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Epithelial cells are the first line of defense against invading pathogens and external threats in the environment. Keratinocytes, often not perceived of as immune cells, release cytokines in response to infection or injury to signal danger to neighboring cells and recruit effector leukocytes to prevent further damage to the host. IL-1 and IL-36 cytokines are a group of closely related proteins that share similarities in structure and function and have been shown to play key roles in inflammatory responses of epithelial tissues. While IL-1, consisting of IL-1α and IL-1β, have been widely studied and recognized as pinnacle cytokines in a variety of inflammatory responses, relatively little is understood about IL-36 cytokines since their discovery more than 15 years ago, and how they differ from their better-known IL-1 relatives. IL-36 cytokines, consisting of IL-36α, IL-36β, and IL-36γ, signal through the same receptor, IL-36R, which is expressed most abundantly on epithelial cells. IL-36 proteins garnered attention when it was discovered that a missense mutation in the gene encoding the naturally occurring receptor antagonist, IL-36Ra, was associated with the deadly form of psoriasis, generalized pustular psoriasis (GPP). This disease is characterized by episodic flares of keratinocyte hyperproliferation leading to red scaly lesions all over the body, excessive neutrophil recruitment to the epidermis resulting in pustule formation, and severe fever. Our data presented here demonstrate that IL-36α, but not IL-36β or IL-36γ is critical for the psoriatic phenotype, including epidermal thickening and neutrophil recruitment, generated during a murine model of psoriasis induced by the drug Imiquimod. Furthermore, IL-36α was found to induce IL-1α expression and vice versa through a signaling feedback loop which perpetuated disease. These data provide insight into mechanisms whereby IL-36 signaling can lead to excessive inflammatory effects in patients with pre-existing regulation deficiencies, which can lead to acute flares of disease. Beyond their association with disease, IL-1 has been shown to contribute to anti-bacterial and anti-viral responses of the immune system by upregulating inflammatory signals and chemoattractants. Herpes Simplex Virus-1 (HSV-1) is a human pathogen that has developed several strategies to manipulate elements of the immune system to avoid detection by the host. One such mechanism is the prevention of activation and release of IL-1β from infected cells thereby blocking its pro-inflammatory responses. Our data show that keratinocytes infected with HSV-1 actively release IL-1α to alert danger to neighboring cells to circumvent this blockage of IL-1β signaling. This release of IL-1α initiates recruitment of leukocytes to early HSV-1 microinfection sites resulting in increased protection against disease, as evident by the increased mortality rate of mice deficient in the IL-1 receptor, IL-1R1. This study, for the first time in vivo, demonstrates the ability of IL-1α to act as an alarmin to initiate an immune response to combat infection. The role of IL-36 cytokines during viral infections has been less defined than that of IL-1. Several studies have shown the upregulation of IL-36 expression during viral infections in epithelial tissues, such as HSV-1 and Influenza, yet a direct link has not been established between these proteins and anti-viral responses. Our research presented within this thesis show that IL-36β, but not IL-36α nor IL-36γ, provides protection against the lethal outcome of cutaneous HSV-1 infection, as demonstrated by IL-36β knockout mice dying earlier and more often than wild type mice. Surprisingly, while previous reports have found IL-36 cytokines to be capable of activating the adaptive immune system, our results found no significant differences in development of HSV-1 specific antibodies or CD8+ T cell development between wild type and IL-36β knockout mice. Furthermore, we found no significant differences in viral copy numbers at infection sites between the two groups. Although our data show that IL-36β clearly plays a critical role in controlling the outcome of HSV-1 infection, further studies are necessary to define the mechanisms behind this protection. The final section of this thesis focuses on the endogenous nature of IL-36 cytokines, specifically IL-36γ, and their potential processing. IL-36 cytokines were originally believed to be synthesized as full-length