Understanding natural mechanisms of protection against HIV infection and disease progression are key priorities for informing vaccine and microbicide design. The research presented in this thesis aimed to characterize mechanisms of defence in HIV-exposed seronegative (HESN) individuals, who naturally resist infection by HIV, and HIV-controllers, who are HIV-infected, but suppress viral replication in the absence of treatment. Previous studies have linked resistance to HIV infection with low basal levels of gene transcription and reduced production of inflammatory mediators, suggesting an overall state of immune quiescence in HESN. Immune quiescence may also be protective in HIV-infected individuals, as immune activation drives disease progression. The central hypothesis of this thesis is that immune quiescence protects against HIV infection and disease progression by limiting the pool of activated target CD4+ T cells susceptible to HIV infection. This hypothesis was addressed by evaluating immune function in HESN from the Pumwani commercial sex worker cohort and HIV-controllers from the Manitoba elite controller cohort. In HESN, immune quiescence was marked by low levels of circulating activated T cells and low levels of the proinflammatory mediators IL-1α and IL-8 in the cervical mucosa. Regulatory T cells (Tregs), which suppress T cell activation, were elevated in HESN, and may represent a driver of immune quiescence. Low T cell activation and elevated Tregs were associated with reduced cellular susceptibility to infection in vitro. These data suggest that immune quiescence protects against infection by limiting the activated target CD4+ T cell pool, in support of the central hypothesis. HIV-controllers expressed low levels of the proinflammatory chemokines IP-10 and MCP-1 and low frequencies of activated T cells. These data demonstrate that immune quiescence is not only protective prior to exposure, but is also beneficial following infection. HIV-controllers also had elevated MIP-1α, reduced TGFβ and HIV-specific T cell proliferation responses, which contribute to protection by mechanisms other than immune quiescence. Taken together, these data support a role for immune quiescence in protection from HIV infection and disease progression. Mechanisms of reducing inflammation and target cell activation should be considered during future HIV vaccine and microbicide development.

Understanding natural mechanisms of protection against HIV infection and disease progression are key priorities for informing vaccine and microbicide design. The research presented in this thesis aimed to characterize mechanisms of defence in HIV-exposed seronegative (HESN) individuals, who naturally resist infection by HIV, and HIV-controllers, who are HIV-infected, but suppress viral replication in the absence of treatment. Previous studies have linked resistance to HIV infection with low basal levels of gene transcription and reduced production of inflammatory mediators, suggesting an overall state of immune quiescence in HESN. Immune quiescence may also be protective in HIV-infected individuals, as immune activation drives disease progression. The central hypothesis of this thesis is that immune quiescence protects against HIV infection and disease progression by limiting the pool of activated target CD4+ T cells susceptible to HIV infection. This hypothesis was addressed by evaluating immune function in HESN from the Pumwani commercial sex worker cohort and HIV-controllers from the Manitoba elite controller cohort. In HESN, immune quiescence was marked by low levels of circulating activated T cells and low levels of the proinflammatory mediators IL-1α and IL-8 in the cervical mucosa. Regulatory T cells (Tregs), which suppress T cell activation, were elevated in HESN, and may represent a driver of immune quiescence. Low T cell activation and elevated Tregs were associated with reduced cellular susceptibility to infection in vitro. These data suggest that immune quiescence protects against infection by limiting the activated target CD4+ T cell pool, in support of the central hypothesis. HIV-controllers expressed low levels of the proinflammatory chemokines IP-10 and MCP-1 and low frequencies of activated T cells. These data demonstrate that immune quiescence is not only protective prior to exposure, but is also beneficial following infection. HIV-controllers also had elevated MIP-1α, reduced TGFβ and HIV-specific T cell proliferation responses, which contribute to protection by mechanisms other than immune quiescence. Taken together, these data support a role for immune quiescence in protection from HIV infection and disease progression. Mechanisms of reducing inflammation and target cell activation should be considered during future HIV vaccine and microbicide development.

