La transmission mère-enfant (TME) est un modèle naturel permettant d’explorer le rôle des anticorps neutralisants dans la protection vis-à-vis de l’infection par le VIH-1 ainsi que les caractéristiques des virus transmis aux enfants malgré la présence d’anticorps maternels. Au cours de mes travaux, nous avons confirmé l’importance de la région V2 de la glycoprotéine de surface (gp120) du VIH-1 dans la résistance du virus à la neutralisation. L’analyse des propriétés de variants transmis à l’enfant et de variants maternels a mis en évidence une grande hétérogénéité de leurs propriétés biologiques sans pour autant identifier de propriétés spécifiques conférant un avantage sélectif aux virus transmis. Cependant, nous avons montré que les virus transmis étaient plus sensibles à la neutralisation par deux anticorps monoclonaux humains largement neutralisants récemment identifiés, PG9 et PG16, une observation qui pourrait avoir des applications intéressantes en termes de prévention de la TME ou de développement vaccinal. Enfin, nous avons identifié deux résidus très conservés de la gp120 impliqués dans la sensibilité à la neutralisation par PG9 et/ou PG16. / Mother-to-child transmission (MTCT) provides a natural model for studying the role of neutralizing antibodies in preventing HIV-1 infection, and the characteristics of the virus transmitted to the infants despite the presence of maternal antibodies. During my thesis works, we confirmed that the V2 domain of the surface envelope glycoprotein (gp120) of HIV-1 plays a major role in resistance to neutralization. The analysis of properties of variants transmitted to infant and maternal variants showed a wide spectrum of their biological properties, but we did not identify any specific property conferring a selective advantage for transmission of the virus to the infant. Nevertheless, we showed that the transmitted variants were more sensitive to neutralization by the two recently described broadly neutralizing monoclonal antibodies PG9 and PG16. This observation should have interesting applications in terms of prevention of MTCT or vaccine development. Finally, we identified two gp120 cross-clade conserved residues involved in neutralization sensitivity to PG9 and/or PG16.

Le virus de l’hépatite C (HCV) est l’agent responsable de l’hépatite C, maladie qui touche environ 3% de lapopulation mondiale. Une des caractéristiques de cette infection est son évolution dans 60 à 90% des casvers des formes chroniques avec des complications sévères telles que la cirrhose et le carcinomehépatocellulaire. Un des handicaps majeurs de la recherche sur le HCV est l’absence de systèmes decultures in vitro efficaces et de modèles animaux adaptés car le HCV n’infecte que l’homme et le chimpanzé.l’anticorps D32.10. Pour cela, nous avons développé un test de cellbindinget nous avons montré quel’interaction des particules virales sériques (HCVsp) radiomarquées à l’Iode 125 avec les celluleshépatocytaires (Huh‐7 et HepaRG) est spécifique et saturable impliquant des sites de haute et faible affinité.De plus, l’anticorps D32.10 est capable d’inhiber spécifiquement et efficacement les interactions de hauteaffinité entre les HCVsp et les cellules HepaRG avec une IC50 ≤ 0,5 μg/ml. Nous avons mis en évidence quel’inhibition est plus efficace lorsque nous utilisons sélectivement une population de particules HCVenveloppées exprimant fortement E1E2. Récemment, nous avons développé un système d’infection originaldes cellules HepaRG qui sont des cellules progénitrices du foie par les HCVsp et avons montré quel’infection, la réplication et la propagation dépendent de l’état de prolifération/différenciation de cescellules. Nous avons aussi démontré que les particules virales produites dans ce système contiennent del’ARN viral, expriment les protéines d’enveloppe E1E2 et sont infectieuses. Des études préliminairesmontrent que l’anticorps D32.10 inhibe fortement l’infection (95% à 80% aux jours 14 et 21 aprèsinfection) vraisemblablement au niveau des étapes précoces du cycle viral.Dans un second temps, nous avons recherché la prévalence des anticorps de même spécificité que le D32.10(anti‐E1E2A,B) dans différents groupes de patients HCV positifs afin de déterminer leur significationbiologique. Par un test ELISA utilisant les peptides biotinylés E1, E2A et E2B dans la phase de capture, nousavons démontré que la réponse