estatisticamente significante entre o método proposto e a titulometria com ácido lático (Pearson=0,304;p=0,032). Porém em relação ao Dentobuff Strip® não foi verificada correlação estatisticamente significante. De acordo com a proposta da metodologia apresentada nesta pesquisa, foi encontrada uma média de neutralização da saliva após o bochecho com o refrigerante, de 23,8% (dp=16,5) até o pH crítico do esmalte (5,5) e considerando o pH fisiológico da saliva em torno de 7,0, a neutralização até este valor foi de 17% (dp=12,4). Diante dos resultados foi possível concluir que o método desenvolvido apresentou-se prático e satisfatório para avaliação da capacidade de neutralização existente na cavidade oral e pode ser utilizado como mais um recurso para predição do risco de cárie do paciente. / SUMMARY Caries prevention remains one of the main goals in dentistry. Since caries is a multifactorial disease, it becomes necessary to obtain all possible information about the patient during anamnesis. Hence, the professional is able to establish an individual preventive treatment for each patient. Saliva bears several functions in the oral cavity; consequently, it is an important host factor that modifies the caries process. Saliva buffering capacity is one of the important factors usually taken into account to predict the individual caries risk. Several tests have been applied to identify this saliva function. Some of them require laboratorial features, whereas others are easy to handle, and can be applied at dental offices. The purpose of the present study was to develop a practical method to assess the neutralizing power inherent of the oral cavity. The methodology was based on a rinse of Coke Ô for 30 seconds, performed by 50 subjects (including children, teenagers and adults), followed by the assessment of pH variation between the initial sample of soft drink and the final mixture (saliva + Coke Ô). Along with this method, two other well known buffer capacity tests were performed – titration with lactic acid and Dentobuff Strip®. We found statistically significant correlation between the proposed method and the titration with lactic acid (Pearson=0.304;p=0.032). On the other hand, there was no significant correlation between the proposed method and the test using Dentobuff Strip®. According to our results, the mean saliva neutralizing power after soft drink rinse, considering the cases of the critical enamel pH (5.5) and physiological saliva pH (7.0), were 23.8% (sd=16.5) and 17.0% (sd=12.4), respectively. The proposed method was practical and reliable to assess the neutralizing power of oral cavity and may be an additional technique to predict caries risk.

The influenza virus hemagglutinin (HA) surface protein is a primary antigenic target for neutralization of viral infection. HA also mediates membrane fusion between the virus and a cell, which is the first critical step during infection. Traditional techniques to study infection neutralization by antibodies or the membrane fusion process rely on ensemble measurements, confounding the precise mechanism of infection neutralization and obscuring transient conformational intermediates. This dissertation describes advances made in a fluorescence microscopy-based single-particle fusion assay to overcome the limitations of ensemble measurements in these types of studies. Virus particles are labeled to visualize lipid mixing between a virus and a target membrane formed upon a glass or polymer support. Optionally, the viral lumen can be labeled to visualize the subsequent release of viral contents.

An effective vaccine to protect against HIV-1/AIDS remains elusive due to the extensive mechanisms employed by the HIV-1 virus to evade immune attack. Highly potent broadly neutralizing antibodies isolated from chronically infected individuals, however, show that such relevant antibodies can be naturally produced, implying that their elicitation through vaccination is a realistic possibility. These broadly neutralizing antibodies target different regions on the trimeric spikes formed by three protomers of the envelope (Env) protein. Each Env protein is comprised of the gp120 surface subunit in non-covalent association with the gp41 transmembrane subunit. Four regions have been identified: the CD4 binding site, the V1/V2 segment and the V3/glycan area all on the gp120 subunit as well as the MPER segment on the gp41 subunit. This dissertation focuses on the gp41 MPER segment given its highly conserved amino acid sequence among all HIV-1 clades and viral strain isolates and essential function in Env-mediated fusion and HIV entry. Of note, the MPER segment contains several adjacent epitopes targeted by broadly neutralizing antibodies, suggesting that the immune system is capable of producing neutralizing antibodies against this specific region. Analysis of both clade B and C MPER segments shows them to be L-shaped, consisting of two  helices separated by a hinge. We have found that the hinge region of the MPER segment provides the conformational flexibility necessary for the Env-mediated hemifusion and fusion processes. A significant reduction in virus infectivity is observed when the hinge region is disrupted by introduction of two amino acid mutations that eliminate -helical capping residues and the tandem hinge joints. The importance of the hinge region of the MPER segment is further supported by the action of four MPER-specific neutralizing antibodies 2F5, 4E10, 10E8 and Z13E1. These neutralizing antibodies block virus infection by disrupting MPER hinge-related function.

The HIV-1 envelope glycoprotein, Env, facilitates the translocation of the viral capsid across the cellular membrane. Env is a trimer of hetero-dimers composed of a gp120 subunit and gp41 transmembrane protein. The gp120 subunit binds the primary receptor, CD4, leading to conformational changes of Env that then promote binding to the coreceptor, principally CCR5 or CXCR4. As the sole protein on the surface of the virion, Env is under continuous pressure from the host's antibody response. Two classes of antibodies target the highly conserved receptor-binding sites of gp120: CD4-binding site (CD4bs) and CD4-induced (CD4i) antibodies.

An immunoglobulin (IgG) consists of two antigen-binding fragments (Fab) connected to a crystallisable fragment through hinge regions. This thesis mainly investigates the production methods of Fabs used in structural studies. A cost effective and time saving protocol has been established for the production of recombinant 2F5 Fab, a HIV-1 broadly neutralizing monoclonal antibody fragment, using an Escherichia coli expression system. The integrity of structure and antigen-binding capability of the produced 2F5 Fab was confirmed by determining the crystal structure of the Fab-antigen peptide complex. In parallel, 3H1 Fab, a fragment of an antibody which is involved in detecting misfolded superoxide dismutase, which is related to familial amyotrophic lateral sclerosis, was produced by papain proteolysis of its parent IgG molecule. Both Fab production methods resulted in high yields of pure Fab samples that are crystallisable and ready to be engaged in structural studies using X-ray crystallography.

An immunoglobulin (IgG) consists of two antigen-binding fragments (Fab) connected to a crystallisable fragment through hinge regions. This thesis mainly investigates the production methods of Fabs used in structural studies. A cost effective and time saving protocol has been established for the production of recombinant 2F5 Fab, a HIV-1 broadly neutralizing monoclonal antibody fragment, using an Escherichia coli expression system. The integrity of structure and antigen-binding capability of the produced 2F5 Fab was confirmed by determining the crystal structure of the Fab-antigen peptide complex. In parallel, 3H1 Fab, a fragment of an antibody which is involved in detecting misfolded superoxide dismutase, which is related to familial amyotrophic lateral sclerosis, was produced by papain proteolysis of its parent IgG molecule. Both Fab production methods resulted in high yields of pure Fab samples that are crystallisable and ready to be engaged in structural studies using X-ray crystallography.