The subunits of IL-12, originating from two distinct cells, can functionally synergize to protect against pathogen dissemination in vivo.

Cytokines are typically single gene products, except for the heterodimeric interleukin (IL)-12 family. The two subunits (IL-12p40 and IL-12p35) of the prototype IL-12 are known to be simultaneously co-expressed in activated myeloid cells, which secrete the fully active heterodimer to promote interferon (IFN)γ production in innate and adaptive cells. We find that chimeric mice containing mixtures of cells that can only express either IL-12p40 or IL-12p35, but not both together, generate functional IL-12. This alternate two-cell pathway requires IL-12p40 from hematopoietic cells to extracellularly associate with IL-12p35 from radiation-resistant cells. The two-cell mechanism is sufficient to propel local T cell differentiation in sites distal to the initial infection and helps control systemic dissemination of a pathogen, although not parasite burden, at the site of infection. Broadly, this suggests that early secretion of IL-12p40 monomers by sentinel cells at the infection site may help prepare distal host tissues for potential pathogen arrival.

To React or Not to React: The Dilemma of Fish Immune Systems Facing Myxozoan Infections.

Myxozoans are microscopic, metazoan, obligate parasites, belonging to the phylum Cnidaria. In contrast to the free-living lifestyle of most members of this taxon, myxozoans have complex life cycles alternating between vertebrate and invertebrate hosts. Vertebrate hosts are primarily fish, although they are also reported from amphibians, reptiles, trematodes, mollusks, birds and mammals. Invertebrate hosts include annelids and bryozoans. Most myxozoans are not overtly pathogenic to fish hosts, but some are responsible for severe economic losses in fisheries and aquaculture. In both scenarios, the interaction between the parasite and the host immune system is key to explain such different outcomes of this relationship. Innate immune responses contribute to the resistance of certain fish strains and species, and the absence or low levels of some innate and regulatory factors explain the high pathogenicity of some infections. In many cases, immune evasion explains the absence of a host response and allows the parasite to proliferate covertly during the first stages of the infection. In some infections, the lack of an appropriate regulatory response results in an excessive inflammatory response, causing immunopathological consequences that are worse than inflicted by the parasite itself. This review will update the available information about the immune responses against Myxozoa, with special focus on T and B lymphocyte and immunoglobulin responses, how these immune effectors are modulated by different biotic and abiotic factors, and on the mechanisms of immune evasion targeting specific immune effectors. The current and future design of control strategies for myxozoan diseases is based on understanding this myxozoan-fish interaction, and immune-based strategies such as improvement of innate and specific factors through diets and additives, host genetic selection, passive immunization and vaccination, are starting to be considered.

Safety and Immunogenicity of ChAd63/MVA Pfs25-IMX313 in a Phase I First-in-Human Trial.

Background: Transmission blocking vaccines targeting the sexual-stages of the malaria parasite could play a major role to achieve elimination and eradication of malaria. The Plasmodium falciparum Pfs25 protein (Pfs25) is the most clinically advanced candidate sexual-stage antigen. IMX313, a complement inhibitor C4b-binding protein that forms heptamers with the antigen fused to it, improve antibody responses. This is the first time that viral vectors have been used to induce antibodies in humans against an antigen that is expressed only in the mosquito vector. Methods: Clinical trial looking at safety and immunogenicity of two recombinant viral vectored vaccines encoding Pfs25-IMX313 in healthy malaria-naive adults. Replication-deficient chimpanzee adenovirus serotype 63 (ChAd63) and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA), encoding Pfs25-IMX313, were delivered by the intramuscular route in a heterologous prime-boost regimen using an 8-week interval. Safety data and samples for immunogenicity assays were taken at various time-points. Results: The reactogenicity of the vaccines was similar to that seen in previous trials using the same viral vectors encoding other antigens. The vaccines were immunogenic and induced both antibody and T cell responses against Pfs25, but significant transmission reducing activity (TRA) was not observed in most volunteers by standard membrane feeding assay. Conclusion: Both vaccines were well tolerated and demonstrated a favorable safety profile in malaria-naive adults. However, the transmission reducing activity of the antibodies generated were weak, suggesting the need for an alternative vaccine formulation. Trial Registration: Clinicaltrials.gov NCT02532049.