fully active proteins; however, large concentrations of the recombinant proteins were required to elicit cellular responses in vitro. Since then, studies have shown that IL-36 cytokines gained up to 1000-fold increases in reactivity following processing at very specific N-terminal locations of each individual cytokine, however this processing has never been shown to occur in vivo. These studies were recently expanded when neutrophil proteases were found to be responsible for processing of these proteins in vitro. Data presented here show, for the first time, that IL-36γ may be endogenously processed by neutrophils in wounded murine skin in vivo, yet, the amino acid processing site appears to be different from that predicted. Although further studies are required to fully characterize the nature of this processing, these data provide valuable insight into the natural mechanisms involved in the potential activation of these cytokines. Taken together, the research presented within this thesis sheds light on the mechanisms whereby IL-1 and IL-36 cytokines enhance immunological defenses against potential threats, and yet, can contribute to disease if unregulated. Furthermore, these studies demonstrate the evolutionary advantage of producing multiple cytokines that appear to have redundant roles within the body, yet can provide multiple levels of protection to the host. This knowledge contributes to our overall understanding of these proteins and their contribution to immunological systems within the body. / Microbiology and Immunology

Herpes Simplex virus (HSV) virions contain the protein vhs (virion host shutoff), which is known to trigger rapid shutoff of host protein synthesis and accelerated decay of viral and cellular mRNAs. HSV-1 strains generally cause weaker shutoff than HSV-2 strains. HSV viruses lacking the VP16 viral transactivator gene show uncontrolled shutoff late in infection (Lam et al., 1996); vhs is known to bind to VP16 (Smibert et al., 1994). In vitro experiments demonstrated that HSV-1 vhs protein (vhsTl) and an intertypic HSV-2 G vhs protein (vhsT2) did not differ in ability to effect mRNA degradation in a rabbit reticulocyte lysate (RRL) assay system, suggesting that virion factors might influence vhs shutoff phenotype. To investigate the possibility that VP 16 influences early shutoff during HSV infection, a virus was constructed containing the HSV-2 strain G VP16 in place of HSV-1 VP16. This virus (8MA2R) grew in a noncomplementing cell line and was unaltered in shutoff phenotype compared to wt strain. Cotransfection assays demonstrated vhsT2 had greater shutoff ability than vhsT1, and this was confirmed in a viral system by constructing intertypic viruses containing portions of the HSV-2 G vhs ORF in a vhs-null HSV-1 tk locus. All intertypic constructs conferred increased shutoff ability relative to vhsT 1, indicating that the strong shutoff ability of HSV -2 vhs is distributed along much of the vhs ORF. Also, to confirm the observation that UL13 null viruses show a lack of shutoff which is not due to lack of vhs in the virion (Overton et al., 1994) a number of wildtype viruses and their UL13-null derivatives were tested for ability to shutoff host translation. All showed near-wt or wt levels of shutoff. / Thesis / Master of Science (MS)

O presente projeto propõe a avaliação de respostas imunológicas induzidas por uma vacina de DNA bivalente para o controle de infecções por HIV e HSV. A vacina genética codifica a proteína gD do vírus HSV-1 geneticamente fusionada à proteína Gag (p24) do HIV, rica em epítopos reconhecidos por células T CD8+. A vacina de DNA pRE4p24 codifica a proteína p24 inserida em região próxima à porção C-terminal da proteína gD-1, permitindo que a proteína recombinante seja expressa na superfície da célula transfectada. A localização da proteína recombinante foi confirmada na superfície das células HEK-293T transfectadas por ensaios de imunofluorescência. Camundongos imunizados com a vacina foram capazes de gerar respostas de anticorpos específicos após três doses administradas pelas vias intramuscular (i.m.), intradérmica com seringa (i.d.) ou intradérmico por biobalística (gg). Foram analisadas as respostas imunológicas mediadas por linfócitos T CD8+ p24-específicas e, em função dos resultados obtidos, sendo a via i.m. escolhida como a mais promissora para os ensaios subsequentes. Na tentativa de aumentar a imunogenicidade da vacina, particularmente, para respostas celulares, foi avaliado o efeito da