Mon laboratoire d’accueil avait montré que la densité de CCR5 à la surface des cellules cibles du VIH détermine très fortement leur infectabilité par les souches virales R5, et que les signaux d’activation cellulaire induits par la fixation virale et transitant par CCR5 favorisent une infection productive des cellules primaires. L’hypothèse de notre travail était que d’autres récepteurs couplés aux protéines G (RCPG) exprimés dans les lymphocytes T CD4+ CCR5+ pourraient également influer sur le cycle de réplication des virus R5. Pour tester cette hypothèse, les RCPGs co-exprimés avec CCR5 dans les cellules T CD4+ ont été identifiés, puis la capacité des plus exprimés d’entre eux à s’hétérodimériser avec CCR5 et à perturber l’infection par le VIH a été déterminée. Au cours de cette thèse, j’ai initié l’étude de deux de ces récepteurs, EBI2 et A2A, et j'ai participé à l’étude d’un troisième, S1P1.J’ai montré que la présence d’EBI2 ou d’A2A qui chacun s’hétérodimérise avec CCR5, augmente la production virale dans des cellules HOS ou MT4, comme c’est le cas pour S1P1. Étudiant les changements au niveau des étapes du cycle viral provoqués par l’expression ou la stimulation de ces RCPGS, j’ai mis en évidence que le principal effet est une augmentation de la transcription virale. De plus, nous avons montré que la signalisation via S1P1 active le facteur de transcription NFKB et par là le promoteur du VIH. De plus, la stimulation de ce récepteur dans un modèle de cellules infectées de façon latente permet la réactivation du virus. Ce résultat ouvre la possibilité de tester la capacité d’agonistes de S1P1 à éliminer les cellules servant de réservoir chez les personnes vivant avec le VIH. / It was shown in the laboratory that CCR5 density at the surface of primary CD4+ T cells strongly determines their R5 HIV-1 productive infectability. This is due to the fact that viral envelope - CCR5 interaction triggers signals that boost the viral life cycle. Our working hypothesis was that other G-protein coupled receptors (GPCR) co-expressed with CCR5 might also modulate HIV replicative cycle. To test this hypothesis, the GPCR coexpressed with CCR5 in CD4+ T cells have been identified, and the ability of the most highly expressed of them to dimerize with CCR5 and to interfere with the infection has been determined. During this thesis, I have initiated the study of two of these GPCR, EBI2 and A2A, and participated in the study of another one, S1P1.I have shown that the presence of EBI2 or A2A that each heterodimerize with CCR5, boosts the viral production in HOS and MT4 cells, similarly to S1P1. Analyzing the stages of HIV life cycle modified by the expression and triggering of these GPCR, I unveiled that the major effect was an increase in HIV genome transcription. Furthermore, we have shown that S1P1 signaling activates the transcription factor NFKB and thereby the HIV promoter. Moreover, triggering S1P1 in an in vitro model of HIV reservoir cells results in the reversion of the viral latency. These findings suggest that S1P1 agonists might be used as latency reversing agents to purge the HIV reservoir.

Peu d'études ont étudié l'impact du traitement antirétroviral sur l'activation et l'inflammation chez les patients infectés par le VIH. Ces études ont inclus des personnes ayant un contrôle virologique variable, ce qui explique leurs résultats discordants. Les objectifs de ce travail étaient d'évaluer l'évolution des marqueurs d'activation et d'inflammation chez les patients initiant un traitement antirétroviral avec un succès virologique rapide et persistante au cours de 2 ans, et d'identifier les facteurs associés à la persistance des niveaux élevés des marqueurs post-traitement. De plus, comparer l'impact des différents traitements antirétroviraux sur l'évolution des marqueurs.Mon travail a permis de montrer qu'avant l'initiation de traitement, les niveaux d'IL-6, IP-10, MIG et sCD14 étaient plus élevés chez les patients que chez les témoins VIH-séronégatifs. Après deux ans, les niveaux d'IL-6, IP-10 et MIG ont diminué significativement alors qu'aucun changement de CRP-us et de sCD14 n'a été observé. Les niveaux élevés d'IP-10 et MIG ont été associées à l'âge. La seule différence observée entre les traitements était une moindre diminution d'IP-10 et MIG avec l'ATV/r qu'avec l'EFV.Ces résultats suggèrent que le contrôle virologique rapide et persistant sous traitement antirétrovirale est nécessaire mais pas suffisant pour atténuer l'activation immunitaire et l'inflammation. L'initiation de traitement antirétroviral avec un meilleur impact sur l'activation immunitaire pourrait être considérée en particulier chez les patients âgés. La mesure des marqueurs d'activation immunitaire pourrait être un critère utile lors de l'évaluation de nouvelles molécules antirétrovirales. / The impact of antiretroviral therapy on immune activation and inflammation markers has been examined in few studies with variable results. These studies included individuals with variable levels of virologic control, explaining partially their variable results. The aims of my work were to evaluate the evolution of immune activation and inflammation markers among patients initiating first-line cART with rapid and persistent virological success through two years and to identify factors associated with elevated levels of these markers post-cART. In addition, the impact of different antiretroviral drugs on these markers was compared in a group of patients who kept their initial therapy over two years.My work showed that at cART initiation, IL-6, IP-10, MIG and sCD14 levels were higher in HIV-infected patients than in HIV-seronegative controls. After two years of effective cART, IL-6, IP-10 and MIG levels declined significantly while no change of sCD14 and hs-CRP levels was observed. Elevated levels of IP-10 and MIG persisted in some patients who tended to be older. The only observed difference between different antiretroviral drugs was a smaller decrease of IP-10 and MIG in patients receiving ATV/r compared to patients receiving EFV.These findings suggest that early and persistent virological control under cART is useful but might not be sufficient to attenuate immune activation and inflammation. Earlier initiation of cART regimens with more potent effect on immune activation and inflammation merits evaluation, in particular, in old patients. The measurement of immune activation markers could be useful criteria when evaluating antiretroviral drugs.