Design of a Novel Multi-Epitope Vaccine Against Echinococcus granulosus in Immunoinformatics.

All the time, echinococcosis is a global zoonotic disease which seriously endangers public health all over the world. In order to speed up the development process of anti-Echinococcus granulosus vaccine, at the same time, it can also save economic cost. In this study, immunoinformatics tools and molecular docking methods were used to predict and screen the antigen epitopes of Echinococcus granulosus, to design a multi-epitope vaccine containing B- and T-cell epitopes. The multi-epitope vaccine could activate B lymphocytes to produce specific antibodies theoretically, which could protect the human body against Echinococcus granulosus infection. It also could activate T lymphocytes and clear the infected parasites in the body. In this study, four CD8+ T-cell epitopes, three CD4+ T-cell epitopes and four B-cell epitopes of Protein EgTeg were identified by immunoinformatics methods. Meanwhile, three CD8+ T-cell epitopes, two CD4+ T-cell epitopes and four B-cell epitopes of Protein EgFABP1 were identified. We constructed the multi-epitope vaccine using linker proteins. The study based on the traditional methods of antigen epitope prediction, further optimized the prediction results combined with molecular docking technology and improved the precision and accuracy of the results. Finally, in vivo and in vitro experiments had verified that the vaccine designed in this study had good antigenicity and immunogenicity.

Leishmanial CpG DNA nanovesicles: A propitious prophylactic approach against visceral leishmaniasis.

Unmethylated CpG motifs with phosphothioate backbone trigger TLR9 to elicit innate immune response characterized by the production of Th1 cytokines. The use of CpG DNA as an adjuvant has established its role in potentiating the humoral and cell mediated vaccine specific immune response. However, none of the synthetic oligodeoxynucleotides (ODNs) know and used till date are associated with the parasite itself. Our group identified a novel CG rich sequence of 14 base pairs from Leishmania donovani genome (Ld CpG ODN) and established it as a TLR9 agonist. The present study was designed to ascertain the adjuvanticity of Ld CpG ODN with soluble leishmanial antigen in experimental model of L. donovani. During the study Schizophyllan (SPG), a fungal polymer was used for encapsulating Ld CpG ODN for efficient endosomal delivery. The synthesized nanovehicles were of nearly 100 nm and localized within endosomes as confirmed by confocal microscopy. Immunization studies displayed the superior ability of synthesized nanovehicles co-administered with parasite antigen in augmenting innate immune response in comparison to ODN, nanoparticles or soluble antigen alone. The response included generation of ROS, NO and iNOS expression followed by proinflammatory cytokine milieu with reduced parasitic load within liver, spleen and bone marrow. These immune-tailored particles in combination with parasitic antigens elicited significant generation of cell mediated response owing to the presence of high levels of CD8+ T-cells and lymphocyte proliferation. Moreover, vaccination regime with synthesized adjuvant also activated humoral immunity by escalating the levels of IgG2 followed by reduced levels of anti-leishmanial IgG and IgG1 antibodies. The findings support the efficacy of Ld CpG ODN as a potential adjuvant against visceral leishmaniasis.

Dendritic cell engineered cTXN as new vaccine prospect against L. donovani.