co-administração da formulação vacinal com outros plasmídeos que expressam citocinas (pIL-2, pIL-12 ou pGM-CSF), o teste de um vetor vacinal baseado no plasmídeo multicópia pVAX (pgDp24) e o emprego de um gene sintético para promover o aumento da expressão da proteína gD em células de mamíferos (pgDhp24). Por fim, desenvolvemos um modelo murino para a avaliação funcional de respostas citotóxicas antígeno-específicas a partir de uma linhagem tumoral capaz de expressar a proteína p24 do HIV. Camundongos Balb/c imunizados com o pgDp24 apresentaram um retardo no crescimento tumoral em relação aos animais não imunizados além de proteção parcial a desafios letais com HSV-1. / The present thesis aims to evaluate the immunological responses induced by a bivalent DNA vaccine to the control HIV and HSV infectious. This genetic vaccine codes the gD protein from the HSV-1 virus envelope genetically fused with HIV Gag (p24) protein, which has various epitopes recognized by human and murine T CD8+ cells. The DNA vaccine, named pRE4p24, codes for p24 protein, inserted close to the C-terminal region of gD-1 protein, leading to the expression of the recombinant protein on the surface of the transfected cells. The location of the recombinant protein was confirmed with transfected HEK-293 cellsby immunofluorescence assays. Mice immunized with the vaccine generated antigen-specific antibody responses after three doses administered intramuscularly (i.m), intradermally with a syringe (i.d) or intradermally by gene gun (bioballistic) (gg). The immunological responses mediated by specific-T CD8+ p24 lymphocytes were evaluated and, according to the data obtained, the i.m administration was chosen for the next assays. Aiming the improvement of the vaccine immunogenicity, particularly for cellular responses, the effect of co-administration with other plasmids was assessed with: plasmids that express cytokines (pIL-2, pIL-12 or pGM-CSF); a vaccine vector based on pVAX (pgDp24); and a vector encoding a synthetic gene capable to increase the expression of gD protein in mammalian cells (pgDhp24). Finally, we developed a murine model for the functional evaluation of antigen-specific cytotoxic responses using a tumor cell line which expresses the p24 protein. Balb/c mice, immunized with pgDp24 had a reduced tumor growth when compared to non-immunized mice. In addition, vaccinated mice showed partial protection to a lethal challenge with HSV-1.

O câncer cervical é considerado um grande problema de saúde pública e um dos maiores causadores de mortes relacionadas a tumores em mulheres. O principal objetivo desta tese foi aumentar a eficácia antitumoral terapêutica da proteína gDE7 por meio da associação de adjuvantes vacinais em formulações testadas em condições experimentais com a linhagem celular tumoral TC-1. A proteína gDE7 foi produzida a partir de uma linhagem de E. coli e associada a diferentes adjuvantes. A proteína gDE7 coadministrada ao poly(I:C) conferiu proteção antitumoral completa aos camundongos previamente desafiados e induziu ativação de linfócitos T CD8+ E7-específicos polifuncionais, citotóxicos e de fenótipo de memória efetora/efetor. Foi demonstrado que a proteína gDE7 ativa de forma específica a subpopulação de células dendríticas especializada na apresentação cruzada de antígenos para linfócitos T CD8+, tanto em camundongos como em seres humanos. Esses resultados abrem perspectivas para o emprego da proteína gD como plataforma vacinal para o controle de tumores induzidos pelo HPV-16. / Cervical cancer is considered a major public health problem and one of the leading causes of cancer death in women. The main goal of this thesis was the improvement of a therapeutic antitumor vaccine based on gDE7 protein in formulations admixed with adjuvants under experimental conditions with the tumor cell line TC-1. The gDE7 protein was expressed and purified from E. coli, and then tested in combination with different vaccine adjuvants. The gDE7 protein admixed with poly(I:C) conferred complete therapeutic antitumor protection to mice previously challenged with TC-1 cells and induced polyfunctional, cytotoxic E7-specific CD8+ T cells with effector/effector memory phenotype. It was also demonstrated that the gDE7 protein activated a specialized dendritic cell subset involved in specific antigen cross-presentation to CD8+ T cells, both in mice and humans. These results open perspectives for the use of the gD protein use as a vaccine platform for the control of HPV-16-induced tumors.