anticorps anti‐E1E2A,B était présente dans 90% des cas chez les patientsqui guérissent spontanément avec des titres élevées (≥ 1/1000). Cette réponse humorale est absente ourare (< 10%) chez les patients porteurs chroniques non traités ou non répondeurs aux traitementsantiviraux. Une étude longitudinale a été réalisée chez des patients non répondeurs ou répondeursdéveloppant une réponse virologique soutenue à une bithérapie standard, interféron pégylé plus ribavirine.L’analyse statistique des résultats a montré que les anticorps anti‐E1E2A,B pouvaient être prédictifs de laréponse au traitement avec une spécificité et une valeur prédictive positive de 100%.La convergence des résultats in vitro et in vivo supporte un rôle neutralisant de l’anticorps monoclonalD32.10, permettant d’envisager son utilisation en immunothérapie. / Hepatitis C Virus (HCV) is the major etiological agent of liver disease in the world with approximately180 million people who are seropositive. The majority (60‐90%) of infected individuals progressesto chronic hepatitis that increases their risk for developing cirrhosis and hepatocellular carcinoma.One of the major limitations of HCV research is the lack of efficient in vitro culture systems andappropriateanimal models. vitro direct cell‐binding assay and an infection system of the human HepaRG cell line were developedby using HCVsp. The HepaRG cells possess potent ability to acquire a mature hepatocyte phenotype.The E1E2‐specific mAb D32.10 was shown to inhibit efficiently and specifically high affinityinteractionsthrough glycosaminoglycans and the CD81 tetraspanin between HCVsp and HepaRGcells with an IC50 = 0.5 μg/ml. This inhibition was more efficient when E1E2‐positive envelopedHCVsp were used selectively for binding studies (IC50 < 0.5 μg/ml). Establishment of infection,replication and propagation of HCVsp were shown to depend on the proliferation/differentiationstage of HepaRG cells. Persistent HCV infection in HepaRG cells could be obtained with production ofE1E2/RNA(+) infectious HCV particles. Preliminary data showed a complete early inhibitory effect ofthe D32.10 mAb on virion RNA production in HepaRG culture supernatants (95% at D14 and 80% atD21 post‐infection).Furthermore, the detection of the anti‐E1E2/D32.10‐binding peptide antibodies during natural HCVinfection demonstrated significant prevalence (90%) of these antibodies: (1) in patients whorecovered spontaneously from HCV infection with high titers compared to patients with chronichepatitis C, and (2) in patients who are complete responders compared to non responders toantivirals. Kinetic analyses revealed that the anti‐E1E2/D32.10‐like humoral response appeared veryearly with high titers (≥ 1/1000) and was associated with complete virus eradication. The positiveand negative predictive values (ROC curve analysis) for achieving or not a sustained viral response toantiviral therapy are 100% and 86%, respectively, reflecting diagnostic accuracy. The anti‐E1E2/D32.10‐binding peptide antibodies may thus predict the outcome of HCV infection andrepresent a new relevant pronostic marker in serum for the HCV diagnosis.Convergence of in vitro and in vivo data strongly support the neutralizing activity of the D32.10 mAb,and thus immunotherapeutic potential of this unique anti‐E1E2 D32.10 mAb.

O Trypanosoma cruzi expressa um grupo de glicoprotcinas de superfície, denominadas Tc-85, que pertencem à superfumília gêmca das gp85/traus-sialidases. Nosso laboratório clonou e caracterizou um membro da fumília Tc85 (Tc85-11), cuja região carboxila tenninal (clone Tc85-1) adere em laminina e em células de mamífero. Usando peptídeos sintéticos, correspondendo em seqüência à Tc85-1, caracterizou-se o motivo mais conservado da superfamilia gênica das gp85/trans-sialidases (VTVxNVFLYNR), o qual não adere em laminina. Esse motivo foi chamado peptídeo J. Por cromatografia de extratos de membrana de cardiomiócitos em coluna de afmidade contendo peptídeo J, foi isolada uma molécula de 30kDa identificada como sendo a subunidade &#946;3 da Na+, K+ ATPase. A porção extracelular da subunidade &#946;3 da Na+, K+ ATPase foi clonada e a interação in vitro desta proteína com peptídeo J foi observada. Deste modo, é sugerido aqui que a subunidade &#946;3 da Na+, K+ ATPase pode ter um papel importante na interação do parasita com a célula hospedeira. / Abstract not available.