Dendritic cells (DCs), as antigen-presenting cells, can reportedly be infected withLeishmaniaparasites and hence provide a better option to trigger T-cell primary immune responses and immunological memory. We consistently primed DCs during culture with purified recombinant cytosolic tryparedoxin (rcTXN) and then evaluated the vaccine prospect of presentation of rcTXN against VL in BALB/c mice. We reported earlier the immunogenic properties of cTXN antigen derived fromL. donovani when anti-cTXN antibody was detected in the sera of kala-azar patients. It was observed that cTXN antigen, when used as an immunogen with murine DCs acting as a vehicle, was able to induce complete protection against VL in an infected group of immunized mice. This vaccination triggered splenic macrophages to produce more IL-12 and GM-CSF, and restricted IL-10 release to a minimum in an immunized group of infected animals. Concomitant changes in T-cell responses against cTXN antigen were also noticed, which increased the release of protective cytokine-like IFN-γ under the influence of NF-κß in the indicated vaccinated group of animals. All cTXN-DCs-vaccinated BALB/c mice survived during the experimental period of 120 days. The results obtained in our study suggest that DCs primed with cTXN can be used as a vaccine prospect for the control of visceral leishmaniasis.

Designing peptide-based vaccine candidates for Plasmodium falciparum erythrocyte binding antigen 175.

Plasmodium falciparum leads to a virulent form of malaria. Progress has been achieved in understanding the mechanisms involved in the malarial infection, still there is no effective vaccine to prevent severe infection. An effective vaccine against malaria should be one which can induce immune responses against multiple epitopes in the context of predominantly occurring HLA alleles. In this study, an integrated approach was employed to identify promiscuous peptides of a well-defined sequence of erythrocyte binding antigen-175 and promiscuous peptides for HLA alleles were designed using bioinformatics tools. A peptide with 15 amino acids (ILAIAIYESRILKRK) was selected based on its high binding affinity score and synthesized. This promiscuous peptide was used as stimulating antigen in lymphoproliferative responses to evaluate the cellular immune response. It was observed this peptide evokes lymphoproliferative and cytokine responses in individuals naturally exposed to the malaria parasite. The intensity of PBMCs proliferation was observed to be higher in sera obtained from P. falciparum exposed as compared to unexposed healthy individuals, suggesting earlier recognition of peptide of this region by T cells. Furthermore, the binding mode of HLA-peptide complex and their interaction may lead to a rational and selective peptide-based vaccine candidate design approach which can be used as a malaria prophylaxis.

Innate immune responses to malaria-infected erythrocytes in pregnant women: Effects of gravidity, malaria infection, and geographic location.

BACKGROUND: Malaria in pregnancy causes maternal, fetal and neonatal morbidity and mortality, and maternal innate immune responses are implicated in pathogenesis of these complications. The effects of malaria exposure and obstetric and demographic factors on the early maternal immune response are poorly understood. METHODS: Peripheral blood mononuclear cell responses to Plasmodium falciparum-infected erythrocytes and phytohemagglutinin were compared between pregnant women from Papua New Guinea (malaria-exposed) with and without current malaria infection and from Australia (unexposed). Elicited levels of inflammatory cytokines at 48 h and 24 h (interferon γ, IFN-γ only) and the cellular sources of IFN-γ were analysed. RESULTS: Among Papua New Guinean women, microscopic malaria at enrolment did not alter peripheral blood mononuclear cell responses. Compared to samples from Australia, cells from Papua New Guinean women secreted more inflammatory cytokines tumor necrosis factor-α, interleukin 1ß, interleukin 6 and IFN-γ; p<0.001 for all assays, and more natural killer cells produced IFN-γ in response to infected erythrocytes and phytohemagglutinin. In both populations, cytokine responses were not affected by gravidity, except that in the Papua New Guinean cohort multigravid women had higher IFN-γ secretion at 24 h (p = 0.029) and an increased proportion of IFN-γ+ Vδ2 γδ T cells (p = 0.003). Cytokine levels elicited by a pregnancy malaria-specific CSA binding parasite line, CS2, were broadly similar to those elicited by CD36-binding line P6A1. CONCLUSIONS: Geographic location and, to some extent, gravidity influence maternal innate immunity to malaria.

Evaluation of the Immune Regulatory Properties of Dendritic Cells During Fasciola hepatica Infection.

Dendritic cells (DCs) are potent antigen-presenting cells that possess the ability to stimulate naïve T cells, initiating the adaptive immune response. Ex vivo DC cultures are useful to evaluate how helminths regulate DC maturation and stimulatory activity. Here, we describe how to isolate CD11c+ from F. hepatica-infected mice to evaluate their activation state, cytokine production and regulatory function in an allogeneic T cell assay.