Le premier objectif a été de sélectionner des promoteurs de gènes cellulaires actifs spécifiquement dans les HCC à l’aide d’une recherche bibliographique puis en utilisant la base de donnée UniGene. Leur activité a été vérifiée par RT-qPCR et CHIP dans des lignées modèles HCC et dans des hépatocytes. Ces promoteurs ont été clonés en amont de la luciférase dans la région intergénique 20 du génome HSV-1 afin d’étudier leur force d’activité, 2 types de cinétiques et leur activité différentielle en fonction du type cellulaire et dans le contexte d’une infection virale. Le deuxième objectif a été de construire des virus oncolytiques ciblés pour l’expression de la protéine Us3, une protéine virale impliquée dans le contrôle de la réponse apoptotique induite par HSV-1. L’expression de la protéine Us3 est placée sous contrôle d’un promoteur cellulaire spécifique d’HCC. L’hypothèse est qu’en l’absence d’activité du promoteur cellulaire dans les cellules non HCC, la protéine Us3 ne sera pas synthétisée et, par conséquent, l’apoptose qui ne sera pas réprimée, inhibera le cycle de réplication et par conséquent, la production virale dans les cellules saines. Dans les cellules HCC, le promoteur actif permettra la réplication virale aboutissant à la destruction de lamasse tumorale. Un virus HSV-1 Us3- a été construit en utilisant la technique de recombinaison en plasmide BAC (Bacterial artificial chromosome), puis 2 virus oncolytiques en réintroduisant le gène Us3 sous contrôle du promoteur ANGPTL3 ou du promoteur HRE (hypoxia responsive element). Leur comportement oncolytique a été étudié en réalisant des courbes de croissance sur lignées cellulaires d’HCC et cellules hepatocyte-like. / Our long-term purpose is to develop transcriptionally targeted oncolytic vectors, derived from herpes simplex virus type 1 (HSV-1), designed to eradicate hepatocellular carcinomas (HCC). We have identified several HCC-specific promoters, as well as other cancer-specific promoters, that maintain their specificity when expressed from the virus genome. More precisely, we have demonstrated that these promoters are able to drive reporter gene (luciferase) expression from the virus genome in HCC-derived cells, both in cultured cells and in nude mice, but not in fresh human hepatocytes or in the WRL38 hepatocyte-like cells. HSV-1 infection induces, but then inhibits, a cellular antiviral apoptotic response, and the early virus protein US3 is a key actor in inhibiting apoptosis. We have hypothesized that inhibition of US3 expression in hepatocytes should led to early apoptotic death of these cells, therefore precluding virus multiplication and spread. In contrast, expression of US3 in cancer cells is expected to block apoptosis, leading to the achievement of the virus life cycle, cell lysis, and virus spread within the tumours. We report in this communication the construction and properties of two different potentially oncolytic HSV-1 vectors. One of them expresses US3 protein under the control of the HCC-specific promoter ANGPTL3, while the second promoter contains 9 repeats of the hypoxia responsive elements of vascular-endothelial growth factor (VEGF) (9xHRE promoter). Growth curves of these viruses were performed on different HCC cell lines to show their oncolytic properties.