No presente trabalho, desenvolvemos uma estratégia vacinal baseada em vacinas de DNA que codificam proteínas do HPV-16, o tipo viral de maior relevância epidemiológica, fusionadas à glicoproteína D (gD) do vírus herpes simplex tipo 1 (HSV-1). A vacina que contêm o gene da E7 de HPV-16 fusionada à gD de HSV-1 (pgDE7), administrada em regime vacinal de quatro doses, foi capaz de gerar significativa ativação de células T CD8+ E7-específicas e apresentar 40% de efeito protetor anti-tumoral terapêutico em camundongos desafiados com células transformadas que expressam as proteínas E6 e E7 do HPV-16 (células TC-1). A partir das evidências geradas, desenvolvemos um novo vetor vacinal que codifica as proteínas E7, E6 e E5 do HPV-16 (pgD-E7E6E5). Em ensaios em modelo murino, apenas uma dose da vacina foi capaz de gerar ativação de células T CD8+ específicas e 70% dos camundongos previamente desafiados com células TC-1 e inoculados com 3 doses da vacina mantiveram-se livres de tumores. Como tentativa de potencializar o efeito protetor terapêutico encontrado, adotamos duas medidas: a co-administração de plasmídeos que codificam citocinas e a otimização de códons da sequência gênica que codifica a proteína quimérica. A combinação das vacinas pgDE7 ou pgD-E7E6E5 com plasmídeos que carregam os genes das citocinas IL-2, IL-12 ou GM-CSF foi capaz de aumentar a proteção terapêutica para 100% em regime vacinal de dose única. A adequação da sequência antigênica ao sistema de expressão humano, aumentou em cerca de 5 vezes o potencial terapêutico do vetor vacinal pgDE7. Em conjunto, os dados apresentados nesta tese demonstram a evolução do desenvolvimento de uma estratégia vacinal contra tumores induzidos por HPV-16 e encorajam seu potencial para uso em futuros ensaios clínicos. / The development of immunotherapeutic strategies against human papillomavirus (HPV) is a priority for the control of HPV-induced lesions and cervical cancer. In this study, we developed DNA vaccines encoding HPV-16 proteins fused to glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) as an approach to control HPV-16 induced tumors. The vaccine encoding HPV-16 E7 fused to HSV-1 gD (pgDE7), when administered in a four doses vaccine regimen, was able to generate significant activation of E7-specific CD8+ T cells and showed 40% of therapeutic anti-tumor effect in mice previously challenged with tumor cells expressing HPV-16 E6 and E7 proteins (TC-1 cells). Following these evidences, we developed a new vaccine vector encoding HPV-16 E7, E6, E5 proteins fused to HSV-1 gD (pgD-E7E6E5). Only one vaccine dose generated antigen-specific CD8+ T cell responses and three doses conferred 70% protection to mice previously challenged with TC-1 cells. As an attempt to enhance the observed therapeutic anti-tumor effects, we tested two approaches: co-administration of cytokine-expressing plasmids and codon optimization of the gene encoding the chimeric protein. The combination of the vaccines pgDE7 or pgD-E7E6E5 with plasmids encoding the cytokines IL-2, IL-12 or GM-CSF increased the therapeutic protection to 100% of the vaccinated animals following a single dose. The gene sequence adaptation increased by a factor of 5 the therapeutic potential of the pgDE7 vaccine. In summary, the data presented in this thesis demonstrated the development of a vaccine strategy against HPV-16 induced tumors and reinforces its potential use in future clinical trials.