In the presence of Trypanosoma cruzi antigens, activated peripheral T lymphocytes retained in the liver induce a proinflammatory phenotypic and functional shift in intrahepatic T lymphocyte.

In secondary lymphoid organs, pathogen-derived and endogenous danger molecules are recognized by pattern recognition receptors, leading to adaptive proinflammatory immune responses. This conceptual rule does not apply directly to the liver, as hepatic immune cells tolerate gut-derived bacterial molecules from the flora. Therefore, the recognition of danger and proinflammatory stimuli differs between the periphery and the liver. However, the tolerant nature of the liver must be overcome in the case of infections or cancer, for example. The central paradigm is the basis for danger recognition and the balance between inflammation and tolerance in the liver. Here, we observed functional integration, with activated peripheral T lymphocytes playing a role in the induction of a proinflammatory environment in the liver in the presence of Trypanosoma cruzi antigens. When only parasite extract was orally administered, it led to the up-regulation of hepatic tolerance markers, but oral treatment plus adoptively transferred activated splenic T lymphocytes led to a proinflammatory response. Moreover, treated/recipient mice showed increased levels of TNF, IFN-γ, IL-6, and CCL2 in the liver and increased numbers of effector and/or effector memory T lymphocytes and F4/80+ cells. There was a reduction in FoxP3+ Treg cells, NKT cells, and γδ T lymphocytes with increased liver damage in the presence of activated peripheral T cells. Our results show that the induction of a proinflammatory liver response against T. cruzi danger molecules is at least partially dependent on cooperation with activated peripheral T cells.

Muscular Sarcocystosis in Sheep Associated With Lymphoplasmacytic Myositis and Expression of Major Histocompatibility Complex Class I and II.

Sarcocystosis is a protozoal disease affecting a wide range of animals. The aims of this study were to characterize the following in sheep: (1) the muscle pathology in Sarcocystis infection, (2) the inflammatory infiltrate and its relationship to severity of infection, and (3) immune markers expressed by parasitized muscle fibers and parasitic cysts. Skeletal muscle samples from 78 sheep slaughtered in southern Italy were snap frozen and analyzed by histopathology, immunohistochemistry, and immunofluorescence. Polymerase chain reaction (PCR) and sequencing were used for Sarcocystis species identification. All 40 muscle samples tested were PCR-positive for Sarcocystis tenella. Histologically, cysts were identified in 76/78 cases (97%), associated with an endomysial infiltrate of lymphocytes and plasma cells. The T cells were predominantly CD8+, with fewer CD4+ or CD79α+ cells. Eosinophils were absent. Notably, sarcolemmal immunopositivity for major histocompatibility complex (MHC) I and II was found in 76/78 cases (97%) and 75/78 cases (96%), respectively, both in samples with and in those without evident inflammatory infiltrate. The number of cysts was positively correlated with inflammation. In addition, MHC I was detected in 55/78 cyst walls (72%), and occasionally co-localized with the membrane-associated protein dystrophin. The findings suggest that muscle fibers respond to the presence of cysts by expression of MHC I and II. The possible role of MHC I and II in the inflammatory response and on the cyst wall is also discussed.

Exhaustion may not be in the human B cell vocabulary, at least not in malaria.

T cells exposed to persistent antigen in the inflammatory environment of chronic infections often show progressive loss of effector functions, high expression of inhibitory receptors and distinct transcriptional programs. T cells in this functional state are termed "exhausted" and T cell exhaustion is associated with inefficient control of infections. A remarkably similar scenario has been described for B cells during chronic infections in humans, including malaria, in which case a subpopulation of atypical memory B cells (MBCs) greatly expands and these MBCs show attenuation of B cell receptor signaling, loss of the B cell effector functions of antibody and cytokine production, high expression of inhibitory receptors and distinct transcriptional profiles. The expansion of these MBCs is also associated with inefficient control of infections. Despite the similarities with exhausted T cells we speculate that at least in malaria, atypical MBCs may not be exhausted but rather may be functional, possibly even beneficial. Our recent results suggest that we simply may not have known how to ask an atypical MBC to function. Thus, exhaustion may not be in the human B cell's vocabulary, at least not in malaria.