O presente projeto propõe a avaliação de respostas imunológicas induzidas por uma vacina de DNA bivalente para o controle de infecções por HIV e HSV. A vacina genética codifica a proteína gD do vírus HSV-1 geneticamente fusionada à proteína Gag (p24) do HIV, rica em epítopos reconhecidos por células T CD8+. A vacina de DNA pRE4p24 codifica a proteína p24 inserida em região próxima à porção C-terminal da proteína gD-1, permitindo que a proteína recombinante seja expressa na superfície da célula transfectada. A localização da proteína recombinante foi confirmada na superfície das células HEK-293T transfectadas por ensaios de imunofluorescência. Camundongos imunizados com a vacina foram capazes de gerar respostas de anticorpos específicos após três doses administradas pelas vias intramuscular (i.m.), intradérmica com seringa (i.d.) ou intradérmico por biobalística (gg). Foram analisadas as respostas imunológicas mediadas por linfócitos T CD8+ p24-específicas e, em função dos resultados obtidos, sendo a via i.m. escolhida como a mais promissora para os ensaios subsequentes. Na tentativa de aumentar a imunogenicidade da vacina, particularmente, para respostas celulares, foi avaliado o efeito da co-administração da formulação vacinal com outros plasmídeos que expressam citocinas (pIL-2, pIL-12 ou pGM-CSF), o teste de um vetor vacinal baseado no plasmídeo multicópia pVAX (pgDp24) e o emprego de um gene sintético para promover o aumento da expressão da proteína gD em células de mamíferos (pgDhp24). Por fim, desenvolvemos um modelo murino para a avaliação funcional de respostas citotóxicas antígeno-específicas a partir de uma linhagem tumoral capaz de expressar a proteína p24 do HIV. Camundongos Balb/c imunizados com o pgDp24 apresentaram um retardo no crescimento tumoral em relação aos animais não imunizados além de proteção parcial a desafios letais com HSV-1. / The present thesis aims to evaluate the immunological responses induced by a bivalent DNA vaccine to the control HIV and HSV infectious. This genetic vaccine codes the gD protein from the HSV-1 virus envelope genetically fused with HIV Gag (p24) protein, which has various epitopes recognized by human and murine T CD8+ cells. The DNA vaccine, named pRE4p24, codes for p24 protein, inserted close to the C-terminal region of gD-1 protein, leading to the expression of the recombinant protein on the surface of the transfected cells. The location of the recombinant protein was confirmed with transfected HEK-293 cellsby immunofluorescence assays. Mice immunized with the vaccine generated antigen-specific antibody responses after three doses administered intramuscularly (i.m), intradermally with a syringe (i.d) or intradermally by gene gun (bioballistic) (gg). The immunological responses mediated by specific-T CD8+ p24 lymphocytes were evaluated and, according to the data obtained, the i.m administration was chosen for the next assays. Aiming the improvement of the vaccine immunogenicity, particularly for cellular responses, the effect of co-administration with other plasmids was assessed with: plasmids that express cytokines (pIL-2, pIL-12 or pGM-CSF); a vaccine vector based on pVAX (pgDp24); and a vector encoding a synthetic gene capable to increase the expression of gD protein in mammalian cells (pgDhp24). Finally, we developed a murine model for the functional evaluation of antigen-specific cytotoxic responses using a tumor cell line which expresses the p24 protein. Balb/c mice, immunized with pgDp24 had a reduced tumor growth when compared to non-immunized mice. In addition, vaccinated mice showed partial protection to a lethal challenge with HSV-1.