Metastasis is the primary cause of mortality in women with breast cancer. Recently, elevated serum levels of a glycoprotein known as chitinase-3 likeprotein- 1 (CHI3L1) has been correlated with poor prognosis and shorter survival of patients with cancer and inflammatory diseases. The biological and physiological functions of CHI3L1 in tumor progression have not yet been elucidated. In this document, we describe the role of CHI3L1 in tumor growth and metastasis and its relationship with inflammation. Using well-established models of breast cancer, we show that CHI3L1 is increased in the serum of tumor bearing mice. We found that CHI3L1 levels are increased at both the “pre-metastatic” and “metastatic stage” and that tumor cells, splenic, alveolar and interstitial macrophages; and myeloid derived population produce CHI3L1. Furthermore, we demonstrated that CHI3L1 has an inhibitory role on the expression of interferon-gamma (IFN γ) by T cells, while enhancing the production of pro-inflammatory mediators by macrophages such as Cchemokine ligand 2 (CCL2/MCP-1), Chemokine CX motif ligand 2 (CXCL2/IL-8) and matrix metalloproteinase-9 (MMP-9), all of which promote tumor growth and metastasis. We demonstrated that in vivo treatment of tumor-bearing mice with chitin microparticles, a TH1 adjuvant and a substrate for CHI3L1, promoted immune effector functions with increased production of IFN-γ but decreased CCL2/MCP-1, CXCL2/IL-8 and MMP-9 expression by splenic and pulmonary macrophages. Significantly, in vivo administration of chitin microparticles decreased tumor growth and pulmonary metastasis in mammary tumor bearing mice. These results suggest that CHI3L1 may play a role in tumor progression. Inflammation plays a pivotal role during tumor progression and metastasis by promoting the production of pro-inflammatory molecules such as CHI3L1. However, little is known about how CHI3L1 expression can affect secondary sites to enhance metastasis. In these studies, we demonstrated that CHI3L1 alters the cellular composition and inflammatory mediators that aid in the establishment of a metastatic niche for the support of infiltrating tumor cells leading to accelerated tumor progression. Since previous studies showed that CHI3L1 modulates inflammation, we determined the role of CHI3L1 in the context of pre-existing inflammation and metastasis. We found that CHI3L1 deficient mice with preexisting inflammation had decreased pro-inflammatory mediators, and significant reduction in tumor volume and metastasis compared to wild type controls. Preexisting inflammation and CHI3L1 may be driving the establishment of a premetastatic milieu in the lungs and aiding in the establishment of metastasis. Understanding the role of CHI3L1 in inflammation during tumor progression could result in the design of targeted therapies for breast cancer patients. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2015. / FAU Electronic Theses and Dissertations Collection

Carbohydrate recognition is one of the most sophisticated recognition processes in biological systems, mediating many important aspects of cell-cell recognition, such as inflammation, cell differentiation, and metastasis. Consequently, lectin-glycan interactions have been intensively studied in order to mimic their actions for potential bioanalytical and biomedical applications. Galectins, a class of ß-galactoside-specific animal lectins, have been strongly implicated in inflammation and cancer. Galectin-3 is involved in carbohydrate-mediated metastatic cell heterotypic and homotypic adhesion via interaction with Thomsen-Friedenreich (TF) antigen on cancer-associated MUC1. However, the precise mechanism by which galectin-3 recognizes TF antigen is poorly understood. Our thermodynamic studies have shown that the presentation of the carbohydrate ligand by MUC1-based peptide scaffolds can have a major impact on recognition, and may facilitate the design of more potent and specific galectin-3 inhibitors that can be used as novel chemical tools in dissecting the precise role of galectin-3 in cancer and inflammatory diseases. Another lectin, odorranalectin (OL), has been recently identified from Odorrana grahami skin secretions as the smallest cyclic peptide lectin, has a particular selectivity for L-fucose and very low toxicity and immunogenicity, rendering OL an excellent candidate for drug delivery to targeted sites, such as: (1) tumor-associated fucosylated antigens implicated in the pathogenesis of several cancers, for overcoming the nonspecificity of most anticancer agents; (2) the olfactory epithelium of nasal mucosa for enhanced delivery of peptide-based drugs to the brain. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2015.