Shared Transcriptional Control of Innate Lymphoid Cell and Dendritic Cell Development.

Innate immunity and adaptive immunity consist of highly specialized immune lineages that depend on transcription factors for both function and development. In this review, we dissect the similarities between two innate lineages, innate lymphoid cells (ILCs) and dendritic cells (DCs), and an adaptive immune lineage, T cells. ILCs, DCs, and T cells make up four functional immune modules and interact in concert to produce a specified immune response. These three immune lineages also share transcriptional networks governing the development of each lineage, and we discuss the similarities between ILCs and DCs in this review.

Thymic Microenvironment Is Modified by Malnutrition and Leishmania infantum Infection.

Detrimental effects of malnutrition on immune responses to pathogens have long been recognized and it is considered a main risk factor for various infectious diseases, including visceral leishmaniasis (VL). Thymus is a target of both malnutrition and infection, but its role in the immune response to Leishmania infantum in malnourished individuals is barely studied. Because we previously observed thymic atrophy and significant reduction in cellularity and chemokine levels in malnourished mice infected with L. infantum, we postulated that the thymic microenvironment is severely compromised in those animals. To test this, we analyzed the microarchitecture of the organ and measured the protein abundance in its interstitial space in malnourished BALB/c mice infected or not with L. infantum. Malnourished-infected animals exhibited a significant reduction of the thymic cortex:medulla ratio and altered abundance of proteins secreted in the thymic interstitial fluid. Eighty-one percent of identified proteins are secreted by exosomes and malnourished-infected mice showed significant decrease in exosomal proteins, suggesting that exosomal carrier system, and therefore intrathymic communication, is dysregulated in those animals. Malnourished-infected mice also exhibited a significant increase in the abundance of proteins involved in lipid metabolism and tricarboxylic acid cycle, suggestive of a non-proliferative microenvironment. Accordingly, flow cytometry analysis revealed decreased proliferation of single positive and double positive T cells in those animals. Together, the reduced cortical area, decreased proliferation, and altered protein abundance suggest a dysfunctional thymic microenvironment where T cell migration, proliferation, and maturation are compromised, contributing for the thymic atrophy observed in malnourished animals. All these alterations could affect the control of the local and systemic infection, resulting in an impaired response to L. infantum infection.

T-cell exhaustion process during chronic infection caused by intracellular trypanosomatids / Proceso de agotamiento de las células T durante la infección crónica causada por tripanosomátidos intracelulares