GB virus C (GBV-C) is a lymphotropic human virus which was recently assigned to a new genus Pegivirus within the Flaviviridae family. GBV-C infection is found worldwide, and viremia prevalence is about 1% to 4% in healthy blood donors and up to 42% in HIV-infected individuals. In clinical studies, GBV-C coinfection is associated with prolonged survival of HIV-infected individuals. GBV-C infection modestly alters T cell homeostasis in vivo through various mechanisms, including modulation of chemokine and cytokine release and receptor expression, and by diminution of T cell activation, proliferation and apoptosis, all of which may contribute to improved HIV clinical outcomes. This thesis explores the interrelationship between GBV-C infection and immunoactivation and identifies potential mechanisms by which GBV-C reduces immunoactivation. Chronic HIV infection is associated with persistent immunoactivation which contributes to the immune dysfunction. In particular, T cell activation supports HIV replication and correlates with HIV viral load (VL). Persistent immunoactivation also contributes to the depletion of uninfected bystander cells by mechanisms of activation induced cell death (AICD). Although treatment with combination antiretroviral therapy (cART) reduces HIV VL, T cell activation does not return to levels found in HIVuninfected individuals. Sustained immunoactivation is also associated with lower virological response to cART suggesting therapies to reduce immunoactivation in combination with cART may benefit HIV-infected individuals. Since GBV-C infection is associated with reduced immunoactivation, understanding mechanisms by which GBV-C modulates these signaling pathways may provide insights into novel approaches to treat HIV infection and chronic immunoactivation. The effect of GBV-C infection on T cell activation and IL-2 signaling pathways were studied in a cohort of HIV-positive individuals. GBV-C viremic HIV positive individuals on cART have reduced T cell activation which was significantly associated with higher percentage of immunomodulatory CD3 +CD4-CD8-T cells. Ex vivo GBV-C infection was associated with reduced lymphocyte proliferation in response to IL-2, lower frequency of reactivation of latent HIV and protection against AICD. In vitro expression of GBV-C envelope glycoprotein E2 in CD4+ T cell lines inhibited T cell receptor (TCR) induced IL-2 secretion and inhibited IL-2 signaling pathways. This effect was mediated at least in part by reducing activation of lymphocyte specific tyrosine kinase (Lck). Through deletion mutagenesis, the inhibitory motif within the viral protein was mapped to a region that contains a predicted Lck substrate, a highly conserved tyrosine at position 87 (Y87). Lck phosphorylated GBV-C E2 protein in vitro and mutation of Y87 residue abolished the inhibitory effects of E2 protein. Synthetic peptides containing this inhibitory motif competed for Lck phosphorylation and inhibited TCR signaling in primary human T cells. The number of GBV-C infected T cells was found to be low in vivo, yet GBV-C infection reduced global TCR signaling. GBV-C RNA and E2 protein were detected in extracellular microvesicles purified from GBV-C infected human serum or the culture supernatant of E2 expressing cells, and these microvesicles inhibited TCR signaling in uninfected bystander T cells. Together, these data identify a novel mechanism by which GBV-C infection leads to global reduction in T cell activation and IL-2 signaling in the infected host, and provide a working model in which the viral envelope glycoprotein serves as a substrate for Lck and competes for Lck phosphorylation in the infected T cells and in uninfected bystander T cells.