In mammals, before fertilisation can occur, sperm have to bind to, and penetrate, the extracellular coat of the oocyte, the zona pellucida (ZP). In the laboratory mouse, which has been used as a model system for fertilization studies, sperm-ZP binding has been found to be mediated by a region near the carboxy terminal, encoded by exon 7 of the Zp3 gene. This region shows considerable interspecific sequence diversity in North American cricetid rodents, with some evidence of adaptive evolution, suggesting that this may contribute to species specific sperm-ZP binding. However, by contrast, in a preliminary study of three species of Australian murine rodents an identical protein sequence of the region encoded by exon 7 of Zp3 was found to be present. The aim of this present study was to determine the pattern of sequence diversity of this region in the most speciose subfamily of mammals, the murine rodents, and to obtain insight into the selective pressures involved in its evolution. For this, DNA was extracted from murine rodents of Africa, Eurasia, South-east Asia, New Guinea and Australia. The nucleotide and predicted amino acid sequence of exons 6 and 7 of Zp3 in 96 murine species from 14 divisions, as recently defined by Musser and Carleton (2005), was determined and compared. Generally, it was found that closely related species shared a highly similar ZP3 sequence. Maximum likelihood analyses of codon substitution models using representatives from 14 murine divisions, suggested that positive selection had occurred within only a few lineages at several different codon sites adjacent to, or within, the putative combining-site for sperm of ZP3. Positive selection was not evident when the analysis was restricted to the Australian taxa which showed low levels of both intra- and inter-generic sequence divergence. There was no good evidence that this region contributes to species specificity of sperm-ZP binding in these species. These findings thus suggest that the selective forces acting on the Zp3 exon 7 region during the evolution of the murine rodents have varied possibly due to a range of selective pressures not necessarily restricted to the prevention of hybridization. It seems unlikely, therefore, that the amino acid sequence of the exon 7 coding region contributes to species specificity of sperm-ZP binding within most of the lineages from this most speciose subfamily of eutherian mammals. / http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1294654 / Thesis(Ph.D.)-- School of Medical Sciences, 2007

Nipah virus (NiV) and Hendra virus (HeV) have been identified as the causes of outbreaks of fatal meningitis, encephalitis, and respiratory disease in Australia, Malaysia, Bangladesh, and India from 1994 until 2004. In order to accommodate the unique genomic characteristics of NiV and HeV, a new genus within the family Paramyxoviridae was created, named Henipavirus. NiV encodes two surface glycoproteins: the attachment glycoprotein (G) binds to the cellular receptor for the virus, while the fusion glycoprotein (F) mediates membrane fusion between the virus and cell membranes. Expression of F and G in the same cell results in cell-cell fusion in transfected cell monolayers, while expression of F and G on their own in cell monolayers does not result in fusion. Co-culture of singly-transfected F and G cells also does not result in fusion. Expression of NiV G in transgenic CRFK cells results in resistance to NiV- and HeV-induced cytopathic effect. Additionally, neither NiV nor HeV nucleic acid could be detected in CRFK-NiV G that had been exposed to NiV or HeV. NiV G expression also prevents NiV F+NiV G-mediated cell-cell fusion, but does not affect cell surface expression of either virus receptor, ephrin-B2 and ephrin-B3. Chimeric glycoproteins derived from NiV G and CDV H were constructed and characterized. None of the chimeric glycoproteins were able to fuse when coexpressed with either NiV F or CDV F. Only one of the chimeric glycoproteins (H145/G458) was detected on the cell surface by immunofluorescence assay (IFA). None of the chimeric glycoproteins altered cell surface expression levels of ephrin-B2 and ephrin-B3. Finally, recombinant NiV genomes (rNiV and rNiV eGFPG) were constructed, as well as chimeric CDV genomes with NiV ORF substitutions (rCDV eGFPH NiVFG and rCDV eGFPH NiVMFG). The only chimeric virus that was generated, rCDV eGFPH NiVFG, was assessed for its release from infected cells. rCDV eGFPH NiVFG was poorly released from infected cells without a freeze-thaw cycle, but was also found to induce the cellsurface down-regulation of the viral receptors ephrin-B2 and ephrin-B3. / October 2007

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Also available online. Includes bibliographical references (leaves 121-140).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 98-113).