Two of the most important neglected tropical diseases, Chagas disease and leishmaniasis, are caused by protozoan intracellular parasites of the Trypanosomatida order. These infections provoke a high social burden and lead to the death of a large number of patients. The host triggers several immune mechanisms, but in the absence of adequate treatment, the infection becomes chronic and in many cases causes the appearance of serious alterations. T lymphocytes are fundamental cells of the adaptive system and are the main immune elements that orchestrate the cell-to-cell response in the context of intracellular infections. Furthermore, it has been described that continuous and persistent stimulation in response to pathogenic antigens causes loss of antigen-specific functional capacities in the T cell subsets. This process is known as exhaustion. This review explores the results to date of the exhaustion process during chronic infections caused by the trypanosomatid parasites Leishmania spp. and Trypanosoma cruzi. A large amount of evidence shows upregulation of the markers of the exhaustion process, namely, the inhibitory receptors, during these chronic infections. This increased expression is observed in both the CD4+ and CD8+ T cell populations. In parallel, with this increased expression of inhibitory receptors, the loss of antigen-specific functional capacity of these T cells is detected, reducing the lymphoproliferative potential and the ability to produce protective molecules against these parasitic infections, such as Th1-like cytokines, among others. Additionally, a positive correlation between the high coexpression of these inhibitory molecules and the severity of the pathology is demonstrated. Furthermore, T cell populations experience a phenotypic fluctuation in the course of these infections toward the predominance of effector memory subsets with a late or terminal differentiation state. This balancing in turn affects the functional capacity of the T cells and enriches the number of cells with senescent and apoptotic characteristics. Thus, it has been demonstrated the existence of an exhaustion process that affects key populations for the parasite control. However, the role of this process in the progression of the severity of these pathologies is still unknown. The current drugs used to treat these neglected diseases seem to partially reverse this exhaustion process, denoting a reduction in the high inhibitory receptor expression observed prior to chemotherapies. An improvement in the functional capacity of these T cell populations is also observed, which could be related to the reversion of the dysfunctional process. However, the efforts made to date to evaluate blocking therapies do not lead us to a promising conclusion. It will probably be necessary to test the simultaneous blockade of several pathways and to continue advancing the knowledge to verify their possible use as immunotherapy. It is therefore necessary to continue investigating how this process is triggered and to what extent it influences the appearance of the symptomatology of patients

La enfermedad de Chagas y la leishmaniasis, causadas por parásitos protozoarios intracelulares del orden Trypanosomatida, son consideradas dos de las enfermedades tropicales desatendidas más importantes. Estas infecciones conllevan un alto desgaste social, provocando el deterioro de la salud de un gran número de pacientes e incluso su muerte. Los linfocitos T son células fundamentales del sistema adaptativo y son los principales elementos inmunitarios para el control de estas infecciones intracelulares. La presente revisión explora los estudios y resultados obtenidos hasta la fecha del proceso de agotamiento celular durante las infecciones causadas por los parásitos Leishmania spp. y Trypanosoma cruzi. Así, se recoge que la persistente estimulación celular en respuesta a antígenos de estos patógenos conduce a un proceso de pérdida de la capacidad funcional antígeno-específica en las poblaciones de células T CD4+ y CD8+. Numerosos estudios muestran la existencia de una correlación directa entre el nivel de la co-expresión de receptores inhibitorios y la gravedad de estas patologías. Paralelamente, se detecta la pérdida de la capacidad funcional específica de antígeno de estas células T, lo que reduce su potencial linfoproliferativo y su capacidad de producir moléculas protectoras contra estas infecciones. Además, durante el curso de estas infecciones se observa un incremento de la frecuencia de células T de memoria efectora con un grado de diferenciación tardía o terminal. Este balanceo fenotípico, a su vez, afecta a la capacidad funcional de las células T aumentando el número de células con características senescentes y apoptóticas. Así, los estudios realizados hasta la fecha demuestran con certeza la existencia de un proceso de agotamiento que afecta a poblaciones clave para el control parasitario. Sin embargo, actualmente se desconoce con precisión el papel que este proceso de agotamiento juega en el agravamiento de estas patologías. Los medicamentos actuales usados para tratar estas enfermedades protozoarias revierten parcialmente este proceso de agotamiento. Así, tras el tratamiento, numerosos pacientes muestra una reducción en la expresión de receptores inhibitorios y co-expresión de los mismos. También se ha observado una mejoría en la capacidad funcional de las distintas poblaciones de células T, que podría estar relacionada con la reversión del proceso disfuncional.Sin embargo, los estudios realizados hasta la fecha en la evaluación de terapias de bloqueo de los receptores inhibitorios no han conducido a resultados prometedores. Algunos autores proponen evaluar terapias de bloqueo simultáneo de varias vías de señalización, con el fin de ampliar el conocimiento sobre esta herramienta como posible inmunoterapia de control de la infección por los mencionados parásitos. Además, se considera necesario continuar investigando sobre cómo se desencadena exactamente este proceso de agotamiento celular y en qué medida influye en la aparición de la sintomatología de los pacientes y ausencia de control de la infección

Trypanosoma brucei gambiense excreted/secreted factors impair lipopolysaccharide-induced maturation and activation of human monocyte-derived dendritic cells.