Indiana University-Purdue University Indianapolis (IUPUI) / Study 1: Background: Insomnia may be a risk factor for cardiovascular disease in HIV (HIV-CVD); however, mechanisms have yet to be elucidated. Methods: We examined cross-sectional associations of insomnia symptoms with biological mechanisms of HIV-CVD (immune activation, systemic inflammation, and coagulation) among 1,542 people living with HIV from the Veterans Aging Cohort Study (VACS) Biomarker Cohort. Past-month insomnia symptoms were assessed by the item, “Difficulty falling or staying asleep?,” with the following response options: “I do not have this symptom” or “I have this symptom and…” “it doesn’t bother me,” “it bothers me a little,” “it bothers me,” “it bothers me a lot.” Circulating levels of the monocyte activation marker soluble CD14 (sCD14), inflammatory marker interleukin-6 (IL-6), and coagulation marker D-dimer were determined from blood specimens. Demographic- and fully-adjusted (CVD risk factors, potential confounders, HIV-related factors) regression models were constructed, with log-transformed biomarker variables as the outcomes. We present the exponentiated regression coefficient (exp[b]) and its 95% confidence interval (CI). Results: For sCD14 and D-dimer, we observed no significant associations. For IL-6, veterans in the “bothers a lot” group had 15% higher IL-6 than veterans in the “I do not have this symptom” group in the demographic-adjusted model (exp[b]=1.15, 95%CI=1.02-1.29, p=.03). This association was nonsignificant in the fully-adjusted model (exp[b]=1.07, 95%CI=0.95-1.19, p=.25). Conclusion: We observed little evidence of relationships between insomnia symptoms and markers of biological mechanisms of HIV-CVD. Other mechanisms may be responsible for the insomnia-CVD relationship in HIV; however, future studies with comprehensive assessments of insomnia symptoms are warranted. Study 2: Background: While insomnia has been identified as a potential risk factor for cardiovascular disease in HIV (HIV-CVD), research on the underlying pathophysiological mechanisms is scarce. Methods: We examined associations between 0-to-12-week changes in sleep disturbance and the concurrent 0-to-12-week changes and the subsequent 12-to-24-week changes in markers of systemic inflammation, coagulation, and endothelial dysfunction among people living with HIV (n = 33-38) enrolled in a depression clinical trial. Sleep disturbance was measured using the Pittsburgh Sleep Quality Index. Inflammatory markers interleukin-6 (IL-6) and C-reactive protein (CRP) and coagulation marker D-dimer were determined from blood specimens; endothelial dysfunction marker brachial flow-mediated dilation (FMD) was determined by ultrasound. 0-to-12-week variables were calculated as 12-week visit minus baseline, and 12-to-24-week variables were calculated as 24-week minus 12-week. We constructed multivariate linear regression models for each outcome adjusting for age, sex, race/ethnicity, Framingham risk score, and baseline depressive symptoms. Results: We did not observe statistically significant associations between 0-to-12-week changes in sleep disturbance and 0-to-12-week or 12-to-24-week changes in IL-6, CRP, D-dimer, or FMD. However, we did observe potentially meaningful associations, likely undetected due to low power. For 0-to-12-weeks, every 1-standard deviation (SD) increase, or worsening, in the sleep disturbance change score was associated with a 0.41 pg/mL and 80 ng/mL decease in IL-6 and D-dimer, respectively. For 12-to-24-weeks, every 1-SD increase in sleep disturbance change score was associated with a 0.63 mg/L, 111 ng/mL, and 0.82% increase in CRP, D-dimer, and FMD, respectively. Conclusion: We observed potentially meaningful, though not statistically significant, associations between changes in sleep disturbance and changes in biological mechanisms underlying HIV-CVD over time. Some associations were in the expected direction, but others were not. Additional studies are needed that utilize larger samples and validated, comprehensive assessments of insomnia.