O câncer cervical é considerado um grande problema de saúde pública e um dos maiores causadores de mortes relacionadas a tumores em mulheres. O principal objetivo desta tese foi aumentar a eficácia antitumoral terapêutica da proteína gDE7 por meio da associação de adjuvantes vacinais em formulações testadas em condições experimentais com a linhagem celular tumoral TC-1. A proteína gDE7 foi produzida a partir de uma linhagem de E. coli e associada a diferentes adjuvantes. A proteína gDE7 coadministrada ao poly(I:C) conferiu proteção antitumoral completa aos camundongos previamente desafiados e induziu ativação de linfócitos T CD8+ E7-específicos polifuncionais, citotóxicos e de fenótipo de memória efetora/efetor. Foi demonstrado que a proteína gDE7 ativa de forma específica a subpopulação de células dendríticas especializada na apresentação cruzada de antígenos para linfócitos T CD8+, tanto em camundongos como em seres humanos. Esses resultados abrem perspectivas para o emprego da proteína gD como plataforma vacinal para o controle de tumores induzidos pelo HPV-16. / Cervical cancer is considered a major public health problem and one of the leading causes of cancer death in women. The main goal of this thesis was the improvement of a therapeutic antitumor vaccine based on gDE7 protein in formulations admixed with adjuvants under experimental conditions with the tumor cell line TC-1. The gDE7 protein was expressed and purified from E. coli, and then tested in combination with different vaccine adjuvants. The gDE7 protein admixed with poly(I:C) conferred complete therapeutic antitumor protection to mice previously challenged with TC-1 cells and induced polyfunctional, cytotoxic E7-specific CD8+ T cells with effector/effector memory phenotype. It was also demonstrated that the gDE7 protein activated a specialized dendritic cell subset involved in specific antigen cross-presentation to CD8+ T cells, both in mice and humans. These results open perspectives for the use of the gD protein use as a vaccine platform for the control of HPV-16-induced tumors.

Le virus de l'herpès simplex de sérotype 1 (HSV-1) est la plus grande cause de l’aveuglement infectieuse dans les pays développés. Les infections cornéennes à HSV-1 ont plusieurs conséquences, comme la difficulté à éliminer l'infection virale et l'inflammation provoquant une plus grande opacité de la cornée. Une infection cornéenne a montré qu'il y a colocalisation des cellules souches et les cellules amplificatrices transitoires avec le virus, mais le HSV-1 était toujours abondant dans le limbe de l'œil. Pour combattre le virus, anciennes publications ont montré que LL37, une cathélicidine humaine, peut réduire la charge virale. Le GF19, un fragment de LL37 modifié pour favoriser la perméabilité, a été capable de réduire la charge virale in vitro et ex vivo, avec la possibilité d'avoir des effets thérapeutique et préventif. Pour combattre les conséquences inflammatoires, un agoniste cannabinoïde CB2r impliqué dans la modulation de la neuroinflammation, TA-A001, a été testé. Des souris ayant reçu des brûlures alcalines pour induire l’inflammation et ils ont montré de meilleurs résultats cliniques avec le TA-A001 qu'avec le véhicule du médicament seul ou un corticostéroïde, la prednisolone. En conclusion, il apparaît que l’HSV-1 infecte rapidement le limbe, que le GF19 a pu réduire la charge virale, et que le TA-A001 a pu réduire l'inflammation en ayant peu d'effets secondaires. Une combinaison des traitements antiviraux et immunosuppresseurs administrés à la cornée pourrait être examinée plus pour combattre contre les infections à HSV-1 dans les yeux. / Herpes Simplex Virus serotype 1 (HSV-1) is the most common cause of infectious blindness in developed countries. Little is known about the early events in HSV-1 ocular infections since clinical symptoms often appear a week after infection. There are two significant consequences of HSV-1 corneal infection: the viral impacts of the disease and the inflammatory impacts. In mouse corneas, after HSV-1 infection, viruses localized in the limbal area of the cornea. However, there was no/little immunohistochemical co-localization with the stem cells or transient amplifying cells. Previous work has shown that LL37, a human cathelicidin, can reduce the viral burden. GF19, a fragment of LL37 with a modification to promote permeability, reduced viral loads in vitro and in ex vivo corneas. To combat the inflammatory consequences of HSV-1 infection, a cannabinoid CB2r agonist implicated in neuroinflammation modulation, TA-A001, was tested. Mice given alkali burns to induce inflammation showed better clinical results with TA-A001 than with the drug’s vehicle alone or a corticosteroid, prednisolone. In conclusion, it appears that HSV-1 quickly infects the limbus, GF19 was able to reduce viral burden, and CB2 agonists such as TA-A001 could reduce inflammation with few side effects. A combination of the anti-viral and immunosuppressant treatments could be a potential HSV-1 treatment.