Trypanosoma brucei gambiense, an extracellular eukaryotic flagellate parasite, is the main etiological agent of human African trypanosomiasis (HAT) or sleeping sickness. Dendritic cells (DCs) play a pivotal role at the interface between innate and adaptive immune response and are implicated during HAT. In this study, we investigated the effects of T gambiense and its excreted/secreted factors (ESF) on the phenotype of human monocyte-derived DCs (Mo-DCs). Mo-DCs were cultured with trypanosomes, lipopolysaccharide (LPS), ESF derived from T gambiense bloodstream strain Biyamina (MHOM/SD/82), or both ESF and LPS. Importantly, ESF reduced the expression of the maturation markers HLA-DR and CD83, as well as the secretion of IL-12, TNF-alpha and IL-10, in LPS-stimulated Mo-DCs. During mixed-leucocyte reactions, LPS- plus ESF-exposed DCs induced a non-significant decrease in the IFN-gamma/IL-10 ratio of CD4 + T-cell cytokines. Based on the results presented here, we raise the hypothesis that T gambiense has developed an immune escape strategy through the secretion of paracrine mediators in order to limit maturation and activation of human DCs. The identification of the factor(s) in the T gambiense ESF and of the DCs signalling pathway(s) involved may be important in the development of new therapeutic targets.

An Animal Model of Acute and Chronic Chagas Disease With the Reticulotropic Y Strain of Trypanosoma cruzi That Depicts the Multifunctionality and Dysfunctionality of T Cells.

Chagas disease (ChD), a complex and persistent parasitosis caused by Trypanosoma cruzi, represents a natural model of chronic infection, in which some people exhibit cardiac or digestive complications that can result in death 20-40 years after the initial infection. Nonetheless, due to unknown mechanisms, some T. cruzi-infected individuals remain asymptomatic throughout their lives. Actually, no vaccine is available to prevent ChD, and treatments for chronic ChD patients are controversial. Chronically T. cruzi-infected individuals exhibit a deterioration of T cell function, an exhaustion state characterized by poor cytokine production and increased inhibitory receptor co-expression, suggesting that these changes are potentially related to ChD progression. Moreover, an effective anti-parasitic treatment appears to reverse this state and improve the T cell response. Taking into account these findings, the functionality state of T cells might provide a potential correlate of protection to detect individuals who will or will not develop the severe forms of ChD. Consequently, we investigated the T cell response, analyzed by flow cytometry with two multicolor immunofluorescence panels, to assess cytokines/cytotoxic molecules and the expression of inhibitory receptors, in a murine model of acute (10 and 30 days) and chronic (100 and 260 days) ChD, characterized by parasite persistence for up to 260 days post-infection and moderate inflammation of the colon and liver of T. cruzi-infected mice. Acute ChD induced a high antigen-specific multifunctional T cell response by producing IFN-γ, TNF-α, IL-2, granzyme B, and perforin; and a high frequency of T cells co-expressed 2B4, CD160, CTLA-4, and PD-1. In contrast, chronically infected mice with moderate inflammatory infiltrate in liver tissue exhibited monofunctional antigen-specific cells, high cytotoxic activity (granzyme B and perforin), and elevated levels of inhibitory receptors (predominantly CTLA-4 and PD-1) co-expressed on T cells. Taken together, these data support our previous results showing that similar to humans, the T. cruzi persistence in mice promotes the dysfunctionality of T cells, and these changes might correlate with ChD progression. Thus, these results constitute a model that will facilitate an in-depth search for immune markers and correlates of protection, as well as long-term studies of new immunotherapy strategies for ChD.

Trypanosoma cruzi-Specific T-Cell Responses to Monitor Treatment Efficacy in Chronic Chagas Disease.