The maternal brain experiences a high level of plasticity during pregnancy which is understood to be largely mediated by changes to the maternal immune, endocrine and nervous systems to prepare for offspring care. Given the prevalence of infections during pregnancy, the effect of gestational immune activation (GIA) on these systems and the maternal brain have not been fully characterized. In this work, GIA is used with respect to immune activation in the dams, instead of the maternal immune activation (MIA) terminology that is typically used for offspring exposure. In addition to this, the protective effects of environmental enrichment against biological stressors such as an infection have only been investigated within the context of MIA offspring. This study examines the effects of lipopolysaccharide (LPS) induced GIA and housing conditions on the postpartum maternal hippocampus, a region of the brain that plays an in-direct role in supporting maternal behaviours. This is achieved through examining the number of ER-α positive cells and active microglia (Iba-1 positive cells) in the different layers of the postpartum maternal rat hippocampus. Nulliparous female Sprague-Dawley rats, housed in either animal care control (ACC) or environmental enrichment (EE) conditions, were treated with intraperitoneal injections of 100μg/kg lipopolysaccharides (LPS) or a pyrogen-free saline solution (VEH). Mothers were sacrificed on postpartum day 22 and brains were collected. Brains were preserved and later prepared for immunohistochemical labelling of ER-α and Iba-1 positive cells in the layers of the hippocampus (CA1, CA3 and Dentate Gyrus (DG): GrDG, MoDG, and PoDG). This project found a statistically significant effect of LPS treatment on the number of ER-α positive cells in the CA3 and in the PoDG layers of the hippocampus; LPS served to reduce the overall number of estrogen receptors in these areas. These findings support the potential for immune challenges to disrupt the adaptations made to maternal neuroendocrine pathways that prepare for post-pregnancy offspring care.

Current sequencing technologies allows researchers unprecedented insight into our biology, and how these biological mechanisms can become distorted and lead to disease. These aberrant mechanisms can be brought about by many causes, but some occur as a result of genetic mutations or external factors through the epigenome. Here, we used our microfluidic technology to profile the epigenome and transcriptome to study such aberrant mechanisms in three different diseases and illnesses: breast cancer, chronic inflammation, and mental illness. We profiled the epigenome of breast tissue from healthy women with the BRCA1 mutation to understand how the mutation may facilitate eventual breast cancer. Epigenomic changes in breast cells suggest that cells in the basal compartment may differentiate into a different cell type, and perhaps become the source of breast cancer. Next, we compared the epigenome and genome of murine immune cells under low-grade inflammation and acute inflammation conditions. We found that low-grade inflammation preferentially utilizes different signaling pathways than in acute inflammation, and this may lead to a non-resolving state. Finally, we analyzed the effect of the maternal immune activation on unborn offspring, and how these changes could cause later mental illness. The insights we made into these diseases may lead to future therapies. / Doctor of Philosophy / Despite advances in medical and scientific research, there is still a dearth of information on how diseases affect the expression of our genes, such as breast cancer, chronic inflammation, and influenza. Mutation in the BRCA1 gene is probably the most well-known mutation that can lead to breast cancer. We know the overarching reason that mutation in BRCA1 can lead to cancer, as BRCA1 is responsible for repairing damage in the DNA, so mutations can compound and create cancerous cells. However, we do not know the exact mechanisms by which this actually happens. Another widespread problem is chronic inflammation, which can promote or lead to diseases such as diabetes, cancer, Alzheimer's, Rheumatoid arthritis, and heart disease. In addition, there are many causes of chronic inflammation that many people have experienced at some point in time, including stress, insomnia, being sedentary, poor eating habits, and obesity. Despite this, we still do not fully understand why chronic inflammation differs from normal inflammation, which is a healthy process, or why it does not resolve. There are also other connections that are surprising, and many are not aware of. If a pregnant woman gets the flu during her second trimester, her baby has much higher odds of developing schizophrenia later in its lifetime. Given the prevalence of the flu, there is a very real chance that an expecting mother will be infected during her pregnancy.

Giardia lamblia is a eukaryotic parasite of the upper small intestine of humans and animals. The infecting trophozoite cells do not invade the epithelium lining of the intestine, but attach to the brush border surface in the intestinal lumen. The giardiasis disease in humans is highly variable. Prior to this study, the molecular mechanisms involved in establishment of infection or cause of disease were largely uncharacterized. In this thesis, the molecular relationship between Giardia and the human host is described. The interaction of the parasite with human epithelial cells was investigated in vitro. Changes in the transcriptome and proteome of the parasite and the host cells, and changes in the micro-environment of the infection have been identified using microarray technology, and 1- and 2-Dimensional SDS-PAGE protein mapping together with mass spectrometry identification. The first large-scale description of cellular activities within host epithelial cells during Giardia infection is included in this thesis (Paper I). We identified a unique activation of the host immune response and induction of apoptosis upon infection by Giardia. Four important virulence factors of the parasite, directly linked to the success of Giardia infection, were characterized and are presented in Papers II and III. The parasite was shown to have immune-modulating capacities, and to release proteins during host-interaction that facilitate the establishment of infection. Additional putative virulence factors were found among Giardia genes transcriptionally up-regulated during early infection (Paper IV). In summary, this thesis provides important insights into the molecular mechanisms of the host-parasite interaction.

A formulação de vacinas efetivas frequentemente requer a adição de adjuvantes capazes de otimizar as respostas imunes humoral e celular. Com o objetivo principal de contribuir para o desenvolvimento de novos adjuvantes, este trabalho foi desenvolvido buscando aprofundar o conhecimento do mecanismo de ação imunoadjuvante de preparações de saponinas de Quillaja brasiliensis e suas formulações em complexos imunoestimulantes do tipo ISCOM. Como a toxicidade das saponinas é um fator crítico para seu uso em preparações vacinais, inicialmente foram realizados ensaios visando comparar a toxicidade in vitro e in vivo de saponinas extraídas de Quillaja brasiliensis com saponinas de ação imunoestimulante reconhecidas, extraídas de Quillaja saponaria (Quil A). O potencial imunoadjuvante das saponinas solúveis de Q. brasiliensis foi avaliado utilizando preparações com dois antígenos: ovalbumina (OVA) e vírus da diarreia viral bovina (BVDV). Numa etapa seguinte, a atividade imunoadjuvante de ISCOMs preparados com saponinas de Q. brasiliensis foram avaliadas em duas vias de administração. O potencial imunomodulador dessas saponinas foi verificado em experimentos de recrutamento celular in vivo e expressão de genes relacionados ao sistema imune. Os resultados mostraram que saponinas de Q. brasiliensis são menos tóxicas que as de Quil A e apresentam atividade adjuvante similar, caracterizada por um perfil Th1/Th2 balanceado. Q. brasiliensis promoveu uma forte resposta imune celular do tipo Th1 caracterizada por uma robusta reação de hipersensibilidade celular tardia (DTH) e pela produção de IFN- e IL-2. A resposta imune induzida pelos ISCOMs produzidos a partir de saponinas de Q. brasiliensis foram superiores às respostas induzidas pelas saponinas solúveis. Os testes in vivo mostraram que as saponinas de Q. brasiliensis promovem um ambiente imunocompetente no local da inoculação e nos linfonodos drenantes. Esse ambiente foi caracterizado pelo intenso influxo celular (neutrófilos, células NK, células dendríticas, linfócitos T e B), além da expressão diferencial de genes relacionados à ativação do sistema imune. Em suma, os resultados mostraram que saponinas de Q. brasiliensis são seguras e seus potencial adjuvante foi equivalente a saponinas com ação imunoadjuvante conhecida de Q. saponaria. / Effective vaccine formulations frequently require addition of adjuvants able to optimize the cellular and humoral immune responses. With the goal to contribute to the development of new classes of adjuvants, this work was developed in order to achieve deep knowledge on the imunoadjuvant mode of action for Quillaja brasiliensis saponins incorporated into immunostimulant complex (ISCOM). The toxicity of saponins is a critical factor for its usage as vaccine preparations. At first, in vivo and in vivo citoxicity assays were carried out to compare to the effects between saponins extracted from Quillaja brasiliensis and the immunostimulant saponins already known from Quillaja saponaria (Quil A). Imunoadjuvant potential of soluble saponins from Q. brasilienis was evaluated using preparations of two antigens: ovoalbumin (OVA) and bovine viral diarrhea (BVD). As a next step, imunoadjuvant activity of ISCOMS prepared with Q. brasiliensis saponins was evaluated using two routes of administration. The immunomodulatory potential of these saponins was tested during in vivo cell recruitment assays and gene expression related to immune system. Our results demonstrated that Q. brasilienis saponins are less toxic than those from Quil A and presenting similar adjuvant activity, characterized by a Th1/Th2 balance profile. Q.brasiliensis induced a strong Th1 cell-mediated immune responses indicated by a robust delayed type hypersensitivity (DTH) as well as IFN-у and IL-2 production. The immune response induced by ISCOMs from Q. brasiliensis saponins was higher than the one induced by soluble saponins. In vivo experiments indicated that saponins from Q. brasiliensis generate an immunocompetent environment at the injection site and draining lymph nodes. This environment was characterized by an intense cell influx (neutrophils, NK cells, dendritic cells, B and T cells) as well as differential gene expression related to immune system activation. In essence, the results showed that saponins from are safe and their adjuvant potential was equivalent to saponins with imunoadjuvant activity of Q. saponaria.