Chagas disease is the highest impact parasitic disease in Latin America. In recent years, the use of immune-related biomarkers to predict diagnostic and treatment efficacy or to monitor diseases has been considered a promising tool. Our group has worked for the past 20 years on the characterization of different immunological aspects of the T-cell responses to T. cruzi antigens. We have shown that monitoring of appropriate immunological responses can provide earlier and robust measures of treatment.The Enzyme-Linked ImmunoSPOT (ELISPOT) assays are powerful tools to evaluate antigen-specific immune responses at the single-cell level. Herein, we describe uses of the ELISPOT assay to determine the T. cruzi-specific T-cell populations in PBMCs from chronic chagasic subjects.

Histamine releasing factor and elongation factor 1 alpha secreted via malaria parasites extracellular vesicles promote immune evasion by inhibiting specific T cell responses.

Protozoan pathogens secrete nanosized particles called extracellular vesicles (EVs) to facilitate their survival and chronic infection. Here, we show the inhibition by Plasmodium berghei NK65 blood stage-derived EVs of the proliferative response of CD4+ T cells in response to antigen presentation. Importantly, these results were confirmed in vivo by the capacity of EVs to diminish the ovalbumin-specific delayed type hypersensitivity response. We identified two proteins associated with EVs, the histamine releasing factor (HRF) and the elongation factor 1α (EF-1α) that were found to have immunosuppressive activities. Interestingly, in contrast to WT parasites, EVs from genetically HRF- and EF-1α-deficient parasites failed to inhibit T cell responses in vitro and in vivo. At the level of T cells, we demonstrated that EVs from WT parasites dephosphorylate key molecules (PLCγ1, Akt, and ERK) of the T cell receptor signalling cascade. Remarkably, immunisation with EF-1α alone or in combination with HRF conferred a long-lasting antiparasite protection and immune memory. In conclusion, we identified a new mechanism by which P. berghei-derived EVs exert their immunosuppressive functions by altering T cell responses. The identification of two highly conserved immune suppressive factors offers new conceptual strategies to overcome EV-mediated immune suppression in malaria-infected individuals.

Cytomegalovirus vectors expressing Plasmodium knowlesi antigens induce immune responses that delay parasitemia upon sporozoite challenge.

The development of a sterilizing vaccine against malaria remains one of the highest priorities for global health research. While sporozoite vaccines targeting the pre-erythrocytic stage show great promise, it has not been possible to maintain efficacy long-term, likely due to an inability of these vaccines to maintain effector memory T cell responses in the liver. Vaccines based on human cytomegalovirus (HCMV) might overcome this limitation since vectors based on rhesus CMV (RhCMV), the homologous virus in rhesus macaques (RM), elicit and indefinitely maintain high frequency, non-exhausted effector memory T cells in extralymphoid tissues, including the liver. Moreover, RhCMV strain 68-1 elicits CD8+ T cells broadly recognizing unconventional epitopes exclusively restricted by MHC-II and MHC-E. To evaluate the potential of these unique immune responses to protect against malaria, we expressed four Plasmodium knowlesi (Pk) antigens (CSP, AMA1, SSP2/TRAP, MSP1c) in RhCMV 68-1 or in Rh189-deleted 68-1, which additionally elicits canonical MHC-Ia-restricted CD8+ T cells. Upon inoculation of RM with either of these Pk Ag expressing RhCMV vaccines, we obtained T cell responses to each of the four Pk antigens. Upon challenge with Pk sporozoites we observed a delayed appearance of blood stage parasites in vaccinated RM consistent with a 75-80% reduction of parasite release from the liver. Moreover, the Rh189-deleted RhCMV/Pk vectors elicited sterile protection in one RM. Once in the blood, parasite growth was not affected. In contrast to T cell responses induced by Pk infection, RhCMV vectors maintained sustained T cell responses to all four malaria antigens in the liver post-challenge. The delayed appearance of blood stage parasites is thus likely due to a T cell-mediated inhibition of liver stage parasite development. As such, this vaccine approach can be used to efficiently test new T cell antigens, improve current vaccines targeting the liver stage and complement vaccines targeting erythrocytic